超支化大分子修饰的磁性纳米颗粒平台的制备及其磁共振成像诊断应用
发布时间：2018-08-12 16:01
【摘要】：癌症已经严重威胁人类健康,而且有呈年轻化、常态化发展趋势。随着分子影像学技术和纳米技术的迅猛发展,基于各种纳米材料的造影剂得到了广大科研工作者的关注。如何将分子影像学与纳米技术更加完美的结合,已经成为提高肿瘤的早期诊断灵敏度和精确性的关键。目前,临床已有多种用于癌症诊断技术,如:Ultrasound(US)、Computed Tomography(CT)、Magnetic Resonance Imaging(MRI)、Positron Emission Tomography(PET)和Single-Photon Emission Computed Tomography(SPECT)。以上技术均有其优点和不足之处。如CT:价格相对低廉、扫描时间短、较高的空间分辨率,但是也存在不足之处:软组织分辨率低、辐射损伤、呼吸运动伪影;MRI技术的费用高、扫描时间长;PET和SPECT成像可获得肿瘤部位的生理生化信息,费用高昂。为了提高正常组织和肿瘤部位的对比度,有必要使用造影剂增强对比度。目前,已有商业化造影剂应用于临床诊断,如用于MR成像的Gd-DTPA和CT成像的Omnipaque等。然而,这些造影剂都存在不足之处:分子量小、血液循环时间短、成像效果较差、具有毒副作用、无组织特异性等。与传统的小分子造影剂相比较,纳米材料具有更长的血液循环时间、表面可功能化等优势。因此,将纳米材料与分子影像学技术相结合、将不同的成像技术相融合,从而构建出更高效、安全的分子探针。本论文借助于超支化大分子(g5.nh2或pei.nh2)其自身独特的物理化学结构做了以下研究工作。一、超小氧化铁纳米颗粒的表面修饰及其靶向肿瘤t2、t1mr成像应用。二、树状大分子协同效应对锰基、钆基造影剂的r1弛豫率和t1加权mr成像性能的影响。三、放射核素锝(99mtc)标记的锰基、钆基螯合物@fe3o4及其肿瘤的spect/mr以及spect/增强mr双模态诊断应用。四、聚乙烯亚胺稳定的mn3o4纳米颗粒的制备和功能化修饰及其肿瘤的靶向mr成像。采用系列技术对制备得到的纳米材料进行了综合表征,并不断完善设计方案。一方面,在增强纳米颗粒的弛豫率和成像性能基础上,提高纳米颗粒的生物相容性,将结构成像与功能成像相结合。另一方面,积极探索可代替树状大分子的聚合物,降低成本,推动产业化。通过系统研究此类造影剂的生物相容性、体外细胞成像以及体内动物成像性能,为开发新型功能化分子影像学造影剂提供新思路、新方法。本论文的主要研究内容如下:1)相比于锰、钆氧化物,fe3o4纳米颗粒具有更高的生物安全性。我们采用一步溶剂热法制备表面柠檬酸稳定的水溶性超小氧化铁纳米颗粒(平均直径约为2.84nm),将其组装到偶联有靶向分子环状多肽rgd的第五代树状大分子(g5.nh2)上,得到具有团簇结构的纳米颗粒,并应用于t2加权mr成像。mtt结果显示纳米颗粒在实验浓度下具有优异的细胞相容性。此外icp-aes测试结果表明偶联rgd多肽后,增加了c6细胞对纳米颗粒的摄入。体外细胞成像和体内动物成像结果也表明修饰rgd后的纳米探针对avb3整合素高表达细胞株系c6细胞及其皮下移植瘤模型具有良好的靶向t2加权mr成像效果。2)除钆、锰造影剂之外,超小氧化铁也可实现t1加权mr成像。前面我们利用g5.nh2得到的团簇结构的纳米颗粒,显示只有t2加权mr成像效应。在此基础上我们改进了设计方案,将一端氨基、一端偶联有环状多肽rgd的peg修饰到fe3o4表面,得到单分散性良好的fe3o4纳米颗粒。其r1弛豫率为1.39mm-1s-1,r2弛豫率为2.79mm-1s-1(r2:r1=2.01),适合作为t1加权mr成像造影剂。体内、体外实验结果表明以peg为桥梁将fe3o4和rgd连接制备得到的纳米颗粒,对avb3整合素高表达细胞株系u87mg肿瘤细胞和皮下移植瘤肿瘤模型具有靶向t1加权mr成像性能。3)为了研究树状大分子的协同效应,我们合成了一系列锰基、钆基螯合物。并分析表面螯合试剂修饰的数量、乙酰化以及包裹纳米金颗粒的量与r1弛豫率之间的关系。结果显示:钆基、锰基螯合物弛豫率均随dota修饰量增加而升高。在dota:g5.nh2=30时钆r1弛豫率为7.69mm-1s-1,乙酰化后为9.77mm-1s-1;锰基螯合物r1弛豫率为2.54mm-1s-1,乙酰化后降低到1.26mm-1s-1。我们选择r1弛豫率最佳的钆基螯合物作为模板,包裹不同比例的纳米金颗粒,得到一系列造影剂。结果显示,随着金含量增加,r1弛豫率从13.12mm-1s-1下降到7.50mm-1s-1。以上结果表明,钆基或者锰基螯合物r1弛豫率与单位g5.nh2上修饰的dota数量成正比例关系,且钆基螯合物mr成像性能优于锰基螯合物;利用树状大分子独特的性质控制钆元素和金元素的比例,可以优化基于树状大分子的ct/mr双模态纳米探针成像性能。4)ct和mr均属于结构成像,仅能得到肿瘤部位解剖学信息。因此,我们设想以g5.nh2为平台,以无肾毒性的锰基螯合物作为mr成像元素,利用dota可标记放射核素99mtc这一特性,将结构成像和功能成像相结合,得到具有spect/mr双模态成像性能的分子探针。结果显示,锰基螯合物的r1弛豫率为3.2mm-1s-1。修饰叶酸(folicacid,fa)后对叶酸受体高表达细胞株系hela细胞及其皮下移植瘤肿瘤模型有明显的靶向成像效果。通过测试mn元素和放射核素99mtc在小鼠体内各脏器的分布。结果显示,我们制备得到的纳米探针能被小鼠代谢出体外。5)前面我们成功地制备得到了具有t1成像性能的超小fe3o4纳米颗粒,但是r1弛豫率较低。另外,单一的mr成像模式很难满足对肿瘤早期诊断的灵敏性和精确度。因此,我们设想以g5.nh2为载体,利用聚乙二醇化和部分乙酰化技术解决团簇效应和胶体稳定性,装载双mr成像元素,实现协同提高复合纳米颗粒的r1弛豫率。此外标记99mtc,得到多功能的造影剂。铁、钆元素摩尔比最终确定为10.4:1。弛豫率结果显示引入适量钆元素后,复合纳米颗粒r1弛豫率从1.39mm-1s-1提高到4.31mm-1s-1,提高了210%。mtt和相差显微镜结果显示制备得到的复合纳米颗粒具有良好的细胞相容性。ICP-AES、普鲁士蓝染色结果表明,偶联RGD多肽后,增加了U87MG细胞对复合纳米颗粒的吞噬。同时,体外细胞成像和体内动物成像结果也表明,偶联RGD多肽可实现对avb3整合素高表达细胞株系U87MG细胞及其皮下移植瘤肿瘤模型的靶向SPECT/MR成像。6)树状大分子成本较高,难以产业化。聚乙烯亚胺因价格低廉易得、易功能化修饰是理想的替代品。基于我们课题组前期工作,我们采用溶剂热法制备得到聚乙烯亚胺稳定的Mn_3O_4纳米颗粒,功能化后应用于体内外MR成像。XRD和TEM结果分别表明制备得到的纳米颗粒为标准的Mn_3O_4晶体结构。直径分布均匀,呈准球形,其平均直径约为8.0 nm。多功能化的Mn_3O_4纳米颗粒r1弛豫率为0.59mM-1s-1;在水、PBS、含血清培养基中均能稳定分散,具有良好的胶体稳定性、细胞相容性和优异的血液相容性。同时,体外细胞成像和体内动物成像结果表明,修饰FA分子后,对于叶酸受体高表达细胞株系KB细胞及其皮下移植瘤肿瘤模型有显著的靶向成像效果。
