Contactless electrotransfer

Abstract

Methods and devices for permeabilizing cell membranes and tissues using electroporation without applying a current are provided herein. The methods allow for electrotransfer of therapeutic agents to cells and tissues and / or tissue ablation within a subject while minimizing pain and muscle twitching.

Description

CONTACTLESS ELECTROTRANSFERCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 609,005, filed December 12, 2023, and U.S. Provisional Application No. 63 / 559,921, filed March 1, 2024, the content of each which is incorporated by reference in its entirety.BACKGROUND

[0002] Electroporation is a physical process utilizing short high-voltage pulses for cell membrane permeabilization. The application of an external electric field coupled with corresponding current, can transiently or permanently destabilize cell membranes. This phenomenon has been exploited in a variety of medical applications for molecular delivery or cell and tissue ablation. In the case of molecular delivery applied electrical potential and current transiently permeabilize the cell membrane to allow for transport across the membrane, followed by resealing of the membrane. Alternatively, the membrane is unable to reseal resulting in cell death and tissue ablation. Standard electrodes used for electroporation in vivo require direct contact with tissue to establish an electric pulse and consequently current to pass between metal electrodes through the tissue. Applied current inadvertently induces action potential activation across the membranes of excitable cells including neurons, smooth and striated muscle cells. Stimulating excitable cells can cause pain, twitching and more serious adverse effects such as ventricular fibrillation depending on which cells were stimulated during pulse application. Strategies to mitigate pain include pharmaceutical injections of nerve blockers and / or anesthesia. Strategies to mitigate ventricular fibrillation risk include pharmaceutical intervention as well as applied pulse synchronization with the absolute refractory period for cardiac cells as observed with an echocardiogram. Alternative strategies for in vivo electroporation that do not stimulate excitable cells are of interest.SUMMARY

[0003] In an aspect, provided herein is a method for permeabilizing a tissue or a portion thereof in a subject, the method comprising applying an electrical field to the tissue using an active electrode and a dissipation electrode; wherein at least one of the active electrode and the dissipation electrode is dielectrically insulated with an insulating material; and wherein applying the electrical field does not apply a current to the subject.

[0004] The method may further comprise administering a therapeutic agent to the subjectbefore applying the electrical field to the tissue. The therapeutic agent may be administered to the subject at an administration site of the tissue, and the electrical field may be applied to the administration site. In embodiments, the electrical field ablates at least a portion of the tissue.

[0005] Administering the therapeutic agent may be performed by subcutaneous, intraperitoneal, or intramuscular injection.

[0006] The electrical field may comprise between about 1 and about 10,00 electrical pulses. Each pulse of the electrical field may be between about 80 and about 4,000 V and between about 0.1 and about 1,000 ps; and the electrical field may comprise an interval of between about 0.1 and about 1,000 ms between each pulse. The electrical field may comprise about 1,000 electrical pulses of about 2000 V for about 100 ps each, and an interval of about 100 ms between each pulse.

[0007] The electrical field may be applied between 1 and 100 times, and the electrical field may comprise between about 1 and about 100 pulses. The electrical field may be applied about 10 times.

[0008] The insulating material may comprise at least one of glass, quartz, silicon dioxide, rubber, neoprene, silicone, titanium dioxide, barium titanate, an insulating ceramic, an insulating polymer, and an insulating composite material.

[0009] In another aspect, a method for permeabilizing a cell membrane or a tissue is provided, the method comprising: suspending the cell or tissue in an electroporation buffer to create a cell suspension or tissue suspension; and applying an electrical field to the cell or tissue suspension using a device comprising an active electrode and a dissipation electrode; wherein at least one of the active electrode and the dissipation electrode is dielectrically insulated with an insulating material; and wherein applying the electrical field does not apply a current to the cell or tissue suspension. When permeabilizing the cell or tissue, the electric field may be sufficient to ablate or destroy the tissue.

[0010] The active electrode may be dielectrically insulated; and the active electrode may be placed between about 0.1 and about 10 mm away from the cell or tissue suspension. The active electrode may be placed about 5 mm away from the cell or tissue suspension. The dielectrically insulated electrode and the cell or tissue suspension may be separated by air, glass, quartz, silicon dioxide, rubber, neoprene, silicone, titanium dioxide, barium titanate, an insulating polymer, an insulating ceramic, or an insulating composite material.

[0011] The electrical field may comprise between about 10 and about 100 electrical pulses, wherein each electrical pulse is between about 80 and about 4,000 V and between about 0.1 and about 1,000 ps; and wherein the electrical field comprises an interval of between about 0.1and about 1,000 ms between each pulse. The electrical pulses may comprise about 130 V or about 400 V and about 50 ps or about 100 ps. The electrical field may comprise an interval of about 250 ms between each pulse.

