The open questions we now address are: 1) to understand the effect of membrane composition and domains, 2) to determine the uptake process of molecules according to their size and charge and 3) to explore the interaction of electromagnetic fields (Pillet et al. 2018 RSC Advance) as well as ns pulsed electric field with membranes.

Investigators: Technician, L. Hellaudais; Engineer : O. Cordier, Project leader: M-P Rols

Collaborators: D Dean (LPT Toulouse), R. Vézinet (CEA, Gramat), P. Levêque (Xlim, Limoges)

Membrane Organization and Dynamics, and intracellular transport

Cells in culture have revealed more complex ways involved in molecule electro-mediated uptake than GUVs can do with the puzzling notion of competent micro-domains for molecule uptake. We indeed observed years ago alteration in membrane organization and dynamics, in particular flip-flop of lipids, which may be involved in molecule uptake (Escoffre et al. 2014 Biochim Biophys Acta).

A key question about therapeutic molecules concerns their subsequent distribution into the cells. This is of high importance in the case of DNA since expression requires traffic, i.e. translocation of DNA across the membrane, migration through the cytoplasm and finally passage through the nuclear envelope. We previously showed actin polymerisation at the membrane sites where DNA interacts with (Rosazza et al. 2011 Mol Ther). We visualized the intracellular traffic of single molecules thanks to a collaboration with a group leader in the field (A. Zumbusch). We attested the evidence for endocytosis and endosomal trafficking of DNA, showing for the first time, the different routes and complexity of the phenomenon of molecules uptake by pulsed electric field according to their size and charge (Rosazza et al. 2016 Mol Ther Nucleic Acids). We showed that gene expression highly depends on electric field parameters (de Caro et al. 2023 Pharmaceutics). The questions we now address aim to improve the efficiency of nucleic acid delivery by the development of new protocols of pulses delivery with the aim to reduce muscle contraction and pain.

We have increased the current knowledge on the mechanisms underlying membrane perturbations via transmembrane voltage modifications and radiofrequencies, by providing a description of the processes at the cellular and tissue levels, including the impact of electric fields membrane receptor functions. We have also used microwave biosensor to monitor and characterize single cells subjected to electroporation and obtain an electronic signature of the treatment efficiency (Tamra et al. 2022 IEEE Trans Biomed Eng).

Last but not least, we now explore the role of extracellular vesicles released following pulses application in molecules electrotransfer. Our expertise in electroporation lead us to collaborate with a physician with the aim to establish the proof of concept of the use of nebulized extracellular vesicles, loaded with mRNA by electroporation, to obtain a cystic fibrosis transmembrane conductance regulator expression in a high challenging pathological pulmonary context (ANR Evade project).

Investigators: Former PhD students: A. Calvel, A. de Caro, C. Rosazza, A. Tamra; Engineer: G. Alberola, F Talmont; Researcher: M. Golzio, Project leader: M-P Rols

Collaborations: A. Zumbush (University of Konstanz, Germany), D. Miklavcic, A. Lekbar (University of Ljubljana, Slovenia), D. Dubuc and K. Grenier (LAAS, Toulouse), J.-B. Leroy (LEROYBiotech, Saint-Orens de Gameville); T. Montier (CHU, Brest), M. Morille (Montpellier)

From Cells to Tissues: 3D cell cultures

Multicellular spheroids are 3D cell culture models that mimic the behavior of cells in complex, organized systems like tissues. They provide a more accurate representation of tissue environments compared to isolated cells, taking into account cell-cell interactions, the extracellular matrix, and the accessibility of molecules. Years ago, we demonstrated that spheroids could be used as an innovative and convenient approach to study the mechanisms of electropermeabilization in tissues (Wasungu et al. 2009, Int J Pharmaceutics; Gibot et al. 2013, J Control Release). We observed, similar to patient outcomes, that normal cells are less sensitive than tumor cells to electrochemotherapy (ECT), which involves the administration of anticancer drugs (such as bleomycin or cisplatin) followed by the local delivery of high-voltage electric pulses (Frandsen et al. 2015, PLOS One).

In the NUMEP project, involving computer scientists (Inria MONC) and radiologists (CHU J. Verdier), we gained new insights into the biological understanding and numerical modeling of electroporation effects on tumors and tumor models. This collaboration aims to develop numerical tools enriched with biological knowledge to enhance the clinical applications of electroporation in cancer treatment (Collin et al. 2022, AIMS Bioengineering). It is crucial to determine the differences between healthy cells, proliferative cancer cells, and quiescent cancer cells, as well as the influence of electric pulses on cell regrowth in 2D and 3D models. We investigate and quantify the efficiency of ECT and gene electrotransfer on spheroids composed of human tumor cells, healthy cells, or both.

Our ongoing project MECI, funded by Plan Cancer, began in February 2022 and will run for three years. This project involves the same partners (Inria MONC, the Interventional Radiology unit of AP-HP, Avicenne) and the Radiology and Nuclear Medicine service of the University Hospital of Poitiers (Tasu et al. Diagn Interv Imaging, 2022).