叶酸介导肿瘤靶向的9-硝基喜树碱胶束递药系统研究
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摘要
恶性肿瘤是威胁人类健康和生命的主要疾病,目前常规的肿瘤治疗方法均有局限性,因此,研制一种能够用于靶向治疗肿瘤的给药系统,具有重要的意义。本文研究的目的在于,构建一种叶酸介导的纳米胶束给药系统,用于主动靶向肿瘤治疗方面的研究,为从制剂角度通过纳米胶束载药系统,来寻求其在肿瘤药物治疗研究方面的新途径提供实验和理论依据,也为难溶性抗肿瘤药物提供一种高效低毒的纳米给药系统。
本研究主要内容包括:制备并表征叶酸共聚物胶束载体材料,叶酸-聚乙二醇-二硬酯酰磷脂酰乙醇胺(FA-PEG-DSPE)和甲氧基-聚乙二醇-二硬酯酰磷脂酰乙醇胺(MPEG-DSPE),并对两种材料对肿瘤细胞的毒性及规避巨噬细胞吞噬的适宜比例进行考察;以9-硝基喜树碱(9-nitrocamptothecin,9-NC)作为难溶性抗肿瘤药物的模型药物,制备叶酸共聚物胶束给药系统,并进行理化性质表征;采用体外细胞学评价方法考察9-NC叶酸共聚物胶束给药体系对肿瘤细胞的生长抑制作用;大鼠体内药物动力学实验,进一步验证载体材料FA-PEG-DSPE所占比例对9-NC叶酸共聚物胶束的体内行为影响。
载体材料的制备,多以羧基和氨基在N,N'-二环己基碳二亚胺的缩合而成,HPLC检测产物纯度均大于95%。采用芘荧光探针法考察两种载体材料的临界胶束浓度(CMC),结果显示FA-PEG-DSPE和MPEG-DSPE的CMC分别为1.0×10~(-5)M和0.97×10~(-5)M,通过两种载体材料混合制备的叶酸共聚物胶束,耐稀释,具有良好的稳定性。载体材料毒性检测结果显示,两种载体材料对细胞的杀伤能力均较小,具有良好的生物相容性。
采用不同叶酸含量的载药胶束对肿瘤细胞生长抑制作用和规避巨噬细胞吞噬能力相结合的方法,确定FA-PEG-DSPE/MPEG-DSPE=1:100作为载体,进行9-NC共聚物胶束的研制。采用成膜水化法制备9-NC叶酸共聚物胶束。以包封率、载药量和胶束溶液药物浓度为指标,在单因素试验初选的基础上,采用星点设计-效应面优化法对胶束处方进行优化。最终确定较优处方:9-NC为0.5 mg,水化介质为13mL(载体量为20mg)。胶束理化性质考察结果显示,胶束呈圆球状,大小较为均匀,平均粒径约为26.8nm。
荧光示踪法考察肿瘤细胞对叶酸共聚物胶束的定性摄取结果显示,肿瘤细胞通过胞吞等方式摄取叶酸共聚物胶束后,将荧光素从内涵体和/或溶酶体中释放到细胞质中,荧光素在释放过程中逐渐扩散至细胞核,因而靠近细胞核处荧光较细胞膜处明显,由此避免药物因存在于细胞膜内表面附近而被细胞膜P-糖蛋白(P-gp)外排至细胞外,而降低药效。
采用9-NC叶酸共聚物胶束(FA-M-9-NC)进行体外细胞学药效试验,同时采用不含叶酸的9-NC共聚物胶束M-9-NC、游离药物9-NC进行对照。结果显示,不同肿瘤细胞对不同抗肿瘤药物的敏感性有所不同,呈剂量、时间依赖关系,对于叶酸受体高表达的肿瘤细胞,叶酸共聚物胶束更能增强抗肿瘤药物对细胞的杀伤能力。
大鼠体内药物动力学实验结果显示,大鼠尾静脉注射不同叶酸含量的9-NC共聚物胶束溶液后,FA-PEG-DSPE/MPEG-DSPE=0.1:100和1:100组比10:100组体内循环时间长。说明FA-PEG-DSPE在载体材料中所占比例过高,会降低胶束制剂的血液循环时间。其中0.1:100组的AUC_(0～t)、AUC_(0～∞)分别是10:100组的2.3倍和1.9倍;1:100组的AUC_(0～t)、AUC_(0～∞)分别是10:100组的2.4倍和2.0倍,提示含叶酸载体材料所占比例高到一定程度,将对9-NC叶酸共聚物胶束在体内的行为产生不利影响。
Malignant tumor is the main disease to threaten the health and life of human being.Till now,all the conventional methods of tumor therapy have limitations.So,it is meaningful to develop a drug delivery system to target tumor cells.In this thesis a folate-mediate nano-micelles drug delivery system was studied,which was used to active target tumor cells.It provided the experimental and theoretical basis to explore a new routine of tumor therapy by drug-loaded nano-micelle system in terms of formulation,and meanwhile,provided a high efficiency and low toxicity nano-drug delivery system for poorly soluble antitumor agents.
