Monitoring the molecular composition of live cells exposed to electric pulses via label-free optical methods

Abstract

the permeabilization of the live cells membrane by the delivery of electric pulses has fundamental interest in medicine, in particular in tumors treatment by electrochemotherapy. Since underlying mechanisms are still not fully understood, we studied the impact of electric pulses on the biochemical composition of live cells thanks to label-free optical methods: confocal Raman microspectroscopy and terahertz microscopy. A dose effect was observed after cells exposure to different field intensities and a major impact on cell peptide/protein content was found. Raman measurements reveal that protein structure and/or environment are modified by the electric pulses while terahertz measurements suggest a leakage of proteins and other intracellular compounds. We show that Raman and terahertz modalities are a particularly attractive complement to fluorescence microscopy which is the reference optical technique in the case of electropermeabilization. finally, we propose an analytical model for the influx and efflux of non-permeant molecules through transiently (electro)permeabilized cell membranes. The main consequence of the delivery of high intensity pulsed electric fields (PEF) of very short duration on biological samples is the permeabilization of the plasma membrane 1. This interaction between PEF and biological samples, termed electropermeabilization or electroporation, has led to many applications in industry or medicine, in particular using 100 microsecond pulses (µsPEF). For instance, electrochemotherapy consists in the combination of tumor cells electropermeabilization and a chemotherapeutic agent such as bleomycin or cisplatin drugs 2,3. Electrochemotherapy is nowadays commonly used for the treatment of many cancer types: skin cancer 4 , breast cancer 5 , head and neck cancer 6 , pancreatic cancer 7 , etc. Although electropermeabilization has been studied for decades, the underlying mechanisms are still not fully understood. It is well established that the electric field induces an additive transmembrane potential to the resting transmembrane potential of cells 8. Molecular dynamics simulations 9,10 have demonstrated that the creation of pores into the membrane is a stochastic phenomenon: the increase of the external field amplitude increases the probability of formation of the pores. Therefore, to ensure that pores occur within the duration of the applied pulses, appropriated field amplitudes must be used, a situation that led to the definition of functional threshold amplitudes by the experimentalists. The destabilization of the cell membrane is also associated with long-term effects on the membrane, such as membrane disorder and a decrease of membrane elasticity, 11,12 that occur minutes after the PEF delivery. Recently, we considered biochemical modifications of the membrane as the major contributor to the electropermeabilization process. Mass spectrometry analysis of the chemical composition of a simple membrane model, Giant Unilamellar Vesicles (GUV), exposed to PEF showed the peroxidation of phospholipids ...

Terahertz Spectroscopy Sheds Light on Real‐Time Exchange Kinetics Occurring through Plasma Membrane during Photodynamic Therapy Treatment

“…Nowadays, THz‐ATR measurements are only performed at a fixed frequency, but thanks to technological developments, broadband terahertz time‐domain spectroscopy systems are now commercially available and can be used for THz‐ATR, so that much more detail about the size and chemical nature of the molecules in the cytosol will be obtained using THz‐TDS‐ATR. THz spectroscopy is, therefore, a very efficient, promising and complementary technique to the established biological tests in the study of short‐term modifications of plasma membrane permeability after chemical [ 8,10 ] or electrical [ 11 ] treatments. This technique could also be applied more generally to monitor the state of cells (cell differentiation, cancer or healthy cells) or to study the adaptation of cells to new environments (cell structure and integrity, hydration imbalance, glycogen storage, ion channel related diseases, etc.)…”

“…Recent publications have demonstrated the possibility of studying complex biological systems such as cells and tissues without altering samples and without staining or specific preparation of the sample, in applications such as cancer tissue imaging and diagnosis, cell identification, and electroporation. [2][3][4][5][6][7][8][9][10][11][12][13][14][15] Understanding permeabilization processes through plasma cell membranes lies at the core of numerous topics in biology, in particular with regard to pathologies and medical treatments. Moreover, the dynamic aspect of such exchanges is essential, as developing efficient therapies implies the control of timing.…”

“…Recent publications have demonstrated the possibility of studying complex biological systems such as cells and tissues without altering samples and without staining or specific preparation of the sample, in applications such as cancer tissue imaging and diagnosis, cell identification, and electroporation. [ 2–15 ]…”

High precision dual-modulation differential terahertz ATR sensor for liquid measurements

“…The detection of small variations in the terahertz properties of solutes would also benefit the study of living cells, which have a terahertz dielectric constant difference of a few percent between the inside of the cell and its surrounding medium. [2,[4][5][6]. However, since water absorption is high, these small variations are often hidden by the instability of the sensor.…”

“…To detect very low concentration levels or very small sample volumes, many types of sensors have been demonstrated: meta-atoms [17], microfluidics [18], gratings [19], chemical sensing plate [20], Fabry-Pérot [21], ring resonators [22], etc. Particular attention must also be paid to long-term stability, especially for studies on biological systems [4][5][6].…”