Laser-Synthesized Bioconjugated Noble Metal Nanoparticles Rational Design and Efficacy against Pathological Protein Aggregation

Transcription

1 Laser-Synthesized Bioconjugated Noble Metal Nanoparticles Rational Design and Efficacy against Pathological Protein Aggregation Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften Dr. rer. nat. vorgelegt von Carmen Streich geboren in Essen Institut für Technische Chemie I der Universität Duisburg-Essen 217

2 Die vorliegende Arbeit wurde im Zeitraum von Januar 214 bis Januar 217 im Arbeitskreis von Prof. Dr.-Ing. Stephan Barcikowski am Institut für Technische Chemie I in Kooperation mit dem Arbeitskreis von Prof. Dr. Thomas Schrader am Institut für Organische Chemie der Universität Duisburg-Essen durchgeführt. Tag der Disputation: Gutachter: Vorsitzende: Prof. Dr.-Ing. Stephan Barcikowski, UDE Prof. Dr. Thomas Schrader, UDE Prof. Dr. Carsten Korth, Heinrich-Heine-Universität Düsseldorf Prof. Dr. Karin Stachelscheid

3 We absolutely must leave room for doubt or there is no progress and there is no learning. There is no learning without having to pose a question. And a question requires doubt. Richard P. Feynman ( )

4 Table of Contents 1. Introduction and Objectives of the Thesis 1 2. Theoretical Background Nanobiomedicine Nanoconjugates for Active Targeting and Multivalent Effects Clinical Nanomedicine Colloidal Gold Nanoparticles Nanoparticle Properties Synthesis of Colloidal Gold Nanoparticles via Pulsed Laser Ablation in Liquids Special Properties of Laser-Generated Colloids and their Bioapplications Tailored Nanoparticle Surface Modifications via Thiolated Ligands In vivo Requirements, Bioresponse and Biodistribution The Role of the Nanoparticle Protein Corona and Properties of Selected Model Serum Proteins Protein Misfolding and Neurodegenerative Diseases Morbus Alzheimer and the Amyloid Cascade Hypothesis Therapies Based on the Amyloid Cascade Hypothesis Small Molecules Targeting Aβ Anti-Amyloidogenic Effects of Nanoparticles Material and Methods Nanoparticle Synthesis and Physicochemical Characterization UV/Vis Extinction Spectrophotometry Analytical Disc Centrifugation (ADC) Analytical Ultracentrifugation (AUC) Transmission Electron Microscopy (TEM) Dynamic Light Scattering (DLS) Zeta Potential Measurements Ligand Conjugation and Characterization of Nanoparticle/Ligand Conjugates Fluorescence Spectrometry Affinity Experiments Gel Electrophoresis Characterization of Aβ aggregation states Preparation of Aβ Circular Dichroism (CD) Spectroscopy Thioflavin T (ThT) Fluorescence Assay Atomic Force Microscopy (AFM) of Aβ Fibrils Transmission Electron Microscopy of Aβ Fibrils Density Gradient Centrifugation (DGC) and Enzyme-Linked Immunosorbent Assay (ELISA) Cellular Aβ Assay Light and Confocal Laser Scanning Microscopy (CLSM) Results and Discussion 8

5 4.1 Nanoparticle Characterization Particle Size Particle Stability Interaction with Small Thiolated Ligands and Serum Proteins Characterization of D3- and Aminopyrazole Trimer-Nanobioconjugates Qualitative Detection of D3 and Aminopyrazole Trimer Binding Nanobioconjugate Stability Stabilization of Nanobioconjugates with Small Thiolated Ligands and BSA Ligand Density on the Nanoparticle Surface Interaction of Nanobioconjugates with Serum Proteins and Aβ Functionality of the Designed Nanobioconjugates Affinity of Nanobioconjugates towards Aβ as Determined from Fluorescence Quenching Influence of Nanobioconjugates on Aβ Secondary Structure Microscopic Analysis of the Effect of Nanobioconjugates on Synthetic Aβ Immunologic Analysis of the Effect of Nanobioconjugates on Synthetic Aβ Effect of Nanobioconjugates on the Cellular Excretion of Aβ Preliminary Summary of the Nanobioconjugate s Interactions with Aβ Outlook Summary References Appendix Lists of Physical Parameters and Abbreviations Supplementary Information, Figures and Tables Curriculum Vitae List of Authored and Coauthored Publications Declarations Danksagung 279

6 1. Introduction and Objectives of the Thesis 1 1. Introduction and Objectives of the Thesis Incurable neurodegenerative diseases such as Alzheimer s disease, Parkinson s disease, Huntington s disease and amyotrophic lateral sclerosis are characterized by various symptoms including impairment of motion, memory, cognition, abstract thinking and emotional feelings. 1 Although these diseases are symptomatically diverse, they share a common feature, which is the misfolding, aggregation and accumulation of certain proteins in the brain. 2 Clinically approved drugs can only help to relieve the symptoms and/or moderately modulate the pathogenesis. However, they fail to stop or reverse the disease progression. Thus, targeting protein aggregates and interfering with their formation processes could represent a new therapeutic strategy for treating certain neurodegenerative diseases. 3 One of the most popular representatives for neurodegenerative diseases is Alzheimer s disease. It is the most common cause of dementia, with an estimate of 46.8 million people affected worldwide, projected to almost double every 2 years, and one new case every 3.2 seconds. 4,5 A multifactorial interplay between different molecular, cellular and genetic imbalances may cause AD. 6 According to the so-called amyloid cascade hypothesis, 7 misfolding, aggregation and accumulation of the amino acid containing amyloid β peptide (Aβ) in the brain is considered to play a central role. Especially, the formation of soluble Aβ oligomers 8,9 and β-sheet rich, insoluble amyloid fibrils 1 may contribute to synaptic dysfunction and neuronal loss. Hence, the molecular interference with aggregated Aβ species could be a novel, disease-modifying therapeutic approach. 11,12 Current research focuses on the identification of highly specific Aβ-antibodies for targeting amyloid aggregates. 13 However, drawbacks of antibodies include their high production costs, potential immunogenicity and high molecular weight, which may prevent crossing of biological barriers such as the blood-brain barrier. 14 Hence, previous works have identified different molecules of lower molecular weight for targeting amyloid aggregates. 15 These include the D-enantiomeric peptide D3 16 and the rationally designed aminopyrazole. 17 Variants of these molecules have shown inhibitory effects on Aβ aggregation in vitro 18 and in vivo. 16,19 Research activities have thus focused on identifying their mode of action. In particular, the D3 peptide has been shown to bind Aβ with micromolar affinity and convert it into nontoxic, amorphous aggregates. 16 Aminopyrazole compounds have been shown to intercalate and break the β-sheet structures typical for Aβ fibrils. 2 Moreover, research

7 1. Introduction and Objectives of the Thesis 2 activities aimed at increasing the compounds efficacies via chemical modifications. For example, aminopyrazole-d3 hybrid compounds with an improved performance and new functionalities could be synthesized via complex chemical linkages. 18 Furthermore, a tandem D3D3 dimer was recently developed. 21 In nanomedicine, new applications for diagnosis, treatment, monitoring and prevention of diseases are being developed based on nanotechnology Since nanoparticles feature sizes similar to the dimensions of biomolecules and biological structures, they are ideally suited to interfere with biological processes on the (sub)cellular level. In particular, the immobilization of therapeutically active agents on nanoparticle surfaces allows a new pharmaceutical formulation of already existing or approved drugs in the form of nanoconjugates. 25 These nanoconjugates may increase the drug s solubility, bioavailability and stability, or faciliate the crossing of biological barriers. 26 Moreover, synergistic effects via ligandligand interaction can result from the immobilization of multiple ligands in close proximity on the particle s surface. 27 These include increased specificity and enhanced target binding. 28 For biomedical applications colloidal gold nanoparticles (AuNPs) are especially well suited since they offer numerous advantageous properties, 29,3 such as a high bioinertness and low toxicity for particle diameters > 2 nm. 31 In addition, they feature special optical properties due to their surface plasmon resonance. 29,3 Moreover, their surface is easily modifiable through the establishment of covalent gold-thiol bonds. 32 With regard to NP synthesis, chemical synthesis routes are scalable and can be carried out in a controlled way with regard to particle size, shape and surface chemistry. 33,34 As chemical AuNP synthesis always involves the reduction of metal salts 35,36 and stabilization with ligands, residual chemical precursors and surface-attached ligands may impede biocompatibility 37 and functionality 38,39 later on. Moreover, their removal is time-consuming and e.g. in the case of citrate not quantitatively possible. 4 To overcome these limitations, pulsed laser ablation in liquids (PLAL), as alternative NP synthesis route, can be employed. 41,42 Since a pulsed laser beam irradiates a bulk gold target immersed in aqueous solution, 43 the resulting colloidal particles can be obtained intrinsically free of ligands and electrostatically stabilized due to a partially oxidized surface. 44 Previous studies concentrated on the combination of the laser-based NP synthesis and bioconjugation in a single step (in situ conjugation 45 ). For this, the process parameters had to

8 1. Introduction and Objectives of the Thesis 3 be optimized to increase productivity and guarantee ligand integrity. Subsequently, the functionalization of AuNP with two or more ligands for a range of applications including cellular uptake, cytotoxicity and immunolabeling could be shown. 46 Moreover, the transferability of the conjugation method to iron-based and silica NP core material has been successfully demonstrated. 46 In this study, NP synthesis and conjugation with ligands are spatially and temporally separated (ex situ conjugation). 41 Particles synthesized via this route are ideally suited starting materials to rationally design nanoconjugates with tailored surface modifications. 47 By employing ligand-free particles, ligand densities can be precisely adjusted from sub-monolayer to monolayer coverages to create a library of nanoconjugates. 48 Especially, multivalent, homo- and heterofunctional nanoconjugates can be systematically fabricated by varying the ligand type and number per nanoparticle. 49,5 Furthermore, the approach allows the direct comparison of (biological) performances of nanoconjugates with that of bare NPs and free ligands. Thereby, synergistic effects of nanoconjugates arising from multivalent inter-ligand interactions can be identified. 27,51 Moreover, the fabrication of Pt-based conjugates will be addressed, for which only a limited number of bioapplications has been reported thus far. 52,53 The present work combines the cross-disciplinary expertise from the fields of technical chemistry, colloid chemistry, organic chemistry and cell biology for the design, synthesis and functional evaluation of nanobioconjugates based on laser-generated, colloidal metal nanoparticles and tailored D3 and aminopyrazole trimer ligands. To get a deeper insight into the structure-function relationship of nanoconjugates, the model system of Aβ misfolding and aggregation is employed. In this regard, it will be addressed how nanoconjugates interfere with neurotoxic Aβ aggregates. Until now, most studies on NP-based targeting of misfolded Aβ are limited with regard to the variability of the nanoconjugate design and/or physicochemical functional readouts. Especially, how nanoconjugates could interfere with Aβ-induced toxicity on cells has only been addressed by few studies. The aim for this thesis was therefore to provide a systematic study addressing the entire process chain, starting with ligand-free particles for tailored nanoconjugate design and synthesis, followed by the comprehensive characterization of the conjugates performances. The focus was laid on the fabrication of different, homo- and heterovalent nanoconjugates, the inclusion of relevant reference samples (bare nanoparticles,

9 1. Introduction and Objectives of the Thesis 4 free ligands) and the conduction of complementary functional readouts, ranging from physicochemical to cell-based assays (Figure 1-1). Laser A) Nanoparticle Fabrication Aqueous solution Nanoparticles Bulk metal 1 nm S - S LA Insulin B) Conjugation with Ligands Stabilizing ligands Aβ-targeting ligands S Aminopyrazole Trimer MUA Transferrin S mpeg-sh S BSA Ligand-free nanoparticle D3 Peptide Bifunctional conjugates 1:1 1:1 1:1 C) Functional assays physicochemical cell-based Ligand charge Specificity Aβ mono Aβ oligo Aβ fibril Ligand density and conjugate charge R5WC PtNP Anionic Neutral Cationic Figure 1-1: Schematic overview of aspects addressed in this thesis, including nanoparticle synthesis via laser ablation (top), conjugation with different ligands (middle) and analysis of the structure-function relationship of nanobioconjugates on the example of the Aβ aggregation process (bottom). Nanoparticles are conjugated to increase their stability (serum proteins or small thiolated ligands) or to equip them with a specific functionality (D3 or aminopyrazole trimer). The ability of differently designed conjugates to interfere with Aβ aggregates is examined via physicochemical techniques and a cell-based assay (LA: lipoic acid, MUA: mercaptoundecanoic acid, mpeg-sh: thiolated polyethylene glycol, BSA: bovine serum albumin). The thesis is divided into three main chapters: At first, the synthesis and characterization of ligand-free colloidal AuNPs will be described (A). Secondly, the design of nanoconjugates will be outlined. The interaction of laser-generated AuNPs with selected small thiolated

10 1. Introduction and Objectives of the Thesis 5 ligands and model serum proteins will be analyzed. On the one hand, basic parameters such as particle size, charge and the ligand concentration, necessary for stabilizing laser-generated NPs against stress conditions (e.g. high ionic strength media) will be determined. This will result in a thorough characterization of the colloidal properties, whereby the value of lasergenerated particles as ligand-free reference material will be emphasized. On the other hand, the design of these conjugates is employed to create reference samples for subsequent tests, as they constitute unspecific, non-aβ targeting particles (B, left). Furthermore, the focus will be laid on the qualitative and quantitative evaluation of the NP surface functionalization with the Aβ-targeting ligands D3 and aminopyrazole trimer. Particle stability is a prerequisite to enable the reproducible processing of the samples in subsequent functionality assays. Hence, special focus will be laid on analyzing the nanobioconjugates stabilities per se and different ways of post-treatments, to increase the colloidal long-term stability afterwards. In this context, the nanobioconjugates interactions with model serum proteins will be analyzed. In cell experiments and in vivo scenarios, the conjugates would be exposed to a complex matrix of biomacromolecules before reaching their actual target in the brain, the Aβ peptide. Thus, the analysis of conjugate/protein interactions is important to give a first indication of the conjugates activity, selectivity and specificity (B, right). While the first two chapters contain important general results on nanoparticle-ligand interactions with respect to bioapplications, the last chapter especially focuses on the characterization of the conjugates interference with Aβ aggregation (C). Different aspects related to the design of the conjugate (e.g. ligand type, ligand loading, homo-/heterofunctional surface coatings) and the type of Aβ species targeted (e.g. monomer/oligomer/fibril, synthetic/natural origin) will be addressed. The interference with synthetic Aβ species is analyzed via different physicochemical methods. Furthermore, an Aβ-cell model enables to evaluate the interaction with naturally secreted Aβ species. A mechanism for NP-induced interference with amyloid species will be proposed. Finally, theoretical considerations which are important for a potential translation into a pharmaceutical formulation of the nanoconjugate are discussed.

11 Number of institutions 2. Theoretical Background 6 2. Theoretical Background 2.1 Nanobiomedicine Nanotechnology, as one of the most promising key enabling technologies in the 21st century, makes use of the unusual physical, chemical, and biological properties of nanoscale materials, which differ significantly from the properties of bulk materials as well as single atoms. Applications employing engineered nanomaterials can already be found in everyday life, 57,58 such as titanium dioxide 59 in sunscreen, antimicrobial silver 6,61,62 in clothing and cosmetics, or gold 63 in pregnancy tests. Research on nanotechnology addresses different areas of application ranging from energy 64 and electronics 65 to the environment 66, nutrition 67, health 68 and medical sector 23 (Figure 2-1) Medicine & Pharma Electronics Energy Chemistry Optics Environment Transport Consumer Products Nutrition Areas involving nanotechnology Figure 2-1: Forecast of the areas of applications which may be impacted the most by nanotechnology in Germany until 223 (adapted from Ref. 57 ). Image references (accessed on ): (medicine), (electronics), (energy), (chemistry), (optics), (environment), (transport), (consumer products), (nutrition). Nanobiomedicine addresses applications for diagnosis, therapy, implantable materials and tissue regeneration by employing nanomaterials. 69 In this regard, a nanomaterial is defined as material containing particles (5% or more of the number size distribution) with any external dimensions in the nanoscale or having internal structure or surface structure in the nanoscale, i.e. between 1 nm and 1 nm. 7 Due to the similar size scales of nanomaterials and biological entities such as proteins, DNA and viruses, nanoscale structures can intrinsically be used to target physiological processes within cells or tissues (Figure 2-2). 71

12 2. Theoretical Background 7 Figure 2-2: Size scales of nanomaterials and biological entities. 71 Copyright 21 by John Wiley Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc. DOI: 1.12/anie In the field of diagnosis, nanotechnology based tools are developed in order to achieve high precision, reliability and sensitivity, e.g. for imaging applications 72 and immunoassays. 73 Therapeutic nanomedical products can be classified according to their underlying mode of action: specific surface effects (e.g. implant-tissue interaction) can be induced with a nanostructure on a medical implant; volume effects (e.g. drug release) can be induced if nanoparticles are embedded in a polymer to result in a functional composite; the nanoparticle itself can be utilized as functional unit if it is decorated with therapeutically active ligands Nanoconjugates for Active Targeting and Multivalent Effects The principle of formulating drugs as nanoparticle-ligand-conjugates for drug delivery is also employed in this work and will therefore be described in more detail. Generally, therapeutic molecules can be immobilized on nanoparticle (NP) surfaces to increase their therapeutic efficacy. 74 Different effects such as high local drug concentrations, increased bioavailability (e.g. by improved solubility), reduced immunogenicity, effective crossing of biological barriers, and controlled drug release can be achieved. 75 Nanoconjugates can either be homofunctional (same type of ligand) or heterofunctional, when combinations of different biomolecules are immobilized on the particle surface to generate multifunctional conjugates (Figure 2-3). 76

13 2. Theoretical Background 8 Figure 2-3: Surface-modification of nanoparticles with different ligands, generating functional conjugates. For example, RNA and DNA can be used for genetic manipulation. Polyethylene glycol (PEG) can increase NP stability, solubility and decrease immunogenicity. Peptides, carbohydrates and other substances can render the nanocarrier therapeutically active. Dyes allow particle tracking and/or optical sensing. Aptamers and antibodies can selectively target specific receptors (left, adapted from Ref. 77, DOI /fchem ). Principle of mono- and multivalent binding (right, Copyright 212 by John 51 Wiley Sons, Inc. adapted from Ref. by permission of John Wiley & Sons, Inc., DOI: 1.12/anie ). The simultaneous interaction of multiple complementary ligands immobilized on the NP surface can increase weak, individual interactions with receptors. Active targeting is a central principle which can be realized with drug delivering nanoconjugates. The principle of active targeting lies is the specific recognition of a target s site of action and the selective enrichment of the NP in one location. For this, NPs require surface modification with ligands which allow strong binding, i.e. high affinity for their complementary receptors. In general, active targeting is realized by decorating functional small molecules, 78 peptides, 79,8 nucleic acids, 81,82 aptamers, 83 or antibodies 84 to the nanoparticle surface for molecular recognition of the target (Figure 2-3, left). However, very specific biomolecules with high affinities such as antibodies and aptamers can sometimes not be employed for NP functionalization due to their cost-expensive synthesis, large size, immunogenicity or chemical instability. In this case, the establishment of high local concentrations of small molecules on the NP surface represents an alternative route for active targeting. Thereby, the lack of affinity of each individual ligand is compensated through the close proximity of multiple ligands on the NP surface, which allows simultaneous binding of multiple ligands to receptors (multivalency, Figure 2-3 right). This results in a collective functional affinity (avidity) through increased binding strengths and cooperative effects. 28 Different examples for the establishment of multiple simultaneous interactions with unique collective properties that are qualitatively different from properties of their monovalent constituents can be found. For example, the influenza virus attaches with multiple

14 2. Theoretical Background 9 hemaglutinin residues to sialic acids residues on the surface of a bronchial epithelial cell. 28 With regard to NP induced avidities, Tassa and colleagues characterized a library of iron oxide nanoparticles conjugated with small molecules in view of their affinity for a single protein target. They reported that weakly binding ligands show significantly enhanced targetspecific affinity when they are immobilized on the nanoparticle surface (up to four orders of magnitude enhancement), which was attributed to multivalent interactions. 78 Hong et al. studied the avidity of folate-coated dendrimers targeting the folate-receptor overexpressed in epithelial cancer cells. They showed that the binding strength could be drastically increased up to 17,-fold as compared to the free compound, by increasing the number of folate molecules bound per dendrimer. 27 Further examples of NP-induced avidity will be given in chapter Generally one can expect that high ligand densities improve the binding capability to receptors, because multiple ligands can interact with numerous target receptors simultaneously. 85 Obviously, the binding sites of the ligands need to be exposed to the target to enable interaction. Hence, dense ligand packing on the nanoparticle surface can also result in reduced binding affinities, if the ligands have low flexibilities or sterically shield each. 86 The careful adjustment of the ligand load per NP and/or the design of spacer molecules (between the NP surface and the functional ligand) may thus be required to optimize ligand disposition for effective target binding Clinical Nanomedicine More than 2 nanomedical products are approved or in various stages of clinical study, including drug delivery systems consisting of organic and inorganic nanomaterials for cancer therapy (Figure 2-4). 69 The first generation, marketed nanomedicines Doxil and Abraxane are based on passive targeting. In contrast to active targeting e.g. via specific ligand-receptor interactions, passive targeting relies on the NP s accumulation in the tumor tissue due to an enhanced permeability and retention in the tumor vs. healthy tissue. 88 Furthermore, the efficiency of passive targeting can be increased by increasing the drug s circulation time. 88 In Doxil, the chemotherapeutic drug Doxorubicin is encapsulated in PEGylated liposomes (PEG, polyethylene glycol). The nanoparticle formulation improves circulation half-life and accumulation in tumor. In Abraxane, the chemotherapeutic drug Paclitaxel is bound to albumin nanoparticles, which enhances solubility. 89 Noteworthy, none of the approved nanoparticulate drugs employs active targeting yet, 9 but some are being clinically investigated. 9 Actively targeting polymeric nanoparticles (BIND-14) were tested in Phase I

15 2. Theoretical Background 1 and II clinical trials. They are composed of a biodegradable and hydrophobic poly-d,l-lactide (PLA) polymeric core and a hydrophilic PEG corona. Their surface is decorated with a peptide targeting a prostate-specific membrane antigen, while the chemotherapeutic drug is encapsulated. 91 Recent results indicate a high tolerability, clinical activity against different tumors and prolonged circulation time of BIND-14 compared to the free chemotherapeutic agent. 92 The results of four other studies employing BIND-14 were completed in 216, but remain to be published (NCT , NCT , NCT228332, NCT , Figure 2-4: Number of EU approved and investigational nanomedical products sorted by their area of application, as of 215. Reproduced from Ref. 22 with permission of Future Medicine Ltd. DOI: /nnm The Nanotherm therapy (by MagForce AG, Berlin, Germany) is an example for a nanotherapeutic treatment of brain tumors, based on inorganic NPs. It was marketed in 211, is approved also in the EU and has ongoing clinical trials. Here, 12 nm superparamagnetic iron oxide nanoparticles coated with aminosilanes are injected into the tumor at a dose of 34 mg/cm³ target tissue. The stimulation by an alternating magnetic field destroys tumor cells through hyperthermia. 93 Wirth regard to gold nanoparticles (AuNPs), colloidal gold was injected in the joints of patients for the treatment of rheumatoid arthritis. The study of Abraham and Himmel from 1997 reports positive effect on joints and no evidence of toxicity in any patient at 3 mg dose, 94 but follow-up studies were not conducted since then. The CYT-691/AurImmune therapy (by CytImmune Science, Rockville, Maryland, USA) consists of 27 nm AuNPs decorated with thiolated PEG for stabilization and tumor necrosis factor (TNF) for the

16 2. Theoretical Background 11 treatment of solid tumors after systemic administration. 95 A phase I clinical trial was successful in showing that a dosage of 1 mg TNF per treatment (which was toxic as free drug) was non-toxic as nanoformulation. 96 The applied NP dose was not stated in the respective publications. However, an AuNP dose of 12 mg per treatment can be recalculated from the dose of TNF, assuming molecular weights of 26 kda (TNF), 1.2x1 8 g/mol (AuNP, 27 nm) and a TNF density of 4 molecules per NP. 95 AuroLase (by Nanospectra Bioscience Inc., Houston, Texas, USA) 97 is a cancer therapy consisting of silica-gold nanoshells which passively accumulate in the tumor after intravenous administration. They are subsequently irradiated with a near-infrared (NIR) laser for photothermal cancer treatment. Clinical trials on lung (NCT167947), head and neck cancer (NCT84842, have been completed, but results have not been published yet. Similarly, silica-gold NPs have been employed for the treatment of coronary atherosclerosis in a phase I/II clinical trial. 98 This therapy is based on the photothermal destruction of coronary plaques in combination with stem cell treatment for functional restoration of the vessel wall. 2.2 Colloidal Gold Nanoparticles The above examples show that AuNPs were already clinically approved and are applied in clinical studies. They offer numerous properties which make them ideally suited materials for biomedical applications. These include their oxidation resistance, resulting high bioinertness, and low acute toxicity (at diameters > 2 nm). Moreover, they can be synthesized in a controlled way with regard to their size, shape and surface chemistry, and this synthesis is scalable. In addition, they feature special optical properties, give high contrast and high photostability in imaging applications. The possibility to quantify AuNPs via analytical techniques such as inductively coupled plasma mass spectrometry and UV/Vis spectrophotometry enables the facile detection at the biological target location. 29, Nanoparticle Properties Colloidal Stability of Nanoparticles Owing to their high surface to volume ratio, nanoparticles feature high surface energies. Hence, colloidal particles are per se thermodynamically unstable systems and tend to form energetically more favorable assemblies with each other. The Derjaguin-Landau-Verwey-

17 2. Theoretical Background 12 Overbeek (DLVO) theory describes the electrostatic stability of nanoparticle dispersions. 99,1 Different forces including van der Waals forces (attraction via fluctuating dipoles), Born repulsion and Coulomb forces (repulsion of electric double layers) exist as a function of the interparticle distance. When two particles approach each other, double layer repulsion predominates at large distances and stabilizes the particles. In contrast, van der Waals attraction prevails at small distances. A secondary minimum in stability exists at intermediate interparticle distances, so that reversible agglomeration can take place. Upon further approximation of two particles, the potential reaches a primary minimum and irreversible aggregation occurs (Figure 2-5). To maintain stable colloids, the interparticle distance should thus be sufficiently large. 11 Practically, particle stabilization can be supported via surface modifications with charged or bulky ligands for electrosteric or steric stabilization, respectively. 12 Alternatively, pure electrostatic NP stabilization can be achieved via the PLAL process. Here, anions adsorb to the NP surface, resulting in AuO - species (ablation in water). 44 Additionally, the NP charge density may be increased in low salinity solutions (e.g. ablation in NaBr, NaCl solution). 13 Figure 2-5: Attractive and repulsive forces as function of the interparticle distances. The combined interaction curve (G T ) with a primary and secondary minimum results from the combination of electrostatic repulsion (G R ), van der Waals attraction (G A ) and Born repulsion (G B, left, with permission of Springer). 11 Electric double layer formation around a colloidal nanoparticle and characteristic potentials related to their distance from the particle surface (right adapted from Ref. 14 ) In solution, electrochemical double layers form around the dispersed particles, which contribute to their stability and apparent charge. They can be described by combining the Helmholtz and Gouy-Chapman models into the Stern double layer model. 15 On the one hand,

18 2. Theoretical Background 13 the Helmholtz layer describes the rigid surface adsorption of electrolyte counter ions compensating the charge of the metal surface, with a linearly decaying concentration gradient from the charged surface into the solution. On the other hand, the Gouy-Chapman model describes the presence of a diffuse layer of electrolyte ions, with an exponentially decaying concentration gradient from the charged surface into the solution. These ions are loosely associated and move under the influence of electric attraction and thermal motion. A measure of the double layer thickness is the Debye length κ -1. It depends on the system s temperature, the solvent dielectric constant and the ionic strength. For 1:1 electrolytes and room temperature, it can be calculated via κ -1 [nm] =.34/(I [mol/l]) 1/2. 16 For example, NPs can be produced in low salinity liquids via pulsed laser ablation (e.g. I = 1 µm NaCl, correlating to a double layer thickness of κ -1 = 3 nm). Notably, the transfer to a biological medium with high ionic strength may strongly reduce their double layer thickness, leading to destabilization of the particles, if no steric stabilizers are present (e.g. κ -1 =.7 nm at I = 2 mm). 41 The zeta potential ζ is the particle s electric potential at the slipping plane of its electric double layer. It arises if the particle is moved in an electric field and is a function of the Debye length, the particle size, the solvent s viscosity and the sample s dielectric constant. Generally, the electrostatic stability of a colloidal system can be evaluated by determining the Zeta potential. Colloids can be regarded as stable if the zeta potential is larger than the thermal voltage of the particles (. 16 The zeta potential is a function of the solution s ph, because protons and hydroxide ions influence the surface chemistry of the colloid. The specific ph, at which the zeta potential approaches zero, is called the isoelectric point. Laser-synthesized gold colloids feature a partially oxidized surface (e.g. 6% for ablation in NaCl). 13 They typically carry zeta potentials around -3 to -4 mv 13 and isoelectric points at ph= and Platinum nanoparticles were shown to have isoelectric points at ph= and are typically higher oxidized (up to 5%), 19 resulting in more negative zeta potential values (around -4 to -5 mv). 18,11 Optical Properties A special feature of noble metal particles is their localized surface plasmon resonance (LSPR). The LSPR results from oscillation of conduction electrons at metal particle interfaces. When the frequency of incident photons matches the frequency of oscillating

19 2. Theoretical Background 14 surface electrons, a distinct absorption peak, often in the visible frequency range, appears. 111 The LSPR is highly sensitive on NP size, shape, surface chemistry and local environment. If the local dielectric environment is changed e.g. by ligand binding, the LSPR peak can be shifted strongly, which is used in sensing applications. 34,111 In 198, Mie presented a solution to Maxwell's equations describing the absorption and scattering of electromagnetic radiation by spherical particles. With the help of Mie theory, the interaction of dielectric absorbing spherical particles with the electromagnetic field can be computed, so that optical properties of a small AuNPs can be predicted. 112 For AuNPs with diameters < 5 nm, the plasmon oscillation becomes damped and AuNPs smaller than ~2 nm do not feature a surface plasmon resonance. 113 In contrast, increasing the particle diameters above 2 nm will lead to SPR peak shifts to wavelengths > 52 nm and increased intensities. 113 Depending on the particle size, absorption and scattering contribute differently to the extinction of a gold colloid. In particular, for AuNPs with diameters of 4 to 2 nm, the total extinction is dominated by absorption, whereas absorption and scattering contribute equally to the extinction of 8 nm AuNPs. 114 Fluorescence quenching is another phenomenon of colloidal AuNPs related to their plasmonic properties. 115,116 Fluorescence quenching is a process which leads to a reduced fluorescence intensity without (chemically) destroying the fluorophore. It can result from dynamic collisions, static complex formation, internal conversion and energy transfer. 115 Thereby the fluorophore s excitation is either prevented in the first place or the transition of the fluorophore into the ground state occurs non-irradiatively. AuNPs influence fluorescence in close proximity to the particle surface due to the strong electromagnetic field generated at the surface of the NP. 117 In this respect, Chhabra et al. reported for 5 nm and 1 nm AuNPs that quenching is proportional to ~1/L 4 (where L is the distance to the gold surface). 118 Within less than 5 nm of the metal NP surface, chromophores donate their excited electrons to the metal through non-radiative pathways, whereby the fluorescence is quenched. In contrast, at a distance larger than 1 nm, the electric field of NPs may enhance the fluorescence probability without direct electronic interactions, so that fluorescence can be enhanced. 111 Chen et al. suggested the following criteria for AuNP quenching: (i) overlap of plasmon resonances with dye emission, (ii) AuNP diameters below 5 nm, (iii) spherical particle shapes, and (iv) AuNP-fluorophore distance below 2 nm. 119 Even without being in close proximity to the fluorophore, AuNPs may absorb the excitation light (primary inner filter effect 12 ) or

20 2. Theoretical Background 15 reabsorb the light emitted by the fluorophore (secondary inner filter effect 12 ). 121 This is due to their SPR peak in the visible spectrum and their high extinction coefficient. For platinum nanoparticles, which do not feature a SPR peak in the visible spectrum, the inner filter effect is less pronounced. Moreover, quenching may occur as concentration quenching. 122 This effect arises from the stacking of fluorophores on the NP surface through hydrophobic interactions, forming non-fluorescent complexes Synthesis of Colloidal Gold Nanoparticles via Pulsed Laser Ablation in Liquids The way of synthesis determines AuNP properties and may consequently influence their functionality. Thus, the following sections give an overview of chemical synthesis and laser ablation procedures, to explain the respective advantages, limitations and resulting colloidal properties, especially with regard to subsequent bioapplications. In general, colloidal metal nanoparticles can be synthesized chemically by assembling atomic precursors (bottom-up approach) or by mechanically breaking down bulk materials (topdown). Chemical NP synthesis procedures involves the reduction of metal salts or decomposition of organometallic precursors. 124 To mediate NP growth and stability, stabilizing agents, which passivate the nanoparticle surface and prevent particle aggregation, are generally added. The first scientific report for the production of colloidal AuNPs was made by Michael Faraday in 1857, by treating aqueous HAuCl 4 with phosphorus dissolved in CS 2 in a two-phase system resulting in a red colloid. 125 Turkevich et al. developed a classical procedure for the synthesis of monodisperse, spherical gold nanoparticles in the aqueous phase by reducing chloroauric acid with trisodium citrate. 35 Thereby citrate anions act both as reduction and stabilizing agents. In contrast, Brust and Schiffrin developed a method to produce AuNPs in organic solvents with sodium borohydride (NaBH 4 ) as reducing and dodecanethiol as capping agent. 36 The NP synthesis in an organic phase offers the advantage of avoiding NP agglomeration associated with ionic interactions. However, for bioapplications, the subsequent transfer of the colloid into an aqueous medium is required. For this, NP surface coatings with amphiphilic, thiolated ligands can be employed. By now, different state-of-the-art chemical synthesis procedures for gold-containing NPs have been developed. They allow the precise control over size and shape. Some prominent examples are shown in Figure

21 2. Theoretical Background 16 Figure 2-6: Plasmonic NPs obtained from advanced chemical synthesis featuring different sizes and shapes. Gold nanospheres (a), gold nanorods (b), gold bipyramids (c), gold nanorods surrounded by silver nanoshells (d), gold-coated iron oxide nanorods (e), SiO 2 /Au nanoshells (f), nanobowls (g), SiO 2 /Au nanostars (h). Reproduced from Ref. 88 with permission of The Royal Society of Chemistry. DOI: 1.139/C1CS15166E Especially for applications in which a high NP purity is desired, pulsed laser-ablation in liquids (PLAL) is a valuable alternative to chemical synthesis. In this method, a pulsed laser beam is focused on a bulk target immersed in liquid to produce surfactant-free colloidal particles. 42,127 The versatility of the method is given by the choice of bulk materials (metal, metal alloys, oxides, semiconductors, ceramics) and liquids (aqueous, organic solvents). The process as well as nanoparticle properties depend on these materials, as well as the laser parameters (wavelength, pulse energy, pulse duration, spot area, repetition rate, ablation time). Different physical and chemical processes are involved in NP generation via PLAL. Hence, pulsed laser ablation in liquids can be regarded as hybrid nanoparticle fabrication technique, because it embodies aspects of both, top-down and bottom-up approaches. 43 Depending on the pulse duration, photoionization (fs- and ps-pulses) or thermal processes such as vaporization, boiling and melting (in the case of ns-pulses) represent the main laser-matter interactions. Explosive boiling by rapidly heating the solid target up to the critical temperature is considered the main mechanism for material detachment by ns-laser pulses, where the pulse duration of the ns-pulse is comparable to the electron lattice thermalization speed. 43

22 2. Theoretical Background 17 Furthermore, in the case of ns-pulses, the laser pulse may spatially and temporally overlap with the plasma plume leading to shielding effects by energy absorption. 128 When the laser pulse reaches the target, the laser energy is absorbed by the material, followed by thermal conduction and expansion of a plasma plume (Figure 2-7). In the liquid phase, ablation takes place from energy transfer directly from the laser pulse to the target and also from the plasma to the target, when it is confined by the liquid. At high laser fluences, this results in greater ablations yields in liquid than in air. 43 While the plasma plume extinguishes, the released energy induces the formation of a cavitation bubble. Its dimensions and lifetime depend on the pulse energy, 129 with a maximum lifetime of hundreds of microseconds and a maximum radius of the order of millimeters. 43 It contains mostly solvent molecucles 13 and, to a lesser extent, ablated material. Furthermore, Figure 2-7: Time scale of processes during PLAL, including laser pulse absorption by the target, evolution of a plasma plume, shockwaves and the cavitation bubble, leading to the generation of colloidal nanoparticles. Reproduced from Ref. 43 with permission from the PCCP Owner Societies. DOI: 1.139/C2CP42895D The exact mechanism for NP formation during PLAL is still investigated. The cavitation bubble was suggested to be the particle reacting factory, confining and re-depositing the ablated material. 131 Recent computational studies by Zhigilei et al. 132 report nanoparticle

23 2. Theoretical Background 18 formation via nucleation and growth during the first 3 ns after the laser pulse hits the target. Experimentally, the dynamics of the cavitation bubble and its role for NP formation and growth were studied by in situ synchrotron, small angle X-ray scattering experiments. 131 In these experiments, it was found that two species, small primary particles and particle agglomerates, are present in a first bubble. Small primary NPs (d 1 nm) were suggested to form via nucleation and growth. 43 At maximum expansion, a temporal maximum mass of primary particles exists, which partially penetrate the bubble and enter the liquid, while a smaller number of agglomerates remains inside the bubble. 133 After the first bubble collapses, NPs are released into the free liquid and a rebound occurs, which confines material, leading to the formation of further agglomerates (Figure 2-8). 131 Upon collapse of the second bubble, outward moving, condensed material was observed in a third, irregularly shaped jet. 134 Figure 2-8: Detailed cavitation bubble dynamics and the development of primary and agglomerated particles during PLAL, as experimentally observed via in situ synchrotron, small angle X-ray scattering experiments. Reproduced from Ref. 131 with permission from the PCCP Owner Societies. DOI: 1.139/C2CP42592K By adjusting the solvent composition, controlled NP size quenching and narrowing of the size distribution width can be achieved during PLAL. For example, the presence of micromolar anion concentrations 135 or larger molecules (sodium dodecyl sulfate, 136 cyclodextrins, 137

24 2. Theoretical Background 19 biopolymers, 138 polyvinylpyrrolidone, 139 cetyltrimethylammonium bromide, 14 peptides, 141 oligonucleotides 142 ) were shown to lower the particle size during laser ablation, by limiting adsorption sites of free atoms and coalescence of NPs amongst each other. In saline aqueous solution, monomodal, number-weighted monodisperse NP colloids can be fabricated, which are free of organic ligands. 48,135 Hence, bioconjugation can be started from scratch, achieving higher conjugation efficiencies than with chemically synthesized NPs. 39 It should be noted that although monomodal, number-weighted monodisperse NP colloids can be fabricated via size quenching in PLAL, byproducts with particle sizes > 1 nm are often observed. Reasons for their appearance are under debate and include the formation of an energy fluence gradient, resulting from the intensity profile of the laser beam. 143 Another point to consider is that a large liquid layer above the target (> 4 mm) is sometimes used during PLAL for practical reasons (e.g. to avoid ablation in air by laser-induced breakdown of the liquid layer). However, the liquid volume, which is irradiated by the laser beam between the chamber window and the metal target, features a conical volume. Hence, a fluence gradients arises, which can lead to post-irradiation effects of already produced nanoparticles, i.e. laser fragmentation (1-5 mj/cm² for AuNP) 41 and laser melting (6-1 mj/cm² for AuNP). 144 Consequently, a subsequent centrifugation step 145 is often required to harvest particles at a certain cut-off radius by exploiting the fast sedimentation of larger NPs. With regard to PLAL productivities, important variables include the laser parameters such as pulse duration, laser power and repetition rate as well as the experimental setup, i.e. target geometry, feeding of the fluid and movement of the laser beam relative to the target. Increased ablation rates can be achieved by automating the system, e.g. ablation of a continuously feeding wire in a flow-through system. 146 Yields in the g/h range were achieved by using high laser fluences, low height of the applied liquid layer, and optimized laser repetition rates. 147 When performing PLAL at high repetition rates (khz-regime), one laser pulse is likely to be scattered at the cavitation bubble generated by the previous laser pulse due to the relatively long lifetime of the bubble. 148 This usually limits the productivity. In order to avoid re-irradiation of the cavitation bubble, the combination of a high-power ps laser, high-repetition-rate, ultrafast scanning system has recently been published. 149 Here, the cavitation bubble is spatially bypassed, which results in productivities up to 4.1 g/h for platinum and 3.8 g/h for gold in continuous operation mode. 15 This productivity is equal to ~4 L colloid per hour at typical concentrations of 1 mg/l.

25 2. Theoretical Background Special Properties of Laser-Generated Colloids and their Bioapplications Laser-generated, colloidal noble metal nanoparticles feature some special properties arising from their fabrication process. Dissolved oxygen or radical species formed during the ablation process may cause the partial surface oxidation of laser-generated noble metal NPs (Au + and Au 3+ : % as measured by surfactant titration, % as determined via X-ray photoelectron spectroscopy 44,13 ). As a consequence, a high colloidal stability in low ionic strength aqueous solution results, which eliminates the need for stabilizing ligands. The production process does not involve any metal salt precursors or reducing agents which may remain after chemical synthesis. Hence, post-synthesis treatment (e.g. dialysis, thermal treatment, oxidation) affecting size, aggregation state or the morphology of the particles 152 can be circumvented. In this regard, the intrinsic ligand-capping of chemically synthesized NPs may diminish their functionality, reactivity and biocompatibility. 42 For example, Brewer et al. showed that the presence of citrate lowers the amount of bovine serum albumin (BSA) that can bind to the surface of gold nanoparticles to 5%. 153 For PLAL-generated ligand-free NPs, functional molecules can directly bind to the particle surface and do not need to replace ligands. Therefore, an up to five-times higher conjugation efficiency can be realized. 39 In contrast, in chemically synthesized NPs, ligand exchange may be incomplete, as was demonstrated by Park et al. 4,154 and Dinkel et al. 155 for the replacement of citrate by thiolated ligands. The remaining ligands from synthesis not only lower the number of bound functional ligands but may also exert toxic effects. Uboldi et al. showed that the presence of citrate impaired the viability and proliferation of human alveolar cells compared to pure gold nanoparticles. 37 Moreover, cytotoxicity was observed for CTABcoated nanorods (cetyltrimethylammonium bromide, CTAB), and a certain cytotoxic effect remained even after removing residual CTAB through washing steps 156 or overcoating the CTAB-layer with a polymer shell. 157 Kinnear and coworkers examined the replacement of the CTAB-layer on Au nanorods by thiolated PEG and found that CTAB could not be replaced completely in a one-step ligand exchange process, so that an advanced treatment was required. 158 With regard to the fabrication of nanobioconjugates, the addition of ligands to the colloidal nanoparticles can in principle be conducted during the ablation process (in situ conjugation 45,46 ) or afterwards (ex situ conjugation 48,135 ). The in situ approach has been

26 2. Theoretical Background 21 established by Petersen and Barchanski and represents a rapid, one-step conjugation method, 45 which can be conducted in a completely sealed system. This enables the nanobioconjugate fabrication in a sterile environment, which is desirable for producing bioactive or pharmaceutical agents under good manufacturing practice. However, the in situ approach requires a careful adjustment of the process window. 45 It means that laser parameters have to be employed, which guarantee both, the integrity of biomolecules and a sufficiently high productivity for NP generation. 45 Moreover, the presence of biomolecules during the ablation process leads to a concentration-dependent size quenching of the nanoparticles in the in situ approach. 45 In contrast, PLAL followed by ex situ conjugation allows the generation of a master batch of ligand-free particles with the exact same properties. From this batch, different conjugate libraries (e.g. with varying ligand densities, varying ligand types) can be easily and systematically fabricated afterwards. 48 This allows the controlled analysis of the structure-function relationship of nanoconjugates, since any difference in functionality can be directly related to the ligand shell. Furthermore, a reference sample containing ligand-free NPs of the same PLAL-batch can always be run in parallel. Regarding the application of laser-generated NPs, only a limited number of studies exist which include both, the conjugate fabrication and the analysis of its biofunctionality. Previous work on laser-generated AuNPs included the design of DNA aptamer nanoconjugates which successfully targeted cancer tissue. 159 Moreover, AuNPs were conjugated with cell penetrating peptides in order to promote cellular uptake. Their biological activity was shown after incubation with bovine endothelial cells and analysis via laser scanning confocal and transmission electron microscopy, which revealed a successful uptake in up to 1% of cells within 2 h. 141 Gamrad et al. designed photodispersible AuNP agglomerates, coated with cell penetrating peptides and BSA. 16 While the cell penetrating peptide triggered cellular uptake of AuNPs via endocytosis, the co-adsorbed BSA sterically stabilized NP agglomerates. 16 Another in vitro study employed laser-generated AuNP conjugates with unspecific oligonucleotides (for stabilization), cell penetrating peptides (for cellular uptake) and small interfering RNA (modulating gene expression). Murine regulatory T cells were incubated with these conjugates. Thereby, successful NP internalization as well as nucleic acid delivery, detected as down-regulation of green fluorescent protein expression, proved the conjugate s functionality. 47

27 2. Theoretical Background 22 With regard to in vivo studies, only few reports on laser-generated NPs exist and most address the biodistribution and/or toxicity of the particles (summarized in the review by Rehbock et al. 41 ). Kabashin and colleagues performed in vivo hyperthermia-based cancer treatment with laser-generated, non-functionalized SiNPs (25-3 nm). For this, colloids were injected intratumorally (.2 ml at a dose of.4 mg/ml) into mice, followed by radio frequency irradiation. This inhibited the growth of the tumor and led to a decrease of the tumor volume. 161 Recently the authors also reported about the in vivo biocompatibility, biodistribution and excretion of laser-generated Si-SiO x NPs (5 nm, -35 mv) in mice. They administered a dose of 2 mg/kg intravenously, and found complete elimination and no signs of toxicity within 7 days. 162 For gold nanoconjugates, many different applications in diagnosis 163 and therapy 3 have been reported, including immunoassays, 73 photothermal therapy, 164 and targeted drug delivery. 165,166 In contrast, for PtNPs or Pt-containing NPs, only few studies related to bioapplications can be found in the literature. Kim et al. reported the conjugation of PtNP with a HIV-1 TAT-fusion peptide and showed that the ligands improved NP internalization into a nematode worm. Thereby, the particles antioxidative effects were increased by a factor of 1 compared to unfunctionalized PtNPs. 52 Xu et al. showed that peptide-functionalization of FePt alloy NPs allows targeting of human ovarian cancer cells, where the FePt NPs exhibited an enhanced cytotoxicity. 53 Pelka et al. examined the ability of PtNPs (< 2 nm, < 1 nm, and > 1 nm) to invade cells of the gastrointestinal tract with the model of human colon carcinoma cells. They showed that NP internalization impaired the cellular redox system and DNA integrity. 167 Recently, the effect of orally-administered PtNPs on lung tumor-bearing mice was published and PtNPs were found to inhibit tumor growth by 66% at 1, mg/kg dose. 168 In a study of Koenen et al., the surface of neural electrodes was nanostructured with laser-generated PtNP via electrophoretic deposition. Subsequently, these modified electrodes were implanted into rat brains for electrostimulation. It was shown that the NP coating affects the electrode s impedance in vivo, with no negative reaction of the surrounding tissue (i.e. reactive gliosis). 169

28 2. Theoretical Background Tailored Nanoparticle Surface Modifications via Thiolated Ligands Nanoparticles can be surface-modified through the immobilization of ligands resulting in nanoparticle-ligand conjugates. Ways of surface-functionalization include adsorption by electrostatic interactions, van der Waals forces and hydrogen bonding or direct conjugation via phosphines or carboxylates, the metal-n or metal-s linkage. 34 Regarding the adsorption enthalpies, the process can be classified in physisorption and chemisorption. Physisorption is a pure physical process without chemical reactions between the particles and ligands, initiated by van der Waals attraction. Multilayers can form during physisorption, because of the relative long range of van der Waals forces. The adsorption enthalpies for physisorption processes are in the range of condensation enthalpies (typically < 4 kj/mol). 12 In contrast to physisorption, chemisorption is characterized by strong interactions and adsorption enthalpies comparable to the energies of chemical bonds (typically > 8 kj/mol). 12 During chemisorption, adsorbed molecules form a monolayer on the NP surface. Hereby, the ligands may experience chemical modifications upon binding to the surface, in particular the ligands anchor groups such as the thiols of cysteines. If ligands bind to the NP surface via terminal groups and extend their tails into the surrounding media, dense packing can be realized. Owing to the surface curvature effect, 17 smaller particles can accommodate a higher ligand number per nm² surface than larger particles. The bonding strength between nanoparticles and ligands follows Pearson s hard-soft acidbase theory 3 with d-metal surfaces as soft Lewis acids (i.e. easily polarizable electron acceptors). 171,172 In the case of AuNPs, covalent gold-thiol bonds represent the strongest linkages with binding energies of 184 kj/mol. 34 Typical alkanethiol grafting densities are 1.5x1 14 molecules/cm². 3 Regarding adsorption kinetics, spontaneous adsorption on a timescale of milliseconds to minutes was reported. 171 However, the structure of the ligand coating can continue to reform and compress over a timescale of 7 to 1 days. 171 Notably, Flynn et al. showed that even for the covalent gold-thiol bond, desorption of ligands can occur under physiological conditions in the presence of high ionic strength medium and serum within 35 days. 173 The bonding strength of alkanethiol-coated AuNPs has also been investigated in terms of their thermal stability. It was found that temperatures between 363 K and 413 K lead to the desorption of short thiols (C 3-5 ), whereas a temperature of 433 K was required to induce desorption of longer thiols (C 6 8 and C 16 ). 32 Moreover, Bhatt et al. showed that thiol-modified DNA may dissociate from AuNPs at elevated ionic strength (1 mm

29 2. Theoretical Background 24 NaCl), high ph, and increased temperature (26 C). 174 In general, minimizing the oxygen concentration in the solution (e.g. by degassing the liquid) can help to limit the oxidation of thiols to sulfonates and other oxygenated species. 171 Another strategy to increase Au-thiol binding strength is the employment of multidentate thiols. With regard to colloidal stability, it was reported that thiolated ligands increase the NP stability as function of their anchor group following the order monothiol (R-SH) < dithiol (HS-R-R-SH) < disulfide (R-S-S-R). 175 However, although dithiols may result in stronger binding avidities per ligand, they are reported to pack less densely and are thus more prone to oxidative desorption. 176 Disulfides can arise as oxidation product of monothiolated compounds and they may feature lower solubilities compared to their monothiolated precursors. 171 Since the adsorption of disulfides on gold surfaces is described to result in the dissociation of S-S bonds and in chemisorption of thiolates, conjugation with (R-S) 2 dimers is conceivable (if their water solubility is sufficiently high). In this respect, a previous study has examined the conjugation of activated penetratin (R-S-S-penetratin) to laser-generated AuNPs. Via matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), it was shown that the ligand adsorbs to the NP surface via cleavage of the S-S bond. 141 Some details for the mechanism of the gold-sulfur bond formation are still discussed, but the main steps are described as follows: at first, the thiol group physisorbs onto the gold surface, during which the relatively strong S-H bond remains intact. Subsequently, the S-H bond breaks and a thiyl radical forms, which builds a covalent bond with the gold surface via chemisorption. The released hydrogen atoms could either be released as H 2, 178 associate with the gold surface to Au-H, 178 or reduce ligand functional groups. 179 Alternatively, thiols may bind to the gold surface with an intact SH-group. 18 Even desorption of the thiolated molecules may occur in this case. Notably, hydrogen dissociation may be inhibited at low ph or at high thiol densities. In the first case, high proton concentrations in solution would hinder the release of further hydrogen. In the second case, the mobilities of hydrogen atoms are reduced by the densely packed ligand shell, so that the formation of molecular H 2 is energetically hindered. 18 With regard to the bioconjugation of PtNPs, only a limited number studies examining the ligand binding exist. However, PtNPs are likely to bind thiolated ligands in a similar way as AuNPs, due to the chemical similarities of the noble metals. 181 In particular, PtFe alloy NPs

