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Biomacromolecules 2009, 10, 2632–2639

Layer-by-Layer Self-Assembly of Modified Hyaluronic Acid/ Chitosan Based on Hydrogen Bonding Uttam Manna, Sri Bharani, and Satish Patil* Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore-560012, India Received May 18, 2009; Revised Manuscript Received July 27, 2009

The fabrication of hydrogen bonded polymer self-assembly for drug delivery has been accomplished via layerby-layer sequential assembly from aqueous solution. In this study, the self-assembly was constructed based on hydrogen bonding between DNA base (adenine and thymine) pairs substituted on the backbone of chitosan and hyaluronic acid. Chitosan was modified with adenine, whereas hyaluronic acid was modified with thymine. Subsequently, these two polymers were sequentially absorbed on flat substrate by taking advantage of interactions of DNA base pairs via hydrogen bonding. Interlayer hydrogen bonding of these two polymers produces stable multilayer film without using any cross-linking agent. Thin film formation on quartz substrate has been monitored with UV-vis spectra and an AFM study. Formation of multilayer hydrogen-bonded thin film has been further confirmed with SEM. Encapsulation and release behavior of the therapeutic drug from the multilayer thin film at different conditions has been illustrated using UV-vis spectra. Cell viability of modified polymers using MTT assay confirmed no cytotoxic effect.

Introduction Chitosan and hyaluronic acid were used extensively in biomedical field,1-4 due to its easy commercial accessibility,5,6 nontoxicity, biodegradability,7-10 and biocompatibility11-13 properties. These materials have great impact on drug delivery application as in form of hydrogel, microgel and composite thin film.14-17 But the insolubility of chitosan in water at neutral pH18-20 possesses a serious restriction to use it and fabricate new tools for drug delivery applications. To overcome these problems different kinds of chemical modifications were employed to facilitate the applications of chitosan for gene delivery,21-23 antimicrobial substances,24-26 and to improve its biodegradable properties at neutral pH.27-29 Chitosan and hyaluronic acid were also extensively explored for fabrication of microcapsules and multilayer thin film using layer-by-layer (LbL) approach.30-32 LbL is a versatile and simple approach for the fabrication of multilayer thin films and it has been widely used for various applications.33-36 It is based on sequential adsorption of materials with complementary functional group and guided by electrostatic interaction and hydrogen and covalent bonding. Recently, fabrication of hydrogen-bonded multilayer thin films and microcapsule using the LbL approach is very fascinating and attractive due to its strong response to environmental stimuli such as pH, temperature, and solvent.37,38 It also plays a very important role in determining the secondary structures of various biological molecules including proteins and nucleic acids.39,40 LbL self-assembly of biodegradable polymers based on hydrogen bonding give rise to numerous remarkable biological properties, such as quick response to pH of the media, although stability of hydrogen-bonded microcapsules is a major issue. As reported by Caruso et al., the H-bonded poly-(acrylic acid/poly(N-isopropylacrylamide) (PAA/PNIPAAM)41 multilayers release their capsule content only at the elevated temperature, this leads to the destruction of multilayers. Because the H-bonded capsules were pH sensitive, the release of the * To whom correspondence should be addressed. Tel.: +91-80- 22932651. Fax: +91-80-23601310. E-mail: [email protected].

