Enhanced Ocular Anti-Aspergillus Activity of Tolnaftate Employing Novel Cosolvent-Modified Spanlastics: Formulation, Statistical Optimization, Kill Kinetics, Ex Vivo Trans-Corneal Permeation, In Vivo Histopathological and Susceptibility Study

Abstract

Tolnaftate (TOL) is a thiocarbamate fungicidal drug used topically in the form of creams and ointments. No ocular formulations of TOL are available for fungal keratitis (FK) treatment due to its poor water solubility and unique ocular barriers. Therefore, this study aimed at developing novel modified spanlastics by modulating spanlastics composition using different glycols for enhancing TOL ocular delivery. To achieve this goal, TOL basic spanlastics were prepared by ethanol injection method using a full 3² factorial design. By applying the desirability function, the optimal formula (BS6) was selected and used as a nucleus for preparing and optimizing TOL-cosolvent spanlastics according to the full 3¹.2¹ factorial design. The optimal formula (MS6) was prepared using 30% propylene glycol and showed entrapment efficiency percent (EE%) of 66.10 ± 0.57%, particle size (PS) of 231.20 ± 0.141 nm, and zeta potential (ZP) of -32.15 ± 0.07 mV. MS6 was compared to BS6 and both nanovesicles significantly increased the corneal permeation potential of TOL than drug suspension. Additionally, in vivo histopathological experiment was accomplished and confirmed the tolerability of MS6 for ocular use. The fungal susceptibility testing using Aspergillus niger confirmed that MS6 displayed more durable growth inhibition than drug suspension. Therefore, MS6 can be a promising option for enhanced TOL ocular delivery.

Keywords: cosolvent; fungal keratitis; kill kinetics; spanlastics; susceptibility; tolnaftate.

Figure 1

Line plots of the significant effect of Span 60 amount (X₁) (a), Tween 80 amount (X₂) (b), response 3D plot for the combined effect of amount of Span 60 (X₁) and amount of Tween 80 (X₂) (c) on EE% of TOL basic spanlastics.

Figure 2

Line plot of the significant effect of Span 60 amount (X₁) (a) and response 3D plot for the combined effect of Span 60 amount (X₁) and Tween 80 amount (X₂) (b) on PS of TOL basic spanlastics.

Figure 3

Line plots of the significant effect of Span 60 amount (X₁) (a), Tween 80 amount (X₂) (b), response 3D plot for the combined effect of Span 60 amount (X₁) and Tween 80 amount (X₂) (c), on ZP of TOL basic spanlastics.

Figure 4

Line plots of the significant effect of cosolvent percentage (X₂) (a) and response 3D plot for the combined effect of cosolvent type (X₁) and cosolvent percentage (X₂) (b), on EE% of TOL-cosolvent spanlastics.

Figure 5

Line plots of the significant effect of type of cosolvent (X₁) (a), percentage of cosolvent (X₂) (b), response 3D plot for the combined effect of type of cosolvent (X₁), and percentage of cosolvent (X₂) (c) on PS of TOL-cosolvent spanlastics.

Figure 6

Transmission electron micrograph of MS6.

Figure 7

DSC thermograms of (a) TOL, (b) Span 60, (c) physical mixture of TOL-cosolvent spanlastics components, and (d) formula MS6.

Figure 8

Ex vivo corneal permeation profile of TOL-cosolvent spanlastics (MS6) and basic spanlastics (BS6) compared to TOL suspension.

Figure 9

The killing kinetics of treatment A (MS6) and treatment B (TOL suspension) tested against Aspergillus niger (ATCC32656). Treatment A tested at concentration of (3.9 µg/mL) (9× MIC), treatment B (TOL suspension) tested at concentration of (125 µg/mL) (64× MIC). Data are represented by means of the number of recovered colonies counted at each time point ± SD, n = 3. The chart was generated using GraphPad Prism (v5) (GraphPad, California, CA, USA).

Figure 10

Histopathological photomicrographs (stained with hematoxylin and eosin) showing control rabbit eye (group I) and MS6-treated eye (group II); (a1,a2) show the cornea, (b1,b2) show the iris, (c1,c2) demonstrate the retina, choroid and sclera with normal histological structure (×40 magnification power). Photomicrographs of Group I ((a1,b1,c1)) were adopted from Aziz et al. [14].

Figure 11

In vivo percentage growth inhibition of MS6 (treatment A) compared to TOL suspension (treatment B) on Aspergillus niger (ATCC32656). * Indicates statistically significant difference between the columns applying one-way ANOVA test (p < 0.05, n = 3).