A Non-Invasive Nanoparticles For Multimodal Imaging Of Ischemic Myocardium In Rats

PART?PREPARATION,CHARACTERIZATION AND BIOSAFETY EVALUATION OF TARGETED PHASE-TRANSFORMATION LIPID NANOPARTICLESObjective:To prepare a novel molecular probe(IMTP-Fe₃O₄-PFH NPs)for ultrasound(US),photoacoustic(PA)and magnetic resonance(MR)imaging of myocardial ischemic,and to evaluate cytotoxicity and biosafety of these nanoparticles(NPs).Methods:(1)IMTP-Fe₃O₄-PFH NPs were prepared by using thin-film dispersion and ultrasound emulsification.(2)The morphology and dispersion were observed by oil microscope,confocal laser scanning microscope(CLSM),and transmission electron microscope(TEM).(3)The particle size,potential,and stability of the NPs were measured by Malvern dynamic light scattering analyzer.(4)The encapsulation efficiency of NPs was obtained by measuring Fe content by atomic absorption spectrophotometry.(5)The changes in the size of NPs irradiated by heating or LIFU were observed under the optical microscope to explore the phase transformation by temperature and acoustic droplet vaporization(ADV)in vitro.(6)CCK-8 assay was used to detect the activity of cells co-incubated with different concentrations of NPs to determine whether they had cytotoxicity.(7)The biosafety of IMTP-Fe₃O₄-PFH NPs in vivo was evaluated by detecting blood routine and blood biochemical indexes at different time points after intravenous injection NPs in rats,and pathological sections of vital organs were taken 14 days later.Results:(1)IMTP-Fe₃O₄-PFH NPs were successfully prepared and their suspension was brown.(2)Under the optical microscope and CLSM,the NPs were small spheres with good dispersion,and Fe₃O₄ particles could be seen distributed in them by TEM.(3)The particle size of IMTP-Fe₃O₄-PFH NPs was 348±1.2 nm,and the zeta potential was-28.4±1.01 m V.(4)Encapsulation efficiency and iron-loading efficiency were64.18±0.26%and 9.44±0.66%,respectively.(5)The temperature of phase transformation caused by heating was about 66?,and the optimal condition of ADV was 3w/cm² after irradiation by LIFU in vitro.(6)The morphology of cells did not change after co-incubation with IMTP-Fe₃O₄-PFH NPs.CCK-8 results also showed that the cell viability could reach 94.27%even at the maximum concentration of IMTP-Fe₃O₄-PFH NPs.(7)No abnormal behavior was observed after the treatment of NPs in the tail vein of rats,and no significant changes were observed in the hematological indexes and pathological results of major organs at different time points.Conclusion:We successfully prepared a novel probe----IMTP-Fe₃O₄-PFH NPs,which had excellent properties such as uniform size,good stability,high encapsulation rate,high biosafety,and liquid-gas phase transformation under certain conditions.PART ? ESTABLISHMENT OF HYPOXIA INJURY MODEL IN VITRO AND IN VIVO AND EVALUATION OF LIPID NANOPARTICLES TARGETING ABILITYObjective: To explore the suitable conditions for the formation of hypoxia injury in H9C2 cells,establish and verify the rat myocardial ischemia model,and on the basis of the above successful modeling,verify the targeting of IMTP-Fe₃O₄-PFH NPs in vivo and in vitro.Methods:(1)The growth curves of H9C2 cells were plotted by CCK-8 method;the survival rates of H9C2 cells were measured at 6h,12 h,24h and 48 h under 5% CO2+1% O2+94% N2 to clarify the treatment time of hypoxic modeling;the appropriate concentration of H₂O₂ to induce modeling was determined by measuring the survival rate of H9C2 cells.(2)Myocardial ischemia model was created by non-invasive transoral tracheal intubation and open-chest ligation of the left anterior descending branch of the rat coronary artery with the support of a ventilator.The successful modeling of rats was verified by intraoperative electrocardiography,postoperative same-day echocardiography,postoperative two-week MRI and histological examination.(3)The distribution of Di I-labeled targeted(IMTP-Fe₃O₄-PFH NPs)and non-targeted(Fe₃O₄-PFH NPs)NPs was observed by CLSM after co-incubation with H9C2 cells treated by hypoxia or H₂O₂.(4)Myocardial ischemia model rats were divided into targeted group(IMTP-Fe₃O₄-PFH NPs)and non-targeted group(Fe₃O₄-PFH NPs), and Di R-labeled NPs of the above two types were injected into the rats via the tail vein.After that,the ex vivo fluorescence imaging of the heart and other important organs was performed at different time points.Results:(1)Combined with the survival rate of H9C2 cells and the operability of the experiment,24 h of hypoxia and 5mol/l of H₂O₂ were selected as the treatment condition of model in the subsequent experiment.