[摘 要] 目的：开发新型溶瘤呼肠孤病毒（Reo）递送系统，以克服中和抗体对Reo的中和作用并提升其肿瘤靶向性。方法： 通过切向流过滤联合超高速离心法制备自然杀伤细胞外泌体（NKexo），叶酸（FA）修饰后采用挤压法包载Reo，构建FA-NKexoReo递送系统；通过透射电镜（TEM）、纳米粒径分析、蛋白质印迹（WB）法、核磁共振氢谱及流式细胞术等技术表征其理化性质； 采用CCK-8、流式细胞术、Transwell实验及激光共聚焦显微镜评估FA-NKexo-Reo递送系统体外细胞毒性及细胞摄取能力；通过 人卵巢癌裸鼠皮下移植瘤模型评价 FA-NKexo-Reo 的肿瘤靶向性、疗效及安全性。结果：FA-NKexo-Reo 粒径为（94.0 ± 28.5） nm，Zeta 电位为（-21.26 ± 1.57）mV，包封率达（49.7 ± 15.6）%；在中和抗体的存在下，FA-NKexo-Reo 对卵巢癌细胞 SKOV3 和 A2780仍可表现出显著的细胞毒性（P < 0.01）；荷瘤鼠活体成像显示FA-NKexo-Reo肿瘤靶向性显著优于NKexo组，肿瘤抑制率 提升60%（P < 0.001）。结论：成功制备FA-NKexo-Reo递送系统，在中和抗体的存在下，FA-NKexo-Reo可保护并靶向递送Reo到 高表达叶酸受体的卵巢癌细胞，从而增强Reo的抗肿瘤作用。

[Abstract] Objective: To develop a novel delivery system for oncolytic reovirus (Reo) to circumvent neutralization by anti-Reo antibodies and enhance tumor-targeting efficiency. Methods: Natural killer cell-derived exosomes (NKexo) were prepared via tangential flow filtration combined with ultracentrifugation. After folic acid (FA) modification, Reo was encapsulated into NKexo using an extrusion method to construct the FA-NKexo-Reo delivery system. The physicochemical properties of FA-NKexo-Reo were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis, Western blot, proton nuclear magnetic resonance spectroscopy, and flow cytometry. The in vitro cytotoxicity and cellular uptake of FA-NKexo-Reo was evaluated using CCK-8 assays, flow cytometry, Transwell assays, and confocal laser microscopy. A human ovarian cancer xenograft model in nude mice was established to assess the tumor-targeting capability, therapeutic efficacy, and treatment safety of FA-NKexo-Reo. Results: FA-NKexoReo exhibited an average particle size of (94.0 ± 28.5) nm and a zeta potential of (?21.26 ± 1.57) mV, with an encapsulation efficiency of (49.7 ± 15.6)% . In the presence of neutralizing antibodies, FA-NKexo-Reo retained significant cytotoxicity against SKOV3 and A2780 ovarian cancer cells (P < 0.01). In vivo fluorescence imaging demonstrated superior tumor-targeting capability of FA-NKexo-Reo compared to NKexo, with a 60% increase in tumor suppression rate (P < 0.001). Conclusion: The FA-NKexo-Reo delivery system was successfully prepared. In the presence of neutralizing antibodies, FA-NKexo-Reo effectively protects and selectively delivers Reo to ovarian cancer cells with high folate receptor expression, significantly enhancing the antitumor efficacy of Reo.

[基金项目] 国家自然科学基金（82060564，82460604）；贵州省科技厅重点科技计划[黔科合基础-ZK（2021）012号]；贵州医科 大学肿瘤免疫治疗技术工程研究中心项目[校工程中心，2024（001）]；贵州省卫生健康委员会科技基金（gzwkj2022-087）