Dual Drug Delivery Microcapsules via Layer-by-Layer Self-Assembly

Jul 14, 2009 - The integration of hydrophobic and hydrophilic drugs in the polymer microcapsule offers the possibility of developing a new drug delive...
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Dual Drug Delivery Microcapsules via Layer-by-Layer Self-Assembly Uttam Manna and Satish Patil* Solid State and Structural Chemistry Unit Indian Institute of Science, Bangalore-560012, India Received April 8, 2009. Revised Manuscript Received June 24, 2009 The integration of hydrophobic and hydrophilic drugs in the polymer microcapsule offers the possibility of developing a new drug delivery system that combines the best features of these two distinct classes of material. Recently, we have reported the encapsulation of an uncharged water-insoluble drug in the polymer membrane. The hydrophobic drug is deposited using a layer-by-layer (LbL) technique, which is based on the sequential adsorption of oppositely charged polyelectrolytes onto a charged substrate. In this paper, we report the encapsulation of two different drugs, which are invariably different in structure and in their solubility in water. We have characterized these dual drug vehicular capsules by confocal laser scanning microscopy, atomic force microscopy, visible microscopy, and transmission electron microscopy. The growth of a thin film on a flat substrate by LbL was monitored by UV-vis spectra. The desorption kinetics of two drugs from the thin film was modeled by a second-order rate model.

Introduction The concept of exploiting oppositely charged polyelectrolytes to assemble in thin films was first reported by Gero Decher.1 It is a general preparative technique for creating multilayer thin films whose structure and composition can be controlled precisely on the nanometer scale. It has been successfully applied to charged spherical particles to develop polymer microcapsules for various applications, including nanomaterial synthesis,2 enhanced immunoessays,3 biomaterial engineering,4 and drug delivery.5 The composition and conformation of polyelectrolytes allow one to control permeability, physicochemical properties, and drug release in the presence of external stimuli.6,7 To tune the properties of microcapsules, large varieties of coating materials have been used, for example, carbon nanotubes,8 dendrimers,9 inorganicorganic composites,10 and inorganic nanoparticles.11 However, microcapsules that efficiently encapsulate water-insoluble drugs are rare. The main problem with the delivery of a hydrophobic drug is its relatively low solubility in water. Large varieties of pharmaceutical drugs have nonpolar hydrophobic backbones and also lack water-soluble functional groups.12 They are also an important class of drugs, so delivery of these drugs is crucial. Several efficient systems have been proposed to address this significant problem: membrane lipids,13 polymer micelles,14 sodium dodecyl sulfate (SDS)-coated hydrophobic materials,15 *To whom correspondence should be addressed. Tel: þ91-80-22932651. Fax: þ91-80-23601310. E-mail: [email protected].

(1) Decher, G.; Hong, J. D. Makromol. Chem. Macromol. Symp. 1991, 46, 321. (2) Decher, G. Science 1997, 277, 1232. (3) Decher, G.; Eckle, M.; Schmitt, J.; Struth, B. Curr. Opin. Colloid Interface Sci. 1998, 3, 32. (4) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Mohwald, H. Angew. Chem., Int. Ed. 1998, 37, 2202. (5) Caruso, F.; Schuler, C.; Kurth, D. G. Chem. Mater. 1999, 11, 3394. (6) Schuler, C.; Caruso, F. Biomacromolecules 2001, 2, 921. (7) Pastoriza-Santos, I.; Schuler, B.; Caruso, F. Adv. Funct. Mater. 2001, 11, 122. (8) Paunov, V. N.; Panhuis, M. Nanotechnology 2005, 16, 1522. (9) Khopade, A. J.; Caruso, F. Nano Lett. 2002, 2, 415. (10) Shckukin, D. G.; Sukhorukov, G. B.; Mohwald, H. Angew. Chem., Int. Ed. 2003, 42, 4472. (11) Caruso, R. A.; Susha, A.; Caruso, F. Chem. Mater. 2001, 13, 400. (12) Liu, J.; Chen, L.; Li, L.; Hu, X.; Cai, Y. Int. J. Pharm. 2004, 287, 13. (13) Zhihua, A.; Mohwald, H.; Junbai, L. Biomacromolecules 2006, 7, 580. (14) Nguyen, P. M.; Zacharia, N. S.; Verploegen, E. V.; Hammond, P. T. Chem. Mater. 2007, 19, 5524. (15) Caruso, F.; Yang, W.; Trau, D.; Renneberg, R. Langmuir 2000, 16, 8932.

