【作者】 刘阳；

【导师】 刘志强；

【作者基本信息】 山东大学 ， 材料学， 2024， 博士

【摘要】 细胞器及其间质的体液环境被称为细胞微环境,细胞内的生理/病理过程通常伴随着细胞微环境的动态变化。细胞微环境参数包括极性、pH值、黏度、温度、质子浓度和氧气速率等,这些参数的细微变动可对细胞器间的交流与互作或对整个细胞的生长与代谢产生巨大影响。当细胞受到病理因素影响、自由基作用等外界刺激时,会导致细胞微环境稳态失衡从而引起细胞行为异常,甚至会诱发糖尿病、脂肪肝、癌症等恶性疾病。因此,对细胞内微环境参数变化的定量监测可及时发现并干预细胞病理过程,还可促进相关疾病的诊断和临床研究。微环境稳态失衡导致相关病理过程的发生/发展可以追溯到细胞器层面的行为和理化性质异常,所以对细胞器微环境的可视化具有重大意义。由于细胞微环境是高度复杂和动态化的系统,多种参数相互交织,荧光信号受多种因素影响,使得分析和结论复杂。利用小分子荧光探针通过荧光成像实时观察和动态监测生理/病理过程中细胞器及其微环境的变化有着广阔的应用前景。为了深入研究不同细胞器微环境参数在生命活动中的变化与规律,探索相关病理过程的发展机制,开发特异性可视化不同细胞器微环境参数变化的荧光探针需求迫切。本文基于芘和四苯乙烯荧光团,通过引入不同的取代基优化结构设计,构建了比率响应型和荧光增强型的微环境敏感小分子荧光探针,通过荧光信号响应实现了对不同细胞器微环境参数变化的监测。1.线粒体通过内膜上的电子传递链产生质子梯度实现细胞内的能量存储,并维持着生命活动的正常进行。鉴于维持线粒体正常功能结构依赖于稳定的质子梯度,本论文提出了构建质子/非质子环境敏感型荧光探针策略,合成了一组比率响应型线粒体荧光探针(PQL-H、PQL-Cl和PPD),以实现线粒体形态和质子环境变化的灵敏监测。PQL-H在质子溶剂和非质子溶剂中荧光信号的差异性表现出对微环境质子浓度监测的巨大潜力。PQL-H的正电荷结构使其能够双通道特异性的靶向线粒体,利用双色成像可监测线粒体在不同病理条件下的形态学信息。在外界环境刺激下,线粒体内部质子分布发生紊乱,膜结构遭受到不可逆的损伤,产生断裂、肿胀和空泡化等形态变化。通过PQL-H的比率成像能够可视化质子环境变化并反映出线粒体的状态,这对线粒体质子环境稳态相关生理过程探索和临床医学诊断具有重要参考意义。2.溶酶体和脂滴之间通过相变接触进行有效的物质交换和信息交流,在一定条件下溶酶体会选择性地降解脂滴引发脂噬过程。为实现多视角研究脂噬,开发了一组比率响应型的脂滴和溶酶体双靶标极性敏感型荧光探针(PTZ、PTZ-OH和PTZ-Me),可同时响应脂噬中的极性变化和相变过程。PTZ具有平衡的亲脂/亲水特性,能够跟踪异质性溶液的液-液相分离过程,并通过比率荧光发射响应溶剂极性变化。利用PTZ双色成像脂滴和溶酶体,成功地追踪了溶酶体和脂滴发生脂噬过程的极性变化和形态学信息,并观察到这两个细胞器相互作用的关键接触模式,从细胞器行为和理化性质两方面阐明了脂噬过程。重要的是发现了一种未知的脂滴与溶酶体之间的均质融合,这为脂噬过程的理解提供了新的研究视角。该成果有望为脂噬相关疾病的临床诊断和治疗提供参考。3.极性作为细胞微环境的重要参数之一,在维持微环境稳态平衡方面发挥着至关重要的作用。许多极性相关疾病的发生和发展不仅反映在特定细胞器上,整个细胞质极性的可视化更有助于理解多细胞器相关病理过程和生命活动。为寻找可同时观察多个细胞器极性的荧光探针,本文提出了工程化分子结构策略,设计了一系列比率响应型的多细胞器靶向极性敏感荧光探针(1P2N、1P3N、4P2N和1PP)。探针的荧光强度比值与溶剂的介电常数之间表现出Logistic函数关系,这种敏感的极性响应使得探针在高极性亲水环境中发出绿色荧光,而在低极性脂质细胞器中表现出红色荧光。探针1P2N可通过比率荧光成像可视化不同生理/病理条件下多个细胞器的极性变化,在细胞凋亡模型中,线粒体和溶酶体等细胞器极性增加;而在细胞内脂质代谢紊乱模型中细胞质极性呈现出总体上升的趋势。总之,这项工作为研究多细胞器极性相关的生物过程提供了一个有效工具,并为微环境极性敏感荧光探针和生物传感器的构建提供了新的设计思路。4.考虑到引入AIE基团是制备在聚集态和高浓度状态下有效发光探针的方法,本论文采用拓展芳环、固化结构策略,构建了一组具有AIE特性的荧光增强型脂滴黏度敏感探针(TPE-BA-H、TPE-BA-OMe、TPE-BA-CN 和 TPE-BA-CF3)。探针的结构和性能与取代基类型密切相关,不同苯胺衍生物取代基极大地影响了分子间的堆积模式,从而影响了探针的光物理特性。由电子给体取代的TPE-BA-OMe表现出最佳的结构稳定性和良好的亲脂性,而电子受体取代的化合物则表现出较差的稳定性。探针TPE-BA-OMe具有较高的量子产率和显著的AIE特性,对溶剂黏度的增加表现出荧光增强信号。得益于上述优点,TPE-BA-OMe能够对活细胞中的脂滴进行特异性生物成像,并对脂滴的黏度升高表现出荧光增强响应。本研究为构建微环境敏感的AIE探针提供了可行的分子设计策略。本论文开发的荧光探针可实现细胞内微环境参数变化的监测、细胞器形态学信息的记录和多种病理过程的长程追踪,促进了对生命活动中微环境参数变化规律的理解,有助于相关疾病的诊断和临床研究。所提出的荧光探针设计策略可为微环境敏感相关探针的构建提供重要参考。更多还原

【Abstract】 The humoral environment of the organelles and their interstation is called the cellular microenvironment,and physiologic/pathologic processes within the cell are typically accompanied by dynamic changes in the cellular microenvironment.Cellular microenvironmental parameters include polarity,pH,viscosity,temperature,proton concentration,and oxygen rate,etc.Even minor variations in these parameters can have a dramatic effect on organelle communication and interactions,or on the growth and metabolism of the entire cell.When cells are affected by pathological factors,free radical action and other external stimuli,there may be an imbalance in the cellular microenvironmental homeostasis thus causing abnormal cellular behavior,and even induce malignant diseases such as diabetes mellitus,fatty liver,and cancer.Therefore,quantitative monitoring of changes in intracellular microenvironmental parameters allows for timely detection and intervention in cellular pathological processes,and also facilitates clinical research and diagnosis of relevant diseases.The imbalance of microenvironmental homeostasis leading to the onset/development of relevant pathological processes can be traced back to abnormalities of behavioral and physicochemical properties at the organelle level,making the visualization of the organelle microenvironment of great significance.Since cellular microenvironments are highly complex and dynamic systems with multiple parameters intertwined,and the fluorescence signals