Fig. 2: CAR^mRNA@aCD206 sEV-mediated CAR-M manufacturing in vitro.

a Representative confocal images showing the targeting and internalization of indicated sEVs by M2 bone marrow-derived macrophages (BMDM) after co-incubation for 4 h and 24 h. sEVs are labeled with DiI (red), M2 BMDMs with DiO (green), and nuclei with DAPI (blue). To ensure consistent fluorescence intensity, sEVs staining with 2 μM DiI dye was strictly controlled for 10 min. Scale bar: 20 μm. b Representative flow cytometry images (left) and statistical analysis (right) depicting the internalization of different sEVs by M2 BMDMs at various time points (n = 5 independent experiments). c Representative confocal microscopy images showing the expression of EGFP in M2 BMDMs treated with the indicated sEVs. Scale bar: 50 μm. d, e Representative flow cytometry images and statistical analysis showing the CAR expression in macrophages after co-incubation with the indicated sEVs (n = 5 independent experiments). f The impact of Cx43-S368A and Lamp2-aCD206 modifications on EV-mediated mRNA delivery and subsequent translation was evaluated by assessing the Nanoluc protein signal in treated cells. EVs without Cx43-S368A and Lamp2-aCD206 modifications served as controls (n = 3 independent experiments). g, h Representative flow cytometry image and statistical analysis of CAR protein levels corresponding to different concentrations of CAR^mRNA@aCD206 sEVs (n = 5 independent experiments). i, j Representative flow cytometry image and statistical analysis showing CAR expression in macrophages over time after co-incubation with the indicated sEVs (n = 5 independent experiments). For (a–f) and (i, j), the dosage of sEVs used was 4 × 10⁶ mRNA copies/mL. UTD: un-treated, Data are presented as mean ± SD. For (a, c), experiment was repeated three times independently with similar results. Data were analyzed using one-way ANOVA with Tukey’s test in (e, f). **** p ≤ 0.0001. Source data are provided as a Source Data file.