Francesco Cardarelli

Paolo Tentori1,2, Luca Pesce1, Fabio Azzarello1, Valentina De Lorenzi1, Giovanni Signore3, Andrea Camposeo4, Annalisa Carretta1, Gianmarco Ferri1, Pasqualantonio Pingue1, Stefano Luin1, Daniela Pozzi5, Enrico Gratton6, Fabio Beltram1,2, Giulio Caracciolo5, Francesco Cardarelli1

1- NEST, Scuola Normale Superiore, Pisa, Italy. 2- Center for Nanotechnology Innovation @NEST, Pisa, Italy. 3- Fondazione Pisana per la Scienza (FPS), Pisa, Italy. 4- NEST, Istituto Nanoscienze-CNR, Pisa, Italy. 5- Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy. 6- Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California at Irvine, USA

Nowadays, Fluorescence Lifetime Imaging Microscopy (FLIM) is an established tool to address molecular questions in vitro and in vivo, due to its exquisite sensitivity to the nanoscale environment of the emitter. Popular applications range from the study of molecular interactions to bio-sensing, from the measurement of the intrinsic live-cell/-tissue autofluorescence lifetime to its interpretation in terms of metabolic switch, differentiation, progression to a pathological state, etc. Based on this background, here we pursue an innovative line of research on nano-encapsulated compounds (e.g. drugs) and their biomedical application which builds on FLIM nanoscale sensitivity. In fact, exploiting the intrinsic fluorescence signal of the encapsulated compound, FLIM can be used to quantitatively extract information on its supramolecular organization within, for instance, a pharmaceutical formulation, from the production phase to the application in living matter. As first case-study, we selected DOXIL®, the liposomal formulation of the well-known anticancer drug Doxorubicin. By the fit-free, fully graphical phasor approach to-FLIM data, the signature of DOXIL® was first resolved into the contribution of three co-existing drug species, each with its characteristic mono-exponential lifetime, namely: crystallized drug, free drug, and drug bound to the liposomal membrane. Then, the exact molar fractions of the three species are determined by combining phasor-FLIM with quantitative absorption/fluorescence spectroscopy on pure standards. Finally, preliminary data on the application of DOXIL® in living cells are presented and interpreted in terms of time evolution of drug bioavailability and supramolecular state. To prove the potency of the proposed approach, additional nano-encapsulated compounds are presented alongside their application in different fields of nanomedicine, from the treatment of Diabetes to neurodegenerative diseases, from cancer to metabolic disorders. This work has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 866127, project CAPTUR3D).