基于肿瘤微环境响应的多级纳米载体构建及其肿瘤渗透性的机制研究

The main obstacle for cancer chemotherapy is the poor solid tumor permeability which blocks the penetration of the conventional nanocarrier into the internal tumor. The smaller size nanocarrier and penetration promoting factor of tumor is a promising approach to overcome the barrier. However, the smaller nanocarrier tends to be cleared in the blood circulation. Our research team previously prepared a gelatin nanoparticle that is degraded by matrix metalloproteinase (MMP-2) in the tumor tissue. Based on this observation, we plan to construct a tumor-microenvironment-responsive multistage nanocarrier, which allows us to prepare the gelatin nanoparticles (GNPs) loading epirubicin micelles and penetration promoting factor hyaluronidase (HAase) and gelatin modified with iRGD for enhancement of the permeability of tumor vessel as the polymer carrier. The larger size GNPs will have better stability in the blood circulation, and the smaller size epirubicin micelles and HAase will be released to easily penetrate into the internal tumor following the degradation of gelatin by the MMP-2 in the tumor tissue. KPAE will be degraded into small molecules at pH5 in lysosome, and the high osmotic pressure in lysosome caused by small molecules will lead to the burst of lysosome, thus the epirubicin will be released from the micelles and lysosome to cytoplasm. The influence of the multistage nanocarrier on the penetration promoting effects and histophysiological characters of tumor will be investigated at the levels of molecular, cellular and animal model. We will focus on the mechanism of the nanocarrier for the enhancement of tumor penetration ability and targeting to internal tumor cells. It is expected to establish an experimental and theoretical basis for the exploitation of nano-delivery system and the clinical therapy of cancer with high efficiency.

实体瘤组织的低渗透性使得常规纳米载体难以进入肿瘤内部，严重影响抗肿瘤纳米制剂的功效。纳米载体的粒径和实体瘤细胞间坚实的细胞外基质、较高的内流体压力是影响药物瘤内渗透的关键因素。在前期研究基础上，本课题拟构建一种肿瘤组织高渗透性的多级纳米载体系统（MGNPs），由聚己内酯-缩酮聚氨基酯（PCL-KPAE）胶束、iRGD修饰的明胶纳米粒（iRGD-GNPs）和透明质酸酶（HAase）通过静电作用组装构成。其优势特点是：HAase可降解肿瘤细胞外基质从而降低内流体压力；GNPs在血液中稳定且可被肿瘤组织丰富的MMP-2降解，释放出小粒径的PCL-KPAE胶束易于进入实体瘤内部；KPAE遇酸降解导致溶酶体胀破使得表柔比星释放，发挥治疗功效。本课题将在分子、细胞和动物水平上，系统研究MGNPs肿瘤渗透作用的影响因素，探索促进纳米载体肿瘤渗透性的有效途径，为抗肿瘤靶向纳米制剂研究提供实验和理论依据。

肿瘤靶向的纳米载体药物是治疗实体瘤的手段之一，可以提高药物在肿瘤组织和肿瘤细胞的分布。然而实体瘤由于坚实的细胞外基质以及较高的肿瘤内流体压力，限制了药物向实体瘤内部的渗透。为解决向实体瘤内部持续输送纳米药物的问题，本研究制备了一种肿瘤微环境响应的多级纳米载体，用以靶向递送抗肿瘤药物表柔比星，其特征为：通过自由基加成断裂链转移(RAFT)聚合反应合成两亲性三嵌段聚合物：mPEG-PDPA-PG（PEDG），其与表柔比星（EPI）形成载药胶束（NPs-EPI）；再以促渗透肽iRGD修饰的明胶 (G-iRGD) 作为载体，通过静电作用与载药胶束和透明质酸酶（HAase）形成多级纳米粒（MGNPs）。.NPs-EPI呈规则的球型，粒径85.10.2nm,电位39.90.6mv；EPI的载药量及包封率分别为4.4%和31.7%；NPs-EPI在pH为7.4和5.5的磷酸缓冲液中累积释放率分别为50%和82%，呈现pH依赖性；MTT结果表明NPs/HAase/G-iRGD无明显细胞毒性；体外肿瘤球渗透性实验以及体内肿瘤冷冻切片结果表明HAase可以降解细胞外基质的透明质酸；胞内分布实验结果表明随着NPs-EPI给药时间的延长，NPs-EPI可以从溶酶体逃逸；流式结果表明NPs-EPI/G-iRGD比NPs-EPI/G更容易被HepG2所摄取，表明iRGD具有一定的主动靶向作用；透射电镜以及流式细胞仪结果证实NPs-EPI主要采用网格蛋白和巨胞饮介导的内吞途径入胞，继而被转运至溶酶体；活体成像结果表明NPs-EPI/G-iRGD相比NPs-EPI、NPs-EPI/G随着给药时间延长在肿瘤内有明显富集；体外3D肿瘤球模型并激光共聚焦显微镜层扫，结果显示随着HAase浓度的增加，NPs-EPI的渗透逐渐增强；此外，NPs-EPI复合低浓度的HAase处理肿瘤球并不显著影响肿瘤球的结构，而复合高浓度HAase肿瘤球结构变得松散；分别用NPs和NPs/HAase处理肿瘤球，标记ECM中的HA，证实HAase可有效降解ECM中的HA增强NPs的渗透效力；NPs-EPI/HAase在降解HA之后，药物能够进入实体瘤抑制肿瘤生长，同时HA的降解还可以增加肿瘤血管密度并诱导肿瘤血管正常化。.本研究所制备的多级纳米载体能够有效的将EPI递送到实体瘤内部，提高实体瘤低渗透性从而发挥抗肿瘤作用。