Preparation Of Multi-functional Magnetic Nanoparticles And Its Application In The Theranostics Of Atherosclerosis

Part Ⅰ:Preparation of RAP@PFN1-CD-MNPs nano-drug delivery systemsObjective:In order to integrate the diagnosis and treatment of atherosclerosis,the multifunctional drug-loaded nanosystem RAP@PFN1-CD-MNPs was successfully constructed.A series of biological activity studies were carried out at the cellular and protein levels to investigate the biocompatibility and drug release of RAP@PFN 1-CD-MNPs.Methods:The RAP@PFN 1-CD-MNPs multifunctional nanosystem was prepared by cyclodextrin as carrier,loaded rapamycin(RAP)and ultrasmall superparamagnetic iron oxide(USPIO),and modified by conjugated profilin-1 antibody.The morphology,particle size,zeta potential and structure were characterized by transmission electron microscopy,Nano-ZS nanometer particle size analyzer,Fourier transform infrared spectrometer and UV-vis absorption spectroscopy analysis.Drug loading,encapsulation rate and controlled release of nanomedicine in different environments were analyzed by drug release.The erythrocyte hemolysis test was used to evaluate its blood compatibility.The biocompatibility of RAP@PFN 1-CD-MNPs and the in vitro targeting effect on murine aortic vascular smooth muscle cells(MOVAS)were analyzed by cell absorption assay,CCK assay and so on.Western blotting was used to detect the expression of profilin-1.Results:The nanoparticles exhibited monodispersity in a dry state with a uniform size distribution,and a spherical morphology with an average particle size of 15-30 nm and a positive zeta potential of 18.37 mV.The saturation magnetization(Ms)was 69.85 emu/g.The RAP content of RAP@PFN 1-CD-MNPs nanoparticles reached 87%,and the hydrolysis of RAP@PFN 1-CD-MNPs was related to the pH of the buffer solution.The lower the pH of the buffer solution,the faster the hydrolysis rate at the same time point.After incubation with different concentrations of PFN1-CD-MNPs,no significant hemolysis was observed at mouse erythrocytes(MRBCs).The nanocarriers were cultured with MOVAS for 1 day,3 days,and 7 days,and the cytotoxicity increased slightly with the prolongation of the culture time,while the cell survival rate was over 80%during the three days of culture.In vitro fluorescence imaging of MOVAS and Prussian blue results showed that PFN1-CD-MNPs had higher cell binding to MOYASConclusions:This study successfully constructed a multifunctional nano-system of PFN1-CD-MNPs,which can be used as an excellent MRI T2 negative contrast agent.The PFN1-CD-MNPs drug-loaded nanosystem has good stability,blood compatibility,biocompatibility and acidic environment responsiveness.The nanosystem conjugated profilin-1 antibody has good targeting ability to MOVAS in vitroPart Ⅱ:Dual-modality imaging study of PFN1-CD-MNPs-Cy5.5Objective:.To study the distribution and the targeting effect of PFN1-CD-MNPs-Cy5.5 nano-system to atherosclerotic plaque by magnetic resonance imaging and near-infrared fluorescence in vivoMethods:The tissue distribution of PFN1-CD-MNPs in various tissues and organs in vivo was detected by near-infrared fluorescence(NIRF).Atherosclerosis model was established by high-fat diet,and serum lipid profiles were determined.The pro filin-1 expression in atherosclerotic plaques was analyzed by western blot.The imaging of PFN1-CD-MNPs-Cy5.5 in carotid atherosclerotic plaques was performed using a 7.0T small animal magnetic resonance imaging system.Ex vivo aortic plaque imaging was performed using NIRF.The tissue distribution of nanoparticles was verified by Prussian blue pathology analysis.Results:The relaxation rate(r2)of the nanoparticles was calculated to be 90.3 mM-1 s-1,meeting the requirements of magnetic resonance T2 negative contrast agent.The circulating time of PFN1-CD-MNPs in the blood is relatively long.After injection 72 hours,iron deposition was relatively more in the liver and spleen than other organs.The ApoE-/-mouse atherosclerosis model was successfully constructed with a high-fat diet for four months.The general Oil red O(ORO)staining for aorta and H&E staining confirmed the presence of plaque.The levels of HDL-C,LDL-C,and TC in the ApoE-/-group were significantly higher than those in the normal control group.And serum TC and lipoprotein levels were higher at 7 months