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STSM Reports Homepage

Below you may find reports of STSMs (Short-Term Scientific Missions) carried out within the scope of the TD1104 COST Action. Some of these reports have also been published in the Action newsletters.

Quick jump-to-STSM menu:

Mihaela Moisescu Nataša Pavšelj Barbara Mali
Hanne Falk Hansen Tudor Savopol Selma Čorović
Justin Teissie Irina Grigorescu Patrizia Lamberti
Mohammed Hamza Bermaki Paolo Marracino Stine Krog Frandsen
Urška Kamenšek Yassine Bellebna Natasa Tesic
Monica Florescu Paola Maresca Tomás García-Sánchez
Urša Lampreht Richard Postma Barbara Zorec
Špela Kos Giuseppe Olivieri Tanja Dolinšek
Daniela Nikolovska Nedelkoska Flavien Pillet Djamel Eddine Chafai
Caterina Merla Lise Pasquet Elisa Luengo
Cristele Delsart Tomás García-Sánchez Aziz Aboulmouhajir
Elena Della Valle Damijan Miklavčič Veselina Uzunova
Lea Rems Stefania Romeo Matej Kranjc
Elisabetta Sieni Bor Kos Aude Silve
Claudia Irene Trainito Emilie Louise Hansen Agnese Denzi
Janja Dermol Noelia Lopez-Giral Quim Castellvi
Shirley Sharabi Gianpiero Pataro Nerijus Lamanauskas
Tomaž Jarm Azadeh Peyman Clair Poignard
Bor Kos Christoph Huber Andraz Polak
Urszula Tylewicz Denitsa Mitkova Brankova Katarzyna Dymek
Tadeja Forjanič Antoine Azan Bostjan Markelc
Barbara Zorec Saša Haberl Meglič

Evaluation of dielectrophoretic behavior of electroporated cells

STSM by Mihaela Moisescu, Early-Stage Researcher

Period of mission: from 15/03/2015 to 09/04/2015

Home institution: Biophysics and Cell Biotechnology Dept., Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

Host institution: UMR 8203 CNRS, University Paris Sud and Gustave Roussy Institute, Villejuif, France

Two types of dielectrophoretic (DEP)-controlled movements are experimentally observed: positive-DEP and negative-DEP (as cell moves respectively, toward or against a high intensity region of the field), each of them occurring in a characteristic range of AC electric field frequencies. By a proper selection of the DEP field frequency, separation of cell categories in a mixture or evaluation of cell dielectric properties becomes possible.

The experimental work carried out during this STSM exploited the 3D DEPtech system recently available in the Host institution by determining the DEP spectra of electroporated cells and correlating the dielectric parameters of the cells with the conditions of electroporation.

By monitoring the changes in optical density of a cell suspension subject to DEP field, the device allowed obtaining a 20-points DEP spectrum since it works with 20 wells energized by different AC field frequencies. This system allowed rapid analysis (within tens of seconds) of thousands of cells simultaneously. DEP spectra of electroporated cells has been acquired as function of exterior medium conductivity (400, 100 and 10 μS/cm) and of time after pulses’ delivery (2 to 30 min).

The single shell dielectric model for a cell in suspension is implemented into the analysis module of the 3D DEPtech system. When a measured DEP spectrum has been analyzed, certain parameters had to be declared, like: exterior medium conductivity and permittivity and cell radius (measured on the actual cell suspension used in the experiments). Dielectric parameters like: specific membrane capacitance and conductance, or cytosol conductivity for electroporated cells could be thus calculated and their evolution in time evaluated.

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Violeta Liuba Calin, Mihaela Georgeta Moisescu, Irina Grigorescu, Liviu Dimitriu and Tudor Savopol
Title Investigation of optical and mechanical characteristics of electroporated cells using optical tweezers

 

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Mihaela G. Moisescu, Mihai Radu, Isabelle Leray, Minodora M. Iordache, Tudor Savopol and Lluis M. Mir
Title Chemical membrane permeabilisation provides insights on the membranes biophysical parameters changes during cell electroporation

 


Study of calcium electroporation and immune modulators

STSM by Hanne Falk Hansen, PhD Student

Period of mission: from 12/04/2015 to 20/05/2015

Home institution: Center for Experimental Drug and Gene Electrotransfer, Department of Oncology, Herlev University Hospital, Herlev, Denmark

Host institution: Cork Cancer Research Center, University College Cork, Cork, Ireland

Calcium electroporation is a new local cancer treatment using electric pulses to internalize calcium in cancer cells, which leads to an intracellular calcium overload and necrosis. Necrosis is often associated with immunogenic cell death, a type of cell death that allows immune cell involvement. If calcium electroporation induce an immune response and create an anti-tumor specific response, it may be possible to enhance the response and create a systemic effect. 

To investigate whether calcium electroporation creates an immune response, Balb/c mice were injected with the murine colon cancer cell line CT26. At a tumor diameter of 0.4-0.8 cm the mice were treated with injection of calcium chloride and electroporation. Post treatment tumors were measured regular for growth and survival, and at day three and seven tumors were collected for histological analysis for infiltrating immune cells, and blood sampled to analyze the pro-inflammatory cytokine profile. Same procedures were performed on control groups (bleomycin and electroporation, electroporation alone, calcium and sonoporation, bleomycin and sonoporation, sonoporation alone, calcium alone, bleomycin alone and untreated).

The four groups with respectively calcium or bleomycin combined with either electroporation or sonoporation, all showed decrease in tumor sizes. Edemas were created around tumors treated with calcium unlike the tumors treated with bleomycin, indicating that the cell death in the two treatments could be different. The other groups showed no effect with increasing tumor size. Further results are pending.


Preparation of the paper on good practice for reporting electroporation research

STSM by Justin Teissié, Emeritus

Period of mission: from 02/06/2015 to 13/06/2015

Home institution: IPBS CNRS Toulouse, France

Host institution: University of Ljubljana, Ljubljana, Slovenia

At the meeting of the COST Steering Committee in Salerno, 2012, some critical issues were raised on many contributions reporting electroporation research. It was decided to start the Preparation of a paper in which good practice for reporting electroporation research is collected, mainly to:

 1. Emphasize on good practice in experimental design,

 2. How to present modeling based on data supplied by some reference groups,

 3. Unifying terminology. 

These issues could be addressed through publishing protocols and what needs to be reported in an experimental report/paper; or by tutorials clarifying what terminology means and what should be most suitable. 

A general rule/guidance in reporting experimental data and most of all exposure conditions was completed along the STSM. This guide point out to report details to the extent that other researchers will be able to repeat, judge and evaluate the experiments and data obtained. 

This kind of reporting is also necessary for future systematic reviews and/or meta-analysis. 

Taking into account the wide field of electroporation, 2 drafts were written, one for Life Science (Basic Science oriented in connection with Health Science applications), the other for research connected with Industrial applications (Food, Biotech). Both drafs were structured in a similar way: a short easy to read set of recommendations and a long list of annexes where all relevant information are provided.

These reports should now be reviewed by experts from the COST Action TD 1104 to get a final consensus version during the World Congress in Portoroz this September.

Editors of relevant journals will then be approached for the publication of these recommendations for good practice in reporting electroporation research.

Publication(s) originating from this mission

Publication ?
Publisher ?
Authors Justin Teissié
Title Recommendations for good practice in reporting electroporation research for Life Sciences
Year 2015
Issue ?
Pages ?
Status Unknown - to be updated

 

Publication ?
Publisher ?
Authors Justin Teissié
Title Recommendations for good practice in reporting electroporation research for Food and  Biotechnologies
Year 2015
Issue ?
Pages ?
Status Unknown - to be updated

 


Development of a versatile Marx generator for electroporation in food processing

STSM by Mohammed Hamza Bermaki, PhD Student

Period of mission: from 01/04/2015 to 31/05/2015

Home institution: APELEC Laboratory, Djillali Liabes University, Sidi Bel Abbes, Algeria

Host institution: University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia

Nowadays many pulse generators are developed, with today’s electronics; it’s possible to generate several Kilovolt pulses with very short duration, every generator has many specifications and its use depends on the kind of application, these generators also differ in circuit design and switch type, also the effectiveness of a pulsed generator depends on its ability to control the different parameters of the pulses (Amplitude, pulse duration, frequency …).

Marx generator is a unique circuit intended for voltage multiplication, its functioning is based on charging capacitors in parallel and discharging them in series into the treatment chamber.

The purpose of the STSM was the realization of a Marx generator with negative pulses (four stages), the circuit design is based on IGBTs, because the IGBTs enable fast switching and therefore a possibility to obtain very short duration pulses.

The Marx generator realized during this mission, is able to deliver high pluses up to 3.6 KV, and 30µs pulse width, and it can be used for electroporation in food processing.

The work done represents a considerable advance; the next step will be to optimize the generator to obtain pulses ranging up to 10 KV, this can be achieved with the addition of other stages, or with using components capable of supporting higher voltage.

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Mohammed Hamza Bermaki, Abdelkader Semmak, Matej Reberšek, Yassine Bellebna, Damijan Miklavčič, Amar Tilmatine
Title Development of a Marx-type PEF generator using IGBT switches

 


Real time monitoring of gene electrotransfer efficiency using different electroporation protocols

STSM by Urška Kamenšek, Early-Stage Researcher

Period of mission: from 02/03/2015 to 29/05/2015

Home institution: Institute of Oncology Ljubljana, Ljubljana, Slovenia

Host institution: Institute of Pharmacology and Structural Biology, Toulouse, France

Gene electrotransfer (GET) is becoming increasingly more recognized method for cancer gene therapy. Although, the method is generally safe, efficient, and can produce good reproducibility and spatial precision, the effective delivery of therapeutic gene to target tissue remains the stumbling block to its effective and broader use. Gene electrotransfer to tumors represent a special challenge due to the heterogeneity of tumors, low transfection efficiency and activation of non-specific antitumor effectiveness, most probably mediated by the induction of immune response. Therefore the purpose of my STSM was to further explore the phenomena associated with gene electrotransfer to tumors with real time evaluation of transfection efficiency as well as cell death following the treatment with noninvasive luminescence monitoring of the effects. Using the light producing tumors, the luminescence technology enabled us the in vivo monitoring of tumor growth, as well as the emergence of hypoxic - necrotic tumor center. Additionally, we were able to visualize the transient transfection after the i.t. GET of the luciferase expressing plasmid. The data obtained during the STSM provided a new insight on some of the phenomena’s associated with gene electrotransfer to tumors, like the vascular lock, the generation of the non-specific antitumor effectiveness and the localization of the source of expression. These can all have an impact on GET efficiency and have to be taken into account when designing improved and more effective cancer gene therapy, allowing the quicker translation of the technology into clinical trials.

Publication(s) originating from this mission

Journal Current Gene Therapy
Impact Factor (according to ISI Thomson) 2,542
Authors Urska Kamensek, Marie-Pierre Rols, Maja Cemazar, Muriel Golzio
Title Visualization of Nonspecific Antitumor Effectiveness and Vascular Effects of Gene Electro-Transfer to Tumors
Year 2015
Issue ?
Pages ?
Status Submitted - to be updated

 


Microfluidics systems for cell electroporation

STSM by Monica Florescu, PhD Student

Period of mission: from 18/05/2015 to 29/05/2015

Home institution: Transilvania University of Brasov, Brasov, Romania

Host institution: University of Twente, Enschede, The Netherlands

During this STSM the grantee has focused on learning about miniaturized and microfluidic devices to be used for cell electroporation. In the microfluidic devices the cells flow in a constricted channel equipped with integrated electrodes where they are exposed to high electric fields (kV/cm). These devices also allow impedance measurements before and after cell electroporation to count the cells and monitor the process of membrane permeabilization. The grantee has learnt about the different steps of the cell electroporation protocol including off-chip cell sample preparation (e.g., cell staining), injection of cell solution in a controlled way in the microchannel using dedicated equipment, delivery of pulses and on-chip detection of pores formation using (fluorescence) microscopy and impedance spectroscopy. The measurements have been conducted using different cancer cell lines, and by varying different experimental parameters. Altogether, these experiments have highlighted the power of microfluidics for cell electroporation and monitoring of the process at the single cell level.

During the STSM the grantee had free access to the bibliographic resources of the University of Twente, and more importantly she acquired practical experience with cell electroporation and microfluidic devices. This STSM has also allowed fostering collaboration between the Transilvania University of Brasov, Romania and the University of Twente, The Netherlands in the field of electroporation or biosensors and microfluidics, collaboration to be materialized in the near future as scientific application for European funding or student exchange.


Electrogene therapy (EGT) with IL-12

STSM by Urša Lampreht, PhD Student

Period of mission: from 15/03/2015 to 31/05/2015

Home institution: Institute of Oncology Ljubljana, Ljubljana, Slovenia

Host institution: National Research Council (CNR), Institute of Translational Pharmacology, University Campus Bio- Medico of Rome, Rome, Italy

Gene electrotransfer (GET) is an effective method to introduce plasmids encoding different genes into cells. GET of plasmid encoding interleukin 12 (IL-12) already showed to have a good antitumor response resulting in significant tumor growth delay and tumor eradication. The mechanism of IL-12 is to elicit a strong immune response and to enhance the functioning of antigen presenting cells that infiltrate the tumors which leads to better recognition of tumor antigens and apoptosis of tumor cells. To visualize this characteristic of IL-12 the method of immunohistochemical staining is an excellent choice. Therefore the purpose of my STSM was evaluate immunohistochemically the involvement of antigen-presenting cells into the tumors following the  intratumoral GET of plasmid encoding IL-12. During STSM we have successfully optimized the protocol for the two antibodies F4/80 and MHCII.The obtained results showed that the electrogene therapy (GET) with both plasmid, therapeutic encoding IL-12 and control plasmid, induced the innate immune response, as there was a high infiltrate of macrophage in both samples regarding to the control group.To determine the M1 macrophages specific staining for MHCII was used. We observed a strong positive staining of cells expressing MHCII in the tumor samples which were treated with therapeutic plasmid. This kind of an observation was not seen in the group treated with control plasmid. Therefore, we can presume that the GET therapy with therapeutic plasmid encoding IL-12 induce the infiltration of M1 macrophages, which has antitumor activity that results in tumor growth delay and complete responses.


Evaluation of novel electrode for the purpose of vaccination and skin gene therapy

STSM by Špela Kos, PhD Student

Period of mission: from 01/03/2015 to 31/05/2015

Home institution: Institute of Oncology Ljubljana, Ljubljana, Slovenia

Host institution: Louvain Drug Research Institute (LDRI), Catholic University of Louvain, Brussels, Belgium

Skin gene electrotransfer (GET) is a safe and efficient method to deliver the genes to skin tissue. Its transfection efficiency depends on different parameters, including the type of electrodes for optimal electric pulses application. So far, different electrodes have been used to deliver the genes to skin, such as plate electrodes, needle electrodes, as well as novel multi-array electrodes (MEA). Based on the previous studies, GET with MEA resulted in minimal skin damages with great reduction of muscle contraction. Therefore, the aim of our study carried out during STSM, was to investigate the novel MEA electrodes for the purposes of DNA vaccination and gene delivery to skin. The transfection efficiency obtained with MAE as compared to plate electrodes was first evaluated in vivo on a C57/BL6 mouse model. Efficiency of GET with MEA was evaluated by following luciferase gene expression after intradermal injection of plasmid DNA (pDNA) and by characterizing the immune response after immunisation with model therapeutic pDNA coding for ovalbumin (OVA).

The results indicate that novel multi-array electrodes promoted higher and longer duration of gene expression compared to luciferase gene delivery by plate electrode. Further on, the results indicate on strong humoral and cellular immune response after MEA application, with a significant delay in tumour growth after immunization. 

The results gained during STSM proved the effectiveness of MEA electrodes for skin gene delivery and characterizes them as a promising tool for gene therapy and vaccinations.

Poster presentation(s) originating from this mission

Event PhD students' day, organized by Louvain Drug Research Institute
Location Brussels/Belgium
Period

21/05/2015

Authors Spela Kos, Tanja Blagus, Kevin Vanvarenberg, Maja Cemazar, Véronique Préat, Gregor Sersa, Gaëlle Vandermeulen
Title Utilization of gene electrotransfer for delivery of plasmid DNA to the mouse skin

 


PEF assisted extraction of bioactive compounds from tomato by-products

STSM by Daniela Nikolovska Nedelkoska, Early-Stage Researcher

Period of mission: from 04/05/2015 to 23/05/2015

Home institution: Faculty of Technology and Technical Sciences, University “St. Kliment Ohridski”, Veles, Republic of Macedonia

Host institution: ProdAl Scarl, University of Salerno, Fisciano, Italy

Tomato by-products, namely peel and seeds, account for about 10-40% of the millions of tons of tomato fruit processed every year to produce different type of products. 

This by-products represent a cheap and reach source of the health-promoting phytonutrients, that are traditionally recovered by extraction with solvents which is a time consuming process and requires large quantities of solvents. 

