Polycationic water soluble copolymer and method for transferring polyanionic macromolecules across biological barriers

• US 7,060,498 B1
• Filed: 11/28/2001
• Issued: 06/13/2006
• Est. Priority Date: 11/28/2001
• Status: Expired due to Fees

• Current Assignee: Genta Salus LLC
• Original Assignee: Genta Salus LLC
• Inventors: Wang, Laixin
• Primary Examiner(s): SCHNIZER, RICHARD A
• Application Number:
  US09/996,507
• Time in Patent Office: 1,658 Days
• Field of Search: 435/455, 435/320.1, 424490- 3, 424/78.18, 424/486, 536/23.1, 536/24.5
• US Class Current: 435/455
• CPC Class Codes: A61K 47/51 the non-active ingredient being a modifying agent
• CPC Class Codes: A61K 47/59 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 47/65 Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C08G 81/00 Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers involving only carbon-to-carbon unsaturated bond reactions C08F299/00 ; polyester-amides C08G69/44; polyester-imides C08G73/16; polyamides-imides C08G73/14; block- or graft polymers containing polysiloxane sequences C08G77/42
• CPC Class Codes: C08L 2205/05 containing polymer components which can react with one another
• CPC Class Codes: C08L 2666/20 Macromolecular compounds having nitrogen in the main chain according to C08L75/00 - C08L79/00; Derivatives thereof
• CPC Class Codes: C08L 2666/26 Natural polymers, natural resins or derivatives thereof according to C08L1/00 - C08L5/00, C08L89/00, C08L93/00, C08L97/00 or C08L99/00
• CPC Class Codes: C08L 71/02 Polyalkylene oxides
• CPC Class Codes: C08L 79/00 Compositions of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen or carbon only, not provided for in groups C08L61/00 - C08L77/00
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle

Abstract

The present provides a carrier molecule for transporting a polyanioic macromolecule such as a nucleic acid across a biological barrier of a cell. The carrier has a biocompatible backbone polymer with two or more polycationic polymer fragments covalently linked. In one embodiment, the backbone polymer is polyethylene glycol (PEG) and the polycationic polymer is polyethylenimine (PEI). The copolymer carrier molecule can be complexed with a polyanionic macromolecule such as a nucleic acid (NA). The NA/copolymer complex is stable in biological conditions by forming a special coreshell-like micelle structure. The nucleic acid can be rapidly released from the complex when biodegradation linker are used to bind the polycationic polymer fragments to the polymer backbone. The carriers and complexes of the invention can be used tin methods of delivering the polycainonic macromolecules to cells both in vitro and in vivo.

Claims

1. A carrier molecule for transporting a polyanionic macromolecule across a membrane of a cell consisting of:

a single polyethylene glycol (PEG) backbone polymer; and

about 4 to about 100 polyethylenimine (PEI) polymers covalently linked to the PEG backbone polymer by linkers.

2. The carrier of claim 1, further comprising at least one targeting moiety connected to the PEG backbone polymer or to one of the about 4 to about 100 PEI polymers.

3. The carrier of claim 2, wherein the at least one targeting moiety is selected from the group consisting of a ligand, an antigen, a hapten, biotin, a lectin, galactose, galactosamine, a protein, a histone, a polypeptide, a lipid, a carbohydrate, a vitamin, and a combination thereof.

4. The carrier of claim 1, further comprising at least one lysis agent connected to the PEG backbone polymer or to one of the about 4 to about 100 PEI polymers.

5. The carrier of claim 4, wherein the at least one lysis agent is selected from the group consisting of a viral peptide, a bacterial toxin, a lytic peptide, alveolysin, bifermentolysin, botulinolysin, cereolysin O, chauveolysin, histolyticolysin O, pneumolysin, seeligerolysin, septicolysin O, sordellilysin, streptolysin O, tenolysin or thuringolysin O, and active fragments thereof.

6. The carrier of claim 1, wherein the linkers are about 2 to about 100 atoms in length.

7. The carrier of claim 6, wherein the linkers are selected from the group consisting of a hydrocarbon chain, a PEG fragment, a polypeptide, a linear polymer containing an ester bond, a linear polymer containing an amide bond, a linear polymer containing a disulfide bond, a linear polymer containing a hydrozone bond, and a linear polymer containing an oxime bond.

8. The carrier of claim 6, wherein the linkers are about 3 atoms to about 30 atoms in length.

9. The carrier of claim 1, wherein the PEG backbone polymer has a molecular weight in the range from about 1,000 to about 1,000,000 daltons and the about 4 to about 100 PEI polymers have a molecular weight in the range from about 100 to about 100,000 daltons.

