Chlorhexidine nanoemulsion: a new antiseptic formulation v1
Authors: NHR Horstmann Risso Natalia , Denise Stopiglia Cheila , Teresa Oliveira Marilia , Elisa Haas Sandra , Ramos Maciel Tamara , Reginato Lazzari Natalia , Luis Kelmer Edilson , Abraão Pinto Vilela Jorge , Vilibaldo Beckmann Diego 
Published: September 2019

Antisepsis is the process that decrease the measure of microorganism present in tissues by the use of antiseptics, such as iodophors, chlorhexidine and alcohol. Chlorhexidine is widely used and recommended as a topical antiseptic but resistance reports to these antiseptic are becoming common and strategies are needed either to maximize the effect or development of new. The nanoparticles solutions are an alternative, since development of these formulations with microbicide or microstatic effect is an option to conventional formulations with free molecule. The aim of this study was the characterization of a 0.25% aqueous-based chlorhexidine nanoemulsion (NM-Cl 0.25%) and to evaluate the antiseptic effect in vitro and in vivo. The NM-Cl 0.25% and a white nanoemulsion (same composition of NM-Cl without the molecule of chlorhexidine) were sintetized by spontaneous emulsification method and physical and chemical properties characterization of the formulations were realized immediately after preparation (mean diameter of the droplet, Zeta potential, pH, quantification and dosing of chlorhexidine by high performance liquid chromatography and encapsulation rate of nanoemulsion). Based on this, two formulations were compared NM-Cl 0.25% (2.5mg/mL) and Chlorhexidine® 0.5% alcohol base (CS-Cl 0,5%). The white nanoemulsion was used as a negative control to ensure that the action against the bacterias was because of the chlorhexidine molecule. Two in vitro studies were performed: Susceptibility of bacteria’s against chlorhexidine formulations and the residual effect of the chlorhexidine formulations. Susceptibility of bacteria’s against the chlorhexidine formulations was evaluated by the broth microdilution method according to Clinical and Laboratory Standards Institute – CLSI, protocol M100-S25.The residual effect of the formulations was evaluated by the kill curve methodology in 24 hours, samples were collected in the following times after inoculation of the formulation: immediatly; two hours; four hours; six hours; eight hours; 12 hours. The in vivo study was performed with 20 male rats (Rattus novergicus albinus -Wistar lineage) that were randomly distributed into control group (G1 and G2) and chlorhexidine group (G3 e G4): G1 - antisepsis out, G2 - white nanoemulsion, G3 - CS-Cl at 0.5% and G4 - NM-Cl 0.25% (test formulation). Samples were collected before antisepsis and after the antisepsis in the following sample times: immediately after, one hour, two hours and three hours, immediately inoculated in blood agar and after 24 hours manual counting of Colony Forming Units (CFU) was performed. Kruskal-Wallis test was used between groups and in the same group, at different sample times. When detected difference Mann Whitney test was performed, comparing the same times between the groups and when detected difference in the same group all the times were tested. The NM-Cl presented adequate physicochemical characteristics for a nanoemulsion and presented antiseptic effect in the microdilution study, having the best effect between the tested formulations. In the Kill curve study there was a slower effect of the nanoemulsions but that was maintained for longer time when compared to chlorhexidine with free molecule. The nanoemulsion groups four-fold of Minimal Inhibitory Concentration were the formulations with the most durable effect of those tested, presenting residual effect until T6 for both bacteria tested. At the in vivo study both formulations (G3 and G4) had a reduction of the microorganisms in the skin of the rats (p <0.0001) and as no difference of action was detected between the formulations in the tested times, showing the effect of the NM-Cl as an antiseptic (p > 0,05). NM-Cl presented satisfactory results at in vitro and in vivo tests despite being a formulation with lower concentration of chlorhexidine than the commercial formulation that is recommended for pre-surgical antisepsis. Therefore, the results show a nanostructured formulation with adequate physicochemical characteristics, antiseptic effect and residual antiseptic effect that are even better than the formulation with free molecule in the in vitro study and similar in vivo assay. Due to different mechanism of action, the NM-Cl has potential to avoid the resistance mechanisms of bacteria, being an alternative for formulation with free molecule. The NM-Cl is a viable antiseptic and potentially cheaper formulation, because of the similar effect with lower concentration of chlorhexidine.