Lacticaseibacillus paracasei-derived extracellular vesicles enhance the in vitro efficacy of methylene blue-mediated photodynamic therapy against multidrug-resistant Staphylococcus aureus.

The increasing incidence of methicillin-resistant Staphylococcus aureus (MRSA) highlights the need for antimicrobial strategies that minimize the use of antibiotics. Although antimicrobial photodynamic therapy (A-PDT) offers a promising non-antibiotic alternative, its clinical application is frequently limited by inadequate localization and delivery of photosensitizers. In this study, we developed a biogenic drug delivery platform utilizing extracellular vesicles (EVs) derived from Lacticaseibacillus paracasei (LPr) to improve the efficacy of methylene blue (MB)-mediated antimicrobial photodynamic therapy (A-PDT). Both active and passive loading strategies were employed to load MB into LPr-EVs with high efficiency (>90%). Upon exposure to 660 nm light-emitting diode irradiation, the resultant MB@LPr-EVs demonstrated an eight-fold reduction in the minimum inhibitory concentration against S. aureus ATCC 25923 and a 16-fold reduction against clinical MRSA strains, compared to free MB controls. This enhancement is attributed to the efficient generation of reactive oxygen species (ROS), which cause irreversible oxidative damage to the bacterial membranes. Additionally, MB@LPr-EVs exhibited antibiofilm activity at concentrations substantially lower than those of free MB. Cytotoxicity analysis using human embryonic kidney-293 (HEK-293) cells indicated that MB@LPr-EVs did not induce significant cytotoxicity compared to free MB. Collectively, these findings establish LPr-EVs as a promising biologically derived carrier that enhances MB-mediated A-PDT against S. aureus including MRSA, through improved targeted delivery, ROS hyperproduction, and biofilm inhibition.