Enhanced corneal retention of hyaluronic acid via a metabolic glycoengineering-based in vivo bioorthogonal reaction

Topical hyaluronic acid (HA) formulations have been extensively employed for managing various ocular diseases, but their therapeutic efficacy is severely compromised by rapid precorneal clearance due to a dense, negatively charged sialic acid-rich glycocalyx. To address this, we report a corneal surface engineering strategy that involves the metabolic incorporation of bioorthogonal azide groups onto the corneal epithelium. This covalent "docking" layer enables site-specific, Click chemistry-mediated immobilization of anionic HA, thereby significantly enhancing its ocular surface retention in vitro and in vivo. First, an alkyne-bearing molecule (DBCO) was labelled with a fluorescent probe (Cy5) to produce DBCO-Cy5. Sequential incubation of human corneal epithelial cells (HCECs) with an unnatural azide-bearing sugar (AAM) and DBCO-Cy5 enhanced the Cy5 retention by more than 10-fold. Then, HA was covalently labelled with DBCO at different conjugation ratios (HA-DBCO), and these conjugates showed negligible cytotoxicity. HA-DBCO binding to AAM-pretreated cells was time- and concentration-dependent; a higher degree of substitution substantially improved the reaction efficiency. Meanwhile, the covalent conjugation of HA to the cell surface did not affect HCEC proliferation but enhanced cell migration. The proof-of-concept was also validated in a mouse model of dry eye disease. AAM was ocularly delivered via a cationic liposome, resulting in the predominant deposition of azide in the corneal epithelium of mice. Subsequent dosing of HA-DBCO induced the in vivo bioorthogonal reaction that enhanced HA retention (up to 6 h) by 2.5-fold compared to the control without AAM pretreatment. The prolonged corneal retention enhanced the therapeutic efficacy at a reduced dosing frequency. This approach offers a clinically translatable strategy to improve the efficacy of topical HA-based therapies for ocular diseases.