Ultrasound/Optical Dual-modality Imaging For Evaluation Of Atherosclerotic Plaques By OPN Targeted Nanoparticles

BackgroundCardiovascular disease is the leading cause of morbidity and mortality worldwide.According to “The report of Chinese cardiovascular disease in 2015”,cerebrovascular disease and coronary atherosclerotic heart disease were the top 2 diseases inducing mortality among Chinese residents.These two diseases are all closely associated with atherosclerosis.Vulnerable atherosclerotic plaque is prone to rupture when triggered by various factors,often resulting in acute cardiovascular events.Due to the acute onset and fast progression of plaque,out-patients often fail to get aid timely,giving rise to irreversible organ damages.Therefore,the prevention of acute cardiovascular events is an urgent challenge for public health.Technologies used in clinical practices like CTA and interventional angiography evaluate plaque risks mainly through examining luminal stenosis anatomically.Generally,a stenosis greater than 75% would be arranged to accept the stenting treatment at present.But an autopsy result manifested that most acute infarctions occurred on the luminal stenosis less than 50% in atherosclerosis plaques.It suggests that the traditional diagnostic imaging based on the structure of vascular anatomy has major limitations in efficiently predicting the risk of plaques ’rupture.Molecular imaging,which is based on pathophysiology,could identify and quantify the biological changes at cellular and molecular levels.Therefore,it exhibits a rosy outlook in precision medicine.The probes of molecular imaging mainly consist of specific target molecules and contrast agents.Osteopontin(OPN),a secreted phosphorylated glycoprotein,was recognized to be closely related to atherosclerotic plaques.Intense macrophage infiltration,phenotype transformation of vascular smooth muscle cells and spotty calcification are strongest predictors and features of vulnerable plaques.OPN is the marker for phenotypic conversion of VSMCs from contractile phenotype to synthetic phenotype.Significant higher OPN expression was observed in foam cells along with the aggravated capacity of recruited macrophages due to the RGD sequence and interaction with CD44 of OPN.Therefore,OPN represents a promising imaging target for recognition of vulnerable plaques.Due to its excellent biocompatibility and modifiability,polymer materials have been widely studied in the field of biological engineering in recent years.Perfluorooctyl bromide,with its preeminent stability and oxygen carrying capacity,can act as ultrasound contrast agent through the aggregation effect for its high density.This study therefore selected OPN as the target molecule and poly lactic acid(PLA)nanoparticles filled with perfluorooctyl bromide(PFOB)as ultrasound contrast agent.Simultaneously,we tagged the antibody with fluorescent dye Cy5.5 to confer optical imaging property for the nanoparticles,hoping to realize the recognition of vulnerable plaques.ObjectiveTo construct the OPN targeted ultrasound/optical double modality nanoparticles(Cy5.5-anti-OPN-PEG-PLA–PFOB)and evaluate its targeting efficiency and imaging capacity as well as cell biocompatibility.Methods1.PLA nanoparticles were constructed by oil-in-water emulsion method,followed by the collection of centrifugal precipitation at 5000 g.Hydrophilic modification of amino polyethylene glycol maleimide(NH2-PEG-mal)attached on the surface was carried out by the catalytic reaction of EDC and NHS.OPN antibody tagged with Cy5.5 fluorescent dye by amide bonds was then conjugated with PEG-PLA-PFOB NPs using click reaction.Characterizations of nanoparticles were experimented by TEM,Fourier infrared spectrometer,dynamic light scattering,and ultrasound imaging in latex tubing.2.Both RAW264.7 cells and MOVAS cells were used in in vitro study.Oxidized low density lipoprotein(ox-LDL)incubation was employed to mimic the insult of atherosclerosis.The expression level of OPN was observed in both RAW264.7 cells and MOVAS cells challenged in the presence or absence of ox-LDL.The biotoxicity of PLA NPs was evaluated in both cells by CCK-8 assay.Sensibility of probes was evaluated by cellular uptake assay under laser scanning confocal microscope in foam cells.Experimental groups including targeted OPN nanoparticles,competitive inhibitor group(with targeted nanoparticles and a huge amount of OPN antibody),and control group(IgG nanoparticles).3.For in vivo study,ApoE-/-mice were fed with high fat diet(containing 15% fat and 0.25% cholesterol)to construct animal models of atherosclerosis.The verification of animal model was made by anatomy and tissue pathological dyeing.We estimated the consistency among the immuno-histochemical distribution of OPN with immuno-histochemical stained CD68 positive macrophages and α-SMA positive smooth muscle cells in continuous pathological sections.Fluorescence imaging was applied to collect signals after injections of tagged antibodies in vivo,and observe the identification capability of tagged antibodies on atherosclerotic plaques.Results1.We successfully constructed Cy5.5-anti-OPN-PEG-PLA–PFOB nanoprobes.After 5000 g centrifugal optimization,the nanoparticles showed uniformed size distribution with the hydrate particle size ranging from 320 nm to 400 nm and Zeta potential at 20.14±0.68 mv.TEM image manifested roundish shape and smooth surface with no obvious reunion and adhesion phenomenon.Ultrasound imaging in latex tubing revealed fine punctuate hyper echo with no attenuation for the rear echo.Spectral results showed Cy5.5 emission wavelength at 705.7 nm.2.Immunofluorescence results illuminated that the OPN expression increased obviously after treatment with ox-LDL.CCK-8 assay showed no significant cytotoxicity.Cell survival rate was still greater than 80% when at the concentration of 0.5 mg/ml.Cellular uptake analysis unveiled fine binding sensibility in foam cells.3.Gross anatomy revealed the atherosclerotic plaques were more likely to form and develop in vascular bifurcation and blood flow shear stress changing area.Tissue pathological staining displayed rich lipid deposition,necrotic core formation and thin fibrous cap in plaques.Immune-histochemical staining showed OPN expression was consistent with macrophages and smooth muscle cells.Optical imaging revealed that the OPN antibodies were accumulated in the atherosclerotic plaques with significantly enhanced fluorescence signal.ConclusionThe OPN expression was up-regulated in ox-LDL-challenged cells and atherosclerotic plaques.OPN targeted PLA nanoparticles exhibited stable property,eminent biocompatibility,and fine imaging ability that can effectively identify targets.