A Subnanosecond Pulsed Electric Field System for Studying Cells Electropermeabilization

Authors: Njomza Ibrahimi, Student Member, IEEE, Leslie Vallet, Franck Andre, Laurent Ariztia, Marc Rivaletto,Antoine Silvestre de Ferron, Bucur M. Novac, Senior Member, IEEE, Lluis M. Mir and Laurent Pécastaing, SeniorMember, IEEE

Monday, December 28th, 2020 Virtual

SEMINARI I PARË SHKENCOR I FAKULTETIT TË INXHINIERISË ELEKTRIKE DHE KOMPJUTERIKEUNIVERSITI I PRISHTINËS

ContentØ Introduction to Pulsed Power, PhD project and the objective of the present study

Ø Experimental arrangement

Ø 3D numerical modelling

Ø Measurement setup: Calibration

Ø Numerical techniques

• Time Domain Reflectometry

• Voltage analysis

• Electric field analysis

• Temperature analysis

Ø Biological methodology

Ø Biological results

Ø Comparison

Ø Conclusion 1

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What is High Pulsed Power ?

Fast energy restitution

Slow energy storage

CONSTANT ENERGY : 1 J

Power

Time100ns 1ms

1 kW

10 MW

Power enhancement by temporal compression

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DC source AmplificationPulse Forming

Network Load

Capacitors chargerPower supply

MARX generator13 stages

Bipolar pulse forming line

Monopolar pulse forming line

Far-field antenna(military application)

Near-field antenna/loop

(biomedical application)

PhD undertaken within ESCAPADE project:Design, development and test of a new ultra wide-band bipolar high power pulse source for civilian and military applications

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View of the main high voltage lab with a 1MV /27kJ Marx generatorHost laboratory: SIAME

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Objective of the present study

• Determination of the PEF parameters to induce permeabilization of biological cells• amplitude• duration• rise time

• Understand their action on cells by firstly using standard electrode-based PEFs driven by subnanosecond pulsed power generators

’’There are basically two types of PEF techniques applied in vivo: invasive, where metallic pairs of penetrating electrodes are used to apply the electric field and noninvasive, where the electric field is generated through electrodes brought in contact with the skin, without penetration of the body. In addition, the contactless term refers here to the approach dealing with the generation of electric fields remotely from the metallic structure of a PEF source (antenna).’’

• Development of a contactless technology, a nonsurgical procedure!

Metallic structure(generation of EM fields)

Biological cells

Distance D

L’Equipe Génie Electrique Experimental arrangement

Pulsed power arrangement for subnanosecond PEF a) Subnanosecond generator and PEF processing load. b) Overall schematic.

Experimental arrangement

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Numerical techniques: 3D modelling3D numerical modelling

a) Experimental arrangement for subnanosecond PEF processing. b) Complete CST model for the PEF processing chamber, including the RG214-U coaxial cable, the two parallel-plate copper

electrodes with the SMA-type V-dot probe attached and the electroporation cuvette, with various compartments filled with paraffin oil and water sample, which may contain bacteria.

a) b)

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Subnanosecond pulse source

Load

3D modelling Input first 10 nsexperimental data

Check load voltage through:

Output of the SMA V-dot probe mounted in the 3D model

CST virtual sensor

! # = 1& '!()*+,- # .#

/

0

Compare and calculate the capacitance C

Oscilloscope with attenuator

SMA typeV-dot probe

Capacitive voltage divider probe

RG214-U 50 Ωcoaxial cable

l=2.5 m

10 dB20 dB

10 dB10 dB

Probe’s coaxial cable l=5 mProbe’s coaxial cable l=5 m

Har

dwar

eSo

ftwar

e

Measurement setup: Calibration algorithm

Output response of the SMA type V-dot probe

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Measurement setup: Calibration

Upper: V-dot probe mounted inside the PEF load, near the cuvette containing the water to be processed. Lower: its corresponding equivalent electric scheme.

Measurement setup: Calibration using 3D modelling

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Integrated Gaussian signal with a rise time of 219 ps anda frequency bandwidth of 4 GHz used to perform the TDR task.

Numerical techniques: Time Domain Reflectometry analysis

Algorithm for TDR using time signals (time domain)

i(t) and o(t)

! " $ %$and! ) $ %$*

+

,

+

- $ = -+ ∫ " $ %$,+ +∫ ) $ %$*

+∫ "($),+ −∫ ) $ %$*

+

Impedance profile along the cable and the processing load.

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Voltage measurement analysis

Full line: experimental signal obtained from the main voltage probe.Dotted line: CST prediction for the voltage at Port 1.

Second comparison between CST model and experimental data.Thick line: time dependence of the integrated voltage signal from V-dot probe.Thin line: CST prediction obtained from a virtual voltage probe placed atlocation B.Dotted line: integrated voltage signal output from Port 2 placedat location A. The two CST generated traces are practically indistinguishable demonstrating that, due to the favorable electric field distribution, the SMA probe acts indeed as a V-dot probe.

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Electric field analysisElectric field analysis

Electric field distribution inside the processing load at the momentthe peak field of 58 kV/cm is generated inside the cuvette water sample.

