The Preparation and Therapeutic Effects of β-Glucan-Specific Nanobodies and Nanobody-Natamycin Conjugates in Fungal Keratitis.

Fungal keratitis (FK) is a severe infectious corneal disease. Since traditional eye drops exhibit poor dissolution and high corneal toxicity, the efficacy of current treatments for FK remains limited. It is needed to develop new approaches to control the cornea damage from FK. In this study, a nanobody (Nb) specific to β-glucan in the fungal cell wall was prepared. The conjugate of the Nb with natamycin (NAT), a traditional antifungal drug, was synthesized. Firstly, we found the Nb specific to β-glucan inhibited fungal growth by disrupting cell wall and biofilm formation.. In addition, the content of β-glucan in the fungal cell wall decreased after Nb treatment. The Nb also reduced the adhesion ability of fungal conidia to human corneal epithelial cells (HCECs). Further, we examined the difference between NAT and Nb-NAT in antifungal growth. Nb-NAT showed better antifungal effects than NAT which was caused by the interaction between Nb and β-glucan. Moreover, Nb concentration below 0.5 mg/mL did not affect the viability of HCECs. Nb-NAT had less cytotoxicity and ocular surface irritation than NAT. Nb specific to β-glucan attenuated Aspergillus fumigatus (A. fumigatus) virulence and relieved inflammatory responses in FK. Nb-NAT treatment of the cornea improved therapeutic effects compared with NAT. It decreased clinical scores and expression level of inflammatory factors. To our knowledge, this study is the first to report a Nb specific to β-glucan and Nb-NAT for the treatment of FK. These unique functions of the Nb specific to β-glucan and Nb-NAT would render it as an alternative molecule to control fungal infections including FK. STATEMENT OF SIGNIFICANCE: Fungal keratitis is a corneal disease with a high rate of blindness. Due to the poor dissolution and high corneal toxicity exhibited by traditional eye drops, the efficacy of current therapeutic treatments for fungal keratitis (FK) remains limited. To enhance the therapeutic effect of natamycin in treating fungal keratitis, this study developed an innovative approach by preparing a β-glucan-specific nanobody and loading it with the antifungal drug natamycin. The β-glucan-specific nanobody has the ability to control both fungal pathogen invasion and inflammation, which can cause damage to the cornea in FK. The conjugation with the β-glucan-specific nanobody significantly increased the antifungal capacity of natamycin and reduced its toxicity. The further application of natamycin conjugated with the β-glucan-specific nanobody could be expanded to other diseases caused by fungal pathogen infections.