“自开关”“可逆性”肿瘤微酸环境响应型近红外比率荧光探针的研究

发布时间：2018-05-09 18:45

  本文选题：肿瘤微环境 + 含硅罗丹明　； 参考：《郑州大学》2017年硕士论文

【摘要】：酸性肿瘤环境与控制肿瘤生长和转移的许多因素密切相关,肿瘤部位的pH检测和pH值变化的监测已在纳米医学领域引起人们广泛关注。本课题通过在含硅元素的罗丹明结构中引入硼酸基团合成pH敏感的近红外(NIR)荧光探针SiRB。将吲哚青绿(ICG)和SiRB荷载到具有良好生物相容性的高聚物聚乳酸-羟基乙酸共聚物(PLGA)中形成PLGA-SiRB-ICG pH响应型近红外比率荧光探针。结果表明,该荧光探针通过增强的渗透性和保留效应可有效地在肿瘤部位蓄积。PLGA在弱酸性环境下加速降解有利于探针的释放,SiRB在pH 5.8~7.6范围内具有良好的pH响应性,而ICG的荧光强度几乎不随pH的变化而改变,两种探针的荧光比值可定量测量出肿瘤部位的pH值及pH值的变化情况,对肿瘤的发生、发展和转移有重要参考价值。本课题主要研究内容包括以下四个部分:一、“自开关”型近红外荧光探针SiRB的合成及其性质考察本文以3-溴-N,N-二甲基苯胺和2-溴苯硼酸为原料,采用四步法成功合成“自开关”型近红外荧光探针SiRB;通过柱层析法对中间体和终产物进行了纯化;利用核磁共振氢谱,紫外-可见光谱和荧光光谱表征了SiRB的分子结构和激发/发射波长,结果证明,SiRB分子结构与预期结构相符,其激发波长和发射波长均位于近红外区;SiRB探针在pH 5.8~7.6范围内具有灵敏的pH响应性,且研究发现该荧光探针的pH响应性具有良好的可逆性,有利于监测肿瘤微环境的pH值变化情况。二、基于SiRB的pH响应型近红外比率荧光探针的制备及其性质考察以SiRB为荧光pH指示剂;以吲哚菁绿(ICG)作为荧光参比试剂(其荧光强度在一定酸碱范围内不受pH值影响),高聚物PLGA为载体,通过溶剂乳化挥发法成功制备pH响应型近红外比率荧光纳米探针PLGA-SiRB-ICG,同法制备了对比试剂PLGA-SiRB和PLGA-ICG。通过透射电镜图和紫外-可见光谱图证实了PLGA将SiRB与ICG荷载,形成了核壳式的结构;激光纳米粒度仪测定结果表明,PLGA-SiRB-ICG的平均粒径和电位分别为95.13±0.21 nm和-8.14±0.13 mV,粒径大小均匀,稳定;通过测定不同pH下的紫外-可见光谱可知:在pH 5.8~7.6之间,PLGA-SiRB-ICG的荧光比值随pH变化而变化。实验排除了人体常见盐类对PLGA-SiRB-ICG探针的干扰,体外释放实验表明PLGA-SiRB-ICG在微酸性环境下具有更高的释放速率,有利于在肿瘤微环境下的释放。三、pH响应型近红外比率荧光探针PLGA-SiRB-ICG的细胞实验以人乳腺癌细胞MCF-7为模型细胞,考察了PLGA-SiRB-ICG的细胞毒性。SRB结果显示,在较低浓度时,PLGA-SiRB-ICG对MCF-7细胞没有明显毒性,当探针浓度大于80μg/mL时,探针对MCF-7细胞具有一定的毒性,且游离的SiRB和ICG的毒性明显高于探针组,说明PLGA的包裹降低了探针对细胞的毒性;激光共聚焦荧光显微镜实验结果表明:探针SiRB可以进入肿瘤细胞内部,并对溶酶体具有特殊靶向性,有望应用于细胞器定位研究。四、pH响应型近红外比率荧光探针PLGA-SiRB-ICG的荧光成像和pH检测研究首先,通过多模式活体成像系统(KODAK)记录PLGA-SiRB-ICG的荧光比值与pH值之间的关系,通过线性回归计算考察两者之间的线性关系并得到线性回归方程。然后,以KM雌性小鼠为模型动物,腹水瘤S180细胞为模型细胞,构建荷瘤鼠模型。通过多模式活体成像系统,成功的对小鼠的肿瘤组织及主要器官进行了pH值检测。通过实验结果发现:PLGA-SiRB-ICG对肿瘤组织具有一定的被动靶向作用,且肿瘤部位的pH数值明显低于其他器官,通过对不同生长天数的肿瘤观察发现:肿瘤生长天数越长,尺寸越大,pH越低,此结果对肿瘤的发展阶段界定与微环境研究具有重要参考价值。
[Abstract]:The acidic tumor environment is closely related to many factors controlling the growth and metastasis of tumor. The pH detection of the tumor site and the monitoring of the change of pH value have aroused widespread concern in the field of nanomedicine. This topic is based on the introduction of the boric acid group of the pH sensitive near infrared (NIR) fluorescence probe SiRB. to the indole in the Luo Danming structure containing silicon elements. Green (ICG) and SiRB loads form a PLGA-SiRB-ICG pH responsive near infrared fluorescence probe into a polymer of polymer polylactic acid copolymer (PLGA) with good biocompatibility. The results show that the fluorescence probe can effectively accumulate.PLGA in the tumor site to accelerate in the weak acid environment through enhanced permeability and retention effect. Degradation is beneficial to the release of the probe, SiRB has good pH responsiveness in the range of pH 5.8~7.6, and the fluorescence intensity of ICG almost does not change with the change of pH. The fluorescence ratio of the two probes can quantitatively measure the changes of the pH value and pH value of the tumor site, and have important reference value for the occurrence, development and metastasis of the tumor. The research contents include the following four parts: first, the synthesis and properties of "self switched" near infrared fluorescence probe SiRB and its properties, the "self switching" near infrared fluorescence probe SiRB is successfully synthesized by four step method using 3- bromine -N, N- two methyl aniline and 2- bromine boric acid as raw materials. The intermediate and final products are purified by column chromatography. The molecular structure and excitation / emission wavelengths of SiRB are characterized by NMR, UV visible and fluorescence spectra. The results show that the molecular structure of SiRB is in accordance with the expected structure, the excitation wavelength and the emission