Patent Grant
10471254
Field of the Invention
The present invention is related to medical therapies involving the administering of electrical treatment energy. Embodiments of the present invention relate to a system and method that produces two treatment zones: a first zone surrounding the electrodes within which cells are killed non-selectively and a second selective treatment zone surrounding the first zone within which cells are killed selectively, such as aberrant cells. In specific embodiments, systems and methods for selectively treating cells, such as cancer cells, through administration of a train of electrical pulses wherein the pulse length and delay between successive pulses is optimized to produce effects on intracellular membrane potentials are provided. Through the systems and methods of the invention, infiltrating tumor cells disposed within a tumor margin can be effectively treated while sparing healthy tissue within the tumor margin.
Description of Related Art
Focal ablation techniques typically attack tumors by destroying cancerous cells within a well-defined region. Typically, these techniques destroy all of the cells and tissue structure within the treated volume, not just the cancerous cells. A major challenge of focal ablation technologies is that there is typically a region surrounding the tumor which contains healthy cells and some infiltrative cancerous cells. These infiltrative cancer cells, if untreated, may lead to recurrence of the tumor. The solution, in traditional surgical resection and focal ablation, is to treat beyond the tumor margin in an attempt to also remove these infiltrative cancer cells. This presents a major challenge for tumors which typically arise near critical structures, such as blood vessels and nerves. Thus, there is a need in the art for new electroporation protocols that overcome these limitations.
The present invention provides a system and method of treating infiltrative cancer cells in a tumor margin. This bimodal enhanced ablation mechanism (BEAM) platform uses burst of high frequency electric fields which have been specifically optimized to enhance the intracellular effects of the pulse while minimizing effects on healthy tissue. In embodiments, an optimal burst contains constitutive pulses with durations approximately equivalent to the charging time of the cell membrane plus the discharge time of the nuclear envelope. The optimal off-time between pulses is approximately equivalent to the charging time of the cell membrane.
In embodiments, certain cells (malignant) cells can be preferentially targeted based on their biophysical subcellular structure. Cells with a larger nucleus-to-cytoplasm ratio, which is an indication of their malignancy, are more susceptible to these pulses. The mechanism affecting the cells is related to disrupting their nucleus. Although cell size is the primary parameter for determining when a cell dies under an applied field for typical IRE, in contrast according to embodiments of the present invention, cell size does not play a dominant role.
Specific embodiments provide a method of selectively treating cells, comprising: applying to a tissue a plurality of electrical pulses with a delay between successive pulses, wherein the length of each pulse and the delay between successive pulses are optimized to produce a first treatment zone and a second treatment zone; wherein in the first treatment zone target cells, such as cancer cells, and non-target cells, such as non-cancer cells, are killed and in the second treatment zone the target cells are killed or inhibited while the non-target cells are spared. In such methods, the applying can be performed in vitro, in vivo, or ex vivo.
According to embodiments, within the second treatment zone target cells, such as cancer cells, are inhibited by way of slowed or arrested cell division, or target cells (e.g., cancer cells) are inhibited by way of slowed or arrested migration, or target cells (e.g., cancer cells) are inhibited by way of reduced transport of blood and nutrients, or target cells (e.g., cancer cells) are killed by apoptosis, or some target cells (e.g., cancer cells) are killed or inhibited in the second treatment zone and some non-target cells (e.g., non-cancer cells) are spared in the second treatment zone. In embodiments, the second treatment zone surrounds a tumor and the target cancer cells are infiltrative cells originating from the tumor. The tissue can be brain tissue, and/or the tumor glioblastoma. The target cells in any embodiment of this disclosure can be any type of cells, including for example cancer cells, infiltrative cells, or any undesired cells. The non-target cells can be any type of cell as well and are typically healthy cells, normal cells, or non-cancer cells.
According to embodiments, within the first treatment zone target cells and non-target cells (e.g., cancer cells and non-cancer cells) are killed by necrosis, or some cancer cells and some non-cancer cells are killed in the first treatment zone.
Likewise, according to embodiments both target and non-target cells (e.g., cancer cells and non-cancer cells) can be killed within the first treatment zone as a result of an increase of their transmembrane potential to a lethal threshold.
In embodiments, cancer cells are killed within the second treatment zone as a result of an increase in their nuclear transmembrane potential to a lethal threshold.
In embodiments, the delay between successive pulses can be greater than the length of each pulse, or the delay between successive pulses can be a fraction of the length of each pulse, or the length of each pulse can be equivalent to the charging time of the cell membrane of the cancer cells plus the discharge time of the nuclear membrane of the cancer cells, while the delay between successive pulses is equivalent to the charging time of the cell membrane of the cancer cells. Likewise, the charging time of the cell membrane of the cancer cells and the discharge time of the nuclear membrane of the cancer cells can be determined through numerical modeling.
In embodiments, the pulse train comprises an electric field waveform which is a rectangular pulse, ramp, decaying exponential, or sine wave. In embodiments, the electric field waveform is unipolar or bipolar, or can be a superimposed, bimodal signal comprising a first frequency harmonic and a second frequency harmonic, wherein the second frequency harmonic has a frequency higher than that of the first frequency harmonic.
In embodiments, the electric field waveform comprises alternating nanosecond-order pulses with microsecond order pulses in succession. Likewise, the electric field waveform can be symmetric or asymmetric. The electric field waveform in embodiments can have a carrier frequency in the range of 100 kHz to 10 MHz. The carrier frequency or pulse duration of the waveforms can be based on the cross-over frequency of the cancer cells.
In embodiments, the length of each pulse and the delay between successive pulses are optimized based on the physical nucleus to cytoplasm size ratio of the cancer cells.
Embodiments of the invention include a method of treating cancer in a patient, comprising identifying a solid tumor in a patient, inserting at least one electrode into or adjacent to the solid tumor, and applying a pulse train comprising a plurality of electrical pulses with a delay between successive pulses. Such methods are also applicable to treating undesired cells or target cells that are not necessarily cancerous. In embodiments, the length of each pulse and the delay between successive pulses are optimized to produce a first treatment zone within a radius of the at least one electrode and a second treatment zone between the first radius and a within a second radius of the electrode, which second treatment zone lies outside of the first treatment zone. In the first treatment zone cancer cells and healthy cells are killed non-selectively while in the second treatment zone cancer cells are selectively killed or inhibited an healthy cells are spared. Methods of treating cancer in a patient can be performed in vivo, ex vivo, or in vitro.
In embodiments of the invention, selective inhibition of the cancer cells in the second treatment zone comprises slowed or arrested cell division. Alternatively or in addition, selective inhibition of the cancer cells in the second treatment zone comprises slowed or arrested migration. Alternatively or in addition, selective inhibition of the cancer cells in the second treatment zone comprises reduced transport of blood and nutrients.
In embodiments of the invention, cancer cells and healthy cells are killed within the first treatment zone by necrosis. Alternatively or in addition, cancer cells are killed within the second treatment zone by apoptosis.
In embodiments of the invention, cancer cells and healthy cells are killed within the first treatment zone as a result of an increase of their transmembrane potential to a lethal threshold. Alternatively or in addition, cancer cells are killed within the second treatment zone as a result of an increase in their nuclear transmembrane potential to a lethal threshold.
Preferred embodiments of the invention may target the transmembrane potential of the nucleus, such that it reaches a lethal threshold as a result of the optimized pulses of the invention. However, other embodiments may target any membrane-bound intracellular organelle, whether through effects on the transmembrane potential or any other mechanism, including without limitation the mitochondria, smooth endoplasmic reticulum, rough endoplasmic reticulum, the golgi apparatus, endosomes, lysosomes, peroxisomes, storage vesicles, and transport vesicles.
In embodiments of the invention, the delay between successive pulses is greater than the length of each pulse. Alternatively, the delay between successive pulses is a fraction of the length of each pulse.
In embodiments of the invention, the length of each pulse is equivalent to the charging time of the cell membrane plus the discharge time of the nuclear membrane, while the delay between successive pulses is equivalent to the charging time of the cell membrane. Likewise, embodiments can comprise a multiple of such timing or even a fraction of such timing. The charging time of the cell membrane and the discharge time of the nuclear membrane may be determined through numerical modeling.
In embodiments of the invention, the pulse train comprises an electric field waveform which is a rectangular pulse, ramp, decaying exponential, or sine wave. The electric field waveform may be unipolar or bipolar. The electric field waveform may be a superimposed, bimodal signal comprising a first frequency harmonic (such as a low frequency harmonic) and a second frequency harmonic (such as a high frequency harmonic). The electric field waveform may comprise alternating nanosecond-order pulses with microsecond order pulses in succession. The electric field waveform may be asymmetric. The electric field waveform may have a carrier frequency in the range of 100 kHz to 10 MHz. The carrier frequency or pulse duration of the waveforms may be based on the cross-over frequency of the cancer cells, undesired cells, or otherwise referred to as the target cells.
In embodiments, the length of each pulse and the delay between successive pulses are optimized based on the physical nucleus to cytoplasm size ratio of the cancer cells.
In embodiments of the invention, the pulses are bipolar square waves and the length of each pulse is between 250 nanoseconds and 50 microseconds.
Embodiments of the invention include a method of treating a cancer in a patient, comprising identifying a solid tumor in a patient, inserting at least one electrode into or adjacent to the solid tumor, and applying a pulse train comprising a plurality of electrical pulses, wherein the pulses are bipolar square waves and the length of each pulse is between 250 nanoseconds and 50 microseconds.
Embodiments of the invention include a method of treating a cancer in a patient, comprising identifying a solid tumor in a patient, inserting at least one electrode into or adjacent to the solid tumor, and applying a pulse train comprising a plurality of electrical pulses, wherein the pulse train has an electric field waveform which is a superimposed, bimodal signal comprising a first frequency harmonic and a second frequency harmonic, such as a low frequency harmonic and a high frequency harmonic.
Embodiments of the invention include a system for treating a cancer in a subject, comprising at least one electrode, and a voltage pulse generator operatively coupled to the electrode and configured to apply a pulse train comprising a plurality of electrical pulses, wherein the pulse train has an electric field waveform which is a superimposed, bimodal signal comprising a low frequency harmonic and a high frequency harmonic. The voltage pulse generator may comprise solid state switching devices arranged in a multi-level, neutral point clamped, or cascaded H-bridge topology.
Also included is a method of selectively treating cells, comprising: applying a plurality of electrical pulses as a treatment to a substance containing cells, wherein the pulses are bipolar square waves and the length of each pulse is between 250 nanoseconds and 50 microseconds, with a delay between pulses of between 250 nanoseconds and 50 microseconds; wherein one type of cell is treated and another type of cell is not treated by the plurality of electrical pulses. In embodiments, the treated cells are killed and untreated cells are not killed. The substance containing cells for example can be a tissue, a non-living object, a solution, a body part, or a living or non-living patient, human, animal, or tissue.
In embodiments, provided is a method of selectively treating cells, comprising: applying a pulse train comprising a plurality of electrical pulses to a substance containing cells, wherein the pulse train has an electric field waveform which is a superimposed, bimodal signal comprising a first frequency harmonic and a second frequency harmonic with a frequency higher than that of the first. In embodiments, the pulse train selectively kills cells of a selected type and spares cells of another type.
Systems of the invention include any system configured to implement one or more methods of the invention. Included is a system for selectively treating cells, comprising: at least one electrode; and a voltage pulse generator coupled to the electrode and configured to apply a pulse train comprising a plurality of electrical pulses, wherein the pulse train has an electric field waveform which is a superimposed, bimodal signal comprising a first frequency harmonic and a second frequency harmonic, wherein the second frequency harmonic has a frequency higher than that of the first frequency harmonic.
In embodiments, the voltage pulse generator is configured to select the bimodal signal such that the pulse train selectively kills cells of a selected type and spares cells of another type, or the voltage pulse generator comprises solid state switching devices arranged in a multi-level, neutral point clamped, or cascaded H-bridge topology.
Additional methods include a method of selectively treating cells, the method comprising: delivering electrical pulses to a substance containing cells in a manner sufficient to kill only cells having a selected biophysical subcellular structure. In embodiments, the cells having the selected biophysical subcellular structure have a nucleus and the cells are killed by disrupting the nucleus of the cells. The cells having the selected biophysical subcellular structure can have a selected nucleus-to-cytoplasm area ratio.
A method of selectively treating cells, comprising: applying a plurality of electrical pulses to a substance containing cells, wherein the plurality of electrical pulses has a frequency, amplitude, and pulse waveform selected to treat target cells of one type of cell and spare non-target cells of another type of cell is also included within the scope of the invention. Such methods of the invention can be a selective method wherein cancer cells are treated and normal cells are spared. Such methods can be a palliative method wherein cancer cells of a more malignant type are treated and cancer cells of a less aggressive type are spared.
Methods of the invention can further comprise determining a nucleus-to-cytoplasm ratio for the target cells; and selecting the frequency, amplitude, and pulse waveform based on the nucleus-to-cytoplasm ratio for the target cells. In embodiments the nucleus-to-cytoplasm ratio is measured or otherwise determined from cells taken from a biopsy of the substance containing cells.
A method of selectively ablating malignant cells is included in the invention, the method comprises: determining a first death threshold for malignant cells present in a tissue region; determining a second death threshold for non-malignant cells present in the tissue region; administering electrical pulses to the tissue region at or above the first death threshold and below the second death threshold to kill the malignant cells. In embodiments of methods of the invention, the non-malignant cells are not killed. In embodiments, the malignant cells each comprise a cell nucleus and are killed by administering the electrical pulses in a manner sufficient to disrupt the cell nucleus.
Included in embodiments is a method of enhancing the transport of material into an organelle, comprising: applying a plurality of electrical pulses to a substance containing cells, wherein the plurality of electrical pulses has a frequency, amplitude, and pulse waveform selected to optimize the transport of molecules into an organelle.
According to embodiments, the plurality of electrical pulses includes positive and negative pulses having different pulse widths, or the plurality of electrical pulses includes positive and negative pulses having different amplitude. In embodiments, the organelle is the nucleus, mitochondria, endoplasmic reticulum, vacuole, lysosome, or chloroplast.
The accompanying drawings illustrate certain aspects of embodiments of the present invention, and should not be used to limit the invention. Together with the written description the drawings serve to explain certain principles of the invention.
Reference will now be made in detail to various exemplary embodiments of the invention. Embodiments described in the description and shown in the figures are illustrative only and are not intended to limit the scope of the invention. Changes may be made in the specific embodiments described in this specification and accompanying drawings that a person of ordinary skill in the art will recognize are within the scope and spirit of the invention.
Throughout the present teachings, any and all of the features and/or components disclosed or suggested herein, explicitly or implicitly, may be practiced and/or implemented in any combination, whenever and wherever appropriate as understood by one of ordinary skill in the art. The various features and/or components disclosed herein are all illustrative for the underlying concepts, and thus are non-limiting to their actual descriptions. Any means for achieving substantially the same functions are considered as foreseeable alternatives and equivalents, and are thus fully described in writing and fully enabled. The various examples, illustrations, and embodiments described herein are by no means, in any degree or extent, limiting the broadest scopes of the claimed inventions presented herein or in any future applications claiming priority to the instant application.
The present inventors have made the surprising discover that high frequency pulsed electric fields can be manipulated and optimized to target intracellular membranes. More particularly, particular protocols of administering high frequency pulsed electric fields can be used to increase intracellular membrane potentials of the cell in organelles such as the nucleus. By targeting intracellular membrane potentials, cancer cells can be selectively targeted over healthy tissue. In one embodiment, high frequency pulsed electric fields are administered to tumors to create two treatment zones: an ablation zone and a selective therapy zone. In the ablation zone, healthy cells and cancer cells die due to necrotic cell death. Outside the ablation zone, only cancer cells die due to programmed cell death as a result of changes in the membrane potential of intracellular organelles, while healthy cells are spared. Thus, the cell membrane is not the primary target of therapy; resonant additive effects target intracellular components such as the nucleus, mitochondria, and other key membrane-bound organelles. In embodiments, these effects are achieved through an optimization of both pulse length and delay time. In an exemplary embodiment, the pulse length is optimized to be approximately equivalent to the charging time of the cell membrane plus the discharge time of the nuclear envelope, while the delay time is optimized to be approximately equivalent to the charging time of the cell membrane. In embodiments, the delay time is a fraction of the pulse length.
Embodiments of the invention include pulses designed to generate a range of field strengths beyond the tumor margin that results in cell death of aberrant cells while preserving healthy cells. Within the tumor margin, the field strengths are sufficient to kill all cell types. Additionally, embodiments of the invention include pulses designed to generate a range of field strengths beyond the tumor margin that results in enhanced nuclear permeability of aberrant cells while not affecting healthy cells. Within the tumor margin, the field strengths are sufficient to enhance the nuclear permeability of all cell types. Additionally, embodiments of the invention include pulses designed to generate a range of field strengths beyond the tumor margin that slow or arrest the division of aberrant cells while not affecting healthy cells. Within the tumor margin, the field strengths are sufficient to slow the growth rate of all cell types. Additionally, embodiments of the invention include pulses designed to generate a range of field strengths beyond the tumor margin that halts the migration of aberrant cells to prevent metastasis while not affecting healthy cells. Within the tumor margin, the field strengths are sufficient to halt migration of all cell types. Additionally, embodiments of the invention include pulses designed to generate a range of field strengths beyond the tumor margin that prevent the transport of blood and nutrients to aberrant cells. Within the tumor margin, transport of blood and nutrients is prevented to all cell types.
In embodiments, the field strengths generated within the tumor margin are selective to aberrant cells while preserving healthy cells. The electric field waveform may be a rectangular pulse, ramp, decaying exponential, or sine wave and may be unipolar or bipolar. In embodiments, the electric field waveform may be a superimposed, bimodal signal consisting of a low frequency component/harmonic and a high frequency component/harmonic. In embodiments, the electric field waveform may consist of alternating short duration, nanosecond-order pulses with long-duration, microsecond order pulse in succession.
In embodiments, the waveforms are asymmetric to electrophoretically drive exogenous agents, chemical agents, DNA molecules, or nanoparticles through permeabilized membranes. The carrier frequency of the waveforms may be in the range of 100 kHz to 10 MHz. In embodiments, the carrier frequency or pulse duration of the waveforms are chosen based on the cross-over frequency of the cell populations. In other embodiments, the pulses are optimized based on the dielectric properties of the cell populations within the targeted zone of therapy to enable selectivity. In other embodiments, the pulses are optimized based on the physical nucleus to cytoplasm size ratio of the cell populations within the targeted zone of therapy to enable selectivity. In other embodiments, the pulses are designed to generate electro fusion within a select population of cells.
In other embodiments, the pulses are designed to generate simultaneous modulation of the nuclear membrane and outer membrane transmembrane potential. The desired modulatory effect may trigger both reversible electroporation of the nuclear and outer membranes. Alternatively, the desired modulatory effect may trigger reversible electroporation of the outer membrane and irreversible electroporation of the nuclear membrane. Alternatively, the desired modulatory effect triggers both necrosis and apoptosis. Alternatively, the desired modulatory effect slows or arrests cell division. Alternatively, the desired modulatory effect is to prevent metastasis of infiltrative cells.
The treatments may be applied in a single session lasting under 1 hr using an external device. The treatments may be applied over multiple days using an external or implantable device. The resting time between pulses may be varied as part of the optimization routine to select for aberrant cells. The pulses may be delivered in a repetitive manner to lower the required effective field strength and enable the use of solid state switching devices.
In embodiments, the required effective field strength is on the order of 100 to 10,000 V/cm. The solid state switching devices may be arranged in a multi-level, neutral point clamped, or cascaded H-bridge topology.
Embodiments of the invention include an electrical pulse designed to generate a range of field strengths beyond the aberrant cell growth region that results in cell death, slowing the growth rate of, halting the migration of, or preventing the transport of blood and nutrients to aberrant cells while preserving healthy cells, and within the aberrant cell growth region, the field strengths are sufficient to kill, slow the growth rate of, halt migration of, or prevent the transport of blood and nutrients to all cell types. Within the tumor margin, the field strengths are sufficient to enhance the nuclear permeability of all cell types.
Embodiments of the invention include a system for treating a subject suffering from an aberrant cell growth, comprising: at least one electrode configured to be introduced into or adjacent the aberrant cell growth region within the body of a subject, a voltage pulse generator coupled to the electrode and configured to applying multiple electrical pulses to generate an electric field within the growth region with field strengths selective to kill, slow the growth rate of, halt migration of, or preventing the transport of blood and nutrients to aberrant cells while preserving healthy cells.
Embodiments of the invention include a method of treating a subject suffering from an aberrant cell growth, comprising: implanting an electrode into or adjacent the aberrant growth region within the body of a subject, and causing multiple electrical pulses to be emitted from the electrode into the aberrant cell growth region to generate an electric field, wherein the electric field strengths generated within the aberrant cell growth region are selective to kill, slow the growth rate of, halt migration of, or prevent the transport of blood and nutrients to aberrant cells while preserving healthy cells. In embodiments of the method, the electric field has unipolar or bipolar wave form of a rectangular pulse, ramp, decaying exponential, or sine wave. The multiple electrical pulses may take the form of a superimposed, bimodal signal consisting of a low frequency component and a high frequency component. Alternatively, the multiple electrical pulses may consist of alternating short duration, nanosecond-order pulses with long-duration, microsecond order pulse in succession. In embodiments of the method, the frequency of multiple electrical pulses may be in the range of 100 kHz to 10 MHz. In embodiments of the method, the pulses are asymmetric to electrophoretically drive exogenous agents, chemical agents, DNA molecules, or nanoparticles through permeabilized membranes.
In embodiments of the method, the required effective field strength is on the order of 100 to 10,000 V/cm. The carrier frequency or pulse duration of the pulses may be chosen based on the cross-over frequency of the cell populations. Alternatively or additionally, the pulses may be optimized based on the dielectric properties, or the physical nucleus to cytoplasm size ratio of the cell populations within the targeted zone of therapy to enable selectivity. In embodiments of the method, the pulses are designed to generate electro fusion within a select population of cells. In embodiments of the method, the pulses are designed to generate simultaneous modulation of the nuclear membrane and outer membrane transmembrane potential. In embodiments, the desired modulatory effect triggers reversible electroporation of the outer membrane, and irreversible or reversible electroporation of the nuclear membrane. Alternatively or in addition, the desired modulatory effect triggers both necrosis and apoptosis.
In embodiments of the method, the treatments are applied by way of at least one session using an external device or implantable device. The resting time between pulses may be varied as part of the optimization routine to select for aberrant cells. In embodiments, the pulses are delivered in a repetitive manner to lower the required effective field strength and enable the use of solid state switching devices. The solid state switching devices may be arranged in a multi-level, neutral point clamped, or cascaded H-bridge topology.
In embodiments, the pulses, systems, and methods of the invention may have applications in biomedical cancer or tumor treatment.
The following figures further illustrate the invention.
In embodiments, the optimized pulse protocol can be used to increase the transport of molecules between the cytoplasm and intracellular organelles. For example, the optimized pulse protocol of the invention can enhance electro-gene and electro-chemo therapy. Additionally, assymetric pulses can enhance electrophoretic transfer. This is shown schematically in
In embodiments, bimodal sinusoidal signals can be used to achieve an amplification effect. Indeed, any signal with two or more different frequency components can be used, such as a signal with two, three, four, five, or six frequency components. For example,
Additionally, embodiments of the invention may include one or more systems capable of performing one or more steps of the method. One embodiment of the present invention is illustrated in
For example, a treatment protocol according to the invention could include one or more of a plurality of electrodes. According to the desired treatment pattern, the plurality of electrodes can be disposed in various positions relative to one another. In a particular example, a plurality of electrodes can be disposed in a relatively circular pattern with a single electrode disposed in the interior of the circle, such as at approximately the center. Any configuration of electrodes is possible and the arrangement need not be circular but any shape periphery can be used depending on the area to be treated, including any regular or irregular polygon shape, including convex or concave polygon shapes. The single centrally located electrode can be a ground electrode while the other electrodes in the plurality can be energized. Any number of electrodes can be in the plurality such as from about 1 to 20. Indeed, even 3 electrodes can form a plurality of electrodes where one ground electrode is disposed between two electrodes capable of being energized, or 4 electrodes can be disposed in a manner to provide two electrode pairs (each pair comprising one ground and one electrode capable of being energized). During treatment, methods of treating can involve energizing the electrodes in any sequence, such as energizing one or more electrode simultaneously, and/or energizing one or more electrode in a particular sequence, such as sequentially, in an alternating pattern, in a skipping pattern, and/or energizing multiple electrodes but less than all electrodes simultaneously, for example.
In the embodiment shown, each probe 22 includes either a monopolar electrode or bipolar electrodes having two electrodes separated by an insulating sleeve. In one embodiment, if the probe includes a monopolar electrode, the amount of exposure of the active portion of the electrode can be adjusted by retracting or advancing an insulating sleeve relative to the electrode. See, for example, U.S. Pat. No. 7,344,533, which is incorporated by reference herein in its entirety. The pulse generator 10 is connected to a treatment control computer 40 having input devices such as keyboard 12 and a pointing device 14, and an output device such as a display device 11 for viewing an image of a target treatment area such as a lesion 300 surrounded by a safety margin 301. The therapeutic energy delivery device 22 is used to treat a lesion 300 inside a patient 15. An imaging device 30 includes a monitor 31 for viewing the lesion 300 inside the patient 15 in real time. Examples of imaging devices 30 include ultrasonic, CT, MRI and fluoroscopic devices as are known in the art.
The present invention includes computer software (treatment planning module 54) which assists a user to plan for, execute, and review the results of a medical treatment procedure, as will be discussed in more detail below. For example, the treatment planning module 54 assists a user to plan for a medical treatment procedure by enabling a user to more accurately position each of the probes 22 of the therapeutic energy delivery device 20 in relation to the lesion 300 in a way that will generate the most effective treatment zone. The treatment planning module 54 can display the anticipated treatment zone based on the position of the probes and the treatment parameters. Additionally, the treatment planning module 54 may have a user interface which allows a user to input one or more parameters for IRE.
The treatment planning module 54 can display the progress of the treatment in real time and can display the results of the treatment procedure after it is completed. This information can be displayed in a manner such that it can be used for example by a treating physician to determine whether the treatment was successful and/or whether it is necessary or desirable to re-treat the patient.
For purposes of this application, the terms “code”, “software”, “program”, “application”, “software code”, “computer readable code”, “software module”, “module” and “software program” are used interchangeably to mean software instructions that are executable by a processor. The “user” can be any human, including for example, a physician or other medical professional. The treatment planning module 54 executed by a processor outputs various data including text and graphical data to the monitor 11 associated with the generator 10.
Referring now to
In embodiments, the user interface may be a graphical user interface which may be used in conjunction with the computer readable code. The user interface may allow a user to enter or input one or more parameters to be used by the treatment planning module 54 in setting a treatment protocol for IRE. The user interface may allow such input through the use of text fields, check boxes, pull-downs, sliders, command buttons, and the like. Based on this input 54, the treatment planning module 54 can calculate a threshold electric field for IRE of the target tissue and one or more parameters of a treatment protocol for administering the IRE in a manner sufficient to produce this threshold electric field.
In embodiments, the treatment planning module 54 provides for numerical modeling capabilities such as those described in the Examples. The model may be used to simulate the nuclear and cellular transmembrane potential of various pulsing parameters prior to treatment. A user interface may allow input of one or more of the parameters listed in the table in
In one embodiment, the computer 40 is built into the voltage generator 10. In another embodiment, the computer 40 is a separate unit which is connected to the voltage generator through the communications link 52. In a preferred embodiment, the communication link 52 is a USB link. In one embodiment, the imaging device 30 is a standalone device which is not connected to the computer 40. In the embodiment as shown in
It should be noted that the software can be used independently of the pulse generator 10. The user can plan the treatment on a different computer as will be explained below and then save the treatment parameters to an external memory device, such as a USB flash drive (not shown). Any non-transitory computer-readable media can be used to store the software and/or the output of the software for a particular treatment protocol. The data from the memory device relating to the treatment parameters can then be downloaded onto the computer 40 to be used with the generator 10 for treatment. Additionally, the software can be used for hypothetical illustration of zones of ablation, temperature thresholds or cutoffs, and electrical field thresholds or cutoffs for training purposes to the user on therapies that deliver electrical energy. For example, the data can be evaluated by a human to determine or estimate favorable treatment protocols for a particular patient rather than programmed into a device for implementing the particular protocol. The treatment protocols can be designed to produce the minimum electrical field threshold for inducing IRE calculated by the treatment planning module 54.
General treatment protocols for the destruction (ablation) of undesirable tissue through electroporation are known. They involve the insertion (bringing) electroporation electrodes to the vicinity of the undesirable tissue and in good electrical contact with the tissue and the application of electrical pulses that cause irreversible electroporation of the cells throughout a region of or the entire area of the undesirable tissue. The cells whose membrane was irreversible permeabilized may be removed or left in situ (not removed) and as such may be gradually removed by the body's immune system. Cell death is produced by inducing the electrical parameters of irreversible electroporation in the undesirable area.
Electroporation protocols involve the generation of electrical fields in tissue and are affected by the Joule heating of the electrical pulses. When designing tissue electroporation protocols it is important to determine the appropriate electrical parameters that will maximize tissue permeabilization without inducing deleterious thermal effects. It has been shown that substantial volumes of tissue can be electroporated with reversible electroporation without inducing damaging thermal effects to cells and these volumes have been quantified (Davalos, R. V., B. Rubinsky, and L. M. Mir, Theoretical analysis of the thermal effects during in vivo tissue electroporation. Bioelectrochemistry, 2003. Vol. 61(1-2): p. 99-107).
The electrical pulses used to induce irreversible electroporation in tissue are typically larger in magnitude and duration from the electrical pulses required for reversible electroporation. Further, the duration and strength of the pulses for irreversible electroporation are different from other methodologies using electrical pulses such as for intracellular electro-manipulation or thermal ablation. The methods are very different even when the intracellular (nano-seconds) electro-manipulation is used to cause cell death, e.g. ablate the tissue of a tumor or when the thermal effects produce damage to cells causing cell death.
Typical values for pulse length for irreversible electroporation are in a range of from about 5 microseconds to about 62,000 milliseconds or about 75 microseconds to about 20,000 milliseconds or about 100 microseconds±10 microseconds. This is significantly longer than the pulse length generally used in intracellular (nano-seconds) electro-manipulation which is 1 microsecond or less—see U.S. Published Patent Application No. 2002/0010491.
The pulse is typically administered at voltage such that the local electric field experienced by the tissue is about 100 V/cm to 7,000 V/cm or 200 V/cm to 2000 V/cm or 300V/cm to 1000 V/cm about 600 V/cm for irreversible electroporation. This is substantially lower than that used for intracellular electro-manipulation which is about 10,000 V/cm—see U.S. Published Patent Application No. 2002/0010491.
The voltage expressed above is the voltage gradient (voltage per centimeter). The electrodes may be different shapes and sizes and may be positioned at different distances from each other. The shape may be circular, oval, square, rectangular or irregular etc. The distance of one electrode to another may be in the range of about 0.5 to 10 cm, 1 to 5 cm, or 2-3 cm, for example. The electrode may have a surface area of 0.1-5 sq. cm or 1-2 sq. cm, for example.
The size, shape and distances of the electrodes can vary and such can change the voltage and pulse duration used. Those skilled in the art will adjust the parameters in accordance with this disclosure to obtain the desired degree of electroporation and avoid thermal damage to surrounding cells.
A primary factor in determining the effect of an electroporation procedure is the electric field to which the tissue is exposed. However, IRE protocols have a variety of electrical pulse parameters that may also affect the toxicity of the treatment. In addition to the electric field, these include pulse shape, number of pulses, pulse length, and repetition rate. The thermal effects of an IRE treatment during a pulse are a direct function of the conductivity of the tissue and the voltage to which it is exposed. Therefore, minimizing the thermal effects for a particular tissue type may be done by finding the minimum required electric field, and thus applied voltage, to kill the cells in the tissue.
