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

【摘要】：近年來(lái),隨著癌癥診斷領(lǐng)域的發(fā)展,生物醫(yī)學(xué)成像下腫瘤標(biāo)志物的檢測(cè)已經(jīng)成為了一種新的趨勢(shì)。熒光成像由于靈敏度高、設(shè)備廉價(jià)、操作簡(jiǎn)便易行等優(yōu)點(diǎn)進(jìn)而對(duì)癌癥的診斷、監(jiān)測(cè)方面產(chǎn)生了極其深遠(yuǎn)的影響。谷胱甘肽(GSH)作為生物體內(nèi)一類含有巰基基團(tuán)的生物分子,其能夠保護(hù)蛋白質(zhì)或者其他細(xì)胞成分免受活性氧(ROS)的氧化的作用,在維持細(xì)胞和生物體內(nèi)氧化還原穩(wěn)態(tài)以及保護(hù)蛋白等方面發(fā)揮著重要的作用。通常,細(xì)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(guān)。研究表明相較于正常組織細(xì)胞,GSH在腫瘤細(xì)胞中的含量遠(yuǎn)遠(yuǎn)高于正常組織,因此,制備能夠特異性地識(shí)別GSH分子的熒光探針對(duì)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(yīng)性的熒光探針?lè)肿佑捎诩ぐl(fā)和發(fā)射波波長(zhǎng)較短,易受到自體熒光的干擾,從而造成背景信號(hào)較強(qiáng),同時(shí)存在靈敏度低、選擇性差以及細(xì)胞組織毒性較強(qiáng)等缺點(diǎn),嚴(yán)重限制了其在生物成像上的應(yīng)用。本文合成了基于羅丹明母核的長(zhǎng)波長(zhǎng)、高靈敏度、高選擇性的“off-on”型熒光探針(probe),并將其負(fù)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(fù)載開(kāi)關(guān)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(kāi)關(guān)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(yīng),自身而顯示弱的熒光(“off”狀態(tài)),當(dāng)其從納米載體中釋放出來(lái)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(qiáng)度增強(qiáng)(“on”狀態(tài)),可通過(guò)熒光的“開(kāi)”、“關(guān)”方式評(píng)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(diǎn)。該熒光探針具有激發(fā)和發(fā)射波長(zhǎng)較長(zhǎng)、量子產(chǎn)率相對(duì)較高、光學(xué)穩(wěn)定性好、熒光壽命長(zhǎng)、檢測(cè)靈敏度及選擇性高等特征,將其應(yīng)用于腫瘤部位診斷能夠充分發(fā)揮其體內(nèi)醫(yī)學(xué)成像以及術(shù)中成像的作用。文中通過(guò)四步化學(xué)合成法制備了具有腫瘤微環(huán)境響應(yīng)的開(kāi)關(guān)型熒光探針,利用紅外、核磁以及紫外等光譜技術(shù)對(duì)其進(jìn)行了表征,并通過(guò)乳化溶劑揮發(fā)法將其負(fù)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(guò)熒光發(fā)射光譜研究探針?lè)肿拥墓鈱W(xué)特性以及對(duì)待檢測(cè)物質(zhì)GSH的響應(yīng)性情況。分別采用激光散射粒度儀對(duì)制備的空白PLGAs納米粒以及probe@PLGA納米粒進(jìn)行粒徑、電位表征。采用透射電子顯微鏡(TEM)來(lái)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(xì)胞(MCF-7)作為細(xì)胞模型,采用SRB法考察游離probe以及probe@PLGA納米粒對(duì)乳腺癌細(xì)胞(MCF-7)的細(xì)胞存活率的影響。使用熒光顯微鏡以及流式細(xì)胞儀對(duì)probe@PLGA納米粒進(jìn)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(xì)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(yùn)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(dòng)力學(xué)行為。探針的熒光光譜實(shí)驗(yàn)表明所合成的熒光探針具有選擇性高、靈敏度強(qiáng)以及光學(xué)穩(wěn)定性較好等優(yōu)點(diǎn)。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(shí)能夠長(zhǎng)期穩(wěn)定存在,且在較長(zhǎng)時(shí)間內(nèi)納米粒子不會(huì)發(fā)生明顯的粒徑變化�？疾靝robe@PLGA納米粒在不同pH下的探針釋放,得出pH=5.3比pH=7.4時(shí)具有更高的體外累積釋放量,這種釋放機(jī)制可能是由于PLGAs納米粒易在腫瘤微酸性條件下降解所導(dǎo)致的。細(xì)胞毒性實(shí)驗(yàn)表明,probe@PLGA納米粒具有良好的生物相容性,細(xì)胞存活率明顯高于游離的探針?lè)肿�。�?xì)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(qiáng)其入胞行為,并且從顯微鏡圖片中可以看出熒光探針?lè)肿幽軌蛱禺愋缘陌邢蛴诩?xì)胞質(zhì)中的線粒體,而沒(méi)有定位于細(xì)胞核。動(dòng)物活體成像實(shí)驗(yàn)表明,制劑納米粒子能夠通過(guò)腫瘤部位的高通透性和滯留(EPR)效應(yīng)定向的靶向于腫瘤部位,并在腫瘤部位顯示強(qiáng)的熒光,從而能用于腫瘤部位的界定。大鼠體內(nèi)的藥代動(dòng)力學(xué)實(shí)驗(yàn)表明,制劑納米粒子具有延長(zhǎng)探針在體內(nèi)的循環(huán)時(shí)間、減少血液清除率等優(yōu)點(diǎn),能夠更加適用于復(fù)雜的體內(nèi)環(huán)境。本文構(gòu)建的腫瘤微環(huán)境響應(yīng)性的開(kāi)關(guān)型熒光探針納米粒子,能夠通過(guò)熒光“開(kāi)”、“關(guān)”的形式來(lái)界定腫瘤部位,具有選擇性好、發(fā)射波長(zhǎng)長(zhǎng)、靈敏度好以及反應(yīng)迅速的特性,具有重要的臨床意義及廣闊的應(yīng)用前景,進(jìn)而用于體內(nèi)熒光標(biāo)識(shí)與熒光手術(shù),為以后的研究奠定了堅(jiān)實(shí)的基礎(chǔ)。
[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(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R943

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本文編號(hào)：1576927