DELIVERY SYSTEMS FOR BIOACTIVE AGENTS
The invention features a composition for the delivery of bioactive agents into cells that includes a delivery matrix, an anionic or zwitterionic compound, and a bioactive agent, e.g. a peptide, protein, or nucleic acid. The compositions of the invention can be used to deliver bioactive compounds, such as nucleic acids encoding immunostimulatory peptides and/or therapeutic proteins.
1 - 36 . (canceled)
37 . A microparticle less than about 100 microns in diameter, comprising:
a polymeric matrix;
a polyethylene glycol distearoyl phosphatidylethanolamine (PEG-DSPE), wherein the PEG-DSPE is an mPEG-DSPE or a PEG2K-DSPE; and
a nucleic acid molecule, wherein the microparticle is not encapsulated in a liposome and the microparticle does not comprise a cell.
38 . The microparticle of claim 37 , wherein the lipid has a pKa of less than about 2.5.
39 . The microparticle of claim 37 , wherein the lipid has a pKa of less than about 2.0.
40 . The microparticle of claim 37 , wherein the lipid has a pKa of less than about 1.8.
41 . The microparticle of claim 37 , wherein the microparticle has a diameter of less than about 50 microns.
42 . The microparticle of claim 37 , wherein the nucleic acid molecule is circular.
43 . The microparticle of claim 37 , wherein the nucleic acid molecule is a plasmid.
44 . The microparticle of claim 37 , wherein the nucleic acid molecule comprises an expression control sequence operatively linked to a coding sequence.
45 . The microparticle of claim 37 , further comprising a targeting molecule.
46 . The microparticle of claim 37 , further comprising a stabilizer.
47 . The microparticle of claim 37 , wherein the microparticle does not comprise a virus.
48 . The microparticle of claim 43 , wherein the plasmid is at least 50% supercoiled.
49 . The microparticle of claim 44 , wherein the coding sequence encodes an expression product that is immunogenic.
50 . The microparticle of claim 44 , wherein the coding sequence encodes an expression product that comprises an amino acid sequence identical to the sequence of an antigenic portion of a tumor antigen.
51 . The microparticle of claim 37 , wherein the polymeric matrix comprises a copolymer of poly-lactic-co-glycolic acid (PLGA).
52 . The microparticle of claim 37 , wherein the polymeric matrix consists of a copolymer of PLGA.
53 . A preparation of microparticles comprising a plurality of the microparticles of claim 37 .
54 . A method of eliciting an immune response in a human, the method comprising administering an effective amount of the microparticle of claim 37 to the human.
55 . A process for preparing microparticles, comprising:
(1) providing a first solution comprising a polymer and a PEG-DSPE, wherein the PEG-DSPE is an mPEG-DSPE or a PEG2K-DSPE;
(2) providing a second solution comprising a nucleic acid dissolved or suspended in a solvent;
(3) mixing the first and second solutions to form a first emulsion; and
(4) mixing the first emulsion with a third solution to form a second emulsion;
wherein both mixing steps are carried out in a manner that minimizes shearing of the nucleic acid while producing microparticles having an average diameter smaller than 100 microns.
56 . The process of claim 55 , further comprising:
(5) washing the microparticles with an aqueous solution to remove the solvent;
(6) concentrating the microparticles; and
(7) lyophilizing the microparticles.