Study Of Targeted MRI/CT Imaging Of Breast Cancer Mediated By The Fe₃O₄@Au Nanoparticles Coupled With Bombesin Like Peptide T-BBN

Objective1. To synthesize multi-core magnetic nano cluesters with different methods, characterize their various physicaochemical properties and screen out a kind of magnetic nano material with superparamagnetism, high transverse relaxivity and controllable particle size distribution.2. To synthesize the Fe3O4@Au nanoparticles with different Au shell thickness by seeded-growth, study the effects of gold shell on the tranverse relaxivity and X-ray attenuation, and select a Fe3O4@Au nanoparticle which have optimal transverse ralaxivity and X-ray absorption capacity for magnetic resonance imaging(MRI) and X-ray computed tomography(CT).3. To fabricate gastrin reeasing peptide receptor targeting molecular probes Fe3O4@Au-PEG-BBN by conjugating the bombesin like peptide t-BBN on the surface of Fe3O4@Au, explore the feasibility of breast cancer targeting MRI/CT dual-modality imaging based on the prepared nano probes.Materials and methods1. Synthesis and characterization of multi-core magnetic nano cluesters: Under the protection of inert gas, synthesize the hydrophobic Fe3O4 nanoparticls using oleic acid and oleylamine as surfactant, 1,2-hexadecanediol as reducing agent and iron acetylacetonate as precursor by thermal decomposition. Then the prepared nanoparticles were treated with tetramethylammonium hydroxide(TMAH) for surface modification to be hydrophilic. After treated with the amphiphilic surfactant sodium dodecyl sulfate(SDS), the hydrophobic Fe3O4 can self-assemble into a multi-core micelle. At the meantime, under the protection of nitrogen, Fe3O4 magnetic nano clusters(MNC) were synthesized via thermal hydrolysis using diethylene glycol as reducing agent and ferric chloride as precursor. The physicochemical property of the prepared nano materials will be fully characterized using transmission electron microscopy(TEM), high resolution transmission electron microscopy(HRTEM), energy disperse spectroscopy(EDS), inductively coupled plasma emission spectrometer(ICP-ES), Zeta laser particle analyzer, vibrating sample magnetometer(VSM), X-ray diffraction(XRD) and MRI.2. Synthesis and characterization of Fe3O4@Au nanoparticles: A thin layer of Si O2 was coated on the surface of MNC via Stober method. After modified with 3-aminopropyl triethoxysilane(APTES), the MNC@Si O2 was functionalized with amino groups on the surface. The gold seed nanoparticls were obtained by the reduction of chlorine acid solution with sodium borohydride as reducing agent. Then the gold seeds were conjugated on the surface of the MNC@Si O2 nanoparticles by the coordination of gold and amino groups. Synthesize Fe3O4@Au nanoparticles with complete gold shell via seeded-growth method and control the thickness of Au shell by regulating the amount of growth solution. The Fe3O4@Au nanoparticles with different thickness gold shell were characterized detailedly, including TEM for observation of morphology, particle size and chemical composition, UV-Vis for determination of absorption spectrum, dynamic light scattering(DLS) for measurement of hydrodynamic diameter, VSM for determination of saturation magnetization(Mp), MRI and CT scanning for measurement of transverse ralaxivity and X-ray absorption capacity.3. Fabrication of Fe3O4@Au-PEG-BBN and its targeting labeling on breast cancer cells T47-D in vitro: The peptide t-BBN were synthesized according to the sequence QWAVGHLM and modified with thiolated PEG. The obtained peptide HS-PEG-t-BBN were then immobilized on the surface of Fe3O4@Au through the coordination of gold and thiol groups and finally the gastrin-releasing peptide receptor targeting molecular probes Fe3O4@Au-PEG-BBN were obtained. Human breast cancer cell lines MDA-MB-231 and T47-D were set as model cells to evaluate the cytotoxicity of Fe3O4@Au-PEG-BBN using CCK-8 kit. Moreover, the specific labeling efficiency and their subcellular localization within the cell of Fe3O4@Au-PEG-BBN was assessed by Prussian blue staining and TEM.4. Targeting MRI and CT dual-modality imaging of breast cancer based on the Fe3O4@Au-PEG-BBN nano probes: Label MDA-MB-231 and T47-D which were both in the logarithmic phase with Fe3O4@Au-PEG-BBN. Then the labeled cells were MRI and CT imaged to evaluate the targeting MRI/CT dual-modality imaging efficiency of Fe3O4@Au-PEG-BBN on T47-D. Both MDA-MB-231 and T47-D cells were injected into nude mice subcutaneously to build breast cancer xenograft models. After introducing Fe3O4@Au-PEG-BBN probes from the tail vein, the mice were scanned by MRI and CT at different time points successively to explore the feasibility of targeting MRI/CT dual-modality imaging of breast cancer in vivo based on the Fe3O4@Au-PEG-BBN molecular probes.Results1. Hydrophobic Fe3O4 nanoparticles with a average size of 7.8 ± 0.8 nm were successfully synthesized by thermal decomposition method and the prepared nanoparticles were spherical, regular morphology and of narrow particle size distribution. After surface modified with SDS, the hydrophobic nanoparticles self-assembled into multi-core micelles which had an average size of 145 ± 13 nm. MNC