Figure 5.

Design, generation, and evaluation of ApoA-I/catalase GIFTed-Exos. (A) Schematic of the design of ApoA-I/catalase GIFTed-Exos. (B) Immunoblot analysis of ApoA-I/catalase GIFTed-Exos and violet light-induced release of catalase. Theoretical molecular weights of fusion proteins are shown. (C) ELISA analysis of binding of ApoA-I/catalase GIFTed-Exos to SR-BI receptor. Data are shown as mean ± SD of duplicates. (D) Representative confocal images of cellular uptake of PKH67-labled GIFTed-Exos. HepG2 cells were treated with catalase GIFTed-Exos or ApoA-I/catalase GIFTed-Exos for 6 hours at 37°C, followed by washing with PBS, permeabilization, and confocal imaging. Blue: nuclei stained with DAPI; green: PKH67-labeled exosomes. Scale bars, 20 μm. (E) Quantitative representation of the fluorescence intensity of PKH67 channel for each group in (D) (6 fields of view per region). Data are shown as mean ± SD. n = 6; ***, p < 0.001. (F) Enzymatic activity of ApoA-I/catalase GIFTed-Exos. The exosomes were exposed to 40 μM of H₂O₂ for 30 minutes at 37°C. The concentrations of H₂O₂ were then detected using Amplex red hydrogen peroxide/peroxidase assay kits. (G) ApoA-I/catalase GIFTed-Exos protect HepG2 cells from H₂O₂-induced cytotoxicity. HepG2 cells were exposed to 500 μM of H₂O₂ in the absence or presence of native exosomes or ApoA-I/catalase GIFTed-Exos without and with 405 nm violet light treatment for 6 hours at 37°C. Cell viability was measured by MTT assays. Data in (F) and (G) are shown as mean ± SD of triplicates. Symbols indicate relative levels of significance compared with the H₂O₂ only-treatment group (ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****,p < 0.0001) or with native exosome-treatment group (#, p < 0.01; ###, p < 0.001; ####, p < 0.0001) or with ApoA-I/catalase GIFTed-Exos-treatment group (ns, not significant; Ω, p < 0.05; ΩΩΩ P < 0.001).