The investigation was conducted to evaluate the effect of pulsed electric field (PEF) treatment for enhancing valuable compounds extraction from tomato by-products. The process was based on the application a PEF pre-treatment at electric field strengths in the range 0.5-5 kV/cm and energy input between 10 and 40 kJ/kg to tomato peels. The effects of the different PEF processing conditions were defined, and the extraction yield of lycopene, total carotenoids and total phenolics was determined. Additionally, the determination of the antioxidant activity of the extracts was performed. 

The results demonstrated that a significantly higher amount of total carotenoids, lycopene and total phenolics was detected in the extracts from PEF pre-treated peels in comparison with those present in the extracts from untreated peels.

The antioxidant capacity of the extracts obtained from tomato peel was correlated with the concentration of the extracted compounds. 

The results indicate that the utilization of PEF technology as pre-treatment stage of the extraction process of valuable bioactive compounds from tomato by-products could add new value to the tomato processing chain, improving economic performance and decreasing waste problems.

Oral presentation(s) originating from this mission

Event 29th EFFoST International Conference - Food Science Research and Innovation
Location Athens/Greece
Period 10/11-12/11/2015
Authors G. Pataro, D. Nikolovska Nedelkoska, D. Carullo, M.M. Capitoli, G. Ferrari
Title PEF treatment for the valorization of tomato by-products

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors D. Nikolovska Nedelkoska, G. Pataro, D. Carullo, M.M. Capitoli, G. Ferrari
Title Pulsed electric field assisted extraction of carotenoids from tomato by­products

 


Experimental assessment of the performance of an antenna prototype to apply nsPEF

STSM by Caterina Merla, Early-Stage Researcher

Period of mission: from 30/04/2015 to 23/05/2015

Home institution: Italian National Agency for New Technology, Energy, Sustainable Economic Development, Radiation Biology and Human Health Unit, Rome, Italy

Host institution: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, US

This STSM focuses on accurate experimental characterization of an EM exposure setup developed (theoretical design, numerical simulations) at the ENEA, Rome, Italy. The setup, built under the scope of a common Italian-American research effort involving the study of nanosecond pulsed electric fields and cancer-related mechanisms, allows the exposure of newborn mice to extremely short (5 ns) and intense (MV/m) electric pulses. Two device prototypes were completely characterized in frequency and time domains. Antenna performance tests, loaded and unloaded with a suitable dielectric, consisting of frequency domain measurements up to 3 GHz, were carried out using a VNA. Then, a high-voltage generator was used to deliver single or multiple 6 ns pulses. A tap-off was used to acquire on an oscilloscope both the input and reflected signals from the device. The electric fields emitted at different distances and positions over the antenna patch (loaded and unloaded) were measured using a D-dot sensor. We obtained a total transmitted voltage into the antenna of nearly 40% of the input voltage. Although the data show rather large reflections; the pulse rise/fall times are not degraded. Good measurement repeatability was obtained. We see now that we need to improve the performance at lower frequencies. However, the dielectric matching slab for holding biological samples increases the amplitude of the radiated electric field.

Despite limitations, the present setup can be used to expose biological targets to shorter pulses, even down to the picosecond regime.

Publication(s) originating from this mission

Journal Medical & Biological Engineering & Computing
Impact Factor (according to ISI Thomson) 1,700
Authors Caterina Merla, Francesca Apollonio, Alessandra Paffi, Carmela Marino, P. Thomas Vernier, M. Liberti
Title Monopole patch antenna for in vivo exposure to nanosecond pulsed electric fields
Year 2015
Issue ?
Pages ?
Status Submitted - to be updated

Conference abstract(s) originating from this mission

Publication IFMBE Proceedings of the 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Publisher Tomaz J, Kramar P (ed), 1st edn. Springer-Verlag, Singapur
Authors Caterina Merla, Alessandra Paffi, Priscilla Monaco, Teresa Calderaro, Francesca Apollonio, Carmela Marino, P. Thomas Vernier, Micaela Liberti
Title Design of an applicator for nsPEF exposure of newborn mice
Year 2015
Issue 53
Pages 228-231
Status Published

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Paolo Marracino, Micaela Liberti, Francesca Apollonio and Andrea Amadei
Title nsPEfs induce irreversible denaturation of sperm whale myoglobin: a thermodynamics analysis

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Caterina Merla, Alessandra Paffi, Priscilla Monaco, Teresa Calderaro, Francesca Apollonio, Carmela Marino, P. Thomas Vernier, Micaela Liberti
Title Design of an applicator for nsPEF exposure of newborn mice

 


Toward a Healthy, Sustainable Food System - Pulsed Electric Fields, a new green technology for sustainable management of agri-food waste

STSM by Cristèle Delsart, Early-Stage Researcher

Period of mission: from 24/04/2015 to 29/05/2015

Home institution: University of Nova Gorica, Nova Gorica, Slovenia

Host institution: University of Salerno, Fisciano, Italy

Two studies have been done during this scientific mission in the laboratories ProdAI (Salerno, Italy).

The first one concerned the valorisation of red grapes wastes obtained after winemaking by the extraction of polyphenols via the use of Pulsed Electric Fields (PEF). The effect of PEF treatments (1, 3 and 5 kV/cm at 10 kJ/kg and 3 kV/cm at 20 kJ/kg) on seeds, skins and a mix of seeds and skins from Merlot grapes pomace followed by a step of diffusion in a hydro-alcoholic solution during 24 hours has been studied. The results have shown that: (1) the phenolics extraction by PEF is better when applied only on skins in comparison with the PEF application on a mix of seeds and skins; (2) the extraction kinetics were not similar according to raw material. In conclusion, with the aim to improve the phenolicsextraction, it would be better to treat separately the seeds and the skins with different modalities of PEF and with different times of diffusion.

The second study concerns the valorization of bran and germ of wheat wastes from flour production by the extraction of carbohydrates, proteins and polyphenols via the use of PEF. Previously, the application of PEF on wheat has never been tested. PEF treatments (same modalities than grape wastes) on these moistened materials have been done. The extraction of proteins, carbohydrates and polyphenols has been measured in extracts after 24 hours of diffusion in a hydro-alcoholic solution. The work is still in progress and is planned to be upgraded in collaboration also in the future.

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Cristèle Delsart, Daniel Carullo, Kajetan Trost, Melita Sternad Lemut, Gianpiero Pataro and Giovanna Ferrari
Title Towards a green and sustainable recovery of phenolic compounds from food by-products

 


Nanosecond pulsed electric fields on liposomes for potential drug delivery applications

STSM by Elena Della Valle, PhD Student

Period of mission: from 09/03/2015 to 09/05/2015

Home institution: Department of Information Engineering, Electronics and Telecommunications, “Sapienza” University of Rome, Italy

Host institution: UMR 8203 CNRS, Univ Paris-Sud and Gustave Roussy, Villejuif, France

The use of nanosecond pulsed electric fields (nsPEF) for potential drug delivery applications is one of the most promising techniques which could allow a controlled release of drugs encapsulated in nano-carriers (i.e.liposomes) by the application of an electrical stimulus. The aim of this STSM was to analyze the capabilities of liposomes to mediate drug delivery through cell membrane under nsPEF exposure without causing any damage to the biological cells. With the microdosimetric models, different thresholds were obtained in terms of the external electric field necessary for the poration of the liposome and cell membranes. However the buffer and the inner liposome conductivity can influence these threshold values; moreover the liposome diameter impacts since the largest liposomes in our study (400 nm of diameter) are electroporated with lower electric field amplitudes than the smaller liposomes.

An experimental activity was carried out in order to verify the feasibility of liposome poration with the electric field intensity predicted by the theoretical models. A field amplitude between 3 and 4 MV/m was applied to the DSPC vesicles containing two different populations of liposomes loaded with a fluorescent dye. The fluorescence value was calculated for the exposed and not exposed samples but only a slightly difference was appreciated and no liposome electroporation occurred. Further experiments will be necessary in order to reach the best conditions in terms of liposome dimension and conductivity values to obtain the liposome membrane poration.

Publication(s) originating from this mission

Journal Journal of Membrane Biology, Special Issue
Impact Factor (according to ISI Thomson) 2,174
Authors Agnese Denzi, Elena della Valle, Francesca Apollonio, Marie Breton, Lluis M. Mir, Micaela Liberti
Title Exploring the Applicability of Nano-poration for Remote Control in Smart Drug Delivery Systems
Year 2015
Issue ?
Pages ?
Status Submitted - to be updated

 


Permeability of peroxidized lipid membranes investigated by molecular dynamics simulations

 STSM by Lea Rems, PhD Student

Period of mission: from 28/02/2015 to 27/05/2015

Home institution: University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia

Host institution: Université de Lorraine, SRSMC, UMR 7565, Vandoeuvre-les-Nancy, France

Lipid peroxidation is a process characterized by modification of lipid fatty acid chains, mediated by reactive oxygen species. Consequently, peroxidized lipid membranes have altered properties, including increased permeability to various molecules (Runas and Malmstadt, Soft Matter 11:499–505, 2015). Lipid peroxidation takes place also during membrane electroporation (Gabriel and Teissié, Eur J Biochem 228:710–718, 1995), and could therefore contribute to the experimentally observed long-lived permeability of cell membranes as a complementary or even alternative mechanism to pore formation. The aim of this STSM project was to quantify the permeability of bilayers formed by 1,2-dilinoleoyl-sn-glycero-3-phosphocholine and various contents of its peroxidized derivatives (Garrec et al., J Phys Chem Lett 5:1653–1658, 2014), to water, sodium, and chloride ions, using molecular dynamics simulations. In order to calculate membrane permeability, we calculated the profiles of the potential of mean force for investigated species along the axis perpendicular to the membrane surface using the recently developed unified free energy dynamics method (Chen et al., J Chem Phys 137:024102, 2012). Our results demonstrate that the permeability to water and ions indeed increases with increasing number of peroxidized lipid species in the bilayer – for ions even up to several orders of magnitude. Similar ratio of increase is expected for membrane conductivity, as the latter is roughly proportional to ion permeability (Vorobyov et al. Biophys J 106:586–597, 2014). Based on this evidence we can conclude that lipid peroxidation should be considered as additional mechanism of increased membrane permeability to water and ions in membrane electroporation.


Cell Electroporation with new Platinum Compounds

STSM by Elisabetta Sieni, Early-Stage Researcher

Period of mission: from 19/03/2015 to 30/03/2015

Home institution: Padova University Department of Industrial Engineering, Padova, Italy

Host institution: University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia

The idea was to use two new platinum compounds with different solubility in the culture media and study their behavior under electroporation conditions.

The cytotoxicity of two platinum compounds (named C, cis-guanidine and D, trans-guanidine) has been tested on B16F1 cells in two different conditions: without and with electroporation. The cytotoxicity without electroporation has been compared with the one of cis-Platin. 

Cytotoxicity without electroporation has been performed in 6 repetitions with different concentration of the compounds in order to evaluate the IC50. Cells have been plated and incubated 24h before adding compounds. The exposure to the compound has been performed for 5 min and 48h. The permeabilization as well as the voltage pulses survival for B16F1 cells has been verified for 6 different voltages applied between electrodes in the range 0 and 320 V using electrodes with 2 plates electrodes at 2mm between them and 8 pulses at 1 Hz (pulse length 100 µs). Permeabilization has been verified using Propidium Iodide (PI) fluorescent indicator and TECAN. From survival and permeabilisation results the suitable voltage intensity for this type of cells has been selected to be 200 V. Finally, cytotoxicity of compounds with and without electroporation has been verified by means of MTS test 72hafter the electroporation/exposition to Platinum compoundof cells seeding. It appears that the compound D shows a reduction of cell survival for a 5 min compound-exposition if voltage pulses have been applied. This difference was not observedin the experiments where the compound C was tested.


Cancer Cell Electrorotation

STSM by Claudia Irene Trainito, PhD Student

Period of mission: from 01/03/2015 to 23/03/2015

Home institution: École Normale Supérieure, Cachan, France

Host institution: Virginia Tech – Wake Forest University School of Biomedical Engineering and Science, Blacksburg, Virginia

Thanks to the support of the COST Action a collaborative project on the characterization of cancer cells at different stages of the illness has been carried on between two research teams: BIOMIS at ENS Cachan (Institut d’Alembert, CNRS UMR8029) and the Bioelectromechanical Systems Laboratory of Virginia Tech-Wake Forest University School of Biomedical Engineering and Science, both specialized on the investigation of the interaction between cells and electric field.

Identification and separation of cells within a complex sample are of prime importance for the cancer diagnosis and treatment.  This project used a combined dielectrophoresis (DEP) and electrorotation (ROT) technique to elucidate the phenotypic differences of sequentially staged cancer cells. First the dielectrophoresis force is applied for cell trapping, and then a rotating electric field through a sample medium is induced. Undergo those two solicitations, the cell experiences a rotational movements and the analysis of its rotational velocity gives as results the dielectric properties such as the permittivity and the conductivity of both membrane and cytoplasm.  Dielectric properties represent electro-physiological traits intrinsic to a cell which are both dependent upon structures both on/within the cell.  Once the electro-physiological fingerprint of the cells is determined, this method can be used as a tool to distinguish malignant cells from healthy ones and determine the patient’s stage of cancer.

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Claudia Irene Trainito, Emilie Bayart, Emilie Bisceglia, Frederic Subra, Olivier Français, Bruno Le Pioufle
Title Electrorotation as a versatile tool to estimate dielectric properties of multiscale biological samples: from single cell to spheroid analysis

 


Electro-induced sensitization of cells

STSM by Janja Dermol, PhD Student

Period of mission: from 18/01/2015 to 20/03/2015

Home institution: University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia

Host institution: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, Virginia, USA

The aim of my STM was to investigate cell sensitization. Cell sensitization is a phenomenon where increasing the duration of the electroporation treatment, for example by splitting the delivered pulses in two shorter trains is reported to significantly increase the cell sensitivity to subsequent pulses. Uptake of different molecules is increased and cell viability is reduced. The mechanisms for occurrence of cell sensitization are not known yet. The suggested possibilities are cell swelling, formation of reactive oxygen species and calcium influx. In this STSM I investigated the connection between cell permeabilization and sensitization. Experiments were performed on human monocyte cell line U937. Pulses were delivered either all in one train or they were split in two trains with a 5 min delay between them. Stable cell sensitization was obtained in a wide range of permeabilizing E-fields. With higher E-fields applied the cell sensitization effect was larger. Cell survival was up to twofold reduced when the same electrical parameters were applied but the second train was delayed for 5 min. In addition it was established that when the delay between the trains is 15 seconds there is already cell sensitization present. Our results are important for clinical applications where lower number of pulses, lower voltages and larger distance between the electrodes can be used if the delivered pulses are split in two parts with a short delay between them.

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Claudia Muratori, Janja Dermol, Shu Xiao, Andrei Pakhomov and Olga Pakhomova
Title The time course of electroporation-induced sensitization

 

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Janja Dermol, Olga N. Pakhomova, Shu Xiao, Andrei G. Pakhomov and Damijan Miklavčič
Title Cell sensitization is induced by a wide range od permeabilizing electric fields

 


Individual electroporation treatment planning for rats brain

STSM by Shirley Sharabi, PhD Student

Period of mission: from 12/04/2015 to 25/04/2015

Home institution: Tel-Aviv University, Sackler Faculty of Medicine, Tel-Aviv / Sheba Medical Center, The Advanced Technology Center, Ramat-Gan, Israel

Host institution: University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia

The overall goal of our research is developing individual treatment planning for brain tumors using local point-source electroporation combined with systemic chemotherapy. The goal of the mission was to develop a mathematical model describing the effect of electrical fields and number of treatment pulses on treatment outcome, including irreversible tissue damage and blood-brain barrier (BBB) disruption which enables efficient drug delivery. It was previously suggested that the Peleg-Fermi statistical model can describe cells survival probability after electroporation. During the STSM we used experimental data obtained from Magnetic Resonance images of naïve rats treated by electroporation with a single intracranial electrode using different number of pulses and fitted the results to the Peleg-Fermi model. The model computes the ratio of surviving cells after electroporation depending on of the applied electric field and number of pulses.

The coeeficients of the equation were computed by optimization methods and the results of the model were compared to experimental data on irreversible electroporation (IRE) the rats' brain. The same model was then adopted to describe the BBB disruption in the brain tissue and new coeeficients were computed from the model.

Electrical field thresholds were calculated for IRE and BBB disruption for different number of pulses. These thresholds can be used for efficient treatment planning for brain tumors, where irreversible electroporation is used for the destruction of the tumor mass and the BBB disruption is used in combination with chemotherapy to assist in drug delivery to the tumor.

