Combination of photodynamic therapy with antimicrobial peptides: effects and mechanisms

Author(s)

Laura Marise de Freitas

• Document type: Doctoral Thesis
• Press: Araraquara. 2018-09-14.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara
• Defense date: 2018-08-23
• Advisor: Carla Raquel Fontana

Abstract

In the past few years, the World Health Organization has been warning that the post-antibiotic era is an increasingly real threat. In addition to the individual resistance of bacterial cells, they may be even more tolerant to antimicrobial agents by growing in biofilms. The higher tolerance to antibiotics observed in biofilms is mainly based on the protection of bacteria by the self-produced extracellular polymeric matrix, and in the various growth phenotypes found in the structure, which may be refractory to the action of conventional drugs. In this scenario of increasing and widespread antimicrobial resistance, the search for new drugs and alternative therapies has become crucial, especially for those that are capable of eliminating resistant microorganisms, preventing the development of new forms of resistance, and are active against biofilms. Antimicrobial photodynamic therapy (aPDT) and antimicrobial peptides (AMP) are highlighted in this context, especially for the treatment of localized infections. However, both approaches have therapeutic limitations when used individually. Therefore, the aim of this study was to evaluate the effects and mechanisms of the combination of the AMPs aurein 1.2 (AU) and its dimer (AU)2K with aPDT mediated by the photosensitizers (PS) methylene blue (MB), chlorine-e6 (Ce6) or curcumin CUR), using the bacteria Enterococcus faecalis as a model, in planktonic phase and biofilm. The dimer (AU)2K did not present antibacterial activity in planktonic phase, so the initial part of the study was conducted only with the monomer. The results show that the combination of aPDT with AU proved to be able to eliminate E. faecalis, in vitro, with low concentrations of PS and peptide and lower light doses, compared to the monotherapies. The synergistic effect was observed only when AU was associated with MB or Ce6-mediated aPDT, and no advantage was found in the combination therapy when the PS was CUR, revealing a PS-dependent mechanism. The synergistic effect between AU and MB-PDT was a result of a greater penetration of the PS in the bacterium in the presence of the peptide, whereas the synergistic effect between AU and Ce6-PDT was due to an interaction between the molecules that led to greater instability of the plasmatic membrane. The combination treatment was also effective against different strains, including a complete elimination of a strain of vancomycin-resistant Enterococcus faecium, an unprecedented result. The combination therapy protocol was tested for its ability to inhibit E. faecalis biofilm formation. Although it did not present activity against planktonic bacteria, the (AU)2K dimer was included in the biofilm studies, showing high capacity to prevent the initial phase of biofilm development, both alone and combined with aPDT. The monomer AU also showed significant activity to prevent E. faecalis from forming biofilm, particularly when combined with Ce6-PDT. Other bacterial species were also evaluated, and the combination protocols were able to prevent biofilm formation by all of them in a significant way. Taken together, the results obtained in this study reveal that the combination of aPDT with an AMP can lead to bacterial death with minimal concentrations of PS and AMP and low light doses, which in turn would minimize adverse effects on host tissue. In addition, the ability of this approach in inhibiting the formation of biofilms reveals its potential to prevent the establishment of chronic infections, strongly related to microbial biofilms. This synergistic approach has the potential to resolve localized infections while minimizing the use of systemic antimicrobials and preventing the development of new resistance profiles.

• FAPESP's process: 14/24581-5 - Evaluation of the role of photodynamic therapy combined with antimicrobial peptides against bacterial resistance
• Grantee: Laura Marise de Freitas
• Support Opportunities: Scholarships in Brazil - Doctorate