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Final Report Summary - FLUMVI (Fluorescent macrocycles as functional viscosity probes in live cells)

Fluorescent macrocycles as functional viscosity probes in live cells

Introduction and Research Results
Viscosity is an important parameter determining the diffusion rate of species in condensed media. In biological systems, it can change as a consequence of disease, malfunction and cell death. In the last decades, several fluorescence-based methods have been developed for probing diffusion on the microscopic scale. Examples include fluorescence recovery after photobleaching, single particle tracking, and, more recently, molecular rotors.
Molecular rotors are a group of fluorophores in which fluorescence depends on intramolecular rotation. In media of high viscosity, intramolecular rotation is inhibited resulting in significant increase of the emission intensity and the fluorescence lifetime. The development of fluorescence lifetime-based sensors is desirable, because they can be used in fluorescence lifetime imaging (FLIM) as concentration independent probes to monitor viscosity in heterogeneous and dynamic systems such as cells. Thus, molecular rotors can be used to monitor viscosity changes under perturbation. For example, changes in viscosity have been observed during light induced cell death in photodynamic therapy (PDT) of cancer utilising a fluorescent molecular rotor.
PDT is used in the treatment of some types of cancer and relies on the photoexcitation of a red-absorbing photosensitizer (drug) and the subsequent production of short-lived cytotoxic species, in particular singlet molecular oxygen. Recent studies on PDT treatment of cells revealed a viscosity-dependent decrease in diffusion of different species in the cell.
The aim of this project was to develop novel classes of functional probes to measure viscosity in cells during normal cell function and PDT. To achieve these objectives, two types of tetrapyrrole macrocycles were selected: porphyrazines and porphycenes as red/NIR PDT sensitizers and emissive molecular rotors based on their fluorescence lifetime.
Porphyrazines have recently emerged as a very important class of tetrapyrroles suitable for PDT of cancer with excellent uptake and retention properties in vivo. One of the important challenges in PDT is the correct light dosimetry to achieve the most favourable treatment outcome. During my fellowship, I explored a new approach to the PDT dosimetry, based on fluorescence lifetime measurements of a new viscosity-sensitive porphyrazine-based dye termed ‘molecular rotor’ pz1.
The photophysical properties of the new porphyrazine pz1 were characterized by steady-state and time-resolved fluorescence spectroscopy in model systems, in a large range of viscosities ranging from 80 to 6000 cP. The results indicate that pz1 can be used as viscosity sensor based on its fluorescence lifetimes. Confocal images reveal a good cellular uptake for the dye and a remarkable increase in the fluorescence signal of the dye in cells. FLIM images were obtained from SKOV-3 cells incubated with pz1. Interestingly, a low correlation was observed between the fluorescence lifetimes obtained in cells and in the model systems. Therefore, the FLIM results could not be used to calculate the cellular viscosity based on the calibration curves obtained in glycerol:MeOH mixtures. However, the overall imaging experiments reveal a remarkable increase in the fluorescence signal and lifetime of pz1 in cells as compared to the weak signal observed in low viscosity solutions. This indicates that pz1 is located in a microenvironment of restricted mobility and it could be potentially used to monitor processes associated to changes in the local rigidity of the media, such as photoinduced cell death.
Thus, we investigated the potential use of the compound as PDT sensitizer in SKOV-3 cells. Selective irradiation of cells loaded with pz1 resulted in dramatic morphological changes in the cells, including cell contraction and detachment along with bubbles formation. A significant irradiation-dependent increase in the fluorescence lifetimes of the dye was observed. These results suggest that the viscosity of the irradiated cells increases due to phoinduced cell death, in agreement with previous results obtained for a porphyrin dimer.1
We conclude that pz1 is an ideal dual agent, which allows a simultaneous PDT treatment and the monitoring of its progress by fluorescence lifetime imaging. Confocal and fluorescence lifetime imaging microscopy (FLIM) experiments show fast uptake of pz1 into cells, as well as excellent photostability, strong fluorescence and negligible dark toxicity, which makes pz1 ideal for live cell imaging. I presented these results at 5 scientific conferences and I am the first author of a manuscript for a scientific journal.2
Next, I set out to study the dual use of porphycenes as molecular rotors and PDT sensitizers. Preliminary experiments indicated that compound TMPc exhibit molecular rotor properties3 and it was selected for this study for the following reasons: i) it is much more apolar than porphyrazines and a different intracellular location is expected, ii) it exhibits much stronger and red-shifted absorption in the visible region, iii) it might be sensible for a different viscosity range than porphyrazines, iv) porphycenes have large two-photon absorption cross-sections.
The photophysical properties of TMPc was measured in solvents of different viscosity. Although the fluorescence intensity is very small and the lifetimes are too short to obtain a viscosity calibration curve, the results in solution were promising to attempt cell experiments and explore the dual functionality of the porphycene as a molecular rotor and a PDT sensitizer. Thus, I developed a protocol to incubate and image SKOV-3 cells with TMPc. Our results confirmed that photoinduced cell death occurs in the presence of the dye using both one and two photon irradiation. However, it was not possible to get good FLIM signals from dying cells, due to significant photobleaching of the dye during PDT. We conclude that TMPc might be a molecular rotor but it is not suitable for monitoring the viscosity in SKOV-3 cells during PDT.
Finally, I investigated the use of macromolecules to increase the biocompatibility of fluorescence molecular rotors. I conducted a series of systematic photophysical experiments to characterize pz1 and other molecular rotors such as BODIPY derivatives in polymer brush. The results indicated that solubilisation of the dyes in aqueous solutions is increased in the presence of polymers and this might be of great interest for biological applications of the dyes. The compounds are currently being tested in vivo in mice cancer models in comparison with more water soluble derivatives (that I have studied during the first year of the Fellowship)4 that do not require polymer brushes to be used for in vivo injections (article in preparation).
Summary of the acquired competences
The excellent environment provided by Dr Marina K. Kuimova´s research group in a world-renowned research institution as Imperial College London is an essential step in my professional development as a scientist.
During my fellowship, I had excellent training opportunities to improve my experimental skills in photophysics, microscopy and cell culture. I have acquired laboratory skills to work with highly viscous systems and I have learnt to measure singlet oxygen, an important technique needed to establish the potential use of new drugs as PDT sensitizers. Very importantly, I have been trained in tissue culture, confocal microscopy and FLIM, including the use of different analysis software. This expertise has allowed me to independently conduct imaging experiments of biological samples and investigate the potential use of porphyrazines, porphycenes and other dyes as viscosity sensors and PDT sensitizers in cells.
Dr Kuimova’s group held one-to-one individual meetings as well as weekly meetings in which each member of the group gave presentations on their reasearch progress. This vibrant environment for learning has allowed me to enhance my scientific communication skills. I have also presented 3 oral communications and 2 posters at conferences, which have greatly contributed to enhance my external visibility.
Imperial College offers an extense selection of courses for staff covering safety matters, English language and professional development, I took courses to improve my communicating skills in English and I also attended several courses delivered by the Post-Doc Development Centre, which have been very helpful for my personal and career development as a scientist.
The Postdoct and Development Centre at Imperial also offers the possibility to participate as pannelist for mock interviews, which help postdocts to prepare for job interviews. I found this very useful for my professional development as I got very useful tips to prepare for the next steps in my career.
For one year, I was nominated group representative for the tissue culture and the wet chemistry labs. Through this responsibility, I learned to work with different services of the department, to troubleshoot problems and to improve my people management skills.
Last but not least, I demonstrated cell imaging experiments to collaborators from the groups of Dr Klapsina, Prof Vilar and Dr Vannier as well as members from the Kuimova group including the supervision of a summer student. This was an excellent practice to manage interpersonal relationships and improve time management and collaborative skills. Thus, along with my main project, I collaborated with Dr Lopez-Duarte and Dr Vu, both from Dr Kuimova’s group on the characterization of different BODIPY derivatives, which can be used to measure the viscosity of the plasma membrane4 and the internal membranes and as red-emitting temperature sensors (article in preparation). I also collaborated with the group of Prof Vilar in the characterization of molecular probes for the intracellular imaging of the G-quadruplex by FLIM.5

