US20060269531A1 - Apparatus for generating electrical pulses and methods of using the same - Google Patents

Apparatus for generating electrical pulses and methods of using the same Download PDF

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US20060269531A1
US20060269531A1 US10/564,994 US56499404A US2006269531A1 US 20060269531 A1 US20060269531 A1 US 20060269531A1 US 56499404 A US56499404 A US 56499404A US 2006269531 A1 US2006269531 A1 US 2006269531A1
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pulse
cell
electric field
nanosecond
duration
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Stephen Beebe
Karl Schoenbach
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Eastern Virginia Medical School
Old Dominion University Research Foundation
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Eastern Virginia Medical School
Old Dominion University
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Assigned to EASTERN VIRGINIA MEDICAL SCHOOL reassignment EASTERN VIRGINIA MEDICAL SCHOOL ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BEEBE, STEPHEN J.
Publication of US20060269531A1 publication Critical patent/US20060269531A1/en
Priority to US13/073,785 priority patent/US8822222B2/en
Priority to US13/593,670 priority patent/US20120315704A1/en
Assigned to OLD DOMINION UNIVERSITY RESEARCH FOUNDATION reassignment OLD DOMINION UNIVERSITY RESEARCH FOUNDATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: OLD DOMINION UNIVERSITY
Assigned to OLD DOMINION UNIVERSITY RESEARCH FOUNDATION reassignment OLD DOMINION UNIVERSITY RESEARCH FOUNDATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: OLD DOMINION UNIVERSITY
Priority to US15/654,625 priority patent/US10653880B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • A61N1/0404—Electrodes for external use
    • A61N1/0408—Use-related aspects
    • A61N1/0412—Specially adapted for transcutaneous electroporation, e.g. including drug reservoirs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/327—Applying electric currents by contact electrodes alternating or intermittent currents for enhancing the absorption properties of tissue, e.g. by electroporation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • Electrocardiography EKG
  • electroencephalography EEG
  • Cardioversion the application of a pulsed electric field to heart muscle, is routinely used to stop, modify, or re-start the heart's beating.
  • Low power electric fields can be applied to bone fractures to stimulate healing.
  • Electromyography the application of measured electrical pulses to muscles or their associated nerves, can be used to measure muscle function and/or judge the degrees of muscle damage.
  • Conditions for optimal electroporation depend on the waveform, the constituents of the media in which the cell is suspended, and the cell type (Weaver, J. C., Electroporation of cells and tissues, in: J. D. Bronzino (Ed.), The Biomedical Engineering Handbook, CRC and IEEE press, Boca Raton, Fla., 1995, pp. 1431-1440; Djuzenova et al., Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells. Biochim Biophys Acta, 1284:143-52, 1996). In any case, the electroporation effects of these millisecond low power applied electric fields occur only at the cell's surface membrane.
  • Enhanced or optimized gene expression has been previously accomplished using classical electroporation pulses by changing the pulse duration of a long pulsed electric field (for example, within the range of 1 microsecond-20 milliseconds), changing the electric field intensity within classical electroporation range (0.1-5 kV/cm), and/or by modifying the conductivity of the buffer or media.
  • enhanced gene expression has been accomplished by changing the concentration of DNA used in the transfection procedure, changing the physical/chemical properties during transfection (pH, ionic strength, etc), using various lipid combinations with different properties, or adding other constituents to the cell culture media or buffers to aid transfection efficiency.
  • the present invention is directed to a method of introducing an agent into a cell comprising the application of nanosecond pulse electric fields (“nsPEF's”).
  • nsPEF's nanosecond pulse electric fields
  • the drug to be an antibiotic or a chemotherapeutic agent selected from the group comprising bleomycin, daunomycin, 5-FU, cytosine arabinoside, colchicine, cytochalasin B, daunorubicin, neocarcinostatin, suramin, doxorubicin, carboplatin, taxol, mitomycin C, vincristine, vinblastine, methotrexate, and cisplatin, and suitable combinations thereof.
  • the agent can be a nucleic acid, wherein the nucleic acid is selected from the group comprising DNA, cDNA, and RNA.
  • these nucleic acids may encode a homologous or heterologous gene product and the cell can be transfected so that this gene product is expressed in the cell.
