Apparatus and method for efficient incorporation of molecules into cells

• US 5,964,726 A
• Filed: 04/16/1997
• Issued: 10/12/1999
• Est. Priority Date: 02/25/1994
• Status: Expired due to Term

• Current Assignee: Ramot University Authority For Applied Research and Industrial Development Limited
• Original Assignee: Ramot University Authority For Applied Research and Industrial Development Limited
• Inventors: Rosemberg, Yosef, Korenstein, Rafi
• Primary Examiner(s): Bockelman, Mark
• Application Number:
  US08/834,340
• Time in Patent Office: 909 Days
• Field of Search: 604/20-21, 604/49, 435/173-1, 435/173.6, 935/52-58, 128/641
• US Class Current: 604/20
• CPC Class Codes: A61N 1/042 Material of the electrode
• CPC Class Codes: A61N 1/325 for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body electrodes for external use A61N1/0428
• CPC Class Codes: A61N 1/327 for enhancing the absorption properties of tissue, e.g. by electroporation
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Abstract

A method and apparatus (20) for introducing molecules and macromolecules into a membrane vesicle, a cell or a tissue by (a) applying a train of low unipolar or alternating voltage pulses to molecules/macromolecules and cells, (b) increase the concentration of the molecules/macromolecules at the surface of the cells, leading to an increased interaction of the molecules/macromolecules with the membrane of the cell while also causing electrophoretic movement of charged proteins and lipids in the cell membrane, and (c) causing the destabilization of the cell membrane whereby the molecules/macromolecules penetrate into the cytosol via an endocytic process and via diffusion through structural defects in the membrane lipid bilayer.

The apparatus (20) includes a support plate (36) having apertures (38) for allowing electrodes (22) to pass therethrough. Support tubes (34) located within the apertures (38) slideably surround the electrodes (22). An electric current supplying mechanism (32) connected to the electrodes (22) supplies sufficient electrical current to a cell for facilitating the introduction of molecules or macromolecules into the cell.

Claims

1. A method for introducing molecules and macromolecules into a membrane vesicle, cell or tissue by

applying a train of unipolar or alternating voltage pulses having an amplitude of voltage in the range of 10V/cm to 150 V/cm at a frequency range of 1 Hz to 50 Mz, and at a pulse width of 20 ns to 20 ms to molecule/macromolecules and to at least one of said membrane vesicle, cell or tissue, and

delivering the train of unipolar or alternating voltage pulses to the membrane vesicle, cell or tissue wherein the molecule/macromolecules are introduced therein.

2. A method of claim 1 further including the steps of preparing a suspension of and molecules/macromolecules to be introduced therein, and applying the train of voltage pulses to the suspension.

3. A method of claim 1 further including the steps of preparing an adherent growth of and molecules/macromolecules to be introduced therein, and applying the train of voltage pulses to the adherent cells.

4. A method of claim 1 further including the steps of introducing the molecules to an area of a population of cells in vivo and applying the train of voltage pulses to the area.

5. An electrode system (20) for use in introducing molecules and macromolecules into a membrane vesicle, cell or tissue, said electrode system (20) comprising:

at least one independently movable needle electrode (22);

support plate means (36) having apertures (38) extending therethrough for supporting said electrodes (22) through said plate means (36); and

electric current supply means (32) operatively connected to said electrodes (22) for supplying sufficient electrical current to a cell to facilitate the instruction of molecules or macromolecules into the cell, said electric current supply needs (32) further including a conductive plate filled with a conducted gel material, said conductive plate (42) conducting electric current through said electrodes (22) when said electrodes (22) contact said conductive plate (42).