摘要
选择天然多糖及其衍生物为基质材料，利用含有相反电荷的聚电解质之间的静电相互作用为原理，在室温和水相的温和条件下，制备了五种新型的纳米载药体系，分别为：羧甲基壳聚糖／壳聚糖纳米粒子、肝素／壳聚糖纳米粒子、羧甲基壳聚糖／磷酸化壳聚糖纳米粒子、肝素／磷酸化壳聚糖纳米粒子和磷酸化壳聚糖／DNA纳米粒子。作为前期研究的探索和比照，还制备了Ca~(2+)交联的羧甲基壳聚糖凝胶珠体系。
使用红外光谱、激光光散射和透射电镜等仪器对各种纳米载药体系的基本性能进行了表征，如纳米粒子复合物的形成、纳米粒子的粒径及其分布、表面电荷性能、纳米粒子的形态结构等。进一步考察了基质材料的相对分子质量、溶液浓度、溶液的加入方式、负载药物或蛋白质的浓度、存放介质的pH值和离子浓度等因素对纳米粒子的粒径及其分布、表面电荷性能、包封率和稳定性的影响。实验结果表明，天然多糖及其衍生物的相对分子质量和其溶液浓度是影响纳米粒子尺寸的重要因素。通过选择适当的相对分子质量的基材和溶液浓度，可以将纳米粒子的粒径有效地控制在100～500nm之间。药物的包封率是衡量纳米载药体系性能的重要指标。同时升高纳米体系二元组分的浓度，能够生成更多的纳米粒子，是提高纳米粒子对药物和蛋白质的包封率的有效途径。
进一步研究了牛血清白蛋白纳米载药体系的释放性能和阿霉素纳米载药体系在体外条件下对HeLa细胞的抑制功能，获得了令人满意的结果。评价了磷酸化壳聚糖对质粒DNA的负载能力、保护作用和转染HeLa细胞的能力，研究结果表明，磷酸化壳聚糖是一种有效的基因载体，具有深入开发的潜力。
In this study, natural polysaccharides and their derivatives were used to prepare nanoparticle drug delivery systems. The preparation was based on the static electric interaction between positive polyelectrolyte and negative polyelectrolyte and was conducted on mild conditions, i.e. at room temperature and in aqueous solutions. The obtained five nanoparticle drug delivery systems were carboxymethyl chitosan/chitosan, heparin/chitosan, carboxymethyl chitosan/phosphonic chitosan, heparin/phosphonic chitosan and phosphonic chitosan/DNA nanoparticles. In preliminary experiments, we prepared calcium-crosslinked carboxymethyl chitosan hydrogel beads.
By means of IR, TEM and zeta potential analyzer etc., the physicochemical characteristics of these drug delivery systems were characterized, including the formation mechanism, the particle size, polydispersity, zeta potential and morphology of the nanoparticles. The pH and ion strength stability of nanoparticles were also evaluated. In addition, we further investigated the effects of the pH of chitosan solution, the molecular weight of chitosan, the concentration of chitosan solution and the concentration of loaded protein on the particle size, polydispersity, zeta potential, protein entrapment efficiency of the nanoparticles. As the results showed, the molecular weight and the concentration of polysaccharides were important factors to affect the size of nanoparticles. Prepared with suitable molecular weight and solution concentration of polysaccharides, the particle size ranged from 100nm to 500nm. The entrapment efficiency of drug is a key factor to evaluate drug delivery systems. We found that, increasing the concentration of both components in nanoparticles could prepare more nanoparticle carriers and hence efficiently improve the entrapment efficiency of drug.
Furthermore, we investigated the effect of doxorubicin-loading nanoparticles on HeLa cells and satisfactory results were obtained. As a potential gene carrier, phosphonic chitosan were evaluated in terms of the entrapment, protection and transfection of plasmid DNA.
引文
1 张阳德，肖志刚，张浩伟，载药纳米粒在肝癌靶向治疗中的研究进展，中国医学工程，2005，13(6)：609—616
2 徐祖顺，易昌凤，聚合物纳米粒子，化学工业出版社，2006，北京，p15—25
3 郝英魁，杨学东，载药壳聚糖纳米粒的研究进展，中国药学杂志，2005，40(17)：1292—1295
4 陈钟，戴新征，壳聚糖及其纳米粒子在组织工程中的应用，国际生物医学工程杂志，2006，29(1)：48—52
5 刘晨光，刘成圣，孟祥红，陈西广，刘万顺，壳聚糖作为药物缓释材料的研究进展，高技术通讯，2003，3：98—102
6 李近，蒋国强，林莹，丁富新，长效载药微粒研究进展，精细化工，2006，23(10)：992—996
7 李和平，阮建明，黄伯云，王亚东，磁性壳聚糖纳米粒子的制备及表征，中南大学学报(自然科学版)，2004，35(2)．175-179
8 Jin Whan Lee, Jae Han Park, Joseph R. Robinson, Bioadhesive-based dosage forms: the next generation, Journal of Pharmaceutical Science, 2000, 89(7): 850-866
9 Abraham Rubinstein, Natural polysaccharides as targeting tools of drugs to the human colon, Drug Development Research, 2000, 50: 435-439
10 V. R. Sinha, Rachna Kumria, Polysaccharides in colon-specific drug delivery, International Journal of Pharmaceutics, 2001, 224: 19-38
11 Th. F. Vandamme, A. Lenourry, C. Charrueau, J-C. Chaumeil, The use of polysaccharides to target drugs to the colon, Carbohydrate Polymers, 2002, 48: 219-231
12 Caroline Lemarchand, Ruxandra Gref, Patrick Couvreur, Polysaccharide-decorated nanoparticles, European Journal of Pharmaceutics and Biopharmaceutics, 2004, 58: 327-341
13 K A Janes, P Calvo, M J Alonso, Polysaccharide colloidal particles as delivery systems for macromolecules, Advanced Drug Delivery Reviews, 2001, 47: 83-97
14 M. Prabaharan, J. F. Mano, Chitosan-based particles as controlled drug delivery systems, Drug Delivery, 2005, 12: 41-57