Publication(s) originating from this mission

Journal Radiology and Oncology
Impact Factor (according to ISI Thomson) 1,912
Authors Shirley Sharabi,  Bor Kos, David Last, David Guez, Dianne Daniels, Sagi Harnof, Yael Mardor, Damijan Miklavcic
Title A statistical model describing combined irreversible electroporation and electroporation-induced blood-brain barrier disruption
Year 2016
Issue 50(1)
Pages 28-38
Status Published

 


Evaluation of the effects of electroporation pulses on implantable cardiac pacemakers

STSM by Tomaž Jarm, Associate Professor

Period of mission: from 03/11/2014 to 31/01/2015

Home institution: University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia

Host institution: University of Zagreb, Faculty of Electrical Engineering and Computing, Zagreb, Croatia

Electrochemotherapy of tumors is an effective treatment also for treatment of internal "deep-seated" tumors. Currently it is contraindicated for patients with implanted pacemakers simply as a precaution measure. Whether the high-voltage electric (electroporation) pulses used in electrochemotherapy can interfere with functioning of pacemakers is actually unknown. A series of experiments was performed to assess voltages and currents that could be induced at a pacemaker's sensing/pacing electrodes during application of electroporation pulses. A container filled with physiological saline was used to mimic the physiological conditions when the electrodes are embedded in an internal organ. Both the treatment and the pacemaker electrodes were placed in the container at varying distances. The minimum distance was 1.5 cm to mimic the worst-case situation when the electroporation pulses are delivered at the outer wall of the heart. In addition to the measurements without the pacemaker, the measurements were performed with the pacemaker connected and working in an asynchronous mode. The atrial pacing pulses were used to trigger the delivery of electroporation pulses during the period of ventricular pacing. Thus we were able to monitor the interference of electroporation pulses with a functioning pacemaker during the period when its susceptibility to electrically induced interference is the greatest due to low impedance of its pacing circuitry. We found no evidence of harmful effects that the electroporation pulses might have on functioning of a pacemaker, even though the induced artefacts during the application of pulses can be considerable and can exceed the amplitude of the pacing pulse itself.

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Tomaz Jarm, Branko Milankovic, Damijan Miklavcic, Ratko Magjarevic
Title Preliminary evaluation of exposure of cardiac pacemakers to electroporation pulses

 


Improvement of IRE models for treatment planning based on clinical data in human patients

STSM by Bor Kos, Early-Stage Researcher

Period of mission: from 15/12/2014 to 19/12/2014

Home institution: University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia

Host institution: Dept of Diagnostic and Interventional Radiology, Universitätsklinikum Leipzig AöR, Leipzig, Germany

Irreversible electroporation is an ablation method, which can be used to treat tumors in organs such as the liver or kidneys in a minimally invasive fashion, when performed under CT guidance. However, irreversible electroporation requires an adequate distribution of electric fields to be achieved during treatment to ensure treatment success. In order to validate the numerical models, it is useful to evaluate well-documented historical cases. The Department for Interventional radiology of the University clinic Leipzig, has performed several IRE therapies of tumors in livers with CT guidance. This allowed us to very accurately reproduce both the patient anatomy and electrode positions during treatment. Coupled with measured current and voltage data logged during pulse delivery, this allowed us to validate a model with coupled electric fields and joule heating. With heating taken into account, the computed currents matched very well with measured currents. The improved models will allow a more precise modelling of heating and heat-related effects during irreversible electroporation and also improve the prediction of ablation regions due occurring achievable with given treatment parameters. The STSM to Leipzig was a great success and a very pleasurable experience of working with motivated and highly skilled scientists.

Publication(s) originating from this mission

Journal Radiology and Oncology
Impact Factor (according to ISI Thomson) 1,912
Authors Kos B, Voigt P, Miklavcic D, Moche M
Title Careful treatment planning enables safe ablation of liver tumors adjacent to major blood vessels by percutaneous irreversible electroporation (IRE)
Year 2015
Issue 49
Pages 234-241
Status Published

 


Low voltage PEF applications on minimally processed fruit

STSM by Urszula Tylewcz, Early-Stage Researcher

Period of mission: from 01/12/2014 to 12/12/2014

Home institution: University of Bologna, Cesena, Italy

Host institution: German Institute of Food Technologies, Quakenbrueck, Germany

Minimally processed (MP) are metabolic active tissues and, as a consequence, they show physiological response to preparation procedures as well as to different pre-treatments. Moreover the freezing process applied further to MP fruits may affect the quality and tissue metabolism, as well as the water mobility in the fruit tissue. In this work, the non-thermal technique such as Pulsed Electric Field (PEF) was used in order to verify the potential application of PEF technique on fruits and vegetables. The preliminary study has been performed to identify the most suitable variety of apples and pears for the PEF treatment. For this purpose the intact apples and apple cylinders were treated with the electric field strength set to 1.23 kV/cm and 0.3 kV/cm, respectively. Pink Lady apple variety and Alexander Lucas pear variety resulted in better preservation of colour after the PEF treatment. Conductivity measurement were performed in order to calculate the cell disintegration index (Zp), showing a high cell disintegration (values of about 0.78-0.85) when high electric field strength was used and low cell disintegration index (of about 0.02-0.03) when low electric field strength was used. Further studies are in progress to understand the effect of PEF pre-treatment on water mobility in frozen apple tissue.

Publication(s) originating from this mission

Journal Innovative Food Science and Emerging Technologies, Special Issue
Impact Factor (according to ISI Thomson) 3,273
Authors Tylewicz U., Aganovic K., Vannini M., Toepfl S., Bortolotti V., Dalla Rosa M, Oey I., Heinz V.
Title Effect of pulsed electric field treatment on water distribution of freeze-dried apple tissue evaluated with DSC and TD-NMR techniques
Year 2015
Issue ?
Pages ?
Status Submitted - to be updated

Oral presentation(s) originating from this mission

Event School on applications of Pulsed Electric Fields for food processing 
Location Fisciano/Italy
Period 07/02 - 12/02/2015
Authors Tylewicz U., Aganovic K., Vannini M., Kiessling M., Toepfl S., Bortolotti V., Dalla Rosa M, Oey I., Heinz V.
Title Changes in water distribution induced by PEF treatment of apple

Poster presentation(s) originating from this mission

Event EFFOST2015
Location Athens/Greece
Period

10/11 - 12/11/2015

Authors Urszula Tylewicz, Kemal Aganovic, Santina Romani, Pietro Rocculi, Stefan Toepfl, Volker Heinz, Marco Dalla Rosaa, Indrawati Oey
Title Effect of low intensity PEF pre-treatment on qualitative characteristics of frozen apple and pear

 

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Tylewicz U., Aganovic K., Vannini M., Toepfl S., Bortolotti V., Dalla Rosa M, Oey I., Heinz V.
Title Effect of PEF pre-treatment prior freeze-drying on water state and mobility in apple tissue

 


Tumor growth modeling based on medical images of electroporation-treated patients

STSM by Tadeja Forjanič, PhD Student

Period of mission: from 01/12/2014 to 10/12/2014

Home institution: University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia

Host institution: INRIA Bordeaux - Sud-Ouest Research Center, Talence, France

Tumor growth modeling is a promising approach for evaluation of response to electroporation based treatments. The aim of my STSM was to explore the possibilities of modeling the growth and inhibition of colorectal liver metastases treated with electrochemotherapy (ECT). Since it was not possible to accurately measure lesion dimensions on available medical images, the time behavior could not be obtained. However, radiooncologist made an interesting observation that in all cases showing the partial response to the treatment, metastases were located in close proximity to big blood vessels. According to his opinion major hepatic vessels can affect and degrade the treatment. This requires further investigation of underlying processes in order to improve the treatment efficacy. Additionally, we were working on modeling the tumor response using the data of tumor growth in mice treated with ECT. We have developed a two-compartment model based on ordinary differential equations that describes the tumor volume changes with time after the treatment. First compartment includes the cells that are not affected by the treatment, so their total volume increases according to the Gompertz law, whereas cells from second compartment eventually die due to damages induced by the treatment. Since described model showed a good approximation to our experimental data, it can be used to study underlying mechanisms in order to get a more complete picture of tumor responses to ECT.

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Tadeja Forjanič, Damijan Miklavčič
Title Modeling of tumor growth inhibition in mice following electrochemotherapy

 


Modification of Franz diffusion cell experimental setup for physical enhancement methods

STSM by Barbara Zorec, PhD Student

Period of mission: from 05/10/2014 to 08/11/2014

Home institution: Iskra Medical d.o.o., Ljubljana, Slovenia

Host institution: University of Canterbury, Department of Mechanical Engineering, Christchurch, New Zealand

Franz diffusion cells represent the gold standard in skin passive diffusion studies, but are not the ideal in vitro setup to be used in transdermal delivery studies with electroporation and other physical enhancement methods. Due to the geometric constraints, delivering electric pulses to the skin inside the diffusion cell is challenging. The electric pulses are delivered through the electrodes placed in the donor and the receiver solution, so most of the delivered voltage rests in the buffer itself and only a small part on the skin. For ultrasound testing the donor compartment of Franz diffusion cells system is way too small. We aimed to modify Franz diffusion cells, to be better suited for experiments with physical enhancement methods of transdermal molecular delivery. We considered parameters that we needed to improve: Ultrasound donor chamber designed especially for sonoporation (minimizes acoustic interference) and 3D printed strain clamp allows for seamless integration of standard cell receiver with novel donor chamber. With this in our mind we developed a modified diffusion cell, which addressed the problems we have outlined. Donor compartment is much larger and with that suitable for other enhancement techniques. The material choices for the donor chamber and clamp were made according to just little reflections that are expected. After the preliminary experiments we confirm that the results of our work have delivered a newly designed donor chamber and clamp for Franz diffusion cells to be suitable for side-by-side electroporation and sonoporation.

Publication(s) originating from this mission

Journal Journal of Biomechanical Engineering
Impact Factor (according to ISI Thomson) 1,780
Authors Nataša Pavšelj, Barbara Zorec, Damijan Miklavčič, Sid Becker
Title Experimental Factors to be Consideredin Electroporation-Mediated Transdermal Diffusion Experiments
Year 2015
Issue ?
Pages ?
Status Accepted for publication - to be updated 

Oral presentation(s) originating from this mission

Event 3rd International Conference on Computational Methods for Thermal Problems (ThermaComp2014)
Location Lake Bled/Slovenia
Period 02/06 - 04/06/2014
Authors Barbara Zorec, ??? (to be updated)
Title Computational modeling of skin electroporation: reconciliation between experimental observations and anticipated physics

 

Event 23rd International Symposium on Transport Phenomena
Location Auckland/New Zeeland
Period 19/11 - 22/11/2012
Authors Sid Becker, Barbara Zorec, Nataša Pavšelj, Damijan Miklavčič
Title Transport Modeling of Skin Electroporation and the Thermal Behavior of the Stratum Corneum

Mechanical phenomena in transdermal drug delivery with electroporation and other physical methods

STSM by Nataša Pavšelj, Researcher

Period of mission: from 05/10/2014 to 08/11/2014

Home institution: University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia

Host institution: University of Canterbury, Department of Mechanical Engineering, Christchurch, New Zealand

In collaboration with Dr. Sid Becker from the Host institution, we have recently added new knowledge into the field of transdermal drug delivery with electroporation, in which we have shown that more pulses do not necessarily mean more transdermal delivery and that the order of the types of pulses administered must also be considered (Zorec at al., International Journal of Pharmaceutics, 2013). We experimented with different combinations of short high-voltage (HV) and long low-voltage (LV) pulses, focusing primarily on the order of pulses. We anticipated that a combination of HV (creation of small aqueous pathways), followed by LV pulses (expansion into bigger local transport regions - LTRs) will work in synergy and will result in better molecular delivery. However, we have shown that the total transport of solute was decreased when LV pulses were preceded by HV pulses. During the STSM we explored this phenomenon further, focusing on analytical description of LTR expansion depending on the initial conditions of the skin, i.e. the size and the density of the preexisting small aqueous pathways. We described the thermodynamics underlying the LTR evolution with an analytical model that shows directly how the heating time required to initiate LTR evolution is dependent upon the pre-existing stratum corneum pathway size and density. The pore size and density strongly influence the pulsing time required to attain sufficient resistive heating to reach lipid phase transition temperatures required for LTR expansion, which agrees with our experimental data.

Publication(s) originating from this mission

Journal Journal of Biomechanical Engineering
Impact Factor (according to ISI Thomson) 1,780
Authors Nataša Pavšelj, Barbara Zorec, Damijan Miklavčič, Sid Becker
Title Experimental Factors to be Consideredin Electroporation-Mediated Transdermal Diffusion Experiments
Year 2015
Issue ?
Pages ?
Status Accepted for publication - to be updated 

Oral presentation(s) originating from this mission

Event 3rd International Conference on Computational Methods for Thermal Problems (ThermaComp2014)
Location Lake Bled/Slovenia
Period 02/06 - 04/06/2014
Authors Barbara Zorec, ??? (to be updated)
Title Computational modeling of skin electroporation: reconciliation between experimental observations and anticipated physics

 

Event 23rd International Symposium on Transport Phenomena
Location Auckland/New Zeeland
Period 19/11 - 22/11/2012
Authors Sid Becker, Barbara Zorec, Nataša Pavšelj, Damijan Miklavčič
Title Transport Modeling of Skin Electroporation and the Thermal Behavior of the Stratum Corneum

Implementation of an optical tweezers based system for the micromanipulation of electroporated cells

STSM by Tudor Savopol, Researcher

Period of mission: from 09/11/2014 to 15/11/2014

Home institution: Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

Host institution: Institute of Pharmacology and Structural Biology (IPBS), CNRS, Toulouse, France

The main purpose of the STSM mission was to start developing a method, based on optical tweezers, which should permit to monitor modifications of optical properties of porated cells with a good time resolution (as compared to cellular events) subsequent to the electroporation. This can be done by measuring the force constant of a trapped cell before electroporation and at different moments after electroporation. In order to do this and to get quantitative robust results, the optical trap should be very well calibrated. The main problem in using optical traps is the calibration of its force. The purpose of the scientific work carried out during STSM was to setup a calibration method which can overcome these disadvantages. The proposed method is based on the use, in the optical path of the laser, before entering in the microscope, of a mirror mounted in the back focal plane of the first telescope of the tweezers, which allows a fast deflection with small well controlled angles, of the laser beam. We tested the method using beads of different materials and diameters, suspended in media with different viscosity. The results were consistent with the characteristics of the beads and proved to be very reproducible. Data collection protocols were established in order to optimize the method and to make it compatible with simultaneous use in electroporation experiments.

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Violeta Liuba Calin, Mihaela Georgeta Moisescu, Irina Grigorescu, Liviu Dimitriu and Tudor Savopol
Title Investigation of optical and mechanical characteristics of electroporated cells using optical tweezers

 


Development of a Matlab-based interface for an optical tweezers system dedicated to electroporated cells

STSM by Irina Grigorescu, MSc Student

Period of mission: from 12/10/2014 to 25/10/2014

Home institution: Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

Host institution: Institut de Pharmacologie et de Biologie Structurale, Toulouse, France

The optical tweezers are a scientific instrument which uses a highly focused laser to provide an attractive or repulsive force to physically move or hold microscopic dielectric objects. I am currently working in performing measurements of the power spectrum of such a device dedicated to studies on electroporated cells. The purpose of this STSM was to develop Matlab software application for acquiring and analyzing the data given by the optical tweezers system and to add features to an existing program. The current software is rigid in parameters outcome and insufficient in providing relevant data since the status of the electroporated cells is changing rapidly in time. The laboratory that hosted me at IPBS is experimenting and using an optical kit similar to the one in Bucharest. Exploiting their experience on correct adjustment of both hard and software components of the device, I gained knowledge in terms of adapting the Matlab interface specialized in controlling the optical tweezers system when single-cell electroporation studies are performed and I applied it when the calibration routines were run.

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Violeta Liuba Calin, Mihaela Georgeta Moisescu, Irina Grigorescu, Liviu Dimitriu and Tudor Savopol
Title Investigation of optical and mechanical characteristics of electroporated cells using optical tweezers

 


Molecular study of transmembrane protein under nanosecond electric signals

STSM by Paolo Marracino, PhD Student

Period of mission: from 12/10/2014 to 25/10/2014

Home institution: Department of Information Engineering, Electronics and Telecommunications, Sapienza, University of Rome, Rome, Italy

Host institution: University College Dublin (UCD), Dublin, Ireland

The use of extremely short and intense electric pulses, allows the manipulation of intracellular membranes and to possibly interact with proteins, both intracellular or embedded in membranes. Full atomistic molecular dynamics simulations aim to shed light to the main mechanisms of interactions between the exogenous electric pulses and these kinds of targets. The present STSM had two major objectives: first of all to perform a feasibility study for the integration of “realistic electric signals” inside a simulation package (NAMD) which does not envisage using this feature; secondly to investigate the possibility to import the topology of a transmembrane protein, the aquaporin (already investigated by the STSM grantee via NAMD simulations), into a GROMACS compatible format. In particular for the first task, the TCL scripting language (Tool Command Language) has been adopted to create a series of scripts that tell NAMD to apply certain forces (for example the ones exerted by external electromagnetic fields) to certain atoms. Therefore specific scripts have been written, each for a particular electric signal (the magnetic components are usually omitted in classical molecular dynamics simulations), specifically a Gaussian pulse, a Bipolar pulse (the Gaussian derivative) and a square wave signal. The scripts were written with the underlying idea to give the user a complete control of signal characteristics in terms of duration, amplitude and frequency content. A series of very short simulations established the right implementation of the TCL script in NAMD. The second task has been tackled by means of some functions embedded in GROMACS package and via the VMD (Visual Molecular Dynamics) program. A series of conversion steps led to a complete topology fully compatible with the GROMACS format.