drug is fast at higher pH and it cannot be controlled. So to achieve a controlled release and stable capsule, the cross-linking is introduced in the thin films. For example, when the H-bonded poly(acrylic acid)/poly(ethyleneoxide)-block-poly(ε-caprolactone) (PAA/PEO-b-PCL)42 thin film is exposed to the physiological condition, the disassembly of the thin films occurred due to the weak nature of the H-bonding in the thin films. To overcome these difficulties a cross-linking is introduced between the COOH group of PAA and PEO by thermal induction. In this case, also, the film is not stable due to the thermal effect. To solve this, the cross-linking based on simple carbodiimide chemistry using ethylenediamine as a cross-linking agent was introduced. Sukhishvili et al. prepared the cross-linked poly (methacrylic acid) (PMAA) hydrogels by using ethylenediamine as a cross-linking agent,43 which leads to swelling of the capsule at higher pH and subsequent release of encapsulated macromolecule achieved at higher salt concentration. Multilayer selfassembly of DNA have been extensively investigated by Caruso et al.44-47 However, using biopolymer fabrication of hollow capsules driven by hydrogen bonding without cross-linking agent still remains a challenge because of the instability in aqueous solution. In this paper, we report the self-assembled H-bonded thin film without using any cross-linking agent. The underlying hypothesis of our approach is to utilize the DNA base pairing model to built multilayer thin film from adenine modified chitosan and thymine modified hyaluronic acid. All chemical modifications have been achieved via simple one step synthesis root. Another robustness of this work is integration of negatively charged (thymine modified) hyaluronic acid and neutral (adenine modified) chitosan biopolymers using LbL approach. In addition, encapsulation and release behavior of anticancer drug doxorubicin (DOX) from multilayer thin film of ACHI and THUA was illustrated in physiological conditions. We have also examined ACHI and THUA, which are highly biocompatible; these materials are almost nontoxic and very similar to native polymers.

10.1021/bm9005535 CCC: $40.75  2009 American Chemical Society Published on Web 08/20/2009

Self-Assembly of Modified Hyaluronic Acid/Chitosan

Biomacromolecules, Vol. 10, No. 9, 2009

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Scheme 1. Fabrication of H-Bonded Multilayer Thin Film Using the Watson-Crick Base Pairing Principle

Experimental Section Materials. Chitosan (MW 120000; degree of deacetylation (DDA) 85%) and hyaluronic acid (MW 160000) were purchased from SigmaAldrich. Adenine, thymine, hydrochloric acid, and 3-mercapto propionic acid was purchased from S. D. fine chemicals. AcOH was obtained from Qualigens, India. All the chemicals were used as such without further purification. Polyphosphate ester was prepared from phosphoruspentoxide (MW 141.94, S. D. fine chemicals) as reported in the literature.48 Ultrapure water (Millipore) having specific resistance around 18MΩ cm was used to prepare the solution for layer-by-layer coating experiments. Preparation of Polyphosphate Ester. Diethyl ether (0.07 mmol) and CHCl3 (0.035 mmol) was added to phosphorus pentoxide (0.035 mmol) with stirring and the mixture was heated under reflux for 12 h at 50 °C to get a clear solution.49 The solvent is then distilled off under vacuum and the colorless viscous residue is used as such without further purification. Synthesis of Nucleotide Modified Biodegradable Polymer. Adenine (0.0037 mmol) was dissolved in 50 mL of dimethylformamide with the addition of 0.25 mL of concentrated HCl with magnetic stirring. Polyphosphate ester was added to chitosan (0.0007 mmol) in 30 mL of dimethylformamide, followed by dropwise addition of adenine solution, and the mixture was heated to 50 °C for 20 h. The reaction was cooled at RT and dimethylformamide is distilled off in vacuo. The moist residue is dissolved in water and kept in an ice-box for about 1 h to precipitate unreacted adenine. To the clean aqueous layer, ammonia is added to bring the pH to 10. Then the ammonia layer was extracted with ethyl acetate and the organic layer is evaporated to get pure (ACHI) product. The final product was characterized by NMR, FT-IR, and UV-vis spectroscopy. The clear solution of thymine (0.00396) was obtained by dissolving in 25 mL of H2O with the addition of 0.25 mL of concentrated HCl solution. Polyphosphate ester was added to hyaluronic acid (0.0007 mmol) in 50 mL of H2O, followed by dropwise addition of thymine, and the mixture was heated to 50 °C for 20 h in an oil-bath and cooled under ice for about 1 h to precipitate unreacted thymine. The acid layer

