Method for combined sequential agent delivery and electroporation for cell structures and use thereof

Abstract

Disclosed is a method for sequential delivery of agents to and/or into a cell structure, wherein an electrolyte-filled tube is provided together with a counter electrode, said tube is connected to a voltage or current generator, at least two agents are introduced in a discrete mode into the electrolyte solution contained in the tube, which is placed close to the cell structure, one agent at the time being transported through the tube to and/or into said cell structure in which a pore has been formed by application of an electric field focused on the cell structure, resulting in electroporation of the cell structure. Also different applications of the method is disclosed, e.g. us of the method in order to transfer cell-impermeant solutes, such as drugs or genes, into the cell structure or out of the cell structure.

1 . A method for sequential delivery of agents to a surface of a cell structure and into the cytoplasm of the cell structure, comprising the following steps: 
 (a) an electrolyte-filled tube is provided together with a ground or counter electrode,    (b) the electrolyte-filled tube is connected to a voltage or current generator,    (c) at least two agents are introduced into the electrolyte solution contained in the electrolyte-filled tube, the agents being arranged in at least two discrete zones or bands each comprising at least one agent,    (d) the electrolyte-filled tube is placed at close distance to the surface of the cell structure,    (e) an agent is transported through the electrolyte-filled tube to the surface of the cell structure,    (f) an electric field of a strength sufficient to obtain electroporation of the surface of the cell structure is focused on the cell structure, resulting in formation of a pore in the membrane surface of the cell structure, and    (g) the agent is transported through the pore formed in step (f) and into the cytoplasm of the cell structure,    wherein steps (a-g) are performed in consecutive order, with the exception that the order of steps (b), (c) and (d) may be altered, and that the order of steps (e) and (f) may be altered, and    wherein steps (e)-(g) are repeated until the desired number of agents have been delivered into the cytoplasm of the cell structure.    
     
     
         2 . A method according to  claim 1 , wherein at least two electrolyte-filled tubes and are used, enabling sequential delivery of agents through more than one pore in a parallel manner.  
     
     
         3 . A method according to  claim 2 , wherein agents are delivered through pores formed in one cell structure.  
     
     
         4 . A method according to  claim 2 , wherein agents are delivered through pores formed in different cell structures.  
     
     
         5 . A method according to any one of the claims  1 - 4 , wherein the electrical field is obtained by applying a voltage between the electrolyte filled tube and the counter or ground electrode using a voltage generator.  
     
     
         6 . A method according to any one of the claims  1 - 4 , wherein the electrical field is obtained by applying a current between the electrolyte filled tube and the counter or ground electrode using a current generator.  
     
     
         7 . A method according to any one of the claims  1 - 6 , wherein the electrolyte-filled tube is an electrolyte-filled capillary.  
     
     
         8 . A method according to any one of the claims  1 - 6 , wherein the electrolyte-filled tube is an electrolyte-filled conically tapered tube.  
     
     
         9 . A method according to any one of the claims  1 - 6 , wherein the electrolyte-filled tube is an electrolyte-filled electrode.  
     
     
         10 . A method according to any one of the claims  1 - 9 , wherein the agents are introduced into the electrolyte solution contained in the electrolyte-filled tube from the tip end using capillary forces or aspiration or suction.  
     
     
         11 . A method according to any one of the claims  1 - 10 , wherein the agents are comprised in the electrolyte in the electrolyte-filled tube.  
     
     
         12 . A method according to any one of the claims  1 - 11 , where the cell structure is contained in a cell bathing medium.  
     
     
         13 . A method according to any one of the claims  1 - 12 , wherein an agent is a drug.  
     
     
         14 . A method according to any one of the claims  1 - 13 , wherein the tube is loaded with the discrete zones or bands containing agents using a microfluidic switch.  
     
     
         15 . A method according to any one of the claims  1 - 14 , wherein the electrolyte-filled tube contains several barrels or channels.  
     
     
         16 . A method according to  claim 15 , wherein the barrels or channels contain different agents.  
     
     
         17 . A method according to  claim 15  or  16 , wherein each barrel or channel is individually connected to an electrode.  
     
     
         18 . A method according to any one of the claims  1 - 17 , wherein the cell structure is a population of cells.  
     
     
         19 . A method according to any one of the claims  1 - 17 , wherein the cell structure is a single cell.  
     
     
         20 . A method according to any one of the claims  1 - 18 , wherein the cell structure is a tissue.  
     
     
         21 . A method according to any one of the claims  1 - 18 , wherein the cell structure is an organ.  
     
     
         22 . A method according to any one of the claims  1 - 18 , wherein the cell structure is an intracellular structure.  
     
     
         23 . A method according to  claim 22 , wherein the intracellular structure is an organelle.  
     
