Acid‐Responsive Polymer Additives Increase RNA Transfection from Lipid Nanoparticles

Abstract Lipid nanoparticles (LNPs) are the most clinically advanced RNA delivery technology, but their efficiency is limited by low RNA release after endosome disruption. To improve RNA release, an acid‐responsive polymer is synthesized with which to formulate LNPs for RNA encapsulation and release. Specifically, three acid‐responsive poly(lactic acid)‐block‐poly(carboxybetaine) zwitterionic derivatives are designed and synthesized that are cationic and complexed with RNA at pH 7.4, but are neutral following cleavage at endosomal pH, thereby having lower affinity to RNA. The polymers are formulated into each of the clinically approved Onpattro, Moderna, or Pfizer LNP formulations to produce hybrid polymer‐lipid nanoparticles (PLNPs). With the PLNPs, the IC₅₀ values of multiple small interfering RNAs (siRNAs) decreased up to 5.4‐fold compared to parent LNPs in several cell lines. Moreover, messenger RNA (mRNA) transfection increased up to two fold. The acid‐responsive polymers in PLNPs accounted for the enhanced RNA transfection as this phenomenon is lost with acid‐inert polymers. Confocal microscopy confirmed that cytosolic RNA concentration increased using the acid‐responsive polymers; conversely, uptake and endosomal escape are identical to existing LNPs. This confirmed that enhanced RNA transfection is due to increased RNA dissociation from its carrier. The novel polymer represents a versatile strategy to increase RNA transfection from LNPs.