Balancing Multivalent Avidity and Receptor Availability Governs mRNA Delivery by Antibody-Functionalized Lipid Nanoparticles.

Abstract

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 nm². 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 <i>in vivo</i> 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.