| Acteoside（ACT）is a kind of phenylethanoid glycosides compounds existing in food or edible plants such as osmanthus,olive and cistanche.Studies have confirmed that ACT has good neuroprotective effect and ameliorative effect on AD.However,due to the blood brain barrier（BBB）,which prevents exogenetic substances from entering the central nervous system,the application of ACT in alleviating AD is limited.Receptor mediated brain targeting delivery is the most commonly used strategy to overcome BBB at present.The commonly used brain targeting receptors include transferrin receptors,low density lipoprotein receptors,insulin receptors,etc.However,there are some deficiencies in the ligands of these receptors.For example,endogenous ligands occupy most of the receptors in the brain,which reduces the brain targeting efficiency of the delivery system,and the monoclonal antibodies used as ligands are expensive.Cannabinoid receptor 1（CBR1）is mainly distributed in the brain,spinal cord and peripheral nervous system.Therefore,the nanodelivery system modified with CBR1 ligand has the potential of delivering neuroactive substances to the brain.At present,there are no reports on increasing the content of ACT in brain to improve its effect on AD,and there are few reports on CBR1mediated brain targeted delivery.Therefore,development of a brain targeted delivery system based on CBR1 and improving the ability of ACT in crossing BBB can provide an important scientific basis and reference value for promoting the clinical application of ACT in alleviating AD.And this strategy can also promote the research on the brain targeted delivery of other neuroactive substances.This study aims to delivery ACT into brain by CBR1 ligand.We used cell models,animal models and molecular docking methods to screen and verify CBR1 ligand with the best brain targeting ability,and then used this ligand to modify LNCs that encapsulated ACT.The CBR1-mediated endocytosis delivered ACT into the brain,and the effect of this delivery system was verified in cells and AD mice.The main contents and results of this research are as follows:1.Preparation and characterization of lipid nanocapsules modified with CBR1 ligand compoundsBlank lipid nanocapsules（LNCs）,LNCs encapsulating fluorescent substances and LNCs modified with 10 CBR1 ligand compounds were prepared by phase inversion temperature method.The 10 CBR1 ligand compounds were:cannabidiol,cannabiterpenol,falcarinol,yangonin,voacamine,olivetol,curcumin,magnolol,sanguinarine and chelerythrine.The results showed that the fluorescent material was encapsulated into the core of LNCs,and the ligand compounds（X）was modified onto the surface of LNCs to form X-LNCs.The different types of LNCs mentioned above all have good stability.The encapsulation rate of fluorescent substance was close to 100%,and the leakage of fluorescent substance in cell culture medium or PBS for 24 h was lower than 3%.At experimental concentrations,X-LNCs showed no obvious cytotoxicity and hemolysis.The cell survival rate was more than 90%and hemolysis rate was less than 3%after 24 h.These results indicated that X-LNCs were successfully prepared with high safety and could be used for targeted screening in vivo and in vitro.2.Screening and validation of brain targeting ability of LNCs modified with CBR1ligand compoundsThe cellular uptake of X-LNCs by h BMECs was qualitatively and quantitatively analyzed by laser confocal microscopy and flow cytometry,respectively.The in vitro BBB model was used to investigate the ability of X-LNCs to cross BBB.X-LNCs with good targeting ability were screened out at the cellular level.The brain targeting ability of screened X-LNCs was further screened and confirmed by fluorescence in vivo imager in mice.The results showed that,at the level of cellular uptake and BBB model in vitro,the brain targeting ability decreased as follows:LNCs modified with falcarinol,voacamine,chelerythrine,yangonin,cannabiterpenol and cannabidiol,among which the cellular uptake of LNCs by h BMECs was increased by 4.1 and 3.9 times after falcarinol and voacamine decoration.Animal experiments in mice showed that the brain targeting ability of VOA was