【作者】 苑振贵；

【导师】 陈大为；

【作者基本信息】 沈阳药科大学 ， 药剂学， 2009， 博士

【摘要】 叶酸受体介导的长循环脂质体是近年来主动靶向制剂研究的重点之一。本文选用多西他赛(DTX)为模型药物,制备了多西他赛长循环脂质体和多西他赛长循环固体脂质纳米粒,并进行了处方、工艺考察,使用效应面法优化了两种长循环纳米制剂的最优处方。在此基础上,制备了叶酸受体介导的多西他赛长循环脂质体,研究了叶酸受体介导的多西他赛长循环脂质体在体内外的靶向性,初步探讨其靶向机理,使用荷瘤裸鼠考察了叶酸受体介导的多西他赛长循环脂质体抑制肿瘤的效果。以多西他赛为模型药物制备了多西他赛长循环脂质体,实验以包封率为指标考察了处方及工艺对制剂包封率的影响,并以效应面法确定优化处方。最终处方为SPC:DTX (20:1)、SPC:Chol(3:1). SPC浓度3%、PEG2000-Chol浓度5%。多西他赛长循环脂质体的包封率为90.42±3.25%,粒径为117.6±5.34 nm；透射电镜下观察脂质体为单室脂质体,呈圆形或类圆形,粒径结果与LS 230激光粒度仪测定结果相符。24 h释放度为62.3±3.12%。在大鼠血浆中,脂质体释放加快。室温放置一个月,制剂稳定性良好。为长期保存多西他赛长循环脂质体,制备了多西他赛长循环冻干脂质体,制剂稳定性良好。以多西他赛为模型药物制备了多西他赛长循环固体脂质纳米粒,实验以包封率、粒径为指标考察了处方及工艺对制剂包封率、粒径的影响,并以效应面法确定优化处方。最终处方为脂质浓度为5%、乳化剂用量为5%(磷脂浓度为2.5%,F68浓度为2.5%)、脂质与药物比为20：1、DOPE-PEG浓度为7%。多西他赛长循环固体脂质纳米粒的包封率为93.1士4.3%,粒径为136.4±11.2 nm；透射电镜下观察纳米粒为类球形实体粒子,粒度分布均匀。在含有0.5%Tween-80的水溶液中,释放前2h,纳米粒有大约30%的药物发生突释,释放24h时纳米粒均未释放完全,表明纳米粒具有缓释能力。合成新型靶向辅料胆固醇-PEG-叶酸(Chol-PEG-Fol),使用IR、NMR等初步鉴定为目的产物。在多西他赛长循环脂质体处方研究基础上,使用Chol-PEG-Fol制备叶酸受体介导的多西他赛长循环脂质体(Fol-PEG-DTXL)。使用荧光标记的Fol-PEG-DTXL定量考察了Fol-PEG-DTXL在MCF-7细胞的结合量。流式细胞仪检测、荧光显微镜观察,推断Fol-PEG-DTXL是通过叶酸介导的细胞内化被细胞内吞。体外细胞毒实验证明、Fol-PEG-DTXL比PEG-DTXL更有效地抑制MCF-7细胞的生长(P<0.01),而Fol-PEG-DTXL与PEG-DTXL对Hela细胞的抑制无显著性差别(P>0.05),加入游离叶酸可抑制Fol-PEG-DTXL对MCF-7细胞的细胞毒作用,而对Hela细胞无效。BALB/c裸鼠体内抑瘤实验发现,Fol-PEG-DTXL可有效抑制体内肿瘤生长,抑瘤率为67.6%,较其它制剂组有显著性差别(P<0.01)。使用wastar大鼠考察了Fol-PEG-DTXL的体内药动学参数。DTX溶液的t1／2为1.157h,DTXL的t1／2为3.08 h,PEG-DTXL的t1／2为7.795 h,为DTX溶液的2.66倍,DTXL的6.74倍。Fol-PEG-DTXL的MRT0-t、MRT0-∞分别为注射剂组的5.5和5.26倍；DTXL的3.32和3.10倍。表明Fol-PEG-DTXL可以显著延长DTX的体内循环时间。此结果提示Fol-PEG-DTXL可有效降低多西他赛的毒副作用,达到安全用药的目的。昆明种小鼠体内组织分布研究发现,多西他赛溶液剂在小鼠体内分布较为迅速、广泛,具有一定透过血脑屏障的能力,且可能对心脏产生急性或慢性毒性。叶酸介导多西他赛长循环脂质体相比多西他赛溶液和多西他赛普通脂质体具有较为明显的增加体内循环时间的作用,同时,能减少药物向心和脑中的分布,降低多西他赛对心、脑的毒性作用。更多还原

