CELLULAR UPTAKE AND INTRACELLULAR TRAFFICKING OF SOLID LIPID NANOPARTICLES FOR ENHANCED DELIVERY OF GLECAPREVIR/PIBRENTASVIR

Abstract

Background: Glecaprevir and pibrentasvir are potent direct-acting antivirals (DAAs) approved for hepatitis C virus (HCV) therapy; however, their clinical utility is constrained by poor aqueous solubility and suboptimal bioavailability. Solid lipid nanoparticles (SLNs) have emerged as promising nanocarriers for enhancing the delivery of lipophilic drugs. While our previous work established the physicochemical characteristics and in vitro release profile of glecaprevir/pibrentasvir-loaded SLNs, the cellular mechanisms governing their uptake and intracellular trafficking remain unexplored.

Objective: This study investigates the cellular uptake mechanisms, intracellular trafficking pathways, and endosomal escape behavior of glecaprevir and pibrentasvir co-loaded SLNs in hepatocyte models to elucidate the enhanced delivery potential of this nanocarrier system.

Methods: Fluorescently labeled SLNs were prepared using the microemulsion technique with rhodamine 123 as a probe. Cellular uptake studies were performed in HepG2 cells using flow cytometry and confocal laser scanning microscopy. Pharmacological inhibitors were employed to delineate endocytic pathways: chlorpromazine (clathrin-mediated), filipin (caveolae/lipid raft-mediated), amiloride (macropinocytosis), and sodium azide (energy-dependent). Intracellular trafficking was tracked using organelle-specific fluorescent markers for early endosomes (Rab5), late endosomes (Rab7), lysosomes (Lamp-1), and endoplasmic reticulum. Endosomal escape was assessed using the pH-sensitive dye LysoTracker and colocalization analysis.

Results: SLN uptake was time- and concentration-dependent, reaching plateau at 4 hours. Mechanistic studies revealed that internalization occurred primarily via clathrin-mediated endocytosis (52.3% inhibition with chlorpromazine, p < 0.01), with minor contributions from caveolae/lipid raft-mediated pathways (31.7% inhibition with filipin) and macropinocytosis (24.8% inhibition with amiloride). Uptake was significantly reduced at 4°C and in the presence of sodium azide, confirming energy dependence. Intracellular trafficking analysis demonstrated that SLNs sequentially localized to early endosomes (15 min), late endosomes (60 min), and lysosomes (120 min), with a significant fraction escaping endosomal compartments and localizing to the cytoplasm by 240 min. Colocalization with LysoTracker showed 68.4 ± 5.4% escape efficiency at 4 hours. The encapsulated drugs exhibited enhanced intracellular accumulation compared to free drug solution (3.2-fold increase, p < 0.001).

Conclusion: Glecaprevir/pibrentasvir-loaded SLNs exploit clathrin-mediated endocytosis as the primary uptake mechanism, followed by efficient endosomal escape and sustained intracellular drug release. These findings establish a mechanistic basis for the enhanced bioavailability and therapeutic potential of SLN-based delivery of DAAs in HCV therapy.