Transfection reagents

• US 7,470,817 B2
• Filed: 12/28/2006
• Issued: 12/30/2008
• Est. Priority Date: 11/12/1998
• Status: Expired due to Fees

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Gebeyehu, Gulilat, Chu, Yongliang, Masoud, Malek
• Primary Examiner(s): Davis; Brian J
• Application Number:
  US11/617,625
• Publication Number:
  US 20070202600A1
• Time in Patent Office: 733 Days
• Field of Search: None
• US Class Current: 564/506
• CPC Class Codes: A61K 31/14 Quaternary ammonium compounds, e.g. edrophonium, choline betaines A61K31/205
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 38/40 Transferrins, e.g. lactoferrins, ovotransferrins
• CPC Class Codes: A61K 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 9/16 Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction A61K9/20 takes precedence if the final form is a tablet; microspheres with drug-free outer coating, microcapsules A61K9/50; mixture of different granules, microcapsules, (coated) microparticles A61K9/5084; nanoparticles A61K9/51
• CPC Class Codes: C07C 211/21 Monoamines
• CPC Class Codes: C07C 211/63 having quaternised nitrogen atoms bound to acyclic carbon atoms
• CPC Class Codes: C07C 211/64 having quaternised nitrogen atoms bound to carbon atoms of six-membered aromatic rings
• CPC Class Codes: C07C 229/26 having more than one amino group bound to the carbon skeleton, e.g. lysine
• CPC Class Codes: C07D 209/48 with oxygen atoms in positions 1 and 3, e.g. phthalimide
• CPC Class Codes: C07D 241/12 with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Disclosed are compounds capable of facilitating transport of biologically active agents or substances into cells having the general structure:

wherein

Q is selected from the group consisting of N, O and S; L is any bivalent organic radical capable of linking each Q, such as C, CH, (CH₂)I, or {(CH₂)i-Y—(CH₂)j}k, wherein Y is selected from the group consisting of CH₂, an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine, a carbonyl, and a secondary amino group and wherein Y is optionally substituted by —X₁-L′-X₂-Z or -Z; R₁-R₆, independently of one another, are selected from the group consisting of H, —(CH₂)p-D-Z, an alkyl, an alkenyl, an aryl, and an alkyl or alkyl ether optionally substituted by one or more of an alcohol, an aminoalcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, a guanidyl, or a carbamoyl group, and wherein at least one of R₁, R₃, R₄ and R₆ is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group; and anyone of R₁, R₃, R₄ and/or R₆ may optionally be covalently linked with each other, with Y or with L when L is C or CH to form a cyclic moiety; Z is selected from the group consisting of amine, spermiyl, carboxyspermiyl, guanidyl, spermidinyl, putricinyl, diaminoalkyl, pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, and protein; X₁ and X₂, independently of one another, are selected from the group consisting of NH, O, S, alkylene, and arylene; L′ is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, alkylene ether, and polyether; D is Q or a bond; A₁ and A₂, independently of one another, are selected from the group consisting of CH₂O, CH₂S, CH₂NH, C(O), C{NH), C(S) and (CH₂)t; X is a physiologically acceptable anion; m, n, r, s, u, v, w and y are 0 or 1, with the proviso that when both m and n are 0 at least one of r, s, u and y is other than 0; i, j, k, l, p and are integers from 0 to about 100; q is an integer from 1 to about 1000; and a is the number of positive charge divided by the valence of the anion.

Claims

1. A compound having the formula:

2. The compound of claim 1, wherein R₅ is an alkyl group substituted with one amine and one or more alcohols.

3. The compound of claim 2, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

4. The compound of claim 3, wherein the amine substituent of the R₅ alkyl group has the structure:

5. The compound of claim 1, wherein R₅ is an alkyl group having 3-30 carbon atoms which is substituted by one amine and one or more alcohols.

6. The compound of claim 5, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

7. The compound of claim 6, wherein the amine substituent of the R₅ alkyl group has the structure:

8. The compound of claim 5, wherein R₅ is a propyl or a butyl group which is substituted by one amine and one or more alcohols.

9. The compound of claim 8, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

10. The compound of claim 9, wherein the amine substituent of the R₅ alkyl group has the structure:

11. The compound of claim 1, wherein R₇ is H.

12. The compound of claim 11, wherein R₄ is a straight-chain alkyl group having 8 to 24 carbon atoms.

13. The compound of claim 12, wherein R₅ is an alkyl group having 3-30 carbon atoms which is substituted with one amine and one or more alcohols.

