effect of pulsed electric fields on biological cells: adding some...

KIT – University of the State of Baden-Wuerttemberg and National Research Center of the Helmholtz Association

Institute for Pulsed Power and Microwave Technology (IHM)

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Effect of pulsed electric fields on biological cells: adding some pieces to the large puzzle

Aude SilveI



www.kit.edu

Thanks to …

Who am I ?

PhDEffects of nanosecond pulsed electric

field on living cells and tissues.

Lluis Mir

Wolfgang Frey

Post-docMeasurement of TMV induced by nanosecond

pulses by means of fluorescence probe

1

+ -E CathodeAnode

Cell

membrane

Membrane Charging

=> high transmembrane voltage

Effect of External Electric Pulses on Biological CellsIntroduction

Modification of membrane propertiesResting state

Impermeable membrane with very

low conductivity

Electroporation or Electropermeabilisation ?

2

NANOPULSE

Magnitude « A » :

1 to 30 kV/mm

Duration « t » :

3 ns to 300 ns

Rising edge « τ » :

500 ps to 10 ns

τ

A

t

ParametersMILLIPULSE / MICROPULSE

Magnitude « A » :

10 to 300 V/mm

Duration « t » :

10 μs to 20 ms

Rising edge « τ » :

1 to 20 μs

Classical versus NanoIntroduction |

3

100 V/mm

20 V/mm

Reversible and IrreversibleImpact of Pulse parameters |

Other determining parameters:- Number of pulses- Shape of pulses- Repetition rate- Type of cells (shape, size, …)- Buffer- Temperature- etc. …

4

Three main observations categoriesWhat can be detected ? |

100 200 300 400 500 600 700 800 900

50

100

1506000

8000

10000

12000

14000

Propidium Iodide

Ca2+

Detecting changes of membrane’s permeability

-> Usually by studying diffusion of normally not permeant ions or molecules

Release of intracellular metabolites (eg: ATP) Release or uptake of fluorescent markers (eg: PI, Lucifer Yellow, Calcein) Uptake of biologically active molecules (eg: cytotoxic drugs, DNA, antibodies)

5

| Z |

f (Hz)103 104 105 106

+

-

Detecting changes of membrane’s conductivity

-> With electrical or opto-electrical measurements

Voltage clamp on lipid bilayers Patch-clamp approach on single cells Bio impedance methods in vivo or in biological tissues Voltage sensitive dyes





5

Detecting the following physiological consequences

Cell death, in vitro Tumor regression in vivo

Detecting changes of membrane’s conductivity

-> With electrical or opto-electrical measurements

Voltage clamp on lipid bilayers Patch-clamp approach on single cells Bio impedance methods in vivo or in biological tissues Voltage sensitive dyes





5

Changes of membrane’s permeability

An example: bleomycin to detect reversible permeabilisationDetection of permeability|

6

Cell survival normalized to control submitted to bleomycin only

Pulses: 4 kV/mm, 10 ns, 10 Hz Medium: SMEM

Detection is not absoluteDetection of permeability|

Even a single 10ns Pulse enables penetration of Bleomycin

7Silve, Leray, Mir, Bioelectrochemistry, 2012

Transposition in vivo

4 min

Injection :1 to 2 M LPB cells

(LPB: murine fibrosarcoma )

J0 : treatment : - Average tumor volume : 30 – 50 mm3

- Retro-orbital injection of Bleomycin(100 µg in 100 µl) or Physiological serum- Application of electric pulses

D-dot sensor output

Pulse generator

Applied pulses computed from D-dot output

Internalization of Bleomycin|

8

What is the life time of permeability ?Detection of permeability|

- Pulse applied at Time t=0.- Survival rates are normalized to the control submitted to the bleomycin only- Bleomycin concentration of 30 nM.- Viability assessed by cloning efficiency test- DC3F cells

1 pulse100 µs

175 V/mm

1 pulse12 ns

9 kV/mm

With this diagnostic : resealing seams to happen in a couple of minutes9

Is there a maximum size of molecules that can be transportedDetection of permeability|

Nesin OM, et al.