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fourth generation poly(amidoamine) dendrimer (4G PAMAM) and poly(styrenesulfonate) (PSS) multilayer films on planar substrates, and colloidal templates.16 Along with this, encapsulation of two different kinds (hydrophilic and hydrophobic) of molecules in a single vehicle is a challenging as well as an important aspect for smart drug delivery.17 The body’s defense mechanisms are very complex, and once triggered, it follows several paths in a cascade.18 Thus, it is difficult to target the entire cascade using single drug molecules. Furthermore, a combined therapy with drugs of different therapeutic effects gives better results in the treatment of diseases and tissue reborn.19,20 So far, very few approaches have been demonstrated to overcome this problem. Fabrication of polymersomes from asymmetric block copolymers is one of the leading approaches where the core of the polymersomes can accumulate hydrophilic molecules and the shell of it can encapsulate hydrophobic molecules. However, these vehicles are sometimes sensitive to external stimuli such as magnetic fields,21,22 ultrasound,23 electric fields,24 temperature,25-27 etc., which demands an external effort to release the encapsulated materials. Furthermore, nondegradability of some of these block copolymers restricts them for use as an effective delivery system.28-31 Another approach is dual drug loaded (16) Khopade, A. J.; Caruso, F. Biomacromolecules 2002, 3, 1154. (17) Meng, F.; Zhong, Z.; Feijen, J. Biomacromolecules 2009, 10, 197. (18) Suthanthiran, M.; Morris, R. E.; Strom, T. B. Am. J. Kidney Dis. 1996, 28, 159. (19) Qiu, L. Y.; Bae, Y. H. Biomaterials 2007, 28, 4132. (20) Lee, J. S.; Bae, J. W.; Joung, Y. K.; Lee, S. J.; Han, D. K.; Park, K. D. Int. J. Pharm. 2008, 346, 57. (21) Krack, M.; Hohenberg, H.; Kornowski, A.; Lindner, P.; Horst Weller, H.; Stephan Forster, S. J. Am. Chem. Soc. 2008, 130, 7315. (22) Yu, T. H.; Li, S. G.; Zhao, J.; Mason, T. J. Technol. Cancer Res. Treat. 2006, 5, 51. (23) Zhou, W.; Meng, F. H.; Engbers, G. H. M.; Feijen, J. J. Controlled Release 2006, 116, e60–e62. (24) Koide, A.; Kishimura, A.; Osada, K.; Jang, W. D.; Yamasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2006, 128, 5988. (25) Li, Y.; Lokitz, B. S.; McCormick, C. L. Angew. Chem., Int. Ed. 2006, 45, 5792. (26) Hales, M.; Barner-Kowollik, C.; Davis, T. P.; Stenzel, M. H. Langmuir 2004, 20, 10809. (27) Chen, X. R.; Ding, X. B.; Zheng, Z. H.; Peng, Y. X. New J. Chem. 2006, 30, 577. (28) Zhou, Y. F.; Yan, D. Y.; Dong, W. Y.; Tian, Y. J. Phys. Chem. B 2007, 111, 1262. (29) Cerritelli, S.; Velluto, D.; Hubbell, J. A. Biomacromolecules 2007, 8, 1966. (30) Tong, X.; Wang, G.; Soldera, A.; Zhao, Y. J. Phys. Chem. B 2005, 109, 20281. (31) Jiang, Y. G.; Wang, Y. P.; Ma, N.; Wang, Z. Q.; Smet, M.; Zhang, X. Langmuir 2007, 23, 4029.

