Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material.

Prosthetic joint infections (PJIs) are becoming a growing public health concern in developed countries as more people undergo arthroplasty for bone fixation or joint replacement. Because a wide range of bacterial strains responsible for PJIs can produce biofilms on prosthetic implants and because the biofilm structure confers elevated bacterial resistance to antibiotic therapy, new drugs and therapies are needed to improve the clinical outcome of treatment of PJIs. Antimicrobial photodynamic therapy (APDT), a non-antibiotic broad-spectrum antimicrobial treatment, is also active against multidrug-resistant micro-organisms such as meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. APDT uses a photosensitiser that targets bacterial cells following exposure to visible light. APDT with RLP068/Cl, a novel photosensitiser, was studied by confocal laser scanning microscopy (CLSM) to evaluate the disruption of MRSA and P. aeruginosa biofilms on prosthetic material. Quantitative CLSM studies showed a reduction in biofilm biomass (biofilm disruption) and a decrease in viable cell numbers, as determined by standard plate counting, in the S. aureus and P. aeruginosa biofilms exposed to APDT with the photosensitiser RLP068/Cl. APDT with RLP068/Cl may be a useful approach to the treatment of PJI-associated biofilms.

Antimicrobial photodynamic therapy with RLP068 kills methicillin-resistant Staphylococcus aureus and improves wound healing in a mouse model of infected skin abrasion PDT with RLP068/Cl in infected mouse skin abrasion.

Photodynamic therapy (PDT) is an alternative treatment for infections that can kill drug resistant bacteria without damaging host-tissue. In this study we used bioluminescent methicillin-resistant Staphylococcus aureus, in a mouse skin abrasion model, to investigate the effect of PDT on bacterial inactivation and wound healing. RLP068/Cl, a tetracationic Zn(II)phthalocyanine derivative and toluidine blue (TBO) were used. The light-dose response of PDT to kill bacteria in vivo and the possible recurrence in the days post-treatment were monitored by real-time bioluminescence imaging, and wound healing by digital photography. The results showed PDT with RLP068/Cl (but not TBO) was able to kill bacteria, to inhibit bacterial re-growth after the treatment and to significantly accelerate the wound healing process (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

In vitro resistance selection studies of RLP068/Cl, a new Zn(II) phthalocyanine suitable for antimicrobial photodynamic therapy.

Resistance to antimicrobial agents is emerging in a wide variety of nosocomial and community-acquired pathogens. The development of alternative therapies against nosocomial infections caused by clinically relevant pathogens represents a major public health concern. RLP068/Cl is a novel Zn(II) phthalocyanine proposed as a photosensitizer suitable for antimicrobial photodynamic therapy (APDT) for localized infections. Its ability, following activation by light, to induce resistance in three major human pathogens after 20 daily passages was studied. Simultaneously for the same strains, the ability of daily sequential subcultures in subinhibitory concentrations of RLP068/Cl to develop resistant mutants without illumination was evaluated. We demonstrate that 20 consecutive APDT treatments with RLP068/Cl did not result in any resistant mutants and that, in dark conditions, only Staphylococcus aureus strains had increased MICs of RLP068/Cl. However, even in this case, the susceptibility of the mutated bacteria to APDT was not affected by their MIC increase.

Photodynamic therapy in the treatment of microbial infections: basic principles and perspective applications.

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) appears to be endowed with several favorable features for the treatment of infections originated by microbial pathogens, including a broad spectrum of action, the efficient inactivation of antibiotic-resistant strains, the low mutagenic potential, and the lack of selection of photoresistant microbial cells. Therefore, intensive studies are being pursued in order to define the scope and field of application of this approach. RESULTS: Optimal cytocidal activity against a large variety of bacterial, fungal, and protozoan pathogens has been found to be typical of photosensitizers that are positively charged at physiological pH values (e.g., for the presence of quaternarized amino groups or the association with polylysine moieties) and are characterized by a moderate hydrophobicity (n-octanol/water partition coefficient around 10). These photosensitizers in a micromolar concentration can induce a >4-5 log decrease in the microbial population after incubation times as short as 5-10 minutes and irradiation under mild experimental conditions, such as fluence-rates around 50 mW/cm2 and irradiation times shorter than 15 minutes. CONCLUSIONS: PDT appears to represent an efficacious alternative modality for the treatment of localized microbial infections through the in situ application of the photosensitizer followed by irradiation of the photosensitizer-loaded infected area. Proposed clinical fields of interest of antimicrobial PDT include the treatment of chronic ulcers, infected burns, acne vulgaris, and a variety of oral infections.

Zn(II)-phthalocyanines as phototherapeutic agents for cutaneous diseases. Photosensitization of fibroblasts and keratinocytes.

Two tetrasubstituted (RLP024 and RLP040) and one monosubstituted (MRLP101) Zn-phthalocyanines were readily accumulated by three skin-derived cell lines (HT-1080 transformed human fibroblasts, 3T3 mouse embryo fibroblasts and HaCaT human keratinocytes) upon 1 h-incubation with 0.5-5 microM phthalocyanine concentrations. The affinity was markedly larger for the tetra- as compared with the mono-substituted phthalocyanine, even though smaller phthalocyanine amounts were generally recovered from keratinocytes. As a consequence, the two tetra-substituted phthalocyanines exhibited a higher phototoxicity against all the three cell lines. Typically, the cell survival decreased by at least 80% after 1 min irradiation with 600-700 nm light at a fluence-rate of 50 mW/cm2 in the presence of 5 microM phthalocyanine. Fluorescence microscopy and caspase-3 activation studies indicate that cell death of fibroblasts largely occurred by a random-necrotic process while the keratinocytes underwent cell death predominantly via apoptosis in spite of a very similar pattern of subcellular distribution of the phthalocyanines.