Biomacromolecules 2005, 6, 2521-2527
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Preparation and Characterization of Chitosan-Based Nanoparticles Magdolna Bodnar,† John F. Hartmann,‡ and Janos Borbely*,†,§ Department of Colloid and Environmental Chemistry, University of Debrecen, H-4010 Debrecen, Hungary, ElizaNor Polymer LLC, Princeton Junction, New Jersey 08550, BBS Nanotechnology Ltd., H-4225 Debrecen, P. O. B. 12, Hungary Received March 25, 2005; Revised Manuscript Received June 13, 2005
The present investigation describes the synthesis and characterization of novel biodegradable nanoparticles based on chitosan for biomedical applications. Natural di- and tricarboxylic acids were used for intramolecular cross-linking of the chitosan linear chains. The condensation reaction of carboxylic groups and pendant amino groups of chitosan was performed by using water-soluble carbodiimide. This method allows the formation of polycations, polyanions, and polyampholyte nanoparticles. The prepared nanosystems were stable in aqueous media at low pH, neutral, and mild alkaline conditions. The structure of products was determined by NMR spectroscopy, and the particle size was identified by laser light scattering (DLS) and transmission electron microscopy (TEM) measurements. It was found that particle size depends on the pH, but at a given pH, it was independent of the ratio of cross-linking and the cross-linking agent. Particle size measured by TEM varied in the range 60-280 nm. In the swollen state, the average size of the particles measured by DLS was in the range 270-370 nm depending on the pH. The biodegradable cross-linked chitosan nanoparticles, as solutions or dispersions in aqueous media, might be useful for various biomedical applications. Introduction Chitosan is a renewable biomaterial, β-[1 f 4]-2-amino2-deoxy-D-glucopyranose, a functional and basic linear polysaccharide, and is prepared from chitin, the second most abundant biopolymer in nature. Chitosan is prepared by N-deacetylation of chitin, resulting in a copolymer of β-[1 f 4]-linked 2-acetamido-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-glucopyranose (Figure 1). The degree of deacetylation (DD) and molecular weight of chitosan have the greatest influence on its physical and chemical properties, including emulsification capacity,1 aggregation activity2, rheological,3 and solution4 and physicochemical properties.5 A limiting factor in the application of chitosan is its low solubility in aqueous media at neutral and alkaline conditions and in most organic solvents. However, a variety of studies have focused on altering the water solubility of chitosan material in aqueous solution by employing water-soluble linkages such as phosphates,6 sulfates,7 cyanates,8 and other agents,9,10 and by degrading high-molecular-weight chitosan. Low-molecular-weight chitosan has been prepared by depolymerization in several ways,11,12 which has resulted in polymers with special physical and physicochemical properties. These include an increased solubility in neutral water or organic solvent and greater biocompatibility13 and biodegradability. Currently, because of its special set of proper* To whom correspondence should be addressed. Fax: +36-52-512938. E-mail:
[email protected]. † University of Debrecen. ‡ ElizaNor Polymer LLC. § BBS Nanotechnology Ltd.
Figure 1. Chemical structure of chitosan.
ties, which include low or no toxicity, biocompatibility, biodegradability, low immunogenicity, and antibacterial properties, chitosan has found wide application in a variety of areas, such as biomedicine,14-18 pharmaceuticals,19,20 metal chelation,21,22 food additives,23 and other industrial applications.24,25 Various methods have been developed for the cross-linking of chitosan, which commonly result in gel formation. Hydrogels can be formed by covalently cross-linking chitosan with itself. In this type of cross-linking reaction, cross-linkers are molecules with at least two reactive functional groups that allow the formation of bridges between polymeric chains. The most common cross-linkers of chitosan are aldehydes,26,27 epoxides,28 cyanates,29 and other agents.30,31 Ionic cross-linking reactions with charged ions32 or molecules14,15,33,34 have also been employed by using ionotropic gelation methods to form hydrogels based on chitosan. Hydrogels have been utilized in a wide range of biomedical application, such as enzyme immobilization,35,36 scaffolds37 and carriers for drugs,38 or implants.14-17 Many recent attempts have been made to create chitosan particulate systems. Ionotropic gelation methods are based on the interaction between chitosan and different anions.39 One type of this method is performed in a w/o emulsion.
