Apo-Transferrin Modified Natamycin-Loaded Liposomes Improve the Prognosis of Fungal Keratitis via Enhanced Antifungal and Anti-Inflammatory Activities.

Background: The proliferation and establishment of infections by pathogenic microorganisms require essential nutrients, such as host-derived iron. The sequestration of iron by the host represents an ancient defense mechanism against microbial invasion. Apo-Transferrin (Apo-Tf) is a promising and novel antimicrobial agent with intrinsic iron sequestration properties and therefore merits clinical investigation. Given the characteristics of natamycin (NAT) such as its low aqueous solubility and significant ocular irritation, we developed apo-Tf decorated liposomes encapsulating NAT for the treatment of fungal keratitis (FK). Methods: NAT was encapsulated into liposomes using the lipid film hydration method. Natamycin-loaded transferrin-conjugated liposomes (NAT@TF-LP) were synthesized via the post-insertion technique and characterized through zeta-sizer analysis, morphology and Fourier transform infrared (FTIR) spectroscopy. The encapsulation efficiency, loading capacity, and in vitro release profile were determined by ultraviolet spectrophotometry. The cytotoxicity, ocular surface irritation, and systemic safety of liposomal formulations were evaluated in corneal epithelial cells and mice. In vitro studies investigated the antifungal properties of NAT@TF-LP, while in vivo experiments assessed eye surface retention and therapeutic efficacy in an FK mouse model. Results: The prepared liposomes exhibited a well-defined nanostructure with uniform dimensions and apo-Tf was effectively conjugated to the surface, as verified by the emergence of characteristic amide bands in FTIR analysis. The loading efficiencies of NAT@LP and NAT@TF-LP were 5.6% and 3.53% respectively, with encapsulation efficiencies above 96%. Both formulations facilitated sustained NAT release and prolonged ocular surface retention. The in vitro release of NAT from NAT@TF-LPs followed a diffusion-controlled profile, exhibiting an excellent fit to both Higuchi and Korsmeyer-Peppas models. Additionally, they exhibited good biosafety profiles in both cytotoxicity and ocular irritation assays. In vivo, NAT@TF-LPs demonstrated superior therapeutic efficacy, resulting in a 15.4-fold reduction in corneal fungal burden (approximately 1.19-log reduction) compared to the free NAT group, alongside a marked decrease in the protein expression of pro-inflammatory cytokines, including IL-1β (65.0%), IL-6 (59.0%), and TNF-α (73.0%), with a 69.3% enhancement in anti-inflammatory IL-10 levels. Conclusion: The apo-Tf functionalized liposomal system improves NAT ocular bioavailability and exhibits augmented antifungal and therapeutic efficacies. This enhancement is attributed to the combined benefits of the liposomal platform and the inherent antimicrobial properties of apo-Tf. This study highlights the promising potential of NAT@TF-LPs for the pharmacotherapy of FK.