is neutralized by the addition of ammonia. The ammonia layer was extracted with ethyl acetate and the evaporation of the organic layer gave the pure (THUA) product. The final product was characterized by NMR, FT-IR, and UV-vis spectroscopy. Preparation of Thin Film. Fresh polymer solutions at 1 mg/mL (Chitosan, HA, ACHI, and THUA) were prepared in 0.15 M NaCl in water. The quartz plate was immersed into 1 mg/mL solution of acidified chitosan solution, followed by adsorption of THUA and ACHI by immersing in respective solutions (0.5 mg/mL) for 1 h. The quartz plate was washed with Millipore water for alternate layer of adsorption. By repeating this cycle, H-bonded multilayer thin film was obtained and growth of thin film was monitored by SEM and UV-vis spectroscopy. (Scheme 1). Preparation of PBS Buffer. To prepare 100 mL of PBS buffer, we have added 0.8 g of NaCl, 0.02 g of KCl, 0.144 g of Na2HPO4, 0.024 g of KH2PO4 in 80 mL of Millipore water, and finally made it 100 mL with Millipore water and HCl to adjust pH to 7.4. Loading and Release of DOX. The multilayer thin film (10 bilayers) of ACHI and THUA was incubated overnight with DOX solution (0.2 mg/mL) at room temperature. The loading was followed by UV-visible spectroscopy. The thin film washed with Millipore water and exposed to the 20 mL of PBS buffer solution (pH of 7.4) and neutral pH solution. The release of DOX from the thin film in PBS buffer solution monitored with UV-vis spectra with time. Cell Culture. Human cervical carcinoma cells (Hela) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) having 10% (v/v) fetal bovine serum (FBS) and 1% penicillin streptomycin under 5% CO2 at 37 °C. Cytotoxicity Measurement. To calculate cytotoxicity of both ACHI and THUA polymers, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay was used.50 Hela cells (10000 cells/well) were placed in 96-well plates in 200 µL of DMEM medium having 10% FBS until it achieved 70-80% confluence (24 h). Media was removed with fresh media (10% FBS) having a different quantity of polymers (ACHI and THUA). After 4 h of incubation, the medium in each well has been removed with 200 µL of fresh complete medium

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Scheme 2. Synthesis of Adenine Functionalized Chitosan (ACHI)

and incubated for 24 h. After that, we have added 25 µL of stock solution of MTT (5 mg/mL in PBS) to each well, followed by 4 h of incubation, and the medium has been replaced with 150 µL of DMSO to dissolve the fomazan crystal formed by live cells. Absorbance was measured at 595 nm using an ELISA microplate reader (Bio-Rad). The cell viability was estimated in the form of percentage of the absorbance with respect to the control experiment without using polymers. Characterization. Spectrometer. UV-Vis spectrometer. The UV-vis spectra of DOX for loading and release studies were obtained with Perkin-Elmer (Lambda 35) spectrometer. All measurements were undertaken at room temperature. Nuclear Magnetic Resonance (NMR) Spectrometer. 1H NMR spectra were recorded using Bruker Avance NMR spectrometer having 400 MHz frequency in D2O as a solvent and TMS as an internal standard. Fourier Transform Infrared (FTIR) Spectrometer. The dried samples were grounded with KBr powder and the mixtures were made into pellets under high pressure. FTIR spectra were recorded on a PerkinElmer FT-IR spectrophotometer, spectrum 1000. Microscopy. Scanning Electron Microscopy (SEM). For SEM measurements, samples were prepared at room temperature and at atmospheric pressure and images were taken using JEOL, JSM-5600LV scanning electron microscope at 5 kV after gold coating. Atomic Force Microscopy (AFM). AFM measurements were carried out using a Digital, Nanoscope IVA AFM, Veeco Instruments, U.S.A. in tapping mode. Thin film was prepared on silicon wafer (cleaned with piranha solution) from THUA and ACHI solutions, and after gentle drying under a nitrogen flow, the film morphology was examined having a different number of layers examined under AFM. Quartz Crystal Microbalance (QCM). Gold-coated QCM electrodes were modified with MPA by immersing it in a MPA-ethanol solution (1 mM) for 24 h.51 Then we washed it several times with Millipore water, and followed by gentle air drying, it has been placed in an ACHI solution for 15 min, then rinsed with Millipore water, dried by a gentle stream of nitrogen gas, and placed in a THUA solution for another 15 min. This cycle was repeated for up to four bilayer coatings. For the control experiment, we have followed similar protocol and we used hyaluronic acid in place of THUA. In both cases the growth of thin film was followed by using difference in the QCM resonance frequency after each deposition step.

