Transfection reagents

• US 7,915,450 B2
• Filed: 12/15/2009
• Issued: 03/29/2011
• 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:
  US12/638,336
• Publication Number:
  US 20100159593A1
• Time in Patent Office: 469 Days
• Field of Search: 564/295
• 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 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 lo prepare liposomes and other lipid aggregates suitable for iransfcclion or delivery of compounds to target cells, cither 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 properly, 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 compound or polycation having the formula:

2. The compound or polycation of claim 1, wherein R₂ and R₅, independently of one another, are H, or an alkyl or alkyl ether substituted by one or more of an amide and an ester.

3. The compound or polycation of claim 1 wherein R₂ and R₅, independently of one another, are an alkyl or alkyl ether substituted by one or more of an amide and an ester.

4. The compound or polycation of claim 1 wherein R₂ and R₅, independently of one another, are H, or an alkyl or alkyl ether substituted by one or more of an amide.

5. The compound or polycation of claim 1 wherein R₂ and R₅, independently of one another, are H, or an alkyl or alkyl ether substituted by one or more of an ester.

9. A composition for transfecting a cell comprising one or more compounds or polycations of claim 1, and a neutral lipid.

10. The composition of claim 9 further comprising a nucleic acid.

11. The composition of claim 10 wherein the nucleic acid is RNA.

15. A kit comprising one or more compounds or polycations of claim 1 and one or more additional components selected from cells, lipid-aggregate forming compounds, and transfection enhancers.

16. A method for introducing a polyanion into a eukaryotic cell, wherein the method comprises:

providing a liposome comprising one or more compounds or polycations of claim 1;

contacting the liposome with the polyanion to form a polyanion-liposome complex; and

incubating the complex with the cell to thereby introduce the polyanion into the cell.

6. A compound or polycation having the formula:

12. A composition for transfecting a cell comprising one or more compounds or polycations of claim 6 and a neutral lipid.

17. A method for introducing a polyanion into a eukaryotic cell, wherein the method comprises:

providing a liposome comprising one or more compounds or polycations of claim 6;

contacting the liposome with the polyanion to form a polyanion-liposome complex; and

incubating the complex with the cell to thereby introduce the polyanion into the cell.

7. A compound or polycation having the formula:

13. A composition for transfecting a cell comprising one or more compounds or polycations of claim 7 and a neutral lipid.

18. A method for introducing a polyanion into a eukaryotic cell, wherein the method comprises:

providing a liposome comprising one or more compounds or polycations of claim 7;

contacting the liposome with the polyanion to form a polyanion-liposome complex; and

incubating the complex with the cell to thereby introduce the polyanion into the cell.

8. A compound or polycation having the formula:

14. A composition for transfecting a cell comprising one or more compounds or polycations of claim 8 and a neutral lipid.

19. A method for introducing a polyanion into a eukaryotic cell, wherein the method comprises:

providing a liposome comprising one or more compounds or polycations of claim 8;

contacting the liposome with the polyanion to form a polyanion-liposome complex; and

incubating the complex with the cell to thereby introduce the polyanion into the cell.