ENHANCED TREATMENT VOLUME AND SELECTIVE ABLATION USING ELECTROPORATION WITH ADJUVANT CALCIUM

Abstract

High-frequency irreversible electroporation (H-FIRE) is an electroporation-based therapy used to ablate cancerous tissue. Treatment consists of delivering short pulses in a series of bursts. Reducing pulse duration leads to reduced treatment volumes compared to traditional IRE, therefore larger voltages are typically applied to generate ablations comparable in size. Administration of adjuvant calcium enhances ablation area in vitro for H-FIRE treatments of several pulse durations. Furthermore, H-FIRE treatment delivered with CaCl.sub.2 results in cell death thresholds higher than that of H-FIRE without calcium and comparable to IRE thresholds without calcium. Quantifying the reversible electroporation threshold revealed that CaCl.sub.2 enhances the permeabilization of cells compared to a NaCl control. H-FIRE treatment with calcium enhances the IRE to thermal cell death ratio, thereby also enhancing the positive immune response from treatment.

Claims

1. A method of treating tissue comprising: applying a plurality of electrical pulses to a tissue region; and exposing the tissue region to one or more agent; wherein the applying of the electrical pulses is performed in a manner sufficient to treat cells of the tissue region with high-frequency irreversible electroporation (H-FIRE); and wherein the agent is capable of protecting cells from, enhancing or increasing, and/or inhibiting, decreasing, or limiting, one or more effects of the H-FIRE.

2. The method of claim 1, wherein one or more of the effects of the H-FIRE is chosen from cell death; quick cell death on the order of seconds or minutes; slow cell death on the order of hours, days, weeks, months or years; apoptosis; necrosis; heat; thermal effects; cell membrane permeabilization; inflammatory response; blood brain barrier disruption; transient blood brain barrier disruption; permanent damage; transient damage; immune response; a reversible electroporation zone and combinations thereof.

3. The method of claim 1, wherein the agent comprises calcium or a non-calcium buffer.

4. The method of claim 1, wherein the agent comprises calcium in an amount sufficient to: increase an area of ablation to a size comparable to that expected from H-FIRE administered using a higher voltage; and/or provide an increased IRE to thermal cell death ratio than without the calcium, such that a lower thermal effect and an enhanced positive immune response are provided.

5. The method of claim 4, wherein the positive immune response is promoted by immune cells present beyond the tissue region to which the plurality of electrical pulses are applied.

6. The method of claim 1, wherein one or more of the agents comprises calcium in an amount capable of increasing one or more effects of the H-FIRE in the tissue region.

7. The method of claim 1, wherein one or more of the agents comprises sucrose capable of protecting cells from, inhibiting, decreasing, or limiting one or more effects of the H-FIRE.

8. The method of claim 1, wherein the exposing of the tissue region to the agent comprises: exposing a first tissue region to an agent comprising calcium in an amount sufficient to enhance one or more effects of the H-FIRE; and exposing a second tissue region to a non-calcium containing buffer in an amount sufficient to limit one or more effects of the H-FIRE.

9. The method of claim 8, wherein the first tissue region comprises cancer cells.

10. The method of claim 9, wherein the second tissue region comprises non-cancerous cells.

11. A method of treating tissue comprising: applying a plurality of electrical pulses to a tissue region; wherein the electrical pulses have a pulse width of less than 100 s; and exposing the tissue region to one or more agent; wherein the applying is performed in a manner sufficient to cause electroporation of cells of at least a portion of the tissue region; and wherein the agent is capable of protecting cells from, enhancing or increasing, and/or inhibiting, decreasing, or limiting, one or more effects of the electroporation in at least a portion of the tissue region.

12. The method of claim 11, wherein the agent comprises calcium or a non-calcium containing buffer.

13. The method of claim 11, wherein the plurality of electrical pulses are capable of electroporation based therapy, electroporation, irreversible electroporation, reversible electroporation, electrochemotherapy, electrogenetherapy, supraporation, and/or high frequency irreversible electroporation, or combinations thereof.

14. The method of claim 11, wherein the applying and the exposing together provide for an increased IRE to thermal cell death ratio in at least a portion of the tissue region, such that a lower thermal effect and an enhanced positive immune response are provided.

15. The method of claim 11, wherein the exposing of the tissue region to the agent comprises exposing the tissue region to an agent comprising calcium capable of enhancing one or more of the effects of the electroporation in at least a portion of the tissue region.

16. The method of claim 11, wherein the exposing of the tissue region to the agent comprises exposing the tissue region to a non-calcium containing buffer capable of limiting one or more of the effects of the electroporation in at least a portion of the tissue region.

17. A method of selectively treating cells, comprising: applying a plurality of electrical pulses to first and second tissue regions; exposing the first tissue region to a first agent in a manner such that more cell death occurs within the first tissue region than without presence of the first agent; and exposing the second tissue region to a second agent in a manner such that: less cell death, or no cell death, occurs within the second tissue region than without presence of the second agent; and/or the second tissue region comprises a zone of reversible electroporation, the zone being enhanced by presence of the second agent.

18. The method of claim 17, wherein the first agent comprises calcium and the second agent comprises a non-calcium buffer.

19. The method of claim 17, wherein the applying and the exposing together provide for an increased IRE to thermal cell death ratio, such that a lower thermal effect and an enhanced positive immune response are provided.

20. The method of claim 17, wherein the second tissue region comprises: vasculature, nerve tissue, and/or tissue near the vasculature or the nerve tissue; and/or tissue near one or more electrodes used in applying the plurality of electrical pulses.

Description

BREIF DESCRIPTION OF THE DRAWINGS

[0080] 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.

[0081] FIG. 1A is a diagram depicting a generic unipolar pulse scheme for electrical energy based therapies, such as IRE.

[0082] FIG. 1B is a diagram depicting representative pulse schemes for H-FIRE comprising a desired number of bursts of bipolar pulses delivered with a desired applied voltage, a desired pulse width (e.g., 1, 2, 5, or 10 s), a desired waveform (e.g., square), a desired inter-pulse delay (e.g., 1 s), a pulse number adjusted for a total on time per burst (e.g., 100 s) (respectively, 100, 50, 20, or 10 pulses), and a desired frequency (such as a frequency of one burst per second).

[0083] FIG. 2 is a schematic depicting custom made electrodes inserted into a collagen scaffold platform prior to pulsing with electrode spacing of 4.0 mm (scale bar 4 mm).

[0084] FIGS. 3A-B are illustrations depicting the electric field distribution (FIG. 3A) and the corresponding temperature distribution (FIG. 3B) in a collagen scaffold.

