Dendrimers. M. Reza Naimi-Jamal Jan. 2013

Transcription

1 Dendrimers M. Reza Naimi-Jamal Jan. 2013

2 Overview What is a dendrimer? How are they formed? What are their properties? What are the applications? How do you characterize them?

3 What is a Dendrimer Highly branched macromolecule Capable of encapsulating High loading capacity Backbone of Carbons or Nitrogens Monodisperse and controllable Highly stable Low immunogenicity and toxicity

4 (05/2007)

5 Tomalia DA, Mardel K, Henderson SA, Holan G, Esfand R. Dendrimers an enabling synthetic science to controlled organic nanostructures. In: Goddard III WA et al, editor. Handbook of nanoscience, engineering and technology. Boca Raton, FL: CRC Press; 2003.

6 Size scale of dendrimers of different generations vs. biological proteins Tomalia DA, Mardel K, Henderson SA, Holan G, Esfand R. Dendrimers an enabling synthetic science to controlled organic nanostructures. In: Goddard III WA et al, editor. Handbook of nanoscience, engineering and technology. Boca Raton, FL: CRC Press; 2003.

7 Dendrimer Components

8 Dendrimer Structure

9 Dendritic polymers Dendritic polymers are highly branched polymer structures, with complex secondary architectures and well-defined spatial location of functional groups. They can be used in applications such as: targeted drug-delivery as macromolecular carriers for catalysis as sensors for light harvesting for surface engineering as biomimetic materials

10 Dendritic polymers exhibit very different properties compared to their linear analogues. Vogtle laboratory in 1978 reported the first concept of branching by repetitive growth The first dendrimers were synthesized by Tomalia and co-workers at Dow Chemical in the early 1980s in parallel with Newkome s arborol systems. In 2008 there were over scientific reports and 1000 patents dealing with dendritic structures.

11 Dendritic materials sub-classes: Dendrimers Dendrones (not shown) hyperbranched polymers dendrigraft polymers dendronized polymers

12 Dendrimers When dendritic polymers are perfectly branched they are either dendrons or dendrimers. The dendrimers comprise a single core that is capped with layers of repeat units which are radially branched. Each layer is called a generation. Much like proteins and natural products, dendrimers are near monodisperse with predictable molecular weights and nano-scale dimensions. The unique properties of dendrimers are attributed to their globular structure, resulting from internal structures in which all bonds emerge radially from a central core, and a large number of end-groups are present at its surface. Due to their globular structures, dendrimers can be seen as nano-particles with the ability of core encapsulation. In contrast to linear polymers, these macromolecules do not entangle, have unusual viscosity behaviors, such as low solution viscosity and functional groups can be either protected or exposed.

13 Dendrons Dendrons represent a structural component of the parent dendrimer, and are monodisperse, wedged-shaped sections of a dendrimer. Hence, a dendrimer is made up of a number of dendrons. Each functional group in a dendritic core gives rise to one dendron. Furthermore, dendrons of higher generations (ZG4) can be seen as dendrimers but with an active core moiety that can be further functionalized.

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15 Hyperbranched polymers Hyperbranched polymers are highly branched polymer structures, much like the dendrimers, but contain imperfections in the branching points due to their traditional onepot synthesis. Hyperbranched polymers contain dendritic sites as well as linear sites with the structure being poorly controlled in comparison to dendrimers Their typical polydispersities are about 2 or higher. Hyperbranched polymers possess many of the properties of dendrimers, in that they are highly branched, have a multitude of end-groups, a globular structure and they show similar viscosity behaviours attributable to significantly reduced entanglements. Hyperbranched polymers have much higher solubility than their linear analogues due to their many end-groups. Examples of hyperbranched polymers: Boltorns (P(bis-MPA) hyperbranched polymer), Hybranet (poly(ester amide)) poly(phenylenes),

