Engineering the Aromaticity of Cationic Helical Polypeptides toward “Self-Activated” DNA/siRNA Delivery

The development of potent yet nontoxic membrane-penetrating materials is in high demand for effective intracellular gene delivery. We have recently developed α-helical polypeptides which afford potent membrane activities to facilitate intracellular DNA delivery via both endocytosis and the nonendocytic “pore formation” mechanism. Endocytosis will cause endosomal entrapment of the DNA cargo, while excessive “pore formation” would cause appreciable cytotoxicity. Additionally, helical polypeptides with stiff, rodlike structure suffer from low siRNA binding affinity. To address such critical issues, we herein incorporated various aromatic domains (benzyl, naphthyl, biphenyl, anthryl, and pyrenyl) into the side-chain terminals of guanidine-rich, helical polypeptides, wherein the flat-rigid shape, π-electronic structures of aromatic motifs “self-activated” the membrane-penetrating capabilities of polypeptides to promote intracellular gene delivery. Benzyl (Bn)- and naphthyl (Naph)-modified polypeptides demonstrated the highest DNA uptake level that outperformed the unmodified polypeptide, P2, by ∼4 fold. More importantly, compared with P2, Bn- and Naph-modified polypeptides allowed more DNA cargos to be internalized via the nonendocytic pathway, which significantly bypassed the endosomal entrapment and accordingly enhanced the transfection efficiency by up to 42 fold, outperforming PEI 25k as the commercial reagent by 3–4 orders of magnitude. The aromatic modification also improved the siRNA condensation capability of polypeptides, achieving notably enhanced gene-silencing efficiency against tumor necrosis factor-α to treat acute hepatic inflammation. Furthermore, we revealed that aromaticity-augmented membrane activity was accompanied by comparable or even significantly reduced “pore formation” capability, thus leading to diminished cytotoxicity at high concentrations. This study therefore provides a promising approach to manipulate the membrane activities and penetration mechanisms of polycations, which overcomes the multiple critical barriers preventing effective and safe gene delivery

Reversibly Cross-Linked Polyplexes Enable Cancer-Targeted Gene Delivery via Self-Promoted DNA Release and Self-Diminished Toxicity

Polycations often suffer from the irreconcilable inconsistency between transfection efficiency and toxicity. Polymers with high molecular weight (MW) and cationic charge feature potent gene delivery capabilities, while in the meantime suffer from strong chemotoxicity, restricted intracellular DNA release, and low stability in vivo. To address these critical challenges, we herein developed pH-responsive, reversibly cross-linked, polyetheleneimine (PEI)-based polyplexes coated with hyaluronic acid (HA) for the effective and targeted gene delivery to cancer cells. Low-MW PEI was cross-linked with the ketal-containing linker, and the obtained high-MW analogue afforded potent gene delivery capabilities during transfection, while rapidly degraded into low-MW segments upon acid treatment in the endosomes, which promoted intracellular DNA release and reduced material toxicity. HA coating of the polyplexes shielded the surface positive charges to enhance their stability under physiological condition and simultaneously reduced the toxicity. Additionally, HA coating allowed active targeting to cancer cells to potentiate the transfection efficiencies in cancer cells in vitro and in vivo. This study therefore provides an effective approach to overcome the efficiency-toxicity inconsistence of nonviral vectors, which contributes insights into the design strategy of effective and safe vectors for cancer gene therapy