[0085] FIG. 4 is a schematic showing the sizes of lesions resulting from various electroporation treatments with calcium chloride and sodium chloride.

[0086] FIG. 5 is a graph showing the calculated lesion area for collagen scaffold experiments demonstrating that calcium chloride treatments lead to larger lesions than NaCl controls.

[0087] FIG. 6 is a graph showing the calculated electric field thresholds for collagen scaffold experiments demonstrating that calcium chloride treatments lead to lower electric field thresholds resulting in cell death than their sodium chloride controls.

[0088] FIG. 7 is a schematic showing the reversible electroporation zones for cells stained immediately following electroporation treatment.

[0089] FIG. 8 is a graph showing the calculated reversible electroporation zone for collagen scaffold experiments demonstrating that calcium chloride treatments result in larger reversible electroporation zones.

[0090] FIG. 9 is a schematic overlaying the reversible electroporation zone (white circle) with the irreversible electroporation zone (darker zone within the white circle) to show the calcium treatments result in smaller reversible:irreversible electroporation zone ratios. The region beyond the white circled area is non-electroporated tissue.

[0091] FIG. 10 is a graph showing the ratio of reversible electroporation to irreversible ablation (IRE) areas for collagen scaffold experiments demonstrating that calcium chloride treatments result in smaller reversible:irreversible electroporation zone ratios.

[0092] FIG. 11 is a schematic showing cell death following H-FIRE treatment for cells treated with solutions of sodium chloride, potassium chloride, calcium chloride, and a mixture of calcium chloride and sodium chloride.

[0093] FIG. 12 is a graph showing the ablation areas for cells treated with sodium chloride, potassium chloride, calcium chloride, and a mixture of calcium chloride and sodium chloride during H-FIRE treatment.

[0094] FIG. 13 is an illustration comparing the areas of thermally damaged cells, electroporated cells, and surviving cells following H-FIRE treatment at various voltages with and without calcium chloride.

[0095] FIGS. 14-18 are flowcharts illustrating various exemplary methods of the invention.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

[0096] Reference will now be made in detail to various exemplary embodiments of the invention. It is to be understood that the following discussion of exemplary embodiments is not intended as a limitation on the invention. Rather the following discussion is provided to give the reader a more detailed understanding of certain aspects and features of the invention.

[0097] 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 inventions presented herein or in any future applications claiming priority to the instant application.

[0098] Embodiments of the present invention provide electrical energy based treatment protocols (e.g., H-FIRE, IRE, RE and/or ECT) with the administration of one or more agent(s) to enhance the zone of electroporation and/or to protect a region from electroporation and/or one or more effects of electroporation.

[0099] In embodiments, the agent administered is adjuvant calcium. In another embodiment, the agent administered is a non-calcium buffer. In another embodiment, both adjuvant calcium and non-calcium buffer are delivered to the tissue region and/or cells receiving electroporation treatment. In another embodiment, the treatment is delivered in a manner promoting an immune response, such as an enhanced positive immune response, by way of immune cells that respond to tissue injury resulting from electroporation.

[0100] Definitions:

[0101] The term pulse refers to an electrical signal with a single phase (monopolar, unipolar) or more than one phase (bi-polar). If bi-polar, there can be a delay between phases or the switch between phases/polarity can be immediate (no intra-pulse delay).

[0102] The term burst refers to a set of pulses, a group of pulses, or a pulse group.

[0103] The term inter-pulse delay refers to the condition where no energy is applied for a period of time between one pulse or set of pulses and another pulse, or between one bi-polar pulse or set of bi-polar pulses and another pulse or bi-polar pulse.

[0104] The term total on time refers to the time associated with energizing an electrode. For example, for a 5-1-5 waveform comprising a bipolar pulse with a 5 s pulse width and a 1 s inter-pulse delay, a burst of 20 pulses would have a total on time of 100 s.

[0105] The term thermal damage refers to damage to a treated tissue caused by an increase in temperature which results in death of the tissue and/or denaturing of proteins.

[0106] The terms selective ablation, electroporation, administration or treatment; selectively ablating, electroporating, treating or administering electrical energy; controlled electroporation, ablation, treatment, or administering of electrical energy; treatment protocols with a dual purpose and the like are used to refer to performing an electrical energy treatment protocol in a manner designed to obtain a desired effect in one tissue area while also obtaining a desired effect in another tissue area. The treatment can have the same type of effect (e.g., H-FIRE) in both tissue regions but with a different level of efficacy (e.g., different size treatment zones), or the treatment can have a different type of effect in each tissue region (e.g., IRE in one region and RE in another region) with a different purpose (e.g., kill cells in one region and spare cells in another, or kill cells in one region and allow for uptake of agents by RE mechanisms in another region).

[0107] The term agent refers to any substance, composition, or solution administered as an adjuvant in combination with administering electrical energy treatment. Agents include calcium adjuvants for enhancing or increasing one or more effects of the electrical energy treatment and buffers, such as non-calcium buffers (such as a buffer comprising sucrose, e.g., an NaCl buffer comprising sucrose) for protecting cells from and/or inhibiting, decreasing, or limiting one or more effects of the electrical energy treatment. The terms agent, adjuvant, buffer, substance, composition, or solution may be used interchangeably in the context of this disclosure.

[0108] In the following examples, the inventors demonstrate that it is possible to increase ablation size and/or treatment areas/zones and/or margins using electroporation with adjuvant calcium, and that using an adjuvant such as a non-calcium containing buffer, such as a sodium chloride and sucrose buffer, offers protection for cells, allowing for selective ablation or treatment, thereby enhancing the safety and efficacy of treatment.

[0109] As the results show for example, using a 10 s H-FIRE pulse with calcium lowers the electric field threshold to a value comparable to an IRE treatment. This would allow the use of clinically available generator to be used and negate the need for custom electronics, making treatment more accessible. Additionally, calcium chloride is non-toxic to cells at the concentrations used, unlike chemotherapeutics that have been used previously in ECT treatments and IRE treatments to increase ablation sizes. Finally, if tumors are located in complex organs such as the pancreas or brain that contain many blood vessels/nerves/neurons, then ablation can be contained/controlled and/or selectively administered to preserve critical structures.

[0110] Scaffold Preparation

[0111] U251 malignant glioma cells (Sigma Aldrich, 09063001) were maintained at 5% CO.sub.2 and 37 C. in Eagle's Minimum Essential Medium (Sigma Aldrich) supplemented with 1% penicillin/streptomycin (Life Technologies), 10% fetal bovine serum (Atlanta Biologicals), 1% non-essential amino acids (Sigma Aldrich) and 1 mM sodium pyruvate (Sigma Aldrich). Cells were routinely passaged at 80-90% confluence.

