Electrospraying apparatus and method for introducing material into cells

• US 6,399,362 B1
• Filed: 05/23/2000
• Issued: 06/04/2002
• Est. Priority Date: 06/12/1997
• Status: Expired due to Term

• Current Assignee: Regents of The University of Minnesota (University of Minnesota)
• Original Assignee: Regents of The University of Minnesota (University of Minnesota)
• Inventors: Chen, Da-Ren, Pui, David Y. H.
• Primary Examiner(s): Beisner, William H.
• Application Number:
  US09/577,747
• Time in Patent Office: 742 Days
• Field of Search: 435/459, 435/470, 435/173.1, 435/173.4, 435/173.5, 435/173.6, 435/285.2, 435/285.3, 435/285.1, 604/68-72, 239/690, 239/704-708
• US Class Current: 435/285.2
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: B01J 2/006 Coating of the granules without description of the process or the device by which the granules are obtained to prevent the granules sticking together B01J2/30
• CPC Class Codes: B01J 2/04 in a gaseous medium if combined with suspending the material in a gas, e.g. fluidised beds B01J2/16
• CPC Class Codes: B01J 2219/00371 comprising electrodes
• CPC Class Codes: B01J 2219/00436 Maskless processes
• CPC Class Codes: B05B 13/04 the spray heads being moved during spraying operation
• CPC Class Codes: B05B 5/025 Discharge apparatus, e.g. electrostatic spray guns
• CPC Class Codes: B05B 5/03 characterised by the use of gas , e.g. electrostatically assisted pneumatic spraying B05B5/04, B05B5/043, B05B5/047 take precedence
• CPC Class Codes: B05B 5/0533 Electrodes specially adapted therefor; Arrangements of electrodes
• CPC Class Codes: B05B 5/081 specially adapted for treating particulate materials
• CPC Class Codes: B05B 5/082 characterised by means for supporting, holding or conveying the objects
• CPC Class Codes: B05B 5/084 the objects lying on, or being supported above conveying means, e.g. conveyor belts
• CPC Class Codes: B05B 5/087 Arrangements of electrodes, e.g. of charging, shielding, collecting electrodes B05B5/12, B05B5/14 take precedence; arrangements of electrodes on the discharge apparatus B05B5/0533
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12N 15/8207 by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/895 using biolistic methods

Abstract

A method of introducing biological material into cells includes providing one or more target cells and establishing a spray of substantially dispersed particles including biological material. The substantially dispersed particles have an electrical charge applied thereto such that one or,more of the substantially dispersed particles of the spray is introduced into one or more of the target cells. The spray of substantially dispersed particles may be established by dispensing a spray of microdroplets suspending particles. The electrical charge is concentrated on the suspended particles as the microdroplet evaporates. The suspended particles may include carrier particles with biological material or the suspended particles may be particles of biological material alone. The space charge effect of the concentrated electrical charge on the substantially dispersed particles of the spray enable one or more of the particles to be introduced into one or more of the target cells. An apparatus for introducing biological material into one or more target cells includes a biological material source including at least biological material and a dispensing device operably connected to the biological material source to receive biological material therefrom. The dispensing device provides a spray of substantially dispersed particles of at least the biological material with the spray of substantially dispersed particles having an electrical charge applied thereto.

Claims

1. An apparatus for introducing biological material into one or more target cells, the apparatus comprising:

a biological material source including at least biological material; and

a dispensing device operably connected to the biological material source to receive at least biological material from the biological material source, wherein the dispensing device provides a spray of substantially dispersed particles of at least the biological material, and further where in the spray of substantially dispersed particles has an electrical charge applied thereto such that a space charge effect from repulsion of the electrically charged substantially dispersed particles is used to provide one or more of the substantially dispersed particles of the spray with a velocity sufficient for introduction of the one or more substantially dispersed particles into one or more of the target cells.

2. The apparatus according to claim 1, wherein the biological material source includes a suspension source, wherein the suspension source includes a suspension of at least biological material, and further wherein the dispensing device receives the suspension and dispenses a spray of microdroplets suspending particles including biological material.

