Biomacromolecules 2005, 6, 2365-2369
Single Component Chitosan Hydrogel Microcapsule from a Layer-by-Layer Approach
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Scheme 1. Preparation Process of Chitosan Hydrogel Microcapsules
Yongjun Zhang, Ying Guan, and Shuiqin Zhou* Department of Chemistry and Center for Engineered Polymeric Materials at College of Staten Island, and The Institute for Macromolecular Assembly, The City University of New York, 2800 Victory Boulevard, Staten Island, New York 10314 Received January 26, 2005 Revised Manuscript Received April 1, 2005
Introduction Smart biomaterials have found broad applications in modern medical technologies.1 However, the development of smart hydrogels with rapid response, good biocompatibility, and controllable degradability is still a formidable challenge. Chitosan, a biodegradable, nontoxic, and renewable linear polysaccharide, has been considered for various biomedical and pharmaceutical applications.2,3 To these ends, microparticles of chitosan in hundreds of micrometers have been prepared in different ways, including coacervation/ precipitation, spray-drying, emulsion cross-linking, emulsiondroplet coalescence, reverse micellization, ionic gelation, and sieving method.3,4 Complexation between chitosan and oppositely charged polysaccharides in solution is another way to synthesize chitosan microparticles; however, this process often leads to the formation of fibers.5 A new strategy, the so-called “layer-by-layer (LbL) assembly”, has been used here to fabricate microcapsules from chitosan. This strategy, based on the electrostatic interaction between oppositely charged polyelectrolytes, was first introduced by Decher et al. to fabricate thin films on planar supports by alternate adsorption of polycation and polyanion.6 Because of its advantages of fine control of the compositions and the thickness of the capsules, the LbL method received more and more attention in recent years.7-9 It was extended to three-dimensional (3D) systems by Mohwald et al. to fabricate core-shell particles and hollow capsules by further removal of the sacrificial core.10 In previous works, we first extended the LbL method based on hydrogen and covalent bonding to 3D systems and synthesized hydrogen or covalent bonded capsules.11,12 Chitosan bears positive charges and is a polycation at low pH. It has been used widely to fabricate LbL films13-19 and capsules20-22 with other polyelectrolytes. For the normal LbL methods, the capsule wall has at least two compositions. In certain applications, one may take advantage of the feasibility of several components by combining their individual material properties. On the other hand, the presence of a second polymer component for LbL assembling may limit the specific functionality of materials. For instance, the biocompatibility and biodegradability of chitosan could be destroyed by a second polymer component. Moya et al. fabricated a nearly single component capsule of * To whom correspondence should be addressed. E-mail: zhoush@ mail.csi.cuny.edu.
polyallylamine hydrochloride (PAH) from the polystyrene sulfonate (PSS)/PAH LbL assembly by using cells as the template.23 The PAH in the capsule wall was oxidized and cross-linked, and then most of PSS was removed while removing the cell core template with sodium hypochlorite. However, this strategy could not be a general method for fabrication of single component capsules. Zhang et al. reported the preparation of single component 2-dimensional films from hydrogen-bonded LbL films, but these films were not cross-linked.24,25 Our new strategy is to selectively crosslink the chitosan chains and remove the second polymer component that was built up in the capsule walls (see Scheme 1). Using this strategy, single component chitosan hydrogel microcapsules were successfully synthesized. The pHresponsive volume phase transition of the chitosan hydrogel microcapsules could be controlled through the cross-linking density. The resultant chitosan gel microcapsules are nearly monodispersed, and the shell thickness is tunable, which is a big advantage to control the releasing kinetics of encapsulated drug or other molecules in response to pH or salt concentration changes. More importantly, although many shell cross-linked capsules have been made from synthetic block copolymers through a strategy of self-assembly of block copolymers with selective cross-linking on the shell block,26-31 it is very challenging, if not impossible, to synthesize well-defined block copolymers from natural polysaccharides. Our novel strategy here can provide a general method to synthesize shell cross-linked microcapsules from natural polysaccharides that have many applications in the pharmaceutical and medical field due to their biocompatibility and degradability. Experimental Section Materials. Chitosan with low molecular weight and poly(acrylic acid) (PAA) with a Mw of 2000 were purchased from Aldrich. SiO2 particles with an average diameter of 306 nm and a polydispersity of µ2/〈Γ〉2 of 0.004 (from dynamic light scattering) were prepared according to the literature.32 Other chemicals are of analytical grade and used as received.
