Reverse protein delivery into cells on coded microparticles

• US 20040146944A1
• Filed: 01/29/2003
• Published: 07/29/2004
• Est. Priority Date: 01/29/2003
• Status: Active Grant

• Current Assignee: Corning Incorporated
• Original Assignee: Corning Incorporated
• Inventors: Webb, Brian L., Fang, Ye
• Granted Patent: US 7,691,580 B2
• Time in Patent Office: Days
• US Class Current: 435/7.2
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5432 Liposomes or microcapsules

Abstract

Systems, methods and kits that utilize uniquely coded microparticles for performing protein assays are provided. The uniquely coded microparticles are used as a substrate for reverse protein delivery into cells. The microparticles and methods offer the possibility of studying the biological functions of either a single protein of interest in multiple cell types per assay or multiple proteins in a single cell type.

Claims

1. A method for multiplexed functional screening of proteins in cells, comprising:

contacting a first microparticle having a unique identification code with a protein-containing mixture under conditions to allow the protein to attach to the microparticle; and

contacting the microparticle having a protein attached thereto with cells under conditions sufficient to deliver protein into the cells.

2. The method of claim 1, wherein the protein-containing mixture includes a carrier reagent.

3. The method of claim 2, wherein the microparticle is contacted with the protein-containing mixture in a well.

4. The method of claim 1, further comprising contacting at least a second microparticle having a unique identification code with a protein-containing mixture under conditions sufficient to allow the protein to attach to the microparticle and contacting the at least second microparticle including a protein bound thereto with cells under conditions sufficient to deliver protein into the cells in contact with the second microparticle.

5. The method of claim 4, wherein the protein-containing mixture includes a carrier reagent.

6. The method of claim 5, wherein the second microparticle is contacted with the protein-containing mixture in a well.

7. The method of claim 4, wherein the code on the microparticles includes at least one rare earth element.

8. The method of claim 7, wherein the at least one rare earth element is patterned on the microparticles.

9. The method of claim 8, wherein the microparticle is porous.

10. The method of claim 4, wherein the particle is encoded with at least one fluorescent molecule.

11. The method of claim 4, further wherein protein-containing mixture on the first microparticle contains a first protein and the protein-containing mixture on the second microparticle contains a second protein different from the first protein.

12. The method of claim 11, wherein the different proteins are functionally related.

13. The method of claim 4, wherein said proteins on the first and second microparticles comprises the same protein of interest but with different point mutations.

14. The method of claim 11, wherein the first and second microparticles are contacted with the same cell type.

15. The method of claim 14, wherein the first and second microparticles are placed in different wells of a multiwell plate.

16. The method of claim 14, wherein the first and second microparticles are screened for drug interactions or ligand binding.

17. The method of claim 1, comprising contacting a plurality of microparticles having the same protein bound thereto with a plurality of different cell types.

18. The method of claim 14, wherein said protein of interest includes a functional peptide, protein mutants, engineered proteins with specific modifications, antibodies, bioactive peptide, protein-domain, intracellular protein, enzyme, cell surface protein, toxin protein, antibody, antibody-nucleic acid conjugate, protein-nucleic acid conjugate, peptide-nucleic acid conjugate, protein-nanoparticle conjugate, protein-polymer conjugate, conjugate between a protein-organic chemical entity or protein-inorganic chemical entity, multi-protein complexes, and any amino-acid containing moiety.

19. The method of claim 14, wherein said protein of interest includes a fusion protein that contains a translocation domain and a protein domain of interest.

20. The method of claim 2, wherein the carrier reagent is a naturally occurring or synthetic translocation peptide, or a liposome.

21. The method of claim 2, wherein said carrier reagent includes:

a bioactive cell membrane-permeable reagent, a membrane-translocating peptide, a peptide containing protein-transduction domains (PTDs), a signal sequence, a bioactive peptide, or a ligand that can bind specifically to and activate cell surface receptors.

22. The method of claim 2, wherein said carrier reagent includes:

Trojan peptides, human immuodeficiency virus (HIV-1) transcriptional activator (TAT) protein or its functional domain peptides, Drosophilia antennapedia homeotic transcription factor, and herpes simplex virus-1 DNA binding protein VP22.

23. The method of claim 1, wherein said protein-containing mixture further comprises a helper reagent.

24. The method of claim 23, wherein said helper reagent includes:

a polymer, a cell adherent-enhancing protein, a sugar-based gelatin, a synthetic or chemical-based gelatin, a RGD peptide, a mixture of a hydrogel and a RGD peptide, or a combination of any the aforementioned molecules.

25. The method of claim 23, wherein said helper reagent is a polymer selected from the group consisting of:

DEAE-dextran, dextran, polylysine, and polyethylamine.

26. The method of claim 23, wherein said helper reagent is a cell adherent-enhancing protein selected from the group comprising a fibronectin and gelatin.

27. The method of claim 23, wherein said helper reagent is either polyethylene glycol or acrylamide.

28. The method of claim 23, wherein said helper reagent is a RGD peptide selected from the group comprising of:

Arg-Gly-Asp-Ser, Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro, and combinations thereof.

29. The method of claim 2, wherein the conditions for delivery include placing the microparticles in a serum-free medium.

30. The method of claim 2, wherein the conditions sufficient for delivery include placing the microparticles in a serum-containing medium.

31. The method of claim 2, wherein the microparticle is encoded with a co-delivered protein separately attached to the microparticle.

32. The method of claim 2, wherein the microparticle is encoded with a fusion protein associated with the protein of interest that uniquely identifies the protein attached to the microparticle.