| Part-Ⅰ Preparation of Targeted Ultrasound and Magnetic Resonance Imaging Contrast Agents Covalently Binding with Vascular Endothelial Growth Factor-2 Antibody and Targeting Study In VitroObjective To construct targeted ultrasound(US)and magnetic resonance imaging(MRI)nanocapsules by covalently binding with vascular endothelial growth factor-2(VEGFR-2)antibody and investigate its characterization,stability and affinity for bovine artery endothelial cells in vitro.Methods The high molecular polymer PLA/PLGA-COOH nanocapsules encapsulating superparamagnetic iron oxide nanoparticles(SPIOs)and perfluorooctyl bromide(PFOB)were produced by the adapted oil-in-water(O/W)emulsion solvent evaporation process.Targeted nanocapsules were prepared by combining PLGACOOH with VEGFR-2 antibody using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC)and N-hydroxysuccinimide(NHS)mediated cross linking agents.To observe its form,particle size,distribution and ability of specific binding to bovine aortic endothelial cells(BAECs)which highly expressed VEGFR-2 under high glucose treatment.Results Targeted nanocapsules became brown emulsion when dissolved in double distilled water and their average diameter was 404.4±153.7nm.They showed welldefined spherical shape with not very smooth surface under transmission electron microscope(TEM).A lot of Fe3O4 particles distributed in the shell structure were observed under TEM.Western blot analysis revealed that VEGFR-2 was highly expressed in BAECs with high glucose treatment,and the targeted nanocapsules conjugated with BAECs tightly.Conclusion VEGFR-2 targeted nanocapsules were successfully constructed by carbodiimide technique and they can specific bind to BAECs effectively in vitro.Part-Ⅱ Imaging Performances of the Nanocapsules and Establishment of A Rat Atherosclerosis Model with Unstable PlaquesObjective To assess the imaging performances of the nanocapsules using ultrasound and magnetic resonance imaging in vitro and in vivo together with construct atherosclerosis model with unstable plaques using SD rats.Methods In vitro US imaging of different concentration of nanocapsules was carried out in the latex tube using Aplio 500 ultrasound system unit with a 12L-5 transducer;In vitro T2-weighted MR imaging of different Fe concentration of nanocapsules was carried out using a 3 Tesla GE Discovery 750 instrument;for in vivo study,the nanocapsules suspension was intravenously injected at a concentration of 0.1ml per kg weight,and the kidney was imaged transabdominally using the transducer in contrast pulse sequencing(CPS)mode.Thirty male SD rats were assigned to either underwent abdominal aorta balloon-injury,vitamin D3 injection and high cholesterol chow(group A,n=22)or fed standard chow(group C,n=8).Before and after 16 weeks,the plasma lipids were measured and the morphological changes of rat abdominal aorta were observed.Results Nanocapsules exhibited excellent increasing contrast enhancement together with the rise of the concentration as shown in the CPS mode in US and T2-weighted MR images.In vivo study showed that the kidney of the rat has obvious enhancement at the CPS mode after bolus intravenous injection of the nanocapsules.At 16 weeks,rat plasma levels of total cholesterol(TC)and low density lipoprotein cholesterol(LDL-C)in group A were higher than the baseline and group C.Unstable atherosclerotic plaques were formed in the rat abdominal aorta.Conclusions Nanocapsules exhibited excellent imaging performance in vitro and in vivo and a rat atherosclerosis model with unstable plaques could be established.Part-Ⅲ Ultrasound and Magnetic Resonance Imaging of Atherosclerotic Neovasculature with Targeted NanocapsulesObjective To investigate the feasibility of ultrasound and magnetic resonance for bimodal molecular imaging of atherosclerotic neovasculture with vascular endothelial growth factor-2 targeted nanocapsules.Methods Rats with vulnerable plaque were assigned to receive either injection of VEGFR-2 targeted nanocapsules(VTNC)(group A1,n=8)or non-targeted nanocapsules(NC)(group A2,n=8),control rats also received injection of VTNC(group C,n=8).US and MR imaging of the abdominal aorta were performed to detect VTNC by measuring of the ultrasonic gray scale intensity(GSI)and MR contrast to noise ratio(CNR)before and at successive time-points after VTNC injection.The percent positive area of CD31(+)or VEGFR-2(+)expression(PPACD31+ or PPAVEGFR-2+)was quantified by immunohistochemical staining.Results The peak GSI of group A1 was higher than that of group A2 and C(69.44±4.45 d B vs 45.24±1.92 d B,p<0.01 and 69.44±4.45 d B vs 44.78±4.59 d B,p<0.01,respectively);the peak CNR of group A1 was higher than that of group A2 and C(87.51±6.63 vs 49.22±3.19,p<0.01 and 87.51±6.63 vs 47.12±5.19,p<0.01,respectively);in the group A1,PPAVEGFR-2+ and PPACD31+ were significantly correlated with GSI(r=0.81,p<0.01 and r=0.73,p<0.05,respectively)and CNR(r=0.89,p<0.01 and r=0.72,p<0.05,respectively);but there was no correlation of PPAVEGFR-2+ and PPACD31+ with GSI(r=0.81,p<0.01 and r=0.73,p<0.05,respectively)and CNR(r=0.89,p<0.01 and r=0.72,p<0.05,respectively)in group A2.Conclusion VTNC with carrying two probes is feasible for bimodal US and MR molecular imaging of atherosclerotic neovasculature,which may offer complementary information for more reliable prediction of plaque vulnerability. |
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