[0012] Step b) may comprise applying the electrical field between about 1 and about 100 times. Step b) may comprise applying the electrical field about 6 times.

[0013] In another aspect, provided herein is an electrotransfer device comprising an active electrode and a dissipation electrode; wherein at least one of the active electrode and the dissipation electrode is dielectrically insulated with an insulating material. In embodiments, the active electrode is dielectrically insulated. In embodiments, both the active electrode and the dissipation electrode are dielectrically insulated. The insulating material may comprise at least one of glass, quartz, silicon dioxide, rubber, neoprene, silicone, titanium dioxide, barium titanate, an insulating polymer, an insulating ceramic, and an insulating composite material.

[0014] These aspects are nonlimiting. Other aspects and features of the systems and methods described herein will be provided below.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGS. 1A-1B. Dielectric isolation eliminates current through mammalian cell suspension, while maintains induced electric potential. Schematic of electrode configuration for cell culture experiment (A), with an electrode in contact with the cell suspension buffer, a dielectric (5mm air gap) between buffer and non-contact electrode. While the applied voltage remained the same, current through the cell suspension was eliminated with isolation of the non-contact electrode (B).

[0016] FIGS. 2A-2D. Induced electric field alone enhances molecular delivery. Fluorescence microscopy merged with phase microscopy of C2C12 cells with Sytox™ (green), PI (red), and both (yellow), for cells exposed to 6 pulses of 130V, lOOus, and 250ms interval (A), 6 pulses of 400V, lOOus, and 250ms interval (B), 6 pulses of 800V, lOOus, and 250ms interval (C). Five fields of view were collected for all groups, and quantified (D), with p<0.05 indicating a significant difference in delivery over no pulse control.

[0017] FIGS. 3 A-E. Induced electric field alone, without current, enhanced irreversible cellular membrane permeabilization. Fluorescence microscopy merged with phase microscopy of C2C12 cells with Sytox™ (green), PI (red), and both (yellow), for cells exposed to 6 pulses of 400V, lOOus, and 250ms interval (A), 6 pulses of 800V, lOOus, and 250ms interval (B), 6 pulses of 130V, lOOus, and 150ms interval (C) and 6 pulses of 130V, lOOus, and 200ms interval (D). Five fields of view were collected for all groups, and quantified €, with p<0.05 indicatinga significant difference in irreversible cell membrane permeabilization over no pulse control.

[0018] FIG. 4. Experimental design for Sytox™ delivery.

[0019] FIG. 5. Square waveforms of pulses for each set of parameters.

[0020] FIG. 6. Electric Field Model.

[0021] FIG. 7. Schematic of cells taking up Sytox™ molecules.

[0022] FIGS. 8A-8H show voltage and current data for each set of parameters.

[0023] FIG. 9 shows Sytox™ delivery for the non-contact (air gap groups).

[0024] FIG. 10 shows ablation for the non-contact (air gap groups).

[0025] FIG. 11 shows glass vs air gap in Sytox™ delivery and ablation.

[0026] FIG. 12. Cell viability for cells pulsed with Bleomycin vs. Bleomycin only.

[0027] FIGS. 13A-13D. TPA eliminates muscle stimulation in vivo. A. Intended pulses delivered to tissue with TPA. B. Muscle contract associated with pulses in TPA group. C. Intended pulse delivered to tissue with contact monopolar electrode. D. Muscle contract associated with pulses in monopolar electrode group.

[0028] FIGS. 14A-14B. TPA enhanced gene delivery to skin and muscle. Gene delivery to the skin (A), gene delivery to skeletal muscle (B).

[0029] FIGS. 15A-15C. Schematics of the electroporation device as contemplated herein, in which (A) the active electrode is insulated, (B) the dissipation electrode is insulated, and (C) both the active electrode and the dissipation electrode are insulated.

[0030] FIGS. 16A-16B. TPA electrode reduces current across tissue in vivo for the duration of the applied pulse. Conventional monopolar pulses maintain current across tissue for the duration of the applied voltage (A), while TPA pulses eliminate current for the duration of the applied voltage (B).DETAILED DESCRIPTION

[0031] This disclosure provides methods and device for permeabilizing cell membranes using electroporation in which the current is eliminated.

[0032] In a first aspect, provided herein is a method for permeabilizing a tissue or a portion thereof in a subject, the method comprising applying an electrical field to the tissue using an active electrode and a dissipation electrode, wherein at least one of the active electrode and the dissipation electrode is dielectrically insulated with an insulating material; and wherein applying the electrical field does not apply a current to the subject. The electrical field may be sufficient for ablation of at least a portion of the tissue.

[0033] In embodiments, the method may further comprise administering a therapeutic agent tothe subject before applying the electrical field to the tissue. In such embodiments, the method may be used to deliver the therapeutic agents to the tissue, and the electrical field may be sufficient for permeabilizing the tissue without ablating the tissue.

