Antimicrobial peptide-porphyrin conjugates: new tools in photodynamic therapy

Antimicrobial PhotoDynamic Therapy (APDT) represents a very promising strategy, particularly for the treatment of localized infectious diseases.[1] PDT involves the use of a non-toxic photosensitizer (PS), f. i. porphyrins, phthalocyanines, in combination with harmless visible light of appropriate wavelength to excite the PS. In the presence of the oxygen, the excited PS transfer energy or electrons to the ground state molecular oxygen, producing reactive oxygen species (ROS), e. g. singlet oxygen and hydroxyl radical, that affecting the integrity and function of different cellular components, e.g. proteins, nucleic acids and lipids, cause cell death. Advantages of APDT over traditional antibiotics include a broad spectrum activity, also against antibiotic-resistant species and the lack of development of resistance mechanisms due to the multi-target process. Gram-positive bacteria can be efficiently killed by light after their incubation with a number of PS. On the contrary Gram-negative bacteria are less susceptible to photodynamic killing and only cationic PS can bind efficiently to this type of bacteria and induce their photoinactivation. An alternative approach to improve the susceptibility of Gram-negative bacteria against neutral PS, involves the covalent attachment of the PS to a polymer molecule containing basic amino groups. Recently we have shown that the conjugation of a neutral porphyrin to the cationic antimicrobial peptide apidaecin 1b (T-api), belonging to the Pro-Arg rich peptide family, afforded a new antibacterial agent with a broader spectrum activity than two individual components or a mixture of them.[2] In the present study we extend our investigation to two new PS-apidaecin conjugates in which either a porphycene (G), a structural porphyrin isomer, or a cationic porphyrin (Y) are covalently linked to the peptide N-terminal end. The bactericidal activity of two new conjugates, in the dark or under light activation, was compared to that of the photosentizers alone and of the coniugate with the neutral porphyrin. PS-apidaecin conjugates did not exhibit any toxicity against both Gram negative (E.coli) and Gram positive (S.aureus) bacteria in the dark. On the contrary, following activation with red light (36 J/cm2), the porphycene conjugate (G-api) caused a significant (3-4 log10) reduction of E. coli survival, comparable to that of T-api, but it was less effective than the porphyrin counterpart against S. aureus. The cationic porphyrin and its apidaecin conjugate (Y-api), following activation with blue light (13 J/cm2), caused complete killing of both Gram-negative and –positive bacteria at lower concentrations than G-api and T-api.