Transfection reagents

• US 8,785,200 B2
• Filed: 03/14/2012
• Issued: 07/22/2014
• 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: Chu, Yongliang, Masoud, Malek, Gebeyehu, Gulilat
• Primary Examiner(s): Davis, Brian J
• Application Number:
  US13/420,091
• Publication Number:
  US 20120238747A1
• Time in Patent Office: 860 Days
• Field of Search: None
• US Class Current: 435/458
• 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 cationic lipid compounds and compositions of lipid aggregates for delivery of macromolecules and other compounds into cells. The compounds can be used alone or in combination with other compounds to prepare liposomes and other lipid aggregates suitable for transfection or delivery of compounds to target cells, either in vitro or in vivo. The compounds are preferably polycationic and preferably form highly stable complexes with various anionic macromolecules, particularly polyanions such as nucleic acids. These compounds have the property, when dispersed in water, of forming lipid aggregates which associate strongly, via their cationic portion, with polyanions. Also disclosed are intermediates for preparing the compound and compositions of the invention and methods of using the compounds to introduce other compounds into cells.

Claims

1. A method for transfecting a target cell with an expressible nucleic acid, said method comprising:

a) obtaining a target cell;

b) forming a liposome complex comprising an expressible nucleic acid, at least one transfection enhancer, and a compound having the structure;

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

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

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

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

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

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

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

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

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

11. The method according to claim 1, wherein R₇ is H.

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

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

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

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

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

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

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

19. The method according to 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 method of claim 19, wherein the amine substituent of the R₅ alkyl group comprises an aminoethanol, an aminopropanol or an aminobutanol group.

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

22. The method according to 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 R₁ is H.

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

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

25. The method according to claim 1, wherein the liposome further comprises one or more neutral lipids.

26. The method according to claim 25, wherein the lipid is DOPE, DOPC or cholesterol.

27. The method according to claim 1, wherein the liposome complex is formed in an aqueous medium.

28. The method according to claim 1, wherein the transfection enhancer is a protein, a peptide or a growth factor that enhances cell-targeting, uptake, internalization, nuclear targeting or expression.