载多西他赛胶束在克服多药耐药及靶向给药方面的研究

发布时间：2018-03-24 23:06

本文选题：PEO-PPO-PCL　切入点：TPGS　出处：《山东大学》2016年硕士论文

【摘要】：多西他赛,又名多西紫杉醇,是新一代紫杉烷类抗肿瘤药,其抗癌谱广,适用于多种癌症的治疗。然而,目前药物化疗效果仍不理想,一方面,由于药物自身理化性质的特点,如水溶性差,使药物不能有效地达到肿瘤组织。另一方面,肿瘤多药耐药性也是化疗失败的重要原因。再一方面,药物缺乏靶向性,引起患者严重的毒副作用,也是临床上化学治疗的主要障碍。因此,本课题制备的新剂型能够提高药物的溶解度、逆转肿瘤细胞的多药耐药,同时提高药物的靶向性,降低毒副作用。本文以PEO-PPO-PCL为主要载体材料来包载多西他赛,主要研究内容如下：本文第一部分主要针对P-糖蛋白在癌细胞膜上过度表达而产生多药耐药的机理,选取TPGS为P-gp抑制剂,制备了PEO-PPO-PCL/TPGS胶束。我们合成了三种不同疏水链段的聚合物,用薄膜水化法制得的三种载多西他赛的PEO-PPO-PCL/TPGS胶束,其平均粒径分别在25-135nm之间,符合EPR效应的粒径要求,形态呈球形,分布均匀。胶束的临界胶束浓度(CMC)低达-10-6gmL-1,在体内不会因为血液的稀释而破坏结构。载药胶束的贮藏稳定性和血浆稳定性均良好,此外,胶束还具有良好的生物相容性,溶血率均低于5%。胶束不仅增加了难溶性药物多西他赛的溶解度,而且能够控制药物的释放,体外释放试验中,载药胶束的释药与原料药相比,具有很明显的缓释作用。体内药动学实验显示,胶束能显著降低药物的消除速度,延长其体内的循环时间和作用时间,有助于药物缓释及提高生物利用度。细胞实验结果表明载药胶束对耐药细胞MCF-7/Adr显示出抑制P-gp的功能,能够逆转肿瘤细胞的多药耐药现象,胶束通过内吞进入细胞,摄取显著提高,细胞毒性比原料药明显增加,IC50实验结果显示三种胶束制剂对乳腺癌细胞MCF-7的抑制效果分别是原料药的能达到原料药的69,82和100倍。本文第二部分主要针对自吞噬引起多药耐药的机理,将自吞噬抑制药氯喹与多西他赛共同包载在胶束,制成共递送给药系统。此制剂是在单载多西他赛胶束制剂基础上的扩展与改进,既抑制P-pg又抑制自吞噬,达到了双重逆转多药耐药。MTT实验筛选出当多西他赛和氯喹质量比为4：1时,协同作用最好。对双载药胶束的载药能力、粒径、形态、溶血性等进行了系统评价。透析法研究了载药胶束在磷酸盐缓冲液(PBS pH 7.4,0.5%吐温80)中的体外释药行为。此外,还评价了胶束制剂的细胞摄取；MTT法研究了双载药胶束对细胞耐药性的逆转情况,实验结果表明：所制备的胶束制剂粒径小,分散性好,溶血度低,具有缓释效果,能显著增强药物摄取,逆转肿瘤细胞多药耐药,两种胶束的逆转指数达到134.16和194.74.本文第三部分主要是在PEO-PPO-PCL胶束的基础上引入叶酸,构建了载多西他赛的FA-PEG2k/PEO-PPO-PCL胶束,叶酸能够赋予胶束对肿瘤的主动识别能力以及增强胶束的跨膜转运能力,实现胶束的主动靶向性。所得胶束制剂分散度良好,呈均一的球形,平均粒径为132 nm,制剂具有良好的血液相容性,溶血率低,包载的多西他赛释放缓慢,具有缓释功能。体外MTT实验结果显示在叶酸受体高表达的MCF-7细胞中,叶酸靶向胶束比普通胶束的抑制作用更显著。由此可见,叶酸修饰明显提高了胶束对叶酸受体过量表达的肿瘤细胞的亲和力,促进了药物的跨膜转运,取得了更好的药效。细胞摄取结果表明,叶酸低表达的A549细胞中,普通胶束与含叶酸靶向胶束荧光强度相近,而在叶酸高表达的MCF-7细胞中,含叶酸介导靶向胶束制剂(FA-PEG2k/P36)可以显著提高其在细胞中的摄取。
[Abstract]:Docetaxel, also known as anticancer drug docetaxel, is a new generation of taxanes, its wide anticancer spectrum, applicable to a variety of cancer treatment. However, the effect of chemotherapy drugs is still not ideal, on the one hand, due to the characteristics of their own drug physicochemical properties, such as water solubility, the drug can not effectively reach the tumor tissue. On the other hand, an important cause of multidrug resistance is the failure of chemotherapy. In another aspect, the lack of targeted drugs, patients caused serious side effects, and clinical chemotherapy in the treatment of the main obstacles. Therefore, the new formulation preparation can improve the solubility of the drug, reversing the multidrug resistance of tumor cells. At the same time improve drug targeting, reduce the side effects. This paper takes PEO-PPO-PCL as the main carrier of material loaded docetaxel, the main research contents are as follows: the first part of this paper focuses on the P- sugar protein in cancer cell membrane Over expression and mechanism of multi drug resistance, TPGS is selected as P-gp inhibitors, PEO-PPO-PCL/TPGS micelles were prepared. We synthesized three different hydrophobic segments of the polymer, three kinds of docetaxel PEO-PPO-PCL/TPGS micelles by thin film hydration method, the average particle size were between 25-135nm, with the effect of EPR particle the size, morphology is spherical and uniform distribution. The critical micelle concentration of micelles (CMC) as low as -10-6gmL-1, the body will not destroy the structure because of blood dilution. The storage stability and plasma stability of drug loaded micelles were good, in addition, the micelle also has good biocompatibility, solubility of the hemolysis rate was lower than that of 5%. micelles