Tunable nonenzymatic degradability of <i>N</i>-substituted polyaspartamide main chain by amine protonation and alkyl spacer length in side chains for enhanced messenger RNA transfection efficiency

Degradability of polycations under physiological conditions is an attractive feature for their use in biomedical applications, such as the delivery of nucleic acids. This study aims to design polycations with tunable nonenzymatic degradability. A series of cationic <i>N</i>-substituted polyaspartamides were prepared to possess primary amine via various lengths of alkyl spacers in side chains. The degradation rate of each polyaspartamide derivative was determined by size exclusion chromatography under different pH conditions. The <i>N</i>-substituted polyaspartamide containing a 2-aminoethyl moiety in the side chain (PAsp(AE)) showed considerable degradability under physiological conditions (pH 7.4, 37 °C). In contrast, the <i>N</i>-substituted polyaspartamides bearing a longer alkyl spacer in the side chain, i.e. the 3-aminopropyl (PAsp(AP)) and 4-aminobutyl moieties (PAsp(AB)), more strongly suppressed degradation. Further, a positive correlation was observed between the degradation rate of <i>N</i>-substituted polyaspartamides and a deprotonation degree of primary amines in their side chains. Therefore, we conclude that the deprotonated primary amine in the side chain of <i>N</i>-substituted polyaspartamides can induce the degradation of the main chain through the activation of amide nitrogen in the side chain. When <i>N</i>-substituted polyaspartamides were utilized as a messenger RNA (mRNA) delivery vehicle via formation of polyion complexes (PICs), degradable PAsp(AE) elicited significantly higher mRNA expression efficiency in cultured cells compared to PAsp(AP) and PAsp(AB). The higher efficiency of PAsp(AE) might be due to the facilitated destabilization of PICs within the cells, directed toward mRNA release. Additionally, degradation of PAsp(AE) considerably reduced its cytotoxicity. Thus, our study highlights a useful design of well-defined cationic poly(amino acid)s with tunable nonenzymatic degradability.聚阳离子在生理条件下的可降解性，是其应用于核酸递送等生物医学领域的极具吸引力的特性。本研究旨在设计具备可调控非酶促降解性能的聚阳离子材料。本研究制备了一系列侧链带有不同长度烷基间隔基、并带有伯胺基团的阳离子<i>N</i>-取代聚天冬酰胺（N-substituted polyaspartamides）。通过尺寸排阻色谱法测定了不同pH条件下各聚天冬酰胺衍生物的降解速率。侧链带有2-氨基乙基基团的<i>N</i>-取代聚天冬酰胺（PAsp(AE)）在生理条件（pH 7.4、37℃）下展现出显著的降解性能。相比之下，侧链带有更长烷基间隔基的<i>N</i>-取代聚天冬酰胺，即带有3-氨基丙基基团的PAsp(AP)与带有4-氨基丁基基团的PAsp(AB)，其降解受到更为显著的抑制。进一步研究发现，<i>N</i>-取代聚天冬酰胺的降解速率与其侧链伯胺的去质子化程度呈正相关。据此我们得出结论：<i>N</i>-取代聚天冬酰胺侧链上的去质子化伯胺，可通过活化侧链的酰胺氮原子，引发主链的降解。当<i>N</i>-取代聚天冬酰胺通过形成聚离子复合物（polyion complexes, PICs）用作信使RNA（messenger RNA, mRNA）递送载体时，可降解的PAsp(AE)在培养细胞中展现出远高于PAsp(AP)与PAsp(AB)的mRNA转染表达效率。PAsp(AE)的更高转染效率，可能源于其介导形成的聚离子复合物在细胞内更易发生解聚，从而促进mRNA的释放。此外，PAsp(AE)的降解可显著降低其细胞毒性。综上，本研究为开发具备可调控非酶促降解性能的结构明确的阳离子聚氨基酸类材料提供了极具价值的设计思路。