Tuning the Formation and Degradation of Layer-by-Layer Assembled

Jul 6, 2009 - Ariga , K., Hill , J. P. and Ji , Q. M. Phys. Chem. Chem. ...... Yan Zhang , Boon M. Teo , Kenneth N. Goldie , and Brigitte Städler. La...
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Tuning the Formation and Degradation of Layer-by-Layer Assembled Polymer Hydrogel Microcapsules† Alisa L. Becker, Alexander N. Zelikin, Angus P. R. Johnston, and Frank Caruso* Centre for Nanoscience and Nanotechnology, Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia Received May 12, 2009. Revised Manuscript Received June 10, 2009 Engineered polymer capsules are finding widespread importance in the delivery of encapsulated toxic or fragile drugs. The effectiveness of polymer capsules as therapeutic delivery vehicles is often dependent on the degradation behavior of the capsules because it is often necessary to release the encapsulated drugs at specific times and in certain locations. Herein we investigate the parameters that govern the formation and degradation of a recently introduced new class of polymer hydrogel capsules based on disulfide cross-linked poly(methacrylic acid). We report a new and efficient method for the synthesis of thiol-functionalized poly(methacrylic acid) (PMASH), the main component of the capsules. Polymeric capsules were synthesized by the layer-by-layer deposition of PMASH and poly(vinylpyrrolidone) (PVPON) on silica particle templates, followed by cross-linking the PMASH layers and removing PVPON and the template particles. The disulfide cross-links provided a redox-active trigger for degradation that was initiated by a cellular concentration of glutathione. We demonstrate that increasing the degree of PMASH thiol modification affords direct control over the thickness of the polymer film and the degradation rate of the polymer capsules. Furthermore, the degradation rate of the PMASH capsules was independent of film thickness, suggesting a bulk erosion process.

Introduction The delivery of encapsulated drugs holds promise for improved therapeutics, from gene therapy to chemotherapy.1-3 Among the important requirements of a viable delivery system is a mechanism to release the cargo at specified times and locations.4 Cargo can be released from the delivery vehicle by diffusion, by an induced change in the properties (e.g., permeability) of the delivery vehicle, or by triggered degradation of the delivery vehicle into its constituent materials. Degradation of the delivery vehicle enables control over cargo release by altering the degradation rate, and using triggered degradation ensures that the cargo is released at a specific time or location for activity. In addition, degradation reduces the delivery vehicle components to smaller units for efficient removal from the body through renal filtration or biliary excretion.5 Polymer capsules prepared by layer-by-layer (LbL) assembly are finding interest in a range of biomedical applications, including therapeutic delivery.4,6-8 Such capsules are prepared through the sequential deposition of polymers through complementary interactions (e.g., electrostatic, hydrogen bonding, and covalent linkages) on template particles, followed by removal of the templates. This facile yet versatile technique can incorporate a large variety of polymers and templates.9 The physiological † Part of the “Langmuir 25th Year: Self-assembled polyelectrolyte multilayers: structure and function” special issue. *Corresponding author. E-mail: [email protected].

(1) Kiick, K. L. Science 2007, 317, 1182–1183. (2) LaVan, D. A.; Lynn, D. M.; Langer, R. Nat. Rev. Drug Discovery 2002, 1, 77–84. (3) Duncan, R. Nat. Rev. Drug Discovery 2003, 2, 347–360. (4) De Geest, B. G.; Sanders, N. N.; Sukhorukov, G. B.; Demeester, J.; De Smedt, S. C. Chem. Soc. Rev. 2007, 36, 636–649. (5) Owens, D. E.; Peppas, N. A. Int. J. Pharm. 2006, 307, 93–102. (6) Johnston, A. P. R.; Cortez, C.; Angelatos, A. S.; Caruso, F. Curr. Opin. Colloid Interface Sci. 2006, 11, 203–209. (7) Ariga, K.; Hill, J. P.; Ji, Q. M. Phys. Chem. Chem. Phys. 2007, 9, 2319–2340. (8) Peyratout, C. S.; Dahne, L. Angew. Chem., Int. Ed. 2004, 43, 3762–3783. (9) Quinn, J. F.; Johnston, A. P. R.; Such, G. K.; Zelikin, A. N.; Caruso, F. Chem. Soc. Rev. 2007, 36, 707–718.