[Abstract]:With the rapid development of molecular imaging and nanotechnology, contrast agents based on various nanomaterials have attracted the attention of researchers. How to combine molecular imaging and nanotechnology more perfectly has become an important way to improve swelling. At present, there are many clinical diagnostic techniques for cancer, such as Ultrasound (US), Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography (SPECT). For example, CT: relatively inexpensive, short scan time, high spatial resolution, but there are also shortcomings: low soft tissue resolution, radiation damage, respiratory motion artifacts; MRI technology is expensive, long scan time; PET and SPECT imaging can obtain physiological and biochemical information of tumor site, high cost. At present, commercial contrast agents have been used in clinical diagnosis, such as Gd-DTPA for MR imaging and Omnipaque for CT imaging. However, these contrast agents have some shortcomings: small molecular weight, short blood circulation time, poor imaging effect, toxic side effects and no tissue specificity. Compared with the traditional small molecule contrast agents, nano-materials have the advantages of longer blood circulation time and functionalization of the surface. Therefore, the combination of nano-materials and molecular imaging technology will be different imaging technology, so as to build a more efficient and safe molecular probe. In this paper, with the help of hyperbranched macromolecule (g5.nh2) Or pei. nh2) has its own unique physical and chemical structure to do the following research work. first, surface modification of ultrasmall iron oxide nanoparticles and their targeting tumor t2, T1 MR imaging applications. second, the synergistic effects of dendrimers on the R1 relaxation rate and T1-weighted MR imaging properties of manganese-based, gadolinium-based contrast agents. third, radionuclide technetium (99mtc) labeled manganese Fourthly, the preparation and functional modification of polyethylenimine-stabilized mn_3o_4 nanoparticles and their tumor-targeting magnetic resonance imaging. A series of techniques were used to synthetically characterize the prepared nanomaterials and constantly improve the design scheme. On the basis of enhancing the relaxation rate and imaging performance of nanoparticles, the biocompatibility of nanoparticles is improved by combining structural imaging with functional imaging. On the other hand, we actively explore polymers that can