羧甲基壳聚糖植入可降解缓释微球的制备工艺及生物医学特性
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摘要
在局部感染的预防和治疗中，抗菌剂的全身应用具有局部有效药物浓度低、血药浓度不稳定及其他诸多限制。目前，局部植入剂型的抗菌效价较低，缓释载体不能被降解吸收，需要二次手术取出。针对以上状况，本研究探索一种抗菌效价较高、低组织修复遮挡、具有良好生物降解性和生物相容性的抗菌剂植入性药物传递系统(IDDS)的制备工艺、剂型的理化性质、体内外的释放行为及生物学特点，试图发现一条局部感染局部治疗和预防的临床规律。
首先根据生物降解、生物相容、高抗菌效价和低组织修复遮挡等标准确定缓释微球(MS)作为剂型、羧甲基壳聚糖(CMC)为缓释辅料、环丙沙星(CPX)为载药。进而根据载药和辅料的特性选择乳化交联作为制备工艺，并根据制备微球的表观等指标，通过正交试验筛选处方构成、优化微球的制备工艺(如交联温度、乳化速度等)，初步确定微球的制备工艺流程。通过多种检测手段，如：扫描电镜(SEM)、红外光谱(IR)、示差热分析(DTA)等对制备样本的理化特性进行检测。建立体外持续流动释放系统，对制备样本的体外释放特性进行检测，明确微球的体外释放模式，并根据其理化和体外释放检测结果再次优化处方构成和制备工艺条件。探索利用HPLC方法检测血液、组织液中的CPX浓度的固相、液相环境以及血液、组织液的净化方法。根据优化后的处方和工艺条件制备CMC/CPX-MS，并将微球植入动物体内，通过多种检测途径，包括：大体观察、病理和透射电镜等手段检测微球的生物降解性和生物相容性等生物学特性。同时使用HPLC法检测动物血液和局部组织中的药物浓度及其变化趋势，明确微球的体内药物释放模式。
通过本实验，我们得到优化后的处方构成：CMC/CPX/NaCl为2/1/1；制备工艺条件：水相／油相为1／4；乳化温度为30℃；交联温度为5℃～8℃；交联度(戊二醛／CMC)为1／1；交联时间为3小时，乳化转速为800rpm。在以上工艺条件下制得的微球形态圆整，表面分布有直径为1μm～3μm的微孔，平均粒径为：200μm，微球的平均载药量为17.5％，微球亲水性良好，溶胀后的微球为凝胶状，溶胀率为65％。DTA和IR结果表明CPX和CMC在微球中无定形均匀分布，并
第二军医大学摘要博士学位论文
州钾甲...，.旦旦.旦旦旦旦旦
形成了较强的分子间作用力。优化后工艺条件制备的微球体外释放曲线表明:药
物的释放时间持续7d以上，无突释现象，微球的累积释放率与时间的平方根呈
线性关系，符合刀心uchi方程。微球植入动物体内后局部大体观察、透射电镜和
病理结果发现CMC/CPX一MS和组织之间具有良好的组织相容性，无明显炎症反
应，微球在体内可通过溶酶体的胞吞作用降解。通过HPLC检测发现:微球植入
体内后局部软组织药物浓度维持在金葡菌MICg。以上约10天，无突释现象;释
放速率和时间成负相关。
因此我们认为:CMC是制备植入可降解缓释微球的理想生物材料。乳化交
联技术是制备CP刀CMC一MS的简单、稳定的方法。CP刃CMC一MS是临床上术
后局部植入预防和治疗感染的安全、有效的剂型。
In the prevention and treatment for local infection, conventional oral and venous administraton of antibiotic have lots of limits, such as low and instable blood concentration, side effects as well. At present, local admistration of antibiotics has low efficiency; the carrier is not be easily degraded and need to be taken out secondly. In our study, we aimed to explore a new kind of implantable drug delivery system (IDDS) , which has better effect, lower prevention for tissue repiring, better biodegradability and biocompatibility.
We adopt microspheres (MS ) as drug form according to the drug efficency of antibiotics and the biodegradability and biocompatibity and lower tissue repare prevention of the carrier, and chose the carboxymethyl chitosan (CMC) as sustained releasing excipient, ciprofloxacin Hydrochloride (CPX) as loaded antibiotics. The emulsification and cross-link was chosed as preparation process according to the characteristics of the carrier and excipient. The formular was choosen throuth orthogonal design acccoding to the MS' apparent characteristics. Then we detected the physiochemical characteristics of the MS sample through scanning electron microscopy ( SEM ) , infrared spectrum ( IR )and differential thermal analysis ( DTA ) . We detected the releasing characteristics of sample and its influencing factors. And the formular was optimized according the detected results.