15 Jia Zhi, Yujun Wang, Guangsheng Luo, Adsorption of diuretic furosemide onto chitosan nanoparticles prepared with a water-in-oil nanoemulsion system, Reactive & Functional Polymers, 2005, 65: 249-257
16 Magdolna Bodnar, John F. Hartmann, Janos Borbely, Preparation and characterization of chitosan-based nanoparticles, Biomacromolecules, 2005, 6: 2521-2527
17 Magdolna Bodnar, John F. Hartmann, Janos Borbely, Nanoparticles from chitosan, Macromolecular Symposia, 2005, 227: 321-326
18 Pilar Calvo, Carmen Remunan-Lopez, Jose Luis Vila-Jato, Maria Jose Alonso, Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers, Journal of Applied Polymer Science, 1997, 63: 125-132
19 Pilaf Calvo, Carmen Remunan-Lopez, Jose Luis Vila-Jato, Maria Jose AIonso, Chitosan and chitosan/ethylene oxide- propylene oxide block copolymer nanoparticles as novel carriers for proteins and vaccines, Pharmaceutical Research, 1997, 14(10): 1431-1436
20 Yongmei Xu, Yumin Du, Ronghua Huang, Leping Gao, Preparation and modification of N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride nanoparticle as a protein carrier, Biomaterials, 2003, 24: 5015-5022
21 徐淑芳，徐咏梅，陈建钢，壳聚糖季铵盐纳米粒子的制备、表征及其缓释蛋白质药物性能，武汉大学学报(理学版)，2004，50(6)：721—725
22 Maryam Amidi, Stefan G. Romeijn, Gerrit Borchard, Hans E. Junginger, Wim E. Hennink, Wim Jiskoot, Preparation and characterization of protein-loaded N-trimethyl chitosan nanoparticles as nasal delivery system, Journal of Controlled Release, 2006, 111: 107-116
23 Xiaowen Shi, Yumin Du, Jianhong Yang, Baozhong Zhang, Liping Sun, Effect of degree of substitution and molecular weight of carboxymethyl chitosan nanoparticles on doxorubicin delivery, Journal of Applied Polymer Science, 2006, 100: 4689-4696
24 Jin-Oh You, Ching-An Peng, Calcium-alginate nanoparticles formed by reverse microemulsion as gene carriers, Macromolecular Symposia, 2005, 219: 147-153
25 A. Zahoor, Sadhna Sharma, G. K. Khuller, Inhalable alginate nanoparticles as antitubercular drug carriers against experimental tuberculosis, International Journal of Antimicrobial Agents, 2005, 26: 298-303
26 Zhengrong Cui, Russell J. Mumper, Chitosan-based nanoparticles for topical genetic immunization, Journal of Controlled Release, 2001, 75: 409-419
27 Yan Chen, Vellore J. Mohanraj, John E. Parkin, Chitosan-dextran sulfate nanoparticles for delivery of an anti-angiogenesis peptide, Letters in Peptide Science, 2003, 10: 621-629
28 Bruno Sarmento, Susana Martins, Antonio Ribeiro, Francisco Veiga, Ronald Neufeld, Domingos Ferreira, Development and comparison of different nanoparticulate polyelectrolyte complexes as insulin carriers, International Journal of Peptide Research and Therapeutics, 2006, 12(2): 131-138
29 俞怡晨，姚炎庆，张亚琼，陆轶业，郭圣荣，范华骅，壳聚糖—海藻酸盐纳米粒子的制备及其对BSA的载药与释放特性，功能高分子学报，2005，18(4)：598—601
30 Maria Alonso-Sande, Margarita Cuna, Carmen Remunan-Lopez, Desiree Teijeiro-Osorio, Jose L. Alonso-Lebrero, Maria J. Alonso, Formation of new glucomannan-chitosan nanoparticles and study of their ability to associate and deliver proteins, Macromolecules, 2006, 39: 4152-4158
31 Jian Du, Rui Sun, Sheng Zhang, Thirumala Govender, Li-Fang Zhang, Cheng-Dong Xiong, Yu-Xing Peng, Novel polyelectrolyte carboxymethyl konjac glucomannan-chitosan nanoparticles for drug delivery, Macromolecules Rapid Communication, 2004, 25: 954-958
32 Jian Du, Rui Sun, Sheng Zhang, Li-Fang Zhang, Cheng-Dong Xiong, Yu-Xing Peng, Novel polyelectrolyte carboxymethyl konjac glucomannan-chitosan nanoparticles for drug delivery. I. Physicochemical characterization of the carboxymethyl konjac glucomannan-chitosan nanoparticles, Biopolymers, 2005, 78:1 - 8
33 Jian Du, Sheng Zhang, Rui Sun, Li-Fang Zhang, Cheng-Dong Xiong, Yu-Xing Peng, Novel polyelectrolyte carboxymethyl konjac glucomannan-chitosan nanoparticles for drug delivery. II. Release of albumin in vitro, Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2005, 72B: 299 - 304
34 Yu-Hsin Lin, Ching-Kuang Chung, Chiung-Tong Chen, Hsiang-Fa Liang, Sung-Ching Chen, Hsing-Wen Sung, Preparation of nanoparticles composed of chitosan/poly-γ-glutamic acid and evaluation of their permeability through Caco-2 cells, Biomacromolecules, 2005, 6:1104 -1112
35 Qi Chen, Yong Hu, Ying Chen, Xiqun Jiang, Yonghua Yang, Microstructure formation and property of chitosan-poly(acrylic acid) nanoparticles prepared by macromolecular complex, Macromolecules Bioscience, 2005, 5: 993 -1000