3. The apparatus according to claim 2, wherein the suspension includes carrier particles and biological material.

4. The apparatus according to claim 3, wherein the microdroplets have a nominal diameter of about 10 nanometers to about 10 microns and the carrier particles have a nominal diameter of about 2 nanometers to about 1 micron.

5. The apparatus according to claim 2, wherein the suspended particles are particles of biological material.

6. The apparatus according to claim 2, wherein the dispensing device receives the suspension and dispenses a continuous spray of dispersed particles.

7. The apparatus according to claim 2, wherein the dispensing device includes:

a dispensing tip from which the spray of microdroplets suspending particles is dispensed; and

an electrode isolated from the dispensing tip, wherein a nonuniform electrical field is created between the dispensing tip and the electrode.

8. The apparatus according to claim 7, wherein the electrode is located at a position relative to the dispensing tip to direct the spray of substantially dispersed particles towards the one or more target cells.

9. The apparatus according to claim 8, wherein the electrode is a ring electrode having a diameter greater than a diameter of a spray orifice of the dispensing tip and positioned such that the center of the ring electrode coincides with a longitudinal axis extending through the spray orifice.

10. The apparatus according to claim 8, wherein the electrode is a conductive target including one or more cells associated therewith positioned forward of the spray orifice, the conductive target having a-surface area greater than a cross section area taken perpendicular to a longitudinal axis extending through the spray orifice.

11. The apparatus according to claim 1, wherein the apparatus further includes a vacuum chamber in which the one or more target cells are located, and further wherein the dispensing device provides the spray of substantially dispersed particles into the vacuum chamber.

12. The apparatus according to claim 1, wherein the apparatus further includes a target including the one or more target cells, and further wherein the target is positioned relative to the dispensing device for allowing introduction of one or more of the particles into one or more of the target cells.

13. The apparatus according to claim 12, wherein at least one of the target and the dispensing device are movable relative to the other.

14. The apparatus according to claim 13, wherein the target is a continuously movable surface having one or more cells thereon, and further wherein the movable surface moves relative to the dispensing device such that one or more different cells are continuously moved past the spray of substantially dispersed particles.

15. The apparatus according to claim 13, wherein the target is a rotatable surface for placement of cells thereon, and further wherein the dispensing device is movable in a plane parallel to the rotatable surface.

16. The apparatus according to claim 12, further including means for adjusting a distance between the dispensing device and the target.

17. The apparatus according to claim 12, wherein the apparatus further comprises a member positioned between the dispensing device and the target, and further wherein the member includes an opening such that only a portion of the spray of particles is allowed through the opening for contact with one or more of the target cells.

18. The apparatus according to claim 1, wherein the apparatus further comprises one or more electrodes positioned about a volume into which the spray is dispensed for accelerating the spray of particles towards the target.

19. An apparatus for introducing biological material into target cells, the apparatus comprising:

a biological material source including a suspension of at least biological material;

a capillary tube electrode, wherein the capillary tube electrode includes a capillary tube having a first open end and a second open end, the capillary tube operatively connected to the biological material source to receive a flow of the suspension of at least biological material at the first open end thereof; and

an electrode isolated from but positioned in proximity to the second open end of the capillary tube, wherein a nonuniform electrical field is created between the capillary tube electrode and the electrode such that a spray of microdroplets suspending particles of at least biological material is provided from the second end of the capillary tube, and further wherein upon evaporation of the microdroplets an electrical charge is concentrated on the suspended particles resulting in a charged spray of substantially dispersed particles such that a space charge effect from repulsion of the electrically charged substantially dispersed particles is used to provide one or more of the substantially dispersed particles of the spray with a velocity sufficient for introduction of the one or more substantially dispersed particles into one or more of the target cells.

20. The apparatus according to claim 19, wherein the suspended particles include carrier particles having biological material associated with such carrier particles.

21. The apparatus according to claim 19, wherein the suspended particles are particles of biological material.

22. The apparatus according to claim 19, wherein the biological material source provides a continuous source of biological material to the capillary tube.

23. The apparatus according to claim 19, wherein the electrode is located at a position relative to the second open end of the capillary tube to direct the spray of substantially dispersed particles towards the target cells.

24. The apparatus according to claim 23, wherein the electrode is a ring electrode having a diameter greater than a diameter of the capillary tube and positioned such that an axis through the center of the ring electrode coincides with an axis extending through the capillary tube.

