Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, 00790 Helsinki, Finland.
J Control Release. 2012 Mar 10;158(2):277-85. doi: 10.1016/j.jconrel.2011.11.007. Epub 2011 Nov 12.
Biomolecules and drug delivery agents, such as liposomes, are often delivered intracellularly with help of cell penetrating peptides (CPPs) and, in particular, Tat peptide. Tat peptide acts as a membrane shuttle; the structural determinants of transport and the manner by which the peptide crosses the lipid bilayer are, however, still unknown. The roles of direct membrane translocation, endocytosis and cell surface proteoglycans, in particular, remain elusive. Our study aimed to explore the relationship between structure and activity of Tat peptide and its uptake mechanism. For this purpose we introduced several modifications (e.g. lipophilic, aromatic, neutral and non-natural amino acids) into the original Tat sequence. We studied the interaction of the peptides with a model lipid membrane and with three cell lines, a phagocytic cell line (human retinal pigment epithelium cell line, ARPE-19), a non-phagocytic cell line (Chinese hamster ovary cells, CHO wt) and a mutant form of the latter cell line deficient in glycosaminoglycans chondroitin sulfate and heparan sulfate (CHO-pgsB 618). The amino acid residues introduced into the original sequence of Tat peptide failed to influence cellular uptake, indicating that the cationic charge alone may be responsible for translocation. Clear discrepancy between permeation activity of the peptides into cells and their interaction with lipid bilayers of liposomes indicated the limited value of the model membrane in predicting cellular peptide delivery. Cell uptake of Tat peptide was unspecific, took place either by phagocytosis or pinocytosis, and was inhibited by cell-surface glycosaminoglycans. The internalized peptides were localized in vesicles and unable to reach the cell nuclei. In conclusion, we show that Tat-related peptides enter cells on the basis of their cationic charge following different endocytosis pathways and that glycosaminoglycans on the cell surface negatively affect their uptake. This lack of specificity should be taken into account when exploiting Tat peptide as vehicle for intracellular delivery of biomacromolecules.
生物分子和药物递送剂，如脂质体，通常在细胞穿透肽（CPPs）的帮助下被递送到细胞内，特别是 Tat 肽。Tat 肽作为一种膜穿梭物；然而，其转运的结构决定因素以及肽穿过脂质双层的方式仍不清楚。直接的膜转位、内吞作用和细胞表面蛋白聚糖的作用，特别是，仍然难以捉摸。我们的研究旨在探索 Tat 肽的结构与活性及其摄取机制之间的关系。为此，我们在原始 Tat 序列中引入了几种修饰（例如亲脂性、芳香族、中性和非天然氨基酸）。我们研究了肽与模型脂质膜和三种细胞系的相互作用，一种吞噬细胞系（人视网膜色素上皮细胞系，ARPE-19）、一种非吞噬细胞系（中国仓鼠卵巢细胞，CHO wt）和后者细胞系的突变形式，该突变形式缺乏糖胺聚糖硫酸软骨素和肝素硫酸（CHO-pgsB 618）。引入 Tat 肽原始序列中的氨基酸残基未能影响细胞摄取，表明阳离子电荷可能是唯一负责转位的因素。肽进入细胞的通透性活性与它们与脂质双层的相互作用之间存在明显差异，这表明模型膜在预测细胞肽递送上的价值有限。Tat 肽的细胞摄取是非特异性的，通过吞噬作用或胞饮作用发生，并且被细胞表面糖胺聚糖抑制。内化的肽定位于囊泡中，无法到达细胞核。总之，我们表明 Tat 相关肽基于其阳离子电荷通过不同的内吞途径进入细胞，并且细胞表面的糖胺聚糖会负影响其摄取。在将 Tat 肽作为生物大分子细胞内递药载体时，应该考虑到这种非特异性。