  • the nucleic acid can also be an expression vector wherein the expression vector contains a homologous or heterologous nucleic acid encoding a gene product operably linked to a suitable promoter sequence.
  • the nucleic acid may also modify the expression of a gene and provide gene therapy, for example.
  • the nucleic acid introduced into the cell may also modulate cell proliferation or elicit an immune response.
  • the agent can be a cytotoxic agent selected from the group comprising ricin, abrin, diphtheria toxin, and saporin.
  • a cytotoxic agent selected from the group comprising ricin, abrin, diphtheria toxin, and saporin.
  • Any type of cell may be used in the present invention including eukaryotic cells, prokaryotic cells, fat cells, bone cells, vascular cells, muscle cells, cartilage cells, stem cells, hematopoeitic cells, lung cells, airway cells, liver cells, intestinal cells, skin cells, nerve cells, cancer cells, bacterial cells, and combinations thereof.
  • a method of enhancing delivery of drugs to tumors or other tissues includes applying nanosecond pulse electric fields to said tumors or other tissues.
  • the nsPEFs can range in time from 1 to 1000 nanoseconds, preferably 1 to 300 nanoseconds.
  • the nsPEFs can also range in electric field intensity from 1 to 1000 kV/cm, preferably 10 to 350 kV/cm.
  • a pulse generator for generating electrical pulses.
  • the pulse generator includes a first circuit, a second circuit, and a control circuit.
  • the first circuit is used to generate a first pulse having a long duration and low voltage amplitude.
  • the second circuit is used to generate a second pulse having a short duration and high voltage amplitude.
  • the control circuit is provided for controlling the timing of the first and second circuits such that the first and second pulses are respectively generated.
  • FIG. 2 shows a similar experiment in which HL-60 cells were exposed to a long pulse of 450 V and 960 uF, a short nsPEF pulse of 60 ns and 60 kV/cm, or a combination of the long and short pulse.
  • the observed geometric mean GFP fluorescence is listed next to the pulsing conditions in the inset of the figure.
  • FIG. 3 shows a similar experiment in which HL-60 cells were exposed to a long pulse of 260 V and 960 uF, a short nsPEF pulse of 60 ns and 60 kV/cm, or a combination of the long and short pulse.
  • the observed geometric mean GFP fluorescence is listed next to the pulsing conditions in the inset of the figure.
  • FIG. 5 shows a similar experiment in which HL-60 cells were exposed to a long pulse of 450 V and 960 uF, a short nsPEF pulse of 10 ns and 150 kV/cm, or a combination of the long and short pulse.
  • the observed geometric mean GFP fluorescence is listed next to the pulsing conditions in the inset of the figure.
  • FIG. 6 shows a similar experiment in which HL-60 cells were exposed to a long pulse of 130 V and 960 uF, a short nsPEF pulse of 10 ns and 150 kV/cm, or a combination of the long and short pulse.
  • the observed geometric mean GFP fluorescence is listed next to the pulsing conditions in the inset of the figure.
  • FIG. 7 is a circuit diagram for a multi-pulse generator according to one or more embodiments of the present invention.
  • nsPEF pulses exhibit energy densities in the millijoules/cc range, with total energies not exceeding 10 J (preferably less than 1 J) and power of about 180 MW. About 90% of the energy contained in a nsPEF pulse is applied in a frequency range up to 60% of the cut-off frequency. In addition to the unique short duration and rapid rise time, nsPEFs are exceptional because they are very low energy and extremely high power.
  • nsPEF pulses can be five to six orders of magnitude shorter, with electric fields and power several orders of magnitude higher, and energy densities considerably lower than electroporation pulses. Even though nsPEF pulses exhibit extremely high power, because their duration is so short, the energy density does not cause significant thermal effects.
  • nsPEF pulses and classic electroporation pulses have dramatically different effects on cells.
  • the cell cytoplasm is a conductive body and the surrounding plasma membrane is a dielectric layer.
  • a unipolar voltage pulse is applied to the electrodes, the resulting current causes accumulation of electrical charges at the cell membrane and, consequently, a voltage across the membrane. If the membrane voltage exceeds a critical value, structural changes in the surface membrane occur with trans-membrane pore formation, a process known as electroporation (Weaver, J. C., Electroporation of cells and tissues, in: J. D.
