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Engineering the Aromaticity of Cationic Helical Polypeptides toward “Self-Activated” DNA/siRNA Delivery Fangfang Li,†,§ Yongjuan Li,†,§ Zhuchao Zhou,‡,§ Shixian Lv,† Qiurong Deng,† Xin Xu,† and Lichen Yin*,† †
Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou 215123, China ‡ Department of General Surgery, Huashan Hospital, Fudan University, Shanghai 200040, China S Supporting Information *
ABSTRACT: 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 sidechain terminals of guanidine-rich, helical polypeptides, wherein the flat-rigid shape, πelectronic 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. KEYWORDS: gene delivery, α-helical polypeptides, aromatic domain, membrane penetration, pore formation, cytotoxicity
1. INTRODUCTION
Cell-penetrating peptides (CPPs), including Pep-1, TP10, and mellitin, are a category of sequence-specific oligopeptides with excellent membrane activities.23 CPPs often adopt inherent helical structure or shape into a helix during membrane penetration, which strengthens the interactions with cell membranes to facilitate cellular internalization.24 However, CPPs often lack sufficient length (often