【Abstract】 Recently, the long-circulating liposomes mediated by folate receptor have attracted considerable attention as drug targeting delivery. In present study, Docetaxel (DTX) was used as a model drug and used to prepare DTX loaded long-circulating liposomes and DTX loaded long-circulating solid lipid nanoparticles. The effect of some factors on the formulations and preparation of these two long-circulating nanocarries was investigated. Furthermore, the response surface methodology was used to optimize these two long-circulating nanocarriers. The long-circulating liposomes mediated by folate receptor were also prepared. The targetability and growth inhibition of immunoliposomes on MCF-7 cells in vitro and Balb/c nude mice in vivo was also studied.The long-circulating liposomes were prepared with DTX and the effect of some factors on the encapsulation efficiency was investigated. Then the formulation was optimized by the response surface methodology. Finally, the long-circulating liposomes were prepared by SPC: DTX (20:10、SPC:Chol(3:1),3% of SPC in water and 5% molar ratio of SPC with the encapsulation efficiency of 90.42±3.25% and the particle distribution of 117.6±5.34 nm. The liposomes were single unilamellar vesicles and spherical under TEM. The cumulative release within 24 h was 62.3±3.12%, and the release rate of liposomes raised in rat plasma. To store for long time, the long-circulating liposomes were freeze-drying.The long-circulating solid lipid nanoparticles were prepared with DTX and the effect of some factors on the encapsulation efficiency and particle size was investigated. Then the formulation was optimized by the response surface methodology. Finally, the long-circulating SLN were prepared by 5% in water,、5% of emulsifier in water (SPC:F68 1:1)、lipid/DTX (20:1)、7% of DOPE-PEG with the encapsulation efficiency of 93.1±4.3% and the particle distribution of 136.4±11.2 nm。The SLNs were spherical under TEM. In water with Tween-80 (0.5%),30% of DTX was released within the first 2h, however, DTX didn’t release completely at the 24th h.Chol-PEG-Folic acid (Chol-PEG-Fol) was synthesized and analyzed with IR, NMR. Using Chol-PEG-Fol, the long-circulating liposome mediated by folate receptor (Fol-PEG-DTXL) was prepared and investigated.Results of flow cytometer and fluorescence microscopy, suggested the liposome internalized in cells mediated by folic acid. Fool-PEG-DTXL was more catatonic on MCF-7cells than PEG-DTXL (P<0.01), when cytotoxicties were compared for Hela cells, no significant differences were observed between Fol-PEG-DTXL and PEG-DTXL (P>0.05), suggesting that Fol-PEG-DTXL showed relative selective cytotoxicity on MCF-7 cells. The additional folic acid reduced the cytotoxicity on MCF-7 cells, but no effect on Hela cells. Using Balb/c nude mice bearing MCF-7 cells, we compared the antitumor efficacy of liposomal DTX and DTX. The treatment with DTX, liposomal DTX inhibited primary tumor growth of MCF-7cells compared with the control group. Furthermore, five injections of Fol-PEG-DTXL resulted in substantial tumor regressions, and tumor regressions in the Fol-PEG-DTXL group were significantly greater than in the non-targeted groups (P<0.05) and far superior to the control PBS treatment (P<0.01). The antitumor efficacy of Fol-PEG-DTXL was 67.6%.The pharmacokinetic parameters of Fol-PEG-DTXL were studied in Wister rat. The t1/2 of DTX, DTXL and Fol-PEG-DTXL were 1.157 h,3.08 h, and 7.795 h, respectively. The t1/2 of Fol-PEG-DTXL was 6.74-fold to DTX, and 2.66-fold to DTXL. MRT0-t and MRT0-∞of Fol-PEG-DTXL were 5.5-fold and 5.26-fold to DTX,3.32-fold and 3.10-fold to DTXL These results suggested Fol-PEG-DTXL reduced the toxicity of DTX. The results of biodistribution in mouse suggested docetaxel solution distributed rapidly and extensively, could somehow penetrate the blood brain barrier, and cause acute and chronic cardiac toxicity. Compared to DTX and DTXL, Fol-PEG-DTXL enhanced the time in blood. DTX distribution in heart、brain and kidney were decreased, which could depress DTX side effects on these organs.更多还原