Construction Of Brain-targeted Nanoreactor System Containing RAGE Antagonist Peptide

ObjectiveRP-1 is an advanced glycosylation terminal product receptor(RAGE)antagonist peptide,which has been shown to be effective in relieving A?-induced neuronal toxicity in vitro.In this experiment,we will prepare PLGA nanoparticles loaded with RP-1,then use transmembrane peptide(TAT)and brain-targeting peptide(Angiopep-2)to modify nanoparticles to construct brain-targeted nanoparticles.We hope to provide experimental basis for the subsequent treatment of Alzheimer's disease(AD)with RP-1.MethodsPrepare the PLGA blank nanoparticles by emulsification solvent evaporation method.The single factor investigation of the preparation conditions of the nanoparticles was performed to determine the four factors affecting the particle size of the nanoparticles.Each factor was set at three levels.The particle size of the nanoparticles was Evaluation indicators,establishment of an orthogonal test of four factors and three levels,orthogonal test,determination of the optimum preparation conditions of nanoparticles by range analysis,and validation of the best process,determination of the size of the blank nanoparticles(NP),Zeta Potential and polydispersity PdI;Nanoparticles were prepared by the best preparation method and observed by transmission electron microscopy(TEM).The transmembrane peptide TAT and the brain-targeting peptide Angiopep-2 were modified on the surface of nanoparticles to obtain TAT-NP and ANG-NP.The thiol detection kit was used to determine the modification efficiency of the transmembrane peptide TAT and the brain-targeting Angiopep-2,and the modified nanoparticles were characterized to determine their particle size and Zeta potential;The HPLC method for the determination of coumarin-6 was established.The coumarin-6 nanoparticles(Cou-6/NP)were prepared by the best preparation process.Entrapping efficiency(EE)and loading were measured by centrifugal sampling method.Drug loading capacity(DLC)and determination of in vitro release characteristics of nanoparticles within 48 h;HPLC in vitro analysis method of RP-1 was established,and RP-1 nanoparticles(RP-1/NP)were prepared using the best preparation process.Determine the entrapment efficiency(EE)and drug loading(DLC);Preparation of NP,TAT-NP,ANG-NP,TAT/ANG-NP four kinds of nanoparticles,CCK-8 method to investigate the nanoparticles on the brain vascular endothelial cells(b.End3 cells)in vitro toxicity.Nanoparticles(DiR/NP)containing near-infrared dye DiR were prepared,and the effects of different doses of DiR on the entrapment efficiency(EE)and drug loading(DLC)of DiR/NP were investigated.The preparation of DiR/NP was investigated by centrifugal sampling.In vitro release characteristics of DiR/NP within 48 h;C57BL/6 mice were selected as experimental animals and three different nanoparticles of DiR/NP,ANG-DiR/NP and TAT/ANG-DiR/NP were injected intravenously at equal doses.In vivo fluorescence imaging was used to investigate the incorporation of different nanoparticles into the brain 1 h later.ResultsThe total amount of PLGA-PEG-Mal and PLGA-MePEG,ultrasonic time,concentration of disperse phase sodium cholate solution,and concentration of sodium cholate solution in the aqueous phase have different effects on the particle size of nanoparticles;Under the optimization of conditions,the nanoparticles prepared by the best process conditions had a particle size of 142.8 nm,a Zeta potential of-11.93 mV,and a polydispersity index(PdI)of 0.161.The morphology of the nanoparticles was observed.Under the transmission electron microscope,the nanoparticles were uniform in size and appearance.The modification rates of TAT-NP and ANG-NP were 65.4 % and 71.7 %,respectively.Zeta potentials of the modified nanoparticles were observed.Change.Three types of nanoparticles,Cou-6/NP,TAT-Cou-6/NP and ANG-Cou-6/NP,were prepared.The entrapment efficiencies of nanoparticles were 38.210 %,27.31 % and 38.377 %,respectively.0.229 %,0.164 %,and 0.230 %;Determination of in vitro release properties of nanoparticles within 48 h,the cumulative release rate of coumarin-6 within 48 h was less than 8 %;Preparation of nanoparticles containing RP-1,the entrapment efficiency was 33.847 %,the drug loading is 0.847 %;The cytotoxicity of NP,TAT-NP,ANG-NP,and TAT/ANG-NP nanoparticles was investigated by CCK-8 method.Nanoparticles had no cytotoxicity in the range of 12.5-800 ug/ml.DiR/NP was prepared according to different doses of DiR.The results showed that when the dosage was 2 %,the drug loading and entrapment efficiency were large;the in vitro release characteristics of DiR/NP were investigated.Within 48 h,DiR/NP The cumulative release rate is approximately 30 %;the incorporation of DiR/NP,ANG-DiR/NP,and TAT/ANG-DiR/NP into the brain at 1 h after administration was observed by in vivo fluorescence imaging experiments.The result was three nanometers at 1 h.The amount of brain pellets were TAT/ANG-DiR/NP>ANG-DiR/NP>DiR/NP.Conclusion1.The single-factor test and orthogonal test were used to screen the preparation conditions of nano-particles.The optimum preparation conditions of nano-particles were as follows: the concentration of sodium cholate in the aqueous phase was 1.5 %,PLGA-MePEG and PLGA-PEG-Mal.The total amount of Mal was 5 mg,the ratio of PLGA-PEG-Mal to PLGA-MePEG was 1:9,and the concentration of sodium cholate in the dispersed phase was 0.1 %.2.Nanoparticles were successfully surface-modified with TAT and Angiopep-2.The modification rates were 65.4 % and 71.7 %,respectively.3.The cumulative release rate of drug-loaded nanoparticles per unit time is low,and its stability is good.4.In the concentration range of 12.5-800 ?g/ml without cytotoxicity,the prepared nanoparticles can be used as a safe brain-targeted drug carrier.5.The nanoparticle brains modified by TAT and Angiopep-2 have better targeting properties and are suitable as a carrier for drug-targeted brain delivery.