10.1021/bm0502258 CCC: $30.25 © 2005 American Chemical Society Published on Web 07/27/2005
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The emulsion cross-linking method utilizes the reactive functional groups of chitosan to cross-link with a crosslinking agent.40 By this method, the size of the particles can be controlled by controlling the size of droplets; however, the size of the particles is large, and they have a broad size range. Preparation of ultrafine polymeric nanoparticles with narrow size distribution could be achieved by using reverse micellar medium. Reverse micelles are thermodynamically stable systems, but they are also a liquid mixture of water, oil, and surfactant. Coacervation is another type of ionotropic gelation method.41 This process avoids the use of toxic organic materials as cross-linking agents; however, the prepared chitosan particles are poorly soluble in aqueous media. Covalently cross-linked chitosan nanoparticles can be prepared by several different methods: emulsion crosslinking,42 reverse micellar,43 solvent evaporation,44 spraydrying,45 or thermal cross-linking.46 The solvent evaporation method can be performed in a w/o emulsion, and the aqueous phase is removed by evaporation at high temperature. Spraydrying is a well-known technique to produce a cross-linked chitosan suspension. This method is based on drying of finely dispersed droplets of chitosan solution in a stream of hot air followed by the addition of a cross-linking agent. Thermal cross-linking is a very simple method for preparing chitosan particles; however, high temperature is necessary to establish cross-linking. Chitosan nano- and microsystems can be employed in a wide range of biomedical application, such as drug-38,39,43 or gene-delivery systems.47,48 The present investigation reports a method for the preparation of nanoparticles based on chitosan by covalently cross-linking via the amino groups of the chitosan chain with natural di- or tricarboxylic acids in aqueous media at room temperature. The solubility, structure, and size of these nanoparticles in the dried and swelled states will be described and discussed. Cross-linked chitosan nanoparticles may dissolve or form stable colloid systems in aqueous media at acidic and neutral pH. Because they are nanosized at neutral pH, they can be attractive candidates for delivery biosystems for a variety of biomedical applications. Experimental Section Materials. Chitosan (degree of deacetylation ) 88%, Mv ) 320 000) was purchased from Sigma-Aldrich Co., Hungary. It was dissolved in 2% aqueous acetic acid solution to give a polymer concentration of 1% w/w and then filtered and dialyzed against distilled water until the pH was neutral. The product was dried by lyophilization to obtain a white chitosan material and used for further experiments. Succinic acid, malic acid, tartaric acid, and citric acid-1-hydrate were purchased from Sigma-Aldrich Co., Hungary, and were used as received without further purification. Water soluble 1-[3(dimethylamino)propyl]-3-ethylcarbodiimide methiodide (CDI) was applied as a condensation agent. Determination of Degree of Deacetylation (DD) and Molecular Weight. The DD of the chitosan material employed has been determined from integral intensities of all protons by its 1H NMR spectrum.49 The measurement
Bodnar et al.
Figure 2. Synthesis of chitosan derivatives.
