Method and device for protein delivery into cells

• US 20060105371A1
• Filed: 10/27/2005
• Published: 05/18/2006
• Est. Priority Date: 07/30/2002
• Status: Active Grant

• Current Assignee: Brian L. Webb, Fang Lai, Laurent A.G. Picard, Ye Fang
• Original Assignee: Brian L. Webb, Fang Lai, Laurent A.G. Picard, Ye Fang
• Inventors: Webb, Brian L., Fang, Ye, Lai, Fang, Picard, Laurent A.G.
• Granted Patent: US 7,829,290 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 2503/00 Use of cells in diagnostics
• CPC Class Codes: C12N 2535/10 Patterned coating
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06

Abstract

Methods for performing surface-mediated protein delivery into living cells, and fabricating protein-transfected cell cluster arrays are provided. The method comprises providing a protein-containing mixture; depositing said protein-containing mixture onto a surface at defined locations; affixing the protein-containing mixture to the surface as microspots; and plating cells onto the surface in sufficient density and under conditions for the proteins to be delivered into the cells. The protein-containing mixture comprises any suitable amino acid sequence, including peptides, proteins, protein-domains, antibodies, or protein-nucleic acid conjugates, etc., with a carrier reagent. Protein-transfected cell arrays may be used for rapid and direct, screening of protein or enzymatic functions or any given intracellular protein interaction in the natural environment of a living cell, as well as for high-throughput screening of other biological and chemical analytes, which affect the functions of these proteins.

Claims

1. A method for performing surface-mediated protein delivery into living cells, the method comprising:

providing a protein-containing mixture;

depositing said protein-containing mixture onto a surface at defined locations;

affixing said protein-containing mixture to the surface; and

plating said cells onto the surface in sufficient density and under conditions for delivering proteins into said cells.

2. The method according to claim 1, wherein said protein-containing mixture comprises a protein of interest with a carrier reagent.

3. The method according to claim 2, wherein said protein of interest includes a 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.

4. The method according to claim 2, wherein said protein of interest includes a fusion protein that contains a translocation domain and a protein domain of interest.

5. The method according to claim 2, wherein the carrier reagent is a naturally occurring or synthetic translocation peptide, or a liposome.

6. The method according to 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.

7. The method according to claim 6, 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.

8. The method according to claim 1, wherein said protein-containing mixture further comprises a helper reagent.

9. The method according to claim 8, 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.

10. The method according to claim 8, wherein said helper reagent is a polymer selected from the group consisting of:

DEAE-dextran, dextran, polylysine, and polyethylamine.

11. The method according to claim 8, wherein said helper reagent is a cell adherent-enhancing protein selected from the group consisting of:

a fibronectin and gelatin.

12. The method according to claim 8, wherein said helper reagent is either polyethylene glycol or acrylamide.

13. The method according to claim 8, wherein said helper reagent is a RGD peptide selected from the group consisting of:

Arg-Gly-Asp-Ser, Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro, and the like.

14. The method according to claim 1, wherein said cell density is at least about 0.3×

10⁴ cells/cm².

15. The method according to claim 1, wherein said cell density is up to about 5×

10⁵ cells/cm².

16. The method according to claim 1, wherein said conditions for delivery is in a serum-free medium.

17. The method according to claim 1, wherein said conditions for delivery is in a serum-containing medium.

18. The method according to claim 1, wherein said surface is that of a microtiter plate well-bottom, a microcolumn, a slide, a strip, a bead, a particle, or nanoparticle.

19. The method according to claim 1, wherein said defined locations comprise an array of microspots on said substrate surface.

20. The method according to claim 19, wherein said defined locations each contains a different protein.

21. The method according to claim 20, wherein each different protein is included on more than one separate microspot on the array.

22. The method according to claim 19, wherein said microspots on the array comprises the same protein of interest but with different point mutations.

23. The method according to claim 20, wherein the different proteins are functionally related.

24. The method according to claim 19, wherein said array form a thematic microarray.

25. The method according to claim 19, wherein said density of the microspots on the surface of the substrate is at least about 1/cm².

26. The method according to claim 19, wherein said density of the microspots on the surface of the substrate of up to about 2000/cm².

27. A reverse delivery method of introducing proteins into living cells comprising:

spotting a protein of interest on a surface in defined and discrete locations;

drying and affixing the protein spot;

treating the protein spot with a carrier reagent;

depositing cells onto the surface; and

transporting said protein into said cells.

