Apparatus for generating electrical pulses and methods of using the same

Abstract

A method and apparatus are provided for delivering an agent into a cell through the application of nanosecond pulse electric fields (“nsPEF's”). The method includes circuitry for delivery of an agent into a cell via known methods followed by the application of nanosecond pulse electric fields to said cell in order to facilitate entry of the agent into the nucleus of the cell. In a preferred embodiment, the present invention is directed to a method of enhancing gene expression in a cell comprising the application of nanosecond pulse electric fields to said cell. An apparatus for generating long and short pulses according to the present invention is also provided. The apparatus includes a pulse generator capable of producing a first pulse having a long duration and low voltage amplitude and a second pulse having a short duration and high voltage amplitude.

1 . A method for introducing an agent into a cell comprising providing a preparation comprising the cell and agent, and applying the nanosecond pulse electric fields to said preparation, which facilitates the entry of the agent into the nucleus. 
     
     
         2 . The method of  claim 1 , wherein the agent is selected from the group comprising drugs, nucleic acids, protein, peptides, and polypeptides. 
     
     
         3 . The method of  claim 2 , wherein the drug is an antibiotic. 
     
     
         4 . The method of  claim 2 , wherein the drug is a chemotherapeutic agent. 
     
     
         5 . The method of  claim 4 , wherein the chemotherapeutic agent is 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. 
     
     
         6 . The method of  claim 2 , wherein the agent is a nucleic acid. 
     
     
         7 . The method of  claim 6 , wherein the nucleic acid is selected from the group comprising DNA, cDNA, and RNA. 
     
     
         8 . The method of  claim 6 , wherein the nucleic acid encodes a homologous or heterologous gene product. 
     
     
         9 . The method of  claim 8 , wherein the cell is transfected with the nucleic acid so that the gene product is expressed in the cell. 
     
     
         10 . The method of  claim 8 , wherein the nucleic acid is an expression vector. 
     
     
         11 . The method of  claim 10 , wherein the expression vector contains a homologous or heterologous nucleic acid encoding a gene product operably linked to a suitable promoter sequence. 
     
     
         12 . The method of  claim 8 , wherein the gene product is expressed in the cell. 
     
     
         13 . The method of  claim 6 , wherein the nucleic acid modulates the expression of a gene. 
     
     
         14 . The method of  claim 13 , wherein the nucleic acid provides gene therapy. 
     
     
         15 . The method of  claim 6 , wherein the nucleic acid modulates cell proliferation. 
     
     
         16 . The method of  claim 6 , wherein the nucleic acid elicits an immune response. 
     
     
         17 . The method of  claim 1 , wherein the agent is a vaccine. 
     
     
         18 . The method of  claim 2 , wherein the polypeptide is a hormone, a cytokine, a lymphokine, a growth factor, or a combination thereof. 
     
     
         19 . The method of  claim 2 , wherein the polypeptide is an antigen. 
     
     
         20 . The method of  claim 2 , wherein the polypeptide is an antibody. 
     
     
         21 . The method of  claim 1 , wherein the agent is a cytotoxic agent. 
     
     
         22 . The method of  claim 21 , wherein the cytotoxic agent is selected from the group comprising ricin, abrin, diphtheria toxin, and saporin. 
     
     
         23 . The method of  claim 1 , wherein the cell is selected from the group comprising eukaryotic cells, prokaryotic cells, fat cells, bone cells, vascular cells, muscle cells, cartilage cells, bacterial cells, and combinations thereof. 
     
     
         24 . The method of  claim 1 , wherein the cell is a cancer cell. 
     
     
         25 . The method of  claim 24 , wherein the cancer cell is selected from the group of cancers comprising adenocarcinoma, squamous carcinoma, carcinoma of the organs, sarcoma, chondrosarcoma, melanosarcoma, leukemia, lymphoma, acute lymphomatic leukemia, acute myelogenous leukemia, non-Hodgkin's lymphoma, Burkitt's lymphoma, B-cell lymphoma, and T-cell lymphoma. 
     
     
         26 . The method of  claim 25 , wherein the cancer cell is a leukemia or fibrosarcoma cell. 
     
     
         27 . The method of  claim 1 , wherein the nanosecond pulse electric field has a pulse duration of about 1 nanosecond to about 1000 nanoseconds. 
     
     
         28 . The method of  claim 27 , wherein the nanosecond pulse electric field has a pulse duration of about 1 nanosecond to about 500 nanoseconds. 
     
     
         29 . The method of  claim 28 , wherein the nanosecond pulse electric field has a pulse duration of about 1 nanosecond to about 300 nanoseconds. 
     
     
         30 . The method of  claim 29 , wherein the nanosecond pulse electric field has a pulse duration of about 10 nanoseconds to about 60 nanoseconds. 
     
     
         31 . The method of  claim 1 , wherein the nanosecond pulse electric field has an electric field intensity from about 1 kV/cm to about 1000 kV/cm. 
     
     
         32 . The method of  claim 31 , wherein the nanosecond pulse electric field has an electric field intensity from about 10 kV/cm to about 350 kV/cm. 
     
     
         33 . The method of  claim 32 , wherein the nanosecond pulse electric field has an electric field intensity from about 10 kV/cm to about 250 kV/cm.