Long term survival of mice with hepatocellular carcinoma after pulse power ablation with nanosecond pulsed electric fields.

Novel therapies are needed for treating hepatocellular carcinoma (HCC) without recurrence in a single procedure. In this work we evaluated anti-neoplastic effects of a pulse power ablation (PPA) with nanosecond pulsed electric fields (nsPEFs), a non-thermal, non-drug, local, regional method and investigated its molecular mechanisms for hepatocellular carcinoma tumor ablation in vivo. An ectopic tumor model was established using C57BL/6 mice with Hepa1-6 hepatocellular carcinoma cells. Pulses with durations of 30 or 100 ns and fast rise times were delivered by a needle or ring electrode with different electric field strengths (33, 50 and 68 kV/cm), and 900 pulses in three treatment sessions (300 pulses each session) or a single 900 pulse treatment. Treated and control tumor volumes were monitored by ultrasound and apoptosis and angiogenesis markers were evaluated by immunohistochemistry. Seventy five percent of primary hepatocellular carcinoma tumors were eradicated with 900 hundred pulses at 100 ns pulses at 68 kV/cm in a single treatment or in three treatment sessions without recurrence within 9 months. Using quantitative analysis, tumors in treated animals showed nsPEF-mediated nuclear condensation (3 h post-pulse), cell shrinkage (1 h), increases in active executioner caspases (caspase-3 > -7 > -6) and terminal deoxynucleotidyl transferase dUTP nick-end-labeling (1 h) with decreases in vascular endothelial growth factor expression (7d) and micro-vessel density (14d). NsPEF ablation eliminated hepatocellular carcinoma tumors by targeting two therapeutic sites, apoptosis induction and inhibition of angiogenesis, both important cancer hallmarks. These data indicate that PPA with nsPEFs is not limited to treating skin cancers and provide a rationale for continuing to investigate pulse power ablation for hepatocellular carcinoma using other models in pre-clinical applications and ultimately in clinical trials. Based on present treatments for specific HCC stages, it is anticipated that nsPEFs could be substituted for or used in combination with ablation therapies using heat, cold or chemicals.

Synergistic effects of local temperature enhancements on cellular responses in the context of high-intensity, ultrashort electric pulses.

Results of self-consistent analyses of cells show the possibility of temperature increases at membranes in response to a single nanosecond, high-voltage pulse, at least over small sections of the membrane. Molecular Dynamics simulations indicate that such a temperature increase could facilitate poration, which is one example of a bio-process at the plasma membrane. Our study thus suggests that the use of repetitive high-intensity voltage pulses could open up possibilities for a host of synergistic bio-responses involving both thermal and electrically driven phenomena.

Bioelectric effects of intense ultrashort pulses.

Models for electric field interactions with biological cells predict that pulses with durations shorter than the charging time of the outer membrane can affect intracellular structures. Experimental studies in which human cells were exposed to pulsed electric fields of up to 300 kV/cm amplitude, with durations as short as 10 ns, have confirmed this hypothesis. The observed effects include the breaching of intracellular granule membranes without permanent damage to the cell membrane, abrupt rises in intracellular free calcium levels, enhanced expression of genes, cytochrome c release, and electroporation for gene transfer and drug delivery. At increased electric fields, the application of nanosecond pulses induces apoptosis (programmed cell death) in biological cells, an effect that has been shown to reduce the growth of tumors. Possible applications of the intracellular electroeffects are enhancing gene delivery to the nucleus, controlling cell functions that depend on calcium release (causing cell immobilization), and treating tumors. Such nanosecond electrical pulses have been shown to successfully treat melanoma tumors by using needle arrays as pulse delivery systems. Reducing the pulse duration of intense electric field pulses even further into the subnanosecond range will allow for the use of wideband antennas to deliver the electromagnetic fields into tissue with a spatial resolution in the centimeter range. This review carefully examines the above concepts, provides a theoretical basis, and modeling results based on both continuum approaches and atomistic molecular dynamics methods. Relevant experimental data are also presented, and some of the many potential bioengineering applications discussed.

Self-consistent analyses for potential conduction block in nerves by an ultrashort high-intensity electric pulse.