10.1021/bm050058b CCC: $30.25 © 2005 American Chemical Society Published on Web 04/28/2005
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Preparation of Single Component Chitosan Films on Planar Silicon Wafers. Chitosan was dissolved in 1% acetic acid under stirring overnight. The solution was filtered to remove the remaining flakes. The chitosan concentration is 0.5%, whereas the PAA concentration is 1%. Silicon wafers were treated in a boiling H2SO4/H2O2 mixture (7:3 v/v) for 30 min and then rinsed with water thoroughly before use. The films were built up by alternately immersing the silicon wafer in chitosan and PAA for 4 min and by washing in water for 1 min. The chitosan-PAA films were cross-linked by treating with 2.5% glutaraldehyde (pH∼4) at room temperature for 2 h. The cross-linked films were brown in color. They were treated with a 0.2 M carbonate buffer of pH ) 9 overnight to remove PAA in the film. The procedure was repeated to guarantee the complete removal of PAA. Preparation of Single Component Chitosan Capsules. SiO2 particles were centrifuged to change the solvent from ethanol to water. HCl was added to neutralize the suspension. The particles were then redispersed in 0.2 M acetate buffer of pH ) 3.5 (about 0.8 g solid content in 30 g solution). A total of 2 mL of 0.5% chitosan in 1% acetic acid was added under stirring. After 8 min, the excess polymer was removed in the supernatant fraction after centrifugation. The particles were washed using a 0.002 M acetate buffer of pH ) 3.8 for 3 times. They were re-dispersed in the 0.2 M acetate buffer of pH ) 3.5, and then 2 mL of 1% PAA solution was added. The excess PAA were removed using the same process. A total of 4 bilayers of chitosan/PAA were coated using this procedure. The resulting core-shell particles were treated with 2.5% glutaraldehyde (pH ∼ 4) for various time length. After washing with water 3 times, they were added to the 0.2 M carbonate buffer of pH ) 9. A decrease in pH value was observed upon the addition of particles. NaOH was added to maintain the pH at 9. After 12 h, they were washed with water 3 times. The procedure was repeated to guarantee the complete removal of PAA. The SiO2 core was removed by adding the core-shell particles to a 10% hydrofluoric acid solution under stirring. The resulting capsules were dialyzed against water to purify them. Characterization. FTIR spectra were recorded on a Nicolet Magna IR 550 spectrometer. TEM measurements were carried out on a Philips CM100 microscopy. Samples were prepared by dropping a suspension onto Formvar-coated copper grids. A standard laser light scattering spectrometer (BI-200SM) equipped with a BI-9000AT digital correlator and a He-Ne laser (35 mW, 633 nm) was used to perform static and dynamic light scattering measurements. Results and Discussion We first confirm if the strategy works on a planar film. The chitosan/PAA film was fabricated on silica wafers by dipping the silica wafers alternately in a chitosan solution and a PAA solution. The film was first treated with glutaraldehyde to selectively cross-link chitosan in the film, followed by treating with a carbonate buffer of pH ) 9 to remove PAA in the film. The FTIR spectra of the glutar-
Notes
Figure 1. FTIR spectra of (a) a 20 bilayer chitosan/PAA film after cross-linked with glutaraldehyde, (b) the same film after further treated with 0.2M carbonate buffer solution of pH ) 9, (c) chitosan gel crosslinked with glutaraldehyde, and (d) single component chitosan microcapsules.
