Nataša Pavšelj

Dependence of induced transmembrane potential on cell density, arrangement, and cell position inside a cell system

A nonuniform transmembrane potential (TMP) is induced on a cell membrane exposed to external electric field. If the induced TMP is above the threshold value, cell membrane becomes permeabilized in a reversible process called electropermeabilization. Studying electric potential distribution on the cell membrane gives us an insight into the effects of the electric field on cells and tissues. Since cells are always surrounded by other cells, we studied how their interactions influence the induced TMP. In the first part of our study, we studied dependence of potential distribution on cell arrangement and density in infinite cell suspensions where cells were organized into simple-cubic, body-centered cubic, and face-centered cubic lattices. In the second part of the study, we examined how induced TMP on a cell membrane is dependent on its position inside a three-dimensional cell cluster. Finally, the results for cells inside the cluster were compared to those in an infinite lattice. We used numerical analysis for the study, specifically the finite-element method (FEM). The results for infinite cell suspensions show that the induced TMP depends on both cell volume fraction and cell arrangement. We established from the results for finite volume cell clusters and layers, that there is no radial dependence of induced TMP for cells inside the cluster.

Efficiency of High- and Low-Voltage Pulse Combinations for Gene Electrotransfer in Muscle, Liver, Tumor, and Skin

Gene electrotransfer is gaining momentum as an efficient methodology for nonviral gene transfer. In skeletal muscle, data suggest that electric pulses play two roles: structurally permeabilizing the muscle fibers and electrophoretically supporting the migration of DNA toward or across the permeabilized membrane. To investigate this further, combinations of permeabilizing short high-voltage pulses (HV; hundreds of V/cm) and mainly electrophoretic long low-voltage pulses (LV; tens of V/cm) were investigated in muscle, liver, tumor, and skin in rodent models. The following observations were made: (1) Striking differences between the various tissues were found, likely related to cell size and tissue organization; (2) gene expression is increased, if there was a time interval between the HV pulse and the LV pulse; (3) the HV pulse was required for high electrotransfer to muscle, tumor, and skin, but not to liver; and (4) efficient gene electrotransfer was achieved with HV field strengths below the detectability thresholds for permeabilization; and (5) the lag time interval between the HV and LV pulses decreased sensitivity to the HV pulses, enabling a wider HV amplitude range. In conclusion, HV plus LV pulses represent an efficient and safe option for future clinical trials and we suggest recommendations for gene transfer to various types of tissues.

The course of tissue permeabilization studied on a mathematical model of a subcutaneous tumor in small animals

One of the ways to potentiate antitumor effectiveness of chemotherapeutic drugs is by local application of short intense electric pulses. This causes an increase of the cell membrane permeability and is called electropermeabilization. In order to study the course of tissue permeabilization of a subcutaneous tumor in small animals, a mathematical model was built with the commercial program EMAS, which uses the finite element method. The model is based on the tissue specific conductivity values found in literature, experimentally determined electric field threshold values of reversible and irreversible tissue permeabilization, and conductivity changes in the tissues. The results obtained with the model were then compared to experimental results from the treatment of subcutaneous tumors in mice and a good agreement was obtained. Our results and the reversible and irreversible thresholds used coincide well with the effectiveness of the electrochemotherapy in real tumors where experiments show antitumor effectiveness for amplitudes higher than 900 V/cm ratio and pronounced antitumor effects at 1300 V/cm ratio.

Hollow Microneedle Arrays for Intradermal Drug Delivery and DNA Electroporation

The association of microneedles with electric pulses causing electroporation could result in an efficient and less painful delivery of drugs and DNA into the skin. Hollow conductive microneedles were used for (1) needle-free intradermal injection and (2) electric pulse application in order to achieve electric field in the superficial layers of the skin sufficient for electroporation. Microneedle array was used in combination with a vibratory inserter to disrupt the stratum corneum, thus piercing the skin. Effective injection of proteins into the skin was achieved, resulting in an immune response directed to the model antigen ovalbumin. However, when used both as microneedles to inject and as electrodes to apply the electric pulses, the setup showed several limitations for DNA electrotransfer. This could be due to the distribution of the electric field in the skin as shown by numerical calculations and/or the low dose of DNA injected. Further investigation of these parameters is needed in order to optimize minimally invasive DNA electrotransfer in the skin.

Numerical modeling in electroporation-based biomedical applications

Numerical modeling in electroporation-based biomedical applications Background. Numerous experiments have to be performed before a biomedical application is put to practical use in clinical environment. As a complementary work to in vitro, in vivo and medical experiments, we can use analytical and numerical models to represent, as realistically as possible, real biological phenomena of, in our case, electroporation. In this way we can evaluate different electrical parameters in advance, such as pulse amplitude, duration, number of pulses, or different electrode geometries. Such numerical models can contribute significantly to the understanding of an experiment and treatment planning as well as to the design of new electroporation devices and electrodes. Methods. We used commercially available modeling software, based on finite element method. We constructed a model of a subcutaneous tumor during electrochemotherapy (EMAS) and a model of skin during gene electrotransfer (COMSOL Multiphysics). Tissue-electrode geometries, pulse parameters and currentvoltage measurements from in vivo experiments were used to develop and validate the models. Results. To describe adequately our in vivo observations, a tissue conductivity increase during electroporation was included in our numerical models. The output currents of the models were compared to the currents and the voltages measured during in vivo experiments and a good agreement was obtained. Also, when comparing the voltages needed for a successful electropermeabilization as suggested by the models, to voltages applied in experiments and achieving a successful electrochemotherapy or in vivo gene electrotransfer, good agreement can be observed. Conclusions. Modeling of electric current and electric field distribution during cell and tissue electroporation proves to be helpful in describing different aspects of the process and allowing us to design electrodes and electroporation protocols as a part of treatment planning.

