MRI And CT Experimental Study Of Breast Cancer Angiogenesis Based On The Synthesized Gold Magnetic Nanoparticles Targeting To CD105

Objective1. To synthesis different Au shell thickness of gold magnetic nanoparticles (Fe2O3@Aunanoparticles), study the effects of Au shell thickness on the magnetic susceptibility andX-ray absorption capacity of Fe2O3@Au nanoparticles, and to prepare a dual-mode contrastagent which can be used in magnetic resonance imaging (MRI) and X-ray computedtomography (CT).2. To prepare a CD105-targeted molecular probe of Fe2O3@Au-PEG-CD105, explorethe feasibility of being applied to assess breast tumor angiogenesis by MRI and CT imaging.Materials and methods1. Synthesis and characterization of Fe2O3@Au nanoparticles: using ferric chloride andferrous chloride as reaction substrates. Fe3O4nanoparticles were synthesized via chemicalco-precipitation method under nitrogen protection. After that Fe3O4nanoparticles wereoxidated in acidic solution to obtain Fe2O3nanoparticles.Using Fe2O3as seeding particles,chloroauric acid as reduction substrate, hydroxylamine as reductant, Au-coated Fe2O3nanoparticles were synthesized by iteratively reducing Au3+onto the Fe2O3surface. Fiveseeding cycles were carried out to synthesis different thicknesses of Au shell of Fe2O3@Aunanoparticles. Characterize the synthesized Fe2O3@Au nanoparticles with different Au shellthicknesses, including inductively coupled plasma atomic emission spectroscopy toquantitate the elemental composition, transmission electron microscopy (TEM) to detect theshape and size, UV-visible spectrophotometer to measure the absorbance curve at400~800nm, Zeta laser particle size analyzer to measure the hydrodynamic diameter and Zetapotential, vibrating sample magnetometer to detect the saturation magnetization, CTimaging to measure the X-ray absorption ability, MRI scanning to detect the magneticsusceptibility of Fe2O3@Au nanoparticles. 2. Surface modification and stability analysis of Fe2O3@Au nanoparticles:polyethylene glycol-thiol (PEG-SH), thiol-polyethylene glycol-carboxyl (HS-PEG-COOH),mercaptopropionic acid (MPA), glutathione (GSH) were selected to modify the surface ofFe2O3@Au nanoparticles with three Au seeding cycles. Spectrophotometer and Zetaanalyzer were used to detect the colloidal stability of Fe2O3@Au nanoparticles at pH5.0~9.0or a salt concentration of0.01~0.10M.3. Synthesis of Fe2O3@Au-PEG-CD105and its cell labeling ability: the molecularprobes of Fe2O3@Au-PEG-CD105were synthesized by coupling anti-CD105antibodieswith Fe2O3@Au nanoparticles modified by HS-PEG-COOH. Utilizing the carboxylterminus provided by HS-PEG-COOH to form an amide bond with anti-CD105antibody.Human umbilical vein endothelial cell (HUVEC) line was set as the cell model to study thecytotoxicity of Fe2O3@Au-PEG-CD105by MTT method, evaluate the specific markingefficiency of Fe2O3@Au-PEG-CD105by prussian blue staining and immunohistochemicalstaining, observe the metabolism of the molecular probe in HUVECs cells.4. MRI and CT target imaging of breast cancer angiogenesis mediated byFe2O3@Au-PEG-CD105: after co-cultured with HUVECs cells, MRI and CT imaging wereperformed to evaluate the targeting efficiency of Fe2O3@Au-PEG-CD105. Human breastcancer cells (MBA-MD-231) were transplanted in nude mice to build breast cancerxenografts models. By introducing Fe2O3@Au-PEG-CD105from the tail vein, mice werescanned by MRI and CT at different time points to observe the targeting and distribution ofthe molecular probe. After the imaging, the tumors were resected and performed CD105immunohistochemical staining and Prussian blue staining to detect tumor microvesseldensity (MVD). The correlation of MVD with the signal intensity of region of interest (ROI)was analyzed.Results1. Fe2O3@Au