To this end, pulse parameters and electrode configurations according to embodiments of the invention can include any combination of any of the following: a pulse length in the range of about 1 μs to 1 ms; a number of pulses ranging from 1 to 10,000; an electric field distribution for each conductive wire pair and/or across a treatment region ranging from about 5-5,000 V/cm; a total electrical charge delivered by way of each conductive wire pair and/or across a treatment region of about 0.1 to about 500 mC; a frequency of pulse application ranging from about 0.001-100 Hz; a frequency of pulse signal ranging from about 0-100 MHz; a pulse shape that is square, exponential decay, sawtooth, sinusoidal, or of alternating polarity although the currently favored pulse shape is a biphasic DC pulse; a positive, negative, and neutral electrical charge pulses (changing polarity within the pulse); a resulting current in the treated tissue ranging from about 0 to about 100 amps; from 1-20 electrodes and/or electrically conductive wires; an electrode and/or electrically conductive wire separation distance ranging from about 0.1 mm to about 5 cm; and multiple sets of pulse/electrode parameters for a single treatment, including changing any of the above parameters within the same treatment, such as removing the electrodes and replacing them in different locations within the tissue or changing the number of electrodes, to specialize/customize outcome.
In embodiments treatment protocols can employ a pulse length in the range of about 250 ns and 50 μs, with a delay between pulses on that order. Pulse lengths ranging from about 1 μs to 1 ms are also possible, such as from about 5 μs to about 0.5 ms, or from about 10 μs to about 0.1 ms, or from about 15 μs to about 95 μs. Pulse lengths of 20 μs, 25 μs, 30 μs, 35 μs, 40 μs, 45 μs, 50 μs, 55 μs, 60 μs, 65 μs, 70 μs, 75 μs, 80 μs, 85 μs, 90 μs, 110 μs, 150 μs, or 200 μs, and so on are also acceptable. In some embodiments, the pulse duration of the electroporation-based therapy can exceed 100 μs. Any length pulse or pulse train can be administered in embodiments according to the invention. For example, pulse lengths of about 1 picosecond to 100 seconds can be used, such as from 10 picoseconds to about 10 seconds, or for example from about 100 picoseconds to about 1 second, or from 1 nanosecond to 100 milliseconds, or from about 10 nanoseconds to about 10 milliseconds, or from about 100 nanoseconds to about 1 millisecond, or from about 1 microsecond or 10 microseconds to about 100 microseconds. Some embodiments may have a pulse length ranging from about 100 microseconds to about 1 second, such as a pulse length of about 110, or 120, or 130, or 140, or 150, or 200, or 300, or 350, or 400, or 500, or 600, or 700, or 800 or 900 microseconds, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 milliseconds, or even 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 milliseconds, or even for example from about 200, 300, 400, 500, 600, 700, 800, or 900 milliseconds and so on.
In exemplary embodiments, the pulses are monopolar or bipolar and the pulse length may range from about 0.25 microseconds to about 100 microseconds, including 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, and 90 μs, or any range in between these values.
In exemplary embodiments, successive pulses, whether monopolar or bipolar may have an interpulse delay between about 0.1 microseconds to about 200 microseconds, including 0.2 microseconds, 0.3 microseconds, 0.4 microseconds, 0.5 microseconds, 0.6 microseconds, 0.7 microseconds, 0.8 microseconds, 0.9 microseconds, 1 microsecond, 1.5 microseconds, 2 microseconds, 2.5 microseconds, 3 microsecond, 3.5 microseconds, 4 microseconds, 4.5 microseconds, 5 microseconds, 5.5 microseconds, 6 microseconds, 6.5 microseconds, 7 microseconds, 7.5 microseconds, 8 microseconds, 8.5 microseconds, 9 microseconds, 9.5 microseconds, 10 microseconds, 20 microseconds, 30 microseconds, 40 microseconds, 50 microseconds, 60 microseconds, 70 microseconds, 80 microseconds, 90 microseconds, 100 microseconds, 120 microseconds, 140 microseconds, 160 microseconds, and 180 microseconds, or any range in between these values.
In exemplary embodiments, the interpulse delay is a portion of the pulse length, including 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% of the pulse length, or any range in between these values. In exemplary embodiments, the interpulse delay exceeds the pulse length, including 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2.0×, 2.2×, 2.4×, 2.6×, 2.8×, 3.0×, 3.2×, 3.4×, 3.6×, 3.8×, 4.0×, 4.2×, 4.4×, 4.6×, 4.8×, 5.0×, 5.5×, 6.0×, 6.5×, 7.0×, 7.5×, 8.5×, 9.0×, 9.5×, and 10.0× the pulse length, or any range in between these values.
The number of pulses can range for example from 5 to 5,000, or from about 10 to 2,000, or from about 20 to 1,000, or from about 30 to 500, or from about 50 to 200, or from about 75 to 150, or from about 90 to 120, or from about 95 to 110, or about 100 pulses. According to other embodiments, the number of pulses can range from about 5 to about 400 pulses, such as from about 10 to about 350 pulses, or for example from about 15 to about 300 pulses, including from about 20 to about 250 pulses, or from about 25 to about 200 pulses, such as from about 30 to about 150 pulses, for example from about 50 to about 125 pulses, such as from about 75 to about 175 pulses, or from about 90 to 110 pulses, such as about 100 pulses.
Typically, the electric field distribution for each conductive wire pair and/or across a treatment region for IRE is performed using voltages ranging for example between 1500 V/cm to 4,000 V/cm, including 1500 V/cm to 2000 V/cm, 2000 V/cm to 3000 V/cm, 3000 V/cm to 4000 V/cm, 2000 V/cm to 4000 V/cm, 2500 V/cm to 4000 V/cm, and so on. Voltages of much lower power can also be used, including using less than about 1500 V/cm. Applied fields of about 500 V/cm to 1000 V/cm can be used, or even of about 10 V/cm to about 750 V/cm, such as from about 50 V/cm to about 200 V/cm, or an electric field distribution of about 75 V/cm to about 100 V/cm. For example, in the treatment of brain tumors, typically, an applied field of less than 1000 V/cm can be used. Electrical pulse generators that can be used include those capable of delivering from 0 to about 5,000 V, such as the NanoKnife® system of AngioDynamics®, which for example can deliver from 0-3,000 V.
In another embodiment, the amplitude of the pulses of the electroporation-based therapy exceeds 2000 V/cm, including an amplitude of about 2200 V/cm, or 2500 V/cm, such as about 3000 V/cm, or 3500 V/cm, or about 4000 V/cm, such as 4500 V/cm, or about 5000 V/cm, such as about 5500 V/cm, or about 6000 V/cm, or about 6500 V/cm, such as about 7000 V/cm, or about 7500 V/cm, such as 8000 V/cm, or about 8500 V/cm, including 9000 V/cm, or about 9500 V/cm, such as about 10,000 V/cm and so on. Amplitude in the context of this specification refers to the magnitude of the electrical energy being applied using electrical pulses and which pulses can be of either positive or negative polarity.
According to methods of the invention, cycle times for pulses are set generally about 1 Hz. Furthermore, it has been found that alternating polarity of adjacent electrodes minimizes charge build up and provides a more uniform treatment zone. More specifically, in experiments performed by the inventors, a superficial focal ablative IRE lesion was created in the cranial aspect of the temporal lobe (ectosylvian gyms) using the NanoKnife® (Angiodynamics, Queensbury, N.Y.) generator, blunt tip bipolar electrode (Angiodynamics, No. 204002XX) by delivering 9 sets of ten 50 μs pulses (voltage-to-distance ratio 2000 V/cm) with alternating polarity between the sets to prevent charge build-up on the stainless steel electrode surfaces. These parameters were determined from ex-vivo experiments on canine brain and they ensured that the charge delivered during the procedure was lower than the charge delivered to the human brain during electroconvulsive therapy (an FDA approved treatment for major depression). Excessive charge delivery to the brain can induce memory loss, and thus is preferably avoided.
Specific method embodiments may employ administering electroporation based therapy using a pulse rate of about 1 Hz to 20 GHz, such as for example from about 10 Hz to 20 GHz, or about 50 Hz to 500 Hz, or 100 Hz to 1 kHz, or 10 kHz to 100 kHz, or from 250 kHz to 10 MHz, or 500 kHz to 1 MHz, such as from 900 kHz to 2 MHz, or from about 100 MHz to about 10 GHz, including from about 200 MHz to about 15 GHz and so on. In an exemplary embodiment, the pulse rate is between 100 kHz and 10 MHz.
In preferred embodiments, a total electrical charge delivered by way of each conductive wire pair and/or across a treatment region of about 0.5 to about 25 mC can be used, such as about 1 mC to about 20 mC, or from about 1.5 mC to about 15 mC, or from about 2 mC to about 10 mC, or from about 5 mC to about 8 mC, and so on. Similarly, in preferred embodiments, the resulting current in the treated tissue can range for example from about 1 A to about 8 A, or from about 2 A to about 6 A, or from about 3 A to about 5 A, such as 4 A. Indeed, for certain applications the total electrical charge delivered can range from about 0.5 to about 500 mC, such as about 10 mC to about 200 mC, or from about 15 mC to about 150 mC, or from about 20 mC to about 100 mC, or from about 50 mC to about 80 mC, and so on. The resulting current in the treated tissue can range for example from about 1 A to about 80 A, or from about 20 A to about 60 A, or from about 30 A to about 50 A, such as 40 A. It is not uncommon for currents for IRE treatments to reach or exceed 40 and 50 amps, and it is further feasible to operate under even higher current with pulse generators capable of operating under such conditions as well. Currents are expected to be high in certain applications, especially when working in an area where the tissue or the medium is highly conductive, such as with blood present in a blood vessel. Pulse width, pulse shape, number of pulses, and the resultant current in the tissue can be adjusted to achieve specific target goals for limiting the total electric charge, and any of the specific values disclosed in this specification can be used to calculate the target expected charge.
Any number of electrically conductive wires or electrodes can also be used. However, in preferred embodiments 3 to about 18 electrodes are used, such as 3 to 16, or from about 3 to 15, or from 4 to 12, or from 5 to 10, or from 6 to 8. Any one or more of the electrodes/wires can be selectively energized to achieve a particular treatment result. Further, the separation distance between electrically conductive surfaces, such as electrically conductive wires and/or electrodes, can range from about 0.2 mm to about 4 mm, such as ranging from about 0.3 mm to about 3 mm, or from about 0.4 mm to about 2 mm, or from about 0.5 mm to about 1 mm, or from about 0.8 mm to about 4 cm, such as from about 0.9 mm to about 3 cm, or from about 1.2 cm to about 2 cm, or from about 1.5 cm to about 1.8 cm, and so on.
Additional parameters of protocols that can be used in embodiments of the invention are provided in U.S. Published Patent Application Nos. US 2007/0043345, 2009/0269317, 2011/0106221, 2012/0109122, 2013/0184702, 2013/0345697, 2014/0039489, and 2015/0088120, as well as in U.S. Pat. Nos. 8,926,606, 8,992,517, 8,814,860, 8,465484, the disclosures of each of which are hereby incorporated by reference in their entireties.
The following Examples serve to further illustrate the invention.
Example 1 presents a bimodal enhanced ablation mechanism (BEAM) platform that uses one or more bursts of high frequency electric fields which have been specifically optimized to modulate intracellular effects in cancer cells while sparing healthy tissue. An optimal burst contains constitutive pulses with durations approximately equivalent to the charging time of the cell membrane plus the discharge time of the nuclear envelope. This novel concept is expanded upon further in the following sections and has implications for targeting specific cancer types without the need of external markers but with similar specificity to pharmaceutical compounds.
Example 2 presents the in-vitro effects of high frequency bi-polar bursts. Individual pulses within the burst are separated by 2 μs and sequential pulses alternate in polarity. The bursts are repeated once per second for 80 seconds and each burst exposes cells to the applied voltages for 100 μs. To demonstrate the effects of these pulses on the cell membrane and intracellular organelles, the inventors present a finite element model of a cell including a nuclear envelope. The charging behavior of the lipid-bilayer and nuclear envelope is evaluated in response to pulses between 250 ns and 50 μs. A parametric analysis is conducted on the intra- and extracellular conductivity, nucleus-to-cytoplasm ratio, and pulse-to-pulse delay time. In-vitro experiments are presented to confirm the non-thermal nature of the protocol and demonstrate irreversible electroporation within this intermediate pulse-width range.
In Example 3, the inventors explored the pulse-duration space between 250 ns and 100 μs and calculated the lethal electric field intensity for specific bimodal enhanced ablation mechanism (BEAM) protocols using a 3D tumor mimic. The inventors found that the nominal lethal thresholds for bursts containing 0.25, 0.5, 1, 2, 5, 10, and 50 μs pulses were 2022, 1687, 1070, 755, 640, 629, and 531 V/cm, respectively. A murine tumor model was used to investigate the effectiveness of BEAM in vivo. Tumors were exposed to 200 bursts, each energized for 100 μs, containing individual pulses 1, 2, or 5 μs in duration. In all treatment groups, average tumor growth was substantially inhibited versus control. 6 of 14 treated mice had no measurable signs of tumors 30 days after treatment and all protocols were able to achieve complete regressions. This work shows the potential for BEAM to be used as a focal therapy and merits its investigation in larger pre-clinical models.
In Example 4, the inventors report a physical treatment method based on electrical disruption of cells, whose action depends strongly on cellular morphology. Interestingly, numerical modeling suggests that while outer lipid bilayer disruption induced by long pulses (˜100 μs) is enhanced for larger cells, short pulses (˜1 μs) preferentially result in high fields within the cell interior, which scale in magnitude with nucleus size. Because enlarged nuclei represent a reliable indicator of malignancy, this presents one method for preferentially targeting malignant cells. While the inventors demonstrate killing of both normal and malignant cells using pulsed electric fields (PEFs) to treat spontaneous canine GBM, properly tuned PEFs can be used to provide targeted ablation based on nuclear size. Using 3D hydrogel models of normal and malignant brain tissues, which permit high-resolution interrogation during treatment testing, the inventors confirmed that PEFs could be tuned to preferentially kill cancerous cells. Finally, the inventors estimated the nuclear envelope electric potential disruption needed for cell death from PEFs. The results may be useful in safely targeting the therapy-resistant cell niches that cause recurrence of GBM tumors.
A numerical model of a cell in suspension was created in Comsol 4.2a. Two schemes were used to model the cell as a membrane covered sphere. In the first model, individual domains were created representing the sample fluid (external to cell), cell membrane, and cytoplasm (internal to cell). The 5 nm thick spherical shell domain representing the cell membrane required significant modification to the default meshing parameters and resulted in a large number of tetrahedral elements. Briefly, the entire geometry was assigned a single mesh with a predefined density of ‘Extremely course’. The values for the default parameters were then changed for minimum element size (0.00025), maximum element growth rate (1.2), resolution of curvature (0.04), and resolution of narrow regions (0.0001) to successfully mesh the geometry with 817,184 tetrahedral elements. A computer with a quad core 3.0 GHz processor and 8 GB of ram required 15 hours of computation time to solve a 14 μs transient model with 1,092,902 degrees of freedom (results shown in
To model the effects of bursts of bipolar square waves and effects on the nuclear membrane, a more efficient impedance boundary condition model was used. In this method, a cubic domain represented the experimental media and two spheres represented the domains for the cytoplasm and nucleoplasm, respectively. For each domain, a separate Electric Currents physics module was used and the dependent voltage variables were defined as Vmedia, Vcyto, Vnuc for the media, cytoplasm, and nucleoplasm domains, respectively. Variables were then defined to calculate the cell membrane (TMP) and nuclear membrane (nTMP) as (Vmedia−Vcyto) and (Vcyto−Vnuc), respectively. In the Electric Currents module, the boundaries representing membranes were defined as impedance boundary conditions with reference voltages prescribed as the voltage in the adjacent domain. In the Media domain, the boundary representing the cell membrane was defined as an impedance boundary with reference voltage of Vcyto. The layer specification was defined as a ‘thin layer’ and the electrical conductivity, relative permittivity, and surface thickness were defined using the values presented in the table in
In the impedance boundary condition model, the mesh was defined as a single Free Tetrahedral group with ‘Normal’ sized elements resulting in 17,825 tetrahedral elements. In a preliminary study of this model, an additional mesh refinement step (Number of refinements=2) was also taken. With refinement, this computation of the same 14 μs simulation was completed in 27 minutes. Without refinement, the computation time was further reduced to 14 minutes. When compared to the physical boundary model, both impedance boundary configurations sufficiently reproduced similar results. The unrefined impedance boundary condition model was used to conduct the remaining parametric studies.
Analytical Modeling
In order to investigate the effects of a bi-modal sine wave on electroporation, an analytical model was implemented that solved the Laplace equation in the frequency domain for a spherical cell with a concentric nucleus (Yao, C. G., et al., Study of transmembrane potentials of inner and outer membranes induced by pulsed-electric-field model and simulation. IEEE Trans Plasma Sci, 2007. 35(5): p. 1541-1549). Each cellular region was characterized by both a dielectric permittivity and conductivity, ensuring TMP and nTMP computational accuracy with frequencies is the MHz range. Briefly, solutions were obtained by merging a low-frequency (250 kHz) and high-frequency (1 MHz) electric field in the time domain, converting the signal to the frequency domain by taking the Laplace Transform, multiplying the signal by a transfer function representing the geometric and dielectric properties of the cell (Kotnik, T. and D. Miklavcic, Theoretical evaluation of voltage inducement on internal membranes of biological cells exposed to electric fields. Biophysical Journal, 2006. 90(2): p. 480-491), and converting the result back to the time domain by taking the inverse Laplace transform. To illustrate the clinical benefits of the optimized burst, the Laplace equation was solved for two needle electrodes (Ø 1 mm) in an infinite tissue domain according to standard techniques. The electrodes were spaced 0.1 cm apart and the applied voltage was set to 20 kV.
Cell Preparation and Experimentation
MDA-MB-231 human breast cancer cells were suspended in buffer with conductivity of 0.1 S/m at a concentration of 2.5×106 cells/ml. A custom pulse generation system capable of delivering 1000 VPeak in each polarity was used to create electric field intensities of approximately 1000, 2000, and 4000 V/cm across cell suspensions in 1 mm or 2 mm electroporation cuvettes. MDA-MB-231 cells were exposed to 90 bursts consisting of 200 bipolar square wave pulses 700 ns wide separated by 1.8 μs of dead time, shown in
Additional experiments were conducted with PPT8182 murine primary pancreatic tumor cells suspended in a buffer at a concentration of 5×106 cells/ml with a media conductivity of 0.2 S/m. 100 μL of cell suspension were added to a 2 mm gap cuvette and 80 bursts with 50 microseconds on time in each polarity (100 μs total) were applied. Within each burst, individual pulses had on times of 250 ns, 500 ns, 1 μs, 2 μs, 5 μs, 10 μs, or 50 μs with a 2 μs delay between the end of a pulse and the beginning of the next pulse in the opposite polarity. The cells were exposed to electric fields with magnitudes of 1500 V/cm, 3000 V/cm, and 4000 V/cm.
Numerical Modeling (Outer Membrane)
Interestingly, previous experimental observations (Sano, M., J. Caldwell, and R. Davalos, A Low Frequency Contactless Dielectrophoresis Platform for Particle Isolation and Enrichment. 2011: USA; Sano, M. B., J. L. Caldwell, and R. V. Davalos, Modeling and Development of a Low Frequency Contactless Dielectrophoresis (cDEP) Platform to Sort Cancer Cells from Dilute Whole Blood Samples. Biosensors & Bioelectronics, 2011; and Sano, M. B., et al., Contactless Dielectrophoretic Spectroscopy: Examination of the Dielectric Properties of Cells Found in Blood. Electrophoresis, 2011. DOI 10.1002/elps.2201100351) showed some degree of electroporation of cells below the first crossover frequency of their Clausius-Mossotti factor, but minimal electroporation above this frequency (i.e. cells were electroporated while experiencing negative DEP, but minimally impacted when experiencing positive DEP). Analysis of the results shown in
As the conductivity of the media is increased, the charging time of the cell membrane decreases until the media conductivity reaches 1.0 S/m. Above this threshold, increases in media conductivity negligibly impact the TMP charging time. At 0.1 S/m, the −3 dB frequency is not reached until approximately 100 kHz. At 1.0 and 10.0 S/m, this frequency is shifted higher to approximately 300 kHz. If higher conductivity buffers are used in in-vitro experiments, then the frequency range should also be shifted to avoid damaging cell membranes.
Numerical Modeling (Nuclear Membrane)
When a cell is exposed to a pulsed electric field, the capacitive nature of the cell membrane blocks the flow of current through the cell when fully charged. However, the membrane cannot charge instantaneously and there is a brief time when ions and molecules are rearranging and current flows through the cytoplasm of the cell. This displacement current increases the transmembrane potential of membranes surrounding the nucleus and organelles. These cellular components are much smaller than the cell and their theoretical maximum TMP within the same electric filed decreases linearly with their effective radius. Additionally, as a fully charged cell membrane blocks the flow of current through the cytoplasm, these internal membranes can charge for a period less than the TMP charging time.
For very short on-time pulses, the nuclear membrane has not fully discharged before the positive pulse returns to zero. This diminishes the maximum negative nTMP achievable. For 500 ns on time pulses, the first positive nTMP reaches 0.35 V while the first negative nTMP reaches −0.47 V. This effect is further enhanced if the positive nTMP is given sufficient time to decay back to zero before the positive pulse is turned off. When the pulse length is increased to 3.5 and 4 μs, the nTMP reaches a maximum magnitude of 0.62 V, nearly double the value achieved by a single mono-polar pulse.
It appears that to maximize the nTMP, the optimal pulse on time is equivalent to the charging time of the cell membrane plus the discharge time of the nuclear membrane. This allows the nTMP to charge up, then return to zero before it is forced negative at the falling edge of the pulse. Similarly, the optimal off time is approximately equivalent to the charging time of the cell membrane. This allows the nTMP to be increased to its maximum opposite polarity value, without decaying, just as the second pulse is initiated.
According to embodiments of the invention, the nucleus-to-cytoplasm ratio can be determined by obtaining the nuclear area and the cytoplasmic area of selected types of cells and determining the ratio of the areas. One way to obtain the nuclear and cytoplasmic areas is to obtain a biopsy of a substance to be treated, such as a tissue, and measure the nuclear area and the cytoplasmic area of selected cells. The ratio of the nuclear and cytoplasmic areas can then be determined for the selected cells. Treatment protocols can be optimized based on the difference between the nucleus-to-cytoplasm ratio of cells selected as targets for the treatment and other non-target cells. For example, treatment protocols can be designed to apply electrical pulses in a manner that would treat or otherwise have an effect on certain cells but not others. The treatment parameters can be selected such as to have a desired effect (e.g., kill such cells) on target cells that have a certain nucleus-to-cytoplasm ratio or higher but have no effect (e.g., no killing) or a different effect on cells that have a nucleus-to-cytoplasm ratio lower than that of the target cells.
Analytical Modeling
The clinical benefit of the bi-modal sine wave presented above is illustrated in
Experimental Results
An extensive parametric study (
Analysis of
Conclusion
The present inventors showed that the frequency of the applied field and the conductivity of the suspending medium play a large role in the buildup of the TMP. In the case where electroporation is not desirable, the lowest conductivity physiologically suitable buffer should be used. Even at 0.01 S/m, the TMP will increase if continuous sine wave voltages are applied between DC and approximately 10 kHz. The extent of electroporation (both reversible and irreversible) diminishes significantly above this frequency if the field strength is held constant. This allows for significant optimization of the temporal properties of the applied electric field to maximize the effect on intracellular components.
If electroporation is desirable, as in the case of tissue ablation, it is advantageous to operate within a sufficiently conductive media. Numerical analysis of the charging times for the cell membrane indicates that 1.0 S/m is a critical conductivity. Above this, the charging time does not increase significantly, while below this, the cell may not reach its maximum TMP for short pulses. However, physiological tissue typically has conductivity between 0.1 and 0.7 S/m. The result is that short duration pulses have a mitigated effect on the cell membrane while having an enhanced effect on intracellular components. Theoretically, bursts of 4 μs pulses with 500 ns off time will result in the largest effect on the nuclear transmembrane potential and may help to further increase the lethality of the high frequency pulses.
Cells form a complex resistor-capacitor (RC) network with the fluid/media surrounding the cell. The capacitive nature of the cell membrane (C) couples with the resistance of the extracellular material (R), limiting the rate at which the cell membrane will charge to its theoretical maximum potential. In general, smaller cells have a smaller net capacitance. This in turn allows them to charge more quickly than larger cells. This charging behavior blocks the flow of current through the intracellular cytoplasm. The smallest cells in a volume of tissue will experience the smallest intracellular effects of any pulsed field. It has been shown that highly metastatic cells have a higher membrane capacitance, due to changes in the morphology of the cell membrane, even when compared to non-cancerous cell of the same size. The result of this biophysical change is that infiltrative cancerous cells will exhibit a larger net capacitance than the surrounding healthy cells. This in turn results in a longer time constant associated with the charging of the cell membranes of infiltrative cells. This lag in cell membrane charging results in an increase in charge build up on intracellular components leading to an amplified electroporation effect on the nucleus and organelles.
The result of this is that a therapeutic electric field can be applied to a volume of tissue containing healthy and cancerous cells such that only the cancerous cells in the tissue will receive the therapeutic effect. This can be selective electroporation of intracellular components for drug, gene, or protein delivery or specific triggering of an apoptotic cascade. These pulses can also be designed such that a specific volume of tissue experiences irreversible electroporation and an additional external volume will experience a targeted cancer-cell-only apoptosis inducing dose. This later scenario allows for selective targeting of infiltrative cells, such as microscopic disease, embedded in healthy tissue surrounding a tumor.
Methods:
Numerical Modeling
A numerical model of a cell in suspension was created in COMSOL 4.2 using an impedance boundary condition scheme (G. Pucihar, T. Kotnik, B. Valič, D. Miklavčič, Numerical determination of transmembrane voltage induced on irregularly shaped cells, Annals of Biomedical Engineering, 34 (2006) 642-652). The solution domain consisted of a three dimensional cube with edge-lengths of 0.1 mm. At the center of this domain, two spheres were created representing the cytoplasm and nucleoplasm. Within the solution domain, the Electric Currents module was used to solve for following equations:
where U is the electric potential, E is the electric field, J is the current density, and Q is the current source. One boundary was assigned a time dependent electrical potential
U=U(t)/V [4]
The opposing boundary was assigned as the relative ground
U=0V [5]
The remaining boundaries were defined as electrical insulation
n·J=0/(A/m) [6]
where n is the normal vector to the surface, J is the electrical current.
For each domain (media, cytoplasm, nucleoplasm), a separate Electric Currents physics module was used and the dependent electric potential variables Umedia, Ucyto, Unuc for the media, cytoplasm, and nucleoplasm domains were defined, respectively. These variables were then defined to calculate the voltage across the cell membrane (Um) and nuclear envelope (Un)
Um=Umedia−Ucyto/V [7]
Un=Ucyto−Unuc/V [8]
In each Electric Currents module, the boundaries representing membranes were defined as impedance boundary conditions with reference voltages prescribed as the electric potential in the adjacent (Uref) domain
where σ is the conductivity, c is the permittivity, and d is the thickness of the cell membrane or nuclear envelope. For example, in the Media domain, the boundary representing the cell membrane was defined as an impedance boundary with reference potential of Ucyto. In the Cytoplasm domain, the same boundary representing the cell membrane was defined as an impedance boundary with a reference potential of Umedia. The boundary was defined as a ‘thin layer’ and the electrical conductivity, relative permittivity, and surface thickness were defined using the values presented in Table 1. The nuclear envelope consists of two individual lipid membranes separated by the perinuclear space. To limit the complexity of the model and avoid improperly assessing the electrical properties of these individual components (not readily available in the literature), the inventors lumped these biological features into a single 40 nm membrane for which electrical properties representing their combined features are available.
The mesh was defined as a single free tetrahedral group with the elements between 1.8 and 10 μm on edge, resulting in 19353 tetrahedral elements. In a preliminary study of this model, finer and courser meshes were used. Simulation times more than doubled between successive refinements. The average deviation between the mesh presented here and the next successive refinement was less than 2.0% and 5.5% for the cell membrane and nuclear envelope potentials, respectively. For each parameter, solutions were found in approximately 22 minutes on a on a quad core 3.0 GHz processor with 8 GB of RAM. Results of the numerical simulations, using the values in the table in
Cell Preparation and Experimentation
In all experiments, cells were suspended in a buffer consisting of a 5.5:1 ratio of culture media (DMEM) to low conductivity sucrose buffer (85 g sucrose, 3.0 g glucose, 7.25 mL RPMI, and 992.75 mL DI water) (L. A. Flanagan, J. Lu, L. Wang, S. A. Marchenko, N. L. Jeon, A. P. Lee, E. S. Monuki, Unique dielectric properties distinguish stem cells and their differentiated progeny, Stem Cells, 26 (2008) 656-665). The electrical conductivity of the cell suspension was measured with a conductivity meter prior to experimentation (Horiba B-173, Cole-Parmer, Vernon Hills, Ill.) to ensure a final conductivity of 0.2 S/m. Clark et al. reported that the conductivity of pancreatic tissue varied between 0.097 and 0.44 S/m for frequencies between 1 kHz and 2 MHz, respectively (D. Clark, J. Greenwell, A. Harper, A. M. Sankey, T. Scratcherd, The electrical properties of resting and secreting pancreas, The Journal of Physiology, 189 (1967) 247-260). A media conductivity of 0.2 S/m was chosen to minimize the current delivered through the sample while maintaining a conductivity value within the range of those found in in-vivo tissue. Due to limitations in the inventors' pulse generation system, higher conductivity buffers would drive the pulse delivery system outside of its safe operating region.
PPT8182 murine primary pancreatic tumor cells (J. von Burstin, S. Eser, M. C. Paul, B. Seidler, M. Brandl, M. Messer, A. von Werder, A. Schmidt, J. Mages, P. Pagel, E-cadherin regulates metastasis of pancreatic cancer in vivo and is suppressed by a SNAIL/HDAC1/HDAC2 repressor complex, Gastroenterology, 137 (2009) 361; von Burstin, 2009) were used in all experiments. These cells have been shown to replicate human pancreatic cancer in terms of histology, metastasis, and genetic alterations (von Burstin, 2009; B. Seidler, A. Schmidt, U. Mayr, H. Nakhai, R. M. Schmid, G. Schneider, D. Saur, A Cre-loxP-based mouse model for conditional somatic gene expression and knockdown in vivo by using avian retroviral vectors, Proceedings of the National Academy of Sciences, 105 (2008) 10137-10142; D. Saur, B. Seidler, G. Schneider, H. Algül, R. Beck, R. Senekowitsch-Schmidtke, M. Schwaiger, R. M. Schmid, CXCR4 expression increases liver and lung metastasis in a mouse model of pancreatic cancer, Gastroenterology, 129 (2005) 1237-1250; and M. J. PaszeK, N. Zahir, K. R. Johnson, J. N. Lakins, G. I. Rozenberg, A. Gefen, C. A. Reinhart-King, S. S. Margulies, M. Dembo, D. Boettiger, Tensional homeostasis and the malignant phenotype, Cancer cell, 8 (2005) 241-254).