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degradation of LbL films and capsules can be triggered by light,10 enzymes,11 and changes in pH,12 salt,13 or redox state.14 Notably, degradation activated by changes in the redox state is physiologically relevant because a redox potential occurs across cellular membranes.15 The cytoplasm is maintained in a reducing environment, whereas extracellular spaces such as the bloodstream are comparatively oxidizing. Incorporating a redox-active bond, such as a disulfide, into a delivery vehicle can achieve triggered degradation or release upon cell entry. Hydrogen-bonded LbL films such as those formed from poly (methacrylic acid) (PMA) and poly(vinylpyrrolidone) (PVPON) can deconstruct in response to a pH trigger.16 To make these films stable at physiological pH and hence potentially useful in drug delivery, cross-links can be introduced to stabilize the films. Previously, bifunctional amine linkers and amine-modified PVPON were used to cross-link and stabilize either PMA or PMA and PVPON in the film respectively.17,18 However, these films were permanently stabilized19 and are not degradable under physiological conditions. Recently, we developed a promising new class of capsules based on a reversibly stabilized, disulfide cross-linked PMA film.20 Thiol-functionalized PMA (PMASH) is deposited alternately with PVPON under conditions designed to promote hydrogen bonding, and the thiol groups are then (10) Radt, B.; Smith, T. A.; Caruso, F. Adv. Mater. 2004, 16, 2184–2189. (11) Serizawa, T.; Yamaguchi, M.; Akashi, M. Macromolecules 2002, 35, 8656– 8658. (12) Vazquez, E.; Dewitt, D. M.; Hammond, P. T.; Lynn, D. M. J. Am. Chem. Soc. 2002, 124, 13992–13993. (13) Schu¨ler, C.; Caruso, F. Biomacromolecules 2001, 2, 921–926. (14) Li, B. Y.; Haynie, D. T. Biomacromolecules 2004, 5, 1667–1670. (15) Schafer, F. Q.; Buettner, G. R. Free Radical Biol. Med. 2001, 30, 1191–1212. (16) Sukhishvili, S. A.; Granick, S. Macromolecules 2002, 35, 301–310. (17) Kozlovskaya, V.; Kharlampieva, E.; Mansfield, M. L.; Sukhishvili, S. A. Chem. Mater. 2006, 18, 328–336. (18) Kozlovskaya, V.; Shamaev, A.; Sukhishvili, S. A. Soft Matter 2008, 4, 1499–1507. (19) Kozlovskaya, V.; Ok, S.; Sousa, A.; Libera, M.; Sukhishvili, S. A. Macromolecules 2003, 36, 8590–8592. (20) Zelikin, A. N.; Quinn, J. F.; Caruso, F. Biomacromolecules 2006, 7, 27–30.

Published on Web 07/06/2009

DOI: 10.1021/la901687a

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oxidized to form cross-linking disulfide bonds between PMA. In addition to providing a mechanism for deconstruction under physiological conditions, the disulfide bonds are essential for film stability because the hydrogen-bonded film is unstable above the pKa of PMA (pH 6.5) (Scheme 1). We have shown that these PMASH hydrogel microcapsules can successfully encapsulate proteins,20 DNA,21,22 oligopeptides,23 and anticancer drugs such as doxorubicin (DOX).24 They have been used for the in vitro delivery of conjugated oligopeptides to dendritic cells for vaccination studies23 and for the delivery of DOX to colon cancer cells for cancer therapy.24 Our initial studies have shown that a PMASH film degrades in a cellular concentration of glutathione (GSH).25 However, the controlled degradation of these PMASH hydrogel capsules remains unexplored. Other groups have also incorporated disulfide cross-links and polymers containing disulfides along the backbone into LbL films.14,26-29 However, these studies have not attempted to control the degradation rate beyond the variation of the concentration of the reducing agent. In addition, the reducing environment has been created with dithiothreitol (DTT), although the most important nonprotein thiol source involved in maintaining the redox potential in cells is GSH.30 In the current work, our objective was to control the rate of degradation of PMASH hydrogel microcapsules under conditions designed to mimic the cytoplasm. We hypothesized that the rate of degradation could be controlled by engineering the film thickness and the cross-linking density via the number of layers deposited and the degree of PMA thiol functionalization, respectively. In this study, we report the controlled degradation of microcapsules by engineering the LbL polymer multilayer film at the molecular level. We also investigated the parameters that govern the formation of the thin films on planar and colloidal substrates. The density of cross-linking in the film was modified by varying the degree of thiol modification on the PMASH and was employed to control the rate of degradation. The effect of the thiol modification on thin film formation, capsule formation, and film degradation was examined. In addition, the effect of film thickness on the degradation rate was investigated. The significance of this work lies in the high degree of control exerted over the capsule properties and in the potential for these microcapsules to be a platform technology for diverse applications.

