Cationic liposomes and methods of use
Highly efficient cationic liposomes are provided as a system for the delivery to cells of agents or compounds, such as, compounds capable of silencing a target protein and enzyme stubstrates. The cationic liposomes can be used in methods of detecting the inhibition activity or apparent activity of a target protein in a cell, and methods of identifying a protein associated with a pathway, such as, a signal transduction pathway in a cell.
1 . A cationic liposome comprising a charge neutral compound and/or a charge neutral mixture of compounds and a cationic phospholipid wherein the molar ratio of charge neutral compound and/or a charge neutral mixture of compounds to cationic phospholipid is greater than about 1:1.
2 - 5 . (canceled)
6 . The liposome of claim 1 in which the cationic phospholipid is a compound of structural Formula (I):
or salts, solvates or hydrates thereof wherein:
each R 1 is independently hydrogen, alkyl or acyl;
n is 0 or 1;
R 2 is hydrogen, —CH 2 CH 2 N(CH 3 ) 3 , —CH 2 CH 2 NH 3 , —CH 2 CH(CO 2 —)NH 3 + , —CH 2 CH(OH)CH 2 OH or
each R 2 is independently hydrogen or —PO 3 H and
with the proviso that at least one R 1 is not hydrogen and is (C 10 -C 30 )alkyl or (C 10 -C 30 )acyl.
7 . The liposome of claim 6 in which n is 1 and R 2 is hydrogen, —CH 2 CH 2 N(CH 3 ) 3 , —CH 2 CH 2 NH 3 , —CH 2 CH(CO 2 —)NH 3 + , or —CH 2 CH(OH)CH 2 OH.
8 - 12 . (canceled)
13 . The liposome of claim 6 in which the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine.
14 - 15 . (canceled)
16 . The liposome of claim 1 in which the cationic phospholipid is a compound of structural formula (II):
or salts, solvates or hydrates thereof wherein:
each R 1 is independently hydrogen, alkyl or acyl;
n is 0 or 1;
R 2 is —CH 2 CH 2 N(CH 3 ) 3 , —CH 2 CH 2 NH 3 , and
R 4 is a protecting group;
with the proviso that at least one R 1 is not hydrogen and is (C 10 -C 30 )alkyl or (C 10 -C 30 )acyl.
17 . The liposome of claim 16 in which the protecting group is —R 5 , —CH 2 OC(O)R 5 or —CH 2 CH 2 SC(O)R 5 wherein R 5 is (C 1 -C 6 )alkyl.
18 - 25 . (canceled)
26 . The liposome of claim 16 in which the cationic phospholipid is 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine.
27 - 32 . (canceled)
33 . The liposome of claim 1 in which the charge neutral compound is 1,2-dioleoyl-sn-glycero-3-phosphocholine.
34 - 37 . (canceled)
38 . A method of delivering an agent to a cell said method comprising contacting the cell with the cationic liposome of claim 1 and the agent.
39 . A liposome comprising
(i) a compound capable of silencing a target protein and
(ii) an enzyme substrate capable of producing a detectable signal when modified by a readout protein, wherein said liposome is capable of delivering said compound and said enzyme substrate into a live cell.
40 - 49 . (canceled)
50 . The liposome of claim 39 wherein said target protein is an enzyme.
51 - 62 . (canceled)
63 . The liposome of claim 39 which comprises a 1,2-diacyl-sn-glycero-3-alkylphosphocholine.
64 . The liposome of claim 63 wherein said 1,2-diacyl-sn-glycero-3-alkylphosphocholine has the formula:
wherein:
R 1 is a saturated or unsaturated alkyl having from 6 to 30 carbon atoms;
R 2 is a saturated or unsaturated alkyl having from 6 to 30 carbon atoms;
R 3 is a saturated or unsaturated alkyl having from 1 to 20 carbon atoms.
65 - 67 . (canceled)
68 . A method of detecting the inhibition of expression of a target protein in a live cell, comprising:
contacting said cell with a liposome comprising (i) a compound capable of silencing a target protein (ii) an enzyme substrate capable of producing a detectable signal when modified by a readout protein, wherein said liposome is capable of delivering said compound and said enzyme substrate said cell; and
detecting whether a detectable signal is produced, wherein a change of detectable signal indicates an inhibition of expression of said target protein in said cell.
69 - 70 . (canceled)
71 . A method of identifying a target protein associated with a signal transduction pathway of interest in a live cell, comprising:
contacting said cell with a liposome comprising (i) a compound capable of silencing a target protein (ii) an enzyme substrate capable of producing a detectable signal when modified by a readout protein, wherein said liposome is capable of delivering said compound and said enzyme substrate into said cell; and
contacting said cell with an agonist of said signal transduction pathway; and
detecting whether a detectable signal is produced, wherein a change of detectable signal indicates said target protein is associated with said signal transduction pathway of interest.
72 - 84 . (canceled)
85 . The method of claim 71 wherein said target protein is an enzyme.
86 - 97 . (canceled)
98 . The liposome of claim 71 wherein said liposome is comprises a 1,2-diacyl-sn-glycero-3-alkylphosphocholine.
99 . The method of claim 98 wherein said 1,2-diacyl-sn-glycero-3-alkylphosphocholine has the formula:
wherein:
R 1 is a saturated or unsaturated alkyl having from 6 to 30 carbon atoms;
R 2 is a saturated or unsaturated alkyl having from 6 to 30 carbon atoms;
R 3 is a saturated or unsaturated alkyl having from 1 to 20 carbon atoms.
100 - 102 . (canceled)
103 . A kit for use in detecting the inhibition of expression of a target protein in a cell, comprising:
a lipid capable of forming a cationic liposome comprising compound capable of silencing a target protein and an enzyme substrate capable of producing a detectable signal when modified by an readout protein; and
instructions to generate a liposome capable of delivering said compound and said enzyme substrate into a live cell.
104 . A kit for use in delivering an agent to a cell, said kit comprising:
a charge neutral compound and/or a charge neutral mixture of compounds and a cationic phospholipid; and
instructions to generate a cationic liposome which delivers the agent to the cell.