目的 筛选外泌体最佳纯化与载药工艺，构建体外血脑屏障（blood brain barrier，BBB）细胞模型和阿尔茨海默病小鼠（Alzheimer’s disease，AD）模型，探究人胚肾293亚系（human embryonic kidney 293T，HEK 293T）细胞外泌体（exosome，Exo）介导人参皂苷Rg₁（ginsenoside Rg₁，Rg₁）跨BBB效率，为脑部疾病治疗提供依据。方法 分别采用超速离心法、切向流法、双耦合谐波振荡技术纯化外泌体，以浓度、粒径及多分散性指数（polydispersity index，PDI）为评价指标，选取最佳纯化方法；在电转电压、脉冲时间、脉冲次数、Rg₁质量浓度单因素筛选的基础上，以药物包封率为评价指标，利用正交设计优化电穿孔法构建Exo-Rg₁复合物的工艺；采用小鼠微脑血管内皮细胞（brain endothelial cell line 3，bEnd.3）构建体外BBB模型，通过测定跨内皮电阻（transendothelial electrical resistance，TEER）、液面渗透及荧光素钠渗透实验验证模型完整性；设置Exo-Rg₁复合物组（实验组）、游离Rg₁组（对照组）、单外泌体组和外泌体（空载）＋游离Rg₁组，采用HPLC检测不同时间点药物透过体外BBB细胞模型的浓度，计算透过效率。通过AD模型小鼠的行为学实验、组织学观察、蛋白与炎症因子检测，评估体内治疗效果。结果 最佳纯化外泌体工艺为双耦合谐波振荡技术，所得外泌体浓度为2.9×10¹¹个/mL，粒径集中在（91.8±1.5）nm，PDI为0.21±0.01；优化后的最佳电穿孔法电转工艺为电转电压100 V、脉冲时间15 ms、脉冲次数5次和Rg₁质量浓度为0.4 mg/mL，此条件下复合物包封率为61.58%，优于传统的超声法；成功构建bEnd.3细胞体外BBB模型，TEER值稳定在300 Ω·cm²以上，并且4 h后体外BBB模型组仍能保持明显的液面差，模型组荧光素钠透过率显著小于空白对照组［（41.34±1.83）% vs（98.87±2.09）%，P＜0.01］，符合屏障功能要求；体外透过实验显示，8 h实验组Rg₁透过效率（15.3%）显著高于对照组（3.3%）（P＜0.05）；动物实验显示，复合物可显著改善模型小鼠学习记忆能力，保护海马神经元，上调胆碱乙酰转移酶（choline acetyltransferase，ChAT）及α7烟碱型乙酰胆碱受体（α7 nicotinic acetylcholine receptor，α7 nAChR）相关蛋白表达，降低白细胞介素-1β（interleukin-1β，IL-1β）、IL-6和肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）炎症因子水平，且效果优于游离Rg₁组与单外泌体组。结论 外泌体可有效提升Rg₁跨越BBB的效率，为改善脑部疾病治疗效果提供实验依据。

Objective To screen the optimal purification and drug-loading processes of exosomes (Exo), construct an in vitro blood-brain barrier (BBB) cell model and an Alzheimer’s disease (AD) mouse model, and investigate the efficiency of human embryonic kidney 293T (HEK 293T) Exo-mediated ginsenoside Rg₁ (Rg₁) across the BBB, so as to provide a basis for the treatment of brain diseases. Methods Exosomes were purified using ultracentrifugation, tangential flow filtration, and dual-coupled harmonic oscillation technology. The optimal purification method was selected based on concentration, particle size, and polydispersity index (PDI). On the basis of single-factor screening of voltage, pulse duration, pulse number, and Rg₁ concentration, the electroporation process for constructing Exo-Rg₁ complexes was optimized using orthogonal design with drug encapsulation efficiency as the evaluation index. An in vitro BBB model was established using brain endothelial cell line 3 (bEnd.3). Model integrity was verified by measuring transendothelial electrical resistance (TEER), liquid level permeability, and sodium fluorescein permeability. The Exo-Rg₁ complex group (experimental group), free Rg₁ group (control group), exosome alone group, and empty exosome + free Rg₁ group were set up. The concentration of Rg₁ penetrating the in vitro BBB model at different time points was detected by HPLC, and the permeability efficiency was calculated. In vivo therapeutic effects were evaluated through behavioral tests, histological observation, and detection of protein and inflammatory factor levels in AD model mice. Results The optimal exosome purification process was dual-coupled harmonic oscillation technology, yielding an exosome concentration of 2.9 × 10¹¹ particles/mL, a particle size of (91.8 ± 1.5) nm, and a PDI of 0.21 ± 0.01. The optimized electroporation conditions were voltage of 100 V, pulse duration of 15 ms, pulse number of four times, and Rg₁ concentration of 0.4 mg/mL. Under these conditions, the encapsulation efficiency of the complexes was 61.58%, which was superior to that of the traditional ultrasonic method. The in vitro BBB model using bEnd.3 cells was successfully established, with TEER values stably above 300 Ω·cm². A significant liquid level difference was maintained in the in vitro BBB model group after 4 h. The sodium fluorescein permeability in the model group was significantly lower than that in the blank control group [(41.34 ± 1.83)% vs (98.87 ± 2.09)%, P < 0.01], meeting the requirements for barrier function. In vitro penetration experiments showed that the penetration efficiency of Rg₁ in the experimental group (15.3%) was significantly higher than that in the control group (3.3%) at 8 h (P < 0.05). Animal experiments demonstrated that the complex significantly improved learning and memory abilities in model mice, protected hippocampal neurons, upregulated the expression of choline acetyltransferase (ChAT) and α7 nicotinic acetylcholine receptor (α7 nAChR) proteins, and reduced the levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α), with effects superior to those of the free Rg₁ group and the exosome-only group. Conclusion Exosomes can effectively enhance the efficiency of Rg₁ in crossing the BBB, providing an experimental basis for improving the therapeutic effect of brain diseases.

R283.6

国家自然科学基金资助项目（82374311）