Desenvolvimento de formulação contendo azul de metileno associado a polímero para terapia fotodinâmica antimicrobiana odontológica

Abstract

Methylene blue (AM) is a cationic phenothiazine compound with interesting properties for application in antimicrobial photodynamic therapy (aPDT). Its aggregation characteristic directs the photochemical action mechanisms and can be favored depending on the medium in which it is transmitted. Additionally, difficulties in maintaining a solution in specific locations in the oral cavity during the pre-irradiation period motivated the development of a formulation with higher viscosity in order to increase the residence time in the location and preserve the photochemical properties of AM. The objective of this research was to develop AM formulation for aPDT associated with mucoadhesive biopolymers of different ionic characters: hydroxypropylmethylcellulose and carboxymethylcellulose (CMC), as well as the anionic surfactant sodium dodecyl sulfate (SDS). The AM aggregation state was evaluated by spectrophotometric determination of the dimer/monomer (D/M) ratio. The viscosity and density of the formulations, the influence of pH and three AM concentrations on the D/M ratio were analyzed. In vitro microbiological tests were conducted on Candida albicans (ATCC 10231) biofilm. In the incorporation experiment, the samples were treated with AM conveyed in water or in a formulation and the pre-incubation time evaluated was 5, 10, 30 and 60 min. Viability was evaluated in water at acidic pH, formula, AM at 0.005% and 0.010%, in water and in formula. After 5 min in the dark, the biofilm was washed and irradiated for 27 or 54 min. Dilutions were incubated and colony forming units per mL (CFU/mL) were counted. The selected formulation had its stability evaluated for one year through organoleptic tests, pH, AM monomer quantification and D/M ratio. Results indicate a lower D/M ratio with increasing concentration of polymers without SDS, however, with the addition of surfactant, there is an even greater reduction in D/M for the two polymers studied, with less aggregation of AM observed in the acidic formulation with CMC and SDS. Lower AM concentrations favor less aggregation, with no statistical difference between 0.002% and 0.005%. AM conveyed in water presents greater incorporation than in formula. Viability in 24h biofilm resulted in a significant reduction only for the formulation containing 0.005% AM. In a 48-hour biofilm, all AM samples, in water or formula, showed a significant reduction, with two successive 27-min irradiations with double treatment being more effective than the single 54-min irradiation, reaching a maximum average reduction of 4.26 Log for the 0.005% AM formula, demonstrating that the lower incorporation did not affect the antimicrobial action for this sample. The optimized formulation remained stable for 1 year in terms of organoleptic characteristics, pH and AM quantification. Between 6 and 12 months, the D/M ratio showed a statistical difference, although it may not be relevant since the values obtained were very close. Therefore, the association of pharmacotechnical adjuvants, the ionic charge of the polymer, the AM concentration and the pH of the medium in which it is conveyed influence its aggregation behavior, making it safe to use the optimized formulation developed for 12 months after manufacturing and effective for aPDT according to in vitro viability assessments in C. albicans biofilm with a pre-irradiation time of 5 min and 2 successive irradiations according to established parameters, without compromising antimicrobial effectiveness and with the advantage of favoring greater retention at the applied site. Additional studies can be carried out, aiming to optimize dosimetry in order to achieve better performance for clinical aPDT.