Transfection reagents

• US 7,601,872 B2
• Filed: 01/21/2005
• Issued: 10/13/2009
• Est. Priority Date: 11/12/1998
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Gebeyehu, Gulilat, Chu, Yongliang, Masoud, Malek
• Primary Examiner(s): Davis; Brian J
• Application Number:
  US11/040,662
• Publication Number:
  US 20050164972A1
• Time in Patent Office: 1,726 Days
• Field of Search: None
• US Class Current: 564/295
• 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)l, 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 method for introducing a nucleic acid into a cell, comprising the steps of contacting the nucleic acid with a polycation, thereby forming a complex;

and incubating the complex with the cell, wherein the polycation has the formula;

2. The method of claim 1, wherein Y is CH₂.

3. The method of claim 1, wherein D is a bond.

4. The method of claim 1, wherein p and k independently are integers from 1 to 25, and i and j independently are integers from 0 to 25.

5. The method of claim 1, wherein p and k independently are integers from 1 to 10, and i and j independently are integers from 0 to 10.

6. The method of claim 1, wherein p and k independently are integers from 1 to 4, i and j independently are integers from 0 to 4, and q is 1.

7. The method of claim 2, wherein R₂ and R₅ are (CH₂)_p-D-Z, wherein (CH₂) )_p is substituted with an —

OH.

8. The method of claim 7, wherein p and k independently are integers from 1 to 4, i and j independently are integers from 0 to 4, and q is 1.

9. The method of claim 7, wherein Z is a diaminoalkyl optionally substituted with —

OH.

10. The method of claim 7, wherein D is a bond.

11. The method of claim 1, wherein R₁, R₃, R₁, and R₆, independent of one another, are selected from H or an alkyl group.

12. The method of claim 1, wherein R₂ and R₅ are an amino alcohol group.

13. The method of claim 1, wherein R₂ and R₅ are independently an aminoalcohol selected from aminoethanol, aminopropanol, or aminobutanol.

14. The method of claim 13, wherein the amino alcohol group has the structure

15. The method of claim 1, wherein the nucleic acid is DNA.

16. The method of claim 1, wherein the nucleic acid is RNA.

17. The method of claim 1, wherein the nucleic acid inhibits expression of nucleic acids in the cell.

18. The method of claim 1, wherein the nucleic acid catalyzes reactions.

19. The method of claim 1, wherein the complex formed is a liposome.

20. The method of claim 1, wherein the complex is a liposome and wherein the method further comprises the step of forming liposomes after contacting the nucleic acid with the polycation.

21. The method of claim 1, wherein a mixture of the polycation and a neutral lipid is formed and the nucleic acid is contacted with the mixture to form the complex.

22. The method of claim 1, wherein a mixture of the polycation and a neutral lipid selected from DOPE, DOPC or cholesterol is formed and the nucleic acid is contacted with the mixture to form the complex.

23. The method of claim 1, wherein a mixture of the nucleic acid and a peptide or a protein is formed and the polycation is contacted with the mixture to form the complex.

38. The method of claim 8, wherein Z is a diaminoalkyl optionally substituted with —

OH.

40. The method of claim 8, wherein i is 0 and j is 1.

41. The method of claim 40, wherein k is 1.

42. The method of claim 41, wherein p is 3.

46. The method of claim 6, wherein k is 1.

24. A method of making a compound having the structure

25. The method of claim 24. wherein epoxyalkyl phthalimide reagent is epoxypropyl phthalimide and the reducing agent is hydrazine.

26. A compound for introducing a nucleic acid into a cell having the formula

27. The compound of claim 26, wherein Y is CH₂.

28. The compound of claim 26, wherein D is a bond.

29. The compound of claim 26, wherein p and k independently are integers from 1 to 4, i and j independently are integers from 0 to 4, and q is 1.

30. The compound of claim 27, wherein R₂ and R₅ are (CH₂)_p-D-Z, wherein (CH₂)_p is substituted with an —

OH.

31. The compound of claim 30, wherein p and k independently are integers from 1 to 4, i and j independently are integers from 0 to 4, and q is 1.

32. The compound of claim 30, wherein Z is a diaminoalkyl optionally substituted with —

OH.

33. The compound of claim 30, wherein D is a bond.

34. The compound of claim 26, wherein R₁, R₃, R₄, and R₆, independent of one another, are selected from H or an alkyl group.

35. The compound of claim 26, wherein R₂ and R₅ are an amino alcohol group.

36. The compound of claim 35, wherein R₂ and R₅ are independently an aminoalcohol group selected from aminoethanol, aminopropanol, or aminobutanol.

37. The compound of claim 35, wherein the amino alcohol group has the structure

39. The compound of claim 31, wherein Z is a diaminoalkyl optionally substituted with —

OH.

43. The compound of claim 31, wherein i is 0 and j is 1.

44. The compound of claim 43, wherein k is 1.

45. The compound of claim 44, wherein p is 3.

47. The compound of claim 29, wherein k is 1.