BBA 2011

Many experiments such as studies of cells osmotic swelling after PEF suggest that membranes are permeable mostly to very small molecules

However, no size limit has been detected:

Orlowsky et al. 1988 – Mir et al. 1988 – Bazile et al. 1989

– Poddevin et al. 1991 – Casabianca-Pignède et al. 1991

Larger molecules still penetrates

Cells: DC3F 8 pulses: 100 µs, 140 V/mm

Molecule Weight (Da) Cint (% of Cext)

Lucifer Yellow 457 100

Bleomycin 1 500 33

Oligonucleotide 12 000 10

Pokeweed Antiviral Toxin 30 000 1

Antibody 150 000 not quantified

10

- PEF induce an increase of membranes permeability to normally non permeant molecules

- Small water soluble molecules can cross the permeable membrane easily but no absolute size limit of the molecule that can be transported could be detected

- After the pulse, a resealing can be observed

Resealing time are in the order of seconds to minutes

Resealing time depends on the pulses parameters

Resealing time depends on temperature and on biological factors

Recovery of membrane´s integrity is at least partially a biologically active

process

11

Changes of membrane’s conductivity

Potato: a powerful biological sampleBioimpedance

Z

Ø = 4 mmh = 5 mm

currents currents

12

Impedance drop due to permeabilisationBioimpedance

Normalized Impedance Drop

NID = 1 : no or very low increase of membrane’s conductivity

NID → 0 : high increase of membrane’s conductivity

13

Micropulses: impact of the repetition rateBioimpedance

Repetition rate (Hz)

Pulses: 100 µs, 80 V/mmDuration between two pulses

T

Repetition rate =1

T

Silve, Guimerà Brunet, Al-Sakere, Ivorra, Mir, BBA 2014 14

Two phenomenon ?Bioimpedance

-50 0 50 100 150 200-20

0

20

40

60

80

100

120

140

160

time /s

Voltage /

V

-50 0 50 100 150 200-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

time /s

Voltage /

A

-50 0 50 100 150 200-20

0

20

40

60

80

100

120

140

160

time /s

Voltage /

V

-50 0 50 100 150 200-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

time /s

Voltage /

A

50 µs

50 µs

10 A

50 V

𝜎𝐷𝐶_𝐴𝑓𝑡𝑒𝑟

𝜎𝐷𝐶_𝐵𝑒𝑓𝑜𝑟𝑒~10

𝜎𝐷𝐶_𝐷𝑢𝑟𝑖𝑛𝑔

𝜎𝐷𝐶_𝐵𝑒𝑓𝑜𝑟𝑒> 100

The high conductivity increase observed during the pulse cannot be detected a few second after the pulseThe conductivity increase detected after the pulses is much lower but persistent 15

Persistent Low Conductivity IncreaseBioimpedance |

- Relaxation of high conductivity state

τ ~ 100 ms

- Additionally to the high conductivity increase, a persistent low conductivity

increase can be detected

Ivorra and Rubinsky, Bioelectrochemistry, 2007

In vivo conductivity of rat liver

16

- Measurement of the conductivity of the complete liver, reveals high increase of the

conductivity on the membranes

Whole-cell

+

-

Patch-clampDetecting conductivity changes|

17

Benefit of patch-clamp in whole cell configuration Patch-clamp |

+ -

++

+

++

---

--

++

+

++

--

-

--

+

++++

++++

++ +

++

--

-

--- -

-

--

-

-

Open FieldWhole cell

Versus

- TMV is directly imposed and can be measured- Current through the membrane can be recorded- Homogeneous changes over the whole membrane surface

=> averaging possible

18

Current-voltage relationPatch-clamp |

VCLAMP

IM IM

RM

CM

VCLAMP

IM

Physiological Response Supraphysiological Response

VCLAMP

160 mV

240 mV

Wegner, Frey, Silve, Biophys J., 2015 19

Threshold for conductivity IncreasePatch-clamp |

Current-voltage relations - 10 ms pulses - DC3F cell

-300 -200 -100 0 100 200 300

-6

-4

-2

0

2

4

6

Curr

ent

(nA

)

Trans-membrane voltage difference (mV)

89 nS

1.7 nS

41 nS

190 mV

-235 mV

DC-3F BY-2 protoplast*

Thresholdpotential (mV)

Depolarisation +201 ± 7 (n=18) +205 ± 7 (n=9)

Hyperpolarisation -231 ± 8 (n=18) +273 ± 7 (n=9)

*data from Wegner et al. (2011)

1rst pulse sequence 2nd pulse sequence

Wegner, Frey, Silve, Biophys J., 2015 20

Relaxation of the high conductivity statePatch-clamp |

10 ms

1 nA

0.1 nA

10 ms

146 mV

360 mV

21

Two phenomenon involved ?Patch-clamp |

22

t=0

-100 0 100

-1500

-1000

-500

0

500

1000

Cu

rren

t (p

A)

Trans-membrane voltage (mV)

prepulse

postpulse 0.775 nS/pF

0.042 nS/pF

- 1:28 + 0:57 +1:41 + 4:56 + 7:29 + 8:53

Voltage pulse

Current response

100 mV

2 ms

20 nA

2 ms

6000 7000 80006000 7000 8000

PI uptake

Correlation with PI uptakePatch-clamp |

23

- PEF induce an increase of membranes conductivity

- This conductivity increase can be detected on lipid bilayer, on single cells and on biological tissue

- Relaxation of the high conductivity states is fast (10-100 ms). Much faster than the resealing of the permeable state

- Some experimental data show after relaxation of the high conductivity state, a persistent low conductivity increase.

