Aude Silve

Transport of siRNA through lipid membranes driven by nanosecond electric pulses: an experimental and computational study.

The use of small interfering RNA (siRNA) is a blossoming technique for gene regulation. However, its therapeutic potential is today severely hampered by the lack of an efficient means of safely delivering these nucleic acids to the intracellular medium. We report here that a single 10 ns high-voltage electric pulse can permeabilize lipid vesicles and allow the delivery of siRNA to the cytoplasm. Combining experiments and molecular dynamics simulations has allowed us to provide the detailed molecular mechanisms of such transport and to give practical guidance for the design of protocols aimed at using nanosecond-pulse siRNA electro-delivery in medical and biotechnological applications.

Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5′ overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi–mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5′-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3′ ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a “cut-and-close” mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics.

Conducting and permeable states of cell membrane submitted to high voltage pulses: mathematical and numerical studies validated by the experiments.

The aim of this paper is to present a new model of in vitro cell electropermeabilization, which describes separately the conducting state and the permeable state of the membrane submitted to high voltage pulses. We first derive the model based on the experimental observations and we present the numerical methods to solve the non-linear partial differential equations. We then present numerical simulations that corroborate qualitatively the experimental data dealing with the uptake of propidium iodide (PI) after millipulses. This tends to justify the validity of our modeling. Forthcoming work will be to calibrate the parameters of the model for quantitative description of the uptake.

Impact of external medium conductivity on cell membrane electropermeabilization by microsecond and nanosecond electric pulses

The impact of external medium conductivity on the efficiency of the reversible permeabilisation caused by pulsed electric fields was investigated. Pulses of 12 ns, 102 ns or 100 μs were investigated. Whenever permeabilisation could be detected after the delivery of one single pulse, media of lower conductivity induced more efficient reversible permeabilisation and thus independently of the medium composition. Effect of medium conductivity can however be hidden by some saturation effects, for example when pulses are cumulated (use of trains of 8 pulses) or when the detection method is not sensitive enough. This explains the contradicting results that can be found in the literature. The new data are complementary to those of one of our previous study in which an opposite effect of the conductivity was highlighted. It stresses that the conductivity of the medium influences the reversible permeabilization by several ways. Moreover, these results clearly indicate that electropermeabilisation does not linearly depend on the energy delivered to the cells.

Cell membrane permeabilization by 12-ns electric pulses: Not a purely dielectric, but a charge-dependent phenomenon.

Electric pulses of a few nanoseconds in duration can induce reversible permeabilization of cell membrane and cell death. Whether these effects are caused by ionic or purely dielectric phenomena is still discussed. We address this question by studying the impact of conductivity of the pulsing buffer on the effect of pulses of 12 ns and 3.2 MV/m on the DC-3F mammalian cell line. When pulses were applied in a high-conductivity medium (1.5 S/m), cells experienced both reversible electropermeabilization and cell death. On the contrary, no effect was observed in the low-conductivity medium (0.1 S/m). Possible artifacts due to differences in viscosity, temperature increase or electrochemical reactions were excluded. The influence of conductivity reported here suggests that charges still play a role, even for 12-ns pulses. All theoretical models agree with this experimental observation, since all suggest that only high-conductivity medium can induce a transmembrane voltage high enough to induce pore creation, in turn. However, most models fail to describe why pulse accumulation is experimentally required to observe biological effects. They mostly show no increase of permeabilization with accumulation of pulses. Currently, only one model properly describes pulse accumulation by modeling diffusion of the altered membrane regions.

Nanosecond-Duration Electric Pulse Delivery In Vitro and In Vivo: Experimental Considerations

Microsecond-duration electric pulses are widely used for research and therapeutic purposes. The most commonly used device for in vitro exposure to electric pulses is a standard electroporation cuvette. The use of nanosecond-duration electric pulses [nanosecond pulsed electromagnetic fields (nsPEFs) or nanopulses] is an emerging technology. For microsecond pulses, some dedicated generators with voltage regulation have been developed and allow one to easily impose a well-known voltage during biological experiments. However, it is not the case for nsPEF. The application of nsPEF thus requires considering the output impedance of the generator used and the impedance of the biological load because both impose the magnitude and the shape of the applied pulses. The model proposed here describes the behavior of the biological sample on a large frequency band (100 Hz to 3 GHz), including or not the exposure device. This model allows predicting the real electric pulses applied to biological samples and highlights the limitations induced by the use of standard electroporation cuvettes. Considering this kind of application device and the use of 10-ns-duration trapezoidal electric pulses, we evaluate the distortion of the pulses and also the impact of the output impedance of the generator on the steepness of the pulse rising and falling edges. This study is then transposed to potato tuber, a model currently used as a homogeneous biological tissue.