(2)The standardized model of myocardial ischemia in rats was established to ensure the consistency of the ischemic site and area in the subsequent experiments.Intraoperatively,the myocardial tissue below the ligation site became pale,and the electrocardiogram showed the typical S-T segment arch-back elevation.Postoperative US showed reduced left ventricular anterior wall motion and limited systolic-diastolic extent.Two weeks after the operation,MRI showed that the anterior wall of the left ventricle was significantly thinner and the left ventricular cavity was dilated.The tissue sections showed that the anterior wall myocardium was atrophied and almost completely replaced by fibrous tissue.(3)In vitro,it was seen that Di I-labeled IMTP-Fe₃O₄-PFH NPs were targeted to aggregate around H9C2 cells in both the hypoxic and H₂O₂ groups,and the aggregation phenomenon became more pronounced with increasing co-incubation time,which was not seen in the non-targeting group.(4)For in vivo,fluorescent signals could be seen in the anterior wall of the left ventricle of rats in the targeting group after the injection of NPs,and the signal reached its peak at 10min,while there was no fluorescent signal in the whole process in the non-targeting group.Conclusion: Based on the successful establishment of hypoxia injury model both in vitro and in vivo,we verified that IMTP-Fe₃O₄-PFH NPs had good targeting performance.PART ? TARGETED PHASE-TRANSFORMATION LIPID NANOPARTICLES FOR ULTRASOUND/PHOTOACOUSTIC/MAGNETIC RESONANCE IMAGING IN VITRO AND IN VIVOObjective: To observe the effects of IMTP-Fe₃O₄-PFH NPs in enhancing US,PA and MR imaging in vitro,and to investigate the imaging effects in three modalities after their application to rats in an ischemic myocardial model.Methods:(1)IMTP-Fe₃O₄-PFH NPs were added to the gel module and excited by LIFU with different irradiation intensities for different times,and observed in B-mode as well as CEUS.IMTP-Fe₃O₄-PFH NPs or Fe₃O₄-PFH NPs were administered via tail vein injection immediately after rat modeling,and irradiated with LIFU 10 min later.During this procedure,two modes of cardiac ultrasound were examined in all rats at four time points: preoperatively,immediately after injection,10 min after injection,and after LIFU irradiation.(2)IMTP-Fe₃O₄-PFH NPs were scanned at different wavelengths ranging from 680 nm to 970 nm to determine the optimal maximum absorbance for PA imaging.According to the concentration of Fe in the nanoparticles,they were diluted to 0.039,0.078,0.156,0.312 and 0.624mg/ml.The PA imaging was performed and the signal intensity was analyzed under the optimal excitation wavelength,meanwhile,saline and IMTP-PFH NPs without Fe₃O₄ as the control.In vivo,model rats were given targeted(IMTP-Fe₃O₄-PFH NPs)and untargeted(Fe₃O₄-PFH NPs)NPs via the tail vein,respectively,and the PA imaging of heart was performed at four time points: preoperative,immediately after injection,10 min and 60 min after injection.(3)IMTP-Fe₃O₄-PFH NPs were prepared with 0.01,0.02,0.04,0.08,0.16,0.32 m M for Fe concentration,saline and IMTP-PFH NPs as the control group.T2-WI and transverse relaxation time quantitative imaging were performed on the above materials,and the relaxation efficiency of IMTP-Fe₃O₄-PFH NPs was calculated.The model rats were divided into three groups: targeted group(IMTP-Fe₃O₄-PFH NPs),non-targeted group(Fe₃O₄-PFH NPs)and iron-free group(IMTP-PFH NPs),and T2*-WI was performed after administration of each NPs.The black and white MR imaging were processed by Matlab software with pseudo-color.After this imaging,the hearts were removed for Prussian blue enhanced staining to verify that the NPs were aggregated in the imaging region.Results:(1)In both B-mode and CEUS,the signal intensity increased with LIFU irradiation intensity and time,peaking at 3w/cm2 for 3min.After given IMTP-Fe₃O₄-PFH NPs and irradiated by LIFU in heart area,a massed hyperechoic signal was detected at the anterior wall of the left ventricle in model rats under two modes.(2)The optimal PA excitation wavelength of IMTP-Fe₃O₄-PFH NPs was 690 nm.IMTP-Fe₃O₄-PFH NPs showed the concentration-dependent contrast enhancement in PA imaging, while no PA signal was observed in the saline group and the IMTP-PFH NPs group.10 min after the injection of IMTP-Fe₃O₄-PFH NPs through the tail vein,the PA signal could be observed in the anterior wall of the left ventricle in model rats' heart,and then gradually weakened.The non-targeted group had no signal throughout.(3)IMTP-Fe₃O₄-PFH NPs resulted in different degrees of negative enhancement of T2-WI imaging,and the relaxation efficiency was calculated to be 152.02 m M-1·s-1.After the injection of different NPs in each group,a signal reduction region was observed in the anterior wall of the left ventricle in the targeted group,but not in the non-targeted group nor in the iron-free group,which was later corroborated by Prussian blue staining of the heart tissue.Conclusion: IMTP-Fe₃O₄-PFH NPs,as a novel molecular probe,could accumulate at the ischemic myocardium region of rats to enhance US,PA and MR imaging.