Post-Conjugation Process for Antibody-Conjugated Lipid Nanoparticles Enabling Tunable Antibody Surface Density for Targeted RNA Delivery

Lipid nanoparticles (LNPs) are established nonviral carriers for RNA therapeutics; however, extrahepatic targeting remains challenging due to liver tropism. Antibody-conjugated LNPs (Ab-LNPs) offer specificity, yet existing methods lack reproducible processes and reliable surface density quantification. We report a postconjugation formulation protocol that enables precise tuning of antibody densities by varying Mal-PEG-lipid molar percentages (0.05%, 0.2%, 0.5%). Using a label-free modeling framework based on orthogonal distance regression and Monte Carlo uncertainty propagation, we quantified the antibody-to-particle ratio (APR) as approximately 340, 760, and 1200, respectively. Flow-through purification minimized particle shear, and the resulting formulations exhibited exceptional colloidal stability for at least one month storage at 4 °C. Comprehensive biophysical characterization revealed APR-dependent changes in hydrodynamic size and surface properties. Noncellular binding assays and cellular uptake studies revealed consistent APR-dependent trends, with low-density Ab-LNP exhibiting the highest binding capacity and internalization efficiency. In vivo biodistribution studies in a disseminated MM1S xenograft mouse model confirmed that low-density Ab-LNP achieved significantly enhanced bone marrow accumulation compared to control LNP, while displaying comparable signals in spleen and kidney and elevated liver accumulation attributable to Fc-mediated sequestration. These results establish APR as a critical quality attribute and demonstrate an optimal antibody density window that balances receptor targeting with minimal off-target hepatic entrapment. The tunable Ab-LNP platform developed in this study provides a rational framework for the design of antibody-conjugated LNPs for targeted RNA delivery.