Simulation studies are presented that probe the possibility of using high-field (> 100 kV/cm) , short-duration ( approximately 50 ns) electrical pulses for nonthermal and reversible cessation of biological electrical signaling pathways. This would have obvious applications in neurophysiology, clinical research, neuromuscular stimulation therapies, and even nonlethal bioweapons development. The concept is based on the creation of a sufficiently high density of pores on the nerve membrane by an electric pulse. This modulates membrane conductance and presents an effective "electrical short" to an incident voltage wave traveling across a nerve. Net blocking of action potential propagation can then result. A continuum approach based on the Smoluchowski equation is used to treat electroporation. This is self-consistently coupled with a distributed circuit representation of the nerve dynamics. Our results indicate that poration at a single neural segment would be sufficient to produce an observable, yet reversible, effect.

Simulations of intracellular calcium release dynamics in response to a high-intensity, ultrashort electric pulse.

Numerical simulations for electrically induced, intracellular calcium release from the endoplasmic reticulum are reported. A two-step model is used for self-consistency. Distributed electrical circuit representation coupled with the Smoluchowski equation yields the ER membrane nanoporation for calcium outflow based on a numerical simulation. This is combined with the continuum Li-Rinzel model and drift diffusion for calcium dynamics. Our results are shown to be in agreement with reported calcium release data. A modest increase (rough doubling) of the cellular calcium is predicted in the absence of extra-cellular calcium. In particular, the applied field of 15 kV/cm with 60 ns pulse duration makes for a strong comparison. No oscillations are predicted and the net recovery period of about 5 min are both in agreement with published experimental results. A quantitative explanation for the lack of such oscillatory behavior, based on the density dependent calcium fluxes, is also provided.

High electrical field effects on cell membranes.

Electrical charging of lipid membranes causes electroporation with sharp membrane conductance increases. Several recent observations, especially at very high field strength, are not compatible with the simple electroporation picture. Here we present several relevant experiments on cell electrical responses to very high external voltages. We hypothesize that, not only are aqueous pores created within the lipid membranes, but that nanoscale membrane fragmentation occurs, possibly with micelle formation. This effect would produce conductivity increases beyond simple electroporation and display a relatively fast turn-off with external voltage. In addition, material loss can be expected at the anode side of cells, in agreement with published experimental reports at high fields. Our hypothesis is qualitatively supported by molecular dynamics simulations. Finally, such cellular responses might temporarily inactivate voltage-gated and ion-pump activity, while not necessarily causing cell death. This hypothesis also supports observations on electrofusion.

Microscopic calculations of local lipid membrane permittivities and diffusion coefficients for application to electroporation analyses.

Interaction of electric fields with biological systems has begun to receive considerable attention for applications that include field-assisted drug delivery, medical interventions, and genetic engineering. External fields induce the strongest effects at membranes with electroporation being a common feature. Membrane transport in this context of poration is often based on continuum approaches utilizing macroscopic parameters such as the permittivity, diffusion coefficients, and mobilities. In such modeling, field dependences, local inhomogeneities, and microscopic details are usually ignored. Here, a molecular dynamics (MD) scheme is used for a more rigorous and physically realistic evaluation of such parameters for potential application to electroporative transport model development. A suitable membrane structure containing a nanopore derived from MD analysis is used as the initial geometric configuration. Both static and frequency dependent diffusion coefficients have been evaluated. Permittivities are also calculated and shown to be dramatically non-uniform in the vicinity of membranes under high external fields. A positive feedback mechanism leading to enhanced membrane fields is discussed.

Plasma membrane voltage changes during nanosecond pulsed electric field exposure.