[0034] In embodiments, the therapeutic agent is administered to the subject at an administration site of the tissue, wherein the electrical field is applied directly to the administration site. In other embodiments, the therapeutic agent is administered to the subject systemically, such as via intravenous administration, wherein the electrical field is applied directly to the desired tissue. The electrical field may be sufficient for ablation of at least a portion of the tissue.

[0035] As used herein, the term "administering" an agent, such as a therapeutic agent, to a tissue or cell, is intended to refer to dispensing, delivering or applying the substance to the intended target by any suitable route for delivery, including delivery by either the parenteral / oral route, intramuscular injection, subcutaneous / intradermal injection, intraperitoneal injection, intravenous injection, retro-orbital injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by the intranasal or respiratory tract route. In preferred embodiments, administering the agent comprises injecting the agent. In exemplary embodiments, administering the agent comprises subcutaneous or intramuscular injection. The therapeutic agent may be administered to the administration site, followed by application of the electrical field to the administration site. Suitable tissues include, but are not limited to, muscles, including skeletal muscle, tumors, and organs. For nucleic acid or delivery (DNA or RNA), the agent may be injected at a site where expression is desired, followed by application of the electrical field to the administration site, where the active electrode is in direct contact with the injection site. For example, for administration to the heart, the nucleic acid may be injected into the myocardium, then the injection site is pulsed. As another non-limiting example, small molecule therapeutics, such as calcium, bleomycin, or other chemotherapeutics, may be injected into a tumor or administered via intravenous injection, followed by application of the electrical field directly to the tumor.

[0036] A “subject” or “subject in need thereof’ refers to a subject in need of the therapeutic agent. The subject may need or be suspected of needing treatment for a disease or disorder. The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects. In preferred embodiments, the subject is a human.

[0037] The therapeutic agent may be any suitable therapeutic agent to achieve the desiredtherapeutic effect. The therapeutic agent may be formulated as a pharmaceutical composition. The term “pharmaceutical composition” refers to a chemical or biological composition suitable for administration to a mammal. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. Examples of compositions appropriate for such therapeutic applications include preparations for intramuscular administration, such as sterile suspensions and emulsions. In some cases, pharmaceutical compositions appropriate for therapeutic applications may be in admixture with one or more pharmaceutically acceptable excipients, diluents, or carriers such as sterile water, physiological saline, glucose or the like.

[0038] The therapeutic agent may be administered in a therapeutically effective amount. As used herein the terms “therapeutically effective amount” and “effective amount” refer to the amount or dose of therapeutic provides the desired effect. In some embodiments, the effective amount is the amount or dose of the agent, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment.

[0039] The step of applying an electrical field to the administration site refers to the application of an electric pulse resulting in induction of a transmembrane voltage across the plasma membrane of cells at the site of application. The transmembrane voltage must be sufficiently strong to render the cell membrane transiently permeable, allowing the entry of the therapeutic agent into the cells.

[0040] The electrical field is applied using at least one dielectrically insulated electrode. The electrical field may be applied using an active electrode and a dissipation or “grounding” electrode, wherein one or both of the active electrode and the dissipation electrode are insulated. The dielectric insulation may be a material having a dielectric constant greater than 1. In embodiments, the dielectrically insulated electrode is coated with glass, quartz, silicon dioxide, rubber, neoprene, silicone, titanium dioxide, barium titanate or other insulating polymers. In exemplary embodiments, the dielectrically insulated electrode is coated with glass. The dielectrically insulated electrode is positioned on the administration site but the active element of the electrode does not directly contact the skin or tissue by virtue of the intervening dielectric.

[0041] The electrical field comprises between 1 and about 10,000 pulses, which can be applied in one or multiple bursts. For example, 100 pulses may be applied in 100 bursts for delivery of10,000 pulses. As another non-limiting example, 1,000 pulses may be applied in 10 bursts for 10,000 pulses.

[0042] In embodiments, the electrical field comprises between about 1 and about 100 electrical pulses, wherein each electrical pulse is between about 100 and about 4,000 V and between about 0.1 and about 1,000 ps; and wherein the electrical field comprises an interval of between about 0.1 and about 1,000 ms between each pulse. In exemplary embodiments, the electrical field comprises about 1,000 electrical pulses of about 2000 V for about 100 ps each, and an interval of about 100 ms between each pulse. The electrical field may be applied between about 1 and about 100 times (bursts). In exemplary embodiments, the electrical field is applied about 10 times.

[0043] The method may be performed more than one time. For example, the method may be performed on a regimen of once every about six months or about every twelve months, or at any interval in between. Each time the method is performed, the administration site may be different. For example, the second and subsequent administrations may be done at a site that is not covered by the electrical field applied at the first administration.

