1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies

Inspired by the work of Sven Carlson, Heinz Helmut Doevenspeck, a German engineer, was starting in 1958 to develop his so-called "Elektroimpulsverfahren", which used for the first time defined discharges of capacitors to generate homogeneous, pulsed electric fields. He applied these fields on dispersed systems of inorganic or organic origin to influence the membranes of plant or animal cells or the surface charges of particles. Cracking of cells, growing and/or killing of microorganisms, acceleration of fermentation processes and treatment of wastewater had been identified, described and patented by Doevenspeck as suitable applications already in the early 1960s. Until mid of the 1980s, working as an independent consultant, he did many trials at technical scale plants without reaching a breakthrough of his technology. From 1985 until 1993 he was cooperating with Krupp Company, Hamburg. In 1988 the first industrial scale plants were built and during this period several applications and process mechanisms were identified, mathematically modeled and presented to the scientific public for the first time.

Pulsed electric fields (PEF) can electroporate eukaryotic and prokaryotic cells which can be used in food processing for product preservation and extraction of intracellular components. In this paper a number of potential processing applications for PEF will be considered. These will include its incorporation in hurdle preservation strategies, its use in the termination of enzyme hydrolysis of proteins, its application in the waste stream valorization through enhancing compound extraction and also its potential for accelerating processes such as meat tenderization or curing. In terms of product pasteurization, the paired combinations of novel technologies examined certainly inactivated more microorganisms than each technology alone but the effect of the combinations was largely additive or overlapping with few examples of synergy. In addition some combinations introduced high specific energy into products (in some cases higher than conventional UHT). In terms of product sterilization, PEF applied at temperatures of 120°C gave a slight though not significant increase in spore inactivation compared to thermal treatments. The use of PEF for endoprotease inactivation in bioactive compound production also proved to be very promising and could have application in hydrolyses termination especially where the resultant bioactives are heat labile. PEF pre-treatments of potato peel and brewers spent grain increased the extraction of glycoalkaloids and

β

-glucans respectively which is promising as conventional methods for the extraction of these compounds are expensive and the market value of the target compounds is high. In terms of its application in meat processing, PEF can have a positive impact on meat tenderization, though ageing/conditioning is still necessary. It has also been shown to accelerate the curing of meat when it is applied as a pre-treatment prior to curing. Overall, these applications are all promising though it remains to be seen which of these niche areas are likely to achieve significant commercial uptake.

Pulsed electric field (PEF) is a technology that causes electroporation of the cell membranes by application of intermittent electric fields of high intensity for short periods of time (

μ

s). The local defects or pores created by the application of an external electric field may lead to the loss of the membrane integrity and uncontrolled molecular transport across microbial membranes. These events may abolish the microbial capacity to maintain the microbial homeostasis causing microbial inactivation at temperatures below those used in thermal processing. Based in this phenomenon PEF provides an excellent alternative to conventional thermal pasteurization of heat sensitive foods.

Cell membranes can be transiently permeabilized under application of electric field pulses. This process, called electropermeabilization or electroporation, allows hydrophilic molecules, such as anticancer drugs and DNA, to enter into cells and tissues. Single cell imaging experiments revealed that the uptake of molecules is a complex multi-step that takes place in well-defined membrane regions under processes that affect the entire membrane, depend on the properties of the molecules (size, charge) and in which the cytoskeleton can play a major role.

Electrochemotherapy is an ablative technique utilizing electroporation for enhanced drug delivery to cells. The predominant mechanism of action is enhanced tumor cell cytotoxicity; however, the vascular disrupting effect is also present and is of significant importance. It is limited to tumor microvasculature, while electrochemotherapy does not affect bigger and normal blood vessels. This clinical indication of electrochemotherapy application is important in the treatment of bleeding metastases, since other ablative techniques lack those mechanisms of action. This was confirmed by the clinical data, where electrochemotherapy proved effective and safe in the treatment of metastases located in the vicinity of the major hepatic vessels, not amenable to surgery or radiofrequency ablation.

Modeling results and an increasing number of experimental results indicate that pulses with durations on the order or less than the charging time constant of the plasma membrane (which is on the order of 100 ns for cells in solution) affect the plasma membrane in a different way than classical electroporation pulses. Such nanosecond Pulsed Electric Fields (nsPEF) have been shown to also target subcellular structures and possibly proteins. When the pulse duration is shortened into the picosecond range (psPEF) the dielectric, rather than the resistive, properties of the outer medium and cytoplasm begin to determine the coupling between electric field and cell membranes. Besides entering a new field of electric field - cell interactions, the use of picosecond pulses allows the application of wideband antennas, rather than invasive electrodes, for the delivery of psPEF to tissue.

Initially diagnosed stage 3 (locally advanced) Pancreatic adenocarcinoma (LAPC) remains an aggressive tumor with an overall poor prognosis (current median survival chemotherapy and radiation therapy of 9-12 months). Improvement in survival can be achieved in the small percentage that can undergo an R0 resection or where all macroscopic tumor can be cleared by local ablation. Long duration (>4-6 months) chemoradiotherapy is not tolerated by all patients and still fails to prolong survival alone. Neoadjuvant treatment also has limited results on pain control or tumor downstaging since most current modalities do not shrink or downsize the tumor. In recent years, there has been a growing interest in the use of local ablative therapy for the treatment of nonresectable tumours in various organs. Ablation techniques are based on direct application of chemical, thermal, or electrical energy to a tumor, which leads to cellular necrosis without removal of the tumor. With ablation, local control, and relief from symptoms can be obtained in the majority of the patients when appropriate patient selection and technique is utilized. LAPC has been treated by various ablation techniques in the last few years with promising results. We present the current status of local ablative therapies in the treatment of LAPC and investigate on the efficiency and future trends.

Endoglin (CD105) is involved in activation and proliferation of tumor endothelial cells. Its pronounced role was recently described in tumors that became resistant to standard anti-VEGF(R) antiangiogenic therapies. In our ongoing studies, the feasibility of targeting endoglin with gene therapy using RNA interference technology was elaborated at the molecular, cellular and organism level. We prepared plasmids encoding shRNA against endoglin under the control of different promoters and evaluated their therapeutic potential in electrogene therapy of different cancers. In TS/A adenocarcinoma tumors, which do not express endoglin, anti-endoglin gene therapy resulted in vascular targeted effect that was more pronounced when plasmid encoding shRNA was used, than when siRNA molecules against endoglin were employed. Furthermore, the plasmid encoding shRNA against endoglin under the control of endothelial cell specific promoter was equally effective as the plasmid with the strong constitutive promoter. In addition, in melanoma tumors, which express endoglin, besides vascular targeted effect, a significant antitumor and anti-metastatic effects were obtained. Collectively, our results demonstrate that endoglin is a suitable target for vascular targeted gene therapy approach and promote further studies with clinically suitable and safe plasmids devoid of antibiotic resistance gene and under the control of tissue specific promoters.

Phylogenetic studies of the last two decades have revealed that horizontal gene transfer (HGT) has been playing a prominent role throughout evolution and contributed importantly to the genetic variability of species inhabiting our planet. Three main biotic mechanisms of HGT – competence for DNA uptake, conjugation, and viral transduction – have been identified and by now rather thoroughly investigated, but it is questionable whether they can account for all natural occurrences of HGT. Namely, most eukaryotes lack ability for either conjugation or competence, while transduction mostly proceeds among organisms that are phylogenetically very close to each other; yet there is mounting evidence of HGT from prokaryotes to eukaryotes, and even between eukaryotes. Here, we posit that of the four laboratory techniques most widely used for artificial genetic transformation – chemical, freeze-and-thaw, microbeads-agitation, and electroporation-based transformation – at least three have analogues in nature that can act as abiotic mechanisms of gene transfer. In particular, we show that these abiotic mechanisms of HGT, while possibly inferior in importance to biotic HGT in those organisms and environments where the latter proceeds efficiently, can also act under circumstances where this is difficult to envision for the three biotic mechanisms.

The aim of this work was to evaluate inactivation of L. monocytogenes cells suspended in a beverage (a mixture of oat milk and orange, papaya, and mango juices) by a hurdle treatment of PEF, Stevia rebaudiana Bertoni (leaf infusion, 8.33% w/v), and refrigerated storage. Beverage samples with and without stevia underwent five PEF treatments (10–40 kV/cm, 50–800

μ

s), with two energy levels (100 and 800 J/mL), and were stored at two temperatures (5 and 10 °C), for a 48 hours period. The results showed the existence of a synergic PEF-stevia effect, achieving inactivation levels exceeding 5 decimal reductions of L. monocytogenes during refrigerated storage of samples after treated by PEF.

These results show that stevia has antimicrobial capacity against L. monocytogenes and that the PEF-stevia combination is a valid strategy to increase the microbiological safety of this type of beverages.

Pulsed electric field (PEF) processing affects the cell permeability of plant tissue. This technology is capable of enhancing mass transfer and releasing valuable compounds (e.g. juice, health-promoting phytochemicals) from plant cells at low electric fields. This study aimed to assess the influence of PEF (1033 square wave bipolar 20

μ

s pulses of 1.4 kV/cm at a frequency of 50 Hz for a total treatment time of 20.66 ms) on the release of anthocyanins from grapes (Vitis vinifera L. cv. Merlot) after 48 h of PEF treatment and on its potential to protect Caco-2 cells from H2O2-induced oxidative stress. Merlot anthocyanins were characterized and quantified using LC-MS. To assess the bioprotective potential, cell viability was measured using 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay that indicates the mitochondrial metabolic activity. A significant increase in the release of anthocyanins was obtained immediately after PEF. After 48 h, PEF had facilitated the release of malvidin, delphinidin and petunidin glucosidic derivatives of anthocyanins. Because of the increase in amount and types of Merlot anthocyanins released due to PEF within 48 h, the obtained grape juice was more effective in protecting Caco-2 cells from oxidative damage. This finding provides evidence that PEF can increase the release of anthocyanins as well as delivering bioactivity important in the field of functional foods.

Pulsed electric field (PEF) processing is a non-thermal food processing technology that applies brief (

μ

s) electrical pulses of high voltage to food products placed between two electrodes. Depending on the process intensity, the process affects cell membrane permeability due to localised structural changes. There are few reports on the use of PEF for the processing of solid foods like meat. Therefore, the current project was designed to assess the impact of PEF processing on beef muscles and its potential use to reduce meat ageing time and cost. Post rigor

Biceps femoris

(a low value beef muscle) cuts were exposed to PEF (electric field strength of 1.7 - 2.0 kV/cm and pulsed electrical energy of 185 kJ/kg) processing and sampled after aging at 4 oC for 3, 7, 14 or 21 days. Samples were assessed for temperature increases, electrical conductivity, pH, purge loss, cooking loss, tenderness and colour stability. The microstructure of PEF treated and untreated meat samples were also investigated. Our results showed that temperature, electrical conductivity, and pH were significantly (P<0.05) affected by PEF treatment conditions. Tenderness, as indicated by a reduced shear force value, significantly (P<0.05) increased following PEF treatment compared to the untreated samples. Cooking loss was not affected by PEF treatment, whilst purge loss significantly (P<0.05) increased after PEF treatment and during ageing. The PEF treated meat samples showed a dramatic increase in the number of myofibrils ruptured along the z-lines compared to the untreated samples during ageing. This resulted in beef muscle with a more porous structure compared to the untreated samples and accounts for the observed increase in electrical conductivity and purge loss. These results suggest that PEF induces changes in the microstructure and texture of meat and could potentially be used to improve tenderness, decrease ageing time or to alter functional properties.