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Paolo Marracino, Micaela Liberti, Francesca Apollonio and Andrea Amadei
Title nsPEfs induce irreversible denaturation of sperm whale myoglobin: a thermodynamics analysis

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Paolo Marracino, Micaela Liberti, Francesca Apollonio and Andrea Amadei
Title nsPEfs induce irreversible denaturation of sperm whale myoglobin in water: a kinetics analysis

 


Realization of solid state modulator for application in PEF-based food processing

STSM by Yassine Bellebna, PhD Student

Period of mission: from 24/04/2014 to 23/06/2014

Home institution: APELEC Laboratory, Djillali Liabes University, Sidi Bel Abbes, Algeria

Host institution: Laboratory of Pulsed Power Advanced Applications Research Group, ISEL (Instituto Superior de Engenharia de Lisboa), Lisbon, Portugal

There are many types of commercially available, high voltage, (>several kilovolts), pulse generators. These generators vary widely in performance and should be chosen according to load requirements. Examples of performance parameters are voltage output, rise time, pulse width, fall time, repetition rate, peak power, and average power. These generators also differ in circuit design and switch type. Circuit designs are categorized into two types of energy storage methods: 1) capacitive and 2) inductive. The most common switch types are reed, spark gap, and solid state. In most cases, the switch type determines the generator performance. The purpose of the STSM was the realization of a pulse generator of several kilo volts (up to 6 kV) by the method of energy storage capacitive. This pulse generator used a fast switch half bridge which formed two MOSFETs in series. A necessary prior study was done according to value of the load to choose the adequate capacity; this study has allowed an optimal functioning of pulse generator. The mission was finished by studying voltage drop of impulsion depending on the load, the pulse duration and frequency of repetition In order to design a high-voltage pulse generator with minimum voltage drop. This pulse generator will use later in applications of food processing.

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Mohammed Hamza Bermaki, Abdelkader Semmak, Matej Reberšek, Yassine Bellebna, Damijan Miklavčič, Amar Tilmatine
Title Development of a Marx-type PEF generator using IGBT switches

 


Allergen structural modifications under PEF by molecular dynamics simulations

STSM by Paola Maresca, Early-Stage Researcher

Period of mission: from 04/06/2014 to 13/06/2014

Home institution: ProdAl Scarl-University of Salerno, Fisciano (SA), Italy

Host institution: CNRS UHP 7565, Université de Lorraine, Vandoeuvre-lès-Nancy, France

In the last years novel technologies, such as PEF (Pulsed Electrical Field), have been studied in order to induce protein modifications without affecting the food quality. Among the food proteins, the allergens represent a very interesting target. The challenge is to design innovative processes able to selectively modify the allergen epitopes (linear or conformational) and spatial conformation and at the same time to preserve the whole quality of the foodstuffs. The STSM aimed to verify the applicability of electro-technologies to induce significant modification of the fold of a food specific allergen. Bovine serum albumin (BSA), a water soluble globular protein, made of two chains of 583 amino acids, organized in alpha-helixes (67% of the whole structure) and joined by 17 S-S groups and one thiol group, was investigated using molecular dynamics (MD) simulations. VMD (Visual Molecular Dynamics) was used to visualize the configuration developed during the simulation steps and the trajectories derived from the simulation at given environmental conditions (temperature ramp, pH shift, Electric field strength). GROMACS was, instead, used to build-up the simulation domain, to perform molecular dynamics simulation and post-simulation analyses. After a preliminary equilibration of the system, specific perturbations were applied: i/ Thermal denaturation at 330 K; ii/ Static Electric field at 0.1 and 1 V/nm; iii/ Effect of the Electric field direction on the protein denaturation. The preliminary results achieved during the STSM were useful to plan the further simulations and to define the conditions to be tested in order to define the unfolding conditions under Electric Fields.


Pulsed Electric Field for protein release from microalgae

STSM by Richard Postma, PhD Student

Period of mission: from 18/05/2014 to 25/05/2014

Home institution: Wageningen University, Wageningen, The Netherlands

Host institution: ProdAl Scarl, Fisciano, Italy

Recently the cultivation and refinery of microalgae biomass has stimulated intensive research due to its relative high content of more than 50% of valuable cell components, like proteins, lipids, polysaccharides, and pigments. Besides, the areal productivity is much higher compared to other agricultural plants. The aim of this STSM was to explore the possibilities to use Pulsed Electric Field (PEF) for extraction of water soluble proteins from microalgae. The microalgae Chlorella vulgaris was cultivated in a 12 L photobioreactor at Wageningen University in Netherlands under both nitrogen replete (N+) and nitrogen deplete (N-) conditions. By means of centrifugation the algal harvest was concentrated and then stabilized as freeze dried. At ProdAl Scarl, Fisciano, Italy the microalgal suspensions were prepared at 25 g/L for both the N+ and N- algae by resuspending the freeze dried material in tap water and rehydrated at 4 °C overnight. Five PEF experiments were performed at different field strengths (20 and 30 kV/cm) and specific energy consumptions (50 and 100 kJ/kg) on both the N+ and N- biomass. The water soluble protein concentration in the supernatant was measured. Unfortunately, the freeze drying caused the cells to break, releasing the water soluble proteins in the supernatant after resuspension. Therefore no significant increase of protein was obtained after PEF. This leads to the conclusion that PEF can only be performed on fresh biomass.

Publication(s) originating from this mission

Journal Bioresource Technology
Impact Factor (according to ISI Thomson) 4,494
Authors PR Postma, G Pataro, M Capitoli, MJ Barbosa, MHM Eppink, RH Wijffels, G Olivieri, G Ferrari
Title Selective extraction of intracellular components from the microalga Chlorella vulgaris by combined Pulsed Electric Field-Temperature treatment
Year 2015
Issue ?
Pages ?
Status Submitted - to be updated

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Richard Postma, Giuseppe Olivieri, Maria Barbosa, Rene Wijffels, Michel Eppink, M Capitoli, Gianpiero Pataro, Giovanna Ferrari
Title Effect of temperature and energy input on PEF processing of the green microalgae Chlorella vulgaris

 


Application of electroporation for microalgal biorefinery

STSM by Giuseppe Olivieri, Assistant Professor

Period of mission: from 18/05/2014 to 25/05/2014

Home institution: Wageningen University, Wageningen, The Netherlands

Host institution: ProdAl Scarl, Fisciano, Italy

Cultivation and biorefinery of microalgae have stimulated intensive research due to the high potential in supply both commodities as food, feed, and biofuels and specialties and antioxidant and poly-unsatured fatty acids. However, several bottlenecks affect the feasibility of the process. One of them is the extraction of these compounds from the biomass. In this frame, Pulsed Electric Field (PEF) technology can be used as a way to enhance the extraction yield of valuable compounds from the inner parts of the cells. The main outcomes of the PEF treatment were:

  • short time scale (less than 1s) -> very small sized units are required in comparison to the large scale of the microalgal cultivation due to the time scale of the growth (days)
  • capacity to treat from diluted to high concentrated stream (from 10 to 100 g/L), provided they are “pumpable” -> preconcentration step of the cultivation is required by flocculation or centrifugation
  • fast temperature increase -> To avoid damage to sensible compound (mainly proteins) an efficient cooling is required together with avoiding too high energy treatment
  • strong sensitivity to initial conductivity which should be lower than 5 mS/cm -> In case of marine algae, it is necessary to carry out a washing step after the harvesting of the culture from the photobioreactor
  • energy cost of less than 1 Euro/kg biomass -> It is still too high considering that the whole microalgal biorefinery should achieve an overall cost (capital+operating) of 2 Euro/kg biomass.

Publication(s) originating from this mission

Journal Bioresource Technology
Impact Factor (according to ISI Thomson) 4,494
Authors PR Postma, G Pataro, M Capitoli, MJ Barbosa, MHM Eppink, RH Wijffels, G Olivieri, G Ferrari
Title Selective extraction of intracellular components from the microalga Chlorella vulgaris by combined Pulsed Electric Field-Temperature treatment
Year 2015
Issue ?
Pages ?
Status Submitted - to be updated

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Richard Postma, Giuseppe Olivieri, Maria Barbosa, Rene Wijffels, Michel Eppink, M Capitoli, Gianpiero Pataro, Giovanna Ferrari
Title Effect of temperature and energy input on PEF processing of the green microalgae Chlorella vulgaris

 


Inactivation of bacterial spores by electric arcs

STSM by Flavien Pillet, Early-Stage Researcher

Period of mission: from 12/05/2014 to 31/05/2014

Home institution: IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France

Host institution: University of Ljubljana, Ljubljana, Slovenia

Bacterial spores are involved in the major part of food contamination and also responsible of grave diseases like Bacillus anthracis which is the etiologic agent of anthrax. The lethality of spores is increased by their highly resistance to chemical, thermal and physical stresses. The inactivation of spores raises a grave issue, particularly because non-thermal inactivation like PEF is inefficient. The purpose of this STSM was to use electric arcs for spore inactivation, by using the system developed by Igor Marjanovic and Tadej Kotnik (Bioelectrochemistry, 2013). This device was previously used for bacteria inactivation in vegetative form. However, electric arcs were not still described for spore inactivation. During this STSM the efficiency of spore inactivation in solid and liquid medium were evaluated according to different parameters (voltage, time and number of electric arc). In liquid, spore inactivation was not observed. By contrast, a high level of inactivation was measured in solid medium. With 1 electric spark at 5 kV during 20 µS, an inactivation rate more than 3 log10 was deduced. The phenomenons responsible of inactivation are probably various, like shock wave, ionization, UV radiation etc. Finally these results are particularly promising for decontamination of solid food and a better understood of phenomena involved would be suitable for improving the technique.

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Matej Reberšek, Igor Marjanovič, Samo Beguš, Flavien Pillet, Marie-Pierre Rols, Damijan Miklavčič and Tadej Kotnik
Title Emulating Exposures of Biological Samples to Lightning Strokes

 


Effect of hyaluronidase on early inflammatory events induced by electrotransfer in mouse skin compared to skeletal muscle

STSM by Lise Pasquet, Early-Stage Researcher

Period of mission: from 05/05/2014 to 30/05/2014

Home institution: IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France

Host institution: CNR-IFT (Institute of Translational Pharmacology) and University Campus Bio-Medico, Rome, Italy

In this STSM we addressed if the use of Hyaluronidase, an enzyme responsible for the degradation of the extracellular matrix, could increase (i) the accessibility of plasmid DNA to target cells in skin submitted to EGT protocols and (ii) the resulted immune inflammation, as it was demonstrated in muscles. Balb/c mice were injected intra-dermally or intramuscularly (both posterior legs) with Hyaluronidase and a combination of IL-12 and Tomato plasmids. The animals underwent to the following electropermeabilisation parameters applied with BTX ECM830 Electroporator (Harvard Apparatus): 4 pulses of 1000V/cm, 100µs + 4 pulses of 250V/cm, 20ms at 1Hz of frequency with 10 mm diameter plate electrodes at 0.1 cm of distance for skin; 10 pulses of 175V/cm, 20ms, 1Hz with 10 mm diameter plate electrodes at 0.4 cm of distance for muscles. Skins, muscles and spleens were collected at 3h, 7 days and 14 days after the treatment. The sample prepared for histological analysis revealed the presence of immune infiltration only in muscles treated with hyaluronidase and EGT, not in skin. By ELISA quantification of cytokine production in tissue, we observed a higher production of inflammatory cytokines IL-6 and TNF-?, from 3h after the treatment both in muscles and in skin.These first results confirm that Hyaluronidase increases the inflammatory response induced by EP in muscles but also reveal interestingly the same effect in skin.

Oral presentation(s) originating from this mission

Event Gordon Research Seminar in Bioelectrics
Location Maine/USA
Period

05/07 - 06/07/2014

Authors Lise Pasquet
Title Currative vaccination against melanoma in mice by IL12

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Muriel Golzio, Lise Pasquet, Justin Teissié, Marie-Pierre Rols and Sophie Chabot
Title Vaccination against melanoma in mice by IL12 electro-geno-immuno-therapy

 


Fast electrical bioimpedance spectroscopy measurements during electroporation (part II)

STSM by Tomás García-Sánchez, PhD Student

Period of mission: from 19/05/2014 to 30/05/2014

Home institution: Technical University of Catalonia (UPC), Electronic and Biomedical Instrumentation Group, Barcelona, Spain

Host institution: Laboratory of Vectorology and Anti-cancer Therapeutics, UMR 8203 CNRS, University Paris Sud and Gustave Roussy, Paris, France

The activity in this STMS was motivated by previous work developed during the first visit to the Institute Gustave Roussy (Paris, France) for five weeks in October 2013. After deep analysis of the results of the first set of experiments we found that some new tests were necessary to improve the understanding of our previous findings. The study of the dynamics of cell permeabilization by means of electrical impedance spectroscopy measurements was extended to new cell lines. In total, five different cell lines were subjected to complete analysis. Electrical impedance of cell monolayers was monitored as a function of field magnitude within a classical electroporation treatment (8 pulses, 100 µs, 1 Hz). Measurements were extended during several minutes before and after pulse application. Additionally the study was repeated using different pulse repetition frequencies. The preliminary analysis of the data shows promising information about the different spectral behavior of the different phenomena occurring during pulse application. This opportunity was useful not only to confirm the analysis made from the previous STSM, but also to provide valuable and novel information. Further work will be focused on the detailed analysis of the very large amount of data collected during the STSMs to generate valuable information for publication.

Publication(s) originating from this mission

Journal Bioelectrochemistry
Impact Factor (according to ISI Thomson) 4,172
Authors Tomás García-Sánchez, Antoine Azan, Isabelle Leray, Javier Rosell-Ferrer, Ramon Bragós, Lluis M. Mir
Title Interpulse multifrequency electrical impedance measurements during electroporation of adherent differentiated myotubes
Year 2015
Issue 105
Pages 123-135
Status Published

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Tomas Garcia-Sanchez, Antoine Azan, Isabelle Leray, Javier Rosell-Ferrer, Lluis M. Mir and Ramon Bragos
Title Modeling dynamic electrical impedance spectroscopy measurements on electroporated cells

 


Linking Biomedicine and Food Processing

STSM by Damijan Miklavčič, Professor

Period of mission: from 11/05/2014 to 17/05/2014

Home institution: University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia

Host institution: ProdAl S.c.a.r.l., Fisciano, Italy

During this STSM, I and professor Giovanna Ferrari who is the member of the Program Committee and organizer of the 2nd BFE Congress worked on integration of the 3rd BFE Congress into the program of 1st World Congress on Electroporation and PEF treatment. We recognized the importance of allowing other electro-technologies than PEF treatment to be included in the program of the world congress to maintain the momentum and audience gained during the 1st and 2nd BFE congresses. This will be achieved through establishing a sub-web page of the BFE congress and providing that BFE specific sessions will not be overlapping with PEF food processing sessions. We also worked on the program of the 2nd PEF School to be held in Salerno in February 7 to 12, 2015. The scheduling and list of potential lecturers was prepared based on 10 years of experience EBTT Workshop held in Ljubljana and 1st PEF Training School held in Zaragoza. A lecture "Electroporation based technologies and treatments" given by D. Miklavcic was organized and attended by doctoral students from informatics, chemistry, food engineering and electrical engineering departments of University of Salerno and members of the ProdAl research group. Furthermore through discussion with members of the ProdAl research group we reviewed current status of modeling PEF treatment in food processing, the chambers and electrodes used in different applications with particular interest to potential needs for improving current models.

Publication(s) originating from this mission

Journal Innovative Food Science & Emerging Technologies
Impact Factor (according to ISI Thomson) 2,997
Authors Javier Raso, Wolfgang Frey, Giovanna Ferrari, d, Gianpiero Pataro, Dietrich Knorr, Justin Teissie, Damijan Miklavčič
Title Recommendations guidelines on the key information to be reported in studies of application of PEF technology in food and biotechnological processes
Year 2016
Issue 37/C
Pages 312-321
Status Published

 


Modeling of electroporation in presence of calcium

STSM by Stefania Romeo, Early-Stage Researcher

Period of mission: from 04/05/2014 to 27/05/2014

Home institution: Institute for Electromagnetic Sensing of the Environment (IREA) – National Research Council of Italy, Naples, Italy

Host institution: Department of Oncology, Copenhagen University Hospital, Herlev, Denmark

Electroporation in presence of calcium has been recently demonstrated by the host research group 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. The first clinical trial using calcium electroporation has recently opened at Herlev Hospital, however better understanding of the mechanism behind calcium electroporation is highly warranted to optimize the effect. The activity carried out during the STSM consisted both in experimental studies on calcium electroporation and in a post-processing analysis of experimental data aimed to define mathematical relations between the experimental parameters. In particular, SW780 bladder cancer cells were exposed to 8, 99 µs 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 the 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, and to assess the dynamics of cell death under different electric fields and calcium concentration conditions. The procedure set up confirm previous in vitro results on calcium electroporation, 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.

