A Strategyto Develop Zirconia Nanoparticle-BindingAntibodies That Can Easily Cross-Link Nanoparticles by Grafting EvenInsoluble Functional Peptides

Inorganic material-binding proteins are valuable tools for conjugating different inorganic materials. The development of efficient methods for obtaining high-affinity inorganic material-binding proteins is desirable. In this study, focusing on ZrO₂, which is available in the medical field as a dental material and a nanocapsule to encapsulate anticancer substances due to its high biocompatibility, we first isolated the peptides ZrO₂BPa and ZrO₂BPn, which bind ZrO₂ nanoparticles using the phage display technique. These peptides are insoluble alone. We prepared the variable domain of the heavy chain of heavy-chain antibodies (VHHs) with low affinity for ZrO₂ nanoparticles by grafting these peptides into the complementary determining region 1 (CDR1) of cAbBCII10 VHH. The affinity for VHH was further improved by optimizing CDR3 using a phage display technique with random mutagenesis. Among the VHHs, ZrO₂N3 VHH showed the highest affinity, with a <i>K</i>_D of 1.2 × 10^–7 M, showing pH-dependent binding. Mixing ZrO₂-binding antibodies with ZrO₂ nanoparticles improved the ZrO₂ nanoparticle dispersibility in phosphate buffer, which is desirable for biological use. We also generated a bispecific antibody by fusing ZrO₂-binding VHH with gold-binding VHH. Unlike chemical conjugation methods, which require complicated multistep reactions, we combined ZrO₂ and Au nanoparticles by simply introducing a bispecific antibody. Thus, we demonstrated an effective method for obtaining high-affinity, inorganic material-binding VHHs and the usefulness of these VHHs as interfacial molecules.