Gels for encapsulation of biological materials

• US 5,529,914 A
• Filed: 10/07/1992
• Issued: 06/25/1996
• Est. Priority Date: 10/15/1990
• Status: Expired due to Term

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Pathak, Chandrashekhar P., Sawhney, Amarpreet S., Desai, Neil P., Hossainy, Syed F. A., Hubbell, Jeffrey A.
• Primary Examiner(s): Lilling, Herbert J.
• Application Number:
  US07/958,870
• Time in Patent Office: 1,357 Days
• Field of Search: 427/213.35, 427/213.3, 427/213.32, 427/23.33, 264/43, 264/4.1, 264/4, 435/177, 435/178, 435/182, 424/93, 424/493, 424/499, 424/489, 424/487, 514/2, 522/87, 522/88, 522/89, 522/149, 522/151, 522/152, 522/154, 522/181
• US Class Current: 435/182
• CPC Class Codes: A61K 2035/128 capsules, e.g. microcapsules
• CPC Class Codes: A61K 38/42 Haemoglobins; Myoglobins
• CPC Class Codes: A61K 38/44 Oxidoreductases (1)
• CPC Class Codes: A61K 47/645 Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• CPC Class Codes: A61K 47/6925 the form being a microcapsule, nanocapsule, microbubble or nanobubble
• CPC Class Codes: A61K 9/1635 obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
• CPC Class Codes: A61K 9/1647 Polyesters, e.g. poly(lactide-co-glycolide)
• CPC Class Codes: A61K 9/5031 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
• CPC Class Codes: A61K 9/5073 having two or more different coatings optionally including drug-containing subcoatings
• CPC Class Codes: A61L 24/0031 Hydrogels or hydrocolloids
• CPC Class Codes: A61L 24/0042 Materials resorbable by the body
• CPC Class Codes: A61L 24/046 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds A61L24/043 takes precedence
• CPC Class Codes: A61L 26/0019 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
• CPC Class Codes: A61L 27/34 Macromolecular materials
• CPC Class Codes: A61L 27/36 containing ingredients of undetermined constitution or reaction products thereof , e.g. transplant tissue, natural bone, extracellular matrix isolated natural compounds, e.g. collagen A61L27/24
• CPC Class Codes: A61L 27/3683 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
• CPC Class Codes: A61L 27/38 containing added animal cells organs or tissue containing native cells A61L27/36
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• CPC Class Codes: A61L 27/52 Hydrogels or hydrocolloids
• CPC Class Codes: A61L 29/085 Macromolecular materials
• CPC Class Codes: A61L 31/10 : Macromolecular materials
• CPC Class Codes: A61L 31/145 : Hydrogels or hydrocolloids
• CPC Class Codes: A61L 31/148 : Materials at least partially resorbable by the body
• CPC Class Codes: C08F 290/00 : Macromolecular compounds obtained by polymerising monomers on to polymers modified by introduction of aliphatic unsaturated end or side groups
• CPC Class Codes: C08F 290/02 : on to polymers modified by introduction of unsaturated end groups
• CPC Class Codes: C08F 290/06 : Polymers provided for in subclass C08G
• CPC Class Codes: C08F 290/062 : Polyethers
• CPC Class Codes: C08F 291/00 : Macromolecular compounds obtained by polymerising monomers on to macromolecular compounds according to more than one of the groups C08F251/00 - C08F289/00
• CPC Class Codes: C08L 71/02 : Polyalkylene oxides
• CPC Class Codes: C09D 153/00 : Coating compositions based on block copolymers containing at least one sequence of a polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds; Coating compositions based on derivatives of such polymers
• CPC Class Codes: C12N 11/04 : entrapped within the carrier, e.g. gel or hollow fibres
• CPC Class Codes: C12N 2533/30 : Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 2533/32 : Polylysine, polyornithine
• CPC Class Codes: C12N 2533/40 : Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
• CPC Class Codes: C12N 2533/74 : Alginate
• CPC Class Codes: C12N 5/0012 : Cell encapsulation
• CPC Class Codes: C12N 5/0677 : Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

