Effects of Lidocaine/Prilocaine mixture in poly-L-lysine/hyaluronic acid films on gingival fibroblasts and in a tail model in mice: in vitro and in vivo study

Abstract

Researches on local anesthetic (LA) seek clinical treatment as close as possible to the "no pain" treatment. Although used for the temporary blockage of the nervous conductivity, LAs also modulate cellular pathways related to inflammatory response, wound healing, apoptosis and cellular necrosis. While the search for the ideal molecule remains a challenge, new anesthetic formulations from known molecules have been developed using controlled release systems. New biopolymers have being studied seeking the improvement in effectiveness and decreased toxicity of local anesthetic agents. The combination of hyaluronic acid (HA) and poly(L-lysine) or PLL produces a multilayer film and it can serve effectively as a platform for drug release. The aim of this project is to observe the effect of films based on PLL/HA in cell cultures and in a tail flick model in mice. Films prepared with PLL/HA of 24 and 96 layers impregnated or not with the mixture of lidocaine and prilocaine, both at 2.5%, will be compared with the EMLA commercial formulation (positive control). After prepared, the films will be submitted to tests to observed their muco-adhesive, retention time and permeation in pig oral mucosa and esophagus. The ALs will be quantified in release, permeation, content and uniformity experiments by liquid chromatography (HPLC). After characterization, the films will be used in cell culture of human immortalized keratinocytes (HaCaT) and human gingival fibroblasts (FGH). Once exposed to the films, these cells will be subjected to cell viability (XTT reduction method) and release of Pro-Inflammatory Cytokines (IL-8, IL1-±, IL-6 and TNF-±), IL-10 and PGE2 tests. Cytotoxicity analysis by fluorescence microscopy with the LIVE/DEAD method will also be used. Then, the films will be submitted to the in vivo test using the tail-flick model in mice (Swiss). The normality of distribution and equivalence of variances of results will be tested and subsequently subjected to parametric or appropriate non-parametric statistical tests. All the analyses will have a significance level of 5%. (AU)