Kazuki Hashiba, Masahiro Fukasaka, Chisa Okuma, Yasuyo Kobayashi-Ooka, Keiko Kajiwara, Akinori Sugiyama, Tomonari Mizuno, Yoshimitsu Shimomura, Yoshiki Maeda, Jumpei Sasaki, Taisuke Enoki, Ryo Hashino, Shintaro Mikuni, Hiroki Nishita, Naoko Urushihara, Hirofumi Ebe, Hiroshi Yamada
High Resolution Image Download MS PowerPoint Slide Expanding the therapeutic scope of mRNA requires delivery systems with precise cell selectivity. Antibody-functionalized lipid nanoparticles (LNPs) enable programmable targeting, but quantitative rules linking surface avidity to delivery remain unresolved. Here, we establish a single-domain antibody (VHH)-LNP platform with controlled orientation and tunable ligand density, combined with an antigen-specific ligand binding fluorescence assay using single-particle nanoflow cytometry to quantify only functional ligands. Using this system, we show that CD8-targeted LNPs exhibit a bell-shaped dependence of delivery efficiency on ligand density, revealing an optimal avidity of ∼0.1 VHH per 100 nm 2 . Mechanistically, excessive ligand density induces receptor degradation, while optimal avidity balances multivalent engagement with receptor preservation. Optimized LNPs achieve selective mRNA expression in CD8 + T cells and enable in vivo CAR-T generation, resulting in dose-dependent B cell depletion at 10–30 μg/kg. Surface avidity thus emerges as a quantitative design parameter, transforming antibody-decorated LNP engineering from empirical to rule-based design.