28. The method according to claim 27, wherein said protein in the spot interacts and complexes with said carrier reagent.

29. The method according to claim 27, wherein said protein of interest includes a 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, or polymer.

30. The method according to claim 27, wherein said protein of interest includes a fusion protein that contains a translocation domain and a protein domain of interest.

31. The method according to claim 27, wherein the carrier reagent is a naturally occurring or synthetic translocation peptide, or a liposome.

32. The method according to claim 27, 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.

33. The method according to claim 27, 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.

34. The method according to claim 27, wherein said protein-containing mixture further comprises a helper reagent.

35. The method according to claim 34, 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.

36. The method according to claim 34, wherein said helper reagent is a polymer selected from the group consisting of:

DEAE-dextran, dextran, polylysine, and polyethylamine.

37. The method according to claim 34, wherein said helper reagent is a cell adherent-enhancing protein selected from the group consisting of:

a fibronectin and gelatin.

38. The method according to claim 34, wherein said helper reagent is either polyethylene glycol or acrylamide.

39. The method according to claim 34, wherein said helper reagent is a RGD peptide selected from the group consisting of:

Arg-Gly-Asp-Ser, Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro, and the like.

40. The method according to claim 27, wherein said conditions for delivery is in a serum-free medium.

41. The method according to claim 27, wherein said conditions for delivery is in a serum-containing medium.

42. The method according to claim 27, wherein said surface is that of a microtiter plate well-bottom, a microcolumn, a slide, a strip, a bead, a particle, or nanoparticle.

43. The method according to claim 27, wherein said defined locations comprise an array of microspots on said substrate surface.

44. The method according to claim 43, wherein said defined locations each contains a different protein.

45. The method according to claim 43, wherein each different protein is included on more than one separate microspot on the array.

46. The method according to claim 43, wherein said microspots on the array comprises the same protein of interest but with different point mutations.

47. The method according to claim 45, wherein the different proteins are functionally related.

48. The method according to claim 43, wherein said array form a thematic microarray.

49. A method of producing an array of cells transfected with a protein on a surface, the method comprising:

spotting a protein-containing mixture onto a surface in discrete, defined locations;

drying said surface bearing the protein-containing mixture such that the spots remain affixed to the surface under array conditions;

adding cells to produce a surface having both proteins and cells; and

transporting of said proteins into said cells.

50. The method according to claim 49, wherein the method further comprises maintaining the surface under conditions that promote protein entry into cells, and producing an array of cells that contain said proteins.

51. The method according to claim 49, wherein said cells are in a medium.

52. The method according to claim 51, wherein said conditions for delivery is in a serum-free medium.

53. The method according to claim 51, wherein said conditions for delivery is in a serum-containing medium.

54. The method according to claim 49, wherein said surface is that of a microtiter plate well-bottom, a microcolumn, a slide, a strip.

55. The method according to claim 49, wherein said defined locations comprise an array of microspots on said substrate surface.

56. The method according to claim 49, wherein said defined locations each contains a different protein.

57. The method according to claim 56, wherein each different protein is included on more than one separate microspot on the array.

58. The method according to claim 55, wherein said microspots on the array comprises the same protein of interest but with different point mutations.

59. The method according to claim 56, wherein the different proteins are functionally related.

60. A high-throughput biological analysis device fabricated according to the method of claim 49.

61. An array produced according to a method comprising:

providing a protein-containing mixture;

depositing said protein-containing mixture onto a surface at defined locations;

affixing said protein-containing mixture to the surface; and

plating said cells onto the surface.

62. The array according to claim 61, wherein said protein containing mixture is a solution of:

a) protein—

of interest;

b) a protein-of-interest complexed with a carrier reagent;

c) a conjugate of a protein-of interest and a carrier reagent;

or a mixture of (b) and (c).

63. The array according to claim 62, wherein said protein-of-interest includes a bioactive peptide, protein-domain, intracellular protein, enzyme, cell surface protein, toxin protein, antibody.

64. The array according to claim 62, wherein said protein-of-interest includes an 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, or any amino-acid containing moiety.

65. The array according to claim 62, wherein said conjugate is a protein-of-interest covalently attached to a carrier reagent.

66. The array according to claim 62, wherein said protein-of-interest includes a fusion protein that contains a translocation domain and a protein domain of interest.

67. The array according to claim 62, wherein the carrier reagent is a naturally occurring or synthetic translocation peptide.

68. The array according to claim 66, 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 targeted ligand that can bind specifically to and activate cell surface receptors.

