微环境响应胞内质子介导释放ZD55-IL-24纳米靶向递送系统抑制肝癌转移作用及机制研究

Hepatocellular carcinoma(HCC) is the fifth prevalent malignant tumor , which is ranked the third of tumor-related death in the world. Numerous approaches, including surgery, chemotherapy, hormonal therapy, immunotherapy, gene therapy, and radiation therapy, have been developed for treating HCC. However, the long-term survival rate of HCC patients remains far from satisfactory. .Therefore, novel therapeutic approaches are desperately needed. One gene therapy approach that has been extensively investigated in cancer is oncolytic adenovirus (Ad) therapy. At supra-physiological levels, MDA-7/IL-24 plays a prominent role in inhibiting tumor growth , invasion, metastasis and angiogenesis. By inserting the interleukin-24 (IL-24) into the oncolytic adenovirus (OncoAd), here is ZD55-IL-24, has much higher anti-tumor effect than that of Ad-IL-24 in vitro, also higher anti-tumor effect than that of ZD55 (OV) . Although the therapeutic efficacy of oncolytic Ad vectors has been successfully and dramatically increased, in vivo application of oncolytic Ad vectors is still limited to local injection, because Ad vectors injected systemically cause acute accumulation in the liver, as well as short blood circulation time. Furthermore, tumor cells have low coxsackie and Ad receptor (CAR) expression, limiting the amount of CAR-mediated tumor cell transduction. Thus, successful cancer gene therapy strategies need to be devised to efficiently deliver Ads systemically to avoid immune clearance, non-specific liver uptake, and poor CAR-mediated viral transduction in a various cancer cell types. Oncolytic Ad surface modification is thus required to enhance safety and therapeutic efficacy. 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-RGD/DOPA/CaP and mPEG was able to be specifically cleaved by MMPs in the extracellular space in tumor tissues. We synthesized ZD55-IL-24-loaded lipid/CaP Nanoparticles and further evaluate the effect of ZD55-IL-24-loaded lipid/CaP Nanoparticles on lung metastasis of HCC and study the underlying mechanisms.

针对溶瘤腺病毒难于直接静脉给药的难题, 本项目选用FDA批准用于人体的经典材料，构建肿瘤微环境响应胞内质子介导释放载IL-24基因溶瘤腺病毒（ZD55-IL-24）的纳米靶向递送系统，研究其静脉注射用于基因治疗的可行性。通过共沉淀自组装法制备磷脂/磷酸钙复合组装载ZD55-IL-24纳米粒，以高转移肝癌为研究模型，研究递送系统的体内外稳定性、安全性；探讨纳米粒组成、结构和理化特性的改变对其入胞过程及胞内运转途径的影响；揭示微环境响应胞内质子介导释放ZD55-IL-24纳米靶向递送系统诱导肿瘤细胞凋亡，抑制肿瘤侵袭、转移机制。本项目研究的主要目的是改善并提高ZD55-IL-24体内肿瘤靶向性，降低免疫原性，通过静脉注射常规给药途径实现靶向给药基因治疗原位与转移瘤。本课题的完成将证实构建递送系统的递送功能和治疗效果，为该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。

溶瘤腺病毒疗法被认为是一种有效的肿瘤治疗策略。目前溶瘤腺病毒仅局限于局部给药，极大地限制了其临床应用。本课题拟构建一个安全有效的溶瘤腺病毒静脉递送系统，即钙磷复合磷脂载“武装”治疗基因的溶瘤腺病毒递送系统。.本课题构建了钙磷复合磷脂载溶瘤腺病毒纳米递送系统，平均粒径为121 nm，电荷为-10.1 mV，分散性良好。性能评价结果显示，PLC-ZD55-IL-24在4℃储存条件下，纳米粒可稳定10 d；血清稳定性检测表明其可以抵抗血清诱导的凝集，延长体内循环时间；静脉递送PLC-ZD55-IL-24后， BALB/c小鼠体内血清白介素-6（IL-6）水平、血清丙氨酸转氨酶（ALT）， 天冬氨酸转氨酶（AST）水平无显著增高，病理分析显示小鼠肝组织无明显肝细胞破坏，且可抵抗小鼠体内预存中和抗体中和作用，在预免疫的荷Hepa1-6细胞移植瘤小鼠体内实现瘤内靶向。.细胞水平实验显示，PLC-ZD55-IL-24 通过抑制Huh-7肿瘤细胞增殖、诱导凋亡发挥抗肿瘤作用。在Huh-7肝癌移植瘤裸鼠体内静脉注射PLC-ZD55-IL-24后， 通过实时荧光定量qPCR 方法检测病毒基因拷贝数显示PLC-ZD55-IL-24注射组显著减少了肝、脾等单核吞噬系统内的病毒基因拷贝数，延长了病毒血液循环时间、实现了有效的瘤内靶向。离体组织荧光成像分析进一步验证了该递送系统的靶向效率。此外，增加给药剂量可增加瘤内靶向效率而对瘤/肝比无显著影响。.Huh-7肝癌移植瘤裸鼠经PLC-ZD55-IL-24治疗后，瘤内目的基因IL-24的mRNA及蛋白表达水平明显高于ZD55-IL-24治疗组，高、低剂量治疗组抑瘤率分别为54.62%、37.40%，提示增加PLC-ZD55-IL-24治疗剂量可显著增加其抗肿瘤疗效。治疗终点血清生化检测及重要脏器切片HE染色结果未见明显脏器功能损害。