replace dendrimers to reduce costs and promote industrialization. The main contents of this paper are as follows: 1) Compared with manganese and gadolinium oxides, fe_3o_4 nanoparticles have higher bio-safety. We prepared water-soluble ultrasmall oxidation of citric acid on the surface by one-step solvothermal method. Iron nanoparticles with an average diameter of about 2.84 nm were assembled onto the fifth generation dendrimers (g5.nh2) coupled with a cyclic polypeptide RGD to obtain clusters of nanoparticles and applied to T2-weighted MR imaging. MTT results showed that the nanoparticles exhibited excellent cytocompatibility at experimental concentrations. in addition, ICP-AES test knot The results showed that the uptake of nanoparticles by C6 cells was increased after coupling with RGD peptides. Cell imaging in vitro and animal imaging in vivo also showed that the modified RGD nanoprobe had a good targeting effect on avb3 integrin overexpression cell line C6 cells and its subcutaneous transplanted tumor model. 2) Except for gadolinium and manganese contrast agent, super-weighted T2 Mr imaging was performed. Small ferric oxide can also be used to achieve T1-weighted magnetic resonance imaging.Previously, clusters of nanoparticles with g5.nh2 structure showed only T2-weighted magnetic resonance imaging effect.On this basis, we improved the design scheme, modifying peg with amino group at one end and cyclic polypeptide RGD at the other end to the surface of fe_3o_4 to obtain well-dispersed fe_3o_4 nanoparticles. R1 relaxation rate was 1.39 mm-1 s-1, R2 relaxation rate was 2.79 mm-1 S-1 (r2:r1 = 2.01), which was suitable for T1-weighted MR imaging contrast agent. In vivo, in vitro experiments showed that the nanoparticles prepared by connecting fe_3o_4 and RGD with PEG as a bridge could target T1 in U87 mg tumor cells and subcutaneous transplanted tumor models with high expression of avb_3 integrin. 3) In order to study the synergistic effect of dendrimers, we synthesized a series of manganese and gadolinium chelates, and analyzed the relationship between the number of chelating agents, acetylation, and the amount of encapsulated gold nanoparticles and the R1 relaxation rate. When dota:g5.nh2=30, the relaxation rate of gadolinium R1 is 7.69mm-1s-1, and the acetylation rate is 9.77mm-1s-1; the relaxation rate of manganese chelate R1 is 2.54mm-1s-1, and the acetylation rate is reduced to 1.26mm-1s-1. With the increase of gold content, the relaxation rate of R1 decreased from 13.12 mm-1 S-1 to 7.50 mm-1 s-1. the results showed that the relaxation rate of gadolinium or manganese chelate R1 was proportional to the number of DOTA modified on unit g5.nh2, and the imaging performance of gadolinium-based chelate was better than that of manganese-based chelate. The ratio can optimize the imaging performance of dual-mode CT/MR nanoprobe based on dendrimers. 