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Surface Fabrication of Hollow Microspheres from N-Methylated Chitosan Cross-Linked with Gultaraldehyde Xianghong Peng†,‡ and Lina Zhang*,† Department of Chemistry, Wuhan University, Wuhan 430072, China, and College of Chemistry and Environmental Engineering, Jianghan University, Wuhan 430056, China Received September 16, 2004 We have successfully prepared biocompatible and biodegradable hollow microspheres with sizes between 2 and 5 µm using cyclohexane droplets as a template and the N-methylated chitosan (NMC) cross-linked with gultaraldehyde (GA) as the shell. The structure, morphology, and formation process of the hollow microspheres were characterized by FT-IR, 1H and 13C NMR, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The results revealed that the microspheres exhibited a very smooth and hollow structure. This work confirmed that the hollow microspheres were accomplished by fabricating on the basis of chemical cross-linking on the surface of the emulsion droplets and by removing cyclohexane as core. The results from SEM and TEM indicated that the emulsion droplets covered with cross-linked NMC in the oil-in-water system aggregated together to form a precipitate of microspheres by coagulating with acetone. Moreover, the cross-linked NMC on the surface of the microspheres continuously cured to form the tight shell, whereas the inner area became a cavity with increase of the aging time, leading to the hollow microspheres. In addition, an anti-infective drug, ofloxacin (Floxin), encapsulated in the microspheres more rapidly released to reach 90 wt % at pH 7.4 within 8 h than at pH 1.2.
Introduction Recently, hollow microspheres as a typical example of colloid species have attracted great attention because of a variety of applications such as delivery vesicles for drugs, dyes, and inks, the microcapsules of artificial cells, and protection of proteins, enzymes, DNA, and catalysis.1-8 Preparation of hollow spheres often needs a template such as polymer beads,9-13 colloids,14 surfactant vesicles,15,16 and emulsions droplets17-20 or requires a self-assembly process using molecules with special structures such as * To whom correspondence may be addressed: e-mail,
[email protected]; tel, +86-27-87219274; fax, +86-2768756661. † Wuhan University. ‡ Jianghan University. (1) Huang, H.; Remsen, E. E.; Kowalewski, T.; Wooley, K. L. J. Am. Chem. Soc. 1999, 121, 3805. (2) Sukhorukov, G.; Da¨hne, L.; Hartmann, J.; Donath, E.; Mohwald, H. Adv. Mater. 2000, 12, 112. (3) Clark, C. G.; Wooley, K. L. In Dendrimers and Other Dendritic Polymers; Frechet, J. M. J., Tomalia, D. A., Eds.; John Wiley & Sons Ltd. Press: New York, 2001; p 166. (4) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev. 2001, 47, 113. (5) Liggins, R. T.; Burt, H. M. Adv. Drug Delivery Rev. 2002, 54, 191. (6) Du, J.; Chen, Y.; Zhang, Y.; Han, C.; Fischer, K.; Schmidt, M. J. Am. Chem. Soc. 2003, 125, 14710. (7) Jang, J.; Oh, J. H. Adv. Mater. 2003, 15, 977. (8) Chu, L.; Yamaguchi, T.; Nakao, S. Adv. Mater. 2002, 14, 386. (9) Caruso, F.; Spasova, M.; Salgueirin˜o-Maceira, V.; Liz-Marza´n, L. M. Adv. Mater. 2001, 13, 1090. (10) Zhang, Y.; Guan, Y.; Yang, S.; Xu, J.; Han, C. Adv. Mater. 2003, 15, 832. (11) Bao, J.; Liang, Y.; Xu, Z.; Si, L. Adv. Mater.2003, 15, 1832. (12) Wei, Z.; Wan, M. Adv. Mater. 2002, 14, 1314. (13) Fujikawa, S.; Kunitake, T. Langmuir 2003, 19, 6545. (14) Caruso, F.; Caruso, R. A.; Mohwald, H. Science 1998, 282, 1111. (15) Hubert, D. H. W.; Jung, M.; German, A. L. Adv. Mater. 2000, 12, 1291. (16) Schmidt, H. T.; Ostafin, A. E. Adv. Mater. 2002, 14, 532. (17) Walsh, D.; Lebeau, B.; Mann, S. Adv. Mater. 1999, 11, 324. (18) Brusinsma, P. J.; Kim, A. Y.; Liu, J.; Baskaran, S. Chem. Mater. 1997, 9, 2507. (19) Velev, O. D.; Furusawa, K.; Nagayama, K. Langmuir 1996, 12, 2374. (20) Tiarks, F.; Landfester, K.; Antonietti, M. Langmuir 2001, 17, 908.