Cells were cultured in DMEM (supplemented with L-glutamine, ATCC, Manassas, Va.) containing 10% fetal bovine serum (Sigma Aldrich, St. Louis, Mo.) and 1% stock solution of penicillin/streptomycin (Invitrogen, Carlsbad, Calif.) at 37° C. in 5% CO2 in a humidified atmosphere. All cells were harvested for experiments by trypsinization at 80% confluence. Suspensions were centrifuged twice and resuspended in an experimental buffer at a concentration of 5×106 cells/mL. 100 μL of cell suspension were injected into a 2 mm gap cuvette (Model 620, Harvard Apparatus, Holliston, Mass.) immediately prior to pulse delivery. A schematic of the experimental setup is shown in
The protocol for all experiments used the waveform presented in
For the in vitro studies, each of the treatment groups was repeated a minimum of three times (n=3) add experiment for each group were conducted on at least two different days. For each treatment, different experimental parameters, including sham exposure, were alternated in a random sequence. After treatment, samples were split into two equal 50 μL samples to be evaluated at 1 and 24 hour time points. The samples were kept at room temperature for approximately 20-30 minutes prior to being placed on ice (1 hour group) or moved to the incubator (24 hour group) while the remaining experimental groups were completed. Approximately one hour post exposure, viability was assessed using a trypan blue exclusion assay. Cells which had been irreversibly electroporated were unable to exclude the dye and were stained blue. Cells were counted visually using a hemocytometer and the percentage viability was determined as
The average viability of sham control samples in the 1 hour time group was greater than 85%. Samples to be analyzed at 24 hours were placed in separate wells in a 12-well pate containing a total of 1 mL of culture media and maintained at room temperature until the well plate was full (approximately 30 minutes). At this point the well plate was placed in an incubator at 37° C. and 5% CO2 for 24 hours. Viability was then assessed using an Alamar blue metabolism assay (Life Technologies, Grand Island, N.Y.) using the manufactures recommended procedure. Briefly, 100 μL/mL stock Alamar blue solution was added to each well. After 4 hours, the samples were read using a spectrophotometer at 570/600 nm wavelengths. For each sample, the absorbance was measured in three separate wells and averaged. Additional measurements were taken for sample media without cells and for control cell samples which were not exposed to an electric field. The percentage viability was determined as
where I is the relative intensity measurement from the spectrophotometer. In general, trypan blue analysis and metabolism assays complement each other quite well. They et al. previously showed that metabolism assays mirrored those from trypan blue analysis after nano-second pulsed electric field exposure (B. L. they, A. G. Pakhomov, B. W. Gregory, V. A. Khorokhorina, C. C. Roth, M. A. Rassokhin, J. A. Bernhard, G. J. Wilmink, O. N. Pakhomova, Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells, Biochimica Et Biophysica Acta-General Subjects, 1800 (2010) 1210-1219). The Alamar blue assay used in this study is well established for measuring cytotoxicity in mammalian cells (J. O'Brien, I. Wilson, T. Orton, F. Pognan, Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity, European Journal of Biochemistry, 267 (2000) 5421-5426). Reduction rates for cells seeded between 2.5×103 and 2×106 cells/mL were measured to ensure that the sham population did not completely reduce the Alamar blue solution (results not shown) and a 4 hour incubation time with 2.5×105 cells/mL was determined to be optimal. Viability data for both the 1 hour and 24 hour groups were normalized to the sham control groups. Statistical analysis of the data was completed using JMP Pro V. 10.0 (SAS Institute Inc., Cary, N.C.).
Electronics
Waveforms were generated using an arbitrary function generator (AFG3021C, Tektronix Inc., Beaverton, Oreg.), which were amplified by a custom built high voltage pulse generator capable of +/−1000V outputs through high impedance loads (Applied Energetics, Tucson, Ariz., USA). Output waveforms were visualized using an oscilloscope (DPO2002B, Tektronix Inc., Beaverton, Oreg.) after the voltage was attenuated using a 50 MHz 1000× high voltage probe (P5210A, Tektronix Inc., Beaverton, Oreg.) and the current was measured using an active clamp on 50 MHz current probe (TCP305, Tektronix Inc., Beaverton, Oreg.). Short circuit protection resistors on the output limited the maximum output voltage through the 2 mm cuvettes to approximately 800 V (4000 V/cm).
Results and Discussion
Numerical Modeling
As shown in
As the positive polarity pulse falls, the cell membrane begins to discharge resulting in a second current flow within the cytoplasm in the opposite direction, as compared to the rising pulse edge. This results in the formation of a negative potential across the nuclear envelope. This negative potential reaches a minimum of −0.28 V and falls below −70 mV in a similar 0.94 μs. The rising edge of the negative polarity pulse creates a similar decrease in Un creating an interesting double peak in the membrane potential of the nuclear envelope. This second peak reaches a value of −0.29 V. Though this peak is only 10 mV different than the maximum achieved by the initial pulse, it suggests that optimization of the pulse length and delay time between pulses could result in an increased effect on intracellular membranes.
In this Example, the inventors elected to disregard the effects of electroporation on the cell membrane to simplify their analysis. However, in the case of electroporation, current would be allowed to flow through the cytoplasm and a sustained potential would be induced across the intracellular membranes.
Analysis of Experimental Parameters
It has been observed that pore formation behavior occurs within 1 μs after Um is elevated above 1V, quenching further increases in potential (K. Kinosita, I. Ashikawa, N. Saita, H. Yoshimura, H. Itoh, K. Nagayama, A. Ikegami, Electroporation of cell membrane visualized under a pulsed-laser fluorescence microscope, Biophysical Journal, 53 (1988) 1015-1019), after which new pore formation is limited and pore expansion takes over as the dominant phenomena (K. Kinosita, T. Y. Tsong, Formation and resealing of pores of controlled sizes in human erythrocyte membrane, (1977); K. Kinosita, T. Y. Tsong, Voltage-induced pore formation and hemolysis of human erythrocytes, Biochimica et Biophysica Acta (BBA)-Biomembranes, 471 (1977) 227-242). At the field strengths presented here, pulses 1 μs in duration and shorter may not efficiently result in pore expansion within the cell membrane (O. M. Nesin, O. N. Pakhomova, S. Xiao, A. G. Pakhomov, Manipulation of cell volume and membrane pore comparison following single cell permeabilization with 60- and 600-ns electric pulses, Biochimica et Biophysica Acta (BBA)-Biomembranes, 1808 (2011) 792-801).
The conductivity of the sample media,
The delay between positive and negative polarity pulses,
The role of DNA damage in the PEF apoptotic cascade is not fully understood and the nucleus is not typically the target for PEF therapy. However, intrinsic and extrinsic apoptotic cell death processes are associated with field strength dependent effects on mitochondria and the endoplasmic reticulum. If waveform optimization can be used to double the increase in the transmembrane potential of these organelles, as shown in
Analysis of Cell Electrical Properties
Electrical properties for the cell membrane, nuclear envelope, cytoplasm, and nucleoplasm are readily available in the literature (B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts, J. D. Watson, Molecular Biology of the Cell, 3rd edition, Garland Science, New York, 1994; P. R. Gascoyne, R. Pethig, J. P. Burt, F. F. Becker, Membrane changes accompanying the induced differentiation of Friend murine erythroleukemia cells studied by dielectrophoresis, Biochimica et Biophysica Acta (BBA)-Biomembranes, 1149 (1993) 119-126; J. Yang, Y. Huang, X. J. Wang, X. B. Wang, F. F. Becker, P. R. C. Gascoyne, Dielectric properties of human leukocyte subpopulations determined by electrorotation as a cell separation criterion, Biophysical Journal, 76 (1999) 3307-3314; I. Ermolina, Y. Polevaya, Y. Feldman, B.-Z. Ginzburg, M. Schlesinger, Study of normal and malignant white blood cells by time domain dielectric spectroscopy, Dielectrics and Electrical Insulation, IEEE Transactions on, 8 (2001) 253-261; J. Gimsa, T. Müller, T. Schnelle, G. Fuhr, Dielectric spectroscopy of single human erythrocytes at physiological ionic strength: dispersion of the cytoplasm, Biophysical Journal, 71 (1996) 495-506; and K. Asami, Y. Takahashi, S. Takashima, Dielectric properties of mouse lymphocytes and erythrocytes, Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1010 (1989) 49-55). Subuncu et al. report that a cytoplasmic conductivity of between 0.3 and 0.6 S/m (A. C. Sabuncu, J. A. Liu, S. J. Beebe, A. Beskok, Dielectrophoretic separation of mouse melanoma clones, Biomicrofluidics, 4 (2010) 021101). Labeed et al. report increases in conductivity from 0.28 S/m to 0.45 S/m as cells begin to undergo apoptosis (F. H. Labeed, H. M. Coley, M. P. Hughes, Differences in the biophysical properties of membrane and cytoplasm of apoptotic cells revealed using dielectrophoresis, Biochimica et Biophysica Acta (BBA)-General Subjects, 1760 (2006) 922-929). Ron et al. report a conductivity of 0.724 S/m and 0.93 S/m for pre-osteoblast cells and normal canine kidney cells, respectively (A. Ron, R. R. Singh, N. Fishelson, I. Shur, R. Socher, D. Benayahu, Y. Shacham-Diamand, Cell-based screening for membranal and cytoplasmatic markers using dielectric spectroscopy, Biophysical chemistry, 135 (2008)) 59-68. Mulhall el al. found cytoplasm conductivities of 0.71, 0.42, 0.26, and 0.25 S/m for normal keratinocytes, abnormal keratinocytes, for two different malignant keratinocytes, respectively (H. Mulhall, F. Labeed, B. Kazmi, D. Costea, M. Hughes, M. Lewis, Cancer, pre-cancer and normal oral cells distinguished by dielectrophoresis, Analytical and Bioanalytical Chemistry, 401 (2011) 2455-2463). Additionally, Chen et al. show that drug resistant cells have a lower cytoplasmic conductivity than non-drug resistant cells (J. Chen, Y. Zheng, Q. Tan, E. Shojaei-Baghini, Y. L. Zhang, J. Li, P. Prasad, L. You, X. Y. Wu, Y. Sun, Classification of cell types using a microfluidic device for mechanical and electrical measurement on single cells, Lab on a Chip, 11 (2011) 3174-3181). These results provide evidence of decreasing cytoplasmic conductivity as cells transition from benign to malignant.
Yuan et al. show an increase in nucleus-to-cytoplasm (NCR) ratio from 0.45 to 0.49 and from 0.40 to 0.49 as cancer cells achieve drug resistance. Similarly, Helczynska et al. show histologically, that the NCR increases from 0.3 to 0.8 as a function of tumor grade, with higher NCRs for increasingly malignant cancers (K. Helczynska, Å. Kronblad, Å. Jögi, E. Nilsson, S. Beckman, G. Landberg, S. Påhlman, Hypoxia promotes a dedifferentiated phenotype in ductal breast carcinoma in situ, Cancer Research, 63 (2003) 1441-1444). Salmanzadeh et al. showed that the specific membrane capacitance of a syngeneic cell line increased from 15.39 mF/m2 to 26.42 mF/m2 as the cells became successively more malignant (A. Salmanzadeh, M. B. Sano, R. C. Gallo-Villanueva, P. C. Roberts, E. M. Schmelz, R. V. Davalos, Investigating dielectric properties of different stages of syngeneic murine ovarian cancer cells, Biomicrofluidics, 7 (2013) 011809). This translates into an increase in relative membrane permittivity from 8.70 to 14.92.
A parametric analysis was conducted using cytoplasmic conductivity values of 0.7, 0.475, and 0.25 S/m, an NCR of 0.3, 0.55, and 0.8, and a membrane permittivity of 9, 12, and 15 to represent this transition from benign to intermediate to metastatic, respectively. The present inventors modeled the response of a ‘benign’ cell having cytoplasmic conductivity of 0.7 S/m, NCR of 0.3, and membrane permittivity of 8.7. A ‘metastatic’ cell was modeled as having cytoplasmic conductivity of 0.25 S/m, NCR of 0.8, and a membrane permittivity of 15. All other values (Table 1) were held constant.
The nucleus-to-cytoplasm ratio (NCR),
ΔU=1.5rE cos θ/V [13]
where r is the radius of the nucleus and E is the electric field which the cell is exposed to. However, other dielectric properties of the nucleus may affect the membrane charging time (T. Kotnik, D. Miklavčič, Theoretical evaluation of voltage inducement on internal membranes of biological cells exposed to electric fields, Biophysical Journal, 90 (2006) 480-491; K. H. Schoenbach, S. J. Beebe, E. S. Buescher, Intracellular effect of ultrashort electrical pulses, Bioelectromagnetics, 22 (2001) 440-448). As the NCR increases in
Numerical Simulation of Experimental Pulses
The simulation results of
For a 1 μs experimental pulse, |Um| reaches a maximum of 1.24 V while |Un| reaches a maximum of 0.32 V. The magnitude of Um for this experimental pulse is approximately equal to the ideal value predicted in
As the pulse length increases, the initial Un response is allowed to fall back towards zero. The result is that for longer pulses, the negative going edge and subsequent ringing have an increased effect. For similar field strengths, a 5 μs pulse results in Un amplitude change from 0.24 V to 0.36 V, a 50% increase (not shown). For these cases, the peak amplitude of the ringing is 46-52% that of the pulse amplitude and lasts for less than 200 ns.
Experimental Results
Experiments were conducted with an initial sample temperature between 22 and 25° C. At 4000 V/cm all experimental groups resulted in a temperature rise less than 3.5° C. Representative temperature profiles for experiments with 50 μs and 250 ns constitutive pulses are shown in
Specifically, at 1500 V/cm, bursts containing 50 μs pulse (2×) resulted in a 1 hour post-treatment viability of 31% which reduced to 3% after 24 hours. The 1500 V/cm bursts containing pulses between 250 ns (400×) and 10 μs (10×) resulted in 1 hour viabilities above 50% and notably, pulses 2 μs (5×) and shorter had viabilities of 85% or greater, similar to sham treatments. In between the 1 and 24 hour time-points, the viability fell by an average of 20% for cells exposed to 1500 V/cm over all constituent pulses. For this field strength, bursts containing 10 μs pulses had the largest change in viability over 24 hours, 49%, while 250 and 500 ns pulses resulted in a negligible change in viability compared to controls. Significant changes in viability occurred between the 1 and 24 hour time points for bursts with pulses 2 μs and longer. It is interesting that 10 and 50 μs pulses resulted in delayed cell death, however, the mechanism of action is unclear.
Cell viability was significantly lower for 3000 V/cm versus 1500 V/cm bursts when the pulse duration was 1 μs or longer. After 24 hours, the viability for 2 to 50 μs pulses reduced to less than 5% at 3000 V/cm. Between 3000 V/cm and 4000 V/cm, the most significant impact on viability occurred for 500 ns pulses. For all field strengths, 250 ns pulses have a minimal impact on cell viability.
For bursts containing 250 ns pulses, the difference in viability after 1500, 3000, and 4000 V/cm treatments was not statistically significant (α≤0.1). All other pulse-widths had a statistically significant difference between the 1500 V/cm and 3000 V/cm treatments at each timepoint (α≤0.06). Between the 3000 and 4000 V/cm treatments, 5 μs (1 hour), 500 ns (1 hour), and 500 ns (24 hour) groups had statistically different viabilities (α≤0.03)
Interestingly, this study shows that viability is not directly correlated to the energy dose delivered. This conforms to the results presented by others that electropermeabilization (A. Maček-Lebar, D. Miklavčič, Cell electropermeabilization to small molecules in vitro: control by pulse parameters, Radiology and Oncology, 35 (2001)) and lethal (B. L. Ibey, A. G. Pakhomov, B. W. Gregory, V. A. Khorokhorina, C. C. Roth, M. A. Rassokhin, J. A. Bernhard, G. J. Wilmink, O. N. Pakhomova, Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells, Biochimica Et Biophysica Acta-General Subjects, 1800 (2010) 1210-1219) effects of mono-polar pulses of different pulse widths exhibit a complex relationship that cannot be correlated to the quantity of energy delivered alone. The inverse correlation between pulse length and toxicity presented may be related to the cell membrane charging time, calculated here as between 1.11 and 7.92 μs.
At 1500 V/cm, none of the pulse durations elevated Un above thresholds of 0.5, 0.75, or 0.9 V (
For all bursts containing pulses 1 μs in duration or longer, the viability at 3000 V/cm after 24 hours is lower than the corresponding viability at 4000 V/cm after one hour. This has interesting implications for in-vivo applications as it indicates that ablation sizes may grow over time and that immediate observation may be inadequate to predict the total volume treated. From the numerical simulations, it is anticipated that cells with a larger cytoplasm-nucleus ratio will achieve higher Un amplitudes than cells of similar size with a smaller ratio. A high nucleus-cytoplasmic ratio (NCR) has been associated with the aggressiveness of malignant cells and is used as a parameter in grading cancers (K. Seibert, S. M. Shafie, T. J. Triche, J. J. Whang-Peng, S. J. O'Brien, J. H. Toney, K. K. Huff, M. E. Lippman, Clonal variation of MCF-7 breast cancer cells in vitro and in athymic nude mice, Cancer research, 43 (1983) 2223-2239; Y. Shimizu, S. Kamoi, S. Amada, F. Akiyama, S. G. Silverberg, Toward the development of a universal grading system for ovarian epithelial carcinoma, Cancer, 82 (1998) 893-901; A. Malpica, M. T. Deavers, K. Lu, D. C. Bodurka, E. N. Atkinson, D. M. Gershenson, E. G. Silva, Grading ovarian serous carcinoma using a two-tier system, The American journal of surgical pathology, 28 (2004) 496-504; and S. G. Silverberg, Histopathologic grading of ovarian carcinoma: A review and proposal, Inter. J. of Gynecological Pathology, 19 (2000) 7-15).
Additionally, it has been shown that an increase in invasiveness and metastatic potential has been correlated to cell membrane ruffling, which leads to higher membrane capacitances in aggressive cells (A. Salmanzadeh, M. B. Sano, R. C. Gallo-Villanueva, P. C. Roberts, E. M. Schmelz, R. V. Davalos, Investigating dielectric properties of different stages of syngeneic murine ovarian cancer cells, Biomicrofluidics, 7 (2013) 011809; A. Salmanzadeh, H. Kittur, M. B. Sano, P. C. Roberts, E. M. Schmelz, R. V. Davalos, Dielectrophoretic differentiation of mouse ovarian surface epithelial cells, macrophages, and fibroblasts using contactless dielectrophoresis, Biomicrofluidics, 6 (2012) 024104; and A. Salmanzadeh, E. S. Elvington, P. C. Roberts, E. M. Schmelz, R. V. Davalos, Sphingolipid Metabolites Modulate Dielectric Characteristics of Cells in a Mouse Ovarian Cancer Progression Model, Integr. Biol., (2013)). In numerical simulations (
Conclusions
The present inventors found, through finite element simulations, that the charge-discharge behavior of the cell membrane impacts the electric field experienced by intracellular components. This simplified model has some limitations. Cells were modeled as simple spheres to reflect the shape of the cells in their non-adhered state. In vivo, cells typically take on more complex, elongated, or spindled shapes which can alter the effects of pulsed electric fields on transmembrane potential. Additionally, cells in tissue are affected by local inhomogeneity and the responses of cells in their immediate vicinity which was not accounted for here.
Cytoplasm-nucleus ratio, cytoplasm conductivity, and cell membrane permittivity play a significant role in the charging characteristics of the nuclear envelope. Experimentally the inventors found that bursts of bi-polar square waves increased the media temperature less than 3.5° C. when the total energized time per burst was held constant at 100 μs and eighty bursts were delivered. The resulting cellular responses are therefore limited to those related directly to non-thermal phenomena. For the bursts of bi-polar pulses presented, there exists an inverse correlation between pulse-width and toxicity despite the delivery of equal quantities of energy. The changes in cellular viability over 24 hours post treatment show presence of both instantaneous and delayed cell death processes, however, the exact mechanisms are unknown.
To the best of the inventors' knowledge, this is the first experimental parametric analysis on the effects of bi-polar square wave bursts with pulses between 0.25 and 50 μs. In the 3000 V/cm treatment groups, cell viability was reduced to 4.0%, 0.5%, 0.3%, and 1.0% for bursts containing 2, 5, 10, and 50 μs pulses, respectively. In the 4000 V/cm treatment groups, cell viability was reduced to 3.8%, 1.4%, 0.9%, 0.8%, and 0.8% for bursts containing 1, 2, 5, 10 and 50 μs pulses, respectively. Rubinsky et al. (J. Rubinsky, G. Onik, P. Mikus, B. Rubinsky, Optimal Parameters for the Destruction of Prostate Cancer Using Irreversible Electroporation, The Journal of Urology, 180 (2008) 2668-2674) showed that ten 100 μs monopolar pulses at 2000 V/cm resulted in a viability of 70%. In the same study, they showed that seventy-five 100 μs monopolar pulses at 250 V/cm resulted in a viability of 10-20% while ninety 100 μs monopolar pulses at 250 V/cm reduced viability to 0-10%. Arena et al. (C. B. Arena, C. S. Szot, P. A. Garcia, M. N. Rylander, R. V. Davalos, A Three-Dimensional In vitro Tumor Platform for Modeling Therapeutic Irreversible Electroporation, Biophysical Journal, 103 (2012) 2033-2042 (“Arena et al., 2012”)) showed that after eighty 100 μs monipolar pulses at 1500 V/cm, cell viability was approximately 8% and this protocols is consistent with those currently being employed successfully in clinical applications of irreversible electroporation in the prostate (G. Onik, B. Rubinsky, Irreversible Electroporation: First Patient Experience Focal Therapy of Prostate Cancer, in: B. Rubinsky (Ed.) Irreversible Electroporation, Springer Berlin Heidelberg, 2010, pp. 235-247), pancreas (R. C. Martin II, K. McFarland, S. Ellis, V. Velanovich, Irreversible electroporation therapy in the management of locally advanced pancreatic adenocarcinoma, Journal of the American College of Surgeons, 215 (2012) 361-369), and liver (R. Cannon, S. Ellis, D. Hayes, G. Narayanan, R. C. Martin, Safety and early efficacy of irreversible electroporation for hepatic tumors in proximity to vital structures, Journal of Surgical Oncology, (2012)). The comparable level of toxicity resulting from the bi-polar burst protocol presented here indicates that it may be advantageous in in-vivo therapies where muscle contractions due to longer duration mono-polar pulses are undesirable.
Materials and Methods
Collagen Hydrogel Tumor Mimics
PPT8182 murine primary pancreatic tumor cells (von Burstin, 2009), shown to replicate human pancreatic cancer in terms of histology, metastasis, and genetic alterations (von Burstin, 2009; Seidler, B., et al. A Cre-loxP-based mouse model for conditional somatic gene expression and knockdown in vivo by using avian retroviral vectors. Proceedings of the National Academy of Sciences 105, 10137-10142 (2008); Saur, D., et al. CXCR4 expression increases liver and lung metastasis in a mouse model of pancreatic cancer. Gastroenterology 129, 1237-1250 (2005); PaszeK, M. J., et al. Tensional homeostasis and the malignant phenotype. Cancer cell 8, 241-254 (2005); and Szot, C. S., Buchanan, C. F., Freeman, J. W. & Rylander, M. N. 3D in vitro bioengineered tumors based on collagen I hydrogels. Biomaterials 32, 7905-7912 (2011)) were used in the 3D tumor platform experiments. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with L-glutamine (ATCC, Manassas, Va.) containing 10% fetal bovine serum (FBS; Sigma Aldrich, St. Louis, Mo.) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, Calif.) at 37° C. in 5% CO2 in a humidified atmosphere. All cells were harvested for experiments by trypsinization at 80% confluence.
Electronics and Protocols
A custom pulse generation system was used to deliver bursts of bi-polar pulses with constitutive pulse widths of 250 ns, 500 ns, 1 μs, 2 μs, 5 μs, 10 μs, and 50 μs. A 500Ω resistor was placed in parallel with the load to ensure proper pulse shaping and to protect against delivering pulses to an open circuit. Custom electrodes were made from hollow 1.27 mm diameter dispensing needles (Howard Electronic Instruments Inc., El Dorado, Kans.) with a 2.0 mm edge-to-edge separation distance.
A pilot study was conducted at 540 Vpeak and a total energized time of 100 μs for all pulse widths. This protocol used 400, 200, 100, 50, 20, 5, or 2 pulses to comprise a burst, with individual pulse durations of 250 ns, 500 ns, 1 μs, 2 μs, 5 μs, 10 μs, or 50 μs, respectively. The ablation zones at 540 Vpeak for bursts containing pulses 1 μs or less were not well formed ovals surrounding the electrodes. Instead, dead cells occupied small triangular zones which extended, but did not connect between the two electrodes. The electric field intensity changed rapidly in this zone resulting in large variations in the calculation of electric field thresholds. To avoid this, a higher voltage of 650 V was used for the 250 ns, 500 ns, 1 μs and 2 μs groups. To facilitate comparison between groups, a simplified electrical dose formula was used.
Dose=V2*Tp*n*N[V2s] [14]
where V is the applied voltage, Tp is the pulse width, n is the number of pulses per burst, and N is the number of bursts per treatment which was held a constant 80. The 540 Vpeak group had an approximate dose of 2300 V2s. At 650 Vpeak, 256, 128, 64, and 32 pulses were used for the 250 ns, 500 ns, 1 μs, and 2 μs groups, respectively. This resulted in an approximate dose of 2200 V2s. An additional 2 μs group at 250 Vpeak with 216 pulses an approximate dose of 2000 V2s was also conducted to compare effects of energy and lethal electric field threshold.
To explore the effect of burst energized time, a set of experiments were conducted with 80 bursts containing 2 μs pulses at 540V. Pulses were repeated 2, 24, or 50 times per burst with a 2 μs inter-pulse delay. To compare ‘diffuse’ and ‘burst’ delivery of pulses an additional group of 50 pulses per second was tested. In this group, one positive and one negative pulse were delivered, with a 2 μs inter-pulse delay, every 20 ms for a total of 80 seconds. This is the only group presented in which a 1 second inter-burst delay was not used.
To explore the effect of treatment time, a set of experiments were conducted with eight bursts. These groups had 2 μs, 50 μs, and 100 μs pulses which were repeated 50, 2, and 1 times per burst, respectively. The experimental parameters are summarized in the table in
Sample Processing
At 24 hours after treatment, normal culture media was replaced with 2.5 mL of media supplemented with 4 μM Calcein AM (live stain, λem=515 nm, Invitrogen, Eugene, Oreg.) and incubated at 37 C for 30 minutes. Five minutes prior to visualization, the media was supplemented with 75 μL of 1.5 mM propidium iodide (PI; dead stain, λem=617 nm, Invitrogen, Eugene, Oreg.) for 5 minutes. Finally, the hydrogels were rinsed with PBS to flush out any unabsorbed dyes and increase the signal to noise ratio. A Leica DMI 6000 fluorescent microscope with a 20× objective (Leica Microsystems Inc., Buffalo Grove, Ill.) was used to tile a set of images and reconstruct an entire plane of the treated scaffolds just under the surface.
Analysis of Electric Field Thresholds in Tissue Mimics
Finite element models were created in COMSOL Multiphysics (Version 4.2a, COMSOL Inc., Burlington, Mass.). The collagen hydrogels were modeled as a 3 mm thick cylinder with a 5 mm radius and conductivity of 1.2 S/m. Cylinders representing the 1.27 mm outer diameter electrodes were offset such that their edge-to-edge distance was equal to 2 mm. Within the solution domain, the Electric Currents module was used to solve for the following equations:
where U is the electric potential, E is the electric field, J is the current density, Q is the current source, a is the conductivity, ∈r is the relative permittivity, and ∈0 is the permittivity of free space. The boundaries surrounding one electrode were assigned a constant electrical potential
U=U[V] [15]
The boundaries of the other electrode were assigned as a relative ground
U=0/V [5]
The remaining boundaries were defined as electrical insulation
n·J=0/(A/m) [6]
where n is the normal vector to the surface, J is the electrical current.
Changes in temperature due to Joule heating were calculated for 540 V and 100 energized time over 80 seconds using a modified duty cycle approach (Arena, C. B., et al. High-Frequency Irreversible Electroporation (H-FIRE) for Non-thermal Ablation without Muscle Contraction. Biomed Eng Online 10(2011); Neal, R. E., 2nd, Garcia, P. A., Robertson, J. L. & Davalos, R. V. Experimental Characterization and Numerical Modeling of Tissue Electrical Conductivity during Pulsed Electric Fields for Irreversible Electroporation Treatment Planning. IEEE Trans Biomed Eng 59, 1076-1085 (2012)). The temperature distribution (T) was obtained by transiently solving a modified heat conduction equation:
where τ is the pulse duration, P is the period of the pulses, k is the thermal conductivity, c is the specific heat at constant pressure, and p is the density. Outer boundaries were treated as convective cooling
with an exterior temperature (Text) of 22° C. and a heat transfer coefficient (h) of 25 (W m−2 K−1). Intermediate time stepping was used to ensure that at least one time step was taken each second. Simulations at 540 V showed that thermal effects resulted in a negligible impact on the electric field distribution and changes in conductivity due to temperature increases were neglected in subsequent models to minimize computational time. Changes in conductivity due to electroporation were similarly neglected due to the low concentration of cells within the scaffold. To replicate the values measured experimentally, the voltage on one electrode was swept between 470-700V, in steps of 10V, and the other was held at ground.
Tiled images near the surface of the hydrogels (representative examples in
Murine Tumor Model
This study was approved by the Virginia Tech Institutional Animal Care and Use Committee. 6-7 week old Hsd:Athymic Nude-Foxn1nu male mice (Harlan, Dublin, Va.) were inoculated subcutaneously in the dorsolateral flank region with human glioblastoma cells (DBTRG-05MG) while anesthetized by inhalation of 3% isoflurane (Abbott Laboratories, Abbott Park, Ill.). Mice were housed in individually ventilated cages in groups of five under specific pathogen free conditions and allowed access to sterilized water and food ad libitum. Prior to inoculation, cells were cultivated using standard techniques in DMEM (High-glucose supplemented with L-glutamine; Thermo Scientific, Logan, Utah) containing 10% FBS and 1% penicillin/streptomycin. Upon reaching 80% confluence, cells were suspended at a concentration of 5×106 cells/mL in an 85/15 mixture of PBS and Matrigel (BD Biosciences, San Jose, Calif.). 200 μL aliquots of this final suspension was used for each injection (1×106 cells total).
Tumor growth was measured over time using calipers, and volumes (v) were calculated according to the modified ellipsoid formula (Jensen, M. M., Jorgensen, J. T., Binderup, T. & Kjaer, A. Tumor volume in subcutaneous mouse xenografts measured by microCT is more accurate and reproducible than determined by 18FFDG-microPET or external caliper. BMC medical imaging 8, 16 (2008)):
where l is the length of the longitudinal diameter and w is the width of the transverse diameter. Tumors were treated when the greatest diameter reached approximately 5 mm; treatment groups are shown in the table in
Following treatment, topical antibiotic ointment was applied to the needle insertion wounds. Mice were removed from anesthesia and provided 5 mg/kg ketoprofen analgesic diluted in 1 mL sterile saline solution for recovery. The mice were euthanized 30 days post-treatment or earlier for humane reasons if the tumor volume reached 800 mm3.
Samples of any present tumor tissue were excised and sectioned for processing. Representative tissues were preserved in 10% neutral buffered formalin and embedded in paraffin. Formalin preserved paraffin embedded samples were sectioned and processed for histology using hematoxylin and eosin (H&E) staining. All photomicrographs were obtained with a Leica DMI 6000 inverted microscope.
Results
BEAM Treatment Pulse Width, Pulse Number, and Total Energized Time Affect the Lethal Electric Field Threshold
Typical IRE treatments involve the delivery of 80 monopolar pulses, each 100 μs in duration at a repetition rate of 1 Hz. Using the PPT8182 cell line and the same tissue mimic, Arena et al (Arena et al., 2012) found that the lethal threshold for this standard protocol is 501 V/cm.
