人参源性囊泡负载栀子苷纳米粒的构建及其体外抗缺血作用研究OA

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 supe-rior blood-brain barrier(BBB)penetration,and to systematically evaluate its effects against cerebral ischemia-reperfusion injury in vi-tro.METHODS G-EVLPs were extracted from ginseng using differential centrifugation combined with ultrafiltration,and their ve-sicular 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 estab-lished 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,immu-nofluorescence,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 en-docytosis 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 me-tabolism.Furthermore,G-EVLPs/Gen promoted the polarization of BV2 microglia towards the anti-inflammatory M2 phenotype,up-regulated the secretion of IL-4 and IL-10,and downregulated the levels of pro-inflammatory factors IL-6 and TNF-α,exerting signifi-cant 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 polar-ization,offering a promising novel strategy for the treatment of ischemic stroke.