丝素载药微球的制备及其性能研究
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摘要
本文以丝素蛋白(SF)为载体,以地塞米松磷酸钠(DSP)为药物模型,采用W/O/W型复乳法,制备丝素微球和丝素载药微球。用扫描电镜观察微球的表面形态,使用激光粒度仪测量粒径大小及分布,采用X-射线衍射法,红外光谱法和DTA差热分析法研究微球的结构。采用MTT法检测丝素微球的细胞毒性,采用药物体外释放法测定微球的释药性能。探讨了乳化剂比例、有机溶剂种类、有机溶剂与丝素比例、丝素浓度、地塞米松磷酸钠与丝素比例对微球制备及其性能的影响。
实验结果表明:微球外观形态近似球型,丝素微球的平均粒径在0.56μm～56.52μm之间,随着乳化剂比例的增加,丝素微球的平径粒径逐渐减小,尤其乳化剂比例大于3%时,减少明显;随着丝素浓度的增加,丝素微球的粒径增加,特别是当丝素浓度大于6%时尤为明显;当有机溶剂与丝素比例由1:1增加到2:1时,丝素微球粒径减小明显,但当有机溶剂与丝素比例大于2:1时,微球粒径变化不明显。丝素载药微球的平均粒径在6.53μm～66.27μm之间。纯丝素的结构主要为无定型。使用不同的有机溶剂、不同的有机溶剂与丝素比例、不同的丝素浓度制得的丝素微球和丝素载药微球,其结构同纯丝素相比均发生了明显的变化,聚集态结构主要为SilkⅠ和SilkⅡ结构,分子构象主要为β-折叠。使用三种不同的有机溶剂制得的丝素微球其细胞毒性均为0级,即无细胞毒性。不同工艺条件下制得丝素载药微球的平均载药率在2.31%～9.01%之间,平均包埋率在31.28%～99.07%之间。微球的释放情况:1h的释药率均在50%左右,当DSP/SF比例在1:20～1:40之间、有机溶剂与丝素比例在4:1～6:1之间和丝素浓度在3%～6%范围内,有一定的缓释效果,尤其当DSP/SF比例在1:20～1:40范围内和有机溶剂与丝素比例为5:1～6:1时,4h开始释药速度减慢明显,96h释放了总药量的85%左右。其他工艺条件下制得的丝素载药微球突释现象较明显。
A w/o/w double emulsion method was developed to prepare silk fibroin microspheres using silk fibroin (SF) as vehicle, dexamethasone sodium phosphate (DSP) as drug model. The surface shape was observed by SEM, the particle diameter’s size and distribution were observed by Laster particle sizer. The structure was studied by X-ray, FTIR and DTA. The cytotoxicity was assessed by MTT assay. The properties of drug release was assessed by in vitro release method. The effect on the preparation of SF microspheres and properties were investigated by the ratio of emulsifier, the variety of organic solvent, the organic solvent :SF ratio, the concentration of SF, the DSP : SF ratio.
The results showed that the microsperes approximatively spherical. The SF microspheres’average size was between 0.56～56.52μm, and the particle sizes decreased with the ratio of emulsifier increased, there was an obviously decrease when the ratio above 3%. The particle size of SF microspheres increased with the concentration of SF increased, especially above 6%. The particle size of SF microspheres decreased obviously when the organic solvent : SF ratio added from 1:1 to 2:1, but there was no obvious change above 2:1. The drug-loading microspheres’average size was between 6.53～66.27μm. The structure of pure silk fibroin was mainly amorphism. The structure of SF microspheres and drug-loading microspheres prepared by different organic solvent, different organic solvent :SF ratio, different concentration of SF had an obviously change compared to pure SF, the state of aggregation was mainly silkⅠand silkⅡand conformation wasβ-sheet. The cytotoxicity rank of SF microspheres prepared by the three different organic solvent were 0, this showed non-poisonous. The average drug-loading efficiency was between 2.31%～9.01% and the average encapsulation efficiency was between 31.28%～99.07%. Drug release of microspheres:the release rate was about 50% for 1h, there had a slow release effect when the DSP : SF ratio was between 1:20～1:40, the organic solvent : SF ratio was between 4: 1～6:1 and the concentration of SF was between 3%～6%, especially the DSP :SF ratio was between 1:20～1:40 and the organic solvent : SF ratio was 5: 1～6:1. After 4h had passed, the release rate obviously decreased, and the quantity of the released drug was about 85% of the total. The SF drug-loading microspheres prepared under other technologies had an obvious suddenly release.