The contents of this thesis were including preparation and characterization of folate-conjugated micelle's carder materials named FA-PEG-DSPE and MPEG-DSPE, investigation of their cytotoxicity to tumor cells and the proper proportion to avoid the phagocytosis of macrophages;preparation and characterization of 9-NC-loaded folate-conjugated micelles;evaluation in vitro;pharmacokinetics experiments in rats to further prove that the ratio of FA-PEG-DSPE could effect the 9-NC-loaded micelles in vivo.
The carrier materials were synthesized by dehydration-condensation reaction of carboxy group and amino group with the catalysis of DCC,and the purity that determined by HPLC was over 95%.CMC was estimated by the Pyrene method and the values of MPEG-DSPE and FA-PEG-DSPE were shown to be as low as 0.97×10 ~(-5)M and 1.0×10~(-5)M,respectively,which suggested that the polymeric micelles had high stability and the ability to maintain the integrity even upon strong dilution.The micelles also had good biocompatibility and produced minimal cytotoxicity to tumor cells.
The suitable molar ratio of FA-PEG-DSPE and MPEG-DSPE was chosen to be 1:100 as the carrier material by flow cytometry(FCM) evaluation on macrophages and active targeting ability evaluation on tumor cells with different folate content in it. The 9-NC-loaded micelles were prepared by film formation method.The formulation optimization based on entrapment efficiency,drug loading and drug concentration. The better formulation was determined as 0.5mg 9-NC and 13mL HBS buffer solution (20mg carrier materials).The micelles had a spherical shape with average size of 26.8nm.
Fluorescence tracing method was applied to show that the dihydroxyfluorane was released from endosome or lysosome after folate-conjugated micelles were uptaken by tumor cells.
The dihydroxyfluorane diffused to the cell nucleus gradually so that the dihydroxyfluorane was more obvious around the cell nucleus than near the cellular membrane,in order to avoid the P-gp pump.
The folate free 9-NC-loaded micelles(M-9-NC) and free 9-NC were used as control preparations when 9-NC-loaded folate-conjugated micelles were studied in vitro.The results showed that the sensitivity of different kinds of tumor cells to different anticancer drugs was not the same.It showed dose and time-dependent relation.The folate-conjugated micelles could enhance the anticancer drugs with the ability of killing folate-receptor overexpressed tumor cells than M-9-NC.
Pharmacokinetics experiments in rats showed that when the molar ratio of FA-PEG-DSPE and MPEG-DSPE was 0.1:100 or 1:100,the cycle time of the folate-conjugated micelles was longer than the group of 10:100.It suggested that if the molar ratio of FA-PEG-DSPE was too high,the blood circulation time would be shortened.The AUC_(0～t) and AUC_(0～∞) of 0.1:100 group and 1:100 group were 2.3,1.9 times and 2.4,2.0 times,respectively,higher than those of 10:100 group.It suggested that if the folate content in the carrier materials exceeded a certain extent,it would produce adverse effect to the 9-NC folate-conjugated micelles in vivo.
