成纤维细胞激活蛋白响应的肿瘤及肿瘤微环境双靶向给药系统的研究

Cancer-associated fibroblasts (CAFs), the predominant cell type in the tumor stroma, form a major barrier that impedes penetration of antitumor drugs into solid tumor. What’s more, the high interstitial fluid pressure in tumor could hinder antitumor drugs penetrating into central regions of tumor. Therefore, targeting drug delivery to CAFs and improving the tumor penetration of antitumor drugs may be a specific and efficient strategy of targeted cancer therapy. CAFs in almost all solid tumors could selectively overexpressed fibroblast-activated protein-α (FAP-α). Peptides contain the amino acid residue sequence Gly-Pro-Ala (GPA) and Gly-Pro (GP) with a N-terminal acetylation could be specifically cleaved by FAP-α. TAT(AcGP) could be obtained by modifying the ε-NH2 of the forth amino acid (diaminocaproic acid) of TAT (a cell penetrating peptide) with AcGP, and the tumor penetrating peptide iRGD could be converted to GAPiRGD by being modified by GAP. The targeting liposomes are prepared by modifying PEGylated liposomes with TAT(AcGP) and GAPiRGD. After being injected into tumor-bearing mice, the targeting liposomes could release iRGD and the TAT(AcGP) on the surface of liposomes would recover to normal TAT upon FAP-α cleavage in tumor stroma. TAT could promote cellular internalization of liposomes by CAFs and tumor cells, and the free iRGD could improve the tumor penetration of drugs and there carriers. Thus the targeting liposomes in this study could target tumors and their microenvironment. targeting liposomes are prepared by modifying PEGylated liposomes with TAT(AcGP) and GAPiRGD. After being injected into tumor-bearing mice, the targeting liposomes could release iRGD and the TAT(AcGP) on the surface of liposomes would recover to normal TAT upon FAP-α cleavage in tumor stroma. TAT could promote cellular internalization of liposomes by CAFs and tumor cells, and the free iRGD could improve the tumor penetration of drugs and there carriers. Thus the targeting liposomes in this study could target tumors and their microenvironment.

肿瘤相关成纤维细胞是肿瘤微环境最主要的基质细胞和药物传递的主要基质障碍；另外，由于肿瘤的高组织间质液压的存在，药物很难进入肿瘤中心区域。因此，以肿瘤相关成纤维细胞作为治疗靶点并提高药物的肿瘤穿透能力对于肿瘤靶向治疗具有重要意义。肿瘤相关成纤维细胞可高表达成纤维细胞激活蛋白α（FAP-α）。以FAP-α的底物AcGP和GPA分别修饰细胞穿膜肽TAT第四个氨基酸的ε-NH2和肿瘤穿透肽iRGD，可得无穿膜活性的TAT(AcGP)和可断裂的iRGD（GPAiRGD）。TAT(AcGP)和GPAiRGD修饰的脂质体进入肿瘤部位后，在FAP-α作用下，可重新暴露TAT并断裂出iRGD。由于TAT的穿膜作用，脂质体可被肿瘤相关成纤维细胞以及肿瘤细胞高效摄取；另外，游离iRGD可提高脂质体的肿瘤穿透能力，使其进入肿瘤中心区域。最终，本项目的药物传递系统可实现肿瘤及肿瘤微环境双靶向。

实体瘤的肿瘤相关成纤维细胞高表达成纤维细胞激活蛋白α（FAP-α）且FAP-α对含有氨基酸序列Gly-Pro-Ala (GPA)的肽段以及N端乙酰化的Gly-Pro二肽（AcGP）具有选择性酶切作用。基于以上事实，本课题设计了一种肿瘤及肿瘤微环境双靶向的递药系统。首先，以AcGP修饰细胞穿膜肽TAT中两个赖氨酸的ε-氨基得到TAT(AcGP)2，以GPA修饰肿瘤穿透肽iRGD得到GPAiRGD，将上述两种肽对脂质体进行修饰得到靶向脂质体。所制得的靶向脂质体粒径为150 nm左右，PDI小于0.3。在没有FAP-α的环境中，靶向脂质体与普通脂质体的细胞摄取相当，当加入FAP-α后，靶向脂质体的细胞摄取与TAT修饰的脂质体相当，为普通脂质体摄取量的2.28倍。经静脉注射后，空白靶向脂质体可以显著提高与之共给药的药物肿瘤分布和穿透，其作用优于游离的iRGD和通过不可断裂肽链连接在脂质体上的iRGD。同时，靶向脂质体可以显著提高药物清除肿瘤相关成纤维细胞的能力和抗肿瘤的作用，该研究为同时以肿瘤相关成纤维细胞和肿瘤细胞为靶点的肿瘤靶向化疗提供了科学基础。