Method of Biomolecule Immobilization On Polymers Using Click-Type Chemistry

• US 20090297609A1
• Filed: 07/06/2006
• Published: 12/03/2009
• Est. Priority Date: 07/06/2005
• Status: Active Grant

• Current Assignee: Jordan Wosnick, Meng Shi, Molly S. Shoichet
• Original Assignee: Jordan Wosnick, Meng Shi, Molly S. Shoichet
• Inventors: Shi, Meng, Yuan, Yumin, Wosnick, Jordan, Shoichet, Molly S.
• Granted Patent: US 9,290,617 B2
• Time in Patent Office: Days
• US Class Current: 424/489
• CPC Class Codes: A61K 47/6935 the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
• CPC Class Codes: C08G 63/64 Polyesters containing both carboxylic ester groups and carbonate groups
• CPC Class Codes: C08G 63/912 derived from hydroxycarboxylic acids
• CPC Class Codes: C08G 64/0241 containing nitrogen
• CPC Class Codes: C08G 64/42 Chemical after-treatment

Abstract

The present invention provides a method for the covalent immobilization of biomolecules on polymers for delivery of the biomolecules, which has the advantage of being simple, highly efficient, environmentally friendly and free of side products relative to traditional immobilization techniques. The invention provides a modified micro/nanoparticle system, which uses a functionalized polymer formed into micro or nanoparticles to bind a molecule to the particles using uses facile chemistry, the Diels-Alder cycloaddition between a diene and a dienophile with the polymer being functionalized with one of them and the molecule with the other, or the Huisgen 1,3-dipolar cycloaddition between a terminal alkyne and an azide to bind the molecule to the particle. The molecules and/or other therapeutic agents may be encapsulated within the polymer particles for intravenous therapeutic delivery. The invention also provides a novel synthetic biodegradable polymer, a furan/alkyne-functionalized poly(trimethylene carbonate) (PTMC)-based polymer, whose composition can be designed to meet the defined physical and chemical property requirements. In one example, the particle system self-aggregates from functionalized PTMC-based copolymers containing poly(ethylene glycol) (PEG) segments. The composition of the copolymers can be designed to meet various particle system requirements, including size, thermodynamic stability, surface PEG density, drug encapsulation capacity and biomolecule immobilization capacity.

Claims

1. A composition for therapeutic delivery of a molecule, comprising:

a polymer nanoparticle or microparticle comprised of a polymer functionalized to include a first unsaturated functional group; and

a molecule functionalized to include a second unsaturated functional group, said first and second functional groups being complementary to each other and being selected such that said first and second unsaturated functional group react with each other by one of Diels-Alder cycloaddition and Huisgen 1,3-dipolar cycloaddition to covalently bind the molecule to said polymer nanoparticle or microparticle to form a delivery vehicle for therapeutic delivery of said molecule.

2. The composition of claim 1 wherein said complementary unsaturated functional groups includes a mixture of two types of pairs of complementary unsaturated functional groups, a first pair in which the two unsaturated functional group reacts with each other by said Diels-Alder cycloaddition, and a second type in which the two unsaturated group reacts with each other by said Huisgen 1,3-dipolar cycloaddition.

3. The composition of claim 1 wherein said complementary unsaturated functional groups which react with each other by said Diels-Alder cycloaddition include a diene and a dienophile, and wherein said complementary unsaturated functional groups which react with each other by said Huisgen 1,3-dipolar cycloaddition include an alkyne and an azide.

4. The composition according to claim 3 wherein said diene is selected from the group consisting of furan and derivatives thereof, cyclopentadiene and derivatives thereof, butadiene and derivatives thereof, and cyclohexadiene and derivatives thereof, and wherein said dienophile is selected from the group consisting of maleimide and derivatives thereof, acrylonitrile and derivatives thereof, acrylamide and derivatives thereof, methyl vinyl ketone and derivatives thereof, esters of maleic acid and derivatives thereof, esters of fumaric acid and derivatives thereof, esters of acrylic acid and derivatives thereof, maleic anhydride and derivatives thereof, esters and amides of but-2-ynedioic acid and derivatives thereof, quinone and derivatives thereof, and substituted acetylenes and derivatives thereof.

