改性壳聚糖纳米粒的制备及其载药释药研究
|
摘要
现代生物技术的飞速发展,导致了大量蛋白质和肽类药物的出现。这类药物在体内、肠道内极易被蛋白水解酶降解,一般不能口服。而且大多数蛋白质和多肽类药物不易通过生物屏障,生物利用度低,只能采取注射或灌注的途径来给药。给药以后,大多数药物成分很快释放,引起体内药物水平的迅速升高,达到峰值后迅速降低。对于药物来讲,其作用同血清中药物的浓度密切相关,剧烈的波动往往引起在峰值时产生不可接受的毒副作用,而后由于血清中药物浓度过低导致不充分的治疗效果。药物缓释系统正是迎合了上述问题而成为目前药学领域的重要发展方向,而对药物载体及缓释材料的选择就成为当前研究的热点。来源广泛、无毒无害、包封率高、缓释效果好的载药材料是人们追求的目标。
本文在研究了壳聚糖(CS)成球条件及其载药释药效果的基础上,对壳聚糖进行亲水改性,得到载药材料羟丙基壳聚糖(HCS),并研究其纳米粒的药物释放效果。之后将具有肿瘤靶向作用的叶酸(FA)分子偶联到羟丙基壳聚糖分子上,制备了叶酸偶联羟丙基壳聚糖(FHCS),并研究了其纳米粒的缓释效果。具体内容和结论主要包括以下几个方面:
1.利用离子凝胶法制备壳聚糖纳米粒,确定成球条件,利用透射电镜对其进行表征。以牛血清蛋白(BSA)作为模型药物,考察壳聚糖纳米粒对药物的包封和释放结果。结果发现,壳聚糖纳米粒对蛋白的包封率和载药量都随壳聚糖初始浓度的增大而增大,而随BSA初始浓度的增大呈现不同趋势,测定的最大包封率和载药量分别达到86%和49%。体外释放现实2h内最少可释放载药量的30%,12h后呈现缓慢而持续的释放。
2.在碱性条件下,利用环氧丙烷与壳聚糖的直接反应,将羟丙基引入壳聚糖分子中,增强其水溶性,得到水溶性的羟丙基壳聚糖,应用红外光谱进行表征。将其制备成纳米粒后进行透射电镜观察,并考察其对牛血清蛋白的包封和缓释效果。结果显示,制备的羟丙基壳聚糖纳米粒外形规则,粒径分布较均匀,包封率和载药量高,对药物具有缓释效果。最大包封率达86%,载药量为46%,在缓释初期2h内平均释放药量的28%,后期均呈现缓慢释放。
3.在中性水溶液、避光条件下,将具有肿瘤靶向作用的叶酸分子偶联到羟丙基壳聚糖上,探讨了反应温度、反应浓度和反应时间对产物产率的影响。结果发现,当羟丙基壳聚糖浓度为2.0mg/ml,叶酸浓度120μg/ml,反应时间70min,反应温度80℃时达到最大产率31.3%。利用离子凝胶法将其制备成纳米粒,于透射电镜下观察,同时考察其载药、释药效果。结果显示,制备的叶酸偶联羟丙基壳聚糖纳米微粒外形规则,粒径小且分布较均匀,最大包封率达90%,载药量为48%,在缓释初期2h内平均释放药量的26%,后期均呈现缓慢释放。
The rapid development of modern biotechnology brings about many protein and peptide drugs, which are degradable by proteolytic enzyme in intestinal and not oral. Moreover, most protein and peptide drugs are hard through the biological barrier, resulting in low bioavailability, and only can be used by injection and perfusion. The most drugs release quickly after administrated, which brings the drug level increases rapidly and decreases after reaching the peak. For drugs, the effect is closely related to the concentrations in serum. So the fluctuation of concentration leads to unaccepted side-effect at the peak, and inadequate treating effect at low drug concentrations. Drug delivery system is catering to said problems, and becoming an important developing in drug field recently, so the selecting and researching of drug carriers or delivery materials become a research focus. The delivery materials with extensive sources, poisonless and harmless, and high encapsulation efficiency are pursued by people.
In this paper, the delivery material of hydroxypropyl-chitosan was prepared by hydrophilic modification of chitosan, basing on researches for the condition of forming chitosan particles and the effect of drug delivery. Then by coupling folate which has the target effect of tumor to hydroxypropyl-chitosan, the folate-hydroxypropyl-chitosan was prepared and the drug delivery result of folate-hydroxypropyl-chitosan particles was studied. The major contents and results include:
1. The chitosan nanoparticle was prepared by ion gel. The condition of chitosan forming sphere was determined and the nanoparticle was characterized by TEM. The results of encapsulation and delivery were studied by taking bovine serum albumin (BSA) as model drug. The results showed that the BSA encapsulation efficiency and the BSA loading were affected by the initial CS concentration and the initial BSA concentration. The higher was the initial chitosan concentration, the higher the BSA encapsulation efficiency and the BSA loading. However, it showed opposite trend when the initial BSA concentration increased. The highest encapsulation efficiency and loading of BSA reached 86% and 49%, respectively. The behavior of chitosan nanoparticles for BSA in vitro release reveals a controlled and continuous release of the entrapped protein after 12 hours and releases 30% of the BSA loading within 2 hours.