was performed in D2O containing a few drops of 20% w/w DCl/D2O and trace DSS as a reference. The viscosity-average molecular weight was determined by measuring relative viscosity with an Ostwald viscometer. The solvent system used was 0.1 M CH3COOH/0.2 M NaCl. Molecular weight was calculated from the intrinsic viscosity based on the Mark-Houwink equation. The values for the constants K and a were 1.81 × 10-3 and 0.93.50 Chitosan Modification. Cross-linked chitosan nanoparticles were prepared by the CDI technique. Di- and tricarboxylic acids (tartaric-, malic-, succinic-, citric acid) were used as cross-linking agents. The overall synthetic route for the modification of chitosan is summarized in Figure 2. Synthesis of Cross-Linked Chitosan Nanoparticles. Carboxylic acid was dissolved in water and then adjusted to pH 6.5 with 0.1 M sodium hydroxide solution. After the addition of water-soluble carbodiimide, the reaction was stirred at 4 °C for 30 min and subsequently mixed with purified chitosan dissolved in water. The reaction mixture was stirred at room temperature for 24 h. The solution containing chitosan nanoparticles was purified by dialysis for 7 days against distilled water and freeze-dried.51 Synthesis of cross-linked chitosan nanoparticles with natural carboxylic acids at diverse stoichiometric crosslinking ratios was accomplished according to the described reaction conditions. The data of the synthesis are summarized in Table 1. Characterization. NMR Spectroscopy. The chitosan and the cross-linked systems were analyzed structurally with NMR spectroscopy. 1H, 1H-1H correlation spectroscopy (COSY), and 1H-13C HETCOR NMR spectra were obtained on a Bruker AM500 MHz instrument. The samples were dissolved in D2O containing a few drops of 20% w/w DCl/ D2O. The chemical shifts were represented in parts per million (ppm) based on the signal for sodium 3-(trimethylsilyl)-propionate-d4 as a reference. Transmission Electron Microscopy (TEM). A JEOL2000 FX-II transmission electron microscope was used to characterize the size and morphology of the dried chitosan nanoparticles. For TEM observation, the chitosan nanoparticles were prepared from the reaction mixture after dialysis at a concentration of 100 µg/mL. The colloid dispersion was sonicated for 3 min to produce better particle dispersion on the copper grid. The sample for TEM analysis
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Chitosan-Based Nanoparticles Table 1. Reaction Condition of Synthesis of Chitosan Nanoparticles stoichiometric ratio of cross-linking agent
bulk of carb. acid (g)
bulk of CDI (g)
succinic acid
25% 50% 100%
0.081 0.161 0.322
0.406 0.811 1.623
malic acid
25% 50% 100%
0.092 0.183 0.366
0.406 0.811 1.623
tartaric acid
25% 50% 100%
0.102 0.205 0.410
0.406 0.811 1.623
citric acid 1-hydrate
25% 50% 100%
0.143 0.287 0.573
0.406 0.811 1.623
carboxylic acid
was obtained by placing a drop of the colloid dispersion containing the chitosan nanoparticles onto a carbon-coated copper grid. It was dried at room temperature and then examined using a TEM without any further modification or coating. Mean diameters and the size distribution of diameters were obtained from measured particles visualized by TEM images and then analyzed by using the SPSS 11.0 program file. Laser Light Scattering (DLS). The hydrodynamic diameters of cross-linked chitosan nanoparticles were gauged by using a BI-200SM Brookhaven Research laser light scattering photometer equipped with a Nd:YAG solid-state laser at an operating wavelength of λo ) 532 nm. Measurements of the average size of nanoparticles were performed at 25 °C with an angle detection of 90° in optically homogeneous quartz cylinder cuvettes. The samples were prepared from the reaction mixture after dialysis. The concentration of the chitosan derivative solutions was 100 µg/mL and was sonicated for 1 min. Each sample was measured three times, and average serial data were calculated. Transmittance. Transmittances of chitosan nanoparticle solutions were measured by using a Unicam SP 1800 ultraviolet spectrophotometer at an operating wavelength of λ ) 480 nm in optically homogeneous quartz cuvettes. Measurement of the samples was performed from the reaction mixture after dialysis at 25 °C. The concentration of the chitosan derivative solutions was 1 mg/mL. Results and Discussion Formation of Nanoparticles. Chitosan is a weak base with a pKa value of the D-glucosamine residue of about 6.27.0 and, therefore, has low solubility at neutral and alkaline pH values. In acidic medium, the amino groups are positively charged, resulting in a highly charged polyelectrolyte polysaccharide. At low charge density, polyelectrolyte chains collapse in a compact globule. At high charge density, polyelectrolytes have an extended coil conformation (I) (Figure 3). The coil-globule transition was influenced by the attraction and repulsion interaction between the polymer segments.
