米托蒽醌聚乳酸纳米粒的制备和细胞摄取研究
|
摘要
本课题制备了米托蒽醌聚乳酸纳米粒(DHAQ-PLA-NP),并用Dextran 70包裹,目的在于提高米托蒽醌(mitoxantrone, DHAQ)的疗效,降低其毒副作用,增加靶细胞的摄取。以人肝癌细胞SMMC-7721和人肝正常细胞L02为模型,探讨DHAQ和DHAQ-PLA-NP的细胞毒性,并评价两种细胞对DHAQ. DHAQ-PLA-NP和Dextran 70包裹DHAQ-PLA-NP的摄取特性,为DHAQ-PLA-NP进一步用于肝癌的临床治疗提供实验依据。
本文首先采用沉淀法制备DHAQ-PLA-NP,对制备工艺与处方中有机溶剂的比例,表面活性剂的浓度等影响因素进行筛选,得到优化的制备工艺和参数。制备的纳米粒粒径在150 nm左右,PDI值在0.1到0.3之间,电位在-40 mv左右。用乳糖作为冻干保护剂制得的DHAQ-PLA-NP冻干针剂外型美观,含量稳定。结果表明,优化的方法可以制备出包封率和载药量均较高的纳米粒,并具有粒径分布均匀,冻干效果良好等特点。
为了降低药物对肝正常细胞的毒性和考察DHAQ-PLA-NP对肝癌化疗的效果,我们采用MTT法进行定量分析,研究DHAQ和DHAQ-PLA-NP对人肝癌细胞SMMC-7721和人肝正常细胞L02的毒性。结果显示,DHAQ和DHAQ-PLA-NP的杀伤效应呈时间依赖性和剂量依赖性。由于纳米粒具有缓释作用,在相同时间点,其IC5o值大于DHAQ的IC5o值,说明DHAQ制成纳米粒后可以减少毒副作用。
为了增加人肝癌细胞SMMC-7721对DHAQ-PLA-NP的摄取和比较其与人肝正常细胞L02对DHAQ-PLA-NP的摄取量的差别,我们进一步采用Dextran 70包裹的DHAQ-PLA-NP,并建立了HPLC检测细胞样品中DHAQ含量和BCA法测定细胞的蛋白含量的方法。结果表明,低浓度时,DHAQ和DHAQ-PLA-NP的药物摄取量没有差别,但在高浓度时,DHAQ-PLA-NP要显著高于DHAQ,并且用2%Dextran70包裹DHAQ-PLA-NP的摄取量更高. DHAQ-PLA-NP的摄取量高于DHAQ, Dextran70包裹DHAQ-PLA-NP摄取量也高于DHAQ-PLA-NP(P<0.05),说明DHAQ制备成用Dextran70包裹的DHAQ-PLA-NP后更容易被细胞摄取。
In this paper, Mitoxantrone polylactic acid nanoparticles (DHAQ-PLA-NP) and the Dextran 70 coating DHAQ-PLA-NP were prepared. The aims are to enhance the therapeutic efficacy of mitoxantrone in treating, reduce the toxicity and increase the uptake amount. Then, the cytotoxicity of DHAQ and DHAQ-PLA-NP on human hepatoma cells (SMMC-7721) and human normal hepatocyte (LO2) were studied. Finally, the uptake of the original drug, DHAQ-PLA-NP and Dextran 70 coating DHAQ-PLA-NP in these two cells was investigated. The study will provide some available information for liver cancer treatment of DHAQ-PLA-NP in clinic.
The method of precipitation was employed to prepare DHAQ-PLA-NP. The factors, such as the ratio of organic solvent and surfactant which influence the preparation were investigated, and the techniques were optimized. The size was about 150 nm; the PDI was between 0.1 to 0.3 and the zeta potential was about -40mv. DHAQ-PLA-NP lyophilization injection was prepared by using lactose as protective agent in freeze drying. The product was of good looks and good quality. The results showed that the embedding ratio and drug loading of the nanoparticles were high. The nanoparticles had the characteristic which was the size well-distributed, freeze-dried good effect and so on.
To reduce the liver toxicity of normal cells and investigate the effect of DHAQ-PLA-NP to liver cancer chemotherapy, the MTT assay was carried out to measure the cytotoxicity of DHAQ and DHAQ-PLA-NP on human hepatoma cells SMMC-7721 and human normal hepatocyte LO2. The results showed that the killing effect of DHAQ and DHAQ-PLA-NP was time- and dose-dependent. It was found that the IC50 of nanoparticles which have slow-releasing property, at the same time, was greater than the IC50 of DHAQ. It was showed that DHAQ-PLA-NP could reduce the toxicity.