In the present paper, a solid-state pulse generator based on magnetic switch is investigated both numerically and experimentally. The generator have potential advantage of high repetitive rate achievability and long life time reliability, which is proper to be used for food treatments by pulsed electric field (PEF). Specially, the pulse generator is designed and the total weight and volume is expected to be 90kg and 0.20m3, approximately. Circuit of the generator is simulated using the P-Spice software. Influence of the impedance of a dummy load is analyzed. The peak voltage over 20kV and pulse duration of 1 s-2.5 s can be achieved on the dummy load. The expected maximum frequency is up to 1kHz. As important parameters, characteristics of the cores used for establishing the magnetic switch were measured at the actual working frequency. Additionally, the magnetic switch with winding number of 30turns and volume of 6000cm3 was tested on an equivalent experimental platform. Typical current with peak amplitude over 520A, duration of 1.52 s was obtained with dummy load of 45 Importantly, the short-circuit termination was also tested for possible flashover in the treatment chamber. Experimental results show reasonable agreement with the numerical analysis.

In the present work, electrical impedance spectroscopy (EIS) measurements were performed during electroporation of C2C12 cells monolayers. The measuring strategy, based on multisine excitations, allows acquiring full impedance spectra at a rate of 1 spectrum/ms during the interval between pulses of a traditional electroporation treatment. The multifrequency information provided by the measuring system was then studied using models. In this study, the Cole model and an electrical equivalent circuit were used. The results show the ability of the system for monitoring the fast impedance dynamics of cells during electroporation. The differences in the information shown at different frequencies suggest the ability of the system to measure different phenomena at the same time, namely: membrane changes and medium conductivity variations. The use of the Cole model reveals the need of more complex models to correctly separate and interpret the results. The use of the proposed circuital model enables studying the dynamics of pore formation independently of the conductivity variations. This work confirms the advantages of performing EIS measurements in order to have a more complete snapshot of the system behavior.

Membrane electropermeabilization is observed when electric pulses over a critical strength are delivered on a cell suspension. This is associated with a time dependent change in the suspension conductance. The magnitude and the kinetics of the conductivity changes can be used to get access to the molecular event supporting the membrane electropermeabilization. In Bioprocesses, where extraction results from electropermeabilization, the control of the conductivity change of the cell suspension allows the definition of optimized safe conditions.

Accurate coverage of tissue with a sufficiently large electric field is one of the key conditions for successful electroporation. Magnetic resonance electrical impedance tomography (MREIT) provides a means to map the electric filed distribution during electroporation. To estimate the electric field strength, the magnetic flux density data induced by the electroporation pulses are measured from MREIT scans during electroporation. Since biological tissues such as skeletal muscle are anisotropic, we propose a novel MREIT technique to map the electric field in anisotropic as well as isotropic regions. We utilize the anisotropic conductivity estimation method based on the lately developed DT-MREIT technique where diffusion tensor imaging is combined with MREIT. To estimate the current density in an optimal way, we adopted the projected current density estimation algorithm. From

ex vivo

experiments using bovine muscle tissues, we found that the new method produces electric field maps with a wider coverage of electroporation than the previous method. The results suggest that it is important to properly handle the effects of the tissue anisotropy for more accurate mapping of electric field during electroporation.

The possibility to use the electric field as a way to interact with the cell or tissue has recently led to many applications in research laboratories and industry: gene therapy, tumour treatment, electrochemiotherapy, cell fusion, cancer diagnosis and characterization of biological tissues. Among the AC electrokinetic techniques, the electrorotation allows identification of the dielectric properties of analysed bio-samples (single prokaryotic or eukaryotic cell, algae, or cell assemblies like spheroids) through the analysis of their rotational velocity when immersed within a rotating electric field.

This paper presents a microfluidic device, dedicated to the determination of the electro-physiological properties of biological samples, which uses the combination of two forces, the negative dielectrophoresis force (nDEP) for the cell trapping and the electrorotation torque for the rotation of the biosample. An estimation of the biosample dielectric parameters is obtained through the analysis of the rotational velocity curve versus the electric field frequency.

An observation set-up includes a fast camera that allows time controlled image sequence acquisition. Frames are then digitalized and from the analysis of the rotational velocity of the sample, its complex permittivity is determined, leading to the estimation of the conductivity and the permittivity of the membrane and the cytoplasm in the case of single cell.

Biological material shows characteristic electrical behavior between 10 kHz and 10 MHz which is mostly governed by cells surrounded by insulating membranes.

Electroporation of cell membranes renders the generally highly resistive lipid structure conductive. This results in dramatic decrease of the low frequency impedance because ions can cross the membrane due to the electrically created aqueous pores. Moreover, there is a pronounced impact on conductivity of the extracellular medium due to an efflux of ions from the cell plasma.

Given the significant impedance changes due to electroporation, electrical characterization is a powerful tool for monitoring this process. Since the time course of electroporation is short compared to conventional impedance measurements and the material is driven into a non-linear range of the current/ voltage characteristics, only dynamic impedance measurements in presence of high dc-offset with sub-millisecond time resolution are suitable for this purpose.

Success of electroporation treatment critically depends on coverage of the target tissue with electric field. The electric field during delivery of the electroporation pulses can be reconstructed by the magnetic resonance electric impedance tomography, a method that uses current density distribution data and electric potentials at the electrodes for reconstruction of electric field in the sample. In this study, two complementary MRI methods for current density imaging during delivery of irreversible electroporation pulses are presented. One of the methods is based on the single-shot rapid acquisition with relaxation enhancement while the other is based on the echo planar imaging MRI method. The methods were compared in terms of their sensitivity and susceptibility to image artifacts by experiments on a liver test sample that were performed on a 2.35 T small bore MRI scanner. In the experiments, a standard protocol for irreversible electroporation where 90 electric pulses of 100

μ

s, 3000 V are delivered at 1 Hz was performed. Results of the study confirmed that both methods have comparable sensitivity. The RARE-based method was found less susceptible to artifacts while the EPI-based method has lower SAR value and may therefore be a better candidate for use in clinics.

The mechanism of electroporation is used in a wide range of medical applications, genetic engineering, and therapies. Measurements of biological cells suspension conductivity have been used to evaluate the effect of electroporation cells. A micro electro-permeabilization system was built to apply 900

μ

s pulses with electric field variations in a cells suspension. The electrodes have distance around 200 micrometers. We observed changing in the cells suspension conductivity as the increased electric field with the micro electro-permeabilization system. During electroporation we conclude that different volume fractions leads different conductivities of cells suspension in same electric field, in low-conductive medium increasing the electric field induce a higher conductivity gradient of cells suspension in lower concentration of cells, and for same cells volume fractions and same electric field results different conductivity of cells suspensions changing external medium conductivity.

Monitoring of electroporation process presents one of the most important aspects towards safe and reliable use of electroporation applications such as irreversible electro-poration (IRE) ablation and electrochemotherapy (ECT). Since electroporation process is related to an induced transmembrane potential, which is directly proportional to the electric field, we proposed a method to determine electric field distribution by means of magnetic resonance electrical impedance tomography (MREIT) and current density imaging (CDI). In our previous studies, CDI was adjusted to acquire current density distribution established by electric pulses with repetition rate in the range of kHz. Therefore we developed a new CDI sequence for lower repetition rates, i.e. in the range of 1 Hz. In this study we evaluated feasibility of new CDI sequence to be applied together with MREIT for electroporation applications such as IRE ablation. We performed experimental evaluation on ex vivo liver tissue during application of electric pulses with the repetition rate of 1 Hz. Results of experiment demonstrated successful reconstruction of electrical conductivity and detection of conductivity increase in the tissue.

Electrochemical reactions at the electrode solution interfaces are unavoidable when typical conditions for PEF processing are applied. The aim of this paper was to set up and validate a mathematical model describing the phenomenon of the metal release from stainless-steel (type AISI 316L) electrodes of a continuous flow parallel plate PEF treatment chamber into model liquid foods. The spatial distribution of the main metallic elements (Fe, Cr and Ni) released from the electrodes in the treatment zone as well as their concentration in the bulk of the product exiting the treatment chamber were simulated. All simulations were performed with the software package COMSOL Multiphysics

TM

.

Electrolytic ablation (EA), a medical treatment increasingly used in solid tumor ablation, consists in the passage of a low direct electric current through two or more electrodes inserted in the tissue thus inducing pH fronts that destroys the tumor. The combined use of EA with a recently introduced one-probe two electrode device (OPTED) results in a minimally invasive tissue ablation technique. Despite its success related to low cost and minimum side effects, EA has drawbacks such as the difficulty in determining the current and time needed to assure total tumor ablation while avoiding healthy tissue intrusion. Here we introduce a realistic dose planning methodology in terms of the coulomb dosage administered and the associated pH tracking, that predicts an optimal EA/OPTED protocol treatment for a given tumor size, that is, the current and exposition time necessary to succeed in eliminating all the tumor mass while minimizing healthy tissue damage.

Electrostimulation of neurons is extensively used in different neuroprosthetic applications. In certain neuroprosthetic applications constant voltage pulses with stimulus amplitudes exceeding 1-2 Volts are used. Here we investigate which stimulus parameters of monophasic or biphasic constant-voltage pulses lead to cell electroporation in blind retinas. Towards this aim we interfaced ex vivo adult blind mouse retina to an electrode array comprising micron-sized iridium oxide electrodes. Our experiments demonstrate cell electroporation in retinal tissue in close vicinity (30

μ

m) to the stimulation electrodes for monophasic voltage pulses of either polarity as low as 1.6 Volts and stimulus durations as short as 1 millisecond. These results will guide safe neuroprosthetic stimulation of blind retina.

This work aimed at providing some novel aspects pertaining to stilbenes (resveratrol, piceid and piceatannol) extraction from grape stems using high voltage electrical discharges (HVED). Treatment time, pH and ethanol concentration affecting the extractability of these compounds were optimized through response surface methodology.

HVED improves significantly the extraction of piceatannol and piceid but are less efficient on resveratrol. The efficiency of HVED is directly correlated with the other processing conditions (pH, ethanol concentration). Prolonged HVED treatment (4 ms) followed by a diffusion with 50 water was a positive combination for piceatannol and piceid recovery.

Unpasteurized fruit juices are perishable products whose microbiological spoilage supposes great economic losses for the food industry. Besides, they can support the survival and growth of human pathogens. Actually, different juices have been involved in major foodborne illnesses outbreaks. Therefore, they should be submitted to treatments that prevent their microbiological spoilage and ensure their microbiological safety. In response to consumer demand for minimally processed foods, without synthetic additives, significant efforts are leading to the development of novel non-thermal technologies, such as the application pulsed electric fields (PEF), to extend juices’ shelf life avoiding thermal processing. The combination of those technologies with preservatives from vegetal, animal and microbial origin could be a convenient strategy to obtain high-quality foods, from farm to fork. Considering

Stevia rebaudiana

Bertoni (

Stevia

)antibacterial and antifungal properties, PEF antimicrobial potential in a juice containing

Stevia

was assessed. With this aim, the effect of PEF and

Stevia

on the microbial load of non-sterile and sterile samples inoculated with

Listeria monocytogenes

was determined. In view of the results obtained, PEF processing reduced the microbial load of the juice under study. Based on cell counts performed before and after different PEF treatments, in presence of different concentrations of

Stevia

, it was observed that the higher the electric field and the treatment time, the higher the inactivation.

