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Photodynamic therapy uses photosensitizers (PS) to kill cancer cells by generating reactive oxygen species – like singlet oxygen (SO) - upon illumination with visible light. PS membrane anchoring augments local SO concentration, which in turn increases photodynamic efficiency. The latter may suffer from SO’s escape into the aqueous solution or premature quenching. Here we determined the time constants of SO escape and quenching by target molecules to be in the nanosecond range, the former being threefold longer. We confined PS and dipolar target molecules either to different membrane monolayers or to the same leaflet and assessed their abundance by fluorescence correlation spectroscopy or membrane surface potential measurements. The rate at which the contribution of the dipolar target molecules to membrane dipole potential vanished, served as a measure of the photo-oxidation rate. The solution of the reaction–diffusion equations did not indicate diffusional rate limitations. Nevertheless, reducing the PS-target distance increased photodynamic efficiency by preventing other SO susceptible moieties from protecting the target. Importantly, our analytical model revealed a fourfold difference between SO generation rates per molecule of the two used PSs. Such analysis of PS quantum yield in a membrane environment may help in designing better PSs.

Porphyrins are well-known photosensitizers (PSs) for antibacterial photodynamic therapy (aPDT), which is still an underestimated antibiotic-free method to kill bacteria, viruses, and fungi. In the present work, we developed a comprehensive tool for predicting the structure and assessment of the photodynamic efficacy of PS molecules for their application in aPDT. We checked it on a series of water-soluble phosphorus(V) porphyrin molecules with OH or ethoxy axial ligands and phenyl/pyridyl peripheral substituents. First, we used biophysical approaches to show the effect of PSs on membrane structure and their photodynamic activity in the lipid environment. Second, we developed a force field for studying phosphorus(V) porphyrins and performed all-atom molecular dynamics simulations of their interactions with bacterial lipid membranes. Finally, we obtained the structure-activity relationship for the antimicrobial activity of PSs and tested our predictions on two models of Gram-negative bacteria, Escherichia coli and Acinetobacter baumannii. Our approach allowed us to propose a new PS molecule, whose MIC50 values after an extremely low light dose of 5 J/cm2 (5.0 ± 0.4 μg/mL for E. coli and 4.9 ± 0.8 μg/mL for A. baumannii) exceeded those for common antibiotics, making it a prospective antimicrobial agent.

Photodynamic therapy (PDT) is a widely used technique for skin cancer treatment and antimicrobial therapy. An improvement in PDT efficiency requires not only an increase in quantum yield of photosensitizer (PS) molecules but also their applicability for biological systems. Recently, we demonstrated that the activity of porphyrin-based PSs in the lipid membrane environment depends on the nature of the cation in the macrocycle due to its interactions with the lipid phosphate moiety, as well as the orientation of the PS molecules inside the membrane. Here, we report the synthesis, membrane binding properties and photodynamic efficiency of novel dicationic free-base, Ni(II) and Zn(II) pyrazinoporphyrins with terminal tetraalkylammonium units (2H-1, Ni-1 and Zn-1), to show the possibility to enhance the membrane binding of PS molecules, regardless of the central cation. All of these substances adsorb at the lipid membrane, while free-base and Zn(II) porphyrins actively generate singlet oxygen (SO) in the membranes. Thus, this study reveals a new way to tune the PDT activity of PSs in biological membranes through designing the structure of the peripheral groups in the macrocyclic photosensitizer.

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