We probe the purifyication technique of blood and tussue fluid and used the method of high performance liquid chromatography (HPLC ) to examine the blood and tissue fluid concentratein of CPX. The CMC/CPXMS samples was prepared according to the optimized formular and preparation processs. We implanted the prepared sample into animal model and detected biocharicteristics such as biodegradability and biocompatibity through general observation, pathology examination and transmission electron microscopy (TFM ) et al. At the same time, we detected in vivo the drug concentrateion and diversification in blood and tissue fluid of animal model and definituded the drug releaseing pattern in vivo.
We got the optimized formular as The ratio of CMC's concentration to CPX's to NaCl's was 2 to 1 to 1; The cubage of water phase to oil phase's in the system of
emulsification was 1 to 4. The temperature of emulsification was 5 to 8 ; The degree of cross-linking impressed by the content ratio of glutaraldehyde ( GD ) to CMC was 1 to 1 . The cross-linking should last for three hour. The rate of emulsificational stiring was 800 rpm. The MS prepared according to these standards was well proportioned and with microbores basing diameter about lum~3um. The diameter of the prepared MS was averagly about 200um; the content of CPX in these MS was about 17.5%. These MS had outstanding hydrophilic capability and would turn into gelatin after swelling. The swelling ratio was 65%. Through IR and DTA examination, it was confirmmed that the CPX and CMC distributed amorphismly in the MS. The releasing test suggested the CPX could last for 7 days and had no phenomena of "sudden release". The accumulative releasing ratio had positive correlation with the square root of time. The releasing behavior could be simulated by the Higuchi equation. In vivo after the MS was implanted into ani
mal model we detected through general observation, pathology examination and TEM and found that it had good biocompatibility, and littlee inflammatory responses were found. The MS could be degraded by lysosome through "swallow"after being degraded into segment throngh lysozyme. The releasing test in vivo indicated that the concentration of CPX in the local tissue could last for 10 days above MICgo of Mlicrococcus aureus. The "sudden release" was not found. The maxium concentration was gained 24 hour after implanted. But the concentration validity could achieve half an hour after implant. The releasing rate had negative correlation with time after the point of the maxium concentration.
We recommended that CMC is an ideal kind of biodegradable biomaterial possessing functi
首先根据生物降解、生物相容、高抗菌效价和低组织修复遮挡等标准确定缓释微球(MS)作为剂型、羧甲基壳聚糖(CMC)为缓释辅料、环丙沙星(CPX)为载药。进而根据载药和辅料的特性选择乳化交联作为制备工艺，并根据制备微球的表观等指标，通过正交试验筛选处方构成、优化微球的制备工艺(如交联温度、乳化速度等)，初步确定微球的制备工艺流程。通过多种检测手段，如：扫描电镜(SEM)、红外光谱(IR)、示差热分析(DTA)等对制备样本的理化特性进行检测。建立体外持续流动释放系统，对制备样本的体外释放特性进行检测，明确微球的体外释放模式，并根据其理化和体外释放检测结果再次优化处方构成和制备工艺条件。探索利用HPLC方法检测血液、组织液中的CPX浓度的固相、液相环境以及血液、组织液的净化方法。根据优化后的处方和工艺条件制备CMC/CPX-MS，并将微球植入动物体内，通过多种检测途径，包括：大体观察、病理和透射电镜等手段检测微球的生物降解性和生物相容性等生物学特性。同时使用HPLC法检测动物血液和局部组织中的药物浓度及其变化趋势，明确微球的体内药物释放模式。
通过本实验，我们得到优化后的处方构成：CMC/CPX/NaCl为2/1/1；制备工艺条件：水相／油相为1／4；乳化温度为30℃；交联温度为5℃～8℃；交联度(戊二醛／CMC)为1／1；交联时间为3小时，乳化转速为800rpm。在以上工艺条件下制得的微球形态圆整，表面分布有直径为1μm～3μm的微孔，平均粒径为：200μm，微球的平均载药量为17.5％，微球亲水性良好，溶胀后的微球为凝胶状，溶胀率为65％。DTA和IR结果表明CPX和CMC在微球中无定形均匀分布，并
第二军医大学摘要博士学位论文
州钾甲...，.旦旦.旦旦旦旦旦
形成了较强的分子间作用力。优化后工艺条件制备的微球体外释放曲线表明:药
物的释放时间持续7d以上，无突释现象，微球的累积释放率与时间的平方根呈
线性关系，符合刀心uchi方程。微球植入动物体内后局部大体观察、透射电镜和
病理结果发现CMC/CPX一MS和组织之间具有良好的组织相容性，无明显炎症反
应，微球在体内可通过溶酶体的胞吞作用降解。通过HPLC检测发现:微球植入
体内后局部软组织药物浓度维持在金葡菌MICg。以上约10天，无突释现象;释
放速率和时间成负相关。
因此我们认为:CMC是制备植入可降解缓释微球的理想生物材料。乳化交
联技术是制备CP刀CMC一MS的简单、稳定的方法。CP刃CMC一MS是临床上术
后局部植入预防和治疗感染的安全、有效的剂型。
