Phosphopantetheinyl Transferase-Catalyzed Formation of Bioactive Hydrogels for Tissue Engineering

Abstract

Synthetic bioactive hydrogels have been widely recognized as key elements of emerging strategies to engineer tissues. However, the current shortage of highly specific and biocompatible methods to form and functionalize these materials hampers their wide pharmaceutical and medical use. In particular, enzymatic reactions are underexplored for the synthesis of bioactive hydrogels. Here, we present an approach by which phosphopantetheinyl transferase (PPTase), a small (16.2 kDa) enzyme that plays a key role in the biosynthesis of many natural products, was employed to catalyze covalent cross-linking of poly(ethylene glycol) (PEG)-based hydrogels. Gels were formed within minutes under physiological conditions by mixing two aqueous precursors containing multiarm PEG macromers end-functionalized with the PPTase substrate Coenzyme A (CoA) and a genetically engineered dimer of a carrier protein. The physicochemical properties of this new class of biomaterials were characterized. Bioactive hydrogels were produced by covalent incorporation of a CoA-functionalized cell adhesion peptide (RGDS), resulting in specific adhesion of primary fibroblasts on the hydrogel surfaces. 3D encapsulation of cells resulted in high cell viability (ca. 95%) and single cell migration over long distances within RGDS-modified gels.

Bioactive factor delivery strategies from engineered polymer hydrogels for therapeutic medicine

“…In addition to HRP/H 2 O 2 and TGase enzymes, other enzymes, such as tyrosinase [170], phosphopantetheinyl transferase [171], an acid phosphatase [172], thermolysin [173], lysyl oxidase [174] and an esterase [175], have been harnessed to catalyze hydrogel formation. However, the instability of some enzymes (such as TGase and tyrosinases) and the presence of remaining enzymes within biomaterials following gelation must be considered when using this gelation approach.…”

PEG-based bioactive hydrogels crosslinked via phosphopantetheinyl transferase

“…PPTase hydrogels show high cell viability, comparable to gels crosslinked by Michael-type addition. Parts of the figure adapted with permission from Reference [14]. Copyright 2010 American Chemical Society.…”

“…c, 1 H NMR of 8-arm PEG-VS macromer (left) and its Coenzyme Amodified version (right) formed via Michael-type addition. Parts of the figure adapted with permission from Reference[14]. Copyright 2010 American Chemical Society.…”

“…a, Scheme of PPTase-mediated cross-linking of PEG hydrogels and obtained 5% hydrogel (stained in blue) after swelling in water; b, time of hydrogel precursors gelation recorded as a function of enzyme concentration; c, oscillatory frequency sweep performed on cross-linked hydrogel shows G'>G'', indicating the presence of a crosslinked elastic polymer network; d, the swelling ratio Q, determined as the swollen gel mass divided by the gel's dry mass.Figure reprintedwith permission from Reference[14]. Copyright 2010 American Chemical Society.…”

A Genetically Modified Protein-Based Hydrogel for 3D Culture of AD293 Cells

“…Moreover, proteins usually serve as cross-linkers via covalent (Michael addition [15], [16], enzyme reaction [17] or site selective conjugation [18], [19]) or non-covalent interactions (specific protein-peptide [20], protein-protein [21], [22] or protein-polysaccharide interactions [23]) in protein-based hydrogels, which require them to have multiple binding sites to their ligands. Some specific amino acid side chain groups such as the lysine’s ε-amine and cysteine’s sulphydryl endow favourable targets for cross-linking reactions [24], [25].…”