36S. Sajeesh, Chandra P. Sharma, Novel pH responsive polymethacrylic acid-chitosan-polyethylene glycol nanoparticles for oral peptide delivery, Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2006, 76B: 298 - 305
37 O. Stoilova, N. Koseva, N. Manolova, I. Rashkov, Polyelectrolyte complex between chitosan and poly(2-acryloylamido-2-methylpropanesulfonic acid), Polymer Bulletin, 1999,43: 67 - 73
38 Yongli Zheng, Yan Wu, Wuli Yang, Changchun Wang, Shoukuan Fu, Xizhong Shen, Preparation, characterization, and drug release in vitro of chitosan-glycyrrhetic acid nanoparticles, Journal of pharmaceutical sciences, 2006, 95:181-191
39 B.I. Florea, M. Thanou, M. Geldof, C. Meaney, H.E. Junginger, G. Borchard, Modified chitosan oligosaccharides as transfection agents for gene therapy of cystic fibrosis, In Proceedings of 27th International Symposium on Controlled Release of Bioactive Materials (Paris), 2000, 27: 846 - 847
40 Rangrong Yoksan, Michiya Matsusaki, Mitsuru Akashi, Suwabun Chirachanchai, Controlled hydrophobic/hydrophilic chitosan: colloidal phenomena and nanosphere formation, Colloid Polymer Science, 2004, 282: 337 - 342
41 Sugimoto Masatoshi, Morimoto Minoru, Sashiwa Hitoshi, Saimoto Hiroyuki, Shigemasa Yoshihiro, Preparation and characterization of water-soluble chitin and chitosan derivatives, Carbohydrate Polymers, 1998, 36(1): 49-59
42 Ouchi Tatsuro, Nishizawa Hidetoshi, Ohya Yuichi, Aggregation phenomenon of PEG-grafted chitosan in aqueous solution, Polymer, 1998, 39(21): 5171 - 5175
43 Xi-Guang Chen, Chong M. Lee, Hyun-Jin Park, O/W emulsification for the self-aggregation and nanoparticle formation of linoleic acids modified chitosan in the aqueous system, Journal of Agriculture Food Chemistry, 2003, 51: 3135 - 3139
44 Chen-Guang Liu, Kashappa Goud H. Desai, Xi-Guang Chen, Hyun-Jin Park, Preparation and characterization of nanoparticles containing trypsin based on hydrophobically modified chitosan, Journal of Agriculture Food Chemistry, 2005, 53:1728-1733
45 Chen-Guang Liu, Kashappa Goud H. Desai, Xi-Guang Chen, Hyun-Jin Park, Linolenic acid-modified chitosan for formation of self-assembled nanoparticles, Journal of Agriculture Food Chemistry, 2005, 53: 437 - 441
46 Gang-Biao Jiang, Daping Quan, Kairong Liao, Haihua Wang, Novel polymer micelles prepared from chitosan grafted hydrophobic palmitoyl groups for drug delivery, Molecular Pharmaceutics, 2006, 3(2): 152-160
47 Ruxandra Gref, Jaqueline Rodrigues, Patrick Couvreur, Polysaccharides grafted with polyesters: novel amphiphilic copolymers for biomedical applications, Macromolecules, 2002, 35: 9861 - 9867
48 Caroline Lemarchand, Patrick Couvreur, Madeleine Besnard, Dominique Costantini, Ruxandra Gref, Novel polyester-polysaccharide nanoparticles, Pharmaceutical Research, 2003, 20(8): 1284 -1292
49 J. S. Rodrigues, N. S. Santos-Magalhaes, L. C. B. B. Coelho, P. Couvreur, G. Ponchel, R. Gref, Novel core(polyester)-shell(polysaccharide) nanoparticles: protein loading and surface modification with lectins, Journal of Controlled Release, 2003, 92:103 -112
50 Caroline Lemarchand, Ruxandra Gref, Catherine Passirani, Emmanuel Garcion, Boris Petri, Rainer Muller, Dominique Costantini, Patrick Couvreur, Influence of polysaccharide coating on the interactions of nanoparticles with biological systems, Biomaterials, 2006, 27:108 -118
51 Seung Ho Choi, Ji-Hwan Lee, Sung-Min Choi, Tae Gwan Park, Thermally reversible pluronic/heparin nanocapsules exhibiting 1000-fold volume transition, Langmuir, 2006, 22:1758 -1762
52 Sung Kyun Han, Jin Ho Lee, Dongmin Kim, Sun Hang Cho, Soon Hong Yuk, Hydrophilized poly(lactide-co-glycolide) nanoparticles with core/shell structure for protein delivery, Science and Technology of Advanced Materials, 2005, 6:468 - 474
53 Inbal Lalush, Hagit Bar, Imad Zakaria, Sigal Eichler, Eyal Shimoni, Utilization of amylose-lipid complexes as molecular nanocapsules for conjugated linoleic acid, Biomacromolecules, 2005, 6:121-130
54 Luc Choisnard, Annabelle Geze, Jean-Luc Putaux, Yung-Sing Wong, Denis Wouessidjewe, Nanoparticles of β-cyclodextrin esters obtained by self-assembling of biotransesterified β-cyclodextrins, Biomacromolecules, 2006, 7: 515-520
