Preparation Of Novel Dendritic Targeting Nanoparticles And Study On Removal Of Cholesterol In Macrophages

In the pathogenesis of atherosclerosis,macrophages phagocytosis a large amount of cholesterol to form foam cells,which accumulate to form plaques,and exacerbates the inflammation of blood vessels and further promotes the development of atherosclerosis.At present,the treatment of atherosclerosis is mainly systemic drug therapy and surgical treatment,and there is no treatment strategy that target the atherosclerotic lesions.In this study,peptide dendrimer polyamide-amine?PAMAM G5.0?was used as the carrier,?-CD and PEG-linked targeting ligand mannose were modified on the surface of PAMAM G5.0 to prepare atherosclerotic plaques nanoparticles target drug carrier system,mannose-specific recognition of macrophages,and bring?-CD to macrophages.?-CD can dissolve cholesterol crystals in macrophage foam cells and removes cholesterol from macrophages.Thereby achieving the effect of inhibiting the progression of atherosclerosis,significant for the treatment of atherosclerosis.Main research contents are as follows:1.Preparation and characterization of dendritic targeting nanoparticles.To modify the surface of dendrimer PAMAM G5.0,first of all,?-CD reacts with TsCl to form the active intermediate 6-OTS-CD.According to the molar ratio of G5:6-OTS-CD=1:100,?-CD modified on G5 surface to obtain CD-G5 nanoparticles by the substitution reaction;Then,through the bridging action of carboxylation at both ends of polyethylene glycol?PEG-?COOH?₂?,one end is connected with a targeting molecule mannose capable of targeting macrophages,and the other end is connected to the surface of PAMAM G5.0,To get targeted nanoparticles CD-G5-PEG-Man;Through a series of characterizations such as FTIR,¹HNMR and TEM,whether it was successfully prepared and the performance of nanoparticles were verified,such as morphology and particle size.The size of the nanoparticles was about 110 nm.2.Cytotoxicity and targeting of dendritic targeting nanoparticles.The effect of CD-G5-PEG-Man on cell viability was evaluated by MTT colorimetry.The results showed that the particles were less toxic and biocompatible.Through in vitro cell experiments to verify its targeting,observation with an inverted fluorescence microscope,the result showed that the mean fluorescence intensity of CD-G5-PEG-Man nanoparticles with targeting molecules is greater than that of CD-G5 nanoparticles without targeting molecules,and have statistical significance?n=3,*p<0.05?.the greater the mean fluorescence intensity,the more it binds to the cell.However,when cells were incubated with free mannose at different concentrations?0?M,5?M,50?M?,and then cells were separately added with CD-G5-PEG-Man to incubate the cells,as the concentration of mannose is increased,the weaker the fluorescence and the smaller the average fluorescence intensity,indicating that fewer nanoparticles are bound to the cells.The above shows that free mannose molecules can competitively inhibit the cellular uptake of CD-G5-PEG-Man,and CD-G5-PEG-Man can specifically target macrophages through mannose.3.Studies on dendritic targeting nanoparticles for removal of cholesterol from macrophages.The BODIPY-cholesterol labeling method was used to study the effect of nanoparticle CD-G5-PEG-Man on intracellular cholesterol efflux,and image J?FIJI?software was used to quantitatively analyze the fluorescence intensity.The results showed that the mean fluorescence intensity of 200 nM CD-G5-PEG-Man was lower than that of blank control DMSO and control group HP-?-CD,indicating that there was more BODIPY-cholesterol outflow in the 200nM CD-G5-PEG-Man group.These results indicate that the CD-G5-PEG-Man?200 nM?group has a certain activity to mobilize cholesterol from macrophages.The above research shows that the CD-G5-PEG-Man nanoparticles prepared by us can selectively target macrophages in vitro cell experiments,and specifically bind to the mannose receptor on the surface of macrophages through the targeting molecule mannose,and remove the cholesterol from the cells according to the dose.This research can provide a new type of nanomaterials for the prevention and treatment of atherosclerosis and provide potential value for atherosclerosis targeted research,so the research is of great significance.