Electropermeabilization of inner and outer cell membranes with microsecond pulsed electric field. Quantitative study with calcium ions

Microsecond pulsed electric fields (mu sPEF) permeabilize the plasma membrane (PM) and are widely used in research, medicine and biotechnology. For internal membranes permeabilization, nanosecond pulsed electric fields (nsPEF) are applied but this technology is complex to use. Here we report that the endoplasmic reticulum (ER) membrane can also be electropermeabilized by one 100 mu s pulse without affecting the cell viability. Indeed, using Ca2+ as a permeabilization marker, we observed cytosolic Ca2+ peaks in two different cell types after one 100 mu s pulse in a medium without Ca2+. Thapsigargin abolished these Ca2+ peaks demonstrating that the calcium is released from the ER. Moreover, IP3R and RyR inhibitors did not modify these peaks showing that they are due to the electropermeabilization of the ER membrane and not to ER Ca2+ channels activation. Finally, the comparison of the two cell types suggests that the PM and the ER permeabilization thresholds are affected by the sizes of the cell and the ER. In conclusion, this study demonstrates that mu sPEF, which are easier to control than nsPEF, can permeabilize internal membranes. Besides, mu sPEF interaction with either the PM or ER, can be an efficient tool to modulate the cytosolic calcium concentration and study Ca2+ roles in cell physiology

Technological and theoretical aspects for testing electroporation on liposomes

Recently, the use of nanometer liposomes as nanocarriers in drug delivery systems mediated by nanoelectroporation has been proposed. This technique takes advantage of the possibility of simultaneously electroporating liposomes and cell membrane with 10-nanosecond pulsed electric fields (nsPEF) facilitating the release of the drug from the liposomes and at the same time its uptake by the cells. In this paper the design and characterization of a 10 nsPEF exposure system is presented, for liposomes electroporation purposes. The design and the characterization of the applicator have been carried out choosing an electroporation cuvette with 1 mm gap between the electrodes. The structure efficiency has been evaluated at different experimental conditions by changing the solution conductivity from 0.25 to 1.6 S/m. With the aim to analyze the influence of device performances on the liposomes electroporation, microdosimetric simulations have been performed considering liposomes of 200 and 400 nm of dimension with different inner and outer conductivity (from 0.05 to 1.6 S/m) in order to identify the voltage needed for their poration

Modelling the positioning of single needle electrodes for the treatment of breast cancer in a clinical case

Background: Breast cancer is the most common cancer in women worldwide and is the second most common cause of cancer death in women. Electrochemotherapy (ECT) used in early-phase clinical trials for the treatment of primary breast cancer resulted in a not complete tumor necrosis in most cases. The present study was undertaken to analyze the feasibility to use ECT to treat patients with histologically proven unifocal ductal breast cancer. In particular, results of ECT treatment in a clinical case are compared with the ones of a simplified 3D dosimetric model. Methods: This clinical study was conducted with the pulse generator Cliniporator Vitae (IGEA, Carpi, Italy). ECT procedures were performed according to ESOPE standard operating procedures. Five single needle electrodes were used with one positioned in the center of the tumor, and the other four distributed around the nodule. Histological images of the resected tumor are compared with the maps of the electric field obtained with a simplified 3D model in Comsol Multiphysics v 4.3. Results: The results of the clinical case demonstrated a reduced efficacy of the ECT treatment described. The proposed simple numerical model of the breast tumor located in a low conductive tissue suggests that this is due to the reduced electric field induced inside the tumor with such 5 electrodes placement. However, where the electric field is predicted higher than the reversible electroporation threshold (E > 400 V/cm), also the histological images confirm the necrosis of the target with a good agreement between the modeled and clinical results. Conclusions: The results suggest the dependence of the effectiveness of the treatment on the careful placement of the electrodes. A detailed planned procedure for the tumor analysis after the treatment is also needed in order to better correlate the single electrode positions and the histological images. Simulation models could be used to identify better electrodes configuration in planning the experimental protocol for ECT treatment of breast tumors

A numerical design of versatile microchambers for nsPEFs experiments

The emergence of nanosecond pulsed electric fields (nsPEFs) for intracellular electro-manipulation experiments implies the application of extremely short (ns) high intensity (MV/m) electric field pulses. Specific pulse generators and miniaturized applicators are necessary to properly deliver this category of voltage signals to biological loads. In this context, we propose the design of a versatile nsPEFs applicator, developed following the guidelines typical of microwave propagating systems. The designed microchamber is suitable for in vitro exposure to undistorted pulses with duration down to 1-3 ns during single and multi cell experiments. Further features are: high efficiency (above 0.95), high cell viability by the integration of microfluidic components, real time monitoring of the biological sample and of the pulse propagation. These features can be considered as designing rules for new nanosecond and sub-nanosecond applicators, to ensure experimental repeatability and reproducibility when the impact of propagation on pulse signals is no more negligibl