Furthermore, in order to increase the uptake of DHAQ-PLA-NP on human hepatoma cells SMMC-7721 and compare the uptake of DHAQ-PLA-NP between human hepatoma cells SMMC-7721 and human normal hepatocyte LO2, Dextran 70 coating DHAQ-PLA-NP was used and the concentration of mitoxantrone in SMMC-7721 cells and LO2 cells was determined by HPLC and the content of protein in cells by BCA method. The results showed that there was no obvious difference between DHAQ and DHAQ-PLA-NP at lower concentration. However, the uptake amount of DHAQ-PLA-NP was much larger than that of DHAQ at higher concentration and the uptake amount of 2% Dextran 70 coating DHAQ-PLA-NP was the most. The uptake amout of DHAQ-PLA-NP was larger than that of DHAQ and the uptake amout of 2% Dextran 70 coating DHAQ-PLA-NP was also higher than that of DHAQ-PLA-NP (P<0.05). It was showed that the uptake of Dextran 70 coating DHAQ-PLA-NP was more easily by cells.
本文首先采用沉淀法制备DHAQ-PLA-NP,对制备工艺与处方中有机溶剂的比例,表面活性剂的浓度等影响因素进行筛选,得到优化的制备工艺和参数。制备的纳米粒粒径在150 nm左右,PDI值在0.1到0.3之间,电位在-40 mv左右。用乳糖作为冻干保护剂制得的DHAQ-PLA-NP冻干针剂外型美观,含量稳定。结果表明,优化的方法可以制备出包封率和载药量均较高的纳米粒,并具有粒径分布均匀,冻干效果良好等特点。
为了降低药物对肝正常细胞的毒性和考察DHAQ-PLA-NP对肝癌化疗的效果,我们采用MTT法进行定量分析,研究DHAQ和DHAQ-PLA-NP对人肝癌细胞SMMC-7721和人肝正常细胞L02的毒性。结果显示,DHAQ和DHAQ-PLA-NP的杀伤效应呈时间依赖性和剂量依赖性。由于纳米粒具有缓释作用,在相同时间点,其IC5o值大于DHAQ的IC5o值,说明DHAQ制成纳米粒后可以减少毒副作用。
为了增加人肝癌细胞SMMC-7721对DHAQ-PLA-NP的摄取和比较其与人肝正常细胞L02对DHAQ-PLA-NP的摄取量的差别,我们进一步采用Dextran 70包裹的DHAQ-PLA-NP,并建立了HPLC检测细胞样品中DHAQ含量和BCA法测定细胞的蛋白含量的方法。结果表明,低浓度时,DHAQ和DHAQ-PLA-NP的药物摄取量没有差别,但在高浓度时,DHAQ-PLA-NP要显著高于DHAQ,并且用2%Dextran70包裹DHAQ-PLA-NP的摄取量更高. DHAQ-PLA-NP的摄取量高于DHAQ, Dextran70包裹DHAQ-PLA-NP摄取量也高于DHAQ-PLA-NP(P<0.05),说明DHAQ制备成用Dextran70包裹的DHAQ-PLA-NP后更容易被细胞摄取。
In this paper, Mitoxantrone polylactic acid nanoparticles (DHAQ-PLA-NP) and the Dextran 70 coating DHAQ-PLA-NP were prepared. The aims are to enhance the therapeutic efficacy of mitoxantrone in treating, reduce the toxicity and increase the uptake amount. Then, the cytotoxicity of DHAQ and DHAQ-PLA-NP on human hepatoma cells (SMMC-7721) and human normal hepatocyte (LO2) were studied. Finally, the uptake of the original drug, DHAQ-PLA-NP and Dextran 70 coating DHAQ-PLA-NP in these two cells was investigated. The study will provide some available information for liver cancer treatment of DHAQ-PLA-NP in clinic.
The method of precipitation was employed to prepare DHAQ-PLA-NP. The factors, such as the ratio of organic solvent and surfactant which influence the preparation were investigated, and the techniques were optimized. The size was about 150 nm; the PDI was between 0.1 to 0.3 and the zeta potential was about -40mv. DHAQ-PLA-NP lyophilization injection was prepared by using lactose as protective agent in freeze drying. The product was of good looks and good quality. The results showed that the embedding ratio and drug loading of the nanoparticles were high. The nanoparticles had the characteristic which was the size well-distributed, freeze-dried good effect and so on.
