Preparation of amphotericin B-loaded hybrid liposomes and the integration of chitin-binding proteins for enhanced antifungal activity.

Amphotericin B (AMB) is a gold standard antifungal drug because of its broad-spectrum activity toward pathogenic yeasts and molds. Because of its low solubility in water and toxicity toward humans, several lipid-based formulations that either increase the aqueous solubility or decrease the side effects have been employed in practical use. In our previous research, we found that the combination of AMB with an artificial palmitoylated chitin-binding domain from Pteris ryukyuensis chitinase (LysM-Pal) resulted in synergistic antifungal action against Trichoderma viride. Herein, we prepared hybrid liposomal formulations by combining a commercially available AMB formulation and liposomes with different surface charges to explore key factors in the antifungal activity. The characterization of AMB-loaded liposomal formulations (AMB-LFs), including particle size distribution and zeta potential, showed that anionic and neutral AMB-LFs could stably encapsulate AMB. The combination of either anionic or neutral AMB-LFs with unmodified LysM decreased the minimum inhibitory concentration of AMB. The combination of neutral AMB-LF with LysM-Pal resulted in a further decrease in the MIC, up to 15-fold compared with that of the neutral AMB-LF alone. Our results demonstrate the potential utility of lipid-based liposomal formulations of AMB combined with lipid-modified proteinaceous binders to tackle fungal infections.

Enhancement of the Antifungal Activity of Chitinase by Palmitoylation and the Synergy of Palmitoylated Chitinase with Amphotericin B.

Combinations of antifungal drugs can have synergistic antifungal activity, achieving high therapeutic efficacy while minimizing the side effects. Amphotericin B (AMB) has been used as a standard antifungal drug for fungal infections; however, because of its high toxicity, new strategies to minimize the required dose are desirable. Chitinases have recently received attention as alternative safe antifungal agents. Herein, we report the combination of palmitoylated chitinase domains with AMB to enhance the antifungal activity. The chitin-binding domain (LysM) from Pteris ryukyuensis chitinase was site-specifically palmitoylated by conjugation reaction catalyzed by microbial transglutaminase. The palmitoylated LysM (LysM-Pal) exhibited strong antifungal activity against Trichoderma viride, inhibiting the growth completely at a concentration of 2 µM. This antifungal effect of LysM-Pal was mainly due to the effect of anchoring of palmitic acid motif to the plasma membrane of fungi. A combination of AMB with LysM-Pal resulted in synergistic enhancement of the antifungal activity. Intriguingly, LysM-Pal exhibited higher level of antifungal activity enhancement than palmitoylated catalytic domain (CatD) and fusion of LysM and CatD. Addition of 0.5 µM LysM-Pal to AMB reduced the minimal inhibition concentration of AMB to 0.31 µM (2.5 µM without LysM-Pal). The possible mechanism of the synergistic effect of AMB and LysM-Pal is destabilization of the plasma membrane by anchoring of palmitic acid and ergosterol extraction by AMB and destabilization of the chitin layer by LysM binding. The combination of LysM-Pal with AMB can drastically reduce the dose of AMB and may be a useful strategy to treat fungal infections.