Transfection reagents

• US 8,158,827 B2
• Filed: 01/14/2009
• Issued: 04/17/2012
• Est. Priority Date: 11/12/1998
• Status: Expired due to Term

• 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:
  US12/353,371
• Publication Number:
  US 20090143583A1
• Time in Patent Office: 1,189 Days
• Field of Search: 564/286
• US Class Current: 564/152
• 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 lipid aggregate comprising a compound or polycation having the formula:

2. The lipid aggregate of claim 1, wherein at least two of R₁, R₃, R₄ and R₆ are a straight-chain, branched, or cyclic alkyl, alkenyl, alkynyl or aryl groups having from about 6 to about 64 carbon atoms.

3. The lipid aggregate of claim 1, wherein at least two of R₁, R₃, R₄ and R₆ are a straight-chain, branched, or cyclic alkyl, alkenyl, alkynyl or aryl groups having from about 8 to about 24 carbon atoms.

4. The lipid aggregate of claim 1, whereinp

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;

i, j, k, p and t are integers from 1 to about 100;

1 is an integer from 3 to about 100; and

q is an integer from 1 to about 1000.

5. The lipid aggregate of claim 1, where L is (CH₂)₁ or {(CH₂)_i—

Y—

(CH₂)_j}_k,pwherein 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, and a secondary amino group;

j and k are integers from 1 to 4 and 1 is 3 or 4.

6. The lipid aggregate of claim 1, wherein at least two of R₁-R₆ are —

(CH₂)_p-D-Z groups.

7. The lipid aggregate of claim 1, where L is (CH₂)₁, where 1 is 3 or 4.

8. The lipid aggregate of claim 1, where R₁ and R₄ are straight-chain alkenyl having 17 carbon atoms.

9. The lipid aggregate of claim 1, where R₂ and R₅ are —

(CH₂)_p-D-Z where p is 1-4, D is a bond and Z is amine.

10. The lipid aggregate of claim 1, where R₃ and R₆ are H.

11. The lipid aggregate of claim 1, where A₁ and A₂ are C(O).

13. The lipid aggregate of claim 1, further comprising at least one neutral lipid.

14. The lipid aggregate of claim 13, wherein the neutral lipid is DOPE, DOPC or cholesterol.

12. A compound or polycation having the formula: