产淀粉酶肺癌靶向的双功能HEA-g-(PCL;PEI)胶束的制备及抗癌活性研究

Lung and bronchus cancers continue to be the leading common causes of global cancer death. paclitaxel (PTX) is a common chemotherapeutic for clinical treatment of lung cancer. The poor water solubility and drug resistance has seriously affected and restricted its clinical application. Some adenocarcinomas lung cancer patients showed hyperamylasemia, which was caused by high level amylase produced by cancer tissue. And down-regulation of microRNA-200b was suggested a main reason of PTX resistance in lung adenocarcinomas. Polyethyleneimine (PEI), the most promising non-viral vector in gene therapy, has disadvantages of high cytotoxicity and short systemic half-life. Therefore, this thesis designed a double-grafted amphiphilic polymer based on hydroxyethyl amylase as backbone, which is sequentially grafted by polycaprolactone (PCL) and straight-chain polyethyleneimine (PEI) using “grafting from” and “grafting onto” techniques respectively. Micelles assembled from the amphiphilic polymer are co-loaded with PTX and microRNA-200b. The coating of HEA can effectively shield the cations of PEI and endue the micelles with long circulation property which leads passive enrichment of paclitaxel and microRNA-200b to tumor tissue through EPR effect. Furthermore, degradation of HEA by amylase in tumor facilitates the releasing of PTX and microRNA-200b into tumor cells. Restoration of microRNA-200b may enhance the sensitivity of PTX by inhibiting the expression of type III beta tubulin, and inhibit drug resistance developing. The project aims to provide new therapeutic approaches for amylase-producing lung adenocarcinoma by targeted delivery of drugs and genes.

肺癌是全球发病率和死亡率最高的癌症，常用化疗药物紫杉醇（PTX）存在水溶性差和易产生耐药性的问题。临床上部分肺腺癌会产生高淀粉酶血症，且对PTX耐药性的产生与下调miR-200b有关。转基因载体聚乙烯亚胺（PEI）细胞毒性大，不利于基因向肿瘤部位的输送。本课题设计了一种以羟乙基直链淀粉（HEA）为骨架，分别采用“grafting from”和“grafting onto”技术接枝聚己内酯和直链PEI，并组装成两亲性胶束，共输送PTX和miR-200b。阐明HEA的屏蔽作用降低PEI细胞毒性和长循环输送PTX和miR-200b的机制，肺癌淀粉酶介导PTX和miR-200b靶向释放的机制，以及miR-200b 通过下调Ⅲ型β微管蛋白增强PTX的敏感性和抑制耐药性产生的机制。本项目有望通过药物和基因的靶向输送为产淀粉酶的肺腺癌提供新的治疗策略。

（1）纳米载药系统亲水化修饰常用的聚乙二醇（PEG）存在“PEG两难”问题，本项目制备了几种不同摩尔取代度的羟乙基直链淀粉（HEA），作为PEG的潜在替代者。采用玉米淀粉作为原料，通过丁醇凝沉法提纯出直链淀粉，采用紫外-可见分光光度计表征了其蓝值，采用乌氏粘度计测定了其粘均分子量。通过在碱性条件下加入不同比例的环氧乙烷对直链淀粉进行醚化，制备了HEA，采用气相色谱色谱法检测了HEA的摩尔取代度。纯化后的直链淀粉蓝值为0.92，粘均分子量为7.6×105，环氧乙烷/直链淀粉葡萄糖单元（mol/mol）分别为4、8、16和32时，制备出的HEA摩尔取代度分别为0.18、0.49、1.00和1.37，且具有良好的水溶性。此结果表明丁醇凝沉法可方便地从玉米淀粉中提纯出直链淀粉，通过不同浓度的环氧乙烷醚化直链淀粉可得到不同摩尔取代度的HEA。.（2）使用1-乙基-3-(3－二甲基氨丙基)-碳化二亚胺( EDC)/N-羟基琥珀酰亚胺(NHS)交联剂合成了一种直链淀粉接枝鹅去氧胆酸聚合物（Amylose–chenodeoxycholic acid conjugates, AMY-CDCA），通过傅里叶变换红外光谱（FTIR）、核磁共振氢谱（1H NMR）和紫外光谱法对聚合物进行表征，结果表明，CDCA成功接枝到了直链淀粉骨架上，接枝度为138.15/100个葡萄糖单元。AMY-CDCA聚合物可通过透析法制备成胶束，动态光散射法测得胶束的平均粒径为224nm，多分散指数为0.110，透射电子显微镜观察到胶束为球形，并具有核壳结构。采用疏水性荧光探针芘和尼罗红研究了胶束的组装行为，结果表明，聚合物的临界胶束浓度（CMC）为2.8×10-3 mg/mL，胶束的疏水核心对尼罗红有增溶作用。