In vitro kinetic release study, antimicrobial activity and in vivo toxicity profile of a kojic acid ester-based nanoemulsion for topical application

Abstract

Nanoemulsions have emerged as novel vehicles for drug delivery that allow sustained or controlled release for topical application. In this study, kojic acid ester-based nanoemulsion (KAE-NA) was analyzed for in vitro permeation evaluation, kinetic release study, in vitro antimicrobial activity and in vivo toxicity profile on embryonic zebrafish (Danio rerio). Based on KAE-NA in vitro permeation evaluation, the percentage of permeation was significantly improved from 4.94% at 1 h to 59.64% at 8 h of application. The permeation rate of KAE-NA at 8 h was 4659.50 μg cm^-2 h^-1 (initial concentration, C ₀ = 2000 μg mL^-1) with a permeability coefficient (K _p) value of 0.48 cm h^-1. The kinetic release analysis showed the Korsmeyer-Peppas model was the best fitted kinetic model with high linearity [R ² = 0.9964]. Antimicrobial activity of KAE-NA was studied against the skin pathogen bacteria Staphylococcus aureus ATCC 43300. The results indicated that the inhibition zone size of the KAE-NA (8.00 ± 0.0 mm) was slightly bigger than that of its active ingredient, kojic acid ester (6.5 ± 0.0 mm). The toxicity profile of KAE-NA on embryonic zebrafish revealed less toxicity with LC₅₀ (50% lethal concentration) more than 500 μg mL^-1. The survival rate of the embryonic zebrafish was more than 80% when treated at doses ranging from 7.81-250 μg mL^-1 and showed normal development throughout the experiment without any observed deformation. Hence, KAE-NA proved to be less toxic on the embryonic zebrafish.

Fig. 1. Calibration curve of kojic acid ester (μg mL⁻¹).

Fig. 2. In vitro permeation study for kojic acid ester based-nanoemulsion (KAE-NA).

Fig. 3. Cumulative kojic acid ester permeation from nanoemulsion (KAE-NA) through cellulose acetate membrane.

Fig. 4. Graph of kinetic models (a) zero order (b) first-order (c) Higuchi (d) Hixson–Crowell (e) Korsmeyer–Peppas.

Fig. 5. Inhibition zone of (a) kojic acid ester and (b) kojic acid ester-based nanoemulsion towards Staphylococcus aureus ATCC 43300.

Fig. 6. The survival rate of embryonic zebrafish treated with kojic acid ester-based nanoemulsion at 0 to 120 h at different sample concentrations.

Fig. 7. Healthy images of zebrafish embryogenesis showing 4 stages of development at different hours of post fertilization (hpf) captured using inverted microscope at 10× digital magnification. (i) Blastula period (4 hpf); (ii) segmentation period (24 hpf); (iii) pharyngula period (48 hpf); (iv) hatching period (72 hpf). A-eye anlarge; An-anus; Bc-blood cells; C-chorda; Ch-chorion; Cb-curved body; F-fin; G-gut; M-melanophores; O-ear bud; P-pericard; Y-yolk sac. Scale bar = 0.5 mm.

Fig. 8. Images of malformation defect in zebrafish embryo and larvae after 120 hours of exposure to a different concentration of KAE-NA captured using microscope 100× digital magnification. (v) Coagulated embryo; (vi) unhatched embryo; (vii) mPe-mild pericardial edema; (viii) Sbl-short body length, Cb-curve body, Ct-curve tail, cPe-chronic pericardial. Scale bar = 0.5 mm.