amphiphilic diblock polymers.21-29 In the template method, the target material is precipitated or polymerized on the surface of the template, and the template was removed to form a cavity, leading to a core-shell structure.30 The hollow spheres are generally fabricated by inorganic material of silica or non-silica oxides directly deposited on the surface of the polystyrene templates.31-33 However, fabrications of biocompatible and biodegradable hollow microspheres from natural polymers and their derivatives have been scarcely published. It is important to develop a new methodology for the preparation of shell cross-linked hollow microsphere using natural materials for a sustainable development and human safety. As biodegradable materials, polysaccharides have been extensively used in medical and pharmaceutical areas, so it would be advantageous to use as drug formulations.34 It is well-known that chitosan, a resource from the ocean, has found wide application in various areas as a result of its biocompatibility and nontoxicity, especially in the pharmaceutical and biomedical fields, such as drug delivery.35-38 N-Methylated chitosan (NMC), a derivative of chitosan, is interesting in view of pharmaceutical (21) Spatz, J. P.; Herzog, T.; Mobmer, S.; Ziemann, P.; Moller, M. Adv. Mater. 1999, 11, 149. (22) Putlitz, B.; Landfester, K.; Fischer, H.; Antonietti, M. Adv. Mater. 2001, 13, 500. (23) Zhang, L.; Eisenberg A. J. Am. Chem. Soc. 1996, 118, 3168. (24) Ro¨sler, A.; Vandermeulen, G. W. M.; Klok, H. A. Adv. Drug Delivery Rev. 2001, 53, 95. (25) Fuhrhop, J. H.; Helfrich, W. Chem. Rev. 1993, 93, 1565. (26) Siu, M.; He, C.; Wu, C. Macromolecules 2003, 36, 6588. (27) Croll, L. M.; Sto¨ver, H. D. H. Langmuir 2003, 19, 10077. (28) Alam, M. M.; Zhu, Y.; Jenekhe, S. A. Langmuir 2003, 19, 8625. (29) Alexandridis, P.; Zhou, D.; Khan, A. Langmuir 1996, 12, 2690. (30) Okahata, Y.; Noguchi, H.; Seki, T. Macromolecules 1986, 19, 493. (31) Jang, J.; Ha, H. Langmuir 2002, 18, 5613. (32) Thurmond, K. B.; Kowalewski, T.; Wooley, K. L. J. Am. Chem. Soc. 1997, 119, 6656. (33) Wu, M.; Wang, G.; Xu, H.; Long, J.; Shek, F. L. Y.; Lo, S. M. F.; Williams, I. D.; Feng, S.; Xu, R. Langmuir 2003, 19, 1362. (34) Qiu, X.; Leporatti, S.; Donath E.; Mo¨hwald, H. Langmuir 2001, 17, 5375. (35) Ravi Kumar, M. N. V. React. Funct. Polym. 2000, 46, 1. (36) Dodane, V.; Vilivalam, V. D. PSTT 1998, 1, 246.
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applications because of the amphiphilic and water-soluble character at physiological pH caused by hydrophilic groups N+(CH3)3 and hydrophobic groups N(CH3)2.39 Moreover, NMCs have been proved to be effective and safe for mucosal delivery of hydrophilic macromolecules such as peptide and protein drugs40,41 and used as gene delivery vectors42 and antibacterial activities.43 In the present paper, we report the preparation and characterization of hollow spheres using the cyclohexane droplets as a template and NMC cross-linked with glutaraldehyde (GA) as the shell. We chose an oil-in-water emulsion system to prepare the microspheres, because it has wide variability in both composition and physical parameters and potential for industrialization. The investigation of structure and the surface-fabrication process of the hollow microspheres may contribute some meaningful information to understand the formation mechanism of a very smooth surface and to establish a methodology of preparation of biocompatible hollow spheres for application in biomedical areas. Experimental Section Materials and Sample Preparation. All the chemical reagents were commercially obtained from China. Chitosan (CS) with weight-average molecular weight (Mw) of 7.08 × 104 and 93% deacetylation degree was purchased from Yuhuan Ocean Biochemistry Co., Ltd., in Zhejiang, China. An anti-infective drug Ofloxacin (Floxin) was a gift from Kunshang Double-Crane Pharm Co. in Jiangsu. Gultaraldehyde (GA), 1-methyl-2-pyrrolidone, and methyl iodide were supplied by Shanghai Chemical Reagent Co. in Shanghai. All chemicals were of analytical grade. N-Methylated chitosan (NMC) was prepared according to the procedure described by