To reduce the liver toxicity of normal cells and investigate the effect of DHAQ-PLA-NP to liver cancer chemotherapy, the MTT assay was carried out to measure the cytotoxicity of DHAQ and DHAQ-PLA-NP on human hepatoma cells SMMC-7721 and human normal hepatocyte LO2. The results showed that the killing effect of DHAQ and DHAQ-PLA-NP was time- and dose-dependent. It was found that the IC50 of nanoparticles which have slow-releasing property, at the same time, was greater than the IC50 of DHAQ. It was showed that DHAQ-PLA-NP could reduce the toxicity.
Furthermore, in order to increase the uptake of DHAQ-PLA-NP on human hepatoma cells SMMC-7721 and compare the uptake of DHAQ-PLA-NP between human hepatoma cells SMMC-7721 and human normal hepatocyte LO2, Dextran 70 coating DHAQ-PLA-NP was used and the concentration of mitoxantrone in SMMC-7721 cells and LO2 cells was determined by HPLC and the content of protein in cells by BCA method. The results showed that there was no obvious difference between DHAQ and DHAQ-PLA-NP at lower concentration. However, the uptake amount of DHAQ-PLA-NP was much larger than that of DHAQ at higher concentration and the uptake amount of 2% Dextran 70 coating DHAQ-PLA-NP was the most. The uptake amout of DHAQ-PLA-NP was larger than that of DHAQ and the uptake amout of 2% Dextran 70 coating DHAQ-PLA-NP was also higher than that of DHAQ-PLA-NP (P<0.05). It was showed that the uptake of Dextran 70 coating DHAQ-PLA-NP was more easily by cells.
引文
[1]项文清,刘伟.HBx在原发性肝癌发生中的作用研究进展[J].浙江大学学报医学版,2010,39(3)：333-338.
[2]吴孟超,陈汉,沈锋.原发性肝癌的外科治疗[J].中华外科杂志,2001,39(1)：25-28.
[3]喻建华,万会平.原发性肝癌的介入治疗及其预后的影响因素[J].肿瘤防治杂志,2004,11(12)：1309-1312.
[4]Youn Jin Kim, Suna Choi, Jae Koh, et al. Controlled release of insulin from injectable biodegradable triblock copolymer[J]. Pharmaceutical Research,2001,18(4):548-550.
[5]栾立标,汤玥.聚乳酸酮洛芬微球的制备及其体外释放度[J].中国药科大学学报,2000,31(2)：99-101.
[6]张海龙,高玲美,邵洪伟.聚乳酸载药微球的制备及应用研究进展[J].西北药学杂志,2010,25(2)：158-160.
[7]徐超群.肝靶向米托蒽醌聚乳酸缓释毫微粒的研究[D].华西医科大学博士学位论文,1998.
[8]Paolo Calicetia, Steefano Salmasoa, Nicola Evassoreb, et al. Effective protein release from PEG/PLA nano-particles produced by compressed gas anti-solvent precipitaion techniques[J]. Journal of Controlled Release,2004,94:195-205.
[9]Alejandro Sanchez, Marya Tobyo, Liba Gonzalez, et al. Biodegradable micro- and nanoparticles as long-term delivery vechiles for interferon-alpha[J]. European Journal of Pharmaceutical Sciences,2003,18:221-229.
[10]thriunala Govender, Trevor Riley, Touraj Entezazi, et al. Defining the drug incoporation properties of PLA-PEG nanoparticles[J]. International Journal of Pharmaceutics,2000,199: 95-110.
[11]何黎黎mPEG-PLGA-mPEG三嵌段共聚物材料及其纳米粒的生物相容性研究[D].四川大学博士学位论文,2009.
[12]徐超群,廖工铁,张志荣.米托蒽醌聚乳酸缓释毫微粒针剂的制备[J].中国医药工业杂志,1999,30(5)：207-209.
[13]徐超群,廖工铁,张志荣.米托蒽醌聚乳酸缓释毫微粒冻干针剂的体外释药特性研究[J].华西药学杂志,1998,13(4)：229-231.
[14]曾丹.多烯磷脂酰胆碱纳米脂质载体的研究[D].四川大学硕士学位论文,2008.
[15]徐颖,周世文.肝靶向载药缓释毫微粒的研究进展[J].重庆医学,2000,29(4)：355-357.
[16]陈萍,张泰松.阿昔洛韦脂质体包封率测定流程的比较[J].山东医药,2005,45(2)34-35.
[17]张强,廖工铁,魏大鹏,等.用毫微球增加体外小鼠腹腔巨噬细胞与大鼠肝细胞对庆大霉素的摄取[J].药学学报,1996,31(5)：375-380.
