*2.3. In Vitro Characterization*

The results of the distribution coefficient logD and protein binding studies are presented in Table 1. All <sup>68</sup>Ga-labeled conjugates showed reasonable hydrophilicity with values ranging from –1.92 for the most lipophilic (SulfoCy7-MAFC-PEG5-Tz) to –2.85 for the most hydrophilic conjugate (SulfoCy5-MAFC-PEG5-Tz). Interestingly, by replacing the acetyl residue by a second PEG5-Tz moiety the hydrophilicity was altered for the SulfoCy5-conjugate while for the corresponding IRDye800CW-conjugate it remained the same. The protein binding of the <sup>68</sup>Ga-labeled FSC-based hybrid imaging agents was consistent over time and ranged from intermediate (30–50%) to high (50–70%). Introducing a SulfoCy5- or -Cy7 residue to the MAFC-Tz scaffold showed a comparable protein binding while the conjugation of the IRDye800CW led to significantly increased values. Interestingly, the replacement of the acetyl moiety by a PEG5-Tz residue did not increase the protein binding compared to the monomeric Tz conjugates. However, this is consistent with the results of a previous study where increasing the number of PEG5-Tz by chelator scaffolding did not increase the protein binding [ *Molecules* **2020**, *25*, x 19]. 4 of 15

**Figure 1.** Representative chromatograms of monomeric FSC-based dual-modality imaging (DMI) agents. (**A**) shows UV-detection at λ = 220 nm, (**B**) represents radio detection of [68Ga]Ga-SulfoCy5- MAFC-PEG5-Tz and [68Ga]Ga-SulfoCy7-MAFC-PEG5-Tz, whereas (**C**) shows radio-ITLC (instant thin layer chromatography) analysis of [68Ga]Ga-IRDye800CW-MAFC-PEG5-Tz using citrate (left) and **Figure 1.** Representative chromatograms of monomeric FSC-based dual-modality imaging (DMI) agents. (**A**) shows UV-detection at λ = 220 nm, (**B**) represents radio detection of [ <sup>68</sup>Ga]Ga-SulfoCy5-MAFC-PEG<sup>5</sup> -Tz and [68Ga]Ga-SulfoCy7-MAFC-PEG<sup>5</sup> -Tz, whereas (**C**) shows radio-ITLC (instant thin layer chromatography) analysis of [68Ga]Ga-IRDye800CW-MAFC-PEG<sup>5</sup> -Tz using citrate (left) and ammonium acetate/ethanol (right) as mobile phase.


ammonium acetate/ethanol (right) as mobile phase. *2.3. In Vitro Characterization*  **Table 1.** Distribution coefficient (logD) and protein binding of <sup>68</sup>Ga-labeled FSC-based Tz-bearing hybrid imaging agents.

Data are presented as mean ± SD (*n* = 3).

the replacement of the acetyl moiety by a PEG5-Tz residue did not increase the protein binding compared to the monomeric Tz conjugates. However, this is consistent with the results of a previous study where increasing the number of PEG5-Tz by chelator scaffolding did not increase the protein binding [19]. The results of cell binding studies of the <sup>68</sup>Ga-labeled FSC-based hybrid imaging agents on CD20-expressing Raji cells pre-treated with Rituximab(RTX)–TCO-modified or non-modified RTX are summarized in Figure 2. All conjugates showed highly specific targeting properties with ratios of specifically to non-specifically bound radioligand ranging from 3 to 5. The binding of the <sup>68</sup>Ga-labeled

**Table 1.** Distribution coefficient (logD) and protein binding of 68Ga-labeled FSC-based Tz-bearing

SulfoCy5-MAFC-PEG5-Tz –2.85 ± 0.08 37.0 ± 0.3 37.4 ± 2.3 37.6 ± 1.2 SulfoCy7-MAFC-PEG5-Tz –1.92 ± 0.05 36.9 ± 0.8 40.4 ± 1.5 41.6 ± 2.7 IRDye800CW-MAFC-PEG5-Tz –2.40 ± 0.05 65.7 ± 1.3 67.3 ± 1.4 67.7 ± 0.2 SulfoCy5-FSC-(PEG5-Tz)2 –2.29 ± 0.10 35.7 ± 0.7 39.1 ± 0.4 40.8 ± 0.6 IRDye800CW-FSC-(PEG5-Tz)2 –2.46 ± 0.09 47.3 ± 0.2 50.3 ± 0.3 54.9 ± 1.1 Data are presented as mean ± SD (*n* = 3).

