Investigating Cellular Mechanisms and Photodynamic Therapy Using Molecular Rotors

许多生物过程都是基于化学反应。粘度决定了分子扩散和反应的速度。因此，在细胞中，粘度可以影响信号传导、运输和药物递送，并且异常粘度与疾病和功能障碍有关。尽管其重要性，但在单个细胞的尺度上测量粘度是一个挑战。传统上使用的机械方法不再适用，必须由光谱方法代替。存在这样的光谱方法，例如单粒子跟踪，监测光漂白后的荧光恢复速率，或监测粘度依赖性光化学反应的速率。然而，上述所有测量都是单点测量，并且在复杂的异构环境中不能提供小区的全部信息。允许粘度成像或绘图的光谱方法将是非常有益的。该提案旨在使用称为分子转子的新型荧光探针以高精度和高空间分辨率测量和映射单个细胞内的粘度。在分子转子中，荧光与分子内旋转竞争。在粘性环境中，旋转减慢，这强烈影响荧光。因此，粘度可以通过检测荧光光谱或寿命的变化来测量。现有技术允许在单个活细胞中以优异的空间分辨率成像荧光光谱或寿命。到目前为止，我们已经使用这种方法在细胞的某些部分绘制了粘度图，并证明这些区域的局部粘度可以比水的粘度高100倍。分子转子方法的重要优点是测量时间非常短。利用这一优势，该建议旨在监测动态生物过程中细胞粘度如何变化，例如细胞扰动、药物给药和细胞死亡时膜结构的变化。光动力疗法（PDT）是癌症治疗的一种形式，其依赖于药物照射后细胞内产生的短寿命毒性剂。这种治疗的有效性关键取决于介质的粘度，细胞毒性剂在其短暂的寿命期间必须通过该介质扩散。该建议将监测细胞粘度和其他重要的生物物理细胞参数在PDT期间如何变化。我们的方法的新奇在于使用细胞内药物的空间分辨辐射。例如，我们可以照射单个细胞器并监测整个细胞的变化。或者，我们可以照射这组细胞并监测其邻居的行为。这种方法是直接探测“旁观者效应”的理想工具，当没有被直接处理的细胞对治疗显示出显著的反应时，这种效应在辐射和PDT癌症治疗中非常重要。这项建议将在伦敦帝国理工学院化学系进行，在那里建立了多学科合作，以确保拟议工作的成功。该项目将解决光化学和细胞生物学中的基本科学问题，并鼓励应用程序的开发，例如测量粘度作为诊断工具和监测治疗进展。

Many biological processes are based on chemical reactions. Viscosity determines how fast molecules can diffuse, and react. Therefore in cells viscosity can affect signalling, transport and drug delivery, and abnormal viscosity has been linked to disease and malfunction. In spite of its importance, measuring viscosity on a scale of a single cell is a challenge. Traditionally used mechanical methods are no longer applicable and must be substituted by a spectroscopic approach. Such spectroscopic approaches exist, e.g. single particle tracking, monitoring the rate of fluorescence recovery after photobleaching, or monitoring the rate of viscosity-dependent photochemical reactions. However all of the above are single point measurements and in a complex heterogeneous environment of a cell can not provide full information. The spectroscopic approach which allows imaging or mapping of viscosity would be of great benefit. This proposal aims to measure and map viscosity inside a single cell with high precision and high spatial resolution using novel fluorescent probes, called molecular rotors. In molecular rotors fluorescence competes with intramolecular rotation. In a viscous environment rotation is slowed down and this strongly affects fluorescence. Thus viscosity can be measured by detecting the change in either the fluorescence spectra or lifetimes. Existing technology allows imaging of either the fluorescent spectra or lifetimes with excellent spatial resolution in single live cells. To date we have produced maps of viscosity in certain parts of cells using this approach and demonstrated that local viscosity in those compartments can be up to 100x higher than that of water.Important advantage of molecular rotor approach is a very short measurement time. Using this advantage, this proposal aims to monitor how viscosity in a cell changes during dynamic biological processes, e.g. change in the membrane structure upon cell perturbation, drug administration and cell death.Photodynamic therapy (PDT) is a form of cancer treatment, which relies on the generation of short-lived toxic agents within a cell upon irradiation of a drug. The efficacy of this treatment critically depends on the viscosity of the medium through which the cytotoxic agent must diffuse during its short life span. This proposal will monitor how cell viscosity and other vital biophysical cell parameters change during PDT. The novelty of our approach is in using spatially resolved irradiation of the drug within cells. E.g. we can irradiate a single organelle and monitor the change in the entire cell. Alternatively, we can irradiate the group of cells and monitor the behaviour of its neighbours. This approach is ideal tool to directly probe the 'bystander effect', when the cells which have not been directly treated show significant response to therapy, the effect which is very important in radiation and PDT cancer treatment. This proposal will be carried out in the Chemistry Department at Imperial College London where multidisciplinary collaborations are established to ensure the success of the work proposed. This project will address both the fundamental scientific issues in photochemistry and cell biology and also encourage the development of applications, such as measuring viscosity as a diagnostic tool and for monitoring the progress of treatments.