Multimodality Molecular Imaging Of The Features Of Atherosclerotic Plaque Via Profilin-1Targeted Multifunctional Nanoprobe

BackgroundCardiovascular diseases (CVD) become the leading cause of mobidity and mortalityworldwide with the changes of human life styles. According to statistics of World HealthOrganization in2008, approximately17.3million individuals died from CVD, up toapproximately30%global death. Prevailing researches showed that thrombosis formationand vascular occlusion induced by rupture of vulnerable plaque frequently leads to majoradverse cardiovascular event (MACE). Although experts already reach to an agree inprevention and therapy of atherosclerosis including lifestyle intervention, medication andPCI therapy, the MACE still in a high level due to onset and sudden rupture of vulnerableplaque. Therefore, how to achieve the goal of early diagnosis and effective interventionfor vulnerable plaque is still an essential clinical issue.Different from current medical imaging, molecular imaging aims to unravel biological and physiology information of diseases from molecular and cellular levels inliving subjects, which can be applied to observe vulnerable plaque characteristicsreal-timely and dynamically as well as evaluate efficacy of preventive and therapeuticapproaches. It is imperative to construct suitable molecular imaging probe, which canimprove specificity and sensitivity of imaging. To select potential specific molecular targetfor the vulnerable plaque is the first key step. Currently, abnormal or high expressionsubstance such as protein, antigen, peptide, receptor or enzymes under pathologyconditions may serve as potential molecular targets.Profilin-1, a type of small actin-binding protein (12-15KD), is widely distributedover various cells, which is involved in the modulation of cytoskeleton polymerisation andreorganisation. Recent researches show that profilin-1overexpression triggers the occurand development of atheroslerosis mainly through regulating vascular smooth muscle cellsproliferation and migraion, which is recognized as a promising potential molecular targetof atherosclerosis.It also needs to choose proper protein carrier into target cells with low cytotoxity,good biocompatibility and suitable retention time in vivo. Super paramagnetic iron oxide(SPIO) holds hardly phagocytosis by macrophage in liver and spleen because of smallvolume with a diameter less than30nm, which can stay in circulation for a long time.Thus, SPIO is thought to be an optimal contrast agent that is frequently appiled in MRIIn the present study, we aim to to utilize profilin-1as molecular target, applyDMSA-Fe3O4nanoparticles as probe carrier to targeted profilin-1for multimodalityMRI/optical imaging, which can be used for assessing vulnerable plaque characteristicsand therapeutic efficacy. These data may provide theory and experimental evidence forearly diagnosis of vulnerable plaque.Aims:1. To construct atherosclerosis model using ApoE-/-mice, investigate the expression levelof profilin-1in atherosclerotic plaque and VSMCs. Further illustrate the relationbetween profilin-1expression and VSMCs proliferation, migration and possible mechanisms.2. Based on chemical synthesis profilin-1targeted multimodality MRI/optical nanoprobe,investigate the characterization, toxicity, biocompatibility and in vivo distribution ofprobe.3. To observe probe accumulation in atherosclerotic plaque from cellular and in vivolevel through MRI and fluorescence imaging, the results were testified by histologyanalysis.Methods:1. Male20g8-week-old apoE-/-mice (C57BL/6J genetic background) were fed westerntype diet (containing15%fat and0.25%cholesterol) for16weeks. Thereafter, thetotal length of carotid artery and desending aorta was harvested, followed by stainedwith oil red and H&E staining to testified whether atherosclerosis animal model wasconstructed sucessfully.2. All animals were randomly allocated into the three groups with n=20each,(1)Control group: mice kept on a standard laboratory chow diet for16weeks;(2) High fatdiet (HFD) group: mice were fed with a western type diet (containing15%fat and0.25%cholesterol);(3) HFD+Atorvastatin group: mice were treated with a westerntype diet and atorvastatin intervention. After16weeks, blood was collected to detectserum levels of TC (total cholesterol), LDL-C and HDL. Vascular tissue was stainedwith Oil red and H&E staining. Immunofluorescence was performed to measure theexpression and distribution of profilin-1and colocalization