[0044] In a second aspect, provided herein is a method for permeabilizing a cell membrane or a tissue, the method comprising: a) suspending the cell or tissue in an electroporation buffer to create a cell suspension or a tissue suspension; and b) applying an electrical field to the cell or tissue suspension using a device comprising a dielectrically insulated electrode and a dissipation electrode. The electroporation buffer contains ingredients used to mimic the impedance of tissues in the human body. Any suitable electroporation buffer known in the art may be used. The electroporation buffer may contain sucrose to modulate the applied electric field’s impact on the suspended cells. The cell may be a population of cells in a petri dish, where the dish is placed on top of the dissipation electrode, and wherein the dielectrically insulated electrode is positioned above the dish. The dielectrically insulated electrode may be placed up to about 10 mm away from the cell or tissue suspension. In exemplary embodiments, the dielectrically insulated electrode is placed 5 mm away from the cell or tissue suspension. The dielectrically insulated electrode and the cell or tissue suspension may be separated by, for example, air, glass, quartz, silicon dioxide, rubber, neoprene, silicone, titanium dioxide, or barium titanate.

[0045] The electrical field may comprise between about 1 and about 100 electrical pulses, wherein each electrical pulse is between about 100 and about 4,000 V and between about 0.1 and about 1,000 ps; and wherein the electrical field comprises an interval of between about 0.1 and about 1,000 ms between each pulse .

[0046] In exemplary embodiments, the electrical pulses comprise about 130 V or about 400 V. In exemplary embodiments, the electrical pulses comprise about 50 ps or about 100 ps. In exemplary embodiments, the electrical field comprises an interval of about 250 ms between each pulse. The electrical field may be applied between about 1 and about 100 times. The electrical field may be applied about 6 times.

[0047] The cell suspension or tissue suspension may further comprise an agent, such as a therapeutic agent or an agent for visualizing the cell or contents of the cell, such as a dye, a probe, etc. In such embodiments, the method results in introduction of the agent into the cell or tissue.

[0048] In a third aspect, provided herein is an electroporation device comprising at least one dielectrically insulated electrode. The device comprises an active electrode and a dissipation electrode, wherein at least one of the electrodes is insulated. The dielectrically insulated electrode is coated with glass, quartz, silicon dioxide, rubber, neoprene, silicone, titanium dioxide, barium titanate, or another insulating polymer, ceramic, or composite material. Schematics of the electroporation device are shown at FIGS. 15A-15B.

[0049] The therapeutic agents described herein include nucleic acid therapeutics, protein therapeutics, and small molecule compounds. In preferred embodiments, the therapeutic agents are nucleic acid and protein therapeutics. Small molecule compounds may include chemotherapy drugs.

[0050] Small molecule drugs are typically comprised of 20 to 100 atoms and have a molecular mass of less than 1000 g / mol or 1 kilodalton [kDa],

[0051] The terms “nucleic acid”, “nucleic acid sequence”, “polynucleotide”, and “polynucleotide sequence” refer to a polymer of nucleotides, an oligonucleotide, a polynucleotide (which terms may be used interchangeably), or any fragment thereof. A polynucleotide may refer to a polydeoxyribonucleotide (containing 2-deoxy-D-ribose), a polyribonucleotide (containing D-ribose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base. There is no intended distinction in length between the terms “nucleic acid”, “oligonucleotide” and “polynucleotide”, and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. For use in the present compositions and methods, an oligonucleotide also can comprise nucleotide analogs in which the base, sugar, or phosphate backbone is modified as well as nonpurine or non-pyrimidine nucleotide analogs. These phrases also refer to DNA or RNA of genomic, natural, or synthetic origin (which may be single-stranded or double-stranded andmay represent the sense or the antisense strand).

[0052] A "therapeutic polynucleotide" as used herein refers to DNA sequence encoding a polypeptide or an RNA that induces a positive therapeutic effect when expressed. A therapeutic polynucleotide may comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding sequence and a 3' non-translated sequence, such as sequence encoding a polyadenylation site. "Expression” of a polynucleotide refers to the process wherein a gene is transcribed into an RNA and / or translated into a protein.

[0053] The nucleic acids may be provided in a construct. The terms “construct” “nucleic acid construct” and “expression construct” are used herein to refer to a recombinant polynucleotide, i.e., a polynucleotide that was formed artificially by combining at least two polynucleotide components from different sources (natural or synthetic). For example, the constructs described herein comprise the coding region of a transgene of interest (a “therapeutic polynucleotide”) operably linked to a promoter that (1) is associated with another gene found within the same genome, (2) is from the genome of a different species, or (3) is synthetic. Constructs can be generated using conventional recombinant DNA methods. A "transgene" refers to a gene that has been introduced into a host cell. The transgene may comprise sequences that are native to the cell, sequences that do not occur naturally in the cell, or combinations thereof. A transgene may contain sequences coding for one or more proteins that may be operably linked to appropriate regulatory sequences for expression of the coding sequences in the cell.