The change in the membrane potential of Jurkat cells in response to nanosecond pulsed electric fields was studied for pulses with a duration of 60 ns and maximum field strengths of approximately 100 kV/cm (100 V/cell diameter). Membranes of Jurkat cells were stained with a fast voltage-sensitive dye, ANNINE-6, which has a subnanosecond voltage response time. A temporal resolution of 5 ns was achieved by the excitation of this dye with a tunable laser pulse. The laser pulse was synchronized with the applied electric field to record images at times before, during, and after exposure. When exposing the Jurkat cells to a pulse, the voltage across the membrane at the anodic pole of the cell reached values of 1.6 V after 15 ns, almost twice the voltage level generally required for electroporation. Voltages across the membrane on the side facing the cathode reached values of only 0.6 V in the same time period, indicating a strong asymmetry in conduction mechanisms in the membranes of the two opposite cell hemispheres. This small voltage drop of 0.6-1.6 V across the plasma membrane demonstrates that nearly the entire imposed electric field of 10 V/mum penetrates into the interior of the cell and every organelle.

Simulations of nanopore formation and phosphatidylserine externalization in lipid membranes subjected to a high-intensity, ultrashort electric pulse.

A combined MD simulator and time dependent Laplace solver are used to analyze the electrically driven phosphatidylserine externalization process in cells. Time dependent details of nanopore formation at cell membranes in response to a high-intensity (100 kV/cm), ultrashort (10 ns) electric pulse are also probed. Our results show that nanosized pores could typically be formed within about 5 ns. These predictions are in very good agreement with recent experimental data. It is also demonstrated that defect formation and PS externalization in membranes should begin on the anode side. Finally, the simulations confirm that PS externalization is a nanopore facilitated event, rather than the result of molecular translocation across the trans-membrane energy barrier.

Simulations of transient membrane behavior in cells subjected to a high-intensity ultrashort electric pulse.

A molecular dynamics (MD) scheme is combined with a distributed circuit model for a self-consistent analysis of the transient membrane response for cells subjected to an ultrashort (nanosecond) high-intensity (approximately 0.01-V/nm spatially averaged field) voltage pulse. The dynamical, stochastic, many-body aspects are treated at the molecular level by resorting to a course-grained representation of the membrane lipid molecules. Coupling the Smoluchowski equation to the distributed electrical model for current flow provides the time-dependent transmembrane fields for the MD simulations. A good match between the simulation results and available experimental data is obtained. Predictions include pore formation times of about 5-6 ns. It is also shown that the pore formation process would tend to begin from the anodic side of an electrically stressed membrane. Furthermore, the present simulations demonstrate that ions could facilitate pore formation. This could be of practical importance and have direct relevance to the recent observations of calcium release from the endoplasmic reticulum in cells subjected to such ultrashort, high-intensity pulses.

Energy-landscape-model analysis for irreversibility and its pulse-width dependence in cells subjected to a high-intensity ultrashort electric pulse.

We provide a simple, but physical analysis for cell irreversibility and apoptosis in response to an ultrashort (nanosecond), high-intensity electric pulse. Our approach is based on an energy landscape model for determining the temporal evolution of the configurational probability function p(q). The primary focus is on obtaining qualitative predictions of a pulse width dependence to apoptotic cell irreversibility that has been observed experimentally. The analysis couples a distributed electrical model for current flow with the Smoluchowski equation to provide self-consistent, time-dependent transmembrane voltages. The model captures the essence of the experimentally observed pulse-width dependence, and provides a possible physical picture that depends only on the electrical trigger. A number of interesting features are predicted.

Improved energy model for membrane electroporation in biological cells subjected to electrical pulses.

A self-consistent model analysis of electroporation in biological cells has been carried out based on an improved energy model. The simple energy model used in the literature is somewhat incorrect and unphysical for a variety of reasons. Our model for the pore formation energy E(r) includes a dependence on pore population and density. It also allows for variable surface tension, incorporates the effects of finite conductivity on the electrostatic correction term, and is dynamic in nature. Self-consistent calculations, based on a coupled scheme involving the Smoluchowski equation and the improved energy model, are presented. It is shown that E(r) becomes self-adjusting with variations in its magnitude and profile, in response to pore population, and inhibits uncontrolled pore growth and expansion. This theory can be augmented to include pore-pore interactions to move beyond the independent pore picture.

Theoretical predictions of electromechanical deformation of cells subjected to high voltages for membrane electroporation.

