DOX和CA4P共载mPEG-PLA聚合物囊泡的制备及其肿瘤抑制作用研究
发布时间：2018-12-13 22:31
【摘要】：阿霉素(DOX)是一种强效抗肿瘤药物,但因其缺乏肿瘤靶向性、毒副作用大,且易产生多药耐药(MDR),临床应用受到很大限制。康普瑞丁A4磷酸酯(CA4P)在1/10最大耐受量(MTD)的剂量下就可以选择性地产生抗肿瘤血管作用,但存在体内消除快、抗肿瘤作用不彻底的问题。鉴于此,我们构建了聚乙二醇单甲醚-聚乳酸(mPEG-PLA)囊泡,共同包载DOX与CA4P,以期通过增强渗透与滞留(EPR)效应使DOX与CA4P同时被动靶向于肿瘤组织,降低药物毒副作用,并着重考察DOX与CA4P共载后对提高肿瘤抑制率及逆转耐药的影响及作用机制。课题研究工作分为以下三部分：第一部分,采用开环聚合反应合成了不同亲／疏水链段比例的两亲性嵌段共聚物聚乙二醇单甲醚-聚乳酸(mPEG-PLA),并优选出具有合适fEO、能自组装形成囊泡,且具有较好载药能力的载体材料mPEG114-PLA162。采用溶剂蒸发法制备了空白聚合物囊泡及DOX和CA4P共载聚合物囊泡(Ps-DOX-CA4P),并对其形态、粒径、包封率及释放行为进行了考察。结果显示,空白自组装体外观呈类球形,形态规整,粒径约为50 nm,具有较大的亲水内核,外周由约10 nm的疏水层环绕,显示出典型的囊泡结构。Ps-DOX-CA4P具有与空白囊泡类似的大小及形态,能够共载DOX和CA4P, DOX的包封率大于90%,CA4P的包封率大于50%。药物释放的实验结果表明,DOX的释放速度略慢于CA4P。第二部分,以KB细胞为模型,考察了Ps-DOX-CA4P对肿瘤抑制的协同增效作用。MTT实验结果显示,空白聚合物囊泡无明显的细胞毒性,而Ps-DOX-CA4P与CA4P单载囊泡(Ps-CA4P)或DOX单载囊泡(Ps-DOX)相比,细胞毒性明显增加。建立裸鼠KB细胞移植瘤模型,考察聚合物囊泡的体内分布及药效。结果显示,聚合物囊泡所载的DOX在肿瘤组织的分布明显高于游离药(p0.05),说明聚合物囊泡能够提高DOX在肿瘤组织的蓄积。Ps-CA4P能快速抑制肿瘤的生长,但作用短暂,Ps-DOX起效慢而作用持久,两者共载的囊泡Ps-DOX-CA4P(1:10)既能迅速起效,又能持续维持抗肿瘤的药效,抑瘤作用显著提高。CD31和Ki67免疫组化实验也显示,DOX与CA4P共载后,不仅能迅速破坏肿瘤血管,而且能持续抑制肿瘤细胞的增殖,产生协同抗肿瘤作用。第三部分,以阿霉素耐药MCF-7/ADR细胞为模型,通过细胞毒实验、细胞凋亡实验、DOX的细胞摄取、ATP酶的活性测定、细胞内ATP测定、胞内ROS测定及体外肿瘤球等实验研究,探讨了Ps-DOX-CA4P逆转耐药的作用机制。结果表明,Ps-DOX-CA4P能提高DOX对MCF-7/ADR细胞的细胞毒性和细胞凋亡率、促进MCF-7/ADR细胞对DOX的摄取、激活ATP酶的活性、降低细胞内ATP、提高细胞内的ROS,最终抑制了药物外排泵P-gp的功能而逆转耐药。同时,通过体外肿瘤球实验发现,Ps-DOX-CA4P能明显促进DOX渗透进入肿瘤球并且抑制肿瘤球的生长,提示Ps-DOX-CA4P有望在动物体内逆转P-gp介导的肿瘤耐药,增加化疗药的敏感性。
[Abstract]:Adriamycin (DOX) is a powerful antitumor drug, but its clinical application is limited because of its lack of tumor targeting, high toxicity and side effects, and easy to produce multidrug resistant (MDR),. Compratine A4 phosphate (CA4P) can selectively produce antiangiogenic effects at the dose of 1 / 10 maximum tolerated dose of (MTD), but it has the problem of quick elimination in vivo and incomplete antitumor effect. In view of this, we constructed polyethylene glycol monomethyl ether-polylactic acid (mPEG-PLA) vesicles and encapsulated DOX and CA4P, in order to make DOX and CA4P target tumor tissues passively by enhancing the effect of permeation and retention of (EPR), so as to reduce the side effects of drugs. The effect of co-loading of DOX and CA4P on increasing tumor inhibition rate and reversing drug resistance was investigated. The research work is divided into the following three parts: in the first part, the amphiphilic block copolymer poly (ethylene glycol monomethyl ether-polylactic acid) (mPEG-PLA) with different ratio of hydrophobic segments was synthesized by ring-opening polymerization, and suitable fEO, was selected. MPEG114-PLA162., a carrier material with good drug loading ability, which can self-assemble to form vesicles Blank polymer vesicles and DOX and CA4P co-loaded polymer vesicles (Ps-DOX-CA4P) were prepared by solvent evaporation method. The morphology, particle size, encapsulation efficiency and release behavior were investigated. The results showed that the blank self-assembly was spherical in appearance, regular in shape, with a large hydrophilic core about 50 nm, in diameter and surrounded by a hydrophobic layer of about 10 nm. The results show that Ps-DOX-CA4P has the same size and shape as the blank vesicle, and the encapsulation efficiency of DOX and CA4P, DOX is larger than 90% of CA4P. The experimental results of drug release