[0054] A "promoter" or "transcription regulatory sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, such as a therapeutic polynucleotide sequence, and is typically located upstream with respect to the direction of transcription of the coding sequence. A promoter is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter, including e.g. attenuators or enhancers, but also silencers. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer.

[0055] The construct may be part of a vector. A “vector” is a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced. Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., lentiviral vectors). Moreover, certain vectors are capable of directing the expression of exogenous genes to which they are operatively linked. Suitable vectors are known in the art and contain the necessary elements in order for the gene encoded within the vector to be expressed as a protein in the host cell. The term "plasmid", and also “minicircle DNA” as well as “nanoplasmid” refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, specifically exogenous DNA segments encoding the mutant a-gal protein. The term “viral vector” is used to describe a virus particle that is used to deliver genetic material (e.g., the constructs of the present invention) into cells, wherein additional DNA segments may be ligated into the viral genome. Viral vectors include replication defective retroviruses (including lentiviruses), adenoviruses, and adeno-associated viruses (AAV), which serve equivalent functions.

[0056] As used herein, the terms “protein” or “polypeptide” or “peptide” may be used interchangeable to refer to a polymer of amino acids. Typically, a “polypeptide” or “protein” is defined as a longer polymer of amino acids, of a length typically of greater than 50, 60, 70, 80, 90, or 100 amino acids. A “peptide” is defined as a short polymer of amino acids, of a length typically of 50, 40, 30, 20 or less amino acids. A protein typically comprises a polymer of naturally or non-naturally occurring amino acids e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).

[0057] Small-molecule drugs are typically comprised of 20 to 100 atoms and have a molecular mass of less than 1000 g / mol or 1 kilodalton [kDa], Small-molecules drugs can typically be administered by a variety of routes (including orally) and can pass through cell membranes to reach intercellular targets.

[0058] Miscellaneous

[0059] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise.

[0060] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0061] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0062] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0063] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0064] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should beunderstood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0065] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0066] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use an aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0067] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0068] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0069] EXAMPLES

[0070] The following examples illustrate uses of the method described herein, and are not intended to be limiting.

[0071] Example 1. Non-contact, Painless Electroporation and Molecular Transfer

[0072] Introduction

[0073] Molecular transfer, the delivery of exogenous molecules into a host cell, can be accomplished by various biochemical, viral, or physical methods. Electrotransfer is a physical method of molecular delivery that utilizes an applied voltage to generate an electric field to induce permeabilization of the cell membrane. In vivo, this applied voltage generates a current, inducing action potentials and stimulating skeletal muscle twitching. With conditions utilizing high applied voltages, this can cause pain or discomfort in the patient or animal. In some cases, it can cause ventricular fibrillation. We hypothesize that if optimized pulsed electric fields are applied without current, then molecular delivery and irreversible electroporation can be accomplished without stimulating excitable cells. Therefore, pain, twitching and ventricular fibrillation can be eliminated, making a safer method and platform technology for clinical applications of electroporation.

[0074] Materials, Device and Methods

[0075] Electrotransfer Procedure

[0076] C2C12 myoblasts (ATCC) were subcultured according to previously reported protocols. Cells were detached with 0.25% Trypsin / ETDA, centrifuged and re-suspended in a sucrose electroporation buffer. Sytox M Green was added to the cell suspension prior to electric pulse application. Cells were then allowed to settle to the bottom of the electroporation dish (FIG. 1), with a direct contact electrode at the bottom of the dish. A dielectrically insulated (dielectric constant >1) "non-contact" electrode was suspended 5mm above electroporation buffer and dish. A BTX-830 electroporator was used to establish desired pulsed electric fields. Current and applied voltage were measured with an oscilloscope (FIG. 2). Experimental groups included varying applied voltage (130, 400, and 800V), pulse length (10, 50 and lOOps), and interval between the 6 pulses (100, 150, 200, and 250ps). After ten minutes, propidium iodide (Pl) was added to the cell suspension and cells were imaged with epifluorescence and phase microscopy to determine molecular delivery, and irreversible electroporation rates.

[0077] Electrotransfer Device

[0078] Our novel electrotransfer device principle establishes a favorable electric field within tissues and mammalian cells while blocking physical current flow across cell membranes. The dissipating electrode is in direct electrical contact with tissue, while the active electrode localized near the treatment site is insulated, protected with a dielectric material of dielectric constant equal to or greater than 1. FIG. 1A shows a diagram of our device, that was used for current in vitro experiments. FIG. IB shows the current and voltage traces that C2C12 cellsexperienced during pulse application with direct contact compared to using an air gap of 5mm as an insulating material. No current was measured, leaving only the applied voltage, and resulting electric field responsible for electroporation, molecular delivery and cellular ablation.