14. The compound of claim 13, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

15. The compound of claim 14, wherein the amine substituent of the R₅ alkyl group has the structure:

16. The compound of claim 13, wherein R₅ is a propyl or a butyl group which is substituted by one amine and one or more alcohols.

17. The compound of claim 16, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

18. The compound of claim 17, wherein the amine substituent of the R₅ alkyl group has the structure:

19. The compound of claim 1, wherein R₄ is a straight-chain alkyl group having 8 to 24 carbon atoms, R₅ is a butyl group substituted with one amine and one or more alcohols, and R₇ is H.

20. The compound of claim 19, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

21. The compound of claim 20, wherein the amine substituent of the R₅ alkyl group has the structure:

22. The compound of claim 1, wherein R₄ is a straight-chain alkyl group having 8 to 24 carbon atoms, R₅ is a propyl group substituted with one amine and one alcohol, and R₇ is H.

23. The compound of claim 22, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

24. The compound of claim 23, wherein the amine substituent of the R₅ alkyl group has the structure:

25. The compound of claim 19, which is a polycation.

26. The compound of claim 1, which is a polycation.

27. A composition comprising one or more cationic compounds of claim 1 solubilized in aqueous medium.

28. The composition of claim 27, further comprising a neutral lipid.

29. The composition of claim 28, wherein the neutral lipid is DOPE, DOPC or cholesterol.

30. The composition of claim 28, wherein the one or more compounds and the neutral lipid are formed into liposomes.

31. The composition of claim 27, further comprising a transfection enhancer.

32. A composition for transfecting a cell which comprises one or more compounds of claim 1 and one or more nucleic acids.

33. The composition of claim 32, wherein the nucleic acid is RNA.

34. The composition of claim 32, further comprising a neutral lipid.

35. The composition of claim 34, wherein the one or more compounds and the neutral lipid are formed into liposomes.

36. The composition of claim 32, further comprising a transfection enhancer.

37. A lipid aggregate made by mixing one or more compounds of claim 1 with a nucleic acid for at least 15 minutes to form a complex.

38. The lipid aggregate of claim 37, further comprising a neutral lipid.

39. The lipid aggregate of claim 38, wherein the neutral lipid is DOPE, DOPC or cholesterol.

40. The lipid aggregate of claim 37, wherein the lipid aggregate is 50 to 200 nm in diameter.

41. The lipid aggregate of claim 37, wherein the lipid aggregate is formed using extrusion, freeze-thaw, or sonication.

42. A kit comprising one or more container means and optionally further comprising one or more additional components selected from the group consisting of a cell, cells, a cell culture media, a nucleic acid and instructions for transfecting a cell or cells wherein said container means comprises one or more cationic compounds of claim 1.

43. The kit of claim 42, wherein the one or more cationic compounds are solubilized in aqueous medium.

44. The kit of claim 42, wherein the one or more cationic compounds are combined with one or more neutral lipids.

45. The kit of claim 44, wherein the neutral lipids are selected from DOPE, DOPC and cholesterol.

46. The kit of claim 42, wherein the one or more cationic compounds are formed into liposomes.

47. A method for introducing a polyanion into a cell or cells, wherein the method comprises the steps of:

(a) forming a liposome from one or more compounds having the formula;

48. The method of claim 47, wherein R₅ is an alkyl group having 3-30 carbon atoms which is substituted with one amine and one or more alcohols.

49. The method of claim 48, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

50. The method of claim 49, wherein the amine substituent of the R₅ alkyl group has the structure:

51. The method of claim 47, wherein R₇ is H.

52. The method of claim 47, wherein the one or more compounds is a polycation.

53. The method of claim 47, wherein the polyanion is a nucleic acid.

54. The method of claim 53, wherein the nucleic acid is RNA.

55. The method of claim 53, wherein the nucleic acid inhibits expression of nucleic acids in the cell or cells.

56. The method of claim 47, wherein the cell or cells are selected from primary cells, tumor cells or cells of immortalized cell lines.

57. The method of claim 47, wherein the cell or cells are selected from epithelial, fibroblastic, neuronal, or hematopoietic cells.

58. The method of claim 47, wherein the cell or cells are those of an animal, fish, insect, fungus or plant.

59. The method of claim 47, wherein the cell or cells are CHO-K1, COS-7, or HEK293 cells.

60. The method of claim 47, wherein the liposome is formed by combining the one or more cationic compounds with a neutral lipid.

61. The method of claim 60, wherein the neutral lipid is selected from DOPE, DOPC or cholesterol.