【摘要】：阿霉素存在靶向性低、毒副作用較強的情況，用聚合物制備的膠束藥物載體可以改善藥物的這些不足，以達到更好的治療效果。本文利用工程菌高密度發(fā)酵并純化了K5多糖(K5PS)，采用脫氧膽酸(DOCA)分子對K5多糖進行修飾，合成了兩親性的K5PS-DOCA (KD)與含雙硫鍵的K5PS-SS-DOCA (KSD)綴合物。利用其在水溶液中自組裝形成膠束的性能包載阿霉素，制備了載藥KD膠束與載藥KSD膠束，并對其理化性質(zhì)進行測試。 (1)載藥KD膠束 通過1H NMR確認了KD綴合物的化學結(jié)構(gòu)并計算出DOCA分子在K5多糖分子上的取代度為0.29，其自組裝形成膠束的臨界膠束濃度為23.4mg/L。在水溶液中的平均粒徑為111nm，載藥后膠束的平均粒徑為211nm，該載體可以抵抗血清吸附，在血液中穩(wěn)定存在。其載藥量為16.71%，包封率為62.66%，有良好的載藥性能。體外釋放結(jié)果表明，載藥KD膠束在pH5.0與β-葡萄糖醛酸酶存在時均表現(xiàn)出了加速釋放行為，在100h時藥物累積釋放率均達90%以上，高于pH7.4條件下的累積釋放率。在細胞抑制實驗中，載藥KD膠束對HeLa細胞的半數(shù)抑制濃度(IC50)也顯著低于對COS7細胞的IC50，同時細胞攝取圖像也驗證了載藥膠束可以快速進入腫瘤細胞，在腫瘤細胞與正常細胞間體現(xiàn)出了顯著的治療效果差異。 (2)載藥KSD膠束 以胱胺為連接臂將DOCA分子通過雙硫鍵偶聯(lián)到K5多糖骨架上制備了取代度分別為0.1與0.16的K5PS-SS-DOCA綴合物(KSD10與KSD16)，其在水溶液中可自組裝形成膠束，臨界膠束濃度分別為69mg/L與84mg/L，平均粒徑為225nm與187nm。用透析法制備了載阿霉素的KSD膠束，形態(tài)為球形，載藥量分別為11.54%與13.14%，包封率為46.17%與54.25%。體外藥物釋放結(jié)果表明，還原環(huán)境可明顯促進阿霉素的釋放，在10mM GSH存在的條件下，兩種載藥膠束在100h的累積釋放率可達85%以上，而pH7.4時僅達60%左右。KSD膠束有良好的生物相容性，載藥KSD膠束對腫瘤細胞的抑制作用與阿霉素相似，對正常細胞的抑制作用小于阿霉素，流式細胞儀測得載藥KSD膠束在同一時間進入HeLa細胞的量大于COS7細胞。通過胞吞抑制劑初步判斷該載體主要是通過網(wǎng)格蛋白介導的內(nèi)吞進入細胞，是一個復雜的耗能過程。 研究結(jié)果表明，由K5多糖經(jīng)過疏水改造制備的膠束載體，有良好的生物相容性和抗血清吸附性。載藥膠束體現(xiàn)出了pH敏感與酶敏感釋放性能，含有雙硫鍵的膠束還表現(xiàn)出了還原響應的藥物釋放行為。對腫瘤細胞的抑制作用明顯強于正常細胞，，在癌癥治療上體現(xiàn)出了靶向性與選擇性。綜上所述，K5多糖納米膠束是一種具有潛在應用價值的藥物載體。
[Abstract]:Doxorubicin has low targeting and strong toxic and side effects. The micellar drug carrier prepared by polymer can improve the drug's deficiency in order to achieve better therapeutic effect. In this paper, K5 polysaccharide (K5PS) was purified by high density fermentation of engineering bacteria and modified by deoxycholic acid (DOCA) molecule to synthesize amphiphilic K5PS-DOCA (KD) conjugates with K5PS-SS-DOCA (KSD) containing disulfide bonds. Drug-loaded KD micelles and drug-loaded KSD micelles were prepared by self-assembly of adriamycin-loaded micelles in aqueous solution, and their physical and chemical properties were tested. The main results are as follows: (1) the chemical structure of KD conjugated compound was confirmed by 1H NMR in drug-loaded KD micelle, and the substitution degree of DOCA molecule on K5 polysaccharide molecule was calculated to be 0.29, and the critical micelle concentration of self-assembled micelle was 23.4 mg 路L ~ (- 1). The average diameter of micelle was 111 nm in aqueous solution and 211 nm in micelle after drug loading. The carrier could resist the adsorption of serum and existed stably in blood. The drug loading rate is 16.71%, the entrapment efficiency is 62.66%, and the drug loading performance is good. The results of in vitro release showed that the drug-loaded KD micelle showed accelerated release behavior in the presence of pH5.0 and 尾-glucuronidase, and the cumulative drug release rate was over 90% at 100 h, which was higher than that under the condition of pH7.4. In the cell inhibition test, the 50% inhibitory concentration (IC50) of drug-loaded KD micelles on HeLa cells was also significantly lower than that on COS7 cells, and the image of cell uptake also confirmed that the drug-loaded micelles could enter tumor cells rapidly. There was significant difference in therapeutic effect between tumor cells and normal cells. (2) K5PS-SS-DOCA conjugates (KSD10 and KSD16) with degree of substitution 0. 1 and 0. 16, respectively, were prepared by coupling DOCA molecules to the backbone of K5 polysaccharides with cystine as the connecting arm in the drug-loaded KSD micelles. The micelle can be self-assembled in aqueous solution. The critical micelle concentration is 69mg/L and 84 mg / L, and the average diameter of micelle is 225nm and 187 nm. Doxorubicin-loaded KSD micelles were prepared by dialysis. The morphology of the micelles was spherical. The drug loading rates were 11.54% and 13.14% respectively. The entrapment efficiency was 46.17% and 54.25% respectively. The results of drug release in vitro showed that the reductive environment could obviously promote the release of doxorubicin. In the presence of 10mM GSH, the cumulative release rate of the two drug-loaded micelles was more than 85% at 100 h. PH7.4 micelles had good biocompatibility. The inhibitory effect of drug-loaded KSD micelles on tumor cells was similar to that of doxorubicin, and the inhibitory effect of KSD micelles on normal cells was less than that of doxorubicin. Flow cytometry showed that the amount of drug-loaded KSD micelle entering HeLa cells at the same time was greater than that of COS7 cells. It is a complex energy consumption process to judge that the vector enters the cell mainly through the endocytosis mediated by grid protein. The results showed that the micellar carrier prepared by hydrophobic modification of K5 polysaccharide had good biocompatibility and antiserum adsorption. The drug-loaded micelles showed pH-sensitive and enzyme-sensitive release properties. The micelles containing disulfide bonds also showed a reduction-responsive drug release behavior. The inhibitory effect on tumor cells was significantly stronger than that of normal cells, which showed targeting and selectivity in cancer treatment. To sum up, K5 polysaccharide nano-micelle is a kind of drug carrier with potential application value.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R943;R917

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