本研究主要内容包括:制备并表征叶酸共聚物胶束载体材料,叶酸-聚乙二醇-二硬酯酰磷脂酰乙醇胺(FA-PEG-DSPE)和甲氧基-聚乙二醇-二硬酯酰磷脂酰乙醇胺(MPEG-DSPE),并对两种材料对肿瘤细胞的毒性及规避巨噬细胞吞噬的适宜比例进行考察;以9-硝基喜树碱(9-nitrocamptothecin,9-NC)作为难溶性抗肿瘤药物的模型药物,制备叶酸共聚物胶束给药系统,并进行理化性质表征;采用体外细胞学评价方法考察9-NC叶酸共聚物胶束给药体系对肿瘤细胞的生长抑制作用;大鼠体内药物动力学实验,进一步验证载体材料FA-PEG-DSPE所占比例对9-NC叶酸共聚物胶束的体内行为影响。
载体材料的制备,多以羧基和氨基在N,N'-二环己基碳二亚胺的缩合而成,HPLC检测产物纯度均大于95%。采用芘荧光探针法考察两种载体材料的临界胶束浓度(CMC),结果显示FA-PEG-DSPE和MPEG-DSPE的CMC分别为1.0×10~(-5)M和0.97×10~(-5)M,通过两种载体材料混合制备的叶酸共聚物胶束,耐稀释,具有良好的稳定性。载体材料毒性检测结果显示,两种载体材料对细胞的杀伤能力均较小,具有良好的生物相容性。
采用不同叶酸含量的载药胶束对肿瘤细胞生长抑制作用和规避巨噬细胞吞噬能力相结合的方法,确定FA-PEG-DSPE/MPEG-DSPE=1:100作为载体,进行9-NC共聚物胶束的研制。采用成膜水化法制备9-NC叶酸共聚物胶束。以包封率、载药量和胶束溶液药物浓度为指标,在单因素试验初选的基础上,采用星点设计-效应面优化法对胶束处方进行优化。最终确定较优处方:9-NC为0.5 mg,水化介质为13mL(载体量为20mg)。胶束理化性质考察结果显示,胶束呈圆球状,大小较为均匀,平均粒径约为26.8nm。
荧光示踪法考察肿瘤细胞对叶酸共聚物胶束的定性摄取结果显示,肿瘤细胞通过胞吞等方式摄取叶酸共聚物胶束后,将荧光素从内涵体和/或溶酶体中释放到细胞质中,荧光素在释放过程中逐渐扩散至细胞核,因而靠近细胞核处荧光较细胞膜处明显,由此避免药物因存在于细胞膜内表面附近而被细胞膜P-糖蛋白(P-gp)外排至细胞外,而降低药效。
采用9-NC叶酸共聚物胶束(FA-M-9-NC)进行体外细胞学药效试验,同时采用不含叶酸的9-NC共聚物胶束M-9-NC、游离药物9-NC进行对照。结果显示,不同肿瘤细胞对不同抗肿瘤药物的敏感性有所不同,呈剂量、时间依赖关系,对于叶酸受体高表达的肿瘤细胞,叶酸共聚物胶束更能增强抗肿瘤药物对细胞的杀伤能力。
大鼠体内药物动力学实验结果显示,大鼠尾静脉注射不同叶酸含量的9-NC共聚物胶束溶液后,FA-PEG-DSPE/MPEG-DSPE=0.1:100和1:100组比10:100组体内循环时间长。说明FA-PEG-DSPE在载体材料中所占比例过高,会降低胶束制剂的血液循环时间。其中0.1:100组的AUC_(0～t)、AUC_(0～∞)分别是10:100组的2.3倍和1.9倍;1:100组的AUC_(0～t)、AUC_(0～∞)分别是10:100组的2.4倍和2.0倍,提示含叶酸载体材料所占比例高到一定程度,将对9-NC叶酸共聚物胶束在体内的行为产生不利影响。
Malignant tumor is the main disease to threaten the health and life of human being.Till now,all the conventional methods of tumor therapy have limitations.So,it is meaningful to develop a drug delivery system to target tumor cells.In this thesis a folate-mediate nano-micelles drug delivery system was studied,which was used to active target tumor cells.It provided the experimental and theoretical basis to explore a new routine of tumor therapy by drug-loaded nano-micelle system in terms of formulation,and meanwhile,provided a high efficiency and low toxicity nano-drug delivery system for poorly soluble antitumor agents.