[0079] Results

[0080] Induced Electric Field Alone Enhances Molecular Delivery to Mammalian Cells

[0081] C2C12 cells were suspended in electroporation buffer with addition of Sytox ™, which can only enter cells if the cell membrane is compromised. Cell suspension was then exposed to an electric field, varying the pulse length, applied voltage, and interval length between pulses. Pl was added ten minutes later to determine which cells we unable to reseal their membrane and were permanently permeabilized / ablated. FIG. 2 shows enhanced Sytox™ delivery and Pl exclusion, indicating feasibility of molecular delivery and transient electroporation, induced with electric fields without current.

[0082] Induced Electric Field Alone Enhances Mammalian Cell Ablation

[0083] C2C12 cells were suspended in electroporation buffer with addition of Sytox ™, which can only enter cells if the cell membrane is compromised. Cell suspension was then exposed to an electric field, varying the pulse length, applied voltage, and interval length between pulses. Pl was added ten minutes later to determine which cells we unable to reseal their membrane and were permanently permeabilized / ablated. FIG. 3 shows enhanced Sytox™ delivery and Pl incorporation, indicating feasibility of permanent mammalian cell permeabilization with induced electric fields, without current.

[0084] Conclusions

[0085] In this work, we have determined for the first time that electrical current stimulation is unnecessary for efficient electroporation of mammalian cells. The significance of this discovery is that electroporation for medical applications can be efficiently accomplished without the traditional concerns associated with current causing action potential stimulation. We, therefore, propose device design and methods that include a dielectric material isolation of one or both electrodes to eliminate current and induce a higher electric field in tissues for efficient painless electroporation. Electroporation pulse sequences can be optimized for gene delivery (gene therapy), drug delivery (electrochemotherapy), and tissue ablation, without the risk of inducing pain, twitching or ventricular fibrillation. To eliminate current, and to achieve favorable induced electric fields for efficient cell membrane permeabilization, a dielectric material with dielectric constant equal to or greater than 1 is needed. Up to date, all electroporation devices do not use dielectric materials, and therefore use the electric field and current, stimulating excitable cells. Our discovery of method and device, yields medicaldevices that can eliminate pain and risk associated with current, predicate electroporation devices used for molecular delivery and tissue ablation.

[0086] In further experiments, C2C12 cells were mixed with Sytox™ green fluorescent nucleic acid dye. A glass insulator was placed between an electrode and a cell surface, or the electrode was hovered 5 mm above the cell surface. The cell sample was zapped with an electric field with applied voltage of 130, 400, or 800V; pulse length of 10, 50 or 100 ps; and intervals of 100, 150, 200, or 250 ms. Cells that were successfully transfected with Sytox™ exhibited green fluorescence. One-way ANOVA comparison and Tukey multiple comparisons test were utilized for quantitative statistical analysis. The experimental design is illustrated in FIG. 4. The square waveforms of pulses for each set of parameters are illustrated in FIG. 5. FIG. 6 is a schematic illustrating cells that become transiently permeabilized by the electric pulse, allowing the Sytox™ (or Bleomycin) to enter the cells via endocytosis (left panel), cells that have uptaken the Sytox™ molecules (middle panel), and a population of cells, where some cells have resealed and others have become irreversibly permeabilized as evidenced by addition and observation of PI.

[0087] Results as shown in FIGS. 8-12

[0088] Sytox™ delivery at 130V was significantly higher than in the No Pulse Control, 400V, and 800V. Pulse lengths of 100 ps and 50 ps both had significantly higher Sytox™ deliverl2y than the No Pulse Control. The 100, 150, 200, and 250 ms interval groups all had significantly higher Sytox™ delivery than the No Pulse control. The 200 ms interval group had significantly higher Sytox™ delivery than the 100 ms interval group.

[0089] For the Air Gap Non-Contact groups, ablation was significantly higher in the 800V than the No Pulse Control. Ablation in the 200 ms interval group was significantly higher than in the 100 ms interval and the No Pulse control. Ablation in the 150 ms interval was also significantly higher compared to the No Pulse control.

[0090] With pulses kept at 6x100 ps and intervals kept at 250 ms, the Glass 130V, No Glass 130V, Glass 40) V, and the No Glass 400V groups all had significantly higher Sytox™ deliver than the No Pulse control. These results show evidence that small molecule electrotransfer can occur without direct contact to the cells.

[0091] The No Glass at 400V (6x100 ps pulses and 250 ms intervals) had significantly higher ablation than the No Pulse control.