The temperature profiles measured were well correlated to those predicted numerically (
Treatments with 8 and 80 bursts were conducted for bursts with 2 and 50 μs pulses. For comparison, treatments with either 8 or 80 monopolar pulses 100 μs in duration were conducted (
To explore the limitations of the inventors' equivalent dose approximation, eighty bursts held constant with 2 μs pulses were delivered at three different voltages: 250, 540, and 650 V. For these cases, each burst contained 216, 50, and 32 pulses, resulting in approximate doses of 2000, 2300, and 2200 V2s, respectively. The threshold for cell death for these treatments were 663, 718, and 822 V/cm (
For bursts with 2 μs pulses, when the voltage was held constant at 540 V, but the energized time per burst was decreased from 100 to 48 or 4 μs, the electric field threshold was found to increase from 718 V/cm to 855 and 1110 V/cm, respectively (
BEAM Treatment Inhibits Tumor Growth In Vivo
At the time of treatment, tumors were on average 91, 101, 45, and 44 mm3 for the sham, 5 μs, 2 μs, and 1 μs groups. Thirty days post-treatment, these averages had changed to 332, 62, 16, and 44 mm3 (
Immediately following in vivo treatment, whitening of the tumor occurred. This is associated with reduced blood flow and the beginning stages of edema (
Discussion
For bursts of bipolar pulses, the electric field threshold required to induce cell death is inversely correlated to the duration of the constitutive pulses (
When the cells are in suspension, they take on a more spherical appearance. In contrast, when grown in the 3D tissue mimics they begin to stretch out and obtain a more natural phenotype. In vivo, IRE is typically observed in regions which are exposed to approximately 500-750 V/cm (Garcia, P. A., et al. Intracranial Nonthermal Irreversible Electroporation: In vivo Analysis. Journal of Membrane Biology 236, 127-136 (2010); Miklavčič, D., Šemrov, D., Mekid, H. & Mir, L. M. A validated model of in vivo electric field distribution in tissues for electrochemotherapy and for DNA electrotransfer for gene therapy. Biochimica et Biophysica Acta (BBA)-General Subjects 1523, 73-83 (2000); and Edd, J. F., Horowitz, L., Davalos, R. V., Mir, L. M. & Rubinsky, B. In vivo results of a new focal tissue ablation technique: irreversible electroporation. Biomedical Engineering, IEEE Transactions on 53, 1409-1415 (2006)) and the field strengths predicted in these 3D tissue mimics are more likely to represent the in vivo thresholds for bipolar bursts. However, extensive in vivo evaluation is still needed to determine how these thresholds compare to those necessary to ablate complex heterogeneous tissues such as pancreatic tumors which contain healthy and malignant cells, vasculature, ductile systems, and connective tissue.
Electro-gene (EGT) and ECT protocols typically employ 8 pulses with the goal of permeabilizing the cell membrane, but not inducing cell death.
Protocols with 1 μs, 500 ns, and 250 ns failed to produce connected lesions in the tissue mimics when the voltage was set to 540 V and the energized time per burst was 100 μs. This made it difficult to accurately calculate the lethal electric field threshold. In the inventors' initial pilot study, the inventors found that increasing the voltage to 650 V while delivering 80 pulses with 100 μs energized time resulted in thermal denaturing of the collagen matrix. Arena et al. (Arena et al., 2012) associated collagen denaturation during IRE with temperatures greater than 45° C. Reducing the energized time to 64 μs at 650 V, a similar dose to 540 V and 100 μs, resulted in well-formed oval shaped lesions for all groups. The present inventors used this higher voltage, equivalent dose protocol for all groups with 1 μs pulses and shorter.
In
It is unclear how far outside this range (250-650 V) the equivalent dose hypothesis is valid. However, clinical IRE systems are currently limited to outputs of 2700 V. At this voltage, a burst energized for 4 μs would have an equivalent dose and a lethal threshold of approximately 750 V/cm (the average of values from
Previous in vivo IRE experiments on murine tumor models required the application of pulses with 1000 Vpeak amplitude or greater to obtain complete regression of similar sized tumors. Neal et al. (Neal II, R. E., et al. Treatment of breast cancer through the application of irreversible electroporation using a novel minimally invasive single needle electrode. Breast cancer research and treatment 123, 295-301 (2010)) achieved complete regression in 5 of 7 mice when 100 monopolar pulses, each 100 μs in duration and 1300 Vpeak (5600 V/cm) were applied through a bi-polar probe with a 2.3 mm electrode spacing. Al-Sakere et al. (Al-Sakere, B., et al. Tumor ablation with irreversible electroporation. PloS one 2, e1135 (2007)) achieved complete regression in 12 of 13 mice when 80 pulses, each 100 μs in duration and 1000 (2500 V/cm) were applied between plate electrodes spaced 4 mm apart.
To mimic the clinical protocol, treatments in this study were applied through two needle electrodes. A spacing of 0.4 mm was used to maximize coverage of the tumors while accounting for the 1000 Vpeak limit of the inventors' pulse generation system. The 0.4 mm diameter electrodes used in these in vivo experiments were significantly smaller than the 1 mm diameter electrodes used clinically and the 1.27 mm electrodes used in the tumor mimics. Electrode diameter is closely linked to the electric field distribution and smaller electrodes will produce a smaller ablation zone. To account for this, the number of bursts delivered was increased to 120 to provide the best possible outcomes while avoiding extensive thermal heating effects. Gross and histological examination did not indicate any scar formation from thermal damage.
In the treated groups, the measured tumor volume increased over the first 1-5 days post treatment. The formation of a scab along with the occurrence of edema may have led to an overestimation of tumor volumes during short-term follow-up. Within two weeks after treatment delivery, scabs resolved and evidence of tumor regression was observable.
This treatment protocol inhibited tumor growth. The average tumor volumes in the treatment groups were significantly smaller than control at the end of the study. Due to the limited time-span of the IACUC protocol, it is unclear if the tumors would have entered an exponential growth period post-treatment and the inventors were unable to obtain Kaplan-Meier survival curves. In total, 6 of 14 treated mice had no measurable signs of tumors 30 days after treatment and all protocols were able to achieve some complete regressions. Future work should include a long-term study to monitor tumor regression over the lifetime of the animals.
Histological examination of some treated animals revealed pockets of neoplastic cells superficial to the muscle fascia in the dermal layers, which is indicative of under treatment. It is possible that better regression results can be obtained by using a protocol with a higher applied voltage, increased number of bursts, and/or higher energized time per burst. It is noted that the work presented by Al-Sakere did not obtain a 100% regression rates, however, their protocol has been successfully adapted to human clinical applications with promising results.
Conclusion
This study shows the differences in lethal threshold for IRE and BEAM protocols. Despite delivering equivalent doses, bursts with shorter constituent pulses typically require higher electric field strengths for ablation. The number of bursts, energized time per burst, and pulse duration are all significant factors affecting the lethal threshold. Using 80 bursts the inventors found that 1, 2, and 5 μs pulses had electric field thresholds of 1070, 755, and 640 V/cm. When 200 bursts were delivered in vivo, these pulses had similar effects on tumor volume. All mice treated with BEAM tolerated the therapy well and experienced a significant reduction in tumor volume when compared to untreated controls. Each group attained at least one complete regression. This study provides strong evidence that BEAM can be used for tumor ablation and future investigation is warranted.
Methods
Cell Culture
U-87 MG primary human glioblastoma cells (ATCC), D1TNC1 rat astrocyte cells (ATCC), and C6 rat glioblastoma cells (ATCC) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS) at 37° C. in 5% CO2 in a humidified incubator. Normal Human Astrocyte (NHA) cells (Lonza) were cultured in Astrocyte Growth Media (Lonza) at 37° C. in 5% CO2 in a humidified incubator. Cells were seeded in hydrogels at a density of 1×106 cells/mL. The hydrogels were submerged in appropriate growth media for the cell type at 37° C. in 5% CO2 in a humidified incubator and cell viability was maintained within hydrogels for up to 7 days (
Construction of 3D Collagen Scaffolds
Stocks of type I collagen were prepared by dissolving rat tail tendon in acetic acid, followed by freezing and lyophilization as described previously (Arena et al. 2012). Two different stock solution concentrations of collagen were created: 4.5 mg/mL and 30 mg/mL. Scaffolds with a final concentration of 2 mg/mL and 20 mg/mL were made from concentrated collagen stocks to create collagen gels of 0.2% (w/w) and 2% (w/w). Neutralized collagen solutions were created by mixing acid-dissolved collagen with 10×DMEM (10% of total collagen solution volume) and sufficient volumes of 1N NaOH until a pH in the range of 7.0-7.4 was achieved. The neutralized collagen was mixed with cells suspended in DMEM to achieve a cell density of 1×106 cells/mL in the final collagen mixture. Solutions were mixed carefully with a spatula to ensure homogenous distribution throughout the gel without damaging cells. Collagen solutions were then dispensed into a polydimethylsiloxane (PDMS) mold with a cut-out of 10 mm diameter and 1 mm depth and molded flat to ensure consistent scaffold geometry. The inventors' previous mathematical modeling and experiments on oxygen (O2) consumption rates by tumor cells (Verbridge, S. S. et al. Oxygen-Controlled Three-Dimensional Cultures to Analyze Tumor Angiogenesis. Tissue Engineering. Part A 16, 2133-2141, doi:10.1089/ten.tea.2009.0670 (2010) (“Verbridge et al., 2010”)) confirms that at this cell density and scaffold thickness, O2 concentration is uniform throughout the scaffold depth. Collagen was allowed to polymerize at 37° C. and 5% CO2 for 45 minutes.
Construction of 3D Alginate Scaffolds
Calcium alginate gels were created using the same PDMS molds as for collagen, creating discs 10 mm in diameter and 1 mm in thickness. Two alginate gel stock concentrations (0.4% and 4.0% (w/v) were prepared using powdered alginate (Protanal LF 10/60, FMC BioPolymer) that was dissolved in buffer, dialyzed, frozen and lyophilized, followed by re-constitution in serum-free DMEM, as the inventors have previously reported (Verbridge, S. S. et al. Oxygen-Controlled Three-Dimensional Cultures to Analyze Tumor Angiogenesis. Tissue Engineering. Part A 16, 2133-2141, doi:10.1089/ten.tea.2009.0670 (2010)). Alginate concentrations were chosen to span a wide range in mechanical stiffness, similar to the collagen concentrations used. Alginate solutions were mixed with cells at a density of 1×106 cells/mL and dispensed into PDMS molds and molded flat with a porous membrane. Alginate hydrogels were cross-linked by submerging under 0.1M CaCl2 dispensed over a porous membrane cover for 45 min. The alginate hydrogels were then cultured in 24 well plates with DMEM supplemented with 10% FBS and 1% PS at 37° C., 5% CO2.
Determination of Shape Factors
U-87, NHA, D1TNC1, and C6 cells were individually seeded in hydrogels of one of the four conditions described previously (0.2%, 2% collagen, 0.4%, 4% alginate). After culturing the cells for 24 hours, the hydrogels were fixed using 4% formalin and blocked and permeabilized using 40 mg/mL bovine serum albumin (BSA) and 0.5% Triton-X. Cellular actin was stained with Alexa Flour 568 phalloidin (Life Technologies, Carlsbad, Calif.) while cell nuclei were stained with diaminophenylindole (DAPI; Sigma-Aldrich, St. Louis, Mo.). Cells were visualized using a Zeiss LSM510 (Carl Zeiss Microscopy LLC, Thornwood, N.Y.) laser scanning confocal microscope. The stained cells were then used to determine cellular shape factors for cells in each of the four conditions. Image analysis was done in Image J (NIH, Bethesda, Md.) to determine the nuclear area, nuclear perimeter, cytoplasmic area, cytoplasmic perimeter, and longest and shortest diameter of the cell. Measurements were made on at least four cells per hydrogel and at least 5 hydrogels were analyzed for each condition.
Live Fluorescent Imaging
U-87 cells were cultured under normal culture conditions and incubated for 16 hours with CellLight Nucleus-RFP, Bacman 2.0 (Molecular Probes, Eugene, Oreg.) and CellLight Tubulin-GFP (Molecular Probes, Eugene, Oreg.) added to the media at a concentration of 10 particles per cell. Cells were then passaged and seeded into hydrogels of a final concentration of 0.2% collagen at a density of 1×106 cells/mL. After cells were cultured in collagen hydrogels for 24 hours, electroporation of hydrogels was performed on the stage of a Zeiss Observer Z1 microscope (Carl Zeiss Microscopy LLC, Thornwood, N.Y.) to allow for imaging during treatment. Images were taken of single cells immediately before pulsing treatments were started and then every 30 seconds for 5 minutes after pulsing began. Cells were imaged upon exposure to IRE treatment or BEAM treatment. Cells that were not exposed to pulses were also imaged as a control.
Electroporation of 3D Scaffolds
Pulsed electroporation experiments were performed in hydrogels with constant electrical properties. The electrical conductivities of each of the gel-cell mixtures were measured with a conductivity meter to ensure similar electrical properties (0.98±0.04 S/m). The IRE pulses were generated using an ECM 830 pulse generator (Harvard apparatus, Holliston, Mass.) and delivered to the tissue through custom electrodes. High-frequency pulses were delivered using a custom-built pulse generation system (INSPIRE 2.0, VoltMed Inc., Blacksburg, Va.). Two solid stainless steel cylinders with diameters of 0.87 mm, separated 3.3 mm edge-to-edge, were used as electrodes.
Treatments were performed delivering a total of 50 square pulses (IRE) or 50 bursts of 1 μs pulses (BEAM). The IRE protocol delivered 100 μs pulses with a repetition rate of 1 pulse per second. In the BEAM protocol, a burst consisting of 100×1 μs pulses with a 5 μs inter-pulse delay was delivered with a repetition rate of 1 burst per second. For IRE treatments, the pulse amplitude was set to 450 Vpeak while for BEAM treatments 700 Vpeak was used to produce ablations of approximately the same volume as the IRE group.
Finite Element Analysis in Hydrogels
Finite element models using COMSOL Multiphysics (Version 4.3, COMSOL Inc., Palo Alto, Calif.) were used to solve the Laplace equation to find the electric field distribution within the hydrogels for each different voltage used. COMSOL Multiphysics was also used to solve the Joule heating equation to calculate the temperature distribution in the hydrogel as a result of each treatment. The simulation geometry was modeled as a 10 mm diameter and 1 mm thick cylinder with two steel electrode cylinders (d=0.87 mm) spanning the depth of the hydrogel. The mesh was refined until error between successive refinements was less than 1%. The final mesh contained 47,438 elements and solutions were found in approximately 3 minutes on a Pentium i3 processor.
Finite Element Analysis of Individual Cells
The transmembrane potentials across the cell membrane and nuclear envelope were modeled using a finite element model with an impedance boundary condition scheme (Sano, M. B., Arena, C. B., DeWitt, M. R., Saur, D. & Davalos, R. V. In-vitro bipolar nano- and microsecond electro pulse bursts for irreversible electroporation therapies. Bioelectrochemistry 100, 69-79, doi:DOI 10.1016/j.bioelechem.2014.07.010 (2014)). These finite element models were used to numerically investigate the response of representative cell geometries to simulated IRE and BEAM pulses. Cell geometry was determined based on average measurements made in ImageJ image analysis software (NIH, Bethesda, Md.) from confocal microscopy images. Geometries for U-87 cells in two different collagen densities (0.2%, 2%) as well as four different cell types (U-87, NHA, C6, D1TNC1) in a 0.2% collagen matrix were used. All models were solved using a 2D-axisymmetric platform in COMSOL Multiphysics. A separate electric currents physics module was used for each domain (media, cytoplasm, nucleoplasm). A large media domain, with sides of 300 μm, was used to avoid any significant boundary effects. The cell and the nucleus were modeled as half-ovals where their lengths and widths were varied according to measurements from confocal microscopy images.
Simulations were solved in the time-domain using an electric currents module. To account for the resistance and capacitance posed by the cell membrane and the nuclear envelope the boundaries of the nucleus and cytoplasm were assigned impedance properties based on the existing literature.
Determination of Lethal Thresholds
The thresholds for cell death were determined by first performing a live-dead stain on the hydrogels 24 hours after delivering treatment. Live cells were stained with Calcein AM (Biotium, Hayward, Calif.) and fluoresced as green while dead cells were stained with ethidium homodimer III (Biotium, Hayward, Calif.) and fluoresced as red. The diameter of the red-stained dead region was measured using ImageJ image analysis software. Geometric measurements of the ablation zones were mapped to a finite element model to calculate the electric field during treatments of the scaffolds (
In Vivo Canine Treatment
All canine in vivo studies were approved by the institutional animal care and use committee (08-218-CVM). IRE treatments were performed in the brains of anesthetized normal canine subjects, and in dogs with spontaneous malignant gliomas according to previously described methods (Edd, J. F. & Davalos, R. V. Mathematical Modeling of Irreversible Electroporation for Treatment Planning. Technology in Cancer Research & Treatment 6, 275-286, doi:10.1177/153303460700600403 (2007) (“Edd and Davalos, 2007”); Garcia, P. A. et al. Non-Thermal Irreversible Electroporation (N-TIRE) and Adjuvant Fractionated Radiotherapeutic Multimodal Therapy for Intracranial Malignant Glioma in a Canine Patient. Technology In Cancer Research & Treatment 10, 73-83 (2011); Rossmeisl, J. H., Garcia, P. A., Roberston, J. L., Ellis, T. L. & Davalos, R. V. Pathology of non-thermal irreversible electroporation (N-TIRE)-induced ablation of the canine brain. Journal of Veterinary Science 14, 433-440, doi:10.4142/jvs.2013.14.4.433 (2013) (“Rossmeisl et al., 2013”)). In tumor-bearing dogs, biopsy of the brain lesion was performed prior to IRE ablation to allow for histopathological diagnosis and grading of tumors, and an additional biopsy of the ablated region obtained within 24 hours of the IRE to characterize the effects of the IRE treatment.
Histomorphological Staining
Archived, paraffin embedded, transversely oriented brain sections from normal and tumor-bearing dogs treated with IRE were retrieved, cut at 5 μm thickness, mounted on positively charged slides, and stained routinely with hematoxylin and eosin (Edd and Davalos, 2007; Rossmeisl et al., 2013). Digital photomicrographs of regions of interest representing IRE ablated regions of cerebral cortex, subcortical white matter, contralateral homologous cortical and white matter controls, and a canine GBM pre- and post-IRE treatment were captured with charge-coupled device digital camera (Nikon DS-Filc, Nikon, Japan) and commercial imaging analysis software system (NIS Elements AR, Nikon, Japan).
Statistical Analysis
Statistical significance was determined by a two-tailed t-test performed in Prism Statistical Software (Version 6, Graphpad, La Jolla, Calif.). A 95% confidence interval was used with significance defined as p<0.05. All numerical results are reported as the mean and the standard deviation of all experimental measurements. No outliers were excluded.
Results
Cell Size Selectivity of Pulsed Electric Fields
Single cell responses to electric field pulses were simulated with finite element modeling. Simulated TMP changes in response to modeled IRE pulses (
To experimentally explore the effect of cell size on electric field thresholds for cell death, the inventors tuned the mechanical and chemical structure of the tumor microenvironment using a three-dimensional GBM hydrogel tumor model (
Cell size and shape within hydrogel scaffolds are functions of scaffold density; by varying collagen density in the tissue model the inventors were able to control cell size and outer membrane perimeter for a single cell type. U-87 MG human GBM cells exhibited a significantly smaller area (p=0.005) in the higher density (2% w/w) collagen (920±249 μm2) as compared with lower density (0.2% w/w) collagen (1572±503 μm2) (
The inventors performed additional experiments in calcium alginate hydrogels, in which cell morphology is relatively constant for different scaffold densities due to the lack of cell-ECM binding sites (
In Vivo Selectivity of IRE
The inventors previously treated canine patients with naturally occurring malignant gliomas using IRE29. Histology from this treatment provides an important comparison point between the inventors' 3D in vitro ablation results presented here, and the in vivo outcome in a context that is highly representative of the human GBM phenotype. When untreated cerebrocortical grey matter (
Intracellular Effect of Pulsed Electric Fields
To examine the potential for BEAM pulses to exert their effect via intracellular localization of electric fields, the inventors performed finite element modeling of field distribution across a single cell. This model predicts that for a simulated IRE pulse with an electric field magnitude of 500 V/cm applied for 100 μs, only 14% of the external electric field traverses the cell membrane and is present in the cytoplasm (
Consistent with model predictions of IRE cell size dependence and nuclear size independence, the four cell types exhibited similar IRE lesions (
Death Mechanisms of IRE and BEAM
To investigate the differences between the mechanism of death with IRE and BEAM the inventors performed single cell imaging upon exposure to each treatment regime. Cell nuclei and tubulin were stained by live fluorescent stain and cultured in 3D collagen hydrogels. Fluorescent imaging in situ within these hydrogels was performed directly before, and then at 30-second intervals after exposure to IRE, revealing an outward diffusion of dye from the cell membrane within 1 minute after pulsing (
Estimate of Lethal Threshold for Nuclear Disruption
The inventors explored the relationship between BEAM lethal thresholds and nuclear size, leveraging the experimental data as input for subsequent mathematical models. Based on this data, it would appear that cell death occurs at a critical nTMP disruption that is independent of cell type, whereas the external field required for this nuclear disruption scales inversely with nucleus size. Using experimental findings for lethal thresholds, nuclear geometries, and idealized cell geometries for glioma cells and astrocytes, the inventors performed finite element modeling of single cell response to minimum lethal electric fields for each cell type. Simulating cell exposure to these lethal conditions, 1006 V/cm for NHA cells and 601 V/cm for U-87 cells, the inventors found a larger increase in TMP for the glioma cell than for the astrocyte (
Discussion
The overall goal was to leverage tissue engineered models of tumor versus normal brain microenvironments, based on previously published methods (Verbridge, S. S. et al. Oxygen-Controlled Three-Dimensional Cultures to Analyze Tumor Angiogenesis. Tissue Engineering. Part A 16, 2133-2141, doi:10.1089/ten.tea.2009.0670 (2010)), to investigate the response of representative cell geometries to IRE and BEAM pulses. These platforms critically provide a three-dimensional physiological tissue context in which to explore the effects of 3D cell morphology on response to electric fields, not possible with 2D experiments, while eliminating other confounding variables found in vivo. Hydrogels have been previously established as a relevant platform to test tissue responses to IRE pulses (Arena et al., 2012), while such models have also been demonstrated to better recapitulate human tumor physiology and therapy response as compared with 2D models (Fischbach, C. et al. Engineering tumors with 3D scaffolds. Nat Meth 4, 855-860, (2007); Fong, E. L. S. et al. Modeling Ewing sarcoma tumors in vitro with 3D scaffolds. Proceedings of the National Academy of Sciences 110, 6500-6505, doi:10.1073/pnas.1221403110 (2013)). With the ability to easily tune targeting parameters and microenvironment, these models provide a valuable tool for measuring the impact of cell morphology and tissue physics on therapy response broadly, and more specifically on response to therapeutic electric fields.
It is important to note that the inventors' work is informed by, and builds on their experience in treating spontaneous GBM in canine patients. Spontaneous, primary brain tumors are only relatively common in two species—dogs and humans. Human and canine brain tumors share many features, including histopathologic and diagnostic imaging characteristics, which allows application of World Health Organization pathologic classification and imaging based therapeutic response assessment schemes used in human clinical practice. Canine and human brain tumors have also been demonstrated to have similar expression patterns of growth factor receptors, chromosomal deletions, and losses of function of tumor suppressor genes. As tumors progress 5- to 7-fold faster in dogs relative to humans, dogs with spontaneous brain tumors are an attractive model for the faithful and rapid evaluation and translation of novel brain tumor therapeutics (Rossmeisl, J. H. New Treatment Modalities for Brain Tumors in Dogs and Cats. Veterinary Clinics of North America: Small Animal Practice 44, 1013-1038, doi:http://dx.doi.org/10.1016/j.cvsm.2014.07.003 (2014)).
Size selective ablation using PEFs has been previously reported for cell suspensions, differentiating tumor from blood cells based on large differences in size (Eppich, H. M. et al. Pulsed electric fields for selection of hematopoietic cells and depletion of tumor cell contaminants. Nature Biotechnology 18, 882-887, doi:http://dx.doi.org/10.1038/78504 (2000) (“Eppich et al., 2000”)), but has yet to be demonstrated for cells cultured in physiologically-relevant tissues. The inventors' experiments support the concept that IRE results in cell size-selective lethal thresholds into 3D tissues. The bulk electrical resistance properties of the cell-seeded hydrogels did not vary as a function of collagen density, and the inventors therefore believe differences measured are a result of cell morphology rather than altered tissue electrical properties. Control experiments performed in alginate further support this hypothesis that the differences observed in collagen resulted from cell size variations rather than additional factors such as direct sensing of matrix density. Although this finding does not eliminate the possibility that variation in binding ligand density may also impact lesion size, this size dependence is consistent with previously published data on cells in solution (Eppich et al., 2000). Furthermore this correlation of threshold with cell size is absent for BEAM. The inventors hypothesize that this is due to the BEAM field primarily interacting with the inner organelles of the cell. The inventors' finite element modeling confirms this hypothesis as a single BEAM burst applied to a single cell model produces a much higher field inside the cell than a simulated IRE burst. BEAM treatment delivers a rapid burst of over 100 of these 1 μs pulses. This allows BEAM pulses to preferentially charge intracellular membranes, which the inventors anticipated would have profound effects on cell death as a function of cell type.
The inventors' in vitro 3D model results demonstrate a statistically significant dependence of field threshold on cell size, however the cell size heterogeneity observed in vivo may prevent this dependence from being leveraged for targeting specificity. A much more obvious difference between cell types, clearly evident in the inventors' H&E staining of tumorous and healthy canine brain samples, is the enlarged nuclei of cancer cells compared to healthy brain tissue. Used as a pathological indicator of cancer, enlarged nuclei compared with their non-malignant counterparts is one of the most reliable distinguishing characteristics of tumor cells (Zink, D., Fischer, A. H. & Nickerson, J. A. Nuclear structure in cancer cells. Nat Rev Cancer 4, 677-687 (2004)), however the targeting of anti-cancer therapy against this hallmark has never been demonstrated.
The nucleus is typically the largest contiguous intracellular feature and a likely target for damage by the high intracellular fields produced by BEAM. To experimentally test the effect of nuclear area on treatments, the inventors chose different cell types, which exhibited differences in nuclear sizes without significant differences in plasma membrane area, eliminating confounding effects due to cell size. Numerical simulations identified increased nuclear size as an important variable for increased nTMP. An increase in nTMP could trigger cell death above a specific threshold, and therefore malignant cells should have a lower BEAM lethal threshold than normal cells, in contrast with IRE, which would not exhibit nuclear selectivity. The similarity of IRE thresholds is consistent with the fact that there was no significant difference in plasma membrane areas. The differences in BEAM lesion sizes supports that BEAM threshold differences are related to nucleus area as opposed to overall cell area, with lower lethal thresholds corresponding to larger nuclei. The intracellular field produced from BEAM seems to affect the intercellular nucleus membrane in a way at least partially analogous to the way IRE affects the plasma membrane, as a larger membrane exposed to the majority of the electric field is easier to affect than a smaller membrane.
Time-course images of single cells exposed to each treatment show a distinct difference in mechanism of killing between BEAM and IRE, consistent with the findings that different cellular characteristics are important variables with the two treatments. The time-course of cell death after IRE treatment strongly implicates the immediate disruption of the cell membrane as a cause of cell death, as tubulin proteins originally confined in the cell by the cell membrane begin diffusing out of the cell upon exposure to IRE. In contrast, cells exposed to BEAM show no diffusion from the outer cell membrane but rather a nuclear collapse while the tubulin is retained within the original cell area. These finding suggest that the outer membrane does not play as much of a role in the mechanism of cell death in BEAM, but rather that the primary effect is on the nucleus.
Given the inventors' results, it appears BEAM is acting on the biophysical structure of the cells in a way that nuclear area becomes a key variable. When glioma and astrocyte cells were simulated at their respective lethal BEAM thresholds (601 V/cm vs. 1006 V/cm), the inventors found similar TMP and nTMP ranges of approximately 150-250 mV and 100-130 mV, respectively. These simulations did interestingly predict a small difference in outer TMP as a function of nuclear size. However the magnitude of this TMP, approximately 150 mV, was significantly lower than the anticipated instantaneous threshold (1 V) for cell death by irreversible electroporation. Thus, it would appear that the primary mechanism of death with BEAM is not an increase in cell TMP, but rather is related to intracellular effects. For glioma and astrocyte cells, the maximum simulated nTMP of 130 mV is also well below the lethal threshold for death resulting from outer membrane disruption, suggesting that small disruptions of nTMP may significantly impact cell survival. It is unclear whether the pathway to cell death is dominated by effects on the nuclear envelope alone, versus in combination with cell membrane disruption, or a separate cascade of intracellular effects. However, the correlation of nTMP values between the two different cell types, at different lethal electric field strengths, indicates that nuclear area impacts the cell death process after BEAM treatment.
The inventors' mathematical model does have limitations, as outer cell membranes are approximated as elliptical, and do not account for the irregular shape of physiological cells, or heterogeneity in electrical properties of individual cells. Inclusion of membrane conductivity changes due to electroporation effects is expected to enhance the accuracy of the inventors' simulations. While experimental evidence also suggests that outer membrane electroporation is occurring during BEAM (at time-points beyond those in
It is important to note, the death mechanism of IRE and BEAM are not based on targeting the highly proliferative phenotype that is leveraged by many current GBM therapies including chemotherapy and tumor treating fields (Kirson, E. D. et al. Alternating electric fields arrest cell proliferation in animal tumor models and human brain tumors. Proceedings of the National Academy of Sciences 104, 10152-10157, doi:10.1073/pnas.0702916104 (2007)). While these therapies leave behind quiescent tumor initiating cells that cause recurrence, IRE and BEAM should elicit a death response through membrane disruption for both bulk tumor cells and tumor initiating cells. It is unlikely that this physical death mechanism would select for the emergence of resistant subpopulations on short timescales, because a large number of genetic mutations would likely be required to render a cell resistant to electric field-induced damage.
Though the exact mechanism of cell killing with BEAM is not yet known, the inventors' modeling and experimental data suggest a mechanism that is different than that of long IRE pulses which target the plasma membrane, and that, unlike for IRE, is cell type dependent among cells of similar size. The BEAM killing mechanism is such that the biophysical structure of malignant cells allows for the selective targeting of these cells using a range of electric field distributions that induce no damage to the healthy cells studied but elicit a death response in malignant cells. Because malignant cells that comprise the bulk tumor have a lower death threshold (˜530-810 V/cm) than normal astrocytes (˜930-1200 V/cm) surrounding the tumor, it follows that a treatment regime delivering a voltage between these two thresholds to the edge of the tumor may result in ablation of tumor cells while sparing healthy astrocytes. A threshold in such a range at the edge of the tumor may be effective at killing the invasive glioblastoma cells that render surgery to be an ineffective treatment for GBM, and infiltrative tumors more broadly.
Individual pulses with durations one to two orders of magnitude shorter than IRE pulses can kills cells in such a way that is less dependent on the outer cell diameter (assuming a similar size nucleus). The individual pulses are applied in alternating polarity to reduce muscle contractions. Additionally, the individual pulses are repeated to form a high-frequency burst, and multiple bursts are typically necessary to induce cell death. This is similar to how multiple, longer duration pulses are applied during an IRE treatment. This form of BEAM treatment typically requires a higher e-field threshold, but there is less dependence on cell size. Therefore, treatment planning is significantly reduced, as different cell types, regardless of their morphology, have the same e-field threshold.
In the theoretical example provided in
The present invention has been described with reference to particular embodiments having various features. In light of the disclosure provided above, it will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. One skilled in the art will recognize that the disclosed features may be used singularly, in any combination, or omitted based on the requirements and specifications of a given application or design. When an embodiment refers to “comprising” certain features, it is to be understood that the embodiments can alternatively “consist of” or “consist essentially of” any one or more of the features. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention.