PEF parameters generated inside the cuvette water sample for the condition presented previously.

E = 58 kV/cm, rise time of 303 ps and FWHM of 773 ps.

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- the electric field is mainly concentrated inside the cuvette. - inside the cuvette the electric field is highly homogeneous allowing a

simple interpretation of the results obtained after PEF processing.

Biological model and methods• E. Coli DH5α, cultured in LB medium for 8 hours (stationary phase)

• Marker used : nucleic acid fluorescent stains(used separately as indicated in experimental designs)

• SYTO®9 (SYTO), (λex 485/ λem 498), enter in all bacteria• YO-PRO™-I iodide (YP), (λex 491/ λem 509), enter only in permeabilized bacteria

• Experimental designs:

SYTOFlow

cytometry

15 min, RT Assessing E.Coli morphological criteria

Adapted from Garrison et al., 2016

YP Flow cytometry

45 min, RT Assessing E.Coli electropermeabilization

PEF Resuspension in LB medium

5-10 min, RT

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Biological results

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Paper containing detailed electrical analysis and preliminary biological results was just published on IEEE Transaction on Plasma Science as follows :

A subnanosecond pulsed electric field system for studying cells electropermeabilization, N. Ibrahimi, L. Vallet, F. Andre, L. Ariztia, M. Rivaletto, A. de Ferron, B.M. Novac, L.M. Mir, L. Pécastaing, IEEE Transactions on Plasma Science, DOI : 10.1109/TPS.2020.3034286, November 2020

• The immediate permeabilization increases with the number of pulses applied.

• Importantly, no increase of the temperature within the samples submitted to PEF treatment was noticed under the conditions tested.

- After applying N = 50000 consecutive shots, the estimated water sample temperature increase is only ∆T = 0.00615 C and therefore negligible.

Condition tested: E = 58 kV/cm, f = 200 Hz

0

20

40

60

80

100

1000 10000 100000

perm

eabi

lizat

ion

perc

enta

ge (%

)

number of pulses

immediate permeabilization

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Cell type System electricalparameters

Electric field Number of pulses

PRF (Hz) Effect observed Reference

B16 (Mouse melanoma)

160 kV200 ps rise time800 ps duration

150-950 kV/cm 125 and 18.000 - 50% of the cells took up the marker right after pulsing, 20% were taking it up after 1 h.

Schoenbachet al., 2008

Hepa (Livercancer cells)

20 kV150 ps rise time200 ps duration

80 kV/cm 2000 7–9 pulses per second, on theorder of 5 min.

30 % permeabilization Camp et al., 2012

GH3, NG108 and CHO

- 160 kV/cm500 ps duration

1 and 100 1000 (for 100 p delivered)

1 p could trigger calcium release on GH3 and NG108,100 p led to no effecton CHO

Semenov et al., 2015

B16 and NG108

- 25 kV/cm200 ps duration

1000 and 10.000 300 No effect observed after18h from exposure

Xiao et al., 2011

B16 and NG108

- 25 kV/cm200 ps duration

1.8 million and2.4 million

10000 20 % and90 % permeabilization

Xiao et al., 2010

E. Coli DH5α 20 kV100 ps rise time600 ps duration

12.3 kV/cm303 ps rise time773 ps duration

50 to 20000 200 37 to 90 % permeabilization

Current study

Conclusion

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• A detailed 3-D model is presented, based on the CST Microwave Studio, a 3-D finite integral time-domain (FITD) electromagnetic simulation software. Ø As the direct measurement of the PEF generated inside a cuvette containing the cell culture is

technically challenging, a comprehensive 3-D modelling and numerical electromagnetic analysis were essential during the experimental studies.

Ø Impedance, temperature, voltage and electric field analysis inside the PEF processing were therefore supported by the numerical analysis.

• We report encouraging biological results that demonstrate that a high percentage of permeabilized bacteria can be achieved using the described arrangement.Ø The permeabilization is increased significantly with the number of pulses applied.

• Results obtained are very promising when compared to the results in the existing literature!

Future work

• Test of the new MARX generator, monopolar and bipolar pulse forming line.• Develop the contactless technology.• Perform experiments in biological cells using the novel pulsed power system.

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EURO-ASIAN PULSED POWER CONFERENCE

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Thank you for your attention !

Contact information: Njomza Ibrahimi, njomza.ibrahimi@univ-pau.frPhD Researcher at: SIAME Laboratory of Applied Sciences to Electrical and Mechanical Engineering, University of Pau, France

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References:

[1] Sersa, G., Miklavcic, D., Cemazar, M., Rudolf, Z., Pucihar, G.Snoj, M. (2008). Electrochemotherapy in treatment of tumours. European journal of surgical oncology : the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology, 34(2), 232–240. https://doi.org/10.1016/j.ejso.2007.05.016[2] Aude Silve. Nouveaux dispositifs pour l’application contrôlée d’impulsions électriques nanosecondes et pour la détection de leurs effets sur les cellules : Nouveaux résultats et hypothèses sur les paramètres contrôlant l’électroperméabilisation des cellules biologiques. Médecine humaine et pathologie. Université Paris Sud - Paris XI, 2011. Français.