Recent studies have demonstrated that enzyme-instructed self-assembly (EISA) in extra- or intracellular environments can serve as a multistep process for controlling cell fate. There is little knowledge, however, about the kinetics of EISA in the complex environments in or around cells. Here, we design and synthesize three dipeptidic precursors (ld-1-SO₃, dl-1-SO₃, dd-1-SO₃), consisting of diphenylalanine (l-Phe-d-Phe, d-Phe-l-Phe, d-Phe-d-Phe, respectively) as the backbone, which are capped by 2-(naphthalen-2-yl)acetic acid at the N-terminal and by 2-(4-(2-aminoethoxy)-4-oxobutanamido)ethane-1-sulfonic acid at the C-terminal. On hydrolysis by carboxylesterases (CES), these precursors result in hydrogelators, which self-assemble in water at different rates. Whereas all three precursors selectively kill cancer cells, especially high-grade serous ovarian carcinoma cells, by undergoing intracellular EISA, dl-1-SO₃ and dd-1-SO₃ exhibit the lowest and the highest activities, respectively, against the cancer cells. This trend inversely correlates with the rates of converting the precursors to the hydrogelators in phosphate-buffered saline. Because CES exists both extra- and intracellularly, we use kinetic modeling to analyze the kinetics of EISA inside cells and to calculate the cytotoxicity of each precursor for killing cancer cells. Our results indicate that (i) the stereochemistry of the precursors affects the morphology of the nanostructures formed by the hydrogelators, as well as the rate of enzymatic conversion; (ii) decreased extracellular hydrolysis of precursors favors intracellular EISA inside the cells; (iii) the inherent features (e.g., self-assembling ability and morphology) of the EISA molecules largely dictate the cytotoxicity of intracellular EISA. As the kinetic analysis of intracellular EISA, this work elucidates how the stereochemistry modulates EISA in the complex extra- and/or intracellular environment for developing anticancer molecular processes. Moreover, it provides insights for understanding the kinetics and cytotoxicity of aggregates of aberrant proteins or peptides formed inside and outside cells.

中文翻译：

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酶指导的针对癌细胞的细胞内组装形成的纳米结构的动力学分析

最近的研究表明，细胞外或细胞内环境中的酶促自组装（EISA）可以作为控制细胞命运的多步骤过程。然而，关于EISA在细胞内或细胞周围复杂环境中动力学的知识很少。在这里，我们设计并合成3二个肽前体（LD - 1 -SO ₃，DL - 1 -SO ₃，DD - 1 -SO ₃），由二苯基（的升-Phe- d -Phe，d -Phe-升-苯丙氨酸，d -Phe- d-Phe分别作为主链，在N端被2-（萘-2-基）乙酸和2-（4-（2-氨基乙氧基）-4-氧杂多酰胺基）乙烷-1-封端C末端的磺酸。在被羧酸酯酶（CES）水解时，这些前体产生了水凝胶化剂，它们在水中以不同的速率自组装。鉴于所有这三种前体均通过细胞内EISA，dl - 1 -SO ₃和dd - 1 -SO ₃选择性杀死癌细胞，尤其是高级浆液性卵巢癌细胞。分别显示出针对癌细胞的最低和最高活性。该趋势与在磷酸盐缓冲盐水中将前体转化为水凝胶剂的速率成反比。由于CES存在于细胞外和细胞内，因此我们使用动力学模型来分析细胞内EISA的动力学，并计算每种前体用于杀死癌细胞的细胞毒性。我们的结果表明：（i）前体的立体化学影响水凝胶化剂形成的纳米结构的形态以及酶转化率；（ii）前体的细胞外水解减少，有利于细胞内的细胞内EISA；（iii）固有特征（例如EISA分子的自组装能力和形态在很大程度上决定了细胞内EISA的细胞毒性。作为细胞内EISA的动力学分析，这项工作阐明了立体化学如何在复杂的细胞外和/或细胞内环境中调节EISA以发展抗癌分子过程。此外，它为理解细胞内部和外部形成的异常蛋白质或肽聚集体的动力学和细胞毒性提供了见识。