Multimodal Molecular Imaging For The Evaluation Of Chronic Cardiotoxicity Of Doxorubicin And Vulnerable Atherosclerotic Plaque

Part Ⅰ Multimodality imaging in the assessment of bone marrow-derived mesenchymal stem cell therapy for doxorubicin-induced cardiomyopathyObjective:Anthracyclines are widely used as first-line chemotherapy for a variety of solid tumors and hematological malignancies due to their broad spectrum and powerful anti-tumor properties.Doxorubicin is the most commonly prescribed anthracyclines in clinical practice.However,dose-cumulative cardiotoxicity caused by anthracyclines,has received increasing clinical attention.Long-term follow-up data from adult survivors of childhood tumors found that up to 30%of patients treated with doxorubicin developed signs of cardiac dysfunction.Anthracyclines induced cardiotoxicity,is mainly manifested as progressive and irreversible cardiac insufficiency even heart failure.Once diagnosed,the survival rate of two years after is only 40%.Cardiovascular disease is currently the second leading cause of longterm morbidity and mortality in survivors of malignant tumours beyond themselves.Several studies have shown that stem cells may be effective against fatal cardiotoxicity caused by anthracyclines.However,the biological behavior of transplanted stem cells,including survival time and differentiation mechanism,remains unclear,which limits the clinical application of stem cells transplantation therapy.Therefore,the in vivo tracing as well as the visualization of transplanted stem cells are the primary scientific problems to be solved at present.In this study,bone marrow derived mesenchymal stem cells(BMSCs)labeled with ultra-small ultra-paramagnetic nanoparticles(USPIO)and Luciferase(luciferase)reporter gene were transplanted into doxorubicin induced cardiotoxicity(DIC)mouse model of mice of the same strain.Multimodality imaging combing cardiac magnetic resonance imaging(CMR)and bioluminescence imaging(BLI)was employed to trace the biological behavior of transplanted BMSCs in vivo.Methods:(1)To establish a mouse model of chronic doxorubicin induced cardiotoxicity.Female Balb/c mice aged 6-8 weeks were intraperitoneally injected adriamycin by the dose of 2mg/kg weekly,totally for 6 weeks.Then wait between 3 months and 6 months until cardiac fibrosis formed subsequently after necrosis of cardiomyocyte.Our study employed cardiac ultrasound,as well as pathological analysis to identify the condition of mouse model,including HE staining,Masson staining and PSR staining.(2)To isolate,purify,amplify and identify the BMSCs derived from Balb/c newborn mice.All femurs and tibias from the new born Balb/c mice were obtained,the bone marrow cavity of which was flushed repeatedly until it turned white.The BMSCs were purified by removing the non-adherent cells by changing the culturing medium frequently.Then the purified BMSCs were identified by inducing them to differentiation into specific drections by Balb/c Mouse Bone Marrow Mesenchymal Stem Cell Osteogenic Differentiation Medium.(3)The Fluc reporter gene was stably expressed in BMSCs(Fluc-BMSCs)by lentiviral transfection and subsequent puromycin screening.CCK8 assay was employed to determine both the optimal concentration of vivotrax and incubation time.The viability of the BMSCs,BMSC-LUC,BMSC-USPIO,and BMSC/LUC-USPIO was determined by CCK-8 assay kits based on the OD values.(4)H9C2 cells were incubated directly with BMSC medium or BMSCs supernatant.DOX was added 12h later into the plate.(5)Bioluminescence imaging(BLI)of the Fluc-BMSCs was performed according to the manufacturer’s instructions.Briefly,200 μl of Fluc-BMSCs were seeded in each well of a 96-well flat-bottom plate at different concentrations(1×106,5×105,2.5×105,1×105,5×104,and 2.5 ×104).After 150 μg/ml D-luciferin was added to each well,a series of bioluminescence images were acquired successively with 60-second exposure times until the BLI signal was attenuated.(6)Labeling of Fluc-BMSCs with USPIO particles(Fluc/USPIO-BMSCs)and magnetic resonance imaging(MRI)in vitro.Fluc-BMSCs were seeded in each well of a 24-well flat-bottom plate at different concentrations(1×106,2.5×105,5×104,and 1×104),then a series of images were acquired by 3.0-T magnetic resonance scanner with T2 and T2*sequence.