This invention provides novel methods for the formation of biocompatible membranes around biological materials using photopolymerization of water soluble molecules. The membranes can be used as a covering to encapsulate biological materials or biomedical devices, as a "glue" to cause more than one biological substance to adhere together, or as carriers for biologically active species. Several methods for forming these membranes are provided. Each of these methods utilizes a polymerization system containing water-soluble macromers, species which are at once polymers and macromolecules capable of further polymerization. The macromers are polymerized using a photoinitiator (such as a dye), optionally a cocatalyst, optionally an accelerator, and radiation in the form of visible or long wavelength UV light. The reaction occurs either by suspension polymerization or by interfacial polymerization. The polymer membrane can be formed directly on the surface of the biological material, or it can be formed on material which is already encapsulated.

Claims

1. A method for encapsulation of biological material comprising the steps of

a) mixing the biological material in an aqueous macromer solution comprising macromer and photoinitiator, said biological material selected from the group consisting of tissue, cells, subcellular organelles and subcellular non-organelle components, said macromer comprising a water soluble polymer having at least two sites of unsaturation;

b) forming small geometric shapes of the mix in (a); and

c) polymerizing the macromer by exposing the geometric shapes to light radiation.

2. The method of claim 1 wherein the biological material is first encapsulated in a microcapsule.

3. The method of claim 1 wherein the macromer is selected from the group consisting of ethylenically unsaturated derivatives of poly(ethylene oxide) (PEO), poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethyloxazoline) (PEOX), poly(amino acids), polysaccharides, and proteins.

4. The method of claim 3 wherein the PEG is PEG multiacrylate.

5. The method of claim 4 wherein the PEG is PEG tetraacrylate which has a molecular weight around 18,500 D.

6. The method of claim 3 wherein the polysaccharides are selected from the group consisting of alginate, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate, heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, water soluble cellulose derivatives and carrageenan.

7. The method of claim 3 wherein the proteins are selected from the group consisting of gelatin, collagen and albumin.

8. The method of claim 1 wherein the macromer is of the formula ##STR4## where F₁ =CONH, COO or NHCOO

X=H, CH₃, C₂ H₅, C₆ H₅, Cl, Br, OH or CH₂ COOH

F₂ =COO, CONH, O or C₆ H₄,

R=CH₂ or -alkyl-,

n≧

5, and

m≧

2.

9. The method of claim 1 wherein the photoinitiator is any dye which absorbs light having frequency between 320 nm and 900 nm, can form free radicals, is at least partially water soluble, and is non-toxic to the biological material at the concentration used for polymerization.

10. The method of claim 9 wherein the photoinitiator is selected from the group consisting of 2,2-dimethoxy,2-phenylacetophenone and 2-methoxy, 2-phenylacetophenone.

11. The method of claim 1 wherein the macromer solution further comprises a cocatalyst and the photoinitiator is selected from the group consisting of ethyl eosin, eosin Y, fluorescein, 2,2-dimethoxy,2-phenylacetophenone,2-methoxy,2-phenylacetophenone, camphorquinone, rose bengal, methylene blue, erythrosin, phloxime, thionine, riboflavin and methylene green.

12. The method of claim 11 wherein the cocatalyst is a nitrogen based compound capable of stimulating a free radical reaction.

13. The method of claim 11 wherein the cocatalyst is a a nitrogen atom-containing electron-rich molecule.

14. The method of claim 11 wherein the cocatalyst is a primary, secondary, tertiary or quaternary amine.

15. The method of claim 14 wherein the cocatalyst is selected from the group consisting of triethanolamine, triethylamine, ethanolamine, N-methyl diethanolamine, N,N-dimethyl benzylamine, dibenzyl amine, N-benzyl ethanolamine, N-isopropyl benzylamine, tetramethyl ethylenediamine, potassium persulfate, tetramethyl ethylenediamine, lysine, ornithine, histidine and arginine.