Publication(s) originating from this mission

Journal Plos One
Impact Factor (according to ISI Thomson) 3,234
Authors Emilie L. Hansen, Esin B. Sozer, Stefania Romeo, Stine K. Frandsen, Paul T. Vernier, Julie Gehl
Title Dose-dependent ATP depletion and Cancer Cell Death following Calcium Electroporation, Relative Effect of Calcium Concentration and Electric Field Strength
Year 2015
Issue 10 (4)
Pages e0122973
Status Published

Conference abstract(s) originating from this mission

Publication IFMBE Proceedings of the 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Publisher Tomaz J, Kramar P (ed), 1st edn. Springer-Verlag, Singapur
Authors Stefania Romeo, Emilie L. Hansen, Stine K. Frandsen, Julie Gehl
Title Electroporation of a Bladder Cancer Cell Line in Presence of Calcium: Efficacy Dependence on Electric Field Strength and Calcium Concentration
Year 2015
Issue 53
Pages 151-154
Status Published

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Stefania Romeo, Emilie L. Hansen, Stine K. Frandsen, Julie Gehl
Title Electroporation of a Bladder Cancer Cell Line in Presence of Calcium: Efficacy Dependence on Electric Field Strength and Calcium Concentration

 


Improving treatment planning for IRE with advanced numerical models

STSM by Bor Kos, Early-Stage Researcher

Period of mission: from 26/04/2014 to 11/05/2014

Home institution: University of Ljubljana, Ljubljana, Slovenia

Host institution: Virginia Tech, Blacksburg, Virgina, USA

Non-thermal irreversible electroporation for the treatment of brain cancer is promising avenue of research according to preliminary studies on veterinary patients. The goal of the STSM was to finalize a user-friendly interface and computational back-end for treatment planning of non-thermal irreversible electroporation of canine brain tumors. We analyzed a group of 5 canine patients, which were treated with non-thermal irreversible electroporation. The patients presented with spontaneous brain tumors of various histologies. During the STSM we implemented a statistical model for cell death based on in-vitro studies in a computational finite-element method model (built in COMSOL Multiphysics v4.3b), and correlated the model predictions with actual treatment outcomes. Additionally, we implemented thermal simulations into the model, which initially only took electric fields into account. The new results for cell viability after electroporation treatment (statistical model) are very promising, since they show characteristics, which are very much in line with experimental results obtained in vivo. This presents one of the first studies, which will be able to directly correlate in-vivo results treatment efficacy in with computed in-situ electric fields, with electrode positions exactly known from intra-operative CT scans and/or stereotactic frame use.

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Garcia, Paulo A., Kos, Bor, Pavliha, Denis, Rossmeisl, John H., Miklavčič, Damijan, Davalos, Rafael V.
Title A comparison between deterministic and statistical treatment planning in irreversible electroporation of spontaneous glioma

 


Cellular ATP content during and after calcium electroporation evaluated by fluorescence microscopy

STSM by Emilie Louise Hansen, Scholarship Student Researcher

Period of mission: from 01/04/2014 to 30/04/2014

Home institution: Department of Oncology, Copenhagen University Hospital Herlev, 2730 Herlev, Denmark

Host institution: Frank Reidy Research Center for Bioelectrics, Norfolk VA, USA

Calcium electroporation is a method used to increase intracellular calcium uptake in cells through permeabilization of the cell membrane by application of electrical pulses. At the Frank Reidy Research center for Bioelectrics it was possible to use scanning confocal imaging via a Leica TCS SP8 microscope for investigation of the hypothesis that ATP depletion caused by calcium electroporation could be partially responsible for inducing necrosis in cancer cells. We used quinacrine as a marker for ATP and imaged the fluorescence in U937 human leukemia cell line 15, 30 and 60 minutes after treatment. Permeabilization marked using propidium iodide was delayed and more irregular after calcium-electroporation compared to electroporation. The quinacrine fluorescence was lower in the electroporated and calcium-electroporated samples than in the controls 15 and 30 minutes after treatment, the fluorescence being lowest for the calcium-electroporated sample. Calcium electroporation caused a short-term decrease in quinacrine fluorescence, which may indicate ATP depletion, and significantly decreased cell viability after 24 and 48 hours. The decrease in quinacrine fluorescence intensity after 1 hour in both electroporated and non-electroporated samples with or without added calcium should be further investigated. More systematic investigations to follow these should use more specific ATP indicators, such as luminescence from luciferin-luciferase.

Publication(s) originating from this mission

Journal Plos One
Impact Factor (according to ISI Thomson) 3,234
Authors Emilie L. Hansen, Esin B. Sozer, Stefania Romeo, Stine K. Frandsen, Paul T. Vernier, Julie Gehl
Title Dose-dependent ATP depletion and Cancer Cell Death following Calcium Electroporation, Relative Effect of Calcium Concentration and Electric Field Strength
Year 2015
Issue 10 (4)
Pages e0122973
Status Published

Conference abstract(s) originating from this mission

Publication IFMBE Proceedings of the 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Publisher Tomaz J, Kramar P (ed), 1st edn. Springer-Verlag, Singapur
Authors Stefania Romeo, Emilie L. Hansen, Stine K. Frandsen, Julie Gehl
Title Electroporation of a Bladder Cancer Cell Line in Presence of Calcium: Efficacy Dependence on Electric Field Strength and Calcium Concentration
Year 2015
Issue 53
Pages 151-154
Status Published

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Stefania Romeo, Emilie L. Hansen, Stine K. Frandsen, Julie Gehl
Title Electroporation of a Bladder Cancer Cell Line in Presence of Calcium: Efficacy Dependence on Electric Field Strength and Calcium Concentration

 


Use of Pulsed Electric Fields for inactivation of bacterial spores of genus Alicyclobacillus

STSM by Noelia Lopez-Giral, Early-Stage Researcher

Period of mission: from 25/03/2014 to 10/05/2014

Home institution: CNTA- National Centre for Food Technology and Food Safety, San Adrián (Navarra), Spain

Host institution: DIL-German Institute of Food Technologies, Quakenbrück, Germany

In recent years, an increasing in the number of organoleptically altered juices has been detected by the juice canning industry. This is due to the presence of the bacterium Alicyclobacillus in the raw material and in the industry. Alicyclobacillus is a genus of Gram posivitve, sporeforming bacteria. It is of special interest to the for fruit juice and beverage industries worldwide due to the bacterium’s acidothermophilic growth capability, heat resistance, and spoilage potential Common pasteurization techniques do not deactivate the spores and when a product is spoiled by Alicyclobacillus, the juice products develop a disinfectant-like odor and/or flavor (due to the production of guaiacol). Specifically one of the most important species is Alicylobacillusacidoterrestris. One possibility to achieve the elimination of spores of this microorganism is the use of pulsed electric fields (PEF) in combination with high temperatures. The main purpose of this STSM which was performed under supervision of Dr. Stefan Toepfl and with the collaboration of Claudia Siemer was to test the influence of different electric field strengths and specific energy in combination with high temperature (> 80°C) to inactivate the spores of the Alicyclobacillusacidoterrestris DSM 3922 in different laboratory media (pH 7 & 4 and 1 & 4 mS/cm), wastewater and orange juice. Finally a comparison with the strain DSM 3522T was performed. We can conclude that the use of PEF in combination with temperature for inactivation of spores offered remarkable results, with more than 5 log10 cycles of inactivation depending on the electric field strength and specific energy applied. The results are very promising for juice canning industry. However, more research is necessary before industrial implementation due to the differences observed between strains, as well as between laboratory media, orange juice and wastewater.

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Noelia López Giral, Carolina González Ferrero, Lucia González Arenzana, Isabel López Alfaro and Teresa Garde Cerdán
Title Use of Pulsed Electric Fields (PEF) in the winemaking industry

 


PEF assisted extraction of food-industry-relevant compounds from microalgae

STSM by Gianpiero Pataro, Researcher

Period of mission: from 27/02/2014 to 10/04/2014

Home institution: Prodal Scarl, Fisciano (SA) - Italy

Host institution: Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany

Microalgae biomass represents a rich source of valuable compounds, like lipids, proteins, polysaccharides, antioxidants and pigments, which are of considerable interest for pharmaceutical and food industry. Therefore, the extraction process of these compounds, which strongly depends on mass transfer phenomena through cell membrane of the microalgae, is a crucial step in order to enhance the yield and purity of the extracts. During this STSM, which was performed under the supervision of Dr. Wolfgang Frey, the influence of PEF pretreatments of different intensities on the extraction of valuable components relevant for food industry from microalgae was investigated. Culture of C. vulgaris strain inoculated in TAP-medium, were cultivated in batch 26 L photo-bioreactor. The algae were harvested after 18-24 days and concentrated up to a final biomass concentration of about 40-50 gdw/kgsus. PEF experiments of different intensities (E=27-35 kV/cm, and WT=50-150 kJ/kg) were carried out in a laboratory scale continuous flow unit. Determinations of time-conductivity profile as well as quantification of dry matter, protein content, total polyphenolics, and antioxidant activity of the supernatant were performed. Results showed a higher increase of the electrical conductivity of PEF treated suspension, when compared to the untreated sample: the higher the PEF treatment intensity, the higher the rate of release of ionic substances. Moreover, the PEF treatment significantly increased the dry matter content as well as the release of proteins from the inner part of the algae cells. Additionally, increments of total phenolics and antioxidant activity were also detected.


Dielectric properties of normal vs. cancerous liver tissue

STSM by Azadeh Peyman, Researcher

Period of mission: from 24/03/2014 to 28/03/2014

Home institution: Public Health England, Didcot, UK

Host institution: University of Ljubljana, Ljubljana, Slovenia

Scientific collaboration was carried out between Physical Dosimetry Department of Public Health England and Faculty of Electrical Engineering University of Ljubljana and Institute of Oncology Ljubljana. The aim of this mission was to characterize the dielectric properties of cancerous human liver tissues. The variation in dielectric properties of tissues as a result of pathological and physiological changes is an area of great interest in particular in distinguishing the differences between normal and diseased tissues. It increases our understanding of how electromagnetic fields interact with tissues of different pathological state. Dielectric measurements were carried out at 50MHz-10GHz on freshly excised liver tumour tissues using an open ended coaxial probe and a vector network analyser. Tumour samples were collected from 6 patients undergoing open surgery to mainly remove metastasis of adenocarcinoma which had spread to liver. The measured data is mapped according to the relevant histological and pathological information. It will provide vital information to enhance understanding of interaction between electric fields and cancerous tissues in particular for electroporation applications (enhanced drug delivery). The dielectric data collected in this experiment covers the microwave frequencies of the electromagnetic field spectrum which is quite useful in understanding the mechanisms involved in electroporation of tissues using nanosecond pulses. It is aimed to extend the measurements to frequencies below 1 MHz, where the majority of electroporation applications (millisecond pulses) are carried out.

Publication(s) originating from this mission

Journal Bioelectromagnetics
Impact Factor (according to ISI Thomson) 1,705
Authors Azadeh Peyman, Bor Kos, Mihajlo Djokić, Blaž Trotovšek, Clara Limbaeck-Stokin, Gregor Serša and Damijan Miklavčič
Title Variation in dielectric properties due to pathological changes in human liver 
Year 2015
Issue 36/8

Pages

603-612
Status Published

Oral presentation(s) originating from this mission

Event BioEM 2015
Location Asilomar Conference Center/California/USA
Period 14/06 - 19/06/2015
Authors Azadeh Peyman
Title Dielectric properties of human liver and liver tumours

 


Artificial hybrid cells created by ns-pulse mediated electrofusion

STSM by Christoph Huber, Early-Stage Researcher

Period of mission: from 14/04/2014 to 18/04/2014

Home institution: Institute of Synthetic Bioarchitectures, Department of Nanobiotechnology, University of Natural Resources and Life Sciences, Vienna, Austria

Host institution: Laboratory of Biocybernetics, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia

Classical electrofusion is considered to be an established technique to fuse cells of similar sizes. It was shown recently that fusion of cells of different sizes is also possible but the application of ns pulses instead of ms- or µs pulses is necessary. This new possibility gave rise to the idea of fusing artificial vesicles, built of amphiphilic block copolymers, with living cells. The so called polymersomes offer a broad variety of physical and chemical properties, determined by the type of polymers used as their building blocks. Polymersomes can work as a shuttle system for molecules filled in their inner compartment, as their permeability is typically lower than the permeability of liposomes and can be precisely tuned by the chemical structure and/or the molecular weight of the used polymers. Moreover polymersomes can work as a support for transmembrane proteins, as these proteins can only maintain their functionality if embedded in the appropriate environment. During the STSM incipient experiments were made to investigate the ability to electroporate polymersomes in principal. It turned out that ns pulses were not strong enough to lead to sufficient pore formation to be investigated in a classical fluorescence microscope. This could be overcome by the application of µs pulses and was monitored by the leakage of fluorophores trapped in the inner compartment of the polymersomes. Moreover it could be demonstrated that close contact between cells and polymersomes can be established by dielectrophoresis, which is an essential prerequisite for successful fusion. To demonstrate fusion between cells and polymersomes further experiments are already planned.


Electrochemical properties of cell-mimetic lipid membranes

STSM by Denitsa Mitkova Brankova, PhD Student

Period of mission: from 02/03/2014 to 01/04/2014

Home institution: Institute of Solid State Physics - Bulgarian Academy of Sciences, Sofia, Bulgaria

Host institution: Max Planck Institute of Colloids and Interfaces, Science Park Golm, Potsdam, Germany

During the STSM, we performed experiments for the measurement of important physical properties of lipid membranes, namely their edge tension and electrical capacitance. The edge tension of phosphatidylcholine membranes was studied by electroporation of giant lipid vesicles (GUVs) at identical sugar solutions inside and outside the vesicles. The effect of sucrose in the concentration range from 0 to 400 mM was explored. Pores in the membranes were induced by applying 30-40 V / 5 ms electrical pulses. Asymmetric conditions were also studied allowing for better visualization of the pore. The closure time of the pore is related to the edge tension of the membrane. The experimental results showed a difference between the time closure of the pore in membranes in symmetric and asymmetric solutions. The capacitance of lipid bilayers was measured by electrodeformation of GUVs exposed to AC fields in the kHz frequency range. The GUVs were in aqueous solutions of sucrose at concentrations between 50 and 400 mM with and without 0.1 mM of sodium chloride. The prolate-oblate transition frequency of GUVs is related to the capacitance of lipid bilayers. The forthcoming analysis of the accumulated data will provide a quantitative evaluation of the edge tension and the capacitance of the studied lipid membranes. Our results will help answering the question whether sugar molecules that are dissolved in the aqueous phase exert any effect on the studied physical properties of lipid membranes.

Poster presentation(s) originating from this mission

Event 18th International School on Condensed Matter Physics
Location Varna/Bulgaria
Period

01/09 - 06/09/2014

Authors Denitsa Mitkova, Rumiana Dimova, Victoria Vitkova
Title Investigation of the electrochemical properties of cell-mimetic membranes using electrodeformation and electroporation of lipid vesicles

 

Event Workshop organized by REGPOT-2012-2013-1 NMP INERA EU project
Location Varna/Bulgaria
Period

04/09 - 06/09/2014

Authors Denitsa Mitkova, Rumiana Dimova, Victoria Vitkova
Title Electrochemical properties of cell-mimetic lipid membranes obtained in cells consisting of indium-tin oxide coated plates and polymer spacers

 


Micro-gap electrodes for electropermeabilization

STSM by Antoine Azan, PhD Student

Period of mission: from 31/03/2014 to 04/04/2014

Home institution: UMR 8203 of the CNRS and University Paris-Sud, Institut Gustave Roussy, Villejuif, France

Host institution: IHM KIT, Karlsruhe, Germany

Although known for decades, the underlying mechanisms of cell electropermeabilization are still not fully understood. One of the most credible hypotheses is the chemical alteration of the membrane phospholipids due to the pulsed electric field. Coherent Anti-stokes Raman Scattering (CARS) microscopy is a powerful tool to acquire image at a single vibrational frequency associated to vibrational mode of chemical bonds. It allows a biochemical decomposition of the sample with a high time resolution (3ns). At UMR8203, a CARS microscope can be coupled with a pulsed generator. The purpose of the STSM is to manufacture micro-gap electrodes on microscope slide in order to be able to pulse the biological sample under the CARS microscope. The laboratory IHM in Karlsruhe Institute of Technology has the technical skills and the background to glue electrodes on substrate with a thin glue layer in order to provide a homogenous field. During the STSM, we successfully manufactured micro-gap electrodes on two different substrates: microscope slide and optical filter. The process is artisanal and has to be performed really carefully due to the fragility of the substrates. With preliminary tests, the process has been optimized for each type of substrate and controlled at several checkpoints. The optical coating has to be checked under the CARS microscope at UMR8203 in order to be sure that it is not damaged. The manufactured gap is really an improvement for the CARS microscope and provides a lot opportunity to work on many topics and investigate the biochemical characterization of electropermeabilization.

