Molecular Determinants of Porphyrin Photosensitizer-Mediated Bacterial Photoinactivation

The widespread and increasing resistance of bacteria to antibiotics creates a demand for development of alternative methods for microbial eradication. Photodynamic inactivation (PDI) is a promising approach because of its demonstrated efficacy against a wide range of drug-resistant microorganisms. The practical application of PDI needs photosensitizers (PSs) with high quantum yield, low toxicity against the host, and potent antimicrobial activity. Design of such PSs requires a detailed understanding of how molecular structure influences PDI efficacy. This work investigated the PDI activity of a series of meso -substituted N -ethylpyridinium-2-yl and pentafluorophenyl substituted porphyrins bearing zero to four positive charges. Results demonstrated that the highest bactericidal activity against Gram-negative ( E. coli ) and Gram-positive ( S. aureus ) microorganisms was displayed by the asymmetric dicationic porphyrin with adjacent positive charges ( cis -H ₂ DiE). Despite having a similar molecular structure, comparable singlet oxygen quantum yield, and similar lipophilicity, its symmetric regioisomer with two opposite positive charges ( trans -H ₂ DiE) was substantially less photoeffective. Pronounced differences were also observed in the PDI sensitivity of Gram-negative and Gram-positive bacteria. Investigations with membrane-impermeable fluorescent agents revealed that membrane permeabilization was the major cause for photo-inactivation of microbes. The combination of moderate lipophilicity and adjacent positive charges facilitates deeper penetration of the PS in the membrane lipid bilayer, positioning the PS in an environment with higher oxygen availability and closer proximity to unsaturated fatty acids, thereby favoring oxidative membrane damage. These findings demonstrate that in addition to lipophilicity and charge number, the molecule symmetry and spatial distribution of charges ( ortho vs meta vs para and adjacent vs opposite), the tridimensional shape of the molecule, its flexibility and bulkiness, should be considered when designing PSs for potential clinical applications.