Fig 2: The identification of Exo and Exo-181b. a TEM was utilized for the observation of the morphology of Exo-NC and Exo-181b. Scale bar = 100 nm. b The identification of Exo-NC and Exo-181b by detecting their surface positive markers (CD9, Alix, and Tsg101) by western blot. c NTA analysis was used for analyzing the diameter distribution of Exo-NC and Exo-181b. d The uptake of Exo by RAW264.7 detected by fluorescence staining. Green, red and blue represent the cytoskeleton (phalloidine), Exo (PKH26) and nucleus (DAPI), respectively. e The gene expression of miR-181b in hBM-MSC and the secreted Exo overexpressing miR-181b. f The gene expression of miR-181b in RAW264.7 after treated by Exo-NC and Exo-181b. (*p < 0.05). Exo exosome without overexpressing miR-181b, Exo-181b exosome overexpressing miR-181b, TEM transmission electron microscopy, NTA nanoparticle tracking analysis

Fig 3: Characterization of ADSC-EVs. ADSC-EVs were isolated from three separate batches of conditioned media (CMs) and characterized. (A) Particle size distribution of ADSC-EVs measured through a nanoparticle tracking analysis (NTA). (B) Mean particle sizes of ADSC-EVs measured through NTA. Data are presented as the mean ± standard deviation (SD; n = 3). (C) Morphology of the ADSC-EVs as observed through transmission electron microscopy. (D) Western blot analysis of the protein levels of CD9, CD81, Alix, and ß-actin in ADSCs and ADSC-EVs.

Fig 4: Validation of the selected proteins using PRM. Exosomes of CON and PCP B cells were isolated and subjected to LC-PRM/MS analysis. Relative protein abundance of eight proteins (ALIX, C3, PTPN6, VIM, CORO1A, LMNA, MYH4, and H1F3) was calculated based on the normalized peak area of peptide fragments. A two-tailed Student's t test was used to detect the difference between two groups (n = 3). *indicates p < 0.05.

Fig 5: Particle population characteristics, zeta potential, and western blot analysis of cell-derived EVs. (A) Categorization of EV populations by size distribution by TRPS. The majority of EVs belong to a size range of 100–250 nm. t-test was performed between their counterparts. (B) pH affects the zeta potential of EVs: Compared to physiological pH (7.4), cyst microenvironmental pH (6.0) showed a drastic change in the zeta potential (measured by TRPS) of EVs derived from T1G cells. (C) Western blot analysis of EVs. Classical EV markers such as Alix, TSG 101, ARL13b, CD63, CD81, and CD9 were present in EVs derived from mIMCD3 and T1G cells. Data (n = 3) were represented as mean ± SD. Significant difference analyzed by t-test (*p < 0.05, **p < 0.01, ***p < 0.001).