Development and Characterization of Nitazoxanide and Quercetin Co-loaded Nanotransfersomal Gel for Topical Treatment of Cutaneous Leishmaniasis

Abstract

Anti-leishmanial medications administered by oral and parenteral routes are less effective for treatment of cutaneous leishmaniasis (CL) and cause toxicity, hence targeted and localized drug delivery methods, are efficient way to improve drug availability for CL. The aim of study was to develop nitazoxanide and quercetin co loaded nanotransfersomes (NTZ-QUR-NT) for topical delivery of drugs against CL. To prepare NTZ-QUR-NT thin film hydration method was used and they were statistically optimized by using Box-Behnken Design in terms of particle size, zeta potential (ZP), polydispersity index (PDI) and entrapment efficiency (EE) of both drugs. Lipid, surfactant and QUR concentrations were taken as independent variables. Transmission Electron Microscopy (TEM) analysis was performed for surface morphology and particle size assessment. Fourier-transform infrared spectroscopy (FTIR) was performed to identify any chemical interaction between components of the formulation. NTZ-QUR-NT were incorporated into 2% chitosan gel to prepare NTZ-QUR-NT gel (NTZ-QUR-NTG). In-vitro release of NTZ-QUR-NT, NTZ-QUR-NTG and NTZ, QUR dispersions were performed to check the release pattern of drugs. To evaluate the penetration behavior of NTZ-QUR-NT and NTZ-QUR-NTG as compared to NTZ and QUR gels (conventional gels), ex-vivo permeation study was performed using Franz diffusion cells. Macrophage uptake, cell viability and toxicity study were performed to investigate the macrophage targeting and potential toxic effects of NTZ-QUR-NT in comparison with NTZ-QUR dispersion. The anti-leishmanial assay was performed to evaluate the anti-leishmanial potential of NTZ-QUR-NT as compared to NTZ-QUR dispersion. The optimized formulation indicated particle size of 210.9 ± 3.67 nm, PDI of 0.155 ± 0.009, ZP of -15.06 ± 1.48 mV and EE of NTZ and QUR 88.04 ± 0.01% and 85.14 ± 0.02%, respectively. TEM analysis of the optimized NTZ-QUR-NT showed spherical and uniform particles. No chemical interaction between components of NTZ-QUR-NT was found after Fourier-transform infrared spectroscopy (FTIR) analysis. In-vitro release of NTZ and QUR from NTZ-QUR-NT and NTZ-QUR-NTG showed more sustained behavior as compared to NTZ, QUR dispersions. Significant increase in skin permeation of NTZ and QUR was observed in case of NTZ-QUR-NT and NTZ-QUR NTG in comparison to conventional gels. To ease the topical delivery the NTZ-QUR NT were dispersed in 2% chitosan gel. Skin deposition studies demonstrated the better viii retention profiles of drugs in case of NTZ-QUR-NT and NTZ-QUR-NTG at dermal level. In-vivo skin irritation and histopathological findings did not show any topical irritation associated with NTZ-QUR-NTG. Macrophage uptake analysis showed that NTZ-QUR-NT cell internalization was almost 10-folds higher in comparison to NTZ QUR dispersion. It was clear from result that NTZ-QUR dispersion was distributed rather than particularly internalized by macrophage. The cytotoxicity result showed that NTZ-QUR-NT have CC50 value of 71.95 ± 3.32 µg/ml and NTZ-QUR dispersion have 49.77 ± 2.15 µg/ml, respectively, which indicates that NTZ-QUR-NT is safer to use as compared to NTZ-QUR dispersion. Moreover, in-vitro anti-leishmanial assay presented that NTZ-QUR-NT have IC50 value of 3.15 ± 0.89 µg/ml against Leishmania tropica promastigotes which was 6.24-times lower than NTZ-QUR dispersion. For the deep seated infections like leishmaniasis, topical application of NTZ-QUR-NTG would be a suitable choice because of its targeted action and efficacy as evident in this study.