are influenced by a variety of factors,complicating analysis and conclusions.Using small molecule fluorescent probes to observe and dynamically monitor the changes of organelles and their microenvironment during physiological/pathological processes in real time by fluorescence imaging has a broad application prospect.In order to deeply study the changes and patterns of unique microenvironmental parameters of different organelles in life activities,and to explore the development mechanism of related pathological processes,there is an urgent need to develop fluorescent probes that specifically visualize the changes of microenvironmental parameters of different organelles.In this dissertation,ratiometric-responsive or fluorescenceenhanced microenvironment sensitive fluorescent probes were constructed based on pyrene and tetraphenylethylene fluorophores by introducing different substituents to optimize the structural design,and monitoring the changes of microenvironmental parameters of different organelles was achieved by fluorescence response.1.Mitochondria realize intracellular energy storage through the generation of a proton gradient by the electron transport chain in the inner membrane and maintain normal life activities.Considering that maintaining the normal functional structure of mitochondria depends on a stable proton gradient,this thesis proposes a strategy for constructing protic/aprotic environment-sensitive ratiometric fluorescent probes,and synthesizes a set of ratiometric-responsive mitochondrial protic environment-sensitive fluorescent probes(PQL-H,PQL-CI,and PPD)for sensitive monitoring of mitochondrial morphology and changes in the protic environment.The different fluorescence signals of PQL-H in protic and aprotic solvents show great potential for monitoring environmental proton concentrations.The positively charged structure of PQL-H enables dual-channel specific targeting of mitochondria,and dualchannel imaging with PQL-H can be utilized to monitor mitochondrial morphology under different pathological conditions.Under external environmental stimuli,the internal proton distribution of mitochondria is disturbed and the membrane structure suffers irreversible damage,leading to morphological changes such as fracture,swelling and vacuolization.Ratiometric imaging by PQL-H is able to visualize changes in the protic environment and reflect the state of mitochondria,which is an important reference for the exploration of physiological processes related to the homeostasis of the mitochondrial protic environment and for clinical medical diagnosis.2.The effective exchange of substances and information between lysosomes and lipid droplets occurs through phase transition contacts,and under certain conditions lysosomes will selectively degrade the lipid droplets to trigger the process of lipophagy.To realize a multiperspective study of the lipophagy process,a set of ratiometric-responsive polarity-sensitive fluorescent probes(PTZ,PTZ-OH,and PTZ-Me)for lipid droplet and lysosomal dualtargeting have been developed,which can respond to both polarity changes and phase transition processes during lipophagy.PTZs with balanced lipophilic/hydrophilic properties are capable of tracking the liquid-liquid phase separation process of heterogeneous solutions and responding to changes in solvent polarity through ratiometric fluorescence emission.Dual-color imaging of lipid droplets and lysosomes using PTZ successfully tracked the polarity changes and morphological alterations of lysosomes and lipid droplets during lipophagy,and observed the key contact patterns of the interaction between these two organelles,elucidating the lipophagy process in terms of organelle behavior and physicochemical properties.Importantly,an unknown