4) Both CT and MR are structural imaging, and only the anatomical information of tumor site can be obtained. The results showed that the R1 relaxation rate of manganese-based chelate was 3.2 mm-1 s-1. Modified folic acid (fa) had obvious targeting effect on HeLa cells with high folic acid receptor expression and their subcutaneous transplanted tumor models. The distribution of Mn and radionuclide 99mTc in the organs of mice was tested. The results showed that the nanoprobe prepared by us could be metabolized out of the body by mice. 5) We successfully prepared ultrasmall fe_3o_4 nanoparticles with T1 imaging properties, but the R1 relaxation rate was low. In addition, single MR imaging mode was difficult to meet the needs of tumor imaging. Therefore, we envisage using g5.nh2 as the carrier, polyethylene glycol and partial acetylation technology to solve the cluster effect and colloid stability, loaded with dual MR imaging elements, to achieve synergistic enhancement of the R1 relaxation rate of the composite nanoparticles. in addition, 99mTc was labeled to obtain a multifunctional contrast agent. mole of iron and gadolinium elements The results showed that the relaxation rate of the composite nanoparticles increased from 1.39mm-1s-1 to 4.31mm-1s-1 by 210%. The results of MTT and phase contrast microscopy showed that the prepared nanoparticles had good cell compatibility. ICP-AES and Prussian blue staining showed that the coupling RGD was more than that of the composite nanoparticles. At the same time, in vitro cell imaging and in vivo animal imaging results also showed that the conjugated RGD peptides could achieve targeted SPECT/MR imaging of U87MG cells and their subcutaneous tumor models with avb3 integrin overexpression. 6) Dendrimers were expensive and difficult to industrialize. Ethylenimine is an ideal substitute for functional modification because of its low cost. Based on our previous work, we prepared polyethylenimine-stabilized Mn_3O_4 nanoparticles by solvothermal method, and applied them to MR imaging in vivo and in vitro. XRD and TEM results showed that the prepared nanoparticles were standard Mn_3O_4. 4 Crystal structure, uniform diameter distribution, quasi-spherical, with an average diameter of about 8.0 nm, multifunctional Mn_3O_4 nanoparticles R1 relaxation rate of 0.59 mM-1s-1; in water, PBS, serum-containing medium can be stably dispersed, with good colloid stability, cell compatibility and excellent blood compatibility. At the same time, in vitro cell imaging and in vivo animal. The imaging results showed that the modified FA molecule had a significant targeting effect on KB cells with high folate receptor expression and their subcutaneous tumor models.