69. The array according to claim 67, 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.

70. The array according to claim 61, wherein said protein-containing mixture further comprises a helper reagent.

71. The array according to claim 69, 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.

72. The array according to claim 69, wherein said helper reagent is a polymer selected from the group consisting of:

DEAE-dextran, dextran, polylysine, and polyethylamine.

73. The array according to claim 69, wherein said helper reagent is a cell adherent-enhancing protein selected from the group consisting of:

a fibronectin and gelatin.

74. The array according to claim 69, wherein said helper reagent is either polyethylene glycol or acrylamide.

75. The array according to claim 69, wherein said helper reagent is a RGD peptide selected from the group consisting of:

Arg-Gly-Asp-Ser, Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro, and the like.

76. The array according to claim 61, wherein said surface is that of a microtiter plate well-bottom, a microcolumn, a slide, a strip.

77. The array according to claim 61, wherein said surface is made of a ceramic, glass, metal, polymer or co-polymer, crystalline, or conductive material, or any combinations thereof.

78. The array according to claim 76, wherein said surface may have a coating of any one of said materials on another.

79. The array according to claim 61, wherein said defined locations comprise an array of microspots on said substrate surface.

80. The array according to claim 61, wherein said defined locations each contains a different protein.

81. The array according to claim 79, wherein each different protein is included on more than one separate microspot on the array.

82. The array according to claim 80, wherein said microspots on the array comprises the same protein of interest but with different point mutations.

83. The array according to claim 79, wherein the different proteins are functionally related.

84. The array according to claim 61, wherein said array is a thematic microarray.

86. The array according to claim 84, wherein said protein synthesizing step is performed as a cell-free reaction.

87. The array according to claim 84, wherein said protein synthesizing step uses coupled transcription and translation processes directly from corresponding DNA templates.

88. The array according to claim 84, wherein said DNA templates include a tag moiety.

89. The array according to claim 84, wherein said DNA templates represent full-length gene clones or gene fragments.

90. The array according to claim 84, wherein said DNA templates is produced either by PCR or RT-PCR using oligonucleotide primers.

91. The array according to claim 84, wherein said method further comprises cleaving at a protein cleavage site in a region of said tag moiety to release said synthesized proteins from said surface for entry into said cells.

92. The array according to claim 84, wherein said array is a thematic array.

93. The array according to claim 84, wherein the carrier reagent is a naturally occurring or synthetic translocation peptide.

94. The array according to claim 84, 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 targeted ligand that can bind specifically to and activate cell surface receptors.

95. The array according to claim 84, 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.

96. The array according to claim 84, wherein said protein-containing mixture further comprises a helper reagent.

97. The array according to claim 95, 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.

98. The array according to claim 95, wherein said helper reagent is a polymer selected from the group consisting of:

DEAE-dextran, dextran, polylysine, and polyethylamine.

99. The array according to claim 95, wherein said helper reagent is a cell adherent-enhancing protein selected from the group consisting of:

a fibronectin and gelatin.

100. The array according to claim 95, wherein said helper reagent is either polyethylene glycol or acrylamide.

101. The array according to claim 95, wherein said helper reagent is a RGD peptide selected from the group consisting of:

Arg-Gly-Asp-Ser, Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro, and the like.

102. The array according to claim 84, wherein said surface is that of a microtiter plate well-bottom, a microcolumn, a slide, a strip, a bead, a particle, or nanoparticle.

103. The array according to claim 84, wherein said surface is made of a ceramic, glass, metal, polymer or co-polymer, crystalline, or conductive material, or any combinations thereof.

85. A transfected-cell cluster array made according to a surface-mediated protein delivery method, the method comprising:

providing DNA templates on a substrate surface;

synthesizing, in situ on said surface, proteins from said DNA templates;

affixing said proteins to said surface;

reacting said synthesized proteins with a carrier reagent;

adding cells to said surface; and

transporting said proteins into said cells.

104. A method of delivering a protein-of-interest into primary cell lines, the method comprises:

providing a first population of cells;

introducing into said first population of cells a DNA-construct of said protein-of interest;

despositing said first population of cells onto a surface;

either lysing in situ said first population of cells, or allowing said protein-of-interest to be excreted by said first population of cells;

capturing said protein-of-interest on said surface;

reacting said protein-of-interest with a carrier reagent;

depositing a second population of cells over said protein-of-interest; and

, transporting said protein-of-interest into said second population of cells.