METHODS AND COMPOSITIONS FOR THE DELIVERY OF BIOACTIVE COMPOUNDS
The present invention provides compositions comprising delivery systems comprising a lipid vehicle, a bioactive compound, and a polyanionic carrier macromolecule that is not a carrier polynucleotide, wherein the lipid vehicle encapsulates the polynucleotide of interest and the polyanionic carrier macromolecule, and wherein the delivery system is essentially free of carrier polynucleotides. Compositions also include delivery systems comprising a lipid vehicle and a bioactive agent, wherein the lipid vehicle encapsulates the bioactive agent, and wherein the lipid vehicle comprises a covalent bilayer or a core supported bilayer comprising polycationic lipids. Other compositions provided herein comprise a delivery system comprising a polypeptide of interest and a means for delivering the polypeptide into a cell. Also provided herein are methods for making the delivery systems, methods for delivering bioactive compounds, methods for treating a disease or unwanted condition in a subject with the delivery systems, and methods for detecting apoptosis in a cell.
1 . A delivery system comprising a cationic liposome, a bioactive compound, and a polyanionic carrier macromolecule that is not a carrier polynucleotide, wherein said cationic liposome encapsulates said bioactive compound and said polyanionic carrier macromolecule, wherein said cationic liposome comprises a lipid bilayer that comprises an inner leaflet and an outer leaflet, and wherein said delivery system is essentially free of carrier polynucleotides.
2 . (canceled)
3 . The delivery system of claim 1 , wherein said polyanionic carrier macromolecule comprises a glycosaminoglycan selected from the group consisting of heparin sulfate, hyaluronic acid, chondroitin sulfate, dermatin sulfate, keratan sulfate, and dextran sulfate.
4 - 22 . (canceled)
23 . The delivery system of claim 1 , wherein said outer leaflet comprises a lipid-PEG conjugate comprising a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol 2000 (DSPE-PEG 2000 ).
24 - 26 . (canceled)
27 . The delivery system of claim 23 , wherein said 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol 2000 (DSPE-PEG 2000 ) is conjugated to anisamide, thereby producing DSPE-PEG 2000 -AA.
28 - 30 . (canceled)
31 . The delivery system of claim 1 , wherein said bioactive compound comprises a polynucleotide of interest comprises a silencing element encoding an siRNA that targets a target polynucleotide selected from the group consisting of an oncogene and an epidermal growth factor receptor.
32 - 36 . (canceled)
37 . The delivery system of claim 1 , wherein said bioactive compound comprises a polypeptide of interest, and further wherein the polypeptide of interest comprises:
(i) the amino acid sequence set forth in one of SEQ ID NO: 3 and SEQ ID NO: 6;
(ii) at least one caspase 3 recognition motif; and
(iii) a donor fluorophore conjugated to a first amino acid residue of said polypeptide of interest and an acceptor fluorophore conjugated to a second amino acid residue of said polypeptide of interest,
wherein said donor fluorophore and said acceptor fluorophore are separated by a distance that allows fluorescence resonance energy transfer to occur between said donor fluorophore and said acceptor fluorophore when said donor fluorophore is excited by a light source.
38 - 42 . (canceled)
43 . The delivery system of claim 37 , wherein said donor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein said acceptor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue; or wherein said acceptor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein said donor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue.
44 - 79 . (canceled)
80 . A method of delivering a bioactive compound to a cell, said method comprising contacting a cell with the delivery system of claim 1 .
81 . A method of treating a disease or unwanted condition in a subject, said method comprising administering a pharmaceutical composition comprising the delivery system of claim 1 and a pharmaceutically acceptable carrier to said subject, wherein said bioactive compound has therapeutic activity against said disease or unwanted condition.
82 . The method of claim 81 , wherein said disease comprises a cancer.
83 - 100 . (canceled)
101 . A method of detecting apoptosis in a cell, said method comprising:
a) contacting said cell with a delivery system comprising a polypeptide of interest, wherein said polypeptide of interest has at least one caspase 3 recognition motif and a donor fluorophore conjugated to a first amino acid residue of said polypeptide of interest and an acceptor fluorophore conjugated to a second amino acid residue of said polypeptide of interest, wherein said donor fluorophore and said acceptor fluorophore are separated by a distance that allows fluorescence resonance energy transfer to occur between said donor fluorophore and said acceptor fluorophore when said donor fluorophore is excited by a light source;
b) exciting said donor fluorophore with a light source; and
c) detecting the emission of said donor fluorophore and said acceptor fluorophore, wherein an increase in the ratio of the emission of said donor fluorophore to said acceptor fluorophore in comparison to a control cell indicates apoptosis in said cell.
102 - 103 . (canceled)
104 . The method of claim 101 , wherein said donor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein said acceptor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue; or said acceptor fluorophore is conjugated to at least one cysteine residue at position 6 and 16 of
SEQ ID NO: 6 through the thiol group of said cysteine residue, and wherein said donor fluorophore is conjugated to at least one lysine residue at position 1 and 11 through the amino group of said lysine residue.
105 . (canceled)
106 . The method of claim 101 , wherein said delivery system comprises a lipid vehicle that encapsulates said polypeptide of interest.
107 . The method of claim 106 , wherein said delivery system further comprises a cytotoxic bioactive compound encapsulated by said lipid vehicle.
108 - 109 . (canceled)
110 . The method of claim 106 , further comprising a polyanionic carrier macromolecule, encapsulated by said lipid vehicle, and further wherein said polyanionic carrier macromolecule is selected from the group consisting of a polyanionic carrier polysaccharide, a polyanionic carrier polypeptide, a glycosaminoglycan, heparin sulfate, hyaluronic acid, chondroitin sulfate, dermatin sulfate, keratan sulfate, dextran sulfate, and combinations thereof.
111 - 117 . (canceled)
118 . The method of claim 106 , wherein said lipid vehicle comprises an exterior surface comprising a polyethylene glycol (PEG) molecule.
119 . (canceled)
120 . The method of claim 118 , wherein:
(i) said PEG molecule comprises a lipid-PEG conjugate comprising a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glyco 2000 (DSPE-PEG 2000 );
(ii) said lipid vehicle comprises a liposome comprising an inner leaflet and an outer leaflet; and wherein
(iii) said outer leaflet comprises the lipid-PEG conjugate at a concentration of about 8 mol % to about 12 mol % of the total lipids.
121 - 123 . (canceled)
124 . The method of claim 106 , wherein said lipid vehicle comprises an exterior surface comprising a targeting ligand that targets said delivery system to a targeted cell.
125 - 126 . (canceled)
127 . The method of claim 124 , wherein said 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol 2000 (DSPE-PEG 2000 ) is conjugated to anisamide, thereby producing DSPE-PEG 2000 -AA.
128 . The method of claim 124 , wherein said targeted cell comprises a cancer cell selected from the group consisting of a bladder cancer, a brain tumor, a breast cancer, a cervical cancer, a colorectal cancer, an esophageal cancer, an endometrial cancer, a hepatocellular carcinoma, a laryngeal cancer, a lung cancer, an osteosarcoma, an ovarian cancer, a pancreatic cancer, a prostate cancer, a renal cancer, and a thyroid cancer.
129 - 130 . (canceled)