Genetic Code Expansion Enables Oriented, Site-Specific Conjugation of DARPins to Lipid Nanoparticles for Selective...

Lipid nanoparticles (LNPs) are an effective platform for delivering therapeutic payloads, but their use for targeting specific cell populations in vivo is limited by a lack of specificity. To address this issue, we developed an innovative approach for creating targeted LNPs based on the post-insertion of protein-lipid conjugates. By expanding the genetic code, a single p-azido-L-phenylalanine residue was specifically incorporated into a DARPin against murine CD8, enabling copper-free click-chemistry with dibenzocyclooctyne (DBCO) to functionalize the particle surface with DARPin conjugates. DARPin-LNP characterization demonstrated preservation of particle size and acquisition of a characteristic negative charge after DARPin insertion, confirming successful post-insertion. In vitro experiments using primary murine splenic T-cells showed that targeted LNPs selectively transfected CD8⁺ T-lymphocytes. These data indicate that the developed platform enables selective in vitro delivery of nucleic acids to cytotoxic lymphocytes, highlighting its potential relevance for designing safer immunotherapeutic strategies.