(7)Balb/c mice were randomly divided into an experimental group(n=20)and a control group(n=10).All mice were subjected to the same experimental procedures,except for mice in the experimental group that received treatment with Fluc/USPIO-BMSCs.BLI of both groups was performed on day 1,day 3,day 5 and day 7 after injection.(8)MRI of both groups was performed on day 1 and day 3 with a T2*relaxome gradient recalled echo(GRE)cine sequence on an Inova 7.0T magnetic resonance scanner(Varian).The electrocardiography,respiration,and core temperature of mice were monitored using an MRIcompatible system(SA Instruments)after the mice were anesthetized with 1.5%isoflurane in 1 L/min oxygen.Ten cardiac phases were acquired for each cine series,which were triggered by the electrocardiogram.Images from successive slices along the short cardiac axis were then acquired with a slice thickness of 1 mm.Five slices were typically required to cover the left ventricle from the apex to the base.Hypointense areas in the myocardium were evaluated to trace the implanted cells.Other MRI parameters included the follwoing:FOV,30×30 mm;matrix,200×200;FA,30;TR,100 ms;NEX,5.0;and TE,6.0 ms.Histologic analysis.Mice in the BMSC-treated group were euthanized after the BLI signal disappeared.The heart tissues were fixed in 4%paraformaldehyde and embedded in paraffin,and the 5-μm-thick sections were stained with hematoxylin-eosin(H&E).Images of the stained sections were obtained using a light microscope(Nikon Eclipse TE2000-U,Japan)at 200×magnification.Results:Picric acid-Sirius scarlet staining and Masson staining both confirmed the successful generation of a chronic cardiotoxicity Balb/c mouse model,which showed connective tissue formed around the myocardial tissues and blood vessels and increased and thickened collagen fibers in the myocardial interstitium.Cardiac ultrasound showed an enlarged left ventricular cavity and decreased ejection fraction.Taken together,these results indicated the successful establishment of cardiac fibrosis in Balb/c mice.BMSCs from Balb/c mice were primarily round mononuclear cells.After 4 to 12 h,BMSCs with a spindle shape were attached to the bottom of culture flasks.BMSCs were purified by passing the cells to passage 4.The 4th passage cells were cultured in Balb/c mouse MSC osteogenic differentiation medium for 2 weeks,and the formed calcium nodules were then stained using alizarin red.The results indicated that BMSCs with differentiation ability were successfully purified from Balb/c mice.the Fluc reporter gene was stably expressed by infection with lentivirus carrying the luciferase reporter gene in BMSCs derived from Balb/c mice.The viability of Balb/c BMSCs incubated with 100 μg/ml Vivotrax for 4 h was significantly different compared with that of BMSCs in the control group.The viability was significantly different between the 80-100 μg/ml group and the control group after incubation with Vivotrax for 12 h.As the Vivotrax incubation time increased,the toxicity of Vivotrax increased.As such,we decided to incubate the cells for 4 h at a concentration of 80 μg/ml.H&E staining.There was no significant difference among the BMSCs,BMSC-LUC,BMSC-USPIO,and BMSC/LUC-USPIO at 24 h,suggesting the safety of USPIO and FLUC labeling.Moreover,the viability of the Flu/USPIOBMSCs was timedependently decreased in comparison with that of the BMSCs after incubation with USPIO or FLUC,indicating that the cell viability and proliferation ability of BMSCs were slightly affected by USPIO and FLUC labeling.H9C2 cells pretreated with DOX were incubated with normal medium and BMSC medium,the cells incubated with BMSC medium showed significantly higher viability,indicating that BMSCs may have the ability to improve the damaged myocardium through paracrine signals.the photo counts measured by Ivis were linearly correlated with the cell number as follows:y=1766.4x+20492(R2=0.9422).A negative correlation between the number of cells and the T2 relaxome value was observed(R2=0.4680).The T2*relaxome value measured by MRI was also linearly correlated with the cell number:y=-18.27x+239.5(R2=0.9342)Fluorescence signal and magnetic resonance signal decrease with the decrease of cell number,indicating that double-labeled stem cells can be tracked quantitatively.Eighteen mice in the BMSC-treated group and 10 mice in the control group survived DOX administration and intramyocardial injection of BMSC/LUC-USPIO.A BLI signal intensity of 1,040,000±1,107,488 counts on day 1 after surgery was detected in the heart region in the BMSCtreated group