5. The composition according to claim 3 wherein said diene is furan and said dienophile is maleimide.

6. The composition according to claim 3 wherein said alkyne is selected from the group consisting of terminal alkynes substituted with alkyl groups and derivatives thereof, ester groups and derivatives thereof, amide groups and derivatives thereof, alkyl and polyoxoalkyl groups and derivatives thereof, aryl and derivatives thereof, phenyl groups and derivatives thereof, and benzyl groups and derivatives thereof, and wherein said azide is selected from the group consisting of alkyl and polyoxoalkyl azides and derivatives thereof, aryl azides and derivatives thereof, and benzyl azides and derivatives thereof.

7. The composition according to claim 6 wherein said alkyne group is an amide of propargylamine, and said azide is a substituted benzyl azide.

8. The composition of claim 1 wherein the polymer is selected from the group consisting of block copolymers, copolymers, terpolymers, graft copolymers, graft terpolymers and amphiphilic copolymers.

9. The composition of claim 1 wherein the polymer is selected from the group consisting of polymers of natural origin, polymers produced by chemical synthesis and polymers produced by biological synthesis, and wherein said polymers of natural origin are selected from the group consisting of proteins, polysialic acids, hyaluronic acid and derivatives thereof, polysaccharides and derivatives thereof, chitosan and derivatives thereof, alginate and derivatives thereof, collagen and derivatives thereof, and aliphatic poly(esters), polycarbonates and derivatives thereof, poly(hydroxyalkanoates) and derivatives thereof, and wherein said polymers produced by biological synthesis include polymers synthesized by fermentation, and wherein said polymers produced by chemical synthesis include polymers produced by ring-open polymerization, polycondensation, free radical polymerization, or ionic polymerization.

10. The composition of claim 1 wherein said polymers are biodegradable polymers selected from the group consisting of homopolymers, copolymers or terpolymers of polyesters, polycarbonates, polyamides, poly(ester-amide)s, poly(anhydride)s, polyurethanes, poly(ester-urethane)s, poly(hydroxyalkanoate)s and combinations thereof.

11. The composition according to claims 1 wherein said microparticles or nanoparticles have a structure with an interior and including said molecule and/or other therapeutic agents encapsulated in said interior.

12. The composition according to claim 11 wherein said molecules encapsulated in said interior of said microparticles or nanoparticles are covalently bound to said microparticles or nanoparticles.

13. The composition according to claim 11 wherein said therapeutic agents encapsulated in said interior are selected from the group consisting of nucleic acids, proteins, small molecules, biomolecules, viruses, phage, cells and cell fragments.

14. The composition according to claim 13 wherein said nucleic acids are selected from the group consisting of DNA, cDNA, RNA, and PNA.

15. The composition according to claim 13 wherein said proteins are selected from the group consisting of antibodies, antibody fragments, enzymes, ligands, receptors, viral vectors and viruses.

16. The composition according to claim 13 wherein said small molecules are selected from the group consisting of polypeptides, peptides, amino acids, metabolites and drugs.

17. The composition according to claim 13 wherein said biomolecules are selected from the group consisting of vitamins, lipids, antibiotics and hormones.

18. The composition according to claim 1 wherein said polymer is one of a liquid polymer, a solid polymer, a hydrogel and a semisolid polymer.

19. The composition according to claim 1 wherein said polymer is poly(trimethylene carbonate) (PTMC)-based polymer.

46. The composition according to claim 1 wherein said Huisgen 1,3-dipolar cycloaddition is Cu(I) catalyzed.

20. A method of delivery of a molecule, comprising:

a) providing nanoparticles or microparticles of a polymer functionalized to include a first unsaturated functional group;

b) providing a molecule functionalized with a second unsaturated functional group, said first and second functional groups being complementary to each other and being selected such that said first and second unsaturated functional groups react with each other by one of Diels-Alder cycloaddition and Huisgen 1,3-dipolar cycloaddition;

c) mixing said nanoparticles or microparticles with said molecule under conditions suitable to react said first unsaturated functional group on said polymer nanoparticle or microparticle with said second unsaturated functional group on said molecule by one of Diels-Alder cycloaddition and Huisgen 1,3-dipolar cycloaddition to covalently bind the molecule to said polymer nanoparticle or microparticle to form a delivery vehicle for said molecule; and

d) introducing said nanoparticles or microparticles having said molecule bound thereto into a biological system.

21. The method of claim 20 wherein said complementary unsaturated functional groups includes a mixture of two types of pairs of complementary unsaturated functional groups, a first type in which each unsaturated functional group reacts with each other by said Diels-Alder cycloaddition, and a second type in which each unsaturated group reacts with each other by said Huisgen 1,3-dipolar cycloaddition.

