The Study of Cells Treated by Electroporation

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When an electric field is applied to living cells, their membrane suffers electrical stretch forces. Depending on the electric field amplitude and duration, their cell shape is deformed and free ions can be transported across the membrane. There are also additional osmotic forces and water leakage through the pore(s) formed. Pores are created when the potential difference across the cell membrane is > 1 volt (Coster and Zimmerman, 1).

Depending on the severity of the membrane deformation, if the pores formed are less than a ‘critical diameter’, the cell’s lipid-free energy mobility can reseal the pore and return normality. But, failure to reseal when the pore exceeds the critical diameter, leads to runaway membrane breakdown. This raises questions:

• How can we detect critical membrane breakdown due to electroporation?

• Under what electric pulse conditions will the pore exceed the critical diameter and cause cell rupture?

• How can the electromechanical processes be identified and quantified?

Since blood can be readily obtained from donors for research purposes, red blood cells (referred to as RBCs or erythrocytes) are commonly used to study membrane electric field effects. This involves obtaining detailed information on the inter-relationships between the electrical and physical parameters for:

• electric field strength;
• the voltage applied across the membrane;
• pulse duration; and
• the number of pulses applied within a very short time.

It’s now recognised that the inter-relationship of these parameters differ with cell type and condition. Whether this is true for normal and cancerous cells is yet to be conclusively demonstrated.

Background

In the late ’80s and early ’90s, a considerable amount of biophysics research was devoted to the study of electric fields (now referred to as electroporation) applied to artificial membranes. Some theoretical models and postulates were published to explain the mechanism of electrical membrane breakdown (see Coster and Zimmerman, 1). The model only considered a very simple bi-membrane structure. It did not describe a true living cell that has quite a complex lipid/protein-membrane structure.

By 1990, electroporation research expanded and focused on optimising chemotherapeutic cell loading. Research fields such as electrochemotherapy (ECT) and electrogene transfer (EGT) were established as new specialist medical applications.

Electroporation of Cancer Cells

In 2005, Davalos and Rubinsky were the first to suggest using electroporation as a form of treatment for destroying targeted cancers [4]. They referred to this process as “irreversible electroporation (IRE)”. In contrast to previous reversible electroporation work, IRE is designed to permanently disrupt cell functions and ‘kill’ the cells when they are electrically treated. 

The thesis work completed in 1990 by Oliver (2) and parts of this work published in 2003 (Oliver and Coster (3)) identified the processes of membrane electrical breakdown. The results are relevant to the 2005 cancer ablation method suggested by Davalos and Rubinsky.

Although the original Oliver and Coster research work was aimed to optimise cellular uptake of chemotherapy drugs, the results provide an understanding of the electromechanical processes leading to reversible electroporation (RE), irreversible electroporation (IRE) and, what is described here as, critical membrane electroporation (CME).

CME is defined as a critical end-point when the membrane breakdown process leads to cell lysis. It’s an end-point for cell death.

Since the Davalos et al paper was published in 2005, many others have extensively researched IRE for cancer therapy. Many clinical trials for the surgical treatment of prostate, pancreas and liver cancers has since followed.

But, despite all the research so far published, the IRE electrical parameters for optimum cancer kill have not been established. Nor is there an adequate explanation of the processes leading to cancer cell death.

The Fundamentals

As described in a previous betterhealthcaretechnology.org article (Cell Membranes – The Beginning of Life), the cell membrane has a lipid molecular structure with a ‘head’ and ‘two legs’ (shown in Figure 1 ). Locked together side-by-side, the lipids form a bilayer chain with extensive elastic properties.

The RBC presents a unique cell for studying the structure and function of the plasma membrane. The RBC looks like a membrane sac or bag which encloses the haemoglobin. There’s an intricate structural relationship between the haemoglobin, membrane proteins and membrane lipids which give the cell excellent flexibility (or plasticity).

Red blood cells have a life span ranging from 100 to 120 days. The cells destroyed in circulation each day are replaced by new cells released from the bone marrow. Therefore, there is a normal distribution of spread in the size, age and condition of the red cells in circulation. Physical measurements of a normal RBC population, gives an average result for the young and old cells in circulation.

How does an electric field produce a membrane pore?

