Cell penetrating peptides can translocate across cell membranes with high efficiency. The TAT protein (Trans-acting Activator of Transcription) of the Human Immunodeficiency Virus (HIV-1) is a good example of this [1,2]. A structure function relationship study of the protein led to the identification of the translocating activity domain, called the Protein Transduction Domain (PTD). The TAT PTD is a 11-amino acid sequence which has six arginine and two lysine residues, making the peptide highly cationic.
The TAT PTD appears to be unstructured by circular dichroism and nuclear magnetic resonance spectroscopy [3]. Covalently binding the TAT peptide to proteins [4], oligonucleotides [5], phages [6], liposomes [7] or even nanoparticles [8], allows these molecules to traverse the cell membrane. The peptide sequence is able to traverse almost all tissue cells including brain [9], and has a wide range of potential delivery applications.
TAT-mediated delivery appears to be independent of cargo size. Proteins in excess of 100000 Da, 40 nm iron nanoparticles and even 200 nm liposomes have been delivered using TAT. Confocal analysis showed that the liposomes were intact inside cells one hour after transduction [7].
TAT-mediated transduction does not appear to involve any disruption of the plasma membrane, as it could not promote the uptake of unrelated non-conjugated peptides present in the incubation media. This observation also demonstrates a key asset of PTD-mediated transduction: it has the ability to deliver therapeutic cargo, while avoiding bringing non-linked molecules into or out of the cell [10].
The mechanism of cellular entry is currently unknown. The large charge of the peptide at physiological pH excludes passive diffusion across the lipid bilayer. It is known that the basic amino acids within the TAT sequence play a central role [11]. Substituting a non-charged glutamine residue with alanine has no effect on cellular uptake, but substitution of any of the basic residues significantly decreases cellular uptake. The rate of cellular uptake also depends on the number of basic residues present, specifically the number of arginine residues [0].
The chirality of the peptide backbone has no affect on the cellular uptake. The inverse and retro forms of TAT peptide, synthesized with D-amino acids or in a reversed linear orientation, are as efficient as the native peptide [11, 12]. The cellular uptake of a branched chain arginine is as efficient as the corresponding linear polymer with the same number of arginines [13]. These findings show that the uptake mechanism is not mediated by a specific receptor and no particular spatial orientation is required. This suggests the exclusive involvement of cationic charges in promoting cellular uptake.
The cationic nature of the amino acids alone, however, is not sufficient. The ability of homopolymers of cationic amino acids to enter cells varies widely. Polymers of arginine are much more efficient at entering cell than similar length polymers composed of lysine, ornithine or histidine. The length of the peptide is also an important factor in its ability to enter cells. Arginine homopolymers with less than five amino acids are not as effective as peptides with six or more amino acids. The uptake efficiency increases as the peptide length is increased up to 15 amino acids. Peptides with more than 15 arginine residue still enter cell but with much less efficiency [14].
The central structural feature of the peptides required for cellular uptake appears to be the guanidinium headgroup of arginine. Its importance is confirmed by the failure of heptamers of citrulline to enter cells. Citrulline is an isostere of arginine, the only difference being the replacement of a nitrogen of the guanidine by an oxygen creating urea. This single substitution removed all transport activity, emphasizing the structural importance of the guanidinium group [14].
Initial studies indicated a very rapid mechanism of cellular uptake of TAT peptide with no diminution at low temperatures. Metabolic inhibitors, too, appeared to have no affect on peptide internalization [15, 16]. This excluded endocytosis as a mechanism of cellular uptake. In a later study [17], the cells were treated with trypsin which can remove the peptide that is bound to the cell surface. After this treatment, low temperature incubation and metabolic inhibitors showed a reduction in cell associated fluorescence.
As yet, no consensus has been reached on whether TAT peptide interacts with cell surface polysaccharides such as heparin sulfate. Cells that are genetically impaired in the biosynthesis of fully sulfonated heparin sulfate showed no uptake of TAT-GFP fusion protein [21]. However, in another study [22], the uptake of shorter TAT peptide did not show such dependence on proteoglycans.