Preparation Of Angiopep-2 Modified Naringin Nanoparticles And Evaluation Of Blood-brain Barrier Permeability

Alzheimer’s disease（AD）is a multi-factorial neurodegenerative disease.The cause of its pathogenesis is very complicated.The accumulation ofβ-amyloid protein,neuronal dysfunction,cholinergic neuron damage,etc.can all lead to AD.Among the 75-84-year-olds,the prevalence rate is 19%,and the morbidity rate is as high as 47%among the elderly over 85,and the mortality rate is as high as 57%.In addition,due to the existence of the blood-brain barrier,it is difficult for most drugs with good in vitro activity to enter the central nervous system to play a role.There is currently no effective drug that can completely cure the disease,but if the amount of drugs into the brain is increased,the treatment greatly improved effect.The traditional Chinese medicine Rhizoma Drynariae also has thousands of theoretical and practical experience in strengthening the brain,strengthening the bones,strengthening the kidneys and filling the essence,etc.Naringin is a representative flavonoid compound with the highest content in Drynaria fortunei.In vitro experiments have confirmed that it has many functions such as protecting neurons,inhibiting enzymes,anti-oxidation,scavenging free radicals,and improving mitochondrial function.However,it has low water solubility and blood-brain barrier permeability,so that less drugs penetrate into the brain.In this study,naringin is combined with a rapidly developing new nano-drug delivery system,and then targeted modification is constructed to construct a targeted drug delivery system,which can increase the delivery efficiency and therapeutic effect.The main content of this article includes the following three parts:（1）Preparation and characterization of naringin nanoparticles（NG-NP）;（2）Preparation of Angiopep-2modified naringin nanoparticles（ANG-NG-NP）And characterization;（3）The influence of nanoparticles on Aβ_25-35-induced injury of PC12 cells and their distribution in vivo.First,naringin nanoparticles（NG-NP）were prepared by the emulsification solvent evaporation method,and the encapsulation efficiency and drug loading were used as indicators,and the preparation process was optimized through orthogonal experiments.The optimal preparation conditions for NG-NP were that the concentration of naringin was 3 mg/m L,the concentration of PEG-PCL was 10 mg/m L,the concentration of sodium cholate was 0.5%,and the volume ratio of oil phase to water phase was 1:5.After determining the best preparation process,the particle size was 96.5±3.85 nm,the potential was-3.28±0.76 m V,the shape and size were uniformly dispersed,the encapsulation efficiency was 78.87±0.45%,and the drug loading was 26.04±0.3%.Subsequently,Angiopep-2 was modified to NG-NP by covalent bonding of maleimide group and sulfhydryl group,and Angiopep-2 modified naringin nanoparticles（ANG-NG-NP）.Characterize it again.ANG-NG-NP is a uniform spherical shape with a particle size of 126.1±4.5 nm,a potential of-2.97±0.29 m V,and an appearance of uniform spherical particles with good dispersion and good visibility.The form of nanoparticle-encapsulated drugs is produced,and both NG-NP and ANG-NG-NP have obvious sustained-release effects,prolonging the drug action time of naringin and improving the bioavailability.Finally,in the in vitro cell experiment,the cytotoxic effects of NG-NP and ANG-NG-NP on PC12 cells were investigated,and the two types of nanoparticles had no obvious toxicity,and the effects of NG-NP and ANG-NG-NP were investigated.Compared with naringin raw materials,the effects of NG-NP and ANG-NG-NP on the proliferation of PC12 cells induced by Aβ_25-35 are more significant,especially ANG-NG-NP can increase the cell proliferation rate.raised dramatically.In addition,by using coumarin-6 as a fluorescent probe to evaluate the cellular uptake of the two types of nanoparticles by PC12 cells injured by Aβ_25-35.Studies have shown that nanoparticles modified Angiopep-2 enhanced its transmembrane penetration ability,enhanced targeting,and enabled more drugs to be transported across the membrane,thereby increasing the intracellular drug concentration.Then,through the in vivo fluorescence imaging technology of small animals,the distribution of nanoparticles in mice was investigated,and DiR was used as a fluorescent probe to evaluate the blood-brain barrier penetration ability of nanoparticles.The results showed that ANG-DiR-NP and DiR-NP have more advantages.Strong targeting and BBB penetration ability,and form aggregation in the brain,staying for a long time is conducive to drug release and drug effect.The test results show that ANG-NG-NP promotes naringin into PC12 cells in a non-invasive way.In recent years,research on the pharmacological effects of naringin has begun to be active,but there are few reports on naringin and nano-targeted drug delivery systems.The Angiopep-2 modified nanoparticles prepared in this experiment have stronger BBB permeability.The enhanced treatment efficiency is an effective medicament that can improve nerve function and provides new ideas and methods for the treatment of AD.In addition,its specific mechanism of action needs to be studied in depth.