functionalization of chitosan by adenine. The chemical modification of chitosan by adenine is resulted into disappearance of characteristic band of adenine and native chitosan at 3288 and 1159 cm-1, respectively. These absorption bands correspond to the N-H vibration of pure adenine and primary alcoholic group of native chitosan at the C-6 position. This together proves the successful formation of C-N bond between the hydroxyl group of chitosan and N-H group of adenine. The characteristic broadband at 3423 cm-1 in ACHI is attributed to free secondary -OH vibrations of chitosan, indicating the lack of replacement of secondary alcoholic group at C-2 position of chitosan polymer. The successful formation of the ACHI was further confirmed by 1H NMR analysis. The appearance of signal at 4.8 and 2.7 ppm is attributed to the -CH protons of both modified and unmodified chitosan and a singlet at 1.99 ppm corresponds to the -CH3 protons of both the types of chitosan. The appearance of the multiplet between 3.93-4.0 ppm is responsible for the hydroxyl and -CH protons of unmodified chitosan and additional multiplet is obtained in the downfield between 3.83-3.94 ppm, due to the attachment of less electronegative nitrogen atom with methylene protons of chitosan. A doublet between 8.16-8.19 ppm, assigned as d and e in 1H NMR is due to the -CH protons of adenine, the disappearance of the singlet at 12.8 ppm confirming that the proton attached to the nitrogen at position 9 of adenine molecules is reacted with the primary -OH group of chitosan. The degree of adenine substitution in chitosan (DS) was found to be 53%. The DS was estimated from the ratio of integral intensity of the adenine proton (assigned as d) to the sum of integral intensities of the chitosan (assigned as f) proton. Synthesis of Thymine Modified Hyaluronic Acid (THUA). Functionalization of hyaluronic acid by thymine is outlined in the Scheme 3. Here, the reaction was carried out in water media, because both of the reactants are soluble in water. As in the case of (THUA), the addition of polyphosphate ester activates the primary alcoholic group of N-acetyl-glu-

Results and Discussion Synthesis of Adenine Modified Chitosan (ACHI). Synthesis of adenine functionalized chitosan (ACHI) was achieved as shown in Scheme 2. Activation of the primary alcoholic group of chitosan is crucial for the modification reactions in a wellcontrolled manner. Polyphosphate ester is known to activate effectively the primary alcoholic group of chitosan in a relatively low pH. Adenine was covalently attached to activated chitosan by a simple condensation reaction between the primary alcoholic group of chitosan and -NH group of adenine. The FT-IR spectra of native chitosan and adenine functionalized chitosan (ACHI) are shown in the Figure 1. A characteristic band at 3197 cm-1 and a transmittance peak at 2975 cm-1 can be assigned to the -NH stretching of adenine and chitosan, respectively. The appearance of a new peak at 1117 cm-1 in ACHI, which corresponds to the C-N stretching vibration, indicating the

Figure 1. FT-IR spectra of chitosan (black line), adenine functionalized chitosan (ACHI; red line) and adenine (green line).

Self-Assembly of Modified Hyaluronic Acid/Chitosan Scheme 3. Synthesis of Thymine Functionalized Hyaluronic Acid (THUA)

