Alleviation of dry eye disease with lyophilized extracellular vesicles.

Dry eye disease (DED), a prevalent ocular condition affecting millions globally, necessitates the development of innovative therapeutic strategies for long-term management. Although extracellular vesicles (EVs) have shown therapeutic potential for DED, their clinical translation has been substantially limited by stability issues during storage. In this study, a novel lyophilization strategy incorporating ectoine - a natural osmolyte with superior bio-stabilization capabilities - was developed to address these challenges. For the first time, an ectoine-enhanced lyophilization protocol was established to preserve the key functional properties of milk-derived EVs (mEVs) during prolonged storage at 4 °C. Comprehensive characterization through hydrodynamic analysis, structural evaluation, β-galactosidase activity assessment, and functional validation demonstrated that mEVs lyophilized with 0.5 %-4 % ectoine maintained critical biological properties for over two months, representing a significant improvement compared to conventional preservation methods. The lyophilized mEVs were shown to effectively protect human corneal epithelial cells (HCECs) against hyperosmolarity-induced damage. Notably, in a benzalkonium chloride (BAC)-induced rabbit model of DED, the optimized formulation exhibited enhanced therapeutic performance, as evidenced by significant improvements in clinical parameters and ocular surface health. These findings established ectoine-based lyophilization as a transformative preservation methodology for EV-based therapeutics, providing a robust solution to persistent stability challenges in ocular drug delivery systems and facilitating the clinical translation of EV-based treatments for DED and related ocular surface disorders.