Fig. 2. Preparation and in vitro evaluation of the microbial nanocomposite.

a TEM and size distribution images of Ads, OMVs@P₂O, and OMVs@P₂O-Ads. Scale bar=100 nm. b CLSM images of the microbial nanocomposite. Ads were stained with DAPI dye (red) and OMVs carried a GFP marker (green). Scale bar = 1 μm. c The expression of P₂O was investigated by the SDS-PAGE method. This experiments in (a–c) were repeated three times independently with similar results. d The ROS level assessment in TC-1 cells by flow cytometry. e TEM images of autophagosomes. Scale bar = 1 μm. f The expression of autophagy-related protein LC3-I and LC3-II by western bolt analyses. g CLSM images of autophagosomes. Cells were stained with EB dye (red) and autophagosomes were stained with MDC dye (blue). Scale bar = 50 μm. This experiments in (e–g) were repeated three times independently with similar results. h The Ads replication in TC-1 cells was quantified using real-time PCR at 0, 24, 36, and 48 h sequentially. 3MA is an autophagy inhibitor: 3-Methyladenine. Data are presented as mean ± SD (n = 3 independent experiments). N.S. (No Significance) P > 0.05, *P < 0.05, **P < 0.01 and ***P < 0.001 by two-tailed Student’s t-test. Source data are provided as a Source Data file. i Cytotoxicity of different formulations in TC-1 cells by CLSM. Living cells were stained with Calcein (green) and dead cells were stained with PI (red). Scale bar = 20 μm. This experiment was repeated three times independently with similar results. j Schematic diagram of bridging ROS with oncolytic Ads replication. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control. G1: PBS, G2: Ads, G3: OMVs, G4: OMVs@P₂O, G5: OMVs-Ads, G6: OMVs@P₂O-Ads.