cosamine unit followed by reaction with thymine. The formation of THUA was confirmed by FT-IR measurement, as shown in Figure 2. Thymine shows characteristic peaks at 3195 and 3079 cm-1 due to the presence of an amide functional group. The appearance of a characteristic band at 3383 cm-1 is attributed to the secondary -OH group of hyaluronic acid, indicating that it did not undergo any reaction with thymine but disappearance of the band at 1314 cm-1, which corresponds to the -OH stretching vibration of primary alcoholic group of hyaluronic acid suggests that only the primary -OH group of N-acetyl-D-glucosamine unit of hyaluronic acid reacted with thymine. Furthermore, the disappearance of the band at 3079 cm-1 in the functionalized hyaluronic acid (THUA; black line) indicates that it undergoes a condensation reaction with the primary -OH group of hyaluronic acid, and the appearance of a new band at 1340 cm-1 is due to the C-N stretching vibration between hyaluronic acid and thymine. The functionalization was further confirmed by NMR. Singlets at 1.75 ppm and at 1.91 ppm are due to the -CH3 protons of thymine and hyaluronic acid, assigned as b and c, respectively, and a singlet at 7.25 ppm corresponds to the -CH protons of thymine, assigned as a of spectra 1 in Figure 3. Between 3.9 and 4.0 ppm, the multiple signals are due to the hydroxyl and -CH protons of the glucuronic acid unit of both modified and unmodified hyaluronic acid, and a multiplet between 3.75 and 3.9 ppm is due to the hydroxyl and -CH protons of the unmodified glucosamine unit of hyaluronic acid. The absence of the singlet at 10.6 ppm, which corresponds to the thymine proton attached to the nitrogen atom, indicates that it undergoes condensation reaction with hyaluronic acid and the appearance of a new multiplet in the upfield between 4.1-4.2 ppm, confirming that the primary -OH group of N-glucosamine unit reacted with the -NH group of thymine. Even though the HA is incorporated with the less electronegative nitrogen atom, it shows the shift in the upfield, due to the presence of the carbonyl group, which is adjacent to the

Figure 2. FT-IR spectra of thymine (red line) and functionalized hyaluronic acid (THUA; black line).


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nitrogen atom of thymine. The degree of substitution (75.19%) was calculated by using NMR integrals of native hyaluronic acid and THUA. We found higher substitution in hyaluronic acid as compared to chitosan; this may be because of better solubility of hyaluronic acid in water, which leads to higher substitution. UV-Vis Study of Modified Polymers. Both the polymers (chitosan and hyaluronic acid) used in this study are UV inactive. But modified polymers (ACHI and THUA) show a UV-vis signature due to the presence of nucleotide bases in the backbone. This provides a platform to monitor the reaction by using UV-vis absorption spectroscopy. Figure 4 represents the UV-vis spectra of ACHI and THUA functionalized by adenine and thymine. As anticipated, the absorption band appearing at 261 nm in ACHI indicates that chitosan is functionalized with adenine. Because chitosan is UV-inactive, the absorption at 261 nm is due to the presence of the π-π* transition of adenine (261 nm). Similarly, the absorption peak at 265 nm corresponds to thymine functionalized hyaluronic acid (THUA). These data indicate that chitosan and hyaluronic acid are successfully functionalized with adenine and thymine, respectively. Layer-by-Layer Growth of Thin Film. The fabrication of the multilayer film of ACHI and THUA is governed by hydrogen bonding on quartz via LbL protocol for flat substrate. Evidence of multilayer formation on flat substrate was obtained from SEM and UV-vis spectroscopy. This multilayer assembly of negatively-charged and neutral biodegradable polymers were based on the Watson and Crick base-pairing principle, more precisely based on hydrogen bonding mainly between adenine and thymine. The very first layer on quartz substrate was coated with charged chitosan at acidic pH and followed by THUA adsorption because it has a negative charge on its backbone. However, the remaining LbL assembly was built based on sequential absorption of THUA and ACHI. So, formation of the first layer was governed by electrostatic attraction. But other than the first layer, multilayer thin film has formed based on hydrogen bonding. This we proved by coating multilayer at neutral pH where ACHI is uncharged. Inset spectrum of Figure 5a shows a clear signature of the adsorption of THUA, whereas for other layers, it is quite difficult to assign peaks of ACHI and THUA as they have merged together in the spectrum as the number of layers increased, but a gradual increase in intensity of these spectra have signified assembly of these two polymers to form a multilayer thin film. Fabrication of the multilayer thin film on quartz substrate has been further verified by SEM image of the thin film, as shown in Figure 5b. To prove the self-assembly of ACHI/THUA is based on hydrogen bonding mainly between adenine and thymine, we have performed a control experiment where we have assembled multilayer film from ACHI and hyaluronic acid (HA). But lack of thymine moiety in hyaluronic acid possessed reluctant behavior toward formation of multilayer thin film via LbL protocol. The growth of thin film was followed by UV-vis spectroscopy and it clearly shows the infinitesimal growth, as shown in Figure 5c. So, multilayer assembly is negligible for this pair (ACHI and HA) of polymers and, thus, it suggests that hydrogen bonding between thymine and adenine plays a momentous role and leads to multilayer growth of THUA followed by ACHI. Further, the growth of thin film for both the system (ACHI/THUA and ACHI/HA) was followed by a QCM study, and these results are similar to that obtained in UV-vis spectroscopy. In the case of the ACHI/THUA pair, a linear increment of frequency