30 2. Theoretical Background 25 have successfully been conjugated with thiol-dopamine mixed-monolayers, in which neutral thiolated PEG ligands bound to Pt atoms. 182 On the one hand, the successful conjugation of the ligands to the PtFeNPs was shown by the improved stability during NP incubation in phosphate buffered saline (PBS) and cell medium. On the other hand, subsequent ligand exchange against charged thiolated PEG was proven via gel electrophoresis. 182 Common small sulfur-containing ligands include lipoic acid, mercaptoundecanoic acid and thiolated polyethylene glycol. Since they were employed to improve AuNP stability in this thesis (Figure 3-4), selected properties will be described in the following paragraphs. Lipoic Acid (LA) Lipoic acid is a small (C 8 ), organosulfur compound, featuring two sulfur atoms connected by a disulfide bond on the one end and a carboxylic acid group on the other end. It is a natural compound, found in mitochondria, acting as cofactor for different enzymes. With regard to AuNPs, LA is widely employed as capping agent for the NP synthesis in organic solvents. 36,183 Moreover, the ionizable carboxylic acid group can also be employed to increase the water solubility of LA-capped AuNPs (ph > 8). 183 When adsorbed to AuNP surfaces, LA is supposed to bind with both sulfur atoms (bidentate). In this regard, it was shown that NPs capped with LA or the reduced form of LA (dihydrolipoic acid, DHLA) are better protected against ligand replacement than MUA-coated NPs. This was attributed to the bidentate binding to the gold surface. 183 Mercaptoundecanoic acid (MUA) Mercaptoundecanoic acid is a short-chained fatty acid made of 11 carbon atoms terminated by a carboxylic acid group on the one side and a thiol group on the other side. Its protonated form is strongly insoluble in water (11 mg/l, estimated from Ref. 184 ). Regarding AuNP surface modification, MUA binds to the gold surface as monodentate ligand. A ligand footprint of.22 nm² corresponding to a packing density of 4.5x1 14 molecules/cm² colloid has been reported. 185 Due to its amphiphilic character, MUA can be employed as phase transfer reagent for AuNP synthesis in organic solvents. The transfer of AuNPs into aqueous solution requires the deprotonation of the carboxylic acids groups of surface-adsorbed MUA ligands. 186

31 2. Theoretical Background 26 Polyethylene glycol (PEG) Polyethylene glycol is the most commonly applied, non-ionic, water soluble polymer with stealth behavior featuring the repeating unit CH 2 CH 2 O (44 g/mol). 187 It is approved as compound in numerous pharmaceutical products. 188 With regard to NP surface modification, different PEG derivates exist varying in size, shape, density, and charge. PEG ligands can bind to the particle via hydrophobic interactions or covalently, if they contain a thiol end group (PEG-SH). PEGylation is commonly applied to render colloids more stable and reduce non-specific interactions with serum proteins. 189 Under in vitro conditions, this typically results in a reduced uptake by cultured cells. In vivo, PEGylation may reduce immune responses and increase NP circulation lifetimes. Notably, although PEGylation can increase the circulation time of NPs in the organism so that they are more likely to reach the target site (e.g. tumor tissue), PEGylation can also hinder the ultimate cellular uptake. 19 Recent investigations on PEGylated polystyrene NPs showed that preincubation in plasma generates a specific protein corona enriched in the glycoprotein clusterin. 191 Only the presence of clusterin and not the PEG layer alone could prevent unspecific NP uptake by cells. This indicates that PEG influences the composition of the protein corona and that this secondary effect influences unspecific cellular uptake. 191 Moreover, there are limitations to the amount of PEG that can be safely administered, 192 due to the presence of toxic side products from its synthesis and its non-biodegradability. 187 With regard to the in vivo stability of ligand coatings, Parak and colleagues recently examined the stability and biodistribution of polymerstabilized AuNPs in rats. They demonstrated that the polymer shell gets partly degraded (possibly by proteolytic enzymes in the liver) and was excreted already during the first hour after intravenous injection In vivo Requirements, Bioresponse and Biodistribution For in vivo applications, the nanoconjugate s properties need to be carefully chosen since they translate directly or indirectly into its functionality. Moreover, colloidal properties such as size, charge, surface modification and stability are closely interrelated (Figure 2-9). Generally conjugates need to be designed in a way that they feature a high colloidal stability under physiological conditions and prolonged circulation time to improve their biodistribution, which may be realized by PEGylation of the particle surface. Alternatively, zwitterionic surface coatings may be advantageous as they extend the NP retention times and enhance cellular uptake at the same time. 194 Moreover, selectivity and specificity, which can be

32 2. Theoretical Background 27 established through the choice of ligands, are required to avoid immune responses and toxic side effects. Figure 2-9: Nanoparticle size 195 (A) and ligand coating 172 (B) critically affect the bioresponse. With regard to NP size, ultrasmall atom cluster induce toxicity. 196 Small NPs can penetrate the blood-brain barrier 197 and be cleared by the kidneys. 34 Intermediate NP sizes faciliate endosomal uptake 195, whereas efficient light scattering 198 and pronounced sedimentation 199 (dominant over diffusion) is typical for large AuNPs. The distribution of ligand charges affects the nanoconjugate s toxicity and ability to penetrate cells. 2 Serum proteins lead to the formation of the protein corona 21 in biological medium. Polymers can be employed as artificial stabilizers and camouflage to modify bioresponses. 19 Image sources (accessed on ): (Toxicity), (Renal excretion), (BBB), (Endosomal uptake), Ref. 198 Copyright 21 Society of Photo Optical Instrumentation Engineers (Light scattering). Referring to toxicity, limited data on the long-term in vivo toxicity of AuNPs exists, but it is suggested that they are bioinert. 22 Cell culture experiments revealed no toxic effects for particles of 3-1 nm, if the in vitro dose did not exceed 1 12 NP/mL. 88 It is noted that a more relevant unit to quantify NP-induced toxicity was recommended by Rehbock et al. in order to improve standardization and comparability between studies. This is the NP surface area per biomass. 31 In vivo, no noticeably toxicity during weeklong administration of daily doses <.5 mg/kg is proposed. 88 Depending on the application, very different doses of AuNPs have

33 2. Theoretical Background 28 been employed in in vivo tests. They mostly range from.1 to 1 mg/kg, 22 but some extremes exist. 23 For computer tomography, very high doses of up to 35 g Au/kg have been employed in mice. 24 In contrast, for photoacoustic imaging doses as low as 22.7 μg Au/kg were reported. 25 Since noble metal NPs are non-degradable, their long-term retention in the body and accumulation may be a concern in clinical use despite their intrinsic bioinertness. Hence, facilitation NP excretion may be a key requirement towards the clinical approval. Studies on the biodistribution of AuNPs reported the accumulation in organs, but no adverse impact on animals within time frames of weeks to one year. 34,26 Ultrasmall AuNPs (d<3 nm, atom cluster size range) may have toxic effects by irreversibly binding to cellular biomolecules 196 and inducing oxidative stress. 27 Particles with diameters smaller than 6 nm may be filtered out by the kidneys, whereas diameters larger than 2 nm are retained in the spleen (Figure 2-9, top). 34,26 Importantly, nanotoxicity and toxicity of the capping agents or residual chemical precursors should be discriminated. 157 In general, a positive particle charge induces higher systemic toxicity (e.g. platelet aggregation, Figure 2-9 bottom). 2 To enhance cellular uptake, particle properties such as charge, size and surface coatings can be adjusted (Figure 2-9). Doorley and Payne revealed that albumin may trigger endocytosis by interacting with cell surface receptors, when they studied the binding of albumin-coated polystyrene NPs to cell surfaces. 28 On the one hand, positively-charged NPs penetrate cells possibly due to interactions with the negatively cell charged membrane. On the other hand negatively charged or neutral NPs have longer circulation half-lifes. 29 Moreover, Parak and colleagues revealed that particle uptake does not only depend on the overall (net negative) particle charge. In particular, they showed that particles with similar zeta potentials (differing only in the distributions of negative and positive charges on their surface) may lead to different cellular uptake rates. 21 Hence, the cellular response is not only triggered by the nanoparticle as a whole, but can be impacted by short-range, sub-np-sized patches, when the nanoenvironment is different from the bulk colloidal properties. 17 Another example that biologic effects can be induced by the local nanoenvironment are the patchy and striped NPs of Stellacci et al.. 211,212 The authors showed that the orientation of the molecules in the ligand shell affects the NP s overall behavior with regard to cellular uptake 213 or protein adsorption. 214 In particular, the membrane penetration of the nanoparticles with sub-nm striations of anionic and hydrophobic ligands penetrated the plasma membrane without bilayer disruption, 211 whereas randomly coated, patchy NPs mostly resided in endosomes. 213

34 2. Theoretical Background 29 To induce receptor-mediated endocytosis, an ideal NP size around 5 nm has been reported. 195 Receptor-mediated endocytosis is initiated by membrane wrapping, which requires the establishment of multiple interactions between NPs and cellular receptors. These can be accomplished by several small NPs interacting simultaneously with receptors in close proximity. Alternatively, the curvature of a single 5 nm particle is ideal to establish a maximum number of interactions between the particle and cell surface receptors to induce wrapping. 215,216 Nanoparticles may not only be employed with the aim of cellular uptake, but also the crossing of macroscopic biological barriers may be desired. The blood-brain barrier (BBB) is a selectively permeable, physiological barrier separating the extracellular fluid in the central nervous system from the blood circulating system to maintain brain homeostasis. The interface is formed by endothelial cells creating tight junctions on the blood side and astrocytes and pericytes on the brain side. 217 It is characterized by a high transendothelial electrical resistance. Therefore, only small (< 4 Da) molecules can penetrate the BBB either paracellularly or transcellularly by passive diffusion (Figure 2-1). 217 In addition, the selective transport of essential molecules such as glucose and amino acids occurs by small transporter molecules. Cationic molecules may also non-specifically induce electrostatic interactions with the negatively charged domains in the endothelial cell membrane for transcytosis across the BBB (absorptive-mediated transcytosis). 14 Receptor-mediated transcytosis enables the transport of larger molecules across the BBB, if they present appropriate binding sites for the receptors expressed at the BBB. Examples for these receptors include the transferrin receptor (TfR), insulin receptor and lipoprotein receptor. In the case of the TfR, one has to consider that this receptor is expressed in other organs besides the BBB, so that potential drugs targeting the TfR could also be taken up in other organs. Secondly the receptor is saturated with endogenous transferrin from the circulating blood stream (c = 25 µm), 218 so one has to select a more affine ligand to replace transferrin or circumvent competition by targeting another epitope. 14,218

35 2. Theoretical Background 3 Figure 2-1: Different mechanisms that can mediate the crossing of the blood-brain barrier. Reprinted by permission from Macmillan Publishers Ltd on behalf of Nature Review Neuroscience: Ref. 217, copyright 26. DOI: 1.138/nrn1824 The use of nanoparticles may faciliate drug transport across the BBB. In this regard, transport efficiency has been suggested to depend on NP size with an upper limit of approximately 2 nm. This size limit may result from the gap of the capillary endothelium and the astrocytes end-feeds. 22 In this regard, De Jong et al. studied the particle size-dependent organ distribution of AuNPs after intravenous administration in rats after 24 h. A dose of 77 µg of 1 nm unfunctionalized AuNPs was injected per rat. After 24 h,.3% of the injected dose were detected in the brain (.13 µg Au/g brain), whereas no particles of bigger size (5, 1, 25 nm) were detected in the brain. 219 Sela et al. report the spontaneous penetration of the BBB by non-functionalized, ultrasmall AuNPs after injection into the abdominal cavity of rats (d = 1.3 nm, µg Au/rat). They found an maximum gold concentrations in the CSF collected 6 h after injection of.16 mg/l and argue that ion channels are involved in the transport mechanism. 22 The delivery of targeting AuNPs into the brain has been demonstrated in different other studies. Shilo et al. developed insulin-coated AuNPs for receptor-mediated BBB transcytosis and injected those into the tail vein of mice (d = 2 nm, 6 mg Au/mouse corresponding to approx..25 g Au/kg). The gold content was analyzed by flame atomic absorption

36 2. Theoretical Background 31 spectroscopy and the maximum amount (5% of injected dose) was found 2 h after the injection. Moreover, the results showed that the insulin-coating improved the NP uptake fivefold compared to PEGylated control AuNPs. 221 Prades et al. injected peptide-functionalized AuNPs into the body cavity of rats (d = 13 nm, 1.98 mg Au/kg) for brain delivery targeting the TfR. Brain uptake was quantified with instrumental neutron activation analysis. Highest gold concentrations were detected 1-2 h after injection (.7% of injected dose). 222 Cheng et al. employed peptide-modified AuNPs which were intravenously injected into glioma bearing mice (d = 5 nm, 1.5 mg Au/kg). NP quantification was carried out after 24 h. 3% of the injected dose was found, corresponding to a five-fold increase over the non-functionalized NP. 223 Besides the examples given above, surface-adsorbed apolipoproteins, 22 polysorbate 8, 224 or glycopeptides 225 have been suggested to facilitate NP transport through the BBB. As an alternative route for NP brain delivery, the intranasal administration has recently been recognized by some groups. 226 In this regard, the direct transfer of polymeric, 227 gold, 228 manganese oxide, carbon-13, iridium-19, iron (II) oxide and titanium dioxide NPs 229 from the nasal cavity to the brain has been reported. Intranasal delivery is possible due to the unique connections of the olfactory nerves between the brain and the environment, 23 so that blood circulation and the challenge to overcome the BBB can be avoided. However, the efficiency of brain delivery by this route remains to be studied more extensively The Role of the Nanoparticle Protein Corona and Properties of Selected Model Serum Proteins For any biologic application, one has to consider that upon administration to a biofluid the nanoparticle surface gets quickly coated with lipids, polysaccharides and proteins to lower the NPs surface energy. Since this corona is the interface between the nanomaterial and living systems, it strongly determines the NP s biological fate and behaviour. 231,232 The formation of an inner, long-lived hard corona and a loosely associated, dynamic soft corona has been suggested. 232 In a newer definition, coronas were described as analytically accessible NPprotein complexes, including those proteins which are strongly attached to the nanoparticle and trigger biologic responses. 233 The composition of this corona was shown to establish on a short timescale of 3 s, which does not change significantly thereafter. 234 Moreover, Mahmoudi et al. and Mailänder et al. have reported that the composition of the protein corona can vary depending on the source of the proteins (e.g. fetal bovine serum vs.

37 2. Theoretical Background 32 human plasma). 235,236 Notably, the incubation with plasma from human individuals carrying different diseases resulted in variable protein compositions on the NPs, indicating the existence of personalized protein coronas. 237 The (unintended) adsorption of proteins to NPs can induce different effects. From the point of view of targeted nanoparticles, the most negative outcome from the protein corona formation could be the masking of the functional ligands, so that the ligands cannot reach their biological target any longer. In contrast, the adsorbed protein corona can also add functionality by increasing NP stability, biodistribution and supporting cellular uptake (e.g. adsorption of transferrin, insulin, apolipoprotein and subsequent binding to their respective receptors). Table 2-1 summarizes some studies which report biological effects (co-) mediated by the NP s protein corona. Table 2-1: Overview of biological effects (co-)mediated by the protein corona of different NPs. NP type AuNPs, 5 nm Polyacrylic acid-coating AuNPs, 4.6 nm + dodecanethiol + polymer-coating polystyrene based NPs, ~1 nm polystyrene based NPs, ~1 nm iron oxide NPs, 11 nm + Oleic acid + PEG silica NPs, + PEG + Transferrin silica NPs, 75 nm Bicyclononyne ligands (targeting azide) Protein corona components fibrinogen BSA, fetal bovine serum ApoA4 and ApoC3 ApoH Transferrin fetal bovine serum fetal bovine serum Biological Effect inflammatory reaction decreased cellular uptake decreased cellular uptake increased cellular uptake increased cellular uptake decreased targeting decreased targeting Reference Deng et al. (211) 238 Hühn et al. (213) 2 Ritz et al. (215) 239 Ritz et al. (215) 239 Bargheer et al. (215) 24 Salvati et al. (213) 241 Mishafiee et al. (213) 242 From the protein s point of view, conformational changes of surface adsorbed proteins can result in altered presentation of active sites and subsequent functional changes. With regard to citrate-coated AuNPs, Halas et al. observed the aggregation of lysozyme upon binding to the NP surface under physiological conditions. The authors discuss that the breakage of intramolecular disulfide bonds and the formation of Au-S bonds lead to altered protein conformations, including protein unfolding and subsequent aggregation. 243 Moreover, the (selective) depletion of proteins from the medium can result in altered protein compositions in the medium vs. on the particle, 191 which can in turn impact protein-protein interactions. 244

38 2. Theoretical Background 33 Especially these last two concepts will be of importance, when considering the processes of pathological protein aggregation, which will be described in chapter 2.3. Albumin, transferrin and insulin are common blood plasma proteins. The analysis of their interactions with laser-generated AuNPs was part of this thesis (Figure 3-4). Therefore, protein properties are described in the following paragraphs. Albumin Albumin is a globular protein and the most abundant protein in human serum, where it acts as transporter of water-insoluble molecules, regulates the ph and is responsible for about 8% of the serum protein osmotic pressure ( oncotic pressure ). Albumins have average molecular weights of 66 kda, consisting of 58 to 59 amino acids and exist in different conformations with different physiological function, depending on the ph (N: native form, predominant at neutral ph; B: basic form, ph > 8; F: fast migrating form, ph < 4.3; E: expanded form, ph < 3.5). 245 A typical model albumin is bovine serum albumin (BSA), with a 76% sequence homology to human serum albumin (HSA). The dimensions of the N-form of BSA have been described as prolate ellipsoid (4 x 4 x 1 nm), 246 characterized by a molecular volume of 88 nm 3 and a hydrodynamic radius of 2.6 nm. 247 For the adsorption of BSA to AuNPs, Brewer et al. showed that the conjugation occurred mostly via electrostatic interactions. 153 On bare AuNP surfaces, BSA may denature to expose hydrophobic groups and form multilayers. 153 In contrast, BSA binding to citrate-coated surfaces was found to be less dense, suggesting that BSA does not replace citrate. Rather, electrostatic interactions between citrate and BSA promote the formation of an additional BSA layer. 153 Tsai et al. proposed that binding occurs via an exposed thiol group, resulting in strong Au-BSA interactions and inducing conformational changes in the BSA secondary structure. 247 In addition, Shang et al. detected a decrease of the helical structure and an increase of β-sheet structures. 245 The strength of BSA binding was also found to depend on the NP surface coating, following the order CH 3 > R-COO - > R-NH + 3 > R-OH > ethylene glycol. 248

39 2. Theoretical Background 34 Insulin Insulin is a 51 amino acid-containing polypeptide synthesized in the pancreas. It is involved in regulating the metabolism of carbohydrates, fatty acids and proteins by promoting the absorption of glucose from the blood. The monomer consists of two peptide chains connected via two disulfide bonds. 249 Inside the body, insulin is produced and stored as highly stable, zinc-containing hexamer, with dimensions of 5 x 5 x 3.5 nm. 25 Zinc-free insulin is present as a dimer at low protein concentrations over the ph range of 2-8 and may transform into a tetramer at protein concentrations of > 1.5 mg/ml. Insulin is conformationally stable under acidic conditions (e.g. monomeric in 2% acetic acid). However, it can aggregate into β- sheet-rich fibrils through conformational changes of the monomers, in which hydrophobic regions become exposed to the solvent. In particular, fibrillation can be induced in response to increased insulin concentrations, temperature, agitation, ph or ionic strength. 251 AuNPs were shown to have a high affinity for insulin fibrils. In fact, insulin conjugation to citrate-coated AuNPs affected the fibril structure, shape, density and delayed the fibril formation dynamics. 252 Significant loss of the secondary structure has been detected. This was attributed to interactions between the gold surface and sulfur-containing amino acids (directly) or between the gold surface and carboxyl groups, which induce intramolecular stress and destabilize the disulfide bonds (indirectly). 253 With regard to the colloidal properties, insulin coating could render the particles extremely stable at salt concentrations up to 1 M NaCl. 254 Recently, insulin has been coated on AuNPs for the treatment of diabetes type 1 in mice, showing prolonged bioactivity over free insulin through the protection against enzymatic degradation. 255 Transferrin Serum transferrin is a glycoprotein mainly synthesized in the liver. 256 It is the fourth most common serum protein (4% of total serum). Transferrin has a molecular weight of approximately 8 kda, consisting of two carbohydrate chains and one polypeptide chain with 679 amino acids. The shape can be described as oblate spheroid with dimensions of 4.7 x 4.7 x 1.6 nm. 257 Its main biologic function is the transport of iron and other metal ions in the blood. Subsequently, cellular uptake occurs via receptor-mediated endocytosis by the TfR.

40 2. Theoretical Background 35 With regard to biomedical applications, transferrin can be employed to support site-specific targeting and efficient cellular uptake of drugs and/or NPs due to the high amounts of transferrin receptors present on cell surfaces. 258 In this regard, transferrin has been employed as NP coating to facilitate their translocation across the blood-brain barrier 259 or internalization by cells Protein Misfolding and Neurodegenerative Diseases Proteins are macromolecules fulfilling different biologic functions, e.g. as hormones, enzymes or structural elements. They consist of multiple amino acids (N > 5), which are linked via peptide bonds. Proteins are zwitterionic macromolecules, because different amino acids carry positively (arginine, histidine, lysine) or negatively charged side chains (aspartic acids, glutamic acid). Hence, the protein s net charge is positive/negative if the ph is below/above the isoelectric point of the protein. Amino acid sequences can form different spatial motifs such as β-sheets, α-helix and random coil exist (secondary structure). Subsequently, further arrangements through disulfide bonds, hydrogen bonds, hydrophobic, ionic and van der Waals interaction build the tertiary structure. The reason for building ordered secondary structures through intramolecular contacts is that the initially unfolded proteins tend to decrease their energy levels (Figure 2-11). An energetic minimum can be reached upon complete folding to self-organized conformations. However, environmental changes (i.e. ph, temperature, agitation, oxidative agents, protein concentration) may lead to a thermodynamically unstable, unfolded conformation. Moreover, some peptides exist natively unfolded. In these cases, proteins tend to reach lower energy levels and higher stability by intermolecular contacts. The self-catalyzed aggregation occurs via nucleation and subsequent growth of aggregate cores. These processes may be initiated by hydrophobic amino acid residues exposed to the surface of the protein, 1 β-sheet formation, and low net charges of protein segments. 261 One specific aggregate form is the filamentous amyloid, which is a thermodynamically stable, structurally organized, highly insoluble aggregate with cross-β motifs consisting of β-strands running perpendicular and hydrogen bonds running parallel to the long axis of the fibril. 262,263

41 2. Theoretical Background 36 Figure 2-11: Funnel-shaped free-energy surface diagram showing the competition between protein folding and aggregation pathway through intra- and intermolecular interactions. Reprinted by permission from Macmillan Publishers Ltd on behalf of Nature: Ref. 264, copyright 211. DOI: 1.138/nature1317 Protein aggregation and inclusion body formation are common cellular and molecular mechanisms co-occurring with the development of different neurodegenerative diseases including Alzheimer s disease (AD), Parkinson s disease (PD), Huntington s disease (HD), amyotrophic lateral sclerosis (ALS), and transmissible spongiform encephalopathies (TSE). 262 In each disease, different proteins form the characteristic aggregates (Table 2-2). 265 Prions are proteinaceous infectious particles, 266 which propagate from one cell to another by imposing their conformation to normally-folded cellular prion proteins. In contrast to AD, protein misfolding and aggregation seems to be a direct cause of the prion diseases, since this diseases can be transmitted from individual to individual by misfolded prion proteins. The fact that prions are transmissible, was formerly considered to be unique. 267 However, experimental findings and clinical observations suggested, that prion-like transmission pathways are likely to occur in other neurodegenerative diseases such as AD, PD and HD. 268 Hence, cell-to-cell transmission may be a more general property of amyloids and may explain the gradual progression of the diseases in the brain over time. 269 Notably, non-pathogenic amyloid formation also occurs under physiological conditions for a few functional proteins such as the melanin-production promoting Pmel This suggests

42 2. Theoretical Background 37 that fibril formation is an evolutionary conserved biological pathway. Moreover, the formation of insoluble aggregates can be regarded as a way to sequester toxic (circulating) protein species. It may thus have a protective role to some extent. 271,272 Table 2-2: Comparison of clinical, pathological and biochemical features of selected neurodegenerative diseases. 1 Disease AD PD HD ALS TSE Mode of transmission Sporadic (95%) Inherited (5%) Mostly sporadic, rarely inherited Inherited Sporadic (9%) Inherited (1%) Sporadic (9%) Inherited (8%) Infectious (2%) Clinical features Dementia Movement disorder Dementia, motor and psychiatric problems Movement disorder Dementia, ataxia, psychiatric problems Involved proteins Aβ and tau α-synuclein Huntingtin Superoxide dismutase Prion protein Cellular location of protein aggregates Extracellular, cytoplasmic Cytoplasmic Nuclear Cytoplasmic Extracellular Affected brain region Hippocampus, Cerebral cortex Substantia nigra, hypothalamus Striatum, cerebral cortex Motor cortex, brain stem Various regions depending on the disease Regarding the role of misfolded proteins for the process of neurodegeneration, three different hypotheses are described and also combinations of these models may be relevant for some diseases (Figure 2-12). 1 In the gain-of-toxicity model, the important species is the misfolded and/or aggregated protein with direct neurotoxic propeties. 273 In contrast, in the loss-ofphysiological-function model, the depletion and lack of activity of the native protein due to the misfolding is the key step. 274 The third model is the inflammation model, in which the activation of astroglial cells induces chronic inflammation reactions, indirectly leading to neuronal death. 1 The protein quality control (PQC) system regulates protein homeostasis by refolding misfolded proteins through chaperons (e.g. heat-shock proteins), initiating degradation by ubiquitination via the ubiquitin-proteasome-system or sequestering protein aggregates within cells. 2 On the one hand, impairment of the PQC system may be an upstream event before the protein aggregation. Hence, adapting protein homeostasis can be an alternative strategy to ameliorate neurodegeneration. 275 On the other hand, protein aggregates may be responsible for inducing failures in the PQC system. 276

43 2. Theoretical Background 38 Figure 2-12: Hypotheses relating the process of protein misfolding to ultimate neurodegeneration. Green: Loss-of-function model, orange: inflammation model, brown: gain-of-toxic-function model. Reprinted by permission from Macmillan Publishers Ltd: Nature Review Neuroscience, Ref. 1, copyright 23. DOI: 1.138/nrn Morbus Alzheimer and the Amyloid Cascade Hypothesis Alzheimer s disease was first described by Alois Alzheimer in the early 2th century. 277 AD as neurodegenerative disease is characterized by progressive tissue loss mainly in the cerebral cortex, which is the outer neural tissue layer in the brain and involved in memory storage, and the hippocampus, which is important for memory retrieval and emotions. This leads to a gradual onset and progression of deficits in more than one area of cognition. 278 AD is classified according to its progression into three stages: preclinical, mild cognitive impairment (MCI), and dementia. While biomarkers but no symptoms are present in preclinical AD, patients show cognitive deficits but no functional impairments in MCI. In dementia, the cognitive or behavioral impairments cause significant interference in the ability to function at work or in usual daily activities. 279 Although most cases of AD occur sporadically, autosomal dominant forms of the disease exist, which cause familial AD. Mutations in three genes (amyloid precursor protein, Presenelin 1 and 2) have been identified and related to the disease. Histopathologically, intracellular aggregates of the hyperphosphorylated tau protein and extracellular plaques mostly consisting of the Aβ peptide can be found in the brains of AD patients. These already form up to 25 years before the onset of clinical symptoms. 28

44 2. Theoretical Background 39 While it is still under debate if the presence of Aβ plaques is a direct cause of AD pathogenesis (classical amyloid cascade hypothesis 7 ), the misfolding and deposition of the Aβ peptide in the brain is suggested to be a key process of a complex, non-linear pathogenic cascade. 12,281,282 Upon sequential cleavage of the integral transmembrane amyloid precursor protein (APP) by β-secretases (BACE1) and γ-secretases, the Aβ peptide arises. It consists of amino acids, with Aβ 1-4 and Aβ 1-42 as most important species. While the 4 amino acid long Aβ 1-4 is the most prevalent species, the 42 amino acid long Aβ 1-42 is more prone to aggregation and represents the majority of plaque-deposited Aβ. 283 The physiological function of Aβ remains unclear, but roles in lipid metabolism 284 and neuronal protection 285,286 have been suggested. The primary sequence of Aβ 1-42 is NH 2 -DAEFR HDSGY EVHHQ KLVFF AEDVG SNKGA IIGLM VGGVV IA-COOH, of which residues 1-27 represent the hydrophilic region (extracellular within APP) and residues represent the hydrophobic domain (located in the cell membrane before cleavage APP). 287 Note that cationic residues are marked in blue, anionic residues in red and hydrophobic areas in bold black (central hydrophobic cluster, C-terminal nucleation site). NMR-studies showed that Aβ 1-42 monomers form a β-turn-β motif and assemble into two intermolecular, parallel, in-register β-sheets consisting of residues and Increased concentrations of the peptide due to overproduction and/or insufficient clearance lead to accumulation and aggregation into oligomers and fibrils (Figure 2-13). Aβ fibrils are formed through a nucleation-dependent polymerization in which the rate limiting step is the formation of a nucleus (lag phase) followed by subsequent incorporation of further Aβ into the oligomers. Of note is that different aggregation conditions may lead to different fibrillar morphologies with different molecular structures and biological effects. 289 Fibrils were shown to induce progressive tau deposition and initiate inflammation, oxidative stress, synaptic dysfunction and neuronal loss. 282 Furthermore, Aβ fibrils were shown to depolarize membranes, alter action potentials 29 and induce neuronal toxicity in vitro. 291 In vivo, it was demonstrated that Aβ fibrils cause neuronal failure, tau phosphorylation and microglial activation when injected in aged rhesus monkeys. 292 However, the role of Aβ fibrils and plaques in AD pathogenesis has been debated over the last decades and the classical amyloid cascade hypothesis has been revised. 293 Specific limitations of the hypothesis include: (i) Overexpression of human APP in mice is not sufficient to induce the full disease pathology, i.e. tangle formation, neurodegeneration and AD-like

45 2. Theoretical Background 4 dementia. (ii) The number of amyloid plaques correlates less with cognitive impairment than the number of neurofibrillary tangles, suggesting that Aβ alone is not sufficient to cause neurodegeneration. (iii) Moreover, Aβ deposits were found in brains of individuals which did not show symptoms of dementia and, vice versa, individuals with substantial plaque burdens showed normal cognition. 6,282 In this regard, one has to keep in mind that amyloid plaques may only represent the end stage of a molecular cascade. Earlier occurring species such as soluble oligomers may impact disease pathogenesis more directly. 265 Nowadays, small, soluble amyloid oligomers are supposed to be the most toxic species responsible for disrupting molecular mechanisms in synapses leading to memory impairment and eventual neuronal degeneration in AD. 294 Toxicity to cultured neurons, 295 inhibition of hippocampal long-term potentiation, 9 impairment of synaptic function, cognition and behavior of mice 296 and rats 297 have been demonstrated for small Aβ oligomers. The detection and interference with soluble Aβ aggregates before plaques and cognitive impairment become evident is thus a desirable aim. 298 Figure 2-13: Dynamic process of Aβ fibrillation. Aβ monomers adopt an abnormal conformation and form small oligomers, protofibrils, fibrils and larger protein deposits. The nucleated polymerization mechanism is characterized by a lag phase followed by rapid growth. Reprinted from Ref. 299 with permisssion of S. Karger AG. Interestingly, recent studies suggest that other disease-related proteins co-occur with Aβ 3 and may trigger or inhibit its aggregation. In this regard, Strittmatter et al. reported that the cellular Prion protein (PrP C ) binds Aβ oligomers with nanomolar affinity and can mediate Aβoligomer-induced synaptic dysfunction. 31,32 Haass et al. showed that the interactions between Aβ and α-synuclein, which constitutes the Lewy body inclusions in Parkinson s

46 2. Theoretical Background 41 disease, inhibit Aβ deposition and reduce plaque formation in mice. 33 Furthermore, Aβ and insulin, linked to Diabetes Mellitus type II, mutually modulate each other s production, function and degradation. In this regard, Luo et al. reported reciprocal molecular interactions, in which one peptide slowed the aggregation of the other. 34 The chaperone protein clusterin has recently been shown to bind Aβ 1-42 oligomers with high affinities (dissociation constant, K D = 1 nm), to interfere with Aβ aggregation and to exert a neuroprotective function in a nematode in vivo model. 35 It should be noted that much research is also focused on the identification of other factors responsible for AD pathogenesis, which might be interrelated with APP, Aβ and tau. These include genetic risk factors and cellular imbalances (e.g. loss of Ca 2+ homeostasis, mitochondrial function or neuroinflammation) as shown in Figure ,36 Figure 2-14: Multiple factors may be responsible for causing AD and dementia. Cellular (light green), genetic (blue) and molecular imbalances (dark green) can result in the symptoms listed in the center. Reprinted by permission from Macmillan Publishers Ltd: Nature Neuroscience, Ref. 6, copyright 215. DOI: 1.138/nn.417

47 2. Theoretical Background Therapies Based on the Amyloid Cascade Hypothesis In the past 2 years, more than 1, different compounds have been studied as potential candidate drugs for the treatment of AD. 37 Since 23, no new drugs have been approved 11 and no therapeutics interfering with or stopping the progression of the disease mechanisms exist in the clinic. Current therapeutic approaches aim at alleviating symptoms of AD, especially to improve cognitive function and retentiveness. For moderately to severely advanced AD, Memantin as (N-methyl-D-aspartate)-receptor-antagonist modulates adverse effects of the neurotransmitter glutamate. The clinically approved drugs Galantamin, Donepezil and Rivastigmin belong to the group of acetylcholinesterase inhibitors, which help to decelerate the degradation of the neurotransmitter acetylcholine and have antiinflammatory effects. 38 Notably, increased acetylcholine concentrations in the peripheral nervous system can lead to the occurrence of side effects, such as nausea, muscular weakness and diarrhea. 39 The major limitation of all approved AD drugs is that they provide only mild symptomatic benefit and have minimal impact on the disease process. 39 Although AD pathology may be caused by multiple factors, targeting Aβ dyshomeostasis represents one possible strategy which has already been extensively studied (compare to Figure 2-14). 14 Cause-oriented therapeutic approaches interfering with or stopping the progression of Aβ aggregation are under investigation, including the reduction of Aβ production, the prevention of Aβ aggregation and the promotion of Aβ clearance (Figure 2-15). 31 Regarding Aβ clearance, Aβ in the brain has been described to be in equilibrium with blood-circulating Aβ through the blood brain barrier (BBB). Hence, plasma Aβ may be an alternative target for AD therapy. 311,312 The sequestration of Aβ in the blood may shift the equilibrium and draw out excess Aβ from the brain ( sink effect ). Brain Blood β-secretase inhibitors γ-secretase inhibitors/ modulators Aβ antibodies Aβ degrading enyzmes Small molecule drugs Aβ antibodies Aβ degrading enyzmes Small molecule drugs Figure 2-15: Potential drug targeting sites related to the amyloid cascade hypothesis, including interference with Aβ production and assembly. Modified from Ref. 313, by permission from Macmillan Publishers Ltd on behalf of Cancer Research UK: Nature Review Neuroscience, copyright 21. DOI: 1.138/nrd2896

48 2. Theoretical Background 43 Enzymatic Aβ Clearance While APP-secretases are potential target molecules for reducing Aβ production a priori (through inhibition of β- and γ-secretases or activation of α-secretase), 314 several naturally occurring enzymes for Aβ clearance in the brain have been identified. These include insulindegrading enzyme (IDE), neprilysin and endothelin-converting enzyme. 315 In particular, IDE is a neutral thiol metalloendoprotease with a molecular weight of 11 kda. For its catalytic activity, it requires both a free thiol and bivalent cations (Zn 2+ ). The active site consists of the sequence histidine-glutamine-aa-aa-histidine (aa: variable amino acids), in which the two histidine residues coordinate the zinc cation and the glutamine is suggested to play an important role for the catalytic reaction. 316 IDE is ubiquitously present in different cells and in extracellular fluids, with the highest expression in the liver, testes, muscle and brain. It plays a crucial role in the degradation of insulin and substrates of similar secondary structure, possibly to prevent the formation of amyloid deposits. Qui et al. reported that microglia cells secrete IDE and degrade Aβ extracellularly under physiological conditions. 317 Moreover, neurons have been reported to regulate extracellular levels of Aβ via IDE. 318 Notably, different substrate affinities may lead to competitive IDE binding and degradation. For example, increasing amounts of insulin (K D =.1 μm) may inhibit the degradation of Aβ (K D > 2 μm). 319 In contrast to neprilysin, IDE is reported to degrade monomeric Aβ only. 32,321 AD Immunotherapy Immunization therapy was pioneered by Schenk et al., who actively immunized transgenic mice with Aβ 1-42 to prevent plaque formation. 322 In contrast, passive immunization is realized employing highly specificity monoclonal antibodies against different Aβ species. They may shift the equilibrium to soluble Aβ species by binding to deposited plaques. Furthermore, they may inhibit the formation of new deposits and/or activate microglia cells that phagocytose plaques and clear them from the brain. 323,278 Examples of clinically tested antibodies include Solanezumab (binding soluble amyloid monomers and recognizing the central region Aβ 13-28) 324, Aducanumab (binding the N-terminal region of aggregates) 325, Crenezumab (binding monomers, oligomers and fibrils in the central region) 326. Notably, a recent AD clinical trial with the antibody Aducanumab suggested a slowing of cognitive decline in a subgroup of patients in the early phase of the disease. 327 If these preliminary finding could be confirmed in

49 2. Theoretical Background 44 ongoing studies, the antibody would be the first successful disease-modifying drug, which can both, lower the amount of Aβ aggregates and influence cognitive function Small Molecules Targeting Aβ Small molecules represent promising alternatives to antibodies for targeting disease-relevant biomarkers such as Aβ. While Aβ-antibodies feature unmatched affinities for Aβ, this high specificity can also cause extensive T-cell activation and adverse immune response. The weaker specificity of small molecules compared to antibodies may be thus lead to a broader therapeutic window in which maximum effectiveness is accompanied by minimal adverse immune response. Furthermore, they are not as costly to produce, structurally versatile and their small dimensions may faciliate BBB delivery. 14 Examples for small molecules targeting Aβ include polyphenolic compounds, whose antioxidative properties may counteract Aβ-induced oxidative stress. Furthermore, they may modulate Aβ production by stimulating the α-secretase or inhibiting β- and γ-secretases. In addition, they may block Aβ self-assembly by competing with aromatic residues. 328 Curcumin, a yellow pigment, belongs to the group of polyphenols and was shown to directly bind small Aβ species, blocking the formation of larger aggregates and disaggregating Aβ in vitro and in vivo. 329 Epigallocatechin gallate (EGCG) is a polyphenol naturally occurring in green tea. Via covalent binding to lysine residues of Aβ, the compound is reported to reduce Aβ aggregation. 33 Moreover, similar to other flavonoids, EGCG was shown to protect neuronal mitochondrial function, modulate signal transduction pathways, expression of genes, and apoptosis. 331 EGCG is tested in Phase II clinical trials. Scyllo-inositol (cyclohexanehexol, ELND-5) is a small aromatic compound binding to the C-terminus of the Aβ monomer. 15 In transgenic animal models, the compound could reduce brain Aβ concentrations, preserve synaptic density, and improve learning deficits. A Phase II trial demonstrated safety, CNS penetration, Aβ reduction, and potential cognitive benefits in patients with mild AD. 332 Tramiprosate (3-amino-1-propanesulfonic acid, Alzhemed) is a small molecule targeting the HHQK subregion at the N-terminus of Aβ involved in oligomerization and fibril propagation. 333 However, the compound failed in the late stages of a Phase III clinical trial. 334 Other non-peptidic compounds include metal-protein attenuating compounds, 335 such as Clioquinol (iodochlorhydroxyquin, PBT-1). Clioquinol was shown to cross the blood-brain barrier, reduce amyloid-beta aggregation, toxicity and memory impairment in mice. The

50 2. Theoretical Background 45 chelator binds copper(ii) and zinc(ii) ions with high affinity and promotes the solubilization of Aβ plaques. 336 The second generation compound PBT-2 has completed phase IIa clinical trials showing safety and tolerance. 337 In contrast to the small molecules listed above, aminopyrazoles are rationally designed peptide receptors. Their specific sequence of hydrogen-bond donors and acceptors complementary to β-sheets allows them to selectively bind to the backbone of misfolded peptides featuring a cross-β-sheet conformation (Figure 2-16). 338,339 Regarding Aβ monomers and oligomers, the binding is suggested to occur to the KLVFF sequence and features low micromolar binding affinities. 339 Different aminopyrazole derivatives have been designed, which induced the transformation of ordered fibrils into high molecular weight aggregates that lost their acute cellular toxicity. 17 Figure 2-16: Aminopyrazole trimer (top) forming hydrogen bonds in a donor-acceptor-donor motif with the peptide backbone of Aβ (bottom). Reprinted with permission from Ref. 17. Copyright (211) American Chemical Society. DOI: 1.138/nrd2896 Typical peptidic compounds for Aβ targeting contain Aβ aggregate-disrupting motifs consisting of charged or methylated amino acids, proline or cholyl-groups. 34 Many of them are derived from the Aβ primary sequence, e.g. the hydrophobic nucleation side KLVFF. 287,341 Particularly, Soto et al. developed the LPFFD sequence which can bind to the hydrophobic core via hydrogen bonds. The peptide was shown to cross the blood-brain barrier and reduce amyloid deposition in transgenic AD models. 342,343 Moreover, Sato et al. developed peptides based on a glycine-aa-phenylalanine-aa-glycine-aa-phenylalanine framework (aa: variable amino acid), which disrupt sheet-to-sheet packing of Aβ fibrils by binding to the surface of the β-strands. Their molecules were shown to reduce Aβ toxicity on cultured rat cortical neurons. 344 Although a number of β-sheet breaking peptides have been developed, only methyl-lvffl (PPI-119) has completed phase I and II human clinical trials. 34

51 2. Theoretical Background 46 To increase peptides short half live in vivo, protease resistant, D-enantiomeric Aβ-binding peptides were developed. The Willbold group identified different candidates through mirror image phage display. 345 Among these, D3 is a 12-residue arginine-rich peptide with affinities of 1.1 µm and.12 µm (K d ) towards two different binding sites within Aβ (Figure 2-17). 346 D3 was shown to inhibit formation of fibrils and reduce Aβ-induced cytotoxicity in a dosedependent manner in vitro. Moreover, D3 reduced plaque load as well as inflammation and improved cognitive function when injected to brains or orally administered to mice, 19,16 indicating that the ligand can cross the blood-brain barrier 347 and is effective in vivo. 348 Figure 2-17: Amino acid sequence (top) and Lewis structure of the D3 peptide (bottom). Reprinted with permission from Ref. 16. Copyright (21) American Chemical Society. DOI: 1.121/cn157j With regard to the mode of action, it is proposed that D3 converts Aβ into nonamyloidogenic, amorphous, non-toxic aggregates. 34,16 In particular, the five arginine residues of D3 may electrostatically interact with the negatively charged glutamyl and aspartyl residues in the N-terminal region of Aβ. Moreover, hydrophobic interactions can arise with the Aβ aggregation nucleation sequence KLVFFA. 349 While inhibitory effects may result from D3 binding and shielding the Aβ-aggregation nucleation site, 35 fibril destruction and conversion into random coil structures may be the result of D3 intercalating with already formed β-strands and breakup of hydrogen bonds. 349,35 Notably, a new generation class of D-peptides has recently been developed by the Willbold group, in which some amino acids of D3 are substituted for an optimized binding to monomeric Aβ. Not only do these peptides bind to Aβ monomers with higher affinities than D3, but they also increased the inhibition of Aβ fibrillation and reduced Aβ-induced cytotoxicity more strongly. 351 Moreover, cyclization

52 2. Theoretical Background 47 and the introduction of an additional arginine residue to increase the overall net charge 352 and D3 dimerization 353 were shown to be strategies to optimize the performance of D3. With regard to heterofunctional hybrid molecules, Masserini et al. linked the peptidic Aβ aggregation inhibitor chemically to a cell penetrating peptide to improve delivery of the molecule to the brain. 354 A bifunctional antibody was designed by Yu et al., targeting the Aβproducing β-secretase and the transferrin receptor for BBB penetration in mice as well as in primates. 355,356 Niewoehner et al. fused an Aβ antibody with a single fragment of a transferrin receptor antibody and detected an increased delivery across the BBB and enhanced in vivo potency in an AD mouse model. 357 Following up the work on D3 and aminopyrazoles, a hybrid molecule was synthesized by the Schrader group to combine the β-sheet breaking properties of aminopyrazole trimers with the molecular recognition of D3 (Figure 2-18). 18 The hybrid showed enhanced performance and synergistic effects on Aβ aggregation. In cells, the complete suppression of Aβ oligomer formation and restoration of long-term potentiation was demonstrated, which were not observed with each compound individually. 18 In a follow-up study, aminopyrazole trimers were subsequently hybridized with an Aβ-specific antibody fragment. 358 The binding properties towards Aβ were evaluated by use of an enzyme-linked immunosorbent assays, showing up to fourfold affinity enhancements compared to the pure antibody fragment. It was hypothesized that the increased affinity resulted from simultaneous binding of the two moieties to different epitopes of Aβ. In this scenario, the antibody may bind to the N-terminal domain of Aβ and the aminopyrazole may bind to the LVFFA hydrophobic core. 358 Figure 2-18: Interaction of the hybrid compound aminopyrazole trimer-teg-d3 with Aβ. Anionic glutamate side chains of Aβ (orange) interact with the cationic D3 peptide (green). Aβ phenylalanines (gray) interact with the aminopyrazole trimer (red). 18 Copyright 21 by John Wiley Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc. DOI: 1.12/anie

53 2. Theoretical Background 48 As shown by the above examples, chemical linkage of two different molecules allows the design of 1:1-heterofunctional hybrids. To overcome the limitation of 1:1-stoichiometry, dendrimers may be employed. Dendrimers are a special class of monodisperse polymers, characterized by well-defined branches which are built in a stepwise manner. 359 These structures are interesting for nanomedical application, because multiple functionalities can be added in precise locations of the dendritic structure. However, their synthesis through the random-statistical approach always leads to mixtures of products and batch-to-batch variations. Hence, non-reproducible pharmacokinetics were observed for multifunctional dendrimers, which restricted their clinical applications until now. 359 As was explained in chapter 2.2, small inorganic nanoparticles can be synthesized with defined geometries and offer high specific surface areas for ligand immobilization. Hence, they represent promising alternatives to dendrimers. For example, a spherical gold nanoparticle with a diameter of 7 nm features a surface area of 154 nm² with 2363 gold atoms exposed at its surface (compare to Table S5). Due to steric hindrance, not every surface atom will be available for ligand binding, but ligand grafting densities of > 15 ligands per NP can be achieved (correlating to typical ligand footprints 1 nm² 39 ). Moreover, these NP-ligand conjugates can be designed to be multivalent and multifunctional, since the number of ligands per NP and the number of different ligand types can be flexibly varied Anti-Amyloidogenic Effects of Nanoparticles Nanoparticulate systems have not only been developed for the Aβ-based diagnosis of AD, 36 but different nanoparticle formulations have already been employed to interfere with Aβ aggregation. Unspecific effects of non-functionalized nanoparticles on protein aggregation have been described. Inhibition as well as acceleration of Aβ fibrillation were observed depending on the physicochemical properties of the nanoparticles (particle size, charge, shape and surface modification). 361,362,363 While Wu et al. report a promoting effect of TiO 2 nanoparticles on Aβ fibril formation, 364 negatively charged AuNPs inhibited fibrillation and induced fibril dissociation. 54 To induce specific Aβ targeting, particles have been tailored with ligands. These ligands include small molecules such as phosphatidic acid 365 and curcumin, 366 the Aβ-oligomer specific antibody NU4, 367 and the Aβ-peptides LPFFD, 368,369 LVFFARK 37 and D1, 371 respectively. 372 More examples for studies which investigated NP/amyloid interactions are listed in (Table 2-3).

54 2. Theoretical Background 49 Table 2-3: Nanoparticulate systems studied for interfering with amyloidogenic proteins. The studies are sorted according to the type of NP-protein interaction: acceleration (A) and inhibition (I) of fibril formation, destruction (D) of preformed fibrils, selective binding (S). Interaction type NP Ligand Target Study type Reference A Inorganic, Au, 5 nm MUA Model peptides physicochemical A Inorganic, Au, 3 nm PEG Lysozyme physicochemical A Inorganic, CdSe QD, ZnS QD, 4 nm A Inorganic, QDs Streptavidin A A A, I A; I D, S D; S Inorganic, TiO 2, 15-2 nm Polymer, Cerium oxide particles, quantum dots, carbon nanotubes, 6-2 nm Polymer, Poly(Nacryloyl-Lphenylalanyl-Lphenylalanine methyl ester), 57 nm Polymer, Polystyrene, nm Polymer, PLGA, 15-2 nm Inorganic, Graphene oxide DHLA HSA physicochemical Biotinylated α-synuclein physicochemical + cell assay - Aβ 1-42 physicochemical - PhePhe, AlaAla dipeptide human β 2 - microglobulin Aβ 1-4 physicochemical physicochemical Amino groups Aβ 1-4 physicochemical Curcumin; Tet-1 peptide Aβ 1-42 physicochemical ThioflavinS Aβ 1-42 physicochemical D; S Inorganic Au, 1 nm CLPFFD Aβ 1-42 physicochemical I I I I I I I I I I Inorganic, Graphene oxide Inorganic, Polyoxymetalates Inorganic, CdSe/ZnS quantum dots, 2.5 nm Inorganic, CdTe Quantum dots, 3-5 nm Inorganic, CdTe quantum dots, 3.5 nm Inorganic, Fe 2 O 3, 15 nm Polymer, polylactid NP, 118 nm Polymer, PLGA, 15 nm Polymer, n- butylcyanoacrylate, 5 nm Nanoliposomes, 143 nm protein corona (fetal calf serum) Aβ Aβ 1-4 physicochemical physicochemical + cell assay DHLA Aβ 1-42 physicochemical N-acetyl-Lcysteine Aβ 1-4 physicochemical + modeling Thioglycolic acid Aβ 1-4 ; Aβ 1-42 physicochemical PHFBA (poly - heptafluorobutyl acrylate) B6 peptide, NAPVSIPQ peptide Chitosan, coenzyme Q1 Polysorbate 8, clioquinol RI-OR2-TAT peptide Insulin Aβ 1-4 Aβ 1-4 ; Aβ 1-42 Aβ 1-42 Aβ 1-4 ; Aβ 1-42 physicochemical in vivo in vivo in vivo in vitro, in vivo Wagner et al. (21) 373 Zhang et al. (29) 243 Vannoy et al (21) 374 Roberti et al. (29) 55 Wu et al. (28) 364 Linse et al. (27) 375 Skaat et al. (212) 376 Cabaleiro- Lago et al. (21) 377 Mathew et al. (212) 378 Li et al. (212) 379 Kogan et al. (26) 368 Mahmoudi et al. (212) 38 Geng et al. (211) 56 Thakur et al. (211) 361 Xiao et al. (21) 381 Yoo et al. (211) 382 Skaat et al. (29) 383 Liu et al. (213) 384 Wang et al. (21) 385 Kulkarni et al. (21) 386 Gregori et al. (216) 387

55 2. Theoretical Background 5 I I Polymer, PLGA, 133 nm PEGylated dendrigraft poly-l-lysines, 97 nm 11 nm tarenflurbil Aβ 1-42 in vivo RVG29 glycoprotein (BBB) D-TJKIVW peptide (tau) BACE1 plasmid (Aβ) Tau, Aβ 1-4 ; Aβ 1-42 in vitro, in vivo I Polymer, 4 nm Aβ 1-4 physicochemical I, D Inorganic, Au, 3 nm Carboxylic acid Aβ 1-4 I, S S Protein, serum albumin, 1.5 µm Inorganic, Au nanoshells, 34 nm KLVFFC peptide Aβ 1-4 physicochemical + cell assay physicochemical + cell assay Sialic acid Aβ 1-4 physicochemical Muntimadugu et al. (216) 227 Liu et al. (216) 388 Cabaleiro- Lago et al. (28) 389 Liao et al. (212) 54 Richman et al. (211) 39 Beier et al. (27) 391 Sophisticated NP surface modifications already led to the development of multifunctional Aβtargeting conjugates. In this regard, Prades et al. designed multifunctional nanoconjugates to target Aβ aggregation. In the AuNP-CLPFFD-THR hybrids, the CLPFFD peptide acted as β- sheet breaker, whereas the THR peptide improved permeation across the BBB by interacting with the TfR. 222 Zhang et al. conjugated ligands for Aβ targeting (D1 peptide, QSH peptide) and the BBB translocation (TGN peptide) to polyethylene glycol-poly lactic acid polymer (PEG-PLA) nanoparticles. 371 In a follow up study they also encapsulated the beta sheet breaking peptide H12 into the bifunctionalized nanoparticles and demonstrated enhanced drug delivery to the AD plaques and a neuroprotective function in mice. 392 Nanoliposomes have been functionalized with phosphatidic acid and with a modified ApoE-derived peptide. While phosphatidic acid strongly bound Aβ (K D =.6 μm) and inhibited its aggregation, the ApoE-peptide faciliated BBB permeation. 393 Polylactic-co-glycolic acid (PLGA) particles have been trifunctionalized with an anti-transferrin-receptor antibody for BBB penetration, an Aβ antibody and a β-sheet breaking peptide for Aβ targeting in a proof-of-principle study by Loureiro et al At the beginning of the project, some examples on NP-based targeting of misfolded proteins were available, but most studies were limited with regard to the variability of the nanoconjugate design and/or the physicochemical functional readouts (Table 2-3, Figure 2-19). Few studies addressed how nanoconjugates could interfere with Aβ-induced toxicity on cells 54 56, but no study employed a cellular AD model (i.e. Aβ-overexpressing cells 9,395 ).

