Mr Study Of PLGA Nanoparticles Modified By CRGD For Targeting Detection Of Thrombosis

Objective: To construct a kind of Fe₃O₄-based molecular probes modified by cRGD（Fe₃O₄-PLGA-cRGD nanoparticles（NPs））to target thrombus and evaluate their physical and chemical characteristics,biological toxicity,biodistribution,MR imaging ability in vivo and ability to target thrombi in vitro and in vivo.Methods: Double emulsion solvent-evaporation technique（water/oil/water,W/O/W）was used to construct Fe₃O₄-based PLGA NPs taking PLGA-COOH as the carrier.cRGD peptides were further linked by carbodiimide-mediated amide bond formation to prepare Fe₃O₄-PLGA-cRGD NPs.The morphology,dispersion,structure,diameter and zeta potential of the NPs were determined by optical microscope,transmission electron microscope（TEM）and laser particle size analyzer.cRGD were labeled by FITC,then laser scanning confocal microscope was used to confirm the link of cRGD and flow cytometry was used to the carrier rate of cRGD.The carrier rate of Fe₃O₄ was measured by atomic absorption spectrometry.The toxicity of NPs in vivo was verified by measuring the liver and kidney function,blood routine and observing main viscera pathological after injection of NPs.The pathological of thrombosis ex vivo confirmed the targeting ability of NPs.7.0T MR scanner was used observe the distribution of NPs in SD rats.The mural thrombus formation of SD rats,the ability and enhancement of NPs targeting thrombus in vivo were observed by 7.0 T MR and pathological examination.Results: The Fe₃O₄-based PLGA NPs were constructed successfully and have a regular shape,a relatively uniform size.The average diameter,PDI and zeta potential of Fe₃O₄-PLGA-cRGD NPs were 368.2±4.5 nm,0.081±0.058 and-9.43±0.86 mV.TEM showed the Fe₃O₄ particles were distributed on the surface of the NPs.Fe₃O₄-PLGA-cRGD NPs was observed as the cricoid green fluorescence using laser scanning confocal microscopy and the carrier rate of cRGD determined by flow cytometry was 99.93±0.06%.The carrier rate of Fe₃O₄ was 64.67±0.9%.Hematological and pathological assays indicated no toxicity of the NPs in vivo.The pathologically frozen section revealed that the NPs readily and selectively accumulated on the surface of the thrombosis and under vascular endothelial cells ex vivo.In the biodistribution of NPs experiment,we observed the signal intensities（SI）of liver and spleen decreased significantly at 10 min after injection of NPs and the SNRs of liver and spleen is significantly lower than before injection.The SNRs of kidneys were slightly lower than before injection.That suggested that the NPs might be internalized by the macrophages of the reticuloendothelial system in the liver and spleen and some elimination by the kindey.The result of in vivo experiment revealed that the abdominal aorta model of wall-adherent thrombosis was successfully constructed and the T2 signal decreased at the mural thrombus 10 min after injection and then gradually increased until 50 min.HE staining of frozen sections showed that the Fe₃O₄-PLGA-cRGD NPs had gathered on the surface of the thrombus and under vascular endothelial cells.This indicate that Fe₃O₄-PLGA-cRGD NPs have good targeting ability and effect of magnetic resonance imaging.Conclusion: Fe₃O₄-PLGA-cRGD NPs were constructed successfully and have a regular shape and relatively uniform size.The carrier rate of cRGD and Fe₃O₄ is higher and there is no obvious toxicity in vivo.The targeting property and MR negative enhancement effect are good.These results suggest that the NPs are suitable for in vivo molecular imaging of thrombosis and represent a very promising MR contrast agent for sensitive and specific detection of thrombosis.