安全高效的递送系统是肿瘤协同治疗的关键。本项目拟通过融合有膜结合序列PB的Cas9蛋白与提取的肿瘤细胞膜有效结合，解决前期实验发现细胞内Cas9蛋白难以在核定位序列存在下结合到细胞膜上的问题，提高其在细胞膜包被载体中的装载量。然后将结合有Cas9蛋白的肿瘤细胞膜通过超声包被在金纳米棒表面形成纳米颗粒，使其具有同源靶向及免疫逃逸功能，构建出用于肿瘤光热/基因协同治疗的仿生纳米颗粒，并通过体内外实验研究其协同疗效。该系统一方面通过核定位序列使Cas9蛋白入核对凋亡抑制基因survivin进行编辑产生疗效；另一方面利用红外光激发金纳米棒产生光热治疗作用，同时促进Cas9蛋白入核，提高编辑效率，间接实现热休克蛋白表达下调，进而反向提高光热疗效。在此基础上，进一步优化Cas9蛋白上PB与核定位序列的比列，提高Cas9蛋白入核能力。本项目对探索高效低毒的药物载体具有重要理论意义和实用价值。

A safe and efficient delivery system is the key for cancer synergistic therapy. Our previous experiment found that Cas9 protein in cells could not be bound on the cell membrane in the presence of nuclear localization sequence. So, in order to increase the loading capacity of Cas9 in drug delivery system, this project intends to bind the Cas9 protein fused with the membrane location sequence PB to the extracted cancer cell membrane. Then, cancer cell membrane bound with Cas9 is coated on the surface of gold nanorods through ultrasound and form nanoparticles, making nanoparticles have homologous targeting and immune escape function, and cancer cell membrane-biomimetic nanoparticles for cancer photothermal-gene editing synergetic therapy are constructed. Furthermore, the synergistic effect of the biomimetic nanoparticles is verified in vivo and in vitro. On the one hand, Cas9 protein can enter the nucleus through nuclear localization sequence and edit the apoptosis suppressor gene (survivin) to produce therapeutic effect. On the other hand, gold nanorods cause remarkable photothermal therapy under the excitation of NIR (800 nm). Meanwhile, Cas9 protein can be facilitated to enter the nucleus to improve the gene editing efficiency, which indirectly down-regulated the expression of heat shock protein and therefore improve the efficacy of photothermal therapy in return. Based on this, the ratio of PB fused on Cas9 protein to the nuclear localization sequence is optimized to further improve the ability of Cas9 protein enter the nucleus. This project has important theoretical significance and practical value for exploring efficient and low-toxicity drug delivery strategies.