超支化大分子修飾的磁性納米顆粒平臺的制備及其磁共振成像診斷應用
發(fā)布時間：2018-08-12 16:01
【摘要】：癌癥已經(jīng)嚴重威脅人類健康,而且有呈年輕化、常態(tài)化發(fā)展趨勢。隨著分子影像學技術和納米技術的迅猛發(fā)展,基于各種納米材料的造影劑得到了廣大科研工作者的關注。如何將分子影像學與納米技術更加完美的結合,已經(jīng)成為提高腫瘤的早期診斷靈敏度和精確性的關鍵。目前,臨床已有多種用于癌癥診斷技術,如:Ultrasound(US)、Computed Tomography(CT)、Magnetic Resonance Imaging(MRI)、Positron Emission Tomography(PET)和Single-Photon Emission Computed Tomography(SPECT)。以上技術均有其優(yōu)點和不足之處。如CT:價格相對低廉、掃描時間短、較高的空間分辨率,但是也存在不足之處:軟組織分辨率低、輻射損傷、呼吸運動偽影;MRI技術的費用高、掃描時間長;PET和SPECT成像可獲得腫瘤部位的生理生化信息,費用高昂。為了提高正常組織和腫瘤部位的對比度,有必要使用造影劑增強對比度。目前,已有商業(yè)化造影劑應用于臨床診斷,如用于MR成像的Gd-DTPA和CT成像的Omnipaque等。然而,這些造影劑都存在不足之處:分子量小、血液循環(huán)時間短、成像效果較差、具有毒副作用、無組織特異性等。與傳統(tǒng)的小分子造影劑相比較,納米材料具有更長的血液循環(huán)時間、表面可功能化等優(yōu)勢。因此,將納米材料與分子影像學技術相結合、將不同的成像技術相融合,從而構建出更高效、安全的分子探針。本論文借助于超支化大分子(g5.nh2或pei.nh2)其自身獨特的物理化學結構做了以下研究工作。一、超小氧化鐵納米顆粒的表面修飾及其靶向腫瘤t2、t1mr成像應用。二、樹狀大分子協(xié)同效應對錳基、釓基造影劑的r1弛豫率和t1加權mr成像性能的影響。三、放射核素锝(99mtc)標記的錳基、釓基螯合物@fe3o4及其腫瘤的spect/mr以及spect/增強mr雙模態(tài)診斷應用。四、聚乙烯亞胺穩(wěn)定的mn3o4納米顆粒的制備和功能化修飾及其腫瘤的靶向mr成像。采用系列技術對制備得到的納米材料進行了綜合表征,并不斷完善設計方案。一方面,在增強納米顆粒的弛豫率和成像性能基礎上,提高納米顆粒的生物相容性,將結構成像與功能成像相結合。另一方面,積極探索可代替樹狀大分子的聚合物,降低成本,推動產(chǎn)業(yè)化。通過系統(tǒng)研究此類造影劑的生物相容性、體外細胞成像以及體內(nèi)動物成像性能,為開發(fā)新型功能化分子影像學造影劑提供新思路、新方法。本論文的主要研究內(nèi)容如下:1)相比于錳、釓氧化物,fe3o4納米顆粒具有更高的生物安全性。我們采用一步溶劑熱法制備表面檸檬酸穩(wěn)定的水溶性超小氧化鐵納米顆粒(平均直徑約為2.84nm),將其組裝到偶聯(lián)有靶向分子環(huán)狀多肽rgd的第五代樹狀大分子(g5.nh2)上,得到具有團簇結構的納米顆粒,并應用于t2加權mr成像。mtt結果顯示納米顆粒在實驗濃度下具有優(yōu)異的細胞相容性。此外icp-aes測試結果表明偶聯(lián)rgd多肽后,增加了c6細胞對納米顆粒的攝入。體外細胞成像和體內(nèi)動物成像結果也表明修飾rgd后的納米探針對avb3整合素高表達細胞株系c6細胞及其皮下移植瘤模型具有良好的靶向t2加權mr成像效果。2)除釓、錳造影劑之外,超小氧化鐵也可實現(xiàn)t1加權mr成像。前面我們利用g5.nh2得到的團簇結構的納米顆粒,顯示只有t2加權mr成像效應。在此基礎上我們改進了設計方案,將一端氨基、一端偶聯(lián)有環(huán)狀多肽rgd的peg修飾到fe3o4表面,得到單分散性良好的fe3o4納米顆粒。其r1弛豫率為1.39mm-1s-1,r2弛豫率為2.79mm-1s-1(r2:r1=2.01),適合作為t1加權mr成像造影劑。體內(nèi)、體外實驗結果表明以peg為橋梁將fe3o4和rgd連接制備得到的納米顆粒,對avb3整合素高表達細胞株系u87mg腫瘤細胞和皮下移植瘤腫瘤模型具有靶向t1加權mr成像性能。3)為了研究樹狀大分子的協(xié)同效應,我們合成了一系列錳基、釓基螯合物。