56 2. Theoretical Background 51 Figure 2-19: Exemplary, published results on the interaction of nano- and microparticles with Aβ, analyzed with typical techniques such as DLS (A 54, Copyright 212 by John Wiley Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc., DOI: 1.12/smll ), TEM (B, reprinted from Ref. 222, Copyright (212), with permission from Elsevier, DOI: 1.116/j.biomaterials ), ThioflavinT fluorescence (C, reprinted with permission from Ref Copyright 26, DOI: 1.121/nl516862) American Chemical Society, ) and cellular toxicity assays (D, Copyright 2 by John Wiley Sons, Inc. Reprinted from Ref. 39 by permission of John Wiley & Sons, Inc., DOI: 1.12/chem ). Most studies are restricted to the analysis of synthetic Aβ aggregation (A-D) and physicochemical characterization techniques (A-C). Only few publications directly compare the performance of pure particles (C), free ligands (C, D) and ligand-functionalized particles (C, D).

57 3. Materials and Methods Material and Methods 3.1 Nanoparticle Synthesis and Physicochemical Characterization For the generation of colloidal metal nanoparticles the technique of pulsed laser ablation in liquids was employed using an Nd:YAG nanosecond laser system at fundamental wavelength (Table 3-1). The pulsed laser beam was focused into a Teflon batch chamber, which contained the bulk metal target foil and a volume of 3 ml sodium chloride solution (c =.1 mm NaCl for AuNP, c = 1 mm NaCl for PtNP). The low ionic strength ablation solution was employed to quench nanoparticle size and electrostatically stabilize the particles. By applying a stirring batch chamber the advantages of higher reproducibility and productivity compared to laser ablation in a conventional batch could be combined. 148 The constant flow ensured that already ablated material was transported away from the laser beam. Table 3-1: Laser parameters used for the fabrication of colloidal AuNPs. Model Manufacturer PowerLine E Rofin Type Nd:YAG Wavelength 164 nm Pulse duration 8 ns Repetition rate 15 khz Pulse energy 367 µj Sport area.95 mm² Fluence 3.86 J/cm² Power A (at outlet) 5.5 W (behind scanner) Focal length 1 cm Working distance (scanner lens to 13.6 cm target) Water column 3 mm The colloid concentration was gravimetrically determined by weighing the metal target before and after laser ablation on an analytical balance. To generate a calibration curve, the mass concentration was correlated to the absorbance at 38 nm (Au) or 4 nm (Pt) via UV/Vis extinction spectroscopy. Within 15 min, approximately 22.5 mg metal nanoparticles were produced corresponding to a colloid concentration of 75 µg/ml (extrapolated productivity of 9 mg/h). 396 To reduce the width of the size distribution and eliminate particles with d>2 nm, colloids were centrifuged. Within the time of the work, centrifugation parameters were adjusted to prepare larger sample volumes (Table 3-2). Centrifugation times were calculated according to Formula (1), which is a combination of the Svedberg equation and the

58 3. Materials and Methods 53 Stokes-Einstein relation. 397 After centrifugation, the pellet was discarded, nanoparticles were taken from the supernatant and the concentration was determined via UV/Vis extinction spectroscopy. (1) 397 η: dynamic viscosity of the solvent [Pa s] ρ NP : particle density [g/cm³] ρ S : solvent density [g/cm³] r min/max : distance from the center of the rotor to the bottom/top of the solution in the centrifuge tube [cm] d NP : particle radius [nm] t: sedimentation time [s] ω: rotational speed, =2π*rpm/6 [rad/s] RCF: relative centrifugal force, =1.12*r[mm]*(rpm/1)² [x g] Table 3-2: Centrifugation parameters to generate a monodisperse size distribution. Time [min] Speed [rpm] Volume per vessel/per run [ml] RCF [x g] Cut-off radius [nm] Rotor radius [cm] Liquid height, r max -r min [cm] 5 4 5/ / / For most experiments, the colloid was diluted with ultrapure water (MilliQ, C, Merck Millipore) to a final concentration of 5 µg/ml. Colloids were characterized according to their optical properties (UV/Vis extinction spectroscopy), size (TEM, AFM, ADC, AUC, DLS) and charge (zeta potential) UV/Vis Extinction Spectrophotometry UV/Vis extinction spectroscopy is a spectroscopic technique in which a sample is irradiated with light of different wavelengths in the ultraviolet-visible spectral region. This induces electronic transitions in the analyte, resulting in a specific absorbance. The transmitted light is then detected relative to the incident light. A spectrophotometer consists of a light source, a sample holder, a monochromator and a detector. According to the Beer-Lambert law (formula 2), 398 the absorbance of a solution is directly proportional to the concentration of the absorbing species and the path length. UV/Vis spectroscopy can therefore be used for the quantification of absorbing species.

59 3. Materials and Methods 54 A: absorbance [-] b: molar absorption coefficient [L*cm/mol] c: concentration [mol/l] I: Intensity of incident (I ) and transmitted light [-] l: path length [cm] (2) UV/Vis spectroscopy measurements were carried out in 1.5 ml quartz cuvettes with 1 mm path length, covering a spectral range from 19 nm to 9 nm with a data interval of 1 nm. The integration time was set to.5 s and the scan speed to 12 nm/min. Stability over time was analyzed for up to eight samples in parallel by using a mechanic cell changer. Spectra were automatically recorded every 15 min for up to 12 h Analytical Disc Centrifugation (ADC) Analytical disc centrifugation relies on the principle of differential centrifugal sedimentation. While the analytes sediment according to their size and shape, the intensity of each analyte fraction (λ=45 nm) reaching the detector is recorded vs. the time. By comparing the sedimentation time of the analytes to standard particles of known size and density, the sedimentation time can be transferred into a particle size distribution by solving equation (1) for the particle diameter, resulting in formula ,4 (3) The ADC consists of an optically clear disc, a light source and a detector (Figure 3-1). After the sample is injected in the center of the disc, the analytes sediment outwards and are detected through absorbance measurements at the outer edge of the disc. For efficient particle separation and prolonged sedimentation times, a saccharose density gradient is employed as solvent. Figure 3-1: Front view and cross-sectional view on an analytical disc centrifuge. 4

60 3. Materials and Methods 55 The raw data is converted into a weight-, number- and surface-related size distribution based on the detected sedimentation time and the absorbance of light. Note that quantitative determination of metal NP size populations is due to the deviation of the scattering intensities of particles < 5 nm from Mie theory. 4 Moreover, for materials other than metals, backdiffusion of small, low-density particles can lead to non-ideal sedimentation. 41 Charged and hybrid particles (i.e. ligand-np conjugates) can make ADC analyses challenging due to the density of the particle (which will be lower than that of the bulk/core material) and has to be specified prior to the measurement. 42 Measurement parameters were set as follows: Calibration Std. Diameter:.237 microns Calibration Std. Density: g/ml Standard Half Width:.15 micros Maximum Diameter:.3 microns Minimum Diameter:.3 or.4 microns Particle Density: 19.3 g/ml Particle Refractive Index: 1.47 Particle Absorption: 1.92 Particle Non-Sphericity: 1 Liquid Density: 1.64 g/ml Liquid Refractive Index: Liquid Viscosity: 1.5 cps The hydrodynamic particle size was analyzed via ADC at a centrifugation speed of 24, rpm with a lower detection limit set to 3-4 nm. A saccharose gradient was established by injecting solutions with different saccharose concentrations into the spinning centrifuge (24 wt%, 22 wt%, 2 wt%, 18 wt%, 16 wt%, 14 wt%, 12 wt%, 1 wt%, 8 wt%, each 1.6 ml), resulting in a continuous gradient. An external standard (PVC particles, d=.237 µm, V=.1 ml) was employed for size calibration and a volume of.1 ml sample was injected afterwards. Generally, the same density gradient was employed for the subsequent analysis of several colloid samples. However, when analyzing the size distribution of ligand-np conjugates containing residual (unbound) ligands, a new gradient was prepared for each sample in order to avoid cross-contaminations (compare to Figure 4-19). Each distribution was analyzed with a log Normal fit. The x C -value of the fit was taken as average particle diameter. The polydispersity index (PDI) was calculated from the resulting x C -value and the standard deviation w² of the distribution:

61 3. Materials and Methods 56 (4) (5) The PDI is a dimensionless parameter which characterizes the width of nanoparticle size distributions. Monodisperse colloids feature PDI values in the range of.3 to.6, whereas polydisperse samples feature PDI values >.5. Narrow size distributions are characterized by PDIs in the range of.1 to.2 and wide size distributions by PDIs in the range of.25 to Analytical Ultracentrifugation (AUC) Similar to analytical disc centrifugation, samples in AUC are exposed to a centrifugal field and sediment according to their size, density and shape. In contrast to ADC, AUC detects the evolution of the sample concentration as function of the time and local position. The movable detector scans the complete cell, so that much more data points and higher resolutions can be obtained. Since AUC is a first principle method, no size calibration is required. Moreover AUC avoids problems arising from matrix interactions in size-exclusion chromatography, as dissolved samples can be analyzed in their respective buffer. 44 The Lamm equation describes the basic principles of AUC, i.e. the analysis of the temporal and spatial change of the analyte concentration, due to its diffusion and sedimentation in the centrifugal field, where D is the diffusion coefficient [m²/s] and s is the sedimentation coefficient [1 S = 1 Svedberg = 1-13 s]: (6) The AUC is composed of an ultracentrifuge, a multicompartment rotor, and an optical detection system. Detection systems can include a fluorescence spectrometer, a UV/Vis spectrophotometer and/or a laser interferometer, which records the refractive index changes. Fluorescence optics are highly sensitive but require the analyte to be labeled with a fluorophore. In contrast, interference optics can be used if the analytes do not absorb light. While the strength of the interference optics lies in its high sensitivity and fast scan time (1-3 s), it cannot distinguish between species if they cause similar changes in the refractive index. In contrast, the absorbance optics has a relative slow scan time (9 s), but can specifically analyze absorbing species at a selected wavelength. 45

62 3. Materials and Methods 57 In principle, two different types of experiments - sedimentation velocity and sedimentation equilibrium - can be performed with AUC. Sedimentation equilibrium experiments are performed at low speed centrifugal fields for the establishment of a steady-state between sedimentation and diffusion. This results in a time-independent concentration profile, which is related to the analyte s molar mass. In contrast, sedimentation velocity experiments are employed at high speed centrifugal fields to gain information about sample shape, molar mass or the size distribution. When centrifugal force, buoyancy and friction are at equilibrium, particles sediment at constant speed. During the centrifugation, the sample becomes depleted, which is detected as distinct solute/solvent boundary ( meniscus ) migrating to the bottom of the cell. This concentration profile is evaluated over the entire sedimentation time to determine the sedimentation coefficient distribution (Figure 3-2). 46 Figure 3-2: AUC cell including reference and sample sector and the resulting absorbance profile as function of the radial position (left, Reproduced from Ref. 46 with permission of The Royal Society of Chemistry, DOI: 1.139/CNR215A). Characteristic profiles of a sedimentation velocity experiment as a function of the centrifugation time (right, reprinted from Ref. 45, Copyright 28, with permission from Elsevier, DOI: 1.116/S91-679X(7)846-4). For known diffusion constants, sedimentation coefficients obtained from sedimentation velocity experiments can be transformed into molecular masses or diameters of spherical particles via the Svedberg equation: 46

63 3. Materials and Methods 58 (7) (8) R: gas constant [J/mol K] T: temperature [K] : partial specific volume, 1/ρ [cm³/g] Sedimentation velocity experiments of colloids are sometimes more complicated than of commonly investigated biomolecules. Reasons for this include the presence of broad particle size distributions, resulting in broad sedimentation coefficient distributions. Hence, more than one centrifugation speed, temperature and/or solvent density may be required for the detection of the different species. Another point to consider is that sedimentation velocity experiments can take more than 8 h so that particle properties may change during the centrifugation, e.g. due to aggregation. In the case of charged particles, electrical double layer repulsion can retard the sedimenting particles. In this case, the addition of salt may be required to screen the inter-particle repulsion. A general problem with plasmonic nanoparticles and their detection via light absorbance is that the particles scatter light not only as function of their concentration but also of their size, which may require correction. 46 Sedimentation velocity experiments were carried out using an analytical ultracentrifuge with an eight-hole rotor (An-5 Ti Rotor). Samples (V=35 µl) were loaded in a double-sector quartz cell and centrifuged against water as reference (V=4 µl) at 3 rpm (724 x g) overnight. Radial scans were recorded at a resolution of.3 cm. Absorbance as well as interference detectors were employed and data was evaluated using the software SEDFIT with the c(s) and ls-g*(s) models Transmission Electron Microscopy (TEM) Transmission electron microscopy enables the direct imaging with a high-voltage, focused electron beam, which transmits thin (1-1 nm) specimen. To prevent electron deflection by air molecules, TEM is conducted in vacuum. As the electron beam passes through the sample, electrons are diffracted by atomic planes of the sample. In this way, transmission electron diffraction patterns are formed, magnified and focused onto an imaging device.

64 3. Materials and Methods 59 Typical electron accelerating voltages range between 8 kv and 4 kv, while the resolution of TEM images can reach the sub-nanometer range. The image contrast is determined by the thickness of the sample and the material-dependent absorption of electrons. Hence, on carboncoated grids as sample holders, metal specimen give good contrasts, while organic samples mostly feature a contrast similar to the sample holder. Consequently, organic ligands cannot be directly visualized in TEM without staining. However, the determination of interparticle distances (IPDs) between ligand-coated nanoparticles can give indications on the presence of adsorbed ligand layers. 47 For nanoparticle size analysis, a droplet of 5-1 µl of the sample was placed on a carboncoated copper grid and air-dried. TEM was performed by Dipl.-Ing. Jurij Jakobi using a Philips CM12 microscope at an acceleration voltage of 8-12 kv Dynamic Light Scattering (DLS) Dynamic light scattering is a noninvasive, quantitative, optical method with which the diffusion coefficients of particles in solution can be determined. In DLS, the scattering of a laser beam entering a liquid sample is detected. Since particles in solution feature Brownian motion, the scattering intensity fluctuates over time and undergoes constructive or destructive interference. These interferences contain information on the velocities of the scattering specimen. Via an autocorrelation function and the determination of a decay rate, the particles diffusion coefficients are derived and calculated into the hydrodynamic sizes via the Stokes- Einstein relation: 399,48 (9) k B : Boltzmann constant (=1.381x1-23 J/K) Since DLS is based on particle diffusion, the measurement is sensitive to changes in temperature and viscosity. Moreover, the analysis of a polydisperse sample can be biased in the presence of large aggregates. In particular, few large specimens will dominate the apparent size distribution, since the intensity of the scattered light is proportional to the sixth power of the particle diameter. Another disadvantage of DLS is its limited resolution. In fact, two size populations can only be resolved if they differ in at least a factor of two. 49

65 3. Materials and Methods 6 DLS measurements were performed at 25 C using a disposable U-shaped capillary cell (DTS 17, Malvern Instruments) with a sample volume of 75 µl. The measurement parameters of aqueous AuNP samples included: Refractive index:.197 Absorption: 3.91 Solvent viscosity:.8872 cp Solvent refractive index: 1.33 Equilibration time: s Measurement angle: 173 (backscatter) Number of runs: 3 Run duration: 1 s Number of measurements: Zeta Potential Measurements Zeta potential measurements are based on electrophoretic light scattering. To measure the zeta potentials of particles in solution, they are exposed to an alternating electric field. This induces particle movement, which is detected by light scattering events and Doppler frequency shifts. By taking into account the scattering angle and the wavelength of the incident light, the frequency shifts can be converted into the particle electrophoretic velocities. Zeta potentials can then be calculated from the electrophoretic velocities using different models. The Smoluchowski model can be employed to determine the zeta potentials of larger particles in high ionic strength medium (formula 11). In contrast, the Hückel model is valid for small particles and low ionic strength solvents (formula 1, Figure 3-3). Because zeta potential measurements are based on light scattering, the results may be dominated by the signals of large, strongly scattering particles. 14,16 Hückel 16 (1) Smoluchowski 16 (11) μ: electrophoretic mobility [µm/s/(v/cm)] η: solvent viscosity (8.9x1-4 Pa s for water at 25 C) κa: ratio of the particle radius and the electrical double layer thickness ε: solvent dielectric constant (8 for water at 25 C) ε : vacuum permittivity (ε 8.854x1-1 C²/Nm²)

66 3. Materials and Methods 61 For each sample, zeta potential measurements were conducted in triplicate. A disposable U- shaped capillary cell (DTS 17, Malvern Instruments) was employed with a sample volume of 75 µl. Results were interpreted by using the Hückel model. For ph-dependent stability analyses, AuNPs were manually titrated with.1 M and 1 M NaOH or HCl and the ph was recorded with a PCE-PHD ph-meter and a microelectrode (Sartorius) at 23 C before zeta potential measurements (Table S1). Figure 3-3: Applicability ranges of the Hückel and Smoluchowski models as function of the particle size and ionic strength of the medium (A). Relationship between the electric double layer thickness and the Hückel and Smoluchowski regimes (B). 16

67 3. Materials and Methods Ligand Conjugation and Characterization of Nanoparticle/Ligand Conjugates To functionalize colloidal NPs, the following small thiolated stabilizers, model serum proteins and Aβ-targeting ligands were employed, which are summarized in Table 3-3 and Table 3-4. An overview of the size scales of typically employed nanoparticles and ligands is given schematically in Figure 3-4. Table 3-3: Small thiolated ligands and serum proteins for nanoparticle conjugation experiments and their characteristic properties (IEP: isoelectric point). Material Manufacturer Mol. weight [g/mol] Purity [%] IEP/ pk A * No. of amino acids/ sum formula * No. of sulfur atoms* Charge (+/-) * Amyloid β 1-42 (Aβ) Bachem 4514 > /3 Amyloid β 1-42, FITClabeled (Aβ-FITC) Bachem /3 Bovine serum albumin (BSA) ChemCruz > /86 Bovine serum albumin, Sigma FITC-labeled (BSA-FITC) Aldrich /86 Insulin Sigma Aldrich 5733 > /2 Insulin, FITC-labeled Sigma (Insulin-FITC) Aldrich /2 Insulin-degrading enzyme (IDE) Abcam /123 Lipoic acid (LA) Sigma Aldrich C 8 H 14 O 2 S 2 2 (-1) Mercaptoundecanoic acid Sigma (MUA) Aldrich C 11 H 22 O 2 S 1 (-1) O-(2-Mercaptoethyl)-O - Sigma methyl-hexa(ethylene Aldrich glycol) 35 (mpeg-sh) 356 >95 - C 15 H 32 O 7 S 1 O-[2-(3- Mercaptopropionylamino)- ethyl]-o -methylpolyethylenglycol 5 (PEG5) Transferrin Sigma Aldrich Sigma Aldrich 7219 >95 - C 6+2n H 13+4n O 2+n NS (n=162) 1 (+1) 775 > /85 *Note that for the protein sequences, data were obtained from the UniProt data base and the ExPASy Bioinformatics Resource Portal.

68 3. Materials and Methods 63 Table 3-4: Functional peptide and aminopyrazole trimer ligands and their characteristic properties. Material Amino acid sequence Mol. weight [g/mol] Net ph=7 IEP Hydrophilic residues Manufacturer CE12W CysGlu 12 Trp % Genosphere D3-Trp TrpArgProArgThrArg- LeuHisThrHisArgAsnArgCys % D3-Trp4 Trp 4 ArgProArgThrArg- LeuHisThrHisArgAsnArgCys % D3-Cou CysArgProArgThrArg- LeuHisThrHisArgAsnArg- Coumarin % Laura Akkari, Department of Organic Chemistry D3_5+ ArgProArgThrArgLeu- (University of % HisThrHisArgAsnArgCys Duisburg-Essen) D3_8+ ArgProArgThrArgLeuHis- ThrHisArgAsnArgLys 3 Cys % D3_1+ ArgProArgThrArgLeu- HisThrHisArgAsnArgLys 5 Cys % NLS GlyTrpGly 3 ProLys 3 - ArgLysValGluAsp % Genosphere NLS-SH CysTrpGly 3 ProLys 3 - ArgLysValGluAsp % Genosphere R5WC Arg 5 TrpCys % ChinaPeptides Trim_5+ Aminopyrazole trimer-lys 5 Cys % Laura Akkari, see Trim_8+ Aminopyrazole trimer-lys 8 Cys % above Figure 3-4: Typical ligands and biomolecules employed in this thesis, approximately drawn to scale relative to a 7 nm AuNP (288 kda): transferrin (77 kda), BSA (66 kda), insulin hexamer (3 kda), Aβ monomer (4.5 kda), D3 peptide (1.7 kda), aminopyrazole trimer (1.1 kda), mpeg-sh (.4 kda), lipoic acid (.2 kda), mercaptoundecanoic acid (.2 kda).

69 3. Materials and Methods 64 To determine ligand-to-np ratios [# ligand/np] and ligand surface concentrations (pmol ligands/cm² colloid], the NP number and surface need to be calculated. By assuming that all particles have a constant average size and a spherical shape, the number of nanoparticles per ml (, ml -1 ) and the total surface of the colloid (, ml -1 ) can be derived from the following equations: 396 : nanoparticle mass[µg] : nanoparticle surface area [cm²] : nanoparticle volume [cm³] : mass concentration [µg/ml] (12) (13) Moreover, when determining the amount of ligands present at the nanoparticle surface, the absolute number of ligands per nanoparticle can be calculated as follows: 396 (14) : Avogadro constant (6.23x1 23 mol -1 ) [1/mol] The number of bound ligands per particle can be quantified if ligands feature a characteristic absorbance peak in the UV/Vis range (e.g. tryptophan-labeled ligands) or a characteristic fluorescence emission (e.g. coumarin-labeled ligands). For the quantification of bound ligands, conjugates were centrifuged at 1, x g for 1 h at 7 C (Optima Max-XP, Beckman Coulter). Under these conditions, nanoparticles with bound ligands formed a pellet, whereas unbound ligands remained in the supernatant. Ligands in the supernatant were quantified spectrometrically against a calibration of the pure ligand solution. The number of bound ligands per nanoparticle was calculated as the difference from the initially applied number of ligands and the number of unbound ligands. Table 3-5 shows the composition of typical conjugates employed for functionality tests. Note that the number of ligands refers to the applied ligand dose and may deviate from the number of ligands, which is actually bound on the particle surface (see chapter 4.2.4).

70 3. Materials and Methods 65 Table 3-5: Applied ligand-to-np ratios and NP surface concentrations of ligands for typical nanobioconjugates employed in functionality tests. The molar ligand concentration refers to a gold concentration of 5 µg/ml. When the gold concentration was changed, the ligand concentration was adjusted by assuming a linear correlation. Molar concentration [µm] Ligand-to-NP ratio Surface concentration [pmol/cm²] cationic cationic cationic neutral anionic anionic Conjugate s net charge (Au/D3_5+) Fluorescence Spectrometry Fluorescence results from a three-stage process described by the Jablonski diagram. It involves the creation of an excited state by the absorption of an external photon, residence in the excited state on a timescale of nanoseconds and subsequent emission of fluorescence (Figure 3-5). During the excited state, the fluorophore can alter its conformation and interact with its environment, by which energy is partially dissipated nonirradiatively. Hence, the energy of the emitted photon is lower than that of the absorbed photon, leading to a characteristic shift in wavelengths from the excitation to the emission spectra ( Stoke s shift ). 41 Figure 3-5: Simplified Jablonski diagram illustrating the fluorophore s excitation through absorption, internal conversion and fluorescence emission. Different (environmental) effects can lower the fluorescence emission (modified from Ref. 41, with permission of Springer). A reduced fluorescence can result from (reversible) quenching or (irreversible) photobleaching. While photobleaching includes the chemical destruction of the fluorophore, quenching processes can result from short-range interactions, either in the excited state (collisional quenching, fluorescence resonance energy transfer, eximer formation) or by the

71 3. Materials and Methods 66 formation of nonfluorescent ground-state species ( static quenching ). The extent to which these processes occur is described by the fluorescence quantum yield, which represents the ratio of the number of emitted to absorbed photons. 41 Fluorescence detection systems consist of an excitation light source, wavelength filters to isolate emission photons from excitation photons and a detector. Important experimental parameters, which determine the fluorescence quantum yield, are the fluorophores concentration, solvent polarity and the ph. Consequently, fluorescence emission depends not only on the parameters given by the Beer-Lambert law for absorbance (i.e. the molar extinction coefficient, the optical path length and the solute concentration), but also on the fluorophore s quantum yield, the excitation intensity and emission collection efficiency. 41 When working with colloids (in particular plasmon-resonant NPs), interferences with fluorescent dyes can occur and such NP-induced quenching effects 115,411 will be discussed in section In this work, a fluorescence spectrometer and a microplate reader were employed. In the fluorescence spectrometer, samples were analyzed in a quartz cuvette. Excitation and emission slits were generally set to 5 nm and 2.5 nm, respectively, while the scan control was set to medium and the detector voltage to high. The competition of serum proteins and Aβ for binding sites on nanoconjugates was investigated by Marcus Hildebrandt (under my cosupervision). For the analysis of the quenching experiments, a Tecan InfiniTe microplate reader was used. For measurements in the microplate reader, samples were prepared in black multiwell plates, using volumes of 9 µl. The data interval to record fluorescence emission was generally set to 1 nm at 1% gain, with 25 flashes and 2 µs integration time. If not noted otherwise, fluorescence excitation and emission wavelengths were 485 nm and 52 nm (FITC-labeled proteins) and 328 nm and 393 nm (coumarin-labeled D3), respectively Affinity Experiments To assess protein binding on the nanoparticle surface, affinity experiments were conducted with FITC-labeled Aβ and BSA. The measurement is based on the fact that AuNPs quench the fluorescence of a fluorophore, if it approaches the NP surface. In particular, the fluorophore s proximity to the AuNP surface determines the extent of fluorescence quenching. 412,413

72 3. Materials and Methods 67 Different effects may reduce fluorescence emission and result in static quenching, including concentration quenching or complex formation (compare to chapter 2.2.1). 41 By assuming that protein binding occurs at equilibrium, the quenching data can be fitted with the Hill formula: (16) 414 B: fluorescence quenching [-] B max : maximum quenching [-] K D : dissociation constant[nm] n: cooperativity factor [-] In this term, the determination of the dissociation constant, K D, allows an estimation of the compound s binding strength for the target. Since K D is reciprocal to the association constant K A, a low dissociation constant correlates to high binding affinities. Kinetically, binding affinity is a measure of the ratio of on-rate (k on ) and off-rate (k off ). For multivalent interactions, the enhancement of binding is more often due to a decreased dissociation rate than to an increase in the association rate. 28 From a thermodynamically point of view, the formation of multivalent ligand-receptor interactions is likely to occur, when they support the formation of energetically favorable, molecular conformations of the ligands. 28 The cooperativity factor n contains information about the impact of already bound compounds to the binding of further ligands. In this respect, cooperativity describes that the number of ligand binding sites is a nonlinear function of the ligand concentration. 415 Specifically, a positive cooperativity factor (n > 1) indicates that preliminary binding of ligands supports the binding of further ligand. In contrast, a negative cooperativity factor (n < 1) indicates that ligands hinder each other so that the binding strength decreases as further ligands adsorbs. 412 To determine K D -values, titration experiments were performed in 384 well plates (Nunclon 384 Flat Bottom Black Polystyrol, Nunc GmbH). A stock solution of gold colloid (c = 5 µg/ml) was conjugated with ligands (c = 1 µm, 3 µm, 1 µm, 1 µm), corresponding to ligand-to-np ratios of 418:1, 125:1, 41:1 and 4:1. A 1:1 colloid dilution series was created with a total volume of 45 µl per well. Equal amounts of labeled protein stock (c =.25 µmol/l Aβ-FITC; V = 45 µl) were added. Fluorescence was measured in a Tecan InfiniTe 2 microplate reader at room temperature. The excitation wavelength was 485 nm for FITC-labeled proteins (emission at 52 nm). Normalized quenching was defined

73 3. Materials and Methods 68 as the ratio between the apparent fluorescence in the presence of gold nanoparticles (I -I) and the fluorescence of the pure fluorophore (I ) Gel Electrophoresis Electrophoresis is the movement of charged species in an electric field. As described in the theory of the zeta potential, the velocities of the analytes depend on their size, shape and charge, the electric field strength, their dielectric constant and the viscosity of the medium. In molecular biology, gel electrophoresis is a routine method for the separation and detection of biomolecules such as proteins 416 and DNA. 417 In agarose gel electrophoresis, the liquid sample is applied to an agarose gel immersed in buffered solution and connected to a direct current voltage source. The analytes migrate through the polymerized agarose network in the direction of the electrode of opposite charge. To adjust the spatial resolution, the agarose pore diameter can be tuned by varying the agarose concentration (typically.5-4%). Moreover, buffer concentrations and voltage have to be adjusted. Gel electrophoresis experiments were conducted by Dennis Brungs Burau (University of Duisburg-Essen, under my co-supervision). In all experiments, native electrophoresis was employed, in which proteins migrated in their native conformation. Gold colloids were typically concentrated by a factor of 3.5 to 15 in Amicon 3, filters (up to 15 consecutive centrifugation steps of 35 s at 32 rpm). The gold concentration was varied between 4 and 22 µg/ml. A volume of 6 µl concentrated AuNPs, 2 µl 87% glycerol and -2 µl protein stock solution were mixed and filled to a final volume of 1 µl. For 1% agarose gels, 1 g agarose was boiled in 1 ml.5x TBE buffer and 4 ml of this suspension were applied per gel. A sample volume of 1 µl was applied per gel pocket. After gel electrophoresis, gels were scanned in an Epson V2 Photo scanner. The image contrast and brightness were adjusted by using the ImageJ software (National Institute of Health, Bethesda, MD, USA). 3.3 Characterization of Aβ aggregation states To characterize the Aβ aggregation process different methods were employed: For the analysis of the Aβ secondary structure, Thioflavin T (ThT) fluorescence assay and circular dichroism (CD) spectroscopy were conducted. For the analysis of the Aβ aggregate morphology, microscopic techniques including atomic force microscopy (AFM) and transmission electron microscopy (TEM) were employed. For the analysis of the Aβ

74 3. Materials and Methods 69 aggregate size distribution, density gradient centrifugation combined with an enzyme-linked immunosorbent assay (DGC/ELISA) and cell-based assay were performed. As will be discussed in this chapter, each technique features specific strengths and weaknesses so that a combination of (partly complementary) methods is required to understand the Aβ aggregation processes. For example, the ThT fluorescence assay and CD spectroscopy give information on the amount of β-sheets and the Aβ secondary structure, respectively, without differentiating Aβ species of different sizes. In contrast, all Aβ species are separated and quantified through DGC/ELISA with respect to their size and shape, independent of their secondary structure. Moreover, the optical, the immunologic and the cellbased assays are performed in solution. In contrast, the microscopic techniques visualize only those Aβ species, which are deposited on substrate surfaces at defined (low) ph. Hence, one needs to keep in mind that this may not represent the species distribution in solution and that the deposition on the substrate surface itself can impact the aggregation of Aβ species Preparation of Aβ The Aβ 1-42 peptide (Bachem, Germany) was pretreated by Laura Akkari in the department of Organic Chemistry (AK Schrader, University of Duisburg-Essen). It was monomerized overnight in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma, Germany) and the lyophilized peptide was stored at -2 C for further use. Proportions of the peptide were taken by dissolving Aβ at a concentration of 1 mg/ml in HFIP, transferring it into new vials and lyophilizing it before further use Circular Dichroism (CD) Spectroscopy In CD spectroscopy, the differential absorption of circularly polarized light by chiral centers of the analyte is measured as a function of the wavelength. For peptides and proteins, CD spectra in the UV range (18 25 nm) give information about the molecules secondary structures, whereas near-uv CD spectra (25 35 nm) are influenced by aromatic side chains and disulfide bonds. Specifically, the appearance of an absorption minimum at nm shows the formation of β-sheets (Figure 3-6). 418 In contrast to dye-based methods, CD can be used over various ph ranges and temperatures. CD is an averaging technique and provides information about the conformational state of the entire sample. Therefore, one cannot distinguish between intra- and intermolecular inhomogeneities in secondary structures. 418

75 3. Materials and Methods 7 CD measurements were performed by Laura Akkari in the department of Organic Chemistry (AK Schrader, University of Duisburg-Essen). For the preparation of CD samples, Aβ was dissolved in HFIP to a concentration of 5 μm. The solution was further diluted with 1 mm potassium phosphate buffer to a final peptide concentration of 1 μm. Each sample was composed of 1 μm, 1 μm ligand, and 5 μg/ml AuNP. Samples were incubated for 1 d in a thermomixer (65 rpm) at room temperature; spectra were recorded after h, 1 h, and 1 d. Circular dichroism measurements were carried out on a J-815 spectropolarimeter (Jasco) at 2 C in a.1 cm cell between 2 and 4 nm (data pitch: 1 nm, response: 1 s., sensitivity: standard, scanning speed: 1 nm/min, accumulation 1). 372 Figure 3-6: CD spectra of protein secondary structures (adapted from Ref. 419 ) Thioflavin T (ThT) Fluorescence Assay Thioflavin T is a fluorescent dye with polar and nonpolar functional groups, forming micelles in aqueous solutions above a critical concentration of 4 µm. 418 Free ThT features excitation and emission wavelengths of 342 nm and 43 nm, respectively. Upon binding to β-sheet structures excitation and emission wavelength of ThT shift to 444 and 482 nm, respectively. 418 The mechanism responsible for fluorescence enhancement upon β-sheet binding is not fully revealed, but may involve ThT self-assembly into micelles and/or conformational changes. 42 In the free ThT molecule, the intramolecular rotation may quench its excited state, causing low fluorescence emission. In contrast, the immobilization of ThT in

76 3. Materials and Methods 71 β-sheets may prevent its rotation, preserve the excited state and result in a stronger fluorescence (Figure 3-7). 42 Figure 3-7: Molecular structure of ThT and scheme of its intramolecular rotation (left). Characteristic fluorescence emission in the absence and presence of amyloid fibrils (right). Adapted from Ref. 42, Copyright (21), with permission from Elsevier. ThT molecules may bind to the grooves in β-sheets and the extent of binding depends on the accessibility of these grooves. Hence, false negative results can also occur when Aβ species assemble in such a way that ThT cannot bind to the surface of the β-sheets. Moreover, multiple reagents including glycerol, PEG and curcumin may inhibit ThT binding through direct competition with the binding sites within the β-sheet structures and lead to false negative results. 42 In addition, it needs to be considered that ThT shows structural similarity to many amyloid inhibitors, so that ThT itself may influence fibril formation. 42 Notably, ThT has also been shown to bind to serum albumin 421 so that (Aβ-free) controls are always required to verify that ThT does not interact with any component other in the sample matrix per se. 422 ThT fluorescence measurements were conducted by Laura Akkari in the department of Organic Chemistry (AK Schrader, University of Duisburg-Essen). Samples were prepared in triplicates or quadruplicates. Each sample (6 µl) was composed of 1 µm Aβ 1-42 in 1 mm phosphate buffered saline, 3.3 µm ThT (Sigma, Germany) and 6 µm of the test compound. After 5 d of incubation in a thermomixer (65 rpm) at 37 C, ThT fluorescence was determined. Each measurement cycle was started by shaking the sample carrier orbitally for 3 s at medium intensity to avoid settling of larger aggregates. Fluorescence intensity was measured in 384-well plates with a Tecan InfiniTe 2 at 37 C, 446 nm excitation wavelength (bandwidth 9 nm) and 49 nm emission wavelength (bandwidth 2 nm). Each data point was averaged over 4 lamp flashes. Each test compound was measured separately, both in 1 mm PBS and 1 mm PBS with ThT to exclude any potential interactions between the ligand and ThT. 372

77 3. Materials and Methods Atomic Force Microscopy (AFM) of Aβ Fibrils Atomic force microscopy is a microscopic technique enabling to study surface topographies under ambient conditions and without staining. Images are gathered by mechanically scanning a surface with a sharp, nanodimensional probe. Hence, in contrast to optical microscopy, the resolution of AFM is not limited to the diffraction limit of light. The probing tip is attached to a cantilever. In response to the force between tip and sample, the cantilever is deflected. These deflections are measured using a laser beam, which is reflected from the top surface of the cantilever into a photodiode. By recording the distance in which the cantilever moves in the z-direction, a topographic image of the sample surface is generated. Feedback mechanisms are employed to maintain a constant force between tip and sample (Figure 3-8). 423 Due to the movement of the AFM tip over the sample surface and its deflection upon approaching the specimen, the analyte s size is better reflected by the height signal (zdirection) than by the signals in the x,y-plane. Electronic and thermal noise limit resolution in the z-direction, so that the height measurement range lies between sub-nanometer to several tens of microns. 424 Besides height signals, phase and error signals can be obtained via AFM. The former is the signal resulting from a phase shift between the driven oscillation of the cantilever and the measured movement. It enables to detect differences in chemical composition, adhesion and friction properties. The latter corresponds to a deflection signal, highlighting edges and producing images with higher resolution. It can be seen as equivalent to the first deviation of the topographic image. 425 Figure 3-8: AFM setup consisting of a cantilever and tip, scanning the sample surface, whereby their deflection is detected to generate a 3D topographical image.

78 3. Materials and Methods 73 One distinguishes between contact, intermittent-contact ( tapping ), and non-contact mode. In non-contact mode, the tip is separated from the sample at a distance of 1 to 1 nm. The cantilever is externally oscillated. Long-range forces, such as van der Waals forces, are detected as changes in oscillation. In contact mode, the distance between tip and sample is in the range of.1 nm, so that the surface topography is directly detected by sensing short-range repulsive forces. Intermittent-contact mode is the most frequently employed mode under ambient conditions, combining the benefits of the other two modes. Here, an oscillating cantilever drives the tip to the sample surface at constant amplitude. As the tip approaches the surface, the amplitude of the oscillation changes due to weak interactions with the sample. A feedback-loop adjusts the height to maintain a fixed cantilever oscillation. Thereby the tip touches the surface only intermittently. Lateral forces and dragging of the sample are avoided by contacting the sample only for short times. 425 Atomic force microscopy was conducted by Jenny Bormann (Chemical Biology, AK Saccà, University of Duisburg-Essen). For AFM experiments, Aβ fibrils were preformed from monomerized, lyophilized Aβ in 1 mm HCl at a monomer concentration of 1 µm at 37 C (shaking at 65 rpm) for 24 h, according to a protocol from Stine et al Insulin fibrils were prepared with slight modifications of the protocol established for Aβ fibrils. Insulin was incubated at a concentration of 2 mg/ml in 4 mm HCl for 5 d at 37 C shaking (65 rpm). After the incubation period, samples of pure AuNPs, pure D3 and AuNP/D3 conjugates were added to the protein fibrils (1:1 volume ratio). Final concentrations were 5 µm Aβ, 125 µg/ml AuNPs and 25 µm D3. The samples were further incubated for 7 d at 37 C (shaking at 65 rpm). AFM analysis was performed directly after addition of pure AuNPs, pure D3 and AuNP-D3 conjugates to Aβ, after 24 h and after 7 d. Briefly, 1 µl aliquots were deposited on freshly cleaned mica substrates (Plano GmbH). After incubation for 5 min, the sample was dried under gentle argon flow and scanned in ScanAsyst Mode using MultiMode TM microscope (Bruker) equipped with a Nanoscope V controller..4 N/m force constant cantilevers with sharpened pyramidal tips (ScanAsyst-Air tips, Bruker) were used for scanning. After engagement, the peak force setpoint was typically.2 V and the scan rates were about 1 Hz. Image processing was performed with the NanoScope Analysis software (version 1.5, Bruker). Fibril length and height were evaluated with Gwyddion software ( ImageJ software (National Institute of Health, Bethesda, MD, USA) was employed to determine the substrate coverage with fibrils. 372

79 3. Materials and Methods Transmission Electron Microscopy of Aβ Fibrils Similar to AFM analyses, surface-deposited Aβ species were analyzed with TEM. In contrast to AFM, TEM analyses are usually conducted in vacuum, which may alter the samples morphology as compared to the hydrated state. Since biomolecules, such as Aβ fibrils, feature a density similar to the density of the carbon film on the grid, samples need to be stained. In negative staining, a heavy metal salt such as ammonium molybdate, uranyl acetate, phosphotungstic acid or osmium tetroxide, is employed. While the background and the sample s profile is stained and thus scatters the electrons, the Aβ fibrils appear bright. 418 Advantages of TEM analysis over other structural methods available for fibril analysis include that samples do not need to be crystalline. Moreover, only small amounts of samples are required and the measurement range is relatively large. Thus, the fibril width (few nm) as well as the fibril length (>1 µm) can be resolved. 427 For TEM analysis, sample compositions were similar to the AFM experiments and contained preincubated Aβ fibrils. Sample staining was conducted by Bertram Hilterhaus (University of Duisburg-Essen, under my co-supervision). 5 µl of the samples (preincubated fibrils and compounds) were placed on carbon coated copper or nickel grids and dried. 5 µl 2% uranyl acetate solution were added to the grid and incubated for 3 min before rinsing the grid in a droplet of 3 µl deionized water and dried. Transmission electron micrographs of Aβ fibrils were recorded by Dr. Markus Heidelmann at the Interdisciplinary Center for Analytics on the Nanoscale (ICAN), Duisburg. A JEOL JEM-22FS electron microscope was employed and images were processed by means of the ImageJ software (National Institute of Health, Bethesda, MD, USA) Density Gradient Centrifugation (DGC) and Enzyme-Linked Immunosorbent Assay (ELISA) The combination of DGC and ELISA allows the quantitative analysis of Aβ aggregate size distributions (Figure 3-9). To fractionate samples according to their size and shape, they are centrifuged in a gradient consisting of different iodixanol sugar solution concentrations. Iodixanol is chosen as solvent due to its high density, with which it reduces the sample diffusion and delays sedimentation for efficient separation. Furthermore, it is water-soluble and does not bind proteins. Hence, it is expected to not impact Aβ aggregation itself. The highest concentrated gradient solution has a density comparable or higher to the protein

80 3. Materials and Methods 75 density, which leads to accumulation of the largest Aβ species in the last fraction, whereas smaller and less dense Aβ species will be detected in earlier fractions. Figure 3-9: Analysis of Aβ size distributions through DGC/ELISA. Samples are centrifuged in a density gradient to fractionate Aβ species according to size and shape. The Aβ content of each fraction is quantified immunologically via an Aβ-specific ELISA. Adapted with permission from Ref Copyright 216 American Chemical Society. DOI: 1.121/acsnano.6b2627 For the preparation of the samples, lyophilized aliquots of 36 µg Aβ 1 42 were dissolved in 1 µl 1mM sodium phosphate buffer, ph 7.4 (c = 8 µm). A volume of 1 µl sample (ligand-coated nanoparticles, nanoparticles or the ligand alone) was added. Typical concentrations were c(aunp) = 4 µg/ml and c(ligand) = 8 µm. As control, 1 µl Aβ solutions were diluted with 1 µl water. The solutions were incubated at 37 C for 9 min or 24 h shaking at 4 rpm. The DGC gradient (Optiprep, 6% w/v, Sigma) was prepared in ultracentrifugation tubes (Quick Seal Polyallomer tubes, 8.9 ml, Beckman Coulter). The following concentrations and volumes were successively overlayed, before 2 µl of sample were added on top: 1155 µl 5%, 1155 µl 4%, 1155 µl 3%, 3455 µl 2%, 115 µl 1%, 442 µl 5% (v/v) Iodixanol in 1 mm sodium phosphate buffer at ph 7.4. Centrifugation was carried out at 7 C for 4.5 h in a Beckman Optima MAX-XP centrifuge (fixed angle rotor MLA-55) at relative centrifugal force of 287, x g. 23 DGC fractions (V=352 µl) were harvested from top to bottom of each tube and further analyzed immunologically. 372 The ELISA was carried out in black 96-well plates (Nunc, Flat-bottom, MaxiSorp, 35 µl, Thermo Fisher Scientific). The principle of an indirect ELISA is employed, consisting of the binding of a primary capture antibody and a secondary reporter antibody to the analyte, an enzymatically catalyzed reaction and the subsequent detection. During the incubation period of the samples in the ELISA plate, Aβ adsorbs through hydrophilic and hydrophobic interactions with the polystyrene surface. Subsequently, the primary, monoclonal antibody

81 3. Materials and Methods 76 6E1 is added and binds to Aβ (K D = 22 nm for monomeric Aβ, K D = 1.4 nm for Aβ fibrils) 428. Specifically, 6E1 interacts with the amino acids 1-16 of the Aβ peptide with its variable fragment, exposing its constant fragment to the secondary antibody. To quantify the binding process, the secondary, polyclonal antibody GAMPO binds with its variable fragment to the constant fragment of the primary antibody. Moreover, the secondary antibody is covalently coupled to a peroxidase. The enzyme catalyzes the redox reaction of the QuantaBlu dye, which results in a fluorescent signal (Figure 3-1). Figure 3-1: Principle of indirect ELISA. The surface adsorbed antigen (Aβ) is detected via binding of the primary and secondary antibodies and enzymatic conversion of a substrate into a fluorescent probe (left). Antibody structure composed of heavy and light chains, interconnected via disulfide bonds and comprised of variable (antigen-specific) and constant regions, respectively (right). Per well, 2 µl of each DGC fraction was mixed with 98 µl water. Four wells containing water were treated as blanks. The plate was incubated over night at 4 C shaking at 65 rpm. ELISA was performed according to the manufacturer protocol of the secondary antibody with minor adaptions. Generally, each treatment step was followed by washing the plate with 2 µl PBS-T (phosphate buffered saline supplemented with.2% TWEEN) per well for 2 min at 4 rpm. After removing the buffer, each well was first blocked with.25% milk powder solution and incubated at room temperature shaking at 4 rpm. After 35 min, the milk powder solution was removed and the plate was washed. 1 µl/well of a 1:5 dilution of the primary antibody 6E1 (Covance) were added. The plate was incubated for 2 h at room temperature shaking at 4 rpm and washed again with PBS-T. Next, 1 µl/well of a 1:6 dilution of the second antibody GAMPO (Dianova) was added and incubated for 2h at room temperature, shaking at 4 rpm. After washing, 1 µl developer solutions from the QuantaBluTM Fluorogenic Peroxidase Substrate Kit (Thermo Scientific, Rockford, Illinois USA) were added per well and incubated at room temperature shaking at 4 rpm. After 3 min, 1 µl/well stop solution was added to the developer solution and the fluorescence was measured using a microplate reader (Tecan InfiniTe 2). The excitation wavelength was set to 325 nm with gain 1, averaging of 5 flashes and 2 µs integration time. The emission

82 3. Materials and Methods 77 was recorded from 419 nm to 421 nm and the average over the three wavelengths and four wells was calculated to determine the Aβ content in each DGC fraction Cellular Aβ Assay The cellular Aβ assay is based on a Chinese hamster ovary (CHO) cell line that expresses a mutant human amyloid precursor protein (APP). It was found that APP is processed into 4 kda Aβ monomers and kda Aβ species, which are excreted into the cell medium. While first reports identified the larger species as Aβ dimers and trimers, 429 they were later reported to be N-terminally elongated Aβ monomers and few noncovalently bound homodimers. 43 Regarding the biologic effect of these Aβ species, disruption of hippocampal long-term potentiation and memory impairment in rodents were shown. 9 While the cell model is useful for analyzing the interference with naturally secreted, bioactive Aβ species, one needs to keep in mind that these species may not be secreted by neurons and may not be the most relevant target for AD. 43 The cellular Aβ assay was conducted by Andreas Müller-Schiffmann (AG Korth) at the Düsseldorf University Hospital. In order to test the influence of the bare or ligand-coated AuNPs on naturally expressed Aβ species, a CHO cell line termed Hennes2, that stably expresses APP751 including the familial Indiana mutation (V717F), was generated and subcloned. A similar cell line, termed 7PA2, has been shown to secrete Aβ oligomers and N- terminal elongated monomers. 429,43 The experiments were performed according to the protocol described by Podlisny et al Briefly, Hennes2 cells were cultured in DMEM with 1% fetal bovine serum supplemented with 1 U/ml penicillin, and 1 µg/ml streptomycin. 5x1 5 cells were seeded into 1 cm cell culture dishes. After 2 h, the settled cells were treated with freshly prepared pure or ligand coated nanoparticles or controls (1:1 dilution, typical final concentrations: c(ligand)=1 µm, c(aunp)=5 µg/ml, corresponding to 2.9x1 8 NPs/cell and.44 mm² NP surface/cell). After 4 d, the medium was replaced with serum-free medium and fresh preparations of nanoparticles or control compounds were added to the cells. After additional 72 h, the conditioned medium (CM) was harvested and cleared by centrifugation (2 x g for 5 min at 4 C). Aβ species were immunoprecipitated from the CM overnight at 4 C with NHS-sepharose coupled mab-ic16, recognizing amino acid 2-8 of Aβ. 18,8 After two washes with PBS, samples were electrophoresed on 1-2% tricine peptide gels (Biorad, Hercules, CA, USA) and transferred to.2 µm nitrocellulose membranes at 4 ma for 2 h. Filters were boiled for 1 min in PBS and blocked overnight at 4 C with 5%

83 3. Materials and Methods 78 skimmed milk (Oxoid, Thermo Scientific, Bonn, Germany) in PBS containing.5% Tween 2 (PBS-T). After three washes in PBS-T, each for 1 min, the membranes were probed with monoclonal 4G8 antibody (Signet, Dedhem, MA, USA; 1:5 in PBS-T) that recognizes amino acid of the Aβ peptide. Bound antibody was detected with horseradish peroxidase conjugated goat anti-mouse Ig (diluted 1:25 in PBS-T, Thermo Scientific, Bonn, Germany) and the Amersham ECL Western Blotting Detection Reagent (GE, Buckinghamshire, UK). Aβ signals were quantified by densitometry using the ImageJ software (National Institute of Health, Bethesda, MD, USA). 372 For detection of APP and Caspase-3 cells were lysed in 75 µl lysis buffer (5 mm Tris ph8, 15 mm NaCl, 1% NP4, 5 mm EDTA) including the Complete protease inhibitor cocktail (Roche, Mannheim, Germany). The protein concentrations of the lysates were quantified using the DC TM protein detection kit (Biorad, Hercules, CA, USA). 3 µg of lysates were separated on a NuPAGE 4-12% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) and transferred to a.2 µm nitrocellulose membrane. After blocking with PBS-T/ 5% skimmed milk, APP and Caspase-3 were detected by CT15 polyclonal rabbit antiserum 431 (diluted 1:3,5 in PBS-T) and Caspase-3 antibody (diluted 1:1, in TBS-T, Tris-buffered saline with TWEEN; #9662, Cell Signalling, Danvers, MA, USA). Detection of tubulin with a monoclonal anti α-tubulin antibody (T926, Sigma-Aldrich, St. Louis, MO, USA; diluted 1:15, in PBS-T) served as internal control. After incubation with 1:25, dilutions of goat anti-mouse IRDye 68RD and goat anti-rabbit IRDye 8CW (LI-COR, Lincoln, NE, USA), signal intensities were analyzed on a LI-COR Odyssey CLX using the corresponding Image Studio Version 2.1 software (LI-COR Biosciences, NE, USA) Light and Confocal Laser Scanning Microscopy (CLSM) Confocal laser scanning microscopy is an optical imaging technique with controlled and highly limited depth of focus. High contrast and high optical resolution are achieved by creating images one depth level at a time through a spatial pinhole, which eliminates out-offocus light (Figure 3-11). By scanning over the sample and collecting images of each confocal plane, three-dimensional images can be reconstructed. CLSM is used in biological applications, since it can provide the direct, noninvasive optical sectioning of 3D living specimens with a minimum of sample preparation. 432,433

84 3. Materials and Methods 79 Figure 3-11: Light paths in a confocal laser scanning microscope. In-focus rays reach the specimen, the pinhole and the photodetector, while out-of-focus rays are excluded (modified from Ref. 433, with permission of Springer). For microscopic analysis of the cells, 2x1 4 cells were seeded on cover glasses (13 mm diameter) that were placed in 24-well plates. After settling of the cells, they were treated with the compounds. At day 4, the cells were washed with PBS and fixed with PBS/4% paraformaldehyd for 3 min at RT. After two washing steps with PBS and one with pure water, cells were mounted with ProLong Gold including DAPI (Invitrogen, Hercules, CA, USA). Images were collected by Andreas Müller-Schiffmann (AG Korth) at the Düsseldorf University Hospital with a Axiovision Apotome.2 (Zeiss) using the differential interference contrast (DIC) filter. Additionally, images were collected by Lisa Gamrad (AG Barcikowski) at the Imaging Center Essen (IMCES, University Hospital Essen) using a Leica SP8 gsted confocal microscope. Three channels were employed: AuNPs were excited close to their SPR maximum at 532 nm (detection channel: nm). 198 DAPI-stained cell nuclei were excited at 45 nm (detection channel: nm) using a white light laser source. The DIC filter was employed for visualization of the cell membrane. 372

