Construction Of Amphiphilic Luminol/Chlorin E6 Conjugated Self-Assembled Nanoparticles And Its Efficacies And Mechanisms For The Treatment Of Atherosclerosis

Cardiovascular disease is the most common cause of death worldwide,with major complications of ischemic stroke,myocardial infarction and peripheral vascular disease.About 50%of deaths related to cardiovascular disease are caused by coronary artery disease.Atherosclerosis（AS）is the pathophysiological basis of coronary artery disease and the most important pathological process in the development of cardiovascular disease.There are many pathogenic factors for atherosclerosis,such as abnormal blood lipids,abnormal blood sugar,high blood pressure,obesity and diabetes.Existing studies have confirmed that atherosclerosis is not only associated with metabolic disorders,but also a chronic inflammatory process of the arterial wall,which is caused by the deposition of lipoproteins in the blood in the damaged endodermis.Monocytes in the blood are affected by inflammation and enter into the intima through activated endothelial cells and differentiate into macrophages.Macrophages oxidized lipid deposits and swallowed them into foam cells to induce the formation of fatty streaks.This is the beginning of atherosclerosis development.In addition,lipoproteins oxidized by macrophages further induce necrosis of foam cells,form necrotic core,promote plaque rupture,induce thrombosis,and accelerate the development of atherosclerosis.At present,the main drugs to treat atherosclerosis are regulating blood lipid,antioxidants,antiplatelet,vasodilator and anti-inflammatory drugs.However,these drugs can lead to liver and kidney dysfunction,gastrointestinal bleeding,diarrhea and hypotension and other side effects,which limit their wide use in clinical.Therefore,targeted drugs have become a hot topic in the treatment of atherosclerosis.The amphiphilic chlorin E6（CE6）,luminol and polyethylene glycol（PEG）conjugates（CLP）were synthesized by our research group in the early stage.It was found that the drug has anti-inflammatory and inhibitory effects on the migration of neutrophils and macrophages.Atherosclerosis is a chronic inflammatory process.Vascular endothelial cells are affected by inflammation,endothelial function is impaired,circulating low-density lipoprotein infiltrates into the vascular intima and is oxidized.Oxidized LDL activates immune cells,resulting in the expression of chemokines and adhesion molecules,and attracts monocytes to gather at the injured site of endothelium,which makes them adhere and migrate to the intima.At this time,monocytes differentiate into macrophages.Macrophages migrate under the endocardium and swallow up oxidized low-density lipoprotein to form foam cells.After accumulation of foam cells necrosis,thrombosis and proinflammatory and necrotic core gradually form,and promote plaque rupture leading to thrombosis,leading to further development of atherosclerosis.Therefore,we assume that CLP nanoparticles have anti-inflammatory effect and inhibit macrophage migration,so whether CLP nanoparticles can target plaque to prevent and treat atherosclerosis.Whether to enhance the efficacy of nanoparticles after adding targeted groups.Methods1.Synthesis and characterization of CLP and CLP-cRGD conjugatesThe Ce6 carboxyl group was activated by n-hydroxysuccinimide（NHS）and N-（3-dimethylaminopropyl）-N’-ethyl carbonimide hydrochloride（EDC）,the amphiphilic CLP conjugate was obtained by condensation with the amino group of Luminol and methoxyamino-polyethylene glycol,respectively.The amphiphilic CLP-MAL conjugate was obtained by using n-hydroxy succinimide（NHS）and N-（3-dimethylaminopropyl）-N’-ethyl carbonimide hydrochloride（EDC）to activation of the Ce6 carboxyl group and condensation of the amino group with luminol and maleimide amino peg,respectively.CLP bonded cRGD polymer（CLP-CRGD）was further obtained on the CLP main chain by maleimide（MAL）and mercapto of arginine-glycine-aspartic acid cyclic peptide（cRGD）.The structures of CLP and CLP-cRGD were confirmed by IR,NMR,MALDI-TOF,UV and fluorescence spectra.2.Synthesis and characterization of CLP nanoparticles and CLP-cRGD nanoparticlesSelf-assembled CLP nanoparticles（CLP NP）and CLP-cRGD nanoparticles（CLP NP）were obtained by dissolving CLP and CLP-cRGD in aqueous solution.The particle size distribution,particle size and zeta potential were measured by transmission electron microscopy and Malvern particle size analyzer.3.Targeting effect and organ distribution of CLP nanoparticles and CLP-cRGD nanoparticles in atherosclerotic plaqueAfter atherosclerotic treatment,the heart,aorta and main organs of mice were taken for in vivo imaging and confocal imaging to investigate the targeting effect of CLP nanoparticles and CLP-cRGD nanoparticles on plaque in heart and blood vessels and organ distribution.4.Pharmacodynamics of CLP nanoparticles in the treatment of atherosclerosisApo E^-/-mice were used to establish atherosclerosis model.After 80 days of treatment with CLP nanoparticles,the plaque areas of