环境响应型聚谷氨酸/脂质复合纳米囊载体递送双硫仑抑制ALDH(+)乳腺肿瘤干细胞的研究

Inhibition of cancer stem cells is essentially important for cancer targeted therapy. Disulfiram (DSF), aldehyde dehydrogenase (ALDH) inhibitor, can inhibit biology functions of ALDH positive cancer stem cells, however its instability in vivo limits its clinical applications. Thus the key to improve cancer treatment is to improve its stability, extend its circulating time and increase its accumulation in tumor site. Our previous results suggested that in vivo stability and tumor targeting efficiency could be significantly improved by encapsulating DSF in pH-sensitive PEG-shedding lipid nanocarriers. Accordingly, in the present study a polyargine modified lipid nanocapsule with strong penetrating to cell membrane is designed to encapsulate DSF to realize the stabilization of DSF-loading and efficient internalization of tumor. pH sensitive polyglutamic acid PEG polymer is then anchored on the surface of the nanocapsule via electrostatic interaction to establish poly glutamic acid/lipid nanocapsule with environmental response for DSF delivery, which may prolong DSF circulating time in vivo, enhance tumor targeting efficiency and inhibitory of cancer stem cells. The tumor cell uptake behaviors affected by arginines can be studied to evaluate the internality of nanocapsule. The tumor microenvironment response and long-circulating properties can be totally assessed and further modified by investigating the effects of polyglutamic acid molecular weight and polymer grafting rate on pH sensitivity and stability. The effects of DSF nanocapsules on ALDH positive breast cancer stem cells in vitro and in vivo are investigated to evaluate the inhibition and mechanism on cancer stem cells. The polyglutamic acid/lipid nanocapsule with environmental response for DSF delivery may provide a new method and stratergy for cancer stem cells targeting therapy.

抑制肿瘤干细胞是肿瘤靶向治疗的重要课题之一。双硫仑(DSF)是乙醛脱氢酶(ALDH)抑制剂，能抑制ALDH(+)肿瘤干细胞功能；DSF不稳定性限制其临床应用，故提高稳定性并增加肿瘤蓄积，是提高肿瘤治疗关键。前期研究显示pH敏感可离去PEG脂质纳米载体可提高DSF稳定性和肿瘤靶向性。基于此，本课题利用精氨酸聚合体的强细胞膜穿透性制备内化型载DSF脂质纳米囊，实现稳定载药和肿瘤高效内化；再将该纳米囊与pH敏感聚谷氨酸PEG聚合物静电结合构建环境响应型聚谷氨酸/脂质复合纳米囊，实现DSF长循环、肿瘤蓄积、肿瘤干细胞抑制。研究精氨酸密度对细胞摄取影响，评价其内化特性；研究聚谷氨酸分子量和聚合物接枝率对纳米囊pH敏感性和稳定性影响，评价其肿瘤微环境响应和长循环特性；考察DSF纳米囊对ALDH(+)乳腺癌干细胞生物学影响，评价其干细胞抑制性并探讨机制。该体系将为肿瘤干细胞靶向治疗提供新方法和新策略。

本课题组在依托单位中国医科大学的支持下，顺利完成了研究计划，成功制备了环境响应型聚谷氨酸脂质复合纳米递药系统，用以装载乙醛脱氢酶（ALDH）抑制剂双硫仑（DSF），实现对乙醛脱氢酶（ALDH）高表达乳腺癌细胞的抑制。该给药系统选用脂质纳米囊对疏水药物进行稳定包载，然后利用聚谷氨酸的pH敏感性实现肿瘤微环境响应的靶向性，并借助富含精氨酸的穿膜肽作用高效入胞到达细胞浆，最后利用DSF与肿瘤细胞内高浓度铜离子结合形成铜复合物实现肿瘤抑制。. 在设计之初，合成12-羟基硬脂酸-聚乙二醇-聚精氨酸多肽聚合物和聚谷氨酸-聚乙二醇嵌段聚合物，通过转相法、后插入法和静电吸附法制备具有“核-壳”结构的电荷可翻转复合纳米囊，筛选两种聚合物配比，对优选的聚谷氨酸/脂质复合纳米囊（PGA-PEG/TAT-DSF-LNCs）进行体内外评价。结果表明，脂质纳米囊对DSF具有高载药性，提高DSF稳定性，延长DSF的半衰期；在聚谷氨酸和聚精氨酸作用下，该纳米囊具有肿瘤微环境响应性，增加肿瘤内的蓄积，增强对ALDH高表达细胞的毒性，提高DSF的抗肿瘤效果。. 本课题进一步合成聚己内酯-聚谷氨酸-聚乙二醇聚合物，构建了具有“核-壳-冠”结构的肿瘤靶向给药系统，在此基础上通过疏水作用力和静电作用力分别将DSF装载在聚己内酯疏水内核和阿霉素（DOX）装载在聚谷氨酸中间壳层，实现双硫仑和阿霉素共输送纳米粒（Co-NPs），并对其进行了体内外评价。结果表明，相比于单载DSF，Co-NPs具有肿瘤抑制协同作用，增加抗肿瘤效果。