Stevia

addition reduced PEF effectiveness against yeast, molds and mesophiles, but increased its effectiveness against

L. monocytogenes

. Anyway, the joint application of PEF and

Stevia

not fulfilled with the microbiological criteria required for this kind of products. Further studies are needed to evaluate

Stevia

effects during post-processing storage. In addition, the study of other strategies should not be neglected for juices’ preservation exploiting the antimicrobial potential of PEF.

The reduction by Pulsed Electric Fields (PEF) of four residual fungicides (pyrimethanil, vinclozolin, cyprodinil and procymidone) in dry white wine was studied in this paper. White wine was treated by PEF with strengths of 5-20 kV/cm and treatment times of 0.5-2 ms (which corresponds to an energy of 10-160 kJ/L). Pesticide residues were extracted by stir bar sorptive extraction (SBSE) and assayed by gas chromatography coupled to mass spectrometry (GC/MS). The results show that the concentrations of the studied pesticides decreased significantly after the PEF treatment and the sensitivity of fungicides concerning PEF was different. The degradation was influenced by strength and energy of PEF treatments. The decrease in levels of pesticides in wine by the application of a pulsed electric field of high intensity seems to be possible.

In recent years, a possible alternative to complement the effectiveness of non-thermal technologies is to combine these technologies with the addition of additives from natural origin with antioxidant properties.

Stevia rebaudiana

Bertoni (

Stevia

) has antioxidant properties but no studies about its potential to enhance the effectiveness of pulsed electric fields (PEF) against some enzymes have been conducted to date.

Response surface methodology (RSM) was used to evaluate the simultaneous effects of electric field strength (from 20 to 40 kV/cm), treatment time (from 100 to 360

μ

s) and

Stevia

concentration (from 0 to 2.50% (w/v) on PPO and POD enzymatic activities of the juice under study.

The results obtained show that, only the highest PEF treatment tested (40 kV/cm; 360

μ

s) induced inactivation of both enzymes in the absence of

Stevia

. However, for any electric field intensity and treatment time combination, the complete inactivation of juice PPO was achieved only in presence of

Stevia

, regardless of the percentage added. On the other hand POD proved to be independent on

Stevia

concentration, but inversely dependent on electric field intensity (E) and treatment time (t). According to this, only in the case of the highest electric field tested (40 kV/cm)

Stevia

enhanced the POD inactivation percentage achieved by PEF.

These results show that

Stevia

could be useful in the food industry as natural antioxidant, preventing oxidative reactions, and its combination with PEF treatment could be a good strategy in reducing enzymatic activity improving at the same time the nutritional and physico-chemical quality of foods.

Salmonella spp

is a foodborne pathogen related with food outbreaks worldwide. To control it, new strategies have been developed to preserve food products, such as the application of non-thermal treatments like pulsed electric fields (PEF) or the addition of antimicrobial compounds from vegetable by-products. The main objective of this study is to evaluate the antimicrobial effect of PEF treatment, combined with several concentrations of cauliflower by-product infusion against S. Typhimurium.

Survival curves of S. Typhimurium, treated and nontreated by PEF, were obtained during their incubation under different cauliflower by-product infusion concentrations (1%, 5%, 10%) at 37°C. The control sample was incubated with buffered peptone water (0.1%). PEF treatment (20 kV/cm - 900

μ

s) reduced 4,5 log cycles of the initial microbial population (108 cfu/mL). Also, both in PEF treated and nontreated samples, cauliflower by-product concentrations of 5% and 10% exerted a bactericidal effect against

S

. Typhimurium, although the time necessary for total inactivation was shorter when combined with PEF (2 hours) than non-PEF treated samples (8 hours).

The results obtained were fitted to a Weibull distribution function, and its kinetic parameters (

b

and

n

) were obtained. Both in PEF treated and non-treated samples,

b

values, which are related with the inactivation rates, were higher when the cauliflower by-product infusion concentration was greater. Also, the

b

values of PEF treated samples were higher than the

b

values of non-treated samples. Therefore, the PEF treatment combined with the addition of cauliflower by-product infusion exerted a higher antimicrobial effect against

S

. Typhimurium than when they were applied separately.

In conclusion, PEF treatment combined with the addition of 5 or 10% of cauliflower by-product infusion could be used as a S. Typhimurium control measure in liquid food products in the future.

Stevia rebaudiana bartoni

due to its high content in steviol glycosides and the ability to serve as a natural antimicrobial to complement the effectiveness of pulsed electric technologies for food preservation, has been used more frequently to improve sensorial properties of liquid foods.

Nevertheless, it remains unknown how steviol glycosides stability is effected after foodstuff is treated by pulse electric treatments and ultrasounds technology. In order to explore this issue, four steviol glycoside were identified and analyzed in an exotic fruit juice blend sweetened with stevia before and after pulsed electric fields (PEF), high voltage electrical discharges (HVED) and ultrasounds (USN) treatments at two equivalent energy inputs (32 and 256 k J/kg ).

The analysis revealed that all the factors had a significant influence in the changes of steviol glycosides (stevioside, rebaudioside A, rebaoudioside F rebaoudioside C) content, although the extent of changes was different.

During the processing of liquid foods, ascorbic acid may be altered, thus reducing beneficial health effects. For this reason, the degradation kinetics of ascorbic acid were determined in a fruit juice mixture (papaya and mango) sweetened with Stevia rebaudiana (SR) infusion after treatment by pulsed electric field (PEF) processing. The variable ranges were 20-40 kV/cm (electric field strength, E) during 100-360

μ

s (time treatment, t). The degradation equation was AA (mg/100 mL) = 26.842 - 0.101·E - 0.003·t- 10.371·%Stevia + 2.865·%Stevia2, demonstrating the use of PEF as an alternative to pasteurization treatments. However, results show the need to optimize treatment conditions whenever there is a change in the matrix (food) or some processing factor.

When a cell is subjected to an external electric field, a voltage establishes on the membrane and adds to the resting transmembrane voltage (TMV). If the intensity and duration of the external field are sufficient, the membrane becomes conductive, which is the first step in the electroporation process. The increase of conductivity results in a collapse of the previously established TMV. Using the fluorescent voltage sensitive dye Annine-6, we investigated the kinetics of TMV on mammalian cells DC3F submitted to pulses of 100

μ

s with field magnitudes ranging from 60 kV.m-1 to 200 kV.m-1. The results contradict the common opinion which suggests that the TMV decreases very quickly as soon as it exceeds a socalled ‘threshold’ of electroporation. On the contrary, at all field strengths tested, experiments show that the TMV remains stable for several microseconds. The duration of the stable phase decreases exponentially as the electric field increases. These results strongly challenge the current models on electro-permeabilisation and provide quantitative data to develop new approaches.

Electroporation in presence of calcium has been recently demonstrated to be capable of inducing cell death, suggesting that calcium might be used as an alternative to bleomycin in the palliative treatment of tumors via electrochemotherapy. In this work, SW780 bladder cancer cells were exposed to eight, 99

μ

s long, 1 Hz repetition rate pulses with variable electric field (0.2 to 1.6 kV/cm) in presence of calcium (0, 1, 3, 5 mM), and cell viability was evaluated by means of MTS assay at 1, 4, 8, 22 and 24 hours after electroporation. A mathematical fitting analysis of the experimental data was then carried out to estimate the electric field amplitudes leading to a desired percentage of cell death at different calcium concentrations. The results here presented confirm previous in vitro findings on calcium electroporation, and highlighted that the presence of extracellular calcium allows to employ lower electric field amplitudes to induce cancer cell death with respect those required in the case of treatments with electric pulses alone. The procedure here adopted can be tailored to different cell types to identify the optimum combinations of electrical conditions and calcium concentrations that maximize cell killing, and can also provide information to optimize application of calcium electroporation in vivo.

The activity here presented was carried out at the University of Copenhagen, Herlev Hospital, Department of Oncology, in the framework of a short term scientific mission funded by the Cost Action TD1104, and included in a recently accepted publication in PlosOne.

The effect of 50 Hz 1 mT sinusoidal electromagnetic field stimulation (EMF) on osteoblast-like MC3T3-E1 cell morphology was studied 24h after single exposure and after 3 weeks of daily stimulation. Scanning electron microscopy (SEM) revealed differences in F-actin-based filopodia and lamellipodia extent between stimulated and control cell cultures. As actin-based structures have been implicated in Ca2+ storage and signaling, these findings suggest that low EMF energies may be able to perturb Ca2+ signaling through affecting the morphology of these extensions. Furthermore, 3 week SEM observations confirm prior findings of EMF-enhanced differentiation of osteoblast-like cells as evidenced by improved spreading and mineralization of stimulated cells.

During the acute phase of injury, factors that are released from damaged tissues recruit blood cells and mesenchymal stem cells (MSCs) to the injury site. Those signals are required to recruit MSCs with high efficiency, which is critical for improving the clinical benefits of MSCs. However, in a variety of clinical conditions, MSCs are administered to damaged tissues at the subchronic or chronic phases of injury, in which the migratory signals for MSCs may be minimal or absent. Thus, exogenous stimuli are necessary to recruit infused MSCs into subchronic or chronic phases of injury for high efficacy of MSC therapy. Evidence shows that electrical stimulation (ES) induces the migration and stimulation of adult cells, including stem cells, improving clinical benefits. In an animal model of spinal cord injury, application of an electrical field (EF) resulted in functional improvement. It has been shown that damaged tissue and wounds generate naturally-occurring endogenous electric fields (EFs) plays a significant role in guiding cell migration. In vitro many cell types respond to applied EFs strengths comparable to endogenous wound EFs in vivo. With the aim of developing novel techniques to guide the migration of stem cells, we tested whether the implanted pulse generator, normally used in deep brain stimulation, directs the migration of MSC cells. Cell motility was assessed by using the deep brain stimulation system. Obtained data could be used as a first step in establishing efficient stem cells therapy for brain injury and neurodegenerative disorders. Supported by grants National Science Center, Poland no 2012/05/D/NZ3/02028; 2012/07/B/NZ4/01427 and UWM 25.610.001-300.

Cell sensitization is a phenomenon where increasing the duration of the electroporation treatment has been reported to increase cell permeabilization and decrease cell survival. Cell sensitization can be obtained by splitting one train of pulses in half and delivering the second half of the pulses after a delay. After the first half of the pulses is applied cells become more sensitive to the following electric pulses. Thus, efficiency of the treatment is sustained even if the second half of the pulses is of lower amplitude. In this study, cell sensitization was investigated in a wide range of electric field amplitudes. Twelve pulses of 100

μ

s were delivered either twelve in one train (one train protocol) or six in the first train and after 5 min another six in the second train (two trains protocol). In both protocols, the electric field of the first six pulses was varied while the electric field of the second six pulses was fixed or vice versa. Stable cell sensitization was obtained with two trains protocol in a wide range of “permeabilizing” electric fields. Cell survival was reduced up to twofold with the two trains protocol in comparison to the one train protocol. Cell sensitization has possible applications in tumor treatment and in soft tissue ablation. In tumor treatment fewer pulses of lower electric field could be applied and electrodes with larger inter-electrode distance could be used while the efficiency of the treatment would be at least as high as obtained now. In the future, research should focus on the still unknown mechanisms of cell sensitization and then extend to 3-dimensional tissue models and tissues.

In this paper, the electric field generated in a conductive medium by needles pairs with inter-needle distance of 1 or 2 cm has been studied by means of 3D numerical models. The computational results have been correlated with experimental ones obtained in vitro on cultured cells. In particular, electroporation effect has been assessed on human osteosarcoma cell line MG63 cultured in monolayer. These results are helpful to evaluate the effect of the needle distance on the electric field distribution in cultured cells in terms of occurrence of reversible or irreversible electroporation.