55 Kenichi Kuroda, Kazushi Fujimoto, Junzo Sunamoto, Kazunari Akiyoshi, Hierarchical self-assembly of hydrophobically modified pullulan in water: gelation by networks of nanoparticles, Langmuir, 2002, 18: 3780 - 3786
56 Kuen Yong Lee, Won Ho Jo, Ick Chan Kwon, Yong-Hee Kim, Seo Young Jeong, Structural determination and interior polarity of self-aggregates prepared from deoxycholic acid-modified chitosan in water, Macromolecules, 1998, 31: 378 - 383
57 K.Y. Lee, I.C. Kwon, Y.-H. Kim, W.H. Jo, S.Y. Jeong, Preparation of chitosan self-aggregates as a gene delivery system, Journal of Controlled Release, 1998, 51: 213-220
58 K. Y. Lee, J. H. Kim, I. C. Kwon, S. Y. Jeong, Self-aggregates of deoxycholic acid-modified chitosan as a novel carrier of adriamycin, Colloid Polymer Science, 2000, 278: 1216-1219
59 Su Young Chae, Sohee Son, Minhyung Lee, Mi-Kyeong Jang, Jae-Woon Nah, Deoxycholic acid-conjugated chitosan oligosaccharide nanoparticles for efficient gene carrier, Journal of Controlled Release, 2005, 109: 330-344
60 Kwangmeyung Kim, Seunglee Kwon, Jae Hyung Park, Hesson Chung, Seo Young Jeong, Ick Chan Kwon, Physicochemical characterizations of self-assembled nanoparticles of glycol chitosan-deoxycholic acid conjugates, Biomacromolecules, 2005, 6: 1154-1158
61 Kyeongsoon Park, Kwangmeyung Kim, Ick Chan Kwon, Sang Kyoon Kim, Seulki Lee, Dong Yun Lee, Youngro Byun, Preparation and characterization of self-assembled nanoparticles of heparin-deoxycholic acid conjugates, Langmuir, 2004, 20: 11726-11731
62 Seunglee Kwon, Jae Hyung Park, Hesson Chung, Ick Chan Kwon, Seo Young Jeong, Physicochemical characteristics of self-assembled nanoparticles based on glycol chitosan bearing 5β-cholanic acid, Langmuir, 2003, 19: 10188-10193
63 Jae Hyung Park, Seunglee Kwon, Ju-Ock Nam, Rang-Woon Park, Hesson Chung, Sang Bong Seo, In-San Kim, Ick Chan Kwon, Seo Young Jeong, Self-assembled nanoparticles based on glycol chitosan bearing 5β-cholanic acid for RGD peptide delivery, Journal of Controlled Release, 2004, 95: 579-588
64 Hyuk Sang Yoo, Jung Eun Lee, Hesson Chung, Ick Chan Kwon, Seo Young Jeong, Self-assembled nanoparticles containing hydrophobically modified glycol chitosan for gene delivery, Journal of Controlled Release, 2005, 103: 235-243
65 Jong-Ho Kim, Yoo-Shin Kim, Sungwon Kim, Jae Hyung Park, Kwangmeyung Kim, Kuiwon Choi, Hesson Chung, Seo Young Jeong, Rang-Woon Park, In-San Kim, Ick Chan Kwon, Hydrophobically modified glycol chitosan nanoparticles as carriers for paclitaxel, Journal of Controlled Release, 2006, 111: 228-234
66 原续波，顾鸣岐，原艳波，李弘，王来好，盛京，胆固醇疏水改性壳聚糖自聚集纳米微粒的制备及其动物体内分布研究，高分子通报，2005，5：122-127
67 Kazunari Akiyoshi, Shigeru Deguchi, Hitoshi Tajima, Takehiro Nishikawa, Junzo Sunamoto, Microscopic structure and thermo-responsiveness of a hydrogel nanoparticle by self-assembly of a hydrophobized polysaccharide, Macromolecules, 1997, 30: 857-861
68 Kazunari Akiyoshi, Seiichi Kobayashi, Shozo Shichibe, Don Mix, Miroslav Baudys, Sung Wan Kim, Junzo Sunamoto, Self-assembled hydrogel nanoparticle of cholesterol-bearing pullulan as a carrier of protein drugs: Complexation and stabilization of insulin, Journal of Controlled Release, 1998, 54: 313-320
69 Kazunari Akiyoshi, Yoshihiro Sasaki, Junzo Sunamoto, Molecular chaperone-like activity of hydrogel nanoparticles of hydrophobized pullulan: Thermal stabilization with refolding of carbonic anhydrase B, Bioconjugate Chemistry, 1999, 10(3): 321-324
70 Kazunari Akiyoshi, Eui-Chul Kang, Satoshi Kurumada, Junzo Sunamoto, Tania Principi, Francoise M. Winnik, Controlled association of amphiphilic polymers in water: Thermo-sensitive nanoparticles formed by self-assembly of hydrophobically modified pullulans and poly(N-isopropylacrylamides), Macromolecules, 2000, 33: 3244 - 3249
71 Minsu Lee, Yong Woo Cho, Jae Hyung Park, Hesson Chung, Seo Young Jeong, Kuiwon Choi, Dae Hyuk Moon, Sang Yoon Kim, In-San Kim, Ick Chan Kwon, Size control of self-assembled nanoparticles by an emulsion/solvent evaporation method, Colloid Polymer Science, 2006, 284(5): 506 -512
72 Yoen Ju Son, Jin-Sung Jang, Yong Woo Cho, Hesson Chung, Rang-Won Park, Ick Chan Kwon, In-San Kim, Jae Yong Park, Sang Bong Seo, Chong Rae Park, Seo Young Jeong, Biodistribution and anti-tumor efficacy of doxorubicin loaded glycol-chitosan nanoaggregates by EPR effect, Journal of Controlled Release, 2003, 91:135 -145