【学位授予单位】：东华大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：TB383.1;TQ421.7
，
本文编号：2179556
[Abstract]:With the rapid development of molecular imaging and nanotechnology, contrast agents based on various nanomaterials have attracted the attention of researchers. How to combine molecular imaging and nanotechnology more perfectly has become an important way to improve swelling. At present, there are many clinical diagnostic techniques for cancer, such as Ultrasound (US), Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography (SPECT). For example, CT: relatively inexpensive, short scan time, high spatial resolution, but there are also shortcomings: low soft tissue resolution, radiation damage, respiratory motion artifacts; MRI technology is expensive, long scan time; PET and SPECT imaging can obtain physiological and biochemical information of tumor site, high cost. At present, commercial contrast agents have been used in clinical diagnosis, such as Gd-DTPA for MR imaging and Omnipaque for CT imaging. However, these contrast agents have some shortcomings: small molecular weight, short blood circulation time, poor imaging effect, toxic side effects and no tissue specificity. Compared with the traditional small molecule contrast agents, nano-materials have the advantages of longer blood circulation time and functionalization of the surface. Therefore, the combination of nano-materials and molecular imaging technology will be different imaging technology, so as to build a more efficient and safe molecular probe. In this paper, with the help of hyperbranched macromolecule (g5.nh2) Or pei. nh2) has its own unique physical and chemical structure to do the following research work. first, surface modification of ultrasmall iron oxide nanoparticles and their targeting tumor t2, T1 MR imaging applications. second, the synergistic effects of dendrimers on the R1 relaxation rate and T1-weighted MR imaging properties of manganese-based, gadolinium-based contrast agents. third, radionuclide technetium (99mtc) labeled manganese Fourthly, the preparation and functional modification of polyethylenimine-stabilized mn_3o_4 nanoparticles and their tumor-targeting magnetic resonance imaging. A series of techniques were used to synthetically characterize the prepared nanomaterials and constantly improve the design scheme. On the basis of enhancing the relaxation rate and imaging performance of nanoparticles, the biocompatibility of nanoparticles is improved by combining structural imaging with functional imaging. On the other hand, we actively explore polymers that can replace dendrimers to reduce costs and promote industrialization. The main contents of this paper are as follows: 1) Compared with manganese and gadolinium oxides, fe_3o_4 nanoparticles have higher bio-safety. We prepared water-soluble ultrasmall oxidation of citric acid on the surface by one-step solvothermal method. Iron nanoparticles with an average diameter of about 2.84 nm were assembled onto the fifth generation dendrimers (g5.nh2) coupled with a cyclic polypeptide RGD to obtain clusters of nanoparticles and applied to T2-weighted MR imaging. MTT results showed that the nanoparticles exhibited excellent cytocompatibility at experimental concentrations. in addition, ICP-AES test knot The results showed that the uptake of nanoparticles by C6 cells was increased after coupling with RGD peptides. Cell imaging in vitro and animal imaging in vivo also showed that the modified RGD nanoprobe had a good targeting effect on avb3 integrin overexpression cell line C6 cells and its subcutaneous transplanted tumor model. 2) Except for gadolinium and manganese contrast agent, super-weighted T2 Mr imaging was performed. Small ferric oxide can also be used to achieve T1-weighted magnetic resonance imaging.Previously, clusters of nanoparticles with g5.nh2 structure showed only T2-weighted magnetic resonance imaging effect.On this basis, we improved the design scheme, modifying peg with amino group at one end and cyclic polypeptide RGD at the other end to the surface of fe_3o_4 to obtain well-dispersed fe_3o_4 nanoparticles. R1 relaxation rate was 1.39 mm-1 s-1, R2 relaxation rate was 2.79 mm-1 S-1 (r2:r1 = 2.01), which was suitable for T1-weighted MR imaging contrast agent. In vivo, in vitro experiments showed that the nanoparticles prepared by connecting fe_3o_4 and RGD with PEG as a bridge could target T1 in U87 mg tumor cells and subcutaneous transplanted tumor models with high expression of avb_3 integrin. 