16 Dendrigraft polymers Dendrigraft polymers, sometimes referred to as arborescent polymers, are a relatively new addition to the dendritic family, combining features of dendrimers and hyperbranched polymers with linear polymers. Dendrigraft polymers are grown in generations, much like dendrimers, but the repeating unit is an oligomer or a polymer chain, rather than a small monomer unit. As dendrigraft polymers, flexible polymers with very high molecular weights are obtained rapidly. Examples of dendrigraft polymers are: Comb-bursts arborescent polybutadiene,

17 Dendronized polymers Dendronized polymers, sometimes termed rod-shaped polymers, are structures having a linear backbone with dendritic sidechains. Dendronized polymers are a sub-class of comb-polymers where the comb s teeth are dendrons instead of linear polymer chains. Depending on the density and size of the attached dendrons, the dendronized polymers can have either random coil or fully stretched out conformation. Fully stretched out dendronized polymers are rod-like cylindrical polymers ( nanotubes ) and are believed to have new and interesting properties, since they have dimensions reminiscent of several biological functional units, such as the mosaic virus (lengths up to 400 nm and diameters up to 6 nm). Dendronized polymers have exceptionally high aspect ratio compared to the globular dendrimers, i.e. they are not only molecular objects; they are also form-anisotropic nanoscopic objects. Examples: PAMAM-based dendronized polymers, P(bis-MPA)-based dendronized polymers, Fre chet-type dendronized polymers, Percec-type dendronized polymers,...

18 Dendrimer Synthesis Two processes used to create dendrimers Divergent synthesis starts at the core and branches out Difficult to remove pure samples from side products Convergent synthesis starts from the end groups, creating dendrons, and ends at the core Advantage of needing less efficiency to run Lower yield and more starting material needed Difficult to create dendrimers in bulk

19 Dendrimer synthesis

20 Synthesis of dendronized polymers

21 Divergent Synthesis (example):

22 Convergent Synthesis Lee J. W., Kim J.H., Kim Byung-Ku. Synthesis of azide-functionalized PAMAM dendrons at the focal point and their application for synthesis of PAMAM-like dendrimers, Tetrahedron Letters, :

23 Structural scheme of growing denrimer generations N. Launay et al, Angew. Chem., Int. Ed. Engl. 1994, 33,

24 Composition and mass of molecules of 11 generations of phosphorus dendrimers, series G i (G i ) 2,0 N t /N r 1,8 1,6 1,4 1,2 1, номер поколения Core Number of repeated unit Number of terminal groups S S S O Generation Molecular mass - O- -C=N-N- P H P P -O - -C H CH 3 (-Сl) G 0 (G 0) 426 (168) G 1 (G1) 1423 (903) G 2 (G2) 417 (2389) G 3 (G3) ) G G G G G G G G Note: in parents presented data for the series with terminal chlorine atoms. 5

25 IR spectra of series G i phosphorus dendrimers S Core P Repeated unit S - O- -C =N-N- P H CH 3 Terminal group -O - O -C H νc=0 νph νc=n δn-ch 3 νph δch δch νpo-ph νc-ph νp-oph νp-nn γch νp=s(n) νp=s(o) G'11 Поглощение G'3 G'2 G'1 δch δc=o G' ν, cм -1 6

26 IR spectra of series Gi phosphorus dendrimers S Core P Repeated unit S - O- -C =N-N- P H CH 3 Terminal group -Cl G3 νph νc=n δn-ch 3 νph δch δch νpo-ph νc-ph νp-oph νp-nn γch νp=s(n) νp=s(o) νp-cl Поглощение G2 G ν, cм -1 7

27 Raman spectra of series G I phosphorus dendrimers G'10 G'8 Поглощение G'6 G'4-1 G'2 G' ν, cм

28 Click chemistry In 2001, Sharpless and co-workers coined the term Click chemistry as a common name for a number of extremely versatile coupling reactions that posses high thermodynamic driving force (420 kcal/mol). The requirements of a Click reaction are: (1) robust and quantitative, i.e., the reactions are highly selective and the yields are near 100%: (2) the reactions are able to proceed in the presence of a variety of solvents such as water, organic solvents, etc., irrespective of their protic/aprotic or polar/non-polar character (3) high tolerance to other functional groups (4) the reaction proceeds at various types of interfaces such as solid/liquid, liquid/liquid or even solid/solid. Another advantage of these reactions is the requirement of only stoichiometric amounts of starting materials with virtually no by-products being formed, which greatly simplifies the purification procedures. Click reactions are often performed at room temperature and are purified by filtration or simple extraction. The use of heat or microwave irradiation can further accelerate the reaction towards completion.