10. The carrier of claim 9, wherein the molecular weight of the PEG backbone polymer is in the range from about 5,000 to about 100,000 daltons.

11. The carrier of claim 9, wherein the molecular weight of the PEG backbone polymer is in the range from about 20,000 to about 40,000 daltons.

12. The carrier of claim 9, wherein the molecular weight of the about 4 to about 100 PEI polymers is in the range from about 200 to about 10,000 daltons.

13. The carrier of claim 9, wherein the molecular weight of the about 4 to about 100 PEI polymers is in the range from about 400 to about 2,000 daltons.

14. The carrier of claim 1, wherein from about 8 to about 15 PEI polymers are covalently linked to the PEG backbone polymer by linkers.

15. The carrier of claim 1, wherein the molecular weight of the PEG backbone polymer is in the range from about 20,000 to about 40,000 daltons.

16. The carrier of claim 1, wherein the molecular weight of the about 4 to about 100 PEI polymers is in the range from about 400 to about 2,000 daltons.

17. The carrier of claim 1, wherein the linkers are selected from the group consisting of a hydrocarbon chain, a PEG fragment, a polypeptide, a linear polymer containing an ester bond, a linear polymer containing an amide bond, a linear polymer containing a disulfide bond, a linear polymer containing a hydrozone bond, and a linear polymer containing an oxime bond.

18. The carrier of claim 1, comprising a linker which is a biodegradable peptide.

19. The carrier of claim 18, wherein the biodegradable peptide is selected from the group consisting of GlyPhePheGly (SEQ ID NO.2) and GlyPheLeuGly (SEQ ID NO.1).

20. A complex for transporting a polyanionic macromolecule across a membrane of a cell comprising:

a carrier molecule for delivering the polyanionic macromolecule to the cell, the carrier molecule consisting of a single poly (N-(2-hydroxypropyl)methacrylamide) (HPMA) backbone polymer and two or more PEI polymers covalently linked to the poly (N-(2-hydroxypropyl)methacrylamide) (HPMA) backbone polymer by linkers; and

a nucleic acid complexed with the carrier molecule.

21. The complex of claim 20, wherein the nucleic acid is selected from the group consisting of genomic DNA, plasmid DNA, synthetic DNA, and RNA.

22. The complex of claim 20, wherein the nucleic acid is selected from the group consisting of an antisense oligonucleotide, a ribozyme, a DNAzyme, a chimeric RNA/DNA, a phosphorothioate oligonucleotide, a 2′

-O-methyl oligonucleotide, a DNA-PNA conjugate, a DNA-morpholino-DNA conjugate, and a combination thereof.

23. The complex of claim 20, wherein the HPMA backbone polymer has a molecular weight in the range from about 1,000 daltons to about 1,000,000 daltons and the PEI polymers have a molecular weight in the range from about 100 daltons to about 100,000 daltons.

24. The complex of claim 23, wherein the molecular weight of the HPMA backbone polymer is in the range from about 20,000 daltons to about 40,000 daltons.

25. The complex of claim 24, wherein the molecular weight of the PEI polymers is in the range from about 400 daltons to about 2,000 daltons.

26. The complex of claim 20, wherein the linkers are selected from the group consisting of a hydrocarbon chain, a PEG fragment, a polypeptide, a linear polymer containing an ester bond, a linear polymer containing an amide bond, a linear polymer containing a disulfide bond, a linear polymer containing a hydrozone bond, and a linear polymer containing an oxime bond.

27. The complex of claim 20, further comprising at least one targeting moiety connected to the HPMA backbone polymer or to one of the two or more PEI polymers, the at least one targeting moiety selected from the group consisting of a ligand, an antigen, a hapten, biotin, a lectin, galactose, galactosamine, a protein, a histone, a polypeptide, a lipid, a carbohydrate, and a combination thereof.

28. The complex of claim 20, further comprising at least one lysis agent connected to the HPMA backbone polymer or to one of the two or more PEI polymers, the at least one lysis agent selected from the group consisting of a viral peptide, a bacterial toxin, a lytic peptide, alveolysin, bifermentolysin, botulinolysin, cereolysin O, chauveolysin, histolyticolysin O, pneumolysin, seeligerolysin, septicolysin O, sordellilysin, streptolysin O, tenolysin or thuringolysin O, and active fragments thereof.

29. The complex of claim 20, wherein from about 4 to about 100 PEI polymers are covalently linked to the HPMA backbone polymer by linkers.