aldehyde cross-linked film before and after treating with carbonate buffer were collected in Figure 1. From Figure 1a, the chitosan/PAA film presents a strong peak at 1704 cm-1, which is associated with the stretching of the uncharged dimerized or associated form of carboxylic groups of PAA, indicating PAA is involved in the film. The peak at 1536 cm-1 is attributed to the COO- group, indicating the carboxylic groups of PAA partially ionized in the film to form ionic bands with chitosan. Significant changes were found after the film was treated with carbonate buffer of pH ) 9 to remove PAA. As shown in Figure 1b, although the two peaks of 1704 and 1536 cm-1 disappeared, a new peak at about 1660 cm-1 appeared, indicating the formation of the Schiff’s base structure.33 For comparison, a pure chitosan gel cross-linked with glutaraldehyde was prepared, and its FTIR spectrum was shown in Figure 1c. The two spectra are almost identical, suggesting that PAA in the film has been removed completely and the film has only one component of chitosan. Cross-linking an LbL film has been applied to a few systems. Usually the approaches rely on condensation reaction of complementary groups located on adjacent layers. By incorporation a photo-cross-linkable polyelectrolyte,34-36 the films can be cross-linked after exposed to UV light. Heating the films containing carboxylic and amino groups at high temperature to produce amide linkages has also been explored.37,38 The condensation reaction can be catalyzed by water-soluble carbodiimide and so can be preformed in a mild condition.39 Although the main purpose of cross-linking is to enhance the film stability, cross-linking may also change the permeability38 and conductivity,37 as well as improve the cell adhesion of films.39 Cross-linking can also improve the stability of hollow capsules fabricated using the LbL method.9,23,40-43 Glutaraldehyde has been widely used to cross-link chitosan for the synthesis of chitosan hydrogels.33,44 It was proposed that the mechanism of cross-linking between glutaraldehyde and the free amine on chitosan follows a Schiff’s base reaction.33 The mobility of a polyelectrolyte in an LbL film during and after its construction is well-known.45-49 The polymer rearrangement can result in phase separation45 and morphology change,46,47 sometimes producing pores.46 The polymer
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Figure 2. Typical TEM graphs of single component chitosan microcapsules made from four bilayers of chitosan/PAA membrane.
mobility is usually enhanced by treatments that minimize interactions between the polyelectrolytes, such as annealing by salt48,49 or pH control for films containing weak polyelectrolyte.49 If the interactions among the film components are completely loosened, the film will dissociate completely.47 Here we use a carbonate buffer to increase PAA mobility in the film, which combines the effects of salt and pH control. We also choose low molecular weight PAA chains for the film and capsule buildup to increase the mobility of PAA chains and make them able to diffuse into the outside solution more easily than high molecular weigh polymers. In the next step, we extended this strategy to 3D systems. First silica particles with a narrow size distribution were synthesized using the literature method.32 Layer-by-layer assembly of chitosan and PAA on the silica surface was done following the previously reported procedure.10-12 A 0.2 M acetate buffer of pH ) 3.5 was used to facilitate the adsorption of polymers onto the silica nanoparticle surface. These particles were then treated with glutaraldehyde for 5 and 42 h, respectively, and then treated with a carbonate buffer of pH ) 9 to remove PAA. The silica core was finally dissolved using hydrofluoric acid. The FTIR spectrum of the dried single component chitosan microcapsules, as shown in Figure 1d, is similar to that of pure chitosan gel, indicating that our strategy for fabrication of single component chitosan microcapsules is successful. Figure 2 shows the typical transmission electronic microscopy (TEM) graphs of the dried chitosan gel microcapsules made from the four-bilayer chitosan/PAA membranes. The dried gel capsules are nonspherical in shape and present a lot of creases with a rough surface, regardless of the pH values that the capsules are dispersed in. This morphology of the dried capsules is understandable. After removing the spherical silica core template, the chitosan gel capsules have a hollow cavity and thin chitosan gel shell. During the gradual drying process in air for the TEM sample preparation, the chitosan gel capsules will collapse and the chitosan shell membrane will shrink and fold in some degree. We speculate that the shape of dried microcapsules with thicker shell and higher cross-linking density will be less distorted from the spherical template. Although the dried thin-shelled microcapsules are distorted in shape from the spherical core template, their average size of about 310 nm is close to the size of the template silica particles.
