Transfection reagents

• US 7,173,154 B2
• Filed: 07/28/2003
• Issued: 02/06/2007
• Est. Priority Date: 11/12/1998
• Status: Expired due to Term

• 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:
  US10/629,522
• Publication Number:
  US 20050260597A1
• Time in Patent Office: 1,289 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)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 compound having the formula:

2. A composition comprising the compound of claim 1 solubilized in aqueous medium.

3. The composition of claim 2 further comprising a neutral lipid.

4. The composition of claim 2 further comprising DOPE, DOPC or cholesterol.

5. A kit comprising a container means which comprises a compound having the formula:

6. The kit of claim 5 wherein the compound is solubilized in aqueous medium.

7. The kit of claim 6 wherein the aqueous medium further comprises a neutral lipid.

8. The kit of claim 6 wherein the aqueous medium further comprises DOPE, DOPC or cholesterol.

9. A method for introducing a nucleic acid into a cell, the method comprising the steps of:

contacting a compound having the formula;

10. The method of claim 9 wherein the contacting step and the incubating step are conducted in physiological medium.

11. A method for introducing a nucleic acid into a cell, the method comprising the steps of contacting a composition comprising a compound having the formula

12. The method of claim 11 wherein the contacting step and the incubating step are conducted in physiological medium.

13. The method of claim 11 wherein the composition comprises a neutral lipid when it contacts the nucleic acid.

14. The method of claim 11 wherein the composition comprises DOPE, DOPC, or cholesterol when it contacts the nucleic acid.

15. A lipid aggregate made by mixing a compound having the formula

16. The lipid aggregate of claim 15, wherein the neutral lipid is DOPE, DOPC or cholesterol.

17. The lipid aggregate of claim 15, wherein the lipid aggregate is 50 to 200 nm in diameter.

18. The lipid aggregate of claim 15, wherein the lipid aggregate is formed using extrusion, freeze-thaw, or sonication.

19. A method for making a lipid aggregate, comprising mixing a compound having the formula

20. The method of claim 19, wherein the lipid aggregate is made by mixing the compound with the neutral lipid DOPE, DOPC or cholesterol.

21. The method of claim 19, wherein the mixing is performed to obtain a lipid aggregate having a diameter of 50 to 200 nm.

22. The method of claim 19, wherein the lipid aggregate is formed using sonication, freeze-thaw, or extrusion.