Phospholipid-Mimetic Amphiphilic Polyion Complexes Enable Intracellular Oligonucleotide Delivery under Charge-Neutral Conditions.

Here, we report phospholipid-mimetic cationic copolymers that facilitate intracellular oligonucleotide delivery at a net charge-neutral polymer/DNA mixing ratio. Conventional nucleic acid delivery nanocarriers are mainly internalized through endocytosis, which often leads to endosomal sequestration and nucleic acid degradation, thereby reducing delivery efficiency and therapeutic efficacy. To address these issues, we developed a cationic phospholipid-mimetic random copolymer designed to physically form polyion complexes with anionic oligomeric DNA. Instead of simply maximizing cationic charge, our approach utilizes the membrane-interactive interfacial amphiphilicity of copolymer/oligonucleotide polyion complexes alongside hydrophobic monomer unit-mediated interactions to regulate interactions with cellular membranes. Consequently, at a net charge-neutral mixing ratio of the cationic polymer and anionic DNA, the polyion complexes showed reduced LysoTracker colocalization, uptake that was partially retained under ATP-depleted and endocytosis-inhibited conditions, and exhibited minimal cytotoxicity and negligible plasma membrane damage in vitro. Functionally, this system enabled antisense oligonucleotide delivery, reducing Bcl-2 mRNA expression by 57% relative to untreated cells in HepG2 cells. These findings suggest that interfacial regulation through phospholipid-mimetic amphiphilic polyion complexes can facilitate intracellular oligonucleotide delivery under net charge-neutral conditions, with uptake showing reduced sensitivity to suppression of classical energy-dependent endocytosis while maintaining negligible plasma membrane damage.