  • the charging time constant ( ⁇ c ) would be 75 ns (Cole, K. S. Electric Impedance of Marine Egg Membranes. Trans Farady Soc 23:966, 1937) [ ⁇ c ( ⁇ c + ⁇ a /2)cmD/2].
  • the charging time constant is a measure of the time during which the cell interior is exposed to the applied pulsed electric field intensity.
  • nsPEF pulses bypass the plasma membrane and target intracellular structures such as the mitochondria and nucleus, leaving the plasma membrane intact. Therefore, nsPEF pulses have effects that are different than those of electroporation pulses because, when the pulse duration is short enough and the electric field intensity is high enough, intracellular structures are targeted.
  • nsPEFs The effects of nsPEFs on cells differ depending on such factors as cell type, pulse duration and rise-time, electric field intensity, and the number of pulses.
  • a desired agent is introduced into a cell using a known technique (i.e., electroporation, lipid vesicles, viral vectors, co-precipitation with calcium phosphate or dextran).
  • the cell is then exposed to one or more nsPEF pulses in order to facilitate transfer of the desired agent into the nucleus of the cell.
  • the nsPEF pulse can range in duration from 1 to 1000 nanoseconds, preferably 1 to 300 nanoseconds.
  • the field amplitude for the nsPEF pulse can range from 1 to 1000 kV/cm, preferably 10 to 350 kV/cm.
  • nsPEFs cause pores in the plasma membrane to open transiently, without permanently damaging the cell.
  • nsPEFs cause pores in the plasma membrane to open transiently, without permanently damaging the cell.
  • Other experiments with nsPEFs have shown that membrane bound organelles in the cell can be opened by the same kind of pulses. (Schoenbach, K. H., Beebe, S. J., Buescher, E. S., Intracellular effect of ultrashort electrical pulses, Bioelectromagnetics 22:440-448, 2001).
  • the term “agent” as used herein refers to drugs (e.g., chemotherapeutic agents), nucleic acids (e.g., polynucleotides), and peptides and polypeptides (including antibodies).
  • the peptide or polypeptide used in the method of the present invention can be an antigen introduced for the purpose of raising an immune response in the subject into whose cells it is introduced.
  • the polypeptide can be a hormone such as calcitonin, parathyroid hormone, erythropoietin, insulin, a cytokine, a lymphokine, a growth hormone, a growth factor, or a combination of any two or more thereof.
  • the “nucleic acid,” “nucleic acid molecule,” “polynucleotide”, or “oligonucleotide” of the present invention includes DNA, cDNA, and RNA sequences of all types.
  • the polynucleotides can be double stranded DNA, single-stranded DNA, complexed DNA, encapsulated DNA, genomic DNA, naked RNA, encapsulated RNA, a DNA-RNA hybrid, a nucleotide polymer, and combinations thereof.
  • Such agents may be introduced into the cell for any purpose.
  • the agents may be used in an amount to modulate cell proliferation or to elicit an immune response, either against the nucleic acid or a protein product encoded by the nucleic acid.
  • the polynucleotides of the present invention can also be DNA constructs, such as expression vectors.
  • Such expression vectors may encode a desired gene product (e.g., a gene product homologous or heterologous to the subject into which it is to be introduced).
  • a therapeutic polypeptide (one encoding a therapeutic gene product) may be operably linked with a regulatory sequence such that the cells of the subject are transfected with the therapeutic polypeptide, which is expressed in cells into which it is introduced according to one aspect of the invention methods.
  • the polynucleotide may further encode a selectable marker polypeptide, such as is known in the art, useful in detecting transformation of cells with agents according to the invention method.
  • drugs contemplated for use in the method of the invention include antibiotics such as are known in the art and chemotherapeutic agents having an antitumor or cytotoxic effect.
  • antibiotics such as are known in the art and chemotherapeutic agents having an antitumor or cytotoxic effect.
  • Such drugs or agents include bleomycin, daunomycin, 5-FU, cytosine arabinoside, colchicine, cytochalasin B, daunorubicin, neocarcinostatin, suramin, doxorubicin, carboplatin, taxol, mitomycin C, vincristine, vinblastine, methotrexate, and cisplatin.
  • membrane-acting agents can also be introduced into cells according to the invention method.
  • Membrane acting agents are a subset of chemotherapeutic agents that act primarily by damaging the cell membrane, such as N-alkylmelamide, and para-chloro mercury benzoate.