24

Pores or not ?

Krassowska and Weaver

Basis of the models

- Pores described using the free-energy of membrane- Pore density obtained using Smoluchowski’s equation

Some aspects still under discussion :

- Size of the predicted pores vs size of molecules transported

- Predicted Life time of pores vs observed life time of permeabilization

- Cumulative effects of pulses and effect of repetition rate

25

Theoretical predictionsPores or not ?

Experimental evidencesPores or not ?

Sengel and Wallace, PNAS, 2016

Tieleman et al, J.AM. CHEM. SOC. 2003

go to : S10-1 and PA-51

BUT … those defects reseal immediately when the voltage on the membrane is removed … 26

𝜕Ω

Traditional models based of description of pores

Pore density Conductivity Sm Permeability

Mathematical approachA two step model |

27

𝜕Ω Two variables to describe the state of the membrane

Sm / Conductivity Pm / Permeability


Conductivity Sm Permeability


New modelling approach

Pore density

Leguèbe, Silve, Mir, Poignard, J. Theor. Biol, 2014 (360:83-94) 27

𝜕Ω Two variables to describe the state of the membrane

Sm / Conductivity Pm / Permeability


Conductivity Sm Permeability

Two phenomenon induced by external electric field

X1 / Something fast (short dynamics)X2 / Something else (?)


New modelling approach

Pore density

Leguèbe, Silve, Mir, Poignard, J. Theor. Biol, 2014 (360:83-94) 27

𝜕Ω


Something ???- Only during the application of

pulse- High conductivity increase

Something else ????- Long lasting phenomenon- Little contribution to

conductivity

Pulse

S0

X1(t,s)S1

X2(t,s)S2

t

Sm

28

𝜕Ω


Contribution of pores ???- Only during the application of

pulse - High conductivity increase

Something else ????- Long lasting phenomenon- Little contribution to

conductivity

Pulse

S0

X1(t,s)S1

X2(t,s)S2

t

Sm

28

Lipid oxidation ?Pores or not ? |

Breton, Silve and Mir, under revision 29

X1

X2

- Need for additional proofsS07-5 (Gailliegue) S09-5 (Mir)

- Need for calibration (E, t, N etc. …)

Voltage Sensitive Dyes

50 100 150 200 250 300

50

100

150

200

250

300

50 100 150 200 250 300

50

100

150

200

250

300

+ -E

sulfonate

headgroup

chromophore

Annine 6

The moleculeVoltage Sensitive Dyes

Fluo Intensity

TMV

- RH-292 (Grinvald 1982, Kinosita 1988, Hibidino 1991, Hibidino 1993)

- di-4-ANEPPS (Gross 1986, Ehrenberg 1987, Lejewska 1989)

- di-8-ANEPPS (Gross 1994, Zhang 1998, Pucihar 2006)

- ANNINE-6 (Frey 2006, Flickinger 2010, Berghoefer 2012)30

Principle of experimentsVoltage Sensitive Dyes

Electric pulse

Laser pulse

t

∆t

TlaserTpulse

F0 F

+ -Before pulse During pulse

- The fluorescence change F/F0 give you information on transmembrane voltage value

- Temporal resolution : Tlaser ~ 5 ns

- To obtain images at different time during an electric pulse, ∆t can be modified

31

Example of resultsVoltage Sensitive Dyes

1µs

32

Analysis, using the « Local equivalent electric field »Voltage Sensitive Dyes

1µs

𝐶𝑚𝜕𝑉

𝜕𝑡+

2𝜎𝑒𝜎𝑖𝑟𝑐 𝜎𝑖 + 2𝜎𝑒

+ 𝑆𝑚 𝑉 =3𝜎𝑒𝜎𝑖

𝜎𝑖 + 2𝜎𝑒𝐸𝑒𝑥𝑡𝑠𝑖𝑛 𝜃

Eext.sinθ

32

Voltage Sensitive Dyes

1µs

Eext.sinθ

Analysis, using the « Local equivalent electric field »

32

𝑉1 =2𝜎𝑒𝜎𝑖

𝑆𝑚𝑟𝑐 2𝜎𝑒 + 𝜎𝑖 + 2𝜎𝑒𝜎𝑖𝑉0

𝑉0 =3

2𝑟𝑐𝐸𝑠𝑖𝑛𝜃

Quasi-static approximation

Voltage Sensitive Dyes What about surface conductance ?

