环境响应型近红外荧光探针在肿瘤诊疗中的应用研究

发布时间：2018-01-08 23:05

  本文关键词：环境响应型近红外荧光探针在肿瘤诊疗中的应用研究　出处：《中国科学院深圳先进技术研究院》2016年硕士论文　论文类型：学位论文

  更多相关文章： 七甲川菁染料 pH探针 近红外荧光成像 光声成像 光稳定性

【摘要】：细胞内的pH值在许多细胞生理过程中起着关键作用。正常生理条件下,细胞液中H+的浓度约为40 nm/L(7.40),变动在0.1-0.2个pH单位就可能导致生理功能紊乱,产生疾病如炎症、阿尔茨海默病、癌症等。研究表明,肿瘤的pH值低于正常组织的pH值。因此,可视化检测肿瘤pH,对实现肿瘤的准确检测和治疗是非常重要的。荧光成像具有如非入侵式、低能量非离子福射、高的灵敏度和选择性等多个优点,而广泛用于活体内pH检测。近红外荧光染料如的最大发射峰位于近红外区(650-900 nm),具有组织穿透深、散射弱并可以可以避免背景荧光的干扰,因此以近红外荧光染料为荧光团的pH探针非常适合用于肿瘤pH检测。本文首先合成七甲川菁染料母体,通过对中位氯的亲核取代反应,我们合成五例中位二元氨取代的以七甲川菁染料为母体的pH荧光探针。然在此基础上,探究其对肿瘤pH微环境检测的应用。基于设计合成的一系列不同的中位氨取代的七甲川菁染料,我们探究这些染料分子在不同pH溶液中的光谱性质,发现其吸收光谱、荧光光谱都随pH值的改变而产生明显的变化,说明可以用作pH探针。所合成的近红外荧光染料1a-5a中探针3a的pKa为6.4,很适合用于肿瘤早起检测和成像,故而筛选出探针3a继续探讨用于肿瘤的诊疗应用研究。由于探针分子3a的吸收光强度表现出明显的pH依赖,所以我们探究其在不同pH溶液中680 nm和760 nm处光声信号。结果表明,760 nm/680 nm光声信号比率表现出明显的pH依赖的光声信号变化性,在pH5.5-7.8范围内表现出很好的线性关系。同时探针分子3a生物组织模型光声成像中,探针分子3a在同一pH值溶液中穿透不同厚度猪肉组织时,其光声信号强度比例没有明显变化,说明可以实现准确的肿瘤微酸环境pH检测,说明探针分子3a是一个很好的光声pH成像探针。动物实验表明,探针分子3a有很好的光热治疗效果。总的来说,我们设计筛选出一例荧光-光声双模pH敏感成像探针,能够实现更准确的肿瘤微环境pH检测。同时,在荧光-光声成像的引导下,实现更准确的光热治疗。众所周知,近红外染料菁染料光稳定性差和水溶性差阻碍其在生物中的应用。针对近红外染料菁染料的缺陷,我们将合成的近红外pH探针包裹在三嵌段多肽聚合物PE G5000-PLL30-PLLeu40中。实验表明近红外探针CP包裹在多肽胶束后,稳定性有了显著提高。染料包裹前后的光漂白特性和光稳定性表明,仅仅光照60s后,游离的近红外探针CP的荧光几乎完全淬灭,可以非常明显地看出溶液的颜色已经完全褪去。而将染料包裹在纳米颗粒(DNPs),在高能量的光照之后,溶液的荧光强度仅仅下降了约25%,而溶液的褪色也几乎无法用肉眼观察到。这些现象再次证实了菁染料的光稳定性差,而包裹到纳米胶束中后,稳定性明显提高。总的来说,我们设计合成了一例pH响应型纳米探针,菁染料的稳定性和水溶性有明显提高,能够用来检测细胞内pH变化,在肿瘤早期诊断和成像有较好的应用前景。
[Abstract]:The intracellular pH plays a key role in many cellular processes. Under normal physiological conditions, the cell sap concentration of H+ is about 40 nm/L (7.40), the change in the 0.1-0.2 pH unit may cause physiological function disorder, cause diseases such as inflammation, Alzheimer's disease, cancer and so on. The study shows that the tumor the pH value is lower than the normal tissue pH value. Therefore, the visual detection of tumor pH, is very important for accurate detection and treatment of tumor. Fluorescence imaging has such as non-invasive, low energy ion radiation, high sensitivity and selectivity and many other advantages, and is widely used for in vivo detection of pH. Infrared fluorescence emission peak as in near infrared region (650-900 nm), with deep tissue penetration, weak scattering and fluorescent background interference can be avoided, so the pH probe near infrared fluorescent dye fluorophore is very suitable for tumor pH Detection. This paper firstly synthesized seven methine cyanine dye precursor, through nucleophilic substitution reaction of chlorinated, we synthesized five cases of two yuan to seven amine substituted methine cyanine dyes as pH fluorescent probes. Then based on the matrix, to explore the application of tumor microenvironment of pH detection. Seven methine cyanine dyes based on the design and synthesis of a series of different substituted in ammonia, we explore the spectral properties of these dyes in solution of different pH, found that the absorption spectra, fluorescence spectra with the change of pH value and produce significant changes that can be used as a pH probe. Near infrared fluorescent dye 1a-5a the synthesis of the probe 3a pKa is 6.4, it is suitable for early tumor detection and imaging, and screened for 3A probe to continue to explore the diagnosis and therapy of tumor research. As the probe molecule absorption of 3A light intensity showed significant pH dependence, so we explore In different pH solution of 680 nm and 760 nm of photoacoustic signal. The results showed that 760 nm/680 nm ratio of photoacoustic signal showing the photoacoustic signal was pH dependent, showed a good linear relationship in the range of pH5.5-7.8. At the same time the probe molecule 3A biological tissue model of photoacoustic imaging, probe molecular 3a in the same pH value through different thickness of pork in the solution, the photoacoustic signal intensity ratio did not change significantly, that can achieve accurate detection of the tumor micro environment of pH, indicating that the probe molecule 3a is a good photoacoustic imaging probe pH. Animal experiments show that the probe molecule 3A photothermal therapeutic effect very good. In general, we screened a case design of photoacoustic pH sensitive fluorescence dual-mode imaging probe, can achieve more accurate detection of tumor microenvironment of pH. At the same time, the fluorescence and photoacoustic imaging guided light heat treatment more accurately . as everyone knows, the near infrared cyanine dye dye light stability and poor water solubility hinder its application in biology. Aiming at the limitation of near infrared dye cyanine dyes, we will probe the synthesis of near infrared pH package in the three block of PE polypeptide polymer G5000-PLL30-PLLeu40. Experiments show that the near infrared probe CP wrapped in polypeptide micelles after and stability are greatly improved. Show that the dye package before and after photobleaching properties and light stability, just after 60s light, near infrared fluorescent probe CP free almost completely quenched, can clearly see the color of the solution has been completely faded. The dye entrapped in nanoparticles (DNPs), after high energy light, fluorescence intensity of the solution only decreased by about 25%, and the solution of the fade is almost impossible to see with the unaided eye. These phenomena proved photostability of cyanine dyes, and the package to nano micelles In the middle, the stability is obviously improved. In general, we have designed and synthesized a pH responsive nanoprobe. The stability and water solubility of cyanine dye has been significantly improved. It can be used to detect intracellular pH changes. It has a good application prospect in early diagnosis and imaging of tumors.

【学位授予单位】：中国科学院深圳先进技术研究院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R730.4;O657.3

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