2. By the reaction of propylene oxide and chitosan under alkaline condition that introducing hydroxypropyl to chitosan, the water-soluble hydroxypropyl-chitosan (HCS) was prepared. The synthesis of hydroxypropyl-chitosan was determined by Fourier Transform Infrared Spectroscope. The hydroxypropyl-chitosan particles were observed by TEM, and the effect of encapsulation and delivery was also researched. The results suggested that the prepared hydroxypropyl-chitosan particle was characterized by regular sphere, uniform distribution of particle size, high encapsulation efficiency, high drug loading and good delivery, and the highest encapsulation efficiency and loading of BSA reached 86% and 46%, respectively. The behavior of HCS nanoparticles releases 28% in average within 2 hours and reveals a continuous release of the entrapped protein.
3. The folate-hydroxypropyl-chitosan (FHCS) was prepared by the folate coupled to hydroxypropyl-chitosan under the condition of neutral aqueous solution and dark environment. The effects of temperature, concentrations and time on FHCS yield were studied. The result suggested that the optimal values of content, time and temperature were FHCS 2.0mg/ml, folate 120μg/ml, 70min and 80℃, respectively. The highest yield is 31.3%. Then the folate-hydroxypropyl-chitosan particles was prepared by ion gel, characterized by TEM,and meanwhile, the effect of encapsulation and delivery were researched. The results showed that the prepared folate-hydroxypropyl-chitosan particle had advantages of regular sphere, uniform distribution of particle size. And the highest encapsulation efficiency and loading of BSA reached 90% and 48%, respectively. The FHCS nanoparticles releases 26% in average within 2 hours and reveals a continuous release of the entrapped protein.
本文在研究了壳聚糖(CS)成球条件及其载药释药效果的基础上,对壳聚糖进行亲水改性,得到载药材料羟丙基壳聚糖(HCS),并研究其纳米粒的药物释放效果。之后将具有肿瘤靶向作用的叶酸(FA)分子偶联到羟丙基壳聚糖分子上,制备了叶酸偶联羟丙基壳聚糖(FHCS),并研究了其纳米粒的缓释效果。具体内容和结论主要包括以下几个方面:
1.利用离子凝胶法制备壳聚糖纳米粒,确定成球条件,利用透射电镜对其进行表征。以牛血清蛋白(BSA)作为模型药物,考察壳聚糖纳米粒对药物的包封和释放结果。结果发现,壳聚糖纳米粒对蛋白的包封率和载药量都随壳聚糖初始浓度的增大而增大,而随BSA初始浓度的增大呈现不同趋势,测定的最大包封率和载药量分别达到86%和49%。体外释放现实2h内最少可释放载药量的30%,12h后呈现缓慢而持续的释放。
2.在碱性条件下,利用环氧丙烷与壳聚糖的直接反应,将羟丙基引入壳聚糖分子中,增强其水溶性,得到水溶性的羟丙基壳聚糖,应用红外光谱进行表征。将其制备成纳米粒后进行透射电镜观察,并考察其对牛血清蛋白的包封和缓释效果。结果显示,制备的羟丙基壳聚糖纳米粒外形规则,粒径分布较均匀,包封率和载药量高,对药物具有缓释效果。最大包封率达86%,载药量为46%,在缓释初期2h内平均释放药量的28%,后期均呈现缓慢释放。
3.在中性水溶液、避光条件下,将具有肿瘤靶向作用的叶酸分子偶联到羟丙基壳聚糖上,探讨了反应温度、反应浓度和反应时间对产物产率的影响。结果发现,当羟丙基壳聚糖浓度为2.0mg/ml,叶酸浓度120μg/ml,反应时间70min,反应温度80℃时达到最大产率31.3%。利用离子凝胶法将其制备成纳米粒,于透射电镜下观察,同时考察其载药、释药效果。结果显示,制备的叶酸偶联羟丙基壳聚糖纳米微粒外形规则,粒径小且分布较均匀,最大包封率达90%,载药量为48%,在缓释初期2h内平均释放药量的26%,后期均呈现缓慢释放。
The rapid development of modern biotechnology brings about many protein and peptide drugs, which are degradable by proteolytic enzyme in intestinal and not oral. Moreover, most protein and peptide drugs are hard through the biological barrier, resulting in low bioavailability, and only can be used by injection and perfusion. The most drugs release quickly after administrated, which brings the drug level increases rapidly and decreases after reaching the peak. For drugs, the effect is closely related to the concentrations in serum. So the fluctuation of concentration leads to unaccepted side-effect at the peak, and inadequate treating effect at low drug concentrations. Drug delivery system is catering to said problems, and becoming an important developing in drug field recently, so the selecting and researching of drug carriers or delivery materials become a research focus. The delivery materials with extensive sources, poisonless and harmless, and high encapsulation efficiency are pursued by people.
