Supporting Information

Transcription

1 Supporting Information Wiley-VCH Weinheim, Germany

2 Supramolecular Aggregates of Dendritic Multishell Architectures as Universal Nanocarriers Michal R. Radowski, Anuj Shukla, Hans v. Berlepsch, Christoph Böttcher, Guillaume Pickaert, Heinz Rehage, and Rainer Haag* [*] M.R. Radowski, Dr. G. Pickaert, Prof. Dr. R. Haag Institut für rganische Chemie und Biochemie Freie Universität Berlin, Takustr. 3, Berlin (Germany) Fax: (+49) Dr. A. Shukla, Prof. Dr. H. Rehage Lehrstuhl für Physikalische Chemie II Universität Dortmund, tto-hahn-str. 6, Dortmund (Germany) Dr. H. v. Berlepsch, Dr. Ch. Böttcher Forschungszentrum für Elektronenmikroskopie Freie Universität Berlin, Fabeckstr. 36a, Berlin (Germany) 1

3 Materials and methods Experimental equipment Dynamic Light Scattering (DSL): The static and dynamic light scattering experiments were performed with a commercial instrument (Zetasizer Nano, Malvern). We used a 4 mw He-Ne laser (633 nm wavelength) with a fixed detector angle of 173. In DLS experiments, we measured the intensity time correlation function g 2 (τ). The hydrodynamic radius was extracted from these measurements by second-order cumulants fitting of g 2 (τ). Critical Aggregation Concentration (CAC): The surface tension was measured using the commercially available pendant drop tensiometer PAT1 SINTECH. This instrument was constructed by Surface & Interface Technologies, Germany. The surface tension γ was calculated by fitting the Gauss-Laplace equation to the coordinates of a drop, using γ as the fitting parameter. CryoTEM: The samples for CryoTEM were prepared at room temperature by placing a droplet (~5 µl) of the solution on a hydrophilized perforated carbon filmed grid (60s Plasma treatment at 8 W using a BALTEC MED 020 device). The excess fluid was blotted off to create an ultra-thin layer (typical thickness of 100 nm) of the solution spanning the holes of the carbon film. The grids were immediately vitrified in liquid ethane at its freezing point (-184 C) using a standard plunging device. Ultra-fast cooling is necessary for an artifact-free thermal fixation (vitrification) of the aqueous solution avoiding crystallization of the solvent or rearrangement of the assemblies. The vitrified samples were transferred under liquid nitrogen into a Philips CM12 transmission electron microscope using the Gatan cryoholder and -stage (Model 626). Microscopy was carried out at -175 C sample temperature using the microscopes low dose protocol at a primary magnification of The defocus was chosen in all cases to be 2.5 µm. 2

4 Negative staining: Aliquots of the aqueous solution (~5 µl) were placed on hydrophilized carbon-coated copper grids and the supernatant fluid was blotted off after incubation of 30s. A droplet of uranyl acetate (1 % w/v) was added for 60 s, subsequently removed, and the sample was allowed to air-dry. Experimental Procedures Typical Synthetic Procedure for the Synthesis of Dendritic Multishell Architectures The first step of synthesis generates an amphiphilic linear 1,6 -; 1,12 -; or 1,18 - alkanoic acid mono-methoxy-[poly(ethylene glycol)] ester with different mono-methoxypoly(ethylene glycol) (mpeg) chain lengths: ~6 glycol units ( ), ~10 glycol units (mpeg 10 ), and ~14 glycol units (mpeg 14 ). To mixture of 1.0 eq of mpeg with 4.0 eq of α,ωdiacid was stired in round-bottom flask under reflux conditions with Dean-Stark trap in toluene for 18 to 24 h with addition of p-toluenesulphonic acid (PTSA) as catalyst (2.5 mol%) (Scheme S1). Purification of the reaction mixture was performed by extracting the product twice with a small amount of cold toluene (50 ml / 10 mmol of mpeg) from the solid residue of α,ω-diacid and removal of solvent by rotary evaporation to obtain crude product. Filtration over silica gel was performed (gradient CHCl 3 CHCl 3 : MeH 10:1) in order to obtain pure monompegdiacid with % yield. For the coupling of amphiphilic building block to the hyperbranched polyethyleneimine core the free carboxyl groups of the amphiphilic molecule were activated as N-hydroxy-succinimide esters.the active ester was created by using dicyclohexyl-carbodiimide (DCC) as the coupling agent. 1.0 eq. of monompegylated α,ωdiacid with 1.05 eq. of N-Hydroxysuccinamide (HNSu) and 1.05 eq. of DCC were dissolved in dry THF (50 ml / 10 mmol of mpeg α,ω-diacid) and stirred for 12 h at 0 C. Purification of the active ester was performed by removal of the insoluble 1,3-dicyclohexyl-urea from THF solution by filtration. The filtrate was concentrated by rotary evaporation to obtained crude 3