It is noted in particular that where a range of values is provided in this specification, each value between the upper and lower limits of that range is also specifically disclosed. The upper and lower limits of these smaller ranges may independently be included or excluded in the range as well. The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is intended that the specification and examples be considered as exemplary in nature and that variations that do not depart from the essence of the invention fall within the scope of the invention. Further, all of the references cited in this disclosure are each individually incorporated by reference herein in their entireties and as such are intended to provide an efficient way of supplementing the enabling disclosure of this invention as well as provide background detailing the level of ordinary skill in the art.
The present application is a National Stage application under 35 U.S.C. § 371 of International Application No. PCT/US15/30429, filed May 12, 2015, which application relies on the disclosure of and claims priority to and the benefit of the filing date of U.S. Provisional Application No. 61/992,023 filed May 12, 2014, the disclosures of each of which are hereby incorporated by reference herein in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2015/030429 | 5/12/2015 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/175570 | 11/19/2015 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 1653819 | Northcott | Dec 1927 | A |
| 3730238 | Butler | May 1973 | A |
| 3746004 | Jankelson | Jul 1973 | A |
| 3871359 | Pacela | Mar 1975 | A |
| 4016886 | Doss et al. | Apr 1977 | A |
| 4037341 | Odle et al. | Jul 1977 | A |
| 4216860 | Heimann | Aug 1980 | A |
| 4226246 | Fragnet | Oct 1980 | A |
| 4262672 | Kief | Apr 1981 | A |
| 4267047 | Henne et al. | May 1981 | A |
| 4278092 | Borsanyi et al. | Jul 1981 | A |
| 4299217 | Sagae et al. | Nov 1981 | A |
| 4311148 | Courtney et al. | Jan 1982 | A |
| 4336881 | Babb et al. | Jun 1982 | A |
| 4344436 | Kubota | Aug 1982 | A |
| 4392855 | Oreopoulos et al. | Jul 1983 | A |
| 4406827 | Carim | Sep 1983 | A |
| 4407943 | Cole et al. | Oct 1983 | A |
| 4416276 | Newton et al. | Nov 1983 | A |
| 4447235 | Clarke | May 1984 | A |
| 4469098 | Davi | Sep 1984 | A |
| 4489535 | Veltman | Dec 1984 | A |
| 4512765 | Muto | Apr 1985 | A |
| 4580572 | Granek et al. | Apr 1986 | A |
| 4636199 | Victor | Jan 1987 | A |
| 4672969 | Dew | Jun 1987 | A |
| 4676258 | Inokuchi et al. | Jun 1987 | A |
| 4676782 | Yamamoto et al. | Jun 1987 | A |
| 4687471 | Twardowski et al. | Aug 1987 | A |
| 4716896 | Ackerman | Jan 1988 | A |
| 4723549 | Wholey et al. | Feb 1988 | A |
| D294519 | Hardy | Mar 1988 | S |
| 4756838 | Veltman | Jul 1988 | A |
| 4772269 | Twardowski et al. | Sep 1988 | A |
| 4798585 | Inoue et al. | Jan 1989 | A |
| 4810963 | Blake-Coleman et al. | Mar 1989 | A |
| 4813929 | Semrad | Mar 1989 | A |
| 4819637 | Dormandy et al. | Apr 1989 | A |
| 4822470 | Chang | Apr 1989 | A |
| 4836204 | Landymore et al. | Jun 1989 | A |
| 4840172 | Augustine et al. | Jun 1989 | A |
| 4863426 | Ferragamo et al. | Sep 1989 | A |
| 4885003 | Hillstead | Dec 1989 | A |
| 4886496 | Conoscenti et al. | Dec 1989 | A |
| 4886502 | Poirier et al. | Dec 1989 | A |
| 4889634 | El-Rashidy | Dec 1989 | A |
| 4907601 | Frick | Mar 1990 | A |
| 4919148 | Muccio | Apr 1990 | A |
| 4920978 | Colvin | May 1990 | A |
| 4921484 | Hillstead | May 1990 | A |
| 4946793 | Marshall, III | Aug 1990 | A |
| 4976709 | Sand | Dec 1990 | A |
| 4981477 | Schon et al. | Jan 1991 | A |
| 4986810 | Semrad | Jan 1991 | A |
| 4987895 | Heimlich | Jan 1991 | A |
| 5019034 | Weaver et al. | May 1991 | A |
| 5031775 | Kane | Jul 1991 | A |
| 5052391 | Silberstone et al. | Oct 1991 | A |
| 5053013 | Ensminger et al. | Oct 1991 | A |
| 5058605 | Slovak | Oct 1991 | A |
| 5071558 | Itoh | Dec 1991 | A |
| 5098843 | Calvin | Mar 1992 | A |
| 5122137 | Lennox | Jun 1992 | A |
| 5134070 | Casnig | Jul 1992 | A |
| 5137517 | Loney et al. | Aug 1992 | A |
| 5141499 | Zappacosta | Aug 1992 | A |
| D329496 | Wotton | Sep 1992 | S |
| 5156597 | Verreet et al. | Oct 1992 | A |
| 5173158 | Schmukler | Dec 1992 | A |
| 5186715 | Phillips et al. | Feb 1993 | A |
| 5186800 | Dower | Feb 1993 | A |
| 5188592 | Hakki | Feb 1993 | A |
| 5190541 | Abele et al. | Mar 1993 | A |
| 5192312 | Orton | Mar 1993 | A |
| 5193537 | Freeman | Mar 1993 | A |
| 5209723 | Twardowski et al. | May 1993 | A |
| 5215530 | Hogan | Jun 1993 | A |
| 5224933 | Bromander | Jul 1993 | A |
| 5227730 | King et al. | Jul 1993 | A |
| 5242415 | Kantrowitz et al. | Sep 1993 | A |
| 5273525 | Hofmann | Dec 1993 | A |
| D343687 | Houghton et al. | Jan 1994 | S |
| 5277201 | Stern | Jan 1994 | A |
| 5279564 | Taylor | Jan 1994 | A |
| 5281213 | Milder | Jan 1994 | A |
| 5283194 | Schmukler | Feb 1994 | A |
| 5290263 | Wigness et al. | Mar 1994 | A |
| 5308325 | Quinn et al. | May 1994 | A |
| 5308338 | Helfrich | May 1994 | A |
| 5318543 | Ross et al. | Jun 1994 | A |
| 5318563 | Malis et al. | Jun 1994 | A |
| 5328451 | Davis et al. | Jul 1994 | A |
| 5334167 | Cocanower | Aug 1994 | A |
| 5348554 | Imran et al. | Sep 1994 | A |
| D351661 | Fischer | Oct 1994 | S |
| 5383917 | Desai et al. | Jan 1995 | A |
| 5389069 | Weaver | Feb 1995 | A |
| 5391158 | Peters | Feb 1995 | A |
| 5403311 | Abele et al. | Apr 1995 | A |
| 5405320 | Twardowski et al. | Apr 1995 | A |
| 5425752 | Vu Nguyen | Jun 1995 | A |
| 5439440 | Hofmann | Aug 1995 | A |
| 5458625 | Kendall | Oct 1995 | A |
| 5484400 | Edwards et al. | Jan 1996 | A |
| 5484401 | Rodriguez et al. | Jan 1996 | A |
| 5533999 | Hood et al. | Jul 1996 | A |
| 5536240 | Edwards et al. | Jul 1996 | A |
| 5536267 | Edwards et al. | Jul 1996 | A |
| 5540737 | Fenn | Jul 1996 | A |
| 5546940 | Panescu et al. | Aug 1996 | A |
| 5562720 | Stern et al. | Oct 1996 | A |
| 5575811 | Reid et al. | Nov 1996 | A |
| D376652 | Hunt et al. | Dec 1996 | S |
| 5582588 | Sakurai et al. | Dec 1996 | A |
| 5586982 | Abela | Dec 1996 | A |
| 5588424 | Insler et al. | Dec 1996 | A |
| 5588960 | Edwards et al. | Dec 1996 | A |
| 5599294 | Edwards et al. | Feb 1997 | A |
| 5599311 | Raulerson | Feb 1997 | A |
| 5616126 | Malekmehr et al. | Apr 1997 | A |
| 5620479 | Diederich | Apr 1997 | A |
| 5626146 | Barber et al. | May 1997 | A |
| D380272 | Partika et al. | Jun 1997 | S |
| 5634899 | Shapland et al. | Jun 1997 | A |
| 5643197 | Brucker et al. | Jul 1997 | A |
| 5645855 | Lorenz | Jul 1997 | A |
| 5672173 | Gough et al. | Sep 1997 | A |
| 5674267 | Mir et al. | Oct 1997 | A |
| 5683384 | Gough et al. | Nov 1997 | A |
| 5687723 | Avitall | Nov 1997 | A |
| 5690620 | Knott | Nov 1997 | A |
| 5697905 | d'Ambrosio | Dec 1997 | A |
| 5700252 | Klingenstein | Dec 1997 | A |
| 5702359 | Hofmann et al. | Dec 1997 | A |
| 5718246 | Vona | Feb 1998 | A |
| 5720921 | Meserol | Feb 1998 | A |
| 5735847 | Gough et al. | Apr 1998 | A |
| 5752939 | Makoto | May 1998 | A |
| 5778894 | Dorogi et al. | Jul 1998 | A |
| 5782882 | Lerman et al. | Jul 1998 | A |
| 5800378 | Edwards et al. | Sep 1998 | A |
| 5800484 | Gough et al. | Sep 1998 | A |
| 5807272 | Kun et al. | Sep 1998 | A |
| 5807306 | Shapland et al. | Sep 1998 | A |
| 5807395 | Mulier et al. | Sep 1998 | A |
| 5810742 | Pearlman | Sep 1998 | A |
| 5810762 | Hofmann | Sep 1998 | A |
| 5830184 | Basta | Nov 1998 | A |
| 5836897 | Sakurai et al. | Nov 1998 | A |
| 5836905 | Lemelson et al. | Nov 1998 | A |
| 5843026 | Edwards et al. | Dec 1998 | A |
| 5843182 | Goldstein | Dec 1998 | A |
| 5865787 | Shapland et al. | Feb 1999 | A |
| 5868708 | Hart et al. | Feb 1999 | A |
| 5873849 | Bernard | Feb 1999 | A |
| 5904648 | Arndt et al. | May 1999 | A |
| 5919142 | Boone et al. | Jul 1999 | A |
| 5919191 | Lennox et al. | Jul 1999 | A |
| 5921982 | Lesh et al. | Jul 1999 | A |
| 5944710 | Dev et al. | Aug 1999 | A |
| 5947284 | Foster | Sep 1999 | A |
| 5947889 | Hehrlein | Sep 1999 | A |
| 5951546 | Lorentzen | Sep 1999 | A |
| 5954745 | Gertler et al. | Sep 1999 | A |
| 5957919 | Laufer | Sep 1999 | A |
| 5957963 | Dobak | Sep 1999 | A |
| 5968006 | Hofmann | Oct 1999 | A |
| 5983131 | Weaver et al. | Nov 1999 | A |
| 5984896 | Boyd | Nov 1999 | A |
| 5991697 | Nelson et al. | Nov 1999 | A |
| 5999847 | Elstrom | Dec 1999 | A |
| 6004339 | Wijay | Dec 1999 | A |
| 6009347 | Hofmann | Dec 1999 | A |
| 6009877 | Edwards | Jan 2000 | A |
| 6010613 | Walters et al. | Jan 2000 | A |
| 6016452 | Kasevich | Jan 2000 | A |
| 6029090 | Herbst | Feb 2000 | A |
| 6041252 | Walker et al. | Mar 2000 | A |
| 6043066 | Mangano et al. | Mar 2000 | A |
| 6050994 | Sherman | Apr 2000 | A |
| 6055453 | Hofmann et al. | Apr 2000 | A |
| 6059780 | Gough et al. | May 2000 | A |
| 6066134 | Eggers et al. | May 2000 | A |
| 6068121 | McGlinch | May 2000 | A |
| 6068650 | Hofmann et al. | May 2000 | A |
| 6071281 | Burnside et al. | Jun 2000 | A |
| 6074374 | Fulton | Jun 2000 | A |
| 6074389 | Levine et al. | Jun 2000 | A |
| 6085115 | Weaver et al. | Jul 2000 | A |
| 6090016 | Kuo | Jul 2000 | A |
| 6090105 | Zepeda et al. | Jul 2000 | A |
| 6090106 | Goble et al. | Jul 2000 | A |
| D430015 | Himbert et al. | Aug 2000 | S |
| 6096035 | Sodhi et al. | Aug 2000 | A |
| 6102885 | Bass | Aug 2000 | A |
| 6106521 | Blewett et al. | Aug 2000 | A |
| 6109270 | Mah et al. | Aug 2000 | A |
| 6110192 | Ravenscroft et al. | Aug 2000 | A |
| 6113593 | Tu et al. | Sep 2000 | A |
| 6116330 | Salyer | Sep 2000 | A |
| 6120493 | Hofmann | Sep 2000 | A |
| 6122599 | Mehta | Sep 2000 | A |
| 6123701 | Nezhat | Sep 2000 | A |
| 6132397 | Davis et al. | Oct 2000 | A |
| 6132419 | Hofmann | Oct 2000 | A |
| 6134460 | Chance | Oct 2000 | A |
| 6139545 | Utley et al. | Oct 2000 | A |
| 6150148 | Nanda et al. | Nov 2000 | A |
| 6159163 | Strauss et al. | Dec 2000 | A |
| 6178354 | Gibson | Jan 2001 | B1 |
| D437941 | Frattini | Feb 2001 | S |
| 6193715 | Wrublewski et al. | Feb 2001 | B1 |
| 6198970 | Freed et al. | Mar 2001 | B1 |
| 6200314 | Sherman | Mar 2001 | B1 |
| 6208893 | Hofmann | Mar 2001 | B1 |
| 6210402 | Olsen et al. | Apr 2001 | B1 |
| 6212433 | Behl | Apr 2001 | B1 |
| 6216034 | Hofmann et al. | Apr 2001 | B1 |
| 6219577 | Brown, III et al. | Apr 2001 | B1 |
| D442697 | Hajianpour | May 2001 | S |
| 6233490 | Kasevich | May 2001 | B1 |
| 6235023 | Lee et al. | May 2001 | B1 |
| D443360 | Haberland | Jun 2001 | S |
| 6241702 | Lundquist et al. | Jun 2001 | B1 |
| 6241725 | Cosman | Jun 2001 | B1 |
| D445198 | Frattini | Jul 2001 | S |
| 6258100 | Alferness et al. | Jul 2001 | B1 |
| 6261831 | Agee | Jul 2001 | B1 |
| 6277114 | Bullivant et al. | Aug 2001 | B1 |
| 6278895 | Bernard | Aug 2001 | B1 |
| 6280441 | Ryan | Aug 2001 | B1 |
| 6283988 | Laufer et al. | Sep 2001 | B1 |
| 6283989 | Laufer et al. | Sep 2001 | B1 |
| 6284140 | Sommermeyer et al. | Sep 2001 | B1 |
| 6287293 | Jones et al. | Sep 2001 | B1 |
| 6287304 | Eggers et al. | Sep 2001 | B1 |
| 6296636 | Cheng et al. | Oct 2001 | B1 |
| 6298726 | Adachi et al. | Oct 2001 | B1 |
| 6299633 | Laufer | Oct 2001 | B1 |
| 6300108 | Rubinsky et al. | Oct 2001 | B1 |
| D450391 | Hunt et al. | Nov 2001 | S |
| 6312428 | Eggers et al. | Nov 2001 | B1 |
| 6326177 | Schoenbach et al. | Dec 2001 | B1 |
| 6327505 | Medhkour et al. | Dec 2001 | B1 |
| 6328689 | Gonzalez et al. | Dec 2001 | B1 |
| 6347247 | Dev et al. | Feb 2002 | B1 |
| 6349233 | Adams | Feb 2002 | B1 |
| 6351674 | Silverstone | Feb 2002 | B2 |
| 6375634 | Carroll et al. | Apr 2002 | B1 |
| 6387671 | Rubinsky et al. | May 2002 | B1 |
| 6398779 | Buysse et al. | Jun 2002 | B1 |
| 6403348 | Rubinsky et al. | Jun 2002 | B1 |
| 6405732 | Edwards et al. | Jun 2002 | B1 |
| 6411852 | Danek et al. | Jun 2002 | B1 |
| 6419674 | Bowser et al. | Jul 2002 | B1 |
| 6428802 | Atala | Aug 2002 | B1 |
| 6443952 | Mulier et al. | Sep 2002 | B1 |
| 6463331 | Edwards | Oct 2002 | B1 |
| 6470211 | Ideker et al. | Oct 2002 | B1 |
| 6482221 | Hebert et al. | Nov 2002 | B1 |
| 6482619 | Rubinsky et al. | Nov 2002 | B1 |
| 6485487 | Sherman | Nov 2002 | B1 |
| 6488673 | Laufer et al. | Dec 2002 | B1 |
| 6488678 | Sherman | Dec 2002 | B2 |
| 6488680 | Francischelli et al. | Dec 2002 | B1 |
| 6491706 | Alferness et al. | Dec 2002 | B1 |
| 6493589 | Medhkour et al. | Dec 2002 | B1 |
| 6493592 | Leonard et al. | Dec 2002 | B1 |
| 6500173 | Underwood et al. | Dec 2002 | B2 |
| 6503248 | Levine | Jan 2003 | B1 |
| 6506189 | Rittman et al. | Jan 2003 | B1 |
| 6514248 | Eggers et al. | Feb 2003 | B1 |
| 6520183 | Amar | Feb 2003 | B2 |
| 6526320 | Mitchell | Feb 2003 | B2 |
| D471640 | McMichael et al. | Mar 2003 | S |
| D471641 | McMichael et al. | Mar 2003 | S |
| 6530922 | Cosman et al. | Mar 2003 | B2 |
| 6533784 | Truckai et al. | Mar 2003 | B2 |
| 6537976 | Gupta | Mar 2003 | B1 |
| 6540695 | Burbank et al. | Apr 2003 | B1 |
| 6558378 | Sherman et al. | May 2003 | B2 |
| 6562604 | Rubinsky et al. | May 2003 | B2 |
| 6569162 | He | May 2003 | B2 |
| 6575969 | Rittman et al. | Jun 2003 | B1 |
| 6589161 | Corcoran | Jul 2003 | B2 |
| 6592594 | Rimbaugh et al. | Jul 2003 | B2 |
| 6607529 | Jones et al. | Aug 2003 | B1 |
| 6610054 | Edwards et al. | Aug 2003 | B1 |
| 6611706 | Avrahami et al. | Aug 2003 | B2 |
| 6613211 | Mccormick et al. | Sep 2003 | B1 |
| 6616657 | Simpson et al. | Sep 2003 | B2 |
| 6627421 | Unger et al. | Sep 2003 | B1 |
| D480816 | McMichael et al. | Oct 2003 | S |
| 6634363 | Danek et al. | Oct 2003 | B1 |
| 6638253 | Breznock | Oct 2003 | B2 |
| 6653091 | Dunn et al. | Nov 2003 | B1 |
| 6666858 | Lafontaine | Dec 2003 | B2 |
| 6669691 | Taimisto | Dec 2003 | B1 |
| 6673070 | Edwards et al. | Jan 2004 | B2 |
| 6678558 | Dimmer et al. | Jan 2004 | B1 |
| 6689096 | Loubens et al. | Feb 2004 | B1 |
| 6692493 | Mcgovern et al. | Feb 2004 | B2 |
| 6694979 | Deem et al. | Feb 2004 | B2 |
| 6694984 | Habib | Feb 2004 | B2 |
| 6695861 | Rosenberg et al. | Feb 2004 | B1 |
| 6697669 | Dev et al. | Feb 2004 | B2 |
| 6697670 | Chomenky et al. | Feb 2004 | B2 |
| 6702808 | Kreindel | Mar 2004 | B1 |
| 6712811 | Underwood et al. | Mar 2004 | B2 |
| D489973 | Root et al. | May 2004 | S |
| 6753171 | Karube et al. | Jun 2004 | B2 |
| 6761716 | Kadhiresan et al. | Jul 2004 | B2 |
| D495807 | Agbodoe et al. | Sep 2004 | S |
| 6795728 | Chornenky et al. | Sep 2004 | B2 |
| 6801804 | Miller et al. | Oct 2004 | B2 |
| 6812204 | McHale et al. | Nov 2004 | B1 |
| 6837886 | Collins et al. | Jan 2005 | B2 |
| 6847848 | Sterzer et al. | Jan 2005 | B2 |
| 6860847 | Alferness et al. | Mar 2005 | B2 |
| 6865416 | Dev et al. | Mar 2005 | B2 |
| 6881213 | Ryan et al. | Apr 2005 | B2 |
| 6892099 | Jaafar et al. | May 2005 | B2 |
| 6895267 | Panescu et al. | May 2005 | B2 |
| 6905480 | McGuckin et al. | Jun 2005 | B2 |
| 6912417 | Bernard et al. | Jun 2005 | B1 |
| 6927049 | Rubinsky et al. | Aug 2005 | B2 |
| 6941950 | Wilson et al. | Sep 2005 | B2 |
| 6942681 | Johnson | Sep 2005 | B2 |
| 6958062 | Gough et al. | Oct 2005 | B1 |
| 6960189 | Bates et al. | Nov 2005 | B2 |
| 6962587 | Johnson et al. | Nov 2005 | B2 |
| 6972013 | Zhang et al. | Dec 2005 | B1 |
| 6972014 | Eum et al. | Dec 2005 | B2 |
| 6989010 | Francischelli et al. | Jan 2006 | B2 |
| 6994689 | Zadno-Azizi et al. | Feb 2006 | B1 |
| 6994706 | Chornenky et al. | Feb 2006 | B2 |
| 7011094 | Rapacki et al. | Mar 2006 | B2 |
| 7012061 | Reiss et al. | Mar 2006 | B1 |
| 7027869 | Danek et al. | Apr 2006 | B2 |
| 7036510 | Zgoda et al. | May 2006 | B2 |
| 7053063 | Rubinsky et al. | May 2006 | B2 |
| 7054685 | Dimmer et al. | May 2006 | B2 |
| 7063698 | Whayne et al. | Jun 2006 | B2 |
| 7087040 | McGuckin et al. | Aug 2006 | B2 |
| 7097612 | Bertolero et al. | Aug 2006 | B2 |
| 7100616 | Springmeyer | Sep 2006 | B2 |
| 7113821 | Sun et al. | Sep 2006 | B1 |
| 7130697 | Chornenky et al. | Oct 2006 | B2 |
| 7211083 | Chornenky et al. | May 2007 | B2 |
| 7232437 | Berman et al. | Jun 2007 | B2 |
| 7250048 | Francischelli et al. | Jul 2007 | B2 |
| D549332 | Matsumoto et al. | Aug 2007 | S |
| 7257450 | Auth et al. | Aug 2007 | B2 |
| 7264002 | Danek et al. | Sep 2007 | B2 |
| 7267676 | Chornenky et al. | Sep 2007 | B2 |
| 7273055 | Danek et al. | Sep 2007 | B2 |
| 7291146 | Steinke et al. | Nov 2007 | B2 |
| 7331940 | Sommerich | Feb 2008 | B2 |
| 7331949 | Marisi | Feb 2008 | B2 |
| 7341558 | Torre et al. | Mar 2008 | B2 |
| 7344533 | Pearson et al. | Mar 2008 | B2 |
| D565743 | Phillips et al. | Apr 2008 | S |
| D571478 | Horacek | Jun 2008 | S |
| 7387626 | Edwards et al. | Jun 2008 | B2 |
| 7399747 | Clair et al. | Jul 2008 | B1 |
| D575399 | Matsumoto et al. | Aug 2008 | S |
| D575402 | Sandor | Aug 2008 | S |
| 7419487 | Johnson et al. | Sep 2008 | B2 |
| 7434578 | Dillard et al. | Oct 2008 | B2 |
| 7449019 | Uchida et al. | Nov 2008 | B2 |
| 7451765 | Adler | Nov 2008 | B2 |
| 7455675 | Schur et al. | Nov 2008 | B2 |
| 7476203 | DeVore et al. | Jan 2009 | B2 |
| 7520877 | Lee et al. | Apr 2009 | B2 |
| 7533671 | Gonzalez et al. | May 2009 | B2 |
| D595422 | Mustapha | Jun 2009 | S |
| 7544301 | Shah et al. | Jun 2009 | B2 |
| 7549984 | Mathis | Jun 2009 | B2 |
| 7565208 | Harris et al. | Jul 2009 | B2 |
| 7571729 | Saadat et al. | Aug 2009 | B2 |
| 7632291 | Stephens et al. | Dec 2009 | B2 |
| 7655004 | Long | Feb 2010 | B2 |
| 7674249 | Ivorra et al. | Mar 2010 | B2 |
| 7680543 | Azure | Mar 2010 | B2 |
| D613418 | Ryan et al. | Apr 2010 | S |
| 7718409 | Rubinsky et al. | May 2010 | B2 |
| 7722606 | Azure | May 2010 | B2 |
| 7742795 | Stone et al. | Jun 2010 | B2 |
| 7765010 | Chornenky et al. | Jul 2010 | B2 |
| 7771401 | Hekmat et al. | Aug 2010 | B2 |
| RE42016 | Chornenky et al. | Dec 2010 | E |
| D630321 | Hamilton | Jan 2011 | S |
| D631154 | Hamilton | Jan 2011 | S |
| RE42277 | Jaafar et al. | Apr 2011 | E |
| 7918852 | Tullis et al. | Apr 2011 | B2 |
| 7937143 | Demarais et al. | May 2011 | B2 |
| 7938824 | Chornenky et al. | May 2011 | B2 |
| 7951582 | Gazit et al. | May 2011 | B2 |
| 7955827 | Rubinsky et al. | Jun 2011 | B2 |
| RE42835 | Chornenky et al. | Oct 2011 | E |
| D647628 | Helfteren | Oct 2011 | S |
| 8048067 | Davalos et al. | Nov 2011 | B2 |
| RE43009 | Chornenky et al. | Dec 2011 | E |
| 8109926 | Azure | Feb 2012 | B2 |
| 8114070 | Rubinsky et al. | Feb 2012 | B2 |
| 8162918 | Ivorra et al. | Apr 2012 | B2 |
| 8187269 | Shadduck et al. | May 2012 | B2 |
| 8221411 | Francischelli et al. | Jul 2012 | B2 |
| 8231603 | Hobbs et al. | Jul 2012 | B2 |
| 8240468 | Wilkinson et al. | Aug 2012 | B2 |
| 8251986 | Chornenky et al. | Aug 2012 | B2 |
| 8267927 | Dalal et al. | Sep 2012 | B2 |
| 8267936 | Hushka et al. | Sep 2012 | B2 |
| 8282631 | Davalos et al. | Oct 2012 | B2 |
| 8298222 | Rubinsky et al. | Oct 2012 | B2 |
| 8348921 | Ivorra et al. | Jan 2013 | B2 |
| 8361066 | Long et al. | Jan 2013 | B2 |
| D677798 | Hart et al. | Mar 2013 | S |
| 8425455 | Nentwick | Apr 2013 | B2 |
| 8425505 | Long | Apr 2013 | B2 |
| 8454594 | Demarais et al. | Jun 2013 | B2 |
| 8465464 | Travis et al. | Jun 2013 | B2 |
| 8465484 | Davalos et al. | Jun 2013 | B2 |
| 8511317 | Thapliyal et al. | Aug 2013 | B2 |
| 8518031 | Boyden et al. | Aug 2013 | B2 |
| 8562588 | Hobbs et al. | Oct 2013 | B2 |
| 8603087 | Rubinsky et al. | Dec 2013 | B2 |
| 8632534 | Pearson et al. | Jan 2014 | B2 |
| 8634929 | Chornenky et al. | Jan 2014 | B2 |
| 8647338 | Chornenky et al. | Feb 2014 | B2 |
| 8715276 | Thompson et al. | May 2014 | B2 |
| 8753335 | Moshe et al. | Jun 2014 | B2 |
| 8814860 | Davalos et al. | Aug 2014 | B2 |
| 8835166 | Phillips et al. | Sep 2014 | B2 |
| 8845635 | Daniel et al. | Sep 2014 | B2 |
| 8880195 | Azure | Nov 2014 | B2 |
| 8903488 | Callas et al. | Dec 2014 | B2 |
| 8906006 | Chornenky et al. | Dec 2014 | B2 |
| 8926606 | Davalos et al. | Jan 2015 | B2 |
| 8958888 | Chornenky et al. | Feb 2015 | B2 |
| 8968542 | Davalos et al. | Mar 2015 | B2 |
| 8992517 | Davalos et al. | Mar 2015 | B2 |
| 9005189 | Davalos et al. | Apr 2015 | B2 |
| 9078665 | Moss et al. | Jul 2015 | B2 |
| 9149331 | Deem et al. | Oct 2015 | B2 |
| 9173704 | Hobbs et al. | Nov 2015 | B2 |
| 9198733 | Neal, II et al. | Dec 2015 | B2 |
| 9283051 | Garcia et al. | Mar 2016 | B2 |
| 9414881 | Callas | Aug 2016 | B2 |
| 9598691 | Davalos | Mar 2017 | B2 |
| 9867652 | Sano et al. | Jan 2018 | B2 |
| 10117701 | Davalos et al. | Nov 2018 | B2 |
| 10117707 | Garcia et al. | Nov 2018 | B2 |
| 10154874 | Davalos et al. | Dec 2018 | B2 |
| 10238447 | Neal et al. | Mar 2019 | B2 |
| 10245098 | Davalos et al. | Apr 2019 | B2 |
| 10245105 | Davalos et al. | Apr 2019 | B2 |
| 10272178 | Davalos et al. | Apr 2019 | B2 |
| 10286108 | Davalos et al. | May 2019 | B2 |
| 10292755 | Davalos et al. | May 2019 | B2 |
| 20010039393 | Mori et al. | Nov 2001 | A1 |
| 20010044596 | Jaafar | Nov 2001 | A1 |
| 20010046706 | Rubinsky et al. | Nov 2001 | A1 |
| 20010047167 | Heggeness | Nov 2001 | A1 |
| 20010051366 | Rubinsky et al. | Dec 2001 | A1 |
| 20020002393 | Mitchell | Jan 2002 | A1 |
| 20020010491 | Schoenbach et al. | Jan 2002 | A1 |
| 20020022864 | Mahvi et al. | Feb 2002 | A1 |
| 20020040204 | Dev et al. | Apr 2002 | A1 |
| 20020049370 | Laufer et al. | Apr 2002 | A1 |
| 20020052601 | Goldberg et al. | May 2002 | A1 |
| 20020055731 | Atala et al. | May 2002 | A1 |
| 20020065541 | Fredricks et al. | May 2002 | A1 |
| 20020072742 | Schaefer et al. | Jun 2002 | A1 |
| 20020077314 | Falk et al. | Jun 2002 | A1 |
| 20020077676 | Schroeppel et al. | Jun 2002 | A1 |
| 20020082543 | Park et al. | Jun 2002 | A1 |
| 20020099323 | Dev et al. | Jul 2002 | A1 |
| 20020111615 | Cosman et al. | Aug 2002 | A1 |
| 20020112729 | DeVore et al. | Aug 2002 | A1 |
| 20020115208 | Mitchell et al. | Aug 2002 | A1 |
| 20020119437 | Grooms et al. | Aug 2002 | A1 |
| 20020133324 | Weaver et al. | Sep 2002 | A1 |
| 20020137121 | Rubinsky et al. | Sep 2002 | A1 |
| 20020138075 | Edwards et al. | Sep 2002 | A1 |
| 20020138117 | Son | Sep 2002 | A1 |
| 20020143365 | Herbst | Oct 2002 | A1 |
| 20020147462 | Mair et al. | Oct 2002 | A1 |
| 20020156472 | Lee et al. | Oct 2002 | A1 |
| 20020161361 | Sherman et al. | Oct 2002 | A1 |
| 20020183684 | Dev et al. | Dec 2002 | A1 |
| 20020183735 | Edwards et al. | Dec 2002 | A1 |
| 20020183740 | Edwards et al. | Dec 2002 | A1 |
| 20020188242 | Wu | Dec 2002 | A1 |
| 20020193784 | McHale et al. | Dec 2002 | A1 |
| 20020193831 | Smith | Dec 2002 | A1 |
| 20030009110 | Tu et al. | Jan 2003 | A1 |
| 20030016168 | Jandrell | Jan 2003 | A1 |
| 20030055220 | Legrain | Mar 2003 | A1 |
| 20030055420 | Kadhiresan et al. | Mar 2003 | A1 |
| 20030059945 | Dzekunov et al. | Mar 2003 | A1 |
| 20030060856 | Chornenky et al. | Mar 2003 | A1 |
| 20030078490 | Damasco et al. | Apr 2003 | A1 |
| 20030088189 | Tu et al. | May 2003 | A1 |
| 20030088199 | Kawaji | May 2003 | A1 |
| 20030096407 | Atala et al. | May 2003 | A1 |
| 20030105454 | Cucin | Jun 2003 | A1 |
| 20030109871 | Johnson et al. | Jun 2003 | A1 |
| 20030127090 | Gifford et al. | Jul 2003 | A1 |
| 20030130711 | Pearson et al. | Jul 2003 | A1 |
| 20030135242 | Mongeon et al. | Jul 2003 | A1 |
| 20030149451 | Chomenky et al. | Aug 2003 | A1 |
| 20030154988 | DeVore et al. | Aug 2003 | A1 |
| 20030159700 | Laufer et al. | Aug 2003 | A1 |
| 20030166181 | Rubinsky et al. | Sep 2003 | A1 |
| 20030170898 | Gundersen et al. | Sep 2003 | A1 |
| 20030194808 | Rubinsky et al. | Oct 2003 | A1 |
| 20030195385 | DeVore | Oct 2003 | A1 |
| 20030195406 | Jenkins et al. | Oct 2003 | A1 |
| 20030199050 | Mangano et al. | Oct 2003 | A1 |
| 20030208200 | Palanker et al. | Nov 2003 | A1 |
| 20030208236 | Heil et al. | Nov 2003 | A1 |
| 20030212394 | Pearson et al. | Nov 2003 | A1 |
| 20030212412 | Dillard et al. | Nov 2003 | A1 |
| 20030225360 | Eppstein et al. | Dec 2003 | A1 |
| 20030228344 | Fields et al. | Dec 2003 | A1 |
| 20040009459 | Anderson et al. | Jan 2004 | A1 |
| 20040019371 | Jaafar et al. | Jan 2004 | A1 |
| 20040055606 | Hendricksen et al. | Mar 2004 | A1 |
| 20040059328 | Daniel et al. | Mar 2004 | A1 |
| 20040059389 | Chornenky et al. | Mar 2004 | A1 |
| 20040068228 | Cunningham | Apr 2004 | A1 |
| 20040116965 | Falkenberg | Jun 2004 | A1 |
| 20040133194 | Eum et al. | Jul 2004 | A1 |
| 20040138715 | Groeningen et al. | Jul 2004 | A1 |
| 20040146877 | Diss et al. | Jul 2004 | A1 |
| 20040153057 | Davison | Aug 2004 | A1 |
| 20040176855 | Badylak | Sep 2004 | A1 |
| 20040193097 | Hofmann et al. | Sep 2004 | A1 |
| 20040199159 | Lee et al. | Oct 2004 | A1 |
| 20040200484 | Springmeyer | Oct 2004 | A1 |
| 20040206349 | Alferness et al. | Oct 2004 | A1 |
| 20040210248 | Gordon et al. | Oct 2004 | A1 |
| 20040230187 | Lee et al. | Nov 2004 | A1 |
| 20040236376 | Miklavcic et al. | Nov 2004 | A1 |
| 20040243107 | Macoviak et al. | Dec 2004 | A1 |
