靶向/促穿膜双功能肽RGDC-TAT介导的肿瘤靶向纳米基因递送系统的构建及其抗肿瘤作用研究

The transfer of gene therapy has been proven difficulty due to the three key problems of gene delivery system, such as the contradiction between transfection efficiency and cytotoxicity, the contradiction between transfection efficiency and the ability of promoting transmembrane, as well as the poor targeting specificity. So in this study, we intend to improve the performance of targeting specificity and penetrating cellular membranes on the basis of gene carrier-Pluronic modified polyethylenimine (Pluronic-PEI), which has high transfection efficiency and low toxicity. The dual-functional peptide (RGDC-TAT) is synthesized to be utilized as the target head. As the RGD peptide could specific binding to the integrin αvβ3 that is expressed highly on tumor new-blood vessels, and the TAT peptide has the effect of promoting the transmembrane abilty of carrier, so the RGDC-TAT has the dual function of active targeting and promoting transmembrane. Afterwards, the dual-functional peptide (RGDC-TAT) is to be coupled to Pluronic-PEI by crosslinking technology, to construct a tumor targeting nano-gene delivery system (RGDC-TAT-.Pluronic-PEI) with high efficiency, low toxicity, well security and stability. Finally, we would like to take the p53 gene with tumor suppressor effect as model drug, to self-assembly into nano drug delivery systems (p53/RGDC-TAT- Pluronic- PEI) with RGDC-TAT-Pluronic-PEI, and evaluate its targeting effect , anti-tumor efficacy as well as safety in vitro and in vivo thoroughly, and also will take a preliminary study on its transmembrane, intracellular transporter and release mechanism. Neither the study on construction of such a novel gene targeting delivery system nor its mechanism of action has been reported at home or abroad. This topic could provides a new research route for the gene targeting therapy of tumor, and provide experimental information and theoretical basis for the design of targeted non-viral gene delivery systems for tumor gene therapy.

针对基因递送系统中存在的三个关键问题：转染效率与细胞毒性相矛盾、稳定性与穿膜能力相矛盾以及靶向性差。本课题拟在构建Pluronic修饰的高效、低毒基因载体Pluronic-PEI基础上，进一步解决靶向及穿膜能力问题，将可与肿瘤新生血管高表达的整合素αvβ3特异结合的RGD短肽，与具促载体穿膜作用的TAT肽连接，合成兼具靶向/促穿膜作用的双功能肽RGDC-TAT，并利用交联技术将其与Pluronic-PEI偶联，构建一种高效、低毒、安全、稳定的肿瘤靶向纳米基因递送系统。然后与具抑癌作用的p53基因自装配成纳米载药系统(p53/RGDC-TAT-Pluronic-PEI)，对其靶向性及抗肿瘤效果和安全性进行体内外评价，并研究其穿膜、胞内转运、释放机制。该新型基因递送系统的构建及作用机制研究国内外尚未见报道。可为肿瘤的基因靶向治疗提供研究方法和思路，并为靶向非病毒基因递送系统设计提供资料和依据。

本研究针对基因递送系统存在的三个突出问题：转染效率与细胞毒性相矛盾、体液环境中的稳定性问题与穿膜能力相矛盾以及靶向性差，在前期研究基础上，采用以下策略构建得到一种兼具高转染效率、肿瘤细胞和组织的特异靶向性及良好稳定性和安全性的转基因载药系统(Pluronic-PEI-DR5-TAT)并进行相应评价。即先采用不同EO/PO比的Pluronic连接低分子量PEI (LMW-PEI)，筛选得到高效、低毒的基因载体材料（Pluronic-PEI）。然后DR5受体在人多数肿瘤细胞表面高表达的特点，选择特异亲和该DR5受体并可有效诱导肿瘤细胞凋亡的的DR5短肽，与细胞穿膜肽TAT连接，合成具有靶向于肿瘤细胞和促载体穿膜作用的双功能肽DR5-TAT，再利用交联技术将DR5-TAT与Pluronic-PEI偶联，构建一种新型肿瘤靶向纳米基因递送系统(Pluronic-PEI-DR5-TAT)，并对其结构特征和生物物理性能进行表征。然后将其与模型基因NC-miRNA自装配成抗恶性黑素瘤靶向纳米基因载药系统(Pluronic-PEI-DR5-TAT/NC-miRNA)，对其转染效率、靶向性及抗肿瘤效果进行体内外评价，并对该系统的细胞穿膜机制进行初步研究。研究结果表明， Pluronic-PEI-DR5-TAT具有良好靶向性、良好的DNA缩合和保护能力，进入细胞后又可及时降解释放DNA，N / P比为15的Pluronic-PEI-DR5-TAT/DNA复合物为球形的纳米粒，平均粒径在122 ± 11.6 左右nm，Zeta电位约为22 ±2.8 mV。体外生物性能评价结果表明，Pluronic-PEI-DR5-TAT具有高转染效率的同时还可大大降低对正常细胞的毒性，有望成为肿瘤靶向基因递送系统的理想载体。在此基础上所构建的Pluronic-PEI-DR5-TAT/NC-miRNA恶性黑素瘤纳米主动靶向给药系统，具有显著优越性，较其他给药系统而言，可显著提高miRAN对恶性黑素瘤的治疗效果并降低毒副作用，有望成为黑素瘤靶向基因治疗理想递药系统，并进一步应用于临床治疗恶性黑素瘤，为恶性黑素瘤的治疗提供新的方法，也可为今后靶向非病毒基因递药系统的设计提供试验资料和理论依据。