5 product with purity of 93-97% and a 95-98% yield. No additional purification was necessary. The 1.05 eq of active ester (for each NH 2 group of hyperbranched core) was next mixed with the respective type of PEI polymer (PEI 3600 or PEI ) and stirred in methanol for 24 h at RT. The purification of the dendritic multishell architectures was performed by dialysis of the product with a benzoylated cellulose membrane (MWC < 1000 Da) for h in methanol and/or water and resulted in analytically pure polymers in % yield. All products (Figure S1) were obtained in % overall yield (3 steps) and were unambiguously characterized by 1 H, 13 C NMR, and IR spectroscopy. NH H 3 C Shell 2 Linker 2 Shell 1 Linker 1 Core symbol core shell 1 shell 2 functionalization (x) PEI 3600 (C 18 ) x PEI 3600 (C 18 mpeg 10 ) x PEI 3600 (C 18 mpeg 14 ) x PEI (C 18 ) x PEI (C 18 mpeg 10 ) x PEI (C 18 mpeg 14 ) x PEI 3600 (C 12 ) x PEI (C 12 ) x PEI 3600 (C 6 ) x PEI (C 6 ) x PEI 3600 ( ) x PEI ( ) x PEI 3600 PEI 3600 PEI 3600 PEI PEI PEI PEI 3600 PEI PEI 3600 PEI PEI 3600 PEI C 18 C 18 C 18 C 18 C 18 C 18 C 12 C 12 C 6 C 6 mpeg 10 mpeg 14 mpeg 10 mpeg 14 70%, 100% 70% 70%, 100% 85%, 90%, 100% 100% 70%, 90%, 100% 90% 90% 90% 90% 100% 100% Figure S1. List of synthesized dendritic multishell architectures. Typical Synthetic Procedure for the Synthesis of a Dendritic Single-shell Architecture To 1.0 eq of -H, 2.0 eq of BrCH 2 CK and 7,0 eq of KH were dissolved in DMF. The mixture was heated to 45 C for 24 h, after which water was added. Heating was continued for additional 16 h to consume excess of bromoacetate. Then the solvent was 4

6 removed by rotary evaporation at reduced pressure and the residue was dissolved in water and washed with 3 times with Dichloromethane. The water phase was acidified to ph 2 with 10 % HCl and extracted 3 times with CH 2 Cl 2. The organic phases were combined and dried over Na 2 S 4, filtered, and the solvent was removed by rotary evaporation at reduced pressure to obtained the crude product. The product was purified by column chromatography (silica, gradient CH 2 Cl 2 : MeH 95 : 5 90 : 10) to obtain pure -carboxylic acid in 54% yield. Next an aqueous solution of mpeg-carboxylic acid (1.0 eq) and EDCI (2.0 eq) were stirred for 1.5 h and transferred to an aqueous solution of PEI (PEI 3600 or PEI )(1.0 eq of NH 2 groups). The mixture was stirred for 24 h in the dark. After solvent removal at reduced pressure, the crude product was dialyzed with benzoylated cellulose membrane (MWC < 1000 Da) in water for 28 h to obtained yellow oil as a pure product (yield %). Typical Encapsulation Procedure and Transport Capacity Determination For the encapsulation experiments the following standard procedure was used: The respective guest molecule in solid state was added to the polymer solution with defined molar/weight concentration. Water was used as a non-solvent for nonpolar molecules and chloroform as a non-solvent for polar compounds. The obtained suspensions were stirred vigorously for 18 h with a magnetic stirrer with 1000 rpm. Next the mixtures were centrifuged for 45 min (15000 rpm) and additionally filtrated via 0.45 µm polytetrafluorethylene (PTFE) filters to remove any trace of unsolubilized drugs or dyes. The clear polymer solutions with nanocompartimented drugs were then analyzed by means of UV-Vis spectroscopy by measuring the characteristics absorption of guests in solution of the nanocarrier. btained absorptions was compared with calibration curve of the guest molecules what allowed to determine the concentration of the guests molecules in solution. To prove a universal 5

7 transport ability of the multishell nanoparticles, the encapsulation capacity for different molecules was determined by UV/VIS spectroscopy (Figure S2). 40 Transport of polar molecules 20 Transport of nonpolar molecules Transport [mg/g of polymer] Transport [mg/g of polymer] Vitamin B2 1 to 6,8 ( n guest/n host) Vitamin B6 1,7 to 1 ( ) Congo red 0 Nimodipin 1 to 3,5 ( ) beta-carotene 1 to 4,2 ( ) Vitamin A 1 to 1,5 ( ) Vitamin D3 1 to 1,9 ( ) Rose bengal 1 to 1,7 ( ) 1 to 1,6 ( ) H N H H Vitamin A H 2 N Nimodipin H Vitamin D 3 β Carotene Vitamin B 6 H N Cl H H H H H H H H H N N N NH Vitamin B 2 K I I Cl Cl Cl S 3 I NH 2 I K Rose bengal Na N N Congo red H N N NH 2 S 3 H Na H Figure S2. Transport of polar and non-polar molecules in their non-solvents with the polymer PEI 3600 (C 18 )

