Antibacterial Activity of Neat and Sonicated Extracellular Vesicles derived from Stromal Vascular Fraction

Background: Small extracellular vesicles (sEVs) promote intercellular communication, and their inherent antibacterial and antibiofilm properties depend on their cellular origin. Sonication is a widely used physical method for loading therapeutic compounds into sEVs, but its impact on vesicle structure and native bioactivity, especially antibacterial efficacy, is not fully understood. This study assessed whether probe sonication disrupts sEVs derived from the stromal vascular fraction of healthy human adipose tissue (hHAT-SVF), causing endogenous antibacterial contents leakage and consequent loss of therapeutic utility. Methods: sEVs were isolated from hHAT-SVF, then characterized before and after sonication, using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blotting (WB) to evaluate morphology, particle size, concentration, and protein profile. The antibacterial and antibiofilm effectiveness of unaltered/intact (neat) versus sonicated SVF-derived small extracellular vesicles (SVF-sEVs) was tested against methicillin-resistant Staphylococcus aureus (MRSA) using colony-forming unit (CFU) counts, time-kill kinetics, and biofilm inhibition assays. Results: Physiochemically, sonicated SVF-sEVs exhibited major structural disruption, showing ~65% fewer vesicles, and >50% higher free protein levels in the supernatant. NTA, TEM and WB confirmed fragmented membranes, reduced vesicle concentration, and leakage of characteristic protein markers. Functionally, neat SVF-sEVs effectively inhibited MRSA, producing 12–13 mm inhibition zones and reducing both bacteria and their biofilm biomass by 80%–90%. In contrast, sonicated vesicles lost >80% of these effects, resembling controls. Conclusion: Probe sonication impairs SVF-sEVs stability and antibacterial capacity by causing cargo leakage. These findings emphasize the importance of preserving vesicle integrity and reassessing their bioactivity after any manipulation for therapeutic use.