Synthesis Of Polypeptide Targeting Nanocarriers Modified Both By Fucose And Cyclodextrin And Induction Of Cholesterol Efflux In Macrophage

Lipid deposition in the arterial lumen wall and promote to form plaques,and monocyte macrophages phagocytize a large amount of lipids transform into foam cells in the diseased region,which induce inflammation in the endometrial lesion area and promote the formation of atherosclerosis.Targeted nanopartiele drugs have great application prospects in the prevention and treatment of atherosclerosis,however there are developing efficient and safe nanomedicine carrier systems still enormous challenge.Polyamide-amine(PAMAM)is one of the most intensively studied dendrimers and has many excellent properties.In this study,the peptide dendrimer polyamide-amine(PAMAM G5.0)was used as the carrier,β-cyclodextrin(β-CD)and the targeting molecule fucose were modified on the surface of PAMAM G5.0.To construct a nano-drug delivery system that targets macrophages in atherosclerotic plaques.Fucose-specific recognize and bind to the CD206 receptor on the surface of macrophages.β-CD as an active molecule can increase the solubility of cholesterol,and dissolve cholesterol crystals in macrophage foam cells,to promote the efflux of cholesterol in macrophages.Therefore,the synergy of the two can make the drug-carrying system specific targeting macrophages and play therapeutic effect on plaques.It provides a new strategy for the targeted therapy of atherosclerosis.The main contents of this research are as follows:1.Construction and characterization of targeting nanoparticles.The dendrimer PAMAM G5.0 is used as a carrier,to modifiy its surface,first,β-CD is activated by CDI,and modified to the surface of G5.0 by substitution reaction to obtain the intermediate product CD-G5nanoparticles.Through the bridging action of mercaptopropionic acid,one end is connected to a fucose molecule with a targeting ligand,and the other end is connected to the surface of G5.0 through a condensation reaction,thereby obtaining targeted nanoparticles CD-G5-S-Fuc.Through FT-IR and ~1HNMR verified the chemical structure of the nanoparticles.The average particle size was measured by Zeta potential and particle size analyzer.The resulting nanoparticles had a mean particle size was about8.97 nm.2.Research on biocompatibility and targeting of targeted nanoparticles.The cell viability of the nanoparticles was tested by the MTT experiment,and the results showed that the toxicity of the nanoparticles both modified byβ-CD and fucose was significantly lower than that before unmodified,indicating that the constructed nanoparticles have good biocompatibility.Through in vitro cell uptake experiments to verify the targeting of nanoparticles to macrophages.The cells were incubated with CD-G5-S-Fuc-FITC and CD-G5-FITC nanoparticles under the same conditions.The fluorescence microscope imaging showed that the fluorescence degree of CD-G5-S-Fuc-FITC nanoparticle connected to the targeting molecule is great than that of the CD-G5-FITC nanoparticles,its larger mean fluorescence intensity with time-dependent,and have statistical significance(n=3,*p<0.05).Through incubated the cells with different concentrations of free Fucose,then continue to incubate the cells with CD-G5-S-Fuc-FITC,the fluorescence microscope imaging shows that with concentration of free fucose increases,the fluorescence becomes weaker,and smaller the mean fluorescence intensity,indicating fewer nanoparticles take up by the cells.It shows that fucose molecules can specifically bind to CD206 receptors on the surface of macrophages,compete with nanoparticle CD-G5-S-Fuc-FITC,and inhibit the uptake of nanoparticles,because of fucose molecules can specifically bind to CD206 receptors on the surface of macrophages.Thereby,the nanoparticle CD-G5-S-Fuc has the function of targeting macrophages.3.Study on targeting nanoparticles to induce cholesterol efflux from macrophages.The BODIPY-cholesterol probe labeling method was used to study the effect of nanoparticle CD-G5-S-Fuc on intracellular cholesterol outflow.The fluorescence imaging was taken by a fluorescence microscope.The results showed that the mena fluorescence intensity of the 200 nM CD-G5-S-Fuc experimental group was lower than that of blank control group and the positive control group of 100μM HP-β-CD,indicating that the lower the average fluorescence intensity,the more BODIPY-cholesterol efflux in the cells.The result indicate that 200nM CD-G5-S-Fuc group has the best performance in inducing cholesterol efflux from macrophages.Combined with the above research,the targeted nanocarrier system CD-G5-S-Fuc has good biocompatibility,can specifically target macrophages and remove cholesterol in macrophages.It is expected that the CD-G5-S-Fuc nano-drugs delivery system can be used in the clinical targeted therapy of atherosclerosis.