Nanostructured lipid carriers dispersed in situ gelling hydrogel for vaginal administration of hypericin associated with photodynamic therapy in the treatment of vulvovaginal candidiasis

Author(s)

Mariana Rillo Sato

• Document type: Doctoral Thesis
• Press: Araraquara. 2020-10-27.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara
• Defense date: 2020-01-30
• Advisor: Marlus Chorilli

Abstract

Vulvovaginal candidiasis (VVC) is a fungal infection typically caused by Candida albicans, which is resistant to the drugs used in the treatment. The hypericin (HYP)- loaded nanostructured lipid carrier (NLC) dispersed in situ hydrogels (HG) proves to be an interesting option for the treatment of VVC, associated to alternative methods such as photodynamic therapy (PDT). This work aims to evaluate the potential of HYPloaded and empty NLC or NLC dispersed in the HG, associated to PDT for vaginal administration. The empty NLC (F6.5) and HYP-loaded NLC (F6.5_0.05, F6.5_0.25 and F6.5_0.5) and were obtained by sonication at 35% amplitude for 5min, containing poloxamer 407 the aqueous phase and in the oil phase, polyoxyethylene stearate, capric/caprylic acid triglycerides, ethoxylated hydrogenated castor oil and/or HYP. The HG were composed of poloxamer 407 and chitosan, being incorporated into HYPloaded and empty NLC in different proportions of NLC:HG giving rise to formulations NLC_HYP-HG1, NLC_HYP-HG2, NLC_HYP-HG3, NLC_HYP-HG4 and NLC_HYPHG5. The F6.5, F6.5_0.05, F6.5_0.25 and F6.5_0.5 systems, characterized by dynamic light scattering, presented nanometric diameters with low polydispersity and showed excellent physical stability. Formulation F6.5 showed unimodal distribution in the nanoparticle screening with predominance of 151 nm particles. Transmission electron microscopy showed particles of practically spherical morphologies and nanometric scales. The HYP encapsulation efficiency was 89.3%. Scanning electron microscopy of the HG and HYP-loaded NLC in different proportions of NLC:HG, revealed average values of surface roughness without statistical difference between the systems. Continuous rheology showed that all systems had Newtonian behavior; while in oscillatory rheology most systems showed G’ values higher than G’’, except of NLC_HYP-HG1. The HG and the NLC_HYP-HG1 and NLC_HYP-HG2 systems showed satisfactory mucoadhesive properties, in addition to syringability values that, compared to reports in the literature, showed their ease of application in the vaginal route. Differential scanning calorimetry showed endothermic events with similar peaks and characteristics to components present in the formulations. Two analytical methods were validated to quantify the HYP (HYP solubilized in methanol and HYP in the receiving solution of phosphate buffered medium with 3% Tween 80), being consistent with the values recommended by ANVISA. The in vitro release profile of HYP released from NLC_HYP-HG1, indicated a sustained drug release behavior during the 12h of analysis, and that the main release mechanisms follow the Peppas and Higuchi models. Such systems showed no permeation in the porcine vaginal mucosa, in addition to low retention values. The NLC with encapsulated HYP dispersed or not in HG, showed potent antifungal action against the strain of C. albicans ATCC 10231 and fungistatic behavior against yeast. The HG incorporated into HYP-loaded and empty NLC in different proportions, treated with PDT, significantly reduced the planktonic cultures of C. albicans ranging from approximately 1 to 2 log10 (CFU/mL) compared to YNB; while the PDT mediated by the F6.5_0.05 and NLC_HYP-HG1 systems showed decreases in the viability of the C. albicans biofilm of 2 and 1.2 log10 (CFU/mL), respectively, in relation to the PBS. In the SOSG analysis, the free HYP solution, as well as the HYP-loaded NLC dispersed in HG were able to effectively supply oxygen to the methanol solution. In the in vivo antifungal analysis associated or not with PDT, the antifungal and groups treated with PDT showed a predominance of fungal cells of different sizes, in addition, NLC_HYP-HG1/PDT stood out among the groups treated with PDT, obtaining a significant improvement in the therapeutic profile compared to the infected group. Thus, the results showed the antifungal potential of in situ systems for the vaginal administration of HYP, demonstrating that they are capable of contributing to the treatment of VVC. (AU)

• FAPESP's process: 16/11198-4 - Nanostructured lipid carriers dispersed in situ gelling hydrogel for vaginal administration of hypericin associated with photodynamic therapy in the treatment of vulvovaginal candidiasis
• Grantee: Mariana Rillo Sato
• Support Opportunities: Scholarships in Brazil - Doctorate