树状大分子杂化的纳米平台的设计及其肿瘤诊疗应用

发布时间：2018-04-26 14:04

  本文选题：树状大分子 + 抗癌药物　； 参考：《东华大学》2016年博士论文

【摘要】：在分子影像学技术及纳米医药学研究不断发展的今天,如何将这两种功能有机地结合起来实现“诊疗”一体化仍然是个巨大的挑战。目前医学造影剂及分子药物均存在许多缺点:造影剂的血液半衰期短、稳定性和生物相容性差,小分子化学药物的疏水性、对正常细胞及组织的毒副作用、对病灶区域无特异性等。因此,需要找到一个合适的纳米载体平台既可以将造影剂的诊断作用和药物的治疗作用有机地结合起来,又可以改善医学造影剂及化学药物的种种缺点,形成多功能的诊疗探针复合体用于癌症的早期诊断及治疗。基于树状大分子独特的理化性质,表面拥有众多的可修饰化功能基团,可用于药物的化学键合及各种功能分子的修饰,内部空腔可用于物理包裹分子药物及纳米金属颗粒。本文以树状大分子作为平台构建具有“诊疗”作用的功能杂化纳米颗粒,对其进行基本表征,研究表明形成的纳米诊疗体系不仅可以提高分子抗癌药物的水溶性、生物相容性还可以延长其在体内的血液循环时间,延长造影成像时间,与此同时在其表面上修饰靶向分子,可以增强其与癌细胞的特异性结合,以实现肿瘤的靶向成像及靶向治疗。因而,基于树状大分子构建的具有诊疗功能的纳米杂化颗粒将为探索新型的集肿瘤诊断与治疗一体化的多功能纳米平台开辟新的思路。研究内容如下所述:1)根据树状大分子独特的理化性质,以第五代聚酰胺胺树状大分子(G5.NH_2)作为纳米诊疗平台,通过表面修饰异硫氰酸荧光素(fluorescein isothiocyanate,FI),聚乙二醇(polyethylene glycol,peg)化的抗癌药物alpha-维生素e琥珀酸酯(a-tocopheryl succinate,α-tos)及peg化的靶向分子叶酸(folicacid,fa),之后在其内部包裹纳米金颗粒构建多功能化树状大分子包裹纳米金颗粒(au DENPs)用于靶向肿瘤ct成像及化学治疗。氢核磁共振(~1hnmr,proton nuclear magnetic resonance spectroscopy)测试结果表明,在形成的au DENPs中,每个树状大分子表面修饰有9.8个α-tos。透射电镜(transmission electron microscope,tem)测试得出形成的au DENPs平均直径约3.3nm,尺寸分布较窄,具有良好的单分散性,且在不同ph条件(ph=5,6,7,8),温度条件(4℃,37℃,50℃)和溶剂条件下具有良好的稳定性。流式细胞仪及激光共聚焦显微镜测试表明,fa的修饰可使形成的功能化au DENPs能与具有fa受体表达的癌细胞特异性结合,实现体外靶向癌细胞ct成像及体内靶向肿瘤ct成像的功能。α-tos的修饰使形成的功能化au DENPs具有体内体外靶向治疗功能,且体外及体内治疗效率比纯α-tos的治疗效率高。由于上述构建的fa靶向的,共价键合α-tos的功能化au DENPs具有比纯α-tos更强的体外癌细胞治疗作用,且为了深入探索其微观治疗机制及了解这一纳米诊疗平台是否适用于靶向其它类型的肿瘤系统,我们以多肽精氨酸-甘氨酸-天冬氨酸(arg-gly-asp,rgd)作为另一靶向分子修饰在α-tos共价键合的功能化au DENPs表面。研究表明形成的au DENPs同样具有较为均一的纳米尺寸及良好的体外稳定性。通过细胞内活性氧(ros)测试表明rgd靶向的,共价键合α-tos的功能化au DENPs能诱导癌细胞产生出比纯α-tos更多的活性氧。fitc标记的重组人annexinv/碘化丙啶(annexinv-fitc/pi)双染实验表明rgd靶向的,共价键合α-tos的功能化au DENPs比纯α-tos能诱导癌细胞产生出更多的早期凋亡及死亡的癌细胞。且经过rgd的修饰,形成的α-tos共价键合的au DENPs同样能够靶向高整合素αvβ3表达的恶性胶质瘤细胞且能够特异性的抑制其生长。形成的该靶向纳米体系比临床应用的基于碘的造影剂欧乃派克具有更高的x-射线衰减效应,且具有体外癌细胞靶向ct成像功能。2)以G5.NH_2为纳米平台,在其表面修饰钆离子螯合剂(2,2',2''-(10-(2-(2,5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-1,4,7,10-tetraaz acyclododecane-1,4,7-triyl)triaceticacid,dota-nhs),peg化的靶向试剂peg-fa,然后在该模板上螯合钆离子并将树状大分子表面的氨基乙酰化,之后利用该纳米平台将其内部包裹抗癌药物阿霉素(dox)形成纳米诊疗体系。研究表明形成的纳米诊疗体系中,每个树状大分子包裹了8.5个dox分子,该纳米体系在不同ph条件下具有良好稳定性,且能够持续缓释dox。fa的修饰使其能够有效的靶向fa受体表达的癌细胞,使其具有体外靶向癌细胞mr成像及化学治疗的作用。3)我们以部分乙酰化且修饰有fa的功能化树状大分子为纳米平台(g5.nhac-fa),以ph敏感顺式乌头酸酐作为连接分子将dox共价键合在其表面。再将形成的g5.nhac-fa-dox作为模板包裹纳米金颗粒形成新的纳米诊疗体系。形成的au DENPs中,每个树状大分子上共价键合了9个dox,形成的金纳米颗粒尺寸为2.76nm且在不同ph及温度条件下具有稳定性。且fa靶向的dox共价键合的au DENPs遵循酸引发药物缓释机制,在越低的ph条件下,dox的缓释速率越快。形成的该纳米诊疗材料除了具有一定的体外癌细胞治疗效果之外,fa的修饰使其能有效的靶向叶酸受体表达的癌细胞用于体外癌细胞ct成像。4)基于前期我们构建的基于树状大分子的纳米诊断及化学治疗体系,我们进一步构建载入放射性碘-131(~(131)I)的放射性化学诊疗体系。基于~(131)I的特性,可同时发射出g射线(7)(18)(21)(19)kev(11)(23)(16)(13)(22)(4)(8)用于单光子发射计算机断层(spect)成像及b射线(7)(15)(13)(21)(15)(21)(44)ev(11)(23)(24)(13)(24)(4)(8)用于放射性治疗,我们将G5.NH_2依次共价键合3-(4-羟基苯基)丙酸n-羟基琥珀酰亚胺酯(hpao)和fa修饰的的peg,之后将树状大分子表面剩余氨基乙酰化并标记放射性碘-131(~(131)I)以构建一个放射性化学诊疗体系。~1hnmr结果表明每个G5.NH_2树状大分子表面修饰有9.4个hpao,且在标记~(131)I之前,该功能化的树状大分子在浓度高达20mm时仍无生物毒性。且由于fa的修饰,该纳米诊疗载体能够靶向fa受体表达的癌细胞。放射性化学纯度测试表明形成的~(131)I标记的功能化树状大分子具有良好的稳定性,直至27h的放射性化学纯度高达92.84%。肿瘤生长曲线及存活率统计表明该~(131)I标记的功能化树状大分子具有良好的放射性治疗效率。体内spect成像表明形成的纳米材料能特异性靶向fa受体表达的肿瘤用于靶向spect成像。
[Abstract]:Today, with the development of molecular imaging technology and nanoscale medicine research, it is still a great challenge to combine the two functions organically to realize the integration of "diagnosis and treatment". There are many shortcomings in the medical contrast agents and molecular drugs: the short half of the blood in the blood of the contrast agent, the poor stability and biocompatibility, and the small molecules The hydrophobicity of chemical drugs, toxic and side effects to normal cells and tissues, and no specificity on the focus area. Therefore, it is necessary to find a suitable nanoscale platform to combine the diagnosis of contrast agents with the therapeutic effect of drugs, and to improve the shortcomings of the medical contrast agents and chemical drugs. The multi-functional diagnosis