85 4. Results and Discussion 8 4. Results and Discussion The following results and their discussion are presented in three subchapters. Laser-generated, ligand-free NPs are characterized at first with respect to their size, charge and their interaction with small thiolated ligands and model serum proteins. Secondly, the surface functionalization of NPs with the Aβ-targeting ligands D3 and aminopyrazole trimer is evaluated. At last, an outline on the conjugates interference with the Aβ aggregation process will be given. 4.1 Nanoparticle Characterization Particle Size Gold nanoparticles were analyzed by employing different techniques in order to get a comprehensive impression on particle size. Knowing the particle size is highly important for further experiments, especially ligand functionalization, since calculations on nanoparticle numbers and the nanoparticle-to-ligand ratios are based on the particle diameter. As has been shown, analyses with different techniques can yield different results. 434 While ADC, AUC and DLS detect hydrodynamic diameters of the colloidal particles, including the Stern and ligand layer, microscopic techniques (AFM and TEM) show the metallic core diameter of the dried particles. For DLS analysis, few larger particles may bias the result due to strong light scattering. Generally, microscopic analyses may be statistically limited due to a limited number of evaluated particles (e.g. TEM: N 1). In contrast, the highly resolving techniques ADC and AUC require prior knowledge of the density of the investigated system. 435 In the case of colloidal citrate-coated AuNPs with diameters below 6 nm, the density was shown to deviate from the bulk material due to the presence of an outer layer of approximately 1.5 nm thickness. 436 This layer was termed hydration layer by the authors, but one has to be aware that it obviously also consists of the citrate ligands. The calculations of particle size distributions can be weighted on the intensity, particle mass (volume), number or surface. Depending on the weighting parameter, few large particles or many small particles may dominate the size distribution, if the particles are polydisperse (following the order: d mass/volume > d surface > d number ). To estimate the average particle size of the distribution, the x C -value of the lognormal distribution or the d 5 -value of the cumulative frequency can be given. While the x C -value represents the particle diameter of the most

86 Gold mass [% of total] Norm. mass frequency 4. Results and Discussion 81 frequent particles, the d 5 -value is the maximum diameter of 5% of all particles. For polymodal size distributions, x C - and d 5 -values may differ widely. Moreover, x C -values have to be calculated for each peak individually, while one d 5- value can represent the whole distribution. However, for narrow monomodal, monodisperse size distributions, the two values will be very similar. 1 8 (water) ablation in.1mm NaCl 1 8 without centrifugation (water) without centrifugation (NaCl) 5min 4RPM 5mL 5min 6RPM 15mL 1min 18RPM 2mL without centrifugation 4% 19% 9% without centrifugation 5min, 4rpm 5min, 6rpm 2% 3% 1min, 18rpm d < 2mn d = 2-5nm Hydrodynamic diameter [nm] 5 nm 5 nm 5 nm Before centrifug. (in water) 25 nm 25 nm Before centrifugation (in NaCl) 25 nm After centrifugation (in NaCl) Figure 4-1: Size analysis with ADC of as-synthesized AuNPs after laser-ablation in pure water and.1 mm NaCl, and of AuNPs in.1 mm NaCl after subsequent centrifugation steps (top). Representative electron micrographs of AuNPs before and after centrifugation (1 min, 18 rpm, cut-off: 8 nm, bottom). Figure 4-1 shows that nanosecond pulsed laser ablation in.1 mm NaCl aqueous solution generates AuNPs with narrower size distributions and smaller particle sizes than ablation in deionized water due to size quenching effects 41 by the electrolyte. As analyzed by ADC, subsequent centrifugation of the as-synthesized colloid can further reduce the number of particles with diameters above 2 nm, which would otherwise contribute to a large mass percentage of the colloid. With regard to subsequent bioapplications, employing nanoparticles with a narrow, monomodal size distribution is desirable. It facilitates calculations of the ligand-to-nanoparticle ratio since one can assume that all particles feature the same average

87 Norm. frequency Norm. frequency 4. Results and Discussion 82 size. Figure 4-1 shows that centrifugation at 4 rpm for 5 min cannot remove larger particles (calculated cut-off radius: 19 nm). However, centrifugation at 6 rpm for 5 min or at 18 rpm for 1 min results in comparably narrow particle size distributions, although the calculated cut-off radii are considerably different (15 nm and 8 nm, respectively). In conclusion, a balance between centrifugation time, volume and speed needs to be found which may limit the usage of the rotor carrying the highest volume due to its speed limitation. When employing optimized centrifugation parameters, particles with d < 2 nm contribute to more than 95% of the total mass. 1 8 AFM mass x C = nm; R²=.663 number x C = nm; R²=.725 surface x C = nm; R²=.695 N=73 1nm Particle height [nm] 2 nm nm 1 µm - 1nm 1 mass 1 x C = nm, R²=.959 number 1 x C = nm, R²= surface x C = nm, R²=.971 N= TEM Feret diameter [nm] 25 nm 1 nm Figure 4-2: AFM (top) and TEM (bottom) analysis of laser-generated, centrifuged gold colloids (18 rpm, 1 min, cut-off: 8 nm). Calculated mass-, number, and surface-weighted size distributions (left) and representative micrographs (right) are shown. Laser-generated, centrifuged AuNPs feature spherical shapes with average diameters of 7 nm, as analyzed by TEM, ADC and AUC (Figure 4-2, Figure 4-3). Few aspherical particles (peanut-shape) are visible, which are well-known TEM artifacts. They result from particle

88 4. Results and Discussion 83 agglomeration upon drying on the TEM grid and subsequent fusion by the electron beam. It is thus recommendable to apply steric ligands to colloidal samples for TEM preparation. 437 Generally, the samples show a very narrow monomodal size distribution, with only little deviations between mass-, number- and surface-weighted distributions, and between x C - and d 5 -values. DLS and AFM analyses show higher average particle diameters of 14 nm and 1 nm, compared to the other techniques. In this regard, AFM analysis requires the surfaceadsorption of the colloid. However, the negative surface charge of laser-generated particles may restrict efficient binding to the negatively charged mica substrate and may explain the limited number of adsorbed particles (N=73). The AFM analysis has thus to be viewed as only semi-quantitative. For DLS analysis, the presence of some large (or aggregated primary) NPs can be seen in the intensity distribution and influences the width of the DLS size distribution (Figure S1). The agreement of TEM with ADC and AUC results indicates that the density of the bulk gold can be employed for calculating the diameter of laser-generated, ligand-free particles in micromolar ionic strength medium from sedimentation velocity experiments. In contrast, Fallabella et al. report that the density of bare AuNPs derived from sedimentation coefficients of AUC measurements varies from that of bulk gold for particle diameters below 6 nm. 436 However, they employed chemically synthesized, citrate-coated colloids as bare particles. They conclude that the hydrate layer around the particles lowers the overall density. In contrast, the influence of the hydrate layer seems to be less pronounced for laser-generated particles. This may be a result of the high colloid purity, the use of a low ionic strength medium and the absence of citrate. Cölfen and colleagues compared different analysis methods to characterize model NPs via AUC. 438 For monodisperse colloids they conclude that all mathematical analysis algorithms give comparable results. In this work, the AUC analyses show very different size distributions depending on whether c(s) or ls-g*(s) are chosen, while the average particle sizes (determined as x C -values) are very similar (Figure 4-3). In the c(s) analysis, broadening of the sedimentation coefficient distribution due to diffusion may be overcorrected by the software. 438 In the ls-g*(s) analysis, diffusion is not taken into account and the s-value distribution can broaden, if diffusion occurs. In fact, in this work, the ls-g*(s) distribution appears broader than the c(s) distribution. However, due to the high density of gold particles,

89 Norm. intensity Norm. frequency Norm. frequency 4. Results and Discussion 84 diffusion is not expected to dominate the sedimentation process (3 rpm correlating to 724 g for 12 h). Notably, ls-g*(s) analysis results are well comparable to TEM and ADC results. Overall, AuNPs have been characterized by five independent techniques, where results from ADC, AUC and TEM perfectly match and give particle diameters of 7 ± 1 nm. At typical concentrations of 5 µg/ml, one ml colloid contains 1.44x1 13 ± 6.62x1^12 NPs with a total surface of 22.2 ± 3.3 cm². Cumulative Frequency [%] Volume Diameter [nm] AUC ADC d 5 =6.9 nm AUC d 5 =6.8 nm DLS d 5 =14.1 nm TEM d 5 =7.1 nm AFM d 5 =1.5 nm ls-g*(s), RMSD=.59 x C = nm, R²=.997 c(s), RMSD=.42 x C,1 = nm, R²=.997 x C,2 = nm, R²= mass 1 x C = 7.1 number 1 x C = surface 1 x C = ADC 1.3 nm, R²= nm, R²= nm, R²= Hydrodynamic diameter [nm] DLS 1 x C = nm, R²=.989 x C = 11.4 x C = nm, R²= nm, R²= Hydrodynamic diameter [nm] 4 2 Volume Number Surface Hydrodynamic diameter [nm] Figure 4-3: AUC, ADC and DLS analysis of laser-generated, centrifuged gold colloids (18 rpm, 1 min, cut-off: 8 nm). Calculated mass-, number, and surface-weighted size distributions and the cumulative volume frequency distribution summarizing the results of all particle sizing techniques are shown (top left, note that AUC results were calculated from the ls-g*(s) analysis).

90 Norm. Absorbance [a.u.] Colloidal stability (PPI) 4. Results and Discussion Particle Stability AuNP stability can be analyzed by UV/Vis extinction spectroscopy due to characteristic properties such as the SPR peak position and the ratio of the absorbances at 38 nm and at 8 nm, which is the Primary Particle Index (PPI), as measure of colloidal stability. 135 As can be seen in the example in Figure 4-4, ligand-free AuNPs feature an SPR maximum at 527 nm and a PPI > 2. Surface adsorption of BSA or a cationic peptide to the particles shifts the SPR peak position to longer wavelengths (because of increased refractive index in the particle s vicinity, Figure 4-4 left) and lowers the PPI (because of charge screening, Figure 4-4 right) nm 517nm 544nm AuNP AuNP + BSA AuNP + cationic peptide Wavelength [nm] AuNP AuNP + BSA AuNP + cationic peptide Figure 4-4: Optical properties of gold colloids. Characteristic features of the UV/Vis extinction spectrum are the wavelength of the SPR peak (insert), the absorbance at 38 nm (proportional to the mass concentration) and at 8 nm (proportional to the number of strongly scattering large particles, aggregates and agglomerates, left). The Primary Particle Index (PPI) as ratio of the absorbance at 38 nm and 8 nm reflects the colloidal stability (right). 135 Gold nanoparticle stability was analyzed over time in order to determine whether the colloid can be stored and used when required. As can be seen from ADC and UV/Vis analyses (Figure 4-5, top), nanoparticle size and concentration remains constant over a period of 5 d. Notably, the diameter seems to increase at the beginning from 6 nm to 7 nm, after which it stays constant. Jendrzej et al. examined growth effects in laser-generated colloids. 11 They found that laserfragmented PtNPs feature two types of growth kinetics: rapid growth is observed during the first hours after synthesis, possibly due to coalescence of nanoparticles and atom clusters, which may arise during laser synthesis and fragmentation processes. This phase is followed by a slow growth phase for t > 1 d due to a combination of ripening and coalescence. 11 For AuNPs, a similar tendency for the formation of atom clusters by laser fragmentation and

91 Counts Zeta Potential [mv] Absorbance [a.u.] Hydrodynamic diameter [nm] Rel. colloid concentration (c/c ) Absorbance [a.u.] 4. Results and Discussion 86 subsequent NP growth was described, although the effect was less pronounced (i.e. Au: 3% extinction increase after h, Pt: 7%). 11 AuNPs in this work were generated by ablation and subsequent centrifugation to avoid the laser fragmentation step which may generate more atom clusters. Hence, ripening of the colloid may occur to a lesser extent. Overall, stable particles with a hydrodynamic diameter constant over 5 d (within error of the measurement) could be generated x x1 8 2.x x1 8 1.x1 8 5.x1 7 mass-weighted number-weighted surface-weighted Time [d] -42 mv Zeta potential [mv] d 3d 35d.4 6d 14d 21d 28d Wavelength [nm] 1d 6d 21d 28d 49d Wavelength [nm] ph IEP =2. Time [d] ph Figure 4-5: Long-term stability of laser-generated, centrifuged AuNPs as determined from nanoparticle size derived from ADC measurements (top, left) and concentration derived from UV/Vis measurements (top, right). Nanoparticle stability, as determined by zeta potential measurements, of the colloid at neutral ph and as a function of ph (bottom). Due to the absence of ligands which could sterically stabilize colloidal nanoparticles, the stability of laser-generated NPs derives from their partial surface oxidation. 13,151 Subsequently, surface hydroxides form 44 and chaotropic anions such as chloride adsorb and further stabilize the particles if present at low salinities. 135 Here,.1 mm chloride are employed to increase the surface charge density. 13 At neutral ph, AuNPs feature a negative zeta potential of -42 mv (Hückel model, Figure 4-5 bottom). By decreasing the ph, the negative nanoparticle charge is compensated by protons. Upon reaching the isoelectric point at ph 2, ligand-free nanoparticles precipitate due to the absence of repulsive forces. 17 Similar

92 Absorbance [a.u.] Colloidal Stability (PPI) SPR peak shift [nm] 4. Results and Discussion 87 isoelectric points were reported for other laser-generated AuNPs (IEP at ph ph ). and For biological applications, buffers of millimolar strength are typically used. Therefore, colloids should ideally be synthesized in these buffers or be transferred into these buffers before bioapplications. However, colloidal stability can be impaired at high ionic strengths (compare to chapter 2.2.1). This effect was analyzed by titrating laser-generated AuNPs with mm-concentrations of NaCl. Increasing the ionic strength with sodium chloride resulted in the destabilization of ligand-free AuNPs, as shown in Figure 4-6. The instability onset lies between 5 mm and 1 mm NaCl at a colloid concentration of 5 µg/ml (corresponding to 2 x1 6 to 4x1 6 ions per NP). Notably, the ionic strength which induces nanoparticles to precipitate is three orders of magnitude higher than that leading to size quenching and stabilization during the ablation process. In this regard, Rehbock et al. reported the that approximately 18 chloride anions per 8 nm nanoparticle are necessary to induce size quenching and stabilization during the laser ablation process mm [NaCl].6 1 cm Ions per Particle [NaCl/NP d=7nm ]. 2.5x1 6 5.x x1 6 1.x x mM NaCl 1mM NaCl 25mM NaCl.2 5mM NaCl 75mM NaCl 1mM NaCl 2mM NaCl 3mM NaCl Wavelength [nm] regime of instability Ionic strength [mm] Figure 4-6: Stability of ligand-free AuNPs generated in.1 mm NaCl via PLAL and incubated in millimolar ionic strength medium afterwards. UV/Vis extinction spectra (left) and evaluation of optical properties (right, c(aunp)=5 µg/ml). Overall, it was demonstrated that laser-generated, ligand-free AuNPs are compatible to a wide ph regime (ph = 3-1) and ionic strength 75 mm.

93 4. Results and Discussion Interaction with Small Thiolated Ligands and Serum Proteins In order to increase particle stability, selected stabilizing molecules can be adsorbed on the nanoparticle surface. In this regard, the initially ligand-free surface allows the establishment of sub-monolayer to above-monolayer coverages solely by tuning the applied ligand dose. Therefore, nanoparticle stabilization with small thiolated ligand and more bulky serum proteins (compare to Figure 3-4) was assessed next. Detection of Ligand Binding To identify which ligand doses can stabilize colloidal AuNPs, ligand-free AuNPs were firstly conjugated with different concentrations of the stabilizer. Zeta potential measurements and UV/Vis extinction spectroscopy showed how the colloidal properties change upon addition of the stabilizer (Figure 4-7). No trend could be observed from zeta potential measurements (Figure S2), possibly because the ligands complicate the measurements, e.g. by decreasing electrophoretic mobilities of the particles. 396,16 Nevertheless, the SPR peak analysis clearly shows shifts to longer wavelengths with increasing stabilizer concentration. Moreover, the onset for the peak shift is specific for each stabilizer, generally following the trend that a higher stabilizer concentration is necessary for smaller stabilizer molecules to induce a peak shift. For all ligands, the saturation peak shift lies between 4 and 7 nm. Mulvaney and coworkers have calculated the SPR peak shifts for 5.2 nm AuNPs as function of the chain length of alkanethiol capping agents. They report peak shifts of 4 nm and 7 nm for 1 nm and 2 nm thick ligand layers. 439 In general, this corresponds well with the values found in this study. Notably, the high molecular weight proteins do not generally induce higher peak shifts compared to the small thiolated ligands. A complementary method such as DLS could be employed to confirm whether the ligand layer thickness is similar for protein-coated AuNPs and AuNPs coated with small thiolated ligands. By evaluating the minimum stabilizer concentration to induce a saturation of the SPR peak shift, one can derive the surface saturation dose, by assuming that all applied stabilizers actually bind to the particle surface. This assumption is reasonable since all ligands carry at least one thiol group. For the small molecules LA, MUA and mpeg-sh, saturation can be achieved with 12 and 8 ligands per NP, respectively. The much larger proteins insulin, transferrin and BSA seem to saturate the nanoparticle surface already at doses of 14 and 13 ligands per NP, respectively.

94 SPR peak shift [nm] Saturation concentration [applied #/NP d=7nm ] 4. Results and Discussion Stabilizer density [applied #/NP d=7nm ] E Stabilizer concentration [µm] LA MUA mpeg-sh Insulin Transferrin BSA LA MUA mpeg-sh Insulin Transferrin BSA Figure 4-7: SPR peak shift of AuNPs (c=5 µg/ml) as function of the applied concentration of thiolated ligands or serum proteins (left). Minimum stabilizer concentration to induce the maximum SPR peak shift (right). When considering the results of BSA as most efficient stabilizer and LA as least efficient stabilizer, one can summarize that NP stabilization can be achieved with approximately 1- times less BSA molecules than LA molecules. However, considering that the stabilization arises from steric effects, one can also take the mass concentration or number of carbon atoms of required stabilizers into account. Following this idea, one can see that LA requires only 6.2 mg/l, whereas transferrin requires 22.6 mg/l. The fact that lower mass doses are required with the small ligands for achieving the SPR peak saturation may be due to their capability of denser packing. The numbers of ligand carbon atoms per NP required to achieve full NP coverage vary between 9185 and It becomes apparent that by evaluating these corrected saturation concentrations, all ligands perform very similar. This can be explained by the fact that the SPR peak shift is altered by changes in the refractive index of the ligand coating. 439 Hence, under saturation concentrations, all ligands seem to bind to the NP core at similar densities, resulting in similar ligand shell volumes and similar changes of the refractive index. Finally, ligand footprints between.12 nm² and 12 nm² can be derived, which will be further discussed together with the results on the salt stress test at the end of this chapter. Table 4-1 summarizes the calculated concentrations of stabilizers required for nanoparticle saturation according to stabilizer mass, number of carbon atoms and ligand footprints.

95 4. Results and Discussion 9 Table 4-1: Concentrations of stabilizers required to achieve the maximum SPR peak shift ( saturation ) according to applied stabilizer mass and number of carbon atoms. LA MUA mpeg- SH Insulin Transferrin BSA Saturation concentration [µm ligands at 5 µg/ml NP] Saturation concentration [ligands/np] Molecular mass [g/mol] Saturation concentration [mg ligands/l] Carbon Atoms per Molecule (two carbon atoms per amino acid are assumed) Saturation concentration [carbon atoms/np] Ligand footprint [nm²] (for AuNP with d=7 nm, surface area=153.9 nm²) The BSA samples feature a slightly different SPR peak shift-profile compared to all other ligands with a maximum peak shift at.8 µm BSA (5.3 mg/l). It is assumed that BSA may cross-link nanoparticles 44 at intermediate concentrations, possibly due to electrostatic interactions and/or binding with the single free exterior cysteine. 247 This would explain the sharp increase of the SPR maximum at ratios of 1 to 5 BSA/NP. By further increasing the BSA/NP ratio, cross-linking is avoided since the number of BSA is sufficiently high to cover individual particles with BSA mono- or multilayers (c >.3 µm). With regard to literature, protein concentration-dependent agglomeration through cross-linking was reported for 7 nm citrate-stabilized AuNPs in the presence of hemoglobin at acidic ph. 441 The authors attributed the stability at low protein-to-np ratios to protein adsorption on NPs, which depletes proteins from solution and is not sufficient for cross-linking. On the other hand, high protein-to-np ratios may induce the formation of electrosterically stabilizing protein multilayers. Interestingly, citrate-coated AuNPs were least stable at a hemoglobin concentration of 3 mg/l comparable to the BSA concentration of 5.3 mg/l for laser-generated AuNPs. The deviation may arise from the different particles concentrations applied (5 µg/ml vs. 16 µg/ml, d= 7 nm) and slightly different molecular weights of BSA (66 kda) and hemoglobin (64.5 kda).

96 4. Results and Discussion 91 Shift of the Isoelectric Point of AuNPs through Binding of Thiolated Ligands Determining the isoelectric point of gold-ligand conjugates can on the one hand be used to analyze the ph stability of the conjugates and on the other hand be interpreted as a proof of binding of the stabilizer to the NP surface. In this regard, the formation of a ligand shell around the particles may shift the isoelectric point of the ligand-free colloid to the pk a (IEP) of the ligand. Figure 4-8 shows that all ligands except mpeg-sh shift the isoelectric point from 2. (ligand-free AuNPs, compare to Figure 4-5) to higher ph values. Indeed, the IEPs of these conjugates are either located between the IEPs of the ligand-free AuNPs and the pure ligands (LA, MUA, insulin, transferrin) or are very similar to the IEP value of the pure ligand (BSA). For BSA adsorbed on oxide NPs, Rezwan et al. observed the same trend and correlated shifts of the zeta potentials with the number of BSA adsorbed per NP. 442,443 In fact, the group also showed that the IEP of Al 2 O 3 NPs was shifted from ph = 9 (Al 2 O 3 NPs without BSA) to ph = 5 (Al 2 O 3 NPs with 1 µg/ml BSA) when increasing the amount of BSA. 443 Since mpeg-sh is a non-charged ligand, effects on the IEP of AuNP would not be expected. In the case of the other ligands, the shift of the IEPs between the IEP of the ligand-free AuNPs and the pure ligands shows that the identity of the conjugate is defined by the surface chemistry of the particles as well as by the pk a (IEP) of the adsorbed ligands. Although the reason for shifting the IEP away from the pk a of the free ligands is not known, it can be speculated that ligand-ligand interactions on the NP surface may be responsible. In this regard, the close ligand proximity on the NP surface may lead to the delocalization of ligand charges. Upon proton addition, multiple NP-bound ligands may share one proton for charge compensation. 444 As a result, overall more protons will remain in solution and contribute to the (low) ph at the IEP. With regard to colloidal stability at different ph, it can been seen that sterically demanding molecules can effectively stabilize AuNPs even at the IEP and at more acidic ph values, where the net charge of the conjugate gets reversed. This is shown in the photograph in Figure 4-8, where all AuNP-BSA conjugates appear red and do not show any signs of aggregation even at ph values of 1-2. This steric stabilization of colloidal AuNPs at their isoelectric point via the adsorption of BSA was also reported by Brewer et al In contrast, the small thiolated ligands LA and MUA fail to stabilize AuNPs when approaching and crossing the IEP at acidic ph (Figure S3), since their small size does not give sufficient steric stability.

97 Zeta potential [mv] Zeta potential [mv] 4. Results and Discussion 92 Notably, at ph values relevant for bioapplications (e.g. ph PBS = 7.4), all ligands allow NP stabilization. However, acidic ph values are employed to stimulate Aβ aggregation, e.g. for AFM analysis (compare to chapter 4.3.3). In these assays, it needs to be considered that colloidal stability may be impaired, if Aβ itself does not (sterically) stabilize the particles. 6 4 AuNP + 1µM LA AuNP + 1µM MUA AuNP + 1µM mpeg-sh AuNP/1µM BSA ph=1 ph= IEP AuNP = ph AuNP + 1µM Insulin AuNP + 1µM Transferrin AuNP + 1µM BSA AuNP + 1µM BSA ph cm pk a of stabilizer AuNP AuNP + 1 µm LA AuNP + 1 µm MUA AuNP + 1µM mpeg-sh AuNP + 1µM Insulin AuNP + 1µM Transferrin AuNP + 1µM BSA AuNP + 1µM BSA Figure 4-8: Zeta potentials of AuNPs stabilized with small thiolated ligands (top, left) and serum proteins as function of ph (c(aunp)=5 µg/ml; bottom, left). Photographs of BSA-coated gold colloids at ph = 1-1 (top, right) and isoelectric points of ligand-stabilized AuNPs compared to the pk a (IEP) of the stabilizer alone (bottom, right).

98 Colloidal stability (PPI) SPR peak maximum [nm] Stabilizing concentration [min. applied #/NP d=7nm ] 4. Results and Discussion 93 Stabilizing Effects upon Salt Stress and Freezing In order to further characterize stabilizer performance, gold-ligand conjugates were exposed to salt stress in two types of experiments. At first, nanoparticles with variable amounts of stabilizing molecules were incubated in 1 mm NaCl medium. Note that human serum contains ions in this concentrations range (chloride: 1-17 mm, sodium: mm). 445 Secondly, nanoparticles conjugated with 418 ligands/np (complete surface coverage) were incubated at different ionic strengths. Figure 4-9 shows the stability of conjugates incubated at constant ionic strength of 1 mm NaCl. A specific threshold concentration correlating with the size of each ligand is required to stabilize NPs against aggregation. In this regard, the number of stabilizers necessary to prevent AuNP aggregation follows the order BSA < mpeg-sh < MUA LA instable Stabilizer density [applied #/NP d=7nm ] Ionic strength: 1mM AuNP+LA AuNP+MUA AuNP+mPEG-SH AuNP+BSA stable Stabilizer concentration [µm] Figure 4-9: Colloidal stability upon salt stress. The SPR peak maximum and PPI of ligand-coated AuNPs are determined as function of stabilizer concentration at constant ionic strength (left). Minimum stabilizer concentration to prevent nanoparticle aggregation at 1 mm NaCl concentration (right). Interestingly, the number of stabilizers necessary to prevent AuNP aggregation is up to 9% lower than that determined to induce the maximum peak shift of the gold colloid without salt stress (which was interpreted as threshold for monolayer coverage, Figure 4-7). Different

99 4. Results and Discussion 94 processes may account for this observation: (i) Full monolayer formation may not be necessary to stabilize the particles. In the case of LA, where 1253 LA/NP (8.1x1 14 LA/cm²) were applied for monolayer coverage and 138 LA/NP (9.x1 13 LA/cm²) enable stabilization, this would mean that a surface coverage of approximately 1% is already sufficient for electrostatic stabilization. (ii) The relatively high ionic strength may enhance ligand binding of less efficiently binding ligands. In the case of the negatively charged LA, ligands and nanoparticles may repulse each other so that the applied ligand dose may not represent the number of actually bound ligands. 446 The presence of NaCl may reduce the opposing forces between particles and ligands by decreasing the Debye lengths between the species and introducing Na + counter ions. This technique of salt aging is routinely applied to conjugate negatively charged AuNPs with anionic DNA, where maximum DNA loading could be achieved with.7 M to 1 M NaCl. 446 Moreover, Volkert et al. analyzed the packing density and stabilizing effect of LA on AuNPs in the absence and presence of up to 16 mm NaCl. They found that the LA packing density can be increased by ~2% in the presence of salt ( LA/cm 2 without NaCl, LA/cm 2 with NaCl), which rendered the NPs more stable. 447 They hypothesized that the ionic strength of the solution leads to a reduction of defects in the ligand arrangement as the electrostatic repulsion between adjacent molecules decreases. Complementary to Table 4-1, Table 4-2 summarizes the ligand concentrations required for nanoparticle stabilization against 1 mm NaCl. By evaluating the stabilizer performance according to the ligand mass concentration required, mpeg-sh appears to be the most efficient stabilizer (c=.5 mg/l). Although mpeg-sh is not the most sterically demanding molecule, it effectively protects AuNPs from agglomeration. The reason for the good performance of mpeg-sh over the other ligands may be that it can be densely packed on the NP surface due to its linear shape (opposed to BSA) and absence of charges (opposed to LA and MUA). Zopes et al. examined the stability of 5.5 nm AuNPs conjugated with different PEG ligands against salt stress and cyanide etching. 448 They found that monothiolated PEG ligands produced most stable colloids in the presence of cyanide compared to di- and trithiolated ligands, possibly due to the high ligand packing on the gold surface. Moreover, AuNPs conjugated with di- and trithiolated PEG exhibited even higher stability at high NaCl concentrations, which may be explained by the stronger binding of multidentate ligands. Liu et al. examined the stability of 1 nm PEG-coated AuNPs in 1 M NaCl. Depending on the PEG size, 1 (PEG 5 ) to 5 (PEG 9 ) molecules/np were required for stabilization. Those

100 4. Results and Discussion 95 PEG stabilizing doses are much higher than the concentrations in the present study, which is also confirmed when considering the surface doses (d=1 nm: 5 PEG 9 /NP 264 pmol/cm² vs. d=7 nm: 57 PEG 35 /NP 63 pmol/cm²). A possibly explanation is that due to the absence of ligands in laser-generated AuNPs, PEG binding may occur more efficiently, so that less PEG needs to be applied for stabilization a priori. 39 Table 4-2: Concentrations of ligands required for nanoparticle stabilization against 1 mm NaCl according to stabilizer mass and number of carbon atoms. LA MUA mpeg-sh BSA Stabilizing concentration [µm ligands at 5 µg/ml NP] Stabilizing concentration [ligands/np] Molecular mass [g/mol] Stabilizing concentration [mg ligands/l] Carbon Atoms per Molecule (two carbon atoms per amino acid are assumed) Stabilizing concentration [carbon atoms/np] Ligand footprint [nm²] (for AuNP with d=7 nm, surface area=153.9 nm²) Figure 4-1 shows the stability of conjugates incubated at different ionic strengths. MUAconjugated particles show an agglomeration tendency similar to ligand-free NPs, which may indicate insufficient ligand binding at the applied dose of 418 ligands/np. For Au/LA conjugates at high salinity (c > 2 mm), some agglomeration tendency can be observed from the PPI analysis (resulting from an increased absorbance at 8 nm), although the SPR peak remains unaffected for all tested NaCl concentrations. Conjugation with mpeg-sh and BSA prevents the particles from agglomeration even at the highest ionic strength analyzed (c=3 mm) supporting previous results obtained under constant ionic strength conditions (Figure 4-9).

101 Absorbance [a.u.] Colloidal stability (PPI) SPR peak maximum [nm] 4. Results and Discussion stabilizers/np d=7nm bare AuNP +LA +MUA +mpeg-sh +BSA Ions per particle [NaCl/NP d=7nm ]. 3.x1 6 6.x1 6 9.x x ( ) 5 Figure 4-1: Colloidal stability upon salt stress. The SPR peak maximum and PPI of ligand-coated AuNPs are determined as function of ionic strength at constant stabilizer concentration (c(aunp)=5 µg7ml, c(ligand)=1 µm, 418 ligands/np). To test the durability of the NP-conjugates, selected samples were frozen or freeze-dried, respectively. Producing a sample which could be frozen and thawed or frozen, dried and resuspended would be ideal for further applications, because this sample could be stored for a prolonged time. 8 As can be seen from Figure 4-11, ligand-free AuNPs cannot be resuspended after one freezing-thawing cycle and aggregate irreversibly. In contrast, mpeg-sh-stabilized AuNPs (7.8 µg mpeg-sh/mg Au) can be frozen and thawed several times without affecting the colloid s optical properties Ionic strength [mm] Ligand-free AuNP freezing (f) thawing (t) 1. AuNP/mPEG-SH AuNP/1µM mpeg-sh (f) (t) (f) (t) (f) (t) (f) (t) 5 cm.4 before 1x freezing.2 2x freezing 3x freezing 4x freezing Wavelength [nm] Figure 4-11: Freezing and thawing of laser-generated, colloidal AuNPs. Comparison of the stability of ligand-free (left, top) and mpeg-sh-stabilized AuNPs after different freezing and thawing cycles (left, bottom and right).

102 Colloidal Stability (PPI) SPR peak maximum [nm] 4. Results and Discussion 97 Since BSA is sterically even more demanding than mpeg-sh and effective BSA-stabilization of freeze-dried gold nanorods has already been reported, 449 a detailed stability analysis of BSA-coated AuNPs was conducted (Figure 4-12). BSA density [applied #/NP d=7nm ] Au+BSA Au+BSA after drying BSA concentration [ M] Figure 4-12: Colloidal stability upon freeze-drying. The SPR peak maximum and PPI of BSA-coated AuNPs as function of BSA concentration are determined before and after freeze-drying and resuspension (c(aunp)=5µg/ml). To completely stabilize AuNPs against agglomeration after freezing, drying and resuspension, a minimum concentration of 3 µm BSA was determined (corresponding to 125 BSA/NP). However, partial stabilization can already be observed for BSA concentrations >.2 µm (corresponding to > 7 BSA/NP and agreeing well with the results from salt stress experiments, Figure 4-9). In the freeze-drying test, generally stronger forces seem to strain the colloids than in the presence of high ionic strength. Hence, higher stabilizer concentration need to be applied to preserve the colloid s properties.

103 7:1 6:1 5:1 4:1 3:1 2:1 8:1 7:1 6:1 5:1 4:1 3:1 2:1 1:1 1:2 1:3 1:4 1:5 1:6 8:1 7:1 6:1 5:1 4:1 3:1 2:1 1:1 1:2 4. Results and Discussion 98 Gel Electrophoresis Gel electrophoresis is commonly used in biology to separate biomolecules according to their size and charge e.g. for sample purification. Here, electrophoretic movement of gold-protein conjugates is used to determine the minimum protein concentration necessary to guarantee colloidal stability (Figure 4-13), their overall charge (i.e. direction of movement to anode or cathode), 45 and relative mobility (i.e. moved distance, Figure 4-14). For this, optimal NP concentrations, voltage and buffer compositions were determined at first (Figure S5, Figure S4). Notably, ligand-free AuNPs or particles conjugated with very low amounts of proteins did not move through the gel, probably due to agglomeration in the high ionic strength gel buffer. Due to the intensive red color of gold colloids, particle movement in the gel can be easily detected. As shown in Figure 4-13, electrophoresis of AuNP/protein conjugates is successful as indicated by intense red bands, moving different distances dependent on the applied protein concentrations. Bands reach a plateau at quite low protein doses > 4 insulin/np, > 1 transferrin/np and > 5 BSA/NP. The broadening of the bands and shorter migrated distance at lower protein doses (e.g. 2-5 BSA/NP) may be caused by NP/protein agglomerates featuring inhomogeneous size and charge distributions. Insulin/NP ratio Transferrin/NP ratio BSA/NP ratio + Figure 4-13: Gel electrophoresis of protein-coated nanoparticles at different protein-to-np ratios. Note that AuNP concentrations were varied within the course of the experiments (insulin and BSA experiments: c = 16 µg/ml, transferrin experiments c = 3 µg/ml). Conditions:.5x TBE buffer, 1% agarose gel, 1 h at 1 V. Figure 4-14 shows that depending on the protein used for NP coating, conjugates saturated with excess amounts of proteins move different distances following the order insulin > BSA > transferrin. The moved distance in an electric field correlates with the electrophoretic mobility, which is a function of sample charge and size. 16 The data indicate

104 saturarted 5:1 4:1 3:1 saturated 2:1 1:1 1:2 saturated 4:1 3:1 2:1 4. Results and Discussion 99 that AuNP/insulin conjugates are smaller and more negatively charged compared to AuNP/BSA and AuNP/transferrin conjugates. This is in agreement with the order of the proteins molecular weights (insulin > BSA > transferrin, Table 3-3), the determined zeta potentials and IEPs (insulin > BSA transferrin, Figure 4-8). Insulin Transferrin BSA + Figure 4-14: Determination of moved distance of protein-coated AuNPs by gel electrophoresis depending on the type of the protein. All samples were run in the same gel. Conditions:.5x TBE buffer, 1% agarose gel, 1 h at 1 V, c(aunp) = 16 µg/ml. Gel electrophoresis with different kinds of gold nanoparticles has been conducted by other groups before Liu et al. report the migration of 2 nm AuNPs coated with different amounts of PEG ligands. 455 Similar to the experiments performed here, they observe an increase in the distance that the particles migrate at increased ligand ratios. Depending on the PEG size, they observe plateau values at applied PEG-to-NP ratios of 1:1 (for PEG 9 g/mol) and 25:1 (for PEG 5 g/mol), which are much higher than the values found in this work, e.g. for Au/insulin conjugates. The nanoparticle size (and available surface area per particle) is much smaller for 7 nm AuNPs of this work compared to 2 nm AuNPs employed by Liu et al., while the size of insulin may be similar to PEG 5. The difference may also arise from the use of initially ligand-free particles, their particular surface chemistry and the employment of sterically more demanding ligands such as serum proteins (in the case of BSA and transferrin) which may adsorb to the laser-generated AuNP surface more efficiently than to chemically synthesized AuNPs.

105 4. Results and Discussion 1 Fluorescence Quenching Adsorption of serum proteins to colloidal AuNPs was further evaluated using fluorophorelabeled proteins. As illustrated in Figure 4-15, BSA-FITC features an excitation maximum around 485 nm and an emission maximum around 52 nm. In preliminary experiments, the number of bound proteins per nanoparticle was quantified. Two different methods were employed: (i) AuNPs were mixed with FITC-labeled proteins and the fluorescence of the colloid was directly determined. In this regard, the reduction of fluorescence may be interpreted as fluorescence quenching induced by the adsorption of proteins to the nanoparticles. 115 In this respect, Chhabra et al. described that quenching is proportional to 1/L 4 (where L is the distance to the gold surface). 118 Figure 4-15 shows that the signal of FITC-labeled BSA decreases by increasing the concentration of AuNPs in the mixture, i.e. lowering the BSA-to- NP ratio. At an applied dose of 2 BSA/NP, the fluorescence of the protein is quenched by 5%. If 5 BSA/NP are applied, the fluorescence signal is quenched by 9%. Hence, one may conclude that tight BSA binding occurs if one applies approximately 5 BSA/NP. Thereby, all FITC dyes seem to be brought in close contact to the gold surface, which almost completely prevents fluorescence. However, also a quenching signal of 5% may indicate binding of almost all BSA molecules. In this regard one has to consider that multiple FITCs are coupled at different sites within one BSA molecule. Due to the large size of the BSA molecule, the distance of FITCs to the gold surface varies. Hence, FITCs which are exposed at the outer end may be quenched less effectively (dimensions of BSA: 4 x 4 x 1 nm 246 ). In this regard, Acuna et al. determined that at a distance of 1.4 nm from the gold surface, fluorescence is quenched by 5%. 413 (ii) Alternatively, the number of bound proteins per NP was determined by separating unbound proteins from Au/protein conjugates via centrifugation, quantifying unbound proteins in the supernatant via fluorescence or UV/Vis-extinction spectroscopy and recalculating the number of NP-bound proteins from the difference of applied and unbound proteins (see also Figure S6, Figure S7). Briefly, the data of the UV/Vis supernatant analysis show that the conjugation efficiency decreases with increasing ratio of applied proteins per NP, similarly to what has been previously reported for DNA binding to laser-generated AuNPs. 39 The conjugation efficiency begins to decrease when applying more than

106 Fluorescence [a.u.] Fluorescence [a.u.] Emission wavelength [nm] Fluorescence [a.u.] Quenching [%] 4. Results and Discussion BSA/NP and 24 insulin/np. It is hypothesized that monolayer coverage may be achieved by applying approximately these numbers of proteins. Notably, the values obtained for Au/BSA conjugates are of the same order of magnitude than determined via SPR-shift measurements (compare to Figure 4-7, Figure 4-9). By applying less BSA, higher conjugation efficiencies may be achieved because the particle surface remains undersaturated. By applying higher concentrations, few additional proteins may bind to the particle surface but some will remain free in solution, resulting in lower conjugation efficiencies. A detailed discussion on the different results of conjugation efficiency experiments and detection of fluorescently labeled ligands will be given in chapter by the example of coumarinlabeled D % : 5 BSA/NP d=7nm %: 2 BSA/NP d=7nm Excitation wavelength [nm] AuNP [nm] % -99% Pt BSA Pt + BSA FITC Pt + FITC -31% -58% BSA + FITC Pt + BSA + FITC BSA-FITC Pt + BSA-FITC 1 Au BSA Au + BSA FITC Au + FITC BSA + FITC Au + BSA + FITC BSA-FITC Au + BSA-FITC Figure 4-15: Quenching experiments with BSA-FITC. Characterization of the BSA-FITC fluorescence via 3D-fluorescence spectroscopy (top, left). Fluorescence and quenching of 1 µm BSA-FITC at varying AuNP concentrations (top, right). Fluorescence quenching experiments with FITC-labeled BSA and PtNPs or AuNPs (bottom, c(np) = 5 µg/ml, c(bsa-fitc, BSA) =.1 µm, c(fitc) = 1.2 µm). To better characterize the quenching effect of colloidal metal NPs, several control samples containing constant particle and protein concentrations were analyzed. Laser-generated PtNPs with similar properties to AuNPs were employed to discriminate between AuNP specific % -99% -93% -76%

107 4. Results and Discussion 12 effects (i.e. the SPR of AuNP and inner filter effect ) and general NP effects (i.e. the dense arrangement of fluorescent molecules on the particle surface). Details on PtNP properties can be found in the appendix (Figure S35). As can be seen from Figure 4-15, BSA-FITC coated PtNPs reduce the fluorescence to a lesser extent than AuNP (58% vs. 76%, purple bars, please note the logarithmic scale). Possible reasons can include that either less BSA binds to the PtNP surface or that the fluorescence quenching by PtNPs is less pronounced. Notably, when conjugating particles with equimolar amounts of BSA and free FITC, the difference between Au and Pt in FITC quenching is even larger (31% vs. 93%, yellow bars). As described before, the quenching by AuNPs is very efficient, if the emission spectrum of the dye overlaps with SPR peak of AuNPs (compare to chapter 2.2). In the presence of PtNPs, a simpler mechanism (i.e. concentration quenching) occurs. In particular, the quenching by PtNPs seems to be less effective over larger distances. AuNPs and PtNPs both quench FITC in the absence of BSA by 99% (light yellow bars), while the quenching is less pronounced for PtNPs and FITC in the presence of BSA, possibly due to BSA acting as spacer between FITC molecules and preventing concentration quenching. Notably, interactions of fluorophores with non-plasmonic nanoparticles have rarely been investigated in the literature. Ariyadasa observed fluorescence quenching of rhodamin B by PdNPs (non-plasmonic in the visible spectrum). The effect was attributed to a NP-induced energy transfer mechanism which does not require a spectral overlap between donor and acceptor. 456 The results of titration experiments employing a constant BSA-FITC or Aβ-FITC concentration and varying the NP concentrations are illustrated in Figure After fitting the data with the Hill fit, dissociations constants can be derived from the turning point of each curve (i.e. effective concentration to induce 5% of maximal quenching). When comparing the quenching of BSA-FITC by AuNPs and PtNPs, the quenching by PtNPs appears less pronounced, supported by 3-4 times higher dissociation constants and supporting the results of Figure Moreover, the cooperativity factors derived from the slope of each titration correspond to negative cooperativity for binding of BSA-FITC on PtNPs (n < 1) and positive cooperativity for binding of BSA-FITC on AuNPs (n > 1). Possible explanations include that (i) the differences arise from measurement artifacts due to different quenching mechanisms of AuNPs and PtNPs or that (ii) the different surface chemistries of laser-generated AuNPs and PtNPs induce protein binding differently. To prove the second hypothesis, further tests e.g.

108 4. Results and Discussion 13 with NPs of the same material but featuring different oxidation states or advanced analytical techniques (analysis of protein binding spatially resolved on a single particle) would be required. In this respect, BSA binding could be favored at the more hydrophobic AuNP surface 44 compared to the higher oxidized surface 19 of PtNPs. Since more efficient adsorption to AuNP over PtNP was also observed for thiolated molecules (compare to Figure S36 and Figure 4-29), this aspect will be further discussed in chapter Quenching (I -I)/I BSA.5uM BSA.25uM BSA.125uM (R²=.996) (R²=.985) (R²=.985) K D =5.8.3nM n= K D =4.6.4nM n= K D =4.2.5nM n=1.6.9 Quenching (I -I)/I BSA.5uM BSA.25uM BSA.125uM (R²=.965) (R²=.945) (R²=.935) K D = nM n=.95.8 K D = nM n=.8.8 K D = nM n= AuNP [nm] PtNP [nm] Quenching (I -I)/I 1. K D =1.6.1nM n= Abeta.5uM Abeta.25uM Abeta.125uM (R²=.984) (R²=.987) (R²=.987) K D =2.9.2nM n= K D =2.1.1nM n= Fluorescence quenching Fluorophor distance from the NP surface AuNP [nm] Figure 4-16: Fluorescence quenching experiments to determine Aβ-FITC and BSA-FITC dissociation constants and cooperativity on ligand-free AuNPs and PtNPs. Schematic illustration of fluorescence quenching of Aβ-FITC through the adsorption to the AuNP surface (not to scale, bottom, left). When comparing the quenching of BSA-FITC and Aβ-FITC to AuNP, it becomes clear that Aβ-FITC adsorbs stronger to the gold surface than BSA-FITC. Thus, the dissociations constants are lower by 37% to 72%. The cooperativity value is up to five times higher for Aβ- FITC than BSA-FITC on AuNPs. This indicates that Aβ-FITC strongly supports binding of further peptides, after the first peptides are adsorbed to the AuNP surface. Effective synergistic binding of Aβ-FITC to AuNPs may be due to the peptide s relatively small size and dense packing capability and/or due to its self-aggregation tendency which may even be

109 4. Results and Discussion 14 enhanced by adsorption to the NP surface. 389,412 Douglas et al. have analyzed the interaction of unfunctionalized, citrate-capped AuNPs with human blood plasma proteins, whereby anticooperative binding was found for the interaction of AuNPs with albumin, fibrinogen, histone and globulin. 412 In contrast, positive cooperativity was observed for insulin, which was attributed to the NP-promoted formation of ordered insulin fiber structures. 412 Cooperative binding of Aβ to AuNPs will be further discussed in a subsequent chapter (4.3.1). In conclusion, stable gold conjugates can be efficiently designed when the minimum ligand dose necessary to achieve NP monolayer coverage is known. Different methods were presented here including the analysis of the SPR peak at different stabilizer doses, exposure to high salinities, freeze-drying, gel electrophoresis, and fluorescence quenching. Taking the example of BSA, one can see that very similar results are obtained with the different methods with the exception of freeze-drying, were approximately 1 times more BSA/NP are necessary. This may be due to the extreme conditions, i.e. the low temperature, removal of the hydrate layer and minimum interparticle distance after drying (Table 4-3). Table 4-3: Minimum BSA-to-NP ratio to maintain colloidal stability (interpreted as minimum BSA-to-NP ratio to achieve monolayer coverage) determined by different methods. Applied BSA per NP SPR peak shift (after freezing) 125 SPR peak shift 13 Gel electrophoresis 6 SPR peak shift (in presence of 1 mm NaCl) 4 Fluorescence quenching (9%) 4-17 By summarizing all results, it becomes apparent that small (<4 g/mol) as well as bulky ligands readily adsorb to laser-generated, ligand-free NPs and stabilize them against agglomeration induced by different external stress factors. The results of decreased stability at increasing salt concentration followed the expected trends from DLVO theory. According to the DLVO theory, the stability of a colloid is determined by the balance between electrostatic repulsion and van der Waals attraction. 99,1 Adding electrolytes to AuNPs will shield the repulsive double-layer charges, reduce electrostatic repulsion and induce particle agglomeration. However, ligands adsorbed to the particle surface can (sterically) stabilize the colloid by preventing the close approach of two particles. Moreover, once chemisorbed to the

110 4. Results and Discussion 15 particle surface, desorption of thiolated ligands and subsequent particle-particle aggregation is unlikely because it would require a large positive free energy input. 457 It was found that the number of ligands required for stabilization was different for each stabilizer. This could be a result of the different ligand sizes, where large ligands saturate the gold surface already at low concentrations. Table 4-4 shows typical footprints for small thiolated ligands. Notably, the footprint of MUA on chemically synthesized AuNPs is the equal to that on laser-generated AuNPs (.18 nm², determined via the shift of the SPR peak maximum, Table 4-1). For LA and mpeg-sh, the footprints on laser-generated AuNPs are smaller than those reported in the literature (.12 nm², determined via the shift of the SPR peak maximum, Table 4-1). Generally one can expect that the conjugation of initially ligandfree NPs enables a more efficient and possibly also denser ligand packing (smaller footprints) compared to conjugation of (pre-coated) chemically synthesized AuNPs. 39 Furthermore, denser ligand packing is faciliated on smaller AuNPs due to their higher surface curvature. 17 Table 4-4: Footprints of exemplary small thiolated molecules on chemically synthesized AuNPs (sorted by the molecular weight of the ligand). AuNP diameter [nm] Ligand type Ligand molecular weight [g/mol] Footprint [nm²] Mercaptopropionic acid Mercaptobenzimidazole LA LA MUA MUA Dodecylthio-3H-1,2- dithiole-3-thione SH-PEG 7 -COOH Method inductively coupled optical emission spectrometry Surface enhanced Raman scattering X-ray photoelectron spectroscopy microscale thermogravimetric analysis inductively coupled plasma mass spectrometry X-ray photoelectron spectroscopy 33.2 SPR peak shift inductively coupled plasma mass spectrometry 42 HS-PEG 5 -NH fluorescence Reference Elzey et al. (212) 458 Zhang et al. (21) 459 Ivanov et al. (212) 46 Sebby & Mansfield (215) 461 Hinterwirth et al. (213) 462 Ivanov et al. (212) 46 Lanterna et al. (212) 463 Hinterwirth et al. (213) 462 Xia et al. (212) 464

111 4. Results and Discussion 16 Alternatively, it could also reflect the different binding affinities of the different ligands. All employed ligands featured at least one thiol group, known to undergo chemisorption on gold. 171 Nevertheless, weaker forces (electrostatic, hydrophobic, hydrogen bonding or van der Waals) may have induced the preliminary interactions before the covalent gold-sulfur bond built. In this regard, the small thiolated ligands mpeg-sh, MUA and LA differed not only in size but also in charge. In contrast to the uncharged, linear mpeg-sh, the adsorption of anionic MUA and LA ligands may not only be electrostatically impeded (repulsion of anionic ligands amongst each other and from the anionic gold surface); their negative charge may also lead to the formation of a hydration layer of the tail groups and steric inter-ligand interferences. 465 The three tested proteins, especially BSA, may have higher affinities to the gold surface than the small thiolated ligands. Despite the overall negative surface charge of serum proteins at neutral ph, the exposure of cationic residues on the protein surface can lead to strong interactions with the anionic NP (e.g. 6 lysine residues in BSA). 466 In the literature, AuNPalbumin dissociation constants between.4 nm and 25 µm have been reported (Table 4-5). Table 4-5: Published dissociation constants for the system BSA/AuNP determined through different methods (adapted from Boulos et al.) 121 and compared with the present study. AuNP AuNP precoating diameter [nm] Analytical method K D [µm] Reference 18 Citrate fluorescence.43 Iosin et al. (29) Citrate fluorescence.33 Shang et al. (27) Citrate circular dichroism.14 Treuel et al. (21) Citrate fluorescence.1 Xie et al. (23) 457 Bulk gold None.2 quartz crystal microbalance surface Citrate.5 Brewer et al. (25) 153 Bulk gold surface MUA quartz crystal microbalance 1.2 Kaufman et al. (27) 248 electrospray-differential 2. 6 Citrate mobility analysis fluorescence 1.6 Tsai et al. (211) 247 infrared spectroscopy MUA analytical ultracentrifugation 5.4 Bekdemir & Stellacci (216) 469 5, 1 Citrate fluorescence 1, 3.3 De Paoli Lacerda et al. (21) Citrate analytical ultracentrifugation 13.6 Bekdemir & Stellacci (216) Citrate scattering correlation spectroscopy 25 7 None fluorescence.49 Dominguez-Medina et al. (212) 47 This study (average value derived from Figure 4-16)

112 4. Results and Discussion 17 As was illustrated in Figure 4-15, NPs feature a high affinity for FITC itself so that BSA binding to the NP surface may be enhanced through interactions between the metal surface and FITC. Moreover, the strong light absorption of AuNPs may significantly interfere with the fluorescence titrations, which will be discussed in more detail in section AuNPs can either absorb protein fluorescence emission even without being in close proximity, or scatter fluorescence excitation light or emission ( inner filter effect ). 121 To analyze if the quenching is a distant-dependent effect, unlabeled proteins or thiolated PEG ligands were employed for preincubation with NPs before adding FITC-labeled proteins. The data show higher dissociation constants in the presence of the spacer molecules, supporting the idea that not BSA-FITC can no longer reach the AuNP surface as effectively. Moreover, BSA-FITC may bind less strongly to preincubated AuNPs (Figure S8,Table S2). With regard to the surface density, maximum surface loads between 3.7x1 12 BSA/cm² and 1.3x1 13 BSA/cm² have been reported before. 153,457 With laser-generated AuNPs, similar or higher BSA packing can be achieved (here: 4 to 125 BSA/NP corresponding to 3.x1 12 to 8.1x1 13 BSA/cm²), possibly due to the high surface curvature of 7 nm AuNPs and the absence of other ligands.

