Recently, much attention has been given to the problem of drug delivery through the cell-membrane in order to treat and manage several diseases. The discovery of cell penetrating peptides (CPPs) represents a major breakthrough for the transport of large-cargo molecules that may be useful in clinical applications. CPPs are rich in basic amino acids such as arginine and lysine and are able to translocate over membranes and gain access to the cell interior. They can deliver large-cargo molecules, such as oligonucleotides, into cells. Endocytosis and direct penetration have been suggested as the two major uptake mechanisms, a subject still under debate. Unresolved questions include the detailed molecular uptake mechanism(s), reasons for cell toxicity, and the delivery efficiency of CPPs for different cargoes. Here, we give a review focused on uptake mechanisms used by CPPs for membrane translocation and certain experimental factors that affect the mechanism(s). 1. Introduction The cell membrane is the structure that protects living cells from the surrounding environment, only allowing the movement of compounds generally with small molecular size across this barrier into the cell. Some drugs are large hydrophilic molecules showing major limitations for their penetration through the cell membrane. A group of short peptides have been discovered that serve as delivery vectors for large molecules. They may have been called by different names such as protein translocation domain, membrane translocating sequence, Trojan peptide, or most commonly, cell-penetrating peptide (CPP). Generally, CPPs are defined as short, water-soluble and partly hydrophobic, and/or polybasic peptides (at most 30–35 amino acids residues) with a net positive charge at physiological pH [1]. The main feature of CPPs is that they are able to penetrate the cell membrane at low micromolar concentrations in vivo and in vitro without using any chiral receptors and without causing significant membrane damage. Furthermore, and even more importantly, these peptides are capable of internalizing electrostatically or covalently bound biologically active cargoes such as drugs with high efficiency and low toxicity [1, 2]. This new class of peptides was introduced in the late 1980s by the discovery of the human immunodeficiency virus type 1 (HIV-1) encoded TAT peptide [3, 4] and the amphiphilic Drosophila Antennapedia homeodomain-derived 16 amino acid penetratin peptide (pAntp), which was discovered a few years later [5–8]. These two peptides are the most extensively studied of all CPPs. The mechanism(s)
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