Investigating Cellular Mechanisms and Photodynamic Therapy Using Molecular Rotors

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 Traditionally used mechanical methods are no longer applicable and must be substituted by a spectroscopic approach.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.

My impact plan is built on the fact that the novel imaging methodology proposed here has the potential to transform the way viscosity monitoring is done with applications across biology, chemistry, engineering and materials science. It has applications in high throughput screening, is compatible with microfluidic technology, and, most importantly, it opens up the possibility of doing completely novel experiments on single cell and tissue levels which can widen the horizons in biological sciences. In addition, since there are correlations established between abnormal levels of viscosity at both cell and tissue levels and diseases, such as atherosclerosis, diabetes, Alzheimer's disease and malignancy, our unique imaging technique could be developed as an important diagnostic tool in medical practice. As always in moving new scientific tools from concept to usage there is a need to provide substantial proof demonstrating reliability and capability and the proposed work will concentrate on these goals to maximise impact of this research. To achieve the highest possible impact of this research we will engage in the following activities: (1) Fostering new links I am pursuing this in three ways: (i) Peer-reviewed academic publications will continue to be the team's gold-standard for dissemination of our scientific research. We will ensure that our research is published in high quality journals with good impact factors; where possible we will publish in journals with a broad readership. I believe that this is a highly effective way to reach potential beneficiaries of our work, academic as well as commercial. I have so far published 6 papers on molecular rotors and viscosity measurements, including a publication in the first edition of Nature Chemistry (2009) and three publications in J. Amer. Chem. Soc. (2008 and 2 x 2009). Several of these papers are already attracting many citations. (ii) I will continue to present the team's research at conferences and workshops. As the community learns more about the opportunities afforded by molecular rotors, so the demand on our expertise increases. This is an excellent way to establish new collaborations and broaden the application base. (iii) I will host academic visitors, and extend it to interested industrial parties when the opportunity arises. As these collaborations mature, opportunities to broaden the research and to commercialise applications will materialise. (2) Managing collaborations Academic partnerships and collaborative projects will be managed by the PI. Regarding possible industrial collaborations we will be assisted in these plans by Imperial Innovations, which has very strong records in technology transfer and is actively engaged with the commercialization of research. As I become aware of any new possibilities in the application of this research I will actively pursue commercialization in partnership with Imperial Innovations. (3) Driving the dissemination The PI will lead the dissemination through lectures at conferences, workshops and invited talks at universities. Wherever appropriate, a post-doctoral researcher and a PhD student will be encouraged to contribute by giving talks and presenting posters at conferences. In conjunction with publication in peer-reviewed journals, we will put out press-releases to accompany publication of significant findings. Because of the breadth of applications stemming from the current Proposal and their potential downstream benefits I am confident that there will be a lot of interest to our research from the broader community. (4) Training The training provided to a postdoctoral researcher and a student in this broad multidisciplinary field is an important outcome of this project, and one that will have a long-term impact, not least because they provide an important route for disseminating and propagating the technology.