25. The apparatus according to claims 23, wherein the electrode is a conductive target including one or more cells associated therewith positioned forward of the second open end of the capillary tube, the conductive target having a surface area greater than a cross section area of the capillary tube taken perpendicular to a longitudinal axis extending through the capillary tube.

26. The apparatus according to claim 19, wherein the capillary tube electrode further includes a casing concentric with at least a portion of the capillary tube between the first and second open ends thereof, the second open end of the capillary tube extending beyond the casing a predetermined distance, and further wherein the apparatus includes a gas source providing a gas to be received between the capillary tube and the concentric casing.

27. An apparatus for introducing biological material into target cells, the apparatus comprising:

a biological material source including a suspension of at least biological material;

a capillary tube electrode, wherein the capillary tube electrode includes;

a capillary tube having a first open end and a second open end, the capillary tube operatively connected to the biological material source to receive a flow of the suspension of at least biological material at the first open end thereof,

a casing concentric with. at least a portion of the capillary tube between the first and second open ends thereof, the second open end of the capillary tube extending beyond the casing a predetermined distance, and

an additional capillary tube concentric with at least a portion of the capillary tube and positioned between the casing and the capillary tube;

a gas source providing a gas to be received between the capillary tube and the concentric casing; and

an electrode isolated from but positioned in proximity to the second open end of the capillary tube, wherein a nonuniform electrical field is created between the capillary tube electrode and the electrode such that a spray of microdroplets suspending particles of at least biological material is provided from the second end of the capillary tube, and further wherein upon evaporation of the microdroplets an electrical charge is concentrated on the suspended particles resulting in a charged spray of substantially dispersed particles such that one or more of the substantially dispersed particles of the spray is introduced into one or more of the target cells.

28. The apparatus according to claim 27, wherein the apparatus further includes a solution source to provide a solution to be received between the additional capillary tube and the capillary tube.

29. An apparatus for introducing biological material into target cells, the apparatus comprising:

a biological material source including a suspension of at least biological material;

an electrolyte source for providing a solution;

a capillary tube electrode having a dispensing tip, wherein the capillary tube electrode includes;

a first capillary tube having a first open end and a second open end, the first capillary tube operatively connected to the biological material source to receive a flow of the suspension of at least biological material at the first open end thereof, and

a second capillary tube concentric with at least a portion of the first capillary tube, wherein the electrolyte solution is received in an annular opening defined between the first and second concentric capillary tubes; and

an electrode isolated from but positioned in proximity to the dispensing tip of the capillary tube electrode, wherein a nonuniform electrical field is created between the capillary tube electrode and the electrode such that a spray of microdroplets suspending particles of at least biological material is provided from the dispensing tip, and further wherein upon evaporation of the microdroplets an electrical charge is concentrated on the suspended particles resulting in a charged spray of substantially dispersed particles.

30. The apparatus according to claim 29, wherein the suspended particles include carrier particles and biological material.

31. The apparatus according to claim 29, wherein the suspended particles are particles of biological material.

32. The apparatus according to claim 29, wherein the biological material source provides a continuous source of at least biological material to the first open end of the first capillary tube.

33. The apparatus according to claim 29, wherein the electrode is located at a position relative to the dispensing tip of the capillary tube electrode to direct the spray of substantially dispersed particles towards the target cells.

34. The apparatus according to claim 33, wherein the electrode is a ring electrode having a diameter greater than a diameter of the first or second capillary tube and positioned such that an axis through the center of the ring electrode coincides with a longitudinal axis extending through the first capillary tube.

35. The apparatus according to claim 33, wherein the electrode is a conductive target associated with one or more cells, wherein the conductive target is positioned forward of the dispensing tip of the capillary tube electrode, and further wherein the conductive target has a surface area greater than a cross section area of the first or second capillary tube taken perpendicular to a longitudinal axis extending therethrough.

36. The apparatus according to claim 33, wherein the capillary tube electrode further includes a casing concentric with at least a portion of the second capillary tube between a first and second open end thereof, the second open end of the second capillary tube extending beyond the casing a predetermined distance, and further wherein the apparatus includes a gas source providing a gas to be received between the second capillary tube and the concentric casing.