In this paper, the delivery material of hydroxypropyl-chitosan was prepared by hydrophilic modification of chitosan, basing on researches for the condition of forming chitosan particles and the effect of drug delivery. Then by coupling folate which has the target effect of tumor to hydroxypropyl-chitosan, the folate-hydroxypropyl-chitosan was prepared and the drug delivery result of folate-hydroxypropyl-chitosan particles was studied. The major contents and results include:
1. The chitosan nanoparticle was prepared by ion gel. The condition of chitosan forming sphere was determined and the nanoparticle was characterized by TEM. The results of encapsulation and delivery were studied by taking bovine serum albumin (BSA) as model drug. The results showed that the BSA encapsulation efficiency and the BSA loading were affected by the initial CS concentration and the initial BSA concentration. The higher was the initial chitosan concentration, the higher the BSA encapsulation efficiency and the BSA loading. However, it showed opposite trend when the initial BSA concentration increased. The highest encapsulation efficiency and loading of BSA reached 86% and 49%, respectively. The behavior of chitosan nanoparticles for BSA in vitro release reveals a controlled and continuous release of the entrapped protein after 12 hours and releases 30% of the BSA loading within 2 hours.
2. By the reaction of propylene oxide and chitosan under alkaline condition that introducing hydroxypropyl to chitosan, the water-soluble hydroxypropyl-chitosan (HCS) was prepared. The synthesis of hydroxypropyl-chitosan was determined by Fourier Transform Infrared Spectroscope. The hydroxypropyl-chitosan particles were observed by TEM, and the effect of encapsulation and delivery was also researched. The results suggested that the prepared hydroxypropyl-chitosan particle was characterized by regular sphere, uniform distribution of particle size, high encapsulation efficiency, high drug loading and good delivery, and the highest encapsulation efficiency and loading of BSA reached 86% and 46%, respectively. The behavior of HCS nanoparticles releases 28% in average within 2 hours and reveals a continuous release of the entrapped protein.
3. The folate-hydroxypropyl-chitosan (FHCS) was prepared by the folate coupled to hydroxypropyl-chitosan under the condition of neutral aqueous solution and dark environment. The effects of temperature, concentrations and time on FHCS yield were studied. The result suggested that the optimal values of content, time and temperature were FHCS 2.0mg/ml, folate 120μg/ml, 70min and 80℃, respectively. The highest yield is 31.3%. Then the folate-hydroxypropyl-chitosan particles was prepared by ion gel, characterized by TEM,and meanwhile, the effect of encapsulation and delivery were researched. The results showed that the prepared folate-hydroxypropyl-chitosan particle had advantages of regular sphere, uniform distribution of particle size. And the highest encapsulation efficiency and loading of BSA reached 90% and 48%, respectively. The FHCS nanoparticles releases 26% in average within 2 hours and reveals a continuous release of the entrapped protein.
引文
[1] C K Rha, D R Sanchez, C K Sterzer. Novel applications of chitosan[J]. Biotechnology of Marine Polysaccharides, 1984: 284-311.
[2]蒋挺大.壳聚糖[M].北京:化学工业出版社,2001: 12-32.
[3] S Nicol. Life after death for empty shells[J]. New Sci, 1991, 129: 46-48.
[4] P C Berscht, B Nies, A Liebendorfer, et al. Incorporation of basic fibroblast growth factor into methylpyrrolidinone chitosan fleeces and determination of the in vitro release characteristics[J]. Biomaterials, 1994, 15: 593-600.
[5] T Sannan, K Kurita, Y Iwakura. Studies on chitin: effect of deacetylation on solubility[J]. Chem. 1976, 177:3589-3600.
[6] H Seo, A Shoji, Y Itoh, et al. Antibacterial fiber blended with chitosan[J]. Chitin World, 1994: 623–631.
[7] D Valérie, D V Vinod. Pharmaceutical applications of chitosan[J].Elsevier Science,1998, 10: 246-253.
[8]孙慎侠,付昌斌,范钦信.壳聚糖的生物活性及其在中医药领域中的应用[J].贵阳中医学院学报, 2003, 25(1): 39-41.
[9] I Genta, F Pavanetto, B Conti, et al. Spray-drying for the preparation of chitosan microspheres[J]. Control Release Bioact.Mater, 1994, 21: 616-617.
[10] E E Hassan, R C Parish, J M Gallo. Optimized formulation of magnetic chitosan microspheres containing the anticancer agent[J]. Pharm. Res, 1992, 9: 390-397.
