Cross-linked gelatin gels and methods of making them

• US 5,834,232 A
• Filed: 05/01/1996
• Issued: 11/10/1998
• Est. Priority Date: 05/01/1996
• Status: Expired due to Term

• Current Assignee: Zymogenetics Incorporated (Bristol-Myers Squibb Company)
• Original Assignee: Zymogenetics Incorporated (Bristol-Myers Squibb Company)
• Inventors: Lasser, Gerald, Bishop, Paul D.
• Primary Examiner(s): Witz, Jean C.
• Assistant Examiner(s): HANLEY, SUSAN MARIE
• Application Number:
  US08/641,463
• Time in Patent Office: 923 Days
• Field of Search: 435/68.1, 435/174, 435/177, 435/180, 435/182, 435/193, 530/356, 530/354
• US Class Current: 435/68.1
• CPC Class Codes: A23J 3/06 Gelatine
• CPC Class Codes: A23L 29/284 Gelatin; Collagen
• CPC Class Codes: A61K 47/6903 the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
• CPC Class Codes: A61K 9/1658 Proteins, e.g. albumin, gelatin
• CPC Class Codes: A61K 9/5057 Gelatin
• CPC Class Codes: A61L 27/34 Macromolecular materials
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: G03C 1/30 Hardeners

Abstract

Enzymatically cross-linked protein gels and methods for preparing them are disclosed. The methods comprise adding a transglutaminase, such as factor XIII, to a composition of a temperature-sensitive gel-forming protein, such as gelatin or collagen, and incubating the composition and transglutaminase under gel-forming conditions. The resulting gels have superior strength and thermal stability, and can be used within a variety of medical and industrial applications.

Claims

1. A method of preparing a cross-linked protein gel comprising:

adding a factor XIII transglutaminase to a composition containing gelatin, while the gelatin is in a sol state at or below about 37°

C. so as to form a transglutaminase and gelatin mixture; and

incubating said mixture at a temperature at which the gelatin undergoes gelation, wherein

a cross-linked gelatin gel is formed by cross-linking of the gelatin by the transglutaminase.

2. A method according to claim 1 wherein said composition is an aqueous solution.

3. A method according to claim 1 wherein said composition is a gel.

4. The method of claim 1 wherein said composition further comprises one or more additional proteins selected from the group consisting of fibronectin, von Willebrand factor, vinculin, and laminin.

5. The method of claim 1 wherein said composition further comprises a cytokine or a hormone.

16. A cross-linked gelatin gel prepared by the method of claim 1.

17. The cross-linked gel of claim 16, further comprising one or more additional proteins selected from the group consisting of fibronectin, von Willebrand factor, and laminin, and vinculin.

18. The cross-linked gel of claim 17, wherein said one or more additional proteins is present in said transglutaminase-containing solution in an amount not exceeding 10% by weight of total protein in said gel.

19. The cross-linked gel of claim 16, further comprising a cytokine or a hormone.

20. The cross-linked gel of claim 16 which is coated on a polymeric or paper support.

21. The cross-linked gel of claim 20 further comprising silver halide grains.

22. The cross-linked gel of claim 20 further comprising a dye-forming coupler compound.

23. The cross-linked gel of claim 16 which is in the form of a microcapsule.

24. A pharmaceutical composition comprising a vehicle and an amine-containing drug, wherein said vehicle comprises a cross-linked gelatin gel prepared by the method of claim 1, and wherein said drug is covalently bonded to said gel.

25. A method of preparing a cross-linked protein gel of claim 1, wherein the said mixture is incubated at a temperature from about 37°

C. and decreased to about 4°

C.

26. A method of preparing a cross-linked protein gel of claim 1, wherein the factor XIII transglutaminase and gelatin mixture is incubated at a temperature from about 37°

C. and decreased to about room temperature.

27. The cross linked gel of claim 16, wherein the cross-linked gelatin gel has a Tm of greater than about 65°

C.

28. The cross linked gel of claim 16, wherein the cross-linked gelatin gel has a Tm of greater than about 100°

C.

6. A method of preparing a cross-linked protein gel comprising:

forming an aqueous solution containing gelatin; and

adding a factor XIII transglutaminase to said solution in an amount sufficient to catalyze cross-linking of the gelatin; and

incubating said transglutaminase and said solution under conditions of time and temperature at which the gelatin undergoes gelation, wherein

a cross-linked gelatin gel is formed by cross-linking of the gelatin by the transglutaminase.

7. The method of claim 6 wherein said aqueous solution is subjected to at least one cycle of gelation and melting before the addition of said transglutaminase.

8. The method of claim 7 wherein the transglutaminase concentration is from 0.125 mg/ml to 2.0 mg/ml.

9. The method of claim 6 wherein said transglutaminase-containing solution is incubated at a temperature of from 4°

C. to 33°

C.

10. The method of claim 6 wherein the gelatin concentration is from 1% to 10% by weight and said transglutaminase-containing solution is incubated at a temperature of from 20°

C. to 32°

C.

11. The method of claim 6 wherein said gelatin and factor XIII transglutaminase are present in a mass ratio from 528:

1 to 66;

1.

12. The method of claim 6 wherein said transglutaminase-containing solution further comprises one or more additional proteins selected from the group consisting of fibronectin, von Willebrand factor, vinculin, and laminin.

13. The method of claim 12 wherein said one or more additional proteins is present in said transglutaminase-containing solution in an amount not exceeding 10% by weight of total protein in said gel.

14. The method of claim 6 wherein said transglutaminase-containing solution further comprises a cytokine or a hormone.

15. A method of preparing a cross-linked protein gel comprising:

adding a factor XIII transglutaminase to a temperature sensitive protein gel containing gelatin, whereby said transglutaminase diffuses into the gel and catalyzes cross-linking of the gelatin.