showed that the release rate of DOX was slightly slower than that of CA4P.. In the second part, using KB cells as a model, the synergistic effect of Ps-DOX-CA4P on tumor inhibition was investigated. The results of MTT experiment showed that blank polymer vesicles had no obvious cytotoxicity. The cytotoxicity of Ps-DOX-CA4P was significantly higher than that of CA4P single vesicles (Ps-CA4P) or DOX single loaded vesicles (Ps-DOX). To investigate the distribution and pharmacodynamics of polymer vesicles in nude mice KB cell xenograft tumor model was established. The results showed that the distribution of DOX in tumor tissue was significantly higher than that in free drug (p0.05), indicating that polymer vesicle could increase the accumulation of DOX in tumor tissue. Ps-CA4P could inhibit the growth of tumor rapidly, but the effect was short. The effect of Ps-DOX was slow and lasting. The vesicle Ps-DOX-CA4P (1:10) loaded with both of them could not only take effect quickly, but also maintain the anti-tumor effect continuously. The anti-tumor effect was significantly increased by CD31 and Ki67 immunohistochemistry. The co-loading of DOX and CA4P can not only destroy tumor blood vessels rapidly, but also inhibit the proliferation of tumor cells and produce synergistic antitumor effect. In the third part, doxorubicin resistant MCF-7/ADR cells were used as the model. The cytotoxicity, apoptosis, DOX uptake, ATP enzyme activity, intracellular ATP, intracellular ROS and tumor balls in vitro were studied. The mechanism of reversal of drug resistance by Ps-DOX-CA4P was discussed. The results showed that Ps-DOX-CA4P could increase the cytotoxicity and apoptosis rate of DOX to MCF-7/ADR cells, promote the uptake of DOX in MCF-7/ADR cells, activate the activity of ATP enzyme, and decrease the ATP, in cells to increase ROS, in cells. The drug resistance was reversed by inhibiting the function of drug efflux pump P-gp. At the same time, it was found that Ps-DOX-CA4P could significantly promote the infiltration of DOX into tumor spheres and inhibit the growth of tumor spheres in vitro, suggesting that Ps-DOX-CA4P could reverse the drug resistance mediated by P-gp in vivo. Increase the sensitivity of chemotherapeutic agents.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R943;R96
本文编号：2377366
[Abstract]:Adriamycin (DOX) is a powerful antitumor drug, but its clinical application is limited because of its lack of tumor targeting, high toxicity and side effects, and easy to produce multidrug resistant (MDR),. Compratine A4 phosphate (CA4P) can selectively produce antiangiogenic effects at the dose of 1 / 10 maximum tolerated dose of (MTD), but it has the problem of quick elimination in vivo and incomplete antitumor effect. In view of this, we constructed polyethylene glycol monomethyl ether-polylactic acid (mPEG-PLA) vesicles and encapsulated DOX and CA4P, in order to make DOX and CA4P target tumor tissues passively by enhancing the effect of permeation and retention of (EPR), so as to reduce the side effects of drugs. The effect of co-loading of DOX and CA4P on increasing tumor inhibition rate and reversing drug resistance was investigated. The research work is divided into the following three parts: in the first part, the amphiphilic block copolymer