[18]王杰,张强,易翔,等.表面修饰对载环孢菌素A聚乳酸纳米粒体外细胞摄取和体内组织分布的影响[J].北京医科大学学报,2000,32(3)：235-238.
[19]Kiss E, Takacs MG, Bertoti I, et al. Surface properties of poly (lactic/ glycolic acid)-Pluronic (R) blend films[J]. Polym Advan Technol,2003,14 (11-12):839.
[20]Fu Yuanfei, Chen Demin, Zhang Jiangzhong. Cytotoxicity of the strontium-substitued hydroyapatite evaluated by MTT colorimertry[J]. Journal of Biomedical Engineering,2001, 18(3):389-390.
[21]蔡文琴.现代实用细胞与分子生物学实验技术.北京：人民军医出版社,2001.
[22]司徒镇强,吴军正.细胞培养.第二版.西安：世界图书出版公司,2007.
[23]Tada H, Shiho O, Kuroshima K, et al. An improved colorimetric assay for Interleukin[J]. J Immunol Methods,1986,93:157.
[24]Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival modifications to the tetrazolium dye procedure giving improved sensitivity and reliability[J]. J Immunol Methods, 1986,89:271.
[25]李海成,何金花,王明,等.晚期糖基化终产物对人肝细胞株L02增殖和凋亡的影响[J].暨南大学学报(医学版),2009,30(2)：143-147.
[26]Wilmer M. J.G., De Graaf-Hess A, Blom H. J., et al. Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells[J]. Biochem Biophys Res Commun,2005,337, 610-614.
[27]杨凯,王昌美,温玉明,等.人口腔鳞癌细胞系对淋巴靶向葫芦素BE聚乳酸纳米微粒的敏感性研究[J].华西口腔医学杂志,2002,20(4)：293-295.
[28]徐超群,廖工铁,张志荣.米托蒽醌聚乳酸缓释毫微粒冻干针剂的体外释药特性研究[J].华西药学杂志,1998,13(4)：229-231.
[29]聂宇,刘文胜,张志荣.米托蒽醌-胰岛素偶联物体内分布和体外药效学评价[J].中国药 学杂志,2006,41(1)：41-45.
[30]刘敏,王朝晖,周桂兰.体外肿瘤细胞培养法对非小细胞肺癌化疗药物敏感性的研究[J].大连医科大学学报,2009,31(1)：64-66,84.
[31]郭平,叶利民,伍朝筼,等HPLC柱切换法测定抗癌药米托蒽醌血浆浓度[J].药学学报,1991,26(5)：367-370.
[32]张志荣,廖工铁,叶利民.柱切换HPLC法研究米托蒽醌毫微粒冻干静脉注射剂的药代动力学[J].1995,30(11)：843-847.
[33]张良珂,侯世祥,卢懿,等.叶酸受体介导米托蒽醌白蛋白纳米粒的体内分布及药效学研究[J].中国药学杂志,2007,42(9)：676-678.
[34]代广会,周卿,尚京川.固相萃取-高效液相色谱法测定细胞内外米托蒽醌的含量[J].药物分析杂志,2007,27(7)：1075-1077.
[35]袁志翔.低分子量壳聚糖作为一种新的肾靶向药物载体的研究[D].四川大学博士学位论文,2010.
[36]Yoo HS, Lee KH, Oh JE, et al. In vitro and in vivo antitumor activities of nanoparticles based on doxorubicin-PLGA conjugates[J]. J Controlled Release,2000,68(3):419-431.
[1]崔福德.药剂学[M].北京：人民卫生出版社,2003.
[2]Kumaresh S. Soppimath, Tejraj M. Aminabhavi, Anandrao R. Kulkarni and et al. Biodegradable polymeric nanoparticles as drug delivery devices[J]. Journal of controlled release,2001,70:1-20
[3]张志清,康英,王育红.聚乳酸纳米粒给药系统研究进展[J].中国医院药学杂志,2002,22(9)：555-557.
[4]Youn Jin Kim, Suna Choi, Jae Koh, et al. Controlled release of insulin from injectable biodegradable triblock copolymer.Pharmaceutical Research,2001,18(4):548-550.
[5]胡洪国,田丰,闫玉华,等.聚乳酸及其共聚物的国内外研究现状和进展[J].国外建材科技,2000,21(1)：1-3.
[6]张宗勇,闫玉华.聚乳酸在生物医学领域中的应用[J].精细与专用化学品,2005,13(14)：7-9.