| 20040267189 | Mavor et al. | Dec 2004 | A1 |
| 20040267340 | Cioanta et al. | Dec 2004 | A1 |
| 20050010209 | Lee et al. | Jan 2005 | A1 |
| 20050010259 | Gerber | Jan 2005 | A1 |
| 20050013870 | Freyman et al. | Jan 2005 | A1 |
| 20050020965 | Rioux et al. | Jan 2005 | A1 |
| 20050043726 | Mchale et al. | Feb 2005 | A1 |
| 20050048651 | Ryttsen | Mar 2005 | A1 |
| 20050049541 | Behar et al. | Mar 2005 | A1 |
| 20050061322 | Freitag | Mar 2005 | A1 |
| 20050066974 | Fields et al. | Mar 2005 | A1 |
| 20050143817 | Hunter et al. | Jun 2005 | A1 |
| 20050165393 | Eppstein | Jul 2005 | A1 |
| 20050171522 | Christopherson | Aug 2005 | A1 |
| 20050171523 | Rubinsky et al. | Aug 2005 | A1 |
| 20050171574 | Rubinsky et al. | Aug 2005 | A1 |
| 20050182462 | Chornenky et al. | Aug 2005 | A1 |
| 20050197619 | Rule et al. | Sep 2005 | A1 |
| 20050261672 | Deem et al. | Nov 2005 | A1 |
| 20050267407 | Goldman | Dec 2005 | A1 |
| 20050282284 | Rubinsky et al. | Dec 2005 | A1 |
| 20050283149 | Thorne et al. | Dec 2005 | A1 |
| 20050288684 | Aronson et al. | Dec 2005 | A1 |
| 20050288702 | McGurk et al. | Dec 2005 | A1 |
| 20050288730 | Deem et al. | Dec 2005 | A1 |
| 20060004356 | Bilski et al. | Jan 2006 | A1 |
| 20060004400 | McGurk et al. | Jan 2006 | A1 |
| 20060009748 | Mathis | Jan 2006 | A1 |
| 20060015147 | Persson et al. | Jan 2006 | A1 |
| 20060020347 | Barrett et al. | Jan 2006 | A1 |
| 20060024359 | Walker et al. | Feb 2006 | A1 |
| 20060025760 | Podhajsky | Feb 2006 | A1 |
| 20060074413 | Behzadian | Apr 2006 | A1 |
| 20060079838 | Walker et al. | Apr 2006 | A1 |
| 20060079845 | Howard et al. | Apr 2006 | A1 |
| 20060079883 | Elmouelhi et al. | Apr 2006 | A1 |
| 20060085054 | Zikorus et al. | Apr 2006 | A1 |
| 20060089635 | Young et al. | Apr 2006 | A1 |
| 20060121610 | Rubinsky et al. | Jun 2006 | A1 |
| 20060142801 | Demarais et al. | Jun 2006 | A1 |
| 20060149123 | Vidlund et al. | Jul 2006 | A1 |
| 20060173490 | Lafontaine et al. | Aug 2006 | A1 |
| 20060182684 | Beliveau | Aug 2006 | A1 |
| 20060195146 | Tracey et al. | Aug 2006 | A1 |
| 20060212032 | Daniel et al. | Sep 2006 | A1 |
| 20060212078 | Demarais et al. | Sep 2006 | A1 |
| 20060217703 | Chornenky et al. | Sep 2006 | A1 |
| 20060224188 | Libbus et al. | Oct 2006 | A1 |
| 20060235474 | Demarais | Oct 2006 | A1 |
| 20060247619 | Kaplan et al. | Nov 2006 | A1 |
| 20060264752 | Rubinsky | Nov 2006 | A1 |
| 20060264807 | Westersten et al. | Nov 2006 | A1 |
| 20060269531 | Beebe et al. | Nov 2006 | A1 |
| 20060276710 | Krishnan | Dec 2006 | A1 |
| 20060283462 | Fields et al. | Dec 2006 | A1 |
| 20060293713 | Rubinsky et al. | Dec 2006 | A1 |
| 20060293725 | Rubinsky et al. | Dec 2006 | A1 |
| 20060293730 | Rubinsky et al. | Dec 2006 | A1 |
| 20060293731 | Rubinsky et al. | Dec 2006 | A1 |
| 20060293734 | Scott et al. | Dec 2006 | A1 |
| 20070010805 | Fedewa et al. | Jan 2007 | A1 |
| 20070016183 | Lee et al. | Jan 2007 | A1 |
| 20070016185 | Tullis et al. | Jan 2007 | A1 |
| 20070021803 | Deem et al. | Jan 2007 | A1 |
| 20070025919 | Deem et al. | Feb 2007 | A1 |
| 20070043345 | Davalos et al. | Feb 2007 | A1 |
| 20070060989 | Deem et al. | Mar 2007 | A1 |
| 20070078391 | Wortley et al. | Apr 2007 | A1 |
| 20070088347 | Young et al. | Apr 2007 | A1 |
| 20070093789 | Smith | Apr 2007 | A1 |
| 20070096048 | Clerc | May 2007 | A1 |
| 20070118069 | Persson et al. | May 2007 | A1 |
| 20070129711 | Altshuler et al. | Jun 2007 | A1 |
| 20070129760 | Demarais et al. | Jun 2007 | A1 |
| 20070156135 | Rubinsky et al. | Jul 2007 | A1 |
| 20070191889 | Lang | Aug 2007 | A1 |
| 20070203486 | Young | Aug 2007 | A1 |
| 20070230757 | Trachtenberg et al. | Oct 2007 | A1 |
| 20070239099 | Goldfarb et al. | Oct 2007 | A1 |
| 20070244521 | Bornzin et al. | Oct 2007 | A1 |
| 20070287950 | Kjeken et al. | Dec 2007 | A1 |
| 20070295336 | Nelson et al. | Dec 2007 | A1 |
| 20070295337 | Nelson et al. | Dec 2007 | A1 |
| 20080015571 | Rubinsky et al. | Jan 2008 | A1 |
| 20080021371 | Rubinsky et al. | Jan 2008 | A1 |
| 20080027314 | Miyazaki et al. | Jan 2008 | A1 |
| 20080027343 | Fields et al. | Jan 2008 | A1 |
| 20080033340 | Heller et al. | Feb 2008 | A1 |
| 20080033417 | Nields et al. | Feb 2008 | A1 |
| 20080045880 | Kjeken et al. | Feb 2008 | A1 |
| 20080052786 | Lin et al. | Feb 2008 | A1 |
| 20080071262 | Azure | Mar 2008 | A1 |
| 20080097139 | Clerc et al. | Apr 2008 | A1 |
| 20080097422 | Edwards et al. | Apr 2008 | A1 |
| 20080103529 | Schoenbach et al. | May 2008 | A1 |
| 20080121375 | Richason et al. | May 2008 | A1 |
| 20080125772 | Stone et al. | May 2008 | A1 |
| 20080132826 | Shadduck et al. | Jun 2008 | A1 |
| 20080132884 | Rubinsky | Jun 2008 | A1 |
| 20080132885 | Rubinsky et al. | Jun 2008 | A1 |
| 20080140064 | Vegesna | Jun 2008 | A1 |
| 20080146934 | Czygan et al. | Jun 2008 | A1 |
| 20080154259 | Gough et al. | Jun 2008 | A1 |
| 20080167649 | Edwards et al. | Jul 2008 | A1 |
| 20080171985 | Karakoca | Jul 2008 | A1 |
| 20080190434 | Wai | Aug 2008 | A1 |
| 20080200911 | Long | Aug 2008 | A1 |
| 20080200912 | Long | Aug 2008 | A1 |
| 20080208052 | LePivert et al. | Aug 2008 | A1 |
| 20080210243 | Clayton et al. | Sep 2008 | A1 |
| 20080214986 | Ivorra et al. | Sep 2008 | A1 |
| 20080236593 | Nelson et al. | Oct 2008 | A1 |
| 20080249503 | Fields et al. | Oct 2008 | A1 |
| 20080262489 | Steinke | Oct 2008 | A1 |
| 20080269586 | Rubinsky et al. | Oct 2008 | A1 |
| 20080269838 | Brighton et al. | Oct 2008 | A1 |
| 20080275465 | Paul et al. | Nov 2008 | A1 |
| 20080281319 | Paul et al. | Nov 2008 | A1 |
| 20080283065 | Chang et al. | Nov 2008 | A1 |
| 20080288038 | Paul et al. | Nov 2008 | A1 |
| 20080300589 | Paul et al. | Dec 2008 | A1 |
| 20080306427 | Bailey | Dec 2008 | A1 |
| 20080312599 | Rosenberg | Dec 2008 | A1 |
| 20090018206 | Barkan et al. | Jan 2009 | A1 |
| 20090024075 | Schroeppel et al. | Jan 2009 | A1 |
| 20090029407 | Gazit et al. | Jan 2009 | A1 |
| 20090038752 | Weng et al. | Feb 2009 | A1 |
| 20090062788 | Long et al. | Mar 2009 | A1 |
| 20090062792 | Vakharia et al. | Mar 2009 | A1 |
| 20090081272 | Clarke et al. | Mar 2009 | A1 |
| 20090105703 | Shadduck | Apr 2009 | A1 |
| 20090114226 | Deem et al. | May 2009 | A1 |
| 20090125009 | Zikorus et al. | May 2009 | A1 |
| 20090138014 | Bonutti | May 2009 | A1 |
| 20090143705 | Danek et al. | Jun 2009 | A1 |
| 20090157166 | Singhal et al. | Jun 2009 | A1 |
| 20090163904 | Miller et al. | Jun 2009 | A1 |
| 20090171280 | Samuel et al. | Jul 2009 | A1 |
| 20090177111 | Miller et al. | Jul 2009 | A1 |
| 20090186850 | Kiribayashi et al. | Jul 2009 | A1 |
| 20090192508 | Laufer et al. | Jul 2009 | A1 |
| 20090198231 | Esser et al. | Aug 2009 | A1 |
| 20090228001 | Pacey | Sep 2009 | A1 |
| 20090247933 | Maor et al. | Oct 2009 | A1 |
| 20090248012 | Maor et al. | Oct 2009 | A1 |
| 20090269317 | Davalos | Oct 2009 | A1 |
| 20090275827 | Aiken et al. | Nov 2009 | A1 |
| 20090281477 | Mikus et al. | Nov 2009 | A1 |
| 20090292342 | Rubinsky et al. | Nov 2009 | A1 |
| 20090301480 | Elsakka et al. | Dec 2009 | A1 |
| 20090306544 | Ng et al. | Dec 2009 | A1 |
| 20090306545 | Elsakka et al. | Dec 2009 | A1 |
| 20090318905 | Bhargav et al. | Dec 2009 | A1 |
| 20090326436 | Rubinsky et al. | Dec 2009 | A1 |
| 20090326570 | Brown | Dec 2009 | A1 |
| 20100004623 | Hamilton et al. | Jan 2010 | A1 |
| 20100023004 | Francischelli et al. | Jan 2010 | A1 |
| 20100030211 | Davalos | Feb 2010 | A1 |
| 20100049190 | Long et al. | Feb 2010 | A1 |
| 20100057074 | Roman et al. | Mar 2010 | A1 |
| 20100069921 | Miller et al. | Mar 2010 | A1 |
| 20100087813 | Long | Apr 2010 | A1 |
| 20100130975 | Long | May 2010 | A1 |
| 20100147701 | Field | Jun 2010 | A1 |
| 20100152725 | Pearson et al. | Jun 2010 | A1 |
| 20100160850 | Ivorra et al. | Jun 2010 | A1 |
| 20100168735 | Deno et al. | Jul 2010 | A1 |
| 20100174282 | Demarais et al. | Jul 2010 | A1 |
| 20100179530 | Long et al. | Jul 2010 | A1 |
| 20100196984 | Rubinsky et al. | Aug 2010 | A1 |
| 20100204560 | Salahieh et al. | Aug 2010 | A1 |
| 20100204638 | Hobbs et al. | Aug 2010 | A1 |
| 20100222677 | Placek et al. | Sep 2010 | A1 |
| 20100228234 | Hyde et al. | Sep 2010 | A1 |
| 20100228247 | Paul et al. | Sep 2010 | A1 |
| 20100241117 | Paul et al. | Sep 2010 | A1 |
| 20100249771 | Pearson et al. | Sep 2010 | A1 |
| 20100250209 | Pearson et al. | Sep 2010 | A1 |
| 20100255795 | Rubinsky et al. | Oct 2010 | A1 |
| 20100256628 | Pearson et al. | Oct 2010 | A1 |
| 20100256630 | Hamilton, Jr. et al. | Oct 2010 | A1 |
| 20100261994 | Davalos | Oct 2010 | A1 |
| 20100286690 | Paul et al. | Nov 2010 | A1 |
| 20100298823 | Cao et al. | Nov 2010 | A1 |
| 20100331758 | Davalos et al. | Dec 2010 | A1 |
| 20110017207 | Hendricksen et al. | Jan 2011 | A1 |
| 20110034209 | Rubinsky et al. | Feb 2011 | A1 |
| 20110064671 | Bynoe | Mar 2011 | A1 |
| 20110106221 | Robert et al. | May 2011 | A1 |
| 20110112531 | Landis et al. | May 2011 | A1 |
| 20110118727 | Fish et al. | May 2011 | A1 |
| 20110118732 | Rubinsky et al. | May 2011 | A1 |
| 20110130834 | Wilson et al. | Jun 2011 | A1 |
| 20110144524 | Fish et al. | Jun 2011 | A1 |
| 20110144635 | Harper et al. | Jun 2011 | A1 |
| 20110144657 | Fish et al. | Jun 2011 | A1 |
| 20110152678 | Aljuri et al. | Jun 2011 | A1 |
| 20110202053 | Moss et al. | Aug 2011 | A1 |
| 20110217730 | Gazit et al. | Sep 2011 | A1 |
| 20110251607 | Kruecker et al. | Oct 2011 | A1 |
| 20110301587 | Deem et al. | Dec 2011 | A1 |
| 20120034131 | Rubinsky et al. | Feb 2012 | A1 |
| 20120059255 | Paul et al. | Mar 2012 | A1 |
| 20120071872 | Rubinsky et al. | Mar 2012 | A1 |
| 20120071874 | Davalos et al. | Mar 2012 | A1 |
| 20120085649 | Sane et al. | Apr 2012 | A1 |
| 20120089009 | Omary et al. | Apr 2012 | A1 |
| 20120090646 | Tanaka et al. | Apr 2012 | A1 |
| 20120095459 | Callas et al. | Apr 2012 | A1 |
| 20120109122 | Arena et al. | May 2012 | A1 |
| 20120130289 | Demarais et al. | May 2012 | A1 |
| 20120150172 | Ortiz et al. | Jun 2012 | A1 |
| 20120165813 | Lee et al. | Jun 2012 | A1 |
| 20120179091 | Ivorra et al. | Jul 2012 | A1 |
| 20120226218 | Phillips et al. | Sep 2012 | A1 |
| 20120226271 | Callas et al. | Sep 2012 | A1 |
| 20120265186 | Burger et al. | Oct 2012 | A1 |
| 20120277741 | Davalos et al. | Nov 2012 | A1 |
| 20120303020 | Chornenky et al. | Nov 2012 | A1 |
| 20120310236 | Placek et al. | Dec 2012 | A1 |
| 20130090646 | Moss | Apr 2013 | A1 |
| 20130108667 | Soikum et al. | May 2013 | A1 |
| 20130110106 | Richardson | May 2013 | A1 |
| 20130184702 | Neal, II et al. | Jul 2013 | A1 |
| 20130196441 | Rubinsky et al. | Aug 2013 | A1 |
| 20130197425 | Golberg et al. | Aug 2013 | A1 |
| 20130202766 | Rubinsky et al. | Aug 2013 | A1 |
| 20130218157 | Callas | Aug 2013 | A1 |
| 20130253415 | Sane et al. | Sep 2013 | A1 |
| 20130281968 | Davalos et al. | Oct 2013 | A1 |
| 20130345697 | Garcia et al. | Dec 2013 | A1 |
| 20130345779 | Maor et al. | Dec 2013 | A1 |
| 20140039489 | Davalos et al. | Feb 2014 | A1 |
| 20140046322 | Callas et al. | Feb 2014 | A1 |
| 20140066913 | Sherman | Mar 2014 | A1 |
| 20140081255 | Johnson et al. | Mar 2014 | A1 |
| 20140088578 | Rubinsky et al. | Mar 2014 | A1 |
| 20140121663 | Pearson et al. | May 2014 | A1 |
| 20140121728 | Dhillon et al. | May 2014 | A1 |
| 20140163551 | Maor et al. | Jun 2014 | A1 |
| 20140207133 | Model et al. | Jul 2014 | A1 |
| 20140296844 | Kevin et al. | Oct 2014 | A1 |
| 20140309579 | Rubinsky et al. | Oct 2014 | A1 |
| 20140378964 | Pearson | Dec 2014 | A1 |
| 20150088120 | Garcia et al. | Mar 2015 | A1 |
| 20150088220 | Callas et al. | Mar 2015 | A1 |
| 20150112333 | Chorenky et al. | Apr 2015 | A1 |
| 20150126922 | Willis | May 2015 | A1 |
| 20150164584 | Davalos et al. | Jun 2015 | A1 |
| 20150173824 | Davalos et al. | Jun 2015 | A1 |
| 20150201996 | Rubinsky et al. | Jul 2015 | A1 |
| 20150265349 | Moss et al. | Sep 2015 | A1 |
| 20150289923 | Davalos et al. | Oct 2015 | A1 |
| 20150320488 | Moshe et al. | Nov 2015 | A1 |
| 20150327944 | Davalos et al. | Nov 2015 | A1 |
| 20160022957 | Hobbs et al. | Jan 2016 | A1 |
| 20160066977 | Neal et al. | Mar 2016 | A1 |
| 20160074114 | Pearson et al. | Mar 2016 | A1 |
| 20160113708 | Moss et al. | Apr 2016 | A1 |
| 20160143698 | Garcia et al. | May 2016 | A1 |
| 20160235470 | Callas et al. | Aug 2016 | A1 |
| 20160287313 | Rubinsky et al. | Oct 2016 | A1 |
| 20160287314 | Arena et al. | Oct 2016 | A1 |
| 20160338761 | Chornenky et al. | Nov 2016 | A1 |
| 20160354142 | Pearson et al. | Dec 2016 | A1 |
| 20170035501 | Chornenky et al. | Feb 2017 | A1 |
| 20170189579 | Davalos | Jul 2017 | A1 |
| 20170209620 | Davalos et al. | Jul 2017 | A1 |
| 20170360326 | Davalos | Dec 2017 | A1 |
| 20180125565 | Sano et al. | May 2018 | A1 |
| 20180161086 | Davalos et al. | Jun 2018 | A1 |
| 20190029749 | Garcia | Jan 2019 | A1 |
| 20190046255 | Davalos et al. | Feb 2019 | A1 |
| 20190069945 | Davalos et al. | Mar 2019 | A1 |
| 20190133671 | Davalos et al. | May 2019 | A1 |
| Number | Date | Country |
|---|---|---|
| 2002315095 | Dec 2002 | AU |
| 2003227960 | Dec 2003 | AU |
| 2005271471 | Feb 2006 | AU |
| 2006321570 | Jun 2007 | AU |
| 2006321574 | Jun 2007 | AU |
| 2006321918 | Jun 2007 | AU |
| 2297846 | Feb 1999 | CA |
| 2378110 | Feb 2001 | CA |
| 2445392 | Nov 2002 | CA |
| 2458676 | Mar 2003 | CA |
| 2487284 | Dec 2003 | CA |
| 2575792 | Feb 2006 | CA |
| 2631940 | Jun 2007 | CA |
| 2631946 | Jun 2007 | CA |
| 2632604 | Jun 2007 | CA |
| 2751462 | Nov 2010 | CA |
| 1525839 | Sep 2004 | CN |
| 101534736 | Sep 2009 | CN |
| 102238921 | Nov 2011 | CN |
| 102421386 | Apr 2012 | CN |
| 863111 | Jan 1953 | DE |
| 4000893 | Jul 1991 | DE |
| 60038026 | Feb 2009 | DE |
| 0218275 | Apr 1987 | EP |
| 0339501 | Nov 1989 | EP |
| 0378132 | Jul 1990 | EP |
| 0533511 | Mar 1993 | EP |
| 0998235 | May 2000 | EP |
| 0528891 | Jul 2000 | EP |
| 1196550 | Apr 2002 | EP |
| 1439792 | Jul 2004 | EP |
| 1442765 | Aug 2004 | EP |
| 1462065 | Sep 2004 | EP |
| 1061983 | Nov 2004 | EP |
| 1493397 | Jan 2005 | EP |
| 1506039 | Feb 2005 | EP |
| 0935482 | May 2005 | EP |
| 1011495 | Nov 2005 | EP |
| 1796568 | Jun 2007 | EP |
| 1207797 | Feb 2008 | EP |
| 1406685 | Jun 2008 | EP |
| 1424970 | Dec 2008 | EP |
| 2381829 | Nov 2011 | EP |
| 2413833 | Feb 2012 | EP |
| 1791485 | Dec 2014 | EP |
| 2373241 | Jan 2015 | EP |
| 1962710 | Aug 2015 | EP |
| 1962708 | Sep 2015 | EP |
| 1962945 | Apr 2016 | EP |
| 2300272 | Jun 2008 | ES |
| 2315493 | Apr 2009 | ES |
| 2001510702 | Aug 2001 | JP |
| 2003505072 | Feb 2003 | JP |
| 2003506064 | Feb 2003 | JP |
| 2004203224 | Jul 2004 | JP |
| 2004525726 | Aug 2004 | JP |
| 2004303590 | Oct 2004 | JP |
| 2005501596 | Jan 2005 | JP |
| 2005526579 | Sep 2005 | JP |
| 2008508946 | Mar 2008 | JP |
| 4252316 | Apr 2009 | JP |
| 2009518130 | May 2009 | JP |
| 2009518150 | May 2009 | JP |
| 2009518151 | May 2009 | JP |
| 2009532077 | Sep 2009 | JP |
| 2010503496 | Feb 2010 | JP |
| 2011137025 | Jul 2011 | JP |
| 2011137025 | Jul 2011 | JP |
| 2012510332 | May 2012 | JP |
| 2012515018 | Jul 2012 | JP |
| 2012521863 | Sep 2012 | JP |
| 101034682 | May 2011 | KR |
| 9104014 | Apr 1991 | WO |
| 9634571 | Nov 1996 | WO |
| 9639531 | Dec 1996 | WO |
| 9810745 | Mar 1998 | WO |
| 9614238 | Apr 1998 | WO |
| 9901076 | Jan 1999 | WO |
| 9904710 | Feb 1999 | WO |
| 0020554 | Apr 2000 | WO |
| 0107583 | Feb 2001 | WO |
| 0107584 | Feb 2001 | WO |
| 0107585 | Feb 2001 | WO |
| 0110319 | Feb 2001 | WO |
| 2001048153 | Jul 2001 | WO |
| 0170114 | Sep 2001 | WO |
| 0181533 | Nov 2001 | WO |
| 02078527 | Oct 2002 | WO |
| 02089686 | Nov 2002 | WO |
| 02100459 | Dec 2002 | WO |
| 2003020144 | Mar 2003 | WO |
| 2003047684 | Jun 2003 | WO |
| 03099382 | Dec 2003 | WO |
| 2004037341 | May 2004 | WO |
| 2004080347 | Sep 2004 | WO |
| 2005065284 | Jul 2005 | WO |
| 2006017666 | Feb 2006 | WO |
| 2006031541 | Mar 2006 | WO |
| 2006130194 | Dec 2006 | WO |
| 2007067628 | Jun 2007 | WO |
| 2007067937 | Jun 2007 | WO |
| 2007067938 | Jun 2007 | WO |
| 2007067939 | Jun 2007 | WO |
| 2007067940 | Jun 2007 | WO |
| 2007067941 | Jun 2007 | WO |
| 2007067943 | Jun 2007 | WO |
| 2007070361 | Jun 2007 | WO |
| 2007100727 | Sep 2007 | WO |
| 2007123690 | Nov 2007 | WO |
| 2008063195 | May 2008 | WO |
| 2008034103 | Nov 2008 | WO |
| 2009046176 | Apr 2009 | WO |
| 2007137303 | Jul 2009 | WO |
| 2009134876 | Nov 2009 | WO |
| 2009135070 | Nov 2009 | WO |
| 2009137800 | Nov 2009 | WO |
| 2010064154 | Jun 2010 | WO |
| 2010080974 | Jul 2010 | WO |
| 2010117806 | Oct 2010 | WO |
| 2010118387 | Oct 2010 | WO |
| 2010132472 | Nov 2010 | WO |
| 2010151277 | Dec 2010 | WO |
| 2011047387 | Apr 2011 | WO |
| 2011062653 | May 2011 | WO |
| 2011072221 | Jun 2011 | WO |
| 2012051433 | Apr 2012 | WO |
| 2012071526 | May 2012 | WO |
| 2012088149 | Jun 2012 | WO |
| 2015175570 | Nov 2015 | WO |
| 2016100325 | Jun 2016 | WO |
| 2016164930 | Oct 2016 | WO |
| Entry |
|---|
| A.I. Daud et al., “Phase I Trial of Interleukin-12 Plasmid Electroporation in Patients With Metastatic Melanoma,” Journal of Clinical Oncology, 26, pp. 5896-5903, 2008. |
| Agerholm-Larsen, B., et al., “Preclinical Validation of Electrochemotherapy as an Effective Treatment for Brain Tumors”, Cancer Research 71: 3753-3762 (2011). |
| Alberts et al., “Molecular Biology of the Cell,” 3rd edition, Garland Science, New York, 1994, 1 page. |
| Al-Sakere et al., “Tumor ablation with irreversible electroporation.” PLoS One, Issue 11, e1135, 8 pages, 2007. |
| Amasha, et al., Quantitative Assessment of Impedance Tomography for Temperature Measurements in Microwave Hyperthermia, Clin. Phys. Physiol. Meas., 1998, Suppl. A, 49-53. |
| Andreason, Electroporation as a Technique for the Transfer of Macromolecules into Mammalian Cell Lines, J. Tiss. Cult. Meth., 15:56-62, 1993. |
| Appelbaum et al., “US Findings after Irreversible Electroporation Ablation: Radiologic-Pathologic Correlation,” Radiology, 262, pp. 117-125, Jan. 1, 2012. |
| Arena et al. “High-Frequency Irreversible Electroporation (H-FIRE) for Non-thermal Ablation without Muscle Contraction.” Biomed. Eng. Online, vol. 10, 20 pages (2011). |
| Arena, C.B., et al., “A three-dimensional in vitro tumor platform for modeling therapeutic irreversible electroporation.” Biophysical Journal, 2012.103(9): p. 2033-2042. |
| Arena, Christopher B., et al., “Towards the development of latent heat storage electrodes for electroporation-based therapies”, Applied Physics Letters, 101, 083902 (2012). |
| Arena, Christopher B., et al.,“Phase Change Electrodes for Reducing Joule Heating During Irreversible Electroporation”. Proceedings of the ASME 2012 Summer Bioengineering Conference, SBC2012, Jun. 20-23, 2012, Fajardo, Puerto Rico. |
| Asami et al., “Dielectric properties of mouse lymphocytes and erythrocytes.” Biochimica et Biophysica Acta (BBA)—Molecular Cell Research, 1010 (1989) pp. 49-55. |
| Bagla, S. and Papadouris, D., “Percutaneous Irreversible Electroporation of Surgically Unresectable Pancreatic Cancer: A Case Report” J. Vascular Int. Radiol. 23(1), 142-145 (2012). |
| Baker, et al., Calcium-Dependent Exocytosis in Bovine Adrenal Medullary Cells with Leaky Plasma Membranes, Nature, vol. 276, pp. 620-622, 1978. |
| Ball, C., K.R. Thomson, and H. Kavnoudias, “Irreversible electroporation: a new challenge in “out of-operating theater” anesthesia.” Anesth Analg, 2010. 110(5): p. 1305-9. |
| Bancroft, et al. Design of a Flow Perfusion Bioreactor System for Bone Tissue-Engineering Applications, Tissue Engineering, vol. 9, No. 3, 2003, p. 549-554. |
| Baptista et al., “The Use of Whole Organ Decellularization for the Generation of a Vascularized Liver Organoid,” Heptatology, vol. 53, No. 2, pp. 604-617 (2011). |
| Barber, Electrical Impedance Tomography Applied Potential Tomography, Advances in Biomedical Engineering, Beneken and Thevenin, eds., IOS Press, pp. 165-173, 1993. |
| Beebe, S.J., et al., “Diverse effects of nanosecond pulsed electric fields on cells and tissues”, DNA and Cell Biology, 22(12): 785-796 (2003). |
| Beebe, S.J., et al., Nanosecond pulsed electric field (nsPEF) effects on cells and tissues: apoptosis induction and tumor growth inhibition. PPPS-2001 Pulsed Power Plasma Science 2001, 28th IEEE International Conference on Plasma Science and 13th IEEE International Pulsed Power Conference, Digest of Technical Papers (Cat. No. 01CH37251). IEEE, Part vol. 1, 2001, pp. 211-215, vol. I, Piscataway, NJ, USA. |
| Ben-David, et al., “Characterization of Irreversible Electroporation Ablation in Vivo Porcine Liver,” Am J Roentgenol, vol. 198, pp. W62-W68, 2012. |
| Blad, et al., Impedance Spectra of Tumour Tissue in Comparison with Normal Tissue; a Possible Clinical Application for Electrical or Electrical Impedance Tomography, Physiol. Meas. 17 (1996) A105-A115. |
| Bolland, F., et al., “Development and characterisation of a full-thickness acellular porcine bladder matrix for tissue engineering”, Biomaterials, Elsevier Science Publishers, Barking, GB, vol. 28, No. 6, Nov. 28, 2006, pp. 1061-1070. |
| Boone, K., Barber, D. & Brown, B. Review—Imaging with electricity: report of the European Concerted Action on Impedance Tomography. J. Med. Eng. Technol. 21, 201-232 (1997). |
| Bower et al., “Irreversible electroporation of the pancreas: definitive local therapy without systemic effects.” Journal of surgical oncology, 2011. 104(1): p. 22-28. |
| BPH Management Strategies: Improving Patient Satisfaction, Urology Times, May 2001, vol. 29, Supplement 1. |
| Brown, et al., Blood Flow Imaging Using Electrical Impedance Tomography, Clin. Phys. Physiol. Meas., 1992, vol. 13, Suppl. A, 175-179. |
| Brown, S.G., Phototherapy of tumors. World J. Surgery, 1983. 7: p. 700-9. |
| Cannon et al., “Safety and early efficacy of irreversible electroporation for hepatic tumors in proximity to vital structures.” Journal of Surgical Oncology, 6 pages (2012). |
| Carpenter A.E. et al., “CellProfiler: image analysis software for identifying and quantifying cell phenotypes.” Genome Biol. 2006; 7(10): R100. Published online Oct. 31, 2006, 11 pages. |
| Cemazar M, Parkins CS, Holder AL, Chaplin DJ, Tozer GM, et al., “Electroporation of human microvascular endothelial cells: evidence for an anti-vascular mechanism of electrochemotherapy”, Br J Cancer 84: 565-570 (2001). |
| Chandrasekar, et al., Transurethral Needle Ablation of the Prostate (TUNA)—a Propsective Study, Six Year Follow Up, (Abstract), Presented at 2001 National Meeting, Anaheim, CA, Jun. 5, 2001. |
| Chang, D.C., “Cell Poration and Cell-Fusion Using an Oscillating Electric-Field”. Biophysical Journal, 56(4): p. 641-652 (1989). |
| Charpentier, K.P., et al., “Irreversible electroporation of the pancreas in swine: a pilot study.” HPB: the official journal of the International Hepato Pancreato Biliary Association, 2010. 12(5): p. 348-351. |
| Chen et al., “Classification of cell types using a microfluidic device for mechanical and electrical measurement on single cells.” Lab on a Chip, vol. 11, pp. 3174-3181 (2011). |
| Chen, M.T., et al., “Two-dimensional nanosecond electric field mapping based on cell electropermeabilization”, PMC Biophys, 2(1):9 (2009). |
| Clark et al., “The electrical properties of resting and secreting pancreas.” The Journal of Physiology, vol. 189, pp. 247-260 (1967). |
| Coates, C.W.,et al., “The Electrical Discharge of the Electric Eel, Electrophorous electricus,” Zoologica, 1937, 22(1), p. 1-32. |
| Cook, et al., ACT3: A High-Speed, High-Precision Electrical Impedance Tomograph, IEEE Transactions on Biomedical Engineering, vol. 41, No. 8, Aug. 1994. |
| Co-Pending U.S. Appl. No. 10/571,162, filed Oct. 18, 2006 (published as 2007/0043345 on Feb. 22, 2007). |
| Co-Pending U.S. Appl. No. 12/432,295, filed Apr. 29, 2009. |
| Co-Pending U.S. Appl. No. 12/609,779, filed Oct. 30, 2009. |
| Co-Pending U.S. Appl. No. 12/751,826, filed Mar. 31, 2010 (published as 2010/0250209 on Sep. 30, 2010). |
| Co-Pending U.S. Appl. No. 12/751,854, filed Mar. 31, 2010 (published as 2010/0249771 on Sep. 30, 2010). |
| Co-Pending U.S. Appl. No. 12/757,901, filed Apr. 9, 2010. |
| Co-Pending U.S. Appl. No. 12/906,923, filed Oct. 18, 2010. |
| Co-Pending U.S. Appl. No. 14/558,631, filed Dec. 2, 2014. |
| Co-Pending U.S. Appl. No. 14/686,380, filed Apr. 14, 2015 and Published as US 2015/0289923 on Oct. 15, 2015. |
| Co-Pending U.S. Appl. No. 14/808,679, filed Jul. 24, 2015 and Published as U.S. Publication No. 2015/0327944 on Nov. 19, 2015. |
| Co-Pending U.S. Appl. No. 15/011,752, filed Feb. 1, 2016. |
| Jarm et al., “Antivascular effects of electrochemotherapy: implications in treatment of bleeding metastases.” Expert Rev Anticancer Ther. vol. 10, pp. 729-746 (2010). |
| Jaroszeski, et al., In Vivo Gene Delivery by Electroporation, Advanced Drug Delivery Review, vol. 35, pp. 131-137, 1999. |