wavelength are in the near infrared region, and the SiRB probe has a sensitive pH responsiveness in the pH 5.8~7.6 enclosure, and the research has found the fluorescein. The pH responsiveness of the light probe has good reversibility, which is beneficial to monitoring the pH value change of the tumor microenvironment. Two, the preparation and properties of the pH responsive near infrared fluorescence probe based on SiRB and its properties are SiRB as the fluorescent pH indicator, and using the indocyanine green (ICG) as the fluorescence reference reagent (its fluorescence intensity is not subject to a certain acid base range. " The effect of pH value), polymer PLGA as the carrier, pH responsive near infrared ratio fluorescent nano probe PLGA-SiRB-ICG was successfully prepared by solvent emulsification and volatilization, and the comparison reagent PLGA-SiRB and PLGA-ICG. were prepared by transmission electron microscopy and UV visible spectrogram to confirm PLGA with SiRB and ICG loads, forming a nuclear shell structure; laser nanoscale. The results showed that the average particle size and potential of PLGA-SiRB-ICG were 95.13 + 0.21 nm and -8.14 + 0.13 mV respectively, and the size of grain was uniform and stable. By measuring the UV visible spectrum under different pH, the fluorescence ratio of PLGA-SiRB-ICG changed with pH in pH 5.8~7.6. The experiment ruled out common salt of human body to PLGA-Si. The interference of RB-ICG probe and in vitro release experiments show that PLGA-SiRB-ICG has a higher release rate in the microacid environment and is beneficial to the release of the tumor microenvironment. Three, the cell experiment of the pH responsive near infrared ratio fluorescence probe PLGA-SiRB-ICG is based on the human breast cancer cell MCF-7 as the model cell, and the cytotoxic.S of PLGA-SiRB-ICG is investigated. RB results showed that PLGA-SiRB-ICG had no obvious toxicity to MCF-7 cells at a lower concentration. When the probe concentration was greater than 80 g/mL, the probe had a certain toxicity to MCF-7 cells, and the toxicity of free SiRB and ICG was significantly higher than that of the probe group. It indicated that the encapsulation of PLGA reduced the toxicity of the probe to the cells; the laser confocal fluorescence microscopy experiment was used. The results show that the probe SiRB can enter the tumor cells and have special targeting to the lysosomes and is expected to be applied to the study of organelles. Four, the fluorescence imaging and pH detection of the pH responsive near infrared ratio fluorescence probe PLGA-SiRB-ICG, first, records the fluorescence ratio of PLGA-SiRB-ICG by multimode living imaging system (KODAK). The relationship between pH values, linear regression and linear regression equation were obtained by linear regression calculation. Then, KM female mice were used as model animals and ascites tumor S180 cells as model cells to construct a tumor bearing rat model. The pH value of the tumor tissues and major organs of mice was successfully carried out by multi mode living imaging system. The test results show that PLGA-SiRB-ICG has a certain passive targeting effect on tumor tissue, and the pH value of the tumor site is obviously lower than that of other organs. Through the observation of the tumor growth days, the longer the growth days, the larger the size and the lower the pH, the result is the definition of the development stage of the tumor and the study of the microenvironment. It has important reference value.

【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

【相似文献】

相关期刊论文 前10条

1 王可;马会民;;对生物环境因素敏感的荧光探针[J];化学进展;2010年08期

2 王姗姗;;小分子荧光探针在硫醇检测中的最新研究进展[J];科技信息;2010年23期

3 向雨秘;龙少波;朱R，

本文编号：1866996