[7]Plard J P, Bazile D. Comparison of the safety profiles of PLA50 and Me.PEG-PLA50 nanoparticles after single dose intravenous administration to rats [J]. Colloid Surface B,1999, 16:173-183.
[8]刘海峰,常津,张爽男,等.明胶-聚乳酸载药纳米微球的制备及其体外释药研究[J].中国生物医学工程学报,2003,22(2)：178-182.
[9]罗颖,李乐,王衍戈,等.透析法制备载羟基喜树碱-聚乳酸纳米粒及其理化性质研究[J].厦门大学学报(自然科学版),2010,49(6)：832-837.
[10]刘明星,董静,杨亚江,等.雷公藤甲素聚乳酸纳米粒的制备及毒性[J].药学学报,2004,39(7)：556-560.
[11]李树珍,欧阳五庆.藤黄酸聚乳酸纳米粒的研制[J].中国生物工程杂志,2008,28(2)：101-105.
[12]Yolles S, Leafe TD, Woodland JH, et al. Long acting delivery systems for narcotic antagonists Ⅱ:release rates of naltrexone from poly(lactic acid) composites[J]. Pharm Sci, 1975,64 (2):348-349.
[13]傅若秋,何凤慈,孟德胜,等.紫杉醇聚乳酸纳米粒的制备[J].第三军医大学学报,2006,28(15)：1573-1574.
[14]Sakai T, Kohno H, Ishihara T, et al. Mizushima eatment of experimental autoimmune uveoretinitis with poly(lactic acid)nanoparticles encapsulating betamethasone phosphate[J]. Exerimental Eye Research,2006,82:657-663.
[15]张继芬,侯世祥,刘惠莲.沉淀法制备2种脂溶性抗癌中药聚乳酸纳米粒的载药过程比较[J].中国中药杂志,2007,32(4)：303-306.
[16]Carino GP, Jacob JS, Mathiowitz E. Nanosphere based oral insulin delivery[J]. J Controlled Release,2000,65(1):261-269.
[17]邓联东,孙多先,董岸杰.载药聚乳酸纳米微粒的制备[J].中国中药杂志,2002,72(7)：496-498.
[18]Allemann E, Leroux J, Gumy R, et al. In witro extended-release properties of drug loaded poly(DL-lactic acid)nanoparticles produced by a salting-out procedure[J]. Pharm Res,1993, 10:1732.
[19]黎洪珊,赵京玲,魏树礼.环孢菌素A聚乳酸纳米粒胶体的制备和大鼠口服吸收[J].中国药学杂志,1999,34(8)：532-535.
[20]陆彬.药物新剂型与新技术[M].北京：人民卫生出版社,1998
[21]袁东锋.肝靶向纳米粒的研究进展[J].医药导报,2003,22(2)：113-114.
[22]Jani P, Halbert GW, Langridge J,et al. Nanoprticle uptake by the ratgastrointestinal mucosa: quantitation and particle size dependency[J]. J Pharm Pharmacol,1990,42 (12):821.
[23]赵秀杰,张鲁明.多肽蛋白的纳米粒口服给药系统研究进展[J].中国药师,2004,7(11)：847-851.
[24]尹宗宁,熊迎新.基于载体材料表面修饰的靶向给药系统[A].首届药用辅料(国际)学 术研讨会论文集[C],2004.
[25]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[J].中国医学工程,2005,13(6)：609-613.
[26]卢剑,常津,赵硕,等.载药纳米微粒表面性能与表面改性[J].化学通报,2003,66：1-6.
[27]王杰,张强,易翔,等.表面修饰对载环孢菌素A聚乳酸纳米粒体外细胞摄取和体内组织分布的影响[J].北京医科大学学报,2000,32(3)：235-238.
[28]Reich G. In vitro stability of poly(D,L-lactide) and poly(D,L-lactide)/poloxamer nanoparticles in gastrointestinal fluids. Drug Dev IndPharm,1997,23 (12):1191.
[29]胡云霞,原续波,张晓金,等.聚乳酸载药纳米微粒的表面修饰及体外评价[J].中国生物医学工程学报,2004,23(1)：30-36.
[30]Tobio M, Sanchez A, Vila A, et al. The rote of Peg on the stability on digestive fluids and in vivo fate of PEG-PLA nanoparticles follewing oral administation [J]. Collids Srug B Biointerfaces,2000,18 (3-4):315.
[31]忻志鸣,叶根深.纳米粒制剂研究进展[J].中国药业,2010,19(16)：15-17.
[32]李卉芳,刘明星,刘清飞,等.表面修饰的灯盏花素聚乳酸纳米粒的制备和大鼠体内药动学[J].中国新药杂志,2007,16(8)：614-618.