with VSMCs markerα-SMA. Western blot further reveal the expressed level of profilin-1in vessel wall.3. Culture and characterization of VSMCs cell line MOVAS, and cells were divided intofour groups:(1) control group;(2) profilin-1siRNA (4μg) treatment group;(3)ox-LDL（20μg/ml）pretreatment group;(4) profilin-1siRNA (4μg)+ox-LDL（20μg/ml） pretreatment group. CCK-8assay was conducted to detect cellsproliferation, transwell assay was performed to observe cells migration.Immunofluorescence and Western blot were carried out to measure profilin-1 expression in the cytoplasm of VSMCs. Western blot was performed to investigate therelated signaling pathway.4. Chemical synthesis of DMSA-Fe3O4-NPs, then conjugated profilin-1antibody andNHS-Cy5.5with DMSA-Fe3O4-NPs via EDC chelation following activation. Theiron oxide core size of Fe3O4-DMSA nanoparticles was measured by transmissionelectron microscopy. The average hydrodynamic particle size and zeta potential (ζ)values of the nanoparticles were characterized using a Zetasizer Nano ZS. Themagnetic properties of the samples were performed by a vibrating samplemagnetometer.5. Macrophage phagocytosis experiment and MTT assay were performed to compare thebiocompatibility and cytoxicity of DMSA-Fe3O4-NPs and Fe3O4-NPs. UV(ultraviolet)-Vis absorption spectroscopy was carried out to confirm whetherprofilin-1antibody was successfully conjugated with Fe3O4-DMSA nanoparticles.BCA assay and ortho-phenathroline photometric methods were performed to analyzethe conjugation efficiency of profilin-1antibody and nanoparticles. To detect themetabolism and distribution of probe in tissues and organs.6. To observe plaque characteristics with9.4T MRI after tail intravenous injection ofprobe, and then perform semiquantitative analysis of probe accumulation in plaquebased on T2WI signal changes. Histology analysis was conducted to confirm probedeposition.Results:1. After fed western type diet for16weeks, apoE-/-mice atherosclerosis model wereconstruct sucessfully.2. ELISA assay showed that serum levels of TC and LDL-C were increased significantly,and HDL was decreased. Atorvastatin therapy lowered TC and LDL-C levels, whilecouldn’t affect HDL level. Immunofluorescence results reveal that expression ofprofilin-1was elevated in plaque, and almost co-localized with VSMCs. Western blotanalysis also confirmed that expression of profilin-1was increased in plaque group, as compared with that in control group.3. Ox-LDL pretreatment could promote MOVAS cells proliferation, migration andprofilin-1expression. However, profilin-1siRNA intervention reversed the effectsinduced by ox-LDL. Western blot analysis further showed that MOVAS cells waspromted proliferation and migration through profilin-1/MEK/ERK1/2signalingpathway.4. The TEM image of DMSA-Fe3O4-NPs showed that the nanoparticles displaypoly-disperse and no aggregation. Malvin size analysis revealed the nanoparticlescontain a fairly unimodal distribution of monodisperse clusters with average sizesaround8nm. Moreover, the particles carried a negative zeta potential of-42.7mv. Thehysteresis curves of DMSA-Fe3O4-NPs showed the saturation magnetization value (Ms)is56.4emu g1.5. The data of UV (ultraviolet)-Vis absorption spectroscopy confirmed that profilin-1antibody was sucessfully conjugated with DMSA-Fe3O4-NPs. BCA assay andortho-phenathroline photometric methods detection indicated that conjugationefficiency is as high as78.6%at PH=8reaction system. Cell immunofluorescenceresults revealed that the highest bound of probe to VSMCs was observed in VSMCspretreated with ox-LDL (20μg/ml), in contrat to other groups.6. MRI data showed that apoE-/-mice exhibited increased wall thickness and pronouncedplaque formation compared to controls. Although almost no signal changes onT2-weighted MRI after administration of non-targeted DMSA-Fe3O4-NPs, significantsignal attenuation was observed24h post PF1-DMSA-Fe3O4-NPs. Perl’s stainingfurther confirmed that more NPs deposited in atherosclerotic lesions in PF1-DMSA-Fe3O4-NPs treated group than that in DMSA-Fe3O4-NPs treated group.Conclusion:1. Profilin-1plays improtant roles in maintaining vessel homeostasis and regulatingatherosclerosis progression by regulating VSMCs proliferation, migration viaProfilin-1/MEK/ERK signaling pathway. 2. Profilin-1is a promising biomarker of AS and vessel homeostasis.3. Profilin-1can serve as a molecular target to consruct molecular imaging probe forearly nonivasive imaging diagnosis of atherosclerosis characteristics.