Antibacterial treatments based on photosensitized production of reactive oxygen species is a promising approach to address local microbial infections. Given the small size of bacterial cells, identification of the sites of binding of the photosensitizing molecules is a difficult issue to address with conventional microscopy. We show that the excited state properties of the naturally occurring photosensitizer hypericin can be exploited to perform STED microscopy on bacteria incubated with the complex between hypericin and apomyoglobin, a self-assembled nanostructure that confers very good bioavailability to the photosensitizer. Hypericin fluorescence is mostly localized at the bacterial wall, and accumulates at the polar regions of the cell and at sites of cell wall growth. While these features are shared by Gram-negative and Gram-positive bacteria, only the latter are effectively photoinactivated by light exposure.

Using a combination of molecular modelling and spectroscopic experiments, the naturally-occurring pharmacologically active hypericin compound is shown to form a stable 1 : 1 complex with apomyoglobin that preserves its fluorescence and singlet oxygen photosensitising properties. The binding equilibrium constant has been determined as K a = (2.4 ¡ 0.5) 6 10 5 M 21 , equivalent to a dissociation constant K d = 4.2 ¡ 0.8 mM. The kinetic details of singlet oxygen production have been characterised and indicate that the protein scaffold protects hypericin from oxygen. The complex is active against Staphylococcus aureus bacteria and shows lower dark toxicity than free hypericin. In view of its superb biocompatibility, apomyoglobin should be considered as a nanovehicle for hypericin in theranostic applications.

Using a combination of molecular modeling and spectroscopic experiments, the naturally occurring, pharmacologically active hypericin compound is shown to form a stable complex with the dimeric form of β-lactoglobulin (β-LG). Binding is predicted to occur at the narrowest cleft found at the interface between monomers in the dimeric β-LG. The complex is able to preserve the fluorescence and singlet oxygen photosensitizing properties of the dye. The equilibrium constant for hypericin binding has been determined as Ka=1.40±0.07µM(-1), equivalent to a dissociation constant, Kd=0.71±0.03µM. The complex is active against Staphylococcus aureus bacteria. Overall, the results are encouraging for pursuing the potential application of the complex between hypericin and β-LG as a nanodevice with bactericidal properties for disinfection.