[0112] Sterile polydimethylsiloxane (PDMS, SYLGARDTM 184, Dow Corning) wells (10 mm diameter, 1 mm height) were placed in a 24 well plate to ensure uniform collagen scaffold geometry and electric field distribution between each replicate. PDMS wells were treated with 1% PEI (Acros Organics) for 10 min, 0.1% glutaraldehyde (Fisher Scientific) for 20 min, and then washed twice with deionized water prior to collagen seeding to ensure collagen adhesion during treatment. Commercial rat tail collagen type I (BD Biosciences) was neutralized using a solution of 10 Dulbecco's Modified Eagle Medium (10% total volume, Sigma Aldrich), 1 N NaOH (2% collagen volume, Sigma Aldrich), and 1 Dulbecco's Modified Eagle Medium (Sigma Aldrich) to a final concentration of 5 mg/mL. U251 cells were detached from flasks using 0.25% trypsin/EDTA (Thermo Fisher Scientific) solution and added to the neutralized collagen solution at a concentration of 110.sup.6 cells/mL. The collagen/cell solution was dispensed into PDMS wells and PDMS tops were used to mold the collagen flat while they polymerized in a cell culture incubator for 20 min. PDMS tops were then removed and cell culture media was added. Collagen scaffolds were maintained in the incubator for 24 hr prior to treatment. For further reference, FIG. 2 shows a representative experimental platform.

[0113] With respect to H-FIRE in particular, embodiments of the invention include a method of treating tissue comprising: applying a plurality of electrical pulses to a tissue region; and exposing the tissue region to one or more agent; wherein the applying of the electrical pulses is performed in a manner sufficient to treat cells of the tissue region with high-frequency irreversible electroporation (H-FIRE); and wherein the agent is capable of protecting cells from, enhancing or increasing, and/or inhibiting, decreasing, or limiting, one or more effects of the H-FIRE. The agent can comprise calcium and/or a non-calcium containing buffer with sucrose.

[0114] Adjuvant and Electroporation Treatment

[0115] In this example, after 24 h, cell culture media was aspirated and replaced with either CaCl.sub.2, NaCl, KCl, or a combination of CaCl.sub.2 and NaCl solutions (1 mM or 5 mM) and allowed to incubate for 30 minutes to saturate the collagen scaffold. All solutions consisted of the same base ingredients: 250 mM sucrose, 1 mM MgCl.sub.2, and 10 mM HEPES buffer in deionized water with a pH in the range of 7.2-7.4. These solutions were then removed and fresh CaCl.sub.2, NaCl, KCl, or CaCl.sub.2/NaCl solutions were allowed to incubate for another 10 minutes to ensure all cell culture media was washed out of the scaffold. Cell culture media contains things such as serum and antibiotics that may affect results. Finally, fresh solutions were added immediately prior to pulsing. All solutions were adjusted to have a pH between 7.2-7.4. The osmolarity and conductivity of the buffers used are shown in Table 1:

TABLE-US-00001 TABLE 1 Properties of solutions used in this invention Conductivity Osmolarity Concentration Solution (S/m) (mOsm/L) 1 mM CaCl.sub.2 0.075 0.004 289 NaCl 0.056 0.001 287 KCl 0.064 289 CaCl.sub.2 + NaCl 0.065 287 5 mM CaCl.sub.2 0.131 300 NaCl 0.089 291

[0116] In embodiments, one or more of the agents comprises calcium in an amount (such as from 0.1 mM to 500 mM) capable of enhancing and/or increasing one or more effects of the H-FIRE in the tissue region, and/or one or more of the agents comprises sucrose, or a combination of NaCl and sucrose, in an amount (such as from 0.1 mM to 500 mM) capable of protecting cells from, inhibiting, decreasing, or limiting one or more effects of the H-FIRE.

[0117] In practice, in vivo, the adjuvant calcium and/or non-calcium containing buffer can be administered in any manner, such as by injection, infusion, or exposure, such as parenteral, intravenous, intraarterial, intradermal, transdermal, intranasal, local or intralesional, intraperitoneal, intramuscular, buccal, oral, or transmucosal administration, depending on the particular tissue or application. Adjuvant calcium and/or non-calcium containing buffer can be administered immediately prior, such as within 5 minutes, 1 minute, less than 30 seconds prior, or up to or more than 5 minutes, 15 minutes, 0.5 hr, 1 hr, 2 hr, 4 hr, 8 hr, or 12 hr before and/or after the applying of the plurality of electrical pulses, and/or the agent can be administered during the applying of the plurality of electrical pulses. In embodiments, the most effective course of action would be to inject the adjuvant directly into the tissue immediately before treatment to limit the amount of diffusion out of the treatment area before the electrical energy treatment begins. Injecting after pulsing may contribute to a loss of some efficacy as reversibly electroporated cells recover within minutes of removing electric field.

[0118] The calcium adjuvant or non-calcium containing adjuvant/buffer can be administered at an amount of about 50% of tissue/tumor volume, such as in the range of about 0.10% to 100% of tissue volume, or more, or from 0.5% to 99%, or from 1% to 95%, or from 20% to 90%, or 30% to 75%, or 25% to 60%, or 40% to 80% of the volume of the tissue region being treated, or any range in between any of these ranges or endpoints, including as endpoints any number encompassed thereby. The calcium adjuvant or non-calcium containing adjuvant/buffer can be administered at concentrations ranging from 0.1 mM to 500 mM, such as from 0.5 mM to 400 mM, or from 1 mM to 300 mM, or from 5 mM to 250 mM, or from 10 mM to 150 mM, or from 20 mM to 100 mM, such as from 2 mM to 15 mM, or 3 mM to 8 mM, or 5 mM to 7 mM, or 4 mM to 12 mM, or any range between any of these ranges or endpoints, including as endpoints any number encompassed thereby.

