Multimodal Targeted Molecular Imaging Of Microthrombus In A Rat Myocardial Ischemia-reperfusion Model

Thrombosis has been well recognized as a primary pathogenesis of acute ischemic cardiovascular diseases, including unstable angina, myocardial infarction (MI), stroke and pulmonary embolism. Recent researches have showed that microthrombus also plays a key role in some cardiovascular diseases. The detection of microthrombus may be beneficial for early recognizing the unstable plaques, understanding the exact mechanisms underlying microvascular ischemia, and determining the optimaltherapeutic regimen. Clinically, the diagnostic regimen for the detection of thrombus excel at localizing thrombi in large and middle-sized vessels, they cannot readily detect clots of microcirculation or provide molecular and/or cellular information about clot composition. Multimodal imaging, providing the combination of sensitivity and spatial resolution, is in theory an ideal candidate for the detection and localization of microthrombus. With the development of nanotechnology and novel nanomaterials (such as superparamagnetic iron-oxide nanoparticles, SPIO), multifunctional nanoparticles holding molecular probes are promising for multimodal imaging of myocardial microthrombus.In this study,we examined the thrombogenicity of 50 nm SPIO in vitro.The SPIO had no effect on platelet aggregation or plasma coagulation in vitro, suggesting little likelihood of SPIO to be thrombogenic in vivo and elucidating the safety of SPIO as drug carrier used in thrombotic diseases. Meanwhile, we found that fibrin deposited in cardiac microvessels in a rat acute myocardial ischemia/reperfusion(MI/R) model. And the present study demonstrated a working strategy for imaging microthrombus with CREKA-SPIO which can specifically and effectively target fibrin. With the targeted nanoagent, the multimodal imaging of microthrombus in the MI/R rat was achieved using in vivo MRI and optical imaging. This technique significantly improves detection of microthrombosis compared to the control agent by near-infrared fluorescence and magnetic resonance imaging and has the potential for clinical translation. This method may provide an experimental tool in studying the molecule formation and mechanism of microthrombosis and the role in the pathophysiology of acute ischemic cardiovascular diseases. Also, it may help in the target delivery of antithrombotic drugs to reduce the risk of bleeding complications. This paper was divided into four parts.PART ONE:Thrombogenicity of Superparamagnetic Iron Oxide Nanoparticles in VitroObjective:To explore the thrombogenicity of 50 nm dextran superparamagnetic iron oxide nanoparticles(SPIO) in vitro, and to deduce the safety of the SPIO as drug carrier.Methods:The characterization of SPIO was measured. Size distrubution and surface charge were determined by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively. Size and morphology were measured by transmission electron microscopy(TEM) and scanning electron microscope(SEM). Different concentrations of SPIO were mixed with test plasma or whole blood. The resulting solution was incubated at 37℃ for 30min or at room temperature for 40min. Platelet aggregation, plasma coagulation time, thrombelastogram and quality of blood clot were analyzed to determine the thrombogenicity of SPIO. The tendency to cause platelet aggregation or perturb plasma coagulation in vitro served as the indication of SPIO’s thrombogenicity in vivo.Results:(1) Platelet aggregation rates in PBS group,0.02 mg/ml SPIO group,0.1 mg/ml SPIO group and 0.5 mg/ml SPIO group were 67.3±5.9%,68.3 ± 4.5%,66.2 ± 5.5% and 69.5 ± 5.9%, suggesting that SPIO did not activate platelets under the tested condition (P>0.05). (2) APTT of the four groups were 28.1±2.7 S,28.5±2.4 S,28.2±2.5 S and 29.1±3.6 S (P>0.05). Also there was no significant difference in PT or TT between groups (P>0.05). (3) For the parameters of thromboelastography (R time presented coagulation time, K time and a angle for function of fibrinogen, MA for platelet function, and coagulation index CI), there was no significant difference between groups (P> 0.05). (4) Quality of blood clots of the four groups were 759.6±38.7 mg,758.8±47.2 mg,769.8±39.2 mg and 766.8±40.8 mg, demonstrating SPIO did not change the quality of blood clots in vitro at concentrations up to 0.5 mg/ml(P>0.05). For different concentrations of SPIO, there was no difference in platelat aggregation, plasma coagulation time, thrombelastogram or quality of blood clot (P<0.05)Conclusion:The SPIO had no effect on platelet aggregation or plasma coagulation in vitro, suggesting little likelihood of SPIO to be thrombogenic in vivo and elucidating the safety of SPIO as drug carrier used in thrombotic diseases.PART TWO:The construction and characterization of SPIO-CREKAObjective:To develop a novel fibrin targeting multimodal system(IR783-R-SPIO-CREKA), which could be uesd to image mocrothrombosis with both MRI and optical imaging, and describe the characterization of SPIO-CREKA.Methods:After labeled with IR.783 and Rhodamine, SPIO was modified with maleimide-poly(ethyleneglycol) (Maleimide-PEG), and covalently conjugated with CREKA via its maleimide functional group. Size distrubution and surface charge were determined by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively. Morphology were measured by transmission electron microscopy(TEM).The connection efficiency of IR783, Rhodamine,PEG and CREKA was determined by visible spectrophotometry method, fluorospectrophotometry method or HPLC method. Different concentrations of SPIO-CREKA were incubated with plasma to test its effects on blood coagulation and platelet aggregation.Results:The particle size was about 100nm for