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Figure 3. 1H NMR spectra of functionalized hyaluronic acid with thymine (1). 1H NMR spectra of functionalized chitosan with adenine (2).

resulting film is 5.75 nm for four bilayers. In contrast, the growth of the thin film for ACHI/HA pair shows a very small change in frequency compared to the ACHI/THUA system, and the thickness of the film for four bilayers is 1.44 nm. These results clearly suggest that the interaction between adenine and thymine guides the multilayer assembly based on hydrogen bonding, and in the absence of THUA, the growth of thin film is very poor and ineffective. Morphology of Thin Film. We have examined the morphological change of the hydrogen-bonded multilayer assembly using an AFM study. With increasing the number of layers, these films undergo a significant change in film morphology as shown in Figure 7. The corresponding Rrms values of these layers are 0.216, 0.821, and 1.0328 nm for 5, 10, and 15 layers, respectively. Figure 4. UV-vis spectrum of functionalized polymers with base pairs. (a) Functionalized chitosan by adenine (solid line). (b) Functionalized hyaluronic acid by thymine (dotted line).

change has been observed with successive adsorption of ACHI followed by THUA, but for the ACHI/HA pair, a very small increment in frequency change was observed on adsorption of ACHI followed by HA, as shown in Figure 6. The thickness of each polymer layer for these two systems was calculated based on the following equation52

d ) -(2.18 × 10-5)

∆F F

where d is the polymer film thickness and F is the polymer film density (in g · m-3). In the literature, the density of the polymer layers has been considered as (1.2 ( 0.1)106 g · m-3,53 and using the average frequency shift, the thickness can be measured with the above equation. As shown in Figure 6, the thickness of the multilayer film from the ACHI/THUA polymer pair increases with an increasing number of layers and the thickness of the

These results were in accordance with the UV-vis, QCM, and SEM results, suggesting that the successful growth of ACHI and THUA thin film based on hydrogen bonding. As we increase the number of layers, it appears that the size of the grain shape as well as roughness of the thin film is dramatically altered, as indicated by a substantial change in grain size and Rrms values of the films. Figure 7a represents the morphology of the thin film having five layers and Figure 7b and c have 10 and 15 layers, respectively. Johnston et al.54 reported two different kinds of adsorption mode for hydrogen-bonded LbL layer self-assembly of DNA; one is the “end-on” mode and another is the “flat” mode of adsorption. In our present study, multilayer thin film morphology shows an almost spherical grain nature, and with increasing the number of layers, the size of the grain also increases. It might happen that during this multilayer thin film assembly, LbL growth leads by an “endon” adsorption mechanism. Encapsulation and Release of Doxorubicin. The thin film of ACHI and THUA (10 bilayers) were incubated in a solution of doxorubicin overnight and washed several times with Millipore water to remove free DOX, as described in Experimental Section. We observed the loading of the DOX in the

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Figure 5. (a) LbL growth of multilayer thin film: all the odd layers (1, 3, 5, 7, 9, 11, 13, and 15) are THUA and all even layers (2, 4, 6, 8, 10, 12, and 14) are ACHI. Inset spectrum shows the signature of THUA; (b) SEM image of multilayer (20 layers) thin film, and the scale bar is 50 µm; and (c) the inefficient LbL growth of ACHI (odd layers 1, 3, 5, 7, 9, 11, 13, and 15) and followed by hyaluronic acid (even layers 2, 4, 6, 8, 10, 12, and 14).

