K5多糖基两亲性药物载体的设计、制备及其药物传递性能研究
发布时间：2019-05-08 18:31
【摘要】：阿霉素存在靶向性低、毒副作用较强的情况，用聚合物制备的胶束药物载体可以改善药物的这些不足，以达到更好的治疗效果。本文利用工程菌高密度发酵并纯化了K5多糖(K5PS)，采用脱氧胆酸(DOCA)分子对K5多糖进行修饰，合成了两亲性的K5PS-DOCA (KD)与含双硫键的K5PS-SS-DOCA (KSD)缀合物。利用其在水溶液中自组装形成胶束的性能包载阿霉素，制备了载药KD胶束与载药KSD胶束，并对其理化性质进行测试。 (1)载药KD胶束 通过1H NMR确认了KD缀合物的化学结构并计算出DOCA分子在K5多糖分子上的取代度为0.29，其自组装形成胶束的临界胶束浓度为23.4mg/L。在水溶液中的平均粒径为111nm，载药后胶束的平均粒径为211nm，该载体可以抵抗血清吸附，在血液中稳定存在。其载药量为16.71%，包封率为62.66%，有良好的载药性能。体外释放结果表明，载药KD胶束在pH5.0与β-葡萄糖醛酸酶存在时均表现出了加速释放行为，在100h时药物累积释放率均达90%以上，高于pH7.4条件下的累积释放率。在细胞抑制实验中，载药KD胶束对HeLa细胞的半数抑制浓度(IC50)也显著低于对COS7细胞的IC50，同时细胞摄取图像也验证了载药胶束可以快速进入肿瘤细胞，在肿瘤细胞与正常细胞间体现出了显著的治疗效果差异。 (2)载药KSD胶束 以胱胺为连接臂将DOCA分子通过双硫键偶联到K5多糖骨架上制备了取代度分别为0.1与0.16的K5PS-SS-DOCA缀合物(KSD10与KSD16)，其在水溶液中可自组装形成胶束，临界胶束浓度分别为69mg/L与84mg/L，平均粒径为225nm与187nm。用透析法制备了载阿霉素的KSD胶束，形态为球形，载药量分别为11.54%与13.14%，包封率为46.17%与54.25%。体外药物释放结果表明，还原环境可明显促进阿霉素的释放，在10mM GSH存在的条件下，两种载药胶束在100h的累积释放率可达85%以上，而pH7.4时仅达60%左右。KSD胶束有良好的生物相容性，载药KSD胶束对肿瘤细胞的抑制作用与阿霉素相似，对正常细胞的抑制作用小于阿霉素，流式细胞仪测得载药KSD胶束在同一时间进入HeLa细胞的量大于COS7细胞。通过胞吞抑制剂初步判断该载体主要是通过网格蛋白介导的内吞进入细胞，是一个复杂的耗能过程。 研究结果表明，由K5多糖经过疏水改造制备的胶束载体，有良好的生物相容性和抗血清吸附性。载药胶束体现出了pH敏感与酶敏感释放性能，含有双硫键的胶束还表现出了还原响应的药物释放行为。对肿瘤细胞的抑制作用明显强于正常细胞，，在癌症治疗上体现出了靶向性与选择性。综上所述，K5多糖纳米胶束是一种具有潜在应用价值的药物载体。
[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.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943;R917
[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.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943;R917
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