Electric pulses: a flexible tool to manipulate cytosolic calcium concentrations and generate spontaneous-like calcium oscillations in mesenchymal stem cells.

Human adipose mesenchymal stem cells (haMSCs) are multipotent adult stem cells of great interest in regenerative medicine or oncology. They present spontaneous calcium oscillations related to cell cycle progression or differentiation but the correlation between these events is still unclear. Indeed, it is difficult to mimic haMSCs spontaneous calcium oscillations with chemical means. Pulsed electric fields (PEFs) can permeabilise plasma and/or organelles membranes depending on the applied pulses and therefore generate cytosolic calcium peaks by recruiting calcium from the external medium or from internal stores. We show that it is possible to mimic haMSCs spontaneous calcium oscillations (same amplitude, duration and shape) using 100 μs PEFs or 10 ns PEFs. We propose a model that explains the experimental situations reported. PEFs can therefore be a flexible tool to manipulate cytosolic calcium concentrations. This tool, that can be switched on and off instantaneously, contrary to chemicals agents, can be very useful to investigate the role of calcium oscillations in cell physiology and/or to manipulate cell fate.

Different Approaches used in Modeling of Cell Membrane Electroporation

Cell electroporation is a complex phenomenon, which consists in the emergence of defects in cell membranes subjected to electric pulses. Since the end of the 90’s biophysical models have been developed to explain and predict the conditions for cell electroporation. However the recent biological data, in particular those dealing with the influence of the repetition rate of the pulses challenge these biophysical models. In this chapter we introduce different biophysical models of electropore formation and we discuss their mathematical basis and their advantages and disadvantages. We also present the phenomenological modeling, which consists in designing the model on an empirical basis thanks to the experience. The aim of the chapter is to introduce the reader to different ways of modeling cell membrane electroporation, and to provide some possible directions to obtain a more reliable theory of electroporation in accordance with the experiments and with a justified theoretical basis.

Dynamical modeling of tissue electroporation

In this paper, we propose a new dynamical model of tissue electroporation. The model is based on equivalent circuit approach at the tissue. Considering two current densities from cells and extracellular matrix, we identify the macroscopic homogenised contribution of the cell membranes. Our approach makes it possible to define a macroscopic homogenised electric field and a macroscopic homogenised transmembrane potential. This provides a direct link between the cell scale electroporation models and the tissue models. Finite element method adapted to the new non-linear model of tissue electroporation is used to compare experiments with simulations. Adapting the phenomenological electroporation model of Leguèbe et al. to the tissue scale, we calibrate the tissue model with experimental data. This makes two steps appear in the tissue electroporation process, as for cells. The new insight of the model lies in the well-established equivalent circuit approach to provide a homogenised version of cell scale models. Our approach is tightly linked to numerical homogenisation strategies adapted to bioelectrical tissue modeling.

Incubation time after pulsed electric field treatment of microalgae enhances the efficiency of extraction processes and enables the reduction of specific treatment energy

Pulsed Electric Field (PEF) pre-treatment, applied on fresh microalgae Auxenochlorella protothecoides, induces spontaneous release of a substantial water fraction and enables subsequent lipid extraction using ethanol-hexane blends. In this study, fresh microalgae suspensions were treated with PEF and incubated under inert conditions. Incubation promotes the release of ions and carbohydrates and increases the yields of subsequent lipid extraction thus enabling a considerable reduction of PEF-treatment energy. With a 20 h incubation period at 25 °C, almost total lipid extraction is achieved with a specific PEF-treatment energy of only 0.25 MJ/kgDW. Incubation on ice remains beneficial but less efficient than at 25 °C. Additionally, incubating microalgae cells in suspension at 100gDW/L or in a dense paste, was almost equally efficient. Correlation between the different results suggests that spontaneous release of ions and carbohydrates facilitates more successful lipid extraction. A direct causality between the two phenomena remains to be demonstrated.