Macrophage Membrane-Coated Nanoparticles for the Delivery of Natamycin Exhibit Increased Antifungal and Anti-Inflammatory Activities in Fungal Keratitis

This study aims to explore the efficacy and safety of macrophage membrane-coated nanoparticles for the delivery of natamycin (NAT) in the therapy of fungal keratitis (FK). Macrophage membranes were isolated and identified by immunofluorescence staining (IFS). NAT was encapsulated into poly(lactic-<i>co</i>-glycolic acid) (PLGA). Fungal stimulated macrophage membranes (M1) or unstimulated membranes (M) were separately mixed and sonicated with PLGA nanoparticles. The biocompatible nanoparticles (PLGA-NAT, PLGA-NAT@M, and PLGA-NAT@M1) were characterized with zeta-sizer analysis, transmission electron microscopy (TEM), and Western blot. Drug encapsulation and loading efficiency and the release of NAT in the nanoparticles were detected by ultraviolet spectrophotometry. The cytotoxicity, ocular surface toxicity and irritability, and systemic safety of nanoparticles with different concentrations were assessed. In vitro, we examined the antifungal properties of the nanoparticles. The eye surface retention time, drug release, and curative effects on FK were evaluated in vitro and in vivo. IFS results showed the separation of the macrophage membrane and nucleus. The prepared nanoparticles had a typical "core-shell" structure and uniform nanometer size, and the membrane proteins were retained on the membrane allowing to exert functional effects of macrophage. The loading efficiencies of PLGA-NAT@M and PLGA-NAT@M1 were 7.6 and 6.7%, respectively. The encapsulation efficiencies of PLGA-NAT@M and PLGA-NAT@M1 were 51.2 and 41.5%, respectively. PLGA-NAT@M and PLGA-NAT@M1 could gradually release NAT and reduce the clearance of the ocular surface. Macrophage membranes enhanced the antifungal activity of PLGA-NAT. Furthermore, the membrane coated with macrophage increased the biocompatibility and decreased the corneal toxicity of nanoparticles. In vivo, PLGA-NAT@M1 significantly alleviated the severity of FK. In vitro, PLGA@M and PLGA@M1 reduced the protein levels of inflammatory cytokines after fungal stimulation. The prepared PLGA-NAT@M1 has good physical properties and biosafety. It could evade ocular surface clearance, release NAT gradually, and achieve high antifungal and anti-inflammatory efficiencies to FK. Macrophage membrane-coated nanoparticles clinically have high application potential to the treatment of FK.