During the very early cell membrane investigations, researchers debated the fundamental physics of how the electric field produced the membrane pore in the cell.

Did the electric force:

– compress the lipid bilayer to eventually ‘punch’ a hole in it?

or

– did the electric field laterally stretch the membrane and eventually part the lipid chain to produce a membrane pore?

In either case, the electric pulse must impart sufficient energy to stretch and ‘deform’ the cell shape which can cause the lipids to separate and form a membrane pore. If the membrane is able to reseal the pore after the electric pulse is applied, then it is referred to as reversible electroporation (RE). If not, it is an irreversible electroporation (IRE) state.

Electromechanical Properties of RBCs

Oliver succeeded in developing a technique that could identify the electric parameters of voltage amplitude and pulse length (or duration) that can cause a fatal pore rupture and cell death within minutes. This was referred to as ‘critical membrane electroporation (CME)’. For information on the experimental method and measurements, click on:

Electrical breakdown of human erthrocytes

The Perspex RBC sample holder is shown in Figure 2. assembled (top) and dis-assembled (bottom). If the RBCs, suspended in the sample holder, receive a pulse of sufficient electric field strength to make a pore large enough for the haemoglobin to leak out of the cytosol and into the supernatant, the resealed intact RBC membrane becomes a transparent ‘ghost’ cell.

The haemoglobin leakage from the RBC mixes as a red solution and is measured as an increase in relative red light intensity. This method is ideal for studying the RBC cells’ continual condition from the time the pulse is applied, to when the cells change size and shape, to when membrane pores are formed.

As far as the author is aware, no other publication has described a suitable method that can:

(i) measure and record electroporation breakdown processes immediately after applying an electric pulse of known amplitude and duration; and

(ii) quantify the RBC’s physical properties under electromechanical forces.

Haemolytic Curves

(i) Varying sample voltage with a fixed pulse duration

Figure 3 (a) shows a family of haemolytic curves recorded over a period of 30 minutes after applying the electric pulse (Oliver 1,2). Each RBC sample was treated with a 5uS square-wave pulse ranging from 440 to 640V amplitude and between electrodes separated 1.9mm (2.2 – 3.2 kV/cm electric field strength).

CME for cell lysis was found to be 570V sample voltage which equates to 2.85kV/cm electric field strength.

Based on their published evidence, Oliver and Coster postulated how the RBCs changed in state after receiving an electrical treatment from 0 to 3kV/cm field strength. To read more on this RBC electromechanical breakdown postulation, click on:

A Postulate Describing Cellular Electromechanical Breakdown by Electroporation

When the suspended blood cells receive a 5µS square wave pulse and increased voltage, important effects identified were:

(a) < 2kV/cm field strength

For less than 2 kV/cm electric field strength, RBC pores are not readily formed because the potential difference across the cells’ membrane is less than the required 1 volt to produce electroporation.

(b) 2.2 to 2.8kV/cm field strength

For a field strength of 2.2 to 2.8kV/cm, pores are formed causing water leakage and osmotic imbalance. But the K⁺ – Na⁺, ATP protein ‘pump’ system is able to ‘fix’ the osmotic imbalance whilst the membrane reseals the pores created.

(c) 3kV/cm field strength

Larger diameter pores are created producing excessive membrane stress. The protein osmotic pump can no longer reinstate the cells’ normal osmotic pressure. Failure to reseal membrane pore leads to haemoglobin leakage before the cell reseals to form ghost cells.

(d) >3kV/cm field strength

For greater than 3kV/cm, the protein pump fails to repair the osmotic imbalance with very few RBCs returning to normality. An excessive number of ghost cells are produced. Hydrolysis also occurs, producing hydrogen bubbles in the blood sample and its resistance decreases significantly.

The production of hydrolysis during surgery produces a similar effect. Surgeons are well aware of this high voltage limitation when carrying out electroporation for prostate cancer patients. Too many pulses applied too frequently at high voltage produces a current that can cause excessive tissue hydrolysis in the patient. Large spark-overs can then occur between the electrodes due to the hydrogen gas collected in the tissue. The effective electrical resistance between the two implant needles is substantially lower and the current exceeds an allowable maximum due to the hydrolysis.