In the prevention and treatment for local infection, conventional oral and venous administraton of antibiotic have lots of limits, such as low and instable blood concentration, side effects as well. At present, local admistration of antibiotics has low efficiency; the carrier is not be easily degraded and need to be taken out secondly. In our study, we aimed to explore a new kind of implantable drug delivery system (IDDS) , which has better effect, lower prevention for tissue repiring, better biodegradability and biocompatibility.
We adopt microspheres (MS ) as drug form according to the drug efficency of antibiotics and the biodegradability and biocompatibity and lower tissue repare prevention of the carrier, and chose the carboxymethyl chitosan (CMC) as sustained releasing excipient, ciprofloxacin Hydrochloride (CPX) as loaded antibiotics. The emulsification and cross-link was chosed as preparation process according to the characteristics of the carrier and excipient. The formular was choosen throuth orthogonal design acccoding to the MS' apparent characteristics. Then we detected the physiochemical characteristics of the MS sample through scanning electron microscopy ( SEM ) , infrared spectrum ( IR )and differential thermal analysis ( DTA ) . We detected the releasing characteristics of sample and its influencing factors. And the formular was optimized according the detected results.
We probe the purifyication technique of blood and tussue fluid and used the method of high performance liquid chromatography (HPLC ) to examine the blood and tissue fluid concentratein of CPX. The CMC/CPXMS samples was prepared according to the optimized formular and preparation processs. We implanted the prepared sample into animal model and detected biocharicteristics such as biodegradability and biocompatibity through general observation, pathology examination and transmission electron microscopy (TFM ) et al. At the same time, we detected in vivo the drug concentrateion and diversification in blood and tissue fluid of animal model and definituded the drug releaseing pattern in vivo.
We got the optimized formular as The ratio of CMC's concentration to CPX's to NaCl's was 2 to 1 to 1; The cubage of water phase to oil phase's in the system of
emulsification was 1 to 4. The temperature of emulsification was 5 to 8 ; The degree of cross-linking impressed by the content ratio of glutaraldehyde ( GD ) to CMC was 1 to 1 . The cross-linking should last for three hour. The rate of emulsificational stiring was 800 rpm. The MS prepared according to these standards was well proportioned and with microbores basing diameter about lum~3um. The diameter of the prepared MS was averagly about 200um; the content of CPX in these MS was about 17.5%. These MS had outstanding hydrophilic capability and would turn into gelatin after swelling. The swelling ratio was 65%. Through IR and DTA examination, it was confirmmed that the CPX and CMC distributed amorphismly in the MS. The releasing test suggested the CPX could last for 7 days and had no phenomena of "sudden release". The accumulative releasing ratio had positive correlation with the square root of time. The releasing behavior could be simulated by the Higuchi equation. In vivo after the MS was implanted into ani
mal model we detected through general observation, pathology examination and TEM and found that it had good biocompatibility, and littlee inflammatory responses were found. The MS could be degraded by lysosome through "swallow"after being degraded into segment throngh lysozyme. The releasing test in vivo indicated that the concentration of CPX in the local tissue could last for 10 days above MICgo of Mlicrococcus aureus. The "sudden release" was not found. The maxium concentration was gained 24 hour after implanted. But the concentration validity could achieve half an hour after implant. The releasing rate had negative correlation with time after the point of the maxium concentration.