Polycation (II), polyanion (V), polyampholyte (IV), or uncharged (III) cross-linked nanoparticles were prepared by chemical modification of chitosan linear chains (I) using diand trifunctional carboxylic acids as cross-linking agents at different ratios. Polycations (II) were obtained by reacting chitosan with dicarboxylic acids. The stoichiometric ratio of cross-linking was less than 100%. In this case, the carboxyl groups were bound covalently, and residual free amino groups of the chitosan chain were available, which can be protonated in acidic medium, and therefore, polycations were formed. Chitosan particles can act as bioadhesive cationic polyelectrolytes. Also, they are good candidates for drug- or genedelivery systems, because the positively charged chitosan can provide a strong electrostatic interaction with negatively charged molecules, such as DNA and RNA, stabilizing and protecting them from degradation. Chitosan cross-linked with dicarboxylic acid at a stoichiometric ratio of 100% resulted in uncharged nanoparticles (III) in aqueous media, because each of the functional amino and carboxyl groups were covalently bound. Polyanion (V) or polyampholyte (IV) cross-linked nanoparticles were constructed from chitosan using citric acid as the cross-linker. Citric acid, a tricarboxylic acid, was used in excess and acts as a difunctional cross-linking agent. Thus, free carboxyl groups were available after cross-linking, which can be deprotonated in neutral and alkaline media, producing partially negatively charged particles. In that case, the stoichiometric ratio of cross-linking was less than 100%. Polyampholyte nanoparticles were obtained, because free amino groups of the chitosan chains were found. Polyanions were prepared by condensation reaction of chitosan and citric acid at a stoichiometric ratio of cross-linking of 100%. This occurs because residual carboxyl groups of citric acid were available and all amino groups of the chitosan chain are bound covalently. Because this colloid dispersion was not stable, it precipitated. Solubility. The solubility of chitosan derivatives was evaluated in deionized water at pH 6.5. Solutions were either clear or opaque aqueous colloid systems and were stable at room temperature for several weeks. Table 2 summarizes the transmittance of chitosan nanoparticles obtained by crosslinking with various carboxylic acids. At lower concentrations of cross-linker, the solubility of the colloid dispersion was greater, and the solutions were either clear or opalescent. This was caused by the protonation of free amino groups of the chitosan chain. An increasing ratio of cross-linker corresponds to increasing opalescence of solutions. As the cross-linking increased, the particles became more compact. Solubility of the chitosan nanoparticles is related to the hydrophilic character of the cross-linking agents and the ratio of free amino groups of the chitosan chain. Thus, the increase in hydrophilic character of carboxylic acids used increased the solubility of the chitosan particles. Nevertheless, chitosan nanoparticles cross-linked with citric acid at a stoichiometric ratio of 100% precipitated in aqueous media, despite the presence of residual free carboxyl groups. This result is probably related to the use of a tricarboxylic acid, which reacts as a difunctional cross-linking agent stoichiometrically.
