Lecture 30, April 17, 2019 ECEN 5341,4341 Electroporation . Frank Barnes

Background Electroporation Chapter 7 Vol. 1 1. The use of short high voltage pulses to punch holes in membrane barriers. 2. Multiple uses including ablation of cancer and inserting molecules in cells. 3. Breaking other biological barriers such as skin.

. Typical Parameters. 1. Creating small holes in membranes with short electrical pulses. Typical pulses are 1µs< τ< 10ms and the required membrane voltages are between 0.2V < Um <1V 2. The membrane concentrates the electric field as it has a low conductivity σ = 10-7 S/m and low dielectric εr ≈2 -4 constant compared to the inside of a cell and the fluid around it. σ = 1S/m, and εr=80

Typical Parameter 3. High voltage to reach up to 200kV/cm and pulses as short as 6ns for tissue segments. 4. Need frequency components less than f<300MHz to concentrate the fields on the membranes. 5. Characteristic charging times for cell membranes are about 1µs

Calculations on the energy to move charges through a membrane. Born energy is the energy to move from the fluid to the center of the membrane Na+ 2rs=0.4nm Membrane thickness d = 5nm

Born Energy The forces are largely local so that Where Km is the relative dielectric constant of the membrane and Kw is that of water Spontaneous crossing of large molecules negligible For rise times faster than moving charges or or f>300 MHz distributed E field For slower rise times most of the voltage is across the membranes The field strength E = 0.1 to 1kV/cm and 10kV/cm for bacteria to get 0.2 to 1.5V in 1μs to 10ms. Models Parallel plate capacitor where Re = resistance of the electrolyte and Cm = membrane capacity The E field across the membrane depends on the length of the pulse

An Electroporation System Gene Pulser Xcell™ Electroporation Systems

Another Electroporation Device 1

Specifications Gene Pulser Xcell™ Total System Includes main unit, CE module, PC module, and ShockPod™ cuvette chamber   Outputs Waveform: Exponential or square Voltage: 10–3,000 V Capacitance 10–500 V, 25–3,275 µF in 25 µF increments 500–3,000 V, 10, 25, 50 µF Resistance (parallel) 50–1,000 Ω in 50 Ω increments, plus infinity Sample resistance 20 Ω minimum at 10–2,500 V 600 Ω minimum at 2,500–3,000 V Square-wave timing  10–500 V: 0.05–10 ms in 0.05 ms increments, 10–100 ms pulse in 1 ms increments, 1–10 pulses, 0.1–10 sec interval 500–3,000 V: 0.05–5 ms in 0.05 ms increments, 1–2 pulses, 5 sec minimum interval

Pulse Characteristics 1. Creating a small hole in the membrane. 2. Pulses 0.2V<V< 1V 1µs<τ<50ms E Membrane εm =2-4εo Cell interior εi =50-80εo

Pulse Characteristics 1. Times greater than 1µs required to concentrate the charges and fields. 2. Fields greater than 0.1KV/cm often greater than 1KV/cm. Field varies with position on the cell surface. 3. High fields and short pulses give the best results

Typical Pulse

Fields for a Spherical Cell

Pore Formation 4 Stages 1. Charging the membrane 2. Constant Voltage Vm , small currents 3. Fluctuating current as transition to long lived excited state,10minutes large σ, low V 4. Increase in pulse length leads to saturation and irreversible damage. log10 τm ~ Vm

Some Theory for Pore Formation

Pore Formation Dynamics

Ion Transport Through a Pore

Some Theories for Electroporation 1. Hydrodynamic Flow 2. Compression 3. Pores that lead to membrane weakness that is local. 4. Pore formation becomes significant from thermal processes plus applied field when Um >~ 0.5 V

A Molecular Dynamics Simulation

Reversible Pore Formation 1. Vm≈ 1V τm≈ 400ns rp≈ 0.8nm 2. R drops by 109 3. Rapid discharge and the membrane reforms. 4. Relaxation times for a fluid τf=εf/σf for saline τ f τ≈ 0.5ns much less than the charging time Vm 50µs 0.4

Energy 1. Energy of a charge in the medium The essential barrier function of cell membranes can be represented by a thin sheet of lipid. To move a charge through the membrane

Forces 1. E Fields on the Membranes are a function of time. Increase and then Decrease 2. Force the opening in the pores that expand with time. 3. E fields drive currents and carry along neutral molecules.

