Peptide hydrogel properties and its applications

Abstract

Peptide hydrogels having a self-assembling, 3-dimensional nanofiber matrix are described. The nanofiber matrix comprises an amphiphilic peptide and optionally albumin. The peptide comprises (consists of) a terminal hydrophobic region, a central turning region, and a terminal hydrophilic region. Methods of making such hydrogels are also described, along with methods of using the hydrogels as scaffolding for tissue engineering, hemostatic agents, as well as 3-dimensional cell cultures, and for drug delivery, encapsulation of active agents (therapeutic cells, molecules, drugs, compounds), cell transplantation, cell storage, virus culture and storage.

1 . A hemostatic agent useful for promoting blood clotting and/or reducing hemorrhaging, said hemostatic agent comprising a peptide, wherein said peptide is amphiphilic and comprises a terminal hydrophobic region, a central turning region, and a terminal hydrophilic region. 
     
     
         2 . The hemostatic agent of  claim 1 , wherein said peptide is in a solution comprising said peptide dispersed, dissolved, or suspended in a solvent system. 
     
     
         3 . The hemostatic agent of  claim 1 , wherein said hydrophilic region is derived from a β-spiral motif of spider flagelliform silk protein, and said hydrophobic and turning regions are derived from the third trans-membrane segment of subunit IV in the dihydropyridine sensitive human muscle L-type calcium channel. 
     
     
         4 . The hemostatic agent of  claim 1 , further comprising albumin and/or calcium. 
     
     
         5 . The hemostatic agent of  claim 1 , wherein said hydrophobic region consists of 2-15 amino acid residues selected from the group consisting of F, L, I, V, A, H, D, P, and G, wherein at least one amino acid residue is I. 
     
     
         6 . The hemostatic agent of  claim 1 , wherein said hydrophilic region consists of 5-20 amino acid residues, wherein at least at least one amino acid residue is G, at least one amino acid residue is P, and at least one amino acid residue is D. 
     
     
         7 . The hemostatic agent of  claim 1 , wherein said turning region consists of 1-12 amino acid residues, wherein at least one amino acid residue is G or S. 
     
     
         8 . A method of treating a wound site or reducing hemorrhaging in a subject having a wound, said method comprising:
 delivering a hemostatic agent to the site of said wound, said hemostatic agent comprising a peptide, wherein said peptide is amphiphilic and comprises a terminal hydrophobic region, a central turning region, and a terminal hydrophilic region.   
     
     
         9 . The method of  claim 8 , wherein said delivering comprises administering hemostatic agent to said subject by injecting said hemostatic agent into said subject at or near the site of the wound. 
     
     
         10 . The method of  claim 8 , wherein said delivering comprises applying said hemostatic agent to the site of the wound. 
     
     
         11 . The method of  claim 8 , wherein said delivering comprises dispensing said hemostatic agent into said wound. 
     
     
         12 . The method of  claim 8 , further comprising obtaining a blood sample from said subject and mixing said peptide with said blood sample prior to said delivering, wherein said peptide self-assembles into a hydrogel after said mixing. 
     
     
         13 . The method of  claim 8 , wherein said peptide is in a solution comprising said peptide dispersed, dissolved, or suspended in a solvent system. 
     
     
         14 . The method of  claim 8 , wherein said hemostatic agent consists of said peptide. 
     
     
         15 . The method of  claim 14 , wherein after said delivering, said peptide self-assembles into a hydrogel in situ at the site of said wound. 
     
     
         16 . The method of  claim 8 , wherein said hemostatic agent consists of said peptide and a source of albumin, said hemostatic agent being in the form of a hydrogel. 
     
     
         17 . A peptide hydrogel comprising a 3-dimensional nanofiber matrix, said nanofiber matrix comprising an amphiphilic peptide and optionally albumin, wherein said peptide consists of a terminal hydrophobic region, a central turning region, and a terminal hydrophilic region, said hydrogel further comprising an extracellular matrix ligand, lipid, protein, or biopolymer attached to said hydrophilic region of said peptide. 
     
     
         18 . The peptide hydrogel of  claim 17 , wherein said extracellular matrix ligand is selected from the group consisting of GRGD (SEQ ID NO:70), RGDS (SEQ ID NO:71), RGD, HAV, RGDSY (SEQ ID NO:72), and KYRGDS (SEQ ID NO:73). 
     
     
         19 . The peptide hydrogel of  claim 17 , wherein said protein is a laminin protein selected from the group consisting of YIGSR (SEQ ID NO:74) and IKVAV (SEQ ID NO:75). 
     
     
         20 . A method of storing and expanding cells, said method comprising
 mixing cells with a self-assembling amphiphilic peptide and a hydrogelation agent, wherein said peptide consists of a terminal hydrophobic region, a central turning region, and a terminal hydrophilic region, to yield a 3-dimensional cell culture comprising said cells embedded in a hydrogel matrix, said hydrogel matrix comprising a 3-dimensional nanofiber matrix comprising said peptide; and   maintaining said cells in said hydrogel matrix under cell culture conditions.   
     
     
         21 . The method of  claim 20 , wherein said hydrogelation agent is a source of albumin or calcium. 
     
     
         22 . The method of  claim 21 , wherein said source of albumin is serum-supplemented cell media comprising said cells. 
     
     
         23 . The method of  claim 20 , wherein said maintaining comprises covering said hydrogel matrix with cell media and incubating said hydrogel containing said cells under said cell culture conditions 
     
     
         24 . The method of  claim 20 , wherein said cells are stem cells, further comprising expanding said cells in said hydrogel matrix for at least about 20 passages after said mixing, wherein said stem cells remain in an undifferentiated state. 
     
     
         25 . The method of  claim 24 , further comprising isolating said cells from said hydrogel. 
     
     
         26 . The method of  claim 25 , wherein said isolating comprises:
 subjecting said hydrogel to a mechanical force to disrupt the hydrogel matrix;   diluting said hydrogel with additional cell media; and   separating said cultured cells from said hydrogel.   
     
     
         27 . The method of  claim 26 , wherein said mechanical force is selected from the group consisting of pipetting, centrifugation, vibration, injection, filtration, spraying, and combinations thereof. 
     
     
         28 . The method of  claim 20 , wherein said cells are red blood cells.