Cationic Polypeptoids with Optimized Molecular Characteristics toward Efficient Nonviral Gene Delivery

The rational design of gene vectors relies on the understanding of their structure–property relationship. Polypeptoids, which are structural isomers of natural polypeptides, hold great potential as gene delivery vectors due to their facile preparation, structural tunability, and most importantly, their desirable proteolytic stability. We herein designed a library of polypeptoids with different cationic side-chain terminal groups, degree of polymerizations (DPs), side-chain lengths, and incorporated aliphatic side chains, to unravel the structure–property relationships so that gene delivery efficiency can be maximized and cytotoxicity can be minimized. In HeLa cells, a polypeptoid bearing a primary amine side-chain terminal group exhibited remarkably higher transfection efficiency than that of its analogues containing secondary, tertiary, or quaternary amine groups. Elongation of the polypeptoid backbone length (from 28 to 251 mer) led to enhanced DNA condensation as well as cellular uptake levels, however it also caused higher cytotoxicity. Upon a proper balance between DNA uptake and cytotoxicity, the polypeptoid with a DP of 46 afforded the highest transfection efficiency. Elongating the aliphatic spacer between the backbone and side amine groups enhanced the hydrophobicity of the side chains, which resulted in notably increased membrane activities and transfection efficiency. Further incorporation of hydrophobic decyl side chains led to an improvement in transfection efficiency of ∼6 fold. The top-performing material identified, <b>P11</b>, mediated successful gene transfection under serum-containing conditions, outperforming the commercial transfection reagent poly­(ethylenimine) by nearly 4 orders of magnitude. Reflecting its excellent serum-resistant properties, <b>P11</b> further enabled effective transfection in vivo following intratumoral injection to melanoma-bearing mice. This study will help the rational design of polypeptoid-based gene delivery materials, and the best-performing material identified may provide a potential supplement to existing gene vectors