Experimental Section Materials. High-purity water with a resistivity greater than 18 MΩ cm was obtained from an inline Millipore RiOs/Origin system. SiO2 particles (1 μm diameter) were purchased from MicroParticles GmbH (Berlin, Germany) as a 50 g L-1 suspension and were used as received. Poly(methacrylic acid, sodium salt) (PMA, 15 000 g mol-1) was purchased from Polysciences (Warrington, PA). Pyridine dithiothylamine hydrochloride (PDA) was purchased from Speed Chemical Corp. (Shanghai, China). (21) Zelikin, A. N.; Becker, A. L.; Johnston, A. P. R.; Wark, K. L.; Turatti, F.; Caruso, F. ACS Nano 2007, 1, 63–69. (22) Zelikin, A. N.; Li, Q.; Caruso, F. Angew. Chem., Int. Ed. 2006, 45, 7743– 7745. (23) De Rose, R.; Zelikin, A. N.; Johnston, A. P. R.; Sexton, A.; Chong, S.-F.; Cortez, C.; Mulholland, W.; Caruso, F.; Kent, S. J. Adv. Mater. 2008, 20, 4698– 4703. (24) Sivakumar, S.; Bansal, V.; Cortez, C.; Chong, S.-F.; Zelikin, A. N.; Caruso, F. Adv. Mater. 2009, 21, 1820–1824. (25) Zelikin, A. N.; Li, Q.; Caruso, F. Chem. Mater. 2008, 20, 2655–2661. (26) Haynie, D. T.; Palath, N.; Liu, Y.; Li, B. Y.; Pargaonkar, N. Langmuir 2005, 21, 1136–1138. (27) Blacklock, J.; Handa, H.; Manickam, D. S.; Mao, G. Z.; Mukhopadhyay, A.; Oupicky, D. Biomaterials 2007, 28, 117–124. (28) Chen, J.; Huang, S. W.; Lin, W. H.; Zhuo, R. X. Small 2007, 3, 636–643. (29) Chen, J.; Xia, X. M.; Huang, S. W.; Zhuo, R. X. Adv. Mater. 2007, 19, 979– 983. (30) Griffith, O. W. Free Radical Biol. Med. 1999, 27, 922–935.

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Scheme 1. PMASH/PVPON Thin Filma

a (a) Under LbL polymer deposition conditions, (b) cross-linked and at physiological pH, and (c) under cellular reducing conditions.

Phosphate-buffered saline (PBS), Alexa Fluor 488 (AF488) maleimide, and AF488-hydrazide were obtained from Invitrogen (Melbourne, Australia). 3-(N-morpholino)-propanesulfonic acid (MOPS) was purchased from Acros Organics (Geel, Belgium). N-Chloro-p-toluenesulfonamide sodium salt (Chloramine T), 2-morpholinoethanesulfonic acid (MES), 4-(4,6-dimethoxy-1,3,5triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), DTT, poly(ethyleneimine) (PEI, 25 000 g mol-1), PVPON (55 000 g mol-1), reduced GSH, hydrofluoric acid, ammonium fluoride, and sodium acetate were purchased from Sigma-Aldrich (Sydney, Australia). Potassium phosphate and sodium hydroxide were obtained from Merck (Australia). All polymer molecular weights are listed as provided by the manufacturer. Preparation of PMASH. A PMA sample with varying thiol content (mol %) was synthesized by the modification of PMA with PDA. Briefly, 40 mg of PMA was diluted into 1 mL of potassium phosphate buffer (0.1 M, pH 7.2), and the PMA was reacted with DMTMM at 4 times molar excess of the target modification for 15 min. PDA (target modifications 20, 15, 10, 5, and 1.7 mol %) was then added to the mixture, and the reaction was allowed to proceed overnight. The resulting mixture was dialyzed extensively against water, and the polymer was isolated via freeze drying. The thiol content in the resulting polymer was characterized by using the absorbance of pendent PDA groups and a PDA standard curve. The degree of thiol modification is denoted as PMASH-1, PMASH-5, PMASH-10, PMASH-15, and PMASH-20 for target modifications of 1.7, 5, 10, 15, and 20%, respectively. Fluorescence labeling of PMA was carried out by incubating 100 mg of PMA (10 g L-1) in potassium phosphate buffer Langmuir 2009, 25(24), 14079–14085