[11] L Illum, N F Farraj, S S Davis. Chitosan as a novel nasal delivery system for peptide drugs[J]. Pharm. Res, 1994, 11: 1186-1189.
[12] H L Luehen, C M Lehr, C O Rentel, et al. Bioadhesive polymers for the peroral delivery of peptide drugs[J]. Control Release, 1994, 29: 329-338.
[13] P A Sanford. Chitosan: commercial uses and potential applications, Chitin and Chitosan–Sources, Chemistry, Biochemistry Physical Properties and Applications[M]. Elsevier, London, 1989: 51–69.
[14]元英进,刘明言,董岸杰.中药现代化生产技术[M].北京:化学工业出版社,2001:227-233.
[15] J R Robinson. Rationale of bioadhesion /mucoadhesion, Possibilities and Future Trends[J].Chem,1990, 8: 13–15.
[16] H Sano, K I Shibasaki, T Matsukubo, et al. Effect of rinsing with phosphorylated chitosan on 4-day plaque regrowth[J]. Coll, 2001, 42: 251–256.
[17]张建林,李平,徐吉光等.盐酸曲马多口腔药膜的制备及质量控制[J].兰州医学院学报, 2001, 4 (27):14-16.
[18] A B Schnǔrch, C E Kast. Chemically modified chitosans as enzyme inhibitors[J]. Adv. Drug Deliv. Rev, 2001, 52: 127-137.
[19] R A Yokel, K M Dickey, A H Goldberg. Selective adherence of a sucralfate-tetracycline complex to gastric ulcers: implications for the treatment of Helicobacter pylori[J]. Bio-pharm Drug Dispos, 1995, 16: 475-479.
[20] S Shah, R Qaqish, V Patel, et al. Evaluation of the factors influencing stomach-specific delivery of antibacterial agents for Helicobacter pylori infection[J]. Pharm. Pharmacol, 1999, 51: 667-672.
[21]董春玲,冷延国,廖进康等.明胶-壳聚糖微胶囊化过程研究[J].明胶科学与技术,1999,19(3):117-123.
[22]徐蔚,曹毅,张纪.壳聚糖微球释药机制的研究[J].昆明医学院学报, 2002,1:7-10.
[23] R Hejazi, M Amiji. Stomach-specific anti-H. pylori therapy:preparation and characterization of tetracycline-loaded chitosan microspheres[J]. Int. Pharm, 2002, 235: 87-94.
[24] C Remuǎan-Lǒpez, A Portero, M Lemos. Chitosan microspheres for the specific delivery of amoxycillin to the gastric cavity[J]. Stp Pharma Sci, 2000, 10: 69-76.
[25]王志国,张敏卿.壳聚糖在靶向制剂中的研究进展[J].化工进展,2004,4(23):375-379.
[26] Y Pan, Y Li, H Zhao, et al. Chitosan nanoparticles improve the intestinal absorption of insulin in vivo[J]. Int. Pharm, 2002, 249: 139-147.
[27] L Ilium, N Farraj, H Critchley, et al. Nasal administration of gentamicin using a novel microsphere delivery system[J].International Journal Pharmaceutics,1988, 46: 261-265.
[28] I. J Gill, A N Fisher, N Farraj, et al. Intranasal absorption of granulocyte-colony stimulating factor (G-CSF) from powder formulations, in sheep European[J]. Journal of Pharmaceutical Sciences, 1998, 6(1): 1-10.
[29] L Illum, I Jabbal-Gill. Chitosan as a novel nasal delivery system for vaccines[J]. Advanced Drug Delivery Reviews, 2001, 51: 81-96.
[30] H Q Mao, K Roy, V L Troung-Le, et al. Chitosan DNA nanoparticles as gene delivery carriers: synthesis, characterization and transfection efficiency[J]. Control Release, 2001, 70: 399- 421.
[31] I C Lee, Y H Kwon, W H Kim, et al. Preparation of chitosan self-aggregates as a gene delivery system[J]. Control Release, 1998, 51: 213-220.
[32] F Zheng, X W Shi, G F Yang. Chitosan nanoparticle as gene therapy vector via gastrointestinal mucosa administration: Results of an in vitro and in vivo study[J]. Life Sciences 2007, 80: 388-396.
[33] Z Cui, R J Mumper. Chitosan-based nanoparticles for topical genetic immunization[J]. Control Release, 2001, 75: 409-419.
[34] T López-León, E L S Carvalho. Physicochemical characterization of chitosan nanoparticles: electrokinetic and stability behavior[J]. Journal of Colloid and Interface Science, 2005, 283: 344-351.
[35] J Akbuga, G Durmaz. Preparation and evaluation of cross-linked chitosan microspheres containing furosemide[J]. Int. Pharm, 1994, 11: 217-222.
[36] S G Kumbar, A R Kulkarni, T M Aminabhavi. Cross-linked chitosan microspheres for encapsulation of diclofenac sodium: effect of cross-linking agent[J]. J. Microencapsulation 2002, 19: 173-180.