| Jensen et al., “Tumor volume in subcutaneous mouse xenografts measured by microCT is more accurate and reproducible than determined by 18FFDG-microPET or external caliper.” BMC medical Imaging vol. 8:16, 9 Pages (2008). |
| Jossinet et al., Electrical Impedance Endo-Tomography: Imaging Tissue From Inside, IEEE Transactions on Medical Imaging, vol. 21, No. 6, Jun. 2002, pp. 560-565. |
| Kingham et al., “Ablation of perivascular hepatic malignant tumors with irreversible electroporation.” Journal of the American College of Surgeons, 2012. 215(3), p. 379-387. |
| Kinosita and Tsong, “Formation and resealing of pores of controlled sizes in human erythrocyte membrane.” Nature, vol. 268 (1977) pp. 438-441. |
| Kinosita and Tsong, “Voltage-induced pore formation and hemolysis of human erythrocytes.” Biochimica et Biophysica Acta (BBA)—Biomembranes, 471 (1977) pp. 227-242. |
| Kinosita et al., “Electroporation of cell membrane visualized under a pulsed-laser fluorescence microscope.” Biophysical Journal, vol. 53, pp. 1015-1019 (1988). |
| Kinosita, et al., Hemolysis of Human Erythrocytes by a Transient Electric Field, Proc. Natl. Acad. Sci. USA, vol. 74, No. 5, pp. 1923-1927, 1977. |
| Kirson et al., “Alternating electric fields arrest cell proliferation in animal tumor models and human brain tumors.” Proceedings of the National Academy of Sciences vol. 104, pp. 10152-10157 (2007). |
| Kotnik and Miklavcic, “Theoretical evaluation of voltage inducement on internal membranes of biological cells exposed to electric fields.” Biophysical Journal, vol. 90(2), pp. 480-491 (2006). |
| Kotnik, T. and D. Miklavcic, “Theoretical evaluation of the distributed power dissipation in biological cells exposed to electric fields”, Bioelectromagnetics, 21(5): p. 385-394 (2000). |
| Kotnik, T., et al., “Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses. Part II. Reduced electrolytic contamination”, Bioelectrochemistry, 54(1): p. 91-5 (2001). |
| Kotnik, T., et al., “Role of pulse shape in cell membrane electropermeabilization”, Biochimica Et Biophysica Acta-Biomembranes, 1614(2): p. 193-200 (2003). |
| Labeed et al., “Differences in the biophysical properties of membrane and cytoplasm of apoptotic cells revealed using dielectrophoresis.” Biochimica et Biophysica Acta (BBA)—General Subjects, vol. 1760, pp. 922-929 (2006). |
| Lackovic, I., et al., “Three-dimensional Finite-element Analysis of Joule Heating in Electrochemotherapy and in vivo Gene Electrotransfer”, Ieee Transactions on Dielectrics and Electrical Insulation, 16(5): p. 1338-1347 (2009). |
| Laufer et al., “Electrical impedance characterization of normal and cancerous human hepatic tissue.” Physiological Measurement, vol. 31, pp. 995-1009 (2010). |
| Lebar et al., “Inter-pulse interval between rectangular voltage pulses affects electroporation threshold of artificial lipid bilayers.” IEEE Transactions on NanoBioscience, vol. 1 (2002) pp. 116-120. |
| Lee, E. W. et al. Advanced Hepatic Ablation Technique for Creating Complete Cell Death : Irreversible Electroporation. Radiology 255, 426-433, doi:10.1148/radiol.10090337 (2010). |
| Lee, E.W., et al., “Imaging guided percutaneous irreversible electroporation: ultrasound and immunohistological correlation”, Technol Cancer Res Treat 6: 287-294 (2007). |
| Li, W., et al., “The Effects of Irreversible Electroporation (IRE) on Nerves” PloS One, Apr. 2011, 6(4), e18831. |
| Liu, et al., Measurement of Pharyngeal Transit Time by Electrical Impedance Tomography, Clin. Phys. Physiol. Meas., 1992, vol. 13, Suppl. A, pp. 197-200. |
| Long, G., et al., “Targeted Tissue Ablation With Nanosecond Pulses”. Ieee Transactions on Biomedical Engineering, 58(8) (2011). |
| Lundqvist et al. Altering the Biochemical State of Individual Cultured Cells and Organelles with Ultramicroelectrodes, Proc. Natl. Acad. Sci. USA, vol. 95, pp. 10356-10360, Sep. 1998. |
| Lurquin, Gene Transfer by Electroporation, Molecular Biotechnology, vol. 7, 1997. |
| Lynn et al., A New Method for the Generation and Use of Focused Ultrasound in Experimental Biology, The Journal of General Physiology, vol. 26, 179-193, 1942. |
| M. Marty et al., “Electrochemotherapy—an easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: Results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study,” European Journal of Cancer Supplements, 4, pp. 3-13, 2006. |
| Mac̆ek Lebar and Miklavc̆ic̆, “Cell electropermeabilization to small molecules in vitro: control by pulse parameters.” Radiology and Oncology, vol. 35(3), pp. 193-202 (2001). |
| Mahmood, F., et al., “Diffusion-Weighted MRI for Verification of Electroporation-Based Treatments”, Journal of Membrane Biology 240: 131-138 (2011). |
| Mahnic-Kalamiza, et al., “Educational application for visualization and analysis of electric field strength in multiple electrode electroporation,” BMC Med Educ, vol. 12:102, 13 pages, 2012. |
| Malpica et al., “Grading ovarian serous carcinoma using a two-tier system.” The American Journal of Surgical Pathology, vol. 28, pp. 496-504 (2004). |
| Maor et al., The Effect of Irreversible Electroporation on Blood Vessels, Tech. in Cancer Res. and Treatment, vol. 6, No. 4, Aug. 2007, pp. 307-312. |
| Maor, E., A. Ivorra, and B. Rubinsky, Non Thermal Irreversible Electroporation: Novel Technology for Vascular Smooth Muscle Cells Ablation, PLoS One, 2009, 4(3): p. e4757. |
| Maor, E., A. Ivorra, J. Leor, and B. Rubinsky, Irreversible electroporation attenuates neointimal formation after angioplasty, IEEE Trans Biomed Eng, Sep. 2008, 55(9): p. 2268-74. |
| Marszalek et al., “Schwan equation and transmembrane potential induced by alternating electric field.” Biophysical Journal, vol. 58, pp. 1053-1058 (1990). |
| Martin, n.R.C.G., et al., “Irreversible electroporation therapy in the management of locally advanced pancreatic adenocarcinoma.” Journal of the American College of Surgeons, 2012. 215(3): p. 361-369. |
| Miklavcic et al., “A validated model of in vivo electric field distribution in tissues for electrochemotherapy and for DNA electrotransfer for gene therapy,” Biochimica et Biophysica Acta, 1523, pp. 73-83, 2000. |
| Miklavc̆ic̆, et al., The Importance of Electric Field Distribution for Effective in Vivo Electroporation of Tissues, Biophysical Journal, vol. 74, May 1998, pp. 2152-2158. |
| Miller, L., et al., Cancer cells ablation with irreversible electroporation, Technology in Cancer Research and Treatment 4 (2005) 699-706. |
| Mir et al., “Mechanisms of Electrochemotherapy” Advanced Drug Delivery Reviews 35:107-118 (1999). |
| Mir, et al., Effective Treatment of Cutaneous and Subcutaneous Malignant Tumours by Electrochemotherapy, British Journal of Cancer, vol. 77, No. 12, pp. 2336-2342, 1998. |
| Mir, et al., Electrochemotherapy Potentiation of Antitumour Effect of Bleomycin by Local Electric Pulses, European Journal of Cancer, vol. 27, No. 1, pp. 68-72, 1991. |
| Mir, et al., Electrochemotherapy, a Novel Antitumor Treatment: First Clinical Trial, C.R. Acad. Sci. Paris, Ser. III, vol. 313, pp. 613-618, 1991. |
| Mir, L.M. and Orlowski, S., The basis of electrochemotherapy, in Electrochemotherapy, electrogenetherapy, and transdermal drug delivery: electrically mediated delivery of molecules to cells, M.J. Jaroszeski, R. Heller, R. Gilbert, Editors, 2000, Humana Press, p. 99-118. |
| Mir, L.M., et al., Electric Pulse-Mediated Gene Delivery to Various Animal Tissues, in Advances in Genetics, Academic Press, 2005, p. 83-114. |
| Mir, Therapeutic Perspectives of in Vivo Cell Electropermeabilization, Bioelectrochemistry, vol. 53, pp. 1-10, 2000. |
| Mulhall et al., “Cancer, pre-cancer and normal oral cells distinguished by dielectrophoresis.” Analytical and Bioanalytical Chemistry, vol. 401, pp. 2455-2463 (2011). |
| Narayan, et al., Establishment and Characterization of a Human Primary Prostatic Adenocarcinoma Cell Line (ND-1), The Journal of Urology, vol. 148, 1600-1604, Nov. 1992. |
| Naslund, Cost-Effectiveness of Minimally Invasive Treatments and Transurethral Resection (TURP) in Benign Prostatic Hyperplasia (BPH), (Abstract), Presented at 2001 AUA National Meeting,, Anaheim, CA, Jun. 5, 2001. |
| Naslund, Michael J., Transurethral Needle Ablation of the Prostate, Urology, vol. 50, No. 2, Aug. 1997. |
| Salmanzadeh et al., “Sphingolipid Metabolites Modulate Dielectric Characteristics of Cells in a Mouse Ovarian Cancer Progression Model.” Integr. Biol., 5(6), pp. 843-852 (2013). |
| Sano et al., “Contactless Dielectrophoretic Spectroscopy: Examination of the Dielectric Properties of Cells Found in Blood.” Electrophoresis, 32, pp. 3164-3171, 2011. |
| Sano et al., “In-vitro bipolar nano- and microsecond electro-pulse bursts for irreversible electroporation therapies.” Bioelectrochemistry vol. 100, pp. 69-79 (2014). |
| Sano et al., “Modeling and Development of a Low Frequency Contactless Dielectrophoresis (cDEP) Platform to Sort Cancer Cells from Dilute Whole Blood Samples.” Biosensors & Bioelectronics, 8 pages (2011). |
| Sano, M. B., et al., “Towards the creation of decellularized organ constructs using irreversible electroporation and active mechanical perfusion”, Biomedical Engineering Online, Biomed Central LTD, London, GB, vol. 9, No. 1, Dec. 10, 2010, p. 83. |
| Saur et al., “CXCR4 expression increases liver and lung metastasis in a mouse model of pancreatic cancer.” Gastroenterology, vol. 129, pp. 1237-1250 (2005). |
| Schmukler, Impedance Spectroscopy of Biological Cells, Engineering in Medicine and Biology Society, Engineering Advances: New Opportunities for Biomedical Engineers, Proceedings of the 16th Annual Internal Conference of the IEEE, vol. 1, p. A74, downloaded from IEEE Xplore website, 1994. |
| Schoenbach et al., “Intracellular effect of ultrashort electrical pulses.” Bioelectromagnetics, 22 (2001) pp. 440-448. |
| Seibert et al., “Clonal variation of MCF-7 breast cancer cells in vitro and in athymic nude mice.” Cancer Research, vol. 43, pp. 2223-2239 (1983). |
| Seidler et al., “A Cre-IoxP-based mouse model for conditional somatic gene expression and knockdown in vivo by using avian retroviral vectors.” Proceedings of the National Academy of Sciences, vol. 105, pp. 10137-10142 (2008). |
| Set, D., Lebar, A. M. & Miklavcic, D. Feasibility of employing model-based optimization of pulse amplitude and electrode distance for effective tumor electropermeabilization. IEEE Trans Biomed Eng 54, 773-781 (2007). |
| Sel, et al., “Sequential finite element model of tissue electropermeabilization,” IEEE Trans Biomed Eng, vol. 52, pp. 816-827, 2005. |
| Sersa, et al., Reduced Blood Flow and Oxygenation in SA-1 Tumours after Electrochemotherapy with Cisplatin, British Journal of Cancer, 87, 1047-1054, 2002. |
| Sersa, et al., Tumour Blood Flow Modifying Effects of Electrochemotherapy: a Potential Vascular Targeted Mechanism, Radiol. Oncol., 37(1): 43-8, 2003. |
| Sharma, A. , et al., “Review on Thermal Energy Storage with Phase Change Materials and Applications”, Renewable Sustainable Energy Rev. 13(2), 318-345 (2009). |
| Sharma, et al., Poloxamer 188 Decreases Susceptibility of Artificial Lipid Membranes to Electroporation, Biophysical Journal, vol. 71, No. 6, pp. 3229-3241, Dec. 1996. |
| Shiina, S., et al, Percutaneous ethanol injection therapy for hepatocellular carcinoma: results in 146 patients. AJR, 1993, 160: p. 1023-8. |
| Szot et al., “3D in vitro bioengineered tumors based on collagen I hydrogels.” Biomaterials vol. 32, pp. 7905-7912 (2011). |
| Talele, S., et al., “Modelling single cell electroporation with bipolar pulse parameters and dynamic pore radii”. Journal of Electrostatics, 68(3): p. 261-274 (2010). |
| Tekle, Ephrem, R. Dean Astumian, and P. Boon Chock, Electroporation by using bipolar oscillating electric field: An Improved method for DNA transfection of NIH 3T3 cells, Proc. Natl. Acad. Sci., vol. 88, pp. 4230-4234, May 1991, Biochemistry. |
| Thompson, et al., To determine whether the temperature of 2% lignocaine gel affects the initial discomfort which may be associated with its instillation into the male urethra, BJU International (1999), 84, 1035-1037. |
| Thomson et al., “Investigation of the safety of irreversible electroporation in humans,” J Vasc Interv Radiol, 22, pp. 611-621, 2011. |
| TUNA—Suggested Local Anesthesia Guidelines, no date available. |
| Verbridge et al., “Oxygen-Controlled Three-Dimensional Cultures to Analyze Tumor Angiogenesis.” Tissue Engineering, Part A vol. 16, pp. 2133-2141 (2010). |
| Vernier, P.T., et al., “Nanoelectropulse-driven membrane perturbation and small molecule permeabilization”, Bmc Cell Biology, 7 (2006). |
| Vidamed, Inc., Transurethral Needle Ablation (TUNA): Highlights from Worldwide Clinical Studies, Vidamed's Office TUNA System, 2001. |
| Weaver et al., “A brief overview of electroporation pulse strength-duration space: A region where additional Intracellular effects are expected.” Bioelectrochemistry vol. 87, pp. 236-243 (2012). |
| Weaver, Electroporation: A General Phenomenon for Manipulating Cells and Tissues, Journal of Cellular Biochemistry, 51: 426-435, 1993. |
| Weaver, et al., Theory of Electroporation: A Review, Bioelectrochemistry and Bioenergetics, vol. 41, pp. 136-160, 1996. |
| Weaver, J. C., Electroporation of biological membranes from multicellular to nano scales, IEEE Tms. Dielectr. Electr. Insul. 10, 754-768 (2003). |
| Weaver, J.C., “Electroporation of cells and tissues”, IEEE Transactions on Plasma Science, 28(1): p. 24-33 (2000). |
| Weisstein: Cassini Ovals. From MathWorld—A. Wolfram Web Resource; Apr. 30, 2010; http://mathworld.wolfram.com/ (updated May 18, 2011). |
| Yang et al., “Dielectric properties of human leukocyte subpopulations determined by electrorotation as a cell separation criterion.” Biophysical Journal, vol. 76, pp. 3307-3314 (1999). |
| Yao et al., “Study of transmembrane potentials of inner and outer membranes induced by pulsed-electric-field model and simulation.” IEEE Trans Plasma Sci, 2007. 35(5): p. 1541-1549. |
| Zhang, Y., et al., MR imaging to assess immediate response to irreversible electroporation for targeted ablation of liver tissues: preclinical feasibility studies in a rodent model. Radiology, 2010. 256(2): p. 424-32. |
| Zimmermann, et al., Dielectric Breakdown of Cell Membranes, Biophysical Journal, vol. 14, No. 11, pp. 881-899, 1974. |
| Zlotta, et al., Long-Term Evaluation of Transurethral Needle Ablation of the Prostate (TUNA) for Treatment of Benign Prostatic Hyperplasia (BPH): Clinical Outcome After 5 Years. (Abstract) Presented at 2001 AUA National Meeting, Anaheim, CA—Jun. 5, 2001. |
| Zlotta, et al., Possible Mechanisms of Action of Transurethral Needle Ablation of the Prostate on Benign Prostatic Hyperplasia Symptoms: a Neurohistochemical Study, Reprinted from Journal of Urology, vol. 157, No. 3, Mar. 1997, pp. 894-899. |
| Co-Pending Application No. AU 2015259303, filed Oct. 24, 2016. |
| Co-Pending Application No. CN 201580025135.6, filed Nov. 14, 2016. |
| Co-Pending Application No. EP 15793361.5, filed Dec. 12, 2016. |
| Co-Pending Application No. JP 2016-567747, filed Nov. 10, 2016. |
| Neal II et al., “A Case Report on the Successful Treatment of a Large Soft-Tissue Sarcoma with Irreversible Electroporation,” Journal of Clinical Oncology, 29, pp. 1-6, 2011. |
| Neal II et al., “Experimental Characterization and Numerical Modeling of Tissue Electrical Conductivity during Pulsed Electric Fields for Irreversible Electroporation Treatment Planning,” Biomedical Engineering, IEEE Transactions on Biomedical Engineering, vol. 59, pp. 1076-1085, 2012. |
| Neal II, R. E., et al., “Successful Treatment of a Large Soft Tissue Sarcoma with Irreversible Electroporation”, Journal of Clinical Oncology, 29:13, e372-e377 (2011). |
| Neal II, R.E., et al., “Treatment of breast cancer through the application of irreversible electroporation using a novel minimally invasive single needle electrode.” Breast Cancer Research and Treatment, 2010. 123(1): p. 295-301. |
| Neal II, Robert E. and R.V. Davalos, The Feasibility of Irreversible Electroporation for the Treatment of Breast Cancer and Other Heterogeneous Systems, Ann Biomed Eng, 2009, 37(12): p. 2615-2625. |
| Nesin et al., “Manipulation of cell volume and membrane pore comparison following single cell permeabilization with 60- and 600-ns electric pulses.” Biochimica et Biophysica Acta (BBA)—Biomembranes, vol. 1808, pp. 792-801 (2011). |
| Neumann, et al., Gene Transfer into Mouse Lyoma Cells by Electroporation in High Electric Fields, J. Embo., vol. 1, No. 7, pp. 841-845, 1982. |
| Neumann, et al., Permeability Changes Induced by Electric Impulses in Vesicular Membranes, J. Membrane Biol., vol. 10, pp. 279-290, 1972. |
| Nikolova, B., et al., “Treatment of Melanoma by Electroporation of Bacillus Calmette-Guerin”. Biotechnology & Biotechnological Equipment, 25(3): p. 2522-2524 (2011). |
| Nuccitelli, R., et al., “A new pulsed electric field therapy for melanoma disrupts the tumor's blood supply and causes complete remission without recurrence”, Int J Cancer, 125(2): p. 438-45 (2009). |
| O'Brien et al., “Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.” European Journal of Biochemistry, vol. 267, pp. 5421-5426 (2000). |
| Okino, et al., Effects of High-Voltage Electrical Impulse and an Anticancer Drug on in Vivo Growing Tumors, Japanese Journal of Cancer Research, vol. 78, pp. 1319-1321, 1987. |
| Onik, et al., Sonographic Monitoring of Hepatic Cryosurgery in an Experimental Animal Model, AJR American J. of Roentgenology, vol. 144, pp. 1043-1047, May 1985. |
| Onik, et al., Ultrasonic Characteristics of Frozen Liver, Cryobiology, vol. 21, pp. 321-328, 1984. |
| Onik, G. and B. Rubinsky, eds. “Irreversible Electroporation: First Patient Experience Focal Therapy of Prostate Cancer. Irreversible Electroporation”, ed. B. Rubinsky 2010, Springer Berlin Heidelberg, pp. 235-247. |
| Onik, G., P. Mikus, and B. Rubinsky, “Irreversible electroporation: implications for prostate ablation.” Technol Cancer Res Treat, 2007. 6(4): p. 295-300. |
| Organ, L.W., Electrophysiological principles of radiofrequency lesion making, Apply. Neurophysiol., 1976. 39: p. 69-76. |
| Ott, H. C., et al., “Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart”, Nature Medicine, Nature Publishing Group, New York, NY, US, vol. 14, No. 2, Feb. 1, 2008, pp. 213-221. |
| Paszek et al., “Tensional homeostasis and the malignant phenotype.” Cancer Cell, vol. 8, pp. 241-254 (2005). |
| Payselj, et al., “The course of tissue permeabilization studied on a mathematical model of a subcutaneous tumor in small animals,” IEEE Trans Biomed Eng, vol. 52, pp. 1373-1381, 2005. |
| PCT International Preliminary Report on Patentability of Corresponding International Application No. PCT/2011/062067, dated May 28, 2013. |
| PCT International Preliminary Report on Patentability of Corresponding International Application No. PCT/2011/066239, dated Jun. 25, 2013. |
| PCT International Search Report (dated Aug. 2, 2011), Written Opinion (dated Aug. 2, 2011), and International Preliminary Report on Patentability (dated Apr. 17, 2012) of PCT/US10/53077. |
| PCT International Search Report (dated Aug. 22, 2012), and Written Opinion (dated Aug. 22, 2012) of PCT/US11/66239. |
| PCT International Search Report (dated Aug. 26, 2005), Written Opinion (dated Aug. 26, 2005), and International Preliminary Report on Patentability (dated Jun. 26, 2006) of PCT/US2004/043477. |
| PCT International Search Report (dated Jan. 19, 2010), Written Opinion (dated Jan. 19, 2010), and International Preliminary Report on Patentability (dated Jan. 4, 2010) of PCT/US09/62806, 15 pgs. |
| PCT International Search Report (dated Jul. 15, 2010), Written Opinion (dated Jul. 15, 2010), and International Preliminary Report on Patentability (dated Oct. 11, 2011) from PCT/US2010/030629. |
| PCT International Search Report (dated Jul. 9, 2009), Written Opinion (dated Jul. 9, 2009), and International Preliminary Report on Patentability (dated Nov. 2, 2010) of PCT/US2009/042100. |
| PCT International Search Report and Written Opinion (dated Jul. 25, 2012) of PCT/US2011/062067. |
| PCT International Search Report, 4 pgs, (dated Jul. 30, 2010), Written Opinion, 7 pgs, (dated Jul. 30, 2010), and International Preliminary Report on Patentability, 8 pgs, (dated Oct. 4, 2011) from PCT/US2010/029243. |
| PCT IPRP for PCT/US15/30429 (WO2015175570), dated Nov. 15, 2016. |
| Phillips, M., Maor, E. & Rubinsky, B. Non-Thermal Irreversible Electroporation for Tissue Decellularization. J. Biomech. Eng, doi:10.1115/1.4001882 (2010). |
| Piñero, et al., Apoptotic and Necrotic Cell Death Are Both Induced by Electroporation in HL60 Human Promyeloid Leukaemia Cells, Apoptosis, vol. 2, No. 3, 330-336, Aug. 1997. |
| Polak et al., “On the Electroporation Thresholds of Lipid Bilayers: Molecular Dynamics Simulation Investigations.” The Journal of Membrane Biology, vol. 246, pp. 843-850 (2013). |
| Precision Office TUNA System, When Patient Satisfaction is Your Goal, VidaMed 2001. |
| Pucihar et al., “Numerical determination of transmembrane voltage induced on irregularly shaped cells.” Annals of Biomedical Engineering, vol. 34, pp. 642-652 (2006). |
| Rajagopal, V. and S.G. Rockson, Coronary restenosis: a review of mechanisms and management, The American Journal of Medicine, 2003, 115(7): p. 547-553. |
| Rebers̆ek, M. and D. Miklavc̆ic̆, “Advantages and Disadvantages of Different Concepts of Electroporation Pulse Generation,” Automatika 52(2011) 1, 12-19. |
| Rols, M.P., et al., Highly Efficient Transfection of Mammalian Cells by Electric Field Pulses: Application to Large Volumes of Cell Culture by Using a Flow System, Eur. J. Biochem. 1992, 206, pp. 115-121. |
| Ron et al., “Cell-based screening for membranal and cytoplasmatic markers using dielectric spectroscopy.” Biophysical chemistry, 135 (2008) pp. 59-68. |
| Rossmeisl et al., “Pathology of non-thermal irreversible electroporation (N-TIRE)-induced ablation of the canine brain.” Journal of Veterinary Science vol. 14, pp. 433-440 (2013). |
| Rossmeisl, “New Treatment Modalities for Brain Tumors in Dogs and Cats.” Veterinary Clinics of North America: Small Animal Practice 44, pp. 1013-1038 (2014). |
| Rubinsky et al., “Optimal Parameters for the Destruction of Prostate Cancer Using Irreversible Electroporation.” The Journal of Urology, 180 (2008) pp. 2668-2674. |
| Rubinsky, B., “Irreversible Electroporation in Medicine”, Technology in Cancer Research and Treatment, vol. 6, No. 4, Aug. 1, 2007, pp. 255-259. |
| Rubinsky, B., ed, Cryosurgery. Annu Rev. Biomed. Eng. vol. 2 2000. 157-187. |
| Rubinsky, B., et al., “Irreversible Electroporation: A New Ablation Modality—Clinical Implications” Technol. Cancer Res. Treatment 6(1), 37-48 (2007). |
| Sabuncu et al., “Dielectrophoretic separation of mouse melanoma clones.” Biomicrofluidics, vol. 4, 7 pages (2010). |
| Salford, L.G., et al., “A new brain tumour therapy combining bleomycin with in vivo electropermeabilization”, Biochem. Biophys. Res. Commun., 194(2): 938-943 (1993). |
| Salmanzadeh et al., “Investigating dielectric properties of different stages of syngeneic murine ovarian cancer cells” Biomicrofiuidics 7, 011809 (2013), 12 pages. |
| Salmanzadeh et al., “Dielectrophoretic differentiation of mouse ovarian surface epithelial cells, macrophages, and fibroblasts using contactless dielectrophoresis.” Biomicrofluidics, vol. 6, 13 Pages (2012). |
| Co-pending U.S. Appl. No. 15/186,653, filed Jun. 20, 2016. |
| Co-Pending Application No. PCT/US04/43477, filed Dec. 21, 2004. |
| Co-Pending Application No. PCT/US09/42100, filed Apr. 29, 2009. |
| Co-Pending Application No. PCT/US09/62806, filed Oct. 30, 2009. |
| Co-Pending Application No. PCT/US10/30629, filed Apr. 9, 2010. |
| Co-Pending Application No. PCT/US10/53077, filed Oct. 18, 2010. |
| Co-Pending Application No. PCT/US11/62067, filed Nov. 23, 2011. |
| Co-Pending Application No. PCT/US11/66239, filed Dec. 20, 2011. |
| Co-Pending Application No. PCT/US15/30429, filed May 12, 2015. |
| Co-Pending Application No. PCT/US15/30429, International Search Report and Written Opinion dated Oct. 16, 2015, 19 pages. |
| Co-pending Application No. PCT/US2010/029243, filed Mar. 30, 2010, published as WO 2010/117806 on Oct. 14, 2010. |
| Co-Pending Application No. PCT/US2015/030429, Published on Nov. 19, 2015 as WO 2015/175570. |
| Co-Pending U.S. Appl. No. 12/491,151, filed Jun. 24, 2009. |
| Co-Pending U.S. Appl. No. 13/332,133, filed Dec. 20, 2011. |
| Co-Pending U.S. Appl. No. 13/550,307, filed Jul. 16, 2012. |
| Co-Pending U.S. Appl. No. 13/919,640, filed Jun. 17, 2013. |
| Co-Pending U.S. Appl. No. 13/958,152, filed Aug. 2, 2013. |