(1) Kuimova, M. K.; Botchway, S. W.; Parker, A. W.; Balaz, M.; Collins, H. A.; Anderson, H. L.; Suhling, K.; Ogilby, P. R. Nature Chem. 2009, 1, 69-73.
(2) Izquierdo, M. A.; Vyšniauskas, A.; Lermontova, S. A.; Grigoryev, I. S.; Shilyagina, N. Y.; Balalaeva, I. V.; Klapshina, L. G.; Kuimova, M. K. J. Mater. Chem. B 2015, 3, 1089-1096.
(3) Gil, M.; Dobkowski, J.; Wiosna-Sałyga, G.; Urbanska, N.; Fita, P.; Radzewicz, C.; Pietraszkiewicz, M.; Borowicz, P.; Marks, D.; Glasbeek, M.; Waluk, J. J. Am. Chem. Soc. 2010, 132, 13472-13485.
(4) López-Duarte, I.; Vu, T. T.; Izquierdo, M. A.; Bull, J. A.; Kuimova, M. K. Chem. Commun. 2014, 50, 5282.
(5) Shivalingam, A.; Izquierdo, M. A.; Le Marois, A.; niauskas, A. V. s.; Suhling, K.; Kuimova, M. K.; Vilar, R. Nat. Commun. 2015, 6, 1-10.

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