Sieval et al.39 The resulting NMC was washed with ethanol and ether and vacuum-dried at 40 °C for 8 h. Mw of the NMC was determined by size exclusion chromatography combined with laser light scatting (SEC-LLS, DAWNDSP, Wyatt Technology Co.) to be 7.36 × 104. The degree of quaterization was 14.1% according to Thanou’s method.40 Microspheres were prepared from NMC by cross-linking with GA on the surface of the emulsion droplets. A 0.5 g portion of NMC powder was dissolved in 18 mL of distilled water, and then 6 mL of cyclohexane as the “oil phase” was added. The mixture was stirred at 700 rpm and at 40 °C for 1 h to form oil-in-water (O/W) emulsion. After addition of 0.5 mL of glutaraldehyde (GA) aqueous solution (25 wt %), the system was stirred at 300 rpm for 30 min to yield the emulsion droplets covered with cross-linked NMC. The resulting products were precipitated by coagulating with acetone, coded as NMC-g. To prepare the microspheres encapsulating Floxin, 1 mL of 15 wt % Floxin acetic acid solution was added to the above system before emulsion, and other steps were the same as those for NMC-g. The one encapsulating Floxin was coded as NMF-g. The precipitate NMC-g was washed immediately twice with acetone to form the solid state and coded as NMC-s. The precipitate NMC without GA was coded as NMC-n. The emulsion containing Floxin and GA was centrifuged for 10 min at 3500 rpm to destroy the droplet and precipitated to get sample without cyclohexane as the core coded as NMC-f. All samples were aged 2-28 days before characterization. The emulsion droplets were aged for 1-3 h for transmission electron microscopy (TEM) measurement. Characterization. FT-IR spectra of Floxin, CS, NMC, NMCg, and NMF-g were recorded with a spectrometer (170SX, Nicolet, (37) Rabea, E. I.; Badawy, M. E. T.; Stevens, C. V.; Smagghe, G.; Steurbaut, W. Biomacromolecules 2003, 24, 1457. (38) Zhang, L.; Guo, J.; Peng, X.; Yong, J. J. Appl. Polym. Sci. 2004, 92, 878. (39) Sieval, A. B.; Thanou, M.; Kotzej, A. F.; Verhoef, J. C.; Brussee, J.; Junginger, H. E. Carbohydr. Polym. 1998, 36, 157. (40) Thanou, M. M.; Kotzej, A. F.; Scharringhausen, T.; Luessen, H. L.; de Boer, A. G.; Verhoef, J. C.; Junginger, H. E. J. Controlled Release 2000, 64, 15. (41) Van der Lubbe, I. M.; Verhoef, J. C.; Borchard, G.; Junginger, H. E. Eur. J. Pharm. Sci. 2001, 14, 201. (42) Tahanou, M.; Florea, B. I.; Geldof, M.; Junginger, H. E.; Borchard, G. Biomaterials 2002, 23, 153. (43) Jia, Z.; Shen, D.; Xu, W. Carbohydr. Res. 2001, 333, 1.
Figure 1. FT-IR spectra of CS, NMC, NMC-g, NMF-g, and Floxin. Minnesota). 1H NMR and 13C NMR spectra for NMC in a mixed solvent of CF3COOD and D2O were recorded on a Mercury 300 and 600 NMR spectrometer (Varian, Inc., USA), respectively. The morphology observation of the samples was carried out on a scanning electron microscope (SEM, S-570, Hitachi, Japan). The samples were coated with gold to be observed and photographed at a desired time. The images of emulsion droplets were observed with a transmission electron microscope (TEM-100CXII, JEOL Ltd, Japan), where the carbon-coated copper grids were directly dipped into the emulsion. TEM images were examined at an accelerating voltage of 80 kV. The ultrastructure of the microspheres was observed with a TEM (H-600, Hitachi, Ibaraki, Japan) with 75 kV as the accelerating voltage. In addition, the microspheres were fixed with 1 wt % OsO4, embedded in Epon812 resin, and then cut into ultrathin sections with thickness of about 70 nm. The ultrathin sections were stained with plumb citrate before TEM observation. Release Experiments. All release experiments were performed in a shaker incubator (SHA-B GuoFA, China) at 100 rpm. The desired quantity of microspheres NMF-g (0.2 g) was suspended in 200 mL of release medium at 37 °C. The phosphate buffer solution (pH 7.4) and 0.1 M HCl solution (pH 1.2) were used as the releasing media, respectively. At the desired time interval, the microsphere dispersions were centrifuged for 10 min at 3000 rpm, and the supernatant was assayed for drug release by UV spectroscopy (2401-PC, Shimadzu Japan) to measure its absorbance at 293 nm.