Figure 3. (a) Apparent hydrodynamic radius (Rh,app) of the chitosan capsules in the presence of 0.017 M acetate (0, cross-linking time 5 h; O, cross-linking time 42 h) at different pH values. (b) Size distribution of the chitosan capsules with cross-linking time of 5 h at pH of 8.6 and 3.5, respectively. (c) Size distribution of the chitosan capsules with cross-linking time of 42 h at pH of 8.6 and 3.5, respectively.
To further illustrate the size and morphology of the chitosan capsules dispersed in aqueous solutions, laser light scattering was employed to characterize a dilute aqueous solution of chitosan microcapsules (cross-linked for 42 h) at pH ) 8.5. The dynamic light scattering (DLS) results reveal that the apparent diffusion coefficients (Dapp) of the chitosan microcapsules show angular dependence. The extrapolation of the linear relationship of Dapp versus sin2(θ/2) (see the Supporting Information) to zero scattering angle produces a D0 of 1.09 × 10-8 cm2/s, corresponding to a hydrodynamic radius (Rh) of 225 ( 10 nm from the StokeEinstein equation for the chitosan microcapsules. On the other hand, the angular dependence of the net absolute scattered intensity of the same chitosan capsules from the static light scattering (SLS) generates a linear Guinier plot (see the Supporting Information), which produces a radius of gyration (Rg) of 228 ( 5 nm. The combination of SLS and DLS results indicates that the ratio of Rg over Rh of the chitosan capsules in the aqueous solution of pH ) 8.5 is close to unity. This result suggests that the chitosan capsules should be hollow after removing the spherical silica core template, although the interface of the chitosan capsule wall
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may not be neat due to possible chitosan chain extension and branching to both the inside and outside solution of the capsules. It is well-known that chitosan gels undergo a volume phase transition at different pH values.50 The single component chitosan gel capsules prepared from our novel strategy show a clear pH-responsive volume phase transition around pH ) 5.7, depending on the cross-linking density of the capsule wall. Figure 3a shows the pH dependence of the apparent hydrodynamic radius (Rh,app) of the single component chitosan capsules with two different cross-linking density, measured at a scattering angle θ ) 30°. To control the pH values, a 0.017 M acetate buffer was used. For chitosan capsules with a cross-linking time of 5 h, the size shrank down when the pH was changed from 3.5 to 5.0, followed by a sharp decrease in the Rh,app when the pH was increased slightly from 5.0 to 5.7 and then remained nearly a constant value when pH is above 7. In contrast, the chitosan capsules with a cross-linking time of 42 h did not show apparent size change at the whole pH range of 3.5-9 that we studied. Obviously, the long cross-linking time of 42 h resulted in a very high cross-linking density of the capsule wall so that the chain network lost the elastic property. It is also important to note that all of the single component chitosan gel capsules prepared from our novel strategy showed a narrow size distribution. As shown in Figure 3, parts b and c, no matter the chitosan capsules are highly or slightly cross-linked, and in swollen or shrunk state, the size distributions of the single component chitosan capsules from LbL method are very narrow. Conclusions In conclusion, a strategy for the fabrication of single component hollow gel capsules from LbL assembly was proposed. It involves an initial selective cross-linking of one component followed by removal of the second component for the LbL assembly. This strategy was confirmed by the successful synthesis of single component chitosan gel microcapsules. The cross-linking of chitosan increased the film stability and make it strong enough to resist osmotic force during the removal of the sacrificial core. The resultant chitosan hydrogel capsules were pH sensitive. They swelled at low pH and shrank at high pH. The cross-linking density of the capsule wall can be controlled and has a significant effect on its pH sensitive behavior. Acknowledgment. S.Z. gratefully acknowledges the partial support of this project from the National Science Foundation (CHM 0316078) and PSC-CUNY research award (60020-33-34). Supporting Information Available. The extrapolation of the linear relationship of Dapp versus sin2(θ/2) and a linear Guinier plot. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Anderson, D. G.; Burdick, J. A.; Langer, R. Science 2004, 305, 1923. (2) Kumar, M. N. V. R. React. Funct. Polym. 2000, 46, 1. (3) Agnihotri, S. A.; Mallikarjuna, N. N.; Aminabhavi, T. M. J. Controlled Release 2004, 100, 5.
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