Transfection reagents
View Patent ↗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, (CH 2 )l, or {(CH 2 )i-Y—(CH 2 )j}k, wherein Y is selected from the group consisting of CH 2 , 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 -X 1 -L′-X 2 -Z or -Z; R 1 -R 6 , independently of one another, are selected from the group consisting of H, —(CH 2 )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 R 1 , R 3 , R 4 and R 6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group; and anyone of R 1 , R 3 , R 4 and/or R 6 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; X 1 and X 2 , 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; A 1 and A 2 , independently of one another, are selected from the group consisting of CH 2 O, CH 2 S, CH 2 NH, C(O), C{NH), C(S) and (CH 2 )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.
1. A cationic compound having the formula:
wherein:
L is {(CH 2 ) i -Y—(CH 2 ) j } k , wherein Y is selected from the group consisting of CH 2 , 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 or a secondary amino group, and wherein a carbon of (CH 2 ) i or a carbon of (CH 2 ) j is optionally substituted with —OH;
R 1 , R 3 , R 4 and R 6 are, independently, H or straight -chain, branched or cyclic alkyl or alkenyl groups having from 8 to 40 carbon atoms;
R 2 is an alkyl substituted by one or more amines and optionally substituted by one or more alcohols;
R 5 is an alkyl substituted by one or more amines and one or more alcohols;
X a is a physiologically acceptable anion; and
a is the number of positive charges divided by the valence of the anion;
i and j are, independently, an integer from 0 to 100, and
is an integer from 1 to 25.
2. The cationic compound of claim 1 , wherein R 1 is a straight-chain alkyl group having from 8 to 24 carbon atoms, and R 4 is H.
3. The cationic compound of claim 1 , wherein at least one carbon of (CH2) i or (CH 2 ) j is substituted with —OH.
4. The cationic compound of claim 1 , wherein Y is CH 2 .
5. The cationic compound of claim 4 , wherein i and j are independently an integer from 0 to 4, and k is an integer from 1 to 4.
6. The cationic compound of claim 5 , wherein i, j and k are 1.
7. The cationic compound of claim 6 , wherein R 2 is an alkyl substituted by one or more amines, and R 5 is an alkyl substituted by one or more amines and one or more alcohols.
8. The cationic compound of claim 7 , wherein R 1 is a straight-chain alkyl group having from 8 to 24 carbon atoms, and R 4 is H.
9. The cationic compound of claim 8 , wherein R 3 and R 6 are H.
10. The cationic compound of claim 4 , wherein at least one carbon of (CH 2 ) i or (CH 2 ) j is substituted with —OH.
11. A composition comprising one or more cationic compounds having the formula:
wherein:
L is {(CH 2 ) i -Y—CH 2 ) j } k , wherein Y is selected from the group consisting of CH 2 , 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 or a secondary amino group, and wherein a carbon of (CH 2 ) i or a carbon of (CH 2 ) j is optionally substituted with —OH;
R 1 , R 3 , R 4 and R 6 are, independently, H or straight-chain, branched or cyclic alkyl or alkenyl groups having from 8 to 40 carbon atoms;
R 2 is an alkyl substituted by one or more amines and optionally substituted by one or more alcohols;
R 5 is an alkyl substituted by one or more amines and one or more alcohols;
X a is a physiologically acceptable anion; and
a is the number of positive charges divided by the valence of the anion;
i and j are, independently, an integer from 0 to 100, and
k is an integer from 1 to 25,
and wherein the one or more cationic compounds are solubilized in aqueous medium.
12. The composition of claim 11 , further comprising a neutral lipid.
13. The composition of claim 12 , wherein the neutral lipid is DOPE, DOPC or cholesterol.
14. The composition of claim 11 , wherein the one or more compounds and the neutral lipid are formed into liposomes.
15. The composition of claim 11 , further comprising a transfection enhancer.
16. A composition for transfecting a cell which comprises one or more nucleic acids and one or more compounds having the formula:
wherein:
L is {(CH 2 ) i -Y—(CH 2 ) j } k , wherein Y is selected from the group consisting of CH 2 , 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 or a secondary amino group, and wherein a carbon of (CH 2 ) i or a carbon of (CH 2 ) j is optionally substituted with —OH;
R 1 , R 3 , R 4 and R 6 are, independently, H or straight -chain, branched or cyclic alkyl or alkenyl groups having from 8 to 40 carbon atoms;
R 2 is an alkyl substituted by one or more amines and optionally substituted by one or more alcohols;
R 5 is an alkyl substituted by one or more amines and one or more alcohols;
X a is a physiologically acceptable anion; and
a is the number of positive charges divided by the valence of the anion;
i and j are, independently, an integer from 0 to 100, and
k is an integer from 1 to 25.
