肿瘤微环境敏感的开关型荧光探针的制备及应用

发布时间：2018-03-06 23:16

本文选题：荧光探针　切入点：生物成像　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：近年来,随着癌症诊断领域的发展,生物医学成像下肿瘤标志物的检测已经成为了一种新的趋势。荧光成像由于灵敏度高、设备廉价、操作简便易行等优点进而对癌症的诊断、监测方面产生了极其深远的影响。谷胱甘肽(GSH)作为生物体内一类含有巯基基团的生物分子,其能够保护蛋白质或者其他细胞成分免受活性氧(ROS)的氧化的作用,在维持细胞和生物体内氧化还原稳态以及保护蛋白等方面发挥着重要的作用。通常,细胞内GSH浓度的改变与体内的生物紊乱(如癌症、获得性免疫缺陷综合征(AIDS)、心脏病)密切相关。研究表明相较于正常组织细胞,GSH在肿瘤细胞中的含量远远高于正常组织,因此,制备能够特异性地识别GSH分子的荧光探针对于癌症的早期诊断显得十分重要。但是,传统地具有生物巯基响应性的荧光探针分子由于激发和发射波波长较短,易受到自体荧光的干扰,从而造成背景信号较强,同时存在灵敏度低、选择性差以及细胞组织毒性较强等缺点,严重限制了其在生物成像上的应用。本文合成了基于罗丹明母核的长波长、高灵敏度、高选择性的“off-on”型荧光探针(probe),并将其负载于生物可降解的高分子聚合物聚乳酸-羟基乙酸(PLGA)共聚物内制备成负载开关型荧光探针的PLGA纳米粒(probe@PLGA)。这种纳米诊断试剂生物相容性良好,由于其结构中存在不稳定的酯键,使得该纳米粒容易在肿瘤微环境条件下降解,从而释放出包裹在其内的开关型荧光探针。荧光探针由于分子内部的光致电子转移(PET)效应,自身而显示弱的荧光(“off”状态),当其从纳米载体中释放出来后能迅速地与肿瘤微环境中的GSH相互作用,发生分子结构的改变从而使探针荧光强度增强(“on”状态),可通过荧光的“开”、“关”方式评估肿瘤部位。probe@PLGA纳米制剂具有生物相容性良好、灵敏度高、以及不易从血液中清除等优点。该荧光探针具有激发和发射波长较长、量子产率相对较高、光学稳定性好、荧光寿命长、检测灵敏度及选择性高等特征,将其应用于肿瘤部位诊断能够充分发挥其体内医学成像以及术中成像的作用。文中通过四步化学合成法制备了具有肿瘤微环境响应的开关型荧光探针,利用红外、核磁以及紫外等光谱技术对其进行了表征,并通过乳化溶剂挥发法将其负载于PLGA高聚物内,从而成功制备了具有纳米尺寸且生物相容性较好的肿瘤微环境诊断试剂(probe@PLGA)。通过荧光发射光谱研究探针分子的光学特性以及对待检测物质GSH的响应性情况。分别采用激光散射粒度仪对制备的空白PLGAs纳米粒以及probe@PLGA纳米粒进行粒径、电位表征。采用透射电子显微镜(TEM)来表征制剂的大小、形态及其粒度分布情况。采用透析袋法分别考察了probe@PLGA纳米粒在pH=5.3以及pH=7.4不同pH条件下体外探针释放行为。选取人源乳腺癌细胞(MCF-7)作为细胞模型,采用SRB法考察游离probe以及probe@PLGA纳米粒对乳腺癌细胞(MCF-7)的细胞存活率的影响。使用荧光显微镜以及流式细胞仪对probe@PLGA纳米粒进行体外摄取性质的研究,使用激光共聚焦显微镜分析probe@PLGA纳米粒在肿瘤细胞水平上的定位情况。采用S180腹水瘤模型考察纳米粒在荷瘤KM小鼠体内的荧光成像以及组织分布情况。运用高效液相色谱仪考察probe@PLGA纳米粒在大鼠体内的药代动力学行为。探针的荧光光谱实验表明所合成的荧光探针具有选择性高、灵敏度强以及光学稳定性较好等优点。经乳化溶剂挥发法制备的probe@PLGA纳米粒的平均粒径为94.49±2.03 nm,Zeta电位为-8.97±1.01 mV。透射电子显微镜图片表明该纳米粒子呈球状,且分布均匀稳定。probe@PLGA纳米粒放置于4℃保存时能够长期稳定存在,且在较长时间内纳米粒子不会发生明显的粒径变化。考察probe@PLGA纳米粒在不同pH下的探针释放,得出pH=5.3比pH=7.4时具有更高的体外累积释放量,这种释放机制可能是由于PLGAs纳米粒易在肿瘤微酸性条件下降解所导致的。细胞毒性实验表明,probe@PLGA纳米粒具有良好的生物相容性,细胞存活率明显高于游离的探针分子。细胞摄取表明,probe@PLGA纳米粒相较于游离的荧光探针能够显著的增强其入胞行为,并且从显微镜图片中可以看出荧光探针分子能够特异性的靶向于细胞质中的线粒体,而没有定位于细胞核。动物活体成像实验表明,制剂纳米粒子能够通过肿瘤部位的高通透性和滞留(EPR)效应定向的靶向于肿瘤部位,并在肿瘤部位显示强的荧光,从而能用于肿瘤部位的界定。大鼠体内的药代动力学实验表明,制剂纳米粒子具有延长探针在体内的循环时间、减少血液清除率等优点,能够更加适用于复杂的体内环境。本文构建的肿瘤微环境响应性的开关型荧光探针纳米粒子,能够通过荧光“开”、“关”的形式来界定肿瘤部位,具有选择性好、发射波长长、灵敏度好以及反应迅速的特性,具有重要的临床意义及广阔的应用前景,进而用于体内荧光标识与荧光手术,为以后的研究奠定了坚实的基础。
[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.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943

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