Membrane Recovery 1. Reported times vary from nanoseconds to minutes or hours. 2. Strongly depends on Temperature 3. Depends on the size of the pore also on what molecules or ions are being transported.

Cell Stress And Survival 1. Cell survival and stress are mainly to exchange of molecules with the environment. Chemical or ion imbalances. 2. Cells can be killed without significant heating. 3. There is a fuzzy threshold for the transport of molecules into or out of the cells and not a large margin to cell death. 4. Transport is not very selective with respect to molecules or ions. 5. Survival seems to go with the ratio of the external volume to the internal volume of the cell. In vitro Vex/Vin is large and favors cell death In vivo it is the reverse with Vex/Vin≈0.15 6. This mean that in vivo cell damage for the same pulses are less likely.

Tissue Electroporation and In Vivo Delivery 1 A purposeful electroporation of tissue in vivo and in vitro has been motivated by therapeutic interventions such as tumor treatment by delivery of anticancer drugs ,gene therapy by delivery of DNA, and other genetic material and delivery of various sized molecules into and across the skin 2. Also tissue electroporation may be relevant to neuromuscular incapacitation (stunning) pulses

Voltage Concentration in Tissue 1. Voltage concentration in tissue needs to be across the membranes. 2. For preferential electroporation, two features should be sought: (I) tissue barriers comprised mainly of lipids (ii) mechanical deformability (compliance) of membranes comprise of the particular lipids so that the electrostatically favored entry of water into a deformable phospholipid-based membrane results in the creation of aqueous pathways.

Current Flows in Tissue. 1. At low fields most of the current flows around the cell membranes. 2. After electroporation much of the current flows through the cell. 3 Tumor tissue is an important example of tissue for which many cells have intercellular aqueous pathways. Even without electroporation there is a significant physiological resistance to entry of anticancer drugs because of limited blood perfusion, elevated interstitial pressure, and relatively large distances to blood vessels

Application to Tumors. 4. Local tissue electroporation should create aqueous pathways that assist drug movement and that may also relieve pressure, but the fourth power dependence of volumetric flow on pathway size implies that significant water flow may be more difficult than diffusion and drift of small drugs.

Applications 1. Electroporation has value largely in cancer treatment for drugs that to not go through the membranes naturally. Bleomycin is an example where electroporation helps. 2. Transdermal Drug delivery through skin. The stratum corneum is the main barrier plus sweat ducts and hair follicles. 3. The double cell lining of sweat ducts should experience electroporation at about Ubarrier =2 to 4 V, but the approximately 100 bilayers of the SC need Ubarrier =50 to 100V for pulses with duration of 100 µs to 1 ms, i.e., about 0.5 to 1V per lipid bilayer

Skin Experiments of this type with human skin show that if exponential pulses with Voltage across SC,0 50 to 300V and time constant, t, pulse 1 ms are applied every 5 s for 1 h, then there is an enhancement by up to a factor of 104 in the flux of charged molecules of up to about 1 kDa Companion electrical impedance measurements show a rapid (25 ms) decrease in skin resistance and both molecular flux and electrical measurements show that either reversible or irreversible behavior occurs, depending on the transdermal pulse amplitude, Voltage SC, 0. Several in vivo experiments show that transdermal delivery can be achieved with minimal damage.

Gene Therapy 1. Gene therapy also requires movement of large molecules through the cell walls. 2. You only need succeed with some of the cells to be effective. Smaller longer pulses. 3. Concerns about damage. 4. Surfactants can improve membrane recovery.

Electroporation of Organelles 1 Short high field pulses (ns) Um = 1.2V E= 106V/m 2. Many small pores in outer membrane as well as in the organelle