The impact of pulse repetition rate (PRR) in modulating the electroporation effect induced by pulsed electric fields (PEF) in mammalian cells has been experimentally analyzed by several groups, that have reported conflicting results. In our previous experiments, Jurkat cells exposed to nsPEFs with variable PRR (2, 5, 10 and 30 Hz) showed increased permeabilization and reduced survival upon reducing the repetition rate. This was in agreement with part of the literature, which reported increased electroporation efficiency of lower PRR pulsing. Recently, experimental evidence has been provided that the efficacy of higher rate nsPEFs can be increased by splitting the total pulsing treatment in separate doses. In this work, the effect of split dose protocols of nsPEFs on a single cell model has been numerically investigated. In particular, the effects of 500, 40 ns, 1.3 MV/m pulses delivered at 30 Hz PRR, were analyzed when delivered as either a single dose of pulses, or a split dose of 250 pulses each, with variable time delay between the first and the second set of pulses. The preliminary results here reported suggest that the overall effect of split nsPEF-exposures might depend on the time delay between the first and the second pulsing sequence. The biological consequences of such pulsing protocols will be verified in future works, where the information gained from this analysis will serve as starting point to drive the experimental design.

Extraction of proteins by electroporation from bacterial cells could provide a basis for significant reduction of downstream processing with low investment costs. Due to avoidance of using harmful chemicals for extraction, a significant improvement of the environmental footprint is also expected. In order to optimize extraction of proteins by electroporation, the optimization of electroporation protocol is needed (e.g. temperature). Thus the aim of our study was to optimize the temperature before and after electroporation in order to affect membrane permeabilization and to obtain maximum amount of extracted proteins, while preserving bacterial cells alive.

Escherichia coli

cells were incubated at different temperatures before and after electroporation, and the amount of extracted proteins and bacterial viability was assessed. Our results show, that the temperature has no effect on bacterial viability, while lower temperature (4 °C) seems to promote better protein extraction. We suggest that lower temperatures decrease lipid fluidity and facilitate slower resealing of electroporated membrane.

The effects of cholesterol on the permeability of soya PC and dipalmitoyl phosphatidylcholine (DPPC) vesicles induced by pulsed electric fields (PEF) treatment with different electric field strength and time were investigated. Results indicated that the electro-permeability of bilayer membrane of vesicles was significantly affected by the addition of cholesterol. Under the PEF treatment at 40 kV/cm for 1.2 ms, it was observed that the 5(6)-Carboxyfluorescein 5(6)-CF release percentage (R%) of soya PC vesicles decreased markedly from 48.13% to 24.11% when the molar ratio of cholesterol to vesicles increased from 0 to 50%. Meanwhile, it is interesting to observe that once the cholesterol was added into DPPC vesicles, the R value was dropped from about 2.83% to almost zero regardless the addition amount of cholesterol. Results from Raman spectroscopy analysis verified that the decrease of electro-permeability of vesicles with the addition of cholesterol was responsible for the incorporation of cholesterol into hydrocarbon partition of lipid bilayer, leading to the order arrangement of acyl chain of phospholipid and the increase of bilayer membrane thickness.

It was observed in preclinical murine tumor models that complete tumor regression can occur when control blank plasmid DNA, not coding for a therapeutic protein, is electrotransfered into the tumor. In our study, the plasmid gWiz Blank was electrotransfered into murine melanoma (B16-F10) and mammary adenocarcinoma (TS/A) cells to determine its cytotoxic effect and expression of pattern recognition receptors (PRRs). Our in vitro results demonstrate that electrotransfer of gWiz Blank is cytotoxic for both cell lines and implicate that gene electrotransfer leads to expression or upregulation of several DNA PRRs. Additionally, increased expression of the proinflammatory cytokine IFN

β

1 was obtained. In conclusion, activation of these sensors in tumor cells may contribute to the cytotoxicity of gene electrotransfer in vitro and inflammation and tumor regression in vivo.

Molecules spontaneously transported inside the cells, like glucose derivatives, can also be used as electropermeabilization markers. In a previous study, we evaluated the uptake of a fluorescent deoxyglucose derivative (2-NBDG) by normal and electropermeabilized Chinese hamster cells. We extend here our previous study to two murine tumor model cells and investigate the effect of rolipram, a selective PDDE4 inhibitor, on 2-NBDG uptake by tumor cells, with or without electric pulses.2-NBDG was added to cell suspensions, and the cells exposed or not to eight square-wave electric pulses of 100-

μ

s duration and of appropriate field amplitude delivered, were incubated at 37 C and uptake was measured by flow cytometry. In rolipram experiments, cells were similarly processed after a 15 min pre-incubation with rolipram. In spite of significant uptake of 2-NBDG, mediated by GLUT transporters into non permeabilized cells, electric pulses significantly increased the 2-NBDG uptake into both murine tumor cells, even though the electrical parameters allowing a maximal uptake were different. Pretreatment with rolipram, only at high concentrations reduced 2-NBDG uptake in non-electropermeabilized cells, affecting more severely the DC-3F cells than the LPB cells. On the contrary, rolipram treatment did not attenuated the uptake of 2-NBDG in the electropermeabilized cells. We extended to other cell lines our previous observation that glucose derivatives can be used to detect cells reversible electropermeabilization. Moreover, our data suggest that rolipram could probably be used as a tool for improving the visualization of tumor using glucose derivatives, by affecting the uptake in the surrounding normal tissue.

Nanosecond pulsed electric fields (nsPEFs) can induce different biological effects in cells, depending on pulse length and field strength. Currently, they are investigated for medical applications such as cancer treatment and wound healing. One particular response that has so far not been investigated is the effect on the communication between cells. Gap junctional intercellular communication (GJIC) is important to maintain the homeostasis in tissues and plays a crucial role in many diseases. For the study of the effects of nsPEFs confluent monolayers of rat liver epithelial cells WB-F344 were exposed to 20 pulses of 100 ns and up to 20 kV/cm and GJIC was tested by scrape loading/dye transfer assay. Gap junctions are built from the protein connexin, therefore changes of connexin 43 (Cx43) distribution in the cells were examined by immunofluorescent staining. Cells showed a field strength dependent decreased communication. In addition less Cx43 was detectable in the membrane after exposure.

Technical development enabled feasible and simple use of 3D cell cultures, spheroids, in cancer research. Although electrochemotherapy (ECT) and gene electrotransfer (GET) are used in preclinical studies, as well as in the clinic, still the efficient and safe delivery of therapeutic molecules is a great challenge. Therefore, human HT-29 colon carcinoma spheroids were used as potential models for evaluating ECT and GET. In the ECT protocol nonpermeant bleomycin (BLM) and poorly permeant cisplatin (CDDP) were used as relevant drugs, where it was determined that ECT led to the reduction of growth and in 4 days also to their complete disintegration. In addition, we demonstrated efficient transfection of plasmid DNA encoding enhanced green fluorescent protein (EGFP) by using GET. The obtained results indicate that spheroids are a reliable model for studying and optimizing ECT with new potential drugs and GET with therapeutic plasmids DNA.

Electrochemotherapy (ECT) is a safe and efficient physical method for the treatment of solid tumors, especially skin cancers. Nowadays it is widely used in human clinics in Europe. After electrochemotherapy, clinicians observed a good tissue healing and an excellent aesthetic and functional recovery at tumor site, meaning that normal tissue located in-between electrodes was not affected by the treatment. Underlying mechanisms explaining this therapeutic window are not understood yet. In order to address this question, we proposed to develop in vitro a relevant human 3D biological model associating cancer and normal cells. Human spheroids were produced with colorectal cancer cells (HCT-116) and/or primary normal dermal fibroblasts. The association of normal cells with cancer cells provides mixed spheroids. Spheroids growth curves were followed after different treatments with drugs alone (cisplatin, bleomycin), electric field alone, or after electrochemotherapy with these drugs. Interestingly, while tumor spheroids were totally destroyed by ECT, normal spheroids were spared as observed in vivo by clinicians. Furthermore, in mixed spheroids we observed that only normal cells remained viable after electrochemotherapy, as confirmed by a staining with calcein-AM. In this study, we developed human mixed spheroids combining tumor and normal cells. With this model, we were able to reproduce in vitro the therapeutic window observed on patients by clinicians. Using this relevant in vitro 3D complex model, we hope will soon be able to study and understand underlying mechanisms involved in this process.

Electroporation of biological tissue is a phenomenon where an electric field above a certain threshold introduces hydrophilic pores into the cell membrane that the drug is able to diffuse through - referred to as cellular drug uptake. The primary motivation of this study is to develop a macroscopic model of drug transport in electroporated tissue to determine the cellular drug uptake, achieved by relating the cell permeability to the extent of electroporation.

Many aspects require consideration when developing a model that couples electroporation to drug transport. The diffusive drug transport in tissue consists of two main aspects: drug diffusion and cellular drug uptake. Additional aspects of electroporation are pore resealing and irreversible electroporation, which influence the cellular drug uptake. Pore resealing results in transient relaxation of the cell membrane permeability after reversible electroporation. Whereas irreversible electroporation, which introduces irreversible pores causes cell death.

The model enables optimization of the desired effects of electroporation. It allows the electrode positions and pulse voltage to be determined to minimize cell death and maximize reversible electroporation for increased drug uptake to living cells. Modeling of an electroporation case is now possible given the appropriate parameters and property values of the system of interest providing valuable insight and predictions.

A dynamic model of electroporation is proposed in order to compute at the tissue scale the distribution of the electric field during the application of the voltage pulse. In this approach, two kinds of density currents are considered: the first one, derived from a model at the cell scale, flows through the cells and the second one flows through the extracellular medium. Simulations have been performed in relation with in vivo experiments made on rabbit livers: they show that the modeling is able to reproduce the chronograms of the current measured through the needles.

The electrochemical treatment (ECT) of solid tumors is an electropermeabilization technique firmly established and widely used. In ECT protocols, pulse intensity as well as tissue electric conductivity are of utmost importance for assessing the final electropermeabilized area. Present ECT mathematical modeling based on the solution of the nonlinear Laplace equation for the electric field with a conductivity coefficient depending on the electric field and the temperature have greatly contributed to ECT protocol optimization. However, experimental results from literature report that a succession of pulses may increase tissue electric conductivity and the extent of tissue permeabilization, a phenomenon that present models fail to describe. Here we present new insights of a recently introduced ECT theoretical model that takes into account the effect of pulse addition on tissue electric conductivity. The model describes the electric field with the nonlinear Laplace equation with a conductivity coefficient depending on the electric field, the temperature and the quantity of pulses applied. ECT theoretical predictions show that the rise in the electric current density during the addition of pulses is due solely to an increment in the tissue electric conductivity with no significant changes in the electric field. A potential consequence of these results is that, under certain conditions, it would be possible to obtain larger electropermeabilized areas with the same pulse amplitude simply by increasing the number of pulses. The theoretical implications of this new model lead to a more realistic description of the EP phenomenon, hopefully providing more accurate predictions of ECT treatment outcomes.