Numerical estimation of a 10 nanosecond pulse effects on non-uniformly distributed liposomes

Nano-systems, often used in biomedical applications for the treatment of a broad category of illnesses, represent one of the nanomedicine approaches recently proposed to target specific drugs only in the region where the disease has been developed. Recently the use of this technique has been proposed with electropulsation, hence taking advantage of the enhanced permeabilization of the cell membrane and simultaneously control the release of the encapsulated drug by the nano-system. In this work, we focus our attention on the study of liposomes nano-systems controlled by the nanosecond pulses electric fields through a microdosimetric approach. The aim is to analyse the electric field necessary to porate a non-uniform distribution of 400 nm liposomes. The work has been carried out by randomly placing 30 liposomes between two electrodes with the application of a 10 nano-second electric field puls

A computational design of a versatile microchamber for in vitro nanosecond pulsed electric fields experiments

The emergence of nanosecond pulsed electric fields (nsPEFs) for intracellular manipulation experiments requires the use of specific miniaturized applicators. We propose the design of a versatile nsPEFs applicator, based on microwave propagating systems, suitable for in vitro exposure to undistorted 1-3 ns pulses in single and multi-cell experiments. Further features of the proposed devices are: high efficiency, microfluidic integration, real time monitoring of the biological sample and of the pulse propagation. Generally, these features can be considered as specific requisites for nanosecond applicators, to ensure experimental repeatability and reproducibility, when propagation related phenomena cannot be considered negligible

Numerical estimation of a 10 nanosecond pulse effects on non-uniformly distributed liposomes

Nano-systems, often used in biomedical applications for the treatment of a broad category of illnesses, represent one of the nanomedicine approaches recently proposed to target specific drugs only in the region where the disease has been developed. Recently the use of this technique has been proposed with electropulsation, hence taking advantage of the enhanced permeabilization of the cell membrane and simultaneously control the release of the encapsulated drug by the nano-system. In this work, we focus our attention on the study of liposomes nano-systems controlled by the nanosecond pulses electric fields through a microdosimetric approach. The aim is to analyse the electric field necessary to porate a nonuniform distribution of 400 nm liposomes. The work has been carried out by randomly placing 30 liposomes between two electrodes with the application of a 10 nano-second electric field pulse

Innovative Flexible Electrodes for Electroporation

Electroporation is one of the most effective and still best promising electric-based techniques in anticancer therapy. In this context the possibility that the tumoral tissue is completely wrapped from the electrode seems strategical to increase the effectiveness of the treatments. In this work the possibility of a flexible electrode, based on MEMS technology and able to reach some millimetres penetration below the electrode surface is shown

Microdosimetry for pulsed e fields in a realistic models of cells and endoplasmic reticulum

Microsecond pulsed electric fields (μsPEFs) with amplitude of tens of kV/m are used to permeabilize the plasma membrane whereas nanosecond pulsed electric fields of MV/m also permeabilize cell internal structures, such as the endoplasmic reticulum. In this work, a numerical realistic model of cell and its reticulum has been realized to study the use of μsPEFs also for the permeabilization of this internal structur

Exploring the applicability of nano-poration for remote control in smart drug delivery systems

Smart drug delivery systems represent an interesting tool to significantly improve the efficiency and the precision in the treatment of a broad category of diseases. In this context, a drug delivery mediated by nanosecond pulsed electric fields seems a promising technique, allowing for a controlled release and uptake of drugs by the synergy between the electropulsation and nanocarriers with encapsulated drugs. The main concern about the use of electroporation for drug delivery applications is the difference in dimension between the liposome (nanometer range) and the cell (micrometer range). The choice of liposome dimension is not trivial. Liposomes larger than 500 nm of diameter could be recognized as pathogen agents by the immune system, while liposomes of smaller size would require external electric field of high amplitudes for the membrane electroporation that could compromise the cell viability. The aim of this work is to theoretically study the possibility of a simultaneous cell and liposomes electroporation. The numerical simulations reported the possibility to electroporate the cell and a significant percentage of liposomes with comparable values of external electric field, when a 12 nsPEF is use