73 Jae Hyung Park, Yong Woo Cho, Yoen Ju Son, Kwangmeyung Kim, Hesson Chung, Seo Young Jeong, Kuiwon Choi, Chong Rae Park, Rang-Woon Park, In-San Kim, Ick Chan Kwon, Preparation and characterization of self-assembled nanoparticles based on glycol chitosan bearing adriamycin, Colloid Polymer Science, 2006, 284(7): 763 - 770
74 Jae Hyung Park, Seunglee Kwon, MinsuLee, Hesson Chung, Ji-Hyun Kim, Yoo-Shin Kim, Rang-Woon Park, In-San Kim, Sang Bong Seo, Ick Chan Kwon, Seo Young Jeong, Self-assembled nanoparticles based on glycol chitosan bearing hydrophobic moieties as carriers for doxorubicin: In vivo biodistribution and anti-tumor activity, Biomaterials, 2006, 27:119 -126
75 Kun Na, Tae Bum Lee, Keun-Hong Park, Eun-Kyung Shin, Yong-Bok Lee, Hoo-Kyun Choi, Self-assembled nanoparticles of hydrophobically-modified polysaccharide bearing vitamin H as a targeted anti-cancer drug delivery system, European Journal of Pharmaceutical Sciences, 2003,18:165 -173
76 S.C. Yang, H.X. Ge, Y. Hu, X.Q. Jiang, C.Z. Yang, Formation of positively charged poly(butyl cyanoacrylate) nanoparticles stabilized with chitosan, Colloid Polymer Science, 2000, 278: 285 - 292
77 Isabelle Bertholon, Christine Vauthier, Denis Labarre, Complement activation by core-shell poly(isobutylcyanoacrylate) polysaccharide nanoparticles: Influences of surface morphology, length, and type of polysaccharide, Pharmaceutical Research, 2006, 23(6): 1313 -1323
78 Irene Bravo-Osuna, Thierry Schmitz, Andreas Bernkop-Schnurch, Christine Vauthier, Gilles Ponchel, Elaboration and characterization of thiolated chitosan-coated acrylic nanoparticles, International Journal of Pharmaceutics, 2006, 316:170-175
79 Cedric Chauvierre, Denis Labarre, Patrick Couvreur, Christine Vauthier, Novel polysaccharide-decorated poly(isobutyl cyanoacrylate) nanoparticles, Pharmaceutical Research, 2003, 20(11):1786-1792
80 C. Chauvierre, C. Vauthier, D. Labarre, H. Hommel, Evaluation of the surface properties of dextran-coated poly(isobutylcyanoacrylate) nanoparticles by spin-labelling coupled with electron resonance spectroscopy, Colloid Polymer Science, 2004, 282:1016 -1025
81 Denis Labarre, Christine Vauthier, Cedric Chauvierre, Boris Petri, Rainer Muller, Mohamed M. Chehimi, Interactions of blood proteins with poly(isobutylcyanoacrylate) nanoparticles decorated with a polysaccharidic brush, Biomaterials, 2005, 26: 5075 - 5084
82 Cedric Chauvierre, Michael C. Marden, Christine Vauthier, Denis Labarre, Patrick Couvreur, Liliane Leclerc, Heparin coated poly(alkylcyanoacrylate) nanoparticles coupled to hemoglobin: a new oxygen carrier, Biomaterials, 2004, 25: 3081 - 3086
83 Catherine Passirani, Gillian Barratt, Jean-Philippe Devissaguet, Denis Labarre, Interactions of nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate) with the complement system, Life Science, 1998, 62(8): 775 - 785
84 Catherine Passirani, Gillian Barratt, Jean-Philippe Devissaguet, Denis Labarre, Long-circulating nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate), Pharmaceutical Research, 1998, 15(7): 1046-1050
85 Fang Zheng, Xiao-Wen Shi, Gui-Fang Yang, Ling-Ling Gong, Hong-Yin Yuan, Ye-Jian Cui, Yan Wang, Yu-Min Du, Yan Li, Chitosan nanoparticle as gene therapy vector via gastrointestinal mucosa administration: Results of an in vitro and in vivo study, Life Sciences, 2007, 80: 388 - 396
86 Hu-Lin Jiang, You-Kyoung Kim, Rohidas Arote, Jae-Woon Nah, Myung-Haing Cho, Yun-Jaie Choi, Toshihiro Akaike, Chong-Su Cho, Chitosan-graft-polyethylenimine as a gene carrier, Journal of Controlled Release, 2007, 117: 273 - 280
87 Yi Yang, Jianlin Chen, Hui Li, Yingyi Wang, Zhao Xie, Mei Wu, Huan Zhang, Zhongzhong Zhao, Qian Chen, Manliang Fu, Kaiyuan Wu, Cheng Chi, Hongning Wang, Rong Gao, Porcine interleukin-2 gene encapsulated in chitosan nanoparticles enhances immune response of mice to piglet paratyphoid vaccine, Comparative Immunology, Microbiology & Infectious Diseases, 2007, 30:19 -32
88 Kenneth A. Howard, Ulrik L. Rahbek, Xiudong Liu, Christian K. Damgaard, Sys Zoffmann Glud, Morten. Andersen, Mads B. Hovgaard, Alexander Schmitz, Jens R. Nyengaard, Flemming Besenbacher, Jbrgen Kjems, RNA interference in vitro and in vivo using a chitosan/siRNA nanoparticle system, Molecular Therapy, 2006, 14(4): 476 - 484