Gene electrotransfer of Plasmid AMEP is a new antiangiogenic approach in cancer treatment. While the local effects after intratumoral administration have been already studied, systemic effects still need to be elucidated. Therefore intramtumoral or intramuscular gene therapy with Plasmid AMEP was performed and effects on distant tumors or metastases were observed and compared. Only 8% of the mice developed distant metastases after Plasmid AMEP gene electrotransfer, compared to 40% in the control groups. Additionally, intramuscular gene elctrotransfer performed before the tumor implantation, has no effect on the tumor outgrowth regardless of the GET protocol used. Study showed that gene therapy with Plasmid AMEP has antimetastatic potential only after intratumoral, but not after intramuscular gene electrotransfer. Most probably circulating AMEP peptide produced in muscle cells may be rapidly biodegraded and could not be able to reach tumor site in high amounts needed for AMEP effectiveness.

Electroporation can be used in living tissues in order to enhance the penetration of drugs or DNA plasmids or to destroy undesirable cells and it is typically performed by applying pulsed high voltages across needle electrodes. When used for ablation, it is often claimed that, in contrast with thermal ablation techniques, electroporation is not significantly impacted by the presence of large blood vessels because the heat sinking characteristic of these is not relevant for the elec-tric field distribution. However, large blood vessels do distort the electric field distribution because of their high inner con-ductivity and should be modeled during treatment planning. For such purpose, vessels may be simply modeled as homoge-neous regions whose conductivity is equal to that of the blood. Nevertheless, vessels are not just blood filled cavities within parenchyma; blood vessels contain a layered wall. The purpose of the present study is to check whether the blood vessel wall needs to be incorporated into the simulations. For that, a vessel wall electrical model has been implemented and it has been incorporated into 2D and 3D simulations in which treatment of a region that comprises a 5 mm thick artery within liver was modeled. The three main layers of a vessel wall (the intima, the media and the adventitia) were modeled as homogeneous ma-terials whose conductivity depends on the electric field magni-tude. The simulations show that the electric field error when the wall model is not incorporated is only marginally signifi-cant at the close vicinity of the vessel for low applied fields. Errors are insignificant beyond 1 or 2 mm. We conclude that in most electroporation scenarios it will not be necessary to simulate the blood vessel wall.

This paper proposes the design of a new in vivo applicator of nsPEFs suitable for whole body exposure of several newborn mice. The system is based on a UWB patch antenna and exhibits a wide operating band (300-1100 MHz).

To improve the system efficiency, a dielectric box, with permittivity similar to that of the mouse has been used as sample holder, placed on the antenna and completely covering it. The system shows a good efficiency and acceptable homogeneity within the samples.

Due to low permeability of the stratum corneum, different enhancement techniques are required to promote transport of larger molecules across the skin. In our present study we focused on skin electroporation with emphasis on the design of the experimental system and experimental protocols. With this approach the results and the conclusions drawn 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 only 6x100 short (100

μ

s) high voltage (200 V) electrical pulses.

Previous work from our laboratory demonstrated significant nanopore formation in cellular membranes following exposure of cells to nanosecond pulsed electric fields (nsPEF). We hypothesize that the sensitivity of cells to nsPEF is dependent on the properties of the plasma membrane, including lipid microdomains. Results show that depletion of membrane cholesterol increases the sensitivity of cells to nsPEF. Cholesterol depletion increases the permeability of cells to small molecules, including propidium iodide and calcium, at shorter nsPEF exposures. In contrast, depletion of caveolin, an important protein component of membrane lipid microdomains, renders the cells less sensitive to nsPEF. The results of the current study suggest that plasma membrane cholesterol and proteins are important determinants in the cellular response to nsPEF.

The purpose of this paper is to try to answer the following questions: Why can electropores in cell membranes sometimes live many hours rather than milliseconds, as theory predicts? Why does such a phenomenon rarely occur in artificial bilayer membranes?

We have used the fact that biological membranes, unlike artificial bilayers, undergo a phase transition from their natural liquid-disordered (fluid) state to solid-ordered (gel) state (phase) at the transition (melting) temperature

T

m

, which is slightly below body or growth temperatures

T

g

:

T

g

–

T

m

≈ 10 – 15

K

.

Lateral pressure produced by the external electric field can shift

T

m

of lipid in the pore wall. We have shown that if the transmembrane voltage U during electroporation of biomembrane exceeds threshold voltage

U

f

s

, then lipid in electropore wall transforms from liquid crystalline phase to gel phase, and long-lived pores are formed. The threshold voltage varies with the square root of

T

–

T

m

. In biomembranes for

T

=

T

g

and for conventional values of parameters

U

f

s

0.44 – 0.54

V

.

If electroporation occurs at

$T \lesssim T_g$

– 15

K

then the biomembrane is already in the gel state.

Resealing of long-lived electropores in solid-state membrane is governed by diffusion of vacancies called diffusion (Nabarro- Herring) creep. The time of pore resealing by diffusive dissolution in the surrounding solid-ordered lipid varies as the cube of pore radius r. Pore lifetime is of the order of milliseconds for r of several nanometers, but it can amount to days for r of the order of microns. Since the latter time exceeds the duration of the experiment, such pores may appear to be stable.

Pore lifetime increases exponentially as the temperature decreases.

In any lipid membrane (including artificial) electropores (if they are formed) can become long-lived, if either

T

 < 

T

m

or

T

and

U

 > 

U

f

s

.

Pulsed electric current can be used to produce irreversible electroporation (IRE) of cell membranes with resulting cell death in a predictable fashion without thermal damage or limitation of site of the ablation. Since 2009 we have used IRE safely in 127 procedures in human subjects. Provided the entire tumour burden is ablated, IRE is as effective as any ablation method and can be applied in situations where open surgery is not advisable. With slow growing tumours, multiple sequential IRE ablations are quite feasible as a treatment for multiple liver lesions or metastatic disease. However it is possible to cause damage with the needle electrodes themselves and there are situations such as metallic biliary stents, where IRE may be inadvisable. The major difference between IRE and other treatment methods for cancer is the rapid recovery following the procedure. This allows combination treatment with cyclic chemotherapy and IRE for effective tumour control.

Background: There is a huge variety of image guided local ablation technique to treat primary and secondary liver cancer. While thermal ablations are most common, these modalities have some limitations like coagulation necrosis to adjacent structures or undesired heat sink near to larger vessels. This indicates the need of alternative techniques to enable ablation of tumors not suitable for thermal procedures. Method: Irreversible electroporation (IRE) is a relatively young image guided ablation method based on ultra-short electricity pulses inducing apoptotic cell death. IRE in the liver is suggested to preserve critical vascular and biliary structures from procedure related coagulation necrosis and is therefore expected to overcome limitations of thermal ablation techniques. We retrospectively assessed our patients treated with IRE regarding procedure safety, short-term complications and local tumor recurrence. Results: We treated 12 patients either with percutaneous CT guided IRE or with open surgical ultrasound guided IRE. The ablation was technically successful in all cases. There were three short-term complications (1 hematoma, 1 bilioma, 1 liver vein thrombosis) and one superficial track metastasis on follow up imaging. Discussion: IRE is a feasible ablation technique for primary and secondary liver cancer and can be the only remaining curative treatment option. However the previously stated preservation of critical vascular structures has to be regarded critically. The missing possibility for track ablation in IRE may indicate a potentially higher risk for inducing track metastases. However further clinical data are necessary to state the value of IRE in comparison to other ablation methods.

Treatments based on electroporation are new and promising approach to treating tumors, especially non-resectable ones. The success of the treatment is, however, heavily dependent on coverage of the entire tumor volume with a sufficiently high electric field. Ensuring complete coverage in the case of deep-seated tumors is not trivial and can in best way be ensured by patient-specific treatment planning. The basis of the treatment planning process consists of two complex tasks: medical image segmentation and numerical optimization. In addition to earlier developing segmentation algorithms for several tissues and the algorithms for numerical optimization of treatment parameters we developed a web-based tool to support the translation of the algorithms and their application in the clinic. The developed web-based tool automatically builds a 3D model of target tissue from the medical images uploaded by the user and then uses this 3D model to optimize treatment parameters. The tool enables the user to validate the results of the automatic segmentation and make corrections if necessary before delivering the final treatment plan.

The software is in constant development to support new target tissues with feedback provided from several clinical studies in which it is currently used.

Introduction: Electrochemotherapy was eveluated as a treatment modality for treatment of colorectal liver metastases. Prospective studies were designed to evaluate the feasibility, safety and efficacy.

Patients and methods: Patients from the prospective pilot trial and phase II study were followed for the response to the treatment and adverse events. During open surgery electrochemotherapy was performed with electrodes with fixed or variable geometry. Electrodes were inserted into and around the tumor to cover the whole tumor area and the margin of normal tissue with a sufficiently high electric field, according to the individualized treatment plan. Pulses were delivered 8-28 minutes after the intravenous administration of bleomycin (15,000 IU/m

2

) and were synchronized with the electrocardiogram.

The results: Pathologic analysis showed a significantly lower percentage of residual vital tumor tissue in electrochemotherapy treated metastases than in non-electrochemotherapy treated metastases, namely 9.9 ± 12.2% and 34.1 ± 22.5% of viable tissue, respectively. Radiological evaluation showed 85% complete responses and 15% partial responses with no statistically significant difference between metastases treated with fixed or variable geometry electrodes. No serious adverse events were reported due to electrochemotherapy.

Conclusion: Electrochemotherapy is feasible, safe and efficient treatment modality for colorectal liver metastases treatment. Further investigations could gain new improvements and knowledge for further application of the method to other internal organs.

Incidence of primary liver tumors is rising across the world. Patients with primary liver tumors can be treated radically with liver resection of the tumors and liver transplantation. When radical treatment is not indicated due to patient condition or tumor extent, transarterialche-moembolization (TACE), radiofrequency ablation (RFA), and other methods provide hope for longer survival of those patients. Patients, in whom those methods are not recommendable, have a stage C disease suitable only for palliative treatment. Therefore, for the treatment of patients with primary liver tumors with electrochemotherapy, a clinical trial was created at our institution, Clinical Department of Abdominal Surgery at the University Clinical Centre Ljubljana, Slovenia. Phase I study is underway in patients who fulfil inclusion criteria.

Electroporation-based treatments provide highly effective local treatment for a variety of tumors. For deep seated tumors in the head and neck region, the complex anatomy and difficult access often warrants the use of single long needle electrodes for which a treatment plan provides the information on optimal voltages and electrode positions in the highly constrained environment. The feasibility of such treatments can be enhanced by use of a navigation system for the positioning of electrodes. Treatment planning is performed using finite element method to compute electric fields in the tumor and surrounding tissues and using this to optimize delivered voltages. Navigation systems consist of optical tracking system, which tracks an instrument in space to introduce it along predefined trajectories. This paper aims to present a technical method for coupling treatment planning with a navigation device and using that to treat tumors in the head and neck area illustrated with a case of electrochemotherapy treatment of tumors in the neck and in the left parotid gland.

Treatment with electrochemotherapy is by far used for treatment of cutaneous cancers. Its potential for treating deep-seated head and neck lesions, but also those in other areas of the body is yet to be fully understood and appreciated. Emphasizing the importance of proper patient and treatment parameters selection, their education about the possible drawbacks of treatment and, last but not least, outcomes are of utmost importance. We present two patients, both with squamous cell carcinomas of head and neck, but with very different outcomes.