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Figure 4. TEM image of microspheres of NMF-g (a) and SEM image of broken microspheres of NMF-g (b) aged for 28 days.
Figure 2. 1H NMR and 13C NMR spectra of NMC in F3CCOOD/ D2O.
Figure 5. TEM images of the emulsion droplets containing Floxin aged for 1-3 h in emulsion.
Figure 3. SEM images of the microspheres NMC-g (a) and NMF-g (b) aged for 28 days.
Results and Discussion Structure and Morphology of Hollow Microspheres. The FT-IR spectra of the samples CS, NMC, NMC-g, NMF-g, and Floxin are shown in Figure 1. The characteristic peaks at 1654 and 1596 cm-1 are assigned to the amide I and II band of CS, respectively. It is worth noting that the peak at 1596 cm-1 for primary amine N-H bending almost disappears in NMC, as a result of methylation at C-2 of glucosamine unit. The peaks at 1481 and 2928 cm-1 (-CH3) indicate that the -N(CH3)2 and -N+(CH3)3 groups exist in the NMC. For the microspheres from NMC cross-linked with GA, peaks at 1713 cm-1 (CdN) and 2940 cm-1 (-CH2) appear, suggesting a crosslinked structure of NMC-g as a result of the reaction between -CHO groups of GA and -NH2 and -NHCH3 groups of NMC. The peaks at 1620, 1475, and 1054 cm-1 of Floxin appear in the FT-IR spectrum of NMF-g, indicating that there is no chemical reaction between crosslinked NMC and Floxin. The 1H NMR and 13C NMR spectra of NMC in F3CCOOD/D2O are shown in Figure 2. The peak at 3.2 ppm is assigned to -N(CH3)2, -NHCH3, and
Figure 6. SEM images of NMF-g samples aged for (a) 2 days, (b) 7 days, (c) 10 days, and (d) 28 days.
-NH2, and a strong single peak at 3.4 ppm is assigned to -N+(CH3)3. The peaks at 44.0 and 50.0 ppm in 13C NMR are assigned to -N(CH3)2 and -N+(CH3)3 groups,39 respectively. These results indicate that amino groups of chitosan have been methylated to changed into -N+(CH3)3, -N(CH3)2, and -NHCH3, and that -NH2 and -NH(CH3) of NMC are converted into Schiff base and enamines
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Figure 7. SEM images of samples NMC-n (a), NMC-s (b), and NMC-f (c) aged for 28 days.
Figure 8. Illustration of the formation of the hollow microspheres.
reacted with GA. In view of the results from IR and NMR, it is believed that the NMC has been cross-linked with GA in the microspheres NMC-g and NMF-g and that the drug Floxin has been encapsulated in NMF-g microspheres. Figure 3 shows SEM images of the microspheres of NMC-g and NMF-g aged for 28 days. Obviously, the microspheres with and without drug all exhibit a very smooth surface, indicating that cross-linked NMC has formed a tight shell. The size of the microspheres lies in the range from 2.0 to 5.0 µm for NMC-g and 2.5 to 5.5 µm for NMF-g, respectively. The average size (3.5 µm) of the microspheres NMC-g without Floxin is smaller than that (4.0 µm) of the microspheres of NMF-g containing the drug. This can be explained that the drug Floxin is dissolved in water phase of emulsion, and has been encapsulated into the cross-linked shell of the microspheres to expand the volume during the formation of the microspheres. To clarify the hollow structure of the microspheres, TEM of a cross section of the NMF-g microspheres aged for 28 days has been observed, and the image is shown in Figure 4a. Obviously, the microsphere NMF-g exhibits a hollow structure. The SEM image of a broken microsphere is shown in Figure 4b. This further confirms the hollow microsphere having a thin wall. To further clarify the formation process of the microspheres, we have observed the TEM images of the emulsion droplets in the emulsion system and the SEM images of the microspheres NMF-g at different aging times. Figure 5 shows the TEM images of the emulsion droplets containing Floxin in the emulsion system. The average
size of the droplets is 3.9 µm, close to that of the hollow microspheres NMF-g. It is regarded that during the preparation, the amphiphilic NMC has been adsorbed on the surface of the cyclohexane droplets to form the coreshell emulsion droplets in the emulsion system. When GA as a bifunctional cross-linker was added, the -NH2 of NMC more easily reacts with GA to from the crosslinked NMC on the surface of emulsion droplets. However, only one aldehyde group of predominant GA is cross-linked with NMC, and the other hydrophilic -CHO group remains untapped.19,44 Therefore, the precipitate of the microspheres first forms aggregates as shown in Figure 6a. With an increase of the aging time, the fully crosslinked NMC formed as a result of continuous reaction of the untapped -CHO groups of GA with NMC on the surface. Moreover, cyclohexane in the inner of the microspheres was evaporated, resulting in a cavity. The moisture and the air gradually enter into the cavity to expand the tight shell, leading to the hollow microspheres as shown in panels b and c of Figure 6. The fully crosslinked NMC was continuously cured to form a more tight shell and a smooth surface during the aging time of 28 days, as shown in Figure 6d. To prove that the cross-linking agent GA is a key factor in the preparation of the hollow microspheres, a sample of NMC-n without GA in the emulsion system has been made, and a SEM image is shown in Figure 7a. There are only porous gels and no microspheres, even if the sample has been aged for 28 days. In addition, emulsion droplets (44) Juang, R.; Wu, F.; Tseng, R. L. Adv. Environ. Res. 2002, 6, 171.