[37] A A Al-Helw, A A Al-Angary, G. M Mahrous, et al.. Preparation and evaluation of sustained release cross-linked chitosan microspheres containing phenobarbitone[J]. Microencapsulation, 1998, 15: 373-382.
[38] K Nishimura, S Nishimura, H Seo, et al. Macrophage activation with multiporous beads prepared from partially deacetylated chitin[J]. Biomed. Mater. Res, 1986, 20: 1359-1372.
[39] S Ozbas-Turan, J Akbuga, C Aral. Controlled release of interleukin-2 from chitosan microspheres[J]. Pharm. Sci, 2002, 91: 124-125.
[40] H Q Mao, K Roy, V L Troung-Le, et al. Chitosan DNA nanoparticles as gene delivery carriers: synthesis, characterization and transfection efficiency[J]. Control Release, 2001, 70:399-421.
[41] P He, S S Davis, L Illum. Chitosan microspheres prepared by spray drying[J]. Int. Pharm, 1999, 187: 53-65.
[42] A G Gonzalez, S A Igea, F J O Espinar, et al. Chitosan and chondroitin microspheres for oral-administration controlled release of metoclopramide[J]. Eur. Pharm. Biopharm, 1999, 48: 149-155.
[43] M L Lorenzo-Lamosa, C Remunan-Lopez, et al. Design of microencapsulated chitosan microspheres for colonic drug delivery[J]. Control Release, 1998, 52:109-118.
[44] R Bodmeier, H G Chen, O Paeratakul. A novel approach to the oral delivery of micro-or nanoparticles[J]. Pharm Res, 1989, 5: 413.
[45] Y Kawashima, T Handa, H Takenaka, et al.. Novel method for the preparation of controlled-release theophylline granules coated with a polyelectrolyte complex of sodium polyphosphate-chitosan[J]. J. Pharm. Sci, 1985, 74: 264-268.
[46] A Polk, B Amsden, K D Yao, et al. Controlled release of albumin from chitosan-alginate microcapsules[J]. Pharm. Sci, 1994, 83: 178-185.
[47] R Fernandez-Urrusuno, P Cavlo, C Remunan-Lopez, et al. Enhancement of nasal absorption of insulin using chitosan nanoparticles[J]. Pharm. Res. 1999, 16: 1576-1581.
[48] Y Xu, Y Du. Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles[J]. Int.Pharm, 2003, 250: 215-226.
[49] H J Ko, S J Park, J B Hwang, et al. Preparation and characterization of chitosan microparticles intended for controlled drug delivery[J]. Int. Pharm, 2002, 249: 165-174.
[50] A Maitra. Determination of size parameters of water-Aerosol OT–oil reverse micelles from their nuclear magnetic resonance data[J]. J. Phys. Chem, 1984, 88: 5122-5125.
[51] S Mitra, U Gaur, P C Ghosh, et al.. Tumor targeted delivery of encapsulated dextran- doxorubicin conjugate using chitosan nanoparticles as carrier[J]. Control Release, 2001, 74: 317-323.
[52]郑化,杜予民. N-酰基壳聚糖的合成及膜的结构和性能[J].武汉大学学报, 2002,48:197- 200.
[53] K Kurita, H Ikeda. Chemoselective Protection of the Amino Groups of Chitosan by Controlled Phthaloylation :Facile Preparation of a Precursor Useful for Chemical Modifications[J]. Biomacromolecules, 2002, 3: 1-4.
[54] Y Tong, S Wang. Synthesis of O,O-dipalmitoyl chitosan and its amphiphilic properties and capability of cholesterol absorption[J]. Carbohydr Polym, 2005, 5:1-5.
[55] F S Kittur, K V H Prashanth, K Udaya Sankar, et al. Characterization of chitin, chitosan and their carboxymethyl derivatives by differential scanning calorimetry Carbohydrate Polymers, 2002, 49(2): 185-193.
[56]陈凌云,杜予民.羧甲基壳聚糖的取代度及保湿性[J].应用化学, 2001, 18: 5-8.
[57] M Thanou, J C Verhoef, P Marbach, et al.. Intestinal absorption of octreotide: N-trimethyl chitosan chloride (TMC) ameliorates the permeability and absorption properties of the somatostatin analogue in vitro and in vivo[J]. Pharm. Sci, 2000, 89: 951– 957.
[58] K Aiedeh, M O Taha. Synthesis of chitosan succinate and chitosan phthalate and their evaluation as suggested matrices in orally administered, colon-specific drug delivery systems[J]. Archiv.der Pharmazie, 1999, 332: 103– 107.
[59] L R Sudimack. Targeted drug delivery via the folate receptor[J]. Adv Drug Deliv Rev, 2000, 41(2): 147.
[60] Y J Lu, S Philip. Folate-mediated delivery of macromolecular anticancer therapeutic agents[J]. Advanced Drug Delivery Reviews, 2002, 54: 675-693.