nanoparticles with different Au seeding cycles were synthesized byco-precipitation and iterative reduction method. Fe2O3@Au nanoparticles with different Aushell thickness showed average diameters of36.5~56.5nm, the absorption peaks of500~600nm range, the saturation magnetization of42.4~59.6emu/g, positive potential inaqueous solution, polydispersity index greater than0.1. Compared Fe2O3@Au nanoparticles with1,3,5Au seeding cycles, the relaxation coefficients decreased from115mM-1S-1to61mM-1S-1as the Au shell thickness increased. However, X-ray absorption capacities of thegold shell were1.65,1.84and2.04times of iodine, respectively. Fe2O3@Au nanoparticlesobtained after three Au seeding cycles had an Fe2O3:Au molar ratio of7.2:26.8, a meandiameter of48.3nm, a UV-vis absorbance peak of550nm, a saturation magnetization of49.0emu/g. The hybrids obtained after three Au seeding cycles are the preferred candidatesfor MRI and CT applications because of their relatively high R2relaxivity (95mM-1s-1) andX-ray attenuation (1.87times that of iodine) compared to those of the other hybridsinvestigated in this study.2. Fe2O3@Au nanoparticles with three Au seeding cycles were modified with theligands containing-SH terminus, including PEG-SH, HS-PEG-COOH, MPA, and GSH.Zeta potentials of the modified nanoparticles were reduced from14.5mV to2.1mV,-19.3mV,-16.7mV, and-22.8mV, respectively. Meanwhile, hydrodynamic diameters of themodified nanoparticles were increased from55.2nm to84.9nm,85.2nm,65.9nm, and70.3nm, respectively. In a pH5.0~9.0or a salt concentration of0.01~0.10M solution,PEG-SH and HS-PEG-COOH modified Fe2O3@Au nanoparticles showed higherdispersibility and stability than the nanoparticles modified by MPA and GSH.3. Using the-COOH terminus provided by HS-PEG-COOH, the nanoparticles werecoupled with anti-CD105antibody by amidation reaction to successfully prepare themolecular probe of Fe2O3@Au-PEG-CD105. The CD105expression ratio of HUVECs cellsidentified by flow cytometry was86.84%. Prussian blue staining, immunofluorescence andimmunocytochemistry proved that the targeting of Fe2O3@Au-PEG-CD105to HUVECscells was specific, and can be blocked by anti-CD105antibody. TEM result suggested thatFe2O3@Au-PEG-CD105was intake by HUVECs cells by the receptor mediatedendocytosis way.4. HUVECs cells labeled by Fe2O3@Au-PEG-CD105were scanned by MRI and CT.With the concentration of the molecule probe increased from0.1mM to1.0mM, MRIsignal intensity of each group of cells decreased gradually. CT values of each group of cellsincreased a little, but the alteration not so noticeable in CT images. Breast cancer xenograftsmodels were successfully established by transplanted human breast cancer cells(MBA-MD-231) in nude mice. MRI signal intensity feature of the tumor ROIs showed "down-up-platform" performance after injection of Fe2O3@Au-PEG-CD105. There weresome minor enhancements of CT values of the tumor ROIs. The enhancement trend wassimilar to MRI result. Both of immunohistochemical staining and Prussian blue stainingresults proved that Fe2O3@Au-PEG-CD105can specifically target to tumor angiogenesis.There was a positive correlation between ROI’s MVD and MRI signal intensity (P<0.05).ConclusionFe2O3@Au nanoparticles with different diameters were synthesized in this study. Weoptimized of the composition of bimetallic core/shell Fe2O3@Au nanoparticles for MRI andCT dual-mode imaging. The molecular probe of Fe2O3@Au-PEG-CD105targeting toCD105was synthesized based on Fe2O3@Au nanoparticles. Mediated by Fe2O3@Au-PEG-CD105, MRI and CT imaging can specifically detect breast cancer xenograftsangiogenesis in vivo. This research is expected to provide a new approach to evaluate tumorangiogenesis via the combination of MRI and CT imaging.