probe complex is used for the early diagnosis and treatment of cancer. Based on the unique physicochemical properties of tree like macromolecules, the surface has many modifiable functional groups, which can be used for chemical bonding of drugs and modification of various functional molecules. The internal cavity can be used in physical encapsulation of molecular drugs and nanoscale particles. As a platform, it constructs the functional hybrid nanoparticles, which has the role of "diagnosis and treatment" as a platform. The research shows that the formed nano diagnosis and treatment system can not only improve the water solubility of molecular anticancer drugs, but also prolong the time of blood circulation in the body and prolong the imaging time. The surface modification of target molecules can enhance the specific binding of the cancer cells to achieve the target imaging and target therapy of the tumor. Therefore, the nano hybrid particles based on the tree like macromolecules with diagnostic and therapeutic functions will open up a new idea for exploring a new type of multi-functional nano platform for tumor diagnosis and treatment. The contents are as follows: 1) according to the unique physicochemical properties of the tree like macromolecules, the fifth generation polyamines tree like macromolecule (G5.NH_2) is used as the nano diagnosis and treatment platform, and the anti-cancer drug alpha- vitamin E succinate (a-to) is modified by the surface of fluorescein isothiocyanate, FI, and polyethylene glycol (polyethylene glycol, PEG). Copheryl succinate, alpha -tos) and PEG targeted molecular folic acid (Folicacid, FA), after which nano gold particles are wrapped in it to construct multi-functional tree like macromolecule encapsulated gold nanoparticles (AU DENPs) for target tumor CT imaging and chemical therapy. It is shown that in the Au DENPs, the surface modification of each tree like macromolecule has 9.8 alpha -tos. transmission electron microscopy (transmission electron microscope, TEM) test that the average diameter of Au DENPs is about 3.3nm, the size distribution is narrow, it has good monodispersity, and in different pH conditions (ph=5,6,7,8), temperature conditions (4, 37, 50 C) and solvent. Under the condition of good stability, flow cytometry and laser confocal microscopy test show that the modification of FA can make the formed functional Au DENPs specific binding to the cancer cells expressed by FA receptor, and realize the function of target cancer cell CT imaging in vitro and target tumor CT imaging in vivo. The modification of alpha -tos makes functional Au DENPs has the target therapy in vitro and in vivo, and the efficiency of treatment in vitro and in vivo is higher than that of pure alpha -tos. Because of the above-mentioned FA targeting, the functional Au DENPs with covalent bond of alpha -tos is more effective than pure alpha -tos in the treatment of cancer cells in vitro, and in order to explore the microscopic treatment mechanism and understand this nano diagnosis and treatment in depth. Whether the platform is suitable for targeting other types of tumor systems, we modify the functional Au DENPs surface with polypeptide arginine glycine aspartic acid (Arg-Gly-Asp, RGD) as another target molecule at the covalent bond of alpha -tos. The study shows that the formed Au DENPs also has a more homogeneous nanoscale size and good in vitro stability. Intracellular reactive oxygen species (ROS) tests showed that RGD targeted, covalently binding alpha -tos functional Au DENPs could induce cancer cells to produce a recombinant human annexinv/ iodized propidium iodide (annexinv-fitc/pi) double staining experiment with more reactive oxygen.Fitc markers than pure alpha -tos, indicating that RGD targeted, covalent bond alpha -tos functional Au The cells produce more