We recommended that CMC is an ideal kind of biodegradable biomaterial possessing functi
引文
1．陆彬．主编．药物新剂型与新技术．第一版．北京：人民卫生出版社．1998：522-526
2．孙效东，曲芬．药物分析杂志．1998；18(6)：380-383
3．张慧，洪有采，余珲等．紫外分光光度法测定盐酸环丙沙星软膏含量．中国药房，1995，6(4)：37-38
4．周秦秦，傅颖君，况荣华．高效液相色谱法测定盐酸环丙沙星血药浓度及其药动学研究．中国医院药学杂志，1998；18(3)：114-116
5．侯春林，卢建熙，包聚良等．几丁质生物学特性研究．中国修复重建外科杂志，1993；7(2) 115-6
6．张建湘，蔡克勤，马卫东等．壳聚糖棒材的组织相容性和安全性评价．生物医学工程学杂志，1996；13(4)：293-7
7．赵瑞芝，王中彦，胡良才．甲壳素及其衍生物作为缓释辅料的研究进展．国外医学，合成药、生化药、制剂分册，1996；17(3)：176-8
8．卢凤琦，曹宗顺，王春香．壳聚糖-海藻酸盐微囊对尼莫地平的缓释作用．中国医学杂志 1996；31(9)：555-6
9．王亚敏，石庭森，蒲永林等．顺铂壳聚糖微粒的制备及特性研究．药学学报，1996；31(4)：300-5
10．张超，胡蕴玉，孙怡群，等．庆大霉素重组异种骨复合体的制备及相关研究．中华外科杂志．1998；36(S)：53-5
11．吕厚山，马迪，丁海明，三种抗生素骨水泥抗菌作用和机械强度的研究．中华外科杂志．1998；36(S)：50-2
12．朱有财，卢琼芳，主编．抗生素临床应用手册．四川科学技术出版社，1987；78
13．黄崇本．局部应用抗生素预防切口感染．重庆医药．1985；14：55-7
14．卢欣，赵玉沛．局部应用抗生素的新进展．中华外科杂志，1998；36：62-3
15．戴自英．实用抗菌药物学．第2版．上海：科学技术出版社，1994：276-289
16．于守汛．环丙沙星与其他抗菌药治疗泛发性腹膜炎的疗效比较．国外医药抗生素分册．1994，15(6)：450-453
17．陈新谦，金有豫．新编药物学，第13版．北京：人民卫生出版社，1995：103-120
18．汪泽．冯萍．喹诺酮类药物对厌氧菌的体外抗活性研究．中国抗生素杂志，1995，20(2)：102-105
19．俞亚花，陆家明．环丙沙星的药动学，药物相互作用和配伍禁忌．现代应用药学，1994，11(4)：50-52
20．章建立，龚正．国产环丙沙星注射液药效与临床研究．中国抗生素杂志，1995，20(2)：113-115
21．姜素椿．喹诺酮类抗菌药物的临床应用．中国抗生素杂志，1995，20(2)：95-98
22．丁国华．氟喹诺酮类药的神经毒性作用．中国医院药学杂志，1995，15(8)：381-383
23．王莹，段保志．氟喹诺酮类药物在临床使用中应使用的问题．药物与临床，1992，8(2)：9-12
24．王睿，吴坚．7种抗菌药物对临床常见致病菌抗菌活性的比较．中国药学杂志，1995，30(3)：140-142
25．叶高峰．绿脓假单胞菌对环丙沙星和泰能等种药物体外交叉耐药研究．中国抗生素杂志，1995，20(1)：54-55
26. Eijer H, Hauke C, Arens S, et al. PC-Fix and local infection resistance-influence of implant design on postoperative infection development, clinical and experimental results. Injury 2001 Sep; 32 Suppl 2:S-B38-43
27. Shu XZ, Zhu KJ. A novel approach to prepare tripolyphosphate/chitosan complex beads for controlled release drug delivery. Int J Pharm. 2000 15:201(1): 51-58
28. Klemm K. The use of antibiotic-containing bead chains in the treatment of chronic bone infections. Clin Microbiol Infect. 2001; 7(1): 28-31
29. Kalkowska M, Kochanska B. Trials of using chitin and its derivatives in medicine and pharmacy. Czas Stomatol. 1990; 43(8): 474-478
30． Burke JF. Preventing bacterial infection by coordinating antibiotic and host activity: a time-dependent relationship. South Med J. 1977; 70 Suppl 1: 24-26,
31． Gomez J, Rodriguez M, Banos V, et al. Infections in joint prostheses: epidemiology and clinical presentation. A prospective study 1992-1999． Enferm Infecc Microbiol Clin 2002; 20 (2) : 74-77
32． Varum-KM; Myhr-MM; Hjerde-RJ, et al. In vitro degradation rates of partially N-acetylated chitosans in human serum. Carbohydr-Res. 1997; 299 (1-2) : 99-101