22. The method of claim 20 wherein said complementary unsaturated functional groups which react with each other by said Diels-Alder cycloaddition include a diene and a dienophile, and wherein said complementary unsaturated functional groups which react with each other by said Huisgen 1,3-dipolar cycloaddition include an alkyne and an azide.

23. The method according to claim 22 wherein said diene is selected from the group consisting of furan and derivatives thereof, cyclopentadiene and derivatives thereof, butadiene and derivatives thereof, and cyclohexadiene and derivatives thereof, and wherein said dienophile is selected from the group consisting of maleimide and derivatives thereof, acrylonitrile and derivatives thereof, acrylamide and derivatives thereof, methyl vinyl ketone and derivatives thereof, esters of maleic acid and derivatives thereof, esters of fumaric acid and derivatives thereof, esters of acrylic acid and derivatives thereof, maleic anhydride and derivatives thereof, esters and amides of but-2-ynedioic acid and derivatives thereof, quinone and derivatives thereof, and substituted acetylenes and derivatives thereof.

24. The method according to claim 22 wherein said diene is furan and said dienophile is maleimide.

25. The method of claim 22 wherein said pair of complementary unsaturated functional groups include an alkyne and an azide which react with each other by said Huisgen 1,3-dipolar cycloaddition.

26. The method according to claim 25 wherein said alkyne is selected from the group consisting of terminal alkynes substituted with alkyl groups and derivatives thereof, ester groups and derivatives thereof, amide groups and derivatives thereof, alkyl and polyoxoalkyl groups and derivatives thereof, aryl and derivatives thereof, phenyl groups and derivatives thereof, and benzyl groups and derivatives thereof, and wherein said azide is selected from the group consisting of alkyl and polyoxoalkyl azides and derivatives thereof, aryl azides and derivatives thereof, benzyl azides and derivatives thereof.

27. The method according to claim 25 wherein said alkyne group is an amide of propargylamine, and said azide is a substituted benzyl azide.

28. The method according to claim 20 wherein the polymer is selected from the group consisting of block copolymers, copolymers, terpolymers, graft copolymers, graft terpolymers and amphiphilic copolymers.

29. The method according to claim 20 wherein the polymer is selected from the group consisting of polymers of natural origin, polymers produced by chemical synthesis and polymers produced by biological synthesis, and wherein said polymers of natural origin are selected from the group consisting of proteins, polysialic acids, hyaluronic acid and derivatives thereof, polysaccharides and derivatives thereof, chitosan and derivatives thereof, alginate and derivatives thereof, collagen and derivatives thereof, and aliphatic poly(esters), polycarbonates and derivatives thereof, poly(hydroxyalkanoates) and derivatives thereof, and wherein said polymers produced by biological synthesis include polymers synthesized by fermentation, and wherein said polymers produced by chemical synthesis include polymers produced by ring-open polymerization, polycondensation, free radical polymerization, or ionic polymerization.

30. The method according to claim 20 wherein said polymers are biodegradable polymers selected from the group consisting of polyesters, polycarbonates, polyamides, poly(ester-amide)s, poly(anhydride)s, polyurethanes, poly(ester-urethane)s, poly(hydroxyalkanoate)s and combinations thereof.

31. The method according to claim 20 wherein said microparticles or nanoparticles have a structure with an interior and including said molecules and/or other therapeutic agents encapsulated in said interior.

32. The method according to claim 31 wherein said molecules encapsulated in said interior of said microparticles or nanoparticles are covalently bound to said microparticles or nanoparticles.

33. The method according to claim 31 wherein said therapeutic agents encapsulated in said interior are selected from the group consisting of nucleic acids, proteins, biomolecules, viruses, small molecules, cells and cell fragments.

34. The method according to claim 33 wherein said nucleic acids are selected from the group consisting of DNA, cDNA, RNA, and PNA.

35. The method according to claim 33 wherein said proteins are selected from the group consisting of antibodies, antibody fragments, enzymes, ligands, receptors, viral vectors and viruses.

36. The method according to claim 33 wherein said small molecules are selected from the group consisting of polypeptides, peptides, amino acids, metabolites and drugs.

37. The method according to claim 33 wherein said biomolecules are selected from the group consisting of vitamins, antibiotics and hormones.

38. The method according to claim 20 wherein said polymer is one of a liquid polymer, a solid polymer, a hydrogel and a semisolid polymer.