Publication(s) originating from this mission

Journal Bioelectrochemistry
Impact Factor (according to ISI Thomson) 4,172
Authors Tomás García-Sánchez, Antoine Azan, Isabelle Leray, Javier Rosell-Ferrer, Ramon Bragós, Lluis M. Mir
Title Interpulse multifrequency electrical impedance measurements during electroporation of adherent differentiated myotubes
Year 2015
Issue 105
Pages 123-135
Status Published

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Tomas Garcia-Sanchez, Antoine Azan, Isabelle Leray, Javier Rosell-Ferrer, Lluis M. Mir and Ramon Bragos
Title Modeling dynamic electrical impedance spectroscopy measurements on electroporated cells

 


The temperature effect on electroextraction of proteins from bacterial cells

STSM by Saša Haberl Meglič, Early-Stage Researcher

Period of mission: from 23/03/2014 to 30/03/2014

Home institution: Laboratory of Biocybernetics, University of Ljubjana, Faculty of Electrical Engineering, Ljubljana, Slovenia

Host institution: University of Zaragoza, Faculty of Veterinary Medicine, Zaragoza, Spain

An increasing number of proteins are being used in medicine, food industry and biotechnology and the need to develop methods for extraction on a large production scale is becoming increasingly important. Different chemical or physical methods are currently used for extraction of proteins from bacterial cells. But on a large scale, a general handicap of all extraction processes is that concentration of contaminants is high, necessitating downstream processing. Extraction of bacterial proteins by means of electroporation has opened a new field for efficient and cost-effective protein production on a large scale and many parameters (e.g. pulse parameters, temperature, etc.) can influence the efficiency the method. Therefore, the main purpose of this STSM which was performed under supervision of Dr. Javier Raso was to test the influence of temperature during exposure of cells to electric pulses (10, 25 and 40°C) and post-pulse incubation temperature (25 and 37°C) and time (from 1 h to 24 h) on extraction of proteins by means of electroporation from bacterial cells. Also the influence of electric pulse parameters (electric field strength and pulse number) on bacterial membrane permeabilization, bacterial inactivation and the concentration of extracted proteins were observed. The model organism for our investigation was E. coli bacteria, strain K12 Top10 and our results showed that with increasing electric field strength, treatment time or treatment temperature also membrane permeabilization increases. Also increased electric field strength or treatment time increases bacteria inactivation. The most tremendous effect on the concentration of extracted proteins by means of electroporation was the post-pulse incubation time and temperature. Namely, when bacterial cells were incubated 24 h at 37°C, the leakage of bacterial proteins was approximately 2 or 3 – times higher compared to 24 h incubation at room temperature or compared to shorter incubation times (1 to 3 hours).
We can conclude that an effect of treatment parameters on bacteria membrane permeabilization does not correlate in the same way as on bacteria inactivation and protein extraction. Additional experiments are needed however to confirm these observations.

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Sasa Haberl Meglic, Eva Levicnik, Elisa Luengo, Javier Raso, Damijan Miklavcic
Title The effect of temperature on protein extraction by electroporation and on bacterial viability

 


Evaluation of functioning of synchronization device during in vivo electrotransfer

STSM by Barbara Mali, Early-Stage Researcher

Period of mission: from 23/01/2014 to 21/02/2014

Home institution: Laboratory of Biocybernetics, University of Ljubjana, Faculty of Electrical Engineering, Ljubljana, Slovenia

Host institution: Old Dominion University, Frank Reidy Research Center for Bioelectrics, Norfolk, Virginia, USA

Electroporation-based clinical treatments that include delivery of pulses close to the heart and/or in internal tissues can affect functioning of the heart. With aim to assure safe delivery of electroporation pulses in such clinical treatments, the algorithm for synchronization of electroporation pulse delivery with electrocardiogram (ECG) was developed and implemented in a stand-alone device (called ECGsync device). The main purpose of this STSM which was performed under supervision of Dr. Richard Heller was to test the functioning of ECGsync device in in vivo conditions. Functioning of ECGsync device was tested during in vivo electrotransfer procedure which included delivery of electroporation pulses directly to the heart muscle of a swine. The electroporation pulses were triggered in real time using ECGsync device. ECG signals and trigger signals from ECGsync device were recorded during the whole procedure and analyzed. The results showed that ECGsync device enables reliable real-time analysis of ECG signals and safe synchronization of electroporation pulse delivery with ECG, i.e., outside the vulnerable period of the heart and in absence of premature beats. The average estimated time reserve for safe electroporation pulse delivery was 45 ms for electrotransfer, and 65 ms for electrochemotherapy and irreversible electroporation. We can conclude that the device can provide effective and reliable synchronization of electroporation pulses with electrocardiogram for use in all medical applications that include delivery of electroporation pulses, regardless of application location. Of course, additional testing on larger number of ECG signals during different conditions is needed.


Developement of a realistic mathematical model for prediction of efficient and safe clinical gene electrotransfer

STSM by Selma Čorović, Early-Stage Researcher

Period of mission: from 19/01/2014 to 30/01/2014

Home institution: Laboratory of Biocybernetics, University of Ljubjana, Faculty of Electrical Engineering, Ljubljana, Slovenia

Host institution: Department of Oncology, Center for Experimental Drug and Gene Electrotransfer, Copenhagen University Hospital Herlev, Herlev, Denmark

During the STSM at the Copenhagen University Hospital Herlev in Denmark we developed a realistic mathematical model that would help in prediction of efficient and safe clinical gene electrotransfer to muscle tissue. The primary objective of the work was to validate the model with clinical data obtained during the phase I clinical study of gene therapy for patients with advanced solid tumors. In addition, we validated the calculated electric field distribution with the spatial distribution of transgenic protein expressed in porcine muscle. We found the perpendicular orientation of electric field with respect to the muscle fibers to be optimal for efficient and safe gene electrotransfer. Due to the anisotropy of muscle tissue other orientations of electric field with respect to the muscle fibers resulted in a higher electric current, larger volume of irreversibly electroporated muscle and lower volume of reversibly electroporatad muscle. These results provide a clearer understanding of the influence of muscle anisotropy on delineation of successfully electroporated muscle area, and subsequently on the efficacy of gene electrotransfer. The precise location of the transfected muscle area is also important for the post-transfection interventions in case of adverse events when the gene expression in the located area needs to be terminated. In addition to gene therapy performed by gene electrotransfer to muscle tissue, our results can be important also in DNA vaccination and local intramuscular gene transfection.


Electromagnetic modeling of biological cells subjected to impulsive electrical fields VS experimental data: the role of the membrane excitability

STSM by Patrizia Lamberti, Researcher

Period of mission: from 02/01/2014 to 11/01/2014

Home institution: DIEM - University of Salerno, Fisciano (SA), Italy

Host institution: Laboratory of Biocybernetics - University of Ljubjana, Ljubljana, Slovenia

"A model is useful? Model is nothing without experiments but model can be used to understand and address experimental results". With this idea in my mind, I proposed myself for a STSM inside this Cost action. The STSM had the scope to exchange experiences concerning the pulsed electric field (PEF) treatment of excitable cell. In particular the hosting laboratory (i.e. Ljubljana’s Laboratory of Biocybernetics, under the supervision of prof. Damijan Miklavčič – "GURU") is performing experimental test on the electrical behaviour of Normal Rat Kidney cell with respect to PEF whereas Dr. Patrizia Lamberti (i.e. me, small and remote Italian researcher) is developing a circuital model of this particular excitable cell. During the STSM the model was improved in order to describe the electromagnetic cell membrane behaviour if the electroporation is taken into account together with the reproduction of the membrane excitability. The obtained ITMV and N behaviours suggest that at the “onset” of the electroporation phenomenon, membrane excitability is not so influent. Moreover it seems that the excitability reduce the pore creation rate and its influence increases if successive pulses are considered. "The experiments represent the reality that model tries to reproduce but the experiments can be affect by neglected sources of noise shadowing the principal. A model is capable to detect the 'main phenomenon' influencing the real behaviour of biological matter by repetition of simplest and cheapest numerical procedures with respect to experimental ones: a model can be useful!". Thanks to the exchange of experiences with the beautiful researchers involved with the Laboratory of Biocybernetics of University of Ljubljana, I restart my research activity at home institution after this STSM with this new idea in my mind!

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Patrizia Lamberti, Stefania Romeo, Maria Rosaria Scarfì, Vincenzo Tucci, Luigi Zeni
Title Numerical analysis of split dose protocols for nsPEFelectroporation

 


Calcium electroporation on 3D multicellular spheroids

STSM by Stine Krog Frandsen, PhD Student

Period of mission: from 18/11/2013 to 13/12/2013

Home institution: Department of Oncology, Copenhagen University Hospital, Herlev, Denmark

Host institution: IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France

IPBS, Toulouse has experience in working with spheroid models and the purpose of the STSM was to perform calcium electroporation on 3D multicellular spheroids. This enables us to further investigate calcium electroporation in vitro in a tumor-like model and maybe in future experiments understand more about the mechanism behind calcium electroporation. To test the effect of calcium electroporation and to compare the treatment with electrochemotherapy we treated 4 different spheroids (SW780, human bladder cancer; HT29, human colon cancer; MDA-MB231, human breast cancer; HDF-n, human normal fibroblast). Size measurements (light microscope images) of each spheroid before and after treatment were performed as a measure of living/dead cells. The cancer cell spheroids treated with either calcium electroporation or bleomycin electroporation decrease in size after treatment whereas, interestingly, spheroids made of the normal fibroblast cell line (HDF-n) were not affected by any of the treatments and all spheroids grew similar to untreated controls. We also measured the intracellular ATP level in spheroids 1, 4, and 24 hours after treatment with calcium electroporation. The results indicate that the intracellular ATP decrease dramatically after treatment with calcium electroporation, in all 4 different spheroids, even in the fibroblast spheroids which are still living after treatment with calcium electroporation. Altogether, the experiments performed during the STSM confirm the results seen previously in vitro and in vivo, and the results of the spheroids made of normal fibroblasts are very interesting and gives us new knowledge in this research area.

Publication(s) originating from this mission

Journal Plos One
Impact Factor (according to ISI Thomson) 3,234
Authors Emilie L. Hansen, Esin B. Sozer, Stefania Romeo, Stine K. Frandsen, Paul T. Vernier, Julie Gehl
Title Dose-dependent ATP depletion and Cancer Cell Death following Calcium Electroporation, Relative Effect of Calcium Concentration and Electric Field Strength
Year 2015
Issue 10 (4)
Pages e0122973
Status Published

 

Journal ?
Impact Factor (according to ISI Thomson) ?
Authors Stine Krog Frandsen, Laure Gibot, Moinecha Madi, Julie Gehl, Marie-Pierre Rols
Title Calcium electroporation: Evidence for differential effects in normal and malignant cell lines, evaluated in a 3D spheroid model
Year ?
Issue ?
Pages ?
Status Under Review - to be updated

Conference abstract(s) originating from this mission

Publication IFMBE Proceedings of the 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Publisher Tomaz J, Kramar P (ed), 1st edn. Springer-Verlag, Singapur
Authors Stefania Romeo, Emilie L. Hansen, Stine K. Frandsen, Julie Gehl
Title Electroporation of a Bladder Cancer Cell Line in Presence of Calcium: Efficacy Dependence on Electric Field Strength and Calcium Concentration
Year 2015
Issue 53
Pages 151-154
Status Published

Oral presentation(s) originating from this mission

Event Bioelectrics 2014
Location Columbia/Missouri/USA
Period 12/10 - 18/10/2014
Authors Stine Krog Frandsen, Laure Gibot, Marie-Pierre Rols, Julie Gehl
Title Calcium electroporation of spheroids induce cell death in cancer cells, not normal cells

 

Event Gordon Research Conference in Bioelectrics
Location Maine/USA
Period 06/07 - 11/07/2014
Authors Stine Krog Frandsen, Laure Gibot, Marie-Pierre Rols, Julie Gehl
Title Calcium electroporation of spheroids induce cell death in cancer cells, not normal cells

 

Event Gordon Research Seminar in Bioelectrics
Location Columbia/Missouri/USA
Period 05/07 - 06/07/2014
Authors Stine Krog Frandsen, Laure Gibot, Marie-Pierre Rols, Julie Gehl
Title Calcium electroporation of spheroids induce cell death in cancer cells, not normal cells

 

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Stefania Romeo, Emilie L. Hansen, Stine K. Frandsen, Julie Gehl
Title Electroporation of a Bladder Cancer Cell Line in Presence of Calcium: Efficacy Dependence on Electric Field Strength and Calcium Concentration

Poster presentation(s) originating from this mission

Event 13th International Meeting of the European Calcium Society
Location Centre de Congrès/ Aix-en-Provence/Frankrig/France
Period

13/09 - 17/09/2014

Authors Stine Krog Frandsen, Laure Gibot, Marie-Pierre Rols, Julie Gehl
Title Calcium electroporation of spheroids induce cell death in cancer cells, not normal cells

 

Event Gordon Research Conference in Bioelectrics
Location Maine/USA
Period

06/07 - 11/07/2014

Authors Stine Krog Frandsen, Laure Gibot, Marie-Pierre Rols, Julie Gehl
Title Calcium electroporation of spheroids induce cell death in cancer cells, not normal cells

 


Antitumor effectiveness of antiangiogenic therapy in murine B16-F1 melanoma

STSM by Natasa Tesic, PhD Student

Period of mission: from 01/09/2013 to 29/11/2013

Home institution: University of Primorska, Faculty of Health Sciences, Izola, Slovenia

Host institution: Université Catholique de Louvain, Louvain Drug Research Institute, Brussels, Belgium

The aims of STSM were (i) to evaluate antitumor effect of antiangiogenic therapy in murine B16 F1 melanoma by intra-tumoral electrotransfer of plasmid encoding endothelial specific promoter-driven short interfering RNA directed to the endoglin (ii) to investigate immune response against endoglin after electrotransfer of DNA vaccines coding for murine and porcine endoglin and to confirm efficacy of immunization by challenging mice with B16 F1 melanoma cells. To determine antitumor effect of antiangiogenic therapy, tumors were transfected with plasmids encoding tissue specific or constitutive eukaryotic promoter-driven siRNA directed to endoglin, using electroporation. Negative control was plasmid encoding constitutive cytomegalovirus promoter-drive luciferase. Tumor growth was followed and tumors were removed at day 7 post-treatment to determine intra-tumoral levels of endoglin mRNA. Two DNA vaccines encoding murine and porcine endoglin were successfully constructed and C57BL/6 mice, were immunized by intramuscular electroporation. For determination of antibodies against endoglin, blood samples were collected 3 days before each vaccine delivery. Two weeks after last boost mice were challenged with B16 F1 melanoma cells. Single electrotransfer of antiangiogenic plasmids in B16 F1 tumors didn’t resulted in significantly prolonged growth delays and survival rates, compared to the negative control. Results of qRT PCR showed that there was no reduction of mRNA endoglin expression level in tumors treated with plasmids compared to the untreated control. For analysis of blood serum, is still necessary to optimize protocol for ELISA assay. Efficacy of immunization after challenge with B16 F1 melanoma cells will be confirmed by following tumor growth and survival rate.

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Maja Čemažar, Tanja Dolinšek, Natasa Tešić, Monika Štimac, Gregor Serša
Title Electrotransfer of antiangiogenic shRNA against endoglin for effective cancer treatment

 


Fast electrical bioimpedance spectroscopy measurements during electroporation

STSM by Tomás García-Sánchez, PhD Student

Period of mission: from 30/09/2013 to 01/11/2013

Home institution: Technical University of Catalonia (UPC), Electronic and Biomedical Instrumentation Group, Barcelona, Spain

Host institution: Laboratory of Vectorology and Anticancer Therapeutics, University Paris-Sud and Gustave Roussy, Paris, France

The study of the dynamics of cell membrane during electropermeabilization is one of the main topics in today’s electroporation research field. Accurate study of pore evolution over time is useful to extract information about the successfulness of electroporation treatment and cell recovering as well as the dynamics of these events. Measurement of the temporal evolution of the electrical impedance spectrum is a label-free technique that can provide valuable information about the state of the system. In this STSM a fast measuring technique (in the range of millisecond) was applied to online monitor the impedance spectra evolution from 5 kHz to 1.31 MHz during electroporation of cell monolayers. Also, as a proof of concept, some in vivo experiences were performed in skin electroporation using the same technique. The goal was to correlate the observed impedance changes with the cell permeabilization detected using a fluorescent dye. The results obtained show promising information on how the study of the complete spectral information can provide additional information about the system. The different effects occurring during the application of pulses such as temperature changes, reactions in the electrode/electrolyte interface, and changes in the conductivity of the extracellular medium due to ion efflux could be differentiated and characterized given the fact that their frequency response is different. Further work will comprise development of theoretical models to adjust the measurements obtained.

