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 [111In] Indium cation ([111In]In3+) 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.
Keywords: PEGylation; SPECT imaging; genetic code expansion; strain-promoted azide−alkyne cycloaddition (SPAAC).