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
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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
- 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
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
- 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