Carboxylated branched poly(β-amino ester) nanoparticles enable robust intracellular protein delivery and crispr/cas9 gene editing

Statement of Purpose: Direct intracellular delivery of protein drugs to target cells is safer than plasmid delivery as it eliminates risks of insertional mutagenesis. In the context of gene editing, there is the added benefit that delivery of CRISPR/Cas9 ribonucleoproteins (RNPs) reduces the probability of off-target editing by reducing RNP persistence time. However, intracellular protein delivery faces many challenges as the large size and hydrophilicity of proteins make them generally membrane impermeable. Poly(β-amino ester)s (PBAEs) are biodegradable, cationic polymers that self-assemble into nanoparticles with nucleic acids via electrostatic interactions and have been developed for effective nucleic acid delivery. In contrast to nucleic acids, different proteins carry different surface charges and nanoparticle encapsulation cannot rely solely on charge interactions. In this study, we modified branched PBAEs with amino acid-like carboxylate ligands and examined their ability to enable intracellular protein delivery. We hypothesized that the carboxylate end-caps facilitate protein encapsulation through hydrogen bonding and salt bridges while the PBAE polymer backbone can enable endosomal escape, resulting in a versatile protein delivery platform.