[61] C P Leamon, P S Low. Folate-mediated targeting: from diagnositics to drug and gene delivery[J]. Drug Discovery Today, 2001, 6(1): 44-51.
[62]张仲男,李睦昭.叶酸在肿瘤治疗中的应用[J].中国综合临床,2004, 20 (12): 1151-1152.
[63]赵建学,刘顺英,沈洪,等.胃粘膜细胞内叶酸水平变化[J].陕西医学检验,2002,31(6): 500-501.
[64]施亦东,季莉,陈衍夏.水溶性羟丙基壳聚糖的制备与结构特征[J].印染助剂.2006, 23 (3): 26-30.
[65]柳时,徐喆,罗智.叶酸偶联壳聚糖纳米粒的制备[J].医药导报, 2006, 25(6): 561-563.
[66] S Y Xiao, C Y Tong. Preparation of folate-conjugated starch nanoparticles and its application to tumor-targeted drug delivery vector[J]. Chinese Science Bulletin, 2006, 51(14): 1693-1697.
[67]张良珂,侯世祥,毛声俊.叶酸偶联白蛋白纳米粒的制备工艺研究[J].生物医学工程学杂志, 2004, 21(2): 225-228.
[68] S Mansouri. Characterization of folate-chitosan-DNA nanoparticles for gene therapy[J]. Biomaterials, 2006, 27: 2060-2065.
[69] P Chan, M Kurisawa. Synthesis and characterization of chitosan-g-poly(ethylene glycol)-folate as a non-viral carrier for tumor-targeted gene delivery[J]. Biomaterials, 2007, 28: 540-549.
[2]蒋挺大.壳聚糖[M].北京:化学工业出版社,2001: 12-32.
[3] S Nicol. Life after death for empty shells[J]. New Sci, 1991, 129: 46-48.
[4] P C Berscht, B Nies, A Liebendorfer, et al. Incorporation of basic fibroblast growth factor into methylpyrrolidinone chitosan fleeces and determination of the in vitro release characteristics[J]. Biomaterials, 1994, 15: 593-600.
[5] T Sannan, K Kurita, Y Iwakura. Studies on chitin: effect of deacetylation on solubility[J]. Chem. 1976, 177:3589-3600.
[6] H Seo, A Shoji, Y Itoh, et al. Antibacterial fiber blended with chitosan[J]. Chitin World, 1994: 623–631.
[7] D Valérie, D V Vinod. Pharmaceutical applications of chitosan[J].Elsevier Science,1998, 10: 246-253.
[8]孙慎侠,付昌斌,范钦信.壳聚糖的生物活性及其在中医药领域中的应用[J].贵阳中医学院学报, 2003, 25(1): 39-41.
[9] I Genta, F Pavanetto, B Conti, et al. Spray-drying for the preparation of chitosan microspheres[J]. Control Release Bioact.Mater, 1994, 21: 616-617.
[10] E E Hassan, R C Parish, J M Gallo. Optimized formulation of magnetic chitosan microspheres containing the anticancer agent[J]. Pharm. Res, 1992, 9: 390-397.
[11] L Illum, N F Farraj, S S Davis. Chitosan as a novel nasal delivery system for peptide drugs[J]. Pharm. Res, 1994, 11: 1186-1189.
[12] H L Luehen, C M Lehr, C O Rentel, et al. Bioadhesive polymers for the peroral delivery of peptide drugs[J]. Control Release, 1994, 29: 329-338.
[13] P A Sanford. Chitosan: commercial uses and potential applications, Chitin and Chitosan–Sources, Chemistry, Biochemistry Physical Properties and Applications[M]. Elsevier, London, 1989: 51–69.
[14]元英进,刘明言,董岸杰.中药现代化生产技术[M].北京:化学工业出版社,2001:227-233.
[15] J R Robinson. Rationale of bioadhesion /mucoadhesion, Possibilities and Future Trends[J].Chem,1990, 8: 13–15.
[16] H Sano, K I Shibasaki, T Matsukubo, et al. Effect of rinsing with phosphorylated chitosan on 4-day plaque regrowth[J]. Coll, 2001, 42: 251–256.
[17]张建林,李平,徐吉光等.盐酸曲马多口腔药膜的制备及质量控制[J].兰州医学院学报, 2001, 4 (27):14-16.
[18] A B Schnǔrch, C E Kast. Chemically modified chitosans as enzyme inhibitors[J]. Adv. Drug Deliv. Rev, 2001, 52: 127-137.
[19] R A Yokel, K M Dickey, A H Goldberg. Selective adherence of a sucralfate-tetracycline complex to gastric ulcers: implications for the treatment of Helicobacter pylori[J]. Bio-pharm Drug Dispos, 1995, 16: 475-479.
