OBJECTIVE To construct a geniposide (Gen)-loaded nanodelivery system based on ginseng-derived extracellular vesicle-like particles (G-EVLPs/Gen), characterized by synergistic efficacy, excellent biocompatibility, cost-effectiveness, and superior blood-brain barrier (BBB) penetration, and to systematically evaluate its effects against cerebral ischemia-reperfusion injury in vitro.

METHODS G-EVLPs were extracted from ginseng using differential centrifugation combined with ultrafiltration, and their vesicular characteristics were verified by detecting surface markers via flow cytometry. G-EVLPs/Gen nanoparticles were prepared using ultrasonication. Their particle size and Zeta potential were measured by dynamic light scattering (DLS), while drug loading capacity and encapsulation efficiency were determined by high-performance liquid chromatography (HPLC). An in vitro BBB model was established using hCMEC/D3 cell monolayers on transwell inserts to assess the BBB-crossing ability of G-EVLPs/Gen. Additionally, an oxygen-glucose deprivation/reoxygenation (OGD/R) model was constructed using PC12 neuron-like cells to establish an in vitro injury model. The cytotoxicity, anti-apoptotic effects, mitochondrial function restoration, and anti-inflammatory properties of G-EVLPs/Gen were comprehensively evaluated using MTT assays, Calcein-AM/PI dual staining, mitochondrial membrane potential detection, immunofluorescence, and ELISA.

RESULTS The extracted G-EVLPs highly expressed characteristic exosomal marker proteins CD9 and CD63, confirming successful isolation. The optimized mass ratio of G-EVLPs to Gen was 2:1. The resulting G-EVLPs/Gen exhibited a drug loading capacity of (9.08±0.07)%, an encapsulation efficiency of (85.67±0.75)%, an average particle size of (120.26±1.22) nm, and a Zeta potential of (-9.64±0.35) mV. MTT assays indicated no significant cytotoxicity of G-EVLPs/Gen at concentrations ≤ 24 μg·mL^-1. A concentration of 3 μg·mL^-1 significantly reversed the OGD/R-induced decrease in PC12 cell viability (P < 0.01). Mechanistic studies revealed that G-EVLPs/Gen were efficiently internalized by hCMEC/D3 cells primarily via caveolin-mediated endocytosis and macropinocytosis, thereby effectively crossing the in vitro BBB model. Further experiments confirmed that G-EVLPs/Gen significantly inhibited OGD/R-induced apoptosis, restored mitochondrial membrane potential, and improved cellular energy metabolism. Furthermore, G-EVLPs/Gen promoted the polarization of BV2 microglia towards the anti-inflammatory M2 phenotype, upregulated the secretion of IL-4 and IL-10, and downregulated the levels of pro-inflammatory factors IL-6 and TNF-α, exerting significant anti-neuroinflammatory effects.

CONCLUSION A G-EVLPs/Gen nanodelivery system with stable physicochemical properties, high drug loading efficiency, and potent BBB penetration is successfully constructed. This system exerts neuroprotective effects through multi-target mechanisms, including inhibiting neuronal apoptosis, improving mitochondrial function, and modulating microglial polarization, offering a promising novel strategy for the treatment of ischemic stroke.