The contents of this thesis were including preparation and characterization of folate-conjugated micelle's carder materials named FA-PEG-DSPE and MPEG-DSPE, investigation of their cytotoxicity to tumor cells and the proper proportion to avoid the phagocytosis of macrophages;preparation and characterization of 9-NC-loaded folate-conjugated micelles;evaluation in vitro;pharmacokinetics experiments in rats to further prove that the ratio of FA-PEG-DSPE could effect the 9-NC-loaded micelles in vivo.
The carrier materials were synthesized by dehydration-condensation reaction of carboxy group and amino group with the catalysis of DCC,and the purity that determined by HPLC was over 95%.CMC was estimated by the Pyrene method and the values of MPEG-DSPE and FA-PEG-DSPE were shown to be as low as 0.97×10 ~(-5)M and 1.0×10~(-5)M,respectively,which suggested that the polymeric micelles had high stability and the ability to maintain the integrity even upon strong dilution.The micelles also had good biocompatibility and produced minimal cytotoxicity to tumor cells.
The suitable molar ratio of FA-PEG-DSPE and MPEG-DSPE was chosen to be 1:100 as the carrier material by flow cytometry(FCM) evaluation on macrophages and active targeting ability evaluation on tumor cells with different folate content in it. The 9-NC-loaded micelles were prepared by film formation method.The formulation optimization based on entrapment efficiency,drug loading and drug concentration. The better formulation was determined as 0.5mg 9-NC and 13mL HBS buffer solution (20mg carrier materials).The micelles had a spherical shape with average size of 26.8nm.
Fluorescence tracing method was applied to show that the dihydroxyfluorane was released from endosome or lysosome after folate-conjugated micelles were uptaken by tumor cells.
The dihydroxyfluorane diffused to the cell nucleus gradually so that the dihydroxyfluorane was more obvious around the cell nucleus than near the cellular membrane,in order to avoid the P-gp pump.
The folate free 9-NC-loaded micelles(M-9-NC) and free 9-NC were used as control preparations when 9-NC-loaded folate-conjugated micelles were studied in vitro.The results showed that the sensitivity of different kinds of tumor cells to different anticancer drugs was not the same.It showed dose and time-dependent relation.The folate-conjugated micelles could enhance the anticancer drugs with the ability of killing folate-receptor overexpressed tumor cells than M-9-NC.
Pharmacokinetics experiments in rats showed that when the molar ratio of FA-PEG-DSPE and MPEG-DSPE was 0.1:100 or 1:100,the cycle time of the folate-conjugated micelles was longer than the group of 10:100.It suggested that if the molar ratio of FA-PEG-DSPE was too high,the blood circulation time would be shortened.The AUC_(0～t) and AUC_(0～∞) of 0.1:100 group and 1:100 group were 2.3,1.9 times and 2.4,2.0 times,respectively,higher than those of 10:100 group.It suggested that if the folate content in the carrier materials exceeded a certain extent,it would produce adverse effect to the 9-NC folate-conjugated micelles in vivo.
引文
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[1]刘敏,叶酸聚乙二醇作为放射性核素和抗肿瘤药物载体用于淋巴转移肿瘤的研究.博士学位论文,2007.
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[1]王彩霞,冯霞,杨军.聚合物胶束型抗癌药物给药系统研究进展.化学研究与应用,2006,18(6):599-602.
[2]韩丽妹,方晓玲.9-硝基喜树碱片剂溶出度方法的考察[J].中国临床药学杂志,2003,12(5):288-291.
[3]李柯,钟大放.高效液相色谱法测定大鼠组织及血浆中9-硝基喜树碱含量[J].药学学报,2003,38(2):124-128.
[4]Kim SC,Kim DW,Shim YH,et al.In vivo evaluation of polymeric micellar paclitaxel formulation:toxicity and efficacy[J].J Control Release,2001,72(1-3):191-202.