Becker et al. (0.1 M, pH 5) with DMTMM for 15 min and then adding 100 μg of AF488 hydrazide (1 g L-1) in water. The reaction between the hydrazide on the fluorescent dye and the carboxyl groups along the polymer was allowed to proceed overnight, after which time the polymer was purified via gel filtration and isolated via freeze drying. PMASH was incubated in a solution of DTT (75 g L-1) in MOPS buffer (10 mM, pH 8) for 15 min at 37 °C and diluted with sodium acetate (50 mM, pH 4) to a working solution of 2 g L-1. Preparation of PVPON-AF488. PVPON-AF488 was prepared using RAFT-synthesized PVPON,31 as described previously. Briefly, the terminal thioester functionality of the RAFT-derived polymer was converted into a terminal thiol group using sodium borohydride and further reacted with AF488 maleimide. The resulting polymer was isolated by chromatography using a NAP-5 column from GE Healthcare (Sydney, Australia) and recovered by freeze drying. The number-average molecular weight was determined to be 12 000 g mol-1. Quartz Crystal Microgravimetry (QCM). Gold-coated 5 MHz AT-cut crystals were cleaned by immersion in piranha solution (70% sulfuric acid/30% hydrogen peroxide) for 20 min followed by extensive rinsing in water. Caution! Piranha solution is very corrosive. Extreme care should be taken when handling piranha solution, and only small quantities should be prepared. The crystals were dried under a stream of nitrogen and placed in a UV ozone cleaner for 20 min. QCM measurements were performed using a QCM-D E4 device with four flow cells (Q-Sense AB, V€astra Fr€ olunda, Sweden). The temperature was kept constant at 23.4 °C during the experiments. All frequency values quoted are for the third overtone. The other overtones measured (fifth and seventh overtones) followed the same trend. After initially depositing a layer of PEI (1 g L-1) in 0.5 M NaCl, PMASH-0 to PMASH-20 and PVPON (1 g L-1) in sodium acetate (50 mM, pH 4) were alternately adsorbed to the film for 10 min. After each adsorption step, the film was washed with sodium acetate (50 mM, pH 4). Cross-linking was performed twice by exposure to the oxidant chloramine T (2 mM) in MES buffer (10 mM, pH 6) for 5 min. Finally, the film was washed with PBS, although all measurements were recorded using the same buffer conditions (sodium acetate, 50 mM, pH 4). Dual Polarization Interferometry (DPI). An AnaChip was cleaned by immersion in piranha solution (70% sulfuric acid/30% hydrogen peroxide) overnight followed by extensive rinsing in water. This procedure was completed twice before drying the chip under a stream of nitrogen. DPI measurements were performed using an Analight Bio200 instrument (Farfield Group Ltd., Cheshire, U.K.). The temperature was kept constant at 24 °C during the experiments. After the initial deposition of a layer of PEI (1 g L-1) in 0.5 M NaCl, PMASH and PVPON (1 g L-1) in sodium acetate (50 mM, pH 4) were alternately adsorbed to the film for 10 min. After each adsorption step, the film was washed with sodium acetate (50 mM, pH 4). All measurements were recorded using the same conditions: sodium acetate (50 mM, pH 4). LbL on Colloidal Particles. Fifty microliters of 1 μm diameter SiO2 particles (0.5 g L-1) in sodium acetate (50 mM, pH 4) was incubated alternately with PVPON and PMASH (1 g L-1) for 15 min. Three centrifugation/wash cycles (900 g for 30 s) with sodium acetate buffer (50 mM, pH 4) were conducted after deposition of each polymer layer. Polymers were added sequentially until 10 layers had been adsorbed. Flow cytometric analysis was performed on the core-shell particles after each layer (laser power 100 mW; FL1 PMT 650 V). The core-shell particles were treated with the oxidant chloramine T (2 mM) in MES (10 mM, pH 6) for 2 min. The particles were suspended in 50 μL of sodium acetate (50 mM, pH 4) to which 250 μL of HF/NH4F (2/8 M, pH 5) was added. Caution! Hydrofluoric acid and ammonium fluoride are very toxic. Extreme care should be taken when handling HF solution, and only small quantities should be prepared. The resulting (31) Zelikin, A. N.; Such, G. K.; Postma, A.; Caruso, F. Biomacromolecules 2007, 8, 2950–2953.

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Article capsules were washed in sodium acetate (50 mM, pH 4) via four centrifugation/wash cycles (4000g for 4 min). For degradation studies, the capsules were postlabeled with 5 μg of AF488maleimide in MES buffer (50 mM, pH 6) overnight and washed extensively. Capsule Degradation. GSH (5 mM) in PBS was prepared by making a 1 M solution of reduced GSH in 1 M NaOH and diluting in PBS. The pH was adjusted as required. The capsule dispersion (∼107 capsules, 2.5 μL) was added to 1 mL of PBS or PBS containing 5 mM GSH and incubated at 37 °C. For flow cytometric analysis, duplicate samples were measured on the flow cytometer and imaged on the fluorescence microscope.