homogeneous fusion between lipid droplets and lysosomes was discovered,which provides a new research perspective for understanding the lipophagy process.The results are expected to provide an important reference for the clinical diagnosis and treatment of lipophagy-related diseases.3.Polarity,as one of the important parameters of the cellular microenvironment,plays a crucial role in maintaining the homeostatic balance of the microenvironment.The occurrence and development of many polarity-associated diseases are not only reflected in specific organelles,but the visualization of whole cytoplasmic polarity is more helpful to understand the pathologies process and life activities associated with multiple organelles.To find appropriate fluorescent probes to monitor the polarity of multiple organelles simultaneously,this dissertation proposes an engineered molecular structure strategy to design a series of ratiometric responsive multicellular organelle targeted polarity-sensitive fluorescent probes(1P2N,1P3N,4P2N,and 1PP).The probes exhibits a Logistic function between the fluorescence intensity ratio and the dielectric constant(ε)of the solvent,and this sensitive polar response allows the probe to emit green fluorescence in highly polar hydrophilic environments and red fluorescence in low-polar lipid organelles.The 1P2N could visualize the polarity changes of multiple organelles under different physiological/pathological conditions by ratiometric fluorescence imaging.In the apoptosis model,the polarity of organelles such as mitochondria and lysosomes was increased;whereas,the cytoplasmic polarity in the intracellular lipid metabolism disorders model showed an overall increasing trend.In conclusion,this work provides an effective tool for the study of cell polarity-related biological processes and a new design idea for the construction of microenvironment-sensitive fluorescent probes and biosensors.4.Considering that the introduction of AIE groups is an effective way to prepare luminescent probes in the aggregated and highly concentrated states,this thesis used the strategy of expanding the aromatic ring and curing the structure to construct a set of fluorescence-enhanced lipid droplet viscosity-sensitive fluorescent probes(TPE-BA-H,TPEBA-OMe,TPE-BA-CN,and TPE-BA-CF3)with AIE properties.The structure and properties of the probes are closely related to the type of substituent,and different aniline derivative substituents greatly affect the intermolecular stacking pattern and thus the photophysical properties of the probes.TPE-BA-OMe substituted by the electron donor exhibits the best structural stability and good lipophilicity,whereas electron acceptor-substituted compounds show poor stability.The TPE-BA-OMe has a high quantum yield and remarkable AIE properties,and exhibits a fluorescence-enhanced signal to the increase of solvent viscosity.Benefiting from these advantages,the TPE-BA-OMe is capable of specific bioimaging of lipid droplets in living cells and exhibits fluorescence-enhanced response to the viscosity increase of lipid droplets.This study provides a feasible molecular design strategy for the construction of microenvironment-sensitive AIE probes.In this dissertation,the fluorescent probes developed can realize the monitoring of intracellular microenvironmental parameter changes,the recording of organelle morphology information and the long-range tracking of multiple pathological processes,which promotes the understanding of the changing law of microenvironmental parameters during life activities,and contributes to the diagnosis and clinical research of related diseases.The proposed fluorescent probes design strategy will provide an important guideline for the construction of microenvironmental sensitive probes.更多还原