The RGD motif is a cell adhesion sequence that binds to integrins, a receptor family for extracellular matrix proteins. We previously reported that the RGDX₁X₂ sequence, where X₁X₂ is VF or NY, is required for integrin αvβ5-mediated cell adhesion. However, the importance and applications of the X₁X₂ combinations and their surrounding sequences of integrin αvβ5-binding RGDX₁X₂-containing peptides have not been comprehensively elucidated. Therefore, we aimed to identify an RGD-containing peptide with enhanced integrin αvβ5 binding activity. We synthesized various peptides based on the RGDVF and RGDNY peptides to optimize the N-terminal, C-terminal, and X₁X₂ combinations of the RGDX₁X₂ sequence. These peptides were immobilized on maleimide-functionalized bovine serum albumin-coated plates via a thiol-maleimide reaction, and cell adhesion was evaluated using HeLa cells and human dermal fibroblasts. Consequently, CPPP-RGDTF and CPPP-RGDTFI were identified as highly active peptides for integrin αvβ5-mediated cell adhesion. CPPP-RGDTF and CPPP-RGDTFI are expected to serve as cell adhesion molecules for developing culture substrates and biomaterials. Furthermore, these findings provide important novel insights into the interaction between the RGD motifs and integrins.

Ultrasound (US) imaging is a widely used imaging technique. The use of US contrast agents such as microbubbles, which consist of phospholipids and are filled with perfluorocarbon gases, has become an indispensable component of clinical US imaging, while molecular US imaging has recently attracted significant attention in combination with efficient diagnostics. The avidin–biotin interaction method is frequently used to tether antibodies to microbubbles, leading to the development of a molecular targeting US imaging agent. However, avidin still has limitations such as immunogenicity. We previously reported that lipid-based nanobubbles (NBs) containing perfluorocarbon gas are suitable for US imaging and gene delivery. In this paper, we report on the development of a novel antibody modification method for NBs using Fc-region-binding polypeptides derived from protein A/G. First, we prepared anti-CD146 antibody-modified NBs using this polypeptide, resulting in high levels of attachment to human umbilical vein endothelial cells expressing CD146. To examine their targeting ability and US imaging capability, the NBs were administered to tumor-bearing mice. The contrast imaging of antibody-modified NBs was shown to be prolonged compared with that of non-labeled NBs. Thus, this antibody modification method using an Fc-binding polypeptide may be a feasible tool for developing a next-generation antibody-modified US imaging agent.