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Figure 3. Schematic structure of cross-linked polyelectrolytes based on chitosan. Table 2. Transmittance of Modified Chitosan Materials stoichiometric ratio of cross-linking agent
result
transmittance
succinic acid
25% 50% 100%
opalescent precipitate precipitate
76%
malic acid
25% 50% 100%
opalescent opalescent opalescent
85% 80% 71%
tartaric acid
25% 50% 100%
clear clear opalescent
99% 92% 88%
citric acid
25% 50% 100%
opalescent opalescent precipitate
38% 38%
carboxylic acid
Chitosan nanoparticles cross-linked with malic acid form colloid dispersions in neutral condition. These colloid dispersions are opalescent systems; the transmittance is between 70% and 85%. It was observed that, by decreasing the pH, the transmittance of cross-linked particles increased, caused by the protonation of free amino groups of chitosan chains (Figure 4.). It can be found that, depending on the pH, cross-linked chitosan nanoparticles can become a macromolecular solution. NMR Results. The structure of chitosan used for the synthesis and the obtained cross-linked nanosystems was characterized by NMR spectroscopy. The assignments and chemical shifts of the 1H and 13C NMR signals of chitosan are given as follows. Chitosan: 1H NMR (DCl/D2O) δ ) 4.9 (1-HD deacylated), δ ) 4.7 (1-HAc acylated with acetic acid), δ ) 3.2 (2-H), δ ) 3.3-4.2 (3-H, 4-H, 5-H, 6-H), δ ) 2.2 (NCOCH3). Chitosan: 13C NMR (DCl/D2O) δ ) 96.80 (1-CAc), δ ) 96.56 (1-CD), δ ) 56.21 (2-C), δ ) 70.80 (3-C), δ ) 76.11 (4-C), δ ) 74.08 (5-C), δ ) 60.05 (6-C).
Figure 4. Transmittance of chitosan nanoparticles cross-linked with malic acid at a stoichiometric ratio of 25% ([) 50% (2)100% (9).
Figure 5. 500 MHz 1H NMR spectrum of chitosan nanoparticles cross-linked with malic acid at 50%.
The assignments and chemical shifts of the 1H signals of chitosan nanoparticles cross-linked with malic acid (Ma) at 50% were determined by 1H, 1H-1H COSY and, 13C and 1 H-13C HETCOR methods. The chemical shift values are in accordance with results published for chitin and chitosan49 and their oligomers.52 Proton chemical shifts are given in Figure 5 and Figure 6. 1H NMR (DCl/D2O): δ ) 4.855.00 (two isomers, 1-HD), δ ) 4.70-4.80 (1-HAc), δ ) 4.604.65 and 4.55-4.65 (two isomers 1-HMa acylated with malic acid), δ ) 3.75-3.85 (2-HMa), δ ) 3.70-3.75 (2-HAc and
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Chitosan-Based Nanoparticles
Table 3. Average Hydrodynamic Diameter of Chitosan Nanoparticles Measured at pH 7.6 in a Buffer Solution cross-linking ratio 25% 50% 100%
Figure 6. 500 MHz 1H-1H COSY NMR spectrum of chitosan nanoparticles cross-linked with malic acid at 50%.
Figure 7. 1H-13C HSQC map of chitosan nanoparticles cross-linked with malic acid at 50% (* ) impurities).
2-HD), δ ) 3.6-4.1 (3-H, 4-H, 5-H, 6-H), δ ) 2.1 (NCOCH3, not shown), δ ) 4.6 (CHMa), and δ ) 2.9 (CH2Ma). Figure 7 shows the 1H-13C hetero single quantum correlation spectrum (HSQC) of chitosan nanoparticles crosslinked with malic acid. The 13C assignment was performed on the basis of 13C. Projection was as follows: δ ) 97.61 (1-CD), δ ) 101.21 (1-CAc and 1-CMa), δ ) 56.15 (2-C), δ ) 70.30 (3-C), δ ) 79.03 (4-C), δ ) 76.80 (5-C), δ ) 60.54 (6-C), and the signs of malic acid δ ) 68.50 (CHMa) and δ ) 39.08 (CH2-MA). The degree of cross-linking was evaluated from the integral intensity of signs by using 1H NMR spectra of cross-linked chitosan nanoparticles. In the case of 25% stoichiometric ratio, the degree of cross-linking was between 20% and 25%. By increasing the stoichiometric ratio, the degree of crosslinking increased. It was between 32% and 44% in the case
succinic acid
malic acid
tartaric acid
citric acid
290
310 300 300
330 290 300
320 340
