Experimental research on fungal inhibition using dissolving microneedles of terbinafine hydrochloride nanoemulsion for beta-1,3-glucanase

Abstract

Objective: Onychomycosis is characterized by high transmission and low recovery rates, leading to a lack of optimal treatments. This study used dissolving microneedles as drug carriers to design dual-loaded DMN patches with β-1,3-glucanase and terbinafine hydrochloride nanoemulsion. Methods: Dissolving microneedles of terbinafine hydrochloride nanoemulsion for beta-1,3-glucanase (Gls-TBH-NE-DMN) were prepared using the centrifugal molding method. Parafilm®, weight method, mouse skin puncture experiments, and mouse skin tissue sections were used to evaluate the mechanical properties of Gls-TBH-NE-DMN. Gls-TBH-NE-DMN was tested for its in vitro anti-Candida albicans susceptibility, ability to inhibit fungal cell wall synthesis, and fungal biofilm penetration and inhibition capabilities. The in vivo inhibitory effect of Gls-TBH-NE-DMN on fungi was studied using a rabbit nail infection model. Results: Gls-TBH-NE-DMN exhibited a penetration rate of 98% on skin simulants and a compressive bending resistance of 12.75%. It was nonirritating to the dorsal skin of mice, with no edema or erythema on the surface of the skin, suggesting a good safety profile. The accumulative release of Gls-TBH-NE-DMN at 72 h was 78.74% ± 0.64%, and that of terbinafine hydrochloride dissolving microneedles was 49.52% ± 0.80%. In the in vitro transdermal permeation experiment, the cumulative transdermal permeation of Gls-TBH-NE-DMN at 72 h was 73.21% ± 0.84% and that of terbinafine hydrochloride (TBH) was 20.57% ± 0.98%. In vitro inhibition of C. albicans showed that the lowest inhibitory concentration in the Gls-TBH-NE-DMN group was 512 μg mL^-1. Furthermore, experiments showed that Gls-TBH-NE-DMN could effectively inhibit fungal cell wall synthesis and disrupt the C. albicans biofilm, inhibiting fungal growth. Conclusions: Gls-TBH-NE-DMN prepared in this study provides new ideas for treating skin fungal diseases and for developing antimicrobial drug formulations.

Fig. 1. Preparation, morphological characterization and mechanical property evaluation of terbinafine hydrochloride nanoemulsion for beta-1,3-glucanase (Gls–TBH-NE–DMN): (a) preparation process of Gls–TBH-NE–DMN, (b)–(e) morphology of Gls–TBH-NE–DMN, (f and g) mechanical strength examination of Gls–TBH-NE–DMN, and (h and i) pressure performance examination of Gls–TBH-NE–DMN.

Fig. 2. Results of experiments on dorsal skin of mice with Gls–TBH-NE–DMN: (a) skin puncture experiments with Gls–TBH-NE–DMN, (b) histological sections of Gls–TBH-NE–DMN pricked mice skin, (c) skin irritation assessments of Gls–TBH-NE–DMN, (d) skin healing experiments with Gls–TBH-NE–DMN, and (e) intradermal dissolution experiments of Gls–TBH-NE–DMN.

Fig. 3. Differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), in vitro release degree and in vitro transdermal permeation experiments of Gls–TBH-NE–DMN: (a) DSC curves of Gls–TBH-NE–DMN, (b) FTIR spectra of Gls–TBH-NE–DMN, (c) in vitro release degree experiments of Gls–TBH-NE–DMN (x̄ ± s, n = 3), (a statistically significant difference was observed between the TBH-DMN and Gls–TBH-NE–DMN groups (*p < 0.05), and (d) in vitro transdermal permeation experiments of Gls–TBH-NE–DMN (x̄ ± s, n = 3), (significant differences were observed among groups (**p < 0.01), with TBH-NE-DMN, Gls–TBH-NE–DMN, and positive controls showing statistically significant variations compared with the TBH group).

Fig. 4. In vitro fungal inhibition experiments of Gls–TBH-NE–DMN: (a) and (b) antifungal susceptibility studies of Gls–TBH-NE–DMN, (c) fungal cell wall synthesis inhibition experiments of Gls–TBH-NE–DMN, and (d and e) fungal biofilm permeation and inhibition experiments of Gls–TBH-NE–DMN.

Fig. 5. In vivo fungal inhibition experiments of Gls–TBH-NE–DMN: (a) microscopic observations of rabbit nail tissues and (b) histopathological results of rabbit nail sections.