Results and Discussion Synthesis of PMASH Using Pyridine Dithiothylamine Hydrochloride (PDA). Reproducible and efficient polymer synthesis and modification is required for the reliable synthesis of a polymer-based drug delivery system. For our system, free thiols are required to cross-link the hydrogen-bonded film after film assembly to stabilize it at physiological pH. Thiols and other functional groups can be incorporated into the polymer either by copolymerization with an appropriate monomer or by the pendent functionalization of an existing polymer. The latter method, although restricted to polymers that contain reactive moieties, is facile and effective. Here we present a new method for PMASH synthesis using PDA (Scheme S1, Supporting Information). The conversion of PMAPDA to PMASH is fast, occurring within 10 min (data not shown). In addition, PDA has strong absorption in the UV region that shifts to 343 nm when it is reduced. Thus, the reduction of PMAPDA to PMASH can be followed spectrophotometrically, and the degree of PMA modification can be measured without reaction with Ellman’s reagent. Previously, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was used to modify the carboxylic acid groups on the PMA. During the reaction between EDC and the carboxylic acids, the O-acylisourea intermediate formed is rapidly hydrolyzed or can undergo rearrangement to form relatively stable N-acylurea groups.32 This can make EDC reaction yields poor and variable. An alternative to carbodiimide is the triazine-based condensing agent DMTMM.33 The use of DMTMM for the aqueous condensation of acids and amines to form amides can produce higher yields than EDC.34-36 When using EDC to couple either cystamine or PDA to PMA, the reaction yield is around 65% (measured with Ellman’s reagent) whereas when using DMTMM it is around 90% (measured against a PDA standard curve). The measured levels of modification for the increasing degrees of thiol functionality are given in Table S1 (Supporting Information). For target modifications of 1.7, 5, 10, 15, and 20%, the modified PMAs are identified as PMASH-1, PMASH-5, PMASH-10, PMASH-15, and PMASH-20, respectively. The use of PDA and DMTMM instead of cystamine and EDC are improvements that simplify and increase the reproducibility of PMASH synthesis and will have a minimal effect on performance, as evidenced by the fact that after reduction the structure of the working polymer is the same. LbL Assembly of PMASH/PVPON Multilayers on Planar Surfaces. To control the properties of the PMASH film, (32) Nakajima, N.; Ikada, Y. Bioconjugate Chem. 1995, 6, 123–130. (33) Thompson, K.; Michielsen, S. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 126–136. (34) Kunishima, M.; Kawachi, C.; Monta, J.; Terao, K.; Iwasaki, F.; Tani, S. Tetrahedron 1999, 55, 13159–13170. (35) Kunishima, M.; Kawachi, C.; Iwasaki, F.; Terao, K.; Tani, S. Tetrahedron Lett. 1999, 40, 5327–5330. (36) Kunishima, M.; Kawachi, C.; Hioki, K.; Terao, R.; Tani, S. Tetrahedron 2001, 57, 1551–1558.

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PMA samples were modified with varying degrees of thiol functionality. We investigated the effect of polymer modification on film formation using QCM. As the degree of thiol modification of PMA increased, the film increased in mass (Figure 1a). By observing the incremental change in the QCM frequency for each polymer, it is clear that although the amount of PMASH incorporated into the film increases, the amount of PVPON in the film remains the same (within experimental error) with increasing PMASH thiol functionality (Figure 1b). That is, the PMASH to PVPON weight ratio increases with increasing PMASH thiol functionality. This suggests the dilution of the hydrogen bonds through the pendent thiol modification, thus requiring more PMASH to hydrogen bond with PVPON. The fact that PVPON incorporation in the film stays the same for each level of PMASH functionalization may also suggest that the PVPON is adsorbed only onto the surface and that there is negligible PVPON interpenetration of the layers within the film. Following formation, the films were cross-linked using the oxidant chloramine T and then transferred into a pH 7 solution to determine their stability above the pKa of PMA (pH 6.5). To account for changes in the solvent with respect to the QCM frequency, the films were restored to pH 4 for the final measurement. After incubation in pH 7, the frequency of the unmodified PMA film increased to the level prior to film growth (Figure 1a). An increase in QCM frequency is indicative of mass loss and represents deconstruction of the film; this is due to the ionization of the PMA and the resulting loss of hydrogen bonding. The PMASH-1 film also deconstructed significantly, indicating that additional cross-links were required to stabilize the film. The PMASH films with thiol modifications of 10% and above remain intact at physiological pH overnight. For the intact films, the frequency measured after cross-linking and washing at physiological pH is a combination of the increased water in the film as the cross-linked PMA becomes a hydrogel and the loss of mass from the film as the PVPON is released when the hydrogen bonding is destroyed.24 DPI was used to determine the absolute thickness and mass of the PMASH/PVPON film. DPI uses dual polarized light to determine the refractive index and thickness of an adsorbed film.37 The mass and density were then calculated using de Feijter’s equation.38 The use of DPI is particularly advantageous for following the growth of PMASH films because this information cannot be gained from QCM using the Sauerbrey equation.39 This is because the dissipation measurements of the PMASH films are relatively high at 0.7  106 per 10 Hz compared with 0.25  106 per 10 Hz for PMA films. For a PMASH-15 film, the thickness measured in situ by DPI was 3.2 nm for each PMASH-15 layer and 1.6 nm for each PVPON layer (Figure 2). This compares with ellipsometric data (4.4 nm per PMA layer and 2.6 nm per PVPON layer) for air-dried PMA/PVPON multilayer films (without thiol modification) assembled at pH 2.16 The ratio of PMASH to PVPON deposited per layer is the same as that obtained by QCM for the same film and corresponds to a 2.6-fold molar excess of PMASH in the film. Because of the dilution of the hydrogen bonding groups, this is slightly higher than the literature value of a 2.1-fold molar excess of PMA for unmodified PMA/PVPON multilayer films.16 The average mass of PMASH-15 deposited per layer was 2.1 mg m-2, and that for PVPON was 1.4 mg m-2. An advantage of using the DPI method is that no assumptions are required about the density or the refractive index of the film. (37) Halthur, T. J.; Bjorklund, A.; Elofsson, U. M. Langmuir 2006, 22, 2227–2234. (38) de Feijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759– 1772. (39) Sauerbrey, G. Z. Phys. 1959, 155, 206–222.

