Biomolecular Hydrogels Formed and Degraded via Site-Specific Enzymatic Reactions

Abstract

We present polymeric hydrogel biomaterials that are biomimetic both in their synthesis and degradation. The design of oligopeptide building blocks with dual enzymatic responsiveness allows us to create polymer networks that are formed and functionalized via enzymatic reactions and are degradable via other enzymatic reactions, both occurring under physiological conditions. The activated transglutaminase enzyme factor XIIIa was utilized for site-specific coupling of prototypical cell adhesion ligands and for simultaneous cross-linking of hydrogel networks from factor XIIIa substrate-modified multiarm poly(ethylene glycol) macromers. Ligand incorporation is nearly quantitative and thus controllable, and does not alter the network's macroscopic properties over a concentration range that elicits specific cell adhesion. Living mammalian cells can be encapsulated in the gels without any noticeable decrease in viability. The degradation of gels can be engineered to occur, for example, via cell-secreted matrix metalloproteinases, thus rendering these gels interesting for biomedical applications such as drug delivery systems or smart implants for in situ tissue engineering.

Heparin-binding domain of fibrin(ogen) binds growth factors and promotes tissue repair when incorporated within a synthetic matrix

“…To investigate this function, we incorporated Fg β15-66 (2) into a multifunctional poly(ethylene glycol) (PEG) matrix designed to mimic fibrin's main functions. The fibrin-mimetic matrix backbone comprised two eight-arm PEG peptide conjugates (at a concentration of 1.75%) crosslinkable by the fibrin stabilizing factor, transglutaminase factor XIIIa (16,17). PEG was conjugated with a peptide containing a factor XIIIa substrate sequence derived from α 2 -plasmin inhibitor (α 2 PI 1-8 , NQEQVSPL) (18) or with a lysine donor peptide containing a substrate sequence for matrix metalloproteases (MMPs) and plasmin (VPMSMRGG) (19), allowing degradation of the matrix in situ.…”

Enzyme Mediated Site-Specific Surface Modification

“…In previous work we have shown that FXIIIa can be employed to crosslink star-shaped PEG polymers that are functionalized with either Lys or TG substrates to form synthetic hydrogels. Moreover, we have shown that the FXIIIa-catalyzed coupling scheme allows for the covalent incorporation of growth factors during the formation of these PEG hydrogels, without compromising the protein's bioactivity [41][42] . Here, we aim to use this enzymatic reaction to site-specifically immobilize growth factors or other bioactive molecules on a surface.…”

“…In a second step, PEG-VS were functionalized with Lys and TG substrates via Michel-type addition to give TG-PEG and Lys-PEG precursors. Functionalization and characterization of these precursors was performed as described elsewhere 42 . In brief, peptides were added to PEG-VS in a 1.2 fold molar excess over VS groups in 0.3 M triethanolamine (pH 8.0) at 37 °C for 2 h, followed by dialysis (Snake Skin, MWCO 10k, PIERCE, Rockford, IL, USA) against ultrapure water for 4 days at 4 °C.…”

“…Small aliquots (200 U/ml) of activated factor XIIIa were stored at -80 °C for further use [41][42] .…”

Hydrogel-Based Platforms for the Regeneration of Osteochondral Tissue and Intervertebral Disc

“…Indeed PEG hydrogels recently approved by the FDA for cosmetics, personal care products and pharmaceuticals, show peculiar properties, such as good biocompatibility, non-immunogenity, and resistance to protein adsorption which make their use satisfactory for different biomedical applications including surface modification, bio-conjugation, drug delivery and tissue engineering [47,48]. Three major crosslinking methods have been used to make PEG hydrogels, including radiation of linear or branched PEG polymers [49,50], free radical polymerization (FRP) of PEG acrylates [51], and specific chemical reactions, such as condensation [52], Michael-type additions [53,54], Click chemistry [55], and enzymatic reactions [56]. The most common approach to make PEG hydrogels is photo-polymerization, which utilizes light to convert liquid PEG macromer solutions into solid hydrogels at physiological temperature and pH.…”