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nanoparticle synthesis,32 but in this case, the release efficiency is less than 50%. Recently, Wei et al.33 introduced a composite of micelle and hydrogel that can encapsulate hydrophobic and hydrophilic molecules, but in this system, the release efficiency is poor above pH 4. Our recent investigation has demonstrated that uncharged water-insoluble drugs can be encapsulated in thin films as well as in polymer membrane capsules by a simple approach containing the assembly of a polyelectrolyte and amphiphilic surfactant.34 Systems containing both polymers and surfactants in aqueous solutions have attracted significant interest due to their unique properties and potential applications in personal care products and food additives.35,36 This novel approach can be used to encapsulate large varieties of water-insoluble drugs with high concentration. It also provides an empty core for loading of another drug in the single capsule. On the basis of the same principle, this work has been extended for encapsulation of two different drugs;37 one preloaded into the shell of the microcapsule and the other in the capsule core via a layer-by-layer (LbL) approach. In our previous study,34 we have shown chitosan/SDS micellar microcapsules that have hydrophobic material (pyrene) preloaded in the shell of the microcapsule. Here, we have incorporated one more different kind of model hydrophilic drug molecule (methylene blue/rhodamine-B/RBBR) into the shell and the vacant core of the microcapsule. So, two different kinds (hydrophilic and hydrophobic) of drugs can be encapsulated in a single microcapsule. The goal of this study is to understand the elasticity and morphological changes of chitosan/SDS micellar microcapsules in the presence of two different drugs. Additionally, we examine the release kinetics of a model drug to establish the drug delivery potential of these microcapsules.

Experimental Section Materials. Melamine formaldehyde (MF) [hydrochloric acid (HCl), soluble] particles with a 3.3-3.5 μm size were procured from Microparticles GmbH (Berlin, Germany). Chitosan (Mw ∼ 200000), perylene, and pyrene were obtained from Aldrich. SDS was obtained from Spectrochem (India). HCl and AcOH were obtained from Qualigens (India). All of the materials have been used without any purification. Ultrapure water (Millipore) with a specific resistance of more than 18 MΩ cm was used. All of the experiments were carried out at room temperature. The other chemicals and solvents were of AR grade and were used as received. Thin Film Preparation. As reported earlier,34 multilayers were deposited by immersing a quartz plate in 1 mg/mL chitosan solution, followed by rinsing with water and drying with a gentle nitrogen gas stream after the deposition of each layer. The film was then dipped in a micellar solution of pyrene (378 ppm) and allowed to adsorb for 2 h followed by a washing and drying step before it was placed into a chitosan solution. Growths of thin films were monitored by UV-vis spectra (not shown here). Fluorescence Resonance Energy Transfer (FRET) Experiment. The bare MF particles (15 μL of 10 wt % dispersion) were incubated in a micellar solution of pyrene for 30 min followed by four washing cycles with water and were dried under nitrogen flow. Then, the coated particles were transferred in 3 mL of water to measure the fluorescence intensity of pyrene (excited at 334 nm). The coated particles were centrifuged for 10 min and incubated in a chitosan solution (1 mL, 1 mg/mL) for 15 min. (32) Song, X.; Zhao, Y.; Hou, S.; Xu, F.; Zhao, R.; He, J.; Cai, Z.; Li, Y.; Chen, Q. Eur. J. Pharm. Biopharm. 2008, 69, 445. (33) Wei, L.; Cai, C.; Lin, J.; Chen, T. Biomaterials 2009, 30, 2606. (34) Manna, U.; Patil, S. J. Phys. Chem. B 2008, 112, 13258. (35) Brackman, J. C.; Engberts, J. B. F. N. Chem. Soc. Rev. 1993, 22, 85. (36) Varga, I.; Gilanyi, T.; Meszaros, R. Prog. Colloid Polym. Sci. 2001, 117, 136. (37) Xia, W.; Chang, J.; Lin, J.; Zhu, J. Eur. J. Pharm. Biopharm. 2008, 69, 546.