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Figure 1. (a) LbL assembly of PMASH with varying thiol modification and PVPON onto a planar surface, measured using QCM-D. Layer 1 is a PEI priming layer; thereafter, the even numbers represent PMASH layers, and the odd numbers represent PVPON layers. (b) Average incremental changes per layer for PMASH (black) and PVPON (gray).

Figure 2. LbL assembly of PMASH-15 and PVPON as monitored in situ by DPI: thickness (b) and mass (O). Layer 1 is a PEI priming layer; thereafter, the even numbers represent PMASH layers, and the odd numbers represent PVPON layers.

The film density without water was 0.79 g cm-3 and was constant after three layers were deposited (excluding the priming PEI layer). After washing into pH 7 solution, the density was reduced to 0.60 g cm-3. This relatively low film density, combined with high dissipation values obtained by QCM (data not shown), suggests that the polymer film is a water-swollen hydrogel. The use of low-molecular-weight polymers allows efficient removal by renal filtration.5 It has been suggested that molecules with a molecular weight of less than 5000 are removed through renal filtration, although there are documented exceptions of up to 100 000 that are also removed.5 PMA of increasing molecular weight, from 4000 to 100 000, was deposited alternately with PVPON of constant molecular weight of 55 000 on a QCM-D electrode. As the size of the PMA increased, the thickness of the thin film increased (Figure S1, Supporting Information). The 15 000 PMA sample was used for all further studies, although Langmuir 2009, 25(24), 14079–14085

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changing the molecular weight provides another variable for control of the degradation rate. Additionally, we investigated the effect of the buffer concentration on thin film formation. The sodium acetate buffer, used to maintain the solution pH, was increased from 0 to 100 mM, and its effect on thin film formation was minimal (Figure S2, Supporting Information). To summarize, on planar substrates we have investigated the formation of the PMASH films containing different degrees of thiol content. We have shown that the thiol content affects the incorporation of PMASH into the film but not that of PVPON, and that the thickness of the thin film can be controlled by varying the thiol content of PMASH. LbL Assembly of PMASH/PVPON Multilayers on Particles. Polymer capsules can be used to transport toxic or vulnerable drugs for use in systemic drug delivery. Thus, the formation of the (precursor) PMASH film on colloidal particles is of interest. The assembly of the PMASH film on microspheres was investigated using flow cytometry and fluorescently labeled polymers. To allow correlation between samples with different degrees of thiol modification, a sample of PMA was labeled prior to thiol modification using a hydrazide-functionalized fluorescent dye (AF488). The PMA was then divided to obtain PMAPDA with varying degrees of modification. For the fluorescent labeling of PVP, we used a RAFT-derived sample of the polymer with an inherent thiocarbonylthio end group that is easily converted into a thiol and subsequently reacted to a maleimide-functionalized fluorescent dye (AF488).30 The increase in fluorescence on the particles after the deposition of each layer was analyzed using flow cytometry. The assembly of the PMASH films was linear up to at least 10 layers, as monitored by PMASH-AF488 (Figure 3a) and PVPON-AF488 (Figure 3b). The amount of PMASH in the film increased as the degree of thiol modification increased (Figure 3a), and the amount of PVPON in the film was independent of the degree of PMASH modification (Figure 3b). These data agree with the results obtained on planar substrates and are likely due to the dilution of the hydrogen bonding groups as a result of thiol functionalization. After the PMASH layers in the PMASH/PVPON film were cross-linked by oxidation, the core was removed and the capsules were transferred to a pH 7 solution. While still in the acidic solution, capsules were observed for every sample, even after only one bilayer had been deposited. This highlights the strength of the hydrogen bonding between PMA and PVPON. As expected, the capsules formed from unmodified PMA deconstructed at pH 7 (Table S2, Supporting Information). The ionization of PMA at physiological pH disrupts the hydrogen bonds. Similarly, PMASH-1 and PMASH-5 capsules were unstable in pH 7 solutions, even after five layers of PMASH had been deposited and the film had been cross-linked (Figure 4). This suggests inefficient cross-linking or lack of interpenetration of cross-links between the layers. At least 10% thiol modification and four PMASH layers were required to obtain stable capsules at pH 7. Stable capsules were formed from only two PMASH layers when higher thiolation modification was used. Because of the ionized PMA, the capsules were swollen at physiological pH compared to their size in acidic solution. For the capsules with higher thiol modification, there is some correlation between increasing layer number and decreasing size at pH 7 (Figure 4). The number of layers deposited in the film and the thiolation degree of PMASH are expected to be the factors governing the degradation of the capsules. Controlling the PMASH Degradation Rate via Tuning the PMASH Thiol Content. The PMASH capsules were designed to degrade in the reducing environment of the cytosol. Capsules Langmuir 2009, 25(24), 14079–14085

