| Objective Alzheimer’s disease(AD)is a progressive and fatal neurodegenerative disease characterized by deteriorating cognitive and memory functions,impaired ability to take care of oneself in daily life,and possible mental and behavioral disorders.At present,there is still no effective treatment.Curcumin(Cur)is an active ingredient extracted from turmeric.It is a polyphenol drug with multiple biological activities and has been proven to have neuroprotective effects.However,its use is limited by a number of disadvantages,including low solubility,poor stability,fast metabolism,and its poor blood-brain barrier(BBB)penetration.At present,exosome has been favored by researchers for its specific advantages.It is an extracellular vesicle of about 40-100 nm,which can carry various substances to transmit various information between cells.Mouse mononuclear macrophage leukemic cells(RAW264.7)have a strong phagocytosis ability for drugs,and exosomes(Exo)derived from the RAW264.7 as a natural carrier,can deliver drugs through BBB.Therefore,the purpose of this study is to prepare curcumin-loaded macrophage-derived exosomes and study its preliminary mechanism to improve cognitive ability of AD model mice.Methods In this study,Cur solution with a suitable concentration was added to the culture medium of RAW264.7 cells by using tetramethylazozolium trace enzyme reaction colorimetric method(MTT method).The culture medium was collected and obtained by ultracentrifugation,then got the Curcumin-loaded macrophage-derived exosomes(Exo-Cur).A fluorescence spectrophotometry method was established for the determination of curcumin content.The morphology,particle size and protein expression of Exo and Exo-Cur were characterized by scanning electron microscope(SEM)and Atomic force microscope(AFM),particle size analysis and Western Blot.The loading efficiency of Exo-Cur to Cur was tested,and the stability,solubility,in vitro release and organization distribution differences of Exo-Cur and free Cur were compared.Kinetic research,H&E staining test to detect the behavior and toxicity of the drug in vivo.In uptake studies,the uptake of brain microvascular endothelial cells(h CMEC/D3)by laser scanning confocal microscope was observed with curcumin spontaneous green fluorescence.HCMEC/D3 cells were used to simulate BBB to explore the possible pathways and mechanisms of Exo-Cur through BBB.The accumulation of Exo-Cur and free Cur in the brain was compared by animal imaging experiments.In the pharmacodynamic experiment,the AD model was established by injecting Okadaic acid(OA)into the lateral ventricle of C57/BL6 mice,then Exo-Cur and free Cur were injected intraperitoneally for 7 consecutive days,applied Morris water maze test to test the learning and memory ability of mice in each group,and immunofluorescence staining was used to investigate the improvement effect of Exo-Cur and free Cur on Neu N damage in the hippocampus.Finally,the mechanism of hippocampus was verified by Western Blot experiment.Results The content of curcumin was analyzed by fluorescence spectrophotometry established in this paper,and its linearity,precision and recovery all met the requirements of methodology.The particle size of Exo-Cur was about 100 nm,the morphology was relatively uniform,and the structure was complete which was not significantly different from Exo.The encapsulation efficiency of Exo-Cur to Cur was 84.8%.Exo-Cur had better stability,higher solubility and slower release profile than free Cur,and Exo-Cur had brain targeting.The results of pharmacokinetic experiments showed that the maximum plasma concentration(Cmax)of Cur in the free Cur treatment group reached a peak value of 0.52 μg/m L after 5 min of administration,and then rapidly decreased;while the Cmax of Cur in the Exo-Cur treatment group was given as it reached a peak of 120 minutes after drug administration,and significantly increased to 0.91 μg/m L.The results of H&E staining experiments showed that Exo was a relatively safe carrier.In uptake studies,Exo-Cur was uptaken more into h CMEC/D3 than free Cur,increased the BBB permeability of Cur through ICAM-1-mediated transcytosis,and had more accumulation of Exo-Cur in the brain than free Cur.In the pharmacodynamic experiment,the Morris water maze experiment found that compared with free Cur,Exo-Cur could improve the learning and memory ability of AD model mice,and Exo-Cur had a better improvement effect on the damaged Neu N in the hippocampus.Western Blot experiments verified that compared with free Cur,Exo-Cur could better inhibit the hyperphosphorylation level of Tau protein,and inhibit the expression of the apoptotic factors Cleaved Caspase-3 and Bax in AD model.Conclusions Exo-Cur had good stability,non-toxicity in vivo,which could increase the permeability of BBB,and had brain targeting,so that more curcumin into the brain.Exo-Cur could significantly improve the damaged Neu N in the hippocampus tissues.Mechanistically,by inhibiting the AKT/GSK-3β/Tau pathway to inhibit the hyperphosphorylation level of Tau protein and the expression of apoptotic factors,it had been proved in vivo that Exo-Cur coulld better improve the learning and memory ability of AD model mice. |
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