[0119] In this example, two hollow, stainless-steel needle electrodes (Howard electronics) were inserted into the scaffolds using a custom designed electrode housing. The electrodes had an outer diameter of 0.914 mm and inner diameter of 0.635 mm and were spaced 4 mm apart (center-to-center) (FIG. 2). H-FIRE pulses were delivered using a high-voltage pulse generator (EPULSUS-FBM1-5, EnergyPulse Systems, Lda). Voltage waveforms were captured using a high voltage probe and oscilloscope (DPO 2012, Tektronix). In embodiments, the plurality of electrical pulses is administered from two or more electrodes (e.g., two or more electrodes disposed in contact with one or another tissue region (such as a needle electrode disposed within a target region and a surface electrode disposed in contact with skin), or two or more electrodes disposed in each or both regions to be treated), and from any number of electrodes, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 electrodes and in any configuration relative to one another. For example, the electrodes can be spaced apart a distance of from 0 cm to 10 cm apart, such as from above 0 cm up to 10 cm apart, or from 0.2 cm to 9 cm, such as from 0.5 cm to 5 cm, or from 1 cm to 4 cm apart, or from 2 cm to 3 cm, or 1.5 cm, or any range in between any of these ranges or endpoints, including as endpoints any number encompassed thereby.

[0120] In any embodiment administering electrical energy described herein, the electrodes can be one or more needle electrodes, plate electrodes, surface electrodes, hollow electrodes, blunt tip electrodes or combinations thereof. For example, in embodiments, the adjuvant(s) can be administered through hollow electrodes and/or electrodes/probes with hollow channels configured for the administration of one or more fluids, such as disclosed in U.S. Pat. No. 10,245,098. In embodiments, the electrodes can have a length (whether the length of the active tip of the electrode or the shaft of the probe) ranging from 1 cm to 30 cm, such as from 10 cm to 20 cm, or from 5 cm to 15 cm, and/or with a length of the active portion of the probe (e.g., energizable region) ranging from 0.5 cm to 10 cm, such as from 1 cm to 5 cm, or from 2 cm to 6 cm, or 1.5 cm to 8 cm, or up to 3 cm or up to 4 cm, or any range in between any of these ranges or endpoints, including as endpoints any number encompassed thereby.

[0121] The pulses delivered were bipolar pulses having a positive pulse duration, inter-pulse delay, and negative pulse duration (see, e.g., FIG. 1B). Inter-pulse delay was kept constant (1 s) while pulse width was varied (1, 2, 5, 10 s). A total of eighty (80) bursts were delivered at 800 V for a total on time of 100 s per burst (respectively, a total number of 100, 50, 20, 10 pulses per burst) at a frequency of one burst per second.

[0122] Depending on the particular application and other protocol parameters, such as electrode spacing, any voltage can be applied, including for example in the range of 0 V to 10,000 V, such as above 0 V or 1 V up to 10,000 V, and/or from 500 V up to 3,000 V, and/or from 1,000 V up to 2,000 V, such as up to 250 V, up to 300 V, up to 350 V, up to 600 V, up to 650 V, up to 800 V, up to 1,200 V, up to 1,500 V, up to 5,000 V, up to 7,500 V, or for example from 100 V to 15,000 V, such as from 500 V up to 3,000 V, and/or from 1,000 V up to 2,000 V, such as up to 250 V, up to 300 V, up to 350 V, up to 600 V, up to 650 V, up to 800 V, up to 1,200 V, up to 1,500 V, up to 15,000 V, up to 7,500 V, from 4,000 V to 12,000 V, such as less than 450 V, or less than 425 V, such as from above 0 V to 400 V, or from about 10 V to 350 V, or about 20 V to about 300 V, or about 30 V to about 250 V, or from about 15 V to about 200 V, or from about 50 V to about 150 V, or about 75 V to 100 V, or from 30 V to 225 V, or from 60 V to 375 V. Additionally, or alternatively, for example, the pulse widths can range from about 1 picosecond to 50 microseconds, such as about 10 ns to about 10 microseconds, or about 10 microseconds or less.

[0123] or any range in between any of these ranges or endpoints, including as endpoints any number encompassed thereby.

[0124] With respect to H-FIRE in particular, one or more pulses of the plurality of electrical pulses can have a pulse length in the picosecond to microsecond range, such as in the nanosecond to microsecond range, including from 1 picosecond to below 10 microseconds, or from 1 picosecond to 1 microsecond, or below 1 microsecond, or from at least 0.1 microsecond up to 5 microseconds, or from 0.5 microseconds up to 2 microseconds or up to 10 microseconds, or any range in between any of these ranges or endpoints, including as endpoints any number encompassed thereby, such as a high-frequency irreversible electroporation burst scheme of pulse width and intra-phase delay ranging from 0.1 s to 10 ms and an inter-pulse delay ranging from 0.1 s to 1 s. The pulses can be unipolar or bi-polar. Any desired waveform can also be used, including square, triangular, trapezoidal, exponential decay, sawtooth, sinusoidal, and/or such waveforms comprising one or more pulses of alternating polarity.

[0125] Additionally, the pulsing schemes can incorporate one or more intra- or inter-pulse delays and/or one or more intra- or inter-burst delays. For example, pulsing schemes of bursts of pulses comprising schemes of 1-1-1 s, 2-1-2 s, 5-1-5 s, or 10-1-10 s with up to a 1-second delay between bursts can be used. In general, for H-FIRE, pulsing schemes conforming to the following formula can be used: (i) administering a pulse with a first polarity and a pulse duration of less than 10 microseconds, (ii) administering a delay with a duration of up to 20 microseconds, (iii) administering a pulse with a second polarity (that can be the same or a different polarity than the first pulse) and a pulse duration of less than 10 microseconds, (iv) administering a delay of up to 1 second, then (v) repeating the administering of (i)-(iv) a desired number of times.

[0126] Any number of pulses can be administered wherein there are a total number of pulses, and/or a total number of pulses per burst, ranging from 1-5,000 pulses, such as from at least 1 up to 3,000 pulses, or at least 2 up to 2,000 pulses, or at least 5 up to 1,000 pulses, or at least 10 up to 500 pulses, or from 10 to 100 pulses, such as from 20 to 75 pulses, or from 30 to 50 pulses, such as 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, or 90 pulses, or the total number of pulses and/or bursts can range from 1 to 5,000 pulses/bursts, such as from at least 1 up to 3,000 pulses/bursts, or at least 2 up to 2,000 pulses/bursts, or at least 5 up to 1,000 pulses/bursts, or at least 10 up to 500 pulses/bursts, or from 10 to 100 pulses/bursts, such as from 20 to 75 pulses/bursts, or from 30 to 50 pulses/bursts, such as 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, or 90 pulses/bursts, or any range in between any of these ranges or endpoints, including as endpoints any number encompassed thereby.

[0127] In embodiments, one or more of the effects of the H-FIRE that is enhanced, limited or prevented is chosen from cell death; quick cell death on the order of seconds or minutes; slow cell death on the order of hours, days, weeks, months or years; apoptosis; necrosis; heat; thermal effects; cell membrane permeabilization; inflammatory response; blood brain barrier disruption; transient blood brain barrier disruption; permanent damage; transient damage; immune response; a reversible electroporation zone and combinations thereof.