29 The most widely studied example of Sharpless-Type Click-reactions are a variant of the Huisgen 1,3-dipolar cycloaddition reaction between C C triple bonds, C N triple bonds, and alkyl-/aryl-/sulfonyl azides.

30 Sharpless and Hawker jointly recognized that the Cu(I)- catalyzed azide alkyne 1,3-dipolar cycloaddition (CuAAC), which results in an aromatic triazole heterocyclic. This is a superior synthetic reaction for preparing complex polymeric structures.

31 Several dendritic polymers have been constructed in recent years as a proof that synthetically challenging macromolecules can be obtained by utilizing the copper(i)-catalyzed azide alkyne cycloaddition (CuAAC) reaction. In reported approaches, the A- and B-functionalities of the ABxmonomer units are azides and acetylenes, respectively, and the resulting triazole ring appears as part of the repeating unit in the final dendritic structure. The significant interest in Click chemistry has allowed new complex dendritic concepts such as unsymmetrical dendrimers, dendritic linear hybrids, layered dendritic structures and dendrimer functionalization to be easily obtained. Recently, Hawker et al. described the utilization of another Clickreaction, thiol ene Click chemistry, for the divergent synthesis of poly(thioether) dendrimers. The synthesis was carried out under mild reaction conditions and without the use of a metal catalyst, making the process even more environmentally friendly.

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34 The synthetic procedure for obtaining dendrimers can be accelerated by utilizing two different kinds of monomers, instead of only a single ABxtype monomer. In this approach monomers of AB2-functionality are coupled to monomers with CD2- functionality. Here, A can selectively react with D and B can only react with C. Due to this, no protection/ deprotection steps are required in the procedure and a dendrimer having a layered structure composed of two alternating repeat units is obtained. This approach was initially reported by Caminade and others.

35 Hybrid structures Polymers containing both linear chains and dendritic parts are often referred to as dendritic linear polymer hybrids or just hybrid materials. (not to be confused with dendronized polymers while they do contain a linear backbone with dendritic side groups, dendronized polymers are usually highly ordered and when the dendron is present on every repeating unit the linearity the polymer backbone is difficult to identify)

36 One approach to obtain hybrid structures is by turning the core or the multiple end-groups of the dendritic polymer into initiating moieties for polymerization. The polymerization rate from the core of sterically hindered high generation dendrons is often decreased as the initiating sites become less available. As a result, long reaction times and elevated temperatures are necessary to obtain the desired hybrids, and it is uncertain if linear chains will grow from each of the chain-ends of the dendritic structure. Another method utilizes a grafting-to strategy, in which pre-formed polymer linear chains are coupled to a dendritic core. Previously, this approach has suffered from low grafting density when traditional synthetic coupling procedures were used. However, if a highly versatile reaction, such as the CuAAc reaction, is utilized in combination with convergent growth, the risk of partial coupling of the active sites in the dendritic core is diminished.

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38 Hybrid structures Structures in which linear chains are attached to dendritic scaffolds have been studied extensively in recent years. These structures are of great interest, combining the uniqueness of both the dendritic and the linear polymer. Frechet et al. in 1998 devised hybrid PEG-unsymmetrical dendrimers with covalently attached cancer drugs and the resulting branched structure affords greater solubility and bioavailability which allows maximized performance in physiological environments. Shabat et al reported on the convergent attachment of PEG-chains to a dendritic core via Click chemistry using only stoichiometric amounts of the linear polymer. Riguera et al. reported on the use of Click chemistry to prepare PEG dendritic block copolymers up to generation three. All reactions were performed at room temperature.