  • the composition can include a deoxyribonucleotide analog, such as azidodeoxythymidine, dideoxyinosine, dideoxycytosine, gancyclovir, acyclovir, vidarabine, ribavirin, or any chemotherapeutic known to those of average skill in the art.
  • DNA immunization a method to induce protective immune responses using “naked” DNA, complexed DNA, or encapsulated DNA, is shown in U.S. Pat. No. 5,589,466.
  • DNA immunization entails the direct, in vivo administration of vector-based DNA or non-vector DNA that encodes the production of defined microbial or cellular antigens, for example, and cytokines (e.g., IL and IFN), for example.
  • cytokines e.g., IL and IFN
  • a polynucleotide that modulates the expression of a gene such as an endogenous gene, in cells.
  • modulate envisions the suppression or augmentation of expression of a gene.
  • nucleic acid sequences that interfere with the gene's expression at the translational level can be used to modulate gene expression.
  • This approach introduces into the cells of a subject active agents capable of interfering with expression, such as antisense nucleic acid sequences, ribozymes, or triplex agents to block transcription or translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or triplex agent, or by cleaving it with a ribozyme.
  • active agents capable of interfering with expression such as antisense nucleic acid sequences, ribozymes, or triplex agents to block transcription or translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or triplex agent, or by cleaving it with a ribozyme.
  • the agent introduced according to the invention methods can also be a therapeutic peptide or polypeptide.
  • immunomodulatory agents and other biological response modifiers can be administered for incorporation by cells.
  • biological response modifiers is meant to encompass substances which are involved in modifying the immune response.
  • immune response modifiers include such compounds as lymphokines. Lymphokines include tumor necrosis factor, interleukins 1, 2, and 3, lymphotoxin, macrophage activating factor, migration inhibition factor, colony stimulating factor, alpha-interferon, beta-interferon, and gamma-interferon, their subtypes and the like.
  • agent of the invention can also be an antibody.
  • antibody as used herein is meant to include intact molecules as well as fragments thereof, such as Fab and F(ab′) 2 .
  • RNA virus such as a retrovirus
  • retroviral vector is a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • a vector such as the gibbon ape leukemia virus (GALV) can be utilized.
  • a number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • the polynucleotides can be introduced into the cell by calcium phosphate and dextran co-precipitation, incorporation of the polynucleotides into lipid vesicles for fusion with the plasma membrane, and electropermeabilization or electroporation using pulsed electric fields to form “pores” in the plasma membrane.
  • the choice of a gene delivery system will be made by those of skill in the art, keeping in mind the objectives of efficient gene transfer, with an appropriate level of gene expression, in a cell-specific manner, and without any adverse effects.
  • the agent introduced into a cell can also include a detectable marker, such as a radioactive label or a fluorescent marker.
  • the composition can include a photoactive modification, such as Psoralin C2.
  • the composition can include a phosphoramidate linkage, such as butylamidate, piperazidate, and morpholidate.
  • the composition can include a phosphothiolate linkage or ribonucleic acid. These linkages decrease the susceptibility of oligonucleotides and polynucleotides to degradation in vivo.
  • the agent of the present invention may be a pharmaceutical agent or pharmaceutically active agent.
  • pharmaceutical agent or pharmaceutically active agent as used herein encompasses any substance that will produce a therapeutically beneficial pharmacological response when administered to a subject, including both humans and animals. More than one pharmaceutically active substance may be included, if desired, in a pharmaceutical composition used in the method of the present invention.
  • the pharmaceutically active agent can be employed in various forms, such as molecular complexes or pharmaceutically acceptable salts.
  • Representative examples of such salts are succinate, hydrochloride, hydrobromide, sulfate, phosphate, nitrate, borate, acetate, maleate, tartrate, salicylate, metal salts (e.g., alkali or alkaline earth), ammonium or amine salts (e.g., quaternary ammonium) and the like.
  • derivatives of the active substances such as esters, amides, and ethers which have desirable retention and release characteristics but which are readily hydrolyzed in vivo by physiological pH or enzymes can also be employed.
  • the term “therapeutically effective amount” or “effective amount” means that the amount of the biologically active or pharmaceutically active substance is of sufficient quantity and activity to induce a desired pharmacological effect.
