In the last years, microdosimetric numerical models of cells including intracellular compartments have been proposed, aiming to investigate the poration induced by the application of nanosecond pulsed electric fields (nsPEFs). A limitation of such models was the extremely approximate cell and organelle shapes, leading to an incorrect estimation of the electric field or transmembrane potential distribution in the studied domain. In order to obtain a reliable model of in vitro experiments and a one-to-one comparison between experimental and simulated results, here, a realistic model of 12 human mesenchymal stem cells was built starting from their optical microscopy images where different cell compartments were highlighted. The microdosimetric analysis of the cells group was quantified in terms of electric field and transmembrane potentials (TMPs) induced by an externally applied 10-ns trapezoidal pulse with rise and fall times of 2 ns, with amplitudes ranging from 2 to 30 MV/m. The obtained results showed that the plasma and endoplasmic reticulum (ER) membrane of each cell respond in a different way to the same electric field amplitude, depending on differences in shape, size, and position of the single cell with respect to the applied electric field direction. Therefore, also the threshold for an efficient electroporation is highly different from cell to cell. This difference was quantitatively estimated through the cumulative distribution function of the pore density for the plasma and ER membrane of each cell, representing the probability that a certain percentage of membrane has reached a specific value of pore density. By comparing the dose-response curves resulted from the simulations and those from the experimental study of De Menorval et al. (2016), we found a very good matching of results for plasma and ER membrane when 2% of the porated area is considered sufficient for permeabilizing the membrane. This result is worth of noting as it highlights the possibility to effectively predict the behavior of a cell (or of a population of cells) exposed to nsPEFs. Therefore, the microdosimetric realistic model described here could represent a valid tool in setting up more efficient and controlled electroporation protocols.

The study of the frequency-dependent response of a biological cell and its organelles to an intense electric field is of growing interest in the bioelectromagnetic area. The possibility offered by continuous waves to uncouple the effects of the electric field during electroporation is employed in this work using a numerical model including the realistic shape of cell and endoplasmic reticulum membranes. The higher efficiency of the microwave signals in electroporating the subcellular structures, in comparison with the radiofrequency signals, is highlighted.

Background: Molecular mechanisms of interaction between cells and extremely low frequency magnetic fields (ELF-MFs) still represent a matter of scientific debate. In this paper, to identify the possible primary source of oxidative stress induced by ELF-MF in SH-SY5Y human neuroblastoma cells, we estimated the induced electric field and current density at the cell level. Methods: We followed a computational multiscale approach, estimating the local electric field and current density from the whole sample down to the single cell level. The procedure takes into account morphological modeling of SH-SY5Y cells, arranged in different topologies. Experimental validation has been carried out: neuroblastoma cells have been treated with Diphenyleneiodonium (DPI) -an inhibitor of the plasma membrane enzyme NADPH oxidase (Nox)- administered 24 h before exposure to 50 Hz (1 mT) MF. Results: Macroscopic and microscopic dosimetric evaluations suggest that increased current densities are induced at the plasma membrane/extra-cellular medium interface; identifying the plasma membrane as the main site of the ELF-neuroblastoma cell interaction. The in vitro results provide an experimental proof that plasma membrane Nox exerts a key role in the redox imbalance elicited by ELF, as DPI treatment reverts the generation of reactive oxygen species induced by ELF exposure. General significance: Microscopic current densities induced at the plasma membrane are likely to play an active physical role in eliciting ELF effects related to redox imbalance. Multiscale computational dosimetry, supported by an in vitro approach for validation, is proposed as the innovative and rigorous paradigm to unveil mechanisms underlying the complex ELF-MF interactions.

When investigating the biophysical effects induced by the interaction between electromagnetic fields and biological cells, it is crucial to estimate the electromagnetic field intensity at the microscopic scale (microdosimetry). This information allows to find a connection between the external applied field and the observed biological event required to establish related biomedical applications. Here, authors present a microdosimetric study based on a 2D realistic model of a cell and its endoplasmic reticulum. The microdosimetric analysis of the cell and endoplasmic reticulum was quantified in terms of electric field and transmembrane potential induced by an externally applied high amplitude 10-ns pulsed electric field. In addition, electroporated local membrane sites and pore densities were also evaluated. This study opens the way to numerically assist experimental applications of nanosecond pulsed electric fields for controlled bio-manipulation of cells and subcellular organelles.

A coplanar broadband and efficient system was developed for real-time exposure of in vitro neuronal samples to electromagnetic fields in the microwave range to test their effectiveness in nervous stimulation.

The increasing interest towards biocompatible nanotechnologies in medicine, combined with electric fields stimulation, is leading to the development of electro-sensitive smart systems for drug delivery applications. Common examples of electro-sensitive materials include phospholipids that can be used to design nano-sized vesicles suitable for external electric actuation. To this regard, recently the use of pulsed electric fields to trigger release across phospholipid membranes has been numerically studied, for a deeper understanding of the phenomena at the molecular scale. Aim of this work is to give an experimental validation of the feasibility of controlling drug release from liposomes mediated by nanosecond pulsed electric fields.