     
         24 . A method according to any one of the claims  1 - 23 , wherein the cell structure is immobilized on a surface.  
     
     
         25 . A method according to any one of the claims  1 - 24 , wherein the cell structure is contained in at least one well on a plate.  
     
     
         26 . A method according to any one of the claims  1 - 25 , wherein the cell structure has been pretreated with a genetic method prior to step (f).  
     
     
         27 . A method according to  claim 26 , wherein the genetic method is a transfection method.  
     
     
         28 . A method according to any one of the claims  1 - 27 , wherein the cell structure has been pretreated with a drug prior to step (f).  
     
     
         29 . A method according to any one of the claims  1 - 28 , wherein the cell structure has been pretreated with an internalized dye or marker prior to step (d).  
     
     
         30 . A method according to any one of the claims  1 - 29 , wherein the tube is additionally connected to a fluid delivery device.  
     
     
         31 . A method according to  claim 30 , wherein the device is a micro-pump.  
     
     
         32 . A method according to  claim 30 , wherein the device is a peristaltic pump.  
     
     
         33 . A method according to  claim 30 , wherein the device is a gravitational pump.  
     
     
         34 . A method according to  claim 30 , wherein the device is a pneumatic pump.  
     
     
         35 . A method according to  claim 30 , wherein the device is a solenoid.  
     
     
         36 . A method according to  claim 30 , wherein the device is a pressure-driven pump.  
     
     
         37 . A method according to any one of the claims  30 - 36 , wherein the fluid delivery device is used for transportation of the agents into the electrolyte-filled tube.  
     
     
         38 . A method according to any one of the claims  30 - 37 , wherein the fluid delivery device is used for transportation of the agents into the cell structure.  
     
     
         39 . A method according to any one of the claims  1 - 38 , wherein the cell structure is a intracellular structure and the electrolyte filled tube(s) and the ground or counter electrode are arranged so that the ends of the tube(s) and the electrode are placed within a host cell containing the intracellular structure.  
     
     
         40 . A method according to any one of the claims  1 - 39 , wherein the electrolyte-filled tube is connected to a voltage generator via at least one electrode.  
     
     
         41 . A method according to any one of the claim  1 - 40 , wherein the voltage generator generates a voltage of from 10 mV to 100 V at the surface of the cell structure.  
     
     
         42 . A method according to  claim 41 , wherein the voltage is from 100 mV to 10 V.  
     
     
         43 . A method according to any one of the claims  1 - 39 , wherein the electrolyte-filled tube is connected to a current generator via at least one electrode.  
     
     
         44 . A method according to any one of the claims  1 - 43 , wherein the current needed for electroporation is carried by an intra-electrodal electrolyte present in the electrolyte-filled tube.  
     
     
         45 . A method according to any one of the claims  1 - 43 , wherein the current needed for electroporation is carried by an electrically conductive layer on the electrolyte-filled tube.  
     
     
         46 . A method according to  claim 5  or  6 , or any one of the claims  7 - 45  when dependent on  claim 5  or  6 , wherein the voltage or current is applied as a pulse.  
     
     
         47 . A method according to  claim 46 , wherein the length of the pulse is from 0.1 μs to several minutes.  
     
     
         48 . A method according to  claim 47 , wherein the length of the pulse is from 1 μs to 5 s.  
     
     
         49 . A method according to any one of the claims  1 - 48 , wherein a programmed electric field varying the strength and/or the wave form is used in step (f).  
     
     
         50 . A method according to any one of the claims  1 - 49 , wherein a pulsed electric field is used in step (f).  
     
     
         51 . A method according to any one of the claims  1 - 50 , wherein the close distance in step (d) is less than 100 μm.  
     
     
         52 . A method according to any one of claims  1 - 51 , wherein the diameter of the electrolyte filled tube at the end closest to the cell structure is from a few nanometers to a few hundred micrometers.  
     
     
         53 . A method according to any one of claims  1 - 52 , wherein the electrolyte-filled tube is positioned by use of at least one micropositioner.  
     
     
         54 . A method according to any one of the claims  1 - 53 , wherein the electrolyte-filled tube is a hollow fused silica electrode.  
     
     
         55 . A method according to any one of the claims  1 - 53 , wherein the electrolyte-filled tube is a polymer electrode.  
     
     
         56 . A method according to any one of the claims  1 - 53 , wherein the electrolyte-filled tube is a fluorocarbon tube such as a Teflon tube.  
     
     
         57 . A method according to any one of claims  1 - 56  wherein at least one agent is a cell-impermeant agent.  
     
     
         58 . A method according to  claim 57 , wherein the cell-impermeant agent comprise a pharmaceutically active compound.  
     
     
         59 . A method according to any one of the claims  1 - 58 , wherein at least one agent is an electrolyte.  
     
