具有可控膜穿透和定位壳脱落功能的阳离子半胱氨酸共聚物载体的研究

To improve the tumor penetration of nanocarriers and reveal the mechanism of interaction between the hierarchical structures of nanovehicles and tumor cells, a class of cationic L-cysteine-based copolymers with controlled membrane activity and on-demand shell-detachment ability were constructed via multiple click reactions. The proton buffering capacity, hydrophobic characteristics, and secondary and tertiary structures of copolymers were controlled by the quaterisation and thioether alkylation of side chains. The hierarchical self-assembly of copolymers were characterized using both computational and experimental techniques. Thereafter, cell experiments were carried out to demonstrate the key role in the penetrability of nanoassemblies during cellular uptake and endosomal escape. Furthermore, a series of poly(ethylene glycol) amines with different pH-sensitivity were designed and synthesized, for the further preparation of a novel cationic nanocarrier capable of shedding its outer corona under extracellular and intracellular environments. The synergistic effect of shell-detachment at various locations on the cell internalization of nanoparticles was investigated. In addition, the nanovehicles were loaded with antitumor drugs or RNA as model therapeutics to verify the controllable penetrability and therapeutic effects of nanoformulations against drug resistant tumors in vitro and in vivo, and to systematically address the fundamental scientific issues between the chemical and assembled structures of polymeric nanocarriers and their tumor penetrating capacity. This work provides guidance and a new strategy for the design of effective intracellular drug delivery systems, and offers a promising nanovehicle for the clinical treatment of cancer, particularly drug-resistant tumors.

为了提高纳米载体的肿瘤渗透性能，揭示其多级结构与细胞之间相互作用的机理，本课题通过多重点击等化学手段构筑一系列具有可控膜穿透和定位壳脱落功能的阳离子半胱氨酸共聚物。利用季铵化和硫醚烷基化调控聚合物的质子缓冲能力、疏水性质和高级结构，使用计算机模拟和实验表征其多级自组装行为，通过细胞实验研究纳米载体在细胞摄取和内体逃逸过程中发挥膜穿透作用的关键因素。在此基础上，使用酸敏感性不同的聚乙二醇胺实现阳离子载体在肿瘤细胞外和细胞内定位脱壳的能力，并探索其在病灶区域不同位置壳脱落行为对细胞内在化的协同作用机理。进一步以抗癌药物或RNA为模型，证明纳米给药系统在体外和动物体内对耐药肿瘤的可控穿透性能和治疗效果，阐明聚合物载体化学结构和多级组装结构与体内外肿瘤穿透性之间的基本科学问题，为高效细胞内药物传递系统的设计提供理论指导和新思路，并为恶性肿瘤，特别是耐药肿瘤的临床化疗提供一种具有应用潜力的纳米载体。

为了提高纳米载体与肿瘤之间的特异性相互作用，揭示高分子多级结构与其自组装及生物学效应之间的科学关系，本课题首先在聚半胱氨酸侧链引入高疏水性胆固醇锁住高级构象，利用活性氧（ROS）作为“门控”触发高分子二级结构由β折叠转变为α螺旋构象，并驱动聚合物自组装体形貌由胶束转变为双分子层囊泡。该聚合物的结构转变赋予其自组装体独特而灵敏的构象功能开关，能够实现良好的ROS响应控释能力，并显示出构象介导的细胞膜相互作用和特异性的高效入胞能力，从而在体内及体外抗肿瘤实验中表现出良好的治疗效果。为了提高自组装体的膜穿透性能并实现功能化，利用硫醚烷基化点击反应在两亲性聚半胱氨酸侧链修饰不同含量的阳离子季铵盐基团，获得具有阳离子α螺旋构象的多孔纳米自组装结构。进一步将此策略拓展到具有α螺旋倾向的阳离子聚赖氨酸体系，通过稳定的酰胺键、pH敏感希夫碱键和静电复合作用在聚赖氨酸侧链修饰胆固醇、肉桂醛和藤黄酸等疏水基团或药物分子，发现疏水侧基的引入显著改变了聚赖氨酸的构象、高级自组装结构及其与细胞之间的相互作用。利用偶联反应改变主链嵌段结构，利用Michael反应对聚赖氨酸侧链进行修饰，合成了系列具有不同质子缓冲能力和二级构象的阳离子聚合物。为了解决隐形和膜活性之间的矛盾，通过阳离子、靶向分子等表面工程原件构筑功能化纳米界面，并利用细胞外微环境触发的聚乙二醇（PEG）外壳开关来适时屏蔽和暴露出活性表面，从而协同增强聚合物纳米粒子与肿瘤细胞相互作用的特异性和高效性。为了进一步提高刺激响应性的时空控制效果，合成系列光敏感壳脱落和自降解的聚合物体系，并研究其光响应降解行为、控制释放及其对细胞内药物传递的影响。本项目研究对理解蛋白质折叠和大分子多级自组装具有重要的理论价值，为高分子多级自组装结构和功能的调控提供了新策略，为克服细胞膜屏障提供了新的解决思路，并在生物传感、疾病治疗和诊断应用方面具有广阔的应用前景。

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