39. The method according to claim 20 wherein said biological system is a human.

40. The method according to claim 20 wherein said biological system is an animal.

41. The method according to claim 20 wherein said biological system is a horticultural product.

42. The method according to claim 20 wherein said Huisgen 1,3-dipolar cycloaddition is Cu(I) catalyzed.

43. The method according to claim 36 wherein said drugs are selected from the group consisting of hydrophobic drugs and hydrophilic drugs.

44. The method according to claim 20 wherein said step a) providing functionalized nanoparticles or microparticles of a polymer includes synthesizing said polymer from a monomer containing said first unsaturated functional group.

45. The method according to claim 20 wherein said step a) providing functionalized nanoparticles or microparticles of a polymer includes synthesizing said polymer from a monomer, followed by incorporation of said first unsaturated functional group into said polymer.

47. A polymer, comprising:

a poly(trimethylene carbonate) (PTMC)-based polymer functionalized to include a first unsaturated functional group which reacts with a second unsaturated functional group, said first and second functional groups being complementary to each other and being selected such that said first and second unsaturated functional groups react with each other by one of Diels-Alder cycloaddition and Huisgen 1,3-dipolar cycloaddition for covalently binding the second unsaturated functional group to the first unsaturated functional group on said polymer.

48. The polymer of claim 47 wherein said complementary unsaturated functional groups includes a mixture of two types of pairs of complementary unsaturated functional groups, a first type in which each unsaturated functional group reacts with each other by said Diels-Alder cycloaddition, and a second type in which each unsaturated group reacts with each other by said Huisgen 1,3-dipolar cycloaddition.

49. The polymer of claim 47 wherein said complementary unsaturated functional groups which react with each other by said Diels-Alder cycloaddition include a diene and a dienophile, and wherein said complementary unsaturated functional groups which react with each other by said Huisgen 1,3-dipolar cycloaddition include an alkyne and an azide.

50. The polymer according to claim 49 wherein said diene is selected from the group consisting of furan and derivatives thereof, cyclopentadiene and derivatives thereof, butadiene and derivatives thereof, and cyclohexadiene and derivatives thereof, and wherein said dienophile is selected from the group consisting of maleimide and derivatives thereof, acrylonitrile and derivatives thereof, acrylamide and derivatives thereof, methyl vinyl ketone and derivatives thereof, esters of maleic acid and derivatives thereof, esters of fumaric acid and derivatives thereof, esters of acrylic acid and derivatives thereof, maleic anhydride and derivatives thereof, esters and amides of but-2-ynedioic acid and derivatives thereof, quinone and derivatives thereof, and wherein said alkyne is selected from the group consisting of substituted acetylenes and derivatives thereof, terminal alkynes substituted with ester groups and derivatives thereof, amide groups and derivatives thereof, alkyl and polyoxoalkyl groups and derivatives thereof, aryl and derivatives thereof, phenyl groups and derivatives thereof, and benzyl groups and derivatives thereof, and wherein said azide is selected from the group consisting of alkyl and polyoxoalkyl azides and derivatives thereof, aryl azides and derivatives thereof, and benzyl azides and derivatives thereof.

51. The polymer according to claim 49 wherein said diene is furan and said dienophile is maleimide.

52. The polymer according to claim 49 wherein said alkyne is an amide of propargylamine, and said azide is a substituted benzyl azide.

53. The polymer according to claim 47 formed as any one of single polymer chains, microparticles, nanoparticles, films, tubes, scaffolds, gels, hydrogels and fibers.

54. The polymer according to claim 47 wherein the functionalized poly(trimethylene carbonate) (PTMC)-based polymer is a homopolymer.

55. The polymer according to claim 47 wherein the functionalized poly(trimethylene carbonate) (PTMC)-based polymer is produced as any one of a copolymer, a block copolymer, a graft copolymer, a terpolymer, graft terpolymer, and an amphiphilic copolymer of poly(trimethylene carbonate) with at least one of a polyester, polycarbonate, polyamide, poly(ester-amide), poly(anhydride), polyurethane, poly(ester-urethane), poly(hydroxyalkanoate), poly(ethylene glycol), and combinations thereof.

56. The polymer according to claim 47 wherein said Huisgen 1,3-dipolar cycloaddition is Cu(I) catalyzed.

57. The polymer according to claim 47 for binding a molecule to said polymer, wherein said molecule includes said second unsaturated functional group of said pair of complementary unsaturated functional groups such that when the first and second functional groups undergo said Diels-Alder cycloaddition or Huisgen 1,3-dipolar cycloaddition said molecule is covalently bound to said polymer.