Development and Characterisation of Liposomes Encapsulating Fluorescent Dyes: Influence of Dye Lipophilicity, Stability, and Purification Methods

This study investigated the formulation, purification, and stability of liposomal nanocarriers loaded with fluorescent dyes of varying physicochemical properties, including Rhodamine 6G, MBA-3, DiR, and IR-806. Liposomes were successfully prepared using the thin-film hydration method, producing nanoscale systems with mean hydrodynamic sizes ranging from approximately 150 to 190 nm and low polydispersity indices (PDI < 0.15), indicating good colloidal homogeneity. Encapsulation efficiency was strongly dependent on dye physicochemical properties. Hydrophilic Rhodamine 6G showed negligible encapsulation, while lipophilic dyes such as MBA-3 (EE of 46.3 ± 6.7%) and DiR (EE of 57.0 ± 6.2%) demonstrated significantly higher incorporation into the lipid bilayer. IR-806 exhibited poor encapsulation (EE of 4.2 ± 1.0%) despite intermediate lipophilicity, indicating limited compatibility with the liposomal system. Purification was performed using centrifugal ultrafiltration (CU) and size exclusion chromatography (SEC). SEC consistently provided higher and more reliable recovery values, with MBA-3 and DiR showing recoveries of 98.78 ± 4.68% and 94.01 ± 3.49%, respectively. In contrast, CU resulted in substantially lower recoveries, particularly for MBA-3 (58.03 ± 4.97%) and DiR (55.14 ± 7.49%), likely due to non-specific adsorption and mechanically induced losses during centrifugation. SEC was also more cost-effective on a per-sample basis compared to single-use ultrafiltration devices. Stability studies demonstrated that empty liposomes remained physically stable over time. DiR-loaded liposomes showed stable size over 21 days with only minor changes in PDI and a modest 1.2-fold decrease in UV–Vis signal. MBA-3 showed excellent stability, while IR-806 exhibited pronounced instability, with complete signal loss over 21 days. Overall, liposomal performance was governed by a combination of carrier stability, dye-lipid interactions, and intrinsic dye stability. SEC proved to be a more reliable and economical purification method than CU, particularly for highly lipophilic fluorescent probes.