Oleanolic acid–hesperetin co-assembled nanoparticles alleviate neurological injury after ischemic stroke via the PI3K/AKT/GSK-3β axis

Abstract Ischemic stroke remains a leading cause of death and long-term disability, while the current clinically applicable adjunctive neuroprotective strategies face substantial limitations. Oleanolic acid and hesperetin exhibit synergistic antioxidant, anti-inflammatory, and anti-apoptotic activities; however, their poor aqueous solubility and limited bioavailability remain major limitations to their therapeutic development. Oleanolic acid and hesperetin were formulated as co-assembled nanoparticles using an emulsion-solvent evaporation method. A 5:2 mass ratio of oleanolic acid to hesperetin produced primarily spherical nanoparticles with a mean hydrodynamic diameter of approximately 282.6 nm, a polydispersity index of 0.197, and a zeta potential of −12.2 mV. Oleanolic acid-hesperetin nanoparticles displayed minimal hemolysis (0.199%) while maintaining a stable hydrodynamic diameter during 30 days of storage. Spectroscopic analyses and a 50 ns molecular dynamics simulation promoted co-assembly via multiple noncovalent-driven interactions. Oleanolic acid-hesperetin nanoparticles effectively reduced oxygen-glucose deprivation/reperfusion-induced viability loss and apoptosis in HT22 cells. In transient middle cerebral artery occlusion mice, Cy5.5-associated fluorescence was observed in brain tissue, whereas oleanolic acid-hesperetin nanoparticle treatment reduced infarct volume and ipsilesional brain water content, preserved neurons, resulting in improved sensorimotor and spatial cognitive outcomes through day 28. Treatment promoted PI3K and AKT phosphorylation, elevated inhibitory phosphorylation of glycogen synthase kinase 3β at Ser9 and BCL-2 expression, and reduced BAX expression. These findings suggest that oleanolic acid-hesperetin co-assembly offers a promising preclinical formulation approach for multifactorial neuroprotection after ischemic stroke and supports further pharmacokinetic, safety, and pathway-causality studies.