aorta,aortic root and brachiocephalic trunk were measured,and the stability of aortic root plaque was investigated to determine the effective administration mode of CLP nanoparticles in the treatment of atherosclerosis.5.Pharmacodynamic study of CLP-cRGD nanoparticles in the treatment of atherosclerosisAfter the successful establishment of atherosclerosis model in Apo E^-/-mice,CLP nanoparticles and CLP-cRGD nanoparticles were administered to the tail vein for 60 days.The plaque area of the aorta,aortic root and brachiocephalic trunk was measured,the stability of the aortic root plaque was investigated,and the efficacy of CLP nanoparticles and CLP-cRGD nanoparticles was evaluated.6.Mechanism of CLP nanoparticles in the treatment of atherosclerosisThe phagocytic effect of CLP nanoparticles was evaluated by using mouse macrophages（RAW 264.7 cells）,mouse aortic endothelial cells（MOVAS cells）and human umbilical vein endothelial cells（HUVEC cells）associated with atherosclerosis.RAW264.7 macrophages were induced by lipopolysaccharide（LPS）to establish an inflammatory cell model.MIP-2protein with the most significant change after treatment with CLP nanoparticles was screened by chemokine protein chip.The results were verified by ELISA kit,fluorescence quantitative PCR and Western blot.The effect of CLP nanoparticles on inflammatory factors was verified by using inflammation model.Monocyte chemoattractant protein-1（MCP-1）and fetal bovine serum（FBS）were used as chemokines to verify the effects of CLP nanoparticles on the migration of RAW 264.7 cells,MOVAS cells and neutrophils.The formation of foam cells was induced by oxidized low density lipoprotein（ox LDL）,and the effect of CLP nanoparticles on the formation of foam cells was verified.7.Safety evaluation of CLP nanoparticles and CLP-cRGD nanoparticlesThe toxic effects of CLP nanoparticles on RAW264.7 cells and MOVAS cells were detected by CCK8 method.After the anti-AS treatment of Apo E^-/-mice,the weight change,the statistics of the organ index,the analysis of blood routine and liver and kidney function,and the observation of H&E staining of the main organs were recorded to investigate the safety of CLP nanoparticles in the treatment of AS.Balb/c mice were given 1000mg/kg,500mg/kg and 250mg/kg CLP nanoparticles at one time respectively in the tail vein,15-day weight change and organ index were counted,blood routine and biochemical tests were performed,and H&E staining of main organs was observed.Investigate the acute toxicity experiment of CLP nanoparticles.Results1.CLP conjugates could be successfully constructed by spectral analysis combined with mass spectrometry.The cyclic peptide of cRGD can be bonded to the main chain of CLP by maleimide,and CLP-cRGD polymer is successfully constructed.The average particle sizes of CLP nanoparticles and CLP-cRGD nanoparticles were 170±4 nm and 156±3 nm,respectively,and the surface potential（Zeta potential）were-19.2±0.2 m V and-16.1±0.3m V,respectively.2.Both CLP nanoparticles and CLP-cRGD nanoparticles can target at the site of vascular plaque.After the introduction of cRGD targeting unit,CLP-cRGD nanoparticles enhanced the plaque targeting effect of nanoparticles.CLP nanoparticles injected intravenously can inhibit the formation of atherosclerotic plaque and maintain plaque stability.CLP-cRGD nanoparticles are more effective than CLP-cRGD nanoparticles.3.CLP nanoparticles can be phagocytic into the cell by RAW264.7 macrophages,MOVAS cells and HUVEC cells.CLP nanoparticles can reduce the content of Macrophage inflammatory protein-2（MIP-2）and inflammatory factors in inflammatory cells.Both CLP nanoparticles and CLP-cRGD nanoparticles could effectively inhibit the migration of RAW264.7 macrophages,MOVAS cells and intraperitoneal neutrophils,and the inhibition effect of CLP-cRGD nanoparticles was stronger.CLP nanoparticles can also effectively inhibit the uptake of ox LDL by RAW264.7 macrophages and inhibit the formation of foam cells.4.CLP nanoparticles and CLP-cRGD nanoparticles have good safety in vitro and in vivo.Conclusions1.CLP conjugate and CLP-cRGD polymer were successfully constructed.2.CLP nanoparticles and CLP-cRGD nanoparticles can target to the plaque site.Compared with CLP nanoparticles,CLP-cRGD nanoparticles are more targeted.Intravenously injected CLP nanoparticles can effectively treat atherosclerosis.CLP-cRGD nanoparticles with additional targeting units have a better therapeutic effect.3.CLP nanoparticles can effectively inhibit the migration of inflammatory cells such as mouse macrophages（RAW264.7 cells）,mouse aortic vascular endothelial cells（MOVAS cells）and abdominal neutrophils,and the targeted nanoparticle inhibition effect is more significant.In addition,CLP nanoparticles can effectively inhibit the formation of foam cells,reduce the content of inflammatory factors and chemokines MIP-2 secreted by inflammatory cells,and inhibit the development of atherosclerosis.4.Preliminary in vitro and in vivo experimental results show that CLP nanoparticles have good tissue and cell safety.