[0092] Conclusions

[0093] Electrotransfer without current can enhance transient cellular permeability and molecular delivery. Electrotransfer without current can also enhance permanent cellularpermeability.

[0094] Example 3. Electrotransfer Procedure in Vivo

[0095] Introduction

[0096] The data in Examples 1 and 2 explored a new platform technology for transiently and permanently permeabilizing cell membranes for molecular delivery and ablation applications focused on in vitro feasibility studies. Classic electroporation in vivo causes unavoidable excitable cell stimulation whether for ablation or drug delivery applications. Here we evaluated a new twitchless pulsing protocol (TP A) for gene delivery in vivo, to skin and muscle without stimulating excitable cells, and therefore accomplishing cell membrane permeabilization in vivo painlessly and efficiently. Applied voltage, and muscle movement were measured with an oscilloscope and accelerometer. Plasmid DNA encoding Firefly luciferase was injected either intradermally for delivery to the skin, and intramuscularly for delivery to skeletal muscle, and TPA or classic monopolar pulses were applied. Gene expression was measured with bioluminescence imaging. TPA delivery enhanced gene delivery over injection only (p<0.005) in skin and muscle. Additionally, TPA completely eliminated muscle twitching, while classic electrotransfer resulted in significant muscle contractions with every pulse. Therefore, TPA is a platform technology can be applied to the field of non-viral gene therapy, in addition to the field of tissue ablation.

[0097] Methods

[0098] Sprague Dawley rats were utilized in this study following an approved IACUC protocol. For delivery to the skin, treatment sites were randomized to plasmid DNA injection only, contactless electrotransfer (TPA), or classic monopolar electrotransfer groups (n=8). A grounding plate electrode was placed under the opposite flank, with conducting gel to ensure good electrical conductivity with the grounding electrode. Each site received an intradermal injection of 50pl of pDNA solution, followed by application of pulses. Treatment sites within the injection only site, received no pulses. TPA assigned sites received 1000 pulses of 2000V via the contactless active electrode for lOOps duration. Sites assigned to the classic monopolar electrotransfer group, received 8 pulses of 90V applied via direct contact with the 10mm platinum monopolar electrode. Voltage, current and movement were recorded with electrical and accelerometer measurements during each applied pulse (FIG. 13). Bioluminescence was measured for the next 2 days (FIG. 14 A).

[0099] For delivery to the skeletal muscle, the grounding plate was placed on the opposite flank as above, and the treatment sites were scrubbed to establish a sterile field. Then a small incision was made with a scalpel followed by blunt dissection to expose skeletal muscle. Treatmentsites were randomly assigned to the TPA or IO groups. As with skin treatments, 50pl of pDNA solution was injected into the exposed muscle followed by TPA pulses of 2000V, delivered in 10 bursts of lOOOpulses of lOOps long. Bioluminescence imaging was performed over the course of 28 days (FIG. 14B). No muscle stimulation was observed.

[0100] Results

[0101] TPA enhances gene delivery to skin and eliminates muscle contractions

[0102] Voltage and accelerometer data, FIG. 13, indicate that intended pulses were delivered to the tissue with the TPA and the contact monopolar electrode (FIG. 13A, 13C). Muscle contraction associated with pulses was observed on both hind legs for the monopolar 90V condition (FIG. 13D), while it was completely absent in the TPA group (FIG. 13B). Gene expression measured on days 1 and 2 were higher for both TPA and monopolar GET over injection only treatment, however not statistically significantly higher (FIG. 14A).

[0103] TPA significantly enhances gene delivery to skeletal muscle and eliminates muscle contractions

[0104] TPA mediated gene delivery resulted in significantly (p<0.005) higher gene expression (~10 fold higher), gene expression than plasmid DNA injection only. Expression was maintained for more than 28 days (FIG. 14B), indicating the feasibility of this approach as a potential gene therapy platform technology. No muscle stimulation was observed.

[0105] TPA mediated delivery also reduces current across tissue in vivo, as shown in FIGS. 16A-16B.

[0106] Conclusions

[0107] In this work, we have determined for the first time that electrical current stimulation is unnecessary for efficient electroporation of mammalian cells and tissue in vivo. The significance of this discovery is that electroporation for medical applications can be efficiently accomplished without the traditional concerns associated with current causing action potential stimulation. This eliminates risk of pain, discomfort as well as risk for ventricular fibrillation. We, therefore, propose device design and methods that include a dielectric material isolation of one or both electrodes to eliminate current and induce a higher electric field in tissues for efficient painless electroporation. Electroporation pulse sequences can be optimized for gene delivery (gene therapy), drug delivery (electrochemotherapy), and tissue ablation, without the risk of inducing pain, twitching or ventricular fibrillation. To eliminate current, and to achieve favorable induced electric fields for efficient cell membrane permeabilization, a dielectric material with dielectric constant equal to or greater than 1 is needed. Up to date, all electroporation devices do not use dielectric materials, and therefore use the electric field andcurrent, stimulating excitable cells. Our discovery of method and device, yields a platform technology that can lead to medical devices that can eliminate pain and risk associated with current, predicate electroporation devices used for tissue ablation such as cardiac ablation, or cancer related ablation, small molecule delivery such as for electro-chemotherapies, and for gene delivery for non-viral gene therapies.