113 Hydrodyn. Diameter [nm] Zeta Potential [mv] 4. Results and Discussion Characterization of D3- and Aminopyrazole Trimer- Nanobioconjugates Qualitative Detection of D3 and Aminopyrazole Trimer Binding To generate nanoconjugates which effectively interact with aggregating Aβ, particles were functionalized with different variants of the D3 peptide and trimeric aminopyrazole ( Trim ). Binding of these functional ligands was qualitatively detected by DLS and zeta potential measurements, including the determination of the isoelectric point of the conjugates. The analyses of hydrodynamic diameters and zeta potentials reveal that all cationic ligands bind to the NP surface (Figure 4-17). All ligands increase the hydrodynamic diameter of the particles by 3-5 nm. Notably, the increase in hydrodynamic diameters does not correlate with the ligand length possibly due to the minor differences of the ligand compositions (e.g. 13, 16, 18 amino acids for D3_5+, 8+, 1+). Moreover, the ligands induce a reversal of net charge from anionic (ligand-free AuNPs) to cationic (nanoconjugate) when applied at a ligand-to-np ratio of 418:1. As expected, the more cationic the ligands are, the stronger is the increase of the zeta potentials (D3_5+ D3_8+ < D3_1+ and Trim_5+ < Trim_8+) AuNP Au/D3_5+ Au/D3_8+ Au/D3_1+ Au/Trim_5+ Au/Trim_8+ -3 AuNP Au/D3_5+ Au/D3_8+ Au/D3_1+ Au/Trim_5+ Au/Trim_8+ Figure 4-17: Increase of hydrodynamic diameter as determined from DLS measurements (number frequency, left) and reversal of net charge as determined from zeta potential measurements (right) upon conjugating AuNPs with 418 ligands per NP (c(aunp) = 5 µg/ml, c(ligand) = 1 µm). Figure 4-18 illustrates that the IEP of the nanobioconjugates shifts away from the isoelectric point of the ligand-free particles (ph = 2) to more neutral and alkaline ph values. The amino acids arginine and lysine, which carry alkaline side chains and feature isoelectric points of and , are part of the employed ligands. Hence, when they are adsorbed to the

114 4. Results and Discussion 19 particle surface, they shift the IEP of the colloids to their pk a, similar to the stabilizing molecules employed in chapter Interestingly, the less lysine residues are linked to the polyarginine peptide sequence, the further the IEP is shifted to neutral ph values. This was demonstrated by comparing AuNPs conjugated with equimolar amounts of D3_5+ (5 arginine residues), D3_8+ (5 arginine and 3 lysine residues) and D3_1+ (5 arginine and 5 lysine residues). Assuming that approximately equal numbers of each ligand bind to the particle, Au/D3_5+ conjugates will carry less charges than those carrying D3_1+. Hence, less hydroxide ions are required to neutralize the nanoconjugate s charge. While this interpretation is valid qualitatively, quantitative statements on the number of ligands bound to the NP surface cannot be extracted. In this regard, the hydroxide concentration to neutralize Au/D3_5+ would be expected to be half of the amount as for Au/D3_1+ (if the same number of ligands bind). However, the experimentally determined concentration is around four orders of magnitude lower. One hypothesis is, that the pk a of D3_5+ is shifted to lower ph due to intramolecular interactions by the relatively high amount of neutral amino acids in D3_5+ (61%). In contrast, the pk a values of D3_8+ and 1+ may be close to the pk a of pure arginine and lysine, possibly because of the high amounts of charged amino acids (5% and 56%). In this regard, it was previously reported that (within proteins in low polar microenvironments) the pk a of lysines gets lowered to favor a more neutral charge at neutral ph. 472 Secondly, the applied number of the same ligand (D3_5+) was varied from under- to oversaturation of the gold surface. As expected, the results show that the isoelectric point of the conjugate shifts towards higher ph by increasing the applied number of ligands and increasing the conjugate s net charge. Notably, a very low ligand concentration (c =.3 µm) corresponding to 12 D3_5+ per NP already induces an IEP shift. It shows that the determination of the colloid s IEP is a very sensitive technique to evaluate the NP surface chemistry, in particular for ligands oppositely charged to the NP. Moreover, this is a first experimental evidence that ligand binding takes place at low applied doses, which will be analyzed in detail in the following chapters. At last, IEPs of monofunctional, as well as bifunctional conjugates were determined. The adsorption of the aminopyrazole trimer ligand (five lysine residues) shifts the IEP of colloidal AuNPs very similar to that of Au/D3_5+ conjugates (125 ligands/np). In contrast, AuNPs

115 Zeta potential [mv] Zeta potential [mv] Zeta potential [mv] 4. Results and Discussion 11 incubated with a mixture of 125 D3_5+ and 125 aminopyrazole trimer_5+ per NP feature a higher IEP than the monofunctional conjugates. Thus, a higher overall ligand number seems to be attached to each particle in the case of bifunctional AuNPs. It indicates that (despite the equal net charges of the two ligand types) one ligand does not prevent the binding of the other. It can be concluded that the negative surface charge of the AuNPs seems to be stronger in attracting the ligands than electrostatic repulsion by already adsorbed cationic ligand. ph 6.3 MilliQ ph 7.3 brain ECF ph 7.4 DMEM PBS blood 4 Au/D3_5+ Au/D3_8+ 3 Au/D3_ AuNP Au/D3_5+ (13) 3 Au/D3_5+ (3) Modell BiDoseResp double span = A2 - A1; 2 double Section1 = span*p/(1+pow(1,(logx Au/D3_5+ (125) ( ) Gleichung Chi-Quadr Reduziert Au/D3_5+ Au/Trim_5+ Au/D3_5+/Trim_5+ 1-x)*h1)); double Section2 = span* (1-p)/(1+pow(1,(LO Gx2-x)*h2)); y=a1 + Section1 +Section2; Kor. R-Quadrat.995 Zeta potential ph Wert Figure 4-18: Isoelectric points of Au-D3 and Au/aminopyrazole trimer conjugates. Shift of the isoelectric points as function of the ligand net charge (top, 3 µm ligand, 5 µg/ml AuNP), the ligand density on the particle surface (middle, ligands/np) and the ligand type (bottom, each 3 µm ligand, 5 µg/ml AuNP). ECF: extracellular fluid, DMEM: Dulbecco's Modified Eagle Medium, PBS: phosphate buffered saline. Standardfehler A A LOGx LOGx h h p ( )

116 4. Results and Discussion 111 Notably, the determination of the ph-dependent zeta potentials does not only give information about the isoelectric point and the influence of ligands thereon. 442 The curves can also be used to determine ph regimes of low and high conjugate net charge, which may correlate with the colloidal stability (Figure S1). When working in biological media or ultimately in the blood or brain, a ph of 7.4 is most common. At this ph, fully-coated, monofunctionalized AuNP/D3_5+ conjugates are almost uncharged and thus unstable. As can be derived from Figure 4-18, a strategy to improve their electrostatic stability includes the reduction of the ligand density (< 125 D3_5+/NP) or replacement of D3_5+ by higher charged D3 variants. Alternatively, AuNP/D3_5+ conjugates can be sterically stabilized, e.g. with an outer BSA coating, as will be discussed in section As will be shown in Figure 4-19 and section 4.2.5, ligand binding (and subsequent interaction with Aβ) can also be monitored via analytical disc centrifugation. In this regard it needs to be considered that ligands can decrease the conjugate s overall density and increase their drag force. This will reduce their sedimentation rate as compared to the bare AuNPs. Since the ADC correlates the time needed for sedimentation with the apparent particle diameter by employing a constant density (19.3 g/cm³ for AuNPs), particles are expected to appear smaller, if their density decreases (as will be shown in detail in Figure 4-2). Figure 4-19 illustrates that at high and low ligand-to-np ratios, the Au/D3 conjugate s diameter is approximately 7-8 nm, which is very similar to the diameter of bare AuNPs (compare to Figure 4-3). However, disc centrifugation reveals that broader size distributions and some larger species exists, when the conjugates are prepared at intermediate ligand-to-np ratios. It is assumed that the cationic ligand induces charge compensation of the anionic NPs in these samples at ligand doses > 2/NP and neutral ph (compare to Figure 4-18, middle, and Figure 4-21). In this regard, particle agglomeration causes the conjugates to appear larger than bare AuNPs. Notably, the size distribution of these Au/D3_5+ conjugates also shows the presence of smaller species (6-7 nm), which may indicate that both agglomerated and few non-agglomerated conjugates are present side by side in the colloid. At high ligand doses, the conjugates recover partly from aggregation and the mode at 6-7 nm becomes dominant again in the mass-weighted size distribution. These observations support the concept of charge balancing 48 of negatively charged NPs by positively charged ligands, which will be introduced in chapter

117 Norm. mass frequency Peak Diameter [nm] 4. Results and Discussion 112 Ligand-to-NP ratio Hydrodyn. diameter [nm] Au/D3_5+ 2:1 4:1 21:1 29:1 835: Ligand-to-NP ratio [# applied/np d=7nm ] 1 1 IEP conjugate bare AuNP Ligand concentration [µm] Figure 4-19: Analysis of the hydrodynamic diameter of Au/D3_5+ conjugates as function of the ligand-to- NP ratio, determined from analytical disc centrifugation at neutral ph. Selected size distributions (left) and peak diameters as function of the applied ligand dose (right). Note that the density of bulk gold was employed for calculating NP diameters from the sedimentation experiment. The diameter of bare AuNPs (red) and the ligand-to-np ratio inducing charge compensation of Au/D3_5+ conjugates (blue) are shown for comparison. Figure 4-2 shows how particle size and density are interrelated and jointly determine the sedimentation behavior of the sample in the centrifugal field. Considering the applied ligand doses of 418 ligands/np (1 µm), the numbers of bound ligands per NP were determined to be 126 (Table 4-7). Figure 4-2 (right) shows that if 126 ligands are attached per particle, the overall density is changed from 19.3 g/cm³ to 8.8 g/cm³, respectively. Bekdemir and Stellacci 469 have recently determined the increase in hydrodynamic diameters and densities of ligand-coated AuNPs via analytical ultracentrifugation. They report that AuNPs with a core diameter of 6.1 nm increased in hydrodynamic diameter due to the binding of MUA and BSA to 9.4 nm. At the same time, the authors report that the conjugates densities were lowered to 5.9 g/cm³. 469 It illustrates that the bulk density of gold cannot be applied for calculations on the sedimentation behavior of Au-ligand conjugates. Moreover, if the overall density decrease resulting from ligand adsorption coincides with a certain increase in overall size, the conjugate cannot be discriminated from the bare NP via analytical centrifugation. In fact, a bare 7 nm AuNPs (ρ= 19.3 g/cm³, (1) in Figure 4-2) sediments in the ADC at the same time as a larger but less dense NP-ligand conjugate (e.g. (1) (2) (3) in Figure 4-2).

118 Time [min] Density of nanoconjugate [g/cm³] 4. Results and Discussion (2) (3) 19.3g/cm³ 18.9g/cm² 15g/cm² 1g/cm² 5g/cm³ 3g/cm² 2.2g/cm³ (1) Diameter [nm] density of bulk gold: 19.3 g/cm³ Number of bound ligands per NP Figure 4-2: Calculated sedimentation behavior of nanoconjugates in a centrifugal field. Correlation of the diameter with the sedimentation time as function of the overall conjugate density. Note that bare, 7 nm AuNPs (1) sediment in the ADC at the same time (2) as conjugates featuring a density of 5 g/cm³ and a size of 14 nm ((3), left). Dependence of the conjugate s density on the number of attached ligands per AuNP. Calculations are based on the partial specific volume of proteins (.73 cm³/g), estimated volume per D3_5+ ligand of 2 nm³, estimated ligand length of 5 nm and a NP core diameter of 7 nm. Further information can be found in the supporting information (Table S4) Nanobioconjugate Stability In order to assess nanobioconjugate stability, colloidal properties were analyzed as function of the applied ligand-to-np ratio directly after conjugation. Figure 4-21 shows how colloidal properties of AuNPs are altered in the presence of different amounts of cationic ligands. As reported by Gamrad et al., 48 a certain range of ligand concentrations will compensate the NPs charge and induce particle agglomeration. On the one hand, titrating ligands to AuNPs to reach the isoelectric point of the particles may be advantageous to generate agglomerates of large size (e.g. to enhance cellular uptake 47,16 ), which feature a red-shifted extinction spectrum (e.g. for NIR excitation 16 ). However, the ligand dose to reach the IEP should be avoided, if colloidally stable particles are required with specific surface areas as high as possible. In particular, two regimes of high colloidal stability exists. At low ligand-to-np doses, the conjugate s properties are mainly determined by the gold particle core (i.e. negative zeta potential). At high ligand doses, the ligand shell around the particles determines their properties (i.e. positive zeta potential), as can be seen schematically in Figure 4-21, top.

119 SPR peak shift [nm] SPR peak shift [nm] Colloidal stability (PPI) Colloidal stability (PPI) Zeta potential [mv] Zeta potential [mv] 4. Results and Discussion 114 Ligand surface density [pmol/cm²] Ligand surface density [pmol/cm²] Au/D3_5+ Au/D3_8+ Au/D3_1+ Ligand-to-NP ratio [#/NP d=7nm ] Trim_5+ Trim_8+ Ligand-to-NP ratio [#/NP d=7nm ] Ligand concentration [µm] Ligand concentration [µm] Figure 4-21: Principle of charge compensation during the conjugation of cationic D3_5+ ligands to negatively-charged AuNPs (top). AuNP zeta potential, colloidal stability and SPR peak shift as function of the ligand-to-np ratio for D3 (bottom, left) and aminopyrazole trimer (bottom, right) variants with different net charges. Adapted with permission from Ref Copyright 216 American Chemical Society. DOI: 1.121/acsnano.6b2627 The ligand concentration with which AuNPs reach the IEP can give (semi-)quantitative information about the number of charges on the AuNP surface (Table 4-6). By assuming that all ligand charges contribute to charge compensation, the NP surface charge can be recalculated. Depending on the ligand (D3 vs. Aminopyrazole trimer) and its net charge (5+ to 1+), the numbers of total charges required to induce the IEP range from 163 to 367 per NP. Assuming that the particles have a diameter of 7 nm and a spherical shape, their surface area is nm². Considering the cross section of gold atoms (A= π*r² = π*(.144 nm)² =.65 nm², see supporting information, Table S5), one can calculate a number of surface atoms of 2363 per NP. If the number of ligand charges required to reach the conjugate s IEP

120 4. Results and Discussion 115 corresponds to the number of charges on the NP surface resulting from Au +, one can conclude that 6.9 to 15.5% of the NPs surface atoms are oxidized. Muto et al. titrated laser-generated AuNPs (d = 11 nm) synthesized in pure water with the surfactant CTAB. By employing similar calculations, they determined the amount of oxidized gold surface atoms to be 3.3 to 6.6%. 44 Merk et al. reported a surface oxidation of 6% for AuNPs generated in.1 mm NaCl. 13 In the present study, it was assumed that the number of applied ligands corresponds to the number of bound ligands. This may not be the case since different ligands can feature different affinities for the gold surface. In the case of D3_1+ and D3_8+, the lowest amounts of ligands are required to reach the IEP. This could be an indicator of a high affinity for the AuNPs and/or highly efficient charge compensation. In this regard, the ligand affinities for compensating the AuNP surface charge seem to be determined by both, the overall ligand charge and ligand type (following the order: D3_1+ > D3_8+ > Trim_8+ > D3_5+ > Trim_5+). With regard to literature, Gamrad et al. 48 examined the charge compensation of laser-generated AuNPs (= 5 nm) with cationic peptides carrying net charges of +3 and +1. Similar to the results shown in Table 4-6, they found that the conjugates IEPs were reached at higher ligand doses for less charged peptides (peptide 3+ : 535 pmol/cm²; peptide 1+ : 225 pmol/cm²). 48 Notably, these values deviate by factors of 3-3 from the results of the present study. The differences may arise from the different peptide ligands used and differences in the AuNP properties. For example, non-centrifuged AuNPs (generated by ps laser ablation and featuring a bimodal surface-weighted size distribution) were employed by Gamrad et al.. 48 Colloids generated by ns-plal and featuring a narrow, monomodal size distribution after centrifugation were employed in this thesis. Table 4-6: Number of cationic ligands and overall charges required to establish the IEP of AuNP. Ligand dose to induce the IEP [No. of applied ligands/np] Ligand dose to induce the IEP [pmol ligands/cm 2 NP] Total ligand charge to induce the IEP [No. of charges/np] D3_ D3_ Trim_ D3_ Trim_ Next, the stability of selected conjugates was analyzed as a function of time (Figure S11, Figure 4-22). At ligand doses above or below the IEP of the colloid, conjugates do not notably change their concentration, wavelength of the SPR peak maximum or PPI over a period of

121 Absorbance [a.u.] SPR peak maximum [nm] Absorbance [a.u.] Absorbance [a.u.] Rel. Colloid Concentration [-] Colloidal stability (PPI) 4. Results and Discussion h (red and blue curves). In contrast, at a ligand dose close to the IEP (42 D3_5+ per NP), the properties of AuNPs strongly change over time. Especially the constantly decreasing colloid concentration illustrates that particle agglomeration takes place due to insufficient electrostatic stabilization. With regard to future applications of nanoconjugates, a high stability over time is desirable to ensure that NPs are accessible for dissolved Aβ and that ligand presentation is always the same. This can be assured by overall anionic nanoconjugates with low ligand coverage or cationic nanoconjugates with high ligand coverage..8 "anionic" 4 D3 per NP "neutral" 42 D3 per NP time.5. 2 anionic neutral cationic time "cationic" 418 D3 per NP time Wavelength [nm] time [h] Figure 4-22: Time-dependent analysis of AuNP colloidal properties after conjugation with D3_5+ at low, intermediate and high ligand doses over a period of 12 h. Note that ligands were added directly after the first measurement. With regard to bifunctionalized AuNPs, Figure 4-23 shows how colloidal properties are altered as a function of the ligand concentration. Note that coumarin-labeled D3 was employed in these experiments. For comparison, the influence of each single ligand type on AuNPs is illustrated. Considering the PPI, the properties of the bifunctional conjugates resemble those of the AuNP/D3-Cou conjugates more closely than the AuNP/Trim_5+ conjugates. Regarding the SPR peak maximum, the maximum of the bifunctional conjugates is located at lower ligand-to-np ratios (each 22 ligands/np) than the maximum of AuNP/D3- Cou (28 ligands/np) and AuNP/Trim_5+ conjugates (46 ligands/np). However, one has to

122 Colloidal stability (PPI) SPR peak maximum [nm] 4. Results and Discussion 117 consider that the total number of ligands corresponds to 44 per NP in the case of bifunctional conjugates. It indicates that both ligands bind to the particle surface in parallel (with slight cooperative effect), resulting in higher overall net charges compared to the monofunctionalized conjugates at similar ligand concentrations. Sanz and colleagues examined the multifunctionalization of AuNPs with TAT peptides (cationic), azide-containing PEG (positively polarized) and oligonucleotides (anionic). 473 They report that the TAT peptide and the PEG synergistically enhance the oligonucleotides loading on AuNPs. In particular, oligonucleotide loading was up to five times higher in the presence of TAT and PEG-azide. This was attributed to electrostatic interactions as well as hydrogen bond formation. 473 As shown by the results in Figure 4-23 and in 4.2.4, enhanced ligand binding can not only be achieved with ligands of opposite charge (oligonucleotide and peptide vs. two cationic ligands). In the case of two cationic ligands one could expect electrostatic repulsion. However, weak forces between ligands (e.g. van der Waals) and attraction to the negatively charged AuNP surface may aid ligand binding. Ligand-to-NP ratio [#/NP d=7nm ] 6 58 Trim_5+ D3-Cou D3-Cou + Trim_ Ligand concentration [µm] Figure 4-23: SPR peak shift and colloidal stability as function of ligand-to-np ratio for mono- and bifunctionalized AuNP conjugates. Bifunctionalized conjugates were prepared by applying a 1:1 mixture of D3-Cou and Trim_5+ to the AuNP colloid with each ligand at the indicated concentration.

123 Zeta Potential [mv] Zeta Potential [mv] Absorbance [a.u.] Absorbance [a.u.] 4. Results and Discussion 118 Since the stoichiometry which is most effective for further applications is yet unknown (compare to chapters and 4.3.5), additional conjugates were designed by applying the two ligands in a 1:1 ratio. By applying the two ligands subsequently starting with a belowmonolayer dosage of the first ligand and adding the second ligand with a 1-times excess, one might expect complete binding of the first ligand. Although the SPR maximum shifts already with the addition of the first ligand, the zeta potential becomes positive only after the addition of the second ligand. Hence, it is concluded that the binding of the second ligand also takes place (Figure 4-24). Whether binding of the second ligand is complete or whether some ligands remain unbound, will be evaluated by determining the ligand density of bifunctional conjugates (see chapter 4.2.4)..8.6 SPR peak maxima nm nm nm nm.8.6 SPR peak maxima nm 52 1 nm 53 1 nm nm.4 Au Au/D3_ Au Au/Trim_5+ 12 Au/D3_ /Trim_ (mix) Au/D3_5+ 12 /Trim_ (successively) Wavelength [nm] Au/Trim_ /D3_ (mix) Au/Trim_5+ 12 /D3_ (successively) Wavelength [nm] AuNP AuNP + D3 AuNP 1. D3 2. Trim AuNP + mix 2-2 AuNP AuNP + Trim AuNP 1. Trim 2. D3 AuNP + mix -4-4 Figure 4-24: Conjugation of D3-Cou and Trim_5+ for the generation of bifunctional 1:1-conjugates. Colloidal properties of 1:1-conjugates are shown for the addition of the two ligands subsequently or as a mixture. Conjugates with 12 D3 and 125 aminopyrazole trimer/np are shown on the right; conjugates with 12 Aminopyrazole trimer and 125 D3 are shown on the left. Note that UV/Vis measurements were performed in triplicate of which one representative spectrum is shown, respectively. In summary, the analysis of colloidal properties as function of the applied ligand-to-np ratio revealed the presence of three stability regimes: high stability and anionic net charge for AuNPs undersaturated with cationic ligands; low stability and zero net charge when crossing the isoelectric point at intermediate ligand-to-np ratios; and high stability and cationic net

124 4. Results and Discussion 119 charge for AuNPs saturated with cationic ligands. 48 The number of ligands required to compensate the NPs charge was specific for each ligand type. It should be considered that these differences might result from different affinities to the gold surface, with D3 ligands generally showing higher affinities than aminopyrazole trimer ligands. Additionally, the titration of AuNPs with the two functional ligands D3 and aminopyrazole trimer as 1:1 or 1:1 mixtures showed that analyzing the SPR peak maxima and zeta potentials are suitable to qualitatively prove the simultaneous binding of both ligands on AuNPs. Regarding the design of bifunctional conjugates, the application of ligand mixtures is recommended for the generation of 1:1 conjugates. If one wants to design 1:1 conjugates, the subsequent addition of the ligands is recommended. Although these conjugates appear less stable (stronger SPR peak shift and peak broadening, Figure 4-24), the subsequent addition can ensure that the less abundant ligand binds to the NP surface. In contrast, the addition as ligand mixture will lead to a competition for binding sites and may reduce the (statistic) probability of binding by the less abundant ligand Stabilization of Nanobioconjugates with Small Thiolated Ligands and BSA As discussed before, in retaining the biological function of the conjugated D3 and aminopyrazole trimer molecules, AuNP stability plays an important role. If AuNPs agglomerate, their specific surface decreases so that less biofunctional ligands will be exposed. As was analyzed in chapter 4.2.2, the colloidal stability may be compromised as function of the ligand-to-np ratio due to charge balancing of the cationic ligands and the anionic gold surface. Hence, additional coating with bulky molecules may help to sterically stabilize the functional conjugates. In this regard, effectively stabilizing ligands should carry functional groups, that bind to the nanoparticle surface strongly. Moreover, charged and/or polymer segments can (electro)sterically stabilize the nanoconjugate. 474 On the other hand, bulky stabilizers may sterically block functional ligands and impair their functionality if they are placed side by side on the AuNP surface. 455 Hence, an optimum stabilizer design needs to be found. In this respect, Au/D3 conjugates were coated with LA, MUA, mpeg-sh and BSA as thiolated dummy ligands with the exclusive function of stabilizing the conjugates. As can be seen in Figure 3-4, LA, MUA and mpeg-sh are relatively small compared to the functional aminopyrazole trimer and D3 ligands, whereas BSA is bulkier than the functional ligands.

125 4. Results and Discussion 12 The long-term stability of cationic AuNP/D3_5+ conjugates was analyzed during 4 d for the incubation in water and in PBS buffer (I 15 mm). As reference, bare AuNPs were analyzed which are electrostatically stabilized intrinsically due to their partially oxidized surface and the adsorption of chloride anions. For the incubation in water, the colloidal stability of the AuNP/D3_5+ conjugates decreased over time contrary to bare AuNPs, where the stability remained constantly high (Figure 4-25, left). The stabilization abilities of the dummy ligands follow the order: LA MUA < mpeg-sh < BSA. After 4 d, the particle concentration of non-stabilized, LA-, MUA- and mpeg-sh-stabilized conjugates dropped by approximately 7%. Notably, BSA was able to effectively prevent particle agglomeration and precipitation even after 4 d. During the incubation in high ionic strength PBS buffer (Figure 4-25, right, close to physiological salinity), the colloidal stability of the AuNP/D3_5+ conjugates decreased even more drastically. Moreover, also bare AuNPs did not remain stable and the high salt concentrations induce particle agglomeration. While the electrostatic stabilizers LA and MUA did not increase particle stability in the buffer medium, the bulkier mpeg-sh sterically stabilizes the conjugates to some extent. Similar to the incubation in pure water, the stabilization with BSA was most effective so that the colloid concentration, PPI and SPR peak maximum remain mainly unaffected over time. Parak and colleagues have recently determined the stability of different polymer-coated AuNPs in biological media at different temperatures. 475 For cationic AuNP-PAH (PAH, polyallylamine hydrochloride, M W = 15 g/mol) incubated in PBS, they observed particle agglomeration at ambient temperature within 2 hours, despite the polymer coating. This shows that high ionic strength media strongly screen the charges on the nanoconjugates surface, which can lead to a loss of colloidal stability even for particles that carry high molecular weight (i.e. sterically stabilizing) ligands. 475

126 SPR peak shift [nm] SPR peak shift [nm] Collidal stability (PPI) Colloidal stability (PPI) Rel. colloid concentration (c/c ) Rel. colloid concentration (c/c ) 4. Results and Discussion 121 In water: In buffer (15 mm ionic strength): bare +D3 +D3+LA +D3+MUA +D3+mPEG-SH +D3+BSA bare +D3 +D3+LA +D3+MUA +D3+mPEG-SH +D3+BSA Incubation time [d] Incubation time [d] Figure 4-25: Long-term stability of bare and cationic Au/D3_5+ conjugates in the absence and presence of small thiolated ligands and BSA. Stability of colloids in pure water (left) and in high ionic strength buffer (right) is analyzed over a period of five days. D3 and stabilizer molecules were applied in a 1:1 ratio with 418 ligands per NP (c(aunp) = 5 µg/ml, c(d3, stabilizer) = 1 µm). Successive addition of D3 and stabilizer. In the next experiment, the order of ligand addition was varied in a way that either the functional D3 was added first and the stabilizer secondly, the stabilizer was added first and the D3 peptide secondly or both molecules were added as mixture (Figure 4-26). The analysis of the SPR peak maximum shows that the least peak shift, i.e. high particle stability, can be achieved by adding stabilizers and peptides as mixture. This is in contrast to findings by Liu et al. who described that the sequential addition of PEG followed by a peptide led to more stable conjugates than simultaneous coadsorption. 455 The difference may arise from the different peptide sequence and PEG molecules used. PEG ligands of Liu s work featured molecular weights of 9-5 g/mol so that their adsorption to the gold surface may have been slower than the adsorption of the smaller mpeg-sh (M W = 35 g/mol) employed in this work. In addition to the optical properties, the conjugates zeta potentials were analyzed. Notably, the addition of any stabilizer lowers the absolute zeta potential values of the AuNP/D3_5+ conjugates. This could indicate that, either less cationic D3 binds to the particles in the

127 SPR peak shift [nm] Zeta Potential [mv] 4. Results and Discussion 122 presence of the stabilizer, or that the additional binding of the stabilizer alters the zeta potential. In particular, stabilizer molecules may increase the conjugate s drag force and friction, hence reduce its electromobility. 16,396 Moreover, anionic stabilizers may compensate the charge of the cationic conjugate. To understand which effect leads to the reduction of the zeta potential values, the D3 density on the AuNP surface will be analyzed in chapter mix peptide first stabilizer first 418 ligands/np d=7nm (each) AuNP +D3 +D3+LA +D3+MUA +D3+mPEG +D3+BSA AuNP D3 D3+LA + D3+MUA + + D3+mPEG D3+BSA Figure 4-26: SPR peak shift and changes in the zeta potential of cationic AuNP/D3_5+ conjugates in the presence of small thiolated ligands and BSA (c(aunp) = 5 µg/ml, c(d3, stabilizer) = 1 µm). For bioapplications, not only the colloidal stability in biological media but also the preparation and storage of the conjugate as lyophilized powder would be advantageous. This could be realized by freeze-drying the conjugate. As shown in Figure 4-27, the AuNP/D3_5+ conjugates cannot be frozen, dried and resuspended without losing their colloidal stability. This may be due to insufficient steric and electrostatic stabilization and is in contrast to the conjugates of Lévy et al.. They designed a peptide sequence (CLANN) for AuNP capping through a combinational library, offering maximum NP stability upon freezing, filtration, chromatography, electrophoresis, and centrifugation. 8 BSA was thus selected as sterically demanding dummy ligand to evaluate the stabilizing ability upon freeze-drying. To determine the minimum number of BSA required for Au/D3 conjugate stabilization, the colloids were frozen, dried and resuspended in the presence of different BSA concentrations (compare to Figure 4-12). The wavelength of the SPR peak

128 Colloidal Stability (PPI) SPR peak maximum [nm] 4. Results and Discussion 123 reaches a minimum at a BSA-to-NP ratio of ~1:1. Notably, colloidal stability (as determined by calculating the PPI) is still relatively low at this BSA-to-NP dose after drying and resuspension. Only for the highest BSA concentration applied (418 molecules/conjugate) effective stabilization occurs, where PPI and SPR peak maximum of dried and resuspended colloids are comparable to the values obtained without freeze-drying (Figure 4-38). 7 6 BSA-to-NP ratio [#/NP d=7nm ] BSA concentration [ M] Figure 4-27: Colloidal stability upon freeze-drying and resuspension of cationic AuNP/D3_5+ conjugates as a function of the BSA concentration. (c(aunp) = 5 µg/ml, c(d3_5+) = 1 µm). Successive addition of D3 and BSA. In summary, improving the colloidal stability of gold-peptide conjugates was most successful employing the bulky mpeg-sh and BSA molecules (in particular for incubation in physiological salinities). Apparently the small thiolated ligands LA and MUA cannot exert sufficient steric effects. In contrast to stabilizing negatively charged ligand-free AuNPs, the negative stabilizer charge rather complicates stabilization in the case of gold-peptide conjugates due to charge balancing effects. 48 The BSA and mpeg-sh concentrations required to stabilize Au/D3 conjugates are generally higher than the concentrations obtained for ligand-free AuNP stabilization (compare to Table 4-2). This can be explained by the increased hydrodynamic size upon conjugating AuNPs with D3, which results in higher numbers of stabilizers to saturate the conjugate s surface.

129 4. Results and Discussion Ligand Density on the Nanoparticle Surface Ligand Density of Monofunctionalized Conjugates The actual density of bound ligands on the NP surface is an important parameter which can later on affect the conjugate s functionality. It needs to be quantified in order to derive rational structure-function-relationships for the nanoconjugates and to elucidate ligand avidity effects. Generally, a dense ligand packing may be advantageous as higher ligand concentrations may induce greater effects, i.e. by presenting more binding sites and higher target avidities. 27 However, also intermediate ligand concentrations with an optimal interligand distance may be advantageous for further applications. Elias et al. 86 found that cells were targeted most effectively at a ligand density below the nanoparticle surface saturation concentration. This effect was attributed to minimized steric interference of the different ligands amongst each other at intermediate packing density. 86 Hence, laser-generated NPs are the ideal starting material to generate colloids with under-saturated to saturated ligand densities, due to the absence of surfactants during synthesis. Since the original sequence of D3 does not carry residues which could be detected via extinction or fluorescence measurement for quantification, special D3 variants carrying tryptophan and coumarin were synthesized for these experiments. The ligand density on the AuNP surface was determined after centrifugation of the conjugate to separate NP-bound from unbound ligands and subsequent analysis of the supernatant, which contained the unbound ligand fraction only. Alternatively, fluorophore-labeled ligands could be conjugated to AuNPs and the fluorescence was analyzed without further treatment of the sample. In this regard, a reduced fluorescence was interpreted as quenching resulting from ligand binding to the gold surface. 115 It is noted that ligand binding can be expressed as conjugation efficiency (relative fraction of applied ligands which actually bind to the NP) or as ligand density (absolute number of bound ligands per NP). Figure 4-28 shows the results of quenching experiments performed with coumarin-labeled D3 and AuNPs. Either the ligand was titrated to a fixed concentration of AuNPs (left) or the particles were titrated to a fixed concentration of the ligand (right). In principle both titration experiments should yield the same results, if the quenching is solely determined by the D3-to- NP ratio. By determining the ligand-to-np ratio of half maximum quenching (Q 5% ), the saturation concentration of D3 on AuNPs can be extrapolated. Since the extent of

130 4. Results and Discussion 125 fluorescence quenching depends on the fluorophore s distance to the NP surface, 118,413 quenching may not be complete, even though the ligands are NP-bound. For example, fluorophores may be exposed to the outer end of the conjugate, similar to results obtained for BSA-FITC (Figure 4-15). Therefore Q 5% was chosen as threshold and interpreted as saturation concentration for ligand monolayer formation on the particle surface. When comparing the ligand-to-np ratios, which induce half maximum quenching, differences become apparent (Figure 4-28). When titrating D3-Cou to 5 µg/ml AuNPs, 71 D3/NP are required to induce fluorescence quenching of 5%. However, when titrating AuNPs to 5, 1 and.5 µm D3-Cou, the corresponding D3-to-NP ratios inducing 5% quenching are 263:1, 114:1 and 116:1, respectively. As discussed previously, when titrating AuNPs, the inner filter effect may bias the result (as a function of the gold concentration, compare to chapter 4.1.3). In contrast, by keeping the AuNP concentration constant, this effect is diminished since the colloid extinction is the same in every sample. Quenching (I -I)/I 1..5 Q 9% of Qmax =19 Ligands/NP (.46 M D3) Q 5% of Qmax =71 Ligands/NP (1.7 M D3) Q 1% of Qmax =334 Ligands/NP (8 M D3) 5 g/ml AuNP c(d3-cou)=variabel. 1E-5 1E-4 1E c(d3-cou) [µm] Figure 4-28: Determination of the ligand density of coumarin-labeled D3 on AuNPs via fluorescence quenching. Note that when titrating D3-Cou to a constant concentration of AuNPs (left), the lowest quenching was 46% and 48% at 12.5 µm and 25 µm applied ligand concentrations. Therefore, all quenching values were interpreted relative to Q max (1.5) and Q % (.45). Titration of AuNPs to a constant concentration of D3-Cou (right). Figure 4-29 shows the ligand density on the AuNP surface determined via centrifugation of the conjugate to separate NP-bound from unbound ligands and analysis of the supernatant. For the D3 variants, the conjugation behavior seems to be influenced by the presence of the labeling residues (Trp 4, Cou, Trp). Maximum ligand loads for D3-conjugated AuNPs are 12±5, 14±26, 183±21 D3 per NP, respectively (Figure 4-29, left). Noteworthy, the number for bound D3-Cou (14 D3/NP) is higher than the value derived from quenching experiments (Q 5% : 71 D3-Cou/NP, Figure 4-28 left). Quenching (I -I)/I , 1,.5 M D3-Cou c(aunp)=variabel Q 9% Q 5% Q 1% 1E c(aunp) [nm]

131 Ligand density [# bound ligands/np d=7nm ] Ligand density [# bound ligands/np d=7nm ] 4. Results and Discussion 126 While the coumarin-labeled D3 is useful to determine the ligand load at low applied ligand concentrations due to a sensitive detection of the fluorophore label, coumarin is a bulky residue which significantly changes the overall structure of the relatively small peptide. In fact, Zanetti-Domingues et al. described that fluorophore tags can change the overall hydrophobicity of the molecule leading to increased non-specific binding. 476 This may explain the overall lower ligand density for coumarin-labeled D3 (i.e. side-on binding) compared to tryptophan-labeled D3. In contrast, the addition of one tryptophan residue to the 13 amino acids of D3 changes its sequence to a lesser degree, so that D3-Trp is expected to resemble the conjugation behavior of D3 more closely. However, due to its low extinction coefficient, its detection is limited to a concentration of 1.8 µm (74 D3-Trp/NP) Au/D3-Trp Au/D3-Trp4 Au/D3-Cou Applied ligand concentration [µm] 5 Au/Trim_5+ Au/Trim_ Applied ligand concentration [µm] Figure 4-29: Ligand density of different D3 and aminopyrazole trimer variants on AuNPs determined by separating NP-bound and free ligands via centrifugation and subsequent analysis of the supernatant via UV/Vis extinction or fluorescence spectroscopy (c(aunp) = 5 µg/ml). The detection of aminopyrazole trimer ligands could be performed without labeling owing to their heterocyclic structures, which feature characteristic extinction maxima in the UV region (Table S3, Figure S14). The maximum ligand densities of aminopyrazole trimer on AuNPs are 148±22 and 216±27 ligands per NP, respectively (Figure 4-29, right). It is noted that although the ligand density was not determined for D3_8+ and D3_1+, similar trends may apply to these ligands. Apparently, the ligand net charge influences the conjugation behavior in a way that higher net charges result in less dense ligand binding. Considering that the lysine residues of aminopyrazole trimer are located next to the cysteine in the sequence, binding to the NP surface may take place with a covalent bond (between cysteine and gold) and electrostatic interactions (between lysine and negatively charged species on the gold

132 4. Results and Discussion 127 surface). Hence, the ligands may partly bind to the particle with a side on conformation. Since Trim_8+ carried three additional lysines compared to Trim_5+, this ligand may take up more space on the particle surface (i.e. higher ligand footprint) and pack less densely. As discussed for thiolated stabilizing ligands before, the formation of sterically demanding hydration layers around the charged groups of ligands was found to limit their adsorption rates to gold surfaces. 465 Not only may the adsorption kinetics be slowed by the hydrate layer, but it may also result in less dense ligand binding. Table 4-7 summarizes ligand densities of nanobioconjugates which were typically employed in functionality tests. Functionality tests will be described in detail in chapter 4.3. Table 4-7: Overview of the ligand densities, surface coverages and conjugate net charges, as determined from centrifugation experiments for nanoconjugates typically employed in subsequent functionality tests. The extent of monolayer formation was calculated as ratio between the number of bound ligands and the maximum number of ligands to achieve surface saturation (n.d.: not detectable in the supernatant). Applied ligands [#/NP] Maximum No. of ligands for surface saturation [#/NP] Bound ligands [#/NP] Extent of monolayer formation Conjugate net charge D3-Cou 14± % Positive D3-Trp 183± % Positive D3-Trp ±5 12 1% Positive Trim_5+ 216± % Positive Trim_8+ 148± % Positive D3-Cou 14± % Positive D3-Trp 183± % Positive D3-Trp ± % Positive Trim_5+ 216± % Positive Trim_8+ 148± % Positive D3-Cou 14±26 13 ~ 1% Negative D3-Trp 183±21 n.d. ~ 1% Negative D3-Trp ±5 n.d. ~ 1% Negative Trim_5+ 216±27 n.d. ~ 1% Negative Trim_8+ 148±22 n.d. ~ 1% Negative Because previous studies have indicated a non-linear behavior between the nanoparticle concentration and colloidal stability in conjugation experiments with cationic peptides, 48 conjugation efficiencies were determined at gold concentration of 25, 5 and 1 µg/ml with coumarin-labeled D3 (Figure 4-3).

133 Conjugation efficiency [%] Ligand denisty [# bound ligands/np d=7nm ] 4. Results and Discussion Conjugation with D3-Cou: 25 µg/ml AuNP 5 µg/ml AuNP 1 µg/ml AuNP Applied ligand concentration [µm] 2 25 µg/ml AuNP 5 µg/ml AuNP 1 µg/ml AuNP exponential fit Applied ligand-to-np ratio [#/NP d=7nm ] Figure 4-3: Influence of the applied AuNP concentration on the determination of the ligand density of coumarin-labeled D3 via centrifugation. In the previous study by Gamrad et al., 48 the colloidal stability was found to be not only a function of the ligand-to-np ratio. In fact, colloidal stability was observed to be generally higher for lower gold concentrations. 48 This was attributed to dilution effects, since the probability of particle collisions and aggregation is lower at lower NP concentrations. 48 In the present study, the conjugation efficiency decreases when applying ligand concentrations >.8 µm for the lowest gold concentration tested (25 µg/ml). In contrast, when applying higher gold concentrations, conjugation efficiencies decrease at ligand concentrations of >.9 µm (5 µg/ml) and > 1 µm (1 µg/ml). When considering the number of bound ligands per particle (Figure 4-3, right), it becomes clear that the conjugation efficiency is only a function of the ratio between applied particles and applied ligands, independent of the gold concentration. Hence, dilution effects may affect colloidal stability, but do not seem to have an impact on the conjugation efficiency. The fact that only the ligand-to-np ratio determines the number of bound ligands per NP is an important finding for further (functionality) tests, where the standard concentration of 5 µg/ml AuNPs cannot always be applied. In fact, most experiments require a certain minimum concentration of ligands to induce effects so that the gold concentration was adjusted accordingly to maintain a certain ligand-to-np ratio (compare to Table 4-11).

134 4. Results and Discussion 129 Removal of Excessive Unbound Ligands from Oversaturated Nanoconjugates Residual, unbound ligands may hinder the quantitative evaluation of the nanoconjugate s functionality, since effects arising from the nanoconjugate cannot be discriminated from the free ligands. One strategy to circumvent this problem is the design of nanoconjugates with 1% conjugation efficiencies, i.e. all applied ligands actually bind to the NP surface. This can be achieved by employing a submonolayer ligand dose to laser-generated ligand-free colloids (e.g. Figure 4-3). Obviously, in this approach ligand densities are generally low, so that effects arising from multivalency may not be fully exploited. Alternatively, conjugates can be created with excess amounts of ligands. Here, unbound ligands need to be removed subsequently through separation techniques such as gel electrophoresis, extraction or chromatography. 477 Recently, the purification of laser-generated AuNPs conjugated with an anionic model peptide was demonstrated via ultrafiltration. 478 The filtration process with a cellulose membrane could remove up to 61% of unbound peptides. 478 Moreover, up to 87% of the purified nanobioconjugate could be recovered. 478 However, this method was limited to overall negatively charged particles for which electrostatic interaction with the negatively charged membrane was minimized. Generally, the removal of excessive unbound ligands from oversaturated (e.g. Au/D3) nanoconjugates is difficult due to the positive surface charge of the ligands. Even when applied at high ligand-to-np ratios to create (electrostatically stabilized) cationic conjugates, the IEP has to be overcome at the expense of colloidal stability. Hence, a washing procedure at intermediate centrifugal force was developed for AuNPs conjugated with 29 D3-Cou ligands and the efficiency of resuspension was analyzed. In this regard, either a long centrifugation time at relatively low centrifugal force, or a short centrifugation time at relatively high centrifugal force can induce NP agglomeration. What was found to be even more critical was the way of resuspending the formed pellet after centrifugation. While repeated aspirating and dispensing with a pipette or constant ultrasonication could not resuspend the pellet, repeated treatment in the ultrasonic bath and manual shaking (Figure S15) could resuspend the colloid. Thereby conjugates with minimal number of unbound ligands were generated. Combining the results from Figure 4-29 and Table 4-8, it can be seen that about 5% (18 D3-Cou) of the applied 29 D3-Cou ligands bind per AuNP. During the washing procedure,

135 4. Results and Discussion 13 9 µl of the supernatant were discarded and the pellet containing AuNPs and bound ligands was resuspended in the remaining volume of 1 µl (1 v/v%). By considering that the residual solution may also contain unbound ligands, a maximum of 1% of the total unbound ligand dose (11 ligands/np) can be present in the remaining volume of 1 µl. In conclusion, at the expense of 22-29% of the initial Au mass concentration, conjugates with 18 (+ 11) attached ligands/np can be obtained after washing, mostly free of unbound ligands. Table 4-8: Assessment of washing procedures to remove unbound ligands from Au/D3 conjugates via ultracentrifugation and the subsequent characterization of the resuspended colloids. Shown are the differences compared to the starting colloid before centrifugation. Centrifuge parameters 1 min, 3, g 6 min, 5, g 3 min, 1, g ΔSPR peak maximum [nm] ΔPPI Relative AuNP concentration [%] (Abs@38nm after/abs@38nm before) ± Ligand Density of Monofunctionalized Conjugates in the Presence of Dummy Ligands Dummy ligands were conjugated to D3-functionalized conjugates to fulfill two different functions: (i) Thiolated ligands can increase the stability of cationic Au/D3 conjugates (see also chapter 4.2.2). (ii) They mimic the presence of the second ligand type (e.g. aminopyrazole trimer). Macroscopically, stabilization of the colloid will generally prevent particle aggregation, preserve the accessible surface area and support the binding of a high number of functional ligands. Simultaneously, adsorption of the dummy ligands may also reduce binding of functional ligands by taking up space on the NP surface. Figure 4-31 shows the conjugation efficiency of D3-conjugated AuNPs in the presence of LA, MUA and mpeg- SH. Two different ligand concentrations were employed and the order of adding D3 and stabilizer was varied. In the case of mpeg-sh, the presence of the stabilizers does not lower the number of D3 attached to the gold surface. This may seem surprising as one might expect that mpeg-sh takes up D3 binding sites on the gold surface. Even more surprising, at the first glance, is that the presence of LA and MUA increases the amount of bound D3. With regard to the order of addition, the addition of D3 and stabilizer as mixture results in similar to higher D3 densities than without stabilizers. This was not expected, as the small

136 D3 Conjugation efficiency [%] D3 Conjugation efficiency [%] 4. Results and Discussion 131 thiolated ligands may compete for binding sites on the NP surface. Moreover, they could even bind to the surface more rapidly than D3, due to lower molecular weights and higher diffusion constants (Table 3-3). When D3 was first mixed with the colloid and LA or MUA were added subsequently, the D3 density on the gold surface could be increased by up to 1% Applied Dose: 29 ligands/np d=7nm 8 Applied dose: 418 ligands/np d=7nm addition as mixture stabilizer as post-treatment D3 D3 + LA D3 + MUA D3 + mpeg D3 D3 + LA D3 + MUA D3 + mpeg Figure 4-31: Conjugation efficiency of D3-Trp 4 on AuNPs in the presence of small thiolated stabilizers as dummy ligands. Schematic illustration of steric and electrostatic effects of dummy ligands to increase the D3 density on AuNPs (top). Different ligand doses of D3 and stabilizers were applied to 5 µg/ml AuNPs (bottom left, addition as mixture: 29 ligands/np, bottom right: 418 ligands/np). To exclude that the results derive from measurement artifacts, the UV/Vis spectra of the pure ligands were recorded. As can be seen in Figure S16, the small thiolated ligands did not feature significant extinctions at 28 nm, so that the detection of D3 was not biased. Hence, it is hypothesized that the non-charged mpeg-sh can increase the overall ligand packing density on the gold surface by acting as spacer between the D3 molecules. This may support head-on binding via the cysteine and avoid side-on binding through interaction between the positively charged arginine residues and AuNPs. Ansar et al. 479 analyzed the binding of mercaptobenzimidazole to AuNPs via surface-enhanced Raman spectroscopy. Depending on the ph, they determined different saturation packing densities. These were attribute to the different binding conformations of the ligand (i.e. monodentate, upright binding at acidic ph and bidentate, tilt-angle binding at alkaline ph). 479 As described before, Sanz et al. 473 studied the functionalization of AuNPs with thiolated, azide-containing PEG, cationic TAT peptides and anionic RNA. They find an enhancement of RNA binding in the presence of the other

137 D3 Conjugation efficiency [%] 4. Results and Discussion 132 ligands. This was ascribed to intra-ligand electrostatic interactions and hydrogen bonds. 473 Considering the effects induced by LA and MUA, the negative charge of the small stabilizers may electrostatically attract cationic D3 ligands and diminish repulsion between different D3 ligands. Moreover, the stabilizers may act as spacer molecules similar to mpeg-sh, increasing the D3 density on the particle surface by supporting head-on binding. The influence of BSA on the conjugation behavior of D3 had to be evaluated by employing coumarin-labeled D3, since the extinction spectra of the two molecules greatly overlap (Figure S16). As shown in Figure 4-32, the presence of BSA reduces the number of bound D3 when low ligand concentrations are applied as mixture. Although BSA features a lower diffusion coefficient than D3, BSA binding seems to compete with D3 binding when applied as a mixture. This may be due to effective interactions between the colloid and different patches of BSA (e.g. one exposed disulfide bond, electrostatic and hydrophobic interactions). In contrast, when low ligand concentrations are applied subsequently (i.e. D3 at first, BSA secondly), the presence of BSA does not impair D3 binding. A reason may be that the time before BSA addition is sufficient for irreversible binding of D3. Note that these results are similar to the results of the conjugation experiments with mpeg-sh and D3 (Figure 4-31). In conclusion, the presence of stabilizing molecules does not necessarily reduce binding of the functional ligand D3. Particularly, when applied to the colloid shortly after the D3 ligand (t 6 sec), bulky dummy ligands stabilize the colloid without reducing the number of bound D3. In fact, small thiolated dummy ligands can even serve to increase the D3 density. D3 with BSA (mix) D3 with BSA (post-treatment) D Applied ligand dose [#/NP d=7nm ] Figure 4-32: Conjugation efficiency of D3-Coumarin on AuNPs in the presence of BSA as dummy ligand (c(bsa) = 1 µm; post-treatment: addition after 6 s).