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Then, the particles were washed four times with water to remove excess chitosan and incubated in a SDS micellar solution of perylene for another 15 min. Then, four centrifugation/wash cycles were performed to remove the excess perylene-loaded SDS micelles, dried under nitrogen flow. The supernatant was removed, and 3 mL of water was added to measure the fluorescence intensity of pyrene as well as perylene (excited at 334 nm). Calibration Curves. A pyrene (0.0405 g) solution was prepared by dissolving in 100 mL of SDS solution by successive ultrasonication and heating around 80-90 °C for 1 h. The asprepared solution was used for a calibration curve at 335 nm. The RBBR and rhodamine-B (100 ppm) solutions were used for calibrations at 590.67 and 554.20 nm, respectively. Capsule Preparation. Bare MF particles (15 μL of 10 wt % dispersion) were incubated with a micellar solution of pyrene for 30 min, followed by three washing cycles with water. A chitosan solution (1 mL, 1 mg/mL) was then added to pyrene-coated MF particles and allowed 10 min for adsorption. Three centrifugation/wash cycles were performed. Alternatively, pyrene and chitosan steps were repeated to construct multilayers on MF particles. Then, by dissolving the MF core in 0.1 N HCl aqueous solutions, hollow capsules were obtained, where pyrene was preloaded into the shell of the microcapsule and the core remained empty. Elasticity of Microcapsules. A change in elasticity of the microcapsule was observed after incubation with 100 ppm rhodamine-B solution, and a change in the morphology with time was studied with confocal laser scanning microscopy (CLSM). Characterization. The UV-vis spectra for the calibration of pyrene, rhodamine-B, and RBBR were obtained with a PerkinElmer (Lambda 35) spectrometer. Microscopy. CLSM. All of the CLSM images were taken using Leica (version SP-V) with 20 water objective. Perylene was excited at 405 nm, and rhodamine-B and RBBR were excited at 561 and 633 nm, respectively. All measurements were done with Millipore water. Atomic Force Microscopy (AFM). AFM measurements were carried out using a Digital, Nanoscope IVA AFM, Veeco Instruments (United States) in tapping mode. Samples were prepared by placing a drop of dilute solution of the particle suspension on a glass plate and were dried under nitrogen flow. Visible Light Microscope. Ringlike capsule images in Millipore water were captured using an Olympus Visible light microscope with 100 air objective at room temperature.

Results and Discussions Encapsulation of Two Different Hydrophobic Molecules. The essential condition for energy transfer to occur is overlap of absorption and emission spectra of the donor and acceptor. Figure 1 shows the absorption and emission spectra of perylene (acceptor) and pyrene (donor), respectively. The absorption spectrum of perylene shows good overlap with the emission spectrum of pyrene in the range of 360-450 nm.38 This provides us a platform to monitor the encapsulation of different model hydrophobic drugs in alternate layers by conducting FRET experiments. On the basis of the same principle, we have encapsulated two different hydrophobic model drugs (pyrene and perylene) with chitosan in between as a spacer as described in the Experimental Section. Emission spectra of pyrene were recorded after coating the micellar solution of pyrene on the MF particles, followed by the coating of chitosan and micellar solution of perylene. It is clearly seen that the emission of pyrene decreases sharply while that of the perylene peaks emerges as shown in Figure 2. This indicates that efficient energy transfer takes place (efficiency of energy transfer; η=0.7313) from pyrene (38) Lindegaard, D.; Madsen, A. S.; Astakhova, I. V.; Malakhov, A. D.; Babu, B. R.; Korshun, V. A.; Wengel, J. Bioorg. Med. Chem. 2008, 16, 94.

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Figure 1. Absorption and emission spectra of perylene (spectra 1) and pyrene (spectra 2). Figure 3. Emission spectra of a mixture of perylene- and pyreneencapsulated micellar solution excited at 334 (pyrene) and 439 nm (perylene), respectively.

Figure 2. (A) Fluorescence emission spectra of pyrene. (B) Evolution of perylene peak as a result of FRET at 334 nm from donor (pyrene) to acceptor (perylene). The efficiency of energy transfer is 0.7313.

to perylene through a chitosan layer. These experiments were conducted on MF particles, and both of the model drugs were excited at 334 nm. Using a FRET experiment, we have shown that the micelle/ polymer multilayer membrane can be used as a multicompartment drug delivery system where each micellar layer of the membrane is efficient at loading different kinds of hydrophobic molecules and the thickness of the bilayer can be determined based on the FRET distance. To examine the effect of the surrounding media and composition, which might variably affect interactions, we have conducted FRET experiments in the micellar solution of aromatic hydrocarbon fluorophores (pyrene and perylene) in the absence and presence of chitosan. The fluorescence spectra for the pyrene and perylene micellar solutions excited at two different wavelengths (334 and 439 nm) for the respective chromophores are shown (Figures S2 and S3 of the Supporting Information) for comparison. Spectral broadening was found in the case of the micellar solution of pyrene and shows a broad emission peak at 479 nm when excited at 334 nm. This peak could be due to the formation of excimer in the micellar solution of pyrene. In a mixture of pyrene- and peryleneencapsulated micellar solutions in the absence of chitosan, the emission signature of perylene appears at excitations of both 334 and 439 nm wavelengths. The signature of emission spectra Langmuir 2009, 25(18), 10515–10522

