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Figure 1. Genetic modification and characterization of PSMA-scFv MSC-EVs. (A) Schematic representation of EV genetic modification. The VH and VL domains of the J591 monoclonal antibody were linked by a peptide linker to generate the PSMA-scFv. The construct was fused to LAMP2B and expressed in HEK293A and 3A6 BM-MSC cells to generate PSMA-scFv displaying EVs; (B and C) Expression of PSMA-scFv in engineered HEK293A and MSC cells and characterization of the corresponding EVs. Western blot analysis showing PSMA-scFv expression in WCL of HEK293A (B) and MSCs (C) and in their respective EV fractions, detected using LAMP2B and FLAG antibodies. Blots were normalized to β-actin for WCL and CD63 for EV fractions. EV marker analysis of Parental, LAMP2B, and PSMA-scFv MSC-derived EVs confirmed the presence of canonical EV markers in accordance with MISEV 2023 guidelines (C); (D) NTA of MSC-EVs. Representative NTA plots showing particle size distribution and concentration of PSMA^scFv-engineered MSC-EVs, compared to Parental and LAMP2B EVs; (E) TEM. Micrographs illustrating the morphology and size of genetically modified MSC-EVs (LAMP2B and PSMA-scFv) and unmodified (Parental). White arrows indicate EV particles. Scale bars: 200 nm (top) and 100 nm (bottom); (F) Surface localization of PSMA-scFv on MSC-EVs. Flow-cytometric analysis following conjugation with Protein L and CD63 magnetic beads, demonstrating the external topology of PSMA-ScFv on the EVs surface. Representative dot plots show protein L-binding profiles for Parental, LAMP2B, and PSMA-scFv MSC-EVs. Three independent EV preparations were analyzed (n = 3). Representative NTA, TEM, and Protein L images are shown, with each analysis performed in triplicate. Immunoblot analyses were performed using three independent biological repeats (n = 3). EVs: Extracellular vesicles; PSMA: prostate-specific membrane antigen; scFv: single-chain variable fragment; MSCs: mesenchymal stem cells; BM-MSC: bone marrow-derived mesenchymal stem cell; LAMP2B: lysosome-associated membrane protein 2B; VH: variable heavy; VL: variable light; WCL: whole-cell lysate; MISEV: Minimal Information for Studies of Extracellular Vesicles; NTA: nanoparticle tracking analysis; TEM: transmission electron microscopy; PE: phycoerythrin; CD9: cluster of differentiation 9; CD63: cluster of differentiation 63; CD81: cluster of differentiation 81; TSG101: tumor susceptibility gene 101; HSP70: heat shock protein 70; BSA: bovine serum albumin; Lamin A/C: lamin A and lamin C; MFI: mean fluorescence intensity; IgG: immunoglobulin G; CD90: cluster of differentiation 90; β-actin: beta-actin; PE-A: phycoerythrin area; FLAG: FLAG epitope tag.