Oligo-Modified Poly(β-Amino Ester)-Mediated mRNA Delivery: A Protocol for Gene Editing Applications

Oligopeptide-modified poly(β-amino ester)s (OM-pBAEs) are polymer-based delivery systems that enable the encapsulation and intracellular transport of nucleic acids, including messenger RNA (mRNA) and mixtures of mRNA with DNA. OM-pBAEs form cationic nanoparticles (NPs) through electrostatic interactions with nucleic acids, protecting them from degradation and enhancing cellular uptake. Terminal oligopeptides composed of lysine, arginine, or histidine enhance polyplex stability, nucleic acid binding, and endosomal escape. This chapter describes a protocol for preparing OM-pBAE nanoparticles, including polymer synthesis, peptide modification, controlled mixing with nucleic acids to form polyplexes, precipitation in RNase-free water, stabilization with excipients, and optional freeze-drying for long-term storage. Nanoparticle formation occurs at a constant temperature of 25°C, with incubation to ensure reproducible self-assembly. Nanoparticles are characterized by using dynamic light scattering (DLS) to determine particle size, polydispersity, and surface charge, while nucleic acid encapsulation efficiency is assessed to evaluate loading capacity. This protocol provides a versatile, nonviral platform for efficient nucleic acid delivery, supporting applications such as gene-editing experiments.