Evaluation of the interplay among the charge of porphyrinic photosensitizers, lipid oxidation and photoinactivation efficiency in Escherichia coli

Abstract

Photodynamic inactivation (PDI) is a simple and controllable method to destroy microorganisms based on the production of reactive oxygen species (ROS) (e.g., free radicals and singlet oxygen), which irreversibly oxidize microorganism’s vital constituents resulting in lethal damage. This process requires the combined action of oxygen, light and a photosensitizer (PS), which absorbs and uses the energy from light to produce ROS. For a better understanding of the photoinactivation process, the knowledge on how some molecular targets are affected by PDI assumes great importance. The aim of this work was to study the relation between the number and position of positive charges on porphyrinic macrocycles and the changes observed on bacterial lipids. For that, five porphyrin derivatives, bearing one to four positive charges, already evaluated as PS on Escherichia coli inactivation, have been tested on lipid extracts from this bacterium, and also on a simple liposome model. The effects were evaluated by the quantification of lipid hydroperoxides and by analysis of the variation of fatty acyl profiles. E. coli suspensions and liposomes were irradiated with white light in the presence of each PS (5.0μM). Afterwards, total E. coli lipids were extracted and quantified by phosphorus assay. Lipid oxidation on bacteria and on liposomes was quantified by ferrous oxidation in xylenol orange (FOX2 assay) and the analysis of the fatty acid profile was done by gas chromatography (GC). As previously observed for E. coli viability, an overall increase in the lipid hydroperoxides content, depending on the PS charge and on its distribution on the macrocycle, was observed. Analysis of the fatty acid profile has shown a decrease of the unsaturated fatty acids, corroborating the relation between lipid oxidation and PDI efficiency. Bacterial membrane phospholipids are important molecular targets of photoinactivation and the number of charges of the PS molecule, as well as their distribution, have a clear effect on the lipid oxidation and on the efficiency of PDI. The distinct extent of the formation of lipid hydroperoxy derivatives, depending on the PS used, is a good indicator of this process. The FOX2 assay allowed the detection of lipid peroxidation of E. coli membrane after PDI with all the five porphyrins, however, it was not the most appropriated method to quantify the relative lipid oxidation caused by PS with different efficiencies. The fatty acid analysis used to quantify the extent of lipid oxidation by the different PS provided better results. The same results were observed for the liposome model. Consequently, the model system based on liposomes is a fast and simple method that can be used for the screening of the efficiency of new PS, before proceeding with the more complex studies on bacterial models.