of cross-linking at a stoichiometric ratio of 50%, and it was 45-70% at a stoichiometric ratio of 100%. Particle Size by TEM. Chitosan material can be prepared as a film; however, the cross-linked chitosan nanoparticles separated into spherical particles in an aqueous environment and in dried states. TEM micrographs (Figure 8) confirmed the nanosize of dried cross-linked chitosan particles and show the distribution of these derivatives. The size of the dried particles varied in the range 60-280 nm. Similar narrow size ranges can be observed in the case of samples A, B, and D. The diameters of chitosan cross-linked with tartaric acid resulting from the TEM experiments were smaller, which can be caused by the third dimensional aspects. The size of dried cross-linked particles was smaller, and the size range was narrower than the swollen particles obtained from DLS. Particle Size by DLS. Solution samples were prepared from the reaction mixture after dialysis. The concentration of the polysaccharide solution was 100 µg/mL. The pH of the samples was adjusted by the addition of hydrochloric acid solution in the presence of 0.2 M sodium acetate buffer. In polydispersed systems, the final results depend on the method of fitting. The average hydrodynamic diameters were calculated by the NNLS (non-negative least squares) method, which separated the different peaks at multimodal distribution and provided more exact results at multimodal systems than those obtained with other methods. The intensity-delay timecorrelation function was evaluated by means of NNLS fit; the called automatic routine was applied to determine the intensity diameter distribution. The effect of dust was canceled by the averaging of numerous simultaneous measurements. Table 3 summarizes the average hydrodynamic diameters of swelled chitosan nanoparticles. Polyelectrolytes were obtained depending on the ratio of cross-linking and on the carboxylic acid used. In case of chitosan cross-linked with dicarboxylic acids at 50%, polycations were produced. The free amino groups of these macromolecules can be protonated in acidic media, and hydrodynamic diameters increase because of the repulsive interaction. Because the conformation of polysaccharide rings also influences the swelling of cross-linked nanoparticles, weak correlations were found. The average hydrodynamic diameter is independent of the hydrophilic character of crosslinking agents or the stoichiometric ratio of cross-linking (Table 3). The average hydrodynamic diameter of single nanoparticles is between 290 and 340 nm. Figure 9 shows a size distribution by intensity of cross-linked chitosan nanoparticles. Chitosan cross-linked with citric acid at a stoichiometric ratio of 50% resulted in a narrow size distribution in aqueous media and formed a polyampholyte. Figure 10 shows the hydrodynamic diameters of crosslinked chitosan nanoparticles measured at different pHs. It
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Figure 9. Size distribution by intensity of chitosan nanoparticles cross-linked with malic acid at 25% (b) and 50% ([), with tartaric acid (9), and with citric acid (2). The size of the nanoparticles ranged from 120 to 650 nm, and the average size is between 290 and 340 nm.
Figure 10. Average hydrodynamic diameter of chitosan nanoparticles cross-linked at a stoichiometric ratio of 50% with tartaric acid (9), malic acid ([), and citric acid (2).
the cross-linker was malic acid. These macromolecules are hydrophilic. The residual amino groups can be protonated, and the repulsive interaction of positive ions increase the size of macromolecules. Polyampholyte macromolecules were obtained from chitosan cross-linked with citric acid at a stoichiometric ratio of 50%. Depending on the pH, the residual amino groups of chitosan chain were protonated or the carboxylic groups of citric acid were deprotonated. The charges of these macromolecules affect the size of the nanoparticles. After the values reported were summarized, the average hydrodynamic diameters of swelled cross-linked chitosan nanoparticles were between 270 and 370 nm. To all appearances, the nanoparticles swell in aqueous media depending on the pH, but the polysaccharide rings established a stable framework, resulting in a conformation that limits the swelling. Conclusions
Figure 8. TEM images and size distribution of chitosan nanoparticles cross-linked with malic acid at 25% (A) and 50% (B), with tartaric acid at 50% (C), and with citric acid at 50% (D).
