Hamano Nobuhito, Murata Masaharu, Kawano Takahito, Piao Jing Shu, Narahara Sayoko, Nakata Ryosuke, Akahoshi Tomohiko, Ikeda Tetsuo, Hashizume Makoto
Center for Advanced Medical Innovation, ‡Department of Advanced Medical Initiatives, Faculty of Medical Sciences, and §Innovation Center for Medical Redox Navigation, Kyushu University , 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
ACS Appl Mater Interfaces. 2016 Mar 2;8(8):5114-23. doi: 10.1021/acsami.5b11902. Epub 2016 Feb 16.
Postoperative pancreatic fistula (POPF) is the most serious and challenging complication following gastroenterological surgery. Activated pancreatic juice leaking from the organ remnant contains proteases that attack the surrounding tissue, potentially leading to severe inflammation, tissue necrosis, and fistula formation. However, it is difficult to observe pancreatic leakage during surgery and to evaluate the protease activity of leaked fluid at the patient's bedside. This report describes a protein nanocage-based protease ratiometric sensor comprising a pancreatic protease-sensitive small heat-shock protein (HSP) 16.5, which is a naturally occurring protein in Methanococcus jannaschii that forms a spherical structure by self-assembly of 24 subunits, and a chemically conjugated donor-acceptor dye pair for Förster resonance energy transfer (FRET). The HSP-FRET probe was constructed by subunit exchange of each dye-labeled engineered HSP, resulting in a spherical nanocage of approximately 10 nm in diameter, which exhibited very high stability against degradation in blood plasma and no remarkable toxicity in mice. The efficiency of FRET was found to depend on both the dye orientation and the acceptor/donor ratio. Pancreatic proteases, including trypsin, α-chymotrypsin, and elastase, were quantitatively analyzed by fluorescence recovery with high specificity using the HSP-FRET nanoprobe. Furthermore, the HSP-FRET nanoprobe was sufficiently sensitive to detect POPF in the pancreatic juice of patients using only the naked eye within 10 min. Thus, this novel nanoprobe is proposed as an effective and convenient tool for the detection of POPF and the visualization of activated pancreatic juice during gastroenterological surgery.
术后胰瘘（POPF）是胃肠外科手术后最严重且最具挑战性的并发症。从胰腺残端渗漏出的活化胰液含有蛋白酶，会攻击周围组织，可能导致严重炎症、组织坏死和瘘管形成。然而，手术过程中很难观察到胰腺渗漏情况，也难以在患者床边评估渗漏液的蛋白酶活性。本报告描述了一种基于蛋白质纳米笼的蛋白酶比率传感器，它由对胰腺蛋白酶敏感的小分子热休克蛋白（HSP）16.5（一种天然存在于詹氏甲烷球菌中的蛋白质，通过24个亚基自组装形成球形结构）和用于Förster共振能量转移（FRET）的化学偶联供体 - 受体染料对组成。HSP - FRET探针通过对每个染料标记的工程化HSP进行亚基交换构建而成，形成直径约10 nm的球形纳米笼，该纳米笼在血浆中对降解具有非常高的稳定性，且在小鼠体内无明显毒性。发现FRET效率取决于染料取向和受体/供体比率。使用HSP - FRET纳米探针通过荧光恢复以高特异性对包括胰蛋白酶、α - 糜蛋白酶和弹性蛋白酶在内的胰腺蛋白酶进行了定量分析。此外，HSP - FRET纳米探针灵敏度足够高，仅用肉眼在10分钟内就能检测出患者胰液中的POPF。因此，这种新型纳米探针被认为是一种有效且便捷的工具，可用于胃肠外科手术中POPF的检测以及活化胰液的可视化。