[20] S Shah, R Qaqish, V Patel, et al. Evaluation of the factors influencing stomach-specific delivery of antibacterial agents for Helicobacter pylori infection[J]. Pharm. Pharmacol, 1999, 51: 667-672.
[21]董春玲,冷延国,廖进康等.明胶-壳聚糖微胶囊化过程研究[J].明胶科学与技术,1999,19(3):117-123.
[22]徐蔚,曹毅,张纪.壳聚糖微球释药机制的研究[J].昆明医学院学报, 2002,1:7-10.
[23] R Hejazi, M Amiji. Stomach-specific anti-H. pylori therapy:preparation and characterization of tetracycline-loaded chitosan microspheres[J]. Int. Pharm, 2002, 235: 87-94.
[24] C Remuǎan-Lǒpez, A Portero, M Lemos. Chitosan microspheres for the specific delivery of amoxycillin to the gastric cavity[J]. Stp Pharma Sci, 2000, 10: 69-76.
[25]王志国,张敏卿.壳聚糖在靶向制剂中的研究进展[J].化工进展,2004,4(23):375-379.
[26] Y Pan, Y Li, H Zhao, et al. Chitosan nanoparticles improve the intestinal absorption of insulin in vivo[J]. Int. Pharm, 2002, 249: 139-147.
[27] L Ilium, N Farraj, H Critchley, et al. Nasal administration of gentamicin using a novel microsphere delivery system[J].International Journal Pharmaceutics,1988, 46: 261-265.
[28] I. J Gill, A N Fisher, N Farraj, et al. Intranasal absorption of granulocyte-colony stimulating factor (G-CSF) from powder formulations, in sheep European[J]. Journal of Pharmaceutical Sciences, 1998, 6(1): 1-10.
[29] L Illum, I Jabbal-Gill. Chitosan as a novel nasal delivery system for vaccines[J]. Advanced Drug Delivery Reviews, 2001, 51: 81-96.
[30] H Q Mao, K Roy, V L Troung-Le, et al. Chitosan DNA nanoparticles as gene delivery carriers: synthesis, characterization and transfection efficiency[J]. Control Release, 2001, 70: 399- 421.
[31] I C Lee, Y H Kwon, W H Kim, et al. Preparation of chitosan self-aggregates as a gene delivery system[J]. Control Release, 1998, 51: 213-220.
[32] F Zheng, X W Shi, G F Yang. Chitosan nanoparticle as gene therapy vector via gastrointestinal mucosa administration: Results of an in vitro and in vivo study[J]. Life Sciences 2007, 80: 388-396.
[33] Z Cui, R J Mumper. Chitosan-based nanoparticles for topical genetic immunization[J]. Control Release, 2001, 75: 409-419.
[34] T López-León, E L S Carvalho. Physicochemical characterization of chitosan nanoparticles: electrokinetic and stability behavior[J]. Journal of Colloid and Interface Science, 2005, 283: 344-351.
[35] J Akbuga, G Durmaz. Preparation and evaluation of cross-linked chitosan microspheres containing furosemide[J]. Int. Pharm, 1994, 11: 217-222.
[36] S G Kumbar, A R Kulkarni, T M Aminabhavi. Cross-linked chitosan microspheres for encapsulation of diclofenac sodium: effect of cross-linking agent[J]. J. Microencapsulation 2002, 19: 173-180.
[37] A A Al-Helw, A A Al-Angary, G. M Mahrous, et al.. Preparation and evaluation of sustained release cross-linked chitosan microspheres containing phenobarbitone[J]. Microencapsulation, 1998, 15: 373-382.
[38] K Nishimura, S Nishimura, H Seo, et al. Macrophage activation with multiporous beads prepared from partially deacetylated chitin[J]. Biomed. Mater. Res, 1986, 20: 1359-1372.
[39] S Ozbas-Turan, J Akbuga, C Aral. Controlled release of interleukin-2 from chitosan microspheres[J]. Pharm. Sci, 2002, 91: 124-125.
[40] H Q Mao, K Roy, V L Troung-Le, et al. Chitosan DNA nanoparticles as gene delivery carriers: synthesis, characterization and transfection efficiency[J]. Control Release, 2001, 70:399-421.
[41] P He, S S Davis, L Illum. Chitosan microspheres prepared by spray drying[J]. Int. Pharm, 1999, 187: 53-65.
[42] A G Gonzalez, S A Igea, F J O Espinar, et al. Chitosan and chondroitin microspheres for oral-administration controlled release of metoclopramide[J]. Eur. Pharm. Biopharm, 1999, 48: 149-155.
[43] M L Lorenzo-Lamosa, C Remunan-Lopez, et al. Design of microencapsulated chitosan microspheres for colonic drug delivery[J]. Control Release, 1998, 52:109-118.
[44] R Bodmeier, H G Chen, O Paeratakul. A novel approach to the oral delivery of micro-or nanoparticles[J]. Pharm Res, 1989, 5: 413.
