Mechanistic Characterization of Norjuziphine-β-Lactoglobulin Nanocomplexes: Protein Conformational Remodeling Governs Encapsulation and Therapeutic Effects in Human Hepatocellular Carcinoma Cells.

The development of protein-based systems capable of delivering bioactive compounds remains an important challenge in pharmaceutical formulation science. In this study, β-lactoglobulin (βLG) was investigated as a biocompatible nanocarrier for norjuziphine, 1-[(4-hydroxyphenyl)methyl]-7-methoxy-1,2,3,4-tetrahydroisoquinolin-8-ol. The binding mechanism, protein structural changes, physicochemical characteristics, drug-release behavior, and biological performance of norjuziphine-βLG nanocomplexes were comprehensively characterized. Fluorescence quenching studies demonstrated spontaneous static complex formation with binding constants in the range of 105-106 L mol-1. Spectroscopic, calorimetric, and computational analyses consistently showed ligand-induced conformational remodeling and stabilization of βLG. The resulting nanocomplexes exhibited spherical morphology with a mean hydrodynamic diameter of 190.1 ± 10.34 nm and an encapsulation efficiency of approximately 79.0%. In vitro release studies demonstrated controlled drug release behavior with moderate pH dependence. Nanoencapsulation increased intracellular norjuziphine accumulation by approximately 2.3-fold relative to free norjuziphine and was associated with enhanced antiproliferative activity in human hepatocellular carcinoma (HCC) cells, a reduced IC50 value (15.3 ± 2.33 μM), and significant regulation of Bax, Bcl-2, and Caspase-3 expression. Collectively, these findings establish a mechanistic relationship between hydrophobic interaction-driven complexation, protein conformational remodeling, and delivery performance, highlighting β-lactoglobulin as a promising protein-based carrier for improving the delivery of bioactive compounds.