[0128] Of particular interest are such methods, wherein the agent comprises calcium in an amount sufficient to: increase an area or margin of ablation to a size comparable to that expected from H-FIRE administered using a higher voltage; and/or provide an increased IRE to thermal cell death ratio than without the calcium, such that a lower thermal effect and an enhanced positive immune response are provided. Additionally, or alternatively, such methods can include promoting a positive immune response, such as by immune cells present beyond the tissue region to which the plurality of electrical pulses are applied. For example, in embodiments, a more positive immune response can be promoted by administering calcium in combination with any one or more of the electroporation methods and protocols, or portions thereof, disclosed in U.S. Patent Application Publication No. 2019/0282294, which is incorporated by reference herein in its entirety. Further, it has been shown that H-FIRE effectively ablates the primary tumor and induces a pro-inflammatory shift in the tumor microenvironment; that local treatment with H-FIRE significantly reduces 4T1 metastases; that H-FIRE kills 4T1 cells through non-thermal mechanisms associated with necrosis and pyroptosis resulting in damage associated molecular pattern signaling in vitro and in vivo; that that the level of tumor ablation correlates with increased activation of cellular immunity; that the decrease in metastatic lesions is dependent on the intact immune system and H-FIRE generates 4T1 neoantigens that engage the adaptive immune system to significantly attenuate tumor progression; and that the non-thermal cell death mechanism of H-FIRE has been shown to elicit a more positive immune response after treatment when compared to modalities that rely on heating such as radiofrequency ablation, microwave ablation, or high-intensity focused ultrasound. See, e.g., Ringel-Scaia, V. et al., High-frequency irreversible electroporation is an effective tumor ablation strategy that induces immunologic cell death and promotes systemic anti-tumor immunity, EBioMedicine, 2019 June; 44: 112-125, which is incorporated by reference herein in its entirety. Thus, combining calcium administration with any one or more of these methods/protocols, or portions thereof, can be used to promote a more positive or enhanced immune response.

[0129] After pulsing, CaCl.sub.2, NaCl, KCl, and CaCl.sub.2/NaCl solutions were removed and cell culture media was added to each scaffold. For irreversible threshold experiments, the scaffolds were returned to the incubator for 24 hr prior to live/dead staining. For reversible threshold experiments, scaffolds were imaged immediately after treatment.

[0130] Analysis of Treatment Areas and Margins, Ablation Size and Efficacy

[0131] To visualize ablations, a live/dead stain was performed to visualize live and dead regions of cells in the scaffold. Scaffolds were incubated with 2 M Calcein AM and 23 M propidium iodide in phosphate buffered saline (PBS) for 30 minutes at room temperature. Scaffolds were then washed 2 with PBS prior to imaging using an inverted microscope (DMI 6000B, Leica Microsystems) with a 5 objective. Lesion areas were quantified for each treatment using a MATLAB algorithm. Since 24 h post treatment is sufficient to allow any reversibly electroporated cells to recover (as pore resealing happens on the order of minutes), staining the cells immediately after treatment and 24 h later allows for quantification of both the reversible zone of electroporation and irreversible zones of ablation.

[0132] To stain the scaffolds, media was removed and replaced with phosphate buffered saline (PBS) containing 2 M Calcein AM (Invitrogen) and 23 M PI (Invitrogen) and incubated at room temperature for 30 min. Scaffolds were then washed twice with PBS prior to imaging using an inverted microscope (DMI 6000B, Leica Microsystems) with a 5 objective, filter cubes, and an EM-CCD camera (Hamamatsu C9100). The appropriate filters were used to image Calcein AM (Ex:460-500, DC: 505, EM: 570-640) and propidium iodide (EX:545/26, DC:565, EM:605/70). To determine the reversible zone of electroporation, treatment followed the same electroporation protocol, but Calcein AM and PI were added to the CaCl.sub.2 and NaCl solutions at all steps prior to pulsing. Scaffolds were then imaged immediately after treatment.

[0133] For analysis, images were separated into two channels (greenCalcein AM, redPI) and ablation areas for the green channel were analyzed for irreversible thresholds and red channels for reversible electroporation thresholds using a custom algorithm written in MATLAB. In cases where the algorithm was unable to accurately measure ablations, coordinate points from the algorithm outlining the ablation area were used as a guide to manually trace and measure the area in ImageJ. Ablation area measurements were mapped to a finite element model of the experimental setup to determine the corresponding electric field threshold.

[0134] Irreversible electroporation experiments were repeated 6-12 times for each condition. Reversible electroporation experiments were repeated at least six times. Discrepancies in the number of replicates between conditions were due to bubbles or other defects that may have changed the electric field distribution or prohibited a reliable measurement of ablation area. These scaffolds were excluded from analysis. Two-way ANOVA was used to test for differences in cell death area due to the different applied solutions and pulse waveforms. Tukey post-hoc comparisons were used to examine differences among treatment groups. Statistical analyses were performed with a confidence level of =0.05 (MP Pro 14). Results are shown as meansstandard deviation.

[0135] Treatments were simulated using the finite element method (COMSOL Multiphysics, Burlington, Mass.) and the electric field and temperature distributions were determined. The mesh was refined until there was <1% change in the electric field and temperature values along a cutline between the electrodes. The mesh consisted of 102,615 total elements. It was determined that temperature only increased 4.4 C. (FIGS. 3A-B) during treatment. From the finite element model of the collagen scaffold, a relationship between lesion area and electric field magnitude is determined by performing numerical integration on the surface of the scaffold for a range of electric field magnitudes (100-3000 V/cm). A curve was constructed and fit to the data using least squares fitting in MATLAB. This resulted in a sixth order polynomial equation. This equation was used to accurately back out the electric field thresholds for each treatment condition without needing to manually determine them using COMSOL. Least squares fitting resulted in a maximum relative error of 4.7%.

[0136] In vitro collagen scaffolds cultured with U251 malignant glioma (MG) cells, treated using H-FIRE with a NaCl control confirms that the field is not high enough to produce a clinically relevant lesion (FIG. 4). Using an applied voltage increased by about 50%, achieves a large ablation volume. Applying a higher energy treatment, however, negates the advantages of H-FIRE because of the risk of introducing deleterious thermal damage as well as increasing the chances of producing muscle contractions, which may in turn increase the possibility of having to administer a neuromuscular block.

