Targeted pH switched europium(III) complexes for monitoring receptor internalisation in living cells
Jack D. Fradgley 1 and David Parker 1 1 Durham University, Lower Mountjoy
The ability to study intracellular pH variations with spatial and temporal control is crucial for the investigation of numerous biological processes, such as internalisation and endosomal uptake. pH is an ideal parameter for monitoring these processes given the varied pH gradient across cellular compartments. Receptor mediated endocytosis is a ubiquitous mode of uptake into cells and can be induced by agonist binding at a membrane surface receptor. 1 Following internalisation, endosomes and phagosomes can age and evolve into lysosomes during their natural cell cycle. Throughout this period, pH tends to fall with time. 2 Such processes can be followed in real-time if the internalised species is labelled with an appropriately responsive luminescent sensor. In this work, we present the design, evaluation, and development of a series of pH responsive europium(III) sensors (from prototype to final probe) for the purpose of monitoring receptor mediated internalisation in real time. 3-4 These pH dependent sensors display dramatic ‘switching on’ of luminescence and emission lifetime on acidification (akin to a light switch). The magnitude of this emission enhancement (the ‘switch-on ratio’) can be increased further still through the use of time-gated measurements. Through appropriate synthetic modifications, we demonstrate how suitable functionality can be incorporated into our sensors to permit conjugation, for example to a targeting vector or a receptor. Finally, we establish the efficacy of these sensors through specific membrane receptor labelling experiments targeting GLP-1R, a receptor protein targeted by anti-diabetic drugs, and monitor its agonist induced internalisation. Comparative analysis against appropriate commercial pH-sensitive dyes reveals the improved signal and specificity of our sensors, indicating the substantial promise of these systems in membrane receptor internalisation assays. In collaboration with industry, pharmacological studies are currently underway to identify the exact context where these bright pH responsive sensors are best applied as a commercial product. 5 References 3. M. Starck, J. D. Fradgley , R. Pal, J. M. Zwier, L. Larmarque and D. Parker, Chem. Eur. J. 2021 , 27, 766-777. 4. J. D. Fradgley , M. Starck, M. Laget, E. Bourrier, E. Dupuis, L. Lamarque, E. Trinquet, J. M. Zwier and D. Parker, Chem. Commun . 2021 , 57, 5814-5817. 5. “Europium complexes as pH sensors”, European Patent Application 20306296.3, Filed Oct 2020. 1. J. L. Goldstein, R. G. W. Anderson and M. S. Brown, Nature 1979 , 279, 679-685. 2. J. R. Casey, S. Grinstein and J. Orlowski, Nat. Rev. Mol. Cell Biol. 2010 , 11,50-61.
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© The Author(s), 2022
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