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.

exosome  /  ginsenoside Rg₁  /  blood-brain barrier  /  drug delivery systems  /  bEnd.3 cells  /  dual-coupled harmonic oscillation technology  /  choline acetyltransferase  /  alpha-7 nicotinic acetylcholine receptor  /  interleukin-1β  /  tumor necrosis factor-α