An electromechanical analysis based on thin-shell theory is presented to analyze cell shape changes in response to external electric fields. This approach can be extended to include osmotic-pressure changes. Our calculations demonstrate that at large fields, the spherical cell geometry can be significantly modified, and even ellipsoidal forms would be inappropriate to account for the deformation. Values of the surface forces obtained from our calculations are in very good agreement with the 1--10 mN/m range for membrane rupture reported in the literature. The results, in keeping with reports in the literature, demonstrate that the final shape depends on membrane thickness. This has direct implications for tissues in which significant molecular restructuring can occur. It is also shown that, at least for the smaller electric fields, both the cellular surface area and volume change roughly in a quadratic manner with the electric field. Finally, it is shown that the bending moments are generally quite small and can be neglected for a simpler analysis.

Mechanism for membrane electroporation irreversibility under high-intensity, ultrashort electrical pulse conditions.

An improved electroporation model is used to address membrane irreversibility under ultrashort electric pulse conditions. It is shown that membranes can survive a strong electric pulse and recover provided the pore distribution has a relatively large spread. If, however, the population consists predominantly of larger radii pores, then irreversibility can result. Physically, such a distribution could arise if pores at adjacent sites coalesce. The requirement of close proximity among the pore sites is more easily satisfied in smaller organelles than in outer cell membranes. Model predictions are in keeping with recent observations of cell damage to intracellular organelles (e.g., mitochondria), without irreversible shock at the outer membranes, by a nanosecond, high-intensity electric pulse. This mechanism also explains the greater damage from multiple electric shocks.

Intracellular effect of ultrashort electrical pulses.

A simple electrical model for biological cells predicts an increasing probability for electric field interactions with cell substructures of prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses with electric field intensities of up to 5.3 MV/m to human eosinophils in vitro. When 3-5 pulses of 60 ns duration were applied to human eosinophils, intracellular granules were modified without permanent disruption of the plasma membrane. In spite of the extreme electrical power levels applied to the cells thermal effects could be neglected because of the ultrashort pulse duration. The intracellular effect extends conventional electroporation to cellular substructures and opens the potential for new applications in apoptosis induction, gene delivery to the nucleus, or altered cell functions, depending on the electrical pulse conditions.

Self-consistent simulations of electroporation dynamics in biological cells subjected to ultrashort electrical pulses.

The temporal dynamics of electroporation of cells subjected to ultrashort voltage pulses are studied based on a coupled scheme involving the Laplace, Nernst-Plank, and Smoluchowski equations. A pore radius dependent energy barrier for ionic transport, accounts for cellular variations. It is shown that a finite time delay exists in pore formation, and leads to a transient overshoot of the transmembrane potential V(mem) beyond 1.0 V. Pore resealing is shown to consist of an initial fast process, a 10(-4) s delay, followed by a much slower closing at a time constant of about 10(-1) s. This establishes a time-window during which the pores are mostly open, and hence, the system is most vulnerable to destruction by a second electric pulse. The existence of such a time window for effective killing by a second pulse is amply supported by our experimental data for E. coli cells. The time constant for the longer process also matches experiments. The study suggests that controlled manipulation of the pore "open times" can be achieved through multiple, ultrashort pulses.

Electroporation dynamics in biological cells subjected to ultrafast electrical pulses: a numerical simulation study.

A model analysis of electroporation dynamics in biological cells has been carried out based on the Smoluchowski equation. Results of the cellular response to short, electric pulses are presented, taking account of the growth and resealing dynamics of transient aqueous pores. It is shown that the application of large voltages alone may not be sufficient to cause irreversible breakdown, if the time duration is too short. Failure to cause irreversible damage at small pulse widths could be attributed to the time inadequacy for pores to grow and expand beyond a critical threshold radius. In agreement with earlier studies, it is shown that irreversible breakdown would lead to the formation of a few large pores, while a large number of smaller pores would appear in the case of reversible breakdown. Finally, a pulse width dependence of the applied voltage for irreversible breakdown has been obtained. It is shown that in the absence of dissipation, the associated energy input necessary reduces with decreasing pulse width to a limiting value. However, with circuit effects taken into account, a local minima in the pulse dependent energy function is predicted, in keeping with previously published experimental reports.