39 Perfecting dendrimers Surface-Engineered Dendrimers Drug molecules can be either chemically conjugated to the dendrimer surface or physically encapsulated inside a dendrimer core. For chemical conjugations, if functional groups are activated prior to coupling, a good coupling efficiency may be achieved. Common functional groups in drug molecules and polymers are: hydroxyl (OH) (can be converted to active intermediates) carboxyl (COOH), primary amine (NH2), thiol (SH) and guanidino. In some cases, direct coupling may lead to the deactivation of drugs, so that the conjugated drugs (i.e., prodrugs) will not have any pharmaceutical effect until they are cleaved from the bonding. A bi-functional spacer can be introduced to the drug delivery system to form bonds that are hydrolysable or enzyme-cleavable in vivo Due to presence of multiple surface sites on the dendrimer surface, moieties of various functionalities can be simultaneously attached to the surface through covalent or non-covalent bonding to make dendrimers multifunctional.

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41 Functionalized Dendrimers Higher generation dendrimers have a greater number of end groups that can be functionalized Longer retention time in blood Targeting ability to attach to certain tissues or active sites Imaging Fluorescence, MRI, or X-ray Drug Carrier Therapeutic used to detect disease while in body

42 Functionalized Dendrimer

43 Functionalized Dendrimers Hydrophobic or hydrophilic Hydrophilic carboxyl, PEO, or sulfonate end group Hydrophobic Hydrocarbons, flurocarbon, silicon end groups Hydrophilic outer surface to the dendrimer, allowing it to penetrate aqueous regions, but inner branches could be made to be hydrophobic to allow the hydrophobic Anionic or Cationic Cationic dendrimers - higher cytotoxicity net negative charge provides better membrane transport net positive charge allows it to attach to positive receptor sites

44 Dendrimer as a drug Antitumor, antiviral, and antibacterial agents PAMAM and Poly(lysine) dendrimers, covalently modified with naphthyl sulfonate, which reside on the surface, have shown antiviral activity verses HIV and Herpes virus, respectively. As antitumor drugs, dendrimers have been used primarily in photodynamic therapy. When used as antibacterial drugs, the dendrimers adhere to the bacterial membrane and damage the anionic membrane causing the cells to burst

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46 Dendrimers Cored dendrimers, like buckyballs, able to carry larger amounts of carrier compounds, while able to be functionalized Dendrimers as homogenous catalysts High activity, high selectivity, good reproducibility, accessibility of the metal Readily recovered after reactions Candidate for ideal catalyst due to persistent and controllable nanoscale dimensions, chemically reactive surface, favorable configurations for migrating reactants, soluble, and can be easily recovered by filtration.

47 Particle formation Process: nucleation, growth, coagulation, and flocculation Dendrimers can alter any step of the particle formation process Lower generation dendrimers- morphology and size control through adsorption of solubles Higher generation dendrimers- inhibitor to crystal formation

48 Example: Rapid, single-step preparation of dendrimer-protected silver nanoparticles through a microwave-based thermal process Third generation poly(propyleneimine) dendrimer in AgNO 3 aqueous solution No reducing or protecting agents Characterized UV spectra and TEM Nobel metals provide novel properties in electronic, optical, catalytic and thermal

49 Silver nanoparticles Dendrimer size 2.4nm Molar ratios of 1:20, 3:20, and 9:20 dendrimer to silver Microwave: 100% power of 600W for 8 min Dendrimer smaller than particles

50 Ag particle size vs Dendrimer molar ratio 1: nm, 25 average Spherical 3:20 Over 300nm x 75nm Spherical and rod shaped 9: nm, 10 average Spherical

51 Particle size vs. Dendrimer molar ratio

52 Example: Hydrothermal synthesis of hydroxyapatite nanorods in the presence of starburst dendrimers Dendrimer, PAMAM (G 5.5), in H2O and NaOH then added to Ca ions (NH 4 ) 2 HPO 4 added, precipitate collected and cleaned and hydrothermal treatment XRD and Scherrer formula used to calculated crystal size TEM to characterize morphology and size

