Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA, 70803, USA.
Craft and Hawkins Department of Petroleum Engineering, Louisiana State University, Baton Rouge, LA, 70803, USA.
Anal Bioanal Chem. 2019 May;411(12):2729-2741. doi: 10.1007/s00216-019-01713-5. Epub 2019 Mar 11.
Cell-penetrating peptides (CPPs) have garnered significant attention as a method to introduce reporters and therapeutics into intact cells. While numerous studies have been performed identifying new CPP sequences, relatively little is known about their uptake efficiency at the single-cell level. Here, a droplet microfluidic trapping array was used to characterize CPP uptake across a population of single intact cells. The microfluidic device allowed for facile and rapid isolation and analysis of single-cell fluorescence in a 787-member overhead trapping array with > 99% droplet trapping efficiency. The permeability efficiencies of four different CPPs were studied and compared in HeLa cells. Population analysis was performed using linkage hierarchical cluster analysis by R programming to bin cells into subpopulations expressing very low to very high peptide uptake efficiencies. CPP uptake was observed to be heterogeneous across the population of cells with peptide concentration and sequence both playing important roles in the diversity of CPP uptake, the overall peptide uptake efficiency, and the intracellular homogeneity of peptide distribution. This microfluidic-based analytical approach finds application in personalized medicine and provides new insight in the heterogeneity of CPP uptake which has the potential to affect both biosensor and drug internalization in intact cells. Graphical abstract .
细胞穿透肽（CPPs）作为一种将报告基因和治疗剂导入完整细胞的方法引起了广泛关注。虽然已经有许多研究确定了新的 CPP 序列，但对于它们在单细胞水平上的摄取效率相对知之甚少。在这里，使用液滴微流控捕获阵列来表征整个单细胞群体中的 CPP 摄取。该微流控装置允许在具有 >99%液滴捕获效率的 787 个成员的头顶式捕获阵列中轻松快速地分离和分析单细胞荧光。在 HeLa 细胞中研究并比较了四种不同 CPP 的渗透率效率。使用 R 编程进行链接层次聚类分析对细胞进行群体分析，将细胞分为表达非常低到非常高肽摄取效率的亚群。在细胞群体中观察到 CPP 摄取具有异质性，肽浓度和序列都在 CPP 摄取的多样性、总体肽摄取效率以及肽分布的细胞内均匀性中起着重要作用。这种基于微流控的分析方法在个性化医学中有应用，并为 CPP 摄取的异质性提供了新的见解，这有可能影响完整细胞中生物传感器和药物内化。
