Augmented glycerosomes as a promising approach against fungal ear infection: Optimization and microbiological, ex vivo and in vivo assessments

Abstract

In the current study, voriconazole (VCZ) augmented glycerosomes were optimized for topical otomycosis management according to a 2³ factorial design, employing a thin film hydration method. By optimizing Glycerol volume, limonene: VCZ ratio and Span® 60: soybean phosphatidyl choline (PC) ratio, glycerosomes with maximum percentage entrapment efficiency (%EE) and zeta potential (ZP) and minimum vesicle size (VS) and polydispersity index (PDI) were to be obtained. An optimal augmented glycerosomal formula (OAG) that contained 10 mg VCZ, 150 mg PC, and 3 mL glycerol, comprising 2.5: and 0.92:1 ratios of the latter two independent variables, was proposed via numerical optimization. OAG exhibited high %EE and ZP values and acceptable low values for VS and PDI (84.3 ± 2.0 %, -38.8 ± 1.8 mV, 191.0 ± 1.1 nm, and 0.192 ± 0.01, respectively). Extensive in vitro testing of OAG revealed the entrapment of VCZ within OAG, biphasic in vitro release profile, stability for up to 3 months at 2-8 °C and spherical morphology of OAG with VS like that obtained via zetasizer. OAG demonstrated higher permeated amounts of VCZ and flux values than VCZ suspension, leading to an enhancement ratio of 2.56 in the ex vivo permeation study. The deeper penetration ability of OAG demonstrated by Confocal Laser Scanning Microscopy and its superior in vitro antifungal activity confirmed the validity of the ex vivo study. Also, the histopathological study confirmed the safety of OAG for topical use, suggesting that VCZ OAG was a promising topical antimycotic formula.

Keywords: Augmented glycerosomes; Factorial design; Minimal fungicidal concentration; Otomycosis; Transmission electron microscopy; Voriconazole.

Graphical abstract

Fig. 1

The effect of different significant formulation variables on: (a and b): percentage entrapment efficiency, (c and d): vesicle size and (e and f): zeta potential of voriconazole augmented glycerosomes.

Fig. 2

(a) Transmission Electron Microscope micrograph of optimal voriconazole augmented glycerosomes and (b) Fourier Transform Infrared spectra of i: pure voriconazole, ii: Span® 60, iii: soybean phosphatidyl choline and iv: lyophilized optimal voriconazole augmented glycerosomes.

Fig. 3

(a) In vitro release profiles of fresh and stored optimal voriconazole augmented glycerosomes, compared to voriconazole suspension and (b) Ex vivo permeation profiles of optimal voriconazole augmented glycerosomes and voriconazole suspension.

Fig. 4

(a) Antifungal activity determination by Kirby–Bauer disk diffusion technique showing inhibition zones upon the loading of 5 μg of i: voriconazole suspension and ii: optimal voriconazole augmented glycerosomes on sterile filter paper disks. (b) Minimal inhibitory concentration values (μg/mL) of both the voriconazole suspension and optimal voriconazole augmented glycerosomes (Statistical significance assessed by independent t-test (n = 9), p = 0.00025).

Fig. 5

Microscopic photographs (16× magnification) showing normal histological structure of rabbit ear skin treated with (a) normal saline (negative control) and (b) optimal voriconazole augmented glycerosomes.

Fig. 6

Confocal Laser Scanning Microscope micrographs showing different penetration depth of rhodamine B through rabbit ear skin from (a) aqueous solution and (b) optimal augmented glycerosomes.