89 Peggy Chan, Motoichi Kurisawa, Joo Eun Chung, Yi-Yan Yang, Synthesis and characterization of chitosan-g-poly(ethylene glycol)-folate as a non-viral carrier for tumor-targeted gene delivery, Biomaterials, 2007, 28: 540 - 549
90 Xiudong Liu, Kenneth A. Howard, Mingdong Dong, Morten Andersen, Ulrik L. Rahbek, Mads G. Johnsen, Ole C. Hansen, Flemming Besenbacher, Jorgen Kjems, The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencing, Biomaterials, 2007, 28:1280 -1288
91 Sania Mansouri, Patrick Lavigne, Karin Corsi, Mohamed Benderdour, Eric Beaumont, Julio C. Fernandes, Chitosan-DNA nanoparticles as non-viral vectors in gene therapy: strategies to improve transfection efficacy, European Journal of Pharmaceutics and Biopharmaceutics, 2004, 57: 1-8
92 李大伟，张海玲，马洁，宋丽萍，郭志义，冷希岗，壳聚糖载基因纳米粒子的研究，生物医学工程学杂志，2005，22(6)：1171-1176
93 蒋挺大，甲壳素，化学工业出版社，2003
94 Yuji Yin, Xiaoyan Luo, Junfeng Cui, Changyong Wang, Ximin Guo, Kangde Yao, A Study on Biomineralization Behavior of N-Methylene Phosphochitosan Scaffolds, Macromolecular Bioscience, 2004, 4: 971-977
95 张灿，丁娅，杨波，沈健，N—亚甲基磷酸盐壳聚糖衍生物的设计、合成和表征，中国天然药物，2004，2(2)：94—98
96 Rigby G. W., 1937, U. S. Patent, 2,072,771
97 吴刚，沈玉华，谢安建，林宏云，郭文豪，N,O—羧甲基壳聚糖的合成和性质研究，化学物理学报，2003，16(6)：499—503
98 Riccardo A. A. Muzzarelli, Fabio Tanfasi, Monica Emanuelli, Sabina Maroitti, N-(carboxymethylidene)-chitosans and N-(carboxymethyl)-chitosans: novel chelating polyampholytes obtained from chitosan glyoxylate, Carbohydrate Research, 1982, 107: 199-214
99 Riccardo A. A. Muzzarelli, Carboxymethylated chitins and chitosans, Carbohydrate Polymers, 1988, 8: 1-21
100 Riccardo A. A. Muzzarelli, Pierluca llari, Solubility and structure of N-carboxymethyl chitosan, International Journal of Biological Macmmolecules, 1994, 16(4): 177-180
101 Pham le Dung, Michel Milas, Marguerite Rinaudo, Jacques Desbrieres, Water soluble derivatives obtained by controlled chemical modifications of chitosan, Carbohydrate Polymer, 1994, 24: 209-214
102 Riccardo A. A. Muzzarellia, Viviana Ramos, Vesna Stanic, Bruno Dubini, Monica Mattioli-Belmonte, Giorgio Tosi, Roberto Giardino, Osteogenesis promoted by calcium phosphate N, N-dicarboxymethyl chitosan, Carbohydrate Polymers, 1998, 36: 267-276
103 Hanno Baumann, Volker Faust, Concepts for improved regioselective placement of O-sulfo, N-sulfo, N-acetyl, and N-carboxymethyl groupsin chitosan derivatives, Carbohydrate Research, 2001, 331: 43-57
104 Xiaohong Wang, Jianbiao Ma, Yinong Wang, Binglin He, Structural characterization of phosphorylated chitosan and their applications as effective additives of calcium phosphate cements, Biomaterials, 2001, 22: 2247-2255
105 H. K. Varma, Y. Yokogawa, F. F. Espinosa, Y. Kawamoto, K. Nishizawa, F. Nagata, T. Kameyama, Porous calcium phosphate coating over phosphorylated chitosan film by a biomimetic method, Biomaterials, 1999, 20: 879-884
106 Norio Nishi, Akira Ebina, Shin-ichiro Nishimura, Akihiro Tsutsumi, Osatoshi Hasegawa, Seiichi Tokura, Highly phosphorylated derivatives of chitin, partially deacetylated chitin and chitosan as new functional polymers: preparation and characterization, International Journal of Biological Macromolecules, 1986, 8, 311-317
107 Ying Wan, Katherine A. M. Creber, Brant Peppley, V. Tam Bui, Synthesis, characterization and ionic conductive properties of phosphorylated chitosan membranes, Macromolecular Chemistry and Physics, 2003, 204: 850-858
108 A. Heras, N. M. Rodriguez, V. M. Ramos, E. Agullo, N-methylene phosphonic chitosan: a novel soluble derivative, Carbohydrate Polymers, 2001, 44: 1-8
109 V. M. Ramos, N. M. Rodriguez, M. F. Diaz, M. S. Rodriguez, A. Heras, E. Agullo, N-methylene phosphonic chitosan. Effect of preparation methods on its properties, Carbohydrate Polymers, 2003, 52: 39-46
110 M. L. Wolfrom, T. M. Shen Han, The sulfonation of chitosan, Journal of American Chemistry Society, 1959, 81: 1764-1766
111 Kurt Moedritzer, Riyad R. Irani, The Direct Synthesis of α-Aminomethylphosphonic Acids. Mannich-Type Reactions with Orthophosphorous Acid, Journal of Organic Chemistry, 1966, 31: 1603-1607
112 Huacai Ge, Dengke Luo, Preparation of carboxymethyl chitosan in aqueous solution under microwave irradiation, Carbohydrate Research, 2005, 340: 1351-1356
