Formulation, characterization, and in vitro antifungal evaluation of liposomal terbinafine prepared by the ethanol injection method.

Abstract

Background and purpose: Fungal infections necessitate advanced delivery systems to improve antifungal therapy. Terbinafine, a potent allylamine antifungal, faces clinical limitations due to poor solubility, low bioavailability, and toxicity. Liposomal encapsulation addresses these challenges by enhancing solubility, enabling controlled release, and reducing toxicity. In this study, a scalable ethanol injection method was used to develop terbinafine-loaded liposomes with optimized physicochemical properties. This study aimed to focus on central nervous system-targeted delivery to combat resistant fungal infections while minimizing systemic toxicity.

Materials and methods: Twenty liposomal formulations were prepared using phospholipids (e.g., dipalmitoylphosphatidylcholine [DPPC], hydrogenated soybean phosphatidylcholine) and characterized for size, zeta potential, polydispersity index, and morphology via dynamic light scattering and transmission electron microscopy. Encapsulation efficiency, drug release kinetics, colloidal stability (3 months), and cytotoxicity (human foreskin fibroblast 2 cells, 48-hour exposure) were evaluated. The M38-A2 method of the Clinical and Laboratory Standards Institute was used to calculate minimum inhibitory concentrations (MICs) of 16 azole-susceptible and -resistant Aspergillus fumigatus and Aspergillus flavus.

Results: Liposomes exhibited sizes of 72-174 nm, zeta potentials between +2 and -15 mV, and a low polydispersity index (<0.3). Moreover, F12 (DPPC-based) demonstrated superior cumulative release, compared to F20, and attributed to the fluid bilayer of DPPC. Both formulations retained stability during storage. Cytotoxicity assays revealed minimal toxicity for free terbinafine (14.73% at 25 mg/mL) and significantly reduced toxicity for liposomal forms (6.77% for F12, p<0.05). The DPPC-based formulation achieved an encapsulation efficiency of 73.48%, ensuring a high drug payload and biocompatibility. The DPPC-based formulation achieved an encapsulation efficiency of 73.48%, ensuring a high drug payload and biocompatibility. Liposomal terbinafine and voriconazole exhibited good in vitro activity against both triazole-susceptible and -resistant Aspergillus isolates (MIC₅₀=0.5 µg/mL).

Conclusion: Based on the results, F12, with its sub-100 nm size, sustained release, and reduced cytotoxicity, emerged as a promising candidate for brain-targeted antifungal therapy. Its stability and high encapsulation efficiency support further evaluation in fungal isolates and in vivo models to optimize central nervous system biodistribution and therapeutic efficacy. In addition, this study underscored the promising in vitro activities of terbinafine and liposomal terbinafine against both triazole-resistant/susceptible A. fumigatus and A. flavus.