Agnese Denzi

Feasibility for Microwaves Energy to Affect Biological Systems Via Nonthermal Mechanisms: A Systematic Approach

The understanding of possible nonthermal bio-effects has been an open question during the last five decades. In this paper, the authors present a critical literature review of the models of the interaction mechanisms, together with an overview of all the publications finding positive results for in vitro and in vivo studies. The systematic approach consisted of pooling together the positive studies on the basis of the endpoints and the biological systems, to identify specific plausible targets of the action of the electromagnetic fields and the related pathways. Such a classification opens the way to the discussion of some hypotheses of interaction mechanisms considered as first transduction step. The authors conclude that only through a multiscale methodology it is possible to perform a comprehensive study of the nonthermal effects, based on affordable and realistic in silico models.

Broadband Electrical Detection of Individual Biological Cells

To resolve the dilemma of cell clogging or solution parasitics encountered by Coulter counters and to evolve a general-purpose electrical detection technique, we used broadband microwave measurements to overcome electrode polarization, ac dielectrophoresis to precisely place cells between narrowly spaced electrodes, and relatively wide microfluidic channels to prevent cell clogging. This unique combination of approaches resulted in reproducible sensing of single Jurkat and HEK cells, both live and dead, of different cultures at different times.

Assessment of Cytoplasm Conductivity by Nanosecond Pulsed Electric Fields

The aim of this paper is to propose a new method for the better assessment of cytoplasm conductivity, which is critical to the development of electroporation protocols as well as insight into fundamental mechanisms underlying electroporation. For this goal, we propose to use nanosecond electrical pulses to bypass the complication of membrane polarization and a single cell to avoid the complication of the application of the “mixing formulas.” Further, by suspending the cell in a low-conductivity medium, it is possible to force most of the sensing current through the cytoplasm for a more direct assessment of its conductivity. For proof of principle, the proposed technique was successfully demonstrated on a Jurkat cell by comparing the measured and modeled currents. The cytoplasm conductivity was best assessed at 0.32 S/m and it is in line with the literature. The cytoplasm conductivity plays a key role in the understanding of the basis mechanism of the electroporation phenomenon, and in particular, a large error in the cytoplasm conductivity determination could result in a correspondingly large error in predicting electroporation. Methods for a good estimation of such parameter become fundamental.

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

BackgroundBreast 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.MethodsThis 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.ResultsThe 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.ConclusionsThe 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 Microdosimetric Study of Electropulsation on Multiple Realistically Shaped Cells: Effect of Neighbours

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.

Single Cell Microdosimetric Studies Comparing Ideal and Measured Nanosecond Pulsed Electric Fields

Recently, the promising effects induced by pulsed electric fields with high intensity and short duration have been highlighted. At the nanosecond time scale, electric pulse targets become both the plasmatic membrane and the sub-cellular structures (possibility of intracellular manipulation). In this paper, a circuit cell model with nucleus is presented, validated and used in order to assess the different cellular and sub-cellular (i.e. nucleus) effects, comparing ideal nanosecond pulsed electric fields (nsPEFs) waveforms with the ones measured from a planar, broadband matched microchamber.

Electropermeabilization of Inner and Outer Cell Membranes with Microsecond Pulsed Electric Fields: Quantitative Study with Calcium Ions

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 Ca2+ as a permeabilization marker, we observed cytosolic Ca2+ peaks in two different cell types after one 100 µs 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 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 Ca2+ roles in cell physiology.

Signal transduction on enzymes: the Effect of electromagnetic field stimuli on superoxide dismutase (SOD)

Protein functions and characteristics can highly differ from physiological conditions in presence of chemical, mechanical or electromagnetic stimuli. In this work we provide a rigorous picture of electric field effects on proteins behavior investigating, at atomistic details, the possible ways in which an external signal can be transduced into biochemical effects. Results from molecular dynamics (MD) simulations of a single superoxidismutase (SOD) enzyme in presence of high exogenous alternate electric fields will be discussed.

Effects of nanosecond pulsed electric fields on the activity of a Hodgkin and Huxley neuron model

The cell membrane poration is one of the main assessed biological effects of nanosecond pulsed electric fields (nsPEF). This structural change of the cell membrane appears soon after the pulse delivery and lasts for a time period long enough to modify the electrical activity of excitable membranes in neurons. Inserting such a phenomenon in a Hodgkin and Huxley neuron model by means of an enhanced time varying conductance resulted in the temporary inhibition of the action potential generation. The inhibition time is a function of the level of poration, the pore resealing time and the background stimulation level of the neuron. Such results suggest that the neuronal activity may be efficiently modulated by the delivery of repeated pulses. This opens the way to the use of nsPEFs as a stimulation technique alternative to the conventional direct electric stimulation for medical applications such as chronic pain treatment.

Smart flexible planar electrodes for electrochemotherapy and biosensing

Electroporation is an effective method to deliver drugs into tumor cells to kill them, by applying a pulsed electric field to the cellular membrane. Existing electrodes consist of clamping claws or arrays of needles and can be effectively applied only to small areas. New electrodes that can treat large areas are sought; flexibility is needed to adapt to irregular tumor shape and, to be folded to enter from small surgical opening. In this work we present the design and test of a 16 cm2 flexible electrode for electroporation with biosensing capabilities, built with standard flexible circuit technologies enclosed in a biocompatible package. The electrode contains electronics to provide cryptography-based identification to the electroporation machine to avoid setup errors and protection against use of counterfeited electrodes. In-vitro tests of the electrode show that electroporation occurs up to a depth of 8 mm with 100% electroporation efficiency over the 30% of electrode area. Temperature rise on the electrode during treatment does not exceed 6 degrees celsius, a value that not causes damage to the cells.