30. The complex of claim 20, wherein from about 8 to about 15 PEI polymers are covalently linked to the HPMA backbone polymer by linkers.

31. A method of transporting a polyanionic macromolecule across a membrane of a cell comprising:

(a) complexing the polyanionic macromolecule to a carrier molecule to create a

complex, the carrier molecule consisting of a single biocompatible hydrophilic backbone polymer and two or more polycationic polymers covalently linked to the biocompatible hydrophilic backbone polymer by a biodegradable peptide linkers which are from about 2 to about 100 atoms in length, wherein the biocompatible hydrophilic backbone polymer is selected from the group consisting of PEG and HPMA; and

(b) contacting the cell with the complex.

32. The method of claim 31, wherein the polycationic polymers are selected from the group consisting of polyalkylamine (PAM), polyethylenimine (PEI), a polylysine (PL), a polypeptide, chitosan, a polysaccharide, and copolymers thereof.

33. The method of claim 31, further comprising at least one targeting moiety connected to the biocompatible hydrophilic backbone or to one of the two or more polycationic polymers, the at least one targeting moiety selected from the group consisting of a ligand, an antigen, a hapten, biotin, a lectin, galactose, galactosamine, a protein, a histone, a polypeptide, a lipid, a carbohydrate, and a combination thereof.

34. The method of claim 31, further comprising at least one lysis agent connected to the biocompatible hydrophilic backbone polymer or to one of the two or more polycationic polymers, the at least one lysis agent selected from the group consisting of a viral peptide, a bacterial toxin, a lytic peptide, aleveolysin, alveolysin, bifermentolysin, botulinolysin, capriciolysin, cereolysin O, chauveolysin, histolyticolysin O, pneumolysin, seeligerolysin septicolysin O, sordellilysin, streptolysin O, tenolysin or thuringolysin O, and active fragments thereof.

35. The method of claim 31, wherein the biodegradable peptide linker is selected from the group consisting of GlyPhePheGly (SEQ ID NO.2) and GlyPheLeuGly (SEQ ID NO.1).

36. The method of claim 31, wherein the biocompatible hydrophilic backbone has a molecular weight in the range from about 1,000 to about 1,000,000 daltons and the polycationic polymers have a molecular weight in the range from about 100 to about 100,000 daltons.

37. The method of claim 36, wherein the molecular weight of the biocompatible hydrophilic backbone is in the range from about 20,000 to about 40,000 daltons.

38. The method of claim 36, wherein the molecular weight of the polycationic polymers is in the range from about 400 to about 2,000 daltons.

39. The method of claim 36, wherein the biocompatible hydrophilic backbone is polyethylene glycol and the polycationic polymers are polyethylenimine.

40. The method of claim 39, wherein the molecular weight of the biocompatible hydrophilic backbone is in the range from about 20,000 to about 40,000 daltons.

41. The method of claim 39, wherein the molecular weight of the polycationic polymers is in the range from about 400 to about 2,000 daltons.

42. The method of claim 31, wherein from about 4 to about 100 polycationic polymers are covalently linked to the biocompatible hydrophilic backbone polymer by the biodegradable peptide linkers.

43. The method of claim 31, wherein from about 8 to about 15 polycationic polymers are covalently linked to the biocompatible hydrophilic backbone polymer by the biodegradable peptide linkers.

44. A carrier for transporting a polyanionic macromolecule across a membrane of a cell comprising:

a PEG biocompatible hydrophilic backbone polymer; and

two or more PEI polycationic polymers covalently linked to the PEG biocompatible hydrophilic backbone polymer by peptide or propionic acid linkers;

wherein the PEI polycationic polymers have a molecular weight of from about 400 to about 2,000 daltons.

45. The carrier of claim 44 wherein the PEI polycationic polymers have a molecular weight of about 2,000 daltons.

46. The carrier of claim 44 wherein the PEI polycationic polymers have a molecular weight of about 1,200 daltons.

47. The carrier of claim 44 wherein the PEI polycationic polymers have a molecular weight of about 800 daltons.

48. The carrier of claim 44 wherein the PEI polycationic polymers have a molecular weight of about 400 daltons.

49. The carrier of claim 44, wherein the linkers are a peptide and the peptide is selected from the group consisting of GlyPhePheGly (SEQ ID NO. 2) and GlyPheLeuGly (SEQ ID NO. 1).

50. The carrier of claim 44, wherein the molecular weight of the PEG biocompatible hydrophilic backbone polymer is in the range from about 20,000 to about 40,000 daltons.