of perylene at 334 nm excitation reveals fluorescence energy transfer from pyrene (donor) to perylene (acceptor) molecules in a micellar-encapsulated state in solution. The residual pyrene emission intensity in a mixture of micelles supports the nontransporting nature of pyrene and perylene molecules within micelles in the mixture of these two micellar solutions, as shown in Figure 3. The repulsion force within negatively charged head groups of SDS molecules at the exterior of the micelles poses a distance barrier between pyrene and perylene molecules to occur FRET within them, and as a consequence, partial quenching of the emission of pyrene molecules has taken place in the mixture. The effect of positively charged chitosan on the micellar solution of pyrene and perylene was observed by following UV-visible and FRET studies. The addition of chitosan (0.5 mg/mL) in the micellar solution of pyrene or perylene (100 ppm) results in a change in absorption as well as FRET efficiency. We have observed that by increasing the amount of chitosan, the solution appears turbid; consequently, the absorption peak intensity of both pyrene and perylene was reduced, and the shape of the peaks was also affected, as shown in Figure S4 of the Supporting Information. These results suggest that the addition of a certain amount of chitosan (0.116 mg/mL) turbidity appears in the mixture and it simply reveals the formation of a complex among positively charged chitosan and negatively charged micelles with pyrene and perylene molecules. The FRET experiment provided a more clear explanation of the interaction among chitosan and micelles in solution. Figure 4 shows the fluorescence intensity values of a FRET pair (pyrene and perylene) excited at 334 nm as a function of chitosan. Initially, the emission intensity of perylene increased in proportion to the amount of chitosan added to the micellar solution. However, after a certain amount of chitosan, the emission intensity of perylene decreases whereas the emission intensity of pyrene increases. These results reveal that at a low concentration of chitosan in the mixture of a micellar solution, it helps to bring closer the micelles (with negatively charged exteriors) that are encapsulated with pyrene and perylene molecules by reducing repulsion forces among micelles through charge compensation. Thus, the FRET efficiency from pyrene to perylene increased with the addition of chitosan. A further increase in the concentration of chitosan compensates for the exterior negative charge of a higher number of micelles and allows a larger number of micelles to come closer DOI: 10.1021/la901243m

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Figure 4. Effect of chitosan in mixture of micelles encapsulated with perylene and pyrene using a FRET study at 334 nm. Scheme 1. Schematic of the FRET Distance between Two Successive Micelle Layers in a Multilayer Membrane

as a result emission intensity of perylene increases. At a higher concentration of chitosan, charge over compensation may take place, and the intermicelle distance increases as the polymer adapts to a different conformation and it keeps micelles apart from each other; as a consequence, fluorescence energy transfer reduces as a result, the emission of pyrene slowly increases, and the emission of perylene decreases as shown in Figure 4. But, we did not observe such an effect in the LbL assembly as shown in Figure 2. The observed difference in FRET efficiency at a higher concentration of chitosan indicates that chitosan separated by an alternate micellar layer in the LbL assembly is low in concentration and adopts the favorable conformation for energy transfer between pyrene and perylene. This experiment gives us a rough idea (some particles must have been lost during the washing steps) about the thickness of the polymer layer in between two successive micellar layers of pyrene and perylene for this system. Here, we have calculated the distance (1.88 nm; R0 =2.23 nm39) between pyrene and perylene molecules based on a FRET experiment. We have shown that the FRET distance can be treated as the thickness of the chitosan polymer layer (approximately) in a multilayer membrane as shown in Scheme 1 and that this polymer (39) Masuko, M.; Ohuchi, S.; Sode, K.; Ohtani, H.; Shimadzu, A. Nucleic Acid Res. 2000, 28, e34.