16. The method of claim 1 wherein the radiation has a wavelength between 320 nm and 900 nm.

17. The method of claim 16 wherein the radiation has a wavelength between 350 nm and 700 nm.

18. The method of claim 2 wherein the microcapsule is comprised of ionically coagulatable or thermally coagulatable polymers which are non-toxic to the encapsulated material.

19. The method of claim 1 wherein the cells are primary cells or established cell lines.

20. The method of claim 1 wherein the biological material is selected from pancreatic islet cells, human foreskin fibroblasts, Chinese hamster ovary cells, beta cell insulomas, lymphoblastic leukemia cells, mouse 3T3 fibroblasts, dopamine secreting ventral mesencephalon cells, neuroblastoid cells, adrenal medulla cells, and T-cells.

21. The method of claim 18 wherein the microcapsule is comprised of alginate.

22. The method of claim 18 wherein the microcapsule is comprised of chitosan.

23. The method of claim 18 wherein the microcapsule is comprised of agarose.

24. The method of claim 18 wherein the microcapsule is comprised of gelatin.

25. The method of claim 1 wherein the geometric shapes are formed by coextrusion of the aqueous macromer solution mixed with the biological material with a non-toxic, non-immunogenic, non-miscible substance capable of maintaining droplet formation.

26. The method of claim 25 wherein the non-miscible substance is oil.

27. The method of claim 26 wherein the oil is mineral oil.

28. The method of claim 1 wherein the geometric shapes are formed by coextrusion of the aqueous macromer solution mixed with the biological material in air.

29. The method of claim 1 wherein the geometric shapes are formed by agitation of the aqueous macromer solution mixed with the biological material with a non-toxic, non-immunogenic, non-miscible substance.

30. The method of claim 29 wherein the non-miscible substance is oil.

31. The method of claim 1 wherein the macromer solution further comprises an accelerator to accelerate the rate of polymerization.

32. The method of claim 31 wherein the accelerator is a small molecule containing an allyl, vinyl or acrylate group.

33. The method of claim 32 wherein the accelerator is selected from the group consisting of N-vinyl pyrolidinone, 2-vinyl pyridine, 1-vinyl imidazole, 9-vinyl carbazole, acrylic acid and 2-allyl,2-methyl,1-3-cyclopentane dione.

34. The method of claim 33 wherein the accelerator is N-vinyl pyrolidinone.

69. The method of claim 31 wherein the macromer is poly(ethylene glycol) (PEG), the biological material is mammalian cells, and the accelerator is N-vinyl pyrrolidinone.

35. A method for encapsulation of biological material comprising the steps of

a) coating the biological material with photoinitiator, said biological material selected from the group consisting of tissue, cells, subcellular organelles, and subcellular non-organelle components;

b) suspending the coated material in an aqueous macromer solution, said macromer comprising a water soluble polymer having at least two sites of unsaturation; and

c) irradiating the suspension with light.

36. The method of claim 35 wherein the macromer is selected from the group consisting of ethylenically unsaturated derivatives of poly(ethylene oxide) (PEO), poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethyloxazoline) (PEOX), poly(amino acids), polysaccharides, and proteins.

37. The method of claim 36 wherein the PEG is PEG multiacrylate.

38. The method of claim 37 wherein the PEG is PEG tetraacrylate which has a molecular weight around 18,500 D.

39. The method of claim 36 wherein the polysaccharides are selected from the group consisting of alginate, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate, heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, water soluble cellulose derivatives and carrageenan.

40. The method of claim 36 wherein the proteins are selected from the group consisting of gelatin, collagen and albumin.

41. The method of claim 35 wherein the macromer is of the formula ##STR5## where F₁ =CONH, COO or NHCOO

X=H, CH₃, C₂ H₅, C₆ H₅, Cl, Br, OH or CH₂ COOH

F₂ =COO, CONH, O or C₆ H₄,

R=CH₂ or -alkyl-,

n≧

5, and

m≧

2.

