This review covers publications on siderophores applied for molecular imaging applications, mainly for radionuclide-based imaging. Siderophores are low molecular weight chelators produced by bacteria and fungi to scavenge essential iron. Research on these molecules has a continuing history over the past 50 years. Many biomedical applications have been developed, most prominently the use of the siderophore desferrioxamine (DFO) to tackle iron overload related diseases. Recent research described the upregulation of siderophore production and transport systems during infection. Replacing iron in siderophores by radionuclides, the most prominent Ga-68 for PET, opens approaches for targeted imaging of infection; the proof of principle has been reported for fungal infections using ⁶⁸Ga-triacetylfusarinine C (TAFC). Additionally, fluorescent siderophores and therapeutic conjugates have been described and may be translated to optical imaging and theranostic applications. Siderophores have also been applied as bifunctional chelators, initially DFO as chelator for Ga-67 and more recently for Zr-89 where it has become the standard chelator in Immuno-PET. Improved DFO constructs and bifunctional chelators based on cyclic siderophores have recently been developed for Ga-68 and Zr-89 and show promising properties for radiopharmaceutical development in PET. A huge potential from basic biomedical research on siderophores still awaits to be utilized for clinical and translational imaging.

Within the last years ⁸⁹Zr has attracted considerable attention as long-lived radionuclide for positron emission tomography (PET) applications. So far desferrioxamine B (DFO) has been mainly used as bifunctional chelating system. Fusarinine C (FSC), having complexing properties comparable to DFO, was expected to be an alternative with potentially higher stability due to its cyclic structure. In this study, as proof of principle, various FSC-RGD conjugates targeting α_vß₃ integrins were synthesized using different conjugation strategies and labeled with ⁸⁹Zr. In vitro stability, biodistribution, and microPET/CT imaging were evaluated using [⁸⁹Zr]FSC-RGD conjugates or [⁸⁹Zr]triacetylfusarinine C (TAFC). Quantitative ⁸⁹Zr labeling was achieved within 90 min at room temperature. The distribution coefficients of the different radioligands indicate hydrophilic character. Compared to [⁸⁹Zr]DFO, [⁸⁹Zr]FSC derivatives showed excellent in vitro stability and resistance against transchelation in phosphate buffered saline (PBS), ethylenediaminetetraacetic acid solution (EDTA), and human serum for up to 7 days. Cell binding studies and biodistribution as well as microPET/CT imaging experiments showed efficient receptor-specific targeting of [⁸⁹Zr]FSC-RGD conjugates. No bone uptake was observed analyzing PET images indicating high in vivo stability. These findings indicate that FSC is a highly promising chelator for the development of ⁸⁹Zr-based PET imaging agents.

Over the last years Gallium-68 ((68)Ga) has received tremendous attention for labeling of radiopharmaceuticals for positron emission tomography (PET). (68)Ga labeling of biomolecules is currently based on bifunctional chelators containing aminocarboxylates (mainly DOTA and NOTA). We have recently shown that cyclic peptide siderophores have very good complexing properties for (68)Ga resulting in high specific activities and excellent metabolic stabilities, in particular triacetylfusarinine-C (TAFC). We postulated, that, starting from its deacetylated form (Fusarinine-C (FSC)) trimeric bioconjugates are directly accessible to develop novel targeting peptide based (68)Ga labeled radiopharmaceuticals. As proof of principle we report on the synthesis and (68)Ga-radiolabeling of a trimeric FSC-RGD conjugate, [(68)Ga]FSC-(RGD)3, targeting αvβ3 integrin, which is highly expressed during tumor-induced angiogenesis. Synthesis of the RGD peptide was carried out applying solid phase peptide synthesis (SPPS), followed by the coupling to the siderophore [Fe]FSC via in situ activation using HATU/HOAt and DIPEA. Subsequent demetalation allowed radiolabeling of FSC-(RGD)3 with (68)Ga. The radiolabeling procedure was optimized regarding peptide amount, reaction time, temperature as well buffer systems. For in vitro evaluation partition coefficient, protein binding, serum stability, αvβ3 integrin binding affinity, and tumor cell uptake were determined. For in vitro tests as well as for the biodistribution studies αvβ3 positive human melanoma M21 and αvβ3 negative M21-L cells were used. [(68)Ga]FSC-(RGD)3 was prepared with high radiochemical yield (>98%). Distribution coefficient was -3.6 revealing a hydrophilic character, and an IC50 value of 1.8±0.6 nM was determined indicating a high binding affinity for αvβ3 integrin. [(68)Ga]FSC-(RGD)3 was stable in PBS (pH7.4), FeCl3- and DTPA-solution as well as in fresh human serum at 37°C for 2hours. Biodistribution assay confirmed the receptor specific uptake found in vitro. Uptake in the αvβ3 positive tumor was 4.3% ID/g 60min p.i. which was 3-fold higher than the monomeric [(68)Ga]NODAGA-RGD. Tumor to blood ratio of approx. 8 and tumor to muscle ratio of approx. 7 were observed. [(68)Ga]FSC-(RGD)3 serves as an example for the feasibility of a novel class of bifunctional chelators based on cyclic peptide siderophores and shows excellent targeting properties for αvβ3 integrin in vivo for imaging tumor-induced neovascularization.

Some [⁶⁸Ga]siderophores show promise in specific and sensitive imaging of infection. Here, we compare the in vitro and in vivo behaviour of selected Ga-68 and Zr-89 labelled siderophores.

Fusarinine C (FSC), a siderophore‐based chelator coupled with the model peptide c(RGDfK) (FSC(succ‐RGD)₃), revealed excellent targeting properties in vivo using positron emission tomography (PET). Here, we report the details of radiolabeling conditions and specific activity as well as selectivity for ⁶⁸Ga.⁶⁸Ga labeling of FSC(succ‐RGD)₃ was optimized regarding peptide concentration, pH, temperature, reaction time, and buffer system. Specific activity (SA) of [⁶⁸Ga]FSC(succ‐RGD)₃ was compared with ⁶⁸Ga‐1,4,7‐triazacyclononane, 1‐glutaric acid‐4,7 acetic acid RGD ([⁶⁸Ga]NODAGA‐RGD). Stability was evaluated in 1000‐fold ethylenediaminetetraacetic acid (EDTA) solution (pH 7) and phosphate‐buffered saline (PBS). Metal competition tests (Fe, Cu, Zn, Al, and Ni) were carried out using [⁶⁸Ga]‐triacetylfusarinine C.High radiochemical yield was achieved within 5 min at room temperature, in particular allowing labeling with ⁶⁸Ga up to pH 8 with excellent stability in 1000‐fold EDTA solution and PBS. The 10‐fold to 20‐fold lower concentrations of FSC(succ‐RGD)₃ led to the same radiochemical yield compared with [⁶⁸Ga]NODAGA‐RGD with SA up to 1.8 TBq/µmol. Metal competition tests showed high selective binding of ⁶⁸Ga to FSC.FSC is a multivalent siderophore‐based bifunctional chelator allowing fast and highly selective labeling with ⁶⁸Ga in a wide pH range and results in stable complexes with high SA. Thus it is exceptionally well suited for the development of new ⁶⁸Ga‐tracers for in vivo molecular imaging with PET.