Publication(s) originating from this mission

Journal Bioelectrochemistry
Impact Factor (according to ISI Thomson) 4,172
Authors Tomás García-Sánchez, Antoine Azan, Isabelle Leray, Javier Rosell-Ferrer, Ramon Bragós, Lluis M. Mir
Title Interpulse multifrequency electrical impedance measurements during electroporation of adherent differentiated myotubes
Year 2015
Issue 105
Pages 123-135
Status Published

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Tomas Garcia-Sanchez, Antoine Azan, Isabelle Leray, Javier Rosell-Ferrer, Lluis M. Mir and Ramon Bragos
Title Modeling dynamic electrical impedance spectroscopy measurements on electroporated cells

 


Skin electroporation for transdermal drug delivery - numerical models of in vitro experiments

Reciprocal STSM by Barbara Zorec, PhD Student

Period of mission: from 06/10/2013 to 30/10/2013

Home institution: University of Ljubljana, Ljubljana, Slovenia

Host institution: University of Canterbury, Christchurch, New Zealand

Electroporation as an active enhancement method for dermal and transdermal drug delivery has been used before, however, the possibilities for further improvement of the method are far from being exhausted. Our in vitro experimental results have shown that longer LV pulses significantly increase the transport of calcein through dermatomed pig skin, while short HV pulses alone result in negligible total calcein transdermal transport. Surprisingly, when the long LV pulses are preceded by short duration HV pulses, the total transport is reduced significantly. In order to make sense of these results, theoretical analysis of the underlying mechanisms was needed. Our main focus during the STSM was oriented towards theoretical comparison of different combinations of square wave short high voltage (HV) and longer low voltage (LV) electroporation pulses in order to understand fundamental physics during electroporation. The theoretical model shows that HV pulses alter the structure of the stratum corneum in such a way that when the LV pulses are applied, insufficient thermal energy is generated to initiate the expansion of transport pathways into larger local transport regions (LTRs). Together, the experimental results and theoretical predictions show that the total pulse energy alone cannot account for total solute transport: that the order and the type of pulses administered must also be considered. During the STSM we also upgraded the description of the thermodynamics underlying the LTR evolution with a simplified analytical model that shows directly how the heating time required to initiate LTR evolution is dependent upon the pre-existing pathway size and density.

Publication(s) originating from this mission

Journal Mathematical Biosciences 
Impact Factor (according to ISI Thomson) 1,303
Authors Sid Becker, Barbara Zorec, Damijan Miklavcic, Natasa Pavselj
Title Transdermal transport pathway creation: electroporation pulse order
Year 2014
Issue 257
Pages 60-68
Status Published

Oral presentation(s) originating from this mission

Event 3rd International Conference on Computational Methods for Thermal Problems (ThermaComp2014)
Location Lake Bled/Slovenia
Period 02/06 - 04/06/2014
Authors Barbara Zorec, ??? (to be updated)
Title Computational modeling of skin electroporation: reconciliation between experimental observations and anticipated physics

 

Event 23rd International Symposium on Transport Phenomena
Location Auckland/New Zeeland
Period 19/11 - 22/11/2012
Authors Sid Becker, Barbara Zorec, Nataša Pavšelj, Damijan Miklavčič
Title Transport Modeling of Skin Electroporation and the Thermal Behavior of the Stratum Corneum

Staining of cell-to-cell junctions after EP - in vitro study

STSM by Tanja Dolinšek, PhD Student

Period of mission: from 09/09/2013 to 04/10/2013

Home institution: Institute of Oncology, Department of Experimental Oncology, Ljubljana, Slovenia

Host institution: Institut of Pharmacology and Structural Biology (IPBS) - CNRS, Toulouse, France

It has been reported that a transient constriction of normal blood vessels occurs immediately after application of electric field pulses (EP). This phenomenon is accompanied by an increase in their permeability. In a previous STSM, Bostjan Markelc observed that the increase of the permeability of blood vessels was most probably due to the transient disruption of the cell-to-cell junctions in endothelial cells. Namely, the fluorescence of AlexaFluor 555 labeled antibody against CD31 became blurred and dispersed after application of EP, suggesting a direct effect on cell-to-cell junctions. However, whether the increased permeability was due to trans- or para- cellular transport was not known. Therefore, the aim of my STSM was to confirm, complement and further elaborate these results obtained in vivo in mice by using an in vitro setting. For that purpose, I stained mouse endothelial cells with cell junctions’ specific antibodies (anti CD31 and VE-cadherin) and observed the changes in cell-to-cell junctions before and at different times after EP. A very specific staining of cell-to-cell junctions has been obtained with VE-cadherin antibodies. However the staining for CD31 was more dispersed and not completely localized to cell junctions. I observed that in the cell monolayer submitted to EP (600 V/cm, 8 pulses, 100 µs, 1 Hz, plate electrodes 7.3 mm), the intensity of staining of cell junctions was immediately decreased and started to recover 10 min after EP. These data therefore confirms the results obtained in vivo, showing that the permeability of blood vessels is due to the transient disruption of cell-to-cell junctions of endothelial cells.

Poster presentation(s) originating from this mission

Event Annual Congress of the French Society of Cell and Gene Therapy (SFTCG)
Location Toulouse/France
Period

24/03 - 26/03/2014

Authors Bostjan Markelc, Elisabeth Bellard, Tanja Dolinsek, Sandrine Pelofy, Gregor Sersa, Marie-Pierre Rols, Justin Teissie, Maja Cemazar, Muriel Golzio
Title Determining the effects of electric pulses on endothelial cells of normal blood vessels with longitudinal biphoton microscopy imaging

 


Contribution of Electrochemical Phenomena to the Electroporation Current of Cell Suspension

STSM by Djamel Eddine Chafai, PhD Student

Period of mission: from 08/07/2013 to 30/09/2013

Home institution: APELEC Laboratory, Djillali Liabes University, Sidi Bel Abbes, Algeria

Host institution: Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia

Electroporation of cells is being successfully used in biology, biotechnology and medicine. Practical problems still arise in electroporation of cells in suspension. For example, determination of cell electroporation is still demanding and time consuming task. Also electric pulses cause contamination of the solution by the metal released from the electrodes and create local enhancements of electric field, leading to the occurrence of electrochemical reactions at the electrode/electrolyte interface. Therefore, an estimation of the current due to electrode/electrolyte interface phenomena is of a great interest to determine and evaluate its contribution to the total electroporation current. The goal of the STSM was to study the contribution of the current due to electrochemical phenomena (electrode/electrolyte interface) to the total electroporation current of cell suspension. The study was performed with and without cells in cuvette made of aluminum parallel plate electrodes. Electrical impedance spectroscopy was used before and after the delivery of electric pulses in different solutions of low and high conductivity. Voltage-current measurements were performed in both cases without and with cells. The study yielded to determine the dominant conduction phenomenon of the electrode/electrolyte interface exposed to pulsed electric field in free medium of cells. In presence of cells in low conductive medium, the contribution of electrode/electrolyte interface phenomena (Electrochemical reactions) is less pronounced compared to the intracellular ionic flux when the cell membrane is permeabilized at high electric field.

Publication(s) originating from this mission

Journal Bioelectrochemistry
Impact Factor (according to ISI Thomson) 4,172
Authors Chafai DE, Mehle A, Tilmatine A, Maouche B, Miklavčič D
Title Assessment of the electrochemical effects of pulsed electric fields in a biological cell suspension
Year 2015
Issue 106
Pages 249-257
Status Published

 


Improving extraction of pigments from microalgae by pulsed electric fields

STSM by Elisa Luengo, Researcher

Period of mission: from 08/07/2013 to 31/07/2013

Home institution: University of Zaragoza, Zaragoza, Spain

Host institution: Institut of Pharmacology and Structural Biology (IPBS) - CNRS, Toulouse, France

Although several studies have demonstrated that electropermeabilization of cells for improving extraction can be achieved with trains of electrical pulses with duration in the range of milliseconds or in the range of microseconds; at the moment, their relative efficacy has not been directly compared. The influence of different protocols of application of pulses, milliseconds and microseconds, on the extraction of pigments from the microalgae Chlorella vulgaris was investigated. Moreover, these studies were combined with observations and measurements performed by microscopy in order to evaluate the PEF treatment effect on C. vulgaris. The application of a PEF treatment of 4.5kV/cm and 60 ms (194 kJ/kg) with a pulse duration between 1 and 3 ms was enough to achieve the maximum carotenoid extraction rate, whereas when C. vulgaris cells were treated with pulses of 3 μs the maximum extraction rate was achieved with a PEF treatment of 20 kV/cm and 150 µs (198 kJ/kg). Batch as well as flow processes were used for extraction. Therefore, in spite of the different pulse duration range, similar energies were needed to achieve the maximum extraction yield. AFM force-volume assay revealed that the application of a PEF treatment of 4.5 kV/cm and 60 ms to C. vulgaris cells induced an increment by 3 times in the stiffness of the envelopes compared with the untreated ones. TEM observations showed a less defined cell wall of the treated cells. However, no differences in the surface were observed between the PEF treated and untreated C. vulgaris cells when they were observed under the SEM.

Publication(s) originating from this mission

Journal Journal of Membrane Biology
Impact Factor (according to ISI Thomson) 2,457
Authors Elisa Luengo, Juan Manuel Martínez, Mathilde Coustets, Ignacio Álvarez, Justin Teissié, Marie-Pierre Rols, Javier Raso
Title A comparative study on the effects of millisecond- and microsecond-pulsed electric field treatments on the permeabilization and extraction of pigments from Chlorella vulgaris
Year 2015
Issue 248
Pages 883-891
Status Published

 


Theoretical investigation of molecular structure and stability of bilayers membranes containing oxidized lipids

STSM by Aziz Aboulmouhajir, Professor

Period of mission: from 29/04/2013 to 02/06/2013

Home institution: Équipe de Chemoinformatique et Spectroscopie, Université Chouaib Doukkali, El Jadida, Morocco

Host institution: Équipe de Chimie et Biochimie Théoriques, CNRS, Université de Lorraine, Nancy, France

The membrane lipids are the primary targets for free radical attack. Furthermore, it was recently suggested (Dr. Luis Mir’s Communication at the EBTT 2012 Meeting in Ljubljana) that one of the main effects of very intense (100 kV/cm) electric fields on lipid membranes is to induce lipid oxidation causing their destabilization even after switching off the electric field. Although the lipids conformations depend on self-assembly structure of the membrane and the external field effects favorable to its alignment, we have used the ab initio computations to determine initially the electronic distribution within isolated phospholipids. We have determined the most stable conformer of POPC and PLPC and quantified the weakest CH bonds prone to abstraction by peroxyl or alkoxyl radicals in initiation phase of oxidation. The bisallylic position is found targeted preferentially by radical abstraction compared to the allylic ones. We have also found that the steric arrangement of the homoconjugated unsaturated carbon-carbon with respect to CH2 which is flanked between them, makes hydrogens not equivalent in terms of abstraction. Furthermore, the possibilities of electron delocalization after H ̇ abstraction and LOO ̇ fragmentation or rearrangement were also studied. We were also interested in the generation of a molecular mechanics energy function of the oxidized forms of these phospholipids by two approaches: either by producing a spectroscopically determined force field (SDFF) - based on scaled ab initio force field parameters - for some specific fragments of oxidized species, or by parameterization of non standard interactions in the oxidized species by creating new covalent links between known model compounds. We agreed with the host laboratory on the possibility of exchange of students.


Effect of the electric field on the organization of the cytoskeleton of endothelial cells

STSM by Veselina Uzunova, Researcher

Period of mission: from 01/03/2013 to 31/05/2013

Home institution: Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Sofia, Bulgaria

Host institution: University of Primorska, Faculty of Health Sciences, Isola, Slovenia

The aim of the experimental work was to study the effect of the electric field, both independently and in combination with bleomycin on the organization of the cytoskeleton of endothelial cells. During this STSM, we studied the degree of cell membrane permeability as a function of the applied high intensity electrical pulses, and how electroporation (EP) affects endothelial cytoskeletal protein reorganisation and cell survival as part of monolayer integrity. Two endothelial cell lines were used (HMEC-1 and HUVEC), that were seeded in 8-chambers slides. Electroporation was performed with plate electrodes perpendicular to the monolayer, without bleomycin or in the presence of 300 nM bleomycin or 100 µM propidium iodide (PI) to determine the degree of cell membrane permeability, after which the treated cells were incubated for 2 or 24 hrs. Next, we performed immunofluorescent staining for cytoskeleton proteins (F-actin, β-tubulin and vimentin) as well as MTS assays for cell viability and survival. We concluded that the EP of an adherent endothelial monolayer, induced changes in cytoskeleton structures. Higher electric fields caused even more rapid disruption of the cytoskeleton and the process was voltage-dependent. At EP ≥ 200V/cm the number of affected cells increased and the monolayers were clearly affected. The cytostatic bleomycin enhanced these effects, most likely because (1) acting on its intracellular target (reached due to electropores produced in the membrane) it induces apoptosis, inhibits cell proliferation and has direct cytotoxic effect, and (2) has specific influence on cell adhesion. Both situations EP/ECT by affecting cell viability and leading to low cell survival may produce in vivo the destruction of the tumor blood vessels.

Oral presentation(s) originating from this mission

Event Scientific session of PhD students and young scientists „Biomedicine and quality of life“, The 145-anniversary of Bulgarian Academy of Sciences
Location Sofia/Bulgaria
Period

02/10/2014

Authors Veselina Uzunova, Cecil Meulenberg, Maja Cemazar, Rumiana Tzoneva
Title Effect of monophasic electric pulses both independently and in combination with bleomycin on the cytoskeleton organization in endothelial cells

 


The XVth International Conference on Electrical Bio-Impedance, Heilbad Heiligenstadt, Germany

ESR Report by Matej Kranjc, Early-Stage Researcher

Period of mission: from 22/04/2013 to 25/04/2013

Home institution: University of Ljubljana, Ljubljana, Slovenia

Host institution: The XVth International Conference on Electrical Bio-Impedance, Heilbad Heiligenstadt, Germany

From 22nd to 25th April 2013 I attended the XV International Conference on Electrical Bio-Impedance for which I received an ESR Conference Grant from COST. The conference took place in the little town called Heilbad Heiligenstadt situated in the middle of Germany. The conference provided important platform for investigators in all aspects of biomedical applications of electrical impedance tomography (EIT) to engage in common areas of interest, whilst also allowed an opportunity for the community to broaden its outlook in the clinical applications and new technologies associated with this area of research. The conference program was focused on the medical applications of electrical impedance tomography, magnetic induction tomography, and magnetic resonance electrical impedance tomography. I was able to learn on latest results and obtained state-of-the-art knowledge on new technologies associated with this area of research from leading experts in the field. I had a presentation entitled Magnetic resonance electrical impedance tomography for determining electric field distribution during electroporation where I explained theoretical background of proposed method and showed experimental results on monitoring electric field distribution during electroporation of agar phantom and ex vivo liver tissue. I showed that MREIT would confirm delivery of sufficiently high electric field in the whole treated tissue and thus enable detection of areas with insufficient electric field coverage during electroporation based treatments like electrochemotherapy and nonthermal irreversible electroporation ablation. In future, this essential near real time information could thus be used to improve the electroporation treatment by setting higher amplitude of electric pulses, changing their duration or number or by electrode repositioning, thus assuring to cover sub treated areas of target tissue and increase effectiveness of electroporation based clinical procedures.


Molecular dynamics study of the voltage sensitive dye ANNINE-6

STSM by Aude Silve, Researcher

Period of mission: from 12/05/2013 to 26/05/2013

Home institution: IHM KIT, Karlsruhe, Germany

Host institution: University of Groningen, Groningen, Netherlands

The Bioelectrics group of the Institute for Pulsed Power and Microwave Technology at KIT possesses a setup designed to measure transmembrane voltage modulation during exposure of cells to nanosecond pulsed electric fields. The measurement is based on the use of a voltage-sensitive fluorescent dye (VSD, ANNINE-6) which incorporates in the membrane of cells. Providing quantitative measurement of transmembrane voltage requires however a calibration of the ANNINE-6 molecule. The STSM was performed with the Molecular Dynamics group of the University of Groningen. We addressed the question of the calibration of ANNINE-6 by means of computation. Molecular dynamic simulations combined with a quantum mechanics (QM) approach were used to provide information for the calibration, especially on the nanosecond timescale. The position, orientation and spectral shift of the ANNINE-6 molecule in a POPC bilayer has been analysed for positive and negative electric field with an absolute value of 0.1 V/nm. Preliminary results show a clear asymmetry in the behaviour of the dye that could explain some asymmetry in the calibration. Additionally, time course of the rearrangement of ANNINE-6 molecule and of its environment has been studied. The preliminary results indicate rearrangements on the nanosecond timescale. This will be further investigated and will inform us on the possibility to transpose long time scale calibration to short time scale measurements.