138 4. Results and Discussion 133 With regard to homofunctionalized bioconjugates published in the literature, different surface densities have been reported and selected studies are summarized in Table 4-9. For peptides conjugated to AuNPs, very high number densities of > 1 peptides per NP were achieved by attaching the functional peptide to BSA which itself bound to the gold surface. 48 For AuNP-DNA conjugates, tunable surface densities ranging from few to multiple strands per particle 481 have been reported e.g. by controlling the ionic strength, incubation time, DNA strand length, 482 sonication or stopping reagents. 446,481 By increasing the NP size to 25 nm, the number of immobilized DNA strand could even be increased to > 24 per NP. 446 However, considering the relative surface area, higher DNA densities could be established on laser-generated AuNPs than on chemically synthesized AuNPs. 39 Table 4-9: Ligand densities on AuNP surfaces for the conjugation with DNA or peptides reported in the literature and compared to the results of this study. Note that ligand densities were recalculated from the number of bound ligands/np and the NP diameters for better comparison. NP and ligand type Laser-generated AuNPs Penetratin peptide Citrate-coated AuNPs ssdna (12 base pairs) Citrate-coated AuNPs ssdna (15 base pairs) AuNPs (in situ reduction of HAuCl 4 in HEPES buffer and peptide) Antimicrobial peptide Laser-generated AuNPs NLS peptide LNA oligonucleotides Citrate-coated AuNPs peptides derived from tumor-associated antigens Laser-generated AuNPs ministrep aptamer penetratin peptide Citrate-coated AuNPs CLPFFD peptide Laser-generated AuNPs ssdna (23 base pairs) Citrate-coated AuNPs Cell penetrating peptide (attached to BSA) Laser-generated AuNPs D3-Trp peptide Trim_5+ NP diameter Ligand density [pmol ligands/cm² colloid] Reference 7 nm 29 Petersen et al. (21) nm 34 Demers et al. (2) nm 59 Hurst et al. (26) nm 65 Rai et al. (216) nm 73 (sum) Gamrad et al. (216) 47 3 nm 76 Lin et al. (213) nm 91 (sum) Barchanski et al. (211) 5 13 nm 156 Olmedo et al. (28) nm nm 7 nm Peptide: 19 BSA: ±23 233±29 Petersen & Barcikowski (29) 39 Tkachenko et al. (23) 48 this study

139 Ligand density [# bound ligands/np d=7nm ] Conjugation efficiency [%] 4. Results and Discussion 134 Ligand Density of Bifunctionalized Conjugates In order to characterize heterofunctional conjugates, nanoparticles were conjugated with D3 and aminopyrazole trimer and the bound ligand density of the two functional ligands on AuNPs was analyzed. At first, the ligand density of conjugates, where D3 and aminopyrazole trimer were applied in a 1:1 ratio, was analyzed. It can be expected that in comparison to the monofunctionalized NPs, the density of each ligand is reduced, when the second ligand is applied to the NPs at the same time. The overall composition of bifunctionalized conjugates is summarized in Figure Generally, an increase in the number of applied ligands also results in an increase in the number of ligands attached to the gold surface, up to a total number of 2 ligands per NP. Although the two ligands were applied at a ratio of 1:1, ligand densities on the NP surface slightly deviate from this stoichiometry. Noteworthy, at intermediate applied ligand doses, D3 seems to bind more effectively, whereas at very low and very high applied ligand doses, a 1:1 stoichiometry can be realized Trim_5+ D3-Cou (2) 1 * * * * Trim_5+ D3-Cou (19) (79) (56) (4) (8) (16) (26) (28) * * * * Applied ligand dose [#/NP d=7nm ] Applied ligand dose [#/NP d=7nm ] Figure 4-33: Ligand density (left) and conjugations efficiency (right) of D3-Cou and Trim_5+ on bifunctional AuNPs after conjugation in a 1:1 ratio. Numbers in brackets show the total number of bound ligands per NP (left). Note that Trim_5+ cannot be quantified reliably below a ligand dose of 21 ligands/np (*). D3 was shown to dominate the colloidal properties of bifunctionalized AuNPs, especially in the concentration range below 1 µm applied ligands (Figure 4-23). Moreover, D3 was shown to have a higher affinity for the gold surface as determined by IEP measurements (Table 4-6). This might explain the high conjugation efficiency at low applied concentrations. On the other hand, quantification of the aminopyrazole trimer ligand is especially difficult at low concentrations. It should be noted that the low extinction at low aminopyrazole trimer concentrations (ε= nm) and a contribution of the D3-Cou extinction

140 Ligand density [# bound ligands/np d=7nm ] Conjugation efficiency [%] 4. Results and Discussion 135 (compare to Figure S16) can bias the results. In conclusion, to achieve bifunctional conjugates with a 1:1 stoichiometry, one should either apply submonolayer ligand doses (i.e. < 21 ligands/np, generating anionic conjugates in which all applied ligands can bind to the NP surface) or very high ligand doses (> 125 ligands/np). In addition to 1:1 conjugates, conjugates synthesized by adding the two ligands in a 1:1 ratio were analyzed according to their ligand density. As shown in Figure 4-34, by adding the first ligand in low amounts and subsequently adding the second ligand at 1-times excess, the complete NP attachment of the first ligand could be realized, whereas only 6% or 74% of the ligand, which is added shortly afterwards, bound. The 1:1 conjugates presented here carry a total number of ligands comparable to the 1:1 bifunctional conjugates and monofunctional conjugates, which were synthesized by adding 125 of the respective ligand (compare to Figure 4-33 and Table 4-7). Hence, if the total number of ligands (or the total number of charges on the NP surface) is the driving force for the conjugate s functionality in bioassays, similar performance can be expected for monofunctional, 1:1 bifunctional and the 1:1 bifunctional conjugates. On the other hand, if differences in the performance are observed, it must be a result of specific ligand-ligand interactions resulting from the ratio of the ligands on the NP surface. This will be further discussed in section (14) 1 D3-Cou Trim_5+ 8 (86) D3 -> 125 Trim 12 Trim -> 125 D3 Applied ligand dose [#/NP d=7nm ] 12 D3 -> 125 Trim 12 Trim -> 125 D3 Applied ligand dose [#/NP d=7nm ] Figure 4-34: Ligand density (left) and conjugations efficiency (right) of D3-Cou and Trim_5+ on bifunctional AuNPs after conjugation in a 1:1-ratio. Numbers in brackets show the total number of bound ligands per NP. With regard to heterofunctionalized bioconjugates, Patel et al. analyzed the attachment of oligonucleotides and peptides on AuNPs. Similar to the results presented here, they did not find a 1:1 correlation between the applied solution stoichiometry and the surface composition,

141 4. Results and Discussion 136 since peptides preferably bound to the gold surface. 76 They noted that by considering the binding preferences and adjusting the applied ligand doses accordingly, the ratio of each component on the AuNP surface can be systematically tuned. For bifunctionalized lasergenerated AuNPs, Barchanski et al. quantified the number of penetratin and ministrep aptamer ligands after fast ex situ conjugation. 5 Ligand coverages of 32 pmol/cm 2 for ministrep and 33 pmol/cm 2 for penetratin were determined, when applying the ligands in a 1.5:1 ratio. By adjusting the ratio to 4.5:1, ligand coverages were altered to 61 pmol ministrep/cm 2 and 3 pmol penetratin/cm 2. 5 As was shown in this chapter, the design of tailored homo- and heterofunctional conjugates has been established, which enables the specific analysis of structure-function-relationships later on. Conjugates are characterized by different ligand loads and net charges, largely determined by the applied ligand type and ligand-to-np ratio. Their ligand load can be further increased by the addition of small thiolated molecules (e.g. by ~1% with LA and MUA, Figure 4-31). Moreover, their stability can be increased by subsequent addition of sterically demanding molecules (e.g. 1 BSA per nanoconjugate, Figure 4-27). Residual ligands can be removed by centrifugation (Table 4-8), if this is required for the application. The corresponding conjugation strategies are summarized in Figure Add small, anionic thiols Net cationic Au/ligand conjugate with increased ligand load Apply abovemonolayer dose of cationic ligands Net cationic Au/ligand conjugate Add bulky serum proteins Net anionic Au/ligand conjugate stable at physiological salinities Laser-generated, ligand-free AuNP Wash conjugate Net cationic Au/ligand conjugate free of unbound ligands Apply belowmonolayer dose of cationic ligands Net anionic Au/ligand conjugate Add second cationic ligand in excess Add bulky serum proteins Net anionic Au/ligand conjugate stable at physiological salinities Figure 4-35: Flow chart summarizing synthesis procedures (gray boxes) for the design of tailored nanoconjugates (white boxes). Nanoconjugates are based on negatively charged, ligand-free AuNPs as starting material, cationic functional ligands (D3: green, aminopyrazole trimer: red), BSA as bulky stabilizer (purple) and MUA as small thiolated dummy ligand (blue). Note that solid lines show the conjugation paths, which will be employed in subsequent functionality tests.

142 4. Results and Discussion Interaction of Nanobioconjugates with Serum Proteins and Aβ After having characterized ligand binding to the nanoparticles and their stabilization with small thiolated ligands, the interaction of the bioconjugates with serum proteins and Aβ were analyzed. When administering nanoparticles systemically to organisms (e.g. for drug delivery applications), the adsorption of proteins can hardly be avoided due to their high abundance in blood plasma. The interaction of NPs with serum proteins has been in the center of research. 486 As reported by Wang et al., serum proteins may alter the cellular uptake of AuNPs as a result of protein binding to the NP surface. 487 However, the influence of the protein corona on the biofunctionality of NPs (i.e. active targeting capability in the presence of serum proteins) has only been examined by few studies (compare to Table 2-1). Interparticle Distances of AuNP/D3 Conjugates in the Absence and Presence of Serum Proteins The analysis of the interparticle distance (IPD) and amount of agglomerated particles via transmission electron microscopy can give qualitative information on the degree of protein adsorption to Au/D3 conjugates (Figure 4-36 and Figure 4-37). For comparison, the IPDs of ligand-free NPs were also determined. It should be noted that the IPDs and the amount of agglomerates of dried particles is only a qualitative indicator for protein binding and may not precisely reflect the colloidal state. As can be seen from Figure 4-36 and Figure 4-37, the IPD increases when ligand-free AuNPs are conjugated with D3_5+. Moreover, when the AuNP/D3_5+ conjugates are incubated with proteins, their IPD increases even further. In this regard, the influence of the proteins on the particle IPD follows the order: Aβ > BSA > Transferrin > Insulin. It can be concluded that the increase in IPD is a result of protein binding to the conjugate s surface which shields two particles from approaching each other. In particular, not only the protein monomer size but also other effects seem to determine the thickness of the resulting protein corona. For the relatively small Aβ peptide, a high affinity for the conjugate s surface due to interactions with D3 may result in the adsorption of Aβ monomers in great quantities. Moreover, high concentrations of already adsorbed Aβ monomers on the NP surface may induce the peptide s self assembly into larger fibrils. 488

143 Relative number frequency [%] Relative number frequency [%] Relative number frequency [%] Relative number frequency [%] 4. Results and Discussion Au/D3_5+ N = 273 x C =1.5 nm 25 2 AuNP N=46 x C =1. nm Interparticle distance [nm] Interparticle distance [nm] Au/D3_5+/A N=571 x C =2.6 nm 16 Au/A 14 N= x C =1.8 nm Interparticle distance [nm] Interparticle distance [nm] Figure 4-36: Interparticle distance of cationic AuNP/D3_5+ conjugates (left) and bare AuNPs (right) in the absence (top) and presence (bottom) of Aβ (c(aunp) = 5 µg/ml, c(d3_5+, Aβ) = 1 µm). Scale bars are 5 nm.

144 Relative number frequency [%] Relative number frequency [%] Relative number frequency [%] 4. Results and Discussion Au/D3_5+/Insulin N=318 x C =1.4 nm Interparticle distance [nm] 1 Au/D3_5+/Transferrin 8 N = 25 x C =1.6 nm Interparticle distance [nm] 1 Au/D3_5+/BSA 8 N = 538 x C =1.8 nm Interparticle distance [nm] Figure 4-37: Interparticle distance of cationic AuNP/D3_5+ conjugates in the presence of serum proteins (c(aunp) = 5 µg/ml, c(d3_5+, protein) = 1 µm). Scale bars are 5 nm. Influence of Serum Proteins and Aβ on Colloidal Properties of AuNP/D3 Conjugates Changes in the colloidal properties of AuNP/D3_5+ conjugates were analyzed as a function of the protein-to-conjugate ratio. By increasing the number of proteins at a constant conjugate concentration, the zeta potential of the initially cationic conjugates decreases (Figure 4-38, left). It should be noted that the zeta potential is calculated from electrophoretic mobilities

145 Zeta potential [mv] SPR peak shift [nm] 4. Results and Discussion 14 and the mobilities of nanobioconjugates depend not only on the surface charge but also on hydrodynamic drag forces. 16,396 Moreover, serum proteins contain amino acids with acidic as well as alkaline side chains, so that their net charge varies with the ph. Notably, all AuNP/D3_5+ conjugates feature at negative zeta potential in the presence of high concentrations of BSA, transferrin and insulin (c(protein) > 1 µm). Due to the covalent Au-S-D3 bond, it is unlikely that the serum proteins replace D3 from the gold surface. The proteins may rather adsorb to the nanoconjugates additionally, as can be also assumed from the IPD analyses (Figure 4-37). The protein concentrations which compensates the conjugate s charge ( IEPs of AuNP/D3_5+/protein conjugates) are.6 BSA/conjugate, 7 transferrin/conjugate, and 22 insulin/conjugate, respectively Protein density [applied #/NP d=7nm ] Insulin +Transferrin +BSA AuNP + 1µM D3_5+ 1E Protein concentration [µm] Protein concentration [µm] Figure 4-38: Interaction of cationic AuNP/D3_5+ conjugates with serum proteins. Influence of the protein concentration on the colloids zeta potentials and SPR peak maxima (c(aunp)= 5 µg/ml, c(d3_5+)=1 µm). Consequently, not only the zeta potential but also the wavelength of the conjugate s SPR maximum is altered during the incubation with serum proteins (Figure 4-38, right). Interestingly, the SPR peaks reach maximum values at protein-to-conjugate ratios of 19:1 (BSA), 1:1 (transferrin), and 41:1 (insulin). At these protein-specific ratios, particle bridging and agglomeration may be induced. On the one hand, the concentrations are too low to saturate the surface of each individual AuNP/D3_5+ conjugate for steric stabilization. On the other hand, concentrations are similar to those inducing to charge compensation (determined by zeta potential measurements, Figure 4-38 left) Protein density [applied #/NP d=7nm ] Au/D3 + Insulin Au/D3 + Transferrin Au/D3 + BSA 1E

146 Zeta Potential [mv] Hydrodyn. Diameter [nm] Colloidal Stability (PPI) SPR peak shift [nm] 4. Results and Discussion 141 With regard to Aβ, changes in the colloidal properties of AuNP/D3_5+ conjugates were analyzed as a function of the Aβ-to-conjugate ratio (Figure 4-39). By increasing the concentration of Aβ monomers at constant conjugate concentration above a threshold of approximately 1 Aβ monomers per conjugate, colloidal properties start to change. Specifically, zeta potential and PPI decrease, whereas the hydrodynamic diameter and SPR peak maxima increase. The results suggest that high concentrations of Aβ monomers lead to the adsorption of the peptide to the nanoconjugate and refractive index changes in the proximity of the AuNP surface. Aβ could also induce particle agglomeration, possibly by the self-association of Aβ monomers with each other and cross-linking of multiple particles. However, one would expect SPR peak shifts >> 2 nm, if interparticle bridging occurs. For example, Sevilla et al. 44 reported the formation of AuNP agglomerates when incubating the particles with myoglobin (M W 17 g/mol) at a protein-to-np ratio of ~.1:1. This resulted in an SPR peak shift of 125 nm. 44 In contrast to the serum proteins, even the highest Aβ concentrations tested in this study did not stabilize the particles. This supports the hypothesis that higher Aβ concentrations promote dynamic Aβ self-association, 364 rather than NP stabilization by surface saturation. 4 A -to-np ratio [# monomer /NP d=7nm ] A -to-np ratio [# monomer /NP d=7nm ] A concentration [ M] A concentration [ M] Figure 4-39: Interaction of cationic AuNP/D3_5+ conjugates with Aβ as a function of the applied Aβ concentration (c(aunp) = 5 µg/ml, c(d3_5+) = 1 µm). Note that Aβ monomers were employed here. Hydrodynamic diameters are derived from the peak of the number frequency distributions. Adapted with permission from Ref Copyright 216 American Chemical Society. DOI: 1.121/acsnano.6b2627 By shaking the Aβ solution at 37 C, fibrillation is promoted. Hence, the colloidal properties of AuNP/D3_5+ conjugates in the absence and presence of 418 Aβ monomers per conjugate were analyzed at 37 C for 49 h (Figure 4-4). Obviously, the colloid concentration decreases

147 SPR peak maximum [nm] Colloidal stability (PPI) Rel. colloid concentration (c/c ) [%] 4. Results and Discussion 142 and the SPR peak maximum shifts to longer wavelengths over time, independent on the presence of Aβ. It shows that the AuNP/D3_5+ conjugates themselves are not sufficiently stabilized under the applied incubation conditions (as no stabilizers such as BSA have been added). However, in the presence of Aβ, the SPR peak shift is even more pronounced. In contrast, the colloid concentration decreases slower when Aβ is present before it reaches the same final value. The results indicate that Aβ fibrils interact with the conjugates. These opposing observations show that Aβ fibril formation can have different impacts on the conjugates: On the one hand, the adsorption of fibrils to the particles may lead to interparticle bridging and red shift in the SPR peak maximum, as has also been observed in previous analyses at high concentrations of Aβ monomers (Figure 4-39). On the other hand, the formation of large oligomers or fibrils may sterically stabilize AuNP/D3_5+/Aβ agglomerates so that particle precipitation and loss of colloid concentration is slowed down. In this regard, Mizutaru et al. recently showed that imbedding agglomerated NPs in a solution of oligopeptide β-sheets can lead to a redispersion of the particles Au/D3_5+/A Au/D3_ Incubation time [h] Figure 4-4: Incubation of cationic AuNP/D3_5+ conjugates and Aβ monomers under fibril formation conditions. In between measurements, samples were incubated shaking with 65 rpm at 37 C (c(aunp)= 25 µg/ml, c(d3_5+)= 5 µm, c(aβ)= 1 µm).

148 Norm. Mass Frequency Norm Mass Frequency 4. Results and Discussion 143 Influence of Aβ on the Sedimentation Behavior of AuNP/D3 Conjugates As was shown for Au/D3_5+ conjugates before (Figure 4-19), the adsorption of biomolecules to the AuNP surface can be detected via ADC and AUC. Hence, it was assessed whether Aβ binding to the conjugate s surface can be detected as well. Analytical disc centrifugation was carried out with cationic conjugates and Aβ (Figure 4-41). By employing.25 µm Aβ (1 Aβ monomers per conjugate), almost no change in the hydrodynamic diameter can be detected via ADC (similar to results from DLS, Figure 4-39). However, by increasing the Aβ concentration to 1 µm (418 Aβ monomers per conjugate), a strong increase in the NP hydrodynamic diameter can be detected. Hence, surface-adsorbed Aβ seems to induce subsequent particle agglomeration above a threshold concentration of Aβ monomer. 1 Au Au/D3_ Au/D3_ A 1 Au Au/D3_ Au/D3_ A Hydrodyn. diameter [nm] Hydrodyn. diameter [nm] Figure 4-41: D3 binding to initially ligand-free AuNPs and interaction of Au/D3_5+ conjugates with Aβ detected via analytical disc centrifugation (c(aunp) = 5 µg/ml, c(d3_5+) = 3 µm or 1 µm, c(aβ monomer ) =.25 or 1 µm, respectively) A complementary experiment was conducted and analyzed via AUC. Compared to the Au/D3_5+conjugate, an increase in NP diameter is observed in the presence of Aβ (Figure 4-42). In this regard, the overall conjugate size may become larger as Aβ induces particle agglomeration due to electrostatic destabilization (charge screening). However, the difference in hydrodynamic diameters is very small. As discussed previously (Figure 4-2), molecule adsorption decreases the overall density of the NP/ligand conjugate, increases the drag force and slows down the sedimentation. Concerning the sedimentation of Au/D3 and Au/D3/Aβ conjugates in the AUC, it is thus possible that the two opposing effects cancel each other out.

149 4. Results and Discussion x1-4 Au/D3_5+, RMSD=.45 Au/D3_5+ and Aß, RMSD=.34 Frequency Rayleigh interference 1.x1-4 5.x1-5 Hydrodyn. diameter [nm] Figure 4-42: Interaction of Au/D3_5+ conjugates with Aβ detected via analytical ultracentrifugation (c(aunp)=5 µg/ml, c(d3_5+)=1 µm, c(aβ)=1 µm, preformed fibrils). Rayleigh interference measurements were conducted during the sedimentation velocity experiment (3 RPM, 724 g) and the diameter was calculated from the c(s)-distribution considering the density of bare AuNPs. Note that the peaks at ~4 nm could be artifacts due to baseline deconvolution problems. 44 The interference size distribution obtained from AUC is approximately equivalent of the mass size distribution obtained from ADC analyses. AUC analysis with laser-generated AuNPs and AuNP-oligonucleotide conjugates have been conducted before. 45 Thereby, a very broad size distribution was obtained for bare AuNPs (- 1 nm), whereas the conjugates featured a narrow, monomodal size distribution (-1 nm). 45 It should be noted that different factors might contribute to the multimodal, polydisperse size distribution obtained by the AUC analysis in the present study. First of all, the starting material (bare AuNPs, Figure 4-3) also featured a bimodal c(s)-size distribution with x C - values at 5.9 nm and 7.4 nm, respectively. This indicates that the laser ablation process might lead to hydrodynamically polydisperse colloids. Since AUC is a high-resolution technique, the presence of distinct size populations (that are statistically not recognized by the comparatively small sampling capability of TEM) can become apparent. 46 Moreover, the binding of D3_5+ ligands to the NP surface may not be uniform on all particles. In fact, if different particles of the same core size carried different numbers of ligands, one can expect a multimodal s-value distribution. 436 Future studies with complementary techniques could be conducted to evaluate if the ligands are distributed homogeneously over all NPs. For example, gel electrophoresis could be a useful (separation) technique, if one could establish conditions by which the samples remain colloidally stable Additionally to the analyses of interactions with Aβ monomers, nanoconjugates have been incubated with preformed Aβ fibrils. The influence of BSA and preformed Aβ fibrils on nanoconjugates carrying different variants of D3 and aminopyrazole trimer is illustrated in Figure Considering the large increase in hydrodynamic diameters in the presence of

150 Zeta Potential [mv] Zeta Potential [mv] Hydrodyn. Diameter [nm] Hydrodyn. Diameter [nm] 4. Results and Discussion 145 BSA of up to 767 nm and the marked changes in the zeta potentials, the net negative protein (ph=6-7) seems to strongly interact with all cationic AuNP conjugates. The least increase in hydrodynamic diameter is observed for D3_5+- and Trim_5+-conjugated AuNPs, indicating that electrostatic interactions may determine the extent of BSA binding. It should be noted that DLS measurements are very sensitive to agglomerate formation. Hence, the strong increase of particle size may indicate the formation of few large AuNP/ligand/BSA agglomerates, although some smaller species exist at the same time. Wang et al. designed anionic gold-peptide conjugates and determined their hydrodynamic diameter before and after incubation with serum. They detected an increase in the conjugate s hydrodynamic diameter from 24 nm to 46 nm. 487 The lower size increase compared to the present study may be due to the initial negative zeta potential of the conjugates of Wang et al.. Hence, their particles may intrinsically interact to a lesser extent with the overall negatively charged serum proteins than the positively charged conjugates tested here. AuNP conjugates + BSA 12 per NP AuNP conjugates + A 12 per NP nm +723nm +517nm +41nm +767nm Au/D3_5+ Au/D3_8+ Au/D3_1+ Au/Trim_5+ Au/Trim_8+ 1 Au/D3_ nm +.6nm +3.9nm -1.7nm -.3nm Au/D3_8+ Au/D3_1+ Au/Trim_5+ Au/Trim_ mV -18mV -2mV -27mV -22mV -.6mV +2.1mV -1.8mV +1.4mV +3.2mV Au/D3_5+ Au/D3_8+ Au/D3_1+ Au/Trim_5+ Au/Trim_8+ Au/D3_5+ Au/D3_8+ Au/D3_1+ Au/Trim_5+ Au/Trim_8+ Figure 4-43: Interaction of cationic AuNP/D3 or AuNP/aminopyrazole trimer conjugates with BSA (left) and Aβ (right) as determined from DLS (number frequency, top) and zeta potential measurements (bottom). The numbers inside the bars illustrate the increase/decrease in hydrodynamic diameters/charge compared to the respective conjugate in the absence of the proteins (c(aunp) = 5 µg/ml, c(ligand) = 3 µm, c(bsa, Aβ) =.3 µm). Note that although preformed Aβ fibrils were employed, the Aβ concentration refers to the monomer concentration. -3

151 4. Results and Discussion 146 In contrast to the incubation with BSA, almost no change of the hydrodynamic diameter and zeta potential of the conjugates is observed in the presence of preformed Aβ fibrils. This may be due to the low concentrations of 12 Aβ monomers per NP, which was applied for direct comparison to the BSA samples. However, greater changes would be expected at higher Aβ concentrations, similar to the results presented in Figure Fluorescence Quenching of BSA-FITC and Insulin-FITC by AuNP/D3 Conjugates in the Presence of Aβ To detect the possible replacement of the serum proteins by Aβ, experiments were conducted with AuNP/D3_5+, FITC-labeled BSA and insulin, and unlabeled Aβ. In particular, a decrease of fluorescence was interpreted to result from quenching of the FITC-labeled proteins upon binding on the NP surface. 115 In contrast, an increased fluorescence was interpreted to result from protein detachment. If Aβ had a higher affinity for the conjugates than BSA or insulin, the peptide could replace the serum protein, which could be detected as increased fluorescence. Hence, competition experiments were carried out by incubating AuNP/D3_5+ conjugates with FITC-labeled proteins and adding Aβ afterwards, and vice versa. Figure 4-44 (top) shows that the fluorescence of insulin-fitc is quenched almost completely by AuNP/D3 conjugates due to effective binding (reduction of fluorescence by 91%). In the presence of equimolar amounts of Aβ monomers, the overall quenching is still very pronounced compared to the signal of insulin-fitc alone (reduction of fluorescence by ~77%). However, the presence of Aβ increases the fluorescence by 136% and 141%, depending on the sequence of the addition. The increased fluorescence could indicate that less insulin binds and/or that the distance between insulin-fitc and the gold surface is increased through the adsorption of Aβ to the conjugate s surface (Scheme a). It should be noted that this effect does not seem to be very specific for the interaction of D3 with Aβ, because a similar enhancement of fluorescence was observed by conjugating AuNPs with a control peptide (R5WC). It was designed as Aβ-unspecific control ligand featuring the same net charge as D3. Details on the properties of Au/R5WC conjugates can be found in the appendix (Figure S34). In fact, also the incubation of Au/D3/insulin conjugates with transferrin led to a comparable increase in fluorescence (+127%), indicating that the interactions between the conjugates and insulin were rather weak. However, replacement of insulin by transferrin is

152 Fluorescence [a.u.] Fluorescence [a.u.] 4. Results and Discussion 147 less pronounced compared to the replacement by Aβ. This could be an indirect evidence, that the conjugate s affinity for Aβ may be higher than for transferrin. 35 (a) % +142% +141% +127% 4 2 Insulin-FITC 75 7 Au/D3 + Insulin-FITC Au/D3 + Aß + Insulin-FITC Au/D3 + Insulin-FITC + Abeta Au/R5WC + Insulin-FITC und Abeta Au/D3 + Insulin-FITC + Transferin Au/D3 + Aß (b) Aß BSA-FITC Au/D3 + BSA-FITC Au/D3 + Aß + BSA-FITC +.3% +1.7% +1.2% +.7% Au/D3 + BSA-FITC + Abeta Au/D3 + BSA-FITC + Transferin Au/R5WC + BSA-FITC und Abeta Au/D3 + Aß Figure 4-44: Fluorescence quenching of FITC-labeled insulin (left, top) and BSA (left, bottom) by D3- conjugated AuNPs in the absence or presence of Aβ and Transferrin (c(aunp) = 5 µg/ml, c(insulin, BSA, Aβ) = 2 µm, c(d3_5+) = 1 µm). The numbers show the increase in fluorescence relative to the fluorescence of Au/D3/protein-FITC complexes. Schematic illustration of the influence of Aβ on the fluorescence of BSA for the adsorption to Au/D3 conjugates (right). Regarding the results of the experiments employing BSA-FITC, generally much smaller increases (.3-1.7%) in fluorescence can be observed when adding a second protein to the Au/D3/BSA-FITC complex. One possible explanation is that changes in the BSA adsorption may generally be more difficult to detect. Due to the presence of 12 FITC per BSA, the BSA fluorescence is much higher at equimolar dose than the fluorescence of insulin. Moreover, (c)

153 4. Results and Discussion 148 BSA has a 11-times higher molecular weight than insulin, so that the number of proteins bound per conjugate are most likely lower. Originally, much higher differences in fluorescence intensities were expected in the competition between Aβ and BSA/insulin for binding sites on the conjugate s surface. However, fluorescence quenching was relatively high in all samples. Thus, either Aβ was not effectively replacing the FITC-labeled protein (Scheme c) or the fluorescence of the FITClabeled protein was quenched although Aβ attached to the conjugate s surface (Scheme b). Taking the small size of Aβ monomers and the relatively low applied concentration into consideration, both hypotheses are conceivable and more sophisticated methods for the analysis of the NP corona composition 49 would be required to better understand the competition between different species for the conjugate. Furthermore, labeling both competing proteins with different fluorophores could enable the parallel analysis of e.g. Aβ and BSA binding to nanoconjugates. As alternative to more sophisticated analytical methods, the functionality of the conjugates in serum can be tested. If the functionality of Au/D3 conjugates is preserved, this would be an indirect prove that the conjugate s surface is accessible for Aβ despite the presence of other biomolecules. Hence, the nanoconjugate s functionality in terms of targeting Aβ will be the topic of the following chapter.

154 4. Results and Discussion Functionality of the Designed Nanobioconjugates As was shown in the previous chapter, laser-generated AuNPs can be surface-functionalized with the thiolated D3 and aminopyrazole trimer ligands. In particular, by employing these cationic ligands one has to carefully choose a ligand-to-np ratio which avoids charge compensation and agglomeration of the conjugate. As straightforward measure, one can add auxiliary, sterically stabilizing molecules. Since bulky proteins are present in biofluids, any NP applied to biological media will build up a stabilizing protein corona anyway. In this chapter, questions concerning the nanobioconjugate s functionality will be addressed by taking up aspects from the previous chapter: i) How effective are mono- and bifunctionalized AuNPs? ii) iii) iv) Do D3- and aminopyrazole trimer-conjugated AuNPs influence Aβ aggregation differently? How do ligand density and conjugate net charge influence the conjugate s functionality? Is it possible to differentiate between the effects of NP-bound and unbound ligands? v) Does the presence of auxiliary stabilizing molecules on the surface impair the vi) vii) conjugate s functionality? Can the design of AuNP conjugates be transferred to another NP core material? How specific is the system, i.e. can similar effects be observed by employing other cationic ligands? Do Au/D3 and Au/Aminopyrazole trimer conjugates have similar effects on other (fibrillar) proteins? Affinity of Nanobioconjugates towards Aβ as Determined from Fluorescence Quenching Fluorescence titration experiments were conducted with nanoconjugates and FITC-labeled proteins to determine their binding strength (expressed as dissociation constant, K D ). The FITC-labeled Aβ was employed at constant concentration and different concentrations of Au/D3 conjugates were titrated. Similar to previous experiments with ligand-free AuNPs (Figure 4-16), a decrease of the Aβ-FITC fluorescence (i.e. increase in quenching) was interpreted to result from the attachment to the nanoparticles. It is noted that the results from titration experiments can be interpreted with regard to the affinity between each individual

155 4. Results and Discussion 15 ligand and Aβ (K D related to the applied ligand concentration, K D,Lig ) or the overall conjugate and Aβ (K D related to the applied NP concentration, K D,NP ). Affinity of BSA and Aβ towards Conjugates with Different D3 Net Charges To characterize the binding of Aβ-FITC and BSA-FITC on Au/D3 conjugates, dissociation constants and cooperativity factors were compared. Moreover, the influence of the D3 net charge was assessed. As can be seen in Figure 4-45, dissociation constants between the conjugates and Aβ are generally lower than between the conjugates and BSA. It shows that D3-functionalized AuNPs have up to two-times higher affinities for Aβ than for BSA. This is in contrast to the finding of the competition experiment in Figure 4-44, where almost no release of BSA-FITC from the conjugate s surface could be detected upon addition of Aβ. However, as discussed before, the arrangement of proteins in the Au/D3/Aβ/BSA-FITC sample is not clear. Even if Aβ binds to the nanoconjugate preferentially, the fluorescence of BSA-FITC may be quenched to some extent (due to the formation of an Au/D3/Aβ/BSA- FITC complex and the inner filter effect ). It should be noted that even though the binding curves for BSA and Aβ appear similar and K D values vary by (only) a factor of 2, cell culture experiments conducted in serum could show the functionality of the conjugates. As will be presented in chapter 4.3.5, this is an indirect prove that the conjugate s surface is accessible for Aβ despite the presence of sterically demanding biomolecules. The dissociation constants derived for BSA adsorbing on Au/D3 conjugates decreases with increasing net charge of the D3 variant, whereas no trend of the D3 net charge can be observed for Aβ adsorbing on Au/D3. This may indicate that the driving forces for BSA binding are electrostatic interactions with the lysine residues of D3. In contrast, Aβ may interact with those residues which are similar in all D3 variants resulting in similar K D values for all samples. With regard to the cooperativity factors, the interactions of the conjugates with Aβ or BSA do not differ greatly. Specifically, AuNP/D3_5+ conjugates seem to support adsorption of Aβ or BSA the most (positive cooperativity). It should be noted that the D3 concentrations employed for NP functionalization were above monolayer coverage of AuNPs (418 D3/NP) and unbound ligands were not removed from the sample before protein addition. Hence, the observed quenching effects may result from adsorption of the proteins to Au/D3 and free D3 at the same time (Figure S9).

156 Cooperativity factor n Dissociation constant [nm NP] 4. Results and Discussion 151 Quenching (I -I)/I 1. A -FITC with: Au/D3_5+ (R²=.99) Au/D3_8+ (R²=.974).8 Au/D3_1+ (R²=.989) Au/D3_5+ Au/D3_8+ Au/D3_ AuNP concentration [nm] + Aß-FITC + BSA-FITC Quenching (I -I)/I 1. BSA-FITC with: Au/D3_5+ (R²=.99).8 Au/D3_8+ (R²=.979) Au/D3_1+ (R²=.987) AuNP concentration [nm] Au/D3_5+ Au/D3_8+ Au/D3_1+ + Aß-FITC + BSA-FITC Figure 4-45: Fluorescence quenching experiments to determine Aβ-FITC and BSA-FITC dissociation constants on D3-conjugated AuNPs as function of the D3 net charge (left, 418 D3/NP). Summary of dissociations constants (right, top) and cooperativity factors (right, bottom) between Aβ-FITC/BSA-FITC and nanoconjugates (c(labeled protein) =.125 µm). Error bars correspond to the standard error given by the Hill fit. To get a better understanding of NP/protein interactions, dissociation constants between Aβ and the free ligand were compared to those of Aβ and NP-bound ligands. Moreover, the number of D3 applied per NP was set to 125 in order to reduce the fraction of unbound ligands. Dissociation constants for the free ligand are in the low micromolar range, comparable to results obtained from SPR measurements for the non-thiolated D3 ligand. 346,35 Notably, the dissociation constant of AuNP-immobilized D3 is 7 times lower than that of the free D3 (Table 4-1). To evaluate nanoparticle quenching independent of plasmonic effects, additional experiments were performed with PtNPs and PtNP/D3_5+ conjugates. In the case of PtNPs, the dominant quenching may be aggregation-induced quenching, 491 if high local concentrations of Aβ- FITC adsorb on the particle surface. As shown in Figure 4-46 and Table 4-1, bare PtNP quench the fluorescence of Aβ-FITC less efficiently than AuNPs. This is possibly due to the missing absorbance/emission spectral overlap and a lower inner filter effect. However, upon

157 4. Results and Discussion 152 functionalization with the D3_5+ ligand, quenching becomes more efficient and the dissociation constant decreases by a factor of 3.5 compared to bare PtNPs. Since the only difference in the two samples is the presence/absence of D3, the increased affinity must result from the ligand coating. With respect to the ligand dissociation constant, the value is two times lower for PtNP-immobilized D3 than for the free ligand. Hence, PtNP immobilization may induce cooperative effects leading to increased affinities of the individual ligands. 372 Table 4-1: Dissociation constants and cooperativity factors for the interaction of NP/D3 conjugates, bare NP or free D3 with FITC-labeled Aβ. Reprinted with permission from Ref Copyright 216 American Chemical Society. DOI: 1.121/acsnano.6b2627 Dissociation constant, K D,Lig [nmol/l, applied D3] Dissociation constant, K D,NP [nmol/l, applied NP] Cooperativity factor, n Correl. Coefficient, R² (Hill fit) D3_5+ with Aβ- FITC 196± ±.7.97 Au/D3_5+ with Aβ- FITC 271± ± ± Pt/D3_5+ with Aβ- FITC 973± ± ± Pt with Aβ-FITC ±6. 1.7± Quenching (I -I)/I Ligand concentration [nm] Au/D3_5+ Pt/D3_5+ Pt D3_ NP concentration [nm] Figure 4-46: Fluorescence quenching experiments to determine the Aβ-FITC dissociation constants on D3- conjugated AuNPs and PtNPs (125 ligands/np).

158 4. Results and Discussion 153 For samples containing NP, positive cooperativity (n > 1) can be derived from the Hill fit, while negative cooperativity (n < 1) is observed for the free ligand. Hence, NP/D3-bound Aβ seems to increase the binding probability as further Aβ adsorbs, possibly due to synergistic effects of multiple ligands on the nanoparticle surface. In contrast, the affinity for Aβ progressively decreases once that Aβ is bound to the free ligand, possible due to saturation of the ligand s binding site. Concerning functionalized nanoparticles, Kogan et al. showed that ligand functionalization with the CLPFFD peptide increased the attached number of NPs to Aβ fibrils. Compared to unfunctionalized AuNPs, peptide-coated AuNPs were attached at four times higher concentrations to the fibrils compared to unfunctionalized NPs. 485 Hence, Kogan et al. could successfully demonstrate an increased performance of the conjugate compared to the unfunctionalized AuNPs. However, they did not compare the performance of NP-bound ligands to free ligands. Affinity of Aβ towards conjugates with different ligand surface coverages If a specific interaction between the functional ligands and Aβ existed, the density of the ligands presented on the NP surface should influence the binding affinities between the conjugate and Aβ. Hence, quenching experiments were conducted with conjugates featuring different surface coverages of D3 (Figure 4-47) and aminopyrazole trimer (Figure 4-48).

159 4. Results and Discussion 154 Quenching (I -I)/I Dissociation constant [nm NP] Cooperativity factor n Dissociation constant [nm ligand] AuNP/D3_5+ and A -FITC 4 D3/NP (R²=.974) 42 D3/NP (R²=.954) 125 D3/NP (R²=.981) 418 D3/NP (R²=.994) AuNP concentration [nm] /NP 42/NP 125/NP 418/NP NP-free Ligand-to-NP ratio /NP 42/NP 125/NP 418/NP Ligand-to-NP ratio Figure 4-47: Fluorescence quenching experiments to determine Aβ-FITC dissociation constants on D3- conjugated AuNPs as function of the D3 density on the NP surface (c(labeled protein) =.125 µm). Error bars correspond to the standard error given by the Hill fit. Concerning the interaction of Aβ-FITC with Au/D3 conjugates, a decrease of the ligand dissociation constants at decreasing NP loads is observed (Figure 4-48, top right). Moreover, the cooperativity factors indicate that cooperativity is more positive at high (125 D3/NP) and low ligand densities (4 D3/NP, Figure 4-48, bottom right). At intermediate ligand density (42 D3/NP), the conjugates are almost uncharged and unstable, which could affect their binding affinity for Aβ. In the case of applying 418 D3 per NP, K D - and n-values lie between the value of the cationic conjugate (AuNPs with 125 D3) and the free ligand. This may be due to the fact that both, free D3 and NP-bound D3, are present in the sample at the same time and bind Aβ with different affinities. Note that the high affinity of anionic conjugates (AuNPs with 4 ligands) will be further discussed and the end of this chapter. 4/NP 42/NP 125/NP 418/NP NP-free Ligand-to-NP ratio

160 4. Results and Discussion 155 Quenching (I -I)/I Dissociation constant [nm NP] Cooperativity factor n Dissociation constant [nm ligand] 1. AuNP/Trim_5+ and A FITC Trim/NP (R²=.979) 42 trim/np (R²=.94) 418 Trim/NP (R²=.965) ( ) ( ) AuNP concentration [nm] /NP 42/NP 418/NP NP-free Ligand-to-NP ratio /NP 42/NP 418/NP 4/NP 42/NP 418/NP NP-free Ligand-to-NP ratio Ligand-to-NP ratio Figure 4-48: Fluorescence quenching experiments to determine Aβ-FITC dissociation constants on aminopyrazole trimer-conjugated AuNP as function of the aminopyrazole trimer density on the NP surface (c(labeled protein) =.125 µm). Error bars correspond to the standard error given by the Hill fit. Similar to the experiments with Au/D3 conjugates, Au/aminopyrazole trimer conjugates were synthesized with different ligand loads and their affinity for Aβ was analyzed. It was shown that the ligand dissociation constants for Aβ decrease when the ligand density on the NP surface is reduced. In contrast, the dissociation constant of the complete conjugate increases, if fewer ligands are bound per NP. The results show that with regard to the complete conjugate (NP + aminopyrazole trimer), affinity for Aβ is higher the more aminopyrazole trimer ligands are presented on the NP surface. Moreover, compared to the interaction of the free ligand with Aβ, the nanoconjugates show up to 7 times higher affinities. This is not surprising, since locally concentrated ligands present multiple binding sites for Aβ. However, in contrast to D3, no positive cooperativity is observed during the binding of Aβ to the conjugates. In a study by Hochdörffer et al., a library of aminopyrazole trimers differing in their C- terminal end group was screened for their potential to inhibit Aβ induced toxicity. IC 5 values (inhibitory concentration leading to 5% reduction in toxicity) between 3 µm (aminopyrazole

161 4. Results and Discussion 156 trimer carrying non-polar alkyl residues) and > 81 µm (aminopyrazole trimer carrying polylysines) were detected. 17 With regard to these previously reported affinities, the affinities of the aminopyrazole trimer ligand for Aβ seem to be increased in the present study by up to three orders of magnitude. This may be a result of the different chemical modifications of the ligands tested (here: addition of five to eight lysine residues and one cysteine to the original molecule) and/or the different read-outs chosen to determine affinities (K D of fluorescence quenching vs. IC 5 of Aβ-induced cellular toxicity). In summary, anionic as well as cationic conjugates were shown to have high affinities for Aβ, featuring K D values in the nanomolar range. In the case of Au/D3 conjugates, uncharged conjugates showed the lowest affinities. Their reduced affinity may be a secondary effect resulting from particle agglomeration and reduction of available NP surface area before Aβ is added. Figure S12 shows that neutral Au/D3_5+ conjugates require an additional sterical stabilizer to prevent particle agglomeration. With regard to the ligands, the lowest K D values determined were 7 nm (AuNPs conjugated with 4 D3 ligands) and 63 nm (AuNPs with 4 aminopyrazole trimer ligands). Notably, when designing anionic conjugates, all applied ligands are expected to bind to the NP surface. Hence, the analysis of Aβ binding to these conjugates will not be biased by free ligands. The high affinities of these conjugates with low ligand densities may appear surprising at the first sight, since one could expect increased binding strength the more ligands are present to act synergistically (according to the principle of multivalency, compare to chapter 2.1.1). On the other hand, in a dense ligand coating, the ligands might shield each other and/or the formation of ligand-target interactions can be prevented due to decreased flexibility. 86 In this regard, a high inter-ligand distance (at low ligand loads per NP) might allow the ligands to adopt a more favorable conformation for Aβ adsorption e.g. by side-on binding 141 to the NP surface. Furthermore, the overall negative net charge of the conjugate may favor interactions with positively charged residues of Aβ (lysines at position 16 and 28, arginine at position 5, compare to sequence in chapter 2.3.1). The functionality of anionic conjugates will thus be analyzed via AFM (Figure S21, Figure S22) and cell tests (Table S6). In a previously published study, Gobbi et al. characterized lipid-based nanoparticles functionalized with phosphatidic acid for Aβ targeting and reported similar affinities. In their study, the immobilization of phosphatidic acid on the liposome resulted in K D -values of 22-

162 4. Results and Discussion nm for Aβ fibrils. 492 Interestingly, they also examined the affinity of liposomes for BSA and did not observe binding, which indicated a certain specificity for Aβ. 492 Even higher affinities were reported for antibody-carrying liposomes. Canovi et al. developed a monoclonal Aβ-antibody which was coupled to the surface of nanoliposomes via biotinstreptavidin interaction. They determined a K D value for surface-immobilized Aβ 1-42 fibrils of.5 nm. 493 The antibody which was employed for Aβ detection via ELISA in this thesis (6E1, compare to chapter 4.3.4) was shown to have affinities in the nm range: K D = 22 nm for monomeric Aβ, K D = 1.4 nm for Aβ fibrils. 428 Despite these remarkable high affinities, the large size of liposomes (~ 13 nm) and antibodies may prevent BBB penetration and limit their application to blood circulating Aβ. 493 In general, all results from fluorescence measurements should be taken with care, since also the unfunctionalized AuNPs seemed to feature high affinities for Aβ (Figure 4-16). Hence, especially in the case of conjugates with below-monolayer ligand coatings, Aβ binding may be governed by the partly bare NP surface as well as attached ligands. To confirm the obtained K D values, complementary measurements such as SPR spectroscopy, isothermal titration calorimetry (ITC) or microscale thermophoresis (MST) 494 should be performed. Moreover, it would be interesting to analyze the binding behavior in competitive assays, in which e.g. serum proteins and Aβ are present at the same time. Another point to consider is that all affinity experiments were carried out with Aβ monomers. Since very low Aβ-FITC concentrations were employed (c =.125 µm), aggregation of the peptide during the titration experiment seemed unlikely. Aggregation was even not observed after incubating a 4 µm Aβ-FITC solution at 37 C for 24 h, as determined from DGC and fluorescence measurements (see chapter 4.3.4). Since the D3 sequence was originally identified as being specific for small Aβ oligomers, 16 the affinity for the binding of Aβ monomers may be lower Influence of Nanobioconjugates on Aβ Secondary Structure ThT fluorescence assay and CD spectroscopy are standard methods to investigate the Aβ secondary structure. In general, the ThT assay indicates the amount of β-sheets, whereas the abundance of different secondary structures can be detected with CD spectroscopy. Thioflavin T Assay

163 4. Results and Discussion 158 Notably, the ThT assay can suffer from quenching effects (e.g. curcumin, resveratrol) and displacement of ThT from the fibrils by the test compound (e.g. EGCG). Moreover competition between the binding of the test compound and Aβ to ThT can occur. 495,496 For colloidal AuNP samples, the strong absorption of AuNPs in the range between 5-55 nm can limit the applicability of the ThT assay. 373 In order to exclude potential interferences between ThT emission and AuNP absorption, control experiments were carried out with PtNPs. As can be observed in Figure 4-49, all PtNP conjugates performed similar to the corresponding AuNP conjugate. This indicates that fluorescence attenuation by plasmon quenching is negligible and AuNP conjugates are applicable under the chosen experimental conditions. In addition, negligible autofluorescence of AuNPs, free ligands and functionalized NPs was detected. The displacement of ThT from Aβ fibrils by the ligands was excluded in previous experiments. 17,372 Figure 4-49 presents the analysis of the Aβ aggregation in the presence of bare and ligandcoated AuNPs and PtNPs. In all experiments, the number of ligands was kept constant for direct comparison (c = 6 µm). With regard to the ligand net charge, free cationic D3 and aminopyrazole trimer derivatives consistently lowered the ThT fluorescence with increasing ligand net charge. This may be due to increased electrostatic interactions between the negatively charged Aβ and cationic ligands. Generally, free aminopyrazole trimer ligands seem to act slightly more effectively following the order: Trim_8+ > Trim_5+ > D3_8+ > D3_1+ > D3_5+. For bare NP, ThT intensity is decreased. It confirms that Aβ may bind to the bare AuNP surface unspecifically, as indicated in previous experiments, including zeta potential measurements, DLS and K D titrations (chapter 4.2.5). Liao et al. observed that 3 nm gold nanoparticles inhibit Aβ 1-4 aggregation, which was attributed to their negative surface charge. 54 In their study the highest applied gold concentration (47 cm² NP surface/ml) reduced ThT fluorescence by 85% after 72 h of coincubation with Aβ 1-4. In the present study, the surface areas of the bare colloids were even higher (3 µg/ml = 139 cm²/ml and 9 µg/ml = 4 cm²/ml), which may explain the strong interaction of negatively charged, bare AuNPs with Aβ resulting in ThT fluorescence reduction of 98% and 1%. 372

164 ThT-Fluoresccence [% of A ] ThT-Fluoresccence [% of A ] 4. Results and Discussion % in the absence and presence of AuNP A 2% % A +Au 53% 1% 1% A +D3_5+ 25% 16% 8+ 2% 32% 16% 2% 17% 5% % 3% A +D3_8+ A +Trim_5+ A +D3_1+ A +Trim_8+ 1 1% in the absence and presence of PtNP Figure 4-49: ThT fluorescence assay of pure Aβ, and Aβ incubated with bare NP, free ligands and NP/ligand conjugates. Experiments were conducted with AuNPs (top) and PtNPs (bottom). Note that in the case of the conjugates, the first column shows the results of the free ligand, the second (and third) columns show the results for the respective conjugates. In the case of AuNPs, two different particle concentrations were employed (second column: c(aunp) = 3 µg/ml, third column: c(aunp) = 9 µg/ml, c(aβ) = 1 µm, c(ligand) = 6 µm, c(ptnp) = 333 µg/ml). Adapted with permission from Ref Copyright 216 American Chemical Society. DOI: 1.121/acsnano.6b2627 Immobilization of the ligands on AuNPs further lowered the amount of β-sheets as determined from ThT fluorescence (decrease to 3-16%, Figure 4-49). It becomes apparent that all ligand derivatives are much more potent if they are presented on the NP surface. In addition, differences between aminopyrazole trimer and D3 derivatives become smaller. For example, for the standard concentration of 3 µg/ml AuNP, the ThT fluorescence is decreased to 3-16% by the AuNP conjugates, with the strongest effect on β-sheets exerted by the Au/Trim_8+ conjugates. A 1% A +Pt 53% 17% A +D3_5+ 25% A +Trim_5+ A +Trim_8+ It was determined that AuNPs are already saturated with each of the three cationic ligands at a ligand dose of approximately 164 ligands/np (Figure 4-29). Thus, when applying 6 µm ligands to 3 µg/ml AuNPs (418 ligands/np), only a minor fraction of about 3% of peptide ligands can bind to the NP surface. As a consequence, the amount of AuNPs was raised to a stoichiometric ratio by applying 6 µm ligands to 9 µg/ml AuNPs (125 ligands/np). In these experiments, almost no ThT fluorescence could be detected in all 9% 8+ 32% 6% A +D3_8+ A +D3_ % 16% 7% 7%

165 4. Results and Discussion 16 cases, indicating that a drastic improvement can be achieved by increasing the fraction of NPbound ligands (Figure 4-49, top). Circular Dichroism Measurements CD measurements were performed to evaluate the effect on Aβ secondary structure. Generally, the results confirm the ThT assay, showing that the formation of β-sheet structures is prevented. As shown in Figure 4-5, Aβ alone displays a pronounced CD band at 22 nm. This corresponds to a rapid formation of β-sheet structures within 24 h. 419 For the incubation of Aβ with bare AuNPs, the β-sheet content is reduced to some extent within 1h. In the presence of free D3_5+, Aβ features a small β-sheet band in the beginning ( h), which disappears within 1 h. Au/D3_5+ conjugates suppress β-sheet formation so that no band is visible within the course of the experiment (Figure 4-5). In conclusion, ThT and CD analyses showed that NP-ligand conjugates as well as the free ligands may redirect the Aβ aggregation towards the formation of unstructured material of lower β-sheet content, unable to form fibrils. The ThT assay revealed that the multivalent presentation of ligands on the AuNP surface results in more effective reduction of β-sheet content (as shown by up to 5 times greater ThT fluorescence reduction). Furthermore, timeresolved CD measurements revealed faster dissolution of β-sheets or prevention of β-sheet formation by the nanoconjugates a priori.