  • the amount of substance can vary greatly according to the effectiveness of a particular active substance, the age, weight, and response of the individual subject as well as the nature and severity of the subject's condition or symptoms. Accordingly, there is no upper or lower critical limitation upon the amount of the active agent introduced into the cells of the subject, but should not be so large as to cause excessive adverse side effects to the cell or tissue containing such cell, such as cytotoxicity, or tissue damage.
  • the amount required for transformation of cells will vary from cell type to cell type and from tissue to tissue and can readily be determined by those of ordinary skill in the art using the teachings herein.
  • the required quantity to be employed in the practice of the invention methods can readily be determined by those skilled in the art.
  • the amount of active agent such as a nucleic acid sequence encoding a gene product introduced into the cells is a “transforming amount.”
  • a transforming amount is an amount of the active agent effective to modify a cell function, such as mitosis or gene expression, or to cause at least some expression of a gene product encoded by the nucleic acid sequence.
  • the agent may be present in an “immunogenic” amount, an “immuno-modulating” amount, or a “therapeutic amount.”
  • An immunogenic amount is an amount of the active agent effective to elicit an immune response.
  • An immuno-modulating amount is an amount of the active agent effective to alter the immune response in some way.
  • a therapeutic amount is an amount of the active agent effective to induce a desired immunological or biological response in order to treat a particular disorder for example.
  • controlled release means that the preparation or formulation requires at least an hour to release a major portion of the active substance into the surrounding medium, for example, about 1-24 hours, or even longer.
  • colloidal dispersion systems which include macromolecular complexes, nanocapsules, microcapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, liposomes, and the like.
  • the controlled release vehicle used to contain the active agent for microinjection is a biodegradable microsphere.
  • Microspheres, wherein a pharmaceutically active agent is encapsulated by a coating of coacervates, is called a “microcapsule.”
  • Liposomes which may typically bear a cationic charge, are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from about 0.2 to 4.0 ⁇ m, can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules, such as DNA.
  • LUV large unilamellar vesicles
  • the composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations, making them suitable vehicles for encapsulating an active agent intended to undergo electrotransport according to the invention methods.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, gangliosides, and the like. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
  • Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoyl-phos-phatidylcholine.
  • Preparations suitable for electrotransport may include the agent with a “pharmaceutically acceptable carrier.”
  • a “pharmaceutically acceptable carrier” Such carriers are known in the art and include sterile aqueous or non-aqueous solutions, suspensions and emulsions.
  • non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, fixed oils, and the like.
  • Vehicles suitable for intercellular or intracellular injection may also include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, for example. Preservatives and other additives may also be present. For example, antimicrobials, antioxidants, chelating agents, and inert gases may also be used.
  • the agent can be introduced into any desired cell or cell type, including eukaryotic and prokaryotic cells.
  • Non-limiting examples include fat cells, bone cells, vascular cells, muscle cells, cartilage cells, adult, fetal and embryonic stem cells, hematopoeitic cells, lung cells, airway cells, liver cells, intestinal cells, skin cells, nerve cells, and bacteria cells.
  • the present invention is directed to a method of enhancing gene expression in a cell using nsPEFs.
  • Gene expression as used herein is defined as the process by which the information encoded in a gene is converted into protein, peptide, or some form of RNA.
  • cells are placed in the presence of polynucleotides being introduced into the cells.
  • the polynucleotide is in a form suitable for introduction into the cell, such as plasmid DNA.
  • the cells and polynucleotides are exposed to relatively long pulses in the millisecond range. These long pulses cause the outer membranes of the cells to open, thereby facilitating the transfer of the polynucleotides into the cell cytoplasm.
  • nsPEF pulses to facilitate transfer of the polynucleotides into the nucleus.
  • field amplitudes are low, on the order of hundreds/low thousands of V/cm.
  • these long pulses can range in duration from 0 . 00 1 to 30 milliseconds, preferably 0.1 to 20 milliseconds.
  • the field amplitudes for the long pulses can range from 0.1 to 5 kV/cm, preferably 0.1 to 1 kV/cm.
  • the free polynucleotides bind reversibly to the plasma membrane and begin their reversible insertion into the electropermeabilized membranes.
  • the nsPEF pulses can range in duration from 1 to 1000 nanoseconds, preferably 1 to 300 nanoseconds.
