(426b) Preparation and Characterization of Polypeptide Hydrogels as Synthetic Extracellular Matrices for Cellular Scaffolds

The synthetic polypeptides, poly(γ-propargyl-L-glutamate) (PPLG), provide great potential for the development of novel hydrogels as extracellular matrix (ECM) for biomedical applications. Incorporating PPLG macromers into conventional step-growth PEG hydrogels results in a hybrid PEG-polypeptide hydrogel platform. This allows for highly clustered adhesion ligands on an α-helical scaffold via side chain modifications and a more diverse spectrum of swelling and mechanical properties than standard PEG gels.

PPLG was synthesized by ring-opening polymerization of the N-carboxy anhydride of γ-propargyl-L-glutamate and adopts a rigid α-helix conformation. The PPLG macromer was synthesized by grafting the “clickable” side chains of PPLG with a mixture of short ethylene glycol chains and norbornene groups. The PPLG hydrogels were prepared by step-growth UV-crosslinking using PPLG macromer and 4-arm polyethylene glycol (PEG) crosslinker bearing thiol end groups. Two control hydrogels were prepared by replacing PPLG macromers with 8-arm PEG crosslinker bearing norbornene end groups or poly(γ-propargyl-D,L-glutamate) (PPDLG) macromer with a flexible random coil conformation, which was a random copolymer produced from a 50:50 mixture of γ-propargyl-L-glutamate and γ-propargyl-D-glutamate monomers.

The physical and mechanical properties of these hydrogels, including swelling ratio, permeability, osmotic pressure, elastic modulus, and rheological properties were characterized by swelling test, fluorescence recovery after photo-bleaching (FRAP), osmotic swelling measurement, atomic force microscopy (AFM) indentation in fluid, and rheometry, respectively. The microstructure of the hydrogels were examined by cryogenic transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS).

The hydrogels were modified with PHSRN-K-RGD cell attachment motif for use as synthetic extracellular matrices. 2D cell culture studies were conducted with human induced pluripotent stem cell-derived endothelial cells (iPS-ECs), envisioning applications involving monolayers of endothelial cells. In addition, peptide-degradable hydrogels were prepared using linear or 4-arm PEG crosslinkers bearing matrix metalloproteinase (MMP)-degradable sequences with PEG, PPLG, or PPDLG macromers. 3D cell culture studies are currently underway on the peptide-degradable hydrogels for cell encapsulation and vascularization.