[5]Xichen Z,Jackson JK,Butt HM.Development of amphiphilic diblockcopolymers as micellar carriers of taxol[J].Int J Pharm,1996,132(1-2):195-206.
[6]Zerrin S,Nilufer Y,Tamer B.Investigation of pluronic and PEG-PE micelles as carriers of meso-tetraphenyl porphine for oral administration[J].Int J Pharm,2007,332:161-167.
[7]韩丽妹.基于逆转肿瘤多药耐药性的共聚物胶束给药体系的研究.博士学位论文,2006年4月.
[8]Dascalescu L,Tilmatine A,Aman F,et al.Optimization of electrostatic separation processes using response surface modeling[J].Ieee T Ind Appl,2004,40(1):53-59.
[9]Gabrielsson J,et al.Multivariate methods in pharmaceutical applications[J].J Chemometr,2002,16(3):141-160.
[10]Xia W,et al.A functional folate receptor is induced during macrophage activation and can be used to target drugs to activated macrophages[J].Blood,2009,113(2):438-446.
[11]Haizheng Zh,Lin Y,Lanry Y.Selectivity of folate conjugated polymer micelles against different tumor cells[J].Int J Pharm,2007,349(1-2):256-268.
[12]杨进.流式细胞仪工作原理及临床应用.医疗设备信息,2002,17(1):15-16.
[13]史桂英,高飞等.流式细胞术检测细胞内活性氧的方法.上海第二医科大学学报,2001,21(2):122-124.
[14]Chow DSL,Gong L,Wolfe MD,et al.Modified lactone/carboxylate salt equilibria in vivo by liposomal delivery of 9-nitro-camptothecin[J].Ann Ny Acad Sci,2000,922:164-174.
[15]Zhong DF,Li K,Xu JH,et al.Pharmacokinetics of 9-nitro-20(S)-camptothecin in rats[J].Acta Pharmacol Sin,2003,24(3):256-262.
[1]钱桂英等,祖师麻总香豆素物理化学常数的测定.中国中医药信息杂志,2006(10):52-53
[2]Haizheng Zh,Lin Y,Lanry Y.Selectivity of folate conjugated polymer micelles against different tumor cells[J].Int J Pharm,2007,349(1-2):256-268.
[3]Shao QL,Nikken W,Shu JG;Yen WT,Yi YY.Bio-functional micelles self-assembled from a folate-conjugated block copolymer for targeted intracellular delivery of anticancer drugs.Biomaterials,2007,28:1423-1433.
[4]Hyuk SY,Tae GP.Folate-receptor-targeted delivery of doxorubicin nano-aggregates stabilized by doxorubicin-PEG-folate conjugate[J].J Controlled Release,2004,100:247-256.
[5]Gottesman MM,Fojo T,Bates SE.Multidrug resistance in cancer:Role of ATP-dependent transporters[J].Nat Rev Cancer,2002,2(1):48-58.
[6]Zgurskaya HI,Nikaido H.Multidrug resistance mechanisms:drug efflux across two membranes[J].Mol Microbiol,2000,37(2):219-225.
[7]Yoo HS,Park TG.Biodegradable polymeric micelles composed of doxorubicin conjugated PLGA-PEG block copolymer[J].J Control Release,2001,70(1/2):63-70.
[8]Allen C,Yu Y,Eisenberg A,et al.Cellular internalization of PCL_(20)-b-PEO_(44)block copolymer micelles[J].Biochim Biophys Acta,1999,1421(1):32-38.
[1]Sands H,Mishra A,Stoeckler JD,et al.Preclinical activity of an i.v.formulation of rubitecan in IDD-P(TM) against human solid tumor xenografts[J].Anti-Cancer Drug,2002,13(9):965-975.
[2]张宏娟,张灿,平其能.聚合物胶束作为药用载体的研究与应用[J].药学进展,2002,26(6):326-329.
[3]Zhao H,Yung LY.Selectivity of folate conjugated polymer micelles against different tumor cells.Int J Pharm.2008,349(1-2):256-68.
[4]Xia W,et al.A functional folate receptor is induced during macrophage activation and can be used to target drugs to activated macrophages[J].Blood,2009,113(2):438-446.
