The Fabrication Of Hyperbranched Macromolecules Modified Magnetic Nanoparticle Platform For Magnetic Resonance Imaging Diagnosis Application

Cancer is a serious threat to human health, and has a younger, normalized development trend. With the fast advances of the molecular imaging and nanotechnology, various nanomaterial-based molecular imaging contrast agents have been attracted more and more attention of scientists. How to make the molecular imaging and nanotechnology merged perfectly to enhance the sensitivity and accuracy is one of the most important key factor and proposition. At present, there are many clinical detection technology has been used in the diagnosis of cancer. Such as: Ultrasonic imaging（US）, Computed Tomography（CT）, Magnetic Resonance Imaging（MRI）, Positron Emission Tomography（PET） or Single-Photon Emission Computed Tomography（SPECT） imaging. However, each imaging mode has its own advantages and disadvantages. For instance, CT imaging with its relatively cheap price, short scanning time, high spatial resolution. But still has some limitation such as low soft-tissue resolution, radiation damage, and respiratory motion artifacts. While, MR imaging with advantages such as high soft-tissue resolution, has some drawbacks like high cost and long scanning time. PET and SPECT imaging as the potential to obtain the physiological and biochemical information of the tumor. But compared with CT or MR imaging, the cost of PET or SPECT is much higher. In order to enhance the contrast of the normal tissue and tumor, various contrast agents have been developed for clinical diagnosis of cancer. Such as: Gd-DTPA for MR imaging and Omnipaque for CT imaging. However, there are many drawbacks for these clinical contrast agents, low molecular weight, short vascular circulation, low imaging sensitivity, side effects and low tissue specificity and so on. Compared with the traditional small molecular contrast agents, nanomaterials possess the advantages such as longer blood circulation time, easy surface modifications to endow the activity targeted ability. Therefore, combined nanomaterials and molecular imaging technology, integrated the different imaging modes to construct more imaging efficiency, biocompatibility, and the perfect combination of structure imaging and the function imaging mode molecular probes are imminent.In this work, hyperbranched polymers generation five Poly（amidoamine）（PAMAM） dendrimers（G5.NH2） or polyethylene imine（PEI.NH2） were employed as platform to form a series contrast agents due to their unique prosperities. Such as highly branch, large number of amine groups, accurate molecular structure, and the internal cavity structure. First, the surface modification of iron oxide NPs contrast agents applied for T2-weighted MR, T1-weighted MR imaging of tumors. Second, the synergistic effect of Mn-based or Gd-based contrast r1 relaxation and the influence of T1-weighted MR imaging performance due to the unique structure of dendrimer. Third, radioactive nuclide ^99mTc-labeled Mn-based or Gd-based@Fe₃O₄ contrast agents applied for targeting SPECT/T1-weighted MR or SPECT/T1 enhanced MR imaging of tumors. Finally, the functional modification of PEI stabled manganese oxide nanoparticles applied for targeting MR imaging of tumors. The formed hybrid NPs were characterized by different techniques. On the one hand, the biomedical application potential of the formed hybrid NPs were improved by keep improving the design strategy. On the other hand, we actively explore other polymer to instead of the dendrimer to reduce the costs, and promote it for industrial production. After system investigated the biocompatibility, in vitro cell imaging and in vivo animal imaging of these contrast agents were studied which shows their potential for clinical multifunctional molecular imaging applications. The main contents and results of this work as fellow:1) Compared with manganese oxide and gadolinium oxide, iron oxide possess better biocompatibility. We adopt one pot solvothermal route to prepare sodium citrate coated and water-soluble ultrasmall iron oxide NPs（with the mean diameter of 2.84 nm）. Fe₃O₄ NPs were used to assembly onto the RGD-targeted G5.NH2 dendrimers to form the cluster structure NPs and applied for T2-weighted MR imaging. MTT results suggests that the developed Fe₃O₄ NPs exhibit remarkably low cytotoxicity over the given Fe concentration upto 100 mg/m L. ICP-AES was used to study the specificity cellular uptake of the RGD-targeted particles by C6 cells. The results show that the enhanced uptake could be due to the high affinity of Fe₃O₄ NPs for avb3 receptors, which are overexpressed on the surfaces of C6 cells. In vitro cell imaging and in vivo animal imaging results suggests that in addition to the passive enhanced permeability and retention（EPR） effect, the RGD mediated targeting pathway plays an important role in the uptake of Fe₃O₄ NPs. It is also indicated that after modified with RGD the Fe₃O₄ NPs have good potential for use as a tumor-targeted negative contrast agent for T2 MR imaging.2) Besides the Gdor Mn-based contrast agents, ultrasmall iron oxide NPs can also achieve the T1-weighted MR imaging. But in our previous work, dendrimer were used as the template to form the cluster structure NPs, the results show that the formed NPs just possess T2-weighted MR imaging performance. Based on this work, we have mended the experiment scheme. In our approach, NH2-PEG-RGD were used as bridge to conjugate with sodium citrate stabilized Fe₃O₄ NPs to form the monodispersed Fe₃O₄ NPs. The r1 and r2 relaxivity of the Fe₃O₄ NPs were measured to be 1.39 m M-1s-1 and 2.79 m M-1s-1, respectively（r2: r1= 2.01）. The T1-weighted MR imaging results of the Fe₃O₄ NPs clear show that Fe₃O₄ NPs display increased MR signal intensity with the Fe concentration. In vitro cell imaging and in