基于乳腺癌氨基酸转运体LAT1和ATB0,+高表达和表达异质性的双靶向纳米药物传递系统的构建和评价

More and more attentions are focused on developing new targets for tumor treatment because the types of receptors expressed in tumor cells are limited and the ligands are poorly stable in vivo. In comparison, simple amino acid modifications are enough to satisfy amino acid transporters targeting. Besides, as breast cancer is a herogenous disease, single-ligand modified nanoparticles are unable to effectively delivery to the full spectrum of breast cancer cells, resulting in unsatisfied antitumor efficacy. Herein, to overcome heterogeneity of breast cancers and improve the antitumor efficiency, LAT1 and ATB0,+ overexpressed in breast cancer cells are selected as targets, then amino acid modified nanoparticles are developed to target the two amino acid transporters (LAT1 and ATB0,+). The qualitative-quantitative relationship between the targeting nanoparticles and targeting efficiency/cytotoxicity are established to optimize substrate and density on the surface nanoparticles. Thereafter, tumor microenvironment pH sensitive dual ligand modified nanoparticles are further designed to improve anticancer efficacy and reduce adverse effects. The activity regulation of LAT1 and ATB0,+ is then studied to illustrate the intracellular fate of the transporters after interaction with the active targeting nanoparticles. The results are meaningful for design of transporters- and tumor heterogeneity-mediated drug delivery systems.

目前可供乳腺癌主动靶向纳米粒选择的受体种类较少，且其配体具有分子量大和体内稳定性差等问题，寻找新的有效靶点是肿瘤主动靶向纳米粒设计中亟待解决的难点。乳腺癌细胞表面特异性高表达氨基酸转运体，简单的氨基酸修饰即可达到靶向目的。此外由于肿瘤异质性，单一配体修饰的纳米粒很难递送到所有肿瘤细胞亚群，导致治疗效果不佳。本项目以乳腺癌高表达的氨基酸转运体LAT1和ATB0,+为靶点，构建双配体修饰的靶向纳米粒，通过建立纳米结构-细胞摄取/活性的定量关系，筛选出具有最优传递效果的双靶向纳米粒。在此基础上，构建智能型“配体包埋式”主动靶向纳米粒，以克服转运体在机体其他部位少量表达所导致的非特异性分布；同时，从蛋白、基因等水平研究靶向纳米粒对转运体活性的调控行为和机制，以主动调控转运体的活性，并解析转运体在介导纳米粒内吞过程中的作用。本课题的研究成果对基于药物转运体和肿瘤异质性的靶向制剂研发具有指导作用。

肿瘤细胞快速增殖对营养物质的需求大大提高，与正常细胞相比，癌细胞膜表面的氨基酸转运体表达量会显著提高，为肿瘤主动靶向提供了良好的靶点。LAT1（SLC7A5）是一种不依赖Na+/Cl-，转运分子量较大的中性氨基酸为主的交换型氨基酸转运体。ATB0,+（SLC6A14）是一种依赖于Na+/Cl-，转运底物很广的单向型氨基酸转运体。这两种氨基酸转运体在乳腺癌、胰腺癌、结肠癌等癌症中均有较高程度的表达上调。但是肿瘤是一种动态的复杂的疾病，单一配体修饰的单靶向制剂很难有效的治疗癌症。而且在正常细胞中也有少量LAT1与ATB0,+转运体的表达，传统的“配体外露式”靶向制剂对正常细胞有潜在的毒性。基于此，本课题设计并合成了LAT1与ATB0,+单靶向和双靶向主动包载伊立替康脂质体并通过细胞水平、动物水平实验筛选出最优的双靶向配体——酪氨酸。接着又设计合成了一种pH敏感的“配体包埋式”双靶向脂质体，该脂质体能同时靶向于LAT1与ATB0,+氨基酸转运体且具有智能响应性，即在正常组织pH7.4的环境中配体被包埋在PEG水化层内部从而阻止了配体与正常细胞受体的识别，在pH6.5的肿瘤微环境中pH敏感键断裂脱去PEG水化层从而暴露出靶头，配体可以与癌细胞受体进行识别结合，并通过体外水平、细胞水平和动物水平一系列实验表征了这种智能响应型脂质体具有最优的抗肿瘤效果和良好的安全性。

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