Delivery tools, including cell-penetrating peptides (CPPs), are often inefficient due to a combination of poor endocytosis and endosomal escape. Aspects that impact the delivery of CPPs are typically characterized using tissue culture models. One problem of using cell culture is that cell culture protocols have the potential to contribute to endosomal uptake and endosomal release of CPPs. Hence, a systematic study to identify which aspects of cell culturing techniques impact the endocytic uptake of a typical CPP, the TMR-TAT peptide (peptide sequence derived from HIV1-TAT with the N-terminus labeled with tetramethylrhodamine), was conducted. Aspects of cell culturing protocols previously found to generally modulate endocytosis, such as cell density, washing steps, and cell aging, did not affect TMR-TAT endocytosis. In contrast, cell dissociation methods, media, temperature, serum starvation, and media composition all contributed to changes in uptake. To establish a range of endocytosis achievable by different cell culture protocols, TMR-TAT uptake was compared among protocols. These protocols led to changes in uptake of more than 13-fold, indicating that differences in cell culturing techniques have a cumulative effect on CPP uptake. Taken together this study highlights how different protocols can influence the amount of endocytic uptake of TMR-TAT. Additionally, parameters that can be exploited to improve CPP accumulation in endosomes were identified. The protocols identified herein have the potential to be paired with other delivery enhancing strategies to improve overall delivery efficiency of CPPs.

由于内吞作用和内体逃逸相结合，包括细胞穿透肽（CPP）在内的递送工具通常效率低下。影响 CPP 递送的方面通常使用组织培养模型来表征。使用细胞培养的一个问题是细胞培养方案有可能促进 CPP 的内体摄取和内体释放。因此，进行了一项系统研究，以确定细胞培养技术的哪些方面影响典型 CPP（TMR-TAT 肽）（源自 HIV1-TAT 的肽序列，N 末端用四甲基罗丹明标记）的内吞摄取。先前发现的细胞培养方案的各个方面通常可以调节内吞作用，例如细胞密度、洗涤步骤和细胞老化，但并不影响 TMR-TAT 内吞作用。相反，细胞解离方法、培养基、温度、血清饥饿和培养基成分都会导致摄取的变化。为了确定不同细胞培养方案可实现的一系列内吞作用，对不同方案之间的 TMR-TAT 摄取进行了比较。这些方案导致摄取量变化超过 13 倍，表明细胞培养技术的差异对 CPP 摄取量具有累积效应。总而言之，这项研究强调了不同的方案如何影响 TMR-TAT 的内吞摄取量。此外，还确定了可用于改善内体中 CPP 积累的参数。本文确定的协议有可能与其他交付增强策略配合使用，以提高 CPP 的整体交付效率。