Programmable Keratinous Bio-adhesives for Recalcitrant Wound Recovery

项目摘要 慢性、顽固性伤口和溃疡给糖尿病、肥胖和老年人的治疗带来了重大挑战 患者需要新的治疗方案来解决发病率上升的问题;需要有针对性的方法来重新启动 正常的愈合级联。组织粘合剂是广泛使用的斯台普斯和缝线的替代品。这些迅速 固化聚合物凝胶，当应用于伤口时，与标准相比，减少疤痕形成、住院时间和感染 缝线，同时消除了针和缝线移除的需要。不幸的是，这些伤口治疗 选项提供很少的生物活性;不适合治疗慢性伤口。细胞外基质（ECM）敷料（例如 角蛋白）是生物活性，但提供的粘合强度很小，且依赖于动物提取物， 生物相容性和生物活性。旨在扩大糖尿病和老年患者的可用治疗选择， 这项研究旨在设计、构建和测试新型的基因功能化重组蛋白， 治疗生物活性作为可配置的药物递送装置的基础;从构建 生物活性、生物相容性组织粘合剂，用于伤口复发风险高的患者的早期伤口护理。 目前，没有工程化ECM蛋白组织粘合剂。作为基础设计，我将采用 已建立的基因组编码生物体聚合物合成技术， 两种非标准氨基酸（nsAAs）、粘膜粘附性L-二羟基苯丙氨酸（L-DOPA） 和光交叉-异戊烯氨基酸（NorAA），每一种转化为单独的上皮化诱导， 重组人头发角蛋白异二聚体亚基，分别为K85和K35。天然和nsAA-角蛋白将 组装成支架，无论是通过缓慢的巯基介导的细丝组装或快速， 交联，并进行结构表征和细胞活力测定。NorAA-DOPA-角蛋白支架 预期可在数秒内快速固化并呈现显著增强的粘合强度， 可用的皮肤粘合剂。将对设计的粘合剂支架变体进行体内表征 C57 BL/6 J糖尿病小鼠;例如愈合率、粘附强度、形态测定分析和组织病理学 测定;将结果与当前可用的组织粘合剂进行比较。我假设将这些新的角蛋白 粘合剂的皮肤伤口将显着提高愈合率，阻止出血，并减少 糖尿病小鼠的疤痕。

PROJECT SUMMARY Chronic, recalcitrant wounds and ulcers pose significant challenges to treating diabetic, obese, and elderly patients. New treatment options are needed to address rising rates; requiring a targeted approach to re-initiate the normal healing cascade. Tissue adhesives are widely used alternatives to staples and sutures. These rapidly curing polymer gels, when applied to wounds, reduce scarring, hospital time, and infection compared to standard sutures, while eliminating the need for needles and suture removal. Unfortunately, these wound treatment options offer little bioactivity; unsuitable for treating chronic wounds. Extracellular matrix (ECM) dressings (e.g. keratin) are bioactive, but offer little adhesive strength and rely on animal extractions that reduce efficacy in biocompatibility and bioactivity. Aimed at broadening available treatment options for diabetic and aging patients, this research seeks to design, build, and test novel genetically functionalized recombinant proteins with innate therapeutic bioactivity as a foundation for configurable drug delivery devices; starting with the construction of a bioactive, biocompatible tissue adhesive for early wound care in patients at high risk of wound recalcitrance. Currently, there are no engineered ECM protein tissue adhesives. As a foundational design, I will employ established genomically recoded organism polymer synthesis technologies for multiple site-specific incorporations of two non-standard amino acids (nsAAs), muco-adhesive L-dihydroxyphenylalanine (L-DOPA) and photo-cross-linkable norbornene amino acid (NorAA), each into separate epithelialization-inducing, recombinant human hair keratin heterodimer subunits, K85 and K35, respectively. Native and nsAA-keratins will be assembled into scaffolds, either via slow thiol-mediated filament assembly or rapid, on-site norbornene crosslinking, and subjected to structural characterization and cell viability assays. NorAA-DOPA-keratin scaffolds are expected to rapidly cure in seconds and present significantly enhanced adhesive strength, comparable to available dermal adhesives. In vivo characterizations of designed adhesive scaffold variants will be performed on C57BL/6J diabetic mice; e.g. healing rates, adhesive strength, morphometric analyses, and histopathological assays; comparing results to currently available tissue adhesives. I hypothesize that applying these novel keratin adhesives to recalcitrant dermal wounds will significantly enhance healing rates, block bleeding, and reduce scarring in diabetic mice.