微环境酶触发诱导基质降解释放OncoAd-shPKM2靶向递送系统抑制胰腺癌转移及机制研究

Pancreatic ductal adenocarcinoma remains extremely difficult to treat, owing to the unique pathobiological barriers that restrict drug delivery to tumour cells: a dense desmoplastic stroma, excessive extracellular matrix deposition, increased interstitial fluid pressure, and compression of blood vessels. The limited drug delivery render most treatment options largely ineffective. .A major component of the extracellular matrix is hyaluronic acid (HA), which raises the interstitial gel fluid pressure within tumors and reduces drug delivery to tumour cells. Reduction of extracellular matrix by depleting the acellular stromal components, hyaluronan, can increase tumour perfusion, vascular permeability and improve the homogeneous distribution in the tumour. Although the tumour perfusion can be improved by using hyaluronidase(HAase), the total drug concentration is limited. The advantages of using Oncolytic adenovirus (OncoAd ) in cancer therapy include cancer cell-specific replication , the infection of neighboring cancer cells and the destruction of infected cells, which dramatically increased the OncoAd concentration in tumours. Furthermore, during the replication of OncoAd , the high expression of inserted therapeutic genes could lead to potent antitumour efficacy. Thus, co-delivery (HAase/OncoAd) drug system have the potential to overcome these pathophysiological barriers of pancreatic cancer. .Oncolytic Ad surface modification is required to enhance safety. mPEG and phospholipids have been given US Food and Drug Administration (FDA) approval for use in a variety of biomaterials applications and phospholipids are one of the main ingredients of cell membranes. Calcium phosphate has excellent biocompatibility, biodegradability and very low toxicity. In this work, we designed an enzymatically cleavable lipid/CaP material that was composed of DPPE-MMP substrate peptide-mPEG / DPPE-MMP substrate peptide–HAase /DOPA/CaP and mPEG/HAase was able to be specifically cleaved by MMPs in the extracellular space in tumour tissues. We synthesized RGD-modified oncolytic adenovirus containing shPKM2 (OncoAd-shPKM2) -loaded lipid/CaP Nanoparticles （HAase/ OncoAd-shPKM2@NPs）. The M2 splice isoform of pyruvate kinase (PKM2), an enzyme that catalyzes the later step of glycolysis, is a key regulator of aerobic glycolysis (known as the Warburg effect) in cancer cells. Knockdown of PKM2 can inhibit proliferation, migration and invasion of tumour cells. The target delivery efficiency and perfusion of HAase/ OncoAd-shPKM2@NPs was examined in pancreatic cancer tumour-bearing nude mice. Further, we evaluated the effects of HAase/ OncoAd-shPKM2@NPs on liver metastasis of pancreatic cancer and the underlying mechanism .

针对胰腺癌病理生理屏障影响药物递送的难题，本项目采用共沉淀自组装制备以磷脂/透明质酸酶（HAase）为壳、磷酸钙/溶瘤腺病毒（OncoAd-shPKM2）为核的复合纳米靶向递送系统，该系统通过肿瘤微环境基质金属蛋白酶触发HAase释放，诱导透明质酸降解，促进纳米粒瘤内渗透；胞内磷酸钙介导质子海绵效应，促使OncoAd-shPKM2逃逸溶酶体，增加转染效率；肿瘤细胞内病毒大量复制、表达靶基因，溶解细胞释放大量子病毒，增加瘤内浓度，达到增强抗肿瘤疗效和抑制转移目的。本研究以胰腺癌为模型，研究递送系统稳定性、安全性及长循环能力；探讨纳米粒诱导肿瘤细胞凋亡，抑制肿瘤侵袭、转移机制；以高转移胰腺癌原位瘤为动物模型，从动物整体水平证实递送系统靶向递送功能、瘤内渗透性能、抑制转移机制及疗效。课题的完成将为载基因纳米靶向递送系统的临床应用提供数据参考和理论支持，对富细胞外基质实体瘤的治疗产生积极指导作用。

溶瘤腺病毒疗法被认为是一种有效的肿瘤治疗策略，胰腺癌肿瘤微环境中的病理生理屏障影响包括溶瘤病毒在内的治疗药物递送，极大限制了其临床运用。本课题拟构建一个安全有效肿瘤微环境响应的静脉递送系统，即肿瘤微环境响应钙磷复合磷脂复合纳米靶向递送系统。.本课题构建了完成了肿瘤微环境基质金属蛋白酶响应的纳米粒材料DPPE-PeptideMMP2 cleavable-mPEG2000合成及表征及前期以siRNA为内核构建的钙磷复合基质金属蛋白酶响应的纳米靶向递送系统（(siRNA@NP2），该系统平均粒径为140 nm，电荷为3+0.31 mV，分散性良好，凝胶阻滞试验显示本递送系统能实现有效包载，防止降解。.细胞水平实验，通过荧光显微镜定性及流式细胞术定量实验显示，siRNA@NP2相比单纯非基质金属蛋白酶响应的靶向递送系统能显著提高肿瘤细胞靶向性。对比低表达基质金属蛋白酶肿瘤细胞Huh7,siRNA@NP2在高表达基质金属蛋白酶细胞SMMC-7721中摄取显著增加，结果显示酶敏感纳米递送系统能增强该系统在肿瘤细胞中的靶向性；通过CCK8方法检测结果显示空载纳米粒对细胞活性无明显影响，显示了良好的安全性。动物水平实验，通过小动物活体成像显示微环境响应靶向递送系统改善递送系统分布，有效实现瘤内靶向。