166 [mdeg] [mdeg] [mdeg] 4. Results and Discussion 161 h 1h 24h A control, 24h 2-2 A + Au 2-2 A + D3_ Wavelength [nm] Figure 4-5: Time-resolved CD spectroscopy of Aβ incubated with bare NP, free ligands, NP/D3_5+ conjugates for 24 h in comparison to the CD signal from pure Aβ (c(aβ) = 1 µm, c(aunp) = 5 µg/ml, c(d3_5+) = 1 µm) Microscopic Analysis of the Effect of Nanobioconjugates on Synthetic Aβ AFM Analysis of the Effects of Monofunctional Conjugates Aβ fibrils, oligomers and nanoparticles were visualized via atomic force microscopy to analyze the fibril length, height and density. For all AFM experiments, Aβ fibrils were preformed at ph = 2, according to a protocol from Stine et al., 426 before free ligands, bare NPs or NP/ligand conjugates were added. It was observed that even after the preincubation period, the density of Aβ species increased over time. Hence, a mixture of different Aβ species seems to be present after the preincubation period, consisting of fibrils and smaller precursors. Hence, all effects observed in AFM may be a result of the inhibition of de novo fibril formation as well as the destruction of already formed fibrils. A + Au/D3_5+ In the first experiment, preformed Aβ fibrils were incubated with D3-functionalized AuNPs (Figure 4-51). With regard to the sample coverage, the free ligands and NP-ligand conjugates

167 4. Results and Discussion 162 strongly lowered the Aβ fibril density compared to the reference samples (at the same time) and also compared to the initially preformed Aβ fibril density (Figure 4-52, top). Analysis of the fibril height and length showed that free D3 and D3-conjugated NPs reduced the fibril size (Figure 4-52, bottom). Figure 4-51: Atomic force micrographs showing the effect of bare AuNPs, free D3_5+ and D3_5+conjugated AuNPs on preformed Aβ fibrils over a period of 7 days. Samples were not diluted before AFM analysis (c(aβ) = 5 µm, c(aunp) = 125 µg/ml, c(d3) = 25 µm). Adapted with permission from Ref Copyright 216 American Chemical Society. DOI: 1.121/acsnano.6b2627

168 Norm. Number Freq. Norm. Number Freq. Surface coverage [%] 4. Results and Discussion 163 Especially after 7 d of coincubation, only few short Aβ fragments were observed in the samples containing D3, whereas a dense peptide layer is present in the samples containing Aβ only and Aβ with ligand-free AuNPs. Hence, one can conclude that both processes, destruction of preformed fibrils and inhibition of the fibril formation, occur in the presence of D3. In contrast, incubation with ligand-free nanoparticles did not stop Aβ aggregation. In previous studies, Thakur et al. analyzed the influence of DHLA-capped quantum dots on the fibrillation of Aβ 1-4. They found that after 7 d of incubation, the overall number of fibrils and fibril heights were strongly reduced in the presence of quantum dots. 361 Moreover, Palmal et al. analyzed the fibril morphology of Aβ 1-4 in the presence of curcumin-functionalized gold nanoparticles via TEM. 366 While average fibril length of 2.3 µm were observed in the control after 7 d of incubation, fibril lengths were restricted to <.2 µm in the presence of functionalized NPs. Similar to the results of Au/D3 conjugates, the authors also report that curcumin-coated AuNPs exert two effects, inhibition of fibrillation and fibril dissociation. 366,372 A A +Au A +D3_5+ A +Au/D3_ Time [d] 1..8 d 1d 7d 1..8 d 1d 7d.6.4 Δt.6.4 Δt Height [nm] Length [ m] Figure 4-52: Calculated surface coverage of Aβ species on the AFM substrate after incubating the samples for, 1 and 7d with AuNPs, D3_5+ and D3_5+-conjugated AuNPs Analysis of fibril height (bottom, left) and length (bottom, right) of Aβ fibrils after incubation with Au/D3 conjugates. Samples were not diluted before the AFM analysis. Adapted with permission from Ref Copyright 216 American Chemical Society. DOI: 1.121/acsnano.6b2627

169 4. Results and Discussion 164 Figure 4-53 shows exemplary micrographs of Aβ fibrils incubated with D3-functionalized AuNPs. It can be observed that particles are in direct contact with the fibrils and seem to attach to or in between the fibrils and break preformed fibrils apart. Similarly, Palmal et al. reported that their nanoparticles (AuNPs coated with histidine-based polymers) attached to Aβ in most cases at the long end of the growing fibrils. They propose that nanoparticles influence the nucleation-and-growth process of Aβ fibrils at the intermediate stage by blocking the fibril elongation. 497 Figure 4-53: Microscopic interactions of D3-conjugated AuNPs with Aβ fibrils as shown by 2D (left) and 3D atomic force micrographs (right). The arrows highlight the sites of NP contact with fibrils. To confirm the results from AFM analysis, transmission electron micrographs were acquired for samples prepared under the same conditions as for AFM (Figure 4-54, Figure S2). It can be seen that Aβ aggregated into fibrils with average diameters of 5 nm and lengths of up to

170 4. Results and Discussion µm in the absence of the nanoconjugates. The addition of AuNP/D3_5+ conjugates resulted in fewer and shorter fibril fragments. Moreover, most AuNP/D3_5+ conjugates were located in close proximity to Aβ aggregates, indicating that intermolecular forces may have attracted the conjugates to the Aβ species. In the future, TEM measurements could be optimized with respect to the staining procedure and the analysis parameters. In fact, less concentrated samples and specific staining may increase the image contrast. Moreover, a lower acceleration voltage may be less destructive for the organic material. However, this may be accompanied by a reduction of the image sharpness. Figure 4-54: TEM analysis of pure Aβ and Aβ incubated with Au/D3_5+ conjugates. Note that samples were produced under AFM sample preparation conditions, but were additionally stained with 2% uranyl acetate to visualize fibril structures prior to the measurements (c(aβ) = 5 µm, c(aunp) = 125 µg/ml, c(d3)= 25 µm). To discriminate better between the effect of unbound and NP-bound ligands, washed conjugates have been employed in a selected experiment. It should be noted that it is difficult to compare the obtained results to a reference sample, because one has to make estimates about the final conjugate composition based on previous tests (compare to Table 4-8). An equal amount of NPs as used in previous experiments was applied to the preformed Aβ fibrils (c(aunp)=125 µg/ml). However, the number of ligands was reduced due to the removal of

171 Norm. Number Frequency Norm. Number Frequency 4. Results and Discussion 166 unbound ligands (applied: 125 D3/NP; bound + residual: D3/NP). Note that the estimated number of NP-bound ligands results from the average surface density of D3-Cou conjugates (Table 4-7). Moreover, 1% of the unbound ligand dose was considered as residual ligands (1% of 36 ligands/np are not removed during washing, Table 4-8). Although the overall number of ligands was reduced in the washed conjugate by 29%, the conjugate s functionality seems to be preserved. As shown by atomic force micrographs, the lengths of preformed Aβ fibrils are greatly reduced after incubating the samples with the washed conjugates for 24 h, similar or even stronger than those results obtained after the incubation with non-washed conjugates (Figure 4-55). 2 nm Aβ Aβ + Au/D3_5+ (washed) Aβ + Au/D3_5+ (non-washed) nm 1 µm 1 µm 1..8 A A Au/D3_5+ (washed) A Au/D3_5+ (non-washed) 1..8 A A Au/D3_5+ (washed) A Au/D3_5+ (non-washed) Length [µm] Height [nm] Figure 4-55: Atomic force micrographs showing the effect of washed and non-washed AuNP/D3_5+ conjugates on preformed Aβ fibrils after an incubation period of 24 h (top). Quantitative analysis of fibril length and height (bottom). Note that washed conjugates contained NP-bound D3 ligands only (c(aβ) = 5 µm, c(aunp) = 125 µg/ml, c(d3 applied) = 7.5 µm). In addition to monofunctionalized Au/D3 conjugates, monofunctionalized Au/aminopyrazole trimer conjugates were incubated with Aβ and the fibril morphology was analyzed via AFM measurements. As can be seen in Figure 4-56, the presence of the aminopyrazole trimer ligand seems to reduce the density of Aβ as compared to the sample of pure Aβ or Aβ incubated with ligand-free NP, similar to D3. The performances of the ligands can only be compared relative to the Aβ sample employed in each experiment due to strong batch-to-

172 Surface coverage [%] Norm. Number Frequency 4. Results and Discussion 167 batch deviations of Aβ. In this regard, Au/D3 conjugates lower the fibrils surface coverage to 51%, whereas Au/Trim conjugates lead to a reduction to 62% relative to the coverage of the Aβ reference sample within 24 h of coincubation. 1. A A + Au.8 A + Trim_5+ A + Au/Trim_5+.6 Aβ Aβ + Au 12 nm Length [µm] Aβ + Trim_5+ Aβ + Au/Trim_5+ nm Aß A Aß+Au A A A + Au Aß+Trim A + Trim A + Au/Trim Aß+Au/Trim Figure 4-56: Effect of aminopyrazole trimer-functionalized AuNPs on preformed Aβ fibrils after incubation for 24 h as analyzed by atomic force microscopy. Samples were diluted 1:2 before AFM analysis (c(aβ) = 5 µm, c(aunp) = 125 µg/ml, c(trim_5+) = 25 µm). AFM Analysis of the Effects of Bifunctional Conjugates 1 µm Moreover, the effect of bifunctionalized conjugates, synthesized by applying a 1:1 ratio of 29 D3 and 29 aminopyrazole trimer per AuNP, was assessed. As reference, equimolar concentrations of the free ligands were incubated with the preformed Aβ fibrils. As is illustrated by Figure 4-57, the analysis of the sample density on the AFM substrate reveals that Aβ fibrils densely cover the AFM surface in the control sample and when incubated with bare AuNPs (surface coverage = 19%). In the presence of the NP-free ligand mixture, the amount of Aβ is reduced to 1%. Notably, in the presence of the bifunctionalized NPs, the amount of Aβ is reduced even to 6%, suggesting that the immobilization of the ligands on the surface of AuNP improves their functionality. By comparing the absolute values of Aβ density achieved with the bifunctionalized nanoparticles to those achieved with monofunctionalized NPs under similar conditions (monofunctional: 418 ligands/np, bifunctional: each 29 ligands/np, incubation for 24 h), one can see that bifunctional NPs seem to have superior functionality. Specifically, D3-conjugated NPs decreased the Aβ

173 Surface coverage [% ] 4. Results and Discussion 168 density to 1.7% (Figures S19 and S2), whereas aminopyrazole trimer-conjugated NPs resulted in a surface coverage of 13.% (Figure 4-56). In contrast, Au/D3/Aminopyrazole trimer conjugates reduced the amount of Aβ approximately twice as much. Synergistic action of the two ligands presented on the NP surface may explain the observed superior effects of bifunctional conjugates. Notably, different Aβ aliquots were employed for each experimental series, which may aggregate differently, even if the same experimental conditions are applied. Moreover, none of the nanoconjugates (Figure 4-51, Figure 4-56, Figure 4-57) was washed to remove unbound ligands. Hence, the difference between free ligands and washed nanoconjugates at equimolar ligand dose could be even higher. This should be investigated in future tests. Aβ Aβ + Au 12 nm 2 19% 19% % 6% Aβ + D3/Trim_5+ Aβ + Au/D3/Trim_5+ nm 5 A Ab A + Au Aß+Au A + D3/Trim Aß+D3/Trim A + Au/D3/Trim Aß+Au/mix 1 µm Figure 4-57: Effect of bifunctionalized AuNP on preformed Aβ fibrils after incubation for 24 h, as analyzed by atomic force microscopy (D3:Aminopyrazole trimer = 1:1, each 29 ligands/np). Samples were diluted 1:2 before AFM analysis (c(aβ) = 5 µm, c(aunp) = 125 µg/ml, c(ligands) = 12.5 µm). AFM Analysis of the Effects on Insulin Fibrils To address the question, whether the anti-amyloidogenic effects of the free ligands and nanobioconjugates are specific for Aβ or can be obtained with other fibrillating proteins, insulin fibrils were formed under similar experimental conditions as Aβ fibrils (in 4 mm HCl). After 24 h of preincubation, free ligands or nanobioconjugates were added and AFM analysis was performed after 24 h of co-incubation (Figure 4-58).

174 Surface coverage [%] 4. Results and Discussion 169 Insulin Insulin + D3_5+ Insulin + Trim_5+ 2 nm 1 nm Insulin + Au Insulin + Au/D3_5+ Insulin + Au/Trim_5+ nm nm 1 µm Insulin Insulin+D3 Insulin+Au/D3 Insulin+Trim Insulin+Au/Trim Figure 4-58: Effect of bare AuNPs, free D3, free aminopyrazole trimer and monofunctional conjugates on preformed insulin fibrils after incubation for 24 h, as analyzed by atomic force microscopy. Samples were not diluted before AFM analysis (c(insulin) = 5 µm, c(aunp) = 125 µg/ml, c(ligands) = 12.5 µm). In the reference sample, one can observe multiple insulin fibrils of linear shape. Compared to Aβ fibrils, insulin fibrils generally appear smaller, with many fibrils being shorter than 1 µm, and thicker. Those samples, in which insulin was incubated with bare AuNPs, could hardly be analyzed via AFM. Large amorphous structures were detected on the mica surface with heights above 1 nm. It is proposed that the negatively charged AuNPs interacted strongly with insulin fibrils, forming overall negatively charged aggregates. These aggregates did not interact well with the negatively charged mica surface. If at all, the aggregates adsorbed on the mica surface inhomogeneously. In contrast, fewer and shorter insulin structures were found in samples treated with the free ligands. Similar to the free ligands, cationic

175 4. Results and Discussion 17 nanobioconjugates seemed to have an anti-amyloidogenic effect on insulin fibrils. The free ligands and nanoconjugates seem to inhibit insulin fibrillation with comparable effectiveness. In conclusion, effective interference and destruction of preformed fibrils consisting of Aβ as well as insulin have been demonstrated for D3- and aminopyrazole trimer-conjugated NPs. For the aminopyrazole trimer, interactions with β-sheet forming proteins can be expected, due to its general ability to break β-sheets. 498 For D3, the ability to interact with insulin fibrils might appear more surprising, as this ligand was originally selected against Aβ monomers to small oligomers. 19 However, rather non-specific (e.g. electrostatic) interactions might account for these interactions. Similar as reported for Aβ, 16,19 D3 might interact via its cationic arginine residues with negatively charged groups of insulin (two negatively charged amino acid side chains, Table 3-3). It should always be kept in mind that micrographs only show selected sections at a single point of time of the dried amyloid aggregates on a surface. On the mica surface itself, artifacts may form and morphologies observed in AFM images may not always accurately reflect the peptide structures in solution. For example, Lin et al. reported the formation of fibrillar structures on the AFM substrate. This was attributed to surface-mediated Aβ adsorption and self-assembly of monomers. 488 To confirm the hypotheses that particles actually attach to Aβ in the aqueous phase, time-resolved AFM measurements 499 would be desirable. Moreover, a fluid cell in which the reaction takes place could be employed. To analyze whether the nanoconjugates can destroy Aβ preferentially over e.g. insulin fibrils, competitive tests could in future be conducted. However, for microscopic analysis, one needs to consider that protein-specific staining will be required to differentiate Aβ species from other proteins Immunologic Analysis of the Effect of Nanobioconjugates on Synthetic Aβ Density gradient centrifugation (DGC) experiments and subsequent analysis of the protein content in each fraction was performed to monitor the Aβ aggregate size distribution. By varying the experimental parameters (e.g. incubation time before centrifugation), the development of smaller and larger Aβ aggregates can be triggered. Hence, snapshots during different stages of the Aβ aggregation process can be taken specifically. The analysis of the

176 4. Results and Discussion 171 Aβ content can be carried out via gel electrophoresis and silver staining, an enzyme-linked immunosorbent assay (ELISA), or high performance liquid chromatography (RP-HPLC). 353 In preliminary experiments, the fractions of the density gradient were characterized by more rapid techniques than ELISA, such as fluorescence or UV/Vis extinction spectroscopy. At first, the gradient was established, centrifuged, and each fraction was analyzed to correlate the absorbance to the relative iodixanol concentration. When comparing to the initially applied density layers, one can clearly see that a more continuous gradient formed after the centrifugation period of 4.5 h, in which the iodixanol content gradually increased from the early to the late fractions. It is expected that the density of the first fraction equals the density of the first applied liquid layer (1.33 g/m³) and the density of fraction 23 equals the density of the last applied liquid layer (1.271 g/cm³). Notably, fractions 6 to 13 appear to have the same iodixanol concentration, which may correspond to the initially applied density of g/cm³ (Figure 4-59, top left). AuNPs, free D3-Cou, FITC-labeled BSA, FITC-labeled Aβ were centrifuged in the gradient. Each solution was divided into 23 fractions and analyzed separately. Thereby, a sedimentation profile for each component could be obtained. For AuNPs, the highest gold concentration (detected as the absorbance at 528 nm) was found in fraction 23. Hence, it is concluded that bare AuNPs sediment to the bottom of the centrifuge tube within 4.5 h due to their high density (19.3 g/cm³, Figure 4-59, top right). FITC-labeled BSA and Aβ spread across the density gradient, with most species present in fraction 1-3 and 1-5, respectively (corresponding to densities of 1.33 to 1.61 g/cm³ for Aβ and 1.33 to g/cm³ for BSA). The fact that decreasing amounts of the FITC-labeled samples are found in each succeeding fractions can be an indication for the propagation of the sample from a previous fraction to the following by the pipetting procedure due to high sample concentrations. In contrast to unlabeled Aβ, Aβ-FITC does not seem to aggregate under the applied conditions (c= 4 µm, incubation for 24 h at 37 C), since no sample was detected in later fractions. Hence, the covalent binding of the extrinsic FITC fluorophore seems to alter the Aβ assembly and function. 5 It can thus be assumed that all experiments conducted with Aβ-FITC majorly contain the monomeric form of Aβ. The fact that BSA sediments further onwards than Aβ agrees well with the difference in their molecular weights, since BSA (66 g/mol) is many times larger than Aβ monomers

177 52nm 393nm Abs@28nm Abs@528nm 4. Results and Discussion 172 (45 g/mol). Moreover, BSA shows a tendency to self-assemble into large macromolecular aggregates, 51 which may explain its presence in fractions > 1. Its secondary structure is predominantly α-helical with one free cysteine and 17 disulfide bridges. These ensure some rigidity within the subdomain of the protein, but modifications in the overall shape and size of serum albumin can occur. It was suggested that BSA may form small ordered aggregates through the conversion from α-helix to β-sheet structures through electrostatic interactions. Moreover, larger aggregates may built via non-specific, hydrophobic interactions. 51 Coumarin-labeled D3 is predominantly found in fraction 1, 2 and 3, indicating that the peptide sediments less than monomeric Aβ-FITC and BSA-FITC, which is in accordance with its smaller size. 2 Iodixanol AuNP Fraction Fraction 35 A -FITC 3 BSA-FITC Fraction Figure 4-59: Characterization of the density gradient. UV/Vis extinction of each DGC fraction, in which the signal at 28 nm correlates with the iodixanol concentration. Note that the dashed lines indicate the boundaries of the six initially applied layers containing different iodixanol concentrations (5-5 vol%) and different densities ( g/cm³). Sedimentation profiles of bare AuNPs, Aβ-FITC (4 µm preincubated 24 h at 37 C), BSA-FITC and D3-Cou obtained after centrifuging the samples for 4.5 h at 286, g. Subsequently, the influence of bare AuNPs, free D3_5+ and Au/D3_5+ conjugates on Aβ species was determined via DGC and ELISA. Because the D3 peptide was originally selected Fraction D3-Cou

178 Fluorescence [RFU] 4. Results and Discussion 173 against Aβ monomers to small oligomers, 19 it was especially interesting to monitor the occurrence of these most neurotoxic species by choosing a short incubation time of 9 min, followed by density gradient centrifugation. As shown in Figure 4-6, small and mediumsized Aβ species are formed in the control sample and in the sample incubated with bare AuNPs after 9 min incubation. The free D3_5+ ligand lowers the amount of medium-sized species. Similarly, AuNP/D3_5+ conjugates carrying equimolar ligand doses retard the development of medium-sized species, but lead to the formation of more high-molecular mass species in fraction 15 to Figure 4-6: Influence of bare AuNPs, free D3_5+ and Au/D3_5+ conjugates on Aβ species as determined from DGC/ELISA. Aggregation occurred for 9 min at 37 C. The ligand-to-np ratio was 418:1 (c(aunp) = 2 µg/ml, c(d3_5+) = 4 µm, c(aβ) = 4 µm). To better discriminate between the effects of free and NP-bound ligands, the ligand-to-np ratio was decreased from 418:1 to 14:1 by tripling the gold concentration (Figure 4-61, left). Notably, also this higher concentration of bare AuNPs did not affect the aggregation profile of Aβ. However, the free and NP-bound D3 ligands changed the Aβ aggregation profile. Incubation with both, free D3 and Au/D3 conjugates strongly lowered the amount of Aβ species in fraction 1 to 15. Moreover, the free D3 ligands increased the amount of Aβ detectable in the fractions 17 and 18, whereas Au/D3 conjugates also increased the amount of Aβ species detectable in the fractions 19 to 23. A with: - + Au + D3_5+ + Au/D3_ Fraction The aim of the next experiment was to determine whether the presence of BSA, which can sterically stabilize the Au/D3 conjugates, affects their interaction with Aβ. As shown in Figure 4-61 (right), the preincubation with BSA strongly alters the sedimentation profile of Aβ. It is assumed that direct interactions of BSA with Aβ arise. Because all sedimentation profiles (Au+BSA, D3+BSA, Au/D3+BSA) look very similar, the influence of the one

179 Fluorescence [RFU] Fluorescence [RFU] 4. Results and Discussion 174 component, which is common in all samples (BSA), seems to dominate all other effects. Previous studies suggested that BSA may have a chaperone-like function, in which it binds aggregating proteins, forms stable, high molecular weight complexes and prevents fibril formation. 52 In contrast, incomplete complexation of Aβ 1-4 at low BSA doses was shown to increase Aβ-induced cell activation. This was possibly due to the presence of residual small Aβ species, acting as seeds for further oligomerization. 53 With regard to the initial idea of steric stabilization of the Au/D3 conjugates (without directly affecting Aβ aggregation), it remains unclear whether the presence of BSA is useful or negatively affects the conjugate s functionality. For future ELISA tests, BSA should only be applied in very low doses so that the number of unbound BSA in the sample remains low. As was determined in the freeze drying assay (Figure 4-27), ~1 BSA per nanoconjugate are required as minimal stabilizer concentration. Moreover, complementary methods are needed to better discriminate between the interactions of (i) BSA and Aβ vs. (ii) Au/D3/BSA and Aβ. Despite the challenges associated with the DGC/ELISA, cell culture experiments will show how the conjugates interact with Aβ in the presence of other serum proteins (chapter 4.3.5) A 1-42 with: x - + Au + D3_5+ + Au/D3_ Fraction Figure 4-61: Effects of unbound and NP-bound ligands in Au/D3_5+ conjugates on the aggregation of Aβ incubated for 9 min at 37 C as determined from DGC/ELISA (c(aunp) = 6 µg/ml, c(d3_5+) = 4 µm, c(aβ) = 4 µm, left). Influence of BSA preincubation on bare AuNPs, free D3_5+ and Au/D3_5+ conjugates for the aggregation of Aβ for 9 min at 37 C as determined from DGC/ELISA (c(aunp) = 2 µg/ml, c(d3_5+) = 4 µm, c(aβ) = 4 µm, c(bsa) = 4 µm, right). Adapted with permission from Ref Copyright 216 American Chemical Society. DOI: 1.121/acsnano.6b2627 Next, the incubation time was increased from 9 min to 24 h in order to allow Aβ protofibril and mature fibril formation. The influence of different D3 variants, Trim_5+, and 1:1 ligand mixtures of D3_5+/Trim_5+ on Aβ fibrils was assessed (Figure 4-62). The sedimentation profile of the reference sample (Aβ) shows that high molecular weight Aβ species are formed A 1-42 with: - AuNP+BSA D3_5+ + BSA AuNP/D3_5+ +BSA Fraction

180 Fluorescence [RFU] Fluorescence [RFU] Norm. Fluorescence [%] Norm. Fluorescence [%] 4. Results and Discussion 175 under the selected incubation conditions, which are mainly located in fractions The incubation with free ligands and Au/ligand conjugates strongly lowers the amount of Aβ in fractions 16-19, with the exception of the free aminopyrazole trimer ligand and Au/Trim_5+ conjugates. Notably, Au/Trim_5+ conjugates seem to function less effectively than the other conjugates, but better than the free aminopyrazole trimer ligand, as evaluated by the abundance of Aβ detected in fractions (Fraction 18) (Fraction 18) % % % 4.8% 3.2% 12.6% % 5.3% 8.6% 12.8% - + Au/D3_5+ + Au/D3_8+ + Au/D3_1+ + Au/Trim_5+ + Au/D3/Trim - + D3_5+ + D3_8+ + D3_1+ + Trim_5+ + D3/Trim Au/D3_5+ + Au/D3_8+ + Au/D3_1+ + Au/Trim_5+ + Au/D3/Trim_ D3_5+ + D3_8+ + D3_1+ + Trim_5+ + D3/Trim_ Fraction Fraction Figure 4-62: Effects of Au/ligand conjugates (left) and free ligands (right) on the aggregation of Aβ incubated for 24 h at 37 C as determined from DGC/ELISA (c(aunp)=2 µg/ml, c(ligands)=4 µm, c(ligands, bifunctional)=2 µm each, c(aβ)=4 µm). Bar diagrams summarize the Aβ content in fraction 18 (top), whereas the complete sedimentation profiles of Aβ species are shown at the bottom. Bifunctional conjugates were generated by applying 29 D3_5+ and Trim_5+ per NP, respectively. For those bifunctional conjugates, an effect similarly strong as the effect of monofunctionalized Au/D3_5+ on Aβ was detected. Since only half of the D3_5+ concentration was applied, one may conclude (i) that D3 dominates the effect of the aminopyrazole trimer (because the conjugate s functionality does not scale linearly with the amount of immobilized D3_5+ ligands) or (ii) that the aminopyrazole trimer ligand acts synergistically with D3_5+. The first hypothesis is supported by the results obtained from the incubation with free ligands (Figure 4-62, right). D3 as free ligand is much more potent than

181 4. Results and Discussion 176 the aminopyrazole trimer. It suggests a dominant role for D3 in lowering the Aβ species when present as 1:1 mixture with the aminopyrazole. In summary, DGC/ELISA experiments showed that bare AuNPs did not noticeably alter the course of Aβ aggregation. In contrast, free and immobilized D3 ligands effectively prevented formation of the most neurotoxic small Aβ oligomers, which arise at an early aggregation stage (9 min). Moreover, both free and AuNP-immobilized D3 ligands eliminate large aggregates, which arise at a late aggregation stage (24 h). Hence, ligand functionality is retained on the AuNP surface. Aminopyrazole trimer ligands were employed in equimolar concentration as D3, but did not show as strong effects, whereas bifunctional AuNP/D3_5+/Trim_5+ conjugates completely suppressed formation of large Aβ species. The question whether the effects towards Aβ are selective and specific remains inconclusive. BSA-coated Au/D3_5+ conjugates lowered the amount of medium-sized Aβ species less efficiently than BSA-free samples. This may either be due to steric shielding of the D3 ligands by the BSA corona or due to the formation of large Aβ-BSA complexes. Control experiments employing the cationic R5WC peptide showed that Au/R5WC conjugates could also effectively reduce Aβ species (Figure S25, left). This result indicates that D3_5+ may not have superior function over other cationic peptides and that the peptide s cationic charge (which is similar in D3_5+ and R5WC) may be the dominant parameter governing the interaction with Aβ. Moreover, by changing the NP core material from gold to platinum, similarly effective NP/D3_5+ conjugates could be generated with respect to their ability to reduce the formation of large Aβ species sedimenting in fractions 15-2 (Figure S25, right). Generally, the calibration of the density gradient with proteins of known s-value and size could be performed in the future. Moreover, the as-prepared samples could be run in an analytical ultracentrifuge to determine their absolute size (i.e. s-value). For example, Aβ monomers feature an s-value of.6 S, while oligomers can range in s-values (e.g. 4.7 S for 12-mers and 6.25 S for 18-mers). 44 Mature Aβ fibrils show s-values > 3 S. 498,54 However, one needs to keep in mind that the question, which Aβ species are the most relevant species involved in AD, is still under debate (Table S9). Hence the results presented here are useful in showing that neither the ligand alone nor the nanoconjugates lead to the formation of high amounts of free Aβ monomers after 24 h of coincubation, which would be detectable in the first fractions. In contrast, high-molecular weight assemblies probably consisting of nanoparticles, ligands and Aβ species seem to form. These can be detected at the bottom of

182 4. Results and Discussion 177 the centrifuge tube after treating the tube with the denaturing agent guanidinium hydrochloride. In addition, one needs to consider that the ELISA setup could be further optimized to guarantee the quantitative determination of Aβ. For example, the amount of Aβ, which can adsorb to the ELISA plate, is limited by the available surface per well. Hence, if all binding sites are occupied, the remaining Aβ will be washed off and will not be detected. Moreover, the antibodies should be applied in success in order to detect all Aβ. Another point to consider is that only those Aβ species can be detected, which expose their binding site to the antibody. Since Aβ is not treated with a denaturing agent after the DGC and before the ELISA, different Aβ aggregates are present. One may expect that antibody binding does not occur per Aβ monomer. Instead, one antibody may bind to an entire Aβ assembly. Thus, especially in the later fractions (containing larger Aβ species), the amount of Aβ may be underestimated and the detection may be only semiquantitative (see Figure S26). Future experiments could address if DGC/ELISA is applicable to analyze samples incubated in cell culture media, blood serum and brain liquor. In principle, the detection of Aβ via high affinity antibodies such as 6E1 (K D = 22 nm for monomeric Aβ, K D = 1.4 nm for Aβ fibrils) 428 should be possible even in a complex matrix. Nevertheless, cross reactions 55 of the antibody with other proteins should be checked. Given the reported interactions between Aβ and other proteins, 35,56 one should also consider the pre-formation of relevant Aβ species (e.g. oligomers, fibrils) before the addition of biofluids and nanoconjugates Effect of Nanobioconjugates on the Cellular Excretion of Aβ Ultimately, cell culture experiments were conducted with a CHO (Chinese hamster ovary) cell line carrying the familial Indiana mutation (V717F) that increases the expression of the amyloid precursor protein and the production of the aggregation prone Aβ It was previously shown that this mutation leads to the secretion of highly synaptotoxic low-n oligomeric and monomeric N-terminal elongated Aβ species into the cell medium. 429,43 In contrast to the previously shown experiments with synthetic Aβ, Aβ peptide is enzymatically cleaved and naturally secreted by the cells in this assay. Cells were incubated with bare AuNPs, free ligands and monofunctionalized particles (Figure 4-63). Aβ was immunoprecipitated and quantified densitometrically by western blot. A serial

183 1-15 kda Aß species [% of mock] 4. Results and Discussion 178 dilution of mock treated cells (negative control) allowed the relative quantification of the Aβ signal intensities. The β-secretase inhibitor LY was employed as positive control, since it completely blocks the production of Aβ ## 51% ### 41% #### 36% * * #### 21% 85% 77% 4% 8% 12,5% mock 25% mock 5% mock 1% mock Au Au/D3_5+ Au/D3_8+ Au/D3_1+ D3_5+ D3_8+ D3_1+ LY Figure 4-63: Cellular excretion of Aβ in the presence of bare AuNPs (c = 5 µg/ml), free D3 ligands and Au/D3 conjugates (c(ligand) = 1 µm, ligand-to-np ratio = 418:1). Immunoprecipitation of Aβ from conditioned medium and densitometric quantification of low-n oligomers or monomeric N-terminal elongated Aβ species (1-15 kda Aβ species). Positive control: 2 nm of the β-secretase inhibitor LY completely block Aβ production (blue bar). Significant differences were derived from Tukey posthoc test between unbound and NP-bound ligands (* p <.5) and samples and mock (##. p <.1, ###. P <.1, ####. P <.1). One way ANOVA, mean ± standard error of the mean, p <,1, n = 4, F(8, 27) = 15,34. Adapted with permission from Ref Copyright 216 American Chemical Society. DOI: 1.121/acsnano.6b2627 All tested samples reduced the amounts of Aβ in the cell medium compared to untreated cells. The incubation with bare AuNPs reduced the amount of Aβ to 51%, which may be due to unspecific binding of Aβ to the AuNP surface. 54 With regard to the ligand effects, it can be seen that the activity of the D3 compounds increases with a higher positive net charge. These results are comparable to the results of the free ligands in the ThT fluorescence assay (Figure 4-49) and may be due to the maximized electrostatic attractions. Moreover, all D3 ligands seem to require nanoparticle attachment for optimal effects, which is in agreement with the results of the ThT assay with synthetic Aβ (Figure 4-49). The most potent AuNP conjugate (Au/D3_1+) lowered the amount of Aβ species by 8%.

184 4. Results and Discussion 179 The microscopic analysis was conducted after 3 d, revealing that the cells grew normally (Figure 4-64). 372 Au/D3_5+ Au/D3_8+ Au/D3_1+ 5μm 5μm 5μm Mock treated Au 1 µm 5μm 5μm Figure 4-64: Light microscopy images of cells monitoring cellular growth and nanoparticle uptake after three days of incubation with bare AuNPs and Au/D3 conjugates. Adapted with permission from Ref Copyright 216 American Chemical Society. DOI: 1.121/acsnano.6b2627 Fluorescence microscopy was employed in order to selectively visualize the nanoparticle aggregates, the outer cell membrane and the cell nucleus. As can be seen in Figure 4-65 and Figure S27, for all tested samples, large nanoparticle aggregates formed in the cell culture medium. These were subsequently endocytosed by cells and formed vesicles in the intracellular space and close to the cells nuclei (Figure 4-65e). 372 Since gold nanoparticles have been described to induce apoptosis in human breast cancer cells, 58 the CHO cells were tested for possible impairment of cell viability (Figure S29). A reduction in cellular viability would also lower the amount of detectable Aβ and thus lead to false positive results. However, none of the compounds used in this assay influenced the overall amount of expressed proteins or APP expression. Moreover, caspase-3 expression was not altered, showing that these AuNPs did not seem to trigger apoptosis. 372

185 4. Results and Discussion 18 Figure 4-65: Confocal microscopy images of CHO cells treated with AuNP/D3_5+ conjugates. Visualization of the cell membrane (a, employing the DIC filter), the nanoparticles (b, excitation at 532 nm), the cell nuclei (c, excitation at 45 nm) and overlaying all filters (d). Enlarged image sections of selected cells (e and f). The functionalities of Au/Trim conjugates and conjugates which were functionalized with D3_5+ and Trim_5+ at the same time were tested next. Corresponding samples were incubated with CHO cells in experiments similar to those conducted with Au/D3 conjugates. Figure 4-66 shows that the free aminopyrazole trimer ligands seem to lower the amount of Aβ more effectively than the free D3_5+, when applied at the same dose. This is in contrast to previous experiments, where only minor effects of aminopyrazole trimer on synthetic Aβ were observed during ELISA (Figure 4-62). When immobilized on AuNPs, the aminopyrazole trimer ligand reduced Aβ species similarly or slightly stronger. The differences between the cell test and ELISA may arise from the different experimental conditions, which may give rise to different Aβ species. With regard to the 1:1 mixture of free D3 and aminopyrazole trimer (Figure 4-66, left), it can be seen that the sum of both ligands does not lower the amount of Aβ species as effectively as each individual component. Here, it should be considered that each ligand is present at half

186 1-15 kda A species [% of mock] 1-15 kda A species [% of mock] 4. Results and Discussion 181 the concentration in the mixture which may reduce the ligands efficiencies as compared to the functionality of each ligand individually. When immobilized on AuNPs, a drastic increase in efficiency can be observed since the amount of Aβ species is lowered twice as much as with the free ligand mixture. Compared to the monofunctional conjugates, the bifunctional conjugates perform similarly well, although each ligand is present at only half the concentration. This is an indication for the dual functionality of the conjugates, since both ligands need to exert their functionality in order to be as effective as the corresponding monofunctional conjugate. 1 1µM 1µM 5µM each 1.3µM + 3µM AuNP D3_5+ Au/D3_5+ Trim_5+ Au/Trim_5+ mix Au/mix Figure 4-66: Cellular excretion of Aβ in the presence of bifunctional 1:1 (left) and 1:1 (right)-conjugates. Ligand-to-NP ratios were 29:1, in the 1:1-conjugates, and 125:1 or 13:1 in the 1:1-conjugates. For comparison the results of monofunctional conjugates are shown. In the case of the 1:1-conjugates, the corresponding monofunctional conjugates containing the more abundant ligand in equimolar dose are illustrated. Assessing the functionality of the 1:1 mixtures of free D3 and aminopyrazole trimer (Figure 4-66, right) one can see that the mixtures function similarly effectively than the single ligand, which constitutes the majority of each mixture (i.e. 3 µm Trim_5+ has the same effect as the mixture of.3 µm D3_5+ and 3 µm Trim_5+). It shows that the more abundant ligand dominates the effect of the 1:1 ligand mixture. The 1:1 ligand mixtures are much more effective in lowering the amount of Aβ when immobilized on AuNPs as compared to the unbound mixtures. Nevertheless, their functionality is also comparable to the effect produced by monofunctional conjugates containing only the more abundant ligand. Thus, adding one ligand at minor dose does not noticeably increase the conjugate s performance. Disproportionate strong reduction of Aβ would be expected from synergistic effects of the two ligands, if they interacted synergistically.

187 1-15 kda A species [% of mock] 1-15 kda A species [% of mock] 4. Results and Discussion 182 The next step included the analysis of the cellular Aβ excretion in the presence of R5WCfunctionalized AuNPs. In Figure 4-67 (left) the performance of Au/R5WC conjugates is shown and compared to the performance of free R5WC and D3_5+ at equimolar dose. While the free R5WC ligand lowered the Aβ content to 61%, Au/R5WC conjugates lowered the Aβ content almost twice as effectively (31%). The results show that comparable reduction of cellularly excreted Aβ species can be achieved with R5WC and D3_5+. Supporting the results from DGC/ELISA (Figure S25 left) and quenching experiments with synthetic Aβ species (Figure 4-44), the effects of D3 do not seem to be sequence specific but rather driven by electrostatic interactions. To examine whether BSA hinders the functionalized conjugates from interacting with cellularly produced Aβ, samples were incubated with and without BSA before adding to the cell medium. As shown in Figure 4-67 (right), more Aβ is detected in the sample with BSAcoated conjugates (45% vs. 32%). Hence, the presence of BSA seems to reduce the ability of the conjugate to interfere with Aβ. It should be noted however, that the functionality of the BSA-coated conjugates still remains quite high. Hence, BSA does not completely block the binding sites of the conjugate D3_5+ (3µM) Au/D3_5+ (3µM) R5WC (3µM) Au/R5WC (3µM) Au/D3_5+/Trim_5+ (1:1) Figure 4-67: Cellular excretion of Aβ in the presence of R5WC-functionalized AuNPs (125 ligands/np, left) and Au/D3/Trim conjugates post-stabilized with BSA (c(bsa) = 4 µm, right). Notably, the cell medium employed for these tests was supplemented with 1% calf serum. Therefore, even unmodified Au/D3_5+ conjugates came into contact with serum proteins. However, the preincubation with BSA seemed to result in a protein corona different of that formed by the medium s serum proteins, since the samples featured different functionalities. This supports the hypothesis that the corona formation occurs rapidly and its composition is dependent on the available proteins, as suggested by Tenzer et al Au/D3_5+/Trim_5+ (1:1) + BSA

188 1-15 kda A species [% of mock] 4. Results and Discussion 183 The last experiment is connected to the question if the nanoparticle core material can be exchanged without affecting the conjugate s functionality. Figure 4-68 shows that Pt/Trim_5+ conjugates reduce Aβ species, but they seem to be less effective than Au/Trim_5+ conjugates. As illustrated in Figure S35, PtNPs and AuNPs featured the same diameter (7 nm). Moreover, the same number of particles were applied in cell experiments by adjusting the mass concentrations to 55 µg/ml PtNPs (considering the ~1% difference in densities Au: 19.3 g/cm³, Pt: g/cm³). From the determination of aminopyrazole densities on PtNPs (Figure S36) it becomes apparent, that ligand binding on PtNPs is not as efficient as on AuNPs. At 3 µm applied aminopyrazole concentration, the conjugation efficiencies are ~3% (PtNP, Figure S36) and ~66% (AuNP, Figure 4-29). The decreased conjugation efficiencies for PtNPs may be a result of their highly oxidized surface. As described before, ~6% 13 of the surface of lasergenerated AuNPs is oxidized, whereas up to 5% 19 of the surface of laser-generated PtNPs is oxidized. This is also supported by the more negative zeta potentials of PtNPs compared to AuNPs (Figure S35). From the theory of gold-sulfur bond formation (compare to chapter 2.2.4) one can conclude that only the non-oxidized metal surface is able to form covalent bonds which enable dense ligand packing. In contrast, electrostatic interactions between surface adsorbed chloride ions and cationic ligands may occur at the oxidized surface, which results in side-on binding and low ligand loads. Due to the surface chemistry of lasergenerated PtNPs, less ligands may thus be able to bind on PtNPs than on AuNPs. Hence, the differences in the cell assay between AuNPs and PtNPs may result from different ligand densities under the experimental conditions Au/Trim_5+ (3µM) Pt/Trim_5+ (3µM) Figure 4-68: Comparison of Au/Trim_5+ and Pt/Trim_5+ conjugate performances on the cellular excretion of Aβ (c(pt) = 55 µg/ml, c(aunp) = 5 µg/ml, 125 ligands/np).

189 4. Results and Discussion 184 As summarized in Table S6, all tested nanoconjugates are more potent than the respective dose of free ligands in the cell assay. The highest improvement of ligand performances can be achieved for submonolayer-coated Au/D3 conjugates (factor 5.2), followed by monolayercoated nanoconjugates (125 D3_5+ per NP, factor 3.9). All cationic conjugates contain a mixture of bound and unbound ligands and their effects cannot be discriminated. Hence, avoiding free ligands may further improve the nanoconjugate s performance and should be examined in future tests Preliminary Summary of the Nanobioconjugate s Interactions with Aβ The model of Aβ aggregation from monomers into oligomers and fibrils has been employed in this thesis to elucidate the structure-function relationship of nanoparticles conjugated with selected Aβ-targeting ligands. Table 4-11 summarizes the experimental conditions employed in the different functional assays. Notably, the different assays did not only address the effect of nanoconjugates on different Aβ species, but also different ligand-to-aβ and NP-to-Aβ ratios have been tested. In this regard, it was shown that Aβ aggregation could be inhibited, when starting from the monomeric form (e.g. ThT, CD, DGC/ELISA assays) and even be reversed, when preincubating Aβ for the formation of fibrils and adding the conjugates subsequently (AFM samples). Moreover, AuNPs were usually applied at substoichiometric doses to Aß (AuNP: nanomolar vs. Aβ micromolar). This shows the high activity of nanoparticles, i.e. through their large specific surface area. Notably, ligand concentrations in AFM experiments were lower than Aβ monomer concentrations, indicating that the nanoconjugates might be effective in substoichiometric doses, which should be examined in more detail through complementary assays.

190 4. Results and Discussion 185 Table 4-11: Comparison of experimental parameters in different assays to examine the nanobioconjugate s functionality in terms of interaction with Aβ. Quenching/ Affinity assay ThT assay CD spectroscopy AFM DGC/ELISA Cell assay c(aβ monomer ).125 µm 1 µm 1 µm 5 µm 4 µm Aβ origin c(ligand) c(aunp) Incubation time Incubation temperature/ conditions Incubation medium Predominant Aβ species (at endpoint) Read-out Synthetic FITCmodified.3-5 µm.1-25 µg/ml (.5-12 nm) Synthetic Synthetic Synthetic Synthetic Not quantified Cultured cells 6 µm 1 µm 25 µm 4 µm 1 µm 3 µg/ml (144 nm) 5 µg/ml (24 nm) d 3 d 24 h Room temperature Deionized water Monomer FITC fluorescence (K D ) 37 C, 65 rpm PBS (137 mm NaCl, 2.7 mm KCl, 13 mm phosphate) Monomer, oligomers, fibrils ThT fluorescence (β-sheet content) Room temperature, 65 rpm 1 mm potassium phosphate buffer Monomer, oligomers, fibrils Absorbance (β-sheet, α- helix, random coil content) Nanoparticle-Induced Effects on Aβ Aggregation 125 µg/ml (6 nm) 24 h (preincubation) +, 1, 7 d 37 C, 65 rpm 1 mm HCl Monomer, oligomers, fibrils Surface topography (fibril morphology) 2 µg/ml (96 nm) 9 min or 24 h (+ 4.5 h centrifugation) 37 C, 65 rpm 1 mm sodium phosphate buffer Monomer, oligomers, fibrils Antibody binding (rel. content of Aβ species) 5 µg/ml (24 nm) 7 d 37 C DMEM with 1% fetal bovine serum Monomer, oligomers, Antibody binding (rel. content of excreted Aβ) Although the ligand-free nanoparticles employed in this study feature an overall negative charge, which may not electrostatically attract the negatively charged Aβ (isoelectric point at ph = ), most of the surface is non-oxidized, uncharged and available for hydrophobic interactions. In contrast, nanobioconjugates featuring cationic charges due to the ligand coating favor electrostatic interaction with Aβ. Even if the surface is not completely covered with ligands and the overall zeta potential of the conjugate is negative, a combination of hydrophobic interactions (with the NP surface) and electrostatic interactions (with positively charges of the ligands) may occur. As suggested by Palmal et al., this combination of

191 Maximum No. of residues forming inter-peptide beta-sheets 4. Results and Discussion 186 electrostatic interactions and hydrophobicity may be optimal for efficient inhibition of amyloid aggregation. 497 Generally, interactions with different types of unfunctionalized nanoparticles have been reported before. 54,382,389 Radic et al. modeled the contrasting effects of NPs in amyloid aggregation as function of the NP-Aβ aggregation strength (Figure 4-69). 51 Aggregation promotion can be the result of an initially increased local peptide concentration on the NP surface due to weak interactions. Subsequently, lateral diffusion may allow the self-assembly of the surface-adsorbed peptides into fibrils. On the other hand, aggregation inhibition may be induced by binding of Aβ species on the NP surface, decreasing their concentration in solution, and shifting the monomer-oligomer-fibril equilibrium away from fibrillation. If the peptides interact stronger with the NP surface than with each other, Aβ self-association will not only be restricted in the solution, but also on the particle surface itself. 51 Furthermore, conformational changes of the peptide can occur on the NP surface, so that binding sites of NP-adsorbed Aβ may be blocked for further monomers. 389 Note that while aggregation promotion and inhibition based on the aforementioned effects can generally take place with bare as well as functionalized nanoparticles, catalytic activity requires the presence of specific ligands on the NP surface (i.e. regeneration of the conjugate s surface for cyclic Aβ degradation, compare to chapter 5 and Figure S33). Promotion (P) Inhibition (I) (P) (I) NP-peptide interaction strength Figure 4-69: Theoretically calculated influence on Aβ aggregation as function of the interatomic interactions between a 1 nm nanoparticle and ten Aβ monomers obtained from discrete molecular dynamics simulations. Adapted from Ref. 51 with permission of The Royal Society of Chemistry. DOI: 1.139/C5RA2182A

192 4. Results and Discussion 187 Effect of D3- and Aminopyrazole Trimer-Conjugated Particles For the adsorption of functional ligands on NP surfaces, a decrease in their activity towards Aβ could occur due to a decreased flexibility. 86 Fortunately, such ligand deactivation was not observed in this work, which supports the hypothesis that binding to the NP surface occurs via the thiol group of the terminal cysteine present in all ligand derivatives. The mode of action of D3-functionalized nanoparticle on Aβ is proposed to be similar to that of free D3, whereby Aβ is converted into non-amyloidogenic, amorphous, non-toxic aggregates (see also section 2.3.2, Figure 4-7). 34,16 While the interaction with D3 may be mainly driven electrostatically through the arginine residues, the aminopyrazole trimer may bind to negative Aβ side chains electrostatically and exert additional interactions with the hydrophobic backbone through donor-acceptor-donor hydrogen bonds. Figure 4-7: Proposed mechanism of the Au/D3 nanoconjugate s interference with Aβ aggregation. Aggregation may be inhibited by binding Aβ monomers (top) and small oligomers (middle). Disaggregation of preformed Aβ fibrils may occur through an intermediate stage, in which short fibrils are attached to the nanoconjugates before the transformation into amorphous aggregates occurs (bottom). Reprinted with permission from Ref Copyright 216 American Chemical Society. DOI: 1.121/acsnano.6b2627 For aminopyrazole trimer conjugated NPs, the mode of action is proposed to be initiated by the binding to β-sheet rich aggregates. Due to its specific molecular structure, the aminopyrazole then intercalates with the aggregates, in which hydrogen-bond donors and acceptors bind to complementary structures within the β-sheets (Figure 2-16, Figure 4-71). These lead to the destruction of the β-sheets via hydrogen bond formation.

193 4. Results and Discussion 188 Figure 4-71: Proposed mechanism of the Au/Trim nanoconjugate s interference with β-sheet rich Aβ aggregates. Aggregates bind to the aminopyrazole trimer via hydrogen bonds, which induces breaking of their secondary structure. 17,511 Notably, all ligands employed in this thesis were modified compared to previously described D3 and aminopyrazole trimer variants. In particular, the addition of lysine residues to the original molecules does not only increase their water solubility and add cationic charge to the nanoconjugate, but it will also increase electrostatic interactions with the negatively charged Aβ peptide. This can be an explanation for the observation of stronger effects of higher charged ligand variants. The importance of electrostatic interactions is also supported by the observations that the cationic peptide R5WC, which was originally employed as unspecific control ligand, did also exert inhibitory effects on Aβ aggregation. Similarly, Assarsson et al. 512 found a correlation between the charge of Aβ ligands (peptides ranging in net charge from +8 to -8) and Aβ aggregation inhibition. They observed that the most cationic peptide inhibited Aβ aggregation most efficiently, whereas a peptide with eight negative charges did not affect the aggregation. It was concluded that electrostatic attraction plays an important role for aggregation inhibition, in which aggregation inhibition occurs due to decreased rates of the Aβ nucleation process. 512 In general, when formulated as nanoconjugate, the ligands effects may be increased through multivalency arising from high local concentrations on the NP surface in close proximity to each other. This may allow stronger binding to a multivalent target. In this respect, the generation of tandem D-peptides has been reported as Aβ targeting ligands by the Willbold group. For example, the synthesis of the DB3DB3 peptide dimer resulted in a 75-fold increase in affinity for Aβ monomers and a 1-fold lower concentration being effective against Aβ aggregation, as compared to the monomeric DB3. The authors attribute the increased potency to the bivalency of the compound. 351 For multivalent ligand presentation, as in the case of nanoconjugates, the increase in potency can be expected to be even higher. Table 4-12 gives an overview of designed nanoconjugates with increased performance over the free ligand in comparison to the conjugate employed in this thesis.