Skin electroporation for transdermal drug delivery: The influence of the order of different square wave electric pulses

Electroporation can be used as an active enhancement method for intra- and transdermal drug delivery. Differences in response of skin to electric pulses depend on their amplitude, duration and number and have been a point of interest in the past. While protocols consisting of the same repetitive, mostly exponentially decaying pulses have been used before, this study is focused on comparing different combinations of square wave short high voltage (HV) and longer low voltage (LV) electroporation pulses. Our in vitro experimental results show that longer LV pulses significantly increase subsequent passive transport of calcein through dermatomed pig skin, while short HV pulses alone result in negligible calcein passive transdermal transport. Surprisingly, when the long LV pulses are preceded by short duration HV pulses, the total calcein transported is reduced significantly. This result is explained using a theoretical physics based model of individual local transport region (LTR) evolution during the applied LV pulse. The theoretical model shows that HV pulses alter the structure of the stratum corneum in such a way that when the LV pulses are applied, insufficient thermal energy is generated to initiate LTR expansion. Together, the experimental results and theoretical predictions show that the total pulse energy alone cannot account for total solute transport: that the order of the types of pulses administered must also be considered. Our findings open a direction for further improvement of the method using new protocols.

Active enhancement methods for intra- and transdermal drug delivery: a review

Transdermal route has some advantages over other drug administration routes. These include avoidance of first pass effect (hepatic metabolism), better pharmacokinetic profile, reduction of side effects and good patient compliance. The greatest obstacle for the drugs to be delivered through the skin is overcoming the impermeable outermost layer of the skin – the stratum corneum. Quite a few enhancement techniques can be used to overcome the stratum corneum barrier and facilitate transdermal drug delivery. These include various passive (penetration enhancers, liposomes) and active approaches (electroporation, iontophoresis, microneedles), which are of prime interest for transdermal drug delivery research area.

Ultrasound and electric pulses for transdermal drug delivery enhancement: Ex vivo assessment of methods with in vivo oriented experimental protocols

In our present study we focus on two physical enhancement methods for transdermal drug delivery: ultrasound and electric pulses either alone or in combination. Great emphasis has been given on the design of the experimental system and protocols, so the results and the conclusions drawn from them would have greater relevance for in vivo use and later translation into clinical practice. Our results show a statistically significant enhancement of calcein delivery (after one hour of passive diffusion following treatment) already after 5 minutes of ultrasound application, or only 6 × 100 short high voltage electrical pulses. We also experimented with combinations of the two enhancement methods hoping for synergistic effects, however, the results showed no evident drastic improvement over single method. Looking closer at physics of both methods, this absence of synergy in our in vivo oriented experimental setting is not surprising. The mechanism of action of both methods is the creation of aqueous pathways in the stratum corneum leading to increased skin permeability. However, when used in combination (regardless of the order of methods), the second method was unsuccessful in adding many new aqueous pathways in the stratum corneum, as it acted preferentially near the sites of the existing ones.

A Numerical Model of Permeabilized Skin With Local Transport Regions

The protective function of skin and hence its low permeability presents a formidable obstacle in therapeutical applications such as transdermal drug delivery and gene delivery in skin. One of the methods to temporarily increase skin permeability is electroporation, creating aqueous pathways across lipid-based structures by means of electric pulses. Also, the application of electric pulses to biological cells causes increased permeability of cell membrane, thus enabling the uptake of larger molecules that otherwise cannot cross the membrane, such as drug molecules or DNA, into the cell. The creation of localized sites of increased molecular transport termed local transport regions (LTRs) can be observed during electroporation, as well as changes in the bulk electric properties of skin layers. We modeled these phenomena with a numerical model and compared the output of the model with our own in vivo experiments and previously published results of skin electroporation and a good agreement was obtained. With the model presented, we used the available data to describe the nonlinear process of skin electropermeabilization from the aspect of tissue conductivity changes and the presence of local transport regions in the permeabilized stratum corneum. The observations derived from various in vivo experiments by different authors were thus confirmed theoretically.

Transdermal transport pathway creation: Electroporation pulse order

In this study we consider the physics underlying electroporation which is administered to skin in order to radically increase transdermal drug delivery. The method involves the application of intense electric fields to alter the structure of the impermeable outer layer, the stratum corneum. A generally held view in the field of skin electroporation is that the skins drop in resistance (to transport) is proportional to the total power of the pulses (which may be inferred by the number of pulses administered). Contrary to this belief, experiments conducted in this study show that the application of high voltage pulses prior to the application of low voltage pulses result in lower transport than when low voltage pulses alone are applied (when less total pulse power is administered). In order to reconcile these unexpected experimental results, a computational model is used to conduct an analysis which shows that the high density distribution of very small aqueous pathways through the stratum corneum associated with high voltage pulses is detrimental to the evolution of larger pathways that are associated with low voltage pulses.