兰州大学机构库 >化学化工学院
基于自毁结构的荧光探针设计合成及其生物应用
Alternative TitleDesign, synthesis and biological application of fluorescent probes based on self-destruct structure
张金龙
Subtype博士
Thesis Advisor张海霞
2023-05-29
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name理学博士
Degree Discipline化学
Keyword硫化氢,荧光探针,自毁化学,毒性评价,诊疗一体,活体成像,可视化 Hydrogen sulfide fluorescent probe self-destruct chemistry toxicity evaluation integrated diagnosis and treatment in vivo imaging visualization
Abstract

在荧光成像技术的发展历程中,荧光探针具有灵敏度高、准确度高、选择性强、检测方便快捷等优点,多用于生命科学领域。大量的研究探索了多种类型的荧光探针并用于检测生物标记物以此实现疾病的监测,但实际上只有少数荧光探针能够实现商业化并广泛的应用于医学临床,荧光探针的研究仍处于拓宽荧光探针的用途和完善实际应用的阶段。自毁化学能够通过响应刺激释放部分结构,其结构通常包含特定的自毁间隔基团,它将部分触发分子共价连接到感兴趣的底物,如荧光基团、药物和其它化合物。本论文以自毁反应为基础,通过改造自毁间隔基团的方式不仅使荧光探针实现检测目的,还增加了其它的功能。开展的研究工作如下:

(1)硫化氢(H2S)作为一种参与自噬的信号分子,被认为在疾病的发生和治疗中至关重要。为了阐明H2S在疾病过程中的作用,迫切需要H2S的动态可视化检测。我们构建了一种水溶性近红外(发射波长为695 nm)自毁型荧光探针CySO3N3,用于H2S的检测。探针与H2S反应后能够发生结构的裂解,不仅能够释放荧光团,而且脂水分布系数大幅改变,这种设计自毁设计策略能够提高探针的代谢能力,进而降低探针的毒性。该探针不仅可用于活细胞和小鼠体内H2S的检测,可以检测炎症和心肌损伤时H2S的变化,也可以通过监测H2S的变化监测细胞自我修复过程。

(2)在生物检测中,探针自身对所检测的生物体造成的毒性往往被忽视,迫切需要在保证检测性能的前提下降低探针的毒性。我们构建了基于1,8-萘酰亚胺结构的分析物补偿荧光探针(NP-SN3)用于H2S的检测。这种补偿型设计策略是将H2S供体结构加载到H2S探针上作为自毁间隔基可以补充检测过程中消耗的H2S。当补偿型探针被H2S触发后,不仅会释放荧光团实现检测目的,还会释放可被碳酸酐酶催化产生H2S的自毁间隔基团,实现H2S消耗-补偿的平衡。通过HepG2细胞、斑马鱼胚胎和幼鱼图像的实验结果发现,新探针与传统探针(NP-N3)在成像性能上没有明显差异。将活化的HSC-T6细胞成像后,新探针显示出较低的检测诱导毒性。在斑马鱼胚胎发育和大鼠连续给药过程中,新探针显示斑马鱼胚胎死亡率低、孵化率高、畸形率低。染色大鼠组织切片同样显示新探针造成的病理症状较轻。

(3)为了更好地了解和实时监测H2S的抗癌作用,设计并合成了一种基于自毁反应释放H2S的近红外荧光诊疗探针YH-NO2。该探针被与肿瘤相关且过表达的硝基还原酶激活后释放近红外荧光团作为检测手段,并且释放包含H2S供体的自毁间隔基团作为治疗手段,实现对癌症的诊疗一体。YH-NO2不仅可以区分不同程度缺氧下的硝基还原酶活性,还可以通过成像识别正常细胞和癌细胞。在细胞中验证YH-NO2可以稳定释放H2S后,在癌细胞和活体研究当中发现YH-NO2不但能够选择性的清除癌细胞,而且还能够准确识别和消除活体中的肿瘤组织。这些结果表明,YH-NO2的诊疗机制不仅可以监测活体当中H2S的抗癌作用,而且对研究H2S与癌症的关系具有重要的推动作用。

(4)根据自毁结构,开发了一种将荧光探针,H2S和非甾体抗炎药(NSAIDs)萘普生结合在一起的新型诊疗探针Nap-NP-NSB。该诊疗剂可被与炎症相关且过表达的ROS激活后发生自毁反应导致结构裂解,同时靶向释放H2S、萘普生和荧光团达到诊疗的目的。通过对细胞和组织上清液的荧光分析,Nap-NP-NSB实现了H2S和NSAIDs之间协同作用的可视化检测。在细胞和活体当中,Nap-NP-NSB均表现出比萘普生更强的抗炎作用和更低的胃黏膜损伤副作用。将荧光探针、H2S和NSAIDs结合在一起展示了原子经济化,提供了研究药物协同作用的新方法,拓展了荧光探针的应用前景。