Cancer is a leading cause of morbidity and mortality worldwide and is growing as indicated by WHO report. In 2012, there were 14M new cases and 8.2M deaths. With over 1M incidences and 500,000 deaths, breast cancer is one of the most common cancers of women. There are several types of breast cancers among which triple negative breast cancer (TNBC) has the worst prognosis due to lack of estrogen, progesterone and HER-2 receptors. About 20% of breast cancers are TNBC. Systemic chemotherapy with normal and high doses is practiced. Considering that these do not serve all patients, the short and long term side effects, and the cost, there is a critical need for alternate therapies and electrochemotherapy (ECT), using electrical pulses for enhanced uptake of chemo drugs has the potential to alleviate the suffering of TNBC patients. In ECT, typically Bleomycin (occasionally Cisplatin) is used. In clinical practice for TNBC, a combination of Gemcitabine and Cisplatin is used. Hence, in this study we explore the efficacy of the Gemcitabine+Cisplatin combination ECT on MDA-MB-231, triple negative, human breast cancer cells. The results indicate cell kill up to 56% at selective doses and pulse conditions. This ECT combination therapy could be optimized and transferred to the clinics.

The paper presents the realization of a prototype of a flexible grid electrode suitable to treat large surface by means of electrochemotherapy. The device has been primarily designed in order to treat breast cancer recurrences appear on chest wall. This case the flexibility of the support allows the adaptability to the surface. In practice, this kind of device can allow the reduction of the treatment time that is limited by the biodisponibility of the drug (i.e. 20 min). The first prototype of the presented device has been preliminary tested in suitable phantoms in order to verify the reliability of electroporation.

Tissue electrical conductivity is correlated to tissue characteristics. In this work some tumor mass excised from patients has been evaluated in terms of histological characteristics (cell size and density) and electrical resistance.

Ablative techniques based on irreversible electroporation (IRE) have emerged as an effective cancer therapy. In these treatments electrical pulses are delivered in order to induce irrecoverable damage to the cell membranes, thus, causing cell death. An interesting feature of electroporation treatments is the capability to predict shape and extension of lesions by means of mathematical simulations. Those prediction methods have been refined thought in-vivo experimental observations. Aiming at minimization of animal experiments, vegetal models have been used to easily observe the IRE outcomes. However, these observations are limited at the surface of the tissue. Here we present an improved method able to observe the inner parts of the tissue and therefore evaluate in three-dimensions the results of IRE using potatoes as vegetal models. The technique consists in using a dye solution to enhance the IRE area in sliced potato tubers. After slice digitalization, the electroporated area is automatically identified and the resulting treated volume is calculated. In addition, a three-dimensional reconstruction of both healthy tissue and IRE volume is generated. The proposed evaluation technique was used to assess different pulse protocols outcomes. Numerical simulations had been carried out to compare the numerical predictions to the experimental observations. The obtained results show a clear match between experimental and simulated volumes confirming the reliability of the proposed method.

Assessment of a tumor response to electrochemotherapy is still limited to the observation of long-term outcomes. Monitoring tumor volume changes can provide additional information required for a more detailed analysis of the tumor response. In this paper, we present a new mathematical model of tumor growth inhibition in mice following electrochemotherapy. The model is based on a single ordinary differential equation describing tumor growth and shrinkage. The growth curve is represented by a function describing initial exponential growth followed by a gradual transition to a linear growth. The opposite process, tumor shrinkage, is modeled by a shifted Gaussian function. Free parameters of the model were estimated using nonlinear least squares fitting. Average value of linear growth rate obtained from the control group was used by fitting the tumor volumes of treated animals. Results show that the model is flexible enough to fit partial as well as complete responses. Additionally, three parameters reflecting the treatment effect were used to calculate the tumor growth delay according to the formula derived from the model. An excellent agreement with observed growth delays confirms that proposed model can serve as a basis for a quantitative assessment of tumor response to electrochemotherapy.

Despite their high potential to replace the time consuming gold standard blood culture for infection diagnosis from whole blood, molecular biology based techniques often lack sensitivity and specificity due to the high human background in complex biological samples such as blood. Concerning the realization of a point-of-care device for sepsis diagnosis which would enable immediate targeted antimicrobial therapy, new, implementable and scalable sample preparation strategies are urgently needed that aim at reducing the human background. In this paper we report a first proof-of-principle for such a novel concept of selective human blood cell depletion preserving bacterial pathogen integrity by using high frequency electric fields. By thin film electrical passivation of the electrode material, ohmic current through the biological sample, which causes unspecific cell lysis, is drastically reduced. With the developed SiO

2

-passivated electrical cell lysis unit (ECLU), cell specific lysis of up to 75.3 % of the total human blood cells could be shown with less than 10 % loss of

E. Coli

viability. Additionally, a small electrode distance of 25

μ

m reduced the required voltage for selective cell lysis to a range of 8-20 V. The results presented here show great potential for the further development of fully automated and integrated cell selective sample preparation.

Cryopreservation is the most effective method for long-term storage of cells. Cryoprotectant solution usually contains 10% dimethyl sulfoxide and fetal bovine serum. Both represent a limiting factor for clinical applications, due cytotoxicity and presence of animal proteins. We have developed an improved method for cryopreservation of human umbilical cord-mesenchymal stem cells using trehalose in combination with reversible electroporation. First, we demonstrated efficient loading of propidium iodide into cells by reversible electroporation. Propidium iodide (100

μ

g/ml) was loaded into cells at 0, 100, 180, 300, 650 V, 8 pulses, 100

μ

s and 1 Hz in 2 mm cuvettes. The highest permeabilization while maintaining high cell viability was reached at 350 V. Second, cells were treated with a combination of 100 mM trehalose and electroporation at 0, 220, 430, 610 V, 8 pulses, 100

μ

s and 1 Hz, before cryopreservation. After thawing, the highest viability of 61 % was obtained at 430 V. As a control standard freezing protocol with 10 % dimethyl sulfoxide in 90% fetal bovine serum (v:v) was preformed, where 85 % after thawing recovery was obtained. In conclusion, according to our preliminary study electroporation seems an efficient method for loading nonpermeable trehalose into human cells and enables successful cryopreservation of human umbilical cord-mesenchymal stem cells.

Gene electrotransfer is becoming increasingly more recognized method for cancer gene therapy, proceeding to clinical trials. It is especially attractive for some indications: like vaccination through the systemic secretion from the transfected muscle or skin, and the utilization of the spatial precision provided by the method for tumor targeting. For the smooth translation of this method in the clinical setting, the appropriate plasmid vector design is of immense importance. Here we present the construction of clinically applicable antibiotic-free therapeutic plasmids for immunotherapy and antiangiogenic therapy of cancer, with

interleukin 12

gene and anti-endoglin shRNA molecule under the transcriptional control of tissue specific promoters for muscle, skin and endothelium as well as a therapy inducible promoter.

An important goal of researchers dealing with gene therapy is the development of safe and efficient systems which will ensure selective and controlled expression of a therapeutic gene in targeted cells and which will also be safe for patients. The aim of the study was to construct a eukaryotic expression plasmid encoding the therapeutic gene for a mouse cytokine interleukin 12 (mIL-12) under the control of

smooth muscle gamma actin

(SMGA) promoter without the antibiotic resistance gene. The final aim was to evaluate the suitability of the SMGA promoter for tissue specific transcriptional targeting by following the mIL-12 expression, and to compare the efficiency of recombinant plasmids with and without the antibiotic resistance gene. Expression of mIL-12 was followed on the mRNA level was followed by a quantitative real time polymerase chain reaction and on the protein concentration was determined by ELISA assay. We demonstrated that expression of mIL-12 on mRNA level was not increased in targeted, for SMGA promoter specific cells compared to the control, for SMGA non-targeted cells. However, higher expression of mIL-12 was demonstrated on the protein level in the targeted cells, specific for the SMGA promoter. Protein concentration, as well as the amount of mRNA of mIL-12 after gene electrotransfer of recombinant therapeutic plasmids with and without antibiotic resistance gene was equal.

To take full advantage of electroporation mediated delivery of therapeutic molecules to skin, it is important to establish an efficient and safe delivery system. Current electrode systems typically induce muscle twitching and pain, which is related to the distance between the electrodes and the applied voltage. Hence, the use of non-invasive electrodes, by which the delivery of electric pulses does not damage the skin, but does successfully permeabilize the skin cells and deliver the desired molecules into them is necessary. For these purposes, a system consisting of an array of electrodes was developed and evaluated in our study. In the scope of our study, the multi-array electrodes combined with electroporation proved to be effective for the delivery of model drugs (dextran and doxorubicin) as well as plasmid DNA to the mouse skin, with minimal skin damage caused by electric pulses. Furthermore, the advantage of the multi-array electrodes is to adjust the number of addressable electrode pairs that are contained within the electrode applicator, which was also demonstrated in our study. The use of multi-arrays with adjustable pins would be a valuable approach for the delivery of genes around the skin area for wound healing as well as for peritumoral delivery for cancer gene therapy. These results suggest that electroporation with multi-array electrodes can be efficient and non-invasive skin delivery method for therapeutic drugs and genes, with less adverse side effects than other electroporation delivery systems, and with promising clinical applications.

Tumor angiogenesis represents a promising target for cancer treatment. Tumor endothelial cells proliferate faster than normal endothelial cells due to activation of specific endothelial cell markers. One of them is endoglin, a Transforming Growth Factor

β

(TGF-

β

) co-receptor, involved predominantly in cellular proliferation and migration. Besides the expression of endoglin in tumor endothelial cells, it is also expressed in some types of tumor cells, especially in melanoma. Therefore, in melanoma, targeting endoglin could provide a beneficial therapeutic effect based on simultaneously inhibiting angiogenesis and melanoma cell proliferation. Therefore, the aim of our study was to determine the level of endoglin expression in endothelial and tumor cell lines (melanoma and adenocarcinoma) and to investigate if endoglin expression is associated with changes in cell viability after endoglin silencing. Our study showed that endothelial and melanoma cells express high levels of endoglin and that after endoglin silencing with gene electrotranfer (GET), cell viability was specifically decreased, whereas in tumor cells with low expression of endoglin, only non-specific decrease in cell viability was observed after GET. Our study is one of the first studies exploring the effect of endoglin on biological properties of melanoma cells and indicates new possibilities for melanoma treatment with targeted gene therapy approach.

This paper discusses the motivations and initial design specifications relating to a custom experimental apparatus for the investigation of ultrasound as an active enhancement method in transdermal drug delivery. The motivations for such an apparatus are: the high standard deviations in published results; the expected discrepancies between inputted parameter values to a specific test setup and the values of those variables at the skin surface during experimentation; and the potentially low validity of empirical relationships due to apparatus inadequacies. Preliminary numerical modelling has indicated that, for ultrasound enhanced transdermal experimentation, an apparatus made of polypropylene and submerged in water will have superior functionality to a standard glass Franz diffusion cell surrounded by air. A novel donor chamber has been made that is capable of accommodating a 50cm diameter ultrasound probe in a Franz diffusion cell setup.

Due to the safety issues of viral vectors, non-viral gene delivery systems, such as chemical carriers, gene electrotransfer, and magnetofection are developing. Magnetofection is a particle mediated transfection method. Our research group developed unique polyacrilyc acid (PAA) coated and polyethlenimine (PEI) functionalized superparamagnetic iron oxide nanoparticles (SPIONs) for the introduction of reporter and therapeutic plasmid DNA (pDNA) into the cells and tumors.

The aim of this study was to compare magnetofection efficiency among tumor and normal cells, and to verify the transferability of magnetofection as non-viral gene delivery method from in vitro to in vivo systems.

The experiments of magnetofection with reporter plasmid DNA encoding enhanced green fluorescent protein (EGFP) were performed on different murine tumor and normal cell lines, spheroid cultures and tumors in mice. The EGFP expression was qualitatively evaluated by fluorescent microscopy 24 h after magnetofection.

