Intracellular Fate of a Dual-Fluorescent Hydrophobic Ion Pair: Comparison of Lipid-Based Nanocarriers.

Effective intracellular trafficking and delivery of hydrophilic drugs remain challenging due to poor membrane permeability and limited encapsulation in lipid-based nanocarriers. To address this, we developed a dual-fluorescent hydrophobic ion pair (HIP) by pairing a model fluorescent hydrophilic drug, Cascade Blue hydrazide, with the lipophilic probe DiA. The HIP was subsequently incorporated into three lipid-based nanocarriers─self-emulsifying drug delivery systems (SEDDS), nanoemulsions, and liposomes─to enable visualization and comparison of how formulation composition influences intracellular uptake and fate of a model hydrophilic drug surrogate delivered as an HIP complex. The complex showed a precipitation efficiency of 95% and an >8130-fold increase in lipophilicity compared to noncomplexed Cascade Blue hydrazide, which enabled incorporation into SEDDS (64.41 ± 0.26 nm), nanoemulsions (92.61 ± 1.27 nm), and liposomes (175.03 ± 3.18 nm). Dissociation studies revealed a strong medium dependence, with <10% release in FaSSGF but ∼60% in phosphate-rich FeSSIF. Cytotoxicity testing demonstrated >90% cell viability at 0.01% for all formulations after 24 h, confirming their biocompatibility under relevant conditions. Hemolysis assays showed negligible membrane disruption for SEDDS, while uptake studies in Caco-2 cells indicated that internalization was mainly energy-dependent, with modest effects observed after inhibition of clathrin- and caveolae-mediated pathways. Confocal laser scanning microscopy highlighted a formulation-dependent intracellular fate: SEDDS confined Cascade Blue to vesicular compartments while redistributing DiA to the plasma membrane, whereas nanoemulsions and liposomes enabled endosomal escape, dispersing Cascade Blue into the cytosol and relocating DiA to perinuclear and plasma membranes. Liposomes also showed residual uptake at 4 °C with membrane colocalization of DiA, supporting fusion as a complementary uptake mechanism.