Hydrogels formed by oxo-ester mediated native chemical ligation

Abstract

Oxo-ester mediated native chemical ligation (OMNCL) is a variation of the more general native chemical ligation (NCL) reaction that is widely employed for chemoselective ligation of peptide fragments. While OMNCL has been used for a variety of peptide ligations and for biomolecular modification of surfaces, it is typically practiced under harsh conditions that are unsuitable for use in a biological context. In this report we describe the use of OMNCL for polymer hydrogel formation, in-vitro cell encapsulation, and in-vivo implantation. Multivalent polymer precursors containing N-hydroxysuccinimide (NHS) activated oxo-esters and N-cysteine (N-Cys) endgroups were chemically synthesized from branched poly(ethylene glycol) (PEG). Hydrogels formed rapidly at physiologic pH upon mixing of aqueous solutions of NHS and N-Cys functionalized PEGs. Quantitative 1H NMR experiments showed that the reaction proceeds through an OMNCL pathway involving thiol capture to form a thioester intermediate, followed by an S-to-N acyl rearrangement to yield an amide cross-link. pH and temperature were found to influence gelation rate, allowing tailoring of gelation times from a few seconds to a few minutes. OMNCL hydrogels initially swelled before contracting to reach an equilibrium increase in relative wet weight of 0%. This unique behavior impacted the gel stiffness and was attributed to latent formation of disulfide cross-links between network-bound Cys residues. OMNCL hydrogels were adhesive to hydrated tissue, generating a lap shear adhesion strength of 46 kPa. Cells encapsulated in OMNCL hydrogels maintained high viability, and in-situ formation of OMNCL hydrogel by subcutaneous injection in mice generated a minimal acute inflammatory response. OMNCL represents a promising strategy for chemical cross-linking of hydrogels in a biological context and is an attractive candidate for in-vivo applications such as wound healing, tissue repair, drug delivery, and tissue engineering.

Injectable polymeric hydrogels for the delivery of therapeutic agents: A review

“…Thereafter, this group applied their NCL cross-linked and peptide-modified hydrogel systems to encapsulate pancreatic islet cells for the treatment of type I diabetes mellitus [69]. Another promising class of NCL is oxo-ester mediated reaction, which has been used to develop biologically compatible hydrogel [70]. This system shared the same biocompatibility property and showed faster gelation rate in physiological pH compared to general NCL chemistry-based hydrogels.…”

Oxygen, Metabolism, and Regeneration: Lessons from Mice

“…However, it was also shown to act on PHDs and to modulate HIF-1α levels (6). Given the results that HIF-1α protein expression appeared to be key in the regenerative response of MRL mice (6), 1,4-DPCA was used in combination with a hydrogel delivery system to slowly release low concentrations of the drug over a 5 day period in mice (92). The amount of drug used was 75-fold less than the dose for soluble drug given orally in rats for prolyl hydroxylation studies in vivo (6,90).…”

“…The amount of drug used was 75-fold less than the dose for soluble drug given orally in rats for prolyl hydroxylation studies in vivo (6,90). Mimicking the kinetics of the MRL- HIF-1α expression response, 1,4-DPCA/hydrogel was injected subcutaneously at the base of the neck into non-regenerative mice and regenerative ear hole responses assessed (92). Non-regenerative (Swiss Webster) mice that received the drug regenerated ear lesions in the same amount of time as regenerative (MRL) mice (92).…”

“…Mimicking the kinetics of the MRL- HIF-1α expression response, 1,4-DPCA/hydrogel was injected subcutaneously at the base of the neck into non-regenerative mice and regenerative ear hole responses assessed (92). Non-regenerative (Swiss Webster) mice that received the drug regenerated ear lesions in the same amount of time as regenerative (MRL) mice (92). The observed phenotypic changes, including remodeling, inflammation, de-differentiation, cell division and migration were replicated in otherwise non-regenerative mice, compared to regenerative mice.…”

Biofabrication of reinforced 3D-scaffolds using two-component hydrogels

“…20 This ligation reaction involves conjugation between an activated ester and an N-terminal cysteine and has been previously demonstrated to be highly compatible with incorporated cells and bioactive molecules. 21 Recently, we reported thermosensitive OMNCL cross-linked hydrogels that showed tunable degradation rates and mechanical properties. 22 Importantly, solutions of relatively low polymer concentrations of 12 wt% were transferred into mechanically stable hydrogels as a result of the combined physical and chemical cross-linking mechanism.…”