(ii) Varying the Pulse duration with fixed sample voltage

Figure 4 demonstrates a similar result as the fixed pulse duration haemolysis test of Figure 3. This time, the pulse duration is varied and the voltage is fixed.

The 30-minute recording of relative red-light intensity for the blood samples treated with a constant 600V electric pulse and a range of 10, 20, 40, 60, 80 and 100uS pulse duration. It’s a similar result to Figure 3 (a).

Identifying Electric membrane Breakdown

For a specific pulse duration and pulse voltage, the haemolytic curves, Figure 3 and 4 do not provide an easy method for quantifying pore formation. This is particularly so for detecting the rate and magnitude of electroporation when it suddenly increases rapidly. This point of inflexion is when critical membrane electroporation occurs.

However, there is an intermediate state when cells rupture, lose their nucleus and reseal as ghost cells. This could be referred to as reversible electroporation, RE but the cells are no longer biologically viable. For other more specialised anatomical cells, this is described as cell death by apoptosis.

Finally, the presence of cell fragments for pulses > 3 kV/cm, the cells suffer more violent membrane breakdown with irreversible membrane damage and cell death (critical membrane electroporation, CME).

Quantifying Critical Membrane Electroporation

To quantify CME, haemolysis at 20 minutes after the pulse is applied were evaluated. Measurements with a 20-minute delay enabled all electrical after-effects to have completed. Relative haemolysis at 20-minutes (taken from Figures 3 and 4) was re-plotted for 3 separate experiments. Figure 5 shows the 20-minute result for samples treated with a 5uS pulse and a voltage ranging from 320V to 640V.

By linearly interpolating a line through the 20-minute haemolysis for (i) lower voltages, 320V to 560V and (ii) higher voltages, 560V to 640V, Figure 5 shows the two lines intercepting at a critical 570V voltage (electric field strength of 2.85 kV/cm) and indicates by this sharp inflexion in relative light intensity, the beginnings of CME total membrane breakdown processes.

Conversely, for samples receiving a constant 600V (3 kV/cm electric field strength), Figure 6 shows a critical pulse duration of 60µS

Relationship of Voltage and Pulse Duration for CME

A plot of the critical voltage and pulse duration for CME (Figure 7.) shows a linear relationship and each normal blood donor was found to have a unique linear relationship.

Other Observations

1. No correlation to the donor’s biochemical blood results was found for their unique CME critical voltage and pulse duration. Substances, such as cholesterol and potassium levels, do not affect CME.

2. The blood from a male donor requires more electrical energy to produce CME than the blood of a female donor (Figure 8).

This infers that the elastic properties of the erythrocyte membrane/protein/cytoskeleton of male blood are different in tensile strength to female blood.

Physical Properties of Cell Membranes

All cells have a cytoskeleton attached to the underside of the membrane’s lipid/protein structure. The cytoskeleton gives the cell membrane extra strength to (I) resist external electric forces from the applied pulse and (ii) internal osmotic forces that come from the RBC pore(s) created by the electric pulse.

Critical membrane breakdown occurs when the sum of the external electric forces and the internal osmotic forces exceed its elastic resistance. When the pore in the membrane exceeds a critical diameter, runaway breakdown and cell lysis (CME) occurs.

The Hypothesis

1. The electromechanical properties of the cell are dependent on:

• the membrane/protein/cytoskeleton structure: and
• the electric pulse’s duration and amplitude.

2. The elastic properties of the membrane/protein/cytoskeleton structure vary with pulse duration and amplitude.

3.  CME (the elastic limit) is dependent on the physical properties of the membrane/protein/cytoskeleton structure.

4. It is thought that the critical elastic limit occurs when the membrane fractures the cytoskeleton at the ankyrin/spectrin site. 

Figure 8. shows the critical pulse amplitude versus duration causing CME for a range of patients with different blood conditions. The result for an average normal donor is substantially different to the blood of a patient diagnosed as having alcoholic hepatitis and a cancer patient receiving cyclosporin chemotherapy. Differences in these results are assumed to be due to variations in the RBC membrane’s elasticity.

Membrane Physical Properties

For those wishing to consider in-depth the theoretical relationship between membrane electrical breakdown and the cell’s physical properties, click on Physical Properties of an RBC Membrane.