[0137] Because H-FIRE treatment results in an irreversibly electroporated zone of a certain size and an associated thermal ablation area within this zone, the inventors have discovered that combining H-FIRE treatment with adjuvant calcium increases/enhances the area and/or margin of ablation without necessitating the use of a higher applied voltage (FIG. 6 and FIG. 13). For example, FIG. 13 shows how the tissue is affected with or without calcium administration and with or without a higher voltage. As illustrated, the region labeled survive are cells that are either reversibly electroporated and/or not affected by the electroporation, while the regions labeled H-FIRE are irreversibly electroporated in response to H-FIRE protocols and the regions labeled thermal are subjected to thermal effects from the electroporation. Additionally, or alternatively, the shape of the area/zone of ablation can also be manipulated and/or controlled with the presence of excess calcium ions during electroporation. For example, different concentrations or amounts of calcium can be administered in different tissue regions for varying levels of effect within the treatment region.

[0138] To induce the same area of cell death without calcium, one could increase the applied voltage, which would in turn lead to an increase in joule heating and thermal damage of tissue, due to electrical properties of tissue. This is because the non-thermal mode of cell death does not denature proteins. Using calcium in combination with H-FIRE treatment will allow clinicians to lower the applied voltage by up to 50% or more, therefore lowering thermal damage while maintaining the same ablation area as H-FIRE treatment without calcium. In addition, clinicians may apply the same voltage to increase/enhance the treatment area/zone and/or margin, without an increase in thermal damage. Calcium H-FIRE treatment enhances the IRE to thermal cell death ratio, therefore enhancing the positive immune response seen after treatment. For example, where a clinician would administer H-FIRE without calcium at voltages ranging from 2,500 V to 10,000 V, the H-FIRE voltage could instead be adjusted/lowered 50% or more to below 5,000 V, such as from 1,200 V to 5,000 V when administering calcium in combination with H-FIRE.

[0139] In an embodiment of the invention, low voltage H-FIRE treatment is combined with calcium to improve the IRE zone, reduce the size of the thermal ablation zone, and induce an immune response by way of immune cells responding to the tissue injury resulting from the IRE.

[0140] In another embodiment of the invention, IRE treatment is combined with calcium to extend the IRE zone while keeping the thermal ablation zone minimal and induce an immune response by way of immune cells responding to the tissue injury. As shown in FIGS. 3A, 3B and 4 these examples have the same thermal zone but the IRE zone is increased with calcium. Therefore, the IRE:thermal ratio and the positive immune response is enhanced.

[0141] Accordingly, the inventors have discovered electroporation treatments using lower voltages and/or shorter pulse lengths and adjuvant calcium that are capable of achieving treatment areas/zones and/or margins comparable to that of similar treatments without the added calcium and which can be used to promote a positive immune response. Such protocols include methods of treating tissue comprising: applying a plurality of electrical pulses to a tissue region at a desired voltage and/or with a pulse width of less than 100 s; and exposing the tissue region to one or more agent; wherein the applying is performed in a manner sufficient to cause electroporation of cells of at least a portion of the tissue region; and wherein the agent is capable of protecting cells from, enhancing or increasing, and/or inhibiting, decreasing, or limiting, one or more effects of the electroporation in at least a portion of the tissue region. In embodiments, the agent administered can comprise calcium to enhance electroporation and/or a non-calcium containing buffer to provide protection to select cells.

[0142] These methods can be used to administer any electroporation based therapy, including electroporation, irreversible electroporation, reversible electroporation, electrochemotherapy, electrogenetherapy, supraporation, and/or high frequency irreversible electroporation, or combinations thereof. Of particular interest, such methods can involve performing the applying and the exposing for an increased IRE to thermal cell death ratio in at least a portion of the tissue region, such that a lower thermal effect and an enhanced positive immune response are provided.

[0143] Additionally, or alternatively, such methods can involve exposing the tissue region to an agent comprising calcium capable of enhancing one or more of the effects of the electroporation in at least a portion of the tissue region. Additionally, or alternatively, such methods can involve exposing the tissue region to a non-calcium containing buffer capable of limiting one or more of the effects of the electroporation in at least a portion of the tissue region.

[0144] With the addition of CaCl.sub.2, larger ablation volumes are obtained without thermal damage and the need for a neuromuscular block is mitigated. FIG. 4 and FIG. 5 confirm that the addition of 5 mM CaCl.sub.2 with the same applied energy leads to the formation of a lesion that is comparable in size or larger than an IRE treatment without calcium for 2, 5 and 10 s pulse widths. This discovery is important because it enhances the efficacy of electroporation treatment, such as H-FIRE treatment, producing lesion sizes that are clinically relevant while also alleviating the limitations found with IRE treatment such as muscle contractions.

[0145] FIG. 5 quantitatively compares ablation area in response to the different treatments administered. For shorter pulse widths, there is no visible lesion (apart from the electrodes) present for NaCl conditions. This indicates that the cells are reversibly electroporated during H-FIRE treatment and that something more is needed to drive cells to undergo cell death, such as the presence of excess calcium. The increase of ablation area for 1 mM CaCl.sub.2 compared to its NaCl control is nearly 2.3 for the 1-1-1 waveform, 4.4 for the 2-1-2 waveform, 6.1 for the 5-1-5 waveform, and 3.42 for the 10-1-10 waveform. For 5 mM CaCl.sub.2 the increase in ablation area is also apparent. For the 1-1-1 waveform, there is a 3.4 increase, for the 2-1-2 waveform there is a 4.37 increase, for the 5-1-5 waveform there is a 5.24 increase, and for the 10-1-10 waveform there is a 2.66 increase. Thus, ablation sizes for calcium groups for all pulse patterns were statistically significantly larger than that of the NaCl groups. As pulse width increases, the ablation areas for both CaCl.sub.2 concentrations also increases since the cells are permeabilized to a greater extent. The TMP of the cell has been shown to spend more time above the critical threshold as the pulse width increases, therefore resulting in more permeabilization.

[0146] From the finite element model, it is possible to determine the electric field threshold required for cell death. FIG. 6 shows that the use of adjuvant calcium reduces the electric field threshold in all experimental conditions. When compared to the NaCl control, 1 mM CaCl.sub.2 reduces the electric field threshold 1.24 for the 1-1-1 waveform, 1.5 for the 2-2-2 waveform, 2.1 for the 5-5-5 waveform, and 2.2 for the 10-1-10 waveform. When comparing 5 mM NaCl to 5 mM CaCl.sub.2, the electric field threshold is reduced 1.46 for the 1-1-1 waveform, 1.77 for the 2-1-2 waveform, 2.49 for the 5-1-5 waveform, and 1.92 for the 10-1-10 waveform. It seems the maximum effect for CaCl.sub.2 is seen with 5 mM CaCl.sub.2 for the 5-1-5 waveform and with 1 mM CaCl.sub.2 for the 10-1-10 waveform.

