本文選題：熒光探針　切入點：生物成像　出處：《鄭州大學》2017年碩士論文　論文類型：學位論文

【摘要】：近年來,隨著癌癥診斷領域的發(fā)展,生物醫(yī)學成像下腫瘤標志物的檢測已經(jīng)成為了一種新的趨勢。熒光成像由于靈敏度高、設備廉價、操作簡便易行等優(yōu)點進而對癌癥的診斷、監(jiān)測方面產(chǎn)生了極其深遠的影響。谷胱甘肽(GSH)作為生物體內(nèi)一類含有巰基基團的生物分子,其能夠保護蛋白質(zhì)或者其他細胞成分免受活性氧(ROS)的氧化的作用,在維持細胞和生物體內(nèi)氧化還原穩(wěn)態(tài)以及保護蛋白等方面發(fā)揮著重要的作用。通常,細胞內(nèi)GSH濃度的改變與體內(nèi)的生物紊亂(如癌癥、獲得性免疫缺陷綜合征(AIDS)、心臟病)密切相關。研究表明相較于正常組織細胞,GSH在腫瘤細胞中的含量遠遠高于正常組織,因此,制備能夠特異性地識別GSH分子的熒光探針對于癌癥的早期診斷顯得十分重要。但是,傳統(tǒng)地具有生物巰基響應性的熒光探針分子由于激發(fā)和發(fā)射波波長較短,易受到自體熒光的干擾,從而造成背景信號較強,同時存在靈敏度低、選擇性差以及細胞組織毒性較強等缺點,嚴重限制了其在生物成像上的應用。本文合成了基于羅丹明母核的長波長、高靈敏度、高選擇性的“off-on”型熒光探針(probe),并將其負載于生物可降解的高分子聚合物聚乳酸-羥基乙酸(PLGA)共聚物內(nèi)制備成負載開關型熒光探針的PLGA納米粒(probe@PLGA)。這種納米診斷試劑生物相容性良好,由于其結(jié)構(gòu)中存在不穩(wěn)定的酯鍵,使得該納米粒容易在腫瘤微環(huán)境條件下降解,從而釋放出包裹在其內(nèi)的開關型熒光探針。熒光探針由于分子內(nèi)部的光致電子轉(zhuǎn)移(PET)效應,自身而顯示弱的熒光(“off”狀態(tài)),當其從納米載體中釋放出來后能迅速地與腫瘤微環(huán)境中的GSH相互作用,發(fā)生分子結(jié)構(gòu)的改變從而使探針熒光強度增強(“on”狀態(tài)),可通過熒光的“開”、“關”方式評估腫瘤部位。probe@PLGA納米制劑具有生物相容性良好、靈敏度高、以及不易從血液中清除等優(yōu)點。該熒光探針具有激發(fā)和發(fā)射波長較長、量子產(chǎn)率相對較高、光學穩(wěn)定性好、熒光壽命長、檢測靈敏度及選擇性高等特征,將其應用于腫瘤部位診斷能夠充分發(fā)揮其體內(nèi)醫(yī)學成像以及術中成像的作用。文中通過四步化學合成法制備了具有腫瘤微環(huán)境響應的開關型熒光探針,利用紅外、核磁以及紫外等光譜技術對其進行了表征,并通過乳化溶劑揮發(fā)法將其負載于PLGA高聚物內(nèi),從而成功制備了具有納米尺寸且生物相容性較好的腫瘤微環(huán)境診斷試劑(probe@PLGA)。通過熒光發(fā)射光譜研究探針分子的光學特性以及對待檢測物質(zhì)GSH的響應性情況。分別采用激光散射粒度儀對制備的空白PLGAs納米粒以及probe@PLGA納米粒進行粒徑、電位表征。采用透射電子顯微鏡(TEM)來表征制劑的大小、形態(tài)及其粒度分布情況。采用透析袋法分別考察了probe@PLGA納米粒在pH=5.3以及pH=7.4不同pH條件下體外探針釋放行為。選取人源乳腺癌細胞(MCF-7)作為細胞模型,采用SRB法考察游離probe以及probe@PLGA納米粒對乳腺癌細胞(MCF-7)的細胞存活率的影響。使用熒光顯微鏡以及流式細胞儀對probe@PLGA納米粒進行體外攝取性質(zhì)的研究,使用激光共聚焦顯微鏡分析probe@PLGA納米粒在腫瘤細胞水平上的定位情況。采用S180腹水瘤模型考察納米粒在荷瘤KM小鼠體內(nèi)的熒光成像以及組織分布情況。運用高效液相色譜儀考察probe@PLGA納米粒在大鼠體內(nèi)的藥代動力學行為。探針的熒光光譜實驗表明所合成的熒光探針具有選擇性高、靈敏度強以及光學穩(wěn)定性較好等優(yōu)點。經(jīng)乳化溶劑揮發(fā)法制備的probe@PLGA納米粒的平均粒徑為94.49±2.03 nm,Zeta電位為-8.97±1.01 mV。透射電子顯微鏡圖片表明該納米粒子呈球狀,且分布均勻穩(wěn)定。probe@PLGA納米粒放置于4℃保存時能夠長期穩(wěn)定存在,且在較長時間內(nèi)納米粒子不會發(fā)生明顯的粒徑變化。考察probe@PLGA納米粒在不同pH下的探針釋放,得出pH=5.3比pH=7.4時具有更高的體外累積釋放量,這種釋放機制可能是由于PLGAs納米粒易在腫瘤微酸性條件下降解所導致的。細胞毒性實驗表明,probe@PLGA納米粒具有良好的生物相容性,細胞存活率明顯高于游離的探針分子。細胞攝取表明,probe@PLGA納米粒相較于游離的熒光探針能夠顯著的增強其入胞行為,并且從顯微鏡圖片中可以看出熒光探針分子能夠特異性的靶向于細胞質(zhì)中的線粒體,而沒有定位于細胞核。動物活體成像實驗表明,制劑納米粒子能夠通過腫瘤部位的高通透性和滯留(EPR)效應定向的靶向于腫瘤部位,并在腫瘤部位顯示強的熒光,從而能用于腫瘤部位的界定。大鼠體內(nèi)的藥代動力學實驗表明,制劑納米粒子具有延長探針在體內(nèi)的循環(huán)時間、減少血液清除率等優(yōu)點,能夠更加適用于復雜的體內(nèi)環(huán)境。本文構(gòu)建的腫瘤微環(huán)境響應性的開關型熒光探針納米粒子,能夠通過熒光“開”、“關”的形式來界定腫瘤部位,具有選擇性好、發(fā)射波長長、靈敏度好以及反應迅速的特性,具有重要的臨床意義及廣闊的應用前景,進而用于體內(nèi)熒光標識與熒光手術,為以后的研究奠定了堅實的基礎。
[Abstract]:In recent years, with the development of cancer diagnostics, biomedical imaging detection of tumor markers has become a new trend. Fluorescence imaging because of its high sensitivity, inexpensive, easy to operate and the advantages of the diagnosis of cancer, had a very profound impact monitoring. Glutathione (GSH) as an organism a class of biological molecules containing thiol groups, which can protect proteins or other cellular components from active oxygen (ROS) oxidation effect plays an important role in maintaining cell and organism redox homeostasis and protect protein. Usually, change and in vivo biological disorder of intracellular GSH concentration (such as cancer, acquired immunodeficiency syndrome (AIDS), heart disease) are closely related. The results show that compared with normal cells, the content of GSH in cancer cells is much higher than that in normal tissues, Therefore, the preparation of fluorescent probe can specifically recognize GSH molecules for the early diagnosis of cancer is very important. However, the traditional biological response with thiol fluorescent probe molecules of the excitation and emission wavelength is short, easy to be disturbed by autofluorescence, resulting in strong background signal, at the same time has low sensitivity and selectivity poor organization and cell toxic and other shortcomings, which severely limits its application in biological imaging. The long wavelength, Luo Danming mother nucleus based on high sensitivity were synthesized in this paper, the high selectivity of the "off-on" type fluorescent probe (probe), and the polymer loaded on the biodegradable poly lactic acid glycolic acid (PLGA) inside the copolymer prepared load switch type fluorescent probe PLGA nanoparticles (probe@PLGA). The nano diagnostic reagent has good biocompatibility. Because its structure does not exist in the stable ester The key to nanoparticles in the tumor microenvironment under conditions of degradation, thereby releasing the switch type fluorescent probe wrapped within the internal molecular fluorescence probe. The photoinduced electron transfer (PET) effect, which shows its weak fluorescence ("off"), when it is released from the nano carrier after the interaction of rapidly and in the tumor microenvironment of GSH, molecular structure changes so that the fluorescence intensity ("on"), the fluorescence of the "open" and "off" to assess the tumor site.Probe@PLGA nano capsule has good biocompatibility, high sensitivity, and is not easy to remove from the blood. The advantages of the fluorescent probe with excitation wavelength and emission wavelength is longer, relatively high quantum yield, good optical stability, long fluorescence lifetime, detection sensitivity and high selectivity characteristics, its application in tumor diagnosis can charge Play in the medical imaging and intraoperative imaging. In this paper, through four steps of chemical synthesis of the switch type fluorescent probe, a tumor microenvironment response by IR, NMR and UV spectroscopy were used to characterize them, and loaded on the polymer PLGA by emulsion solvent evaporation method. It was successfully prepared with nanometer size and good biocompatibility of tumor microenvironment diagnostic kit (probe@PLGA). The optical properties of the fluorescence emission spectra of probe molecules and treatment response to detect substance GSH respectively. The laser scattering particle size analyzer for the preparation of blank PLGAs nanoparticles and probe@PLGA nanoparticles were particle size potential characterized by transmission electron microscopy (TEM) to characterize the preparation of size, morphology and particle size distribution were investigated. Probe@PLGA nanoparticles by dialysis bag method The release behavior in