[33]梁海峰,李成植,徐肖肖,等.纳米中药研究进展[J].辽宁中医学院学报,2005,7(1)：25-27.
[34]许洁.RGD修饰冬凌草甲素聚乳酸载药纳米粒的研究[D].上海中医药大学,2009.
[35]章怡彬,梁文权,高建青.纳米粒作为抗肿瘤药物载体的研究进展[J].医学导报,2006,25(6)：551-553.
[36]Pan J, Feng SS. Targeted delivery of paclitaxel using folate-decorated poly(lactide)-vitamin E TPGS namoparticles[J]. Biomaterials,2008,29(17):2663-2672.
[37]裘迪,朱宏.聚乳酸类载紫杉醇纳米粒表面修饰的研究进展[J].化工进展,2009,28(7)：1198-1201.
[2]吴孟超,陈汉,沈锋.原发性肝癌的外科治疗[J].中华外科杂志,2001,39(1)：25-28.
[3]喻建华,万会平.原发性肝癌的介入治疗及其预后的影响因素[J].肿瘤防治杂志,2004,11(12)：1309-1312.
[4]Youn Jin Kim, Suna Choi, Jae Koh, et al. Controlled release of insulin from injectable biodegradable triblock copolymer[J]. Pharmaceutical Research,2001,18(4):548-550.
[5]栾立标,汤玥.聚乳酸酮洛芬微球的制备及其体外释放度[J].中国药科大学学报,2000,31(2)：99-101.
[6]张海龙,高玲美,邵洪伟.聚乳酸载药微球的制备及应用研究进展[J].西北药学杂志,2010,25(2)：158-160.
[7]徐超群.肝靶向米托蒽醌聚乳酸缓释毫微粒的研究[D].华西医科大学博士学位论文,1998.
[8]Paolo Calicetia, Steefano Salmasoa, Nicola Evassoreb, et al. Effective protein release from PEG/PLA nano-particles produced by compressed gas anti-solvent precipitaion techniques[J]. Journal of Controlled Release,2004,94:195-205.
[9]Alejandro Sanchez, Marya Tobyo, Liba Gonzalez, et al. Biodegradable micro- and nanoparticles as long-term delivery vechiles for interferon-alpha[J]. European Journal of Pharmaceutical Sciences,2003,18:221-229.
[10]thriunala Govender, Trevor Riley, Touraj Entezazi, et al. Defining the drug incoporation properties of PLA-PEG nanoparticles[J]. International Journal of Pharmaceutics,2000,199: 95-110.
[11]何黎黎mPEG-PLGA-mPEG三嵌段共聚物材料及其纳米粒的生物相容性研究[D].四川大学博士学位论文,2009.
[12]徐超群,廖工铁,张志荣.米托蒽醌聚乳酸缓释毫微粒针剂的制备[J].中国医药工业杂志,1999,30(5)：207-209.
[13]徐超群,廖工铁,张志荣.米托蒽醌聚乳酸缓释毫微粒冻干针剂的体外释药特性研究[J].华西药学杂志,1998,13(4)：229-231.
[14]曾丹.多烯磷脂酰胆碱纳米脂质载体的研究[D].四川大学硕士学位论文,2008.
[15]徐颖,周世文.肝靶向载药缓释毫微粒的研究进展[J].重庆医学,2000,29(4)：355-357.
[16]陈萍,张泰松.阿昔洛韦脂质体包封率测定流程的比较[J].山东医药,2005,45(2)34-35.
[17]张强,廖工铁,魏大鹏,等.用毫微球增加体外小鼠腹腔巨噬细胞与大鼠肝细胞对庆大霉素的摄取[J].药学学报,1996,31(5)：375-380.
[18]王杰,张强,易翔,等.表面修饰对载环孢菌素A聚乳酸纳米粒体外细胞摄取和体内组织分布的影响[J].北京医科大学学报,2000,32(3)：235-238.
[19]Kiss E, Takacs MG, Bertoti I, et al. Surface properties of poly (lactic/ glycolic acid)-Pluronic (R) blend films[J]. Polym Advan Technol,2003,14 (11-12):839.
[20]Fu Yuanfei, Chen Demin, Zhang Jiangzhong. Cytotoxicity of the strontium-substitued hydroyapatite evaluated by MTT colorimertry[J]. Journal of Biomedical Engineering,2001, 18(3):389-390.
[21]蔡文琴.现代实用细胞与分子生物学实验技术.北京：人民军医出版社,2001.
