Reverse protein delivery into cells on coded microparticles

• US 7,691,580 B2
• Filed: 01/29/2003
• Issued: 04/06/2010
• Est. Priority Date: 01/29/2003
• Status: Expired due to Fees

• Current Assignee: Corning Incorporated
• Original Assignee: Corning Incorporated
• Inventors: Webb, Brian L., Fang, Ye
• Primary Examiner(s): CHEU, CHANGHWA J
• Application Number:
  US10/353,496
• Publication Number:
  US 20040146944A1
• Time in Patent Office: 2,624 Days
• Field of Search: 435/7.1, 435/7.2, 435/92
• US Class Current: 435/7.1
• 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 delivering proteins in cells, comprising:

contacting a first micro-carrier substrate having a unique identification code with a first protein-containing mixture under conditions to allow the protein to attach to the micro-carrier substrate, wherein the first protein-containing mixture comprises a protein of interest, a carrier reagent, and a helper reagent; and

contacting the first micro-carrier substrate having the protein of interest attached thereto with cells under conditions sufficient for the cells to adhere to the surface of the micro-carrier substrate and for the cells to uptake the protein of interest, wherein the code on the first micro-carrier substrate includes at least one rare earth element.

2. The method of claim 1, wherein the micro-carrier substrate is contacted with the protein-containing mixture in a well.

3. The method of claim 1, further comprising contacting at least a second micro-carrier substrate having a unique identification code with a second protein-containing mixture under conditions sufficient to allow the second protein to attach to the second micro-carrier substrate and contacting the at least second micro-carrier substrate including a second protein bound thereto with cells under conditions sufficient to deliver the second protein into the cells in contact with the second micro-carrier substrate, the second protein-containing mixture comprises the second protein of interest, a carrier reagent, and a helper reagent.

4. The method of claim 3, wherein the second micro-carrier substrate is contacted with the second protein-containing mixture in a well.

5. The method of claim 1, wherein the at least one rare earth element is patterned on the first micro-carrier substrate.

6. The method of claim 5, wherein the first micro-carrier substrate is porous.

7. The method of claim 3, further wherein protein-containing mixture on the first micro-carrier substrate contains a first protein and the protein-containing mixture on the second micro-carrier substrate contains a second protein different from the first protein.

8. The method of claim 7, wherein the different proteins are functionally related.

9. The method of claim 3, wherein said proteins on the first and second micro-carrier substrates comprise the same protein of interest but with different point mutations.

10. The method of claim 7, wherein the first and second micro-carrier substrates are contacted with the same cell type.

11. The method of claim 10, wherein the first and second micro-carrier substrates are placed in different wells of a multiwell plate.

12. The method of claim 10, wherein the first and second micro-carrier substrates are screened for drug interactions or ligand binding.

13. The method of claim 1, comprising contacting a plurality of first micro-carrier substrates having the same protein bound thereto with a plurality of different cell types.

14. The method of claim 10, 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.

15. The method of claim 10, wherein said protein of interest includes a fusion protein that contains a translocation domain and a protein domain of interest.

16. The method of claim 1, wherein the carrier reagent is a naturally occurring or synthetic translocation peptide, or a liposome.

17. The method of claim 1, 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.

18. The method of claim 1, 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.

19. The method of claim 1, 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 Arg-Gly-Asp (RGD) peptide, or a combination of any the aforementioned molecules.

20. The method of claim 1, wherein said helper reagent is a polymer selected from the group consisting of:

DEAE-dextran, dextran, polylysine, and polyethylamine.

21. The method of claim 1, wherein said helper reagent is a cell adherent-enhancing protein selected from the group comprising a fibronectin and gelatin.

22. The method of claim 1, wherein said helper reagent is either polyethylene glycol or acrylamide.

23. The method of claim 1, wherein said helper reagent is a RGD peptide selected from the group comprising of:

(SEQ ID NO;

     1), (SEQ ID NO;

     2), and combinations thereof.

24. The method of claim 1, wherein the conditions for delivery include placing the first micro-carrier substrates in a serum-free medium.

25. The method of claim 1, wherein the conditions sufficient for delivery include placing a plurality of first micro-carrier substrates in a serum-containing medium.

26. The method of claim 1, wherein the first micro-carrier substrate is encoded with a co-delivered protein separately attached to the first micro-carrier substrate.

27. The method of claim 1, wherein the first micro-carrier substrate is encoded with a fusion protein associated with the protein of interest that uniquely identifies the protein attached to the first micro-carrier substrate.