本文選題：樹(shù)狀大分子 + 抗癌藥物��； 參考：《東華大學(xué)》2016年博士論文

【摘要】：在分子影像學(xué)技術(shù)及納米醫(yī)藥學(xué)研究不斷發(fā)展的今天,如何將這兩種功能有機(jī)地結(jié)合起來(lái)實(shí)現(xiàn)“診療”一體化仍然是個(gè)巨大的挑戰(zhàn)。目前醫(yī)學(xué)造影劑及分子藥物均存在許多缺點(diǎn):造影劑的血液半衰期短、穩(wěn)定性和生物相容性差,小分子化學(xué)藥物的疏水性、對(duì)正常細(xì)胞及組織的毒副作用、對(duì)病灶區(qū)域無(wú)特異性等。因此,需要找到一個(gè)合適的納米載體平臺(tái)既可以將造影劑的診斷作用和藥物的治療作用有機(jī)地結(jié)合起來(lái),又可以改善醫(yī)學(xué)造影劑及化學(xué)藥物的種種缺點(diǎn),形成多功能的診療探針復(fù)合體用于癌癥的早期診斷及治療�；跇�(shù)狀大分子獨(dú)特的理化性質(zhì),表面擁有眾多的可修飾化功能基團(tuán),可用于藥物的化學(xué)鍵合及各種功能分子的修飾,內(nèi)部空腔可用于物理包裹分子藥物及納米金屬顆粒。本文以樹(shù)狀大分子作為平臺(tái)構(gòu)建具有“診療”作用的功能雜化納米顆粒,對(duì)其進(jìn)行基本表征,研究表明形成的納米診療體系不僅可以提高分子抗癌藥物的水溶性、生物相容性還可以延長(zhǎng)其在體內(nèi)的血液循環(huán)時(shí)間,延長(zhǎng)造影成像時(shí)間,與此同時(shí)在其表面上修飾靶向分子,可以增強(qiáng)其與癌細(xì)胞的特異性結(jié)合,以實(shí)現(xiàn)腫瘤的靶向成像及靶向治療。因而,基于樹(shù)狀大分子構(gòu)建的具有診療功能的納米雜化顆粒將為探索新型的集腫瘤診斷與治療一體化的多功能納米平臺(tái)開(kāi)辟新的思路。研究?jī)?nèi)容如下所述:1)根據(jù)樹(shù)狀大分子獨(dú)特的理化性質(zhì),以第五代聚酰胺胺樹(shù)狀大分子(G5.NH_2)作為納米診療平臺(tái),通過(guò)表面修飾異硫氰酸熒光素(fluorescein isothiocyanate,FI),聚乙二醇(polyethylene glycol,peg)化的抗癌藥物alpha-維生素e琥珀酸酯(a-tocopheryl succinate,α-tos)及peg化的靶向分子葉酸(folicacid,fa),之后在其內(nèi)部包裹納米金顆粒構(gòu)建多功能化樹(shù)狀大分子包裹納米金顆粒(au DENPs)用于靶向腫瘤ct成像及化學(xué)治療。氫核磁共振(~1hnmr,proton nuclear magnetic resonance spectroscopy)測(cè)試結(jié)果表明,在形成的au DENPs中,每個(gè)樹(shù)狀大分子表面修飾有9.8個(gè)α-tos。透射電鏡(transmission electron microscope,tem)測(cè)試得出形成的au DENPs平均直徑約3.3nm,尺寸分布較窄,具有良好的單分散性,且在不同ph條件(ph=5,6,7,8),溫度條件(4℃,37℃,50℃)和溶劑條件下具有良好的穩(wěn)定性。流式細(xì)胞儀及激光共聚焦顯微鏡測(cè)試表明,fa的修飾可使形成的功能化au DENPs能與具有fa受體表達(dá)的癌細(xì)胞特異性結(jié)合,實(shí)現(xiàn)體外靶向癌細(xì)胞ct成像及體內(nèi)靶向腫瘤ct成像的功能。α-tos的修飾使形成的功能化au DENPs具有體內(nèi)體外靶向治療功能,且體外及體內(nèi)治療效率比純?chǔ)?tos的治療效率高。由于上述構(gòu)建的fa靶向的,共價(jià)鍵合α-tos的功能化au DENPs具有比純?chǔ)?tos更強(qiáng)的體外癌細(xì)胞治療作用,且為了深入探索其微觀(guān)治療機(jī)制及了解這一納米診療平臺(tái)是否適用于靶向其它類(lèi)型的腫瘤系統(tǒng),我們以多肽精氨酸-甘氨酸-天冬氨酸(arg-gly-asp,rgd)作為另一靶向分子修飾在α-tos共價(jià)鍵合的功能化au DENPs表面。研究表明形成的au DENPs同樣具有較為均一的納米尺寸及良好的體外穩(wěn)定性。通過(guò)細(xì)胞內(nèi)活性氧(ros)測(cè)試表明rgd靶向的,共價(jià)鍵合α-tos的功能化au DENPs能誘導(dǎo)癌細(xì)胞產(chǎn)生出比純?chǔ)?tos更多的活性氧。