can be seen that, by increasing the pH, average size decreased in the case of tartaric acid, but no effect was found when
We have shown that biocompatible chitosan has been successfully modified by condensation reaction using natural di- and tricarboxylic acids as cross-linking agents to form particles. Clear or opalescent stable colloid systems based on chitosan were fabricated in aqueous medium at room temperature. The size of colloid dispersion ranges from 270 to 370 nm. The average size of swelled cross-linked chitosan macromolecules was about 300 nm. The size was independent of the hydrophilic character of the cross-linker used or
Chitosan-Based Nanoparticles
the stoichiometric ratio of cross-linking, but depended on the pH exclusively. Acknowledgment. This work was supported by RET (Grant of Regional University Knowledge Center) contract number (RET-06/432/2004) and by ElizaNor Polymer LLC, U.S.A. The authors would like to thank L. Daro´czi for TEM micrographs. References and Notes (1) Rodrı´guez, M. S.; Albertengo, L. A.; Agullo´, E. Carbohydr. Polym. 2002, 48, 271-276. (2) Shimojoh, M.; Fukushima, K.; Kurita, K. Carbohydr. Polym. 1998, 35, 223-231. (3) Wang, W.; Xu, D. Int. J. Biol. Macromol. 1994, 16, 149-152. (4) Sorlier, P.; Viton, C.; Domard, A. Biomacromolecules 2002, 3, 13361342. (5) Berth, G.; Dautzenberg, H.; Peter, M. G. Carbohydr. Polym. 1998, 36, 205-216. (6) Heras, A.; Rodrı´guez, N. M.; Ramos, V. M.; Agullo´, E. Carbohydr. Polym. 2001, 44, 1-8. (7) Holme, K. R.; Perlin, A. S. Carbohydr. Res. 1997, 302, 7-12. (8) Welsh, E. R.; Schauer, C. L.; Qadri, S. B.; Price, R. R. Biomacromolecules 2002, 3, 1370-1374. (9) Sugimoto, M.; Morimoto, M.; Sashiwa, H.; Saimoto, H.; Shigemasa, Y. Carbohydr. Polym. 1998, 36, 49-59. (10) Xie, W.; Xu, P.; Wang, W.; Liu, Q. Carbohydr. Polym. 2002, 50, 35-40. (11) Tian, F.; Liu, Y.; Hu, K.; Zhao, B. Carbohydr. Polym. 2004, 57, 31-37. (12) Mao, S.; Shuai, X.; Unger, F.; Simon, M.; Bi, D.; Kissel, T. Int. J. Pharm. 2004, 281, 45-54. (13) Richardson, S. C. W.; Kolbe, H. V. J.; Duncan, R. Int. J. Pharm. 1999, 178, 231-243. (14) Berger, J.; Reist, M.; Mayer, J. M.; Felt, O.; Gurny, R. Eur. J. Pharm. Biopharm. 2004, 57, 35-52. (15) Berger, J.; Reist, M.; Mayer, J. M.; Felt, O.; Peppas, N.; A.; Gurny, R. Eur. J. Pharm. Biopharm. 2004, 57, 19-34. (16) Khor, E.; Lim, L. Y. Biomaterials 2003, 24, 2339-2349. (17) Suh, J. K. F.; Matthew, H. W. T. Biomaterials 2000, 21, 25892598. (18) Langer, R.; Tirrell, D. A. Nature (London) 2004, 428, 487-492. (19) Dodane, V.; Vilivalam, V. D. Pharm. Sci. Technol. Today 1998, 1, 246-253. (20) Rabea, E. I.; Badawy, M. E.-T.; Stevens, C. V.; Smagghe, G.; Steurbaut, W. Biomacromolecules 2003, 4, 1457-1465. (21) Guibal, E.; Von Offenberg Sweeney, N.; Zikan, M. C.; Vincent, T.; Tobin, J. M. Int. J. Biol. Macromol. 2001, 28, 401-408.
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BM0502258
