What question did this study set out to answer?

The research aims to compare various thiol bioconjugation strategies for attaching zirconium-89 to human serum albumin.

April 23, 2026ACS Bio & Med Chem Au0 citationsOpen Access

Side-by-Side Comparison of Different Thiol Bioconjugation Strategies for the Chemoselective Radiolabeling of Human Serum Albumin with Zirconium-89

JKJulia KronbergerUniversity of Vienna BNBarbora NeužilováInstitute of Molecular and Translational Medicine AFAnja FederaUniversity of Vienna

Key Points

The research aims to compare various thiol bioconjugation strategies for attaching zirconium-89 to human serum albumin.
Comparison of maleimides, phenyloxadiazole methyl sulfone, and vinylketone for thiol-specific functionalization of HSA.
Synthesis and conjugation of bifunctional chelating agents to HSA, followed by radiolabeling with zirconium-89.
Assessment of stability and imaging performance of the resulting conjugates in biological models.
Conjugation efficiency ranked as maleimides > phenyloxadiazole > vinylketone.
[89Zr]Zr-DFO*-POD-HSA demonstrated high tumor uptake and visualization effectiveness in PET imaging.
Unexpected uptake of radioactivity was noted in bones for the POD-HSA conjugate.

Abstract

Different bioconjugation strategies are available for the cysteine (Cys)-specific functionalization of proteins with different payloads, including imaging probes and (pro-)drugs. Most commonly applied linkers include maleimides (mal); however, because of the sometimes-observed instability of the formed thiosuccinimidyl linkage, its suitability for in vivo applications has been challenged. Consequently, several alternatives have been developed and compared to mal as a benchmark, yet examples of a direct comparison among new methodologies are scarce. We herein report a comparison of the use of mal, phenyloxadiazole methyl sulfone (PODS), and vinylketone (VK) as functional groups for the thiol-specific functionalization of human serum albumin (HSA) via its available free Cys34. Bifunctional chelating agents (BFCA) based on DFO*, identical in all regards but the functional group for bioconjugation, were prepared and conjugated to HSA and the obtained DFO*-HSA conjugates were radiolabeled with Zirconium-89 (89Zr) for positron emission tomography (PET). The efficiency of the conjugation of DFO*-X (X = mal, PODS, VK) to HSA differed significantly, with mal > PODS > VK. Stability studies of the 89Zr-labeled HSA-conjugates indicated good stability for 89ZrZr-DFO*malHSA 11 and 89ZrZr-DFO*-POD-HSA 12 in blood serum but only the latter was found stable in cell culture medium. 89ZrZr-DFO*VK-HSA 13 was excluded from biological experiments due to its surprisingly low stability in all media tested. 89ZrZr-DFO*-POD-HSA 12 was further investigated in CT26-tumor-bearing mice by PET/CT imaging and biodistribution studies. Specific uptake of radioactivity in tumors was high (up to 17% ID/g) and the tumors could be clearly visualized by PET at all time points with excellent tumor-to-background signal (tumor-to-blood ratio 3.2 ± 1.0 after 48 h p.i.). Unexpectedly, the uptake of radioactivity in bones was observed for 89ZrZr-DFO*-POD-HSA 12. Overall, the in vivo performance of 89ZrZr-DFO*-labeled HSA obtained by mal chemistry is the most promising candidate as a companion diagnostic PET imaging probe for the stratification of patients for therapies based on HSA-binding (pro-)drugs.

Ask AI

Helpful

Bookmark

View Full Paper