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Figure 5. Visible light microscopic image of a hollow microcapsule in water.

layer thickness (1.88 nm) is very similar to earlier reports in the literature.40 SDS Micelles/Chitosan Hollow Capsules. The hollow capsules were obtained by dissolving MF particles in 0.1 N HCl solutions as reported earlier.34 Figure 5 shows a visible light microscope image of the resulting hollow capsules in solution. The capsules were monodisperse, and the size of the capsule reflected more or less the original size of the core. The spherical shape of the initial MF particles was retained in the hollow capsule in aqueous solution. As shown previously, this property depends upon the interaction of polyelectrolytes on the core and its backbone chemistry.41 Nevertheless, it clearly indicates that stable hollow capsules can be obtained by depositing SDS micelles and chitosan by a versatile LbL approach. These capsules preloaded with model drug (pyrene/perylene) in the shell were further used for the encapsulation of another hydrophilic drug inside the core. Loading of Hydrophilic and Hydrophobic Model Drug. We have already demonstrated that a hydrophobic model drug can be loaded into the shell by the LbL approach.34 To demonstrate the utility of these capsules as a dual drug delivery vehicle, we have encapsulated a model hydrophilic drug inside the hollow capsules as well as in the shell. Encapsulation of rhodamine-B was achieved in an aqueous solution (1 mg/mL-1). These experiments were extended to other hydrophobic and hydrophilic model drugs, namely, perylene and RBBR. The chemical structures of model drugs used for encapsulations are shown in Figure 6. The loading of hydrophilic drug was followed by CLSM. Figure 7a,b shows the encapsulation of perylene and rhodamine-B in a multilayer membrane onto the MF particles. Perylene was loaded via the LbL approach and SDS/chitosan provide pores for the encapsulation of rhodamine-B as well. As shown in Figure 7a,b, when perylene was excited selectively, MF particles displayed a characteristic fluorescent ring under a confocal microscope. Whereas MF particles appeared red in color after the excitation of rhodamine-B, similar experiments have been carried out on capsules as well. The loading of rhodamine-B inside the capsules deformed the shape due to osmotic stress (discussed later). The CLSM images (Figure 7c,d) clearly show the evidence of (40) Dong, W.; Ferri, J. K.; Adalsteinsson, T.; Schonhoff, M.; Sukhorukov, G. B.; Mohwald, H. Chem. Mater. 2005, 17, 2603. (41) Caruso, F. Adv. Mater. 2001, 13, 11.

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Figure 6. Chemical structures of perylene (a), rhodamine-B (b), and RBBR (c), respectively.

encapsulation of both drugs in a single capsule. Interestingly, capsules are very stable after the loading of rhodamine-B or RBBR inside the core. Elastic Nature of Micelles/Chitosan Capsules. Elasticity is one of the most interesting mechanical properties of the microcapsules, and this property certainly depends upon the composition of multilayers and loading of charged drug inside the core. To investigate the elasticity of SDS micelle/chitosan microcapsules, we have incubated them in an aqueous solution of rhodamine-B (100 ppm) for 24 h. This high-concentration loading of charged model drug leads to a decrease in the diameter of the capsule from 4 to 1.0 μm due to an osmotic stress to the capsule membrane (Figure 8). Interestingly, after incubation for 72 h in the same solution, the capsules reswell to 2 μm. Initially, when the microcapsules are in aqueous solution, an osmotic pressure is in equilibrium due to the presence of water (solvent) both inside and outside of the capsule, so the size of the microcapsule is 4 μm. However, loading of Rh-B leads to a change in the local osmotic pressure of the microcapsule. In other words, the inside and outside of the capsule has an osmotic pressure gradient, as inside the capsule has only water (solvent) whereas outside has hydrophilic molecules (solute), so this leads to a reduction in the diameter of the microcapsule. However, with continuous incubation in same solution, the concentration of the solute (Rh-B) will be greater inside the capsule core than the outside of the capsules; as a consequence, the capsule will start to expand and regain its size. Time-dependent expansion and contraction of the microcapsule is shown in a schematic representation (Scheme 2). The SDS/chitosan microcapsule membranes are different from conventional microcapsules, which lead to such interesting behavior. Morphology of Micelle/Polymer Multilayer Thin Film. LbL growth of the thin film has been noticed by UV-vis spectral analysis, as we have already reported in our earlier study.34 Here, we have shown the morphological change of this multilayer thin film with a number of deposition layers of coating materials (chitosan and SDS). Multilayers prepared by oppositely charged polyelectrolytes have a low degree of internal organization.42,43 As a consequence, Bragg reflections corresponding to the internal layer structure of the film are usually not observed.44 However, the ordering of the internal structure within the multilayer thin (42) Kellogg, G. J.; Mayer, A. M.; Stockton, W. B.; Ferreira, M.; Rubner, M. F.; Satija, S. K. Langmuir 1996, 12, 5109. (43) Losche, M.; Schmitt, J.; Decher, G.; Bouwman, W. G.; Kjaer, K. Macromolecules 1998, 31, 8893. (44) Arys, X.; Laschewsky, A.; Jonas, A. M. Macromolecules 2001, 34, 3318.