IR783-R-SPIO-CREKA. The zeta potential was about -2.2mv. TEM photograph showed that the CREKA-SPIO had the magnetite (Fe3O4) core that was 10-20 nm surrounded by a layer of dextran Under our experimental conditions,300 IR dyes,1500 rhodamine,6000 PEG and 2000 CREKA were given to each nanoparticle.Conclusion:We developed a novel fibrin targeting multimodal system(IR783-R-SPIO-CREKA), with three elements:SPIO as a MRI contrast agent and carrier, IR783 as a near-infrared fluorescent probe and CREKA as fibrin-targeting molecules. This nanosystem had high surface density of IR783 and CREKA,which enhanced targeting specificity and efficiency of the imaging agent. Meanwhile, SPIO-CREKA had no effect on platelet aggregation or plasma coagulation in vitro, suggesting its safety as imaging agent used in thrombotic diseases.PART THREE:The expression of microthrombus in a rat myocardial ischemia/reperfusionObjective:To detect whether microthrombus was expressed in microcirculation of a rat model with acute myocardial ischemia/reperfusion (MI/R),and preliminarily explore its mechanism.Methods:The rat myocardial ischemia-reperfusion model was used,and the rat was allowed to reperfuse for 24 hours. ECG, echocardiography and cardiac tissue histopathology were used to evaluation model. The fibrin deposition was detected by immunofluorescence staining. The transmission electron microscopy to used to detect main components of microthrombus. Immunohistochemical staining of TF and PAI-1 were performed to explore the possible mechanism of the formation of microthrombus.Results:Twenty-four hours after the operation, cardiac microthrombus was observed by immunofluorescence in cardiac microvessels of LVAW where the left anterior descending artery suppled blood in rats with acute MI/R, whereas no cardiac microthrombosis was shown in the nonischemia-reperfusion section and the sham group. Laser scanning confocal microscopy showed fibrin deposition with the blood cells. TEM observation revealed the main components of microthrombus were fibrin,platelets and red blood cells. Expression of TF and PAI-1 were increased in the ischemic myocardium compared with the sham group.Conclusion:After reperfusion for 24 hours in a rat MI/R modal, local microthrombosis was detected and its main components included fibrin, platelets and red blood cells. The imbalance of coagulation and fibrinolytic system in local ischemia area may be involved in the possible mechanism of the formation of microthrombus. Further study is needed to explore the exact role and the underlying mechanism of the microthrombus during MI/R injury, and the therapeutic penitential in the treatment of MI/R injury also merits further investigation.PART FOUR:Multimodal molecular imaging of microthrombus with SPIO-CREKAObjective:To evaluate the multimodal imaging capability to microthrombosis of SPIO-CREKA both in vitro and in a rat model of myocardial ischemia-reperfusion (MI/R).Methods:Fibrin clots were incubated with SPIO-CREKA and were imaged by fluorescence microscope, optical imaging and MRI to test the fibrin-targeting ability of SPIO-CREKA in vitro. We used a rat myocardial ischemia-reperfusion model to test the multimodal imgaing capability in vivo. After 24-h reperfusion, the survived MI/R rats were injected with SPIO-CREKA via the tail vein. After4-h injection, the animals were analyzed for particle homing by both MRI and optical imaging. Histological examination and TEM of the heart were performed to confirm the binding SPIO-CREKA with microthrombus.Results:(1) In vitro, for imaging of fluorescence microscope, fibrin clots which were incubated with fibrin-targeted nanoagents (SPIO-CREKA) revealed fluorescence on the surface of clots, whereas there was no significant binding to the control clots(SPIO-PEG or PBS). In optical imaging, clots incubated with SPIO-CREKA demonstrated considerably greater radiant efficiency than clots incubated with SPIO-PEG. The radiant efficiency of SPIO-CREKA clots (1.29± 0.25×1010) was more than 7.5-fold the radiant efficiency of control SPIO-PEG (0.17± 0.07×1010, P<0.001 compared to SPIO-CREKA group). For MRI, SPIO-CREKA demonstrated strong MRI signal loss(66.4±13.7) on T2WI-flash images. Clots incubated with SPIO-PEG demonstrated negligible signal changes as compared to PBS group(201.8±11. vs.210.9±17.2). (2) The targeting ability of the nanoagents was tested in the rat model of MI/R induced microthrombus. To investigate the microthrombus-targeted MRI capability of SPIO-CREKA, SPIO-CREKA demonstrated strong signal loss on T2 fl2dimages(relative signal intensity of LVAM 0.65 ± 0.07) in the anterior wall of left ventricle where LAD supplied.In contrast, the rats that received SPIO-PEG and PBS demonstrated no significant signal changes (relative signal intensity of LVAM 0.95 ± 0.08 and 0.99 ± 0.11, respectively; P<0.01 compared to SPIO-CREKA group). And the relative signal intensity of LVAM of sham group that received SPIO-CREKA was 0.92 ± 0.06. For optical imaging, SPIO-CREKA displayed a 1.95-fold greater radiant efficiency than the control particle, SPIO (radiant efficiency 1.88 ± 0.5×107 vs.0.96 ± 0.09×107, P<0.001), while the radiant efficiency for PBS and sham-operated group that received SPIO-CREKA was 0.99 ± 0.1×107 and 0.94 ± 0.1×107. As to other major organs, SPIO-CREKA distribution was almost the same as SPIO mainly distributed in the organs including liver and kidney. Histological examination of the heart from rats injected with SPIO-CREKA revealed fluorescence localized with microthrombus whereas there was no significant thrombus binding in rats that received SPIO. This binding was further confirmed by transmission electron microscope (TEM).Conclusion:The present study demonstrated a working strategy for imaging...