【摘要】：阿霉素(DOX)是一種強效抗腫瘤藥物,但因其缺乏腫瘤靶向性、毒副作用大,且易產(chǎn)生多藥耐藥(MDR),臨床應用受到很大限制�？灯杖鸲4磷酸酯(CA4P)在1/10最大耐受量(MTD)的劑量下就可以選擇性地產(chǎn)生抗腫瘤血管作用,但存在體內(nèi)消除快、抗腫瘤作用不徹底的問題。鑒于此,我們構(gòu)建了聚乙二醇單甲醚-聚乳酸(mPEG-PLA)囊泡,共同包載DOX與CA4P,以期通過增強滲透與滯留(EPR)效應使DOX與CA4P同時被動靶向于腫瘤組織,降低藥物毒副作用,并著重考察DOX與CA4P共載后對提高腫瘤抑制率及逆轉(zhuǎn)耐藥的影響及作用機制。課題研究工作分為以下三部分：第一部分,采用開環(huán)聚合反應合成了不同親／疏水鏈段比例的兩親性嵌段共聚物聚乙二醇單甲醚-聚乳酸(mPEG-PLA),并優(yōu)選出具有合適fEO、能自組裝形成囊泡,且具有較好載藥能力的載體材料mPEG114-PLA162。采用溶劑蒸發(fā)法制備了空白聚合物囊泡及DOX和CA4P共載聚合物囊泡(Ps-DOX-CA4P),并對其形態(tài)、粒徑、包封率及釋放行為進行了考察。結(jié)果顯示,空白自組裝體外觀呈類球形,形態(tài)規(guī)整,粒徑約為50 nm,具有較大的親水內(nèi)核,外周由約10 nm的疏水層環(huán)繞,顯示出典型的囊泡結(jié)構(gòu)。Ps-DOX-CA4P具有與空白囊泡類似的大小及形態(tài),能夠共載DOX和CA4P, DOX的包封率大于90%,CA4P的包封率大于50%。藥物釋放的實驗結(jié)果表明,DOX的釋放速度略慢于CA4P。第二部分,以KB細胞為模型,考察了Ps-DOX-CA4P對腫瘤抑制的協(xié)同增效作用。MTT實驗結(jié)果顯示,空白聚合物囊泡無明顯的細胞毒性,而Ps-DOX-CA4P與CA4P單載囊泡(Ps-CA4P)或DOX單載囊泡(Ps-DOX)相比,細胞毒性明顯增加。建立裸鼠KB細胞移植瘤模型,考察聚合物囊泡的體內(nèi)分布及藥效。結(jié)果顯示,聚合物囊泡所載的DOX在腫瘤組織的分布明顯高于游離藥(p0.05),說明聚合物囊泡能夠提高DOX在腫瘤組織的蓄積。Ps-CA4P能快速抑制腫瘤的生長,但作用短暫,Ps-DOX起效慢而作用持久,兩者共載的囊泡Ps-DOX-CA4P(1:10)既能迅速起效,又能持續(xù)維持抗腫瘤的藥效,抑瘤作用顯著提高。CD31和Ki67免疫組化實驗也顯示,DOX與CA4P共載后,不僅能迅速破壞腫瘤血管,而且能持續(xù)抑制腫瘤細胞的增殖,產(chǎn)生協(xié)同抗腫瘤作用。第三部分,以阿霉素耐藥MCF-7/ADR細胞為模型,通過細胞毒實驗、細胞凋亡實驗、DOX的細胞攝取、ATP酶的活性測定、細胞內(nèi)ATP測定、胞內(nèi)ROS測定及體外腫瘤球等實驗研究,探討了Ps-DOX-CA4P逆轉(zhuǎn)耐藥的作用機制。結(jié)果表明,Ps-DOX-CA4P能提高DOX對MCF-7/ADR細胞的細胞毒性和細胞凋亡率、促進MCF-7/ADR細胞對DOX的攝取、激活ATP酶的活性、降低細胞內(nèi)ATP、提高細胞內(nèi)的ROS,最終抑制了藥物外排泵P-gp的功能而逆轉(zhuǎn)耐藥。同時,通過體外腫瘤球?qū)嶒灠l(fā)現(xiàn),Ps-DOX-CA4P能明顯促進DOX滲透進入腫瘤球并且抑制腫瘤球的生長,提示Ps-DOX-CA4P有望在動物體內(nèi)逆轉(zhuǎn)P-gp介導的腫瘤耐藥,增加化療藥的敏感性。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R943;R96

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