pH=5.3 and pH=7.4 under different pH conditions. The probe selected human breast cancer cells (MCF-7) as a cell model, SRB method was used to examine the free probe and probe@PLGA nanoparticles on human breast cancer cells (MCF-7) affected the rate of survival cells. Studies using fluorescence microscopy and in vitro uptake properties of probe@PLGA nanoparticles by flow cytometry the instrument, using laser confocal microscopy analysis of probe@PLGA nanoparticles on the level of localization in tumor cells. Using S180 ascites tumor model were investigated in vivo fluorescence imaging of tumor bearing KM mice and tissue distribution. By using high performance liquid chromatography of probe@PLGA nanoparticles in rats in vivo pharmacokinetic behavior. Fluorescence spectroscopy experimental probe show that the synthesized fluorescent probe has the advantages of high selectivity, high sensitivity and good optical stability by emulsion solution. The average granule prepared by evaporation of probe@PLGA nanoparticles diameter was 94.49 + 2.03 nm, Zeta potential is -8.97 + 1.01 mV. transmission electron microscope images showed that the nanoparticles were spherical, stable and uniform distribution of.Probe@PLGA nanoparticles placed at 4 DEG C when saving can exist stably in a long time, and the nanoparticles were not obvious size change probe. Effects of probe@PLGA nanoparticles in different pH release, that pH=5.3 has higher in vitro cumulative release amount is pH=7.4, this may be due to the release mechanism of PLGAs nanoparticles in tumor acidic conditions degradation caused by cytotoxicity assays showed that probe@PLGA nanoparticles have good biocompatibility, cell survival rate significantly higher than the probe molecule. Free cell uptake showed that the fluorescent probe of probe@PLGA nanoparticles compared to free can enhance the endocytosis significantly The behavior, and can be seen from the microscope images of fluorescent probe molecules to specific mitochondrial targeting in the cytoplasm, but not localized in the nucleus. Imaging of animal experiments, preparation of nanoparticles can through the tumor site of high permeability and retention (EPR) target tumor effect oriented, and show strong the fluorescence in the tumor site, which can be used to define the location of tumor. It shows that the pharmacokinetics in rats in vivo, preparation of nanoparticles with a prolonged probe in vivo circulation time, reduce the blood clearance rate and other advantages, can be more suitable for the complex in vivo environment. The tumor microenvironment response switch type fluorescent probes of nanoparticles the fluorescence through "open" and "off" to define the tumor site, has good selectivity, long wave emission, good sensitivity and rapid response The characteristics, which have important clinical significance and broad application prospect, are applied to the fluorescence labeling and fluorescence operation in vivo, which lays a solid foundation for future research.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R943

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本文編號：1576927