Institute for Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institute, 5232 Villigen, Switzerland.
ACS Biomater Sci Eng. 2020 Jan 13;6(1):425-432. doi: 10.1021/acsbiomaterials.9b01248. Epub 2019 Dec 13.
Fibroblast growth factor 2 (FGF-2) is a small 18 kDa protein with clinical potential for ischemic heart disease, wound healing, and spinal cord injury. However, the therapeutic potential of systemic FGF-2 administration is challenged by its fast elimination. Therefore, we deployed genetic codon expansion to integrate an azide functionality to the FGF-2 N-terminus, which was site-directly decorated with poly(ethylene glycol) (PEG) through bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC). PEGylated FGF-2 was as bioactive as wild-type FGF-2 as demonstrated by cell proliferation and Erk phosphorylation of fibroblasts. The PEGylated FGF-2 conjugate was radiolabeled with [In] Indium cation ([In]In) to study its biodistribution through noninvasive imaging by single-photon emission computed tomography (SPECT) and by quantitative activity analysis of the respective organs in healthy mice. This study details the biodistribution pattern of site-specific PEGylated FGF-2 in tissues after intravenous (iv) administration compared to the unconjugated protein. Low accumulation of the PEGylated FGF-2 variant in the kidney and the liver was demonstrated, whereas specific uptake of PEGylated FGF-2 into the retina was significantly diminished. In conclusion, site-specific PEGylation of FGF-2 by SPAAC resulted in a superior outcome for the synthesis yield and in conjugates with excellent biological performances with a gain of half-life but reduced tissue access in vivo.
成纤维细胞生长因子 2（FGF-2）是一种 18kDa 的小蛋白，具有治疗缺血性心脏病、伤口愈合和脊髓损伤的临床潜力。然而，全身给予 FGF-2 的治疗潜力受到其快速消除的挑战。因此，我们采用遗传密码扩展将叠氮化物功能整合到 FGF-2 N 端，通过生物正交应变促进的叠氮化物-炔烃环加成（SPAAC）将其位点特异性修饰聚乙二醇（PEG）。PEGylated FGF-2 与野生型 FGF-2 一样具有生物活性，如成纤维细胞的增殖和 Erk 磷酸化所示。PEGylated FGF-2 缀合物用 [In]铟阳离子（[In]In）放射性标记，通过单光子发射计算机断层扫描（SPECT）进行非侵入性成像研究其体内分布，并通过对健康小鼠相应器官的定量活性分析研究其体内分布。这项研究详细描述了与未缀合蛋白相比，静脉内（iv）给予后组织中定点 PEGylated FGF-2 的分布模式。证明 PEGylated FGF-2 变体在肾脏和肝脏中的积累较低，而 PEGylated FGF-2 对视网膜的特异性摄取明显减少。总之，SPAAC 对 FGF-2 的定点 PEGylation导致合成产率提高，并且获得了半衰期延长但体内组织通透性降低的优异生物性能的缀合物。
