Cardiff School of Pharmacy and Pharmaceutical Sciences, Redwood Building, Cardiff University, Cardiff, Wales CF10 3NB, United Kingdom.
J Control Release. 2012 Jul 10;161(1):132-41. doi: 10.1016/j.jconrel.2012.03.015. Epub 2012 Mar 24.
Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic macromolecules. It is widely accepted that they can enter cells directly across the plasma membrane but also gain access through endocytic pathways that are yet to be fully defined. Here we developed siRNA methods in epithelial cell lines, HeLa and A431, to inhibit endocytic pathways regulated by clathrin heavy chain, flotillin-1, caveolin-1, dynamin-2 and Pak-1. In each case, functional uptake assays were developed to characterize the requirement for these proteins, and the pathways they regulate, in the internalisation of defined endocytic probes and also the CPPs octaarginine and HIV-Tat. Peptide uptake was only inhibited in A431 cells depleted of the macropinocytosis regulator Pak-1, but experimental variables including choice of cell line, pharmacological inhibitor, macropinocytic probe and serum starvation significantly influence our ability to assess and assign this pathway as an important route for CPP uptake. Actin disruption with Cytochalasin D inhibited peptide entry in both cell lines but the effects of this agent on dextran uptake was cell line dependent, reducing uptake in HeLa cells and increasing uptake in A431 cells. This was further supported in experiments inducing actin stabilisation by Jasplakinolide, emphasising that the actin cytoskeleton can both promote and hinder endocytosis. Overall the data identify important aspects regarding the comparative mechanisms of CPP uptake and macropinocytosis, and accentuate the significant methodological challenges of studying this pathway as an endocytic portal and an entry route for drug delivery vectors.
细胞穿透肽 (CPP) 已被广泛研究作为治疗性大分子细胞内递呈的载体。人们普遍认为，它们可以直接穿过质膜进入细胞，但也可以通过尚未完全定义的内吞途径进入细胞。在这里，我们在上皮细胞系 HeLa 和 A431 中开发了 siRNA 方法，以抑制网格蛋白重链、 flotillin-1、caveolin-1、dynamin-2 和 Pak-1 调节的内吞途径。在每种情况下，都开发了功能摄取测定来表征这些蛋白质在特定内吞探针和 CPP 八聚精氨酸和 HIV-Tat 的内化过程中的作用，以及它们调节的途径。只有在 Pak-1 被耗尽的 A431 细胞中，肽摄取才被抑制，但包括细胞系选择、药理学抑制剂、巨胞饮探针和血清饥饿在内的实验变量显著影响了我们评估和分配该途径作为 CPP 摄取的重要途径的能力。细胞松弛素 D 破坏肌动蛋白抑制了两种细胞系中的肽进入，但该试剂对葡聚糖摄取的影响依赖于细胞系，减少了 HeLa 细胞中的摄取，增加了 A431 细胞中的摄取。用 Jasplakinolide 诱导肌动蛋白稳定化的实验进一步支持了这一点，强调了肌动蛋白细胞骨架既可以促进也可以阻碍内吞作用。总体而言，该数据确定了 CPP 摄取和巨胞饮的比较机制的重要方面，并强调了作为内吞门户和药物递送载体进入途径研究该途径的重要方法学挑战。
