He Hongjian, Guo Jiaqi, Xu Jiashu, Wang Jiaqing, Liu Shuang, Xu Bing
Department of Chemistry, Brandeis University, 415 South Street, Waltham, Massachusetts 02453, United States.
Nano Lett. 2021 May 12;21(9):4078-4085. doi: 10.1021/acs.nanolett.1c01029. Epub 2021 May 3.
Alkaline phosphatase (ALP) enables intracellular targeting by peptide assemblies, but how the ALP substrates enter cells remains elusive. Here we show that nanoscale phosphopeptide assemblies cluster ALP to enable caveolae-mediated endocytosis (CME) and endosomal escape. Specifically, fluorescent phosphopeptides undergo enzyme-catalyzed self-assembly to form nanofibers. Live cell imaging unveils that phosphopeptides nanoparticles, coincubated with HEK293 cells overexpressing red fluorescent protein-tagged tissue-nonspecific ALP (TNAP-RFP), cluster TNAP-RFP in lipid rafts to enable CME. Further dephosphorylation of the phosphopeptides produces peptidic nanofibers for endosomal escape. Inhibiting TNAP, cleaving the membrane anchored TNAP, or disrupting lipid rafts abolishes the endocytosis. Decreasing the transformation to nanofibers prevents the endosomal escape. As the first study establishing a dynamic continuum of nanoscale assemblies for cellular uptake, this work illustrates an effective design for enzyme-responsive supramolecular therapeutics and provides mechanism insights for understanding the dynamics of cellular uptake of proteins or exogenous peptide aggregates.
碱性磷酸酶（ALP）可通过肽组装实现细胞内靶向，但ALP底物如何进入细胞仍不清楚。在此，我们表明纳米级磷酸肽组装可使ALP聚集，从而实现小窝介导的内吞作用（CME）和内体逃逸。具体而言，荧光磷酸肽会发生酶催化的自组装形成纳米纤维。活细胞成像显示，与过表达红色荧光蛋白标记的组织非特异性ALP（TNAP-RFP）的HEK293细胞共孵育的磷酸肽纳米颗粒，会在脂筏中聚集TNAP-RFP以实现CME。磷酸肽的进一步去磷酸化产生用于内体逃逸的肽纳米纤维。抑制TNAP、切割膜锚定的TNAP或破坏脂筏会消除内吞作用。减少向纳米纤维的转变会阻止内体逃逸。作为第一项建立用于细胞摄取的纳米级组装动态连续体的研究，这项工作阐明了酶响应性超分子疗法的有效设计，并为理解蛋白质或外源性肽聚集体的细胞摄取动力学提供了机制见解。