| Co-Pending U.S. Appl. No. 13/989,175, filed May 23, 2013. |
| Co-Pending U.S. Appl. No. 14/012,832, filed Aug. 28, 2013. |
| Co-Pending U.S. Appl. No. 14/017,210, filed Sep. 3, 2013. |
| Co-Pending U.S. Appl. No. 14/627,046, filed Feb. 20, 2015. |
| Co-Pending U.S. Appl. No. 14/686,380, filed Apr. 14, 2015. |
| Co-Pending U.S. Appl. No. 14/940,863, filed Nov. 13, 2015 and Published as US 2016/0066977 on Mar. 10, 2016. |
| Corovic et al., “Analytical and numerical quantification and comparison of the local electric field in the tissue for different electrode configurations,” Biomed Eng Online, 6, 14 pages, 2007. |
| Cowley, Good News for Boomers, Newsweek, Dec. 30, 1996/Jan. 6, 1997. |
| Cox, et al., Surgical Treatment of Atrial Fibrillation: A Review, Europace (2004) 5, S20-S-29. |
| Crowley, Electrical Breakdown of Biomolecular Lipid Membranes as an Electromechanical Instability, Biophysical Journal, vol. 13, pp. 711-724, 1973. |
| Dahl et al., “Nuclear shape, mechanics, and mechanotransduction.” Circulation Research vol. 102, pp. 1307-1318 (2008). |
| Daud, A.I., et al., “Phase I Trial of Interleukin-12 Plasmid Electroporation in Patients With Metastatic Melanoma,” Journal of Clinical Oncology, 26, 5896-5903, Dec. 20, 2008. |
| Davalos et al., “Electrical impedance tomography for imaging tissue electroporation,” IEEE Transactions on Biomedical Engineering, 51, pp. 761-767, 2004. |
| Davalos et al., “Theoretical analysis of the thermal effects during in vivo tissue electroporation.” Bioelectrochemistry, vol. 61(1-2): pp. 99-107, 2003. |
| Davalos et al., “Tissue ablation with irreversible electroporation.” Annals of Biomedical Engineering, vol. 33, No. 2, pp. 223-231 (Feb. 2005). |
| Davalos, et al., A Feasibility Study for Electrical Impedance Tomography as a Means to Monitor T issue Electroporation for Molecular Medicine, IEEE Transactions on Biomedical Engineering, vol. 49, No. 4, Apr. 2002. |
| Davalos, R. V. & Rubinsky, B. Temperature considerations during irreversible electroporation. International Journal of Heat and Mass Transfer 51, 5617-5622, doi:10.1016/j.ijheatmasstransfer.2008.04.046 (2008). |
| Davalos, Real-Time Imaging for Molecular Medicine through Electrical Impedance Tomography of Electroporation, Dissertation for Ph.D. in Engineering-Mechanical Engineering, Graduate Division of University of California, Berkeley, 2002. |
| De Vuyst, E., et al., “In situ bipolar Electroporation for localized cell loading with reporter dyes and investigating gap functional coupling”, Biophysical Journal, 94(2): p. 469-479 (2008). |
| Dean, Nonviral Gene Transfer to Skeletal, Smooth, and Cardiac Muscle in Living Animals, Am J. Physiol Cell Physiol 289: 233-245, 2005. |
| Demirbas, M. F., “Thermal Energy Storage and Phase Change Materials: An Overview” Energy Sources Part B 1(1), 85-95 (2006). |
| Dev, et al., Medical Applications of Electroporation, IEEE Transactions of Plasma Science, vol. 28, No. 1, pp. 206-223, Feb. 2000. |
| Dev, et al., Sustained Local Delivery of Heparin to the Rabbit Arterial Wall with an Electroporation Catheter, Catheterization and Cardiovascular Diagnosis, Nov. 1998, vol. 45, No. 3, pp. 337-343. |
| Duraiswami, et al., Boundary Element Techniques for Efficient 2-D and 3-D Electrical Impedance Tomography, Chemical Engineering Science, vol. 52, No. 13, pp. 2185-2196, 1997. |
| Duraiswami, et al., Efficient 2D and 3D Electrical Impedance Tomography Using Dual Reciprocity Boundary Element Techniques, Engineering Analysis with Boundary Elements 22, (1998) 13-31. |
| Duraiswami, et al., Solution of Electrical Impedance Tomography Equations Using Boundary Element Methods, Boundary Element Technology XII, 1997, pp. 226-237. |
| Edd et al., “Mathematical modeling of irreversible electroporation for treatment planning.” Technology in Cancer Research and Treatment, vol. 6, No. 4, pp. 275-286 (2007). |
| Edd, J. et al., In-Vivo Results of a New Focal Tissue Ablation Technique: Irreversible Electroporaton, IEEE Trans. Biomed. Eng. 53 (2006) p. 1409-1415. |
| Ellis TL, Garcia PA, Rossmeisl JH, Jr., Henao-Guerrero N, Robertson J, et al., “Nonthermal irreversible electroporation for intracranial surgical applications. Laboratory investigation”, J Neurosurg 114: 681-688 (2011). |
| Eppich et al., “Pulsed electric fields for selection of hematopoietic cells and depletion of tumor cell contaminants.” Nature Biotechnology 18, pp. 882-887 (2000). |
| Erez, et al., Controlled Destruction and Temperature Distributions in Biological Tissues Subjected to Monoactive Electrocoagulation, Transactions of the ASME: Journal of Mechanical Design, vol. 102, Feb. 1980. |
| Ermolina et al., “Study of normal and malignant white blood cells by time domain dielectric spectroscopy.” IEEE Transactions on Dielectrics and Electrical Insulation, 8 (2001) pp. 253-261. |
| Esser, A.T., et al., “Towards solid tumor treatment by irreversible electroporation: intrinsic redistribution of fields and currents in tissue”. Technol Cancer Res Treat, 6(4): p. 261-74 (2007). |
| Esser, A.T., et al., “Towards Solid Tumor Treatment by Nanosecond Pulsed Electric Fields”. Technology in Cancer Research & Treatment, 8(4): p. 289-306 (2009). |
| Faroja, M., et al., “Irreversible Electroporation Ablation: Is the entire Damage Nonthermal?”, Radiology, 266(2), 462-470 (2013). |
| Fischbach et al., “Engineering tumors with 3D scaffolds.” Nat Meth 4, pp. 855-860 (2007). |
| Flanagan et al., “Unique dielectric properties distinguish stem cells and their differentiated progeny.” Stem Cells, vol. 26, pp. 656-665 (2008). |
| Fong et al., “Modeling Ewing sarcoma tumors in vitro with 3D scaffolds.” Proceedings of the National Academy of Sciences vol. 110, pp. 6500-6505 (2013). |
| Foster RS, “High-intensity focused ultrasound in the treatment of prostatic disease”, European Urology, 1993, vol. 23 Suppl 1, pp. 29-33. |
| Foster, R.S., et al., Production of Prostatic Lesions in Canines Using Transrectally Administered High-Intensity Focused Ultrasound. Eur. Urol., 1993; 23: 330-336. |
| Fox, et al., Sampling Conductivity Images via MCMC, Mathematics Department, Auckland University, New Zealand, May 1997. |
| Freeman, S.A., et al., Theory of Electroporation of Planar Bilayer-Membranes—Predictions of the Aqueous Area, Change in Capacitance, and Pore-Pore Separation. Biophysical Journal, 67(1): p. 42-56 (1994). |
| Garcia et al., “Irreversible electroporation (IRE) to treat brain cancer.” ASME Summer Bioengineering Conference, Marco Island, FL, Jun. 25-29, 2008, 2 pages. |
| Garcia et al., “Non-thermal irreversible electroporation (N-TIRE) and adjuvant fractionated radiotherapeutic multimodal therapy for intracranial malignant glioma in a canine patient,” Technol Cancer Res Treat, 10, pp. 73-83, 2011. |
| Garcia et al., “Towards a Predictive Model of Electroporation-Based Therapies using Pre-Pulse Electrical Measurements” Abstract presented in the IEEE Engineering in Medicine and Biology Conference in Aug. 28, 2012 in San Diego, California, 4 pages. |
| Garcia P.A., et al., “7.0-T Magnetic Resonance Imaging Characterization of Acute Blood-Brain-Barrier Disruption Achieved with Intracranial Irreversible Electroporation”, PLOS One, Nov. 2012, 7:11, e50482. |
| Garcia P.A., et al., “Pilot study of irreversible electroporation for intracranial surgery”, Conf Proc IEEE Eng Med Biol Soc, 2009:6513-6516, 2009. |
| Garcia, et al., “A Parametric Study Delineating Irreversible Electroporation from Thermal Damage Based on a Minimally Invasive Intracranial Procedure,” Biomed Eng Online, vol. 10:34, 22 pages, 2011. |
| Garcia, P. et al. Intracranial nonthermal irreversible electroporation: in vivo analysis. J Membr Biol 236, 127-136 (2010). |
| Gascoyne et al., “Membrane changes accompanying the induced differentiation of Friend murine erythroleukemia cells studied by dielectrophoresis.” Biochimica et Biophysica Acta (BBA)—Biomembranes, vol. 1149, pp. 119-126 (1993). |
| Gauger, et al., A Study of Dielectric Membrane Breakdown in the Fucus Egg, J. Membrane Biol., vol. 48, No. 3, pp. 249-264, 1979. |
| Gehl, et al., In Vivo Electroporation of Skeletal Muscle: Threshold, Efficacy and Relation to Electric Field Distribution, Biochimica et Biphysica Acta 1428, 1999, pp. 233-240. |
| Gençer, et al., Electrical Impedance Tomography: Induced-Current Imaging Achieved with a Multiple Coil System, IEEE Transactions on Biomedical Engineering, vol. 43, No. 2, Feb. 1996. |
| Gilbert, et al., Novel Electrode Designs for Electrochemotherapy, Biochimica et Biophysica Acta 1334, 1997, pp. 9-14. |
| Gilbert, et al., The Use of Ultrasound Imaging for Monitoring Cryosurgery, Proceedings 6th Annual Conference, IEEE Engineering in Medicine and Biology, 107-111, 1984. |
| Gilbert, T. W., et al., “Decellularization of tissues and organs”, Biomaterials, Elsevier Science Publishers, Barking, GB, vol. 27, No. 19, Jul. 1, 2006, pp. 3675-3683. |
| Gimsa et al., “Dielectric spectroscopy of single human erythrocytes at physiological ionic strength: dispersion of the cytoplasm.” Biophysical Journal, vol. 71, pp. 495-506 (1996). |
| Glidewell, et al., The Use of Magnetic Resonance Imaging Data and the Inclusion of Anisotropic Regions in Electrical Impedance Tomography, Biomed, Sci. Instrum. 1993; 29: 251-7. |
| Solberg, A. and Rubinsky, B., “A statistical model for multidimensional irreversible electroporation cell death in tissue.” Biomed Eng Online, 9, 13 pages, 2010. |
| Gothelf, et al., Electrochemotherapy: Results of Cancer Treatment Using Enhanced Delivery of Bleomycin by Electroporation, Cancer Treatment Reviews 2003: 29: 371-387. |
| Gowrishankar T.R., et al., “Microdosimetry for conventional and supra-electroporation in cells with organelles”. Biochem Biophys Res Commun, 341(4): p. 1266-76 (2006). |
| Griffiths, et al., A Dual-Frequency Electrical Impedance Tomography System, Phys. Med. Biol., 1989, vol. 34, No. 10, pp. 1465-1476. |
| Griffiths, The Importance of Phase Measurement in Electrical Impedance Tomography, Phys. Med. Biol., 1987, vol. 32, No. 11, pp. 1435-1444. |
| Griffiths, Tissue Spectroscopy with Electrical Impedance Tomography: Computer Simulations, IEEE Transactions on Biomedical Engineering, vol. 42, No. 9, Sep. 1995. |
| Gumerov, et al., The Dipole Approximation Method and Its Coupling with the Regular Boundary Element Method for Efficient Electrical Impedance Tomography, Boundary Element Technology XIII, 1999. |
| Hapala, Breaking the Barrier: Methods for Reversible Permeabilization of Cellular Membranes, Critical Reviews in Biotechnology, 17(2): 105-122, 1997. |
| Helczynska et al., “Hypoxia promotes a dedifferentiated phenotype in ductal breast carcinoma in situ.” Cancer Research, vol. 63, pp. 1441-1444 (2003). |
| Heller, et al., Clinical Applications of Electrochemotherapy, Advanced Drug Delivery Reviews, vol. 35, pp. 119-129, 1999. |
| Hjouj, M., et al., “Electroporation-Induced BBB Disruption and Tissue Damage Depicted by MRI”, Neuro-Oncology 13: Issue suppl 3, abstract ET-32 (2011). |
| Hjouj, M., et al., “MRI Study on Reversible and Irreversible Electroporation Induced Blood Brain Barrier Disruption”, PLOS One, Aug. 2012, 7:8, e42817. |
| Hjouj, Mohammad et al., “Electroporation-Induced BBB Disruption and Tissue Damage Depicted by MRI,” Abstracts from 16th Annual Scientific Meeting of the Society for Neuro-Oncology in Conjunction with the AANS/CNS Section on Tumors, Nov. 17-20, 2011, Orange County California, Neuro-Oncology Supplement, vol. 13, Supplement 3, p. iii114. |
| Ho, et al., Electroporation of Cell Membranes: A Review, Critical Reviews in Biotechnology, 16(4): 349-362, 1996. |
| Holder, et al., Assessment and Calibration of a Low-Frequency System for Electrical Impedance Tomography (EIT), Optimized for Use in Imaging Brain Function in Ambulant Human Subjects, Annals of the New York Academy of Science, vol. 873, Issue 1, Electrical BI, pp. 512-519, 1999. |
| Huang, et al., Micro-Electroporation: Improving the Efficiency and Understanding of Electrical Permeabilization of Cells, Biomedical Microdevices, vol. 2, pp. 145-150, 1999. |
| Hughes, et al., An Analysis of Studies Comparing Electrical Impedance Tomography with X-Ray Videofluoroscopy in the Assessment of Swallowing, Physiol. Meas. 15, 1994, pp. A199-A209. |
| Ibey et al., “Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells.” Biochimica Et Biophysica Acta-General Subjects, vol. 1800, pp. 1210-1219 (2010). |
| Issa, et al., The TUNA Procedure for BPH: Review of the Technology: The TUNA Procedure for BPH: Basic Procedure and Clinical Results, Reprinted from Infections in Urology, Jul./Aug. 1998 and Sep./Oct. 1998. |
| Ivanus̆a, et al., MRI Macromolecular Contrast Agents as Indicators of Changed Tumor Blood Flow, Radiol. Oncol. 2001; 35(2): 139-47. |
| Ivorra et al., “In vivo electric impedance measurements during and after electroporation of rat live.” Bioelectrochemistry, vol. 70, pp. 287-295 (2007). |
| Ivorra et al., “In vivo electrical conductivity measurements during and after tumor electroporation: conductivity changes reflect the treatment outcome.” Physics in Medicine and Biology, vol. 54, pp. 5949-5963 (2009). |
| Ivorra, “Bioimpedance monitoring for physicians: an overview.” Biomedical Applications Group, 35 pages (2002). |
| Co-pending U.S. Appl. No. 15/423,986, filed Feb. 3, 2017. |
| Co-pending U.S. Appl. No. 15/424,335, filed Feb. 3, 2017. |
| Co-pending U.S. Appl. No. 16/404,392, Petition for Priority, filed Jun. 4, 2019, 2 pages. |
| Co-pending U.S. Appl. No. 16/404,392, Preliminary Amendment, filed Jun. 4, 2019, 9 pages. |
| Co-Pending Application No. JP 2016-567747, First Office Action (Translation) dated Feb. 21, 2019, 4 pages. |
| Co-Pending Application No. JP 2016-567747, First Office Action dated Feb. 21, 2019, 4 pages. |
| Co-Pending U.S. Appl. No. 13/332,133, Amendment with RCE after Board Decision, dated Mar. 29, 2019, 16 pages. |
| Co-Pending U.S. Appl. No. 13/332,133, Board Decision dated Jan. 29, 2019, 13 pages. |
| Co-Pending U.S. Appl. No. 13/332,133, Notice of Allowance, dated May 31, 2019, 5 pages. |
| Co-Pending U.S. Appl. No. 13/332,133, Office Actions and Responses through Mar. 2018, 221 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Acceptance of 312 Amendment dated Sep. 12, 2018, 1 page. |
| Co-Pending U.S. Appl. No. 14/017,210, AFCP dated Aug. 13, 2018, 9 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Final Office Action dated Aug. 30, 2016, 11 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Notice of Allowance (dated Dec. 12, 2018 RCE) dated Jan. 9, 2019, 5 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Notice of Allowance dated Sep. 12, 2018, 7 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Petition dated Dec. 11, 2015, 5 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Petition Decision dated Aug. 12, 2016, 9 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Petition Decision dated Aug. 2, 2016, 5 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, RCE dated Aug. 1, 2017, 13 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, RCE dated Nov. 30, 2016, 13 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Response to Dec. 15, 2016 Non-Final Office Action dated Mar. 20, 2017, 9 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Response to May 1, 2017 Final Office Action dated Aug. 1, 2017, 10 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Response to Non-Final Office Action dated Mar. 8, 2016, 16 pages. |
| Co-Pending U.S. Appl. No. 14/627,046, Amendment dated Jun. 29, 2017, 8 pages. |
| Co-Pending U.S. Appl. No. 14/627,046, Final Office Action dated Sep. 14, 2017, 11 pages. |
| Co-Pending U.S. Appl. No. 14/627,046, Interview Summary dated dated Apr. 27, 2018, 3 pages. |
| Co-Pending U.S. Appl. No. 14/627,046, Non-Final Office Action dated Feb. 15, 2018, 12 pages. |
| Co-Pending U.S. Appl. No. 14/627,046, Non-Final Office Action dated Mar. 29, 2017, 9 pages. |
| Co-Pending U.S. Appl. No. 14/627,046, Notice of Allowance dated Sep. 19, 2018, 7 pages. |
| Co-Pending U.S. Appl. No. 14/627,046, Response to Sep. 14, 2017 Final Office Action dated Dec. 14, 2017, 7 pages. |
| Co-Pending U.S. Appl. No. 14/627,046, Rule 132 Affidavit and Response to Feb. 15, 2018 Non-Final Office Action, dated Jun. 15, 2018, 13 pages. |
| Co-Pending U.S. Appl. No. 16/152,743, filed Oct. 5, 2018. |
| Co-Pending U.S. Appl. No. 16/232,962, filed Dec. 26, 2018. |
| Co-pending U.S. Appl. No. 16/404,392, Preliminary Amendment, filed Jun. 6, 2019, 5 pages. |
| Co-Pending U.S. Appl. No. 14/686,380, Final Office Action dated May 9, 2018, 14 pages. |
| Co-Pending U.S. Appl. No. 14/686,380, Non-Final Office Action dated May 1, 2019, 26 pages. |
| Co-Pending U.S. Appl. No. 14/686,380, Non-Final Office Action dated Nov. 22, 2017, 11 pages. |
| Co-Pending U.S. Appl. No. 14/686,380, Response to Jul. 19, 2017 Restriction Requirement, dated Sep. 15, 2017, 2 pages. |
| Co-Pending U.S. Appl. No. 14/686,380, Response to May 9, 2018 Final Office Action with RCE, dated Aug. 30, 2018, 14 pages. |
| Co-Pending U.S. Appl. No. 14/686,380, Response to Nov. 22, 2017 Non-Final Office Action dated Mar. 28, 2018, 11 pages. |
| Co-Pending U.S. Appl. No. 14/686,380, Restriction Requirement Jul. 19, 2017, 7 pages. |
| Co-pending U.S. Appl. No. 15/843,888, filed Dec. 15, 2017. |
| Co-pending U.S. Appl. No. 15/881,414, filed Jan. 26, 2018. |
| Co-Pending U.S. Appl. No. 14/017,210, Final Office Action dated Apr. 11, 2018, 10 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Final Office Action dated May 1, 2017, 11 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Non-Final Office Action dated Dec. 15, 2016, 8 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Non-Final Office Action dated Oct. 25, 2017, 9 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Non-Final Office Action, dated Sep. 8, 2015, 8 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Priority Petition Dec. 11, 2015, 5 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, RCE filed Aug. 1, 2017, 13 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Response to Aug. 30, 2016 Final Office Action, dated Nov. 30, 2016, 10 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Response to Oct. 25, 2017 Non-Final Office Action dated Jan. 25, 2018, 11 pages. |
| Co-Pending U.S. Appl. No. 14/017,210, Response to Sep. 8, 2015 Non-Final Office Action, dated Mar. 8, 2016, 57 pages. |
| Garcia, Paulo A., Robert E. Neal II and Rafael V. Davalos, Chapter 3, Non-Thermal Irreversible Electroporation for Tissue Ablation, In: Electroporation in Laboratory and Clinical Investigations ISBN 978-1-61668-327-6 Editors: Enrico P. Spugnini and Alfonso Baldi, 2010, 22 pages. |
| Kotnik et al., “Sensitivity of transmembrane voltage induced by applied electric fields—a theoretical analysis”, Bioelectrochemistry and Bioenergetics,vol. 43, Issue 2, 1997, pp. 285-291. |
| Neal RE II, et al. (2013) Improved Local and Systemic Anti-Tumor Efficacy for Irreversible Electroporation in Immunocompetent versus Immunodeficient Mice. PLoS One 8(5): e64559. https://doi.org/10.1371/journal.pone.0064559. |
| Tibbitt et al., “Hydrogels as Extracellular Matrix Mimics for 3D Cell Culture”, Jul. 2009, Biotechnol Bioeng, 103(4),655-663. |
| Wimmer, Thomas, et al., “Planning Irreversible Electroporation (IRE) in the Porcine Kidney: Are Numerical Simulations Reliable for Predicting Empiric Ablation Outcomes?”, Cardiovasc Intervent Radiol. Feb. 2015; 38(1): 182-190. doi:10.1007/s00270-014-0905-2. |
| Co-Pending U.S. Appl. No. 12/491,151, Final Rejection dated Apr. 20, 2012, 8 pages. |
| Co-Pending U.S. Appl. No. 12/491,151, Non-Final Rejection dated Apr. 4, 2014, 12 pages. |
| Co-Pending U.S. Appl. No. 12/491,151, Non-Final Rejection dated Dec. 28, 2011, 7 pages. |
| Co-Pending U.S. Appl. No. 12/491,151, Official Notice of Allowance dated Feb. 12, 2015, 2 pages. |
| Co-Pending U.S. Appl. No. 12/491,151, Official Notice of Allowance dated Nov. 6, 2014, 15 pages. |
| Co-Pending U.S. Appl. No. 12/491,151, Official Notice of Allowance dated Oct. 15, 2014, 5 pages. |
| Co-Pending U.S. Appl. No. 12/491,151, Requirement for Restriction/Election dated Dec. 2, 2011, 6 pages. |
| Co-Pending U.S. Appl. No. 12/491,151, Response to Apr. 4, 2014 Non-Final Rejection dated Aug. 22, 2014, 12 pages. |
| Co-Pending U.S. Appl. No. 12/491,151, Response to Non-Final Rejection dated Mar. 28, 2012, 10 pages. |
| Co-Pending U.S. Appl. No. 12/491,151, Response to Requirement for Restriction/Election dated Dec. 13, 2011, 2 pages. |
| Co-Pending U.S. Appl. No. 12/491,151, Response with RCE to Final Rejection dated Aug. 20, 2012, 14 pages. |
| Co-Pending U.S. Appl. No. 12/491,151, Supplemental Amendment dated Dec. 17, 2012, 6 pages. |
| Co-Pending U.S. Appl. No. 12/757,901, File History 2018. |
| Co-Pending U.S. Appl. No. 12/906,923, Office Actions and Responses Jul. 2017, 55 pages. |
| Co-Pending U.S. Appl. No. 14/558,631, Final Office Action dated Sep. 1, 2017, 9 pages. |
| Co-Pending U.S. Appl. No. 14/558,631, Non-Final Office Action dated Jan. 8, 2018, 5 pages. |
| Co-Pending U.S. Appl. No. 14/558,631, Non-Final Office Action dated Mar. 13, 2017, 10 pages. |
| Co-Pending U.S. Appl. No. 14/558,631, Notice of Allowance dated Jul. 17, 2018, 2 pages. |
| Co-Pending U.S. Appl. No. 14/558,631, Notice of Allowance dated Jun. 21, 2018, 7 pages. |
| Co-Pending U.S. Appl. No. 14/558,631, Response to Jan. 8, 2018 Non-Final Office Action dated Apr. 9, 2018, 8 pages. |
| Co-Pending U.S. Appl. No. 14/558,631, Response to Mar. 13, 2017 Non-Final Office Action dated Jul. 13, 2017, 10 pages. |
| Co-Pending U.S. Appl. No. 14/558,631, Response to Sep. 1, 2017 Final Office Action dated Dec. 1, 2017, 7 pages. |
| Co-Pending U.S. Appl. No. 14/808,679, Restriction Requirement dated Mar. 19, 2018, 7 pages. |
| Co-Pending U.S. Appl. No. 14/808,679, Final Office Action dated Jan. 11, 2019, 12 pages. |
| Co-Pending U.S. Appl. No. 14/808,679, Interview Summary dated Apr. 26, 2019, 3 pages. |
| Co-Pending U.S. Appl. No. 14/808,679, Non-Final Office Action dated Sep. 10, 2018, 12 pages. |
| Co-Pending U.S. Appl. No. 14/808,679, Petition for Priority and Supplemental Response, filed May 8, 2019, 25 pages. |
| Co-Pending U.S. Appl. No. 14/808,679, Preliminary Amendment, filed Jul. 27, 2015, 9 pages. |
| Co-Pending U.S. Appl. No. 14/808,679, RCE filed Apr. 11, 2019, 8 pages. |
| Co-Pending U.S. Appl. No. 14/808,679, Response to Mar. 19, 2018 Restriction Requirement dated May 21, 2018, 2 pages. |
| Co-Pending U.S. Appl. No. 14/808,679, Response to Sep. 10, 2018 Non-Final Office Action dated Dec. 10, 2018, 9 pages. |
| Co-pending U.S. Appl. No. 15/186,653, Notice of Allowance dated Aug 1, 2018, 7 pages. |
| Co-pending U.S. Appl. No. 15/186,653, Notice of Allowance dated Mar. 20, 2019, 9 pages. |
| Co-pending U.S. Appl. No. 15/424,335 Examiner Interview Request (date Jul. 2, 2018), 1 page. |
| Co-pending U.S. Appl. No. 15/424,335 Examiner Interview Summary (dated Jul. 20, 2018), 3 pages. |
| Co-pending U.S. Appl. No. 15/424,335 Non-Final Office Action dated Apr. 26, 2018, 9 pages. |
| Co-pending U.S. Appl. No. 15/424,335 Notice of Allowance dated Sep. 21, 2018, 7 pages. |
| Co-pending U.S. Appl. No. 15/424,335 Preliminary Amendment filed Apr. 17, 2017, 17 pages. |
| Co-pending U.S. Appl. No. 15/424,335 Response to Non-Final Office Action dated Jul. 26, 2018, 13 pages. |
| Co-pending U.S. Appl. No. 15/881,414 Amendment and Petition for Priority Claim dated Jul. 26, 2018, 26 pages. |
| Co-pending U.S. Appl. No. 15/881,414 Apr. 26, 2018 Non-Final Office Action, 8 pages. |
| Co-pending U.S. Appl. No. 15/881,414 Notice of Allowance dated Oct. 24, 2018, 7 pages. |
| Co-pending U.S. Appl. No. 15/881,414 Petition Decision dated Oct. 9, 2018, 9 pages. |
| Co-pending U.S. Appl. No. 16/177,745, filed Nov. 1, 2018. |
| Co-pending U.S. Appl. No. 16/177,745, Preliminary Amendment dated Dec. 19, 2018, 7 pages. |
| Co-pending U.S. Appl. No. 16/275,429, filed Feb. 14, 2019. |
| Co-pending U.S. Appl. No. 16/280,511, filed Feb. 20, 2019. |
| Co-pending U.S. Appl. No. 16/372,520, filed Apr. 2, 2019. |
| Co-pending U.S. Appl. No. 16/375,878, filed Apr. 5, 2019. |
| Co-pending U.S. Appl. No. 16/404,392, filed May 6, 2019. |
| Co-pending Application No. JP 2016-567747 Amendment filed Jul. 18, 2019, 7 pgs. |
| Co-pending Application No. JP 2016-567747 English version of amended claims filed Jul. 18, 2019, 6 pgs. |
| Co-pending Application No. JP 2019-133057 filed Jul. 18, 2019, 155 pgs. |
| Co-pending Application No. JP 2019-133057 Filing receipt for application filed Jul. 18, 2019, 2 pg. |
| Number | Date | Country | |
|---|---|---|---|
| 20170266438 A1 | Sep 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61992023 | May 2014 | US |
