Current Protein and Peptide Science - Volume 4, Issue 2, 2003

Peptide-mediated protein delivery into living cells has been attracting our attention. Among the peptides that have been reported to have carrier activity, the one from the human immunodeficient virus (HIV)-1 Tat has been most often used for the introduction of exogenous macromolecules into cells. We have shown that not only the Tat peptide, but also various arginine-rich peptides showed very similar characteristics in translocation, and the possible presence of ubiquitous internalization mechanisms among the arginine-rich peptides has also been suggested. These arginine-rich peptides includes ones derived from HIV-1 Rev and flock house virus coat proteins. The linear- and branched-chain peptides containing ∼8 residues of arginine also show a similar ability. In this review, we present the structural variety of membrane permeable peptides and provide a survey of the findings on the translocation of these peptides through the cell membranes.

Due to the barrier imposed by the cell membrane, delivery of macromolecules in excess of 500 Daltons directly into cells remains problematic. However, proteins, which have been evolutionarily selected to perform specific functions, are therefore an attractive therapeutic agent to treat a variety of human diseases. In practice, the direct intracellular delivery of these proteins has, until recently, been difficult to achieve due primarily to the bioavailability barrier of the plasma membrane, which effectively prevents the uptake of the majority of peptides and prote ins by limiting their passive entry. However, recent wor k using small c ationic pe ptide s, termed protein transduction domains (PTDs), derived from nucleic acid binding proteins, such as HIV TAT protein or the Dros. m. transcription factor Antp. or synthetic poly-Arginine, have now been shown to deliver a myriad of molecules, including synthetic small molecules, peptides and proteins, into animal models in vivo. Here, we focus on the delivery of biologically active, full length proteins to treat pre-clinical disease models.

The use of peptide or peptidomimetic transporters to enable or enhance the uptake of drugs or probe molecules into cells and tissues has received increasing research attention and clinical interest over the past 10 years. This review summarizes a class of transporters that have been studied and focuses on the variation and use of guanidinium based transporters to facilitate the uptake of various types of molecules into cells and tissues. Lead conjugates in this area are currently in clinical trials.

During the last decade several peptides have been extensively studied for their ability to translocate across the plasma membrane. These peptides have been called “cell penetrating peptides” (CPP) or “protein transduction domains” (PTD). These peptides also promote the cellular uptake of various cargo molecules. Their mechanism of cellular entry appeared very intriguing since most publications in the field highlighted an energyindependent process. Indeed, cellular uptake of these peptides was still observed by fluorescence microscopy at low temperature or in the presence of several drugs known to inhibit active transport. In addition, internalization was reported to be much faster than known endocytic processes. However the involvement of a specific cellular component responsible for this uptake process appeared unlikely following intensive structure activity relationship studies using a wide panel of Tat analogues.Several reports about a possible artefactual redistribution of CPPs, and their associated cargos, during the cell fixation step commonly used for fluorescence microscopy have recently emerged in the literature. Moreover strong ionic interactions of CPPs with the cell surface also led to an overestimation of the recorded cell-associated fluorescent signal.It now seems well established that arginine-rich peptides are internalized by an energy dependant process involving endocytosis. Whatever the case, however, an increasing number of data indicate that the conjugation of non-permeant molecules to these CPPs allows their cellular uptake and leads to the expected biological responses, thus pointing to the interest of this delivery strategy. However, initial structure activity relationship studies of these CPPs will have to be reconsidered and the relative potency of each peptide (and their analogues) to vectorize the cargos to their most appropriate subcellular compartment will require careful re-evaluation.

TAT peptide was attached to the surface of plain and PEGylated liposomes. These TAT peptide-modified liposomes have been shown to translocate into a variety of normal and cancer cells if a non-hindered interaction between the cell surface and liposome-attached TAT peptide was made possible. TAT peptide-liposomes translocated into cells remain intact within first few hours as proved by a co-localization of fluorescent markers entrapped inside liposomes and incorporated into the liposomal membrane. After 2 hours liposomes had slowly migrating towards cell nuclei. Liposomes had completely disintegrated with their inner marker released by approximately 9 hours. TAT peptide-liposomes were made slightly cationic by adding up to 10 mol % of a cationic lipid (DOTAP). These slightly cationic liposomes were non-toxic towards cells, formed firm complexes with DNA (plasmid encoding for the formation of the Green Fluorescent Protein), and efficiently transfected a variety of cells. TAT peptideliposomes can be considered as promising carriers for the non-endocytotic intracellular delivery of drugs and DNA.

Improving the performance of non-viral gene-delivery vehicles that consist of synthetic compounds and nucleic acids is a key to successful gene therapy. Supplementing synthetic vehicles with various biological functions by using natural or artificial peptides is a promising approach with which to achieve this goal. One of the obstacles hindering this effort is that some of the potentially useful peptides, especially those with many basic amino acid residues, interfere with the formation of the complex owing to strong electrostatic interactions with the nucleic acid. In this review, we describe our recent work in examining the potential of these peptides in gene delivery, using a recombinant lambda phage particle as the model for the gene-delivery complex. Lambda phage encapsulates large duplex DNA in a rigid polyplex-like shell with a diameter of 55 nm, and can display various peptides on this capsid, independently of particle formation. By examining the expression of marker genes encapsulated in the phage capsid, we have demonstrated that the protein transduction domain of HIV Tat protein and the nuclear localization signal derived from SV40 T antigen can remarkably facilitate the delivery of these marker genes across the two major barriers, the cell membrane and the nuclear membrane, respectively. Our results indicate that these basic peptides can constitute effective components of synthetic gene-transfer complexes, as long as sufficient copies are displayed on the outer surface of the complex.

Protein Therapy is a newly developed method, which allows proteins, peptides and biologically active compounds to penetrate across the plasma membrane of eukaryotic cells by a polyarginine (most efficiently by 11-arginine, 11R) protein transduction domain [1]. This method enables us to control the localization of targeted substances in subcellular compartments, such as the nuclei, mitochondria and post-synaptic density. The method is very efficient and applicable not only to c ultur ed ce lls but also to tissue slic e s and the whole a nimal. Bra in, hea r t, skeleta l muscle, liver, pancreas and lymphocytes are efficient target organs and tissues for Protein Therapy [1, 2]. The method is therefore a very useful strategy in the post-genomic era. In this mini-review, the development of Protein Therapy and its application for cancer cells and neuroscience study will be shown.