Avaliação da atividade antimicrobiana in vitro do peptídeo Lrot3.6 nanoencapsulado frente às bactérias E. coli, S. aureus e S. epidermidis

Abstract

Bacterial infections are a threat to human health and a serious problem for health systems due to an increasing incidence of infections caused by multiresistant bacteria. Among important bacteria that cause infections are Staphylococcus aureus, a species of major medical interest, being the most common cause of nosocomial and community infections; Staphylococcus epidermidis, the most frequent isolate found in human epithelia, being the main responsible for infections related to medical devices; and Escherichia coli among gram-negative bacteria is the most common cause of nosocomial and community-acquired infections. Antimicrobial peptides (AMPs) are a diverse and abundant group of compounds produced by multicellular organisms, both animal and plant, and are major components of innate immunity against infections and may be alternatives in the treatment of infectious diseases. In this context, the association between AMPs and nanotechnology can be used as a strategy to combat bacterial infections. Among the existing nanomaterials are polymer nanoparticles, which are biodegradable and biocompatible. These nanomaterials may participate in formulations containing usual antibiotics or novel bioactive molecules, such as AMPs, and may be promising alternatives for the treatment of bacterial infections. The objective of this work was to evaluate the antimicrobial potential of the peptide Lrot3.6 nanoencapsulated against gram-positive and gram-negative bacteria, as well as the cytotoxic effects in vitro culture of HEK293 cells. Free chitosan nanoparticles were synthesized and structured with the Lrot3.6 peptide by the ionic gelation method. The synthesized nanoparticles were characterized by size, polydispersity index, Zeta potential and the peptide encapsulation efficiency was determined. After the synthesis, antimicrobial assays were performed by microdilution in a 96-well plate using chloramphenicol, the free and nanoencapsulated peptide, and the free nanoparticle at the concentrations of 8, 16, 32, 64 and 128 μg/mL, as well as assay for analysis of the bactericidal or bacteriostatic action of the treatments and evaluation of the cytotoxicity of the treatments by MTT. Statistical analyzes were performed using the Tukey test (p<0.05). The polydispersity indices were 0.356 (free nanoparticle) and 0.398 (nanoencapsulated peptide). Zeta potentials were 36.5 ± 2.1 mV (free nanoparticle) and 35.7 ± 2.2 mV (nanoencapsulated peptide). The nanoencapsulated peptide showed an encapsulation efficiency of 93.55%. Regarding the antimicrobial assays, the free peptide showed MIC of 8 μg/mL for all the bacteria tested. The free nanoparticle also showed antimicrobial effect, with MIC of 64 μg/mL for all bacteria. Nanoencapsulation of the peptide mainly favored the inhibition of the growth of E. coli, a bacterium in which the treatment was most highlighted by a possible synergistic effect between the chitosan and the peptide Lrot3.6. In general, prolonged release of the peptide was also observed within 48 hours. In addition, the nanosystem at the lowest concentration, 8 μg/mL, was not cytotoxic within 48 hours.