Strehin Iossif, Gourevitch Dmitri, Zhang Yong, Heber-Katz Ellen, Messersmith Phillip B
Northwestern University, Evanston, IL 60208, Biomedical Engineering Department, Materials Science and Engineering Department, Chemical and Biological Engineering Department, Chemistry of Life Processes Institute, Institute for Bionanotechnology in Medicine, Robert H. Lurie Comprehensive Cancer Center.
Biomater Sci. 2013 Jun 1;1(6):603-613. doi: 10.1039/C3BM00201B.
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 -hydroxysuccinimide (NHS) activated oxo-esters and -cysteine (-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 -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.
氧代酯介导的天然化学连接（OMNCL）是更通用的天然化学连接（NCL）反应的一种变体，NCL反应被广泛用于肽片段的化学选择性连接。虽然OMNCL已用于各种肽连接和表面生物分子修饰，但它通常在不适用于生物学环境的苛刻条件下进行。在本报告中，我们描述了OMNCL在聚合物水凝胶形成、体外细胞封装和体内植入中的应用。含有N-羟基琥珀酰亚胺（NHS）活化的氧代酯和巯基（-Cys）端基的多价聚合物前体由支化聚乙二醇（PEG）化学合成。在生理pH值下，将NHS和-Cys功能化的PEG水溶液混合后，水凝胶迅速形成。定量1H NMR实验表明，反应通过OMNCL途径进行，包括硫醇捕获形成硫酯中间体，随后进行S到N的酰基重排以产生酰胺交联。发现pH值和温度会影响凝胶化速率，从而可以将凝胶化时间从几秒调整到几分钟。OMNCL水凝胶最初会膨胀，然后收缩，最终相对湿重增加至0%并达到平衡。这种独特的行为影响了凝胶的硬度，这归因于网络结合的Cys残基之间潜在的二硫键交联形成。OMNCL水凝胶对水合组织具有粘附性，产生的搭接剪切粘附强度为46 kPa。封装在OMNCL水凝胶中的细胞保持高活力，并且通过在小鼠皮下注射原位形成OMNCL水凝胶产生的急性炎症反应最小。OMNCL是一种在生物学环境中进行水凝胶化学交联的有前途的策略，并且是伤口愈合、组织修复、药物递送和组织工程等体内应用的有吸引力的候选者。
