基于粒径智能化调控及程序性释放自噬抑制剂和化疗药物的纳米传递系统的构建及评价

Autophagy is one of the key reasons involved in tumor therapeutic resistance, and autophagy inhibitors can improve the sensitivity of tumor cells to chemotherapeutics. But the non-selectivity, severe side effects and incapability of penetrating into tumor center of both of these two classes of drugs overshadowed their combination therapy. More importantly, antitumor efficacy of the combination of autophagy inhibitors and chemotherapeutics was directly related to their administration sequence. Thus, to simultaneously achieve high specificity, enhanced retention and penetration effect and sequential release in tumor tissues, an enzyme and pH dual-sensitive drug delivery system with smartly changeable particle size was established. The system had a crosslinked core, consisting of PSA with pH sensitivity and corbelled materials, along with a metalloproteinase-2 sensitive gelatin shell which was modified with tumor targeting iNGR. Chemotherapeutics and autophagy inhibitors were respectively entrapped in its core and shell. After the particles reached tumor tissue mediated by iNGR, the shell was degraded by metalloproteinase-2 to release autophagy inhibitor firstly for reducing tumor cells' autophagy level and exposing the core with smaller size for deep tumor penetration. Then core was delivered to endosomes and lysosomes, leading to a reversible swelling-shrinking change in response to outside pH variation for a sequential intra-intercellular system delivery. The swelling of the core resulted in the releasing of chemotherapeutics and the killing of host tumor cells. After escaping from the killed cells, the core could re-enter neighboring tumor cells, killing other tumor cells in a chain-like reaction. In a word, the system designed in this project could simultaneously achieve sequential drug release and deep tumor drug delivery to exert effective and synergistic antitumor therapy.

自噬是导致肿瘤细胞耐药的一种重要因素，自噬抑制剂能提高肿瘤细胞对化疗药物的敏感性。但这两类药物均存在特异性差、毒性较强、无法深入肿瘤深部的缺陷，并且药物作用顺序与疗效有直接关系。为实现高特异性、滞留性、渗透性和程序性释药，本项目拟将具pH敏感性的PSA和支撑材料交联形成粒径随pH可逆变化的小粒径纳米核心，其外包覆基质金属蛋白酶敏感的明胶壳层，并修饰靶向分子iNGR，得到一种酶与pH双敏感的粒径可智能化调控的纳米递药体系。将化疗药物和自噬抑制剂分别包载于体系核心和壳层。iNGR介导体系靶向至肿瘤，壳层酶解释放自噬抑制剂，预先降低肿瘤细胞自噬水平并实现体系粒径变小，以实现肿瘤深部渗透，而后纳米核心进入细胞内涵体与溶酶体，发生pH响应性的可逆膨胀收缩，释放化疗药物杀死其所在细胞，纳米粒逃逸出死亡细胞，继续进入下一个细胞发挥作用。本项目所构建体系能够实现程序性释药和肿瘤深部递送，发挥高效协同治疗。

自噬在肿瘤的生长和转移过程中发挥着至关重要的作用，抑制肿瘤自噬是一种颇具潜力的治疗策略。本项目聚焦于自噬抑制剂的肿瘤深部递送，通过对肿瘤自噬的抑制，增强化疗药物的抗肿瘤效果。.本项目首先从扰乱肿瘤细胞“自噬平衡”的角度出发，通过自噬初期诱导剂联合自噬终端抑制剂实现了肿瘤细胞内的“自噬体堆积效应”。证明“自噬体堆积效应”本身既会抑制肿瘤。随后证明自噬抑制剂可以显著增强化疗药物的抗肿瘤效果，为自噬抑制剂与化疗药物的联用策略提供了理论基础。.对于实体瘤而言，远离血管的深部肿瘤细胞是肿瘤药物耐受、转移和复发的根源地，此区域肿瘤细胞对自噬有着更强的依赖性。为了将自噬抑制剂递送到肿瘤深部区域，我们设计了具有肿瘤深部穿透功能的，且粒径多级变化的“壳-核”结构纳米粒D/PSP@M/GNP。D/PSP@M/GN具有MMP-2酶和pH双响应性，在靶向至肿瘤部位后，其壳层先行被MMP-2酶降解并释放自噬抑制剂，抑制肿瘤细胞自噬，暴露出的纳米核心可以实现肿瘤深层穿透并在逃离溶酶体时释放DOX，杀死自噬受抑制的肿瘤细胞，该体系实现了自噬抑制剂和化疗药物的肿瘤深部递送和程序性释放，达到了最佳的协同治疗效果。.由于D/PSP@M/GNP中自噬抑制剂的释放依赖于肿瘤MMP-2酶的表达，限制了纳米载体普适性，上述系统中自噬抑制剂在肿瘤细胞外释放，不利于在细胞内发挥自噬抑制效果，我们以上述D/PSP为母核，进一步制备了外层为氯喹磷酸钙纳米沉淀的壳核结构纳米粒D/PSP@CQ/CaP。该纳米粒可经由磷酸钙与细胞膜的亲和力而介导入胞，并在溶酶体中释放出氯喹，从而优化了自噬抑制剂的胞内递送。.在达到项目预期研究目的的基础上，本项目进行了进一步拓展研究，考察了自噬对肿瘤转移和肿瘤相关成纤维细胞的调控作用。.在该自然基金的资助下，发表学术论文8篇，包括Autophagy（IF=11.059）2篇，Biomaterials（IF=10.273）1篇，Journal of Controlled Release（IF=7.901）1篇，Acta Biomaterialia（IF=6.638）2篇，International Journal of Pharmaceutics （IF=4.213）1篇，Pharmaceutical Research（IF=3.896）1篇。培养博士生1名和硕士生4名。作学术报告10次。

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