Protein‐Mediated Photodynamic Inactivation of Gram‐Negative Bacteria Enabled by Nano‐Delivered TSPO

ABSTRACT Antimicrobial photodynamic therapy (aPDT) offers a promising alternative to conventional antibiotics, yet its application to Gram‐negative bacteria remains limited by the poor membrane association and rapid diffusion of most photosensitizers. Here, we report a protein‐mediated strategy that overcomes this intrinsic barrier by nano‐delivering the porphyrin‐binding translocator protein (TSPO) to the outer membrane of Escherichia coli . TSPO was reconstituted into lipid nanodiscs and transferred to bacterial membranes without genetic manipulation of the host cells. Membrane‐localized TSPO retained high‐affinity binding to protoporphyrin IX (PpIX), enabling stable photosensitizer anchoring at the bacterial surface. Upon light irradiation, the TSPO‐PpIX complex generated localized reactive oxygen species, resulting in pronounced photodynamic inhibition of bacterial growth in both wild‐type and lipopolysaccharide‐deficient E. coli strains. Complementary in vivo assays leveraging endogenous PpIX biosynthesis further confirmed that TSPO‐mediated photosensitizer localization is sufficient to drive photodynamic inactivation. Together, these results establish nano‐delivered TSPO as a versatile protein‐based platform for enhancing aPDT against Gram‐negative bacteria and provide a generalizable framework for overcoming membrane‐associated resistance in antimicrobial phototherapy.