fitc標(biāo)記的重組人annexinv/碘化丙啶(annexinv-fitc/pi)雙染實(shí)驗(yàn)表明rgd靶向的,共價(jià)鍵合α-tos的功能化au DENPs比純?chǔ)?tos能誘導(dǎo)癌細(xì)胞產(chǎn)生出更多的早期凋亡及死亡的癌細(xì)胞。且經(jīng)過(guò)rgd的修飾,形成的α-tos共價(jià)鍵合的au DENPs同樣能夠靶向高整合素αvβ3表達(dá)的惡性膠質(zhì)瘤細(xì)胞且能夠特異性的抑制其生長(zhǎng)。形成的該靶向納米體系比臨床應(yīng)用的基于碘的造影劑歐乃派克具有更高的x-射線(xiàn)衰減效應(yīng),且具有體外癌細(xì)胞靶向ct成像功能。2)以G5.NH_2為納米平臺(tái),在其表面修飾釓離子螯合劑(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化的靶向試劑peg-fa,然后在該模板上螯合釓離子并將樹(shù)狀大分子表面的氨基乙�；�,之后利用該納米平臺(tái)將其內(nèi)部包裹抗癌藥物阿霉素(dox)形成納米診療體系。研究表明形成的納米診療體系中,每個(gè)樹(shù)狀大分子包裹了8.5個(gè)dox分子,該納米體系在不同ph條件下具有良好穩(wěn)定性,且能夠持續(xù)緩釋dox。fa的修飾使其能夠有效的靶向fa受體表達(dá)的癌細(xì)胞,使其具有體外靶向癌細(xì)胞mr成像及化學(xué)治療的作用。3)我們以部分乙�；倚揎椨衒a的功能化樹(shù)狀大分子為納米平臺(tái)(g5.nhac-fa),以ph敏感順式烏頭酸酐作為連接分子將dox共價(jià)鍵合在其表面。再將形成的g5.nhac-fa-dox作為模板包裹納米金顆粒形成新的納米診療體系。形成的au DENPs中,每個(gè)樹(shù)狀大分子上共價(jià)鍵合了9個(gè)dox,形成的金納米顆粒尺寸為2.76nm且在不同ph及溫度條件下具有穩(wěn)定性。且fa靶向的dox共價(jià)鍵合的au DENPs遵循酸引發(fā)藥物緩釋機(jī)制,在越低的ph條件下,dox的緩釋速率越快。形成的該納米診療材料除了具有一定的體外癌細(xì)胞治療效果之外,fa的修飾使其能有效的靶向葉酸受體表達(dá)的癌細(xì)胞用于體外癌細(xì)胞ct成像。4)基于前期我們構(gòu)建的基于樹(shù)狀大分子的納米診斷及化學(xué)治療體系,我們進(jìn)一步構(gòu)建載入放射性碘-131(~(131)I)的放射性化學(xué)診療體系�；趡(131)I的特性,可同時(shí)發(fā)射出g射線(xiàn)(7)(18)(21)(19)kev(11)(23)(16)(13)(22)(4)(8)用于單光子發(fā)射計(jì)算機(jī)斷層(spect)成像及b射線(xiàn)(7)(15)(13)(21)(15)(21)(44)ev(11)(23)(24)(13)(24)(4)(8)用于放射性治療,我們將G5.NH_2依次共價(jià)鍵合3-(4-羥基苯基)丙酸n-羥基琥珀酰亞胺酯(hpao)和fa修飾的的peg,之后將樹(shù)狀大分子表面剩余氨基乙�；�(biāo)記放射性碘-131(~(131)I)以構(gòu)建一個(gè)放射性化學(xué)診療體系。~1hnmr結(jié)果表明每個(gè)G5.NH_2樹(shù)狀大分子表面修飾有9.4個(gè)hpao,且在標(biāo)記~(131)I之前,該功能化的樹(shù)狀大分子在濃度高達(dá)20mm時(shí)仍無(wú)生物毒性。且由于fa的修飾,該納米診療載體能夠靶向fa受體表達(dá)的癌細(xì)胞。放射性化學(xué)純度測(cè)試表明形成的~(131)I標(biāo)記的功能化樹(shù)狀大分子具有良好的穩(wěn)定性,直至27h的放射性化學(xué)純度高達(dá)92.84%。腫瘤生長(zhǎng)曲線(xiàn)及存活率統(tǒng)計(jì)表明該~(131)I標(biāo)記的功能化樹(shù)狀大分子具有良好的放射性治療效率。體內(nèi)spect成像表明形成的納米材料能特異性靶向fa受體表達(dá)的腫瘤用于靶向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.

【學(xué)位授予單位】：東華大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R730.4;TB383.1
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本文編號(hào)：1806327