Fig 2: Characterisation of fluorescent mEmerald-CD81 MCF7 EVs. (a) Nanoparticle tracking analysis (NTA) was performed on MCF7/ mEmerald-CD81 MCF7 EVs. The graph shows the size distribution of the EVs. N = 5 (biological), error bars = SEM. (b) An electron micrograph of negatively stained mEmerald-CD81 MCF7 EVs. Arrows indicate EVs. (c) MCF7 and mEmerald-CD81 MCF7 EVs were incubated with MACsPlex immunocapture beads, the beads were then washed and incubated with an antibody cocktail containing APC conjugated antibodies against CD9/CD63/CD81. The histogram shows the APC mean fluorescence intensity for immunobeads CD9, CD29, CD63, CD81, CD209, and negative controls REA and mIgG1. N = 3 (biological), error bars = SEM. (d) EV lysate was used in an immunoblot using antibodies for EV positive markers Alix, TSG101, CD81 and CD9, and EV negative marker GM130, with cell lysate as a positive control. Arrows indicate approximate sizes of the bands in kDa. N = 3 (separate batches). (e) Lysates from MCF7 or mEmerald-CD81 MCF7s and their EVs were used in immunoblots using antibodies against CD81 and Beta actin. Blots were cropped to make (d) and (e). (f) mEmerald-CD81 MCF7 EVs were placed in 1% (w/v) agarose gel and a coverslip placed on top. (g) mEmerald-CD81 MCF7 EVs (left) treated with Proteinase K (100 µg/mL) (middle) or with Proteinase K (100 µg/mL) and Triton X-100 (1% v/v) (right) were placed in 1% (w/v) agarose gel and a coverslip placed on top. Images captured on a Zeiss LSM880 confocal using 100 × objective. N = 3, (biological). Scale bar is 10 µm.

Fig 3: Successful isolation and characterization of engineered EVs displaying peptides of interest. (A) Representative size distribution of the naïve, RDG, and E626 display EVs determined by nanoparticle tracking analysis. The peak particle sizes were 106.2 nm, 98.2 nm, and 102.6 nm, respectively. (B) Western blot analysis of engineered EVs (RDG and E626 fusion peptide 55kDa) for the presence of EV biomarkers CD63 (30–60 kDa) and TSG101 (44 kDa), Alix (95 kDa), and peptide HA-tag. The analysis of cell lysate and engineered EVs for cellular biomarker calnexin (67KDa) is also shown. (C) Representative immuno-transmission electron microscopy images of naïve, RDG, and E626 EVs showing gold-labeled HA on engineered EV (RDG and E626) surfaces and CD63 EV surface markers on all the EVs.

Fig 4: Extraction method and characterization of milk exosomes. (A) Schematic diagram of a method for extracting exosomes from milk. (B) Size distribution diagram and representative TEM image of Col M-exo and Mat M-exo. (C) Expression level of exosomal marker proteins, Alix, Tsg101, and MFG-E8, in various exosomes. (D) Comparison of exosome yield. Exosomes were extracted with an equal volume of cell-culture medium and milk. n = 5; *** p < 0.001 versus cell-culture medium.

Fig 5: Characterization of extracellular vesicles (EVs) from HEK AMY3 and HEK Wt cells.A, Western blot detection of the Alix and TSG101, two commonly used EV markers. Molecular weights are presented in kDa. B, Transmission electron microscopy of EVs derived from AMY3 cells using a negative staining method. EVs appear as closed vesicles (Scale bar = 200 nm). C, Dynamic light scattering (DLS) showing the relative size of HEKAMY3 and Wt EVs. D. Dot blots (in triplicate from each EV preparation) show amylin receptor heterodimeric components CTR (green) and RAMP3 (red) proteins are more abundant in EVs derived from HEK-AMY3 than HEK Wt cells. Histogram showing the relative quantification of CTR and RAMP3intensity (from dot blots) in AMY3 compared to Wt EVs. Data are presented as mean ± SEM, n = 3, and an average from three independent EV preparations, ** p<0.01, * p<0.05).