Other Abstract

In the development of fluorescence imaging technology, fluorescent probes have the advantages of high sensitivity, high accuracy, strong selectivity, convenient and fast detection, etc., and are mostly used in the field of life sciences. A large number of studies have explored various types of fluorescent probes and used them to detect biomarkers to achieve disease monitoring, but in fact only a few fluorescent probes can be commercialized and widely used in clinical medicine. The research of fluorescent probes Still in the stage of broadening the use of fluorescent probes and perfecting practical applications. Self-destruct chemistry enables the release of moiety structures in response to stimuli, often containing specific self-destruct spacers, which covalently link moiety-triggering molecules to substrates of interest, such as fluorophores, drugs, and other compounds. In this thesis, based on the self-destruct reaction, the fluorescent probe not only achieves the purpose of detection, but also adds other functions by modifying the self-destruct spacer. The research work carried out is as follows:

(1) Hydrogen sulfide (H2S), as a signaling molecule involved in autophagy, is considered to be crucial in the occurrence and treatment of diseases. To elucidate the role of H2S in disease processes, the dynamic visual detection of H2S is urgently needed. We constructed a water-soluble near-infrared (emission wavelength of 695 nm) self-destructing fluorescent probe CySO3N3 for the detection of H2S. It not only verified the ability of the probe to detect H2S, but also improved the metabolic ability of the probe and reduced the toxicity of the probe. The probe can not only be used to detect H2S in living cells and mice, but also detect changes in H2S during inflammation and myocardial injury, and monitor cell self-repair processes by monitoring changes in H2S.

(2) In biological detection, the toxicity of the probe itself to the detected organism is often ignored, and it is urgent to reduce the toxicity of the probe under the premise of ensuring the detection performance. We constructed an analyte-compensated fluorescent probe (NP-SN3) based on the structure of 1,8-naphthoimide for the detection of H2S. Through the experimental results of HepG2 cells, zebrafish embryos and juvenile images, it was found that there was no significant difference in imaging performance between the new probe and the traditional probe (NP-N3). After imaging activated HSC-T6 cells, the new probe showed lower detection-induced toxicity. During zebrafish embryo development and continuous dosing in rats, the new probe showed low embryonic mortality, high hatchability, and low malformation rates in zebrafish embryos. Stained tissue sections of rats also showed less pathological symptoms caused by the new probe.

(3) In order to better understand and monitor the anticancer effect of H2S in real time, a near-infrared fluorescent diagnostic probe YH-NO2 based on the self-destruct reaction to release H2S was designed and synthesized. After the probe is activated by the overexpressed nitroreductase in the tumor, it releases near-infrared fluorophore and H2S simultaneously to realize the function of diagnosis and treatment of cancer. YH-NO2 can not only distinguish nitroreductase activity under different degrees of hypoxia, but also identify normal cells and cancer cells by imaging. After verifying that YH-NO2 can stably release H2S in cells, it was found that YH-NO2 can not only selectively eliminate cancer cells, but also accurately identify and eliminate tumor tissues in vivo. These results indicate that the diagnosis and treatment mechanism of YH-NO2 can not only monitor the anticancer effect of H2S in vivo, but also play an important role in promoting the relationship between H2S and cancer.

(4) According to the self-destruct structure, a novel diagnostic probe Nap-NP-NSB combining fluorescent probe, H2S and non-steroidal anti-inflammatory drug (NSAIDs) naproxen was developed, which can be associated with inflammation Moreover, the overexpressed ROS releases H2S, naproxen and fluorophores at the same time after activation to achieve the purpose of diagnosis and treatment. Through fluorescence analysis of cell and tissue supernatants, Nap-NP-NSB enables the visual detection of synergy between H2S and NSAIDs. In cells and in vivo, Nap-NP-NSB showed stronger anti-inflammatory effect and lower gastric mucosal damage side effects than naproxen. Combining fluorescent probes, H2S, and NSAIDs demonstrates atom economy, provides a new way to study drug synergy, and expands the application prospects of fluorescent probes.

Subject Area荧光分析
MOST Discipline Catalogue理学 - 化学 - 分析化学
URL查看原文
Language中文
Other Code262010_120200904701
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/538948
Collection化学化工学院
Affiliation
兰州大学化学化工学院
Recommended Citation
GB/T 7714
张金龙. 基于自毁结构的荧光探针设计合成及其生物应用[D]. 兰州. 兰州大学,2023.
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