any detectable release. So, the release experiments were conducted at higher pH 7.4 in PBS buffer at room temperature. We found the release rates were accelerated at pH 7.4 and a burst release of ∼48-50% was observed as shown in Figure 8. Percentage of release of DOX has been determined with the following equation

% release of DOX )

Figure 6. Layer-by-layer QCM frequency change on sequential adsorption of two complementary polymers for both ACHI/THUA and ACHI/HA systems. All odd layers (1, 3, 5, and 7) are ACHI for both systems and all even layers (2, 4, 6, and 8) are THUA and HA for the former and latter systems, respectively.

self-assembly of ACHI and THUA by the reddish color to the thin film as well as its characteristic peak at 490-500 nm in UV-visible spectroscopy (not shown here). Drug release experiments were performed to demonstrate the release of entrapped DOX and determine duration and extent of drug release from a hydrogen-bonded multilayer thin film. The DOX loaded thin film was found stable at neutral pH in water without

(c0 - ct)100 co

where co and ct are the concentration of corresponding molecules in thin film at time zero and t, respectively. This trend of pHdependent release suggests that the loaded doxorubicin remain intact in the self-assembly of ACHI/THUA at neutral pH. This interaction provides desirable environment for doxorubicin to remain intact in the self-assembly of ACHI/THUA at neutral pH. The increase in pH neutralizes electrostatic interaction between DOX and THUA, which leads to a higher percentage of DOX release. Cytotoxicity of ACHI and THUA. The modified polymers were employed in drug delivery as described earlier. It was important to retain low cytotoxicity as of its starting materials. The biocompatibility of ACHI and THUA was evaluated in vitro by MTT and live/dead staining assays. As shown in Figure 9, the viability of the human cervical carcinoma cells was close to 100% at the tested concentration of polymers after 4 h of incubation. These results demonstrate the nontoxicity of modified polymers.

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Figure 7. AFM images (3D image) obtained by tapping mode showing the morphology of hydrogen-bonded thin film; (a-c) morphology of thin films having 5, 10, and 15 layers.

Figure 8. Release profile of DOX in PBS buffer solution (pH 7.4) at room temperature. There could be a prospect of electrostatic interaction between DOX and negatively-charged THUA.

Conclusion In conclusion, this study demonstrates that modified polymers (ACHI and THUA) can interact with a hydrogen bonding to form a self-assembly by the LbL process. We have introduced a simple and facile one-step root for the modification of biodegradable and biocompatible polymers (hyaluronic acid and chitosan) with (adenine and thymine) nucleobases. This modification provided us a driving force for the fabrication of multilayer thin films based on hydrogen bonding. The multilayer thin film exhibited efficient drug loading and release in physiological conditions, rendering the potential as a drug carrier for systematic drug delivery. The pH-dependent drug delivery could be exploited for controlled drug delivery applications. Thus, the simple modification of chitosan and hyaluronic acid

Figure 9. Cell viability of human cervical carcinoma cells incubated with ACHI and THUA after 4 h of incubation.

represents an easy and novel routes for the fabrication of selfassembly based on hydrogen bonding. This multilayer thin film can be used as a carrier for delivering important pharmaceutical drugs. Acknowledgment. The work was supported by research grants from the Department of Science and Technology, India. The authors thank Nano centre and Veeco-India Nanotechnology Laboratory for assistance with SEM and AFM measurements. Mr. Sounik Saha is thanked for assistance in cytotoxicity measurements.

References and Notes (1) Ojha, S. S.; Stevens, D. R.; Hoffman, T. J.; Stano, K.; Klossner, R.; Scdt, M. C.; Krause, W.; Clarke, L. I.; Gorga, R. E. Biomacromolecules 2008, 9, 2523.

Self-Assembly of Modified Hyaluronic Acid/Chitosan (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29)

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