  • the field amplitudes for the nsPEF pulses can range from 1 to 1000 kV/cm, preferably 10 to 350 kV/cm. The application of the nsPEF pulses results in enhanced gene expression.
  • nsPEF pulses allow an increased flux of genes into the nucleus by opening the nuclear membrane.
  • the nsPEFs could promote the expression of genes through other undefined mechanisms such as enhanced transcription efficiency and/or enhanced transcription of RNA, and/or enhanced translation of protein by mechanisms related or not to calcium mobilization.
  • the nsPEF pulses alone can be used to enhance gene expression. Because nsPEFs may lead to enhanced transcription efficiency, and/or enhanced RNA transcription, and/or enhanced protein translation, nsPEFs alone may be applied to a cell in order to enhance gene expression in that cell.
  • the gene or genes enhanced by the present invention may be native to the cell and need not necessarily be transfected into the cell.
  • the nsPEF pulses can be used to enhance gene expression in cells that have already been transfected with DNA using any commonly known method described above including lipid transfer, DNA precipitation with calcium phosphate or dextran, and viral vectors. Following transfection, the nsPEF pulses further facilitate the transport of DNA into the nucleus of the cells.
  • nsPEFs may enhance gene expression by activating transcription and/or translation machinery.
  • intracellular electromanipulation Intracellular electromanipulation, “IEM”) requires potential differences across such membranes on the order of 1 V, electric fields on the order of kV/cm will be needed for poration of intracellular structures with characteristic dimensions of 1 ⁇ m.
  • IEM intracellular electromanipulation
  • Most of the unipolar pulse generators that have been used in bioelectric experiments produce microsecond to millisecond duration pulses with a rise time too slow to generate measurable intracellular effects.
  • the preferred embodiment of gene delivery described above utilizes a pulse generator that can provide both classical electroporation pulses (to open the plasma membrane) and nsPEF pulses (to open the nucleus). Therefore, in one embodiment, the present invention is directed to a pulse generator that is capable of delivering two different pulse types in succession in the same apparatus. This pulse generator may also be able to vary the pulse durations, electric fields, intervals between pulses, and order of the pulses.
  • One pulse type has a duration in the range of a classical electroporation pulse in the microsecond or the millisecond range (1 microsecond to 20 milliseconds). Such a pulse type is defined herein as a long pulse.
  • the second type of pulse has a duration in the nanosecond range (1 to 300 nanoseconds), and defined herein as a short pulse.
  • the time between the long and short pulses in each set can vary between 0 . 1 second to several minutes or hours. Either the long or the short pulse can precede the other.
  • the electric field intensity (kV/cm) of the long and/or the short pulse in the set can vary.
  • the apparatus of the present invention can deliver dual pulses differing by these magnitudes in a single apparatus.
  • the optimum time between pulses will be determined by the diffusion of the agent from the outer membrane to the nucleus, and is expected to be in the ms range or longer. Determination of the diffusion time is within the capabilities of a skilled artisan.
  • the dual-pulse generator may deliver pulses variable in amplitude and duration, as well as in time difference between delivery of the pulses, in order to optimize the system for transfer of the agent into various cells or tissues.
  • the delivery could be, for example, a cuvette (for cells in suspension) or two or multiple metal electrodes for tissue treatment. Other methods of delivery, for example, in vivo deliver of the pulses, are also envisioned by the present invention.
  • FIG. 7 is a circuit diagram illustrating an arrangement for a pulse generator 100 according to one or more embodiments of the present invention.
  • the pulse generator 100 of FIG. 7 is designed to delivers a set of multiple pulse types in succession within the same apparatus.
  • the pulse generator 100 can be configured to deliver two different pulse types.
  • the pulse generator 100 of such embodiments can be considered a dual-pulse generator 100 .
  • Other embodiments of the invention can allow the pulse generator 100 to deliver more than two pulse types, if and when necessary.
  • the pulse generator 100 delivers a first pulse type having a duration in the microsecond or millisecond range, and having a low voltage, as will be discussed in greater detail below. This is considered a long pulse.
  • the second pulse type can have a duration in the nanosecond range (e.g., 1 to 1000 nanoseconds).
  • the length of the second pulse type (or pulse) can also be increased or decreased by, for example, up to thirty percent (30% ).