**logD (pH 7.4) 1 h 2 h 4 h** 

hybrid imaging agents.

monomeric DMI agents was 3.09 <sup>±</sup> 0.58% for [68Ga]Ga-SulfoCy5-MAFC-PEG5-Tz, 4.12 <sup>±</sup> 0.88% for [ <sup>68</sup>Ga]Ga-SulfoCy7-MAFC-PEG5-Tz and 2.88 <sup>±</sup> 0.53% for [68Ga]Ga-IRDye800CW-MAFC-PEG5-Tz which was comparable to the non-fluorescent conjugate [68Ga]Ga-DAFC-PEG5-Tz (4.01 <sup>±</sup> 0.36%) [19]. The binding of the dimeric DMI agents radiolabeled with gallium-68 was significantly higher (*p* < 0.005) in comparison to their monomeric counterparts and resulted to be 5.91 ± 1.62% for [ <sup>68</sup>Ga]Ga-SulfoCy5-FSC-(PEG5-Tz)<sup>2</sup> and 4.59 <sup>±</sup> 1.45% for [68Ga]Ga-IRDye800CW-FSC-(PEG5-Tz)<sup>2</sup> but was significantly lower (*p* < 0.0005) compared to the <sup>68</sup>Ga-labeled non-fluorescent conjugate [ <sup>68</sup>Ga]Ga-MAFC-(PEG5-Tz)<sup>2</sup> (7.35 <sup>±</sup> 0.50%) [19]. Furthermore, the unspecific binding was significantly increased (*p* < 0.005) also when comparing <sup>68</sup>Ga-labeled mono- and dimeric FSC-based DMI agents. specifically to non-specifically bound radioligand ranging from 3 to 5. The binding of the 68Ga-labeled monomeric DMI agents was 3.09 ± 0.58% for [68Ga]Ga-SulfoCy5-MAFC-PEG5-Tz, 4.12 ± 0.88% for [68Ga]Ga-SulfoCy7-MAFC-PEG5-Tz and 2.88 ± 0.53% for [68Ga]Ga-IRDye800CW-MAFC-PEG5-Tz which was comparable to the non-fluorescent conjugate [68Ga]Ga-DAFC-PEG5-Tz (4.01 ± 0.36%) [19]. The binding of the dimeric DMI agents radiolabeled with gallium-68 was significantly higher (*p* < 0.005) in comparison to their monomeric counterparts and resulted to be 5.91 ± 1.62% for [68Ga]Ga-SulfoCy5-FSC-(PEG5-Tz)2 and 4.59 ± 1.45% for [68Ga]Ga-IRDye800CW-FSC-(PEG5-Tz)2 but was significantly lower (*p* < 0.0005) compared to the 68Ga-labeled non-fluorescent conjugate [68Ga]Ga-MAFC-(PEG5-Tz)2 (7.35 ± 0.50%) [19]. Furthermore, the unspecific binding was significantly increased (*p* < 0.005) also when comparing 68Ga-labeled mono- and dimeric FSC-based DMI agents.