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form fully cross-linked networks as the shell. Moreover, the cyclohexane inside has been removed to form the cavity. With an increase of the aging time, moisture and air enter the cavity to expand the shell, resulting in the hollow microspheres with a very smooth surface, as shown in Figure 8c, corresponding to the SEM images in Figures 3a,b, 4b, and 6c,d and the TEM image in Figure 4a. Release of Drug. The in vitro release behavior of Floxin from the microspheres NMF-g is shown in Figure 9. Interestingly, the drug in the microspheres more speedily releases in pH 7.4, to reach 90 wt % within 8 h, than in pH 1.2 media. Only 46 wt % of the drug is released in pH 1.2 media within 4 h, corresponding to the gastric emptying time. Obviously, the drug encapsulated in the microspheres has sustained release properties, and the release of the microspheres is pH sensitive. This work provides hollow microspheres that possess potential applications in the biomedical and biomaterial fields. Figure 9. Time dependence of Floxin release from microspheres in a phosphate buffer solution (9, pH 7.4) and 0.1 M HCl solution (b, pH 1.2)
containing cyclohexane are also an important factor. Fresh NMC-g precipitate was washed twice with acetone to remove cyclohexane from the core, or the emulsion droplets were destroyed by centrifugation for the samples NMC-s and NMC-f. There are no hollow microspheres for NMC-s and NMC-f even after 28 days of aging time, as shown in panels b and c of Figure 7. These results further confirm that the technology of fabricating with cross-linked NMC on the surface of emulsion droplets is feasible. The crosslinked NMC as the shell on the emulsion droplets and the cyclohexane as the core play an important role in the formation of the hollow microspheres. On the basis of the information from FT-IR, 1H NMR, 13C NMR, SEM, and TEM, a schematic illustration of the formation of the hollow microspheres is presented in Figure 8. With the cross-linked NMC as the shell and the cyclohexane as the core, droplets in the O/W emulsion have formed, as shown in Figure 8a, corresponding to the TEM image in Figure 5. With addition of acetone, the emulsion droplets aggregated together to form a precipitate of microspheres, as shown in Figure 8b, corresponding to the SEM image in Figure 6a. The cross-linked NMC on the surface of the microspheres continuously reacted to
Conclusions Novel hollow microspheres were successfully prepared using cyclohexane droplets in water as a template and cross-linked NMC as the shell. The cross-linked reaction between NMC and GA on the surface of emulsion droplets plays an important role in the formation of hollow microspheres with a very smooth surface. Results from SEM and TEM revealed the formation process of hollow microspheres. The emulsion droplets covered with NMC cross-linked with only one aldehyde groups of predominant GA first aggregated to form the gels of microspheres. With an increase of the aging time, a fully cross-linked NMC formed on the surface and the cyclohexane inside was removed to create the cavity. Moreover, moisture and air gradually entered the cavity resulting in shell expansion to form hollow microspheres with a tight and smooth shell. Such biocompatible and biodegradable hollow microspheres exhibited certain release behavior and pH sensitivity. Acknowledgment. This work was supported by the National Natural Science Foundation of China (20074025) and the Foundation of Science and Technology Bureau of Wuhan (20015007090). LA047689W