实验结果表明:微球外观形态近似球型,丝素微球的平均粒径在0.56μm～56.52μm之间,随着乳化剂比例的增加,丝素微球的平径粒径逐渐减小,尤其乳化剂比例大于3%时,减少明显;随着丝素浓度的增加,丝素微球的粒径增加,特别是当丝素浓度大于6%时尤为明显;当有机溶剂与丝素比例由1:1增加到2:1时,丝素微球粒径减小明显,但当有机溶剂与丝素比例大于2:1时,微球粒径变化不明显。丝素载药微球的平均粒径在6.53μm～66.27μm之间。纯丝素的结构主要为无定型。使用不同的有机溶剂、不同的有机溶剂与丝素比例、不同的丝素浓度制得的丝素微球和丝素载药微球,其结构同纯丝素相比均发生了明显的变化,聚集态结构主要为SilkⅠ和SilkⅡ结构,分子构象主要为β-折叠。使用三种不同的有机溶剂制得的丝素微球其细胞毒性均为0级,即无细胞毒性。不同工艺条件下制得丝素载药微球的平均载药率在2.31%～9.01%之间,平均包埋率在31.28%～99.07%之间。微球的释放情况:1h的释药率均在50%左右,当DSP/SF比例在1:20～1:40之间、有机溶剂与丝素比例在4:1～6:1之间和丝素浓度在3%～6%范围内,有一定的缓释效果,尤其当DSP/SF比例在1:20～1:40范围内和有机溶剂与丝素比例为5:1～6:1时,4h开始释药速度减慢明显,96h释放了总药量的85%左右。其他工艺条件下制得的丝素载药微球突释现象较明显。
A w/o/w double emulsion method was developed to prepare silk fibroin microspheres using silk fibroin (SF) as vehicle, dexamethasone sodium phosphate (DSP) as drug model. The surface shape was observed by SEM, the particle diameter’s size and distribution were observed by Laster particle sizer. The structure was studied by X-ray, FTIR and DTA. The cytotoxicity was assessed by MTT assay. The properties of drug release was assessed by in vitro release method. The effect on the preparation of SF microspheres and properties were investigated by the ratio of emulsifier, the variety of organic solvent, the organic solvent :SF ratio, the concentration of SF, the DSP : SF ratio.
The results showed that the microsperes approximatively spherical. The SF microspheres’average size was between 0.56～56.52μm, and the particle sizes decreased with the ratio of emulsifier increased, there was an obviously decrease when the ratio above 3%. The particle size of SF microspheres increased with the concentration of SF increased, especially above 6%. The particle size of SF microspheres decreased obviously when the organic solvent : SF ratio added from 1:1 to 2:1, but there was no obvious change above 2:1. The drug-loading microspheres’average size was between 6.53～66.27μm. The structure of pure silk fibroin was mainly amorphism. The structure of SF microspheres and drug-loading microspheres prepared by different organic solvent, different organic solvent :SF ratio, different concentration of SF had an obviously change compared to pure SF, the state of aggregation was mainly silkⅠand silkⅡand conformation wasβ-sheet. The cytotoxicity rank of SF microspheres prepared by the three different organic solvent were 0, this showed non-poisonous. The average drug-loading efficiency was between 2.31%～9.01% and the average encapsulation efficiency was between 31.28%～99.07%. Drug release of microspheres:the release rate was about 50% for 1h, there had a slow release effect when the DSP : SF ratio was between 1:20～1:40, the organic solvent : SF ratio was between 4: 1～6:1 and the concentration of SF was between 3%～6%, especially the DSP :SF ratio was between 1:20～1:40 and the organic solvent : SF ratio was 5: 1～6:1. After 4h had passed, the release rate obviously decreased, and the quantity of the released drug was about 85% of the total. The SF drug-loading microspheres prepared under other technologies had an obvious suddenly release.