but decreased to 221,000±159,578.4 on day 3 and 67,700±106,890.5 on day 5.The signal was beyond the threshold of detection on day 7 in all mice of the experimental group.Corresponding with the injection sites,the signal of the anterior wall of the left ventricle was decreased significantly.These hypointense regions were not found in the control group on day 1.Visualization of BMSC/LUC-USPIO stem cells by MRI confirmed their successful intramyocardial delivery and survival,which was in agreement with the results of BLI stem cell tracing.Prussian blue staining revealed positive cells with blue USPIO particles,which also proved the successful intramyocardial injection of BMSCs/LUC-USPIO.Conclusions:In the current study,we labeled Fluc-expressing mouse BMSCs with USPIO particles and applied two molecular imaging techniques(BLI and MRI)to monitor the biological behaviors of transplanted BMSCs in vivo in a chronic DIC mouse model.Since Balb/c mice are an inbred strain,we transplanted BMSCs derived from Balb/c mice into the myocardium of the same strain to avoid immune rejection.Therefore,this study reflects the real biological behaviors of BMSCs transplanted into the damaged hearts of a chronic DIC mouse model.The results showed that the signal of dual-labeled BMSCs derived from Balb/c mice and transplanted into DIC model mice(the same mouse strain)lasted less than 1 week,which is identical to the findings of our previous study.As the study showed,even without immune rejection,the signals of transplanted BMSCs did not survive over 1 week,indicating that patches loaded with BMSCs or other beneficial biomaterials may be superior in improving the outcome of cardiovascular diseases characterized by the loss of myocardial fibers and cardiac fibrosisPart Ⅱ A peptide-based multimodal magnetic nanoprobe for the imaging of vulnerable atherosclerotic plaquesObjective:Atherosclerosis is the most common form of vascular disease and a major cause of death worldwide.Extensive effort has been invested in the identification of atherosclerotic plaques that are vulnerable(i.e.,likely to rupture),and there is an urgent need for early diagnosis of such plaques.The cluster of differentiation(CD)137 protein is absent from stable atherosclerotic plaques and normal blood vessels,but it is overexpressed by cells present within vulnerable atherosclerotic plaques.Therefore,CD137 is a potential target for high-specificity imaging of vulnerable atherosclerotic plaques.We aimed to develop a novel peptide-based multimodal magnetic nanoprobe,CD137@superparamagnetic iron oxide(Fe3O4)/indocyanine green(ICG),to facilitate the noninvasive detection of vulnerable atherosclerotic plaques by magnetic resonance imaging and fluorescence imaging.Methods:C57BL/6J apolipoprotein E(ApoE)-/-mice fed a high-fat diet were used to establish a mouse model of vulnerable atherosclerotic plaques.The detailed protocol of development of the multimodality imaging probe CD137@Fe304/ICG included establishment of a CD137targeted peptide screening system,synthesis of hydrophobic Fe3O4 nanoparticles,and preparation of CD137@Fe304/ICG by Fmoc strategy SPPS.Then the morphology and size of CD137@Fe3O4/ICG nanoparticles was detected by transmission electron microscopy.The Relaxivity(R2)was detected by MRI.The total radiant efficiency with increasing concentration of CD137@Fe304/ICG was evaluated by IVIS spectrum.The Cell viability of HUVECs treated with an increasing concentration of CD137@Fe3O4/ICG was detected by CCK8 assay.Mice were subjected to in vivo fluorescence image acquisition before and after(at 1h)the injection of CD137@Fe3O4/ICG through the tail vein.The carotid and abdominal aorta arteries were collected from HFD mice at 48 h after the completion of in vivo imaging.MRI data were acquired before and 1 h after the injection of CD137@Fe304/ICG.Results:The level of CD137 expression was significantly higher in vulnerable atherosclerotic plaques than in stable plaques or normal arterial tissue from experimental mice(P<0.05).The CD137@Fe3O4/ICG multimodal magnetic nanoprobe exhibited robust contrast enhancement on T2-weighted magnetic resonance imaging,along with high specificity for the detection of vulnerable atherosclerotic plaques via fluorescence imaging.Conclusion:Our novel peptide-based multimodal magnetic nanoprobe,CD137@Fe304/ICG,can detect vulnerable atherosclerotic plaques with high sensitivity and high specificity.