Our in vitro results demonstrated high magnetofection efficiency in tumor cells, especially melanoma, and poor in normal cells. Both, the number of cells expressing EGFP and the intensity of EGFP fluorescence, were higher in tumor cells. Further results, obtained after magnetofection of spheroids and tumors in mice, proved its feasibility in 3D cell culture as well as in in vivo system. In the case of spheroids, only the outer layer of cells expressed high levels of EGFP, while in the center of the spheroid no transfection was noticed. On frozen section of subcutaneous tumor in mice, randomly distributed and small EGFP fluorescent dots after three consecutive magnetofections were noticed.

The encouraging outcomes of this study demonstrate the effectiveness, selectivity, usefulness, feasibility and transferability of magnetofection as non-viral gene delivery method from in vitro to in vivo systems.

We developed a generator for controlled exposure of small biological samples to conditions they experience when lightnings strike their habitats. We tested the generator with our exposure chamber described previously. Recording at 341 thousand frames per second with Vision Research Phantom® v2010, the fastest commercially available camera, we found that the initial arc descended vertically into the sample, but then became accompanied by arcs descending increasingly sideways; after 8–12

μ

s, as the first of these arcs formed direct contact with the receiving electrode. We eliminated this artefact by a Plexiglas® cylinder positioned concentrically between the electrodes. While bacterial spores are highly resistant to electric pulses delivered through direct contact, we show that with discharges delivered as arcs through an air gap and thus accompanied by acoustic shock wave, spore inactivation is readily obtained.

Sonoporation is a technique in which ultrasound induces permanent or transient increase in cell membrane permeability, allowing various molecules to cross membrane barrier. Sonoporation efficiency in MHz frequency range in in vitro conditions is enhanced by introducing contrast agents - bubbles into cell medium. Cavitation of these bubbles is considered as the main mechanism for sonoporation phenomenon. In this study we focused on sonoporation using low intensity ultrasound in low frequency range (29.6 kHz). To improve the sonoporation efficiency we used carbon dioxide (CO

2

) bubbles. The saturation of the cell medium with CO

2

causes the drop in medium pH, therefore cell viability after 60 s sonoporation by continuous ultrasound exposure in CO

2

saturated medium and in acidic medium without addition of CO

2

was followed.

The results showed that the experimental procedure and the presence of B16-F1 cells in acidic medium or in CO

2

saturated medium for 60 s has no effect on cell viability. Also the exposure of B16-F1 cells to ultrasound for 60 s in culture medium or in acidic medium did not change the cell viability. But the exposure of the cells to ultrasound for 60 s in CO

2

saturated medium decreased cell viability to 8%±11%.

CO

2

bubbles enhance sonoporation performed by low intensity ultrasound in kHz range. The results of our study show that decrease in cell viability is a consequence of the presence of the bubbles and not the acidity of the cell medium.

The use of nonthermal plasma in the clinic has gained recent interest, as the need for alternative or supplementary strategies are necessary for preventing multi-drug resistant infections. The purpose of this study was to evaluate the antibacterial efficacy of a novel plasma reactor based on a high current version of sliding discharge and operated by nanosecond voltage pulses without an applied gas flow. This modification is advantageous for both portability and convenience. Bacterial inactivation was determined by direct quantification of colony forming units. The plasma was bactericidal against

Escherichia coli

and

Staphylococcus epidermidis

seeded on culture media. Plasma exposure significantly inhibited the growth of both model organisms following a 1-minute application (p<0.001). Results indicate that

S. epidermidis

was more susceptible to the plasma after a 5-minute exposure, compared to

E. coli

. Temperature measurements taken immediately before and after plasma exposure determined that heat does not play a role in bacterial inactivation. These findings suggest the current plasma has potential application for surface bacterial decontamination.

Pulsed electric fields (PEF) technology is a promising innovative non-thermal process to improve mass transfer in food sector. PEF treatments induce a partial cell membranes electroporation which extent depends on electric field strength, number, duration and shape of the pulses and application time. The present work aimed at highlighting the effect of the application of PEF on mass transfer phenomena in apple parenchyma tissue, by evaluating the water distribution across cell compartments by means of NMR relaxometry. Pulsed electric fields treatments were carried out using near-rectangular shaped pulses with fixed 100

μ

s pulse width and 10 ms repetition time at three different specific voltage (100, 250 and 400 V cm

− 1

) and two different pulse number series (n=20 and n=60).

Results showed different trends according to the applied voltage. The lowest (100 V cm

− 1

) was not able to induce significant changes in plasma membranes, so that no water redistribution was achieved between cytoplasm and extracellular space. At the opposite, a marked redistribution was registered inside the cellular compartments, namely vacuole and cytoplasm, showing an alteration of the tonoplast. The total number of pulses was found to influence the amount of water migrating, from vacuole to cytoplasm, from 15 % with 20 pulses to 40 % with 60 pulses.

Medium and high voltage (250 and 400 V cm

− 1

, respectively) removed the possibility to distinguish the different cell compartments, probably due to intense damage of both plasma membrane and tonoplast. By observing water transverse relaxation time an additive effect of both voltage and total number of pulses was demonstrated. Interestingly, by considering water distribution during 120 minutes after PEF treatment time-dependent trends were found in the effects of each experimented protocol.

Pulsed electric fields (PEF) processing has been explored as a way to enhance extraction of bioactive compounds owing to its ability to alter cell membrane permeability. In wine making PEF has been used to improve the effectiveness of maceration process, however, little is known about how PEF affects the subsequent composition of the wild yeast population. Such information is required as wild yeast growth, during maceration and early stages of fermentation, can modify the flavor profile of wine. This study investigated the impact of PEF on

Saccharomyces cerevisiae, Picchia kluyveri and Hanseniaspora uvarum

. Each yeast species (4.3-4.8 Log CFU /mL) was suspended in defined grape juice medium (DM) of pH 3.5 and subjected to PEF treatments of either 15 , 60 or 160 kJ/kg at a field strength of 1.0-1.1 kV/cm. Yeast cell numbers were estimated immediately after PEF treatment and after 1, 2, 3, or 8 days under simulated maceration conditions.

S. cerevisiae

was the most sensitive species tested, to pH in DM and to the PEF treatments (P<0.05). After an initial decrease in numbers, cells in the control and 15 kJ/kg PEF treatments resumed growth, while 60 or 160 kJ/kg PEF treatments maintained the cell numbers below the initial levels for at least 8 days. PEF treatments and low pH of DM had no effect on

P. kluyveri

numbers or their subsequent growth (p>0.05). The numbers of spoilage yeast

H. uvarum

decreased to below the detection limit of 2.5 log CFU/mL after high PEF treatment and the degree of sub lethal injury was dependent on maceration time and PEF intensity (P<0.05). This study suggests that application of PEF during winemaking can enhance extraction and inhibit spoilage yeast while having a limited effect on the beneficial natural wine yeast.

The aim of the study was to investigate the influence of PEF pre-treatment (1, 3, 5 kV/cm and 10 kJ/kg) of blueberry fruits on the extraction yield, anthocyanins content and antioxidant activity of the juice obtained by pressing (1.32 bar for 8 min). Additionally, the extraction with solvent of anthocyanins from the berry by-products (pressed cakes) was also evaluated. Blueberry juice obtained from PEF treated berries at 1 and 3 kV/cm showed the highest increase in juice yield (+32%), total anthocyanins content (+55%) and DPPH-RSC (+ 40%), compared with the untreated sample. The extracts from press cakes obtained after PEF assisted-pressing of berry fruits showed a significant higher content of total anthocyanins (+95%) and DPPH-RSC (+138%) when compared with the control sample. The higher the field strength applied the greater the recovery of antioxidant compound in the extracts. Significant differences in the contents of anthocyanin glycosides were detected between blueberry juice and press cake extracts. The results obtained from this study demonstrate that PEF-assisted pressing is promising for production of higher quality juices and for the valorization of berry by-products.

Bacterial viability can be affected by pulsed electric fields (PEFs) offering a new approach for decontamination of liquids for the food industry and for waste water treatment. The molecular mechanisms remain poorly elucidated. One question is the control by the pulsing solution and the role of its conductance. Escherichia coli (E. coli) was chosen as a model system. The strain E. coli BL21(DE3) was grown to its stationary growth phase, washed and re-suspended in a saline solution at a well-controlled conductivity. Survival was evaluated by plating after a proper dilution.

Accumulation of microsecond pulses were efficient for electro-eradication as previously reported but, in most cases a capacitive discharge pulse generator was used where the pulse duration might be affected by the buffer composition and undergo changes during the pulse. This was not the case with our approach using a square wave electropulsator. The main conclusion was that eradication was more efficient in a buffer with a significant conductivity. A tentative explanation taking into account the different physical properties of a bacteria is provided.

The efficiency of PEF treatments in the range of milliseconds (0.4 to 0.6 kV/cm; 10 to 60 ms) and in the range of microseconds (4 to 6 kV/cm; 30 to 150

μ

s) has been compared for the extraction of betanine from red beet. Both PEF-treatments in the milliseconds and in the microseconds range improved the extraction of betanine in comparison to the control. Similar betanine extraction was obtained with pulses of milliseconds and microseconds under the PEF conditions that resulted in the maximum betanine extraction. The more intense treatment conditions applied in the ms range (0.6 kV/cm; 40 ms) and the

μ

s range (6 kV/cm; 150

μ

s) increased the BEYmax 6.7 and 7.2 times, respectively, compared with the control. However, lower specific energy was involved in the PEF-treatments applied in the range of

μ

s (28.8 kJ/kg) than in the PEF-treatments applied in the ms range (43.2 kJ/kg).

Preliminary study with extraction by electroporation of non-polar molecules from green alga

Chlorella vulgaris

showed very promising results. For successful extraction by electroporation from above mentioned alga takes at least 3.8 kJ of energy input/L dense algae, the best extraction is carried out around 7.2 kJ of energy input/L dense algae (at the amplitude of the electric field from 0,5 to 2,7 kV/cm). A sufficient quantity of algal biomass (dense algal suspension has > 10

7

cells/L in our experimental conditions). Indeed, when more cells are present in the suspension, the less likely the pulse will avoid cells due to the conductivity of the medium, resulting in better extraction by electroporation. It is also important to have optimal conductivity of the medium (1 to 1.3 mS/cm in our experimental conditions). Studied extraction by electroporation parameters for chosen alga (voltage, pulse length and different number of pulses delivered to biomass) and algal growth regeneration after different electroporation settings are presented.

Extraction of sugar solution from sugar beet cossettes has been studied at different parameters of electric pulses, with electric field intensities varying from 0.52 to 1.52 kV/cm. Sugar beet has been exposed to different number of electric pulses of different duration. Experiments where the only treatment was compressive load gave us maximum juice yield of 30.64 %, with PEF treatment added juice yield rose to a maximum of 80.32%. The whole process lasted 30 minutes. The first 5 minutes we pressed with pressure of 9.254 bars, then electric pulses were applied, followed by additional 25 minutes of pressing. To compare juice yields and specific energy consumption, we also performed experiments replacing PEF treatment with more established thermal treatment at temperatures (30, 40, 50, 60, 72) °C.

The complexity of cancer immunobiology imposes the use of reliable and strongly predictive preclinical animal models to successfully translate immunotherapeutic approaches into the clinical practice. In line with this, increasingly complex and comprehensive experimental mouse models have been developed. As an alternative scenario, comparative oncology proposes the investigation of naturally occurring cancers in pet animals for cancer immunotherapy studies. Veterinary clinical trials are highly predictive of what happens in the human setting, providing excellent translational “avatars” for the study of human cancer and of innovative strategies for its prevention and cure.

