Photoacoustic Imaging Of Myocardial Infarction Region Using Fibrin Targeted Nanoparticles

PartⅠPREPARATION,CHARACTERISTICS AND SAFETY OF FIBRIN TARGETED NANOPARTICLESObjective:ICG NPs modified with CREKA were prepared(CREKA-ICG-LIP NPs).Then,the fundamental characteristics,stability,and safety of the NPs were detected.Methods:The CREKA-ICG-LIP NPs were prepared by one step method to encapsulate ICG into the lipid shell while simultaneously carrying CREKA on the surface.The morphology and nanostructure of the CREKA-ICG-LIPNPs were observed by optical microscopy and transmission electron microscopy(TEM).The mean particle size,zeta potential and stability of the CREKA-ICG-LIP NPs were measured by a Zetasizer Nano ZS unit.The UV-vis-NIR absorption spectrums of free ICG,CREKA-LIP NPs,and CREKA-ICG-LIP NPs were obtained by UV-vis-NIR spectroscopy.The standard curve of ICG was established by determining the absorption values at 780nm.The EE was calculated.The cytotoxicity of CREKA-ICG-LIP NPs was determined by the CCK-8 assay.Blood samples were collected 1 d,3 d,7 d,and 14 d postinjection for the routine blood test and serum biochemical indexes.The major organs(heart,liver,spleen,lung,and kidney)were harvested for HE staining and observation of pathological changes.Results:The NPs were successfully prepared.Bright-field optical microscopy and TEM showed that the CREKA-ICG-LIP NPs featured a uniform size/shape morphology and high dispersity.The average size of the CREKA-ICG-LIP NPs was 260.77±9.00nm(PDI=0.138).The average zeta potential of the CREKA-ICG-LIP NPs was-30.50±0.78 m V.The standard curve was Y=0.1538X+0.0607(R~2=0.9957).The EE of ICG in CREKA-ICG-LIP NPs was above 86.72%.These results of the CCK-8assay indicated that there was no obvious cytotoxicity to H9C2 cells and cell viability remained above 90%.The serum biochemistry profiles showed no statistically significant increase of biomarkers.HE staining of the major organs showed no significant acute or chronic physiological toxicity compared with the control group.Conclusion:In this study,CREKA-ICG-LIP NPs were successfully constructed and showed uniform size,good dispersion,strong stability,and high biosafety.PARTⅡ EVALUATION ON TARGET ABILITY AND DISTRIBUTION OF FIBRIN TARGETED NANOPARTICLESObjective:IR model was established and assessed.The targeting ability in vivo and vitro and the distribution in vivo of CREKA-ICG-LIP NPs were evaluated.Methods:IR model was established by ligating the left anterior descending coronary artery(LAD)of SD rats and removing the ligation suture.The establishment was assessed by ECG,TTC staining,HE staining and Masson staining;In vitro targeting experiments,slides with fibrin clots were incubated with respective solutions of CREKA-ICG-LIP/Di I NPs,ICG-LIP/Di I NPs,and PBS solution.Fluorescence signals were observed.In vivo targeting experiments,six IR model rats and six normal SD rats were respectively randomly divided into: NC target group,NC non-target group,IR target group,IR non-target group(n = 3/group).Rats in each group were injected with 1 ml of CREKA-ICG-LIP NPs/Di I solution(4 mg/ml)or ICG-LIP NPs/Di I solution(4 mg/ml)through the tail vein.The heart was harvested and prepared for heart frozen sections.The distribution and co-localization of the NPs and fibrin were observed.In vivo distribution experiment,major organs(heart,liver,spleen,lung,kidney)of normal SD rats 1 h,6 h,and 24 h after injection of CREKA-ICG-LIP/Di I NPs were collected to observe the fluorescence distribution in vivo(n = 3/group).Results: Intraoperative ECG showed and ST segment elevation of Ⅲ,postoperative TTC staining showed myocardial ischemia area,and HE staining and Masson staining 3 weeks after operation showed myocardial fibrosis.The above results indicated that IR model was successfully established.The slides in the CREKA-ICG-LIP NPs group incubated with fibrin-targeted NPs showed obvious fluorescence signals on the surface of the fibrin clots.The fibrin(green)signal in IR group was significantly stronger than in NC group.The red signal(NPs)in the IR target group was significantly stronger than that in the NC target group and that in the IR non-target group,which obviously colocalized with fibrin.An obvious fluorescence signal(red)was observed in the liver,spleen,and kidney at 6 h and 24 h after injection,weak fluorescence signal was observed in the liver,spleen at 1 h,and no fluorescence signal was observed in the heart and lung,all the timeConclusion:The targeted nanoparticles(CREKA-ICG-LIP NPs)have a strong targeting ability to bind to fibrin in vitro,and can effectively bind to fibrin in the injury area of myocardium through vascular endothelial space in vivo,which proves that CREKA-ICG-LIP NPS nanoparticles have good targeting ability in vivo and vitro.Nanoparticles are mainly distributed in the liver,spleen,and kidney,dedicating that the NPs could be degraded by the liver-spleen system and kidney excretion.PARTⅢ PHOTOACOUSTIC IMAGING OF MYOCARDIAL INFARCTION REGION USING FIBRIN TARGETED NANOPARTICLESObjective:The fibrin targeting nanoparticles(CREKA-ICG-LIP NPs)were prepared and evaluated the performance of the NPs in detecting the infarct region on PA imaging.Methods:In vitro PA imaging,the CREKA-ICG-LIP NPs diluent solution was scanned at different excitation wavelengths ranging from 680 nm to 970 nm to determine the optimal absorbance spectrum for PA imaging.CREKA-ICG-LIP NPs(ICG,0-400 μg/ml),CREKA-LIP NPs(2 mg/ml)and PBS solutions were added to an agar gel phantom to acquire the corresponding PA images.PA images of the CREKA-ICG-LIP NPs solution(containing 200 μg/ml ICG)over time(0,4,8,12h)were recorded for PA stability analysis.In vivo PA imaging,6 IR models were prepared and randomly divided: target group and non-target group(n=3/group).Nanoparticles(CREKA-ICG-LIP NPs or ICG-LIP NPs,1 m L,4 mg/ m L)were injected through tail vein.Photoacoustic images of the heart region before surgery,after surgery,0 min after injection,and 60 min after injection were collected and the photoacoustic signal intensity was measured.After PA imaging,the hearts of the CREKA-ICG-LIP NPs group and the ICG-LIP NPs group were harvested for TTC staining to identify the injured region.Results:835 nm was determined to be the optical excitation wavelength for PA imaging.The PA signal from the CREKA-ICG-LIP NPs solution was brighter than that from the CREKA-LIP NPs solution and PBS solution.Moreover,the PA signal intensity and the ICG concentration showed a positive linear correlation(Y=0.0068X+0.0983,R~2=0.9943).PA images of CREKA-ICG-LIP NPs(ICG,200μg/ml)over time were obtained and the PA signal intensity showed no obvious change.PA images immediately after injection showed no PA signal in either group.After one hour of blood circulation,no PA signal was found in the ICG-LIP NPs group.However,in contrast,a strong PA signal was observed in the anterior wall of the left ventricle in the CREKA-ICG-LIP NPs group.The TTC staining of the CREKA-ICG-LIP NPs group showed the injury region.Conclusion:Fibrin-targeted nanoparticles(CREKA-ICG-LIP NPs)generated photoacoustic signals and had good photoacoustic stability in vitro.After injection,the targeted NPs reached the injury region through the vascular endothelial space and combined with fibrin to perform real-time specific photoacoustic imaging of the infarcted region.