纳米级革兰氏阴性菌分泌的细胞外囊泡（OMVs）因其免疫刺激性能，已成为一种新型的抗肿瘤纳米医学试剂。通过在产生OMVs的菌株上操作生物工程技术来编辑OMVs中的细菌成分，我们可以设计一个巧妙的抗肿瘤平台，将Polybia-mastoparan I（MPI）融合肽装载到OMVs中。通过对重组质粒转化的工程大肠杆菌进行操作，获得了含有MPI融合肽的OMVs。通过对MB49和UMUC3细胞分别进行细胞存活、创口愈合和凋亡实验，验证了生物工程OMVs的抗肿瘤功效。使用皮下MB49肿瘤小鼠进行研究，以调查生物工程OMVs的肿瘤抑制能力。此外，详细评估了肿瘤中的激活免疫反应和生物安全性。结果表明，OMVs成功地封装了MPI融合肽，并进行了形态、大小和Zeta电位的物理表征。当与生物工程OMVs一起孵育时，膀胱癌细胞（包括MB49和UMUC3）的细胞存活率下降，而非癌性细胞系(bEnd.3)的细胞存活率没有下降。此外，生物工程OMVs抑制了癌细胞的迁移并诱导了其凋亡。通过皮内注射生物工程OMVs，显著抑制了皮下MB49肿瘤的生长。OMV的先天免疫刺激被证明可以触发树突状细胞(DCs)的成熟、巨噬细胞的招募和细胞毒性T淋巴细胞(CTLs)的浸润，从而增加了促炎细胞因子(IL-6，TNF-α和IFN-γ)的分泌。同时，多种证据也表明，生物工程OMVs具有令人满意的生物安全性。本研究建立的生物工程OMVs具有强烈的膀胱癌抑制和良好的生物相容性，为临床治疗膀胱癌提供了一条新途径。版权所有©2023 Ren，Li，Cong，Li，Xie和Wu。

Nanosized bacterial outer membrane vesicles (OMVs) secreted by Gram-negative bacteria have emerged as a novel antitumor nanomedicine reagent due to their immunostimulatory properties. The encapsulated bacterial composition in OMVs can be edited via manipulating bioengineering technology on paternal bacteria, allowing us to design an ingenious antitumor platform by loading the Polybia-mastoparan I (MPI) fusion peptide into OMVs.OMVs containing the MPI fusion peptide were obtained from bioengineered Escherichia coli transformed with recombinant plasmid. The antitumor efficacy of bioengineered OMVs in vitro was verified by performing cell viability and wound-healing and apoptosis assays using MB49 and UMUC3 cells, respectively. Subcutaneous MB49 tumor-bearing mice were involved to investigate the tumor inhibition ability of bioengineered OMVs. Moreover, the activated immune response in tumor and the biosafety were also evaluated in detail.The resulting OMVs had the successful encapsulation of MPI fusion peptides and were subjected to physical characterization for morphology, size, and zeta potential. Cell viabilities of bladder cancer cells including MB49 and UMUC3 rather than a non-carcinomatous cell line (bEnd.3) were decreased when incubated with bioengineered OMVs. In addition, bioengineered OMVs restrained migration and induced apoptosis of bladder cancer cells. With intratumor injection of bioengineered OMVs, growths of subcutaneous MB49 tumors were significantly restricted. The inherent immunostimulation of OMVs was demonstrated to trigger maturation of dendritic cells (DCs), recruitment of macrophages, and infiltration of cytotoxic T lymphocytes (CTLs), resulting in the increased secretion of pro-inflammatory cytokines (IL-6, TNF-α, and IFN-γ). Meanwhile, several lines of evidence also indicated that bioengineered OMVs had satisfactory biosafety.Bioengineered OMVs fabricated in the present study were characterized by strong bladder cancer suppression and great biocompatibility, providing a new avenue for clinical bladder cancer therapy.Copyright © 2023 Ren, Li, Cong, Li, Xie and Wu.