[22]司徒镇强,吴军正.细胞培养.第二版.西安：世界图书出版公司,2007.
[23]Tada H, Shiho O, Kuroshima K, et al. An improved colorimetric assay for Interleukin[J]. J Immunol Methods,1986,93:157.
[24]Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival modifications to the tetrazolium dye procedure giving improved sensitivity and reliability[J]. J Immunol Methods, 1986,89:271.
[25]李海成,何金花,王明,等.晚期糖基化终产物对人肝细胞株L02增殖和凋亡的影响[J].暨南大学学报(医学版),2009,30(2)：143-147.
[26]Wilmer M. J.G., De Graaf-Hess A, Blom H. J., et al. Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells[J]. Biochem Biophys Res Commun,2005,337, 610-614.
[27]杨凯,王昌美,温玉明,等.人口腔鳞癌细胞系对淋巴靶向葫芦素BE聚乳酸纳米微粒的敏感性研究[J].华西口腔医学杂志,2002,20(4)：293-295.
[28]徐超群,廖工铁,张志荣.米托蒽醌聚乳酸缓释毫微粒冻干针剂的体外释药特性研究[J].华西药学杂志,1998,13(4)：229-231.
[29]聂宇,刘文胜,张志荣.米托蒽醌-胰岛素偶联物体内分布和体外药效学评价[J].中国药 学杂志,2006,41(1)：41-45.
[30]刘敏,王朝晖,周桂兰.体外肿瘤细胞培养法对非小细胞肺癌化疗药物敏感性的研究[J].大连医科大学学报,2009,31(1)：64-66,84.
[31]郭平,叶利民,伍朝筼,等HPLC柱切换法测定抗癌药米托蒽醌血浆浓度[J].药学学报,1991,26(5)：367-370.
[32]张志荣,廖工铁,叶利民.柱切换HPLC法研究米托蒽醌毫微粒冻干静脉注射剂的药代动力学[J].1995,30(11)：843-847.
[33]张良珂,侯世祥,卢懿,等.叶酸受体介导米托蒽醌白蛋白纳米粒的体内分布及药效学研究[J].中国药学杂志,2007,42(9)：676-678.
[34]代广会,周卿,尚京川.固相萃取-高效液相色谱法测定细胞内外米托蒽醌的含量[J].药物分析杂志,2007,27(7)：1075-1077.
[35]袁志翔.低分子量壳聚糖作为一种新的肾靶向药物载体的研究[D].四川大学博士学位论文,2010.
[36]Yoo HS, Lee KH, Oh JE, et al. In vitro and in vivo antitumor activities of nanoparticles based on doxorubicin-PLGA conjugates[J]. J Controlled Release,2000,68(3):419-431.
[1]崔福德.药剂学[M].北京：人民卫生出版社,2003.
[2]Kumaresh S. Soppimath, Tejraj M. Aminabhavi, Anandrao R. Kulkarni and et al. Biodegradable polymeric nanoparticles as drug delivery devices[J]. Journal of controlled release,2001,70:1-20
[3]张志清,康英,王育红.聚乳酸纳米粒给药系统研究进展[J].中国医院药学杂志,2002,22(9)：555-557.
[4]Youn Jin Kim, Suna Choi, Jae Koh, et al. Controlled release of insulin from injectable biodegradable triblock copolymer.Pharmaceutical Research,2001,18(4):548-550.
[5]胡洪国,田丰,闫玉华,等.聚乳酸及其共聚物的国内外研究现状和进展[J].国外建材科技,2000,21(1)：1-3.
[6]张宗勇,闫玉华.聚乳酸在生物医学领域中的应用[J].精细与专用化学品,2005,13(14)：7-9.
[7]Plard J P, Bazile D. Comparison of the safety profiles of PLA50 and Me.PEG-PLA50 nanoparticles after single dose intravenous administration to rats [J]. Colloid Surface B,1999, 16:173-183.
[8]刘海峰,常津,张爽男,等.明胶-聚乳酸载药纳米微球的制备及其体外释药研究[J].中国生物医学工程学报,2003,22(2)：178-182.
[9]罗颖,李乐,王衍戈,等.透析法制备载羟基喜树碱-聚乳酸纳米粒及其理化性质研究[J].厦门大学学报(自然科学版),2010,49(6)：832-837.
[10]刘明星,董静,杨亚江,等.雷公藤甲素聚乳酸纳米粒的制备及毒性[J].药学学报,2004,39(7)：556-560.
[11]李树珍,欧阳五庆.藤黄酸聚乳酸纳米粒的研制[J].中国生物工程杂志,2008,28(2)：101-105.
