Investigating Cellular Mechanisms and Photodynamic Therapy Using Molecular Rotors

许多生物过程都是以化学反应为基础的。粘度决定了分子扩散和反应的速度。因此，在细胞中，粘度会影响信号传递、运输和药物输送，而异常粘度与疾病和功能障碍有关。尽管它很重要，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，必须用光谱方法取而代之。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、或监测依赖于粘度的光化学反应的速率。然而，以上都是单点测量，在复杂的细胞异质环境中不能提供完整的信息。允许成像或绘制粘度的光谱方法将是非常有益的。这项提议旨在使用称为分子旋转器的新型荧光探针，以高精度和高空间分辨率测量和绘制单个细胞内的粘度。在分子旋转体中，荧光与分子内旋转竞争。在粘性环境中，自转速度变慢，这对荧光有很大影响。因此，可以通过检测荧光光谱或寿命的变化来测量粘度。现有技术允许在单个活细胞中以优异的空间分辨率对荧光光谱或寿命进行成像。到目前为止，我们已经用这种方法绘制了细胞某些部分的粘度图，并证明了这些隔室中的局部粘度可以比水高出100倍。分子旋转法的重要优势是测量时间非常短。利用这一优势，这一建议旨在监测细胞在动态生物过程中粘度的变化，例如细胞扰动、给药和细胞死亡时膜结构的变化。光动力疗法(PDT)是一种癌症治疗形式，它依赖于药物照射后细胞内产生短暂的有毒物质。这种治疗的效果关键取决于介质的粘度，细胞毒剂在其短暂的生命周期内必须通过介质扩散。这项提议将监测细胞粘度和其他重要生物物理细胞参数在光动力疗法期间的变化。我们方法的新奇之处在于使用了细胞内药物的空间分辨照射。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，我们可以对这组细胞进行辐射，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，当未直接治疗的细胞对治疗表现出显著的反应时，这种效应在放射治疗和光动力疗法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，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，并鼓励开发应用程序，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 Traditionally used mechanical methods are no longer applicable and must be substituted by a spectroscopic approach.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.