3) In order to study the synergistic effect of dendrimers, we synthesized a series of manganese and gadolinium chelates, and analyzed the relationship between the number of chelating agents, acetylation, and the amount of encapsulated gold nanoparticles and the R1 relaxation rate. When dota:g5.nh2=30, the relaxation rate of gadolinium R1 is 7.69mm-1s-1, and the acetylation rate is 9.77mm-1s-1; the relaxation rate of manganese chelate R1 is 2.54mm-1s-1, and the acetylation rate is reduced to 1.26mm-1s-1. With the increase of gold content, the relaxation rate of R1 decreased from 13.12 mm-1 S-1 to 7.50 mm-1 s-1. the results showed that the relaxation rate of gadolinium or manganese chelate R1 was proportional to the number of DOTA modified on unit g5.nh2, and the imaging performance of gadolinium-based chelate was better than that of manganese-based chelate. The ratio can optimize the imaging performance of dual-mode CT/MR nanoprobe based on dendrimers. 4) Both CT and MR are structural imaging, and only the anatomical information of tumor site can be obtained. The results showed that the R1 relaxation rate of manganese-based chelate was 3.2 mm-1 s-1. Modified folic acid (fa) had obvious targeting effect on HeLa cells with high folic acid receptor expression and their subcutaneous transplanted tumor models. The distribution of Mn and radionuclide 99mTc in the organs of mice was tested. The results showed that the nanoprobe prepared by us could be metabolized out of the body by mice. 5) We successfully prepared ultrasmall fe_3o_4 nanoparticles with T1 imaging properties, but the R1 relaxation rate was low. In addition, single MR imaging mode was difficult to meet the needs of tumor imaging. Therefore, we envisage using g5.nh2 as the carrier, polyethylene glycol and partial acetylation technology to solve the cluster effect and colloid stability, loaded with dual MR imaging elements, to achieve synergistic enhancement of the R1 relaxation rate of the composite nanoparticles. in addition, 99mTc was labeled to obtain a multifunctional contrast agent. mole of iron and gadolinium elements The results showed that the relaxation rate of the composite nanoparticles increased from 1.39mm-1s-1 to 4.31mm-1s-1 by 210%. The results of MTT and phase contrast microscopy showed that the prepared nanoparticles had good cell compatibility. ICP-AES and Prussian blue staining showed that the coupling RGD was more than that of the composite nanoparticles. At the same time, in vitro cell imaging and in vivo animal imaging results also showed that the conjugated RGD peptides could achieve targeted SPECT/MR imaging of U87MG cells and their subcutaneous tumor models with avb3 integrin overexpression. 6) Dendrimers were expensive and difficult to industrialize. Ethylenimine is an ideal substitute for functional modification because of its low cost. Based on our previous work, we prepared polyethylenimine-stabilized Mn_3O_4 nanoparticles by solvothermal method, and applied them to MR imaging in vivo and in vitro. XRD and TEM results showed that the prepared nanoparticles were standard Mn_3O_4. 4 Crystal structure, uniform diameter distribution, quasi-spherical, with an average diameter of about 8.0 nm, multifunctional Mn_3O_4 nanoparticles R1 relaxation rate of 0.59 mM-1s-1; in water, PBS, serum-containing medium can be stably dispersed, with good colloid stability, cell compatibility and excellent blood compatibility. At the same time, in vitro cell imaging and in vivo animal. The imaging results showed that the modified FA molecule had a significant targeting effect on KB cells with high folate receptor expression and their subcutaneous tumor models.
【学位授予单位】：东华大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：TB383.1;TQ421.7
，
本文编号：2179556
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