[5]韩丽妹.基于逆转肿瘤多药耐药性的共聚物胶束给药体系的研究.博士学位论文,2006年4月.
[6]Zhong DF,Li K,Xu JH,et al.Pharmacokinetics of 9-nitro-20(S)-camptothecin in rats[J].Acta Pharmacol Sin,2003,24(3):256-262.
[7]Stehlin J,Giovanella B,Natelson E,et al.A study of 9-nitrocamptothecin (RFS-2000) in patients with advanced pancreatic cancer[J].Int J Oncol,1999,14:821-833.
[8]梁文权等.生物药剂学与药物动力学,第2版[M].北京:人民卫生出版社,2005.
[9]Xia W,et al.A functional folate receptor is induced during macrophage activation and can be used to target drugs to activated macrophages[J].Blood,2009,113(2):438-446.
[10]应晓英等.大鼠口服黄体酮脂质纳米粒的血药浓度测定.浙江大学学报(医学版),2008,37(2):146:149.
[1]逯敏飞,程永清,李丽君,伍建军.被动靶向药物载体的研究进展.材料导报,2005,19(9):108-110.
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[5]Sledge G,Miller K.Exploiting the hallmarks of cancer:the future conquest of breast cancer[J].Eur J Cancer,2003,39(12):1668-1675.
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[1]刘敏,叶酸聚乙二醇作为放射性核素和抗肿瘤药物载体用于淋巴转移肿瘤的研究.博士学位论文,2007.
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[7]徐建业等.加温诱导肿瘤细胞凋亡与逆转多药耐药性的实验研究.http://www.eti.com.cn/view.asp?id=58&classid=6.
[1]王彩霞,冯霞,杨军.聚合物胶束型抗癌药物给药系统研究进展.化学研究与应用,2006,18(6):599-602.
[2]韩丽妹,方晓玲.9-硝基喜树碱片剂溶出度方法的考察[J].中国临床药学杂志,2003,12(5):288-291.
[3]李柯,钟大放.高效液相色谱法测定大鼠组织及血浆中9-硝基喜树碱含量[J].药学学报,2003,38(2):124-128.
[4]Kim SC,Kim DW,Shim YH,et al.In vivo evaluation of polymeric micellar paclitaxel formulation:toxicity and efficacy[J].J Control Release,2001,72(1-3):191-202.
[5]Xichen Z,Jackson JK,Butt HM.Development of amphiphilic diblockcopolymers as micellar carriers of taxol[J].Int J Pharm,1996,132(1-2):195-206.
[6]Zerrin S,Nilufer Y,Tamer B.Investigation of pluronic and PEG-PE micelles as carriers of meso-tetraphenyl porphine for oral administration[J].Int J Pharm,2007,332:161-167.
[7]韩丽妹.基于逆转肿瘤多药耐药性的共聚物胶束给药体系的研究.博士学位论文,2006年4月.
[8]Dascalescu L,Tilmatine A,Aman F,et al.Optimization of electrostatic separation processes using response surface modeling[J].Ieee T Ind Appl,2004,40(1):53-59.
[9]Gabrielsson J,et al.Multivariate methods in pharmaceutical applications[J].J Chemometr,2002,16(3):141-160.
[10]Xia W,et al.A functional folate receptor is induced during macrophage activation and can be used to target drugs to activated macrophages[J].Blood,2009,113(2):438-446.
[11]Haizheng Zh,Lin Y,Lanry Y.Selectivity of folate conjugated polymer micelles against different tumor cells[J].Int J Pharm,2007,349(1-2):256-268.
[12]杨进.流式细胞仪工作原理及临床应用.医疗设备信息,2002,17(1):15-16.
[13]史桂英,高飞等.流式细胞术检测细胞内活性氧的方法.上海第二医科大学学报,2001,21(2):122-124.
[14]Chow DSL,Gong L,Wolfe MD,et al.Modified lactone/carboxylate salt equilibria in vivo by liposomal delivery of 9-nitro-camptothecin[J].Ann Ny Acad Sci,2000,922:164-174.
