Branched Macromolecule-assisted Synthesis Of Functional Hybrid Nanoparticles For Molecular Imaging Applications

With the fast advances of nanotechnology, nanoparticles（NPs） have been widely developed as imaging contrast agents for various molecular imaging applications, especially for early cancer diagnosis. However, there are many drawbacks for clinical contrast agents, such as rapid clearance rates from vascular circulation, fast renal excretion, relatively low imaging sensitivity, and non-specificity. Besides, each imaging mode has its own advantages and limitations. For example, computed tomography（CT） imaging with its high hard-tissue imaging contrast, high spatial and density resolution, and low cost, still has some limitation, such as low soft-tissue imaging contrast and radioactivity, while magnetic resonance（MR） imaging with advantages such as high soft-tissue imaging contrast, high spatial resolution（⁵0 μm）, and high tomographic capabilities, has some drawbacks like high cost and long scanning time. Therefore, in order to detect the cancer diagnosis as early and accurately as possible, it is important to form a nanoparticle（NP） system incorporating different radiodense elements（Fe, Gd, Au） for effective dual mode or multi mode imaging applications. With the unique properties of dendrimers and branched polyethyleneimine（PEI）, both of which possesses abundant amino groups, we have used them as ideal templates or stabilizers to entrap, stabilize or assemble the inorganic NPs, generating a series of novel organic/inorganic hybrid NPs for dual modal（MR/CT） imaging. The fabricated hybrid NPs were characterized via different techniques. Then we further optimize the ratio of different radiodense elements in hybrid NPs to achieve better MR relaxivity and X-ray attenuation property. Finally, we investigate the imaging efficiency of hybrid NPs in vitro and in vivo. The main methods and results are as follows:1) In chapter 2, with the combination of electrostatic layer-by-layer（Lb L） self-assembly technique and dendrimer chemistry, we formed the Fe₃O₄@Au nanocomposite particles（NCPs） with the Au/Fe₃O₄ molar ratio of 0.152 and with non-uniform core-shell structure. Thestability, 3-（4,5-cimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide（MTT） assay and hemolysis assay results show that the formed Fe₃O₄@Au NCPs are colloidally stable, hemocompatible, and biocompatible in the given concentration range. Besides, due to the synergistic effect of Fe and Au element, the Fe₃O₄@Au NCPs displayed good R2 relaxivity（R2 = 71.55 m M-1s-1） and enhanced X-ray attenuation intensity compared with the pure Fe₃O₄（R2 = 277.81 m M-1s-1）, Fe₃O₄@G5（R2 = 68.98 m M-1s-1） without Au NPs and Au DENPs. The in vitro and in vivo MR/CT imaging results showed that Fe₃O₄@Au NCPs can be used for MR imaging of liver and CT imaging of a subcutaneous tissue. These results show that dendrimer-assembled Fe₃O₄@Au NCPs are potential contrast agents for dual mode MR/CT imaging.2) Fe₃O₄@Au NCPs with a low molar ratio of Au/Fe₃O₄（0.152） is not desirable for CT imaging due to the low sensitivity of CT. We next attempted to tune the Fe/Au composition to optimize the R2 relaxivity and X-ray attenuation intensity for better MR/CT imaging. Therefore, based on chapter 2, we further synthesized the nontargeted Fe₃O₄/Aun NCPs with tunable Au/Fe₃O₄ molar ratio（0.2-2.02） by Au seed-mediated iterative growth of Au shell and targeted Fe₃O₄/Aun-FA NCPs by modifying the dendrimers with targeting ligands（FA）. The acetylated（Ac） Fe₃O₄/Au5.Ac-FA NCPs with the Au/Fe₃O₄ molar ratio of 2.02 were hemocompatible, and biocompatible in the given concentration. The optimization of the R2 relaxivity and X-ray attenuation intensity of the NCPs show that, compared with the nontargeted