*2.5. Imaging Studies*

procedures.

*2.5. Imaging Studies*  A simple pretargeting model using bone targeting TCO-alendronate was chosen for a straight forward proof of concept *in vivo*. PET/CT images revealed specific uptake of both IRDye800CW A simple pretargeting model using bone targeting TCO-alendronate was chosen for a straight forward proof of concept in vivo. PET/CT images revealed specific uptake of both IRDye800CW conjugates in bone structures of TCO-alendronate pre-treated mice, whereas no uptake was seen in

conjugates in bone structures of TCO-alendronate pre-treated mice, whereas no uptake was seen in mice treated with alendronate alone, proving the *in vivo* specificity of both constructs. Both conjugates revealed predominant renal excretion and good contrast 1 h p.i. Figure 5 shows images of [68Ga]Ga-

confirmed by optical imaging of excised bones with a high accumulation in the joint area, which is not seen in the alendronate control animal. Comparable images of [68Ga]Ga- IRDye800CW-MAFC-PEG5-Tz are shown in supplementary data (Figure S1) indicating less pronounced uptake in comparison with its monomeric counterpart both in PET/CT and optical images, however without being statistically significant. This is in line with the higher *in vitro* binding (Figure 2) and our findings when comparing non derivatized mono- and dimeric Tetrazine-FSC conjugates [19], showing that dimerization has a positive effect on *in vivo* targeting properties. Overall, this proves that dyeconjugated tetrazines based on the FSC scaffold can be used for pretargeting applications combining PET and optical imaging. Further studies, ideally in appropriate pretargeting tumor models, are required to judge the full potential of these compounds for tumor imaging and image-guided mice treated with alendronate alone, proving the in vivo specificity of both constructs. Both conjugates revealed predominant renal excretion and good contrast 1 h p.i. Figure 5 shows images of [ <sup>68</sup>Ga]Ga-IRDdye800CW-FSC-(PEG5-Tz)<sup>2</sup> revealing high accumulation in joints and spine with excellent contrast and no uptake in the alendronate control images. This specific accumulation could be confirmed by optical imaging of excised bones with a high accumulation in the joint area, which is not seen in the alendronate control animal. Comparable images of [68Ga]Ga- IRDye800CW-MAFC-PEG5-Tz are shown in supplementary data (Figure S1) indicating less pronounced uptake in comparison with its monomeric counterpart both in PET/CT and optical images, however without being statistically significant. This is in line with the higher in vitro binding (Figure 2) and our findings when comparing non derivatized mono- and dimeric Tetrazine-FSC conjugates [19], showing that dimerization has a positive effect on in vivo targeting properties. Overall, this proves that dye-conjugated tetrazines based on the FSC scaffold can be used for pretargeting applications combining PET and optical imaging. Further studies, ideally in appropriate pretargeting tumor models, are required to judge the full potential of these compounds for tumor imaging and image-guided procedures. *Molecules* **2020**, *25*, x 8 of 15

**Figure 5.** Imaging of 68Ga-labeled IRDdye800CW-FSC-(PEG5-Tz)2 in mice receiving alendronate alone (left row) or alendronate-TCO (right), PET/CT images: transverse slices (top) and sagittal slices (middle), yellow arrows indicate uptake in joints and spine, white arrow indicates the bladder. Bottom image: Optical image of excised bones of the lower limbs. Both PET and OI indicate higher accumulation in bone of alendronate-TCO pre-treated mice as compared to controls. **Figure 5.** Imaging of <sup>68</sup>Ga-labeled IRDdye800CW-FSC-(PEG<sup>5</sup> -Tz)<sup>2</sup> in mice receiving alendronate alone (left row) or alendronate-TCO (right), PET/CT images: transverse slices (top) and sagittal slices (middle), yellow arrows indicate uptake in joints and spine, white arrow indicates the bladder. Bottom image: Optical image of excised bones of the lower limbs. Both PET and OI indicate higher accumulation in bone of alendronate-TCO pre-treated mice as compared to controls.

This is to our knowledge the first report on combining PET and NIRF imaging based on a single tetrazine based pretargeting vector. The FSC scaffold, which is a versatile chelator for Gallium-68, was well suited to develop this approach. In contrast to other attempts, where the dye was conjugated to an antibody [16,17], our approach allows a DMI application without modifying the actual targeting vector, thereby avoiding separate development and characterization of the two versions with and without the dye conjugate. This is to our knowledge the first report on combining PET and NIRF imaging based on a single tetrazine based pretargeting vector. The FSC scaffold, which is a versatile chelator for Gallium-68, was well suited to develop this approach. In contrast to other attempts, where the dye was conjugated to an antibody [16,17], our approach allows a DMI application without modifying the actual targeting vector, thereby avoiding separate development and characterization of the two versions with and without the dye conjugate.

UltiMate 3000 autosampler, Ultimate 3000 column compartment (25 °C oven temperature), UltiMate 3000 Variable Wavelength Detector (Dionex, Germering, Germany; UV detection at λ = 220 nm) a radio detector (GabiStar, Raytest; Straubenhardt, Germany), Jupiter 5µm C18 300 Å 150 × 4.6 mm (Phenomenex Ltd., Aschaffenburg, Germany) column with acetonitrile (ACN)/H2O/0.1% trifluoroacetic acid (TFA) as mobile phase; flow rate of 1 mL/min; gradient: 0.0–1.0 min 10% ACN, 1.0–12.0 min 10–60% ACN, 13.0–15.0 min 60–80% ACN, 15.0–16.0 min 80–10% ACN, and 16.0–20.0

Preparative RP-HPLC*.* Sample purification via RP-HPLC was carried out as follows: Gilson 322 Pump with a Gilson UV/VIS-155 detector (UV detection at λ = 220 nm) using a PrepFC™ automatic

**3. Materials and Methods** 

*3.1. Analytics* 

min 10% ACN.