33

𝑉1 =2𝜎𝑒𝜎𝑖

𝑆𝑚𝑟𝑐 2𝜎𝑒 + 𝜎𝑖 + 2𝜎𝑒𝜎𝑖𝑉0

𝑉0 =3

2𝑟𝑐𝐸𝑠𝑖𝑛𝜃

Quasi-static approximation

Voltage Sensitive Dyes What about surface conductance ?

0 50 100 15060

90

120

150

ER [kV

.m-1]

tR [s]

Model ExpDec1

Equation y = A1*exp(-x/t1) + y0

Reduced Chi-Sqr

0.18657

Adj. R-Square 0.96947

Value Standard Error

Field of rupture

y0 75.48881 8.43636

A1 58.42325 7.55108

t1 49.19312 18.9021

k 0.02033 0.00781

tau 34.09807 13.10194

Model Allometric1

Equation y = a*x^b

Reduced Chi-Sqr

0.17384


Value

Field of rupturea 167.39671

b -0.14659

Model Log3P1

Equation y = a - b*ln(x+c)

Reduced Chi-Sqr

0.09014


Value

Field of rupture

a 175.9346

b 19.86705

c 5.23059

Pulse parameters that generate the same conductivity increase of the membrane

= Pulse parameters inducing the same pore density ?

33

The applications

What kind of applications ?

Lluis MirWolfgang Frey

Medical applications

Industrial applications

34

Example of clinical trial:

- Efficient - No side effect- Local treatment- Treatment specific to cancerous cells- Cheap- No hospitalization is necessary- Simple procedure- Structures like vessels or nerves are preserved

Benefits of the method :

CR : Complete Response, PR : Partial response (↓ volume >50%), NC : No significant change, PD : Progression

From ESOPE European clinical trial

Response after a single treatment(total 171 nodules)

CR PR NC

NC

0

20

40

60

80

%

73.7

11.1 10.54.7

Permeabilisation of the cell

Penetration of anticancerous drugs

Medical Application / Electrochemotherapy

Marty et al (2006)

35

Medical Application / Electrochemotherapy

Pulse voltage: 100 bis 1000 VoltsNumber of pulses: 1 à 20Duration of pulses: 100 μsRepetition frequency: 1 à 5000 Hz

Electrodes for medical application

Design and Realization of the “Cliniporator” (IGEA – 2004)

Countries Centers

Italy 41

Germany 46

Great Britain 16

France 10

Spain 6

Total EUROPA 139

3 000 in 2013

~11 000 by end

of 2014

Treated PatientECT Centers in Europe

36

Applications in the food industry Sugar industry

37

2 µs - 500 kV/m - 5 kJ/kg


Throughput: ~ 10 t / h

Sack M, Schultheiss C, and Bluhm H (2005) . IEEE TIA, Vol. 41, No 3, May-June 2005, pp. 725-733. Dr. Martin SACK (KIT, IHM) 38

2 µs - 500 kV/m - 5 kJ/kg

Dr. Martin SACK (KIT, IHM)


Advantages:

• Better extraction of juice enables a decrease of the extraction temperature

•Better water extraction in the pulp press saves evaporation energy for drying

• Over all up to 30 % less energy required

Throughput: ~ 10 t / h

39

Applications in the food industry PEF treatment of crushed grapes

5 µm 5 µm

Microscopic view of peel tissue from Lemberger grapes

before and after PEF-treatment

Advantages:

Improved extraction

Fast processing

No use of enzymes

Schmidt O, Schick A, Sack M, Sigler J, Das Deutsche Weinmagazin, 6 / 24. März 2012, S. 32-38.

Electroporation device “KEA-WEIN”

40

Future applications … Bioeconomy Microalgae, a concentrate of valuable components

light

lipidsoligo/polysaccharides

- alginate

- agar

- carrageenan

- glucans

lipids, fatty acids:

primary target for energetic use

Biomass

H2O CO2

nutrients

pigments

proteins

O2

Adapted from I. Hariskos, C. Posten: Biotechnology Journal, 2014, 9 (with permission)

colorants, antioxidants,

vitamins:

- astaxanthin,

- ß-carotene

Two main challenges :- Reduction of cultivation costs- Extraction

Many potential Market - Food industry- Feed- Pharmaceutical industry- Biofuel

41



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effect of pulsed electric fields on biological cells: adding some...

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