[0147] Although the H-FIRE thresholds are higher than they are for IRE, using calcium significantly lowers the threshold needed to produce a lesion comparable in size to an IRE treatment. Using a 10-1-10 waveform resulted in an electric field threshold (784107 V/cm) that is comparable to IRE treatment without CaCl.sub.2 (698103 V/cm) and close to IRE treatment with CaCl.sub.2 (46767 V/cm). Utilizing 10-1-10 H-FIRE waveforms in combination with adjuvant calcium lowers the required electric field threshold for cell death to a level comparable to IRE treatment. In addition, these waveforms may eliminate the need for custom built generators with complex electronics while avoiding muscle contractions and thermal damage.

[0148] For the NaCl controls, the H-FIRE treatment resulted in smaller lesions, again demonstrating that a much higher applied voltage would be needed to produce an ablation without the addition of CaCl.sub.2. The inventors have harnessed this protective function of the non-calcium containing buffer (such as a buffer comprising sucrose, e.g., an NaCl buffer comprising sucrose) as a way of protecting cells present in one or more non-target treatment zones during treatment.

[0149] Accordingly, additional methods of selectively treating cells are included that involve applying a plurality of electrical pulses to a tissue region; and exposing the tissue region to one or more agent; wherein the applying of the electrical pulses is performed in a manner sufficient to treat cells of the tissue region with high-frequency irreversible electroporation (H-FIRE); and wherein the agent is capable of protecting cells from, enhancing or increasing, and/or inhibiting, decreasing, or limiting, one or more effects of the H-FIRE and that involve exposing a first tissue region to an agent comprising calcium in an amount sufficient to enhance one or more effects of the H-FIRE; and exposing a second tissue region to a non-calcium containing buffer in an amount sufficient to limit one or more effects of the H-FIRE. Such methods can provide for selective treatment of tissue/cells by administering one type of treatment in a first region and a second type of treatment in a second region. In embodiments, selectively treating cells/tissue according to the invention can be used to treat cancer cells in a first tissue region and non-cancerous and/or non-target cells in a second tissue region.

[0150] To investigate how CaCl.sub.2 and NaCl treatments affect permeabilization of the cells, the areas for reversible electroporation and their corresponding reversible electric field thresholds were characterized. Reversible electroporation thresholds for H-FIRE treatment have not been extensively characterized, making this work one of the first to quantify reversible thresholds for a range of unexplored pulse durations.

[0151] FIGS. 7-8 show that both NaCl solutions had smaller reversible areas (i.e., the darker interior regions of the images) than their CaCl.sub.2 counterparts, therefore the buffers used seemed to affect the extent that the cells were being permeabilized. In addition, it appeared that the difference between NaCl and CaCl.sub.2 reversibly electroporated zones was similar for all waveforms. To compare the reversible zones more quantitatively, area was measured using the custom MATLAB algorithm.

[0152] FIG. 8 shows that for 1 mM CaCl.sub.2, the reversible zone was 1.56 larger than the 1 mM NaCl zone for the 2-1-2 waveform, 1.7 larger for the 5-1-5 waveform, and 1.42 larger for the 10-1-10 waveform. For 5 mM CaCl.sub.2, the reversible zone was 1.66 larger than the 1 mM NaCl zone for the 2-1-2 waveform, 1.73 for the 5-1-5 waveform, and 1.46 larger for the 10-1-10 waveform. There was no statistically significant difference between 1 mM and 5 mM treatments for the reversible case, although, after 24 h, the 5 mM CaCl.sub.2 areas are larger than the 1 mM CaCl.sub.2 for shorter pulse durations suggesting that cells exposed to lower levels of calcium recover to some extent.

[0153] Thus, the inventors have discovered that the presence of excess or added calcium in combination with H-FIRE can provide not only for an enhanced irreversible electroporation zone but also can provide for an enhanced surrounding reversible electroporation zone. Accordingly, additional methods of selectively treating cells are included that involve applying a plurality of electrical pulses to a tissue region; and exposing the tissue region to one or more agent comprising calcium; wherein the applying of the electrical pulses is performed in a manner sufficient to treat cells of the tissue region with high-frequency irreversible electroporation (H-FIRE); and wherein the agent is capable of protecting cells from, enhancing or increasing, and/or inhibiting, decreasing, or limiting, one or more effects of the H-FIRE, such as providing for an enhanced RE zone. Such methods are useful for administering IRE to undesirable tissue (e.g., tumors) in a first zone, while also increasing the amount of RE in a second zone for either an enhanced immune response and/or to provide for a better opportunity for the administration of other agents such as chemotherapy agents or gene/DNA delivery in the RE zone, both of which could be used to complement/enhance the IRE treatment.

[0154] For example, FIG. 9 shows that reversible electroporation zones (white circled regions) were larger than irreversible zones (darker interior regions) for all conditions tested. For the 2-1-2 waveform, reversible zones were 4.68 larger than irreversible zones for 1 mM NaCl, 3.88 for 5 mM NaCl, 2.62 for 1 mM CaCl.sub.2, and 1.47 for 5 mM CaCl.sub.2. For the 5-1-5 waveform reversible zones were 5.58 larger than irreversible zones for 1 mM NaCl, 3.94 for 5 mM NaCl, 1.67 for 1 mM CaCl.sub.2, and 1.3 for 5 mM CaCl.sub.2. For the 10-1-10 waveform, reversible zones were 3.17 larger for 1 mM NaCl, 2.94 larger for 5 mM NaCl, 1.32 larger for 1 mM CaCl.sub.2, and 1.59 larger for 5 mM CaCl.sub.2 when compared to irreversible zones. It is noted that in FIG. 9 there are some dark areas beyond the RE zone circled in white because sometimes there are bubbles of media that block the light from the microscope. Cells that are dead will be stained with propidium iodide which is darker in color here. It is not possible to have both the reversible and irreversible zones characterized on the same hydrogel because it requires different procedures, therefore, the RE zones overlaid here are zones that best represent the mean area that was measured.

[0155] In FIG. 10, the difference between reversible and irreversible electroporation areas was largest for NaCl solutions. When CaCl.sub.2 was used, the difference between reversible and irreversible electroporation areas decreased because the cells that were reversibly electroporated are driven instead to undergo cell death (IRE) due to uptake of excess calcium. Generally, as pulse duration increased, the difference between reversible and irreversible electroporation areas also decreased.