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film can be improved simply by employing two different types of polycations/polyanions. Here, we describe the multilayers of an amphiphilic surfactant (SDS micelles) and chitosan induces different kinds of morphological changes on the thin film depending on the outermost layer in the LbL assembly.45 It is well-known that the interaction of a surfactant with a macromolecule leads to various interesting features like micelle formation in the presence of a macromolecule, which occurs at a lower critical micellar concentration, and the average size of the micelle is smaller when compared to a polymer-free surfactant solution.46 Small angle X-ray scattering experiments showed the existence of nanoordering in the SDS/chitosan complex.47 Different kinds of other approaches have been used to understand this interesting complex.48,49 So, it is interesting to see the morphology in this system as well. The morphology of the thin film has been investigated by AFM. AFM images of two successive layers (10th and 11th layers) of the thin film are shown in Figure 9c,d. The surface morphology of the film undergoes a change from a smooth to a globular shape when the SDS micellar layer is deposited over chitosan. In other words, the surface roughness of the film with the SDS micelle is higher than the chitosan layer. The root-meansquare (rms) roughness of the chitosan layer (11th layer) is 7.8 nm, whereas for SDS layers, they are 3.62, 4.17, and 15.2 nm for the 6th, 8th, and 10th layers, respectively. Loading of Dual Drugs in Thin Film. The loading of hydrophobic drug (pyrene or perylene) was achieved by using a LbL approach with alternate adsorption of pyrene/perylene in the SDS micellar solution and chitosan. After the formation of a thin film (seven bilayers) from chitosan followed by pyrene-incorporated SDS micelles, the film was dipped in an aqueous solution of hydrophilic drug (rhodamine-B or RBBR) to encapsulate into the pyrene preloaded multilayer thin film. The presence of the model drugs in the SDS/chitosan membrane was confirmed from the UV-vis spectra. Figure 10 compares the UV-vis spectra of the pyrene and Rh-B with encapsulated model drugs (pyrene and rhodamine-B) in a multilayer thin film. A typical UV-vis spectrum of rhodamine-B has distinct absorption bands (as shown in the inset in Figure 10). The strongest π-π* transition absorption peak occurs at 562 nm. In comparison to rhodamine-B spectra, the dual drug-containing thin film shows additional peaks at 240, 273, and 336 nm, corresponding to the π-π* transition of pyrene. This provides additional evidence of encapsulation of both hydrophobic and hydrophilic drugs in a single multilayer polymer/micelle membrane. Release of Hydrophilic and Hydrophobic Molecules. The release experiments were conducted in saline solution (0.155 M NaCl, pH 6.5) at room temperature. A percentage of release for corresponding molecules (hydrophilic or hydrophobic) has been calculated using following equation % release of pyrene ¼

ðC 0 -C t Þ100 C0

where C0 and Ct are the concentrations of corresponding molecules in thin films at times zero and t, respectively. It clearly shows that the release of pyrene is similar as shown previously. (45) Lobo, R. F. M.; Pereira-da-Silva, M. A.; Raposo, M.; Faria, R. M.; Oliveira, O. N., Jr. Nanotechnology 2003, 14, 101. (46) Barreiro-Iglesias, R.; Alvarez-Lorenzo, C.; Concheiro, A. J. Therm. Anal. Calorim. 2005, 82, 499. (47) Babak, V. G.; Merkovich, E. A.; Desbrieres, J.; Rinaudo, O. M. Polym. Bull. 2000, 45, 77. (48) Onesippe, C.; Lagerge, S. Colloids Surf., A 2008, 317, 100. (49) Thongngam, M.; McClements, D. J. Langmuir 2005, 21, 79.