8 Supporting Results on the Aggegate Sizes The aggregate size for PEI cores was approximately two times larger (diameter ~ 80 nm) than for PEI 3600 cores in water as determined by DSL. In chloroform the corresponding relationship was 1.5 times (~ 40 nm). A similar trend in size change with drug incorporation was observed in both cases. Incorporation of Nimodipine decreased the aggregate size up to ~ 20 nm, while incorporation of Congo red and β-carotene increased aggregate size above nm. From DLS no significant change was seen in the initial decay of correlation functions for C 12 and C 18 polymers. Departure from the single-exponential decay at higher time scales in the case of polymers with C 12 in comparison to C 18 suggested that polymers with C 12 were more polydisperse or formed larger aggregates. It is interesting to note that tests below the CAC did not show any transport phenomena of polar molecules (see main article) and nonpolar guest molecules. With increasing polymer concentration (above the CAC), we only observed a very low transport capacity of β-carotene inside the nanocarriers. There measurements were performed at a fixed polymer concentration (6.0 x 10-6 M) and we observed that 75 host molecules were present for only one guest molecule (transport ratio 1 to 75 (n guest to n host )). Further increase of the nanotransporters concentration to 1.2 x 10-5 M resulted in a dramatic increase of the transport capacity to a ratio 1 to 10. This specific relation remained almost constant up to the highest tested concentration (ratio max 1 to 7) (Figure 3). Similar phenomena were also observed for the Nimodipine transport, however, low intensity of UV absorption in combination with the poor solubility of the drug in water did lead to an unfavorable signal-to-noise ratio. This problem caused sometimes unreliable numeric data. Transport was also not observed for polar molecules, i.e. Congo red, what was described in main article. 7

9 Changes in CAC as a Function of the Chemical Structure Surface tension measurements have shown that changes of the core sizes from PEI 3600 to PEI (both C 18 ) 0,9 ) cause a significant decrease of the CAC from 8.0 x 10-7 M to 1.7 x 10-7 M. By modification of the alkyl chain length from C 12 to C 18 a slightly decrease of the CAC was observed for both core sizes: for PEI 3600 (C 12 ) 0.9 and PEI 3600 (C 18 ) 0.9 from CAC = 1.2 x 10-6 M to CAC = 8.0 x 10-7 M respectively and for PEI (C 12 ) 0.9 and PEI (C 18 ) 0.9 from CAC = 5.6 x 10-7 M to CAC = 1.7 x 10-7 M. Changes in the functionalization level from 70% to 90% cause a decrease of CAC value from 6.5 x 10-6 to 8.0 x 10-7 M for PEI 3600 (C 18 ) X. With prolongation of the mpeg chains the CAC of polymers decreases from 8.0 x 10-7 M for PEI 3600 (C 18 ) 0,9 to the CAC = 1.8 x 10-7 M for PEI 3600 (C 18 mpeg 14 ) 0,9. Small differences between CAC of the pure nanotransporter solutions and the critical concentrations at which transport occurred was observed. By encapsulation of Nimodipine CAC changed from 6.5 x 10-6 M to 5.0 x 10-6 M and for encapsulated β-carotene CAC increased from 6.5 x 10-6 M to 1.5 x 10-5 M for PEI 3600 (C 18 ) 0,7. These shifts may have been caused by the presence of the encapsulated guest molecules, which can act as a co-surfactant and therefore, possibly shift CAC concentrations. 8

10 Figure S3. Molecular modeling of the unimolecular dendritic multishell architecture [PEI 3600 (C 18 ) 0.7 ] with HyperChem release 6.0. The unimer size is approximately 6 nm in diameter. Calculations were performed in periodic water box. 9

11 Congo Red with PEI 3600 ( C 18 PEG 6 ) 0,7 Congo Red without polymer Figure S4. Size exclusion chromatography on Sephadex LH-20. The Lengths of the SEC columns are 6 cm. Both columns were loaded with 0.2 ml of solutions: (right) 0.4 g L -1 Congo red in water, (left) identical solution with addition of PEI 3600 (C 18 ) 0,7 (concentration1.0 g L -1 ). Both columns were washed with 0.3 ml of pure water. 10

12 Figure S5. TEM micrograph (the contrast has not been inverted for better visibility) of pure PEI 3600 (C 18 ) 0.7, which was deposited and dried on a carbon film after staining with uranyl acetate (1 % w/v). The image shows monomolecular species with a typical size of 8-9 nm and one large aggregate. To permit the imaging of isolated particles the polymer solution (of 1.0 g/l) was diluted to 0.01 g/l prior to sample preparation. 11