Preparation of targeted lipid nanoparticles for precision nucleic acid delivery

Intravenous administration of lipid nanoparticles for the delivery of nucleic acid therapeutics remains constrained by passive uptake mechanisms in the liver, often necessitating high doses to achieve meaningful transfection in specific cells of interest. Targeted LNPs (tLNPs) can overcome these challenges by (i) enabling receptor-mediated endocytosis in difficult-to-transfect cells, thereby reducing passive clearance; (ii) increasing the proportion of LNPs reaching their intended target; and (iii) enabling comparable protein expression at lower doses. Here, we provide a step-by-step guide for formulating tLNPs functionalized with whole antibodies or antibody fragments using traditional laboratory equipment. We outline procedures for antibody preparation and labeling (0.5-1 d), antibody-LNP conjugation (1-2 d), tLNP purification and characterization (1 d) and in vivo and ex vivo targeting evaluation (3-4 d). To demonstrate the versatility of this protocol, we validate in vivo targeting to two mouse tissues: we show that anti-platelet endothelial cell adhesion molecule 1 antibody conjugation to lung-tropic LNPs enhances lung transfection by five times compared to nontargeted LNPs, and anti-epidermal growth factor receptor antibody conjugation to liver-tropic LNPs enhances liver transfection by 20 times. We also demonstrate ex vivo targeting to primary human T cells, where anti-CD5 antibody conjugation to LNPs boosts uptake by 4.5 times and significantly increases mRNA transfection. Importantly, this modular strategy is compatible with any LNP formulation or antibody. In outlining these procedures, we seek to deliver a robust and reproducible workflow for the manufacturing of tLNPs, with the ultimate goal of advancing their therapeutic potential and facilitating clinical translation.