Stable macroscopic membranes formed by self-assembly of amphiphilic peptides and uses therefor

• US 5,670,483 A
• Filed: 11/30/1994
• Issued: 09/23/1997
• Est. Priority Date: 12/28/1992
• Status: Expired due to Term

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Zhang, Shuguang, Lockshin, Curtis, Rich, Alexander, Holmes, Todd
• Primary Examiner(s): GRIMES, ERIC BURTON
• Application Number:
  US08/346,849
• Time in Patent Office: 1,028 Days
• Field of Search: 530/300, 530/350, 514/12, 514/13, 514/14
• US Class Current: 530/300
• CPC Class Codes: A61K 9/009 Sachets, pouches characterised by the material or function of the envelope with gastric retention A61K9/0065; sachets which are not administered but function merely as a container are classified according to the content, e.g. sachets comprising powder for reconstitution of a drink A61K9/0095
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 14/395 from Saccharomyces
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 2533/50 Proteins
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06

Abstract

Described herein is the self-assembly of amphiphilic peptides, i.e., peptides with alternating hydrophobic and hydrophilic residues, into macroscopic membranes. The membrane-forming peptides are greater than 12 amino acids in length, and preferably at least 16 amino acids, are complementary and are structurally compatible. Specifically, two peptides, (AEAEAKAK)₂ (ARARADAD)₂, were shown to self-assemble into macroscopic membranes. Conditions under which the peptides self-assemble into macroscopic membranes and methods for producing the membranes are also described. The macroscopic membranes have several interesting properties: they are stable in aqueous solution, serum, and ethanol, are highly resistant to heat, alkaline and acidic pH, chemical denaturants, and proteolytic digestion, and are non-cytotoxic. The membranes are potentially useful in biomaterial applications such as slow-diffusion drug delivery systems, artificial skin, and separation matrices, and as experimental models for Alzheimer'"'"'s disease and scrapie infection. The sequence of the peptide, EAK16, was derived from a putative Z-DNA binding protein from yeast, called zuotin. The cloning and characterization of the ZUO1 gene are also described.

Claims

1. A macroscopic membrane which is formed by self-assembly of amphiphilic peptides in an aqueous solution containing monovalent metal cations, wherein the peptides contain 12 or more amino acids, have alternating hydrophobic and hydrophilic amino acids and are complementary and structurally compatible.

2. The membrane of claim 1 wherein the peptides are homogeneous.

3. The membrane of claim 1 which is non-cytotoxic to mammalian cells.

4. The membrane of claim 1 wherein the hydrophilic amino acids are acidic and basic amino acids.

5. The membrane of claim 4 wherein the acidic amino acids are independently selected from the group consisting of aspartic acid and glutamic acid.

6. The membrane of claim 5 wherein the basic amino acids are independently selected from the group consisting of arginine, lysine, histidine and ornithine.

7. The membrane of claim 6 wherein the hydrophobic amino acids are selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine and glycine.

8. The membrane of claim 7 wherein the hydrophobic amino acids are alanine.

9. The membrane of claim 1 wherein the peptides are 12 to about 200 amino acids in length.

10. The membrane of claim 9 wherein the peptides are greater than 16 and less than about 200 amino acids.

11. The membrane of claim 1 wherein the peptides are soluble in aqueous solution in the absence of monovalent metal cations.

12. The membrane of claim 1 which is composed of β

-sheets.

13. The membrane of claim 1 wherein the difference in interpeptide distance of the complementary peptides upon self-assembly is less than 3Å

.

14. The membrane of claim 13 wherein the interpeptide distance is constant.

15. The membrane of claim 1 wherein the peptides are chemically synthesized.

16. The membrane of claim 1 wherein the monovalent metal cations are selected from the group consisting of Li⁺, Na⁺, K⁺ and Cs⁺.

17. The membrane of claim 16 wherein the monovalent metal cation is Na⁺.

18. The membrane of claim 17 wherein the monovalent metal cation is added at a concentration of at least 5 mM.

19. The membrane of claim 1 wherein the peptides are present in the aqueous solution at a concentration of at least 1 mg/ml.

20. The membrane of claim 19 wherein the peptides are present in the aqueous solution at a concentration of at least about 10 mg/ml.

21. The membrane of claim 19 wherein the peptide is added to an aqueous solution containing monovalent metal cations.

22. The membrane of claim 1 wherein the membrane is stable in water at a temperature below the boiling point of the water.

23. The membrane of claim 22 wherein the CD spectra of the membrane is unaffected in water at a temperature below the boiling point of water.

24. The membrane of claim 1 wherein the membrane is unaffected at a pH from 1.5 to about 11.

25. The membrane of claim 24 wherein the pH profile of the membrane shows less than 10% decrease in ellipticity of the CD spectra by the treatment.

26. The membrane of claim 25 wherein the membrane is stable in aqueous solution containing less than about 10% sodium dodecyl sulfate, less than 7M guanidine hydrochloride, or less than 8M urea.

27. The membrane of claim 26 wherein the CD spectra is unaffected when the membrane is in aqueous solution containing less than about 10% sodium dodecyl sulfate, less than 7M guanidine hydrochloride, or less than 8M urea.

28. The membrane of claim 1 wherein the membrane is not degraded by trysin, α

-chymotrypsin, papain, protease K or pronase.

