Hydrogel Comprising A Scaffold Macromer Crosslinked With A Peptide And A Recognition Motif

Patent metadata

Field	Value
Publication number	US-2018010091-A1
Application number	US-201615543866-A
Country	US
Kind code	A1
Filing date	Jan 15, 2016
Priority date	Jan 15, 2015
Publication date	Jan 11, 2018
Grant date	—

Abstract

Methods of forming, dissolving, and functionalizing an extracellular matrix gel on demand based on cross-linking, modification, and dissolution of hydrogels using transpeptidase (e.g. sortase) are disclosed. Also provided are hydrogels comprising one or more macromers crosslinked to a mixture of peptides, wherein all or a portion of the peptides in the mixture comprise a recognition motif cleavable by a transpeptidase (e.g., sortase).

First claim

1 . A hydrogel comprising one or more scaffold macromers crosslinked to a mixture of peptides, wherein all or a portion of the peptides in the mixture comprise a recognition motif cleavable by a transpeptidase.

2 . The hydrogel of claim 1 , wherein the transpeptidase is a sortase or a sortase variant.

3 . The hydrogel of claim 1 , wherein the recognition sequence comprises a motif selected from the group consisting of: LPXSG, LPXTG, and LAXTG.

4 . The hydrogel of any of claim 1 , wherein 0.001% to 80% of the peptides in the mixture comprise a recognition motif cleavable by a first transpeptidase.

5 . The hydrogel of claim 4 , wherein each peptide that comprises a first recognition sequence cleavable by the first transpeptidase does not comprise a second recognition motif cleavable by a second transpeptidase.

6 . The hydrogel of any of claim 1 , wherein the peptide further comprises a sequence cleavable by a protease.

7 . The hydrogel of claim 6 , wherein the protease is an endopeptidase or a metalloprotease.

8 . The hydrogel of claim 1 , wherein the peptide comprises the amino acid sequence GCRDLPRTGGPQGIWGQDRCG.

9 . The hydrogel of claim 1 , wherein a portion of the peptides in the mixture is crosslinked to a macromer at its N-terminus, and is free at its C-terminus.

10 . The hydrogel of claim 1 , wherein the hydrogel encapsulates a cell, a tissue, or an organ.

11 . The hydrogel of claim 1 , wherein the scaffold macromer is selected from any one or more of polyethyleneglycol (PEG), a dextran, hyaluronic acid, nipaam, alginate, polyacrylic acid, polyhydroxymethacrylate, elastin polypeptide, silk polypeptide, water-soluble polypeptide, chitosan, agarose, heparin sulfate, or heparin.

12 . The hydrogel of claim 11 , wherein the PEG is a linear or a branched PEG.

13 . The hydrogel of claim 11 , wherein the water-soluble polypeptide is a branched polypeptide.

14 . A method of forming a hydrogel dissolvable by a transpeptidase, said method comprising:
combining 1) a mixture of peptides, wherein all or a portion of the peptides in the mixture comprise a recognition motif cleavable by a transpeptidase, each peptide having a first crosslinking moiety; 2) one or more scaffold macromers having a second crosslinking moiety; and 3) a suitable crosslinking agent under suitable conditions that promote crosslinking of the first and second crosslinking moieties, thereby forming a hydrogel dissolvable by a transpeptidase.

15 . The method of claim 14 , wherein the transpeptidase is a sortase or a sortase variant.

16 . The method of claim 14 , wherein the recognition motif comprises a sequence selected from the group consisting of: LPXSG, LPXTG, and LAXTG.

17 . The method of claim 14 , wherein 0.001% to 80% of the peptides in the mixture comprise a recognition motif cleavable by a first transpeptidase.

18 . The method of claim 17 , wherein a peptide that comprises a first recognition motif cleavable by a first transpeptidase does not comprise a second recognition motif cleavable by a second transpeptidase.

19 . The method of claim 14 , wherein the peptide further comprises a sequence cleavable by a protease.

20 . The method of claim 19 , wherein the protease is an endoprotease or a metalloprotease.

21 . The method of claim 14 , wherein the peptide comprises the amino acid sequence GCRDLPRTGGPQGIWGQDRCG.

