[摘 要] 目的：构建程序性死亡受体1（PD-1）高表达的细菌质膜纳米囊泡（BMV）BMV-PD-1，评估其对小鼠肺癌移植瘤组织 的靶向性。方法：通过质粒转化将PD-1与膜孔蛋白细胞溶素A（ClyA）融合质粒ClyA-PD-1-EGFP转入大肠杆菌BL21 Codonplus，使用激光共聚焦显微镜、SDS-PAGE和WB法检测融合蛋白ClyA-PD-1-EGFP的表达。提取质膜并采用挤出法，利用 挤出器制备BMV-PD-1。采用透射电子显微镜（TEM）、纳米粒子跟踪分析（NTA）技术分别对BMV-PD-1的形态、粒径和膜电位进 行检测，用WB鉴定PD-1蛋白的携带情况。采用激光共聚焦成像检测Lewis肺癌LLC细胞对BMV-PD-1的摄取。建立肺癌LLC 细胞C57BL/6J小鼠皮下移植瘤模型，采用小动物活体成像系统评估BMV-PD-1的肿瘤靶向性。结果：激光共聚焦显微成像结果 显示，质粒ClyA-PD-1-EGFP 被转入BL21-Codonplus 并成功表达蛋白。SDS-PAGE结果表明，ClyA-PD-1-EGFP在BL21 Codonplus中过表达。WB分析表明，PD-1在细菌中表达且在BMV-PD-1上呈高表达（P < 0.001）状态。NTA和TEM分析表明， BMV-PD-1是一种粒径为（145 ± 14） nm、表面呈负电性的球状囊泡。激光共聚焦成像显示，PD-1高表达能显著提升肺癌细胞对 BMV-PD-1的摄取（P < 0.01），小动物活体成像也进一步证实PD-1高表达能有效提升BMV-PD-1的肿瘤靶向性（P < 0.01）。结 论：本研究成功构建了PD-1高表达的细菌纳米囊泡BMV-PD-1，发现PD-1高表达可显著提高BMV-PD-1的肺癌LLC细胞移植瘤 组织的靶向性，为进一步开发以BMV-PD-1为载体的肿瘤靶向药物递送系统奠定基础。

[Abstract] Objective: To construct bacterial cytoplasmic membrane nanovesicles (BMV) with overexpressing programmed death 1 (PD-1), denoted as BMV-PD-1 and evaluate the targeting efficacy of BMV-PD-1 towards transplanted lung tumor tissues in mice. Methods: The fusion plasmid ClyA-PD-1-EGFP fused by PD-1 and Cytolysin A (ClyA) was transferred into Escherichia coli BL21 Codonplus through plasmid transformation. Laser confocal microscopy, SDS-PAGE, and WB were used to detect the expression of the fusion protein ClyA-PD-1-EGFP. Bacterial membranes were extracted and processed with an extruder to generate BMV-PD-1. TEM and NTA were utilized to assess the morphology, size distribution, and zeta potential of BMV-PD-1, while WB was used to verify the presence of PD-1 protein. Laser confocal imaging was conducted to monitor the uptake of BMV-PD-1 by Lewis lung cancer cells. A C57BL/6J mouse subcutaneous transplant tumor model of LLC lung cancer cells was constructed, and the tumor targeting of BMV-PD-1 was evaluated by small animal imaging system. Results: Laser confocal microscopy images demonstrated that the plasmid ClyA-PD-1 EGFP was transferred into BL21-Codonplus and successfully expressed into protein. SDS-PAGE results suggested that ClyA-PD-1 EGFP was overexpressed in BL21-Codonplus. WB analysis indicated that PD-1 was expressed in bacteria and highly expressed in BMV-PD-1 (P < 0.001). NTA and TEM analyses revealed that BMV-PD-1 were spherical vesicles with a diameter of (145 ± 14) nm and a negative surface charge. Laser confocal imaging showed that the high expression of PD-1 significantly increased the uptake of BMV PD-1 by lung cancer cells (P < 0.01). In vivo imaging of small animals further confirmed that the high expression of PD-1 can effectively improve cancer targeting of BMV-PD-1 (P < 0.01). Conclusion: In this study, bacterial plasma membrane nanovesicles BMV-PD-1 with high PD-1 expression are successfully constructed, and it is found that PD-1 overexpression markedly improve the mouse lung cancer xenograft tissue targeting specificity of BMV-PD-1, laying the groundwork for further development of BMV-PD-1 as a carrier for targeted drug delivery systems in tumors.

厦门医学院科技计划项目学校-医院联合资助项目（No. K2023-04）；厦门医学院大学生创新创业训练计划资助项目（No. 202412631001）