| Part 1 Preparation and characterization of USPIO-cy5.5-c RGDOBJECTIVE: To develope an magnetic resonance imaging(MRI)/ near-infrared fluorescence(NIRF)dual-modality probe for integrin αvβ3 targeting,and to study its particle size,stability,fluorescence coupling conditions and magnetic effects.METHODS: Fe3O4 magnatic nanoparticles(USPIO)were synthesized by high-temperature pyrolysis,coated with PEG and cy5.5,then coupled with cyclic RGD peptide,a integrin αvβ3 targeted ligand,resulting in dual-modality probe USPIO-cy5.5–c RGD.Its particle size was tested by transmission electron microscopy(TEM),hydrodynamic size and zeta potential by dynamic light scattering(DLS),fluorescence coupling conditions by UV spectrophotometry,and magnetic effects by 3.0T MRI.RESULTS: The dual-modality probe for integrin αvβ3 targeting was constructed successfully,with a maximum wavelength absorption peak at 690 nm,,and its T2 WI signal intensity decreased gradually with the probe concentration rising,and its r2 was 251.55 by linear fitting.Nanoparticle diameter of Fe3O4 was 10.08 ± 0.34nm;hydrodynamic size of USPIO-cy5.5 –c RGD was 46.66 ± 16.31 nm,zeta potential was-53.87 ± 1.99 m V.CONCLUSION: USPIO-cy5.5-c RGD molecule may be a promising dual-modality probe for imaging integrin αvβ3 by MRI and NIRF.Part II: A preliminary experiment of USPIO-cy5.5-c RGD in vitroOBJECTIVE: To investigate the effect on growth and migration ability of HUVEC cells and the imaging performance of USPIO-cy5.5-c RGD,and provide the basis for further imaging in vivo.METHODS: Human umbilical vein endothelial cells(HUVEC)were seeded in 96-well plates and incubated with USPIO-cy5.5-c RGD solution,Fe concentration were 0,10,20,40,80,160 μg / ml respectively,co-cultured for 24,48 and 72 hours.The effect of USPIO-cy5.5-c RGD on cell proliferation was detected by CCK-8.HUVEC cells were seeded in the transwell chamber,and Fe concentration of USPIO-cy5.5-c RGD in the lower chamber were 0,5,and 25μg / ml,respectively.The effects on the migration ability of the cells were observed after 24 hours of incubation.0,5,25,50,100 μg / ml USPIO-cy5.5-c RGD and 100 μg / ml USPIO were incubated with HUVEC cells in vitro for 2h,then the incubated HUVEC cells were took for MRI and NIRF.Results: CCK-8 results showed that cell viability was higher than 90% when cells were incubated with the probe for 24 hours and concentration of Fe in the USPIO-cy5.5-c RGD solution was lower than 80μg / ml.The concentration of Fe was 160 μg / ml,the cell survival rate was 44.2%.When the concentration of Fe was 10,20,40 μg / ml for 48 hours and 10,20 μg / ml for 72 hours,the cell viability was 50% ~ 75%.When the concentration of Fe was 80,160 μg / ml for 48 hours and 40,80 μg / ml for 72 hours,the cell viability was 1% ~ 24%.When incubated for 72 hours,the concentration of Fe was 160 μg / ml,cell viability <1%.Transwell results showed that the cells in the lower chamber were 90 ± 5.27 / vision in group without the USPIO-cy5.5-c RGD,75 ± 3.33 / vision in the 20 μg / ml group and 63 ± 4.15 / vision in the 40 μg / ml group.The results of MRI showed that when the concentration of USPIO-cy5.5-c RGD was less than 100 μg / ml,the signal of the cell incubated was increased with the concentration of co-incubation probe on the T2 WI image.Group of USPIO-cy5.5-c RGD was lower than group of USPIO at the same concentration.The results of fluorescence imaging showed that the fluorescence intensity of the cell incubated with USPIO-cy5.5-c RGD increased with the concentration of co-incubation probe.Conclusion: USPIO-cy5.5-c RGD showed no inhibitory when the concentration of Fe was less than 40 μg / ml incubated with cells for 24 hours in vitro;The inhibitory was increased with the drug concentration rised and co-incubation time prolonged.The effect of USPIO-cy5.5-c RGD on the migration of HUVEC cells was inhibited in vitro,and the inhibition rate was increased with the increase of drug concentration.The binding rate of probe and cell increased with the increase of probe concentration when the concentration of Fe was less than 100 μg / ml.Results of MRI and fluorescence imaging showed good imaging performance.The binding rate of probe and cell was increased with the increase of probe concentration.At the same concentration,the ability of USPIO-cy5.5-c RGD to bind to cells was stronger than that of USPIO. |
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