Purpose: Local hotspots of elevated tear hyperosmolarity (exceeding 900 mOsM) are predicted in dry eye disease (DED) but have not been measured. This study aims to develop, characterize, and evaluate the suitability of fluorescent nanoliposomes for noninvasive sensing of the local osmolarity of the tear film. Methods: Fluorescent nanoliposomes, loaded with calcein (susceptible to self-quenching; sensor dye) and sulforhodamine 101 (SR101; reference dye), were produced by the thin-film hydration method. Results: Dynamic light scattering measurements and Cryo-TEM showed that liposomes were negatively charged (-23.7 ± 1.5 mV), spherical (diameter = 117.9 ± 6.4 nm), and uniform in size (polydispersity index = 0.15 ± 0.02). These properties were unaffected by cold storage (4°C; 14 days), but dye leakage was significant after 3 days. Swelling and shrinkage of the liposomes by exposure to hypoosmotic and hyperosmotic media led to rapid dequenching and quenching of calcein fluorescence (FGreen), with no effect on SR101 fluorescence (FRed). The ratio FGreen/FRed decreased with increasing osmolarity and vice versa, obeying the Boyle van't Hoff relationship. When liposomes were dispersed in a gelatin film with dynamic radial sucrose gradients, local FGreen/FRed decreased with increasing hyperosmolarity as predicted. When instilled on the hydrophilic surface of contact lenses or ex vivo corneas, nanoliposomes dispersed evenly as thin films. Importantly, the measured FGreen/FRed declined continuously with evaporation and consequent increase in their osmolarities. Conclusions: The study provides proof of principle for noninvasive measurement of local tear film osmolarity based on osmosensitive fluorescent nanoliposomes. The strategy can potentially advance our understanding of the pathophysiology of DED.
Keywords: calcein; dry eye disease; fluorescence quenching; local osmolarity; nanoliposomes; tear film.