Fe₃O₄/Au5.Ac with Au/Fe₃O₄ molar ratio of 1.59（R2 = 112.25 m M-1s-1）, the targeted Fe₃O₄/Au5.Ac-FA NCPs with the highest Au loading display the best X-ray attenuation intensity for CT imaging and do not significantly compromise its R2 relaxivity（92.67 m M-1s-1） because the non-uniform Au NP coating onto the surface of Fe₃O₄ NPs is beneficial for the water molecule to access the surface of Fe₃O₄ cores. In vitro and in vivo CT and MR images of tumors show that the targeted Fe₃O₄/Au5.Ac-FA NCPs can be used for targeted MR/CT imaging of FAR-overexpressing tumors.3) With the abundant primary amino groups, branched PEI is not only used as ideal stabilizers to stabilize Au NPs, but also used for modifying Fe₃O₄ NPs. Besides, it can be further functionalized via the active amine groups for different biomedical applications. Therefore, compared with the high cost of G5 dendrimers, cheaper PEI may be more potential for practical biomedical applications. Thus in chapter 4, we used PEI as a stabilizer and synthesized PEI-coated Fe₃O₄ NPs（Fe₃O₄-PEI NPs） with tunable size via a one-pot hydrothermal method. The diameter of Fe₃O₄-PEI NPs（16.7-21.8 nm） synthesized using PEI as stabilizer was smaller than the pure Fe₃O₄ NPs（31.1 nm） synthesized under the same conditions in the absence of PEI, showing that PEI can inhibit the crystal growth of Fe₃O₄NPs. In addition, Fe₃O₄-PEI NPs were further functionalized with polyethylene glycol（PEG）, acetic anhydride, and succinic anhydride（SAH） to form Fe₃O₄-PEI.Ac, Fe₃O₄-PEI.SAH, and Fe₃O₄-PEI.PEG, respectively. Fe₃O₄-PEI and Fe₃O₄-PEI.PEG NPs with the good R2 relaxivity（137.11-156.23 m M-1s-1） are desirable for MR imaging. The MTT, hemolysis and the macrophage cellualr uptake assay results show that compared with the Fe₃O₄-PEI NPs, functionalized Fe₃O₄-PEI.Ac and Fe₃O₄-PEI.PEG NPs have improved cytocompatbility and hemocompatibility, and decreased non-specific macrophage uptake.4) Based on chapter 4, using PEI as stabilizer, in chapter 5 we further synthesized PEI-coated Fe₃O₄-Gd（OH）3 NPs（Fe₃O₄-Gd（OH）3-PEI NPs） via a modified hydrothermal method in the presence of gadolinium nitrate hexahydrate. The formed Fe₃O₄-Gd（OH）3-PEI-PEG NPs（14.4 nm） with the Gd/Fe molar ratio of 0.25:1 are colloidally stable and biocompatible in a given concentration range. Besides, the Fe₃O₄-Gd（OH）3-PEI-PEG NPs not only display the relatively high R2 of 151.37 m M-1s-1, but also show the high R1 of 5.63 m M-1s-1, which suggests that they can be used as a unique contrast agent for T1/T2-weighted MR imaging. Combined with their high R1/R2 relaxivity and good biocompatibility, the prepared Fe₃O₄-Gd（OH）3-PEI-PEG NPs can be used for dual mode T1/T2-weighted MR imaging of rat liver through rat’s mesenteric vein injection of the NPs by interventional operation and mouse liver through intravenous injection of the NPs. In vivo T1/T2-weighted MR imaging show that Fe₃O₄-Gd（OH）3-PEI-PEG NPs are potential contrast agents.5) Based on chapters 4 and 5, in chapter 6 we formed a series of Gd（OH）3 nanomaterials with tunable size and morphology via a hydrothermal method by tuning the reaction time（6, 24, 40 h）, adding the crystal inhibitor of Na3 Cit or stabilizer of PEI, and changing the reaction solvent（diethylene glycol（DEG） or water）. Transmission electron microscope（TEM） results show that Gd（OH）3 nanorods change to a rice shape after adding the Na3 Cit, proving that Na3 Cit can change the morphology of Gd（OH）3. By changing the reactants from Na3 Cit to PEI, the morphology of Gd（OH）3 nanorods did not change, but the size of the Gd（OH）3 nanorods became shorter and wider. In addition, we further explored the synthesis, characterization, and photothermal property of Gd（OH）3@Au-PEI nanorod with Au stars on the surface of Gd（OH）3 nanorods.