33． F-L Mi, S-S Shyu, C-T Chen et al. Porous chitosin microsphere for controlling the antigen release of Newcastle disease vaccine: preparation of antigen-adsorbed microsphere and in vitro release.biomaterials. 1999; 20:1603-1612
34． Felt-O; Buri-P; Gurny-R Chitosan: a unique polysaccharide for drug delivery. Drug-Dev-Ind-Pharm. 1998;24 (11) :979-93
35． Sabnis-S; Rege-P; Block-LH. Use of chitosan in compressed tablets of diclofenac sodium: inhibition of drug release in an acidic environment. Pharm-Dev-Technol. 1997; 2(3) : 243-55
36． Gaserod-O; Smidsrod-O; Skjak-Braek-G Microcapsules of alginate-chitosan-I. A quantitative study of the interaction between alginate and chitosan. Biomaterials. 1998; 19 (20) : 1815-25
37． Kas-HS. Chitosan: properties, preparations and application to microparticulate systems J-Microencapsul. 1997; 14 (6) : 689-711
38． Genta-I; Perugini-P; Pavanetto-F. Different molecular weight chitosan microspheres: influence on drug loading and drug release. Drug-Dev-Ind-Pharm. 1998; 24 (8) : 779-84
39． Huang-RG; Schwartz-JB; Ofner-CM-3rd. Microencapsulation of chlorpheniramine maleate-resin particles with crosslinked chitosan for sustained release. Pharm-Dev-Technol. 1999; 4 (1) : 107-15
40． Singh-UV; Udupa-N Methorcxate loaded chbosan and chitin microsphercs-in vitro characterization and pharmacokinetics in mice bearing Ehrlich ascitcs carcinoma. J-Microencapsul. 1998; 15 (5) : 581-94
41． Al-Helvv-AA; Al-Angary-AA; Mahrous-GM et al Preparation and evaluation of sustained release cross-linked chitosan microspheres containing phenobarbitone. J-Microencapsul.
1998;15 (3) :373-82
42． Chen Aimin, hou chunlin, bao juliang, et al. Antibiotic loaded chitosan bar. Clinical orthoaedics and related research. 1999,366:239-47
43． Tasker-RA; Connell-BJ; Ross-SJ, et al Development of an injectable sustained-release formulation of morphine: antinociceptive properties in rats. Lab-Anim. 1998; 32 (3) : 270-5
44． Genta-I; Conti-B; Perugini-P. Bioadhesive microspheres for ophthalmic administration of acyclovir. J-Pharm-Pharmacol. 1997; 49 (8) : 737-42
45． Thacharodi-D; Rao-KP. Development and in vitro evaluation of chitosan-based transdermal drug delivery systems for the controlled delivery of propranolol hydrochloride. Biomaterials. 1995;16 (2) :145-8
46． Stemberger .A, Scherer J, Ascherl R, et al. A biological drug-carrier in soft tissue infections. First Mediterranean congress of angiology, 1988; 275-277．
47． Smith AJ, Daniels T, Bohnen Jm. Soft tissue infections and the diabetic foot. Am J Surg, 1996; 172 (6A) :7s-12s.
