Photosensitizers against pathogens: Model membranes characterization with hypericin.

Enveloped viruses, i.e., comprising a phospholipidic membrane encapsulating their genetic material, are pathogens responsible for many diseases, often severe (e.g., COVID-19 and AIDS). Since the viral envelope has a fundamental role in their infectivity, it may represent a promising target for broad-spectrum antivirals. We are proposing the use of photosensitizers (PS), molecules that, in the presence of absorbable light, react with O2 triggering the production of reactive oxygen species, very strong oxidants capable of damaging biological structures, such as membranes. A specific PS called hypericin (hyp) exhibits not only a useful fluorescence in the orange spectral range, but also a strong antiviral activity toward enveloped viruses. In a recent article, we demonstrated it to be virucidal against SARS-CoV-2 not only in the presence of light, in accordance with its photodynamic properties, but also in dark conditions, revealing a multimodal mechanism of action. To characterize further the effects of hyp on coronavirus envelopes, we used phase-separated supported lipid bilayers (SLBs), an excellent model system to simulate biological membranes. We studied their morphology with Atomic Force Microscopy (AFM) and discovered that a substantial rearrangement occurs in the presence of hyp and light, and also in the dark at higher hyp concentrations; the effect is primarily visible in the ordered phase, the lipid rafts. We aim to address if hyp changes the membrane's mechanical properties (e.g., stiffness and fluidity) using fluorescence techniques such as FCS and FLIM in correlation with AFM-Force Spectroscopy. Additionally, since lipid rafts and phosphatidylserine (a lipid on the envelope) are believed to play a crucial role in viral infection and immune escape of SARS-CoV-2, respectively, we investigate if hyp multimodal antiviral activity is also related to a preferential interaction with specific lipids, by means of correlative AFM-fluorescence microscopy.