113 Shin-lchiro Nishimura, Norio Nishi, Seiichi Tokura, Bioactive chitin derivatives: activation of mouse peritoneal macrophages by o-carboxymethyl chitins, Carbohydrate Research, 1986, 146: 251-258
114 Xiguang Chen, Hyun-Jin Park, Chemical characteristics of O-carboxymethyl chitosans related to the preparation conditions, Carbohydrate Polymers, 2003, 53: 355-359
115 Kittur FS, Vishu Kumar AB, Tharanathan RN. Low molecular weight chitosans preparation by depolymerization with aspergillus niger pectinase, and characterization. Carbohydr Res 2003; 338: 1283-1290
116 刘峻，原续波，安超，侯信，盛京，离子凝胶反应法制备壳聚糖／N，O—羧甲基壳聚糖微球，高分子通报，2005，6：84—89
117 Kennedy R, Costain D J, McAlister VC, Lee TDG, Prevention of experimental postoperative peritoneal adhesions by N, O-carboxymethyl chitosan, Surgery, 1996, 120: 866-870
118 Feng Tian, Yu Liu, Keao Hu, Binyuan Zhao, Study of the depolymerization behavior of chitosan by hydrogen peroxide, Carbohydydrate Polymers, 2004, 57: 31-37
119 Roberts GAF, Domszy JG, Determination of the viscometric constants for chitosan, International Journal of Biological macromolecules, 1982, 4: 374-377
120 Donini C, Robinson D N, Colombo P, Giordano F, Peppas N A, Preparation of P(MAA-g-EG) nanospheres for pharmaceutical. Applications, International Journal of Pharmaceutics, 2002, 245, 83-91
121 Manee Luangtana-anan, P. Opanasopit, T. Ngawhirunpat, J. Nunthanid, P. Sriamornsak, S. Limmatvapirat, Effect of chitosan salts and molecular weight on a nano-particulate carrier for. therapeutic protein, Pharmaceutical Development and Technology, 2005, 10, 189-196
122 Yongmei Xu, Yumin Du, .Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles, International Journal of Pharmaceutics, 2003, 250(1): 215-226
123 Lu, B.; Xiong, S.; Yang, H.; Yin, X.; Zhao, R. Mitoxantrone-loaded BSA nanospheres and chitosan nanospheres for local injection against breast cancer and its lymph node metastases Ⅰ: Formulation and in vitro characterization, International Journal of Pharmaceutics, 2006, 307, 168-174
124 De Campos A M, Sanchez A, Alonso M J, Chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to the ocular surface. Application to cyclosporin A, International Journal of Pharmaceutics, 2001, 224: 159-168
125 Yan Wu, Wuli Yang, Changchun Wang, Jianhua Hu, Shoukuan Fu, International Journal of Pharmaceutics: Pharmaceutical Nanotechnology, 2005, 295: 235-245
126 Luppi E, Cesaretti M, Volpi N. Purification and characterization of heparin from the Italian clam Callista chione. Biomacromolecules, 2005, 6(3): 1672-1678
127 Suk JY, Zhang FM, Balch WE, Linhardt R J, Kelly JW. Heparin accelerates gelsolin amyloidogenesis. Biochemistry, 2006, 45(7): 2234-2242
128 Jee KS, Park HD, Park KD, Kim YH, Shin JW. Heparin conjugated polylactide as a blood compatible material. Biomacromolecules, 2004, 5(5): 1877-1881
129 Linhardt RJ. Claude S. Hudson Award address in carbohydrate chemistry. Heparin: structure and activity. Journal of Medical Chemistry, 2003, 46(13): 2551-2564
130 Xu F J, Li YL, Kang ET, Neoh KG Heparin-coupled poly(poly(ethylene glycol) monomethacrylate)-Si(111) hybrids and their blood compatible surfaces. Biomacromolecules, 2005, 6(3): 1759-1768
131 Feyerabend TB, Li JP, Lindahl U, Rodewald HR. Heparin sulfate C5-epimerase is essential for heparin biosynthesis in mast cells. Nat Chem Bio, 2006, 2(4): 195-196
132 Lever R, Page CP. Novel drug development opportunities for heparin. Nature Reviews Drug Discovery, 2002, 1(2): 140-148
133 Bertini S, Bisio A, Torri G, Bensi D, Terbojevich M. Molecular weight determination of heparin and dermatan sulfate by size exclusion chromatography with a triple detector array. Biomacromolecules, 2005, 6(1): 168-173
134 王斌，蒋珍菊，段明，胡星琪，水溶性磺化壳聚糖的研制，应用化工，2004，33(1)：21—23
135 Zhang H, Megan O, Christine A, Eugenia K. Monodisperse chitosan nanoparticles for mucosal drug delivery. Biomacromolecules, 2004, 5(6): 2461-2468
136 汤爱国，张阳德，O-羧甲基乳糖酰化壳聚糖-聚乳酸阿霉素纳米粒的制备及其物理化学性质的测定，中国现代医学杂志，2006，16(12)：1786—1788
137 陈汝福，王捷，李志花，陈积圣，硬脂酸阿霉素纳米粒的制备及在抗肝癌中的应用，中华肝胆外科杂志，2005，11(2)：125—127
138 于美丽，王勇，舒贵明，朱争艳，方叔昌，王黎，卵磷脂和氢化卵磷脂长循环阿霉素脂质体的制备及缓释研究，北京生物医学工程，2006，25(6)：654—657
139 董兰凤，赵兴茹，冯凤莲，范美菊，杨丽，阿霉素长循环热敏脂质体的研制及其靶向治疗肿瘤作用的研究，中国肿瘤生物治疗杂志，2005，12(1)：52—56
140 王旭波，徐希明，葛亮，丁妍，张钧寿，半乳糖化阿霉素白蛋白纳米粒的制备及其质量评价，中国新药杂志，2005，14(7)：870—872