nanoparticles synthesized through thermal hydrolysis also showed spherical morphology and narrow particle size distribution, which had an average size of 110 ± 10 nm. The HRTEM showed that TMAH-Fe3O4 and MNC had the similar lattice fringes, and the spacing between the lattices fringes were correspond to the planes of(111) of bulk Fe3O4 very well. Moreover, the measured interplanar spacing based on the diffraction rings in the selected area electron diffraction(SAED) was perfectly in agreement with the respective hkl indexes of bulk Fe3O4 from the JCPDS database, indicating that the synthetic NPs are Fe3O4 nanocrystals. The Fe and O peaks showed in the results of EDS indicated the presence of Fe and O in the nanoparticle, meanwhile the Cu signal is derived from the TEM grid. The results from XRD showed that the prepared nanoparticles had good crystallinity and were consistent to the standard X-ray diffraction spectra of Fe3O4 nanocrystals. The hydrodynamic diameter of TMAH-Fe3O4, SDS-Fe3O4 and MNC were 9.7 ± 1.2 nm、164 ± 18 nm and 123 ± 13 nm and their Zeta potential were-45.2 m V、-52.3 m V and-55.1 m V, respectively. The results from VSM indicated that all the three kinds of magnetic nanoparticles were superparamagnetic and had a similar Mp which was 71 emu g-1, 69 emu g-1 and 65 emu g-1. However, the results of MRI showed that SDS-Fe3O4 and MNC had much higher r2 than TMAH-Fe3O4, which were 67 m M-1S-1, 256 m M-1S-1 and 245 m M-1S-1, respectively.2. A thin silica shell was successfully coated on the surface of MNC and the Fe3O4@Si O2 core-shell nanoparticles were successfully functionalized with APTES. Through the coordination of Au and amino, the prepared gold seeds were immobilized on the surface of Fe3O4@Si O2, and a gold shell was obtained by seeded-growth. The thickness of gold shell was showed various from each other when different amount growth solution were added, and the diameters were 118 ± 12 nm(Fe3O4@Au-10), 125 ± 13 nm(Fe3O4@Au-20) and 130 ± 11 nm(Fe3O4@Au-30) when 10 m L, 20 m L and 30 m L growth solution were added, and the obtained nanoparticles were termed as Fe3O4@Au-10, Fe3O4@Au-20 and Fe3O4@Au-30, respectively. The UV-Vis showed that the absorption peak varied when the thickness of gold shell was different. The absorption peak of gold seeds located at 520 nm and it showed a notable red-shift with the increase of thickness of gold shell. The Mp of Fe3O4@Au decreased gradually from 63 emu g-1 to 32.5 emu g-1 when the thickness of gold shell increase and the r2 decreased gradually from 235 m M-1S-1 to 168 m M-1S-1 accordingly. On the contrary, the X-ray absorption capacity of these nanoparticles increased with the increase of thickness of gold shell and the X-ray absorption capacity of Fe3O4@Au-10, Fe3O4@Au-20 and Fe3O4@Au-30 were 1.72, 1.91 and 2.12 times higher than that of iodine solution with same concentration.3. Through the strong coordination of gold and thiol group, HS-PEG-t-BBN were successfully immobilized on the surface of Fe3O4@Au and finally the targeting MRI/CT dual-modality molecular probes Fe3O4@Au-PEG-BBN were obtained. The results of CCK-8 analysis showed that the prepared Fe3O4@Au-PEG-BBN had no acute cytotoxicity and were of good biocompatibility. The results of Prussian blue staining and TEM indicated that higher uptake of Fe3O4@Au-PEG-BBN probed were engulfed into T47-D cells than that engulfed into MDA-MB-231. Blocking GRPR with the free t-BBN peptides effectively reduced the amount of blue granules in the cytoplasm of T47-D, indicating that the internalization of Fe3O4@Au-PEG-BBN was specifically mediated by t-BBN peptides. Moreover, the results of TEM clearly showed that the internalized Fe3O4@Au-PEG-BBN probes mainly distributed in the lysosomes around nucleus.4. The MRI and CT of labeled T47-D cells showed that the signal intensity of cells decreased with the increase of concentration in T2 WI. On the contrary, the CT value of the labeled cells increased with the increase of concentration. Breast cancer xenograft models were successfully established by injecting T47-D and MDA-MB-231 cells into nude mice subcutaneously. After injecting Fe3O4@Au-PEG-BBN nano probes, the signal intensity of tumor region in MR T2 WI decreased significantly and can last for a long time. Then, the signal intensity recovered gradually over time. Meanwhile, the CT value increased firstly and then decreased gradually over time.ConclusionIn this study, we had synthesized a kind of magnetic nanoparticles which were of superparamagnetism and high r2. Through the seeded-growth method, we grew a complete gold shell on the surface of magnetic nanoparticle, and obtained a kind of composite nanoparticle which had both the optimal r2 and X-ray absorption capacity and suitable for MRI/CT dual-modality imaging by finely control of the thickness of gold shell. Based on the strong coordination between gold and thiol groups, we successfully immobilized the peptide t-BBN on the surface of Fe3O4@Au and made them can target human breast cancer. Mediated by the molecular probes Fe3O4@Au-PEG-BBN, human breast cancer had been successfully targeting MRI and CT imaged in vivo, which provide a new path for early diagnosis of breast cancer.