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Aude Silve, Clair Poignard, Martin Sack, Ralf Straessner and Wolfgang Frey
Title Study of transmembrane voltage kinetics during 100 μs pulse using voltage sensitive dyes

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Aude Silve, Isabelle Leray, Clair Poignard and Lluis M Mir
Title Impact of external medium conductivity on cell membrane electropermeabilization by microsecond and nanosecond electric pulses

 


Nanosecond pulses effects on cells: microdosimetry supporting experiments

STSM by Agnese Denzi, PhD student

Period of mission: from 15/04/2013 to 24/05/2013

Home institution: Department of Information Engineering, Electronics and Telecommunications, Sapienza, University of Rome, Rome, Italy

Host institution: UMR 8203 of the CNRS and University Paris-Sud, Institut Gustave Roussy, Villejuif, France

Recently the use of electric pulses always more intense and shorter, allowed the manipulation of intracellular membranes. The aim of the STSM was to analyze the possibility to couple theoretical and experimental aspects in order to understand the electropermeabilization phenomenon for both the outer and the inner membranes of the cells. The main experimental activity was on human adipose mesenchymal stem cells with one 10 nanoseconds pulse with different amplitudes (30 kV/cm-350 kV/cm) to analyze the value of the external electric field necessary to obtain the permeabilization of the plasma and the endoplasmic reticulum (ER) membranes. The experiments were carried out with two different external buffer solutions with (DMEM) and without (SMEM) calcium ion (Ca2+). The first allows to obtain the threshold for the plasma membrane whereas the second one the threshold for the ER membranes. The main result of the host laboratory was the finding that there are different thresholds in terms of the external electric field necessary to electropermeabilize the different membranes of the cells. With the microdosimetric model, different thresholds were obtained in terms of the external electric field for the permeabilization of the membranes. However the dielectric parameters of the organelle can greatly influence these threshold values up to the point that in some conditions the permeabilization for the inner membrane appears with electric field values lower than for the outer one. The microdosimetric model can be useful to understand the particular conditions that determine the different thresholds between the inner and the outer membranes.

Publication(s) originating from this mission

Journal Journal of Membrane Biology
Impact Factor (according to ISI Thomson) 2,174
Authors Agnese Denzi, Caterina Merla, Paola Camilleri, Alessandra Paffi, Guglielmo d’Inzeo, Francesca Apollonio, Micaela Liberti
Title Microdosimetric Study for Nanosecond Pulsed Electric Fields on a Cell Circuit Model with Nucleus
Year 2013
Issue 246
Pages 761-767
Status Published

 

Journal Journal of Membrane Biology, Special Issue
Impact Factor (according to ISI Thomson) 2,174
Authors Agnese Denzi, Elena della Valle, Francesca Apollonio, Marie Breton, Lluis M. Mir, Micaela Liberti
Title Exploring the Applicability of Nano-poration for Remote Control in Smart Drug Delivery Systems
Year 2015
Issue ?
Pages ?
Status Submitted - to be updated

 


In vivo behaviour of novel electrode designs

STSM by Quim Castellvi, PhD Student

Period of mission: from 03/03/2013 to 23/03/2013

Home institution: Universitat Pompeu Fabra, Barcelona, Spain

Host institution: 8203 CNRS, Institut Gustave Roussy, Villejuif, France

Precise control over the electric field magnitude that develops in tissues is crucial for electroporation therapies and thus optimum electric field distributions are sought for. With such objective in mind, numerical models and tools are being developed for helping in the process of specifying the location of the electrodes and the voltages to be applied between those electrodes. Sophisticated treatment planning tools are being developed for indicating where the electrodes must be placed. Therefore, deployment accuracy and mechanical stability of the electrodes will be key features for optimum treatment, particularly in the case of internal organs. Without mechanical stability – which is particularly compromised due to involuntary movements caused by the electric pulses required for electroporation – fine pre-treatment planning employing advanced numerical methods will be of little use. The aim of the STSM was to study the mechanical performance of novel electrode setups in in vivo environments. Specifically, the novel electrode arrays were tried in rat liver and skeletal muscle under ultrasound imaging guidance. Tissue samples were collected for posterior histological analysis in which damage caused by electrode insertion, by electroporation treatment and by electrode extraction will be assessed. In addition, extraction forces were registered with a custom-developed force measurement system. Interestingly, it turns out that extraction forces are consistently higher after performing electroporation, thus revealing some sort of modification of mechanical properties of tissue or of the mechanical properties of the interface between the tissue and the electrode. We are now performing further experiments for elucidating the cause, or causes, of such increase in extraction force.

This report is also featured in the July 2013 newsletter available here.

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Quim Castellví, Patricia Sánchez-Velázquez, Alberto Villanueva, Enrique Berjano, Fernando Burdío and Antoni Ivorra
Title Sudden Death of Mice after Irreversible Electroporation of a Large Portion of the Liver: Probable Role of Hyperkalemia

 


Blueberries, black currant and sea buckthorn berries cell disintegration depending on PEF parameters

STSM by Nerijus Lamanauskas, Early-Stage Researcher

Period of mission: from 04/03/2013 to 17/03/2013

Home institution: Vytautas Magnus University, Kaunas, Lithuania

Host institution: ProdAl S.c.a.r.l. & University of Salerno, Fisciano, Italy

Pulsed electric field (PEF) is used to improve extraction of bioactive molecules, such as antioxidants, colorants, flavours and others (Donsi et al., LWT - Food Science and Technology, 2010). Many investigations are carried out with grapes, but much less work is done on other types of berries. Thus the aim of STSM was to investigate cell disintegration of frozen/thawed blueberries, black currant and sea buckthorn berries under different PEF conditions. Complex impedance Z and phase angle were measured and cell disintegration index Zp was calculated. PEF experiments were carried out at 1-5 kV/cm electric field strenght, 1-10 kJ/kg total electric energy, 20 μs pulse width and 20 Hz frequency. Zp values were similar for blueberries and sea buckthorn. Zp depended more on electric field, and not so much on total electric energy. Therefore, 5 kV/cm and 1 kJ/kg were found as optimal conditions. Under these PEF conditions, Zp = 0.49 for blueberries, Zp = 0.58 for sea buckthorn and Zp = 0.29 for black currant was found. In conclusion, with the same PEF parameters applied, cell disintegration was similar for blueberries and sea buckthorn, maximal Zp = 0.5-0.6. Black currant berries were less affected by the same parameters, maximal Zp = 0.29. It should be also stressed, that all the berries used in this work were frozen/thawed and this can explain the low increase of Zp value detected. It can be expected that the application of PEF pre-treatment to fresh products will guarantee a greater increase of cell membrane permeabilization.

This report is also featured in the July 2013 newsletter available here.

Publication(s) originating from this mission

Journal Zemdirbyste-Agriculture
Impact Factor (according to ISI Thomson) 0,470
Authors Lamanauskas Nerijus, Bobinaite Ramune, Satkauskas Saulius, Viskelis Pranas, Pataro Gianpiero, Ferrari Giovanna
Title Pulsed Electric Field-Assisted Juice Extraction of frozen/thawed blueberries
Year 2015
Issue 102 (1)
Pages 59-66
Status Published

 

Journal Innovative Food Science and Emerging Technologies - Special Issue
Impact Factor (according to ISI Thomson) 3,273
Authors R. Bobinaitė , G. Pataro , N. Lamanauskas, S. Šatkauskas, P. Viškelis , G. Ferrari Giovanna
Title Pulsed electric field-assisted pressing and extraction of juice and bioactive compounds from blueberry fruits and their by-products
Year 2014
Issue ?
Pages ?
Status Unknown - to be updated

Oral presentation(s) originating from this mission

Event School on applications of Pulsed Electric Fields for food processing
Location Fisciano/Italy
Period 07/02 - 12/02/2015
Authors N. Lamanauskas, R. Bobinaite, S. Satkauskas, G. Pataro, G. Ferrari
Title PEF application for cell disintegration of Black Currant and Sea Buckthorn berries

 


Modelling both conducting and permeable states of cell membranes submitted to electric pulses

STSM by Clair Poignard, Researcher

Period of mission: from 04/02/2013 to 09/02/2013

Home institution: INRIA, Bordeaux, France

Host institution: Bioelectrics Group, IHM KIT, Karlsruhe, Germany

Current models of electroporation describe electroporation as an increase of membrane conductivity. Such an approach seemed to us rather inappropriate since most experimental observations indicate that the conducting state of the membrane and the permeabilised state cannot be identified. Experiments of Zimmerman et al. (J Membr Biol, 1979) at the end of the seventies suggested that the conducting state lasts only few microseconds after the pulse delivery, while the membrane remains permeabilised several minutes afterwards. This is the reason why the team MC2 of INRIA is developing new models that describe almost separately the two phenomena. Experiments from KIT have provided most significant information on the conducting state. The first important result obtained during the STSM relies on the link between high transmembrane voltage and pore creation. In the most highly developed models of Krassowska et al. (Biophys J, 1999) the dynamics of the pore creation is proportional to the factor: exp((1-q)(V/Vep)2).
Such dynamics implies that as soon as the transmembrane threshold value (Vep) is reached, the pores almost immediately open, leading to a drop of the transmembrane voltage. On the contrary, the experiments of KIT on transmembrane potential measurement showed a quite smooth behaviour of the transmembrane voltage during the pulse delivery, suggesting that the membrane response to the pulse has its intrinsic dynamics. The second important result concerns the link between the data obtained by patch-clamp and the modelling. By applying low voltage pulses in the physiological range to cell membranes, patch-clamp data provide estimates of the membrane conductivity and capacity at rest, i.e. before the electroporation. For higher voltage pulses, measurement of the current increase makes it possible to estimate the increase of the membrane conductivity due to the pore creation. Such data can thus directly be used to calibrate our models.

This report is also featured in the May 2013 newsletter available here.

Oral presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period 06/09 - 10/09/2015
Authors Aude Silve, Clair Poignard, Martin Sack, Ralf Straessner and Wolfgang Frey
Title Study of transmembrane voltage kinetics during 100 μs pulse using voltage sensitive dyes

Poster presentation(s) originating from this mission

Event 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies
Location Portorož/Slovenia
Period

06/09 - 10/09/2015

Authors Aude Silve, Isabelle Leray, Clair Poignard and Lluis M Mir
Title Impact of external medium conductivity on cell membrane electropermeabilization by microsecond and nanosecond electric pulses

 


Molecular dynamics simulation of archaeal lipid membranes

STSM by Andraž Polak, PhD student

Period of mission: from 07/01/2013 to 01/02/2013

Home institution: University of Ljubljana, Ljubljana, Slovenia

Host institution: University of Lorraine, Nancy, France

The aim of current research is to investigate a novel delivery system for various biologically active substances by encapsulating them in artificial liposomes made of archaeal lipids, delivering the liposomes into cells and then controlling the substance release into the cytosol by electroporation. Compared to simple phosphatidylcholine lipids, archaeal lipids have a special head-group formed of sugar moieties, but also methyl branches in the lipid tails and ether linkages instead of ester linkages between the head group and the carbonyl region. During this STSM, we have first studied the response of DPPC, DPhPC-ester and DPhPC-ether based lipid bilayers to applied electric fields. We have first characterized the dipole potential and the capacitance of each bilayer. Then, comparing between lipids with acyl chains (DPPC) and methyl branched chains (DPhPC), and between lipids with ether and ester linkages, we find that in both cases the electroporation threshold differs substantially. Following this, we performed similar studies of the more complex membranes composed of archaeal lipids from Aeropyrum pernix and DPPC lipids that are simultaneously under study experimentally at our home institution, with the aim of revealing (1) how the electric field affects these novel membrane models; and (2) how the temperature affects their electroporation. The results of the simulations are being now analyzed, however preliminary results show again that archaeal lipids have a much higher electric field threshold for electroporation than simple phosphatidylcholine lipids.

This report is also featured in the May 2013 newsletter available here.

Publication(s) originating from this mission

Journal Journal of  Membrane Biology
Impact Factor (according to ISI Thomson) 2,174
Authors Andraz Polak, Daniel Bonhenry, Francois Dehez, Peter Kramar, Damijan Miklavcic, Mounir Tarek
Title On the Electroporation Thresholds of Lipid Bilayers: Molecular Dynamics Simulation Investigations
Year 2013
Issue 246
Pages 843-850
Status Published

 


Electroporation of spinach leaf: theoretical and experimental investigation

STSM by Katarzyna Dymek, PhD student

Period of mission: from 01/09/2012 to 30/11/2012

Home institution: Department of Food Technology, Engineering and Nutrition, Lund University, Sweden

Host institution: Faculty of Electrical Engineering, University of Ljubljana, Slovenia

One of the promising applications of reversible electroporation of plant tissue is the improvement of freezing tolerance of spinach leaves after electroporation and vacuum impregnation treatment (Phoon et al. 2008). Spinach leaf possesses heterogeneous internal structure. To investigate the electroporation of such complex structure a numerical model of the leaf cross section was built. The model represents the 3D reconstruction of plant tissues. It is based on the microscopic images of the spinach leaf cross section and it includes four layers: single cell layer of epidermal cells, double cell layer of palisade mesophyll and multi cell layer of spongy mesophyll cells. The air fraction is located in the spongy mesophyll. Cells are surrounded by cell walls, which are connected with each other. Moreover, the model representing the vacuum impregnated leaf was also built. This model does not contain the air fraction, which is removed during the vacuum impregnation process. Mathematical equations describing the electroporation of the cellular membrane are based on DeBruin et al. 1999 and the method for calculating the induced transmembrane voltage is adopted from Pucihar et al. 2009. COMSOL Multiphysics 3.5a (COMSOL AB, Stockholm, Sweden) commercial finite element software package was used to perform the calculations. Models were adjusted and validated by comparison with the experimental data. Measurements of the impedance at 20 Hz-100 kHz frequency range were performed in the Franz cells before and after application of high voltage electric pulse to the samples.

This report is also featured in the February 2013 newsletter available here.

Publication(s) originating from this mission

Journal Innovative Food Science and Emerging Technologies, Special Issue
Impact Factor (according to ISI Thomson) 3,273
Authors Katarzyna Dymek; Lea Rems; Barbara Zorec; Petr Dejmek; Federico Gomez Galindo; Damijan Miklavcic
Title Modeling electroporation of the non-treated and vacuum impregnated heterogeneous tissue of spinach leaves
Year 2015
Issue 29
Pages 55-64
Status Published

Oral presentation(s) originating from this mission

Event XXIII International Symposium on Bioelectrochemistry and Bioenergetics of the Bioelectrochemical Society
Location Malmö, Sweden
Period 14/06 - 18/06/2015
Authors Katarzyna Dymek; Lea Rems; Barbara Zorec; Petr Dejmek; Federico Gomez Galindo; Damijan Miklavcic
Title Modeling electroporation of the non-treated and vacuum impregnated heterogeneous tissue of spinach leaves

 


The in vivo effects of electroporation on the endothelial cells of blood vessels

STSM by Bostjan Markelc, PhD student

Period of mission: from 15/10/2012 to 30/11/2012

Home institution: Institute of Oncology, Dept. of Experimental Oncology, Ljubljana, Slovenia

Host institution: Institute of Pharmacology and Structural Biology – CNRS, Dept. of Cellular Biophysics, Toulouse, France – Dr. Muriel Golzio

In the STSM we addressed the blood flow modifying effects of electroporation/electropermeabilization (EP) which have been observed in vivo in muscles and tumors. Recently it has been shown that a transient constriction of normal blood vessels occurs immediately after EP, which is accompanied by an increase in their permeability. The aim of the STSM was to determine the effects of EP on the integrity of the blood vessels wall and the endothelial cells in vivo. In the project we used C57Bl/6 mice strain in which we surgically fitted the dorsal window chamber. For visualization of endothelial cells in blood vessels we used Alexa Fluor® 555 labeled CD31 antibodies and to visualize the increased permeability of blood vessels we used fluorescently labelled dextrans (FD). Mice were subjected to EP (8 pulses, 520 V/4 mm, 100 μs, 1 Hz). Image acquisition was performed with a multiphoton microscope and the subsequent image processing and analysis by Imaris software. The results show that the labeling of the endothelial cells with fluorescent antibodies enables direct observation of the effects of EP at the level of endothelial cells. We determined that after EP, there is an increase of permeability in blood vessels for FD and that this increase is due to the transient disruption of the cell-to-cell junctions in the endothelial cells of blood vessels, which however started to recover already within the first hour after EP. This was in vivo confirmation of the observed in vitro effects of EP.

This report is also featured in the February 2013 newsletter available here.

Poster presentation(s) originating from this mission

Event Annual Congress of the French Society of Cell and Gene Therapy (SFTCG)
Location Toulouse/France
Period

24/03 - 26/03/2014

Authors Bostjan Markelc, Elisabeth Bellard, Tanja Dolinsek, Sandrine Pelofy, Gregor Sersa, Marie-Pierre Rols, Justin Teissie, Maja Cemazar, Muriel Golzio
Title Determining the effects of electric pulses on endothelial cells of normal blood vessels with longitudinal biphoton microscopy imaging

 


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