| Parkinson’s disease(PD)is a neurodegenerative disease caused by extensive progressive loss of dopaminergic neurons in the substantia nigra and striatum.The incidence of PD in males over 65 years old was 1.7%,and the incidence o f PD in females was about1.6%.Especially with the aging of the population,the impact of PD on human health and social economy has become increasingly prominent.Therefore,its treatment has become a major scientific problem to be solved in the field of biomedicine.It has been reported that the pathogenesis of PD is related to oxidative stress,that is,the imbalance between oxidation and anti-oxidation.At present,PD treatment is mainly based on drug treatment,but 98%of the drugs cannot achieve the desired therapeutic effect because they cannot penetrate the Blood Brain Barrier(BBB).Therefore,developing an effective delivery method to improve the permeability of BBB and constructing drug carriers targeting midbrain dopamine neurons has become a hot and difficult point in the treatment of PD.Curcumin is a polyphenolic compound with important economic value and extensive pharmacological action,especially its antioxidant effect is believed to be useful for the prevention and treatment of PD.However,curcumin is almost insoluble in water,has a short half-life in the body,is fast in metabolism,and has low bioavailability,which makes it limited in clinical application.Nanovesicle(NV)is formed by membrane fusion after mechanical disruption of cells.NV,20-130 nm in size,is protected from being cleared by the mononuclear phagocytic system and protects drug activity in the in vivo circulatory system.NV has the advantages of safety,stability,drug loading capacity,targeting and immuno-inertity,as well as high yield and preparation processes,making it a huge potential for drug loading.In this study,NV was used as an effective carrier of curcumin to target midbrain dopamine neurons to achieve therapeutic effects on PD.Objective:1.Construction of a nanovesicle delivery system with targeted delivery capabilities.2.Using the nano-vesicle system to achieve PD drug curcumin targeting nigral dopaminergic neurons and enhancing the neuroprotective effect of curcumin.Method:Firstly,the isolation and identification of the midbrain neurons in newborn SD rats were performed,and the in vitro PD model was prepared using1-methyl-4-phenylpyridine(MPP~+).Next,the mouse RAW264.7 macrophages were sequentially extruded through a 10,5,1 um polycarbonate membrane filter using a cell extruder to prepare nano-vesicles,which were purified by differential centrifugation.Then,curcumin was loaded into the nano-vesicles(NV-CUR)by means of co-incubation,repeated freeze-thaw method and ultrasonic method,and its morpho logy was observed to evaluate its encapsulation efficiency and drug loading.Finally,NV-CUR was co-incubated with midbrain neurons,and half of the lethal dose of MPP+was used to prepare rat in vitro PD model.The NSA-CUR cells were observed by laser confocal microscope(LSCM).The protective effect of curcumin nanocapsules on PD model in vitro was preliminarily evaluated,which laid a foundation for further study on the efficacy of curcumin nano-vesicles on PD model.Results:In order to prepare the in vitro PD model,MPP~+was incubated with mature midbrain neurons for 48 hours in vitro,and the model was successfully confirmed by LSCM and CCK-8 kits.The nano-vesicles were prepared by extrusion and purified by differential centrifugation.Quantitative analysis of nano-vesicles using the BCA protein quantification kit.Nanoparticle tracking analysis,transmission electron microscopy,and atomic force microscopy were used to characterize the size and morphology of nano-vesicles.Dynamic light scattering instrument was used to monitor the change of cytoplasmic particle size of nanocapsules with time to investigate the stability of nanovesicles in dispersion medium.Nano-vesicles as carriers were successfully prepared.Ultraviolet spectrophotometry was used to compare the loading of curcumin by nanovesicles under different treatment methods such as co-incubation,repeated freeze-thaw and sonication.The efficiency was followed by ultrasonic method>co-incubation method>repeated freezing and thawing method.Unloaded curcumin was removed by centrifugation,and the purified NV-CUR was co-incubated with primary neurons.After 2 h,MPP~+was administered for 48 h.Compared with the MPP~+group(65.37±6.37)%,the neuron survival rate in the NV-CUR group was significantly increased to(90.91±3.18)%.In the present study,we found that NV-CUR has a protective effect on MPP~+-induced primary neurons.Conclusion:nano-vesicle with double-layer lipid membrane can load the fat-soluble drug curcumin,which successfully targets mid-brain neurons and enhances the therapeutic effect.Compared with traditional PD treatment,nano-vesicle system improves the bioavailability of the drug.It is expected to become a new and effective drug delivery system,and is further applied to the field of therapeutic drugs for the delivery of various system diseases in the whole body. |
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