[45] Y Kawashima, T Handa, H Takenaka, et al.. Novel method for the preparation of controlled-release theophylline granules coated with a polyelectrolyte complex of sodium polyphosphate-chitosan[J]. J. Pharm. Sci, 1985, 74: 264-268.
[46] A Polk, B Amsden, K D Yao, et al. Controlled release of albumin from chitosan-alginate microcapsules[J]. Pharm. Sci, 1994, 83: 178-185.
[47] R Fernandez-Urrusuno, P Cavlo, C Remunan-Lopez, et al. Enhancement of nasal absorption of insulin using chitosan nanoparticles[J]. Pharm. Res. 1999, 16: 1576-1581.
[48] Y Xu, Y Du. Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles[J]. Int.Pharm, 2003, 250: 215-226.
[49] H J Ko, S J Park, J B Hwang, et al. Preparation and characterization of chitosan microparticles intended for controlled drug delivery[J]. Int. Pharm, 2002, 249: 165-174.
[50] A Maitra. Determination of size parameters of water-Aerosol OT–oil reverse micelles from their nuclear magnetic resonance data[J]. J. Phys. Chem, 1984, 88: 5122-5125.
[51] S Mitra, U Gaur, P C Ghosh, et al.. Tumor targeted delivery of encapsulated dextran- doxorubicin conjugate using chitosan nanoparticles as carrier[J]. Control Release, 2001, 74: 317-323.
[52]郑化,杜予民. N-酰基壳聚糖的合成及膜的结构和性能[J].武汉大学学报, 2002,48:197- 200.
[53] K Kurita, H Ikeda. Chemoselective Protection of the Amino Groups of Chitosan by Controlled Phthaloylation :Facile Preparation of a Precursor Useful for Chemical Modifications[J]. Biomacromolecules, 2002, 3: 1-4.
[54] Y Tong, S Wang. Synthesis of O,O-dipalmitoyl chitosan and its amphiphilic properties and capability of cholesterol absorption[J]. Carbohydr Polym, 2005, 5:1-5.
[55] F S Kittur, K V H Prashanth, K Udaya Sankar, et al. Characterization of chitin, chitosan and their carboxymethyl derivatives by differential scanning calorimetry Carbohydrate Polymers, 2002, 49(2): 185-193.
[56]陈凌云,杜予民.羧甲基壳聚糖的取代度及保湿性[J].应用化学, 2001, 18: 5-8.
[57] M Thanou, J C Verhoef, P Marbach, et al.. Intestinal absorption of octreotide: N-trimethyl chitosan chloride (TMC) ameliorates the permeability and absorption properties of the somatostatin analogue in vitro and in vivo[J]. Pharm. Sci, 2000, 89: 951– 957.
[58] K Aiedeh, M O Taha. Synthesis of chitosan succinate and chitosan phthalate and their evaluation as suggested matrices in orally administered, colon-specific drug delivery systems[J]. Archiv.der Pharmazie, 1999, 332: 103– 107.
[59] L R Sudimack. Targeted drug delivery via the folate receptor[J]. Adv Drug Deliv Rev, 2000, 41(2): 147.
[60] Y J Lu, S Philip. Folate-mediated delivery of macromolecular anticancer therapeutic agents[J]. Advanced Drug Delivery Reviews, 2002, 54: 675-693.
[61] C P Leamon, P S Low. Folate-mediated targeting: from diagnositics to drug and gene delivery[J]. Drug Discovery Today, 2001, 6(1): 44-51.
[62]张仲男,李睦昭.叶酸在肿瘤治疗中的应用[J].中国综合临床,2004, 20 (12): 1151-1152.
[63]赵建学,刘顺英,沈洪,等.胃粘膜细胞内叶酸水平变化[J].陕西医学检验,2002,31(6): 500-501.
[64]施亦东,季莉,陈衍夏.水溶性羟丙基壳聚糖的制备与结构特征[J].印染助剂.2006, 23 (3): 26-30.
[65]柳时,徐喆,罗智.叶酸偶联壳聚糖纳米粒的制备[J].医药导报, 2006, 25(6): 561-563.
[66] S Y Xiao, C Y Tong. Preparation of folate-conjugated starch nanoparticles and its application to tumor-targeted drug delivery vector[J]. Chinese Science Bulletin, 2006, 51(14): 1693-1697.
[67]张良珂,侯世祥,毛声俊.叶酸偶联白蛋白纳米粒的制备工艺研究[J].生物医学工程学杂志, 2004, 21(2): 225-228.
[68] S Mansouri. Characterization of folate-chitosan-DNA nanoparticles for gene therapy[J]. Biomaterials, 2006, 27: 2060-2065.
[69] P Chan, M Kurisawa. Synthesis and characterization of chitosan-g-poly(ethylene glycol)-folate as a non-viral carrier for tumor-targeted gene delivery[J]. Biomaterials, 2007, 28: 540-549.