Porphyrin-loaded hydrogels: influence of polymer concentration on bacterial photoinactivation.

Rosario

Congreso; Ricifa 2023; 2023

RICIFA

Photodynamic inactivation of bacteria (PDI) is a powerful strategy that may support the treatment of colonized or infected skin. It was already demonstrated that photosensitizers can improve the healing of both acute and chronic wounds. TMPyP is a cationic porphyrin extensively studied as a photosensitizer. When irradiated with light of appropriate wavelength, it generates reactive oxygen species (ROS) that react with a variety of biological substrates, inducing a decrease in biological functions that lead to cell inactivation. A challenge in using antimicrobial photodynamic therapy for wound management is the lack of a convenient carrier system that releases the compounds and that is transparent for activating by light. Considering this point, hydrogels based on ionic complexes between the anionic polymer Carbomer-974P and TMPyP (C974-TMPyP) were designed. As expected, the hydrogels exhibited adequate pH and viscosity for topical application and modulation of TMPyP release. However, a lower amount of singlet oxygen was detected, compared to TMPyP in solution evaluated with the same light doses, and preliminary studies showed delay in the photoinactivation of P. aeruginosa mediated by TMPyP when it is loaded on carbomer hydrogels. Therefore, the objective of this work is to investigate the influence of the carrier polymer on the photoinactivation process of P. aeruginosa mediated by TMPyP. Hydrogels with different polymer concentrations (range 0.1, 0.25, 0.5, 0.75 and 1%) were prepared and loaded with the cationic porphyrin (5 µM). PDI of P. aeruginosa mediated by TMPyP free solution and from hidrogels C974-TMPyP was evaluated in vitro by viable count on solid medium after irradiating with white light (300?800 nm) up to 60 min (fluence rate 28.7 mW/cm2) in the absence and presence of two suppressors of ROS, sodium azide (50 mM) and D-mannitol (10 mM). Furthermore, the absorbance spectra of TMPyP as well as the transmittance of the hydrogels C974-TMPyP were investigated. The range studied was established between 300 and 800 nm.The C974-TMPyP hydrogels exhibited a reduction in the PDI of P. aeruginosa mediated by TMPyP dependent on the polymer concentration, requiring a higher dose of light to eradicate the culture compared to that required by free TMPyP. Concentrations > 0.5% showed no differences with the untreated control, evaluated up to 60 min of irradiation. The addition of sodium azide produced complete photoprotection of bacterial survival, while only a partial photoprotective effect was induced by D-mannitol, both in cultures treated with TMPyP and with hydrogels with low polymer concentration. This indicates that the photodamage mechanism is mainly mediated by 1O2 (type II reaction), although there would also be some contribution of the type I pathway. Similar performance with the scavengers of ROS was observed in the absence and presence of the polymer. In addition, reduction in the transmittance of the hydrogels was observed as polymer concentration increased. PDI results showed that hydrogels with transmittance <70% were not photoactive against P. aeruginosa.Hydrogels must have high transmittance to ensure that light can penetrate them and consequently the photosensitizer can be excited and produce ROS. Then, the integrated analysis of results from previous studies of these hydrogels (pH, viscosity, release modulation, etc.) with the gained knowledge concerning the polymer effect on the PDI contributes to the optimization of these hydrogels, in order to initiate future studies of prevention or treatment of wound infections.