vivo animal imaging results suggests that the Fe₃O₄ NPs can be used as a nanoprobe for targeted T1-weighted positive MR imaging of overexpressing avb3 integrin U87 MG cells or tumors in vitro or in vivo thanks to the RGD-mediated active targeting pathway.3) In order to investigate the synergies generated by the unique structureof dendrimer, we synthesized a series of Mn-based, Gd-based chelates. Subsequently, we study the relationship between the number of the chelates onto the G5 dendrimer, acetylation and the amout of gold. The r1 relaxivity results shown that the r1 relaxivity of Gd based contrast agents was increased with the number of DOTA. When the ratio of G5:DOTA upto 1:30, the r1 relaxivity of the Gd based NPs arrived to 7.69 m M-1s-1 and changed to 9.77 m M-1s-1 after the acetylated reaction. The r1 relaxivity of the DOTA（Mn） arrived to 2.54 m M-1s-1 with increased the number of DOTA-Mn from 5 to 30 per G5.NH2. After acetylation, the r1 relaxivity of the DOTA-Mn obviously decreased to 1.26 m M-1s-1. We chose the highest Gd based chelates as the template to entrapped gold nanoparticles to form the CT/MR dual mode contrast agents. The r1 relaxivity test results shows that with increased the amount of gold atom per G5.NH2, the r1 relaxivity decreased from 13.11 to 7.50 m M-1s-1. This due to that more gold nanoparticles entrapped into the interior of G5.NH2 the primary cavity structure changed and the G5.NH2 molecule became tight leads to the water exchange rate between the inner and external slow. These results indicated that with the appropriate tuning of the number of ion per G5 and Gd/Au composition, the formed Gd based or Gd/Au NPs may be able to be applied for dual mode MR/CT imaging and diagnosis of a particular disease（e.g., cancer） with high accuracy.4) CT and MR imaging both belong to structure imaging which only get the anatomic information of tumor. Therefore, we hypothesis that use the G5.NH2 as the template to conjugate the no renal toxicity Mn（II） based chelates and combined the functional imaging elements 99 mTc to construct SPECT/MR dual mode nanoprobes. The r1 relaxivity test results shows that when increased the number of DOTA to 35, the r1 relaxivity of the nanoprobe arrived to 3.2 m M-1s-1. After modified the folic acid（FA） the formed multifunctional nanoprobes can well perform the targeting SPECT/MR dual mode imaging of the FA receptor overexpression cell line（He La cell） and xenografted tumors. The biodistribution of Mn and 99 mTc in the major organs of the mice were investigated by ICP-AES. The results indicated that the nanoprobe can be excreted by the liver and kidney metabolic pathways.5) Although we successful get the Fe₃O₄ based T1-weighted MR imaging contrast agents. But compared with the clinical using of contrast agent, the r1 relaxivity of the Fe₃O₄ NPs is lower than Gd-DTPA（ ≈ 4 m M-1s-1）. Therefore, in order to improve the clinical use value of the Fe₃O₄ based contrast agents. We hypothesis that use the G5 dendrimer as a carrier to load two MR imaging elements, combine the PEGylation and partially acetylated technique to synergetic enhance the r1 relaxivity and T1-weighted MR imaging performance. In addition, we also hope that combine the structure imaging with functional imaging to form the multifunction contrast agents. We fabricated the G5 based Gd@Fe₃O₄ NPs with tunable the Fe:Gd molar ratio as 10.4:1. The r1 relaxivity test results indicated that after combined with Gd-DOTA, the r1 relaxivity of the Fe₃O₄ NPs increased from 1.39 m M-1s-1 to 4.31 m M-1s-1（210%）. The MTT results and cell morphology observation display that the G5 based Gd@Fe₃O₄ NPs have good cytocompatibility in the studied concentration range. The cellular uptake of G5 based Gd@Fe₃O₄ NPs for U87 MG cells was investigated by ICP-AES and prussian blue staining. The results show that the enhanced uptake of RGD-targeted G5 based Gd@Fe₃O₄ NPs could be due to the high affinity of the RGD for avb3 receptors, which are overexpressed on the surfaces of U87 MG cells. Meanwhile, In vitro cell imaging and in vivo animal imaging results suggests that labeled with 99 mTc and conjugated with RGD endow the G5 based Gd@Fe₃O₄ NPs possess the ability of targeted SPECT/MR dual mode imaging of the overexpressing avb3 integrin U87 MG cells or tumors.6) Because of the higher cost of dendrimer, it is difficult to commericialistion. PEI with lower costs and easy to modify then it is the ideal polymer to instead of dendrimer. In our previuous work, we have prepared the PEI stabilized Fe₃O₄ NPs. Based on our previous work, we adopt the similar pathway to fabricate the stable PEI-coated Mn₃O₄ NPs by a solvothermal route. And sequentially functionalized to form the multifunctional Mn₃O₄ NPs and applied for in vitro and in vivo MR imaging. The XRD and TEM results shown that the crystal form of the formed manganese oxide is the standard Mn₃O₄ crystal texture and both particles possess a spherical or quasi-spherical shape with a quite uniform size distribution. The mean diameter of the multifunctional FA-targeted Mn₃O₄ NPs were measured to be nearly 8.0 nm. The r1 relaxivity of the FA-targeted Mn₃O₄ NPs was measured to be 0.590 m M-1s-1. The multifunctional Mn₃O₄ NPs are stable in water, PBS, and cell culture medium and no sedimentation occurs for at least two weeks, further demonstrating the stability of the particles. The MTT results and hemolysis assay indicate that the multifunctional Mn₃O₄ NPs have good cytocompatibility and hemocompatibility in the studied concentration range. The in vitro and in vivo results indicated that with the targeting specificity to FAR-overexpressing cancer cells rendered by the modification of PEGylated FA, the formed multifunctional Mn₃O₄ NPs were able to be used as a nanoprobe for efficient specific MR imaging of cancer cells in vitro and a xenografted tumor model in vivo.