Development of antifungal fibrous ocular insert using freeze-drying technique

Abstract

Candida species is one of the pathogenic fungi of the eye responsible for keratitis that frequently causes vision impairment and blindness. Effective treatment requires long-term use of antifungal drugs, which is opposed by the defensive mechanisms of the eye and inadequate corneal penetration. The objective of this study was to develop a carrier for prolonged ocular application of fluconazole (FLZ) to treat keratitis. FLZ was encapsulated into chitosan fibrous matrices (F1-F4) using different chitosan concentrations (0.02, 0.1, 0.5, and 1%w/v, respectively) by freeze-drying as a single-step technique. Studying the morphology and surface properties of the inserts revealed a porous matrix with fibrous features with a large surface area. Thermal stability and chemical compatibility were confirmed by DSC/TGA/DTA and FT-IR, respectively. Loading capacity (LC) and entrapment efficiency (EE) were determined. According to the in vitro release study, F4 (0.11 mg mg^-1 LC and 87.53% EE) was selected as the optimum insert because it had the most sustained release, with 15.85% burst release followed by 75.62% release within 12 h. Ex vivo corneal permeation study revealed a 1.2-fold increase in FLZ permeation from F4 compared to FLZ aqueous solution. Also, in the in vivo pharmacokinetic study in rabbits, F4 increased the AUC_0-8 of FLZ by 9.3-fold and its concentration in aqueous humor was maintained above the MIC through the experimentation time. Studies on cytotoxicity (MTT assay) provide evidence for the safety and biocompatibility of F4. Therefore, the freeze-dried FLZ-loaded chitosan fibrous insert could be a promising candidate for treating ocular keratitis.

Keywords: Candida; Chitosan; Fluconazole; Freeze-drying; Insert; Ocular delivery.

Fig. 1

Morphological characterization of the fibrous ocular inserts using SEM techniques: (a) CS insert (0.02 wt% CS), (b) CS insert (0.1 wt% CS), (c) CS insert (0.5 wt% CS), (d) CS insert (1 wt% CS), and (e) F1 ocular insert (0.02 wt% CS and FLZ)

Fig. 2

Brunauer–Emmett–Teller analysis of fluconazole-loaded freeze-dried ocular inserts (F1, F2, F3, and F4) with different concentrations of CS with different CS concentrations (0.02, 0.1, 0.5, and 1%), respectively. a Surface area, b pore size, c pore volume, and d swelling degree (mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001)

Fig. 3

a Loading capacity (LC, mg mg⁻¹), b entrapment efficiency (EE%) of FLZ-loaded ocular inserts, and c the drug release profile of the FLZ-loaded CS ocular inserts with different concentrations of polymer (CS) (mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001)

Fig. 4

FT-IR spectra of chitosan (CS), fluconazole (FLZ), plain CS insert, and FLZ-loaded CS inserts

Fig. 5

Thermal analysis: a DSC thermograms of pure chitosan (CS), pure fluconazole (FLZ), CS-FLZ physical mixture (1:1), and FLZ-CS freeze-dried insert. b TGA, c DTGA, and d DTA of plain CS-Fs and FLZ-loaded CS inserts

Fig. 6

a Cumulative amount permeated per unit area, b permeation flux, J_ss, and c permeability coefficient, PC, of FLZ from freeze-dried FLZ-loaded nanofiber insert (F4) and FLZ solution through the excised bovine cornea (mean ± SD, n = 3, **p < 0.01)

Fig. 7

a Cell viability (%) obtained in MTT assays using L929 (fibroblast cells). b The antifungal inhibition zone of various formulations. The antifungal inhibition zone against Candida albicans of (c) FLZ aqueous solution, (d) plain CS ocular insert, and (e) FLZ-loaded CS ocular insert (mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001)

Fig. 8

Concentration of FLZ in aqueous humor of rabbit’s eyes with time from the fibrous insert (F4) compared to FLZ eye drops (aqueous solution)

Fig. 9

Histological examination: section of the excised corneal tissues treated with F4