Solid surface with immobilized degradable cationic polymer for transfecting eukaryotic cells

• US 20060134790A1
• Filed: 12/14/2005
• Published: 06/22/2006
• Est. Priority Date: 01/13/2003
• Status: Abandoned Application

• Current Assignee: Chris P. Castello, Lei Yu, Yasunobu Tanaka
• Original Assignee: Chris P. Castello, Lei Yu, Yasunobu Tanaka
• Inventors: Castello, Chris P., Yu, Lei, Tanaka, Yasunobu
• Application Number:
  US11/303,082
• Publication Number:
  US 20060134790A1
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C12M 23/20 Material Coatings immunocoatings C12M25/00
• CPC Class Codes: C12M 35/00 Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion machines for extracting juice from animal or plant tissue by electroplasmolysis A23N1/006, processes employing electric or wave energy B01J19/08; treatment of microorganisms or enzymes with electrical or wave energy C12N13/00; methods for cell fusion C12N15/02; introduction of foreign genetic material C12N15/87
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2533/30 Synthetic polymers
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06

Abstract

A cell transfection/culture device is disclosed which includes a solid support coated with a degradable polymer cation as a transfection reagent. The transfection/culture device is conveniently stored at room temperature until use. Cell transfection is accomplished easily by adding the nucleic acid of interest and the cells to be transfected to the transfection/culture device. Cell transfection is completed in less than one hour by using the transfection/culture device described herein.

Claims

1. A device comprising a solid support coated with a composition comprising a transfection reagent which is not complexed to a biomolecule.

2. The device of claim 1, wherein the solid support is selected from the group consisting of polystyrene resin, epoxy resin and glass.

3. The device of claim 1, wherein the coating is on the surface of the solid support.

4. The device of claim 3, wherein the coating amount of the transfection reagent is from about 0.1 to about 100 μ

g/cm².

5. The device of claim 1, wherein the transfection agent is a polymer.

6. The device of claim 5, wherein the polymer is a cationic polymer.

7. The device of claim 1, wherein the transfection agent comprises a degradable cationic polymer.

8. The device of claim 7, wherein the degradable cationic polymer comprises cationic compounds or oligomers linked together by one or more degradable linkers.

9. The device of claim 7, wherein the transfection agent further comprises a non-degradable cationic polymer.

10. The device of claim 9, wherein the ratio of the non-degradable cationic polymer to the degradable cationic polymer is from 1:

0.5 to 1;

20 by weight.

11. The device of claim 1, wherein the transfection reagent comprises a plurality of cationic molecules and at least one degradable linker molecule connecting said cationic molecules in a branched arrangement, wherein said cationic molecules are selected from the group consisting of:

(i) a cationic compound of formula (A) or (B) or a combination thereof;

wherein R¹ is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, another Formula A, or Formula B;

R² is a straight chain alkylene group of the formula;

—

(CH₂)_a—

wherein a is an integer number from 2 to 10;

R³ is a straight chain alkylene group of the formula;

—

(C_bH_2b)—

wherein b is an integer number from 2 to 10;

R⁴ is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, another Formula A, or Formula B;

R⁵ is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, another Formula A, or Formula B;

R⁶ is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, Formula A, or another Formula B;

R⁷ is a straight chain alkylene group of the formula;

—

(C_cH_2c)—

in which c is an integer number from 2 to 10; and

R⁸ is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, Formula A, or another Formula B;

(ii) a cationic dendritic or branched polyamidoamine (PAMAM) with terminated primary or secondary amino groups;

(iii) a cationic polyamino acid; and

(iv) a cationic polycarbohydrate;

and wherein said degradable linker molecule is represented by the formula;

A(Z)_d

wherein A is a spacer molecule having at least one degradable bond, Z is a reactive residue which reacts with amino group, and d is an integer equal to or more than two and wherein A and Z are bound covalently.

12. The device of claim 8, wherein the cationic compound or oligomer is selected from the group consisting of poly(L-lysine) (PLL), polyethyleneimine (PEI), polypropyleneimine (PPI), pentaethyleneamine, N,N′

-bis(2-aminoethyl)-1,3-propanediamine, N,N′

-bis(2-aminopropyl)-ethylenediamine, spermine, spermidine, N-(2-aminoethyl)- 1,3-propanediamine, N-(3-aminopropyl)-1,3-propanediamine, tri(2-aminoethyl)amine, 1,4-bis(3-aminopropyl)piperazine, N-(2-aminoethyl)piperazine, dendritic polyamidoanine (PAMAM), chitosan, and poly(2-dimethylamino)ethyl methacrylate (PDMAEMA).

13. The device of claim 8, wherein the linker molecule is selected from the group consisting of di- and multi-acrylates, di- and multi-acrylamides, di- and multi-isothiocyanates, di- and multi-isocyanates, di- and multi-epoxides, di- and multi-aldehydes, di-and multi-acyl chlorides, di- and multi-sulfonyl chlorides, di- and multi-halides, di- and multi-anhydrides, di- and multi-maleimides, di- and multi-N-hydroxysuccinimide esters, di- and multi-carboxylic acids, and di-and multi-a-haloacetyl groups.

14. The device of claim 8, wherein the linker molecule is selected from the group consisting of 1,3-butanediol diacrylate, 1,4-butanediol diacrylate, 1,6-hexanediol diacrylate, 2,4-pentanediol diacrylate, 2-methyl-2,4-pentanediol diacrylate, 2,5-dimethyl-2,5-hexanediol diacrylate, poly(ethylene glycol) diacrylate, trimethylolpropane triacrylate, pentaerythritol tetraacrylate, di(trimethylolpropane) tetraacrylate, dipentaerythritol pentaacrylate, and a polyester with at least three acrylate or acrylamide side groups.

15. The device of claim 8, wherein the molecular weight of the polymer is from 500 da to 1,000,000 da.

16. The device of claim 8, wherein the molecular weight of the polymer is from 2000 da to 200,000 da.

17. The device of claim 8, wherein the molecular weight of the cationic compound or oligomer is from 50 da to 10,000 da.

18. The device of claim 8, wherein the molecular weight of the linker molecule is from 100 da to 40,000 da.

19. The device of claim 1 wherein the solid support is a dish bottom, a multi-well plate, or a continuous surface.

20. The device of claim 1, which can be stored at room temperature for at least 5 months without significant loss of transfection activity.

21. A method of cell transfection comprising:

adding a solution comprising a nucleic acid to be transfected to the device of claim 1;

adding eukaryotic cells to the device; and

incubating the cells and the nucleic acid solution to allow cell transfection.

22. The method of claim 21, wherein the incubation is for 5 min. to 3 hours.

23. The method of claim 21, wherein the incubation is for 10 min. to 90 min.

24. The method of claim 21, wherein the nucleic acid is selected from the group consisting of DNA, RNA, DNA/RNA hybrid and chemically-modified nucleic acid.

25. The method of claim 24, wherein the DNA is circular (plasmid), linear, fragment or single strand oligonucleotide (ODN).

26. The method of claim 24, wherein the RNA is single strand (ribozyme) or double strand (siRNA).

27. The method of claim 21, wherein the cell is a mammalian cell.

28. The method of claim 21, wherein at least some of the cells undergo cell division.

29. The method of claim 21, wherein the cell is a transformed or primary cell.

30. The method of claim 21, wherein the cell is a somatic or stem cell.

31. The method of claim 21, wherein the cell is a plant cell.