53 Hydrothermal treatment vs. time Samples treated for 3, 6, 10, and 15 hours 3 and 6 hrs Contained rods, aggregates, and fibers 10 and 15 hrs Uniform nanorods

54 Nanorods Room temperature, untreated Grain size 40nm length 200nm x <15nm Clongated fibers Nanocrystal of very small size Treated Nanorods 80nm with aspect ratio ~8 Crystal nanorods with well crystallization

55 Untreated vs. Treated

56 Dendrimer assistance to particle formation The role of the dendrimer is not completely known There are mechanisms in particle formation Much more research needs to be done to understand the process

57 Surface-Engineered Dendrimers Modification of functional groups on the periphery of a dendritic structure offers a convenient route to new dendrimers with a structure of the external generation different from that of the first generations. Important classes of surface-engineered dendrimers are: 1) Dendritic boxes 2) PEGylated Dendrimers 3) Glycodendrimers 4) Peptide-Functionalized Dendrimers 5) Galabiose-Functionalized Dendrimers

58 Surface-Engineered Dendrimers PEGylated Dendrimers

59 Surface-Engineered Dendrimers Glycodendrimers

60 Lagnoux, D.; Darbre, T.; Schmitz, M.L.; Reymond, J.-L. Inhibition of mitosis by glycopeptide dendrimers conjugates of colchicine. Chem. Eur. J. 2005, 11,

61 Toxicological Aspects of Surface- Engineered Dendrimers Prior to use it is necessary to estimate the biocompatibility, cytotoxicity and biodistribution of dendrimers. Many tests can be used focusing on cell viability, morphology, haemolysis, protein content and other cellular parameters in cells exposed to dendrimers. In animals pharmacokinetics, biodistribution and fate of dendrimers are key issues that, together with the nature of the disease to be treated (acute or chronic, life-threatening), ultimately define whether the riskbenefit (in terms of potential safety/efficacy) of any novel technology can be justified.

62 Toxicological Aspects of Surface- Engineered Dendrimers Cytotoxicity is a measure of non-specific, unwanted harm it may elicit toward cells, organs, or the patient as a multi-organ system. In vivo aspect of polymer toxicity is profoundly influenced by its pharmacokinetics and biodistribution hence biodistribution studies form an important part of the early polymer screening. Features such as polymer molecular weight, charge and hydrophobicity and more subtle aspects of physico-chemical solution behavior can have a profound effect on biodistribution particularly if a polymer is carrying a surface disposed drug payload. Biocompatibility can be described as the capability of a material to execute an appropriate host response during a specific application. It highlights the need to consider the suitability of a material both in respect of its potential harmful effect in the body (toxicity), and in respect of the potential detrimental or beneficial effect of the physiological environment on the performance of the material.

63 Toxicological Aspects of Surface- Engineered Dendrimers If a dendrimer or dendron is used as a component of a biomedical material, e.g., a tissue-engineering application, the biocompatibility of the material must be defined. As a general rule, for any polymeric carrier to be suitable for parenteral application it is essential that the carrier is non-toxic and non-immunogenic and it should preferably be biodegradable. It must display an inherent body distribution that will allow appropriate tissue targeting to the desired site but away from sites of toxicity. Simple in vitro assays such as cytotoxicity against a panel of cell lines (72 h; MTT assay) and red blood cell haemolysis assays (1 h, 5 h and 24 h) were established to judge whether preliminary in vivo evaluation is ethically acceptable. Usually dextran (approx. 70 kda) is used as a negative control and polyethyleneimine (PEI) or poly(l-lysine) as positive reference controls for these studies.