not only the increase of insoluble drug docetaxel, and can control the release of drug in vitro release test, compared with the raw material drug release of drug loaded micelles, sustained release effect obviously. In the pharmacokinetic experiment showed that micelles could significantly reduce the drug elimination rate, cycle time and prolong the duration of action in vivo, contribute to the sustained release of the drug and improve the bioavailability of cells. The experimental results show that the micelles of MCF-7/Adr cells showed inhibition of P-gp function, can reverse the multidrug resistance of tumor cells enter the cell through endocytosis, micelles, uptake increased significantly, significantly increased cytotoxicity than raw material medicine, IC50 experimental results showed that the inhibition effect of three kinds of micelle preparation on MCF-7 breast cancer cells are the raw material medicine can reach the medicine raw materials of 69,82 and 100 times. The second part mainly for autophagy mechanism of multidrug resistance will cause, autophagy inhibitor chloroquine and docetaxel loaded micelles were made in and delivered to the delivery system. This formulation is extended and improved in single carrier micelle preparation based on docetaxel, Both the inhibition of P-pg and inhibition of autophagy, both to the reversal of multidrug resistance in.MTT screening experiments when docetaxel and chloroquine ratio was 4:1, the best synergism. The drug loading ability of double micelles, particle size, morphology, hemolytic were evaluated. Dialysis of micelles in phosphate buffer (PBS pH, 7.4,0.5% Twain 80) release behavior in vitro. In addition, also evaluated the cellular uptake of micelles; MTT was studied by reverse micelles of double cell resistance, the experimental results show that the prepared micelles prepared by particle size, good dispersion, low degree of hemolysis that has sustained release effect, can significantly enhance the drug uptake, reversing the multidrug resistance of tumor cells, reverse micelle index of two to 134.16 and 194.74. the third part is the introduction of folic acid based on PEO-PPO-PCL micelles, constructs carrying docetaxel FA-PEG2k/PEO-PPO-PCL micelles, folic acid to give micelles on the active tumor recognition ability and enhance the ability of transmembrane transport of micelles, micelles to achieve active target. The micelle preparation good dispersion, a uniform sphere, mean diameter of 132 nm. The preparation has good blood compatibility, low hemolytic rate, docetaxel the package slowly released, with slow release function. The experimental results showed that the expression of MTT in vitro in folate receptor MCF-7 cells, folate targeted micelles inhibition ordinary micelles were more significant. Thus, significantly improve the folate modified micellar folate receptor overexpression on tumor cell affinity, promote the transmembrane transport of drugs the better the efficacy. Cell uptake results showed that low expression of folic acid in A549 cells, normal micelles and folate targeted micelles containing similar fluorescence intensity, and high in folic acid In MCF-7 cells, folic acid mediated targeting micellar preparation (FA-PEG2k/P36) can significantly increase its uptake in cells.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R943

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