*Molecules* **2020**, *25*, x 5 of 15

summarized in Figure 2. All conjugates showed highly specific targeting properties with ratios of

The results of cell binding studies of the 68Ga-labeled FSC-based hybrid imaging agents on CD20-

**Figure 2.** Cell binding studies of [68Ga]Ga-SulfoCy5-MAFC-PEG5-Tz (A), [68Ga]Ga-SulfoCy7-MAFC-PEG5-Tz (B), [68Ga]Ga-IRDye800CW-MAFC-PEG5-Tz (C), [68Ga]Ga-SulfoCy5-FSC-(PEG5-Tz)2 (D), and [68Ga]Ga-IRDye800CW-FSC-(PEG5-Tz)2 (E) on CD20-expressing Raji cells pre-treated with *trans*-**Figure 2.** Cell binding studies of [68Ga]Ga-SulfoCy5-MAFC-PEG<sup>5</sup> -Tz (A), [68Ga]Ga-SulfoCy7-MAFC-PEG<sup>5</sup> -Tz (B), [68Ga]Ga-IRDye800CW-MAFC-PEG<sup>5</sup> -Tz (C), [68Ga]Ga-SulfoCy5-FSC-(PEG<sup>5</sup> -Tz)<sup>2</sup> (D), and [68Ga]Ga-IRDye800CW-FSC-(PEG<sup>5</sup> -Tz)<sup>2</sup> (E) on CD20-expressing Raji cells pre-treated with *trans*-cyclooctene (TCO) modified rituximab (white bars) and non-modified antibody (black bars).

cyclooctene (TCO) modified rituximab (white bars) and non-modified antibody (black bars). Fluorescence microscopy of SulfoCy5-conjugates is presented in Figure 3 and clearly shows the high target specificity as the RTX–TCO pre-treated Raji cells revealed SulfoCy5-specific signaling Fluorescence microscopy of SulfoCy5-conjugates is presented in Figure 3 and clearly shows the high target specificity as the RTX–TCO pre-treated Raji cells revealed SulfoCy5-specific signaling whereas the negative control, lacking the TCO functionality, did not.

#### whereas the negative control, lacking the TCO functionality, did not. *2.4. Biodistribution Studies*

The ex vivo biodistribution profile of the <sup>68</sup>Ga-labeled FSC-based hybrid imaging agents in healthy BALB/C mice 1 h post injection (p.i.) is summarized in Figure 4. Except from the SulfoCy7-conjugate all imaging probes showed relatively fast blood clearance and low retention in muscle and bone indicating suitable properties for imaging applications matching the short half-life of Gallium-68. The SulfoCy7-conjugate also revealed higher activities in all organs compared to their SulfoCy5 and IRDye800CW counterparts, matching the highest lipophilicity of this conjugate, which was therefore not further explored in imaging studies. Comparable tissue activity was found comparing Sulfo-Cy5 and IRdye800CW conjugates for most organs, except for kidneys where values for IRDye800CW were considerably higher (>20%IA/g). Monomeric MAFC-PEG5-Tz conjugates showed comparable biodistribution to non-dye conjugated FSC-based tetrazines [19] and considerably lower activities especially in liver and spleen (~2%IA/g) as compared to FSC-(PEG5-Tz)<sup>2</sup> conjugates with around 10%IA/g. Lung retention was relatively high for all compounds (~5–10%IA/g), no specific reason for this phenomenon could be found.

**Figure 3.** Fluorescence microscopy of CD20-expressing Raji cells pre-treated with RTX (upper lane) and with RTX–TCO (lower lane, negative control) prior to incubation with SulfoCy5-MAFC-PEG5-Tz. Each lane shows the fluorescence signal of the FSC-based conjugate (red), optical signal from AlexaFlour488-WGA labeled cells (green) and both **Figure 3.** Fluorescence microscopy of CD20-expressing Raji cells pre-treated with RTX (upper lane) and with RTX–TCO (lower lane, negative control) prior to incubation with SulfoCy5-MAFC-PEG<sup>5</sup> -Tz. Each lane shows the fluorescence signal of the FSC-based conjugate (red), optical signal from AlexaFlour488-WGA labeled cells (green) and both images merged (from left to right). *Molecules* **2020**, *25*, x 7 of 15

**Figure 4.** Ex vivo biodistribution profile studies of 68Ga-labeled FSC-based Tz-bearing dual-modality imaging agents in healthy BALB/c mice 1 h after administration. **Figure 4.** Ex vivo biodistribution profile studies of <sup>68</sup>Ga-labeled FSC-based Tz-bearing dual-modality imaging agents in healthy BALB/c mice 1 h after administration.