48． Rushton N. Applications of local antibiotic therapy. Eur J Surg Suppl, 1997; 578: 27-30．
49． Vogel P, Lenz J. Prospective randomised atudy of gentacoll plus metronidazole versus dalacin in the treatment of infected pilonidal sinus. Internal Report. 1992; 63: 748-753．
50． Rahn HD. Abdominoperineal rectum excision. Chir Praxis, 1989; 41: 637-643．
51． Jorgensen LG, SorensenTS, Lorentzen JE. Clinical and pharmacokinetic evaluation of gentamicin containing collagen in groin wound infections after vascular reconstruction. Eur J Vasc, Surg, 1991; 5: 87-91．
52． Stmberger A, Grimm H, Bader F, et al. Local treatment of bone and soft tissue infections with the collagen-gentamicin sponge. Eur J Surge, 1997; 587 (s) : 17-26
2．孙效东，曲芬．药物分析杂志．1998；18(6)：380-383
3．张慧，洪有采，余珲等．紫外分光光度法测定盐酸环丙沙星软膏含量．中国药房，1995，6(4)：37-38
4．周秦秦，傅颖君，况荣华．高效液相色谱法测定盐酸环丙沙星血药浓度及其药动学研究．中国医院药学杂志，1998；18(3)：114-116
5．侯春林，卢建熙，包聚良等．几丁质生物学特性研究．中国修复重建外科杂志，1993；7(2) 115-6
6．张建湘，蔡克勤，马卫东等．壳聚糖棒材的组织相容性和安全性评价．生物医学工程学杂志，1996；13(4)：293-7
7．赵瑞芝，王中彦，胡良才．甲壳素及其衍生物作为缓释辅料的研究进展．国外医学，合成药、生化药、制剂分册，1996；17(3)：176-8
8．卢凤琦，曹宗顺，王春香．壳聚糖-海藻酸盐微囊对尼莫地平的缓释作用．中国医学杂志 1996；31(9)：555-6
9．王亚敏，石庭森，蒲永林等．顺铂壳聚糖微粒的制备及特性研究．药学学报，1996；31(4)：300-5
10．张超，胡蕴玉，孙怡群，等．庆大霉素重组异种骨复合体的制备及相关研究．中华外科杂志．1998；36(S)：53-5
11．吕厚山，马迪，丁海明，三种抗生素骨水泥抗菌作用和机械强度的研究．中华外科杂志．1998；36(S)：50-2
12．朱有财，卢琼芳，主编．抗生素临床应用手册．四川科学技术出版社，1987；78
13．黄崇本．局部应用抗生素预防切口感染．重庆医药．1985；14：55-7
14．卢欣，赵玉沛．局部应用抗生素的新进展．中华外科杂志，1998；36：62-3
15．戴自英．实用抗菌药物学．第2版．上海：科学技术出版社，1994：276-289
16．于守汛．环丙沙星与其他抗菌药治疗泛发性腹膜炎的疗效比较．国外医药抗生素分册．1994，15(6)：450-453
17．陈新谦，金有豫．新编药物学，第13版．北京：人民卫生出版社，1995：103-120
18．汪泽．冯萍．喹诺酮类药物对厌氧菌的体外抗活性研究．中国抗生素杂志，1995，20(2)：102-105
19．俞亚花，陆家明．环丙沙星的药动学，药物相互作用和配伍禁忌．现代应用药学，1994，11(4)：50-52
20．章建立，龚正．国产环丙沙星注射液药效与临床研究．中国抗生素杂志，1995，20(2)：113-115
21．姜素椿．喹诺酮类抗菌药物的临床应用．中国抗生素杂志，1995，20(2)：95-98
22．丁国华．氟喹诺酮类药的神经毒性作用．中国医院药学杂志，1995，15(8)：381-383
23．王莹，段保志．氟喹诺酮类药物在临床使用中应使用的问题．药物与临床，1992，8(2)：9-12
24．王睿，吴坚．7种抗菌药物对临床常见致病菌抗菌活性的比较．中国药学杂志，1995，30(3)：140-142
25．叶高峰．绿脓假单胞菌对环丙沙星和泰能等种药物体外交叉耐药研究．中国抗生素杂志，1995，20(1)：54-55
26. Eijer H, Hauke C, Arens S, et al. PC-Fix and local infection resistance-influence of implant design on postoperative infection development, clinical and experimental results. Injury 2001 Sep; 32 Suppl 2:S-B38-43
27. Shu XZ, Zhu KJ. A novel approach to prepare tripolyphosphate/chitosan complex beads for controlled release drug delivery. Int J Pharm. 2000 15:201(1): 51-58
28. Klemm K. The use of antibiotic-containing bead chains in the treatment of chronic bone infections. Clin Microbiol Infect. 2001; 7(1): 28-31
29. Kalkowska M, Kochanska B. Trials of using chitin and its derivatives in medicine and pharmacy. Czas Stomatol. 1990; 43(8): 474-478
30． Burke JF. Preventing bacterial infection by coordinating antibiotic and host activity: a time-dependent relationship. South Med J. 1977; 70 Suppl 1: 24-26,
31． Gomez J, Rodriguez M, Banos V, et al. Infections in joint prostheses: epidemiology and clinical presentation. A prospective study 1992-1999． Enferm Infecc Microbiol Clin 2002; 20 (2) : 74-77
32． Varum-KM; Myhr-MM; Hjerde-RJ, et al. In vitro degradation rates of partially N-acetylated chitosans in human serum. Carbohydr-Res. 1997; 299 (1-2) : 99-101
