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

## TL;DR

This study compares how different lipid-based nanocarriers affect the intracellular delivery and fate of a hydrophilic drug using a dual-fluorescent complex.

## Contribution

A dual-fluorescent hydrophobic ion pair was developed to visualize and compare intracellular drug delivery across lipid-based nanocarriers.

## Key findings

- The HIP complex showed high precipitation efficiency and significantly increased lipophilicity, enabling incorporation into various nanocarriers.
- Intracellular fate varied by formulation, with SEDDS confining the drug to vesicles and nanoemulsions/liposomes enabling endosomal escape.
- Liposomes showed residual uptake at 4°C, suggesting fusion as a complementary uptake mechanism.

## Abstract

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.