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Figure 3. LbL assembly of (a) PVPON and PMASH-AF488 or (b)

PVPON-AF488 and PMASH onto 1 μm diameter silica particles. PMA (b), PMASH-1 (O), PMASH-5 (9), PMASH-10 (0), PMASH15 (2) or PMASH-20 (Δ). The even numbers represent PMASH layers; the odd numbers represent PVPON layers.

Figure 4. Capsule volume at pH 7 as a function of degree of PMASH thiolation and number of PMASH/PVPON bilayers deposited. Capsules were formed from 1 μm diameter silica particles. The capsules measured at pH 4 were formed using PMASH-15.

formed from an inherently unstable PMA film were held together by reversible disulfide cross-links. At physiological pH, the PVPON is released from the film20 and the PMA is ionized, so the cross-linking disulfide bonds hold the film together. These disulfide bonds can be reduced to thiols under reducing conditions. In a cell, the cytosol is maintained in a reducing environment by the enzymes thioredoxin and glutaredoxin and by glutathione.13 To mimic the environment of the cytosol, degradation experiments were performed in 5 mM reduced GSH in PBS solution at 37 °C. Capsules were fluorescently labeled via the residual free thiol groups after cross-linking, and the fluorescence of the capsules was monitored by flow cytometry over 24 h. Capsules were degraded in GSH but remained intact in PBS (Figure 5). The degradation rate of the capsules was controlled by the degree of PMASH thiol modification (Figure 6). The increase in fluorescence observed for PMASH-20 is probably due to a reduction in the self-quenching of closely spaced dye molecules as the capsules swell prior to degradation.25 Capsules formed from DOI: 10.1021/la901687a

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Figure 5. Fluorescence microscopy images of five-bilayer PMASH-20 capsules after 24 h of incubation in (a) PBS or (b) 5 mM GSH in PBS; and five-bilayer PMASH-15 capsules after 24 h of incubation in (c) PBS or (d) 5 mM GSH in PBS. Images b and d were acquired at the edge of a droplet to highlight the solution fluorescence.

Figure 6. Flow cytometry analysis of five-bilayer capsules synthesized using PMASH-10 (0), PMASH-15 (2), and PMASH-20 (4) in 5 mM GSH in PBS. These data were normalized to control samples in PBS (b). The capsules were labeled with AF488.

PMASH-10 degraded within approximately 5 h, whereas the PMASH-20 capsules were still intact at that time. Such a variation in the degradation rate may be exploited to improve the delivery of therapeutics. Furthermore, a mixture of two or more types of capsules that vary in the level of film cross-linking could be administered simultaneously to give two or more release profiles. One administration of the mixture could provide several doses of a drug that is metabolized quickly, which might otherwise require administration several times. Another way to achieve this is to incorporate polymers with different thiolation percentages into the same capsule. This can be used to release therapeutics trapped in the wall at different rates. Release profiles are expected to be cargo-dependent because many drugs will be released from a partially degraded capsule. An investigation into the release of different sizes of DNA from these capsules is currently underway. Effect of Film Thickness on the Degradation Rate of PMASH Capsules. A distinct advantage of LbL assembly is the high degree of control afforded over the film thickness at the nanometer scale. Altering the film thickness can affect capsule properties; for example, increasing the thickness of polymer 14084 DOI: 10.1021/la901687a

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Figure 7. Flow cytometry analysis of PMASH-15 capsules made from 5

bilayers (2, 4), 10 bilayers (9, 0), and 15 bilayers (b, O) exposed to 5 mM GSH in PBS (2,9,b) or exposed to neat PBS (4,0,O). (a) Unprocessed data and (b) data that has been normalized to control samples in PBS. The capsules were labeled with AF488.

