A hydroxamic acid-methacrylated collagen conjugate for the modulation of inflammation-related MMP upregulation.

He Liang,Stephen J Russell,Giuseppe Tronci,David J Wood

doi:10.1039/c7tb03035e

Abstract

Medical devices with matrix metalloproteinase (MMP)-modulating functionality are highly desirable to restore tissue homeostasis in critical inflammation states, such as chronic wounds, rotator cuff tears and cancer. The introduction of MMP-modulating functionality in such devices is typically achieved via loading of either rapidly diffusing chelating factors, e.g. EDTA, or MMP-cleavable substrates, raising issues in terms of non-controllable pharmacokinetics and enzymatic degradability, respectively. Aiming to accomplish inherent, long-term, device-induced MMP regulation, this study investigated the synthesis of a hydroxamic acid (HA)-methacrylated collagen conjugate as the building block of a soluble factor-free MMP-modulating hydrogel network with controlled enzymatic degradability. This was realised via a two-step synthetic route: (i) type I collagen was functionalised with photonetwork-inducing methacrylic anhydride (MA) adducts in the presence of triethylamine (TEA); (ii) this methacrylated product was activated with a water-soluble carbodiimide prior to reaction with hydroxylamine, resulting in MMP-chelating HA functions. Nearly-quantitative methacrylation of collagen amines was observed via 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay; this was key to avoiding intramolecular crosslinking side reactions during the carbodiimide-mediated activation of collagen carboxyl groups. The molar content of HA adducts was indirectly quantified via the conversion of remaining carboxyl functions into ethylenediamine (EDA), so that 12-16 mol% HA was revealed in the conjugate by both TNBS and Ninhydrin assays. Resulting UV-cured, HA-bearing collagen hydrogels proved to induce up to ∼13 and ∼32 RFU% activity reduction of MMP-9 and MMP-3, respectively, following 4-day incubation in vitro, whilst displaying an averaged mass loss in the range of 8-21 wt%. Dichroic and electrophoretic patterns of native type I collagen could still be observed following the introduction of HA adducts, suggesting preserved triple helix architecture and chemical sequence in respective HA-methacrylated collagen conjugate. No hydrogel-induced toxic response was observed following the 4-day culture of G292 cells, whilst a lower compression modulus and gel content were measured in HA-bearing compared to methacrylated hydrogels, likely related to HA radical scavenging activity. The novel synthetic strategies described in this work provide a new insight into the systematic chemical manipulation of collagen materials aiming at the design of biomimetic, inflammation-responsive medical devices.

Highlights

Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent proteinases, which play a key role in breaking down extracellular matrix (ECM) proteins
The medical device is realised at the molecular scale via the synthesis of a UV-cured hydrogel network of methacrylated collagen triple helices, in which the MMP-modulating capability is achieved via the introduction of hydroxamic acid (HA) adducts to carboxyl groups of methacrylated collagen
The TEA-catalysed reaction of methacrylic anhydride (MA) with collagen proceeds via an amine-initiated nucleophilic addition/elimination mechanism and was carried out prior to coupling with HA. This reaction with MA was exploited for two purposes (Scheme 1): (i) to introduce photoactive functions on to free amino terminations of collagen, e.g.lysines and amino termini, responsible for the subsequent formation of a hydrogel network;[36,40] (ii) to protect highly-reactive amino terminations prior to selective derivatisation of carboxyl into MMP-chelating functional groups, so that intra-crosslinking side reactions could be minimised

Summary

Introduction

Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent proteinases, which play a key role in breaking down extracellular matrix (ECM) proteins. The levels of MMPs are precisely regulated and are responsible for key physiological events such as homeostasis, tissue remodelling, wound healing and angiogenesis.[1,2,3,4,5,6,7] Together with their beneficial role, numerous clinical studies have indicated that the overexpression of MMPs a Clothworkers’ Centre for Textile Materials Innovation for Healthcare, School of Design, University of Leeds, UK Paper. The proteolytic removal of the pro-domain for MMP activation is mediated by other proteinases, including MMPs, whilst MMP regulation can be further achieved by complexation with TIMPs, which block access to the active site.[24]

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