22 . The method of claim 14 , wherein the mixture of peptides further comprises a terminal peptide having a recognition motif cleavable by a transpeptidase, said terminal peptide having a crosslinking moiety on one end.

23 . The method of claim 14 , further comprising combining a cell, a tissue, or an organ.

24 . The method of claim 14 , wherein the scaffold macromer is selected from any one or more of polyethylene glycol (PEG), a dextran, hyaluronic acid, nipaam, alginate, polyacrylic acid, polyhydroxymethacrylate, elastin polypeptide, silk polypeptide, water-soluble polypeptide, chitosan, agarose, heparin sulfate, or heparin.

25 . The method of claim 24 , wherein the PEG is a linear or branched PEG.

26 . The method of claim 24 , wherein the water-soluble polypeptide is a branched polypeptide.

27 . A method of dissolving the hydrogel of claim 1 , said method comprising treating the hydrogel with a first transpeptidase and a peptide comprising an acceptor substrate sequence of the first transpeptidase under conditions that promote dissolution of the hydrogel, thereby dissolving the hydrogel.

28 . The method of claim 27 , wherein the acceptor substrate sequence comprises NH 2 -(G) n , wherein n is equal to or greater than 1.

29 . A method of dissolving a hydrogel, said method comprising:
treating a hydrogel comprising a transpeptidase recognition motif with a transpeptidase and a peptide comprising an acceptor substrate sequence under conditions that promote dissolution of the hydrogel, thereby dissolving the hydrogel.

30 . The method of claim 29 , wherein the transpeptidase is a sortase or a sortase variant.

31 . The method of claim 29 , wherein the transpeptidase recognition motif comprises a sequence selected from the group consisting of: LPXSG, LPXTG, and LAXTG.

32 . The method of claim 29 , wherein the acceptor substrate sequence comprises NH 2 -(G) n , wherein n is equal to or greater than 1.

33 . The method of claim 29 , wherein the acceptor substrate sequence comprises NH 2 -triglycine (GGG).

34 . The method of claim 29 , wherein the hydrogel encapsulates a cell, a tissue, or an organ.

35 . The method of claim 29 , wherein the hydrogel is pretreated with sortase prior to treatment with the peptide.

36 . The method of claim 29 , wherein the hydrogel is sufficiently dissolved to release the cell, tissue, or organ.

37 . A method of forming a hydrogel comprising a pendant transpeptidase recognition motif, said method comprising:
combining one or more scaffold macromers having a first crosslinking moiety, a peptide comprising a transpeptidase recognition motif having a second crosslinking moiety at its N-terminal end, and a suitable crosslinking agent under conditions that promote crosslinking of the first and second crosslinking moieties, thereby forming a hydrogel comprising a pendant transpeptidase recognition motif.

38 . The method of claim 37 , wherein the transpeptidase is a sortase.

39 . The method of claim 38 , wherein the transpeptidase recognition motif comprises a sequence selected from the group consisting of: LPXSG, LPXTG, and LAXTG.

40 . The method of claim 39 , wherein the transpeptidase recognition motif comprises LPRTG.

41 . The method of claim 37 further comprising treating the hydrogel with a biomolecule having an acceptor substrate sequence that comprises NH 2 -(G) n , where n is equal to or greater than 1.

42 . The method of claim 41 , wherein the acceptor substrate sequence comprises NH 2 -triglycine (GGG).

43 . The method of claim 41 , wherein the biomolecule is a growth factor or an adhesion factor.

44 . The method of claim 37 , wherein the scaffold macro

Assignees

• Massachusetts Inst Technology

Inventors

• Griffith Linda G
• Imperiali Barbara
• Cook Christi D
• Ahrens Caroline Chopko
• Renggli Kasper
• Valdez Macias Jorge L

Classifications

• G01N33/575

  for cancer · CPC title

• C08G2650/20

  Cross-linking · CPC title

• C08G65/33396

  having oxygen in addition to nitrogen · CPC title

• G01N33/574

  Physics · mapped topic

• C12N2533/30

  Synthetic polymers · CPC title