Cell-penetrating Peptide Synthesis

The process of introducing drugs into cells has always proved a major challenge for scientists. However, cell-penetrating peptides (CPPs) have the ability to enter a cell's plasma membrane independent of a membrane receptor. They are usually small peptides at 10–30 residues in length. The sequences of amino acids are often positively charged.

Tat, the transcription activator of the human immunodeficiency virus type 1 (HIV-1) viral genome was shown to enter cells in a non-toxic and highly efficient manner. Tat became known as the first cell-penetrating peptide.

CPPs have demonstrated themselves to be capable of delivering biologically active cargo to the cell interior. Attached to a CPP, therapeutic cargo could be delivered to an intracellular target, thus overcoming the entry restrictions set by the plasma membrane.

There are three proposed routes of CPP entry: Model 1: The inverted micelle model. Model 2: The direct penetration (pore formation) mechanism. Model 3: An endocytic mechanism of uptake. Source: Cell-penetrating peptides and their therapeutic applications, Victoria Sebbage, BioscienceHorizons, Volume 2, Number 1, March 2009.

Since the discovery of Tat, the number of known peptides with cell-penetrating capabilities has grown. The following table shows a selection of currently known CPPs, their origins and sequences.

Amino acid composition of cell-penetrating peptides (CPPs)

Cell-penetrating peptides (CPPs) such as the HIV TAT peptides are able to enter cell by direct translocation and endocytosis. HIV TAT or even simple poly-arginines can be effectively designed for drug delivery. However how cell-penetrating peptides, HIV TAT peptide for example, accomplish these cellular molecular transfer has so far been a mystery.

How does simple HIV TAT peptide facilitate mechanisms like direct translocation and multiple endocytotic processes? Researchers from Gerard Wong’s lab found how HIV TAT peptides can have multiple interactions with the cell membrane, the actin cytoskeleton and specific cell-surface receptors to produce multiple pathways of translocation under different conditions. Click here for the publication from Gerard Wong’s lab: http://bit.ly/zQrH6t.

Interestingly, TAT peptide can multiplex different interactions with the same sequence, thus interacting with the membrane, the actin cytoskeleton, and specific receptors to produce multiple pathways of translocation under different conditions.

CPPs entry mechanism is sensitive to the peptide sequence. The addition of a single hydrophobic residue to purely hydrophilic CPPs can drastically modify the translocation mechanism. For example, polyarginine (polyR), the simplest prototypical CPP, can induce the cell membrane pore formation. Hydrophobic amino acids create positive curvature by inserting into the membrane. Arginine simultaneously creates positive and negative curvatures, whereas lysine creates negative curvature along one direction only. This implies a compensatory relation between arginines and lysines/hydrophobes.

Why is the hydrophobic content of the TAT peptide relatively low if hydrophobicity can help generate negative Gaussian curvature? CPPs use less hydrophobic residues to generate saddle-splay curvature. This difference in sequences can potentially only induce transient pore-like translocation structures in the membrane and thus lead to shorter pore lifetimes for CPPs. Because of the amino acid composition for CPPs, the TAT peptide can mediate endocytosis with or without receptors.