29. The membrane of claim 1 wherein the membrane is stable in serum or ethanol.

30. The membrane of claim 1 wherein the peptides are selected from the group consisting of:

AKAKAEAEAKAKAEAE, (SEQ ID NO;

     32);

AKAEAKAEAKAEAKAE, (SEQ ID NO;

     33);

EAKAEAKAEAKAEAKA, (SEQ ID NO;

     34);

KAEAKAEAKAEAKAEA, (SEQ ID NO;

     35);

AEAKAEAKAEAKAEAK, (SEQ ID NO;

     36);

ADADARARADADARAR, (SEQ ID NO;

     37);

ARADARADARADARAD, (SEQ ID NO;

     38);

DARADARADARADARA, (SEQ ID NO;

     39);

RADARADARADARADA, (SEQ ID NO;

     40);

ADARADARADARADAR, (SEQ ID NO;

     41);

ARADAKAEARADAKAE, (SEQ ID NO;

     42);

AKAEARADAKAEARAD, (SEQ ID NO;

     43);

ARAKADAEARAKADAE, (SEQ ID NO;

     44);

AKARAEADAKARADAE, (SEQ ID NO;

     45);

AQAQAQAQAQAQAQAQ, (SEQ ID NO;

     46);

VQVQVQVQVQVQVQVQ, (SEQ ID NO;

     47);

YQYQYQYQYQYQYQYQ, (SEQ ID NO;

     48);

HQHQHQHQHQHQHQHQ, (SEQ ID NO;

     49);

ANANANANANANANAN, (SEQ ID NO;

     50);

VNVNVNVNVNVNVNVN, (SEQ ID NO;

     51);

YNYNYNYNYNYNYNYN, (SEQ ID NO;

     52);

HNHNHNHNHNHNHNHN, (SEQ ID NO;

     53),

ANAQANAQANAQANAQ, (SEQ ID NO;

     54);

AQANAQANAQANAQAN, (SEQ ID NO;

     55);

VNVQVNVQVNVQVNVQ, (SEQ ID NO;

     56);

VQVNVQVNVQVNVQVN, (SEQ ID NO;

     57);

YNYQYNYQYNYQYNYQ, (SEQ ID NO;

     58);

YQYNYQYNYQYNYQYN, (SEQ ID NO;

     59);

HNHQHNHQHNHQHNHQ, (SEQ ID NO;

     60);

HQHNHQHNHQHNHQHN, (SEQ ID NO;

     61);

AKAQADAKAQADAKAQAD, (SEQ ID NO;

     19);

VKVQVDVKVQVDVKVQVD, (SEQ ID NO;

     62);

YKYQYDYKYQYDYKYQYD, (SEQ ID NO;

63 ); and

HKHQHDHKHQHDHKHQHD, (SEQ ID NO;

     64).

31. The membrane of claim 1 wherein the peptides are (AEAEAKAK)_n (amino acids 310-317 of SEQ ID NO:

2) and n is greater than or equal to 2.

32. The membrane of claim 1 wherein the peptides are (ARARADAD)_n (amino acids 1-8 of SEQ ID No:

3) and n is greater than or equal to 2.

33. The membrane of claim 1 wherein the peptides are (RADARADA)_n (amino acids 1-8 of SEQ ID NO:

40) and n is greater than or equal to 2.

34. A macroscopic membrane which is formed by self assembly of amphiphilic peptides in an aqueous solution containing monovalent metal cations;

wherein the amphiphilic peptides are water-soluble, 12 to 200 amino acids in length, have alternating hydrophobic and hydrophilic amino acids, and are complementary and structurally compatible; and

wherein the hydrophilic amino acids are complementary acidic and basic amino acids and the difference in interpeptide distance of the complementary peptides upon self-assembly is less than 3Å

.

35. A macroscopic membrane formed by self-assembly of a peptide having the sequence (amino acid numbers 310 to 317 of SEQ ID NO:

2) (Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)_n, where n is greater than or equal to 2.

36. A macroscopic membrane formed by a peptide having the sequence (amino acid numbers 1 to 8 of SEQ ID NO:

40) (Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala)_n, where n is greater than or equal to 2.

37. A method for forming a macroscopic membrane comprising forming an aqueous mixture of peptides, which are 12 or more amino acids in length, have alternating nonpolar and hydrophilic amino acids, and are complementary and structurally compatible, and monovalent metal cations under conditions suitable for self-assembly of the peptide into the macroscopic membrane and allowing the membrane to be formed.

38. The method of claim 37 wherein the peptides are homogeneous.

39. The method of claim 38 wherein the peptides have a sequence (amino acid numbers 310 to 317 of SEQ ID NO:

2) (Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)_n, where n is greater than or equal to 2.

40. The method of claim 38 wherein the peptides have a sequence amino acid numbers 1 to 8 of SEQ ID NO:

3) (Ala-Arg-Ala-Arg-Ala-Asp-Ala-Asp)_n, where n is greater than or equal to 2.

41. The method of claim 38 wherein the peptide is chemically synthesized.

42. The method of claim 38 wherein the monovalent metal cations are selected from Li⁺, Na⁺, and K⁺.

43. The method of claim 38 wherein the peptides are added to an aqueous solution containing the monovalent metal cations.

44. The method of claim 43 wherein the aqueous solution is phosphate-buffered saline.

45. The method of claim 38 wherein the suitable conditions comprise the absence of an inhibitor of the self-assembly of the peptides into the macroscopic membrane.

46. The method of claim 45 wherein the inhibitor is a divalent metal cation.

47. The method of claim 45 wherein the inhibitor is sodium dodecyl sulfate.

48. The method of claim 38 wherein the suitable conditions comprise a pH of less than 12.