42. The method of claim 35 wherein the photoinitiator is any dye which absorbs light having frequency between 320 nm and 900 nm, can form free radicals, is at least partially water soluble, and is non-toxic to the biological material at the concentration used for polymerization.

43. The method of claim 42 wherein the photoinitiator is selected from the group consisting of 2,2-dimethoxy,2-phenylacetophenone and 2-methoxy,2-phenylacetophenone.

44. The method of claim 35 wherein the macromer solution further comprises a cocatalyst and the photoinitiator is selected from the group consisting of ethyl eosin, eosin Y, fluorescein,2,2-dimethoxy,2-phenylacetophenone,2-methoxy,2-phenylacetophenone, camphorquinone, rose bengal, methylene blue, erythrosin, phloxime, thionine, riboflavin and methylene green.

45. The method of claim 44 wherein the cocatalyst is a nitrogen based compound capable of stimulating a free radical reaction.

46. The method of claim 44 wherein the cocatalyst is a a nitrogen atom-containing electron-rich molecule.

47. The method of claim 44 wherein the cocatalyst is a primary, secondary, tertiary or quaternary amine.

48. The method of claim 47 wherein the cocatalyst is selected from the group consisting of triethanolamine, triethylamine, ethanolamine, N-methyl diethanolamine, N,N-dimethyl benzylamine, dibenzyl amine, N-benzyl ethanolamine, N-isopropyl benzylamine, tetramethyl ethylenediamine, potassium persulfate, tetramethyl ethylenediamine, lysine, ornithine, histidine and arginine.

49. The method of claim 35 wherein the radiation has a wavelength between 320 nm and 900 nm.

50. The method of claim 49 wherein the radiation has a wavelength between 350 nm and 700 nm.

51. The method of claim 35 wherein the cells are primary cells or established cell lines.

52. The method of claim 35 wherein the biological material is selected from pancreatic islet cells, human foreskin fibroblasts, Chinese hamster ovary cells, beta cell insulomas, lymphoblastic leukemia cells, mouse 3T3 fibroblasts, dopamine secreting ventral mesencephalon cells, neuroblastoid cells, adrenal medulla cells, and T-cells.

53. The method of claim 35 wherein the geometric shapes are formed by coextrusion of the aqueous macromer solution mixed with the biological material with a non-toxic, non-immunogenic, non-miscible substance capable of maintaining droplet formation.

54. The method of claim 53 wherein the non-miscible substance is oil.

55. The method of claim 54 wherein the oil is mineral oil.

56. The method of claim 35 wherein the geometric shapes are formed by coextrusion of the aqueous macromer solution mixed with the biological material in air.

57. The method of claim 35 wherein the geometric shapes are formed by agitation of the aqueous macromer solution mixed with the biological material with a non-toxic, non-immunogenic, non-miscible substance.

58. The method of claim 57 wherein the non-miscible substance is oil.

59. The method of claim 35 wherein the biological material is first encapsulated in a microcapsule.

60. The method of claim 59 wherein the microcapsule is comprised of ionically coagulatable or thermally coagulatable polymers which are non-toxic to the encapsulated material.

61. The method of claim 60 wherein the microcapsule is comprised of alginate.

62. The method of claim 60 wherein the microcapsule is comprised of chitosan.

63. The method of claim 60 wherein the microcapsule is comprised of agarose.

64. The method of claim 60 wherein the microcapsule is comprised of gelatin.

65. The method of claim 35 wherein the macromer solution further comprises an accelerator to accelerate the rate of polymerization.

66. The method of claim 65 wherein the accelerator is a small molecule containing an allyl, vinyl or acrylate group.

67. The method of claim 66 wherein the accelerator is selected from the group consisting of N-vinyl pyrolidinone, 2-vinyl pyridine, 1-vinyl imidazole, 9-vinyl carbazole, acrylic acid and 2-allyl,2-methyl,1-3-cyclopentane dione.

68. The method of claim 67 wherein the accelerator is N-vinyl pyrolidinone.