than 4 months on a high-fat diet.Compared with the C57BL/6 normal group,the profilin-1 protein expression on the arterial wall of the ApoE-/-group was significantly increased.Significant T2-weighted signal intensity attenuation was observed in the carotid of ApoE-/-mice after injection of PFN1-CD-MNPs for 24 h compared with pre-injection,whereas there was almost no signal change in the carotid wall of the CD-MNPs group Compared with the CD-MNPs-Cy5.5 injection group,the fluorescence signal intensity of the aorta in the PFN1-CD-MNPs-Cy5.5 injection group was significantly higherConclusions:PFN1-CD-MNPs were relatively increased in uptake by the reticuloendothelial system.A high-fat diet for 4 months can successfully construct an atherosclerosis model with elevated blood lipid levels as the high-fat diet increases.Arterial plaque imaging can be achieved by both in vivo MRI and ex vivo NIRF.The profilin-1 protein was highly expressed in the atherosclerotic plaque region,and the nanoparticles coupled with the PFN1 antibody molecule can better target atherosclerotic plaquePart Ⅲ:Therapeutic effect study of RAP@PFN1-CD-MNPs nanosystems against atherosclerosis in vivoObjective:To evaluate the safety and therapeutic effect of multi-functional drug delivery system(RAP@PFN1-CD-MNPs)against atherosclerosis in vivoMethods:After 8 weeks of high-fat feeding,ApoE-/-mice were randomized and given different treatments with targeted nanomedicine,non-targeted nanomedicine,and saline respectively,while continuing high-fat feeding until 16 weeks.The extent of aortic plaque lesions between different groups was observed by gross ex vivo fluorescence imaging and ORO staining.Masson trichrome staining and immunohistochemical analysis were performed on different groups of ApoE-/-mice aorta.The relative content of collagen,CD68 and α-SMA were analyzed to further evaluate the plaque stability after treatment.The potential toxicity of the RAP@PFN1-CD-MNPs nano-system was evaluated by H&E staining of the main organs and observation of blood biochemical indicatorsResults:Fluorescence imaging of aorta showed that the RAP@PFN1-CD-MNPs treatment group had the weakest fluorescence signal intensity,and the saline group had the strongest aortic fluorescence signal intensity.From the representative stereomicrographs of the ORO-stained aorta,the most extensive atherosclerotic lesions were observed in the saline control group.After treatment with RAP@PFN1-CD-MNPs,the area of aortic plaque was significantly reduced.And ORO staining of the aortic arch cross-section showed a significant reduction in lesions.Masson trichrome staining result showed that after nanomedicine intervention,the collagen content around the plaque increased significantly,and the RAP@PFN1-CD-MNPs group provided better effect.According to the results of immunohistochemical staining,the relative content of CD68 decreased accompanied with the relative content of α-SMA increased in the RAP-loaded nanosystem group.H&E results showed no significant organic damage in the main organs of ApoE-/-mice,while a few renal function indicators(creatinine(CREA),uric acid(UA))and one cardiac function index(creatine kinase(CK))in the RAP@CD-MNPs group was significantly higher than the control and RAP@PFN1-CD-MNPs groups.There were no significant differences in blood lipids(CHOL,HDL,LDL-C,and TG)between the control and nanomedicine groups(RAP@CD-MNPs and RAP@PFN1-CD-MNPs)Conclusions:Treatment with RAP@PFN1-CD-MNPs by intraperitoneal administration significantly alleviated the progression of atherosclerosis and significantly reduced lesion area.Compared with the counterpart RAP@-CD-MNPs without active target molecule,the pH-responsive nanosystem RAP@PFN 1-CD-MNPs achieve better therapeutic effect.Rapamycin exerted the effect of stabilizing plaques by reducing macrophages and increasing SMCs in plaques.In the long-term treatment,RAP@PFN1-CD-MNPs nanosystem alleviated kidney and myocardial function damage to some extent.Rapamycin effectively reduced inflammation,inhibited progression,and enhanced the stability of atherosclerotic plaques without altering serum lipid levels.The RAP@PFN1-CD-MNPs nanosystem synthesized in this study did not show any obvious side effects,so it may be a safe multifunctional nanosystem for the treatment of atherosclerosis.