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Archive > Volume 32 Issue 3 > 2025,32(3):239-246. DOI:10.3872/j.issn.1007-385X.2025.03.002 Prev Next
Basic Research
Construction of PD-1 overexpressing bacterial cytoplasmic membrane vesicles and evaluation of its targeting efficacy of mouse lung cancer xenograft tissue
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Abstract:
[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.
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