33． F-L Mi, S-S Shyu, C-T Chen et al. Porous chitosin microsphere for controlling the antigen release of Newcastle disease vaccine: preparation of antigen-adsorbed microsphere and in vitro release.biomaterials. 1999; 20:1603-1612
34． Felt-O; Buri-P; Gurny-R Chitosan: a unique polysaccharide for drug delivery. Drug-Dev-Ind-Pharm. 1998;24 (11) :979-93
35． Sabnis-S; Rege-P; Block-LH. Use of chitosan in compressed tablets of diclofenac sodium: inhibition of drug release in an acidic environment. Pharm-Dev-Technol. 1997; 2(3) : 243-55
36． Gaserod-O; Smidsrod-O; Skjak-Braek-G Microcapsules of alginate-chitosan-I. A quantitative study of the interaction between alginate and chitosan. Biomaterials. 1998; 19 (20) : 1815-25
37． Kas-HS. Chitosan: properties, preparations and application to microparticulate systems J-Microencapsul. 1997; 14 (6) : 689-711
38． Genta-I; Perugini-P; Pavanetto-F. Different molecular weight chitosan microspheres: influence on drug loading and drug release. Drug-Dev-Ind-Pharm. 1998; 24 (8) : 779-84
39． Huang-RG; Schwartz-JB; Ofner-CM-3rd. Microencapsulation of chlorpheniramine maleate-resin particles with crosslinked chitosan for sustained release. Pharm-Dev-Technol. 1999; 4 (1) : 107-15
40． Singh-UV; Udupa-N Methorcxate loaded chbosan and chitin microsphercs-in vitro characterization and pharmacokinetics in mice bearing Ehrlich ascitcs carcinoma. J-Microencapsul. 1998; 15 (5) : 581-94
41． Al-Helvv-AA; Al-Angary-AA; Mahrous-GM et al Preparation and evaluation of sustained release cross-linked chitosan microspheres containing phenobarbitone. J-Microencapsul.
1998;15 (3) :373-82
42． Chen Aimin, hou chunlin, bao juliang, et al. Antibiotic loaded chitosan bar. Clinical orthoaedics and related research. 1999,366:239-47
43． Tasker-RA; Connell-BJ; Ross-SJ, et al Development of an injectable sustained-release formulation of morphine: antinociceptive properties in rats. Lab-Anim. 1998; 32 (3) : 270-5
44． Genta-I; Conti-B; Perugini-P. Bioadhesive microspheres for ophthalmic administration of acyclovir. J-Pharm-Pharmacol. 1997; 49 (8) : 737-42
45． Thacharodi-D; Rao-KP. Development and in vitro evaluation of chitosan-based transdermal drug delivery systems for the controlled delivery of propranolol hydrochloride. Biomaterials. 1995;16 (2) :145-8
46． Stemberger .A, Scherer J, Ascherl R, et al. A biological drug-carrier in soft tissue infections. First Mediterranean congress of angiology, 1988; 275-277．
47． Smith AJ, Daniels T, Bohnen Jm. Soft tissue infections and the diabetic foot. Am J Surg, 1996; 172 (6A) :7s-12s.
48． Rushton N. Applications of local antibiotic therapy. Eur J Surg Suppl, 1997; 578: 27-30．
49． Vogel P, Lenz J. Prospective randomised atudy of gentacoll plus metronidazole versus dalacin in the treatment of infected pilonidal sinus. Internal Report. 1992; 63: 748-753．
50． Rahn HD. Abdominoperineal rectum excision. Chir Praxis, 1989; 41: 637-643．
51． Jorgensen LG, SorensenTS, Lorentzen JE. Clinical and pharmacokinetic evaluation of gentamicin containing collagen in groin wound infections after vascular reconstruction. Eur J Vasc, Surg, 1991; 5: 87-91．
52． Stmberger A, Grimm H, Bader F, et al. Local treatment of bone and soft tissue infections with the collagen-gentamicin sponge. Eur J Surge, 1997; 587 (s) : 17-26