[0156] Using the finite element model of H-FIRE treatment in the scaffold, the corresponding electric field thresholds for each ablation area were characterized. Table 2 shows that using adjuvant calcium reduces the electric field threshold (compared to the controls) in all experimental conditions.

[0157] Boxes highlight that 10-1-10 H-FIRE treatment with calcium results in comparable electric field thresholds to standard IRE treatment. When compared to the NaCl control, 1 mM CaCl.sub.2 reduces the electric field threshold 1.24 for the 1-1-1 waveform, 1.48 for the 2-1-2 waveform, 2.05 for the 5-1-5 waveform, and 2.19 for the 10-1-10 waveform. When comparing 5 mM NaCl to 5 mM CaCl.sub.2, the electric field threshold is reduced 1.46 for the 1-1-1 waveform, 1.91 for the 2-1-2 waveform, 2.43 for the 5-1-5 waveform, and 1.83 for the 10-1-10 waveform. It seems the maximum effect for CaCl.sub.2 is seen with 5 mM CaCl.sub.2 for the 5-1-5 waveform and with 1 mM CaCl.sub.2 for the 10-1-10 waveform. Using a 10-1-10 waveform with 1 mM CaCl.sub.2 results in an electric field threshold of 771129 V/cm, reducing the threshold to less than half its value with NaCl (1641159 V/cm). It is important to note that 1 mM CaCl.sub.2 also reduces the threshold to a level that is comparable to an IRE treatment with NaCl (698103 V/cm).

[0158] It should be noted that despite most tissues having extracellular calcium concentrations around 1 mM, calcium ions are often bound by other macromolecules and only a small fraction are free in the extracellular fluid. Therefore, when treating tumors in vivo using calcium electroporation, administration of exogenous calcium is likely needed to provide the desired effect. Ensuring a desired distribution of calcium in the tissue region (e.g., uniform distribution) to be treated may be difficult to achieve due to one or more of leaky vasculature, high interstitial pressure and convective forces that drive fluid out of the tumor, however, one remedy could be to co-administer the adjuvant and/or buffer during electroporation, and/or administer immediately prior to treatment, and/or in addition to (or alternatively to) administering prior to treatment. Embodiments can include injecting calcium, such as calcium chloride, directly into a tumor and delivering electroporation, such as H-FIRE pulses, through electrodes inserted into the tumor. For protecting or sparing tissue, sucrose buffer could be injected into blood vessels near tumors prior to treatment to prevent electroporation of the endothelial cells in the vessel. Electrodes may be designed to inject CaCl.sub.2 into the tumor during treatment while also delivering a non-calcium buffer (such as a buffer comprising sucrose, e.g., an NaCl buffer comprising sucrose) on the tumor borders to protect surrounding tissue. Venofer is an Iron Sucrose solution (300 mg/ml sucrose w/v) that is administered intravenously and utilized to treat anemia. Such solutions could also be used to protect certain tissues from effects of electroporation. Most side effects are associated with the speed of administration and include dizziness, nausea, vomiting, and muscle cramps. Indeed, any composition comprising sucrose can be used as the non-calcium containing agent for protecting cells against one or more effects of electroporation.

[0159] There are several ion channels in the plasma membrane that act to pump calcium out of the cell. One of these pumps is the Na.sup.+-Ca.sup.2+ exchanger. The exchanger works to allow Na.sup.+ to be transported into the cell while pumping Ca.sup.2+ out of the cell. To investigate whether NaCl would be able to aid the cells in pumping the excess Ca.sup.2+ out, a solution that contained 1 mM of both ions was tested. A solution of KCl (potassium chloride) was also tested to determine whether the enhanced cell death effect is unique to calcium. FIGS. 11-12 show that the CaCl.sub.2 solution and the combined solution of NaCl and CaCl.sub.2 did not result in significantly different ablation sizes (p<0.001), therefore, NaCl does not rescue the cells from the effect of excess Ca.sup.2+. In addition, testing a 1 mM KCl solution did not result in statistically significant ablation size relative to the NaCl control, again confirming that the enhanced ablation areas were unique to the presence of excess calcium ions.

[0160] The inventors have thus discovered and provide a method of selectively treating cells, comprising: applying a plurality of electrical pulses to first and second tissue regions; exposing the first tissue region to a first agent in a manner such that more cell death occurs within the first tissue region than without presence of the first agent; and exposing the second tissue region to a second agent in a manner such that: less cell death, or no cell death, occurs within the second tissue region than without presence of the second agent; and/or the second tissue region comprises a zone of reversible electroporation, the zone being enhanced by presence of the second agent.

[0161] In such embodiments, the plurality of electrical pulses the first and/or second tissue region are capable of electroporation based therapy, electroporation, irreversible electroporation, reversible electroporation, electrochemotherapy, electrogenetherapy, supraporation, and/or high frequency irreversible electroporation, or combinations thereof.

[0162] For example, ablation of tissue can be performed in the first tissue region by administering IRE or H-FIRE along with an adjuvant comprising calcium. Addition of the calcium can be in an amount sufficient to increase/enhance the treatment area/zone and/or margin and result in a larger ablation volume in the first tissue region. In embodiments, CaCl.sub.2 is used. The calcium adjuvant can be administered before and/or during the electroporation treatment, such as by injection into the tissue region as discussed above and/or in the amounts/concentrations provided.

[0163] A concurrent part of the treatment in selectively treating cells according to this embodiment, entails administering the same or similar or a different type of electroporation treatment in the second tissue region. A non-calcium buffer is administered into the second tissue region to protect the cells in that region from ablation, or limit the amount of ablation. In embodiments, a buffer comprising sucrose, e.g., an NaCl buffer comprising sucrose, is used as the non-calcium containing buffer. Thus, using calcium adjuvant in the first tissue region with a selected electroporation modality, a desired ablation volume can be achieved, while using the non-calcium buffer in the second tissue region, a protective effect such as less or no cell death will occur in the second tissue region.

[0164] Such selective ablation techniques are useful for achieving an increased IRE to thermal cell death ratio in the first tissue region, such that a lower thermal effect and an enhanced positive immune response are provided. Additionally, or alternatively, cells in the second tissue region can be spared, which is useful in the context of preserving vasculature, nerve tissue, and/or tissue near the vasculature or the nerve tissue and/or tissue near one or more electrodes used in applying the plurality of electrical pulses, especially from Joule heating.

[0165] Additionally, various exemplary method embodiments of the invention are illustrated in FIGS. 14-18. According to embodiments, one or more features of the methods described in this specification and corresponding figures can be used to achieve one or more goals outlined herein.

[0166] 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.

[0167] 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.