引文
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[2] Wada R,Hyon SH,Ikada Y. Lactic acid oligomer microspheres containing hydrophilic drugs[J].J Pharm Sci,1990,79(10):919-924
[3] 胡一桥,郭建新,郑梁元等.胰岛素聚乳酸微球处方筛选[J].中国药学杂志,1999,34(12):822-826
[4] 张万国,胡晋红,蒋雪涛等.聚乳酸相对分子量对利福平微球及其药物分布状态的影响[J].解放军药学学报,2001,17(2):59-61
[5] 韩永涛,黄桂华,席延卫等.阿奇霉素聚乳酸微球的制备及其体外释药特性的研究[J].山东大学学报,2006,44(8):853-856
[6] 覃宇悦,程春生,樊建等.替硝唑聚乳酸微球的制备及其体外释药性能[J].中国医院药学杂志,2007,27(6):735-737
[7] Wang YM,Sato H,Adachi I,et al. Preparation and characterization of poly(lactic-co-glycolic acid) microspheres for targeted delivery of a novel anticancer agent,taxol[J]. Chem Pharm Bull,1996,44(10):1935-1940
[8] 李学明,涂家生,何飞.生物降解型尼索地平微球的研究[J].中国药科大学学报,2001,32(5):346-349
[9] T. Hickey,D. Kreutzer,et al. In vivo evaluation of a dexamethasone /PLGA microsphere system designed to suppress the inflammatory tissue response to implantable medical devices[J]. BiomedMater Res,2002,61:180–187
[10] Carrascosa C,Torres C. Microspheres containing insulin-like growth fatorI for treatment of chronic neurodegeneration[J]. Biomaterials,2004,25(8):707-714
[11] 段宏,沈彬,何勤等.缓释bFGF/PLGA 微球制备及其体外释药性质和生物活性的研究[J]. 中国药学杂志,2004,39(3):196-198
[12] 马晓东,郭小明,张纪等. PLGA/地塞米松缓释剂的合成及脑内局部应用研究[J]. 中国微侵袭神经外科杂志,2006,11(7):324-326
[13] 王思铭.紫杉醇长效注射缓释微球的体内外评价[J].长春中医药大学学报,2007,23(3):15-16
[14] 杨千帆.聚乳酸(PLA)在药物控释体系中的应用及其前景[J].中国西部科技,2003,6:18-19
[15] Sandow J,Seidel HR. Microcapsule of regulatory peptides with controlled release: manufactureand injection preparation. Eur Pat Appl,172422[P],1985
[16] Abe H,Yamamoto Y,Doi Y,et al. Preparation of poly(3-hydroxybutyrate) microspheres containing lastet of an anticancerdrug and its application to drug delivery systems[J].Kvbunshi Rvnshu,1992,19(1):61-67
[17] Kassab AC,Xu K,Denkbas EB,et al. Rifampicin carrying polyhydroxybutyrate microspheres as a potential chemoembolization agent[J]. J Biomater Sci Polym Ed,1997,8(12):947-961
[18] 陈建海,陈志良,侯连兵等.聚羟基丁酸酯缓释微球的制备与性能[J].功能高分子学报,2000,13(1):61-64
[19] 胡弢,吴宝剑,吴伟.多柔比星聚羟基丁酸酯微球的制备和体外性质[J].复旦学报,2005,32(4):407-410
[20] 陆彬,张景勍，杨红.肺靶向卡铂明胶微球的研究[J].药学学报,1999,34 (10):786-789
[21] 丁红,邢桂琴,谢茵.阿霉素明胶微球的制备与特性研究[J].中国医院药学杂志,2000,20(7):387-389
[22] 吴红,吴道澄,于开涛等.平阳霉素明胶微球的制备及其释药特性[J].生物医学工程学杂志,2003,20(4):646-649
[23] 苏小妹,杨志文,付达华.对乙酰氨基酚明胶微球的制备及缓释效果研究[J]. 江西医学院学报,2005,45(6):43-45
[24] 王忆娟,刘守信,房喻等.作为细胞微载体的明胶基缓释微球的制备[J].高等学校化学学报,2007,28:1776-1780
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