     
         60 . A method according to any one of the claims  1 - 59 , wherein at least one agent is a substance that activates receptors on the cell plasma membrane.  
     
     
         61 . A method according to any one of the claims  1 - 60 , wherein at least one agent is an agent that affects intracellular chemistry.  
     
     
         62 . A method according to any one of the claims  1 - 61 , wherein at least one agent is an agent that affects cellular physics.  
     
     
         63 . A method according to any one of the claims  1 - 62 , wherein the agents independently are selected from the group consisting of genes, gene analogs, RNA, RNA analogs, DNA, DNA analogs, colloidal particles, receptors, receptor ligands, receptor antagonists, receptor blockers, enzymes, enzyme substrates, enzyme inhibitors, enzyme modulators, proteins, protein analogs, amino acids, amino acid analogs, peptides, peptide analogs, metabolites, metabolite analogs, oligonucleotides, oligonucleotide analogs, antigens, antigen analogs, haptens, hapten analogs, antibodies, antibody analogs, organelles, organelle analogs, cell nuclei, bacteria, viruses, gametes, inorganic ions, metal ions, metal clusters, polymers, and any combinations thereof.  
     
     
         64 . A method according to any one of the claims  1 - 63 , wherein the agents are delivered into the cell structure by electrophoresis or electroosmosis.  
     
     
         65 . A method according to any one of the claims  1 - 64 , wherein the electrolyte-filled tube is one tube in a one-dimensional array or a two-dimensional array of more than one electrolyte-filled tubes.  
     
     
         66 . A method according  claim 65 , wherein the array is microfabricated from a solid substrate into a chip device, the surface of which having several openings each constituted by a tip end of an individual electrolyte-filled tube.  
     
     
         67 . A method according to any one of the claims  1 - 66 , wherein the electrolyte-filled tube is individually controlled.  
     
     
         68 . A method according to any one of the claims  1 - 66 , wherein more than one electrolyte-filled tube is used and the electrolyte-filled tubes are population-wise controlled.  
     
     
         69 . A method according to  claim 67  or  68 , wherein the electrolyte-filled tube(s) is (are) controlled by a robotic device.  
     
     
         70 . A method according to any one of the claims  1 - 69 , wherein said cell structure can be translated in relation to the outlet end of the electrolyte-filled tube(s).  
     
     
         71 . A method according to  claim 70 , wherein the cell structure is translated using a movable stage.  
     
     
         72 . A method according to  claim 70  or  claim 71 , wherein the cell structure is translated using a motorized stage.  
     
     
         73 . A method according to  claim 71  or  claim 72 , wherein the stage is a microscope stage.  
     
     
         74 . A method according to any one of the claims  1 - 73 , comprising a further step (h) performed after step (g) wherein a response evoked by at least one of the agents in the cell structure is measured by detection of fluorescence.  
     
     
         75 . Use of a method according to any one of the claims  1 - 74  for gene transfection.  
     
     
         76 . Use of a method according to any one of the claims  1 - 74  for gene identification.  
     
     
         77 . Use of a method according to any one of the claims  1 - 74  for enzyme identification.  
     
     
         78 . Use of a method according to any one of the claims  1 - 74  for protein identification.  
     
     
         79 . Use of a method according to any one of the claims  1 - 74  for receptor identification.  
     
     
         80 . Use of a method according to any one of the claims  1 - 74  in binding assays, enzyme assays, receptor assays, viral assays, bacterial assays, drug assays, and/or kinetic assays.  
     
     
         81 . Use of a method according to any one of the claims  1 - 74  in pharmacokinetics.  
     
     
         82 . Use of a method according to any one of the claims  1 - 74  in pharmacology.  
     
     
         83 . Use of a method according to any one of the claims  1 - 74  for modification of a metabolic pathway and/or a signaling pathway.  
     
     
         84 . Use of a method according to any one of the claims  1 - 74  for in vitro fertilization.  
     
     
         85 . Use of a method according to any one of the claims  1 - 74  for nuclear and/or organelle transfer.  
     
     
         86 . Use of a method according to any one of the claims  1 - 74  for screening for receptors on the surface of said cell structure.  
     
     
         87 . Use of a method according to any one of the claims  1 - 74  for screening for receptors on the inside of said cell structure.  
     
     
         88 . Use of a method according to any one of the claims  1 - 74  in study of signaling systems inside said cell structure.  
     
     
         89 . Use of a method according to any one of the claims  1 - 74  in a sensor.  
     
     
         90 . Use of a method according to any one of the claims  1 - 74  in robotics.  
     
     
         91 . Use of a method according to any one of the claims  1 - 74  in a chemical computer.  
     
     
         92 . Use of a method according to any one of the claims  1 - 74  in a biological computer.