194 4. Results and Discussion 189 Table 4-12: Multivalent, homofunctional nanoconjugates sorted by their increase in target avidity as compared to the free ligand. Enhancement factors derived from different assays of the present study are shown for comparison (cationic Au/D3_5+ conjugates). NP Ligand Target Enhancement factor compared to free ligand AuNP antibiotics bacteria Up to 3 AuNP iron oxide NP oligonucleotide complementary nucleic acids RGD peptide integrin 38 AuNP D3 Aβ 1-42 iron oxide NP Small molecules (synthetic derivatives of the natural product FK56) FK56-binding protein (ThT assay) 7. (Aβ-FITC quenching) 3.9 (cell assay) 1.3 (DGC/ELISA) Reference Saha et al. (27) 513 Rosi et al. (26) 514 Montet et al. (26) 515 This work 372 Figure 4-49 Table 4-1 Table S6 Figure Tassa et al. (21) 78 AuNP carbohydrates lectin 1 5 Wang et al. (211) 516 dendrimers folate folate-receptor 1 5 Hong et al. (27) 27 AuNP Small molecule (SDC- 1721) CCR5 co-receptor of HI virus active only in multivalent presentation on AuNP surface Bowman et al. (28) 74 Generally, ligand functionalization through thiol bonds does not seem to be limited to AuNPs. PtNPs could also be ligand functionalized and these nanoconjugates featured similar performances to AuNP/D3 conjugates with regard to Aβ aggregation inhibition (compare to Figure 4-68 and the corresponding discussion). The employment of Pt-based nanoconjugates is useful when one wants to circumvent certain effects arising from the plasmonic properties of AuNPs, i.e. quenching of fluorophores or absorbance overlap with red dyes. Additionally, different gold and platinum nanoconjugates may be applied in parallel. Their effects can be analyzed simultaneously and differentiated with multiplex techniques due to the different core materials. For example, AuNPs could be conjugated with one type of ligand and PtNPs with another. After in vivo administration, their biodistribution can be monitored via ICP-MS and effects arising from the different ligands can be differentiated based on the simultaneous quantification of each element. 517

195 4. Results and Discussion 19 Strategies to Stabilize Nanoconjugates To determine the influence of auxiliary stabilizing molecules on the nanoconjugate s functionality, BSA was added. BSA is an effective electrosteric NP stabilizer which quickly adsorbs on the NP surface and hardly gets replaced afterwards. For example, Xie et al. have shown that the NP-adsorbed BSA does not get replaced by physiologically relevant concentrations of glutathione. 457 Moreover, after systemic administration, the conjugate would, in any case, be exposed to serum proteins before reaching Aβ as a target. As shown in Table 4-13, albumin constitutes to the major fraction of proteins in serum and CSF, so that albumin-coating represents a facile and efficient way to sterically stabilize nanoconjugates with a naturally occurring biomacromolecule. Table 4-13: Comparison of protein concentrations in the brain and blood (CSF: cerebrospinal fluid). Serum (blood) CSF (brain) Reference Total proteins 75 g/l mg/l Felgenhauer et al. 518 Albumin 36.6 g/l 155 mg/l 53 µm 2.24 µm Felgenhauer et al. 518 Transferrin 2.4 g/l 14.4 mg/l 25 µm.17 µm Felgenhauer et al. 518 Insulin.26 µg/l.18 µg/l 45 pm 3.2 pm Craft et al. 519 Aβ soluble ng/l ( nm) 435 ng/l (.93 nm) Spies et al. 52 Toledo et al. 521 With regard to Aβ binding, albumin itself has a micromolar affinity for Aβ (5 µm for Aβ 1-4 ) and in blood serum, > 9% of Aβ 1-4 and Aβ 1-42 are bound to albumin. 56 As observed in the cellular assay, BSA seems to compete with D3 and aminopyrazole trimer for Aβ binding. Moreover, ligand binding sites may be sterically shielded, rendering the D3/aminopyrazole trimer coating less effective. Thus, NP stabilization with BSA occurs at the expense of partial impairment of the conjugate s functionality. Similarly, Palmal et al. described that nanoparticles coated with stabilizing PEG layers featured lower fibril inhibition potentials. 497 Hence, replacement of BSA by PEG for stabilization does not seem to be a promising alternative. In the future, one could consider designing ligands with tailored spacer lengths in order to achieve stable particles, while the active sites reach beyond the stabilizer coating. Moreover, the design of colloidally stable, anionic conjugates featuring a submonolayer ligand coverage (compare to Figure 4-21) could be another alternative. It is easily executable via PLAL, due to the production of bare NPs as starting material. The values of submonolayer-coated, net anionic nanoconjugates lie in their highly efficient ligand binding

196 4. Results and Discussion 191 capability (1% conjugation efficiencies, Figure 4-3), their charge-twisted nanoenvironment ( zwitterionic character ) and their net anionic charge (with good colloidal stability and potentially higher biocompatbility 2 ). Considerations with Regard to the Medical Applicability of Nanoconjugates For the clinical application of any type of nanoconjugate, different aspects regarding quality, safety and efficacy would need to be considered in the future. A potential pharmaceutical product comprised of nanoparticles and ligands needs to be synthesized sterilely. In principle, a completely sealed process could be installed, since the laser can be installed outside and focused into a sealed ablation chamber. Furthermore, NP synthesis via PLAL can be conducted with a flow-through chamber (employing only sterile water and a pure gold target). Automatically, the sterile ligand solution can be fed into the liquid flow downstream of the PLAL process ( fast ex situ conjugation 41,5 ). Furthermore, it would be advantageous to formulate the drug as solid powder for increased storage time and convenient handling. This was the motivation for proving the compatibility of BSA-stabilized nanoconjugates with the freeze-drying and resuspension process (Figure 4-12, Figure 4-27). In addition, the conjugate s therapeutic window, i.e. the dose inducing the desired biologic effect while exerting minimum side effect, needs to be determined. From preliminary experiments, data on the effective in vivo D3 ligand dose exist. 16 Hence, the following considerations give a rough indication on the AuNP amount possibly required to immobilize D3 on NPs in medically relevant doses: For D3, doses of.1 to 1 mg per mouse and day were given orally for 7 weeks, corresponding to.625 to.625 µmol per day (M = g/mol). Considering a ligand-to-np ratio of 2, daily NP number doses of.3125 to nmol would be required (corresponding to NP mass doses of.65 to 6.5 mg for 7 nm particles). These doses correspond to mg Au per kg body weight and are quite high, when considering previously published in vivo studies. Since published studies show controversial results about the toxicity of AuNPs, the report of Chen et al. should be regarded as one example amongst others. It was chosen because it represents a long-term (t > 5 d) in vivo study. They administered a dose of 8 mg/kg/week of 5 nm AuNPs to mice and did not observe lethality within 5 days. 522 In clinical trials, doses of 12 mg AuNPs have been applied in humans as chemotherapeutic agent (AurImmune therapy, see section 2.1.2). 95

197 5. Outlook Outlook There may still be a long way to go until a therapy is available, which modifies molecular and/or cellular mechanisms underlying Alzheimer s and other protein misfolding diseases. Hence, this work should be rather regarded as a contribution to better understand the structure-function relationship of nanoconjugates, illustrated on the model of Aβ aggregation, than as development of a pharmaceutical product. Within the Aβ aggregation project, further experiments should be conducted with fluorophore-labeled ligands. These will allow the simultaneous quantification of different ligand types on the NP surface in order to better characterize heterofunctional conjugates. Moreover, labeled ligands will allow to track NPs inside cells and locate them in different compartments. It should be taken into account that fluorophore labels significantly contribute to the chemical identity of small molecule ligands, as their molecular weights may lie in the same range. Hence, chemical propeties 476 and binding to Aβ may be different for labeled and non-labeled variants of the same ligand. When testing the functionality of fluorophore-labeled ligands, one should thus consider to mix them as a sub-fraction (e.g. 1%) with unlabeled ligands (e.g. 9%). The physicochemical characterization, on how NPs interact with Aβ, could be extended to AUC experiments. Preliminary experiments, in which Aβ species were analyzed via the interference detector, remained unsuccessful, possibly due to too low Aβ concentrations. However the employment of an AUC with a UV or fluorescence detector may represent an alternative and has been employed for the analysis of Aβ aggregation before. 44 Another physicochemical technique could be fluorescence lifetime measurements to better understand the fluorescence quenching processes on AuNPs and PtNPs. This could help to differentiate between static and dynamic quenching during the formation of complexes between FITClabeled Aβ and nanoconjugates. In this regard, static quenching involves the formation of a non-fluorescent complex in the ground state. 223 Subsequently, the fraction of un-complexed fluorophores would be detected. Their lifetime would remain the same, regardless of the presence or absence of the NP quencher. In contrary, in dynamic quenching, the excited fluorophore is brought to the ground state by non-radiative decay. This would reduce the lifetime of all fluorophores present in the sample relative to the lifetime in the absence of the NP quencher. 223

198 5. Outlook 193 With regard to the design of nanoconjugates, the development of catalytically active compounds could be addressed. In this regard a third type of ligand could be immobilized on the NP surface, consisting of a natural or synthetic protease. After binding Aβ aggregates to the NP via D3 and breaking their β-sheets via the aminopyrazole trimer, the third ligand could enzymatically digest the remaining Aβ fragments. This could lead to the regeneration of Aβ binding sites on the NP for a repetitive, cyclic process. Obviously, ligand design with regard to the spatial orientation of the ligands towards each other would need to be carefully optimized. Moreover, the protease should neither degrade the peptide ligands on the NP surface nor unspecifically degrade any (serum or liquor) protein which accumulates around the NP. Preliminary experiments were conducted with trifunctionalized AuNPs containing D3, aminopyrazole trimer and the insulin degrading enzyme. In cell tests, the trifunctional conjugate showed superior function over the mixture of free ligands (see appendix, Figure S32). This indicates that immobilization of the ligands on the NPs occurred, which changed their mode of action and allowed synergistic function. However, to understand the mode of action, further tests need to be conducted. It is noted that IDE served as model protease in these preliminary experiments and could be replaced in later experiments by smaller, artificial proteases. Ideally, these should feature a higher chemical stability than IDE and higher specificity for Aβ. For example, Co(III) and Cu(II) complexes of cyclen were developed in the group of Suh et al. (Figure 5-1). 523,524 They were shown to catalyze the cleavage of peptide bonds via hydrolysis. For Aβ 1-42 oligomers, cleavage was shown to occur at submicromolar concentrations of Co(III) cyclen. 523 With regard to the design of nanoconjugates, the modification of cyclens with thiol groups could enable their immobilization to AuNP surfaces. Figure 5-1: Lewis structure of Co(III) cyclen complexes. Adapted from Ref. 524 with permission of The Royal Society of Chemistry. DOI: 1.139/B71345J Intermediate-term goals could include the scale-up of nanoconjugate synthesis. High amounts of nanoconjugates would be required, e.g. if the samples should be tested in vivo. In this regard, NP synthesis should be conducted continuously. For example, the laser ablation

199 5. Outlook 194 process should be transferred from a batch reactor to a flow-through reactor. A high power laser system recently proved g/h productivities in continuous flow mode, equivalent to several liters of colloid per hour. 15 Before in vivo tests can be performed, pretesting should include analysis of the blood-brain barrier permeability of the nanoconjugates for which different cell-based BBB models exist. 525,526 These could give an indication if the conjugates parameters such as size and shape need to be adjusted to improve their BBB permeability. 527 In parallel, the toxicity of the conjugates needs to be evaluated. Here, anionic submonolayer-coated nanoconjugates should be further included in in vitro as well as in vivo assays. In the long term, one could think of transferring and adapting the conjugate design for interfering with the tau protein. It is scientifically discussed that combination therapies or multitarget compounds 528 may be better suited for the treatment of a complex, multifactorial disease, such as AD, than compounds which interfere with Aβ aggregation only. 529 In addition, conjugates could be designed for treating other protein misfolding disease. In this regard, targeting Huntington s disease could be considered. HD is an inheritably, progressive, neurodegenerative disease caused by a genetic mutation. This results in the expansion of the huntingtin protein with polyglutamin, leading to the subsequent protein aggregation and inclusion body formation. 53 A strong correlation exists between the threshold for the in vitro aggregation of huntingtin and in vivo neurodegeneration. Hence, protein aggregation seems to correlate strongly with disease pathogenesis and targeting huntingtin could directly interfere with the disease mechanism. 262 In contrast to Aβ, results obtained from in vitro tests on huntingtin may thus be better transferable to in vivo test. Moreover, it could be interesting to employ NPs to a glaucoma model. Glaucoma is the second leading cause of blindness worldwide. It comprises a heterogeneous group of disorders affecting the optic nerve function, with Primary Open Angle Glaucoma (POAG) being the most prevalent form. 41 In particular, the accumulation of the glycoprotein myocilin has been suggested to be linked to POAG. Genetic mutations may lead to an increased expression of myocilin and to alterations in the protein s three-dimensional structure. These may prevent protein degradation and normal function. Increased amounts of myocilin may then cause an elevated intraocular pressure by increasing the outflow resistance. 41 In contrast to neurodegenerative diseases, interfering with myocilin would not require the crossing of the BBB. Instead, a nanoparticulate compound could be administered directly to the eye.

200 6. Summary Summary Neurodegenerative diseases such as Morbus Alzheimer, Morbus Parkinson and Chorea Huntington are considered incurable so far and will affect a higher number of people due to continuing population growth and increase in life expectancies worldwide in the next decades. For cellular toxicity and disease pathology, protein accumulation, misfolding and aggregation seem to be common molecular mechanisms. Since only symptom-relieving drugs are clinically approved to date, the development of disease-modifying therapeutics is investigated intensively. Following the concept that aggregation of the amyloid β peptide (Aβ) in the human brain may play a central role in the pathogenesis of Alzheimer s disease (AD), compounds that interfere with Aβ could potentially be of therapeutic value against AD and also serve as starting points to target other protein misfolding diseases. In the field of nanomedicine, the combination of bioinert nanoparticles and bioactive ligands leads to the formulation of conjugates with biomedical function, with nanogold being already clinically investigated. In particular, nanoparticles may serve to increase ligand potencies and induce synergistic effects by accumulating multiple ligands in close proximity. Moreover, they act as carriers by transporting the compounds across biological barriers. The aim of the following work was to systematically design, fabricate and characterize nanobioconjugates and to contribute to a better understanding of their structure-function relationship, using the example of the Aβ aggregation model relevant for AD pathogenesis. In particular, nanoconjugates comprised of gold nanoparticles, Aβ-targeting D3 peptide and β- sheet breaking aminopyrazole ligands were fabricated and characterized with respect to their potential to hinder and/or reverse Aβ aggregation into oligomers and β-sheet-rich amyloid fibrils. In this work, small colloidal gold nanoparticles (AuNPs, d = 7 nm) were generated via pulsed laser ablation in liquids and subsequent centrifugation. The technique enables the generation of monodisperse, biocompatible, ligand-free colloids with negative charge in aqueous solution. This starting material proved to be ideally suited for the subsequent conjugation with different cationic variants of the Aβ-targeting D3 and aminopyrazole trimer ligands via covalent gold-thiol bonds. Thereby tailored ligand coverages from submonolayer to monolayer could be created on the NP surface, which resulted in overall anionic conjugates

201 6. Summary 196 (with a cationic nanoenvironment ) as well as overall cationic conjugates. Monofunctional conjugates with maximum numbers of 198 ± 23 and 233 ± 29 pmol ligands/cm² could be obtained, respectively. Moreover, bifunctional conjugates with stoichiometries of 1:1, 1:1 and 1:1 were designed. It was demonstrated that the cationic charge of the ligands enabled effective electrostatic interaction with the anionic AuNPs. However, a specific intermediate ligand-to-np ratio resulted in neutral conjugates with low colloidal stability and impaired colloidal properties. Hence, steric particle stabilization with small thiolated ligands was assessed by employing the model molecules lipoic acid (LA), mercaptoundecanoic acid (MUA) and thiolated polyethylene glycol (mpeg-sh). Moreover, interactions with the model serum proteins transferrin, insulin and bovine serum albumin (BSA) were analyzed. Ligand attachment was qualitatively verified by analyzing the alterations in the particles optical properties, their size and charge. Stabilization efficacy was generally found to correlate with the molecular size of the stabilizers, following the order: LA < MUA < mpeg-sh < BSA. In high ionic strength medium 4 BSA per NP were sufficient to stabilize AuNPs against aggregation, whereas > 1 LA and MUA ligands were required. Besides salt stress, colloidal stability was analyzed upon freeze-drying and resuspension and a minimum amount of 125 BSA per NP was identified to guarantee particle stability. During gel electrophoresis of Au/protein conjugates, minimum numbers of 5 insulin, 2 transferrin and 6 BSA per NP were found to be necessary to enable NP migration in the gel. In addition to stabilizing ligand-free AuNPs, the stabilization of cationic Au/D3 conjugates was assessed. It was found that the negative charge of LA and MUA complicates stabilization due to charge balancing effects. In the presence of serum proteins, the conjugate s net charge was compensated at protein-to-conjugate ratios of.6:1 (BSA), 7:1 (transferrin) and 22:1 (insulin). BSA and mpeg-sh concentrations required for Au/D3 stabilization were found to be higher than the concentrations for ligand-free AuNPs, which may be due to the increased hydrodynamic sizes of the conjugates. Interestingly, the presence of additional stabilizer molecules did not decrease the amount of D3 bound to the NP surface in most cases and even led to an increased amount of bound D3 under some conditions. Finally, the conjugates abilities to interfere with Aβ aggregation was investigated with regard to the influence on the Aβ secondary structure, the morphology of Aβ fibrils and the Aβ aggregate size distribution. Ligand immobilization on the NP surface was shown to increase their affinities for Aβ, as determined from the dissociation constants (K D ) of fluorescence

202 6. Summary 197 quenching experiments. Dissociation constants were in the nanomolar range, with anionic conjugates featuring exceptionally high affinities for Aβ (Au/D3_5+ anionic : K D,AuNP = 1.9 nmol/l, K D,D3 = 7.8 nm). Moreover, it was found that monofunctionalized Au/D3 and Au/Trim conjugates affected synthetic Aβ aggregation by decreasing the amounts of β-sheets and converting Aβ into short fibril fragments and large, non-fibrillar aggregates. In particular, the Thioflavin T assay revealed that the reduction of β-sheets was up to 53 times greater with nanoconjugates than with free ligands in equimolar doses. Finally, a cellular assay, based on the natural secretion of neurotoxic Aβ species, was employed. The assay revealed the conjugate s effectiveness under in vitro conditions. In particular, optimal ligand performances required their immobilization on the NP surface. Preliminary experiments employing a Au/D3/Trim/protease conjugate in the cellular assay indicated that complex, trifunctional NPs can be designed successfully. These reduced the amounts of cellularly excreted Aβ species more effectively than the mixture of the free ligands. In conclusion, the immobilization of Aβ-binding, β-sheet breaking and proteolytic ligands on NP surfaces represent a promising research approach for the generation of multifunctional, bioactive nanoconjugates. These may not only interact with the Aβ aggregation process, but their design could also be adjusted to target other protein misfolding diseases in the future.

203 6. Summary 198 Zusammenfassung Neurodegenerative Erkrankungen wie Morbus Alzheimer, Morbus Parkinson und Chorea Huntington sind bisher unheilbar und werden in den kommenden Jahrzehnten durch ein weltweites Bevölkerungswachstum und die steigende Lebenserwartung immer mehr Menschen betreffen. Die Ansammlung, Fehlfaltung und Aggregation bestimmter Proteine im Gehirn scheint ein gemeinsamer molekularer Mechanismus für die Entstehung der Krankheiten und die zugrunde liegende Zelltoxizität zu sein. Da bisher nur symptomlindernde Medikamente klinisch zugelassen sind, wird die Entwicklung neuer Wirkstoffe, die aktiv in den Krankheitsmechanismus eingreifen, intensiv erforscht. In Anlehnung an die Hypothese, dass die Aggregation des Amyloid-β-Peptids (Aβ) eine zentrale Rolle für die Entstehung der Alzheimererkrankung spielt (Alzheimer-Demenz, AD), können Moleküle, die mit Aβ wechselwirken, potentielle Wirkstoffe darstellen. Im Bereich der Nanomedizin führt die Kombination bioinerter Nanopartikel mit bioaktiven Wirkstoffen zur Formulierung von Konjugaten mit biomedizinischer Funktion, wobei Nanogold-basierte Formulierungen bereits in klinischen Studien untersucht werden. Dabei erfüllen die Nanopartikel die Aufgabe, die Wirksamkeit der Liganden zu erhöhen und synergistische Effekte zu ermöglichen, indem sie mehrere Moleküle in räumliche Nähe zueinander bringen. Außerdem können sie den Wirkstoffen als Transportmittel dienen, um biologische Barrieren zu überwinden. Das Ziel dieser Arbeit lag im systematischen Design, der Herstellung und Charakterisierung verschiedener Nanobiokonjugate. Zudem sollte ein Beitrag zum besseren Verständnis ihrer Struktur-Funktion-Beziehung am Beispiel des Modells der Aβ-Aggregation geleistet werden. Insbesondere sollten Konjugate bestehend aus Goldnanopartikeln und mit Aβ wechselwirkenden D3-Peptid- und Aminopyrazoltrimer-Liganden hergestellt werden. Besonderes Augenmerk sollte darauf gelegt werden, ob und auf welche Weise die Konjugate auf die Aβ-Aggregation wirken. Kleine, kolloidale Goldnanopartikel (AuNP, d = 7 nm) wurden mittels gepulster Laserablation in Flüssigkeiten und anschließender Zentrifugation generiert. Diese Methode ermöglicht die Herstellung monodisperser, biokompatibler, ligandenfreier Kolloide in wässriger Lösung. Die gezielte Funktionalisierung des AuNP-Ausgangsmaterials erfolgte über die Konjugation mit

204 6. Summary 199 Liganden durch Thiolbindungen. Verschiedene kationische Derivate der D3-Peptid- und Aminopyrazoltrimer-Liganden wurden herangezogen, um monofunktionale Konjugate mit maximalen Ligandenbedeckungen von 198 ± 23 und 233 ±29 pmol/cm² zu generieren. Zudem wurden bifunktionale Konjugate durch Mischen der beiden Ligandentypen in den Verhältnissen 1:1, 1:1 und 1:1 hergestellt. Die positive Ladung der Liganden führte dabei zu einer effektiven elektrostatischen Wechselwirkung mit den anionischen Nanopartikeln, sodass je nach eingesetztem Ligand-zu-NP-Verhältnis sowohl insgesamt negativ geladene als auch insgesamt positiv geladene Konjugate hergestellt werden konnten. Ein mittleres Ligandzu-NP-Verhältnis führte jedoch zur Ladungsneutralisierung und Beeinträchtigung der Kolloideigenschaften. Deshalb wurde untersucht, inwiefern die Zugabe von kleinen Thiolliganden wie Liponsäure (LA), Mercaptoundecansäure (MUA) und thiolisiertem Polyethylenglycol (mpeg-sh) zu einer sterischen Stabilisierung beitragen kann. Zusätzlich wurde die Wechselwirkung mit Modellserumproteinen wie Insulin, Transferrin und Albumin (Rinderserumalbumin, BSA) analysiert. Die Anbindung der Liganden wurde qualitativ über die Analyse der physikochemischen Partikeleigenschaften (optische Eigenschaften, Partikelgröße und ladung) verifiziert. Im Allgemeinen wurde eine Korrelation zwischen der Größe des eingesetzten Stabilisators und der Stabilisierungseffizienz entdeckt, wonach die Stabilisatoren in der Reihe LA < MUA < mpeg-sh < BSA eingeordnet werden können. Während in hochkonzentrierten Salzlösungen beispielsweise 4 BSA je NP zur Stabilisierung ausreichen, werden von LA und MUA mehr als 1 Moleküle je NP benötigt. Neben der Durchführung von Salz-Stresstests wurde die kolloidale Stabilität nach Gefriertrocknung und Resuspension untersucht. Dabei wurde eine minimale Anzahl von 125 BSA-Molekülen je NP zum Erhalt der Partikelstabilität ermittelt. Gelelektrophorese-Experimente mit Au/Protein- Konjugaten zeigten, dass mindestens 5 Insulin-, 2 Transferrin- und 6 BSA-Moleküle je NP eingesetzt werden müssen, damit die Partikel im Gel separiert werden können. Die Charakterisierung der Stabilisierung kationischer Au/D3-Konjugate mit Thiolliganden machte deutlich, dass LA und MUA aufgrund ihrer negativen Ladung und dem daraus resultierenden Ladungsausgleich weniger geeignet sind als das neutrale mpeg-sh und das sterisch anspruchsvolle BSA. Weiterhin konnten spezifische Verhältnisse von Serumproteinen zu Nanokonjugaten bestimmt werden (.6:1 für BSA, 7:1 für Transferrin, 22:1 für Insulin), die zu einem Ladungsausgleich führten. Die zur Stabilisierung nötigen Konzentrationen an mpeg-sh und BSA waren im Fall der Konjugate höher als für ligandenfreie NP, was auf die erhöhte hydrodynamische Größe der Konjugate zurückzuführen

205 6. Summary 2 sein könnte. Interessanterweise zeigten Experimente zur Bestimmung der Konjugationseffizienz am NP, dass die Anwesenheit von (zusätzlichen) Stabilisatormolekülen generell nicht zu einer Verringerung der Anzahl gebundener Liganden führt und sie in manchen Fällen sogar erhöht. Zuletzt wurde der Einfluss der Konjugate auf die Aβ-Sekundärstruktur, die Morphologie und die Aβ-Größenverteilung untersucht. Über Fluoreszenzquenching-Titrationen konnte gezeigt werden, dass die Immobilisierung der Liganden auf der NP-Oberfläche ihre Affinität gegenüber Aβ erhöht. Dissoziationskonstanten (K D ) für die Interaktion der Nanokonjugate mit Aβ lagen typischerweise im nanomolaren Bereich, wobei anionische Konjugate auffallend hohe Affinitäten aufwiesen (Au/D3_5+ anionisch : K D,AuNP = 1.9 nmol/l, K D,D3 = 7.8 nm). Monofunktionale Au/D3- und Au/Trim-Konjugate beeinflussten die Aβ-Aggregation, indem sie den Anteil der β-faltblattstruktur herabsetzten und synthetisches Aβ in kurze Fibrillenfragmente und große, amorphe, nicht-fibrilläre Aggregate konvertierten. Durch Messung der ThioflavinT-Fluoreszenz konnte eine Reduktion des β-faltblattgehalts durch Nanokonjugate nachgewiesen werden, die bis zu 53-Mal stärker war als die der freien Liganden in äquimolarer Dosis. Ein Zelltest basierend auf der Sekretion natürlicher, neurotoxischer Aβ-Spezies untermauerte, dass die Konjugate auch unter in vitro-bedingungen wirken und eine optimale Ligandenwirkung vor allem durch deren Immobilisierung auf der NP-Oberfläche hervorgerufen wird. Vorläufige Zelltests, die mit Au/D3/Trim/Protease- Konjugaten durchgeführt wurden, deuteten darauf hin, dass auch komplexe, trifunktionale Konjugate erfolgreich hergestellt werden können. Diese reduzierten das durch Zellen ausgeschiedene Aβ effektiver als die Mischung der freien Liganden. Zusammenfassend kann festgestellt werden, dass die Immobilisierung einer Aβ- Erkennungseinheit, eines β-faltblattbrechers und eines proteolytischen Liganden auf NP- Oberflächen einen vielversprechenden Ansatz zur Herstellung multifunktionaler, bioaktiver Nanokonjugate darstellt. Diese Konjugate sind nicht auf das Modell der Aβ-Aggregation beschränkt. Ihr Design könnte in Zukunft so modifiziert werden, dass sie auch in andere Proteinfehlfaltungsprozesse eingreifen können.

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244 8. Appendix Appendix 8.1 Lists of Physical Parameters and Abbreviations Table 8-1: Physical parameters employed in the formulas within this thesis Acronym Description Unit A absorbance [ ] - B fluorescence quenching - B max maximum quenching - I Intensity of incident (I) and transmitted light - n cooperativity factor - κa ratio of the particle radius and the electrical double layer thickness - mass concentration µg/ml μ electrophoretic mobility µm/s/(v/cm) s sedimentation coefficient 1 Svedberg =1 S =1-13 s k B Boltzmann constant 1.381x1-23 J/K NP surface concentration 1/mL NP number concentration 1/mL Avogadro constant 6.23x1 23 1/mol ε vacuum permittivity 8.854x1-12 C²/Nm² η dynamic viscosity 8.9x1-4 Pa s for water at 25 C ε solvent dielectric constant 8 for water at 25 C l path length of light cm r min/max distance from the centre of the rotor to the bottom/ top of the solution in the centrifuge tube cm nanoparticle surface area cm² nanoparticle volume cm³ partial specific volume, 1/ρ cm³/g nanoparticle mass g ρ NP particle density g/cm³ ρ S solvent density g/cm³ R gas constant J/mol K T temperature K ε molar absorption coefficient L*cm/mol d diffusion coefficient m²/s c concentration mol/l r NP particle radius nm K D dissociation constant nmol/l ω rotational speed, =2π*rpm/6 rad/s

245 8. Appendix 24 Table 8-2: Abbreviations employed in this thesis AD Alzheimer s disease ADC Analytical disc centrifuge AD Alzheimer s disease AFM Atomic force microscope ALS Amyotrophic lateral sclerosis APP Amyloid precursor protein AUC Analytical ultracentrifuge AuNP Gold nanoparticle Aβ Amyloid β BBB Blood-brain barrier BSA Bovine serum albumin CD Circular dichroism CM Conditioned medium CPP Cell penetrating peptide CTAB Cetyltrimethylammonium bromide DGC Density gradient centrifugation DHLA Dihydrolipoic acid DLS Dynamic light scattering DLVO theory Derjaguin-Landau-Verwey-Overbeek theory DNA Deoxyribonucleic acid EDTA Ethylene diamine tetraacetic acid EGCG Epigallactocatechin gallate ELISA Enzyme-linked immunosorbent assay FITC Fluoresceine isothiocyanate HD Huntington s disease HFIP Hexafluoroisopropanol HSA Human serum albumin IDE Insulin degrading enzyme IEP Isoelectric point IPD Interparticle distance LA Lipoic acid mpeg-sh Thiolated PEG, Polyethylene glycol methyl ether thiol MALDI Matrix-assisted laser desorption ionization MS Mass spectrometry MUA Mercaptoundecanoic acid NP Nanoparticle PBS Phosphate buffered saline PBS-T Phosphate buffered saline with TWEEN2 PD Parkinson s disease PDI Polydispersity index PEG Polyethylene glycol PLA Poly-D,L-lactide PLAL Pulsed Laser Ablation in Liquids PLGA Polylactic-co-glycolic acid PPI Primary Particle Index PtNP Platinum nanoparticle RP-HPLC Reversed phase high performance liquid chromatography SD Standard deviation SDS Sodium dodecyl sulfate SEM Scanning electron microscope SPR Surface plasmon resonance TAE TRIS acetate EDTA TEM Transmission electron microscope TfR Transferrin receptor Trim Aminopyrazole trimer TRIS Tris-(hydroxymethyl)-aminomethane TSE Transmissible spongiform encephalopathies UV/Vis Ultraviolet/visible

246 Zeta potential [mv] Intensity [a.u.] 8. Appendix Supplementary Information, Figures and Tables Characterization of AuNPs and the Influence of Small Thiolated Ligands and Serum Proteins Hydrodynamic Diameter [nm] Figure S1: DLS intensity distributions of laser-generated centrifuged AuNP. Shown are data from three different samples Stabilizer density [applied #/NP d=7nm ] LA +MUA +mpeg-sh +Insulin +Transferrin +BSA E Stabilizer concentration [µm] Figure S2: Zeta potentials of AuNP as function of the concentration of small thiolated ligands or serum proteins Au/LA Au/MUA Au/mPEG-SH ph 1 ph 1 1 cm Figure S3: Photographs of colloidal AuNP conjugated with the small thiolated ligands LA, MUA and mpeg-sh and incubated at different ph values.

247 8. Appendix 242 Table S1: Added volumes of HCl and NaOH for the titration of 1 ml AuNP to establish ph values between 1 and 1. ph Addition of acid/base µl 1 M HCl µl 1 M HCl µl 1 M HCl µl 1 M HCl µl.1 M HCl µl.1 M HCl µl.1 M HCl µl.1 M NaOH µl 1 M NaOH TAE.5x TAE 1x TAE 2x TAE 4x _ 75V 1V 125V 15V + _ Figure S4: Gel electrophoresis employing 1 to 6 µg/ml AuNP and 2.5 g/l BSA in 1% agarose for 1 h, varying the buffer concentration (top) or the voltage (bottom). +

248 Fluorescence [a.u.] Fluorescence [a.u.] Emission wavelength [nm] Conjugation efficiency [%] 6 µg/ml 55 µg/ml 5 µg/ml 45 µg/ml 4 µg/ml 35 µg/ml 3 µg/ml 25 µg/ml 2 µg/ml 15 µg/ml 1 µg/ml 8. Appendix Figure S5: Gel electrophoresis employing 1 to 6 µg/ml AuNP and 2.5 g/l BSA in 1% agarose and 1x TAE buffer for 1 h at 15 V. Quenching Supernatant (UV/Vis) Supernatant (Fluorescence) Insulin-FITC dose [# applied/np d=7nm ] Excitation wavelength [nm].1 1 Applied Insulin-FITC [µm] 5 4 Ex: 485 nm Em: 52 nm acidic neutral alkaline.5.4 acidic: y=(.22.) x, R²=.996 neutral: y=(.5.1) x, R²=.997 alkaline: y=(.95.7) x, R²= ( ) ( ) Insulin-FITC [µm] Insulin-FITC [µm] Figure S6: Quenching experiments with insulin-fitc. Characterization of the insulin-fitc fluorescence via 3D-fluorescence spectroscopy (top, left). Conjugation efficiencies determined via analysis of AuNP quenching or separating NP-bound from unbound insulin and quantifying the unbound fraction in the supernatant (top, right). The optical properties of insulin-fitc are sensitive to changes in ph (bottom).

249 Fluorescence [a.u.] nm [a.u.] Conjugation efficiency [%] 8. Appendix 244 Quenching Supernatant (UV/Vis) Supernatant (Fluorescence) 1 BSA-FITC dose [# applied/np d=7nm ] Applied BSA-FITC [µm] 5 4 Ex: 485 nm Em: 52 nm ( ) acidic: y= (.271.5) x, R²=.996 neutral: y= (.348.1) x, R²=.991 alkaline: y= (.388.6) x, R²= acidic neutral alkaline BSA-FITC [µm] ( ) BSA-FITC [µm] Figure S7: Quenching experiments with BSA-FITC. Conjugation efficiencies determined via analysis of AuNP quenching or separating NP-bound from unbound BSA and quantifying the unbound fraction in the supernatant (top). The optical properties of BSA-FITC are sensitive to changes in ph (bottom).

250 8. Appendix 245 Fluorescence Quenching Experiments to Determine Binding Affinities Table S2: Dissociation constants (K D ) and cooperativity factors (n) for the binding of Aβ-FITC and BSA- FITC to free ligands, bare NPs and NP/ligand conjugates (derived from Hill fits of fluorescence quenching titration experiments). Ligand affinity NP affinity Cooperativity Fit correl. K D [nm] SD Free ligand + Aβ K D [nm] SD n SD R² D3_5+/Aβ-FITC.125µM D3_8+/Aβ-FITC.125µM D3_1+/Aβ-FITC.125µM Trim_5+/Aβ-FITC.125µM Trim_8+/Aβ-FITC.125µM Variation of BSA- or Aβ-FITC concentrations Au/BSA-FITC.5µM Au/Aβ-FITC.5µM Au/BSA-FITC.25µM Au/Aβ-FITC.25µM Au/BSA-FITC.125µM Au/Aβ-FITC.125µM Preincubation with unlabeled spacer molecules Au/BSA (4)/BSA-FITC.125µM Au/BSA (42)/BSA-FITC.125µM Au/BSA (418)/BSA-FITC.125µM Au/PEG (418)/BSA-FITC.125µM Au/PEG (418)/Aβ-FITC.125µM NP/ligand conjugates (418 ligands per NP) + Aβ Au/D3_5+/Aβ-FITC.125µM Au/D3_8+/Aβ-FITC.125µM Au/D3_1+/Aβ-FITC.125µM Au/Trim_5+/Aβ-FITC.125µM Au/D3_5+/Trim_5+/Aβ-FITC.125µM NP/ligand conjugates (418 ligands per NP) + BSA Au/D3_5+/BSA-FITC.5µM Au/D3_8+/BSA-FITC.5µM Au/D3_1+/BSA-FITC.5µM Au/Trim_5+/BSA-FITC.5µM Au/D3_5+/Trim_5+/BSA-FITC.5µM Variation of ligand densities Au/D3_5+ (4)/Aβ-FITC.125µM Au/Trim_5+ (4)/Aβ-FITC.125µM Au/D3_5+ (42)/Aβ-FITC.125µM Au/Trim_5+ (42)/Aβ-FITC.125µM Au/D3_5+ (125)/Aβ-FITC.125µM

251 8. Appendix 246 PtNP Pt/Aβ-FITC.125µM Pt/D3_5+ (125)/Aβ-FITC.125µM Pt/BSA-FITC.5µM Pt/BSA-FITC.25µM Pt/BSA-FITC.125µM Pt/PEG (418)/BSA-FITC.125µM Pt/PEG (418)/Aβ-FITC.125µM Quenching (I -I)/I AuNP/BSA + BSA-FITC +4 BSA/NP +42 BSA/NP +418 BSA/NP Quenching (I -I)/I 1. Pt/PEG+BSA.125µM Pt/PEG+A.125µM.8 Au/PEG+BSA.125µM Au/PEG+A.125µM AuNP [nm] NP [nm] Figure S8: Titration experiments to determine quenching of BSA-FITC with AuNP, which were preincubated with unlabeled BSA (left). Determination quenching of Aβ- and BSA-FITC with AuNP and PtNP, which were pre-incubated with unlabeled PEG-SH (5 g/mol, 418 ligands/np).. Quenching (I -I)/I Trim_5+ R5WC D3_5+ D3_8+ D3_ Ligand concentration [nm] Figure S9: Exemplary titration curves showing the quenching of Aβ-FITC with the free derivates of D3 and trimer (in the absence of NP).

252 SPR peak maximum Colloidal stability (PPI) SPR peak maximum [nm] Colloidal stability (PPI) SPR peak maximum [nm] Colloidal Stability (PPI) 8. Appendix 247 Characterization of Au/D3 and Au/Trimer Conjugates Au/D3_5+ (125) Au/D3_8+ (125) Au/D3_1+ (125) ph ph AuNP Au/D3_5+ (13) Au/D3_5+ (3) Au/D3_5+ (125) ph ph ph Au/D3_5+ Au/Trim_5+ Au/D3_5+/Trim_ ph Figure S1: Influence of the ph on the colloidal properties of Au/D3 and Au/Trimer conjugates to determine the isoelectric point.

253 Colloidal stability (PPI) SPR peak maximum [nm] SPRmax [nm] SPRmax [nm] SPRmax [nm] SPRmax [nm] 8. Appendix AuNP +.1uM D3_8+ AuNP + 1uM D3_8+ AuNP + 1uM D3_ AuNP +.1µM Trim_5+ AuNP + 1µM Trim_5+ AuNP + 1µM Trim_ time [h] time [h] 62 6 AuNP +.5µM D3_1+ AuNP +.5µM D3_1+ AuNP + 5µM D3_ AuNP +.4µM Trim-K8-Cys AuNP +.4µM Trim-K5-Cys AuNP + 4µM Trim-K5-Cys time [h] time [h] Figure S11: Time-dependent analysis of AuNP colloidal properties after conjugation with different D3 and Trimer derivates at low, intermediate and high ligand doses over a period of 12 h. 3 2 bare AuNP AuNP+1µM D3 AuNP + 1µM D3 + 5µM LA AuNP + 1µM D3 + 5µM MUA AuNP + 1µM D3 + 5µM mpeg-sh AuNP + 1µM D3 + 5µM BSA bare AuNP AuNP + 1µM D3_5+ AuNP + 1µM D3 + 5µM LA AuNP + 1µM D3 + 5µM MUA AuNP + 1µM D3 + 5µM mpeg-sh AuNP + 1µM D3 + 5µM BSA min 24 h 48 h 72 h 96 h Incubation time Figure S12: Long-term stability of bare and neutral Au/D3_5+ conjugates in the absence and presence of small thiolated ligands and BSA in pure water. Successive addition of D3 and stabilizer. 1 min 24 h 48 h 72 h 96 h

254 Emission wavelength [nm] 8. Appendix Excitation wavelength [nm] Figure S13: Characterization of the D3-Cou fluorescence via 3D-fluorescence spectroscopy Table S3: Summary of the results from calibration curves for the determination of ligand concentrations. Limit of quantification, slope of calibration curves and calculated maximum ligand density per AuNP. Note that the limit of quantification was defined as 1 times the standard deviation of the blank. Limit of quantification Slope of calibration curve Correl. Coefficient (R²) D3-Trp 1.8µM.328 D3-Trp4.4µM.1359 D3-Cou.7µM 5495 a.u./µm (Ex: 328nm, Em: 393nm).998 Trim_5+.3µM.1989 Trim_8+.3µM.1788

255 [a.u.] [a.u.] [a.u.] [a.u.] Fluorescence [a.u.] 8. Appendix Gleichung Gewichtung Fehler der Summe der Quadrate y = a + b*x Keine Gewichtu ng E7 Pearson R.9991 Kor. R-Quadrat F Schnittpunkt mit der Y-Achse Wert D3-Coumarin [µm] Standardfehler -- F Steigung D3-Trp-Cys [ M] D3-Trp 4 -Cys [µm] Trim_5+ [µm] Trim_8+ [µm] Figure S14: Calibration curves correlating the absorbance or fluorescence of the ligand molecules with their molar concentration.

256 Absorbance [a.u.] Norm. Absorbance [a.u.] Absorbance [a.u.] Absorbance [a.u.] Absorbance [a.u.] 8. Appendix Au/D3_5+, 6min, 5xg before after (pipette) after (3s ultrasonic bath) after (3x1s ultrasonic bath) min, 5xg, 3x1s ultrasonic bath Au/BSA before Au/BSA after Au before Au after Au/D3_5+ before Au/D3_5+ after Wavelength [nm] Au/D3_5+, 3x1s ultrasonic bath Wavelength [nm] before after 3min, 1xg after 6min, 5xg after 1min, 3g % Au/BSA before Au/BSA after Au before -97% Au after Au/D3_5+ before Au/D3_5+ after 6min, 5g, 3x1s ultrasonic bath Wavelength [nm] -2% -29% -2% -22% before after 3min, 1xg after 6min, 5xg after 1min, 3g -8% -92% -2% before after (pipette) after (3s ultrasonic bath) after (3x1s ultrasonic bath) Figure S15: Assessment of the ability to resuspend Au/D3_5+ conjugates after ultracentrifugation in order to remove excessive unbound ligands from oversaturated conjugates. Variation of the way of resuspending the pellet after centrifuging the colloid and removing the supernatant (top left). Bare AuNP and Au/BSA conjugates were employed as reference (top right). Variation of the centrifugation parameters (bottom, left). Summary of colloid concentrations before and after centrifugation (~Abs@38 nm) and relative mass losses, given in % (bottom, right)..3.2 D3-Trp4 25µM D3-Cou 1µM BSA 5µM LA 1µM MUA 1µM mpeg-sh 1µM Trim_5+ 1µM nm 266nm 328nm D3-Trp4 D3-Cou Trim_ Wavelength [nm] Wavelength [nm] Figure S16: Exemplary UV/Vis extinction spectra of aqueous ligand solutions (left). Peak-normalized spectra of D3 and trimer derivates illustrating the overlap of the extinction peaks of D3-Trp 4 and Trim_5+ (right).

257 Absorbance [a.u.] Absorbance [a.u.] Conjugation efficiency [%] Conjugation efficiency [%] 8. Appendix 252 Insulin-FITC dose [# applied/np d=7nm ] BSA-FITC dose [# applied/np d=7nm ] Quenching Supernatant (UV/Vis) Supernatant (Fluorescence).1 1 Applied Insulin-FITC [µm] 2 Quenching Supernatant (UV/Vis) Supernatant (Fluorescence).1 1 Applied BSA-FITC [µm] Figure S17. Conjugation efficiencies of insulin-fitc (left) and BSA-FITC to cationic Au/D3 conjugates (c(aunp) = 5 µg/ml, c(d3_5+) = 1 µm) nm Medium AuNP Mix d 1d 2d 3d 4d nm Wavelength [nm].2 Au/D3_ Wavelength [nm] Figure S18: UV/Vis spectra of cell culture medium supplemented with 1% fetal bovine serum (left). Note the spectral overall of the medium with the SPR peak of colloidal AuNPs. Stability of Au/D3_5+ conjugates incubated in cell culture medium, analyzed via UV/Vis measurements over a period of 4 days (c(aunp) = 5 µg/ml, c(d3_5+) = 1 µm, right). Despite the spectral overlap, colloidal stability seems to remain high over the entire course of the experiment (i.e. constant absorbance at 38 nm, arrow).

258 colloidal stability (PPI) Peak maximum [nm] Absorbance [a.u.] Absorbance [a.u.] Absorbance [a.u.] Absorbance [a.u.] Absorbance [a.u.] 8. Appendix AuNP Temperature [ C] AuNP/D3_5+ Temperature [ C] AuNP/Aβ Wavelength [nm] Wavelength [nm] Wavelength [nm] Temperature [ C] Wavelength [nm] AuNP/D3_5+/Aβ Temperature [ C] Wavelength [nm] AuNP AuNP-D3 AuNP-D3/Abeta AuNP/Abeta Temperature [ C] AuNP AuNP-D3 AuNP-D3/Abeta AuNP/Abeta Temperature [ C] Figure S19: Temperature stability of bare AuNP (left) and Au/D3 conjugates (right) in the absence (top) and presence of Aβ (bottom). Employed concentrations were c(aunp) = 5 µg/ml, c(d3_5+, Aβ) = 1 µm. Samples were automatically heated within the UV/Vis cell holder (1 C per cycle: 12 min hold, 3 min heat). UV/Vis spectra were automatically recorded every 15 min.

259 8. Appendix 254 Calculations on the Sedimentation Behavior of Nanoconjugates in a Centrifugal Field The sedimentation time vs. size diagrams were obtained for the Svedberg equation by estimating the centrifuge parameters of ADC, i.e. detector position at 3 mm liquid column height, 6 cm rotor radius, 24 rpm centrifugal speed (corresponding to x g),.998 g/cm² solvent density and 1 mpa s solvent viscosity. The actual gradient of densities is neglected for the calculations. Table S4: Boundary conditions for calculating the sedimentation behavior of Au/D3_5+ conjugates in a centrifugal field NP core diameter NP core density NP volume ( Ligand length Ligand density Ligand volume 7 nm 19.3 g/cm³ nm³ 5 nm (13 amino acids, bond angle 19 ) 1.37 g/cm³ (derived from the partial specific volume of proteins, i.e..73 cm³/g) 2 nm³ (molecular weight: 173 g/mol) Note that the volume of the ligand layer is calculated from the product of the number of NPbound ligands and the ligand volume. Calculations on AuNP atoms and surface atoms as function of the NP size Assuming that the particles feature a spherical shape, their surface area can be calculated as A NP,surf = π*d² and their volume as V NP = 1/6*π*d³. Considering the covalent atomic radius for gold (r =.144 nm, the cross section of gold atoms can be calculated as A atom,cros s = π*r² = π*(.144 nm)² =.65 nm². Note that these values are comparable to those reported by Mei et al. (d = 1 nm, N total = 38, N surface = 4412). The authors performed calculations differently, i.e. based on n layers of gold atoms per NP (surface atoms = 1n²+2), 531 which is especially useful when considering small NPs (d < 2 nm).

260 8. Appendix 255 Table S5: Fraction of surface atoms on spherical gold nanoparticle of diameters between 2 and 2 nm. NP diameter NP volume # Atoms/NP (*74% packing) NP surface # atoms/np surface surface atoms nm nm³ # nm² % Microscopy of Aβ fibrils Figure S2: TEM analysis of pure Aβ and Aβ incubated with bare AuNP or free D3_5+. Note that samples were produced under AFM sample preparation conditions, but were additionally stained with 2% uranyl acetate to visualize fibril structures prior to the measurements (c(aβ)= 5 µm, c(aunp)= 125 µg/ml, c(d3)= 25 µm).

261 Surface coverage [%] 8. Appendix 256 1d 3d 7d Au/D3 13 +Aβ anionic 1 nm Au/D Aβ cationic nm Au/D Aβ cationic Aβ 6 μm Figure S21: Atomic force micrographs illustrating the influence of the ligand density on the AuNP surface on Aβ fibril formation (c(aβ) = 5 µm, c(aunp) = 125 µg/ml, c(d3) =.75, 7.5, 25 µm) Aß Aß+Au/D3 418 Aß+Au/D3 125 Aß+Au/D Time [d] Figure S22: Quantification of the Aβ fibril density on the AFM substrate in the presence of anionic and cationic Au/D3_5+ conjugates. Note that Au/D3 418 and Au/D3 125 are both cationic conjugates, but application of 125 D3 will result in less unbound ligands in the sample than application of 418 D3.

262 8. Appendix 257 Aβ Aβ, D3_5+ Aβ, Trim_5+ Aβ, D3_5+, Trim_5+ 2 nm 1d nm 7d 1µm Figure S23: Atomic force micrographs illustrating the influence of the free ligands (top) and mono- and bifunctional conjugates (bottom) on Aβ fibril formation after 1 and 7 days of co-incubation (c(aβ) = 5 µm, c(aunp) = 125 µg/ml, c(ligand) = 7.5 µm). Bifunctional conjugates were generated by mixing 7.5 µm D3_5+ with 7.5 µm Trim_5+.

263 Length [µm] Height [nm] 8. Appendix 258 1) Aβ 1 2) +D3_5+ 3) + Trim_5+ 4) +D3_5+ & Trim_5+ 5) +Au/D3_5+ 6) +Au/D3_5+/Trim_5+ 7) +Au/Trim_5+ 8) Aβ d 7 d Figure S24: Quantification of fibril lengths and heights for Aβ species analyzed with AFM after 1 and 7 days of co-incubation (c(aβ) = 5 µm, c(aunp) = 125 µg/ml, c(ligand) = 7.5 µm). Bifunctional conjugates were generated by mixing 7.5 µm D3_5+ with 7.5 µm Trim_5+. Note that different Aβ samples aggregated differently (Aβ 1, Aβ 2).

264 Fluorescence [RFU] Fluorescence [RFU] Fluorescence [RFU] Fluorescence [RFU] 8. Appendix 259 DGC/ELISA - +R5WC +Au/R5WC +D3_5+ +Au/D3_ Fraction Figure S25: Effects of Au/R5WC conjugates (left) and Pt/D3_5+ conjugates (right) on the aggregation of Aβ as determined from DGZ/ELISA (Incubation for 9 min at 37 C, c(aunp) =2 µg/ml, c(ptnp) = 44 µg/ml, c(ligand) =4 µm, c(aβ) =4 µm) Pt + D3_5+ + Pt/D3_5+ Fraction A in density gradient + ELISA 9min, 37 C, 1 9min, 37 C, 2 6h, 37 C 24h, 37 C % 25% 1% Fraction v/v% of sample Figure S26: Typical sedimentation profiles of Aβ species incubated under different experimental conditions. Note that a lot-to-lot variability exists between Aβ samples 1 and 2 (left). Dilution series of a DGC fraction and subsequent Aβ detection via ELISA. Notably, the fluorescence signal does not decrease linearly with decreasing amount of sample. This indicates that the ELISA detects Aβ only semiquantitatively, possibly due to too high concentrations of Aβ (saturation effect, right).

265 8. Appendix 26 Cellular Aβ Assay

266 protein [mg/ml] 8. Appendix 261 Figure S27: Confocal microscopy images of CHO cells treated with Au/D3_8+ (1), Au/D3_1+ (2) and bare AuNPs (3). Visualization of the cell membrane (A, employing the DIC filter), the nanoparticles (B, excitation at 532 nm), the cell nuclei (C, excitation at 45 nm) and overlying all filters (D) % +Au + BSA bi 1:1 bi 1:1 D3_5+ Trim_5+ R5WC +Au +Au +Au +Au +Pt +Au LY Figure S28: Analysis of cellular viability measured as protein content of CHO cells after incubation with free ligands and NP conjugates (composition of bifunctional mixtures: 1:1 = 3 µm D3_5+ and 3 µm Trim_5+, 1:1 = 3 µm D3_5+ and 3 µm Trim_5+; the single ligands were applied with a concentration of 3 µm). Note that the compounds of samples 2, 3 and 11 impaired cell viability to some extent (brown bars).

267 APP expression [% of mock] Caspase-3/tubulin [% of mock] Mock Au Au/D3_5+ Au/D3_8+ Au/D3_1+ D3_5+ D3_8+ D3_1+ LY Mock Au Au/D3_5+ Au/D3_8+ Au/D3_1+ D3_5+ D3_8+ D3_1+ LY Au Au/D3_5+ Au/D3_8+ Au/D3_1+ D3_5+ D3_8+ D3_1+ LY Mock1 Mock2 8. Appendix 262 (a) (b) (c) Figure S29: Analysis of cellular viability upon incubation of CHO cells with bare AuNP, free ligands and Au/D3 conjugates. Quantification of the overall protein content (a), level of APP expression (b), and caspase-3 activity.

268 8. Appendix 263 Figure S3: Cell assay performed with different doses of bare AuNP, Au/D3_1+ nanoconjugates and free D3_1+, quantification of secreted Aβ species (top), APP expression (middle) and protein concentrations in cell lysates (bottom).

269 8. Appendix 264 Figure S31: Cell assay performed with the free D3-aminopyrazole-hybrid compound and the corresponding nanoconjugate (c(aunp)=5 µg/ml, c(hybrid) = 5 µm, 29 ligands/np), anionic Au/D3_5+ conjugates (c(aunp) = 5 µg/ml, c(d3_5+) =.1µM, 4 D3_5+ per NP), Au/Trim_8+ conjugates (c(aunp) = 5 µg/ml, c(trim_8+) = 3µM, 125 Trim_8+ per NP) and equimolar doses of the free ligands. Shown are the quantification of secreted Aβ species (top), APP expression (middle) and protein concentrations in cell lysates (bottom). Design of Trifunctional Nanoconjugates and Analysis of their Functionality Trifunctional conjugates were fabricated by mixing equal amounts of D3 and Trimer (5 µm) with AuNP (5 µg/ml), corresponding to applied doses of 29 of each ligand per NP. Afterwards, 21 µg IDE were added, corresponding to a NP:IDE ratio of 1:1. NP-free reference samples were prepared with equimolar ligand concentrations. The samples were incubated with Aβ-secreting CHO cells to assess their interaction with cellularly produced Aβ species. The results show that trifunctional conjugates effectively lower the amount of secreted Aβ species, whereas the mixture of free ligands does not have an effect. In comparison to nanoparticles which were monofunctionalized with one IDE per NP, the trifunctional conjugates lead to the formation of additional Aβ species which may correlate to Aβ monomers (bottom band in the gel). Neither incubation with the ligand mixture, nor pure

270 8. Appendix 265 IDE nor Au/IDE conjugates results in the formation of these Aβ species. This new functionality can be seen as indirect evidence for the binding of all three ligand types on the NP surface and should be further investigated. Figure S32: Quantification of Aβ species secreted from CHO cells via immunoprecipitation and gel electrophoresis followed by subsequent densitometric analysis. CHO cells were incubated with mixtures of free D3, aminopyrazole trimer and IDE, with IDE alone and with the corresponding Au-nanoconjugates. Figure S33: Schematic drawing of the hypothesized interaction of nanoconjugates comprised of AuNPs (yellow), D3 peptides (green), aminopyrazole trimers (red) and IDE (grey) with Aβ aggregates (black, top). IDE is supposed to be only loosely associated to the nanoconjugate (physisorption, indicated by ~ symbols) due to its large size and the presaturation of AuNP with D3 and aminopyrazole trimers. The role of D3 is supposed to include the binding of Aβ and its transformation into amorphous aggregates, whereas aminopyrazole trimers may break the β-sheet structures (top right). IDE hydrolyses Aβ and regenerates the binding sites of the conjugate (bottom).

271 8. Appendix 266 Table S6: Summary of Aβ-cell tests employing free ligands and nanoconjugates with ligands in equimolar dose. Data are sorted by the enhancement factor, which expresses how NP-binding increases the potency of the tested ligands. Note that all cationic conjugates contain a mixture of bound and unbound ligands which may underestimate the enhancement factor. Ligand type Reduction of cellularly excreted Aβ species (1-15 kda) [%] unbound nanoconjugates ligands Enhancement factor Sample description D3_ ligands per AuNP (anionic) D3_ ligands per AuNP (cationic) IDE IDE per AuNP (anionic) Hybrid_ ligands per AuNP (cationic) 1:1 Bifunctional 125 D3 and 12 Trim per (D3_5+, Trim_5+) AuNP (cationic) Trim_ ligands per AuNP(cationic) Trifunctional (D3_5+, each 29 ligands + 1 IDE per Trim_5+, IDE) AuNP (cationic) 1:1 Bifunctional 12 D3 and 125 Trim per (D3_5+, Trim_5+) AuNP (cationic) Trim_ ligands per AuNP (cationic) D3_5+ 85±12 41± ligands per AuNP (cationic) D3_8+ 77±6 36± ligands per AuNP (cationic) D3_1+ 4±1 21± ligands per AuNP (cationic) 1:1 Bifunctional each 29 ligands per AuNP 54±18 29±8 1.9 (D3_5+, Trim_5+) (cationic) 1:1 Bifunctional (D3_5+, Trim_5+) + BSA D3 and 125 Trim per AuNP + BSA coating (anionic) Trim_ ligands per PtNP (cationic) Trim_5+ 36±18 28± ligands per AuNP (cationic)

272 Absorbance [a.u.] Counts norm. mass frequency Zeta potential [mv] Colloidal stability (PPI) 8. Appendix 267 Characterization of AuNP/R5WC Conjugates Ligand particle density [#/NPd=7nm] Ligand concentration [µm] Figure S34: Colloidal properties of AuNPs as function of the applied R5WC dose (left) and Lewis structure of the R5WC ligand (right). Characterization of ligand-free PtNP and Pt/Ligand Conjugates 1 8 d Peak =7nm AuNP PtNP 5 nm hydrodynamic diameter [nm] λ Peak =517nm AuNP PtNP 3x1 8 2x1 8 AuNP PtNP Pt = -51 mv Au = -42 mv.4.2 1x Wavelength [nm] Zeta potential [mv] Figure S35: Characterization of laser-generated PtNP via ADC (top left), TEM (top right), UV/Vis extinction spectroscopy (bottom left) and zeta potential measurements (bottom right). Results are compared to the colloidal properties of AuNPs.