RGERPPR介导的DOX/miRNA靶向递送系统的构建及其对肿瘤异质性作用机制研究

NRP-1 is a receptor overexpressed on both the tumour cells and the tumor endothelium. RGERPPR peptide, is a tumor-penetrating peptide, the specific ligand of NRP-1, is able to penetrate through tumor vessels and tumor tissues. It could act as the carrier of the micellar delivery system for penetrating tumor vessels barrier and matrix to achieve the synergistic antitumor effect. In order to challenge the tumor heterogeneity, we adopt the “RGERPPR mediated targeting” strategy. In our study, the new copolymers: methyl polyethylene glycol-S-S-poly-ε-caprolactone-I-polyethylenimine(mPEG-S-S-PCL-IPEI) and TPGS2K-RGERPPR, that was first synthesized by us, were used to prepare the DOX/miRNA-140 mimics micelle co-delivery system. The study was conducted to verify our hypothesis that, after the intravenous administration, the prepared micelle would specifically bind to NRP-1 and fracture disulfide bond through the reduction effect to reverse the drug releasing process and the process of gene regulation. The aim of this study is to discover a new way of explaining the tumor heterogeneity, such as MDR and gene mutation, and to provide experimental evidence for the study of tumor gene therapy delivery system and the molecular therapy of miRNA.

RGERPPR多肽，是肿瘤细胞和肿瘤血管内皮细胞表面均高表达受体NRP-1特异性配体，具有穿透肿瘤血管壁和肿瘤组织的能力，可携载多功能胶束递送系统突破肿瘤血管屏障和基质屏障，发挥多角度协同抗肿瘤作用。项目基于挑战肿瘤异质性，采用“RGERPPR介导靶向”策略，首次合成共聚物“甲基聚乙二醇-S-S-聚己内酯-线性聚乙烯亚胺(mPEG-S-S-PCL-IPEI)”与“TPGS2K-多肽”，构建一种多功能胶束递送系统，靶向输送化疗药物阿霉素（DOX）和肿瘤抑制基因上调因子miR-140 mimics。以期达到：静脉注射后，该系统在血液中稳定存在，靶向渗入到整个肿瘤组织，在高浓度的GSH作用下，还原敏感释放药物与调控基因，功能性载体（TPGS）发挥逆转多药耐药（MDR）作用。为解决肿瘤MDR和基因突变等异质性问题提供新途径，为肿瘤基因治疗递送系统研究及以miRNA为靶点的肿瘤分子治疗提供实验依据。

项目基于挑战肿瘤异质性，首次合成共聚物“甲基聚乙二醇-S-S-聚己内酯-线性聚乙烯亚胺(mPEG-S-S-PCL-IPEI，PSSPP)”，构建一种多功能胶束递送系统，靶向输送化疗药物阿霉素(DOX)和肿瘤抑制基因上调因子miR-140 mimics。以期达到:静脉注射后，该系统在血液中稳定存在，靶向渗入到整个肿瘤组织，在高浓度的GSH作用下，还原敏感释放药物与调控基因，发挥逆转多药耐药(MDR)作用，为解决肿瘤MDR和基因突变等异质性问题提供途径。成功合成了阳离子基因载体PSSPP2.5K和PSSPP25K，且选出适合的载体PSSPP2.5K进行了有效的基因递送。探究出miR-140-5p与游离阿霉素在MCF-7细胞中可通过降低Wnt、SOX2以及SOX9的表达来协同抑制肿瘤生长与转移。通过琼脂糖凝胶电泳验证其负载基因的能力，采用MTT方法验证两个空白载体胶束对MCF-7的细胞毒性最后进行载体综合性筛选，采用细胞划痕的方法研究其协同抗转移作用，通过Westrn Blot技术研究miR-140-5p与游离阿霉素在MCF-7细胞中对相关信号通路Wnt1、SOX2、SOX9及P-糖蛋白的表达影响。对合成载体的表征分析，通过核磁共振氢普特征峰以及红外光谱特征峰（C-S-：1102cm-1、C=O-：1735cm-1）可判断目的载体合成成功。PSSPP2.5K粒径为160.8nm在细胞内吞粒子的粒径50-200nm范围内，则可有效进入细胞。电镜图显示呈分布均匀且均一的球形状态。琼脂糖凝胶电泳检测出PSSPP2.5K的最佳N/P为18。使用PSSPP2.5K将miR-140-5p递送至体内且与游离阿霉素共同作用后，其MTT结果显示，miR-140-5P + Free Dox组相对于Control组、Free Dox组、Control RNA + Free Dox组具有明显更高的抑制肿瘤生长作用。细胞划痕结果显示miR-140-5P + Free Dox组相对于另外三组，其细胞愈合率显著低于其他组。Western Blot结果显示，miR-140-5P + Free Dox组相对于其他三组可明显降低Wnt1、SOX2以及SOX9在细胞内的表达。