目的 制备经济高效、安全性高且具有强血脑屏障（blood-brain barrier，BBB）穿透能力的葡萄柚囊泡（grapefruit exosome-like vesicles，GEVs）载红景天苷（salidroside，Sal）仿生纳米粒（GEVs/Sal），并考察其体外抗脑缺血损伤作用。方法 采用差速离心法和超滤法提取GEVs，流式细胞术检测其特征性膜蛋白。采用超声破碎法制备GEVs/Sal，纳米颗粒跟踪分析对其粒径进行表征，HPLC法测定载药量和包封率；采用Transwell法考察BBB穿透及保护能力，采用MTT法、免疫荧光染色、qRT-PCR等技术考察GEVs/Sal对受损PC12细胞的保护作用。结果 GEVs表达CD81、CD9特征性膜蛋白，表明囊泡成功提取，且HPLC显示GEVs中含有特征组分柚皮苷（naringin，NAR），有望与中药发挥协同增效作用；GEVs/Sal的最佳处方比例为m_GEVs∶m_Sal＝10∶1，载药量、包封率、粒径和Zeta电位分别为（8.07±0.23）%、（91.08±2.65）%、（173.9±3.8）nm、（−11.49±0.48）mV；MTT结果显示药物浓度在100 μmol/L以内时，GEVs/Sal对PC12细胞没有明显毒性作用，且50 μmol/L的GEVs/Sal能够显著改善氧糖剥夺/复氧（oxygen glucose deprivation/reoxygenation，OGD/R）诱导的PC12细胞活力；进一步证明PC12细胞通过网格蛋白和脂筏作用介导了GEVs/DiO的内化；通过构建体外BBB模型证明GEVs/Sal能有效穿过BBB，进一步确认hCMEC/D3细胞通过网格蛋白和巨胞饮作用介导GEVs/DiO的内化；FITC-Dextran渗漏实验结果表明GEVs/Sal减少OGD/R损伤后BBB的通透性；Calcein-AM/PI染色检测及线粒体功能考察发现GEVs/Sal具有抗细胞凋亡、改善线粒体膜电位、恢复细胞能量代谢过程的作用。通过qRT-PCR分析表明，GEVs/Sal升高了B淋巴细胞瘤-2（B cell lymphoma 2，Bcl-2）的基因表达，降低了Bcl-2相关X蛋白（Bcl-2-associated X protein，Bax）和半胱氨酸天冬氨酸蛋白酶-3（cysteine containing cysteinyl aspartate-3，Caspase-3）的基因表达，从而发挥抑制细胞凋亡的作用。此外，免疫荧光染色表明GEVs/Sal干预可诱导BV2细胞向M2型极化，并介导抗炎作用。结论 成功制备高效载药、性质稳定的仿生纳米药物递送平台GEVs/Sal，并证明其对OGD/R引发的神经元损伤具有良好神经保护效果，有望为缺血性脑卒中提出了一种新的治疗策略。

Objective To prepare inexpensive, safe and non-toxic grapefruit exosome-like vesicles (GEVs) loaded with salidroside (Sal) bionanoparticles (GEVs/Sal) with strong blood-brain barrier (BBB) penetration capacity, and to investigate their anti-ischemic injury effects in vitro. Methods GEVs were extracted by differential centrifugation and ultrafiltration, and their characteristic membrane proteins were detected by flow cytometry. GEVs/Sal were prepared by ultrasonic fragmentation, characterized by nanoparticle tracking analysis for particle size, and HPLC for drug loading and encapsulation rates; Transwell method was used to investigate the BBB penetration and protective ability; MTT method, immunofluorescence staining and qRT-PCR were used to investigate the protective effect of GEVs/Sal on damaged PC12 cells. Results GEVs expressed CD81 and CD9 characteristic membrane proteins, indicating that the vesicles were successfully extracted, and HPLC showed that GEVs contained the characteristic component naringin (NAR), which may play a synergistic role with the traditional Chinese medicine; The optimal preparation prescription ratio for GEVs/Sal was m_GEVs∶m_Sal= 10∶1, drug loading, encapsulation efficiency, particle size and zeta potential were (8.07 ± 0.23)%, (91.08 ± 2.65)%, (173.9 ± 3.8) nm, (−11.49 ± 0.48) mV, respectively; MTT assay result showed that concentration within 100 μmol/L of GEVs/Sal had no significant cytotoxicity on PC12 cells, and 50 μmol/L of GEVs/Sal significantly improved the viability of PC12 cells induced by oxygen glucose deprivation/reoxygenation (OGD/R); It was further demonstrated that PC12 cells mediated the internalization of GEVs/DiO through clathrin and lipid raft interaction; It was demonstrated by constructing an in vitro BBB model that GEVs/Sal could effectively cross the BBB, and it was further confirmed that hCMEC/D3 cells mediated the internalization of GEVs/DiO through clathrin and macropinocytosis interaction; The results of FITC-Dextran leakage assay showed that GEVs/Sal reduced the permeability of the BBB after OGD/R injury; Calcein AM/PI staining and mitochondrial function investigation revealed that GEVs/Sal could inhibit cell apoptosis, improve mitochondrial membrane potential and restore cellular energy metabolism processes. qRT-PCR analysis showed that GEVs/Sal increased the gene expression of B cell lymphoma-2 (Bcl-2), decreased the gene expressions of Bcl-2 associated X protein (Bax) and cysteine containing cysteine aspartate-3 (Caspase-3), thereby exerting an inhibitory effect on cell apoptosis. In addition, immunofluorescence staining showed that GEVs/Sal intervention could induce polarization of BV2 cells towards M2 type and mediate anti-inflammatory effects. Conclusion GEVs/Sal, a highly efficient drug-carrying and stable biomimetic nanomedicine delivery platform, has been successfully prepared and has been demonstrated to have good neuroprotective effects against OGD/R-induced neuronal damage, suggesting a new therapeutic strategy for ischemic stroke.

R285.5

国家自然科学基金资助项目（82274104）；国家自然科学基金资助项目（82074024）；国家自然科学基金资助项目（82374042）；中药制药过程控制与智能制造技术国家重点实验室创新项目（NZYSKL240103）；江苏省优秀青年基金项目（BK20240144）；南京中医药大学优青培育项目（RC202407）；大学生创新创业项目（202210315056Y，202310315055Z）