Membrane Elasticity

When the cell’s membrane ruptures, the model assumes:

1. The total kinetic energy (E_KE) can be derived from the critical pulse voltage and duration;
2. there’s a critical level of cell membrane deformation when cell lysis occurs after electroporation; and
3. the external electric field intensity (E_c) applied across the electrodes is proportional to the potential difference induced across the cell’s membrane (E_m).

The model assumes that the ‘Deformation Energy’ required to stretch the cell membrane to a critical cell rupture point is produced by a critical pressure (P_c) after stretching to a critical volume (Vol_c). There’s a proportional relationship between deformation energy D_KE to the electrical energy E_KE necessary for cell lysis.

Figure 9. graphs the relative E_KE versus sample voltage for the blood of normal male, normal female, alcohol hepatitis and cyclosporin donors. The variation of  E_KE for each sample is shown across the voltage range from 300V to 800V (1500 to 4000V/cm electric field strength). 

As expected, the higher voltage and shorter pulse duration for the cell lysis endpoint is around 700V. This infers the elastic limit for alcoholic hepatitis and cyclosporin patients is much less than for normal donors (740V female and 800V male donor).

But, conversely, the blood of a cyclosporin treated patient exhibited maximum membrane elastic strength at 440V (2.2 kV/cm) and 500V (2.5 kV/cm) for the hepatitis patient and female donor. Whereas the male donor maximum elastic strength is 560V (2.8 kV/cm).

The inferred stronger cell membrane of the male donor (as compared to the female donor) and the much higher kinetic energy requirement for cell lysis for the cyclosporin patient’s blood, needs further research to understand the biological complexities causing the variations in cell membrane mechanical strength and elasticity.

The voltage pulse, expressed as a function of pulse duration causing critical cell lysis, is approximately linear (Figure 7 and 8). 

Unfortunately, the experimental range for these results had insufficient data to support the linear assumption when the pulse width approaches zero. It could be linear or parabolic for pulses < 5µS duration. Nevertheless, the sample’s voltage extrapolated to zero pulse duration is a tempting parameter to use for the existing range investigated.

The results can be used to identify fundamental mechanical properties of the membrane/protein/cytoskeleton structure. Extrapolation of the line-of-best-fit in Figure 8., shows an imaginary voltage for zero pulse duration (E_Inst). The hypothesis suggests that this could possibly be due to cytoskeleton damage when the pulse duration, E_Inst, is infinitely small. Normal and abnormal blood samples can be compared using this method.

To investigate the elastic properties of the membrane/protein/cytoskeleton in different blood samples, the slope of the line-of-best-fit (K = volts/microseconds) and E_Inst are suitable parameters to compare.

The results found wide differences in the average voltage causing cytoskeleton damage:

• female blood cells:                 = 740V and a K slope of -1.9V/µS
• cyclosporin treated blood:    = 673V and a  K slope of -1.07 V/µS
• alcoholic hepatitis blood:      = 703V, -1.73 V/µS), are quite different results. 

Bulk Modulus Properties

Using the electric properties for cell lysis, the slope of the line (K = ∆E_c/∆τ_c) and E_Inst in Figure 8, the bulk modulus properties can be investigated.

A comparative study of the characteristic cell lysis line of best fit in these results provides a convenient method to investigate the elastic properties (such as the mechanical stress and elastic limit) of the erythrocyte’s membrane/protein structure attached to its cytoskeleton.

Using data from Figure 9, the Bulk Modulus ratio for normal and abnormal blood samples are shown in Figure 10. When comparing a normal blood sample to an abnormal sample, the relationship is:

E_KE1/E_KE2 = β₁ K₂ / β₂ K₁

A comparison of normal and abnormal blood for  β⁄_K (Figure 10.) shows the blood result of:

(a) normal female versus male donor;

(b) normal versus an alcoholic hepatitis female donor; and

(c) normal versus a cyclosporin male donor.

The cyclosporin blood indicates a much greater β⁄_K in the 300 – 500V (1500 – 2500 V/cm field intensity) range but then decreases significantly after 600V (3000 V/cm).