better than other compounds.After 24 h,the fluorescence intensity in the brain of mice in the group of VOA-LNCs was 3.8 times higher than that of pure fluorescent substance.The amount of VOA-LNCs in the brain of mice achieved the highest at 2 h,and each gram of brain tissue accounted for 0.82%of the injected dose.3.Verification the mechanism of the brain targeting ability of VOA-LNCsDifferent temperatures and inhibitors were used to investigate the cellular uptake of VOA-LNCs by h BMECs and the ability of VOA-LNCs in crossing the BBB model.Meanwhile,CBR1 inhibitor was used to verify the brain targeting ability of VOA-LNCs.Finally,the binding sites of VOA and CBR1 was investigated by molecular docking.The results showed that:Firstly,VOA-LNCs can cross BBB through receptor-mediated clathrin endocytosis,and the cellular uptake of VOA-LNCs by h BMECs was significantly reduced by low temperature（4°C）,ATP inhibitors and clathrin inhibitors.Secondly,VOA-LNCs cross the BBB through CBR1-mediated endocytosis.The CBR1 inhibitor significantly reduced the the cellular uptake of VOA-LNCs by h BMECs.In the presence of CBR1inhibitor,the penetration of VOA-LNCs decreased from 32.1%to 13.2%in the in vitro BBB model,and the content in the brain decreased by 37.5%.The types of interactions between VOA and CBR1 include hydrophobic interactions,hydrogen bonding,and Pi-Pi stacking.4.Preparation and safety evaluation of VOA-LNCs-ACTA reversed-phase micell method was used to optimize the preparation of VOA-LNCs-ACT.The stability and release pattern of VOA-LNCs-ACT were investigated,and the safety of VOA-LNCs-ACT was preliminatively evaluated at the cellular and animal levels.The results showed that ACT could be encapsulated into LNCs by reversed-phase micelles,and the encapsulation rate reached about 85%.After 90 days of storage at 20°C,the degradation rates of ACT in the solution and the degradation rates of ACT in the VOA-LNCs-ACT were 31.1%and 14.5%,respectively.Within the experimental concentration range,VOA-LNCs-ACT had no obvious toxicity to nerve cells,and the cell survival rate was more than 90%after 24 h.There was no obvious toxic and side effects on mice in the VOA-LNCs-ACT group.The blood biochemical life indexes,hepatorenal toxicity indexes,body weight and pathological sections of mice in the VOA-LNCs-ACT group showed no different with those in the normal saline control group.5.Study on the neuroprotective effect of VOA-LNCs-ACT on cell modelAβ25-35 was used to induce cell injury model in SH-SY5Y cells,and the neuroprotective effect and mechanism of VOA-LNCs-ACT on SH-SY5Y cells were studied.The results showed that VOA-LNCs-ACT improved the neuroprotective effect of ACT on AD cells.Specifically,VOA-LNCs-ACT further increased the activity and antioxidant enzyme activity of AD cells,decreased the content of Aβ1-42 and the level of reactive oxygen species,and reduced the production of lipid peroxide.Studies on mechanisms related to signaling pathways have found that VOA-LNCs-ACT promotes redox balance and improves AD symptoms by activating Nrf2 signaling pathway.6.Study on the neuroprotective effect of VOA-LNCs-ACT on AD mouse modelThe neuroprotective effect of VOA-LNCs-ACT on AD mice induced by Aβ25-35 was investigated.The results showed that VOA-LNCs-ACT improved the neuroprotective effect of ACT on AD mice.Specifically,VOA-LNCs-ACT further improved the learning and memory ability of AD mice,and decreased the content of Aβ1-42 in the brain of AD mice.At the same time,VOA-LNCs-ACT reduced the level of reactive oxygen species in the brain of AD mice,increased the activity of related antioxidant enzymes,reduced the production of lipid peroxides,reduced synaptic damage in the brain of AD mice,and decreased the activation levels of astrocytes and microglia.In conclusion,this study developed a novel brain targeting delivery system based on CBR1,which improved the ability of ACT in crossing BBB and elucidated its role in alleviating AD.This study can provide an important scientific basis and reference value for improving the clinical application of ACT in alleviating AD and the targeted delivery of other neuroprotective substances to the brain. |