Gene electro transfer to the left ventricular myocardium is a promising technique for delivery of therapeutic genes for treatment of ischemia, myocardial infarction and heart failure directly to the effected myocardium. There are multiple variables to consider for efficient gene delivery and gene expression modulation, including electro transfer parameters, timing and duration of pulses relative to the electrocardiogram and ischemic state. Here we report establishing a small animal model and a large animal model for gene delivery to non-ischemic and ischemic left ventricular myocardium. Gene expression was evaluated histologically for location of expression within the myocardium as well as quantitatively via ELISA. We developed electro transfer electrodes and protocols that are safe and result robust gene expression. These animal models allow for evaluation of therapeutic potential of particular gene delivery as well as translation to clinical settings.

Degeneration of intervertebral discs can lead to various health problems, among which low back pain is the most common. Studies concerning methods of dealing with that problem are being conducted and magnetic resonance imaging is one of the most popular methods of assessing their progress. Many of these studies are based on animal models. The paper links these two facts and so it contains the description of methodology that can be used for assessing the degree of degeneration of the porcine intervertebral discs based on scans obtained in MRI study. Proposed method uses Otsu method of automatic thresholding and next basic operators of morphological image processing. The outcome is binary mask giving direct information about locations of intervertebral discs in MRI scans. The obtained mask can be also used to separate locations of intervertebral discs in following MRI studies of the same animals, in cases where they are much harder to delineate due to their degeneration. The paper shows also exemplary results demonstrating usefulness of proposed methodology for the evaluation of discs treatment using mesenchymal stem cells.

Classical methods, used for large scale treatments such as mechanical or chemical extractions, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on batch and flow processes electro-extraction on yeasts proved that pulsed electric field technology was highly effective and preserve the activity of released cytosolic proteins. As induction of electro-permeabilization is under the control of target vesicle size, protocols were chosen to affect only the plasma membrane and not vacuole membranes. Furthermore, large cell culture volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electro-extract total cytoplasmic proteins from microalgae (Nannochloropsis salina, Chlorella vulgaris). Due to the size selective effect of electro-permeabilization, as the mean diameter for N. salina is only 2.5

μ

m, we used stronger field strengths than for C. Vulgaris. A train of repetitive 2 ms long pulses of alternating polarities was delivered. The electric treatment was followed by a 24 h incubation period in a salty buffer containing DTT at room temperature. The amount of total proteins release was observed by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris and N. Salina under optimized electrical conditions.

Thanks to their high fatty acids content when cultivated in stress conditions (high light, no nitrogen), algae are considered as a renewable source of lipid-rich biomass feedstock for biofuels. The application of Pulsed Electric Field (PEF) to electroporate the algae, is a promising alternative to the use of solvents for lipid droplets extraction from these cells. In order to study the effects of PEF applied to oleaginous algae cells, we developed a microfluidic device, allowing real time visualization. Multiple closed electroporation chambers are designed on this biochip to characterize in real-time Propidium Iodide penetration on a population of algae, submitted to the PEF, and confined within each chamber. The method developed allows (1) to find the permeabilization threshold considering the treatment parameters (pulse width, amplitude and frequency) varying electric field, (2) to distinguish cell sensitivity in different conditions of growth, or different cell strains. Lipid droplets visualization in fluorescence and bright field during treatment was also performed on the device. We investigated the effects of PEF on the possible displacement or merging of droplets within the cytoplasm. While optimized electroporation parameters could be found for the different algae strains with different lipid content that we investigated, the possible extraction of lipids remains challenging and strain dependent, due to the cellulosic cell wall.

The adoption of pulsed electric field technologies is currently limited by the lack of application-specific knowledge as well as the availability of reliable and validated full-scale reactors. In this paper, the progress made towards the design, optimization and scale-up of the OpenCEL

TM

technology applied to high-flow, heterogeneous matrices are illustrated. Specific challenges included engineering systems capable of sustained long-term operation in harsh environments operating with complex non-Newtonian fluids. The paper also describes the importance of adopting standard operating procedures for evaluating and validating equipment performance. Robust experimentation coupled with numerical model was also instrumental in guiding the optimization of the reactor chamber in a way that mass and energy transport could simultaneously be taken into account.

During exotic fruits processing, a large amount of by-products, especially seeds and peels, are produced and discarded into the environment causing organic pollution. However, it is recognized that these by-products are a good source of high-added value compounds, which can be used for different purposes (i.e. food additives and/or nutraceuticals). At this stage of development, there is a lack of information about the different extraction methods that can be used for the recovery of high-added value compounds from papaya and mango by-products. In this line, the extraction assisted by pulsed electric technologies seems to be rather promising. In this research study, the exponential decay pulses with initial electric field strengths of 13.3 kV/cm and 40 kV/cm for PEF and HVED treatments, respectively, were used. The impacts of temperature and pH on extraction efficiency of different components (proteins, total phenolic compounds, carbohydrates, isothiocyanates) and antioxidant capacity were evaluated. The highest values of nutritionally valuable and antioxidant compounds were obtained for HVED-assisted extraction. However, the application of HVED-treatment may produce undesirable contaminants (chemical products of electrolysis, free reactive radicals, etc.) and the obtained extracts were unstable and cloudy. Application of the two-stage method (PEF + supplementary aqueous extraction at 50 °C) allowed a significant enhancement of the yields 200%) and antioxidant capacities 20%) of the extracted components from papaya and mango by-products even at neutral pH.

Electrochemotherapy (ECT) is combined local or systemic chemotherapy with electroporation that permeabilize cell membranes to locally potentiate the antitumor effectiveness of chemotherapeutic drugs. ECT became a standard method for the specific type and location of tumors (cutaneous, subcutaneous and oral tumors) in dogs, cats and horses. It can be used as a single therapy or as an adjuvant therapy to surgery. The results of the clinical trials conducted at the Clinic for surgery and small animals of Veterinary Faculty in collaboration with the Institute of Oncology Ljubljana are presented.

Forty-eight dogs, 12 cats and 32 horses were included in the clinical studies. 70% of the CR (complete response) was obtained in dogs with mastocytoma in different clinical stages. In dogs with different type of perianal tumors the OR (objective response) was > 90% (65-87.9% CR). 11 cats with cutaneous or mucosal squamous cell carcinoma (SCC) responded with 70% of OR (50% CR, according to the location). 100% OR was obtained in horses with sarcoidosis (92.5% CR, only in one case the recurrence was noted just after 60 months). The results of our clinical trials confirmed that ECT is an effective local treatment, comparable with the classic local treatments in veterinary medicine. In addition, ECT is simple, safe and cheap.

The aim of presented study was to determine efficacy and safety of combined electrochemotherapy and electrogene therapy in treatment of canine cutaneous mast cell tumors. 18 tumor nodules in 18 patients were treated with combination of intratumoral ECT using cisplatin and peritumoral intradermal IL-12 EGT. Complete response was achieved in 72% of patients (objective response in 78%), with 100% complete response rate in smaller tumors (<2cm

3

). In patients which presented without metastases before treatment (14/18), the disease did not progress and tumors did not metastatise despite 6 of them being higher grade tumors. Poor response to therapy (progression of disease) was observed in only 3 patients with pre-existing metastases and higher clinical stage of disease. However, one patient with metastatic disease was completely cured and remained tumor free for over 2 years. Systemic release of hIL-12 was detected in 88% of examined patients and 70% of patients responded with elevation of IFN-

γ

up to 3 months after therapy. The treatment didn’t cause any local or systemic side effects. In conclusion, electrochemogene therapy with a combination of IL-12 and cisplatin is highly effective and safe antitumor treatment in canine cutaneous mast cell tumors.

The electrochemotherapy (ECT) is a cancer treatment based on joint action between chemotherapy drugs and high electric fields over the tissue. A relevant parameter to ECT efficiency is the complete coverage of target tissue by a sufficiently high electric field E

REV

. The induced transmembrane voltage for the reversible threshold open pores in the cells and macromolecules go through. Strong electric fields produce cell instability and necrosis. We present a case study of a cutaneous tumor in veterinary oncology, which was used to validate the model. We used 3D finite element numerical models with an ellipsoidal tumor, a skin of three layers and needle electrodes (type II and III). The goal of this work was vary such parameters as distance between needles, electrode polarities and arrangement to evaluate the distribution of electric field. We observed that increasing the rows distance, or reducing the space between needles in same row reduces the minimum voltage necessary to cover the entire tumor with electric field higher than E

REV

. Our results allowed us to conclude that type II electrodes are adequate to treatment of cutaneous ellipsoidal tumor.

Electrochemotherapy (ECT) was used to treat 176 client owned dogs and cats with a variety of, mainly, superficial tumours in sites where surgical treatment would be likely to produce cosmetic or functional problems. ECT was applied either as sole modality, during surgery or after wound healing. Analysis of follow-up data from these cases was used retrospectively to assess efficacy in different species, tumour types and locations. Local tissue damage associated with ECT was assessed and related to pulse frequency. The incidence of second malignancies in ECT treated patients was compared controls.

Clinical studies using gene electrotransfer of plasmid encoding human interleukin 12 (IL-12) have already shown good results when treating tumors in canine patients. Nevertheless, the use of a plasmid encoding canine IL-12 is essential for further clinical trials. Therefore, the aim of this study was to evaluate the effect of the plasmid encoding canine IL-12 in in vitro and in vivo studies and compare its activity to the plasmid encoding the human IL-12. Our results showed that after in vitro gene electrotransfer plasmid encoding canine IL-12 had similar or better IL-12 expression capacity than plasmid encoding human IL-12. Further on, the in vivo experiment in nude mice revealed that the therapeutic effect of plasmid encoding canine IL-12 after gene electrotransfer was comparable to the therapeutic effect of plasmid encoding human IL-12 after gene electrotransfer. Also, the effect on tumor growth delay was equivalent after first and second therapy, which shows that the gene electrotherapy of plasmid encoding canine IL-12 is repeatable. Therefore, our study suggests that the plasmid encoding canine IL-12 is suitable for further clinical studies in dogs.

Electrochemotherapy (ECT), a medical treatment widely used in human tumor treatment, consists in the administration of bleomycin either systemically or locally followed by the application of an electric field. This procedure increases the toxicity of the bleomycin by 1000 fold in the treated area with an objective response rate of around 80%. Despite its success related to efficiency, low cost and minimum side effects, there is still a 20% of cases in which the ECT treatment is not responding. This could be ascribed to the fact that bleomycin, administered in the preferred way, that is, systemically for large tumors, is not properly reaching the whole tumor mass. The main cause is poor tumor vascularization, in which case local administration could cover areas unreachable with systemic administration. To address this problem here we propose the combined administration of bleomycin, systemic and locally, using companion animals as models for ECT tumor treating. Accordingly, we selected 7 canine and 2 feline patients with a single tumor with poor or no response to ECT, and then we repeat the treatment with ECT but now with a combined administration of bleomycin, systemic and local. The results show, according to an evaluation using the WHO criteria of tumor response, that from the 9 cases, 5 achieved a Complete Response, 3 a Partial Response, and 1 a Stable Disease after 30 days from the combined treatment date. In conclusion, the combined administration of bleomycin, systemic and local, in ECT could provide a good response in tumors that previously showed an unsatisfactory response. It is expected that these results could hopefully increase ECT efficiency.