Claims

CLAIMSWe claim:

1. A method for permeabilizing a tissue or a portion thereof in a subject, the method comprising applying an electrical field to the tissue using an active electrode and a dissipation electrode; wherein at least one of the active electrode and the dissipation electrode is dielectrically insulated with an insulating material; and wherein applying the electrical field does not apply a current to the subject.

2. The method of claim 1, further comprising administering a therapeutic agent to the subject before applying the electrical field to the tissue.

3. The method of claim 2, wherein the therapeutic agent is administered to the subject at an administration site of the tissue, and wherein the electrical field is applied to the administration site.

4. The method of any one of claims 1-3, wherein the electrical field ablates at least a portion of the tissue.

5. The method of any one of claims 2-4, wherein administering the therapeutic agent is performed by subcutaneous, intraperitoneal, or intramuscular injection.

6. The method of any one of claims 1-5, wherein the electrical field comprises between about 1 and about 10,000 electrical pulses.

7. The method of claim 6, wherein each electrical pulse is between about 80 and about 4,000 V and between about 0.1 and about 1,000 ps; and wherein the electrical field comprises an interval of between about 0.1 and about 1,000 ms between each pulse.

8. The method of claim 7, wherein the electrical field comprises about 1,000 electrical pulses of about 2000 V for about 100 ps each, and an interval of about 100 ms between each pulse.

9. The method of any one of claims 6-8, wherein the electrical field is applied between 1 and 100 times, and wherein the electrical field comprises between about 1 and about 100 pulses.

10. The method of claim 9, wherein the electrical field is applied about 10 times.

11. The method of any one of claims 1-10, wherein the insulating material comprises at least one of glass, quartz, silicon dioxide, rubber, neoprene, silicone, titanium dioxide, barium titanate, an insulating ceramic, an insulating polymer, and an insulating composite material.

12. A method for permeabilizing a cell membrane or a tissue, the method comprising: a) suspending the cell or the tissue in an electroporation buffer to create a cell or a tissue suspension; and b) applying an electrical field to the cell suspension or tissue suspension using a device comprising an active electrode and a dissipation electrode; wherein at least one of the active electrode and the dissipation electrode is dielectrically insulated with an insulating material; and wherein applying the electrical field does not apply a current to the cell suspension or tissue suspension.

13. The method of claim 12, wherein the active electrode is dielectrically insulated; and wherein the active electrode is placed between about 0.1 and about 10 mm away from the cell suspension or tissue suspension.

14. The method of claim 13, wherein the active electrode is placed about 5 mm away from the cell suspension or tissue suspension.

15. The method of any one of claims 12-14, wherein the dielectrically insulated electrode and the cell suspension or tissue suspension are separated by air, glass, quartz, silicon dioxide, rubber, neoprene, silicone, titanium dioxide, barium titanate, an insulating polymer, an insulating ceramic, or an insulating composite material.

16. The method of any one of claims 12-15, wherein the electrical field comprises between about 10 and about 100 electrical pulses, wherein each electrical pulse is between about 80 and about 4,000 V and between about 0.1 and about 1,000 ps; and wherein the electrical field comprises an interval of between about 0.1 and about 1,000 ms between each pulse.

17. The method of claim 16, wherein the electrical pulses comprise about 130 V or about 400 V; and wherein the electrical pulses comprise about 50 ps or about 100 ps.

18. The method of any one of claims 16-17, wherein the electrical field comprises an interval of about 250 ms between each pulse.

19. The method of any one of claims 12-18, wherein step b) comprises applying the electrical field between about 1 and about 100 times.

20. The method of claim 19, wherein step b) comprises applying the electrical field about 6 times.

21. An electrotransfer device comprising an active electrode and a dissipation electrode; wherein at least one of the active electrode and the dissipation electrode is dielectrically insulated with an insulating material.

22. The device of claim 21, wherein the active electrode is dielectrically insulated.

23. The device of claim 21, wherein both the active electrode and the dissipation electrode are dielectrically insulated.

24. The device of any one of claims 21-23, wherein the insulating material comprises at least one of glass, quartz, silicon dioxide, rubber, neoprene, silicone, titanium dioxide, barium titanate, an insulating polymer, an insulating ceramic, and an insulating composite material.

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