64 Toxicological Aspects of Surface- Engineered Dendrimers Examination of antigenticity (IgG, IgM production) and cellular immunogenicity (cytokine and chemokine induction) are essential for any novel polymer/dendrimer destined for parenteral use. In addition, a non-immunogenic carrier is likely to produce a hapten effect when it presents surface moieties such as the drug, peptide, or saccharide. Inherent immunogenicity can preclude future use for many applications. A cytotoxicity study of a new dendrimer needs physiologically meaningful incubation time (exposure could be days, weeks or months if the dendrimer is to be administered parenterally) and high enough concentrations to define the inhibitory concentration diminishing viability by 50% (IC50 value). The cationic dendrimers (with amine end groups) are responsible for haemolysis and cytotoxicity. Cationic PAMAM dendrimers may be accepted for biological application as long as the surface is attached to anionic or neutral groups.

65 Applications of Dendrimers in Tissue Engineering The highly branched, multivalent nature of dendrimers makes them ideal candidates for a variety of tissue engineering applications, including as crosslinking agents, modulators of surface charge and surface chemistry, and as primary components in scaffolds that mimic natural extracellular matrices. Compared to linear polymers, the multiple end groups of dendrimers may potentially offer more control over factors such as cell proliferation rates and biodegradation profiles through systematic variation of generation size, concentration, and end group chemistry. The combination of dendrimers and other traditional scaffold polymers, such as proteins, carbohydrates, and linear synthetic polymers has led to the creation of hybrid scaffolds with new physical, mechanical, and biochemical properties.

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73 Cancer therapy with dendrimers Functionalization of dendrimers with chemotherapeutic drugs, radiopharmaceuticals or radiosensitizers combined with functionalization with targeting moieties increases therapeutic activity of these therapies

74 PAMAM dendritic polymers <5 nm in diameter were used as carriers. Acetylated dendrimers were conjugated to folic acid as a targeting agent and then coupled to either methotrexate or tritium and either fluorescein or 6 carboxytetramethylrhodamine. These conjugates were injected i.v. into immunodeficient mice bearing human KB tumors that overexpress the folic acid receptor. In contrast to nontargeted polymer, folate-conjugated nanoparticles concentrated in the tumor and liver tissue over 4 days after administration. The tumor tissue localization of the folate-targeted polymer could be attenuated by prior i.v. injection of free folic acid. Confocal microscopy confirmed the internalization of the drug conjugates into the tumor cells. Targeting methotrexate increased its antitumor activity and markedly decreased its toxicity, allowing therapeutic responses not possible with a free drug.

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80 We describe the simple fabrication of poly({198au}) radioactive gold dendrimer composite nanodevices in distinct sizes (diameter between 10 nm and 29 nm) for targeted radiopharmaceutical dose delivery to tumors in vivo. Irradiation of aqueous solutions of 197Au containing poly(amidoamine) dendrimer tetrachloroaurate salts or {197Au0} gold dendrimer nanocomposites in a nuclear reactor resulted in the formation of positively charged and soluble poly{198au0} radioactive composite nanodevices (CNDs). A mouse melanoma tumor model was used to test whether the poly{198au0} CNDs can deliver a therapeutic dose. A single intratumoral injection of poly{198au0}d=22nm CNDs in phosphate-buffered saline delivering a dose of 74 μci resulted after 8 days in a statistically significant 45% reduction in tumor volume, when compared with untreated groups and those injected with the cold nanodevice. No clinical toxicity was observed during the experiments. This study provides the first proof of principle that radioactive CNDs can deliver therapeutic doses to tumors.

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82 Figure 5. Tumor growth of radioactive {Au} CND-treated mice versus PBS-treated control mice. Error bars represent standard deviation. In the first experiment, groups of mice received PBS, 35 μci of {198Au} (CND-4*), and 74 μci of {198Au} (CND- 4**). In the second experiment, mice received PBS and a CND-4** sample that has been radioactively decayed. Preliminary analysis of these data was shown in our recent book review chapter;43 here we present the full data set and analysis.

83 Summary Dendritic polymers in biology/medicine are used in applications such as targeted drug-delivery targeted radiosensitizer delivery as macromolecular carriers for surface engineering as biomimetic materials