[15]Zhong DF,Li K,Xu JH,et al.Pharmacokinetics of 9-nitro-20(S)-camptothecin in rats[J].Acta Pharmacol Sin,2003,24(3):256-262.
[1]钱桂英等,祖师麻总香豆素物理化学常数的测定.中国中医药信息杂志,2006(10):52-53
[2]Haizheng Zh,Lin Y,Lanry Y.Selectivity of folate conjugated polymer micelles against different tumor cells[J].Int J Pharm,2007,349(1-2):256-268.
[3]Shao QL,Nikken W,Shu JG;Yen WT,Yi YY.Bio-functional micelles self-assembled from a folate-conjugated block copolymer for targeted intracellular delivery of anticancer drugs.Biomaterials,2007,28:1423-1433.
[4]Hyuk SY,Tae GP.Folate-receptor-targeted delivery of doxorubicin nano-aggregates stabilized by doxorubicin-PEG-folate conjugate[J].J Controlled Release,2004,100:247-256.
[5]Gottesman MM,Fojo T,Bates SE.Multidrug resistance in cancer:Role of ATP-dependent transporters[J].Nat Rev Cancer,2002,2(1):48-58.
[6]Zgurskaya HI,Nikaido H.Multidrug resistance mechanisms:drug efflux across two membranes[J].Mol Microbiol,2000,37(2):219-225.
[7]Yoo HS,Park TG.Biodegradable polymeric micelles composed of doxorubicin conjugated PLGA-PEG block copolymer[J].J Control Release,2001,70(1/2):63-70.
[8]Allen C,Yu Y,Eisenberg A,et al.Cellular internalization of PCL_(20)-b-PEO_(44)block copolymer micelles[J].Biochim Biophys Acta,1999,1421(1):32-38.
[1]Sands H,Mishra A,Stoeckler JD,et al.Preclinical activity of an i.v.formulation of rubitecan in IDD-P(TM) against human solid tumor xenografts[J].Anti-Cancer Drug,2002,13(9):965-975.
[2]张宏娟,张灿,平其能.聚合物胶束作为药用载体的研究与应用[J].药学进展,2002,26(6):326-329.
[3]Zhao H,Yung LY.Selectivity of folate conjugated polymer micelles against different tumor cells.Int J Pharm.2008,349(1-2):256-68.
[4]Xia W,et al.A functional folate receptor is induced during macrophage activation and can be used to target drugs to activated macrophages[J].Blood,2009,113(2):438-446.
[5]韩丽妹.基于逆转肿瘤多药耐药性的共聚物胶束给药体系的研究.博士学位论文,2006年4月.
[6]Zhong DF,Li K,Xu JH,et al.Pharmacokinetics of 9-nitro-20(S)-camptothecin in rats[J].Acta Pharmacol Sin,2003,24(3):256-262.
[7]Stehlin J,Giovanella B,Natelson E,et al.A study of 9-nitrocamptothecin (RFS-2000) in patients with advanced pancreatic cancer[J].Int J Oncol,1999,14:821-833.
[8]梁文权等.生物药剂学与药物动力学,第2版[M].北京:人民卫生出版社,2005.
[9]Xia W,et al.A functional folate receptor is induced during macrophage activation and can be used to target drugs to activated macrophages[J].Blood,2009,113(2):438-446.
[10]应晓英等.大鼠口服黄体酮脂质纳米粒的血药浓度测定.浙江大学学报(医学版),2008,37(2):146:149.
[1]逯敏飞,程永清,李丽君,伍建军.被动靶向药物载体的研究进展.材料导报,2005,19(9):108-110.
[2]王亚敏.靶向药物载体的研究现状及展望[J].国外医学药学分册,1997,24(4):216-218.
[3]吴琼珠,王冬燕.靶向给药系统[J].海军医高专学报,1995,17(2):128-130.
[4]李赛.高分子纳米粒子在靶向药物载体中的研究进展[J].生物医学工程学杂志,2004,21(3):495-497.
[5]Sledge G,Miller K.Exploiting the hallmarks of cancer:the future conquest of breast cancer[J].Eur J Cancer,2003,39(12):1668-1675.
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