The use of dielectric response as a tool for studying molecular and cellular parameters is a well-established approach on continuing attention. A valuable approach, which is used to gather inside mechanisms of dielectric response and to provide the average electrical parameters of both the cell membrane and the intra-cellular and extracellular medium, is represented by the modeling one. In this paper we will describe some advances in models able to describe the dielectric behavior of different bio-systems, in particular: molecular models for Na+ and Cl-ion in water and water in proximity of charged surfaces and cell continuum microdosimetric models.

The electromagnetic fields application as a way to enhance biomedical techniques has recently drawn the attention of the scientific community, together with the possibility to use biocompatible nanocarriers to safely transport therapeutic substances along the body. For this reason, microdosimetric studies represent a fundamental tool to deeply understand biological effects and improve drug delivery nanosystems methods. With this regard, the behaviour of liposomes, biological vesicles with a bilayered membrane similar to cell membrane, are often under investigation in order to both use them as electromagnetic fields triggerable nanocarriers and as simple model able to mimick cellular membrane response. In this work, 2D microdosimetric analyses on single liposomal vesicles, with different dimensions from the nanometer to the micrometer scale, have been carried out studying the feasibility to increase biological membrane permeability through radiofrequency fields signals. At the end, the response of a 2D model of 142 randomly placed nanosized liposomes has been proposed as a more realistic drug delivery systems suspension.

In the study of the biophysical mechanisms at the basis of the EM field interaction leading to biomedical applications, one needs also to know the field strength at the microscopic scale to establish a quantitative relation between the field and the observed effect. Here authors will show the results on the TMP of the plasma membrane and the endoplasmic reticulum (ER) due a 2D realistic shaping of the ER itself, both analysed in the frequency domain and for an electric pulsed field of 10 ns.

Microsecond pulsed electric fields (μsPEF) 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 µs pulse without affecting the cell viability. Indeed, using Ca 2+ as a permeabilization marker, we observed cytosolic Ca 2+ peaks in two different cell types after one 100 µs pulse in a medium without Ca 2+ . Thapsigargin abolished these Ca 2+ 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 Ca 2+ 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 µsPEF, which are easier to control than nsPEF, can permeabilize internal membranes. Besides, μsPEF interaction with either the PM or ER, can be an efficient tool to modulate the cytosolic calcium concentration and study Ca 2+ roles in cell physiology.

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 1–3ns 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.

Electroporation of Jurkat T-lymphoma human cells was investigated using 10-MHz continuous waves and bench-marked against that at 100 kHz. Both cell poration and cell death were simultaneously monitored by fluorescence microscopy, and found to occur under approximately four times higher voltages at 10 MHz than that at 100 kHz. This weaker-than-expected increase in poration threshold could be explained by detailed analysis of the distributed effect often ignored in electroporation studies.

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 structure.

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 pulse.

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.

Over the past decades, the effects of ultrashort-pulsed electric fields have been used to investigate their action in many medical applications (e.g. cancer, gene electrotransfer, drug delivery, electrofusion). Promising aspects of these pulses has led to several in vitro and in vivo experiments to clarify their action. Since the basic mechanisms of these pulses have not yet been fully clarified, scientific interest has focused on the development of numerical models at different levels of complexity: atomic (molecular dynamic simulations), microscopic (microdosimetry) and macroscopic (dosimetry). The aim of this work is to demonstrate that, in order to predict results at the cellular level, an accurate microdosimetry model is needed using a realistic cell shape, and with their position and packaging (cell density) characterised inside the medium.

In the last decades, the effects of ultrashort pulsed electric fields have been investigated demonstrating their capability to be involved in a great number of medical applications (e.g. cancer, gene electrotransfer, drug delivery, electrofusion). In particular, experiments in literature demonstrate that internal structures can be involved when pulse duration is reduced. Up to now, the mechanism that permits the electroporation phenomenon has not been completely understood and hence atomistic, microdosimetry and dosimetry models have been developed to help in this field. Aim of this work is to demonstrate the importance of realistically model also the internal organelles to obtain predictive results of effects at sub-cellular level with a microdosimetry model.

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 used.

In the last decades, the advances in the micro and nano fabrication techniques have led to the development of microdevices that improved the possibility of analysis at cell level. These devices can be used in different applications (e.g., cell detection and identification, manipulation, cell treatments). The requisites, that are necessary to achieve, are different for various applications and represent the starting point of the project. The numerical multiphysics models can be very advantageous to analyze the performances of such devices and to predict their operation. Aim of this work is to give a look of the design rules of microchamber devices in particular for their application in electric field exposure. Two different applications for cell discrimination and characterization are reported considering time and frequency domain measurements.

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 negligible.