[12]Yolles S, Leafe TD, Woodland JH, et al. Long acting delivery systems for narcotic antagonists Ⅱ:release rates of naltrexone from poly(lactic acid) composites[J]. Pharm Sci, 1975,64 (2):348-349.
[13]傅若秋,何凤慈,孟德胜,等.紫杉醇聚乳酸纳米粒的制备[J].第三军医大学学报,2006,28(15)：1573-1574.
[14]Sakai T, Kohno H, Ishihara T, et al. Mizushima eatment of experimental autoimmune uveoretinitis with poly(lactic acid)nanoparticles encapsulating betamethasone phosphate[J]. Exerimental Eye Research,2006,82:657-663.
[15]张继芬,侯世祥,刘惠莲.沉淀法制备2种脂溶性抗癌中药聚乳酸纳米粒的载药过程比较[J].中国中药杂志,2007,32(4)：303-306.
[16]Carino GP, Jacob JS, Mathiowitz E. Nanosphere based oral insulin delivery[J]. J Controlled Release,2000,65(1):261-269.
[17]邓联东,孙多先,董岸杰.载药聚乳酸纳米微粒的制备[J].中国中药杂志,2002,72(7)：496-498.
[18]Allemann E, Leroux J, Gumy R, et al. In witro extended-release properties of drug loaded poly(DL-lactic acid)nanoparticles produced by a salting-out procedure[J]. Pharm Res,1993, 10:1732.
[19]黎洪珊,赵京玲,魏树礼.环孢菌素A聚乳酸纳米粒胶体的制备和大鼠口服吸收[J].中国药学杂志,1999,34(8)：532-535.
[20]陆彬.药物新剂型与新技术[M].北京：人民卫生出版社,1998
[21]袁东锋.肝靶向纳米粒的研究进展[J].医药导报,2003,22(2)：113-114.
[22]Jani P, Halbert GW, Langridge J,et al. Nanoprticle uptake by the ratgastrointestinal mucosa: quantitation and particle size dependency[J]. J Pharm Pharmacol,1990,42 (12):821.
[23]赵秀杰,张鲁明.多肽蛋白的纳米粒口服给药系统研究进展[J].中国药师,2004,7(11)：847-851.
[24]尹宗宁,熊迎新.基于载体材料表面修饰的靶向给药系统[A].首届药用辅料(国际)学 术研讨会论文集[C],2004.
[25]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[J].中国医学工程,2005,13(6)：609-613.
[26]卢剑,常津,赵硕,等.载药纳米微粒表面性能与表面改性[J].化学通报,2003,66：1-6.
[27]王杰,张强,易翔,等.表面修饰对载环孢菌素A聚乳酸纳米粒体外细胞摄取和体内组织分布的影响[J].北京医科大学学报,2000,32(3)：235-238.
[28]Reich G. In vitro stability of poly(D,L-lactide) and poly(D,L-lactide)/poloxamer nanoparticles in gastrointestinal fluids. Drug Dev IndPharm,1997,23 (12):1191.
[29]胡云霞,原续波,张晓金,等.聚乳酸载药纳米微粒的表面修饰及体外评价[J].中国生物医学工程学报,2004,23(1)：30-36.
[30]Tobio M, Sanchez A, Vila A, et al. The rote of Peg on the stability on digestive fluids and in vivo fate of PEG-PLA nanoparticles follewing oral administation [J]. Collids Srug B Biointerfaces,2000,18 (3-4):315.
[31]忻志鸣,叶根深.纳米粒制剂研究进展[J].中国药业,2010,19(16)：15-17.
[32]李卉芳,刘明星,刘清飞,等.表面修饰的灯盏花素聚乳酸纳米粒的制备和大鼠体内药动学[J].中国新药杂志,2007,16(8)：614-618.
[33]梁海峰,李成植,徐肖肖,等.纳米中药研究进展[J].辽宁中医学院学报,2005,7(1)：25-27.
[34]许洁.RGD修饰冬凌草甲素聚乳酸载药纳米粒的研究[D].上海中医药大学,2009.
[35]章怡彬,梁文权,高建青.纳米粒作为抗肿瘤药物载体的研究进展[J].医学导报,2006,25(6)：551-553.
[36]Pan J, Feng SS. Targeted delivery of paclitaxel using folate-decorated poly(lactide)-vitamin E TPGS namoparticles[J]. Biomaterials,2008,29(17):2663-2672.
[37]裘迪,朱宏.聚乳酸类载紫杉醇纳米粒表面修饰的研究进展[J].化工进展,2009,28(7)：1198-1201.