  • a pause i.e., time between the pulse types
  • the pause between each set of pulses can vary between 0.1 second to several minutes or hours. Either the long or the short pulse can precede the other. Furthermore, any number of either the long or short pulses can be applied.
  • the electric field intensity (kV/cm) of the long and/or the short pulse in the set can vary, as necessary for various applications.
  • the time between pulses can be determined by the diffusion of the agent from the outer membrane to the nucleus. Typically, this time interval is expected to be in the millisecond range or longer, although physical measurements of the diffusion would provide better guidance in determining the length of the pause.
  • the dual-pulse generator 100 can deliver pulses having variable amplitude and duration. The time difference between delivery of the pulses can also be varied in order to configure the system for gene transfer into different cells or tissues.
  • the pulses can be delivered in various ways including, for example, a cuvette for cells in suspension, two or more metal electrodes for tissue treatment, etc. Other methods of delivery such as, for example, in vivo delivery of the pulses, are also envisioned by the present invention.
  • antennas can be used independently or in conjunction with an electrode or cuvette to deliver the pulse.
  • the antennas can be, for example, wide-band antenna, which are used to superimpose a plurality of asymmetrical, unipolar pulses to create a single pulse of the desired duration. This type of antenna arrangement can be used to deliver a short pulse.
  • the long pulse is generated in a first, low voltage circuit, shown in the upper left corner of the diagram and generally referenced by the numeral 110 .
  • a capacitor 112 for example with a capacitance on the order of 1 mF, is charged by a charging resistor, 114 , using a high voltage power supply 116 (HV).
  • HV high voltage power supply
  • FIG. 7 shows a capacitance of 1 mF
  • other embodiments of the invention can utilize capacitors having a capacitance ranging from 0.1 mF to 10 mF.
  • Various other capacitance ratings can be used with the capacitor 112 depending on the specific application.
  • the resistor 114 can have a resistance, for example, of 10 kohms to 10 MOhms, depending on the choice of capacitor. Preferably, the resistor 114 is rated at 1 MOhms to 300 kOhms.
  • the capacitor 112 is subsequently discharged into the load 118 , which is schematically represented by its resistance (R L ).
  • the load 118 could be, for example, a cuvette filled with cells in suspension, tissue between electrodes, or an apparatus that enables in vitro delivery of the pulse.
  • the load resistance may vary according to how the biological sample to be treated is presented and the apparatus used to deliver the load.
  • the closing and opening of the transistor 120 which acts as a switch, is centrally controlled by means of a control system 122 (or control circuit).
  • the control circuit can be, for example, a delay generator, microcontroller, microprocessor, computer controlled circuit, etc.
  • the second pulse type is generated in a second, high voltage, circuit shown in the lower left corner of the diagram and generally indicated by the numeral 124 .
  • the second circuit 124 can be designed, for example, in a Blumlein configuration as shown in FIG. 7 .
  • Two transmission lines 126 or two parallel plates, can be used as an energy reservoir, similar to a capacitor.
  • the transmission lines 126 are charged through a charging resistor 128 to a high voltage, for example, 50 kV, by means of a dc power supply 130 (HV).
  • the resistor 128 can have a resistance, for example, of 10 MOhms to 400 MOhms.
  • the length of the transmission line 126 determines the duration of the short pulse.
  • the duration can be calculated as the length of the transmission line 126 divided by the speed of light in the dielectric of the transmission line 126 .
  • the impedance of the transmission lines 126 can be, for example, half of the load resistance. For example, in the case of a 10-Ohm load, the impedance would be 5 Ohms.
  • the dual-line structure of the Blumlein configuration enables full delivery of charge to the load. If, for example, only one cable were used as the transmission line 126 , only half of the voltage from the power source would be applied. Thus, to deliver 50 kV across the load resistance, the transmission line 126 would need to be charged to 100 kV, which may cause technical difficulties. Using the dual-line transmission lines 126 enables maximum charge delivery to the load. Any other double line-type transmission line 126 could be used in place of the Blumlein configuration.
  • the system illustrated in FIG. 7 enables generation of two pulses of variable duration and amplitude.
  • the time between the long and short pulses can also be varied.
  • the pulse generator of the present invention is also capable of providing multiple pulses, both long and short pulses, etc.
  • the pulses can be controlled based on programming instructions received by the control circuit 122 .
  • Other embodiments of the invention can provide different methods of controlling pulse delivery.
