Liang He, Russell Stephen J, Wood David J, Tronci Giuseppe
Clothworkers' Centre for Textile Materials Innovation for Healthcare, School of Design, University of Leeds, UK.
J Mater Chem B. 2018 Jun 14;6(22):3703-3715. doi: 10.1039/c7tb03035e. Epub 2018 May 22.
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.
具有基质金属蛋白酶（MMP）调节功能的医疗器械对于恢复诸如慢性伤口、肩袖撕裂和癌症等严重炎症状态下的组织稳态非常必要。在这类器械中引入MMP调节功能通常是通过加载快速扩散的螯合因子（如乙二胺四乙酸（EDTA））或MMP可裂解底物来实现的，这分别在不可控的药代动力学和酶促降解性方面引发了问题。为了实现固有的、长期的、由器械诱导的MMP调节，本研究调查了一种异羟肟酸（HA）-甲基丙烯酸化胶原共轭物的合成，作为一种具有可控酶促降解性的无可溶性因子MMP调节水凝胶网络的构建模块。这是通过两步合成路线实现的：（i）在三乙胺（TEA）存在下，用诱导光子网络的甲基丙烯酸酐（MA）加合物对I型胶原进行功能化；（ii）在与羟胺反应之前，用一种水溶性碳二亚胺激活该甲基丙烯酸化产物，从而产生MMP螯合HA功能。通过2,4,6-三硝基苯磺酸（TNBS）测定法观察到胶原胺几乎定量的甲基丙烯酸化；这是避免在碳二亚胺介导的胶原羧基激活过程中发生分子内交联副反应的关键。通过将剩余羧基功能转化为乙二胺（EDA）间接定量HA加合物的摩尔含量，因此通过TNBS和茚三酮测定法在共轭物中均显示出12 - 16 mol%的HA。所得的紫外线固化的含HA胶原水凝胶在体外培养4天后，分别证明可使MMP - 9和MMP - 3的活性降低高达约13 RFU%和约32 RFU%，同时显示出平均质量损失在8 - 21 wt%范围内。在引入HA加合物后，仍可观察到天然I型胶原的二向色性和电泳图谱，这表明在相应的HA - 甲基丙烯酸化胶原共轭物中保留了三螺旋结构和化学序列。在G292细胞培养4天后未观察到水凝胶诱导的毒性反应，而与甲基丙烯酸化水凝胶相比，含HA水凝胶的压缩模量和凝胶含量较低，这可能与HA的自由基清除活性有关。本工作中描述的新型合成策略为旨在设计仿生、炎症响应性医疗器械的胶原材料的系统化学操作提供了新的见解。
