Methods for efficient intracellular delivery using anisotropic magnetic particles

Patent metadata

Field	Value
Publication number	US-10787657-B2
Application number	US-201615387230-A
Country	US
Kind code	B2
Filing date	Dec 21, 2016
Priority date	Dec 21, 2015
Publication date	Sep 29, 2020
Grant date	Sep 29, 2020

Abstract

In various embodiments methods are provided for delivering an agent of interest (e.g., protein, antibody, nucleic acid) into cells. In certain embodiments the method comprises contacting the cells with anisotropic magnetic particles in the presence of the agent; and applying a substantially uniform magnetic field to said magnetic particles where movement of said particles induced by said magnetic field introduces transient openings into said cell facilitating entry of said agent of interest into said cells.

First claim

What is claimed is:

1. A method of delivering an agent of interest into cells, said method comprising:
providing a plurality of cells in a cell culture medium containing said agent;
introducing shape-anisotropic magnetic particles that are not conjugated to said agent into said culture medium; and
applying a substantially uniform magnetic field to said magnetic particles where movement of said particles induced by said magnetic field introduces transient openings into said cell facilitating entry of said agent of interest into said cells.

2. The method of claim 1 , wherein said substantially uniform magnetic field is created by one or more magnets disposed in proximity to said cells.

3. The method of claim 2 , wherein said magnet comprises a magnet selected from the group consisting of a neodymium magnet, a samarium cobalt (SmCo) magnet, an alnico magnet, and a ceramic or ferrite magnet.

4. The method of claim 2 , wherein said cells are disposed in a vessel and said applying a substantially uniform magnetic field comprises disposing said vessel over said magnet.

5. The method of claim 2 , wherein said magnet is disposed to provide a field strength at said cells ranging from about 0.01 tesla up to about 0.1 tesla.

6. The method of claim 1 , wherein said substantially uniform magnetic field is created by an electromagnet.

7. The method of claim 1 , wherein said cells are disposed in a vessel and said applying a substantially uniform magnetic field comprises placing said vessel over a disc-shaped magnet.

8. The method of claim 1 , wherein said substantially uniform magnetic field is applied for a period of time ranging from about 0.5 sec up to about 30 sec.

9. The method of claim 1 , wherein said method further comprises using a magnet to remove the magnetic particles from the medium in which the cells are disposed.

10. The method of claim 1 , wherein said shape-anisotropic magnetic particles comprise a material selected from the group consisting of nickel, and cobalt, and alloys thereof.

11. The method of claim 1 , wherein said shape-anisotropic magnetic particles range in average or median size from about 40 μm up to about 200 μm.

12. The method of claim 1 , wherein said magnetic particles are of a size that prevents internalization into said cells.

13. The method of claim 1 , wherein said magnetic particles are sterile before application to said cells.

14. The method of claim 1 , wherein said magnetic particles are disposed in a flexible membrane that is applied to said cells.

15. The method of claim 14 , wherein said flexible membrane comprises PDMS or another material used in soft lithography.

16. The method of claim 1 , wherein said magnetic particles are applied to said cells at a density ranging from about 1×10 3 particles/cm up to about 3×10 6 particles/cm.

17. The method of claim 1 , wherein said agent of interest comprises an agent selected from the group consisting of a nucleic acid, and a protein or a peptide.

18. The method of claim 1 , wherein said agent of interest is selected from the group consisting of Cas9 from Streptococcus pyogenes (SpCas9), a nucleic acid encoding Cas9 from Streptococcus pyogenes (SpCas9), Cas9 from Streptococcus aureus (SaCas9), a nucleic acid encoding, Cas9 from Streptococcus aureus (SaCas9), a Cpf1 nuclease, a nucleic acid encoding the Cpf1 nuclease, and a guide RNA.

19. The method of claim 1 , wherein said cells are prokaryotic cells.

20. The method of claim 1 , wherein said cells comprise eukaryotic cells.

21. The method of claim 20 , wherein said cells comprise mammalian cells.

22. The method of claim 21 , wherein said cells comprises human cells.

23. The method of claim 20 , wherein said cells comprise stem cells selected from the group consisting of fetal stem cells, adult stem cells, cord blood stem cells, and induced pluripotent stem cells.

24. The method of claim 1 , wherein said method is configured for a high throughput format.

25. The method of claim 24 , wherein said method is configured to perform at least 2, or at least 4, or at least 8, or at least 16, or at least 32, or at least 64, or at least 128 different transfections simultaneously.

26. The method of claim 1 , wherein:
said substantially uniform magnetic field is not provided by a component of a magnetic stirrer; and/or
said substantially uniform magnetic field is not provided as an element of a magnetic cell isolation or cell component isolation.

27. The method of claim 22 , wherein said cells comprise cells selected from the group consisting of fibroblasts, neural cells, A549 cells, HeLa cells, primary human mammary epithelial cells (HMECs), red blood cells, white blood cells, and stem cells.

28. The method of claim 1 , wherein said agent of interest comprises one or more agents selected from the group consisting of an enzyme, a plasmid, a viral vector, a cosmid, an artificial chromosome, an antibody, an RNAi, a component of a CRISPR/Cas9 system, a color dye, and calcium.

Assignees

• Univ California

Inventors

• Chiou Pei-Yu E
• Teitell Michael A
• Lin Ming-Yu
• Wu Yi-Chien
• Zhou Jessica

Classifications

• C12N13/00 Primary

  Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves · CPC title

• C12N15/895

  using biolistic methods · CPC title

• C12N15/87

  Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation · CPC title

• B82Y5/00

  Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery · CPC title