  • the pulse generator 100 can be controlled by a sequence of instructions from the control circuit 122 shown in FIG. 7 .
  • the capacitor 112 can be initially charged to a voltage determined by its capacitance rating. The voltage can vary up to 1.7 kV through the high voltage power supply 116 .
  • the transistor 120 is triggered to close. During this time, the capacitor 112 is discharged. If the magnetic switch 136 is also closed, current will flow through the load 118 (R L ).
  • the long pulse can be either exponentially decaying or a square wave, depending on the value of the capacitor 112 . For example, a square wave can be used for higher capacitance values.
  • the long pulse is terminated by triggering the transistor 120 to stop discharge of the capacitor 112 . Therefore, the time interval (t 2 ⁇ t 1 ) determines the duration of the long pulse.
  • the amplitude of the pulse can be controlled by various means, including through the charging circuit, the voltage source, the capacitor, and the charging resistor.
  • the trigger unit 138 coupled to the magnetic switch 136 is actuated, thus opening the magnetic switch 136 . This action decouples the first circuit 110 (low voltage) from the second circuit 124 (high voltage).
  • trigger unit 134 actuates the closing (or spark gap) switch 132 .
  • the closing switch 132 causes the Blumlein transmission lines 126 to discharge into the load 118 .
  • the transmission lines 126 are charged by the power supply 130 and the charging resistor 128 . This process is similar to charging the capacitor 112 of the first circuit 110 , as described above.
  • the ultra-short close time of the closing switch 132 enables delivery of the short, high voltage pulse to the load 118 .
  • the cycle of pulse deliveries can be repeated as often as desired. Additionally, the order of pulse discharged can be altered, for example, such that the short pulse is delivered first. This alternating order of pulse delivery requires only that the magnetic switch 136 initially be in an open position. Nevertheless, as stated previously, the various embodiments of the invention can allow for any number of pulses to be delivered in any order using the pulse generator of the present invention (i.e., two short, one long; two long, one short; three short, one long; two long, two short; etc).
  • User interfaces can be developed in connection with an HTML display format. Although HTML is utilized in the illustrated examples, it is possible to utilize alternative technology (e.g., XML) for displaying information, obtaining user instructions and for providing user interfaces.
  • XML e.g., XML
  • the system used in connection with the invention may rely on the integration of various components including, as appropriate and/or if desired, hardware and software servers, database engines, and/or other content providers.
  • the configuration may be, preferably, network-based and uses the Internet as a primary interface with the at least one user.
  • the system may store collected information and/or indexes to information in one or more databases.
  • An appropriate database can be maintained on a standard server, for example, a small SunTM SparcTM or other remote location.
  • Any presently available or future developed computer software language and/or hardware components can be employed in the various embodiments of the present invention. For example, at least some of the functionality mentioned above could be implemented using Visual Basic, C, C++, C#, or any assembly language appropriate in view of the processor being used. It could also be written in an interpretive environment such as Java and transported to multiple destinations to various users.
  • HL-60 cells were removed from growth media, washed, and re-suspended in low conductivity buffer (LCB) containing 0.85 mM K 2 HPO 4 , 0.3 mM K 2 HPO 4 (pH 7.2), and 10 mM KCl (conductivity 1.5 mS/cm at 22° C.). Osmolality was adjusted to 290 mOsm by the addition of inositol.
  • Cell suspensions (10 6 cells/ml) were loaded into the BioRad gene Pulser® cuvettes (Bio-Rad Laboratories, Hercules, Calif.) prior to nsPEF pulsing.
  • a cable pulse generator was use to deliver the NsPEF pulses.
  • NsPEFs were delievered by means of a cable pulse generator to cells suspended in a cuvette with parallel plate electrodes separated by 0.1, 0.2, or 0.4 cm.
  • the generator consists of 10 ⁇ pulse-forming network (five 50 ⁇ cables in parallel) and a spark gap in atmospheric air as a nanosecond closing switch. Post/pulse the cell suspension was removed from the pulsing cuvette and assayed.
  • control cells had a mean GFP fluorescence of 3.73
  • the cells exposed to the short pulse had a mean GFP fluorescence of 3.58
  • the cells exposed to the long pulse had a mean GFP fluorescence of 9.67
  • the cells exposed to the combination of pulses had a mean GFP fluorescence of 33.58.

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