Photoactive dye doped polymeric nanoparticles: an efficient toolbox in photodynamic inactivation of pathogens

MARTINEZ, SOL R.; WENDEL, ANA; PORPORATO, CARINA ; PALACIOS, RODRIGO E.; PONZIO, RODRIGO; FORCONE, VIRGINIA; SPESIA, MARIANA B.; IBARRA, LUIS; CAGNETTA, GONZALO; CHESTA, CARLOS A.

Viña del Mar

Encuentro; XIV Encuentros Latinoamericanos de Fotoquímica y Fotobiología; 2019

Microorganisms, particularly bacteria, have been natives of the earth for 3.5 millions of years and their evolution demonstrates that they can adapt to many different environments. The antibiotic development has seemed to give battle against pathogens. However, in 2017 the World Health Organization (WHO) published its first-ever list of antibiotic-resistant priority pathogens. The most critical group of multidrug resistant bacteria were Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and the methicillin-resistant Staphylococcus aureus (MRSA). In response to this latent threat, scientists developed alternative therapies such as photodynamic inactivation (PDI), which emerged late in the 20th century as a promising treatment to kill pathogens. In this work, we efficiently combine photoactive dye doped polymeric nanoparticles (NPs) with blue light to kill some multidrug pathogens included in the WHO list. Initial screening with different NPs concentrations (0.3 to 6.5 ppm) and irradiation doses was performed in nine pathogenic strains to explore the efficiency of the PDI treatment in planktonic cells. Bactericidal effect was observed in MRSA strains using short irradiation period/ low light dose (10-15 min/ 24-36 J/cm2). The Gram-negative species required higher light (72 J/cm2) and NPs (16.5 ppm) doses. Bacterial NPs uptake was initially studied using bright-field and fluorescent microscopy taking advantage of the intrinsic NPs fluorescence. Further studies on NPs uptake for different bacterial genders(species) were performed using flow cytometry. Restults from both techniques indicate that NPs superficially bind and slightly penetrate the bacterial envelope. To further explore PDI treatment strategies using our NPs we tested their performance on mature bacterial biofilms. In both Gram-positive and negative bacterial strains, the crystal violet assay displayed a similar antibiofilm trend after PDI treatment. At the same time, the effect achieved on the metabolic activity of the cellular cluster after PDI treatment was larger in Gram-negative cells than Gram-positive strains; showing a drop in the activity between 67 to 84 % respect to the control. To further test the overall effect on the biofilm matrix and the eradication effect of our PDI therapy we used confocal microscopy. For these experiments E. coli and S. aureus were selected and both strains exhibited substantial matrix disruption. Notably, almost complete biofilm eradication was achieved for the E. coli strain. Overall, our results demonstrated that PDI protocols using NPs plus blue light are an efficient tool not only to kill superbugs as sessile cells but also to disrupt and eradicate mature biofilms of relevant bacterial species.