并分析表面螯合試劑修飾的數(shù)量、乙�；约鞍{米金顆粒的量與r1弛豫率之間的關系。結果顯示:釓基、錳基螯合物弛豫率均隨dota修飾量增加而升高。在dota:g5.nh2=30時釓r1弛豫率為7.69mm-1s-1,乙�；鬄�9.77mm-1s-1;錳基螯合物r1弛豫率為2.54mm-1s-1,乙�；蠼档偷�1.26mm-1s-1。我們選擇r1弛豫率最佳的釓基螯合物作為模板,包裹不同比例的納米金顆粒,得到一系列造影劑。結果顯示,隨著金含量增加,r1弛豫率從13.12mm-1s-1下降到7.50mm-1s-1。以上結果表明,釓基或者錳基螯合物r1弛豫率與單位g5.nh2上修飾的dota數(shù)量成正比例關系,且釓基螯合物mr成像性能優(yōu)于錳基螯合物;利用樹狀大分子獨特的性質(zhì)控制釓元素和金元素的比例,可以優(yōu)化基于樹狀大分子的ct/mr雙模態(tài)納米探針成像性能。4)ct和mr均屬于結構成像,僅能得到腫瘤部位解剖學信息。因此,我們設想以g5.nh2為平臺,以無腎毒性的錳基螯合物作為mr成像元素,利用dota可標記放射核素99mtc這一特性,將結構成像和功能成像相結合,得到具有spect/mr雙模態(tài)成像性能的分子探針。結果顯示,錳基螯合物的r1弛豫率為3.2mm-1s-1。修飾葉酸(folicacid,fa)后對葉酸受體高表達細胞株系hela細胞及其皮下移植瘤腫瘤模型有明顯的靶向成像效果。通過測試mn元素和放射核素99mtc在小鼠體內(nèi)各臟器的分布。結果顯示,我們制備得到的納米探針能被小鼠代謝出體外。5)前面我們成功地制備得到了具有t1成像性能的超小fe3o4納米顆粒,但是r1弛豫率較低。另外,單一的mr成像模式很難滿足對腫瘤早期診斷的靈敏性和精確度。因此,我們設想以g5.nh2為載體,利用聚乙二醇化和部分乙酰化技術解決團簇效應和膠體穩(wěn)定性,裝載雙mr成像元素,實現(xiàn)協(xié)同提高復合納米顆粒的r1弛豫率。此外標記99mtc,得到多功能的造影劑。鐵、釓元素摩爾比最終確定為10.4:1。弛豫率結果顯示引入適量釓元素后,復合納米顆粒r1弛豫率從1.39mm-1s-1提高到4.31mm-1s-1,提高了210%。mtt和相差顯微鏡結果顯示制備得到的復合納米顆粒具有良好的細胞相容性。ICP-AES、普魯士藍染色結果表明,偶聯(lián)RGD多肽后,增加了U87MG細胞對復合納米顆粒的吞噬。同時,體外細胞成像和體內(nèi)動物成像結果也表明,偶聯(lián)RGD多肽可實現(xiàn)對avb3整合素高表達細胞株系U87MG細胞及其皮下移植瘤腫瘤模型的靶向SPECT/MR成像。6)樹狀大分子成本較高,難以產(chǎn)業(yè)化。聚乙烯亞胺因價格低廉易得、易功能化修飾是理想的替代品�；谖覀冋n題組前期工作,我們采用溶劑熱法制備得到聚乙烯亞胺穩(wěn)定的Mn_3O_4納米顆粒,功能化后應用于體內(nèi)外MR成像。XRD和TEM結果分別表明制備得到的納米顆粒為標準的Mn_3O_4晶體結構。直徑分布均勻,呈準球形,其平均直徑約為8.0 nm。多功能化的Mn_3O_4納米顆粒r1弛豫率為0.59mM-1s-1;在水、PBS、含血清培養(yǎng)基中均能穩(wěn)定分散,具有良好的膠體穩(wěn)定性、細胞相容性和優(yōu)異的血液相容性。同時,體外細胞成像和體內(nèi)動物成像結果表明,修飾FA分子后,對于葉酸受體高表達細胞株系KB細胞及其皮下移植瘤腫瘤模型有顯著的靶向成像效果。
[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.
【學位授予單位】：東華大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：TB383.1;TQ421.7
，
本文編號：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.
【學位授予單位】：東華大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：TB383.1;TQ421.7
，
本文編號：2179556
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