multilayer films decreases its permeability to oligonucleotides.40 The effect of film thickness on the degradation rate of the capsules was investigated. Capsules formed from 5, 10, and 15 bilayers of PMASH-15/PVPON were degraded in 5 mM GSH, and the fluorescently labeled capsule wall was monitored by flow cytometry. Despite the difference in thickness (indicated by the increased fluorescence of the capsule), each sample was degraded completely within 6 h (Figure 7a). Normalizing the fluorescence of the capsule wall to the fluorescence of the nondegraded control sample in PBS (Figure 7b) indicates that the degradation rate of the capsules was largely independent of the thickness. GSH is a small molecule of 307 g mol-1 and can therefore permeate the thin film and reduce the disulfides at the surface or in the center equally. Whereas these observations suggest that the degradation mechanism is mainly homogeneous bulk erosion, some combination of surface erosion or other mechanisms cannot be excluded. The films investigated here were all less than 100 nm thick, and surface erosion may become apparent at increased thickness. Other LbL assembled films report surface erosion, bulk erosion, and other complex mechanisms. Films created with the hydrolytically degradable poly( β-amino ester)s principally display surface-type erosion,41,42 although when alternated with plasmid DNA a more complex mechanism involving polyelectrolyte complexes is also involved.42,43 The salt-induced degradation of poly(acrylic acid) and poly(allylamine hydrochloride) films also included the release of polyelectrolyte complexes.44 Bulk erosion was observed in the rapid degradation of a PSS/protein film.45 (40) Angelatos, A. S.; Johnston, A. P. R.; Wang, Y.; Caruso, F. Langmuir 2007, 23, 4554–4562. (41) Zhang, J. T.; Montanez, S. I.; Jewell, C. M.; Lynn, D. M. Langmuir 2007, 23, 11139–11146. (42) Fredin, N. J.; Zhang, J.; Lynn, D. M. Langmuir 2005, 21, 5803–5811. (43) Fredin, N. J.; Zhang, J.; Lynn, D. M. Langmuir 2007, 23, 2273–2276. (44) Nolte, A. J.; Takane, N.; Hindman, E.; Gaynor, W.; Rubner, M. F.; Cohen, R. E. Macromolecules 2007, 40, 5479–5486. (45) Jewell, C. M.; Fuchs, S. M.; Flessner, R. M.; Raines, R. T.; Lynn, D. M. Biomacromolecules 2007, 8, 857–863.

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Although changing the film thickness does not influence the degradation rate, the permeability of the films is different,40,46 and this will affect cargo release. For example, after 4 h, the 15-bilayer film contains significantly more material than the 5-bilayer film (Figure 7a), although they have both lost a similar percentage of material compared to the nondegraded control (Figure 7b). The cargo may be able to leak out of the 5-bilayer capsule but still be impermeable to the 15-bilayer capsule. Thus, increasing the film thickness may delay the onset of cargo release but will not change the total time for cargo release.

Conclusions We have investigated several parameters of PMASH thin film formation and degradation with particular reference to their future application as delivery vehicles. Increasing the degree of thiol modification of PMASH increased the amount of PMASH in the film during LbL assembly as a result of the dilution of the hydrogen bonding groups. Cross-linked films were formed on planar and colloidal substrates, and we determined that a 10% degree of thiolation was required to form stable films at physiological pH. The degradation rate of disulfide cross-linked capsules was investigated under conditions designed to mimic the cytosol. Changing the cross-linking density of the capsules by changing (46) Chong S.-F.; Sexton, A.; De Rose, R.; Kent, S. J.; Zelikin, A. N.; Caruso, F. Biomaterials 2009, doi:10.1016/j.biomaterials.2009.05.078.

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Article

the degree of thiol functionality on PMA provides control over the degradation rate. Increasing the degree of thiol groups by 5% prolonged complete degradation by 2 h, as monitored by flow cytometry. Altering the thickness of the capsule wall did not change the degradation rate but may have an effect on cargo release because films of different thickness have different permeability. These capsules hold promise for the encapsulated delivery of therapeutics because capsule properties such as size, film thickness, and degradability can be readily engineered with defined specifications. Acknowledgment. This work was supported by the Australian Research Council via the Australian Postdoctoral Fellowship (A.N.Z., A.P.R.J.), Discovery Project (A.N.Z., A.P.R.J., F.C.), and Federation Fellowship (F.C.) schemes. The Particulate Fluids Processing Centre is acknowledged for infrastructure support. We thank Prof. David Putnam (Cornell University) for valuable scientific discussions. Supporting Information Available: PMA modification reaction scheme, degree of modification of PMASH, QCMD analysis of the effect of PMA molecular weight on PMA/ PVPON thin film formation, and the effect of sodium acetate buffer concentration on PMA/PVPON thin film formation, and measured capsule diameter after core removal. This material is available free of charge via the Internet at http://pubs.acs.org.

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