141 殷香保，黎洪浩，陈汝福，叶华，任山，王捷，区庆嘉，聚乳酸阿霉素纳米微粒的制备及体内外释药的研究，中华实验外科杂志，2006，23(2)：227—229
142 RJ Mumper, J Wang, JM Claspell, AP Rolland, Novel polymeric condensing carriers for gene delivery, Proc lnt Symp Control Rel Bioact Mater, 1995, 22: 178-179
143 Zhengrong Cui, Russell J. Mumper, Chitosan-based nanoparticles for topical genetic immunization, Journal of Controlled Release, 2001, 75: 409-419
144 Hai-Quan Mao, Krishnendu Roy, Vu L. Troung-Le, Kevin A. Janes, Kevin Y. Lin, Yan Wang, J. Thomas August, Kam W. Leong, Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency, Journal of Controlled Release, 2001, 70: 399-421
145 刘文广，姚康德，戚务勤，水凝胶研究的最新进展，高分子材料科学与工程，2002，18(5)：54-57
146 李方，赵峰，尹玉姬，姚康德，壳聚糖基智能凝胶材料及其应用，化学通报，2001，3：129—134
147 孙立苹，杜予民，陈凌云，黄荣华，陈效，羧甲基壳聚糖水凝胶制备及其在药物控释中的应用，高分子学报，2004，2：191—195
148 张新房，智能水凝胶，食品与药品，2005，7(3A)：17—22
149 李亚娜，霍东霞，王红英，钱庆文，水凝胶作为胰岛素控释系统载体的研究进展，中国医药工业杂志，2005，36(7)：438—441
150 Berger J, Reist M, Mayer JM, Felt O, Peppas NA, Gurny R. Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications. European Journal of Pharmaceutics and Biopharmaceutics, 2004; 57: 19-34
151 Berger J, Reist M, Mayer JM, Felt O, Gurny R. Structure and interactions in chitosan hydrogels formed by complexation or aggregation for biomedical applications. European Journal of Pharmaceutics and Biopharmaceutics, 2004; 57: 35-52
152 Wheatley MA, Chang M, Park E, Langer R. Coated alginate microspheres: factors influencing controlled delivery of macromolecules. Journal of Applied Polymer Science, 1991; 43: 2123-2135
153 Hari PR, Chandy T, Sharma CP. Chitosan/calcium-alginate beads for oral delivery of insulin. Journal of Applied Polymer Science, 1996; 59: 1795-1801
154 Lee KY, Park WH, Ha WS. Polyelectrolyte complexes of sodium alginate with chitosan or its derivatives for microcapsules. Journal of Applied Polymer Science, 1997; 63: 425-432
155 Dashevsky A, Protein loss by the microencapsulation of an enzyme (lactase) in alginate beads. International Journal of Pharmaceutics, 1998; 161: 1-5
156 Zhang L, Guo J, Peng X, Jin Y. Preparation and release behavior of carboxymethylated chitosan/alginate microspheres encapsulating bovine berum albumin. Journal of Applied Polymer Science, 2004; 92: 878-882
157 Lin YH, Liang HF, Chung CK, Chen MC, Sung HW. Physically crosslinked alginate/N, O-carboxymethyl chitosan hydrogels with calcium for oral delivery of protein drugs. Biomaterials, 2005; 26: 2105-2113
158 黄荣华，杜予民，陈凌云，樊李红，孙立苹，pH值对壳聚糖／羧甲基壳聚糖水凝胶溶胀行为的影响，武汉大学学报(理学版)，2003，49(4)：465—469
159 Alsarra IA, Betigeri SS, Zhang H, Evans BA, Neau SH. Molecular weight and degree of deacetylation effects on lipase-loaded chitosan bead characteristic. Biomaterials, 2002; 23: 3637-3644
160 Nishimura SI, Ikeuchi Y, Tokura S. The adsorption of bovine blood proteins onto the surface of O-(carboxymethyl)chitin. Carbohydrate Research, 1984; 134: 305-312
161 Chen L, Tian Z, Du Y. Synthesis and pH sensitivity of carboxymethyl chitosan-based polyampholyte hydrogels for protein carrier matrices. Biomaterials, 2004; 25: 3725-3732
162 Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson B J, Klenk DC. Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 1985; 150: 76-85
163 Alsarra IA, Neau SH, Howard MA. Effects of preparative parameters on the properties of chitosan hydrogel beads containing candida rugosa lipase. Biomaterials, 2004; 25: 2645-2655
164 Busto MD, Ortega N, Perez-Mateos M. Characterization of microbial endo-β-glucanase immobilized in alginate beads. Acta Biotechnology, 1998; 18: 189-200
165 Patil RT, Speaker TJ. Retention of trypsin activity in spermine alginate microcapsules. Journal of Microencapsulation, 1997; 14: 469-474
166 Huguet ML, Groboillot A, Neufeld R J, Poncelet D, Dellacherie E. Hemolglobin encapsulation in chitosan/calcium alginate beads. Journal of Applied Polymer Science, 1994; 51: 1427-1432
167 Xiao Huang, Christopher S. Brazel, On the importance and mechanisms of burst release in matrix-controlled drug delivery systems, Journal of Controlled Release, 2001; 73: 121-136
168 Xiao Huang, Bdgitta L. Chestang, Christopher S. Brazel, Minimization of initial burst in poly(vinyl alcohol) hydrogels by surface extraction and surface-preferential crosslinking, International Journal of Pharmaceutics, 2002; 248: 183-192