early apoptotic and dead cancer cells. And the modified alpha -tos covalently bonded Au DENPs, formed by RGD modification, can also target malignant glioma cells expressed by high integrin alpha v beta 3 and can specifically inhibit its growth. It has a higher x- ray attenuation effect, and has the target CT imaging function.2 in vitro cancer cells. G5.NH_2 is the nanometer platform, and the gadolinium ion chelating agent (2,2', 2''- (2- (2,5-dioxopyrrolidin-1-yloxy) -2-oxoethyl) is modified on its surface. -fa, then chelating gadolinium ions on the template and acetylation of the amino group on the surface of a tree like macromolecule, then use the nanoplatform to encapsulate the anticancer drug adriamycin (DOX) in the nano diagnosis and treatment system. The study shows that in the nanoscale system, 8.5 DOX molecules are encapsulated in each tree, and the nano system is in different pH. Conditions with good stability and sustained release of dox.fa make it able to target cancer cells expressed by FA receptor effectively, so that it has the role of MR imaging and chemical therapy in target cancer cells in vitro.3). We use partial acetylation and modified FA functional tree like macromolecules as g5.nhac-fa, pH sensitive CIS Aconitine is used as a connecting molecule to combine the covalent bond of DOX on its surface. Then the formed g5.nhac-fa-dox is used as a template to wrap nano gold particles into a new nano diagnosis and treatment system. In the form of Au DENPs, each tree like macromolecule covalently bonds 9 DOX, and the gold nanoparticle inch is 2.76nm and has a different pH and temperature conditions. The FA targeting DOX covalently bonded Au DENPs follows the slow release mechanism of acid induced drug, and the faster the slow release rate of DOX under the lower pH conditions. The nano diagnosis and treatment material, in addition to a certain therapeutic effect of cancer cells in vitro, can effectively target the cancer cells expressed by folic acid receptor in vitro for cancer cells in vitro. Cell CT imaging.4) based on the early phase of the nanoscale diagnosis and chemical therapy based on tree like macromolecules, we further construct a radioactive chemical diagnosis system carrying radioactive iodine -131 (~ (131) I). Based on the characteristics of ~ (131) I, we can simultaneously emit g rays (7) (21) (19) keV (11) (23) (16) (13) (22) (4)) for single photon emission Computed tomography (SPECT) imaging and B ray (7) (21) (21) (44) ev (11) (23) (24) (24) (24)) (24) (24) (24) (24) (24) (24) (4) are used for radiotherapy. We covalently covalently bond G5.NH_2 to 3- (4- hydroxy phenyl) propionate n- hydroxysuccinimide (hPAO) and FA modified PEG, after which the residual amino acetylation of the dendrimer surface and radioactive iodine are marked. 131 (~ (131) I) to construct a radiochemical diagnosis system.~1hnmr results showed that each G5.NH_2 dendrimer surface modified with 9.4 hPAO, and before labeling ~ (131) I, the functionalized tree like macromolecules still had no biotoxicity when the concentration was up to 20mm. And the nano diagnosis and treatment carrier could target the cancer expressed by FA receptor because of the modification of FA. Cell. Radiochemical purity tests showed that the formation of ~ (131) I labeled functional tree like macromolecules had good stability until the 27h radiochemical purity was up to 92.84%. tumor growth curve and the survival rate statistics showed that the ~ (131) I labeled functional tree like macromolecules had good radioactivity efficiency. In vivo SPECT imaging The results show that the nanomaterials can specifically target the FA receptor expressed tumor for targeting SPECT imaging.

【学位授予单位】：东华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.4;TB383.1
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本文编号：1806327