Hyaluronic Acid-functionalized Gadolinium-doped Iron Oxide Nanoparticles As T₁-T₂ Dual-model MR Contrast Agents For Active Targeting Atheroscerosis

Objective:1.HA-Gd IO NPs were prepared by a facile one-pot polyol approach and their applications as T₁-T₂ dual modal contrast agents for enhanced MRI in vitro and in vivo image after being coated with HA.The characterization,toxicity and biocompatibility of the particles were tested.2.To establish an Apo E-/--deficient mice model of atherosclerosis,vascular smooth muscle cells and macrophages were cultured in vitro,and the expression of CD44 in different cells was elucidated from the cell level.3.MRI T₁/T₂ imaging was used to verify the distribution of nanoparticles from the cell level and the body level,and to verify the histological toxicity,and to observe the nature and location of atherosclerotic plaques by in vivo imaging.Methods:1.HA-Gd IO NPs were prepared by a facile one-pot polyol approach;The chemical structures of HA-Gd IO NPs were analyzed using a Fourier transform infrared?FT-IR?spectrometer;The morphologies of the HA-Gd IO NPs were examined by high-resolution transmission electron microscopy;The particle size of HA-Gd IO NPs were measured by Nanosight;The composition and structure of HA-Gd IO NPs were detected by X-ray diffraction;The elemental composition was determined by elemental analysis performed on X-ray photo electron spectroscopy.2.The relaxation effect of HA-Gd IO NPs were verified by MRI with in vitro and in vivo.3.The cytotoxicity of HA-Gd IO NPs were evaluated on VSMC,MCF-7 and RAW264.7 cells using the CCK8 assay.4.Male 20 g 8-week-old Apo E-/-mice were fed western type diet?containing 10% lard?4% milk?0.5% sodium cholate?for 4 month.Thereafter,the artery was harvested,followed by stained with oil red and HE staining to testified whether atherosclerosis animal modal was constructed successfully.Results:1.After fed western type diet for 4 month,Apo E-/-mice atherosclerosis model were construce successfully.2.These FT-IR results showed that the functional groups of HA-Gd IO NPs mainly contained certain plentiful C=O,-COOH,and-OH groups.The presence of these functional groups located at their surface endowed the HA-Gd IO NPs with excellent solubility and dispersibility in water.TEM/HRTEM imaging showed that The HA-Gd IO NPs all exhibited uniform dispersion and discrete quasi-spherical shape without apparent aggregation.HA-Gd IO NPs had a well-defined structure of ²5 nm diameter.These diffraction peaks matched well with the characteristic peaks of cubic Gd2O3?JCPDS card No.2004 15-0432?.XPS spectra of the HA-Gd IO NPs.were composed of Gd2O3 spectrum,Fe3O4 spectrum,O1 s spectrum and C1 s spectrum.Magnetic test indicating that HA-Gd IO NPs possessed strongly magnetic performance.3.The HA-Gd IO NPs has excellent hemocompatibility?1?Cell viability: All the HA-Gd IO NPs at different concentration exhibited negligible cytotoxicity.Even at the concentration of 400 ?g/m L,the cell viability was approximately 90%;?2?Hemolysis assay: The results showed that the hemolysis percentages of the HA-Gd IO NPs were all less than 7% in the studied concentration range from 25 to 200 ?g/m L,which verified their excellent hemocompatibility;?3?HA-Gd IO NPs with the Prussian blue stain can be clearly seen in VSMC and Raw264.7 cells,which further confirmed Gd IO NPs internalization process;?4?we conducted the histological assessment of the mouse susceptible organs?liver,heart,spleen,lung,kidney and brain?.No obvious adverse effect,such as inflammation,or tissue damage,associated with the administration of the HA-Gd IO NPs was observed compared with the control group.4.HA-Gd IO NPs have been demonstrated to be capable of accumulating in the atherosclerotic plaque due to enhanced permeability and positive targeting.The T₁-image signal intensity increased with the HA-Gd IO NPs concentration.On the contrary,T₂-image signal intensity displayed the opposite weakening tendency.Further quantitative analysis data showed that T₁-and T₂-dual modalimaging capacity of the HA-Gd IO NPs have good imaging effect.T₁-and T₂-weighted MR images of vessel regions at time points of pre-injection,30 min post-injection of HA-Gd IO NPs at a dose of 20 mg/kg through tail vein into the rats were obtained.Therefore,these results demonstrated that HA-Gd IO NPs might be used as T₁-positive and T₂-negative dual modal MR contrast agent.Conclusion:1.The successfully constructed HA-Gd IO NPs nanoparticles have the characteristics of moderate size,good stability,good water solubility and superparamagnetic properties.2.These results clearly indicated that the as-synthesized HA-Gd IO NPs had very low cytotoxicity and good hemobiocompatibility.HA can be used to achieve active targeting of atherosclerosis3.The treatment of HUK for the progressing ischemic stroke has better efficacy in the anterior circulation infarction than that in the posterior circulation infarction.4.In MR imaging HA-Gd IO NPs showed good relaxation effect,whether on T₁ or T₂ weighted phase.Which can be used as T₁ and T₂ dual-mode MRI contrast agent for atherosclerosis imaging.