Extended Data Fig. 3: Molecular characterization, assembly validation, and functional stability of engineered iNV-GOV nanovesicles.
a, Western blot analysis of B2M, CIITA, HLA-E, and CD47 in donor cells across increasing passages (p0 to p20), with GAPDH as loading control. Blot is representative of n = 2 independent experiments. b, Flow cytometry histograms and quantification of HER2 expression levels in donor cells across passages (n = 3 biological independent samples). c, Flow cytometry analysis of iNV-GOV-1 to -3 of HER2 expression across batches (n = 3 biological independent samples). d, Western blot analysis of protein levels in iNV-GOV-1 to -3 vesicles. Blot is representative of n = 3 independent experiments. e, Imaging flow cytometry images of co-localized Cy5-labeled iNVs and Cy7-labeled GOVs across three iNV-GOV formulations. Images are representative of n = 3 independent experiments. f, qRT-PCR assay of relative virus replication ability to naked GOV and iNV-GOV in Capan-1 cells with indicated treatments (n = 5 biological independent samples). g, Representative fluorescence imaging and flow cytometry quantification of GFP expression in Capan-1 cells treated with different iNV-GOV batches on day 6. GOVs used in this study expressed GFP (n = 5 biological independent samples). h, qPCR quantification of viral genome copy number in Capan-1 cells infected with GOV or iNV-GOV different batches (n = 5 biological independent samples). i, Dot blot assay (upper), and the quantitative analysis (lower) of iNV-GOV stored at different temperatures and +/- serum for 24 h (n = 5 biological independent samples). j, Dot blot assay (upper), and the quantitative analysis (lower) of iNV-GOV after incubation with or without stirring (100 rpm) at 37 °C for 24 h, GOV as a control (n = 5 biological independent samples). k, Adenovirus pol gene copies were quantified using qRT-PCR and adenovirus dot blot assay in Capan-1 cells incubated with indicated treatments (n = 5 biological independent samples). l, Western blot analysis of GSDMDNT expression after treated with GOVUS, iNV-GOV and iNV-GOVUS at 96 h, ultrasound induces localized activation of GOV at 72 h after indicated treatment. The concentration of GOV is 20 VP/cell. Blot is representative of n = 3 independent experiments. m, n, Stability of iNV-GOV under storage at different temperatures (m, n = 5 biological independent samples or n = 3 biological independent samples) or different days (n, n = 5 biological independent samples). o, Schematic representation, and timeline of an experiment investigating the antiviral response in peripheral blood from healthy HLA-A2+ donors + incubation using GOV or iNV-GOV. p, Concentration of Ad5-neutralizing antibodies (NAbs) in human donors (left), n = 40 biological independent individuals. The infection efficiency of GOV-CMV-mCherry in Capan-1 cells was assessed in serum-free DMEM buffer (middle), n = 5 biological independent samples, or in donor serum containing DMEM buffer (right, n = 40 biological independent individuals). q, The infection efficiency of Ad5-mCherry, GOV-CMV-mCherry, or iNV-GOV-CMV-mCherry to Capan-1 cells were evaluated against varying concentrations of standard ad5 NAbs (n = 3 biological independent samples). The data is presented as the means ± S.D. One-way ANOVA with Tukey’s post hoc test was used for (b, c, f, g, h, m, n), with P values indicated on the graphs. Schematics in o was created with BioRender.com.