poly (ethylene glycol monomethyl ether-polylactic acid) (mPEG-PLA) with different ratio of hydrophobic segments was synthesized by ring-opening polymerization, and suitable fEO, was selected. MPEG114-PLA162., a carrier material with good drug loading ability, which can self-assemble to form vesicles Blank polymer vesicles and DOX and CA4P co-loaded polymer vesicles (Ps-DOX-CA4P) were prepared by solvent evaporation method. The morphology, particle size, encapsulation efficiency and release behavior were investigated. The results showed that the blank self-assembly was spherical in appearance, regular in shape, with a large hydrophilic core about 50 nm, in diameter and surrounded by a hydrophobic layer of about 10 nm. The results show that Ps-DOX-CA4P has the same size and shape as the blank vesicle, and the encapsulation efficiency of DOX and CA4P, DOX is larger than 90% of CA4P. The experimental results of drug release showed that the release rate of DOX was slightly slower than that of CA4P.. In the second part, using KB cells as a model, the synergistic effect of Ps-DOX-CA4P on tumor inhibition was investigated. The results of MTT experiment showed that blank polymer vesicles had no obvious cytotoxicity. The cytotoxicity of Ps-DOX-CA4P was significantly higher than that of CA4P single vesicles (Ps-CA4P) or DOX single loaded vesicles (Ps-DOX). To investigate the distribution and pharmacodynamics of polymer vesicles in nude mice KB cell xenograft tumor model was established. The results showed that the distribution of DOX in tumor tissue was significantly higher than that in free drug (p0.05), indicating that polymer vesicle could increase the accumulation of DOX in tumor tissue. Ps-CA4P could inhibit the growth of tumor rapidly, but the effect was short. The effect of Ps-DOX was slow and lasting. The vesicle Ps-DOX-CA4P (1:10) loaded with both of them could not only take effect quickly, but also maintain the anti-tumor effect continuously. The anti-tumor effect was significantly increased by CD31 and Ki67 immunohistochemistry. The co-loading of DOX and CA4P can not only destroy tumor blood vessels rapidly, but also inhibit the proliferation of tumor cells and produce synergistic antitumor effect. In the third part, doxorubicin resistant MCF-7/ADR cells were used as the model. The cytotoxicity, apoptosis, DOX uptake, ATP enzyme activity, intracellular ATP, intracellular ROS and tumor balls in vitro were studied. The mechanism of reversal of drug resistance by Ps-DOX-CA4P was discussed. The results showed that Ps-DOX-CA4P could increase the cytotoxicity and apoptosis rate of DOX to MCF-7/ADR cells, promote the uptake of DOX in MCF-7/ADR cells, activate the activity of ATP enzyme, and decrease the ATP, in cells to increase ROS, in cells. The drug resistance was reversed by inhibiting the function of drug efflux pump P-gp. At the same time, it was found that Ps-DOX-CA4P could significantly promote the infiltration of DOX into tumor spheres and inhibit the growth of tumor spheres in vitro, suggesting that Ps-DOX-CA4P could reverse the drug resistance mediated by P-gp in vivo. Increase the sensitivity of chemotherapeutic agents.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R943;R96
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