靶向Tie2的多肽纳米药物设计及变构增效肿瘤治疗研究

Breast cancer is one of the most common tumor types, and metastasis greatly increases the risk of death from this disease. By studying the process of intravasation or entry of cells into the vasculature, it has been discovered that, in addition to killing tumor cells, chemotherapy treatment can also increase intravasation. Groups of cells collectively known as tumor microenvironment of metastasis can serve as gateways for tumor cells entering the vasculature, including tumor cells, Tie2 associated macrophages (TAMs) and vascular endothelial cells. Vascular reconstruction is a key step for tumor relapse after chemotherapy, and previous studies demonstrated that Tie2 expressed by macrophages was necessary and sufficient to promote the reconstruction of blood vessels after chemotherapy. .Blocking or interfering with the Tie2 signal pathway, either by introducing soluble Tie2 gene, antisense or RNA interference, antibodies or small molecular compounds could effectively inhibit angiogenesis and tumor growth as a result. As far as we are concerned, small molecular inhibitor can be eliminated easily in blood circulation, and systemical usage of Tie2 inhibitor may induce severe vasculature dysfunction. So Tie2 inhibitors are usually used by intratumoral administration. Therefore, it is urgent to develop systemical usage of them that local disturbing Tie2-receptor signaling in TAMs and endothelial cells to inhibit tumor vessel reconstruction and delay tumor relapse after chemotherapy. .A short peptide (sequence: NLLMAAS) could block Tie2 receptor signaling to induce strong anti-tumor and anti-angiogenic effects through stereotactic administration. Moreover, it is water-insoluble, with low bioavailability. The major obstacle to peptide application is the short half-life due to the enzymatic degradation and rapid excretion. Strategies for increasing the stability and activity of peptide drugs are required to improve their therapeutic outcome. Nanostructures with adequate size, shape, and surface properties can improve the pharmacokinetics and bioavailability of drugs and realize combination therapy. Amphiphilic peptides with distinct hydrophobic and hydrophilic segments can self-assemble into particular nanostructures in aqueous solutions. Peptide self-assemblies have been widely reported as nanocarriers to deliver antitumor drugs. Here, inspired by the peptide self-assembly concept, we sought to develop a tumor-responsive nanoformulation for this Tie2 blocking peptide as a building block and other functional moieties as responsive and assembling elements. Such agents would have intrinsic antitumor activity and simultaneously serve as nanomedicine. This peptide nanomedicine would disassemble to locally activate the Tie2 blocking peptide in tumor site. The strategies will be effective to decrease the reconstruction of blood vessels, inhibit the tumor relapse and metastasis after chemotherapy treatment, and reduce side-effects of chemotherapeutic drugs. The nanomedicine might be also potentially applicable for the delivery of a broad spectrum of poorly soluble agents, being able to greatly enhance tumor targeting and drug delivery efficacy, in establishing an efficient treatment of a wide range of solid tumors. Furthermore, our study will give a new insight for precise control of peptide self-assembly. This concept, therefore, has significant implications in understanding the interactions of nanostructures with biological systems and assisting in the nanomedicine construction mediated by peptide self-assembly, with great potential for the development of clinical therapeutics based on nanotechnology.

本项目以化疗后期肿瘤复发和转移率增多的临床问题为导向，选择Tie2为治疗靶点。为解决具有抑制Tie2功能的抗肿瘤活性短肽的体内血液循环时间短、生物利用度差和副作用风险等限制其临床应用的瓶颈问题，基于模块化设计的理念，从抗肿瘤活性肽的结构和功能特性出发，设计并构建对肿瘤微环境生物特性双响应的多肽纳米药物。可提高疏水性抗肿瘤活性肽在血液循环中的稳定性和生物利用度，实现对肿瘤的精准靶向和高特异性解聚变构激活抗肿瘤活性肽。系统研究并揭示多肽纳米药物的模块化设计、组装动力学、理化性质等纳米特性与其肿瘤精准靶向、智能响应、可控变构等集成功能的构效关系。以期通过特异性抑制Tie2功能，减少化疗后血管重建和破裂等异常，降低乳腺癌的复发和转移率，并减少对机体的副作用。从而削弱和有效控制肿瘤的恶性发展，为实现更加高效、长效、安全的肿瘤治疗的关键科学目标奠定实验与理论基础。

化疗是抗肿瘤治疗的主要手段之一,化疗药物触发血管重建与破裂是导致肿瘤复发和转移的关键原因。经化疗后的肿瘤复发血管需要巨噬细胞和内皮细胞表达促血管生成素受体酪氨酸激酶、免疫球蛋白和同源性表皮生长因子-2 (Tie2)。因此，可以靶向并阻断Tie2活性的小分子肽有望成为预防化疗后肿瘤复发的有效手段。在众多具有Tie2抑制功能的抑制剂中，短肽分子NLLMAAS能显著阻断Tie2-ANG 通路，从而显示出强大的抗癌活性。然而，这些小分子肽往往是疏水的，生物利用度很低，会快速地被酶降解从而表现出短的循环半衰期，严重增加对其改造并提高成药性的难度，治疗效果也很差。.基于此，综合考虑短肽分子NLLMAAS的结构和功能特点，以及Tie2在正常血管内皮细胞上的关键作用，本工作设计了一种双响应性的两亲性肽(mPEG1000-K(DEAP)-AAN-NLLMAAS)来修饰Tie2抑制剂T4 (NLLMAAS)，将疏水性短肽（T4）序列共轭上豆荚蛋白酶酶切底物AAN序列，mPEG1000作为亲水端能够减少体内网状内皮系统的内吞来延长体内半衰期，为了尽可能的暴露豆荚蛋白酶的酶切位点，将微酸响应的疏水性分子二乙氨基丙基异硫氰酸酯（DEAP）共轭到两亲性分子上，使其可以在生理条件下自组装构成纳米药物，延长其循环半衰期并使其可特异性靶向肿瘤组织。最终的纳米药物P-T4在酸性肿瘤微环境和肿瘤组织中过表达的豆荚蛋白的共同作用下会释放T4，作用在内皮细胞和巨噬细胞的tie2受体上。该短肽T4将在肿瘤微环境内局部干扰内皮细胞和巨噬细胞上的tie2信号来抑制血管重建进而抑制化疗后复发和转移。与游离的T4相比，P-T4可以更加明显地降低血管密度并延缓化疗后肿瘤的再生，同时也减少了转移肿瘤的形成。