17. The composition of claim 16 , wherein the nucleic acid is RNA.
18. The composition of claim 16 , wherein the nucleic acid is DNA.
19. The composition of claim 16 , further comprising a neutral lipid.
20. The composition of claim 19 , wherein the neutral lipid is DOPE, DOPC or cholesterol.
21. The composition of claim 16 , wherein the one or more compounds is formed into a liposome.
22. The composition of claim 16 , further comprising a transfection enhancer.
23. A lipid aggregate made by mixing a nucleic acid with one or more compounds having the formula:
wherein:
L is {(CH 2 ) i -Y—(CH 2 ) j } k , wherein Y is selected from the group consisting of CH 2 , 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 or a secondary amino group, and wherein a carbon of (CH 2 ) i or a carbon of (CH 2 ) j is optionally substituted with —OH;
R 1 , R 3 , R 4 and R 6 are, independently, H or straight -chain, branched or cyclic alkyl or alkenyl groups having from 8 to 40 carbon atoms;
R 2 is an alkyl substituted by one or more amines and optionally substituted by one or more alcohols;
R 5 is an alkyl substituted by one or more amines and one or more alcohols;
X a is a physiologically acceptable anion; and
a is the number of positive charges divided by the valence of the anion;
i and j are, independently, an integer from 0 to 100, and
k is an integer from 1 to 25,
wherein the nucleic acid and the one or more compounds are mixed for at least 15 minutes to form a complex.
24. The lipid aggregate of claim 23 , further comprising a neutral lipid.
25. The lipid aggregate of claim 24 , wherein the neutral lipid is DOPE, DOPC or cholesterol.
26. The lipid aggregate of claim 23 , wherein the lipid aggregate is 50 to 200 nm in diameter.
27. The lipid aggregate of claim 23 , wherein the lipid aggregate is formed using extrusion, freeze-thaw, or sonication.
28. A kit comprising one or more container means and optionally further comprising one or more additional components selected from the group consisting of a cell, cells, a cell culture media, a nucleic acid and instructions for transfecting a cell or cells wherein said container means comprises one or more cationic compounds having the formula:
wherein:
L is {(CH 2 ) i -Y—CH 2 ) j } k , wherein Y is selected from the group consisting of CH 2 , 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 or a secondary amino group, and wherein a carbon of (CH 2 ) i or a carbon of (CH 2 ) j is optionally substituted with —OH;
R 1 , R 3 , R 4 and R 6 are, independently, H or straight -chain, branched or cyclic alkyl or alkenyl groups having from 8 to 40 carbon atoms;
R 2 is an alkyl substituted by one or more amines and optionally substituted by one or more alcohols;
R 5 is an alkyl substituted by one or more amines and one or more alcohols;
X a is a physiologically acceptable anion; and
a is the number of positive charges divided by the valence of the anion;
i and j are, independently, an integer from 0 to 100, and
k is an integer from 1 to 25.
29. The kit of claim 28 , wherein the one or more cationic compounds are solubilized in aqueous medium.
30. The kit of claim 28 , wherein the one or more cationic compounds are solubilized in aqueous medium.
31. The kit of claim 28 , wherein the one or more cationic compounds are combined with one or more neutral lipids.
32. The kit of claim 31 , wherein the neutral lipids are selected from DOPE, DOPC or cholesterol.
33. The kit of claim 28 , wherein the one or more cationic compounds are formed into liposomes.
34. A method for introducing a polyanion into a cell or cells, wherein the method comprises the steps of:
(a) forming a liposome from one or more cationic compounds having the formula:
wherein:
L is {(CH 2 ) i -Y—(CH 2 ) j } k , wherein Y is selected from the group consisting of CH 2 , 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 or a secondary amino group, and wherein a carbon of (CH 2 ) i or a carbon of (CH 2 ) j is optionally substituted with —OH;
R 1 , R 3 , R 4 and R 6 are, independently, H or straight-chain, branched or cyclic alkyl or alkenyl groups having from 8 to 40 carbon atoms;
R 2 is an alkyl substituted by one or more amines and optionally substituted by one or more alcohols;
R 5 is an alkyl substituted by one or more amines and one or more alcohols;
X a is a physiologically acceptable anion; and
a is the number of positive charges divided by the valence of the anion;
i and j are, independently, an integer from 0 to 100, and
k is an integer from 1 to 25;
(b) contacting the liposome with the polyanion to form a positively-charged polyanion-liposome complex; and
(c) incubating the complex with a cell or cells to thereby introduce the polyanion into the cell or cells.
35. The method of claim 34 , wherein the polyanion is a nucleic acid.
36. The method of claim 34 , wherein the nucleic acid is RNA.
37. The method of claim 34 , wherein the nucleic acid is DNA.
38. The method of claim 34 , wherein the nucleic acid inhibits expression of nucleic acids in the cell or cells.
39. The method of claim 34 , wherein the cell or cells are selected from primary cells, tumor cells or cells of immortalized cell lines.
40. The method of claim 34 , wherein the cell or cells are selected from epithelial, fibroblastic, neuronal, or hematopoietic cells.
41. The method of claim 34 , wherein the cell or cells are those of an animal, fish, insect, fungus or plant.
42. The method of claim 34 , wherein the cell or cells are CHO-K1, COS-7 or HEK293 cells.
43. The method of claim 34 , wherein the liposome is formed by combining the one or more cationic compounds with a neutral lipid.
44. The method of claim 43 , wherein the neutral lipid is selected from DOPE, DOPC or cholesterol.