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Figure 7. CLSM images (a and b) of coated MF particles (loaded with perylene and Rh-B), whereas panels c and d represent loaded microcapsules with perylene and Rh-B. Perylene was excited at 405 nm, and Rh-B was excited at 561 nm. Yellowish green represents perylene molecule emission, and red stands for Rh-B molecules.

Figure 8. (a) Morphology of microcapsule after 24 h and (b) morphology of microcapsule after 72 h. Scheme 2. Shows the Mechanism of Contraction and Elongation of the Microcapsule

philic drug is similar (Rh-B and RBBR) as shown in Figure 11. In comparison, the release rate of hydrophobic and hydrophilic rate is different in isotonic solution. As shown in Figure 11, pyrene showed a burst release of ∼70% within 3 h, whereas Rh-B or RBBR showed a release of ∼50% for the same period of time. It may due to the ionic interaction between the polyelectrolytes and the corresponding hydrophilic molecules, which lead to tightly bound ionic complexes; as a consequence, low amounts (40-45%) of hydrophilic molecules have released from the thin film, whereas there is no such kind of interaction between hydrophobic (pyrene) molecules and polyelectrolytes. So, such a system can be very applicable as a sustained release tool. Desorption Kinetics of Hydrophilic and Hydrophobic Molecules. To determine the desorption kinetics for both hydrophilic and hydrophobic molecules from multilayer SDS micelle/ chitosan thin films, a pseudo second-order model was used50 dqt ¼ ks ðqe - qt Þ2 dt where qe = [(C0 - Ce)V]/W, V represents the volume of the saline solution, W represents the weight of the thin film, ks is the rate

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(50) Ho, Y. S.; McKay, G. J. Environ. Sci. Health A 1999, 34, 1179.

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Figure 9. AFM images of the thin film: Panels a-c are the 6th, 8th, and 10th SDS micellar layers, respectively, and panel d is a chitosan layer.

Figure 10. UV-vis spectra of encapsulated model drugs (pyrene and Rhodamine-B) in thin film. The inset shows the UV-vis spectra of Rhodamine-B and pyrene in solution for comparison.

constant of the desorption kinetics, and Ce is the concentration of RBBR/pyrene in the thin film at equilibrium. Integration of the above equation with q=qt at time t gives t 1 1 ¼ þ t qt ks qe 2 qe Langmuir 2009, 25(18), 10515–10522

Figure 11. Release profile of pyrene, Rhodamine-B, and RBBR in SDS micelle/chitosan thin film (seven bilayers).

Thus, a plot of t/qt with time is linear. Both hydrophilic (RBBR and Rh-B) and hydrophobic (pyrene) molecules follow the pseudo second-order desorption kinetics (Figure 12). Values of qe for pyrene, Rh-B, and RBBR are 0.058, 0.016, and 0.03, respectively, and can be obtained from the saturation value (inset of Figure 12), which matches the value of qe obtained from the slope of the linear plot. DOI: 10.1021/la901243m

10521

Article

Manna and Patil

open up a general avenue for the fabrication of dual drug vehicular microcapsules. It can be further extended to load a wide range of uncharged water-insoluble drugs, as demonstrated in this work. We have also described a new approach to determine the thickness of a single polymer layer using a FRET experiment and have discussed the elastic behavior of the microcapsules, which is studied with CLSM. The desorption of both hydrophilic and hydrophobic molecules has been demonstrated in the presence of one another. The effects of pH, temperature, and UV radiation on this novel microcapsule are under investigation in our laboratory. Acknowledgment. We thank Manish Kumar and Veeco-India Nanotechnology Laboratory for assistance with CLSM and AFM measurements. We greatly acknowledged the Department of Science and Technology, India, for financial support. Figure 12. Desorption kinetics of RBBR, Rh-B and pyrene molecules and inset shows qt vs time.

Conclusion In summary, a novel methodology has been developed for the encapsulation of both hydrophilic and hydrophobic molecules in hollow microcapsules as well as in thin films. This approach will

10522 DOI: 10.1021/la901243m

Supporting Information Available: UV-visible and fluorescence spectra of pyrene and perylene excited at 334 and 439 nm and UV-visible spectra of micellar solution of pyrene and pyrelene in the presence of chitosan. This material is available free of charge via the Internet at http://pubs. acs.org.

Langmuir 2009, 25(18), 10515–10522