分级激活型聚肽抑制肿瘤细胞自噬及其增强化疗疗效的研究

Autophagy is a tolerance mechanism of tumor cells against chemotherapy. The inhibition of autophagy can enhance the efficacy of chemotherapy. However, it is still a challenge to effectively inhibit autophagy of tumor cells in the tumor microenvironment. We previously developed lysosomal acidity-activated polypeptide, which could potentially inhibit autophagy by destroying lysosomal membrane. But this polypeptide showed low cell uptake by tumor cells due to its zwitterionic property, limiting its efficacy. In this project, we aim to develop grading-activated polypeptides (GAP) that effectively inhibit tumor cells’ autophagy in tumor microenvironment by taking advantage of the special tumor acidic microenvironment (pH 6.8) and lysosome acidic microenvironment (pH 4.5-5.5). We will synthesize cationically helical polypeptides and modify the side-chain amines of polypeptides with 1-cyclohexene-1,2-dicarboxylic anhydride (CDA) and 2-methylmaleic anhydride (MMA), generating pH-sensitive amide bonds that can be cleaved under tumor acidity and lysosomal acidity, respectively. The GAPs can be activated in two steps. At normal tissues (pH 7.4), GAPs would exhibit a nonhelical structure with low cytotoxicity, and their zwitterionic property would prevent them from cell uptake; in the tumor acidic microenvironment (pH 6.8), GAPs would transit from electroneutrality to electropositivity and thus would be easily uptake by cells after the degradation of side-chain amide groups that generated from amines and CDA; in the lysosome (pH 4.5-5.5), GAPs would transit into helical structure after the degradation of amide groups that generated from amines and MMA. After conformational transition, GAPs would exhibit high membrane activity against lysosomal membrane, and thus inhibit the autophagy of tumor cells. We will further study the mechanism of the inhibitory of autophagy by polypeptides and the therapeutic effect of the combination of GAPs and chemotherapy drugs. The project will provide a new strategy for the inhibition of autophagy in tumor cells.

自噬的抑制可有效增强肿瘤化疗疗效，但如何在肿瘤微环境下高效抑制肿瘤细胞自噬仍是重要挑战。项目前期发展了溶酶体酸性环境可激活的聚肽，可通过破坏溶酶体膜抑制细胞自噬，但这类聚肽细胞摄取效率低，限制了其疗效。在此基础上，项目拟利用肿瘤组织及溶酶体酸性环境，发展分级激活型聚肽。使用1-环己烯-1,2-二羧酸酐（CDA）及柠康酸酐（MMA）修饰聚肽侧基氨基，分别生成对肿瘤酸度及溶酶体酸度响应的酰胺键。在中性条件下，聚肽呈低毒的非螺旋结构，细胞摄取效率低。而在肿瘤组织，氨基与CDA反应生成的酰胺键断裂，聚肽转变为正电性，易被细胞摄取；进入细胞溶酶体后，氨基与MMA反应生成的酰胺键断裂，聚肽转变为螺旋结构，破坏溶酶体膜，抑制细胞自噬。项目拟在不同pH下，研究聚肽被细胞摄取情况及破膜活性的激活，及其抑制细胞自噬的机制；在体内外评估聚肽与化疗药物的联合抗癌疗效。项目将为肿瘤组织内细胞自噬的抑制提供新策略。

针对肿瘤耐药问题，我们通过以下两个方面来研究：.1、自噬的抑制可有效增强肿瘤化疗疗效，但如何在肿瘤微环境下高效抑制肿瘤细胞自噬仍是重要挑战。项目利用肿瘤组织及溶酶体酸性环境，发展分级激活型聚肽。使用1-环己烯-1,2-二羧酸酐（CDA）及柠康酸酐（MMA）修饰聚肽侧基氨基，分别生成对肿瘤酸度及溶酶体酸度响应的酰胺键。在中性条件下，聚肽呈低毒的非螺旋结构，细胞摄取效率低。而在肿瘤组织，氨基与CDA反应生成的酰胺键断裂，聚肽转变为正电性，易被细胞摄取；进入细胞溶酶体后，氨基与MMA反应生成的酰胺键断裂，聚肽转变为螺旋结构，破坏溶酶体膜，抑制细胞自噬。项目在不同pH下，研究聚肽被细胞摄取情况及破膜活性的激活，及其抑制细胞自噬的机制；在体外评估聚肽与化疗药物的联合抗癌疗效。项目将为肿瘤组织内细胞自噬的抑制提供新策略。.2、通过破坏细胞膜来诱导细胞死亡可以绕过靶细胞的胞内信号通路，忽略肿瘤细胞耐药和代谢异质性，为治疗耐药肿瘤开辟一种有前景的策略。然而，在肿瘤和正常组织之间细微差异的背景下实现高选择性破膜仍是一个挑战。在这里，我们开发了一种“质子晶体管”纳米洗涤剂（pTNTs），它可以放大和转换肿瘤组织的微小的pH扰动信号，将之转化为破膜活性的信号，用于选择性癌症治疗。我们筛选得到了性能最好的pTNT P(C6-Bn20)在0.1 pH转变条件下，其细胞毒性变化可达32倍。P(C6-Bn20)由聚乙二醇（PEG）和pH响应的破膜区域组成（叔胺段（乙基哌啶（C6））和疏水段（苄基））。在生理pH值（pH 7.4）下，P(C6-Bn20)自组装成中性纳米粒子，其疏水内核被PEG壳屏蔽，为无活性状态，在800 μg/mL孵育24小时后表现出最小的毒性。在肿瘤酸度下，C6质子化的急剧转变诱导P(C6-Bn20)转变为具有活性的阳离子纳米颗粒，从而产生了强大的破膜活性。P(C6-Bn20)在小鼠肿瘤模型显示出很高的抗肿瘤疗效，并在小鼠中具有良好的耐受性。因而我们的破膜材料设计将会是一种有前途的选择性癌症治疗策略。

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