本文選題：PEO-PPO-PCL　切入點(diǎn)：TPGS　出處：《山東大學(xué)》2016年碩士論文

【摘要】：多西他賽,又名多西紫杉醇,是新一代紫杉烷類抗腫瘤藥,其抗癌譜廣,適用于多種癌癥的治療。然而,目前藥物化療效果仍不理想,一方面,由于藥物自身理化性質(zhì)的特點(diǎn),如水溶性差,使藥物不能有效地達(dá)到腫瘤組織。另一方面,腫瘤多藥耐藥性也是化療失敗的重要原因。再一方面,藥物缺乏靶向性,引起患者嚴(yán)重的毒副作用,也是臨床上化學(xué)治療的主要障礙。因此,本課題制備的新劑型能夠提高藥物的溶解度、逆轉(zhuǎn)腫瘤細(xì)胞的多藥耐藥,同時(shí)提高藥物的靶向性,降低毒副作用。本文以PEO-PPO-PCL為主要載體材料來包載多西他賽,主要研究內(nèi)容如下：本文第一部分主要針對P-糖蛋白在癌細(xì)胞膜上過度表達(dá)而產(chǎn)生多藥耐藥的機(jī)理,選取TPGS為P-gp抑制劑,制備了PEO-PPO-PCL/TPGS膠束。我們合成了三種不同疏水鏈段的聚合物,用薄膜水化法制得的三種載多西他賽的PEO-PPO-PCL/TPGS膠束,其平均粒徑分別在25-135nm之間,符合EPR效應(yīng)的粒徑要求,形態(tài)呈球形,分布均勻。膠束的臨界膠束濃度(CMC)低達(dá)-10-6gmL-1,在體內(nèi)不會因?yàn)檠旱南♂尪茐慕Y(jié)構(gòu)。載藥膠束的貯藏穩(wěn)定性和血漿穩(wěn)定性均良好,此外,膠束還具有良好的生物相容性,溶血率均低于5%。膠束不僅增加了難溶性藥物多西他賽的溶解度,而且能夠控制藥物的釋放,體外釋放試驗(yàn)中,載藥膠束的釋藥與原料藥相比,具有很明顯的緩釋作用。體內(nèi)藥動學(xué)實(shí)驗(yàn)顯示,膠束能顯著降低藥物的消除速度,延長其體內(nèi)的循環(huán)時(shí)間和作用時(shí)間,有助于藥物緩釋及提高生物利用度。細(xì)胞實(shí)驗(yàn)結(jié)果表明載藥膠束對耐藥細(xì)胞MCF-7/Adr顯示出抑制P-gp的功能,能夠逆轉(zhuǎn)腫瘤細(xì)胞的多藥耐藥現(xiàn)象,膠束通過內(nèi)吞進(jìn)入細(xì)胞,攝取顯著提高,細(xì)胞毒性比原料藥明顯增加,IC50實(shí)驗(yàn)結(jié)果顯示三種膠束制劑對乳腺癌細(xì)胞MCF-7的抑制效果分別是原料藥的能達(dá)到原料藥的69,82和100倍。本文第二部分主要針對自吞噬引起多藥耐藥的機(jī)理,將自吞噬抑制藥氯喹與多西他賽共同包載在膠束,制成共遞送給藥系統(tǒng)。此制劑是在單載多西他賽膠束制劑基礎(chǔ)上的擴(kuò)展與改進(jìn),既抑制P-pg又抑制自吞噬,達(dá)到了雙重逆轉(zhuǎn)多藥耐藥。MTT實(shí)驗(yàn)篩選出當(dāng)多西他賽和氯喹質(zhì)量比為4：1時(shí),協(xié)同作用最好。對雙載藥膠束的載藥能力、粒徑、形態(tài)、溶血性等進(jìn)行了系統(tǒng)評價(jià)。透析法研究了載藥膠束在磷酸鹽緩沖液(PBS pH 7.4,0.5%吐溫80)中的體外釋藥行為。此外,還評價(jià)了膠束制劑的細(xì)胞攝��；MTT法研究了雙載藥膠束對細(xì)胞耐藥性的逆轉(zhuǎn)情況,實(shí)驗(yàn)結(jié)果表明：所制備的膠束制劑粒徑小,分散性好,溶血度低,具有緩釋效果,能顯著增強(qiáng)藥物攝取,逆轉(zhuǎn)腫瘤細(xì)胞多藥耐藥,兩種膠束的逆轉(zhuǎn)指數(shù)達(dá)到134.16和194.74.本文第三部分主要是在PEO-PPO-PCL膠束的基礎(chǔ)上引入葉酸,構(gòu)建了載多西他賽的FA-PEG2k/PEO-PPO-PCL膠束,葉酸能夠賦予膠束對腫瘤的主動識別能力以及增強(qiáng)膠束的跨膜轉(zhuǎn)運(yùn)能力,實(shí)現(xiàn)膠束的主動靶向性。所得膠束制劑分散度良好,呈均一的球形,平均粒徑為132 nm,制劑具有良好的血液相容性,溶血率低,包載的多西他賽釋放緩慢,具有緩釋功能。體外MTT實(shí)驗(yàn)結(jié)果顯示在葉酸受體高表達(dá)的MCF-7細(xì)胞中,葉酸靶向膠束比普通膠束的抑制作用更顯著。由此可見,葉酸修飾明顯提高了膠束對葉酸受體過量表達(dá)的腫瘤細(xì)胞的親和力,促進(jìn)了藥物的跨膜轉(zhuǎn)運(yùn),取得了更好的藥效。細(xì)胞攝取結(jié)果表明,葉酸低表達(dá)的A549細(xì)胞中,普通膠束與含葉酸靶向膠束熒光強(qiáng)度相近,而在葉酸高表達(dá)的MCF-7細(xì)胞中,含葉酸介導(dǎo)靶向膠束制劑(FA-PEG2k/P36)可以顯著提高其在細(xì)胞中的攝取。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R943

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本文編號：1660443