Development of chitosan-g-poly(N-vinylcaprolactam) nanoparticles as a carrier of agents for extracellular DNA degradation and therapy of bacteria-infected oral epithelial cells for application in peri-implant disease

Abstract

The use of nanoparticles (NPs) to encapsulate, carry and deliver drugs is a strategy that can be used targeting the treatment of peri-implant diseases in the therapy of oral epithelial cells infected by bacteria, as well as in the degradation of extracellular DNA present in the extracellular matrix of the biofilm. The aim of this study is to develop biodegradable and thermosensitive chitosan-g-poly(N-vinylcaprolactam) NPs as carriers of agents for the degradation of extracellular DNA and therapy of oral epithelial cells infected by bacteria for application in peri-implant disease. Initially, the minimum inhibitory concentration and the minimum bactericidal concentration of metronidazole (MTZ) and the extracellular DNA degrading agent deoxyribonuclease (DNase) will be determined against human salivary microbiota in order to establish the initial concentration of the drugs to be incorporated into the NPs. The study will include the following groups: (1) NPs without drug encapsulation as a control; (2) NPs + MTZ; (3) NPs + DNase; (4) NPs + MTZ + DNase. The NPs will be characterized by critical transition temperature analysis, dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), zeta potential, degradation by TG/DTA, drug loading and release efficiency using ultraviolet-visible spectroscopy (UV/VIS), cytotoxicity when in contact with human keratinocyte and gingival fibroblast cells and visualization of the NPs in an intracellular environment using fluorescence microscopy. An in vitro microbiological test will be carried out to verify the antibacterial effectiveness and DNA degradation of the NPs by quantifying colony-forming units (CFU) using monospecies (Porphyromonas gingivalis) and multispecies biofilms (microcosm model), dry weight, microbial composition by DNA-DNA checkerboard, exopolysaccharide dosage, extracellular DNA dosage, scanning electron microscopy for structural organization and laser confocal microscopy to verify the presence of live, dead and matrix cells. In addition, this new material will be investigated for its intra- and extracellular antimicrobial potential and its effect on the inflammatory response, using an organotypic oral mucosa tissue model. To this end, the tissue will be contaminated with monospecies (P. gingivalis) and multispecies (microcosm) biofilm and exposed to the NPs. The analyses will include CFU counting, lactate dehydrogenase release, expression of the cytokines IL-1², IL-6, IL-8 and TNF±, gene expression of E-cad, histological analysis and immunofluorescence. Quantitative data will be subjected to appropriate statistical analysis with a significance level of 5%.