effect of pulsed electric fields on biological cells: adding some...
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KIT – University of the State of Baden-Wuerttemberg and National Research Center of the Helmholtz Association
Institute for Pulsed Power and Microwave Technology (IHM)
www.kit.edu
Effect of pulsed electric fields on biological cells: adding some pieces to the large puzzle
Aude SilveI
KIT – University of the State of Baden-Wuerttemberg and National Research Center of the Helmholtz Association
Institute for Pulsed Power and Microwave Technology (IHM)
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
Three main observations categoriesWhat can be detected ? |
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
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
Three main observations categoriesWhat can be detected ? |
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
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
Traditional models based of description of pores
Conductivity Sm Permeability
Mathematical approachA two step model |
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
Traditional models based of description of pores
Conductivity Sm Permeability
Two phenomenon induced by external electric field
X1 / Something fast (short dynamics)X2 / Something else (?)
Mathematical approachA two step model |
New modelling approach
Pore density
Leguèbe, Silve, Mir, Poignard, J. Theor. Biol, 2014 (360:83-94) 27
𝜕Ω
Mathematical approachA two step model |
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
Mathematical approachA two step model |
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
Adj. R-Square 0.97155
Value
Field of rupturea 167.39671
b -0.14659
Model Log3P1
Equation y = a - b*ln(x+c)
Reduced Chi-Sqr
0.09014
Adj. R-Square 0.98525
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
Applications in the food industry Sugar industry
37
2 µs - 500 kV/m - 5 kJ/kg
Applications in the food industry Sugar industry
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)
Applications in the food industry Sugar industry
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
KIT – University of the State of Baden-Wuerttemberg and National Research Center of the Helmholtz Association
Institute for Pulsed Power and Microwave Technology (IHM)
www.kit.edu
Thanks to …