Development and In Vitro Characterization of Apigenin-Loaded Nanoemulsion for Improved Cellular Antioxidant Activity

Abstract

Background: Apigenin is a potent natural antioxidant with considerable therapeutic potential; however, its clinical application is limited by poor aqueous solubility, high crystalline lattice energy, and low cellular permeability, resulting in poor bioavailability.

Objective: This study aimed to develop, optimize, and evaluate an isotropic apigenin-loaded nanoemulsion (A-NE) to enhance intracellular drug delivery and improve antioxidant efficacy.

Methods and Results: The optimized A-NE was formulated using medium-chain triglycerides, Tween 80, and Transcutol HP at a fixed Smix ratio of 2:1. Dynamic light scattering and electron microscopy confirmed the formation of highly uniform, monodisperse nanodroplets with an average size of 65.4 nm, a polydispersity index below 0.2, and a zeta potential of −28.6 mV, indicating excellent electrosteric stability. FTIR and XRD analyses confirmed the complete amorphization of apigenin and its successful encapsulation within the lipid core through strong intermolecular hydrogen-bonding interactions, achieving an encapsulation efficiency of 91.8 ± 1.4%. In vitro release studies demonstrated a controlled biphasic drug release profile with more than 85% cumulative release over 24 hours, following a Fickian diffusion mechanism. MTT assay results confirmed excellent biocompatibility, maintaining cell viability above 92% in HepG2 cells. Flow cytometric analysis demonstrated rapid endocytosis-mediated intracellular uptake of the nanoemulsion. Furthermore, the optimized A-NE reduced H₂O₂-induced intracellular reactive oxygen species by approximately 78%, exhibiting significantly greater antioxidant activity than the free apigenin suspension.

Conclusion: The developed nanoemulsion effectively addressed the major biopharmaceutical limitations of apigenin by improving its solubility, intracellular delivery, and antioxidant efficacy. These findings establish A-NE as a stable and efficient nanocarrier platform with considerable potential for advanced intracellular antioxidant therapy.