Diblock Copolymer Targeted Lipid Nanoparticles: Next-Generation Nucleic Acid Delivery System Produced by Confined Impinging Jet Mixers

Despite the recent advances and clinical demonstration of lipid nanoparticles (LNPs) for therapeutic and prophylactic applications, the extrahepatic delivery of nucleic acids remains a significant challenge in the field. This limitation arises from the rapid desorption of lipid-PEG in the bloodstream and clearance to the liver, which hinders extrahepatic delivery. In response, we explore the substitution of lipid-PEG with biodegradable block copolymers (BCPs), specifically poly(ε-caprolactone)-block-poly(ethylene glycol) (PCL-b-PEG). BCPs offer strong anchoring for large macromolecules, potentially enhancing cell-specific targeting. To develop and optimize BCP-stabilized LNPs (BCP-LNPs), we employed a Design of Experiment (DOE) approach. Through a systematic exploration, we identified optimal formulations for BCP-LNPs, achieving desirable physicochemical properties and encapsulation efficiency. Notably, BCP-LNPs exhibit surprising trends in transfection efficiency, with certain formulations showing up to a 40-fold increase in transfection in Hela cells, while maintaining minimal cytotoxicity. The lipid compositions that optimized PCL-b-PEG LNP transfection were different from the compositions that optimized PEG-lipid LNP transfection. Furthermore, our study confirms the versatility of BCP-LNPs in encapsulating and delivering both mRNA and pDNA, demonstrating their cargo-agnostic nature. Lastly, we showcased the targeted BCP-LNPs using a Cetuximab-conjugated formulation. These targeted LNPs show significant promise in delivering cargo specific to EGFR-overexpressing cells (A549 cells), with up to 2.4 times higher transfection compared to nontargeted LNPs. This finding underscores the potential of BCP-LNPs in targeted gene therapy, especially in challenging scenarios such as tumor targeting. Overall, our study establishes the viability of BCP-LNPs as a versatile, efficient, and targeted delivery platform for nucleic acids, opening avenues for advanced therapeutic applications.

Block copolymers; Design of Experiments; Flash NanoPrecipitation; Lipid Nanoparticles; mRNA; plasmid DNA.