In two tests at different times, the β⁄_K ratio for the blood from the two normal donors, was unity. Any variations from this value would indicate a change in the deformation constant, K, and/or a change in the membrane bulk modulus, β.

Membrane Properties of Cyclosporin Treated Blood

1. A  β⁄_K ratio greater than one indicates that more work is required to lyse the cyclosporin blood cells.
2. Regardless of the blood under investigation, the current flowing through the sample remains constant for a given sample voltage. So, the additional work required to lyse the more resilient cells containing cyclosporin in the membrane could be achieved by increasing the pulse length.
3. Conversely, less work was necessary for the cyclosporin blood when the sample voltage was >600V (3000 V/cm field intensity) and the β⁄_K ratio became much smaller than unity.
4. For such a high voltage and short pulse length, the excessive deformation effects of the induced intra-membrane potential appeared to reduce the cyclosporin treated blood cell’s ability to remain intact.
5. For 3000 V/cm field intensity, the membrane/protein cytoskeleton elastic properties had changed.

These results show that the physiological state of the cell’s membrane influences the choice of optimum parameters for electroporation treatment. For instance, cancer patients undergoing chemotherapy at the time of surgery may affect the optimum electrical parameters necessary for tumour ablation by electroporation.

Discussion

Although these results are based on electroporation measurements of red blood cells, the information gathered for how:

• the membrane pores are formed;
• the cell recovers from electrical treatment;
• electrical breakdown eventually occurs; and
• critical membrane electroporation leads to cell lysis,

is pertinent information for those involved in this avenue of cell biology research. But, it should be noted that the electrical resistance of red blood cells suspended in normal saline will be slightly different to cells in the patient’s muscle tissue or other structures.

Even though studies have shown that 5µs pulses of approximately 3 kV/cm electric field strength would ablate the treated cells, it’s an impractical method to use in a surgical operation. The ability to accurately insert the electrode needles into the patient’s tumour and ensure adequate electrical safety for the patient and surgical staff are major difficulties to overcome.

Consequently, Davalos³ and Rubinsky⁴ chose to copy the 10 pulses applied at a low voltage previously developed for reversible electroporation. By using a much larger number of 60µs pulses of up to 2 kV/cm field strength, they claimed that the modified reversible electroporation technique could irreversibly kill cancer cells.

This confusing issue in terminology between reversible and irreversible cellular breakdown will be discussed in more detail in the next article.

See Part 2, Non-Thermal Irreversible Electroporation (to be published soon).

References

1. Lyn Oliver, Electromechanical Study of Blood Cells, PhD Thesis (UNSW), 1991. 

2. Lyn D Oliver and Hans G. L. Coster, Electrical breakdown of human erythrocytes: a technique for the study of electro-haemolysis, Bioelectrochemistry, Volume 61, Issues 1–2, October 2003, Pages 9-19

3. R. V. Davalos et al., “Tissue ablation with irreversible electroporation,” Ann. Biomed. Eng., vol. 33, no. 2, pp. 223–231, Feb. 2005.

4. Rubinsky B, Onik G, Mikus P. Irreversible electroporation: a new ablation modality–clinical implications. Technol Cancer Res Treat. 2007;6:37–48. 

5. Claudio Bertacchini, Pier Mauro Margotti, Enrico Bergamini, Andrea Lodi, Mattia Ronchetti and Ruggero Cadossi, Electroporation System for Clinical Use, Technology in Cancer Research and Treatment, Volume 6, Number 4, August 2007.

6. Onik G, Rubinsky B. Irreversible electroporation: first patient experience focal therapy of prostate cancer. In: Rubinsky B, editor. Irreversible electroporation, series in biomedical engineering. Berlin: Chennai Springer-Verlag; 2010. pp. 235–247.

7. Kenneth N. Aycock, BS, and Rafael V. Davalos, PhD Irreversible Electroporation: Background, Theory, and Review of Recent Developments in Clinical Oncology, BIOELECTRICITY, Volume 1, Number 4, 2019 Mary Ann Liebert, Inc. DOI: 10.1089/bioe.2019.0029

Please Note: This is a scientific and technically based article. It is not intended to provide medical advice and is for information only. If you have any health problems or questions related to your health, then please consult your doctor.

Lyn Oliver Am MSc PhD 1 May 2021

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