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      Mechanisms of Cellular Uptake of Cell-Penetrating Peptides

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          Abstract

          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).

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          Most cited references76

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          Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis.

          The TAT protein transduction domain (PTD) has been used to deliver a wide variety of biologically active cargo for the treatment of multiple preclinical disease models, including cancer and stroke. However, the mechanism of transduction remains unknown. Because of the TAT PTD's strong cell-surface binding, early assumptions regarding cellular uptake suggested a direct penetration mechanism across the lipid bilayer by a temperature- and energy-independent process. Here we show, using a transducible TAT-Cre recombinase reporter assay on live cells, that after an initial ionic cell-surface interaction, TAT-fusion proteins are rapidly internalized by lipid raft-dependent macropinocytosis. Transduction was independent of interleukin-2 receptor/raft-, caveolar- and clathrin-mediated endocytosis and phagocytosis. Using this information, we developed a transducible, pH-sensitive, fusogenic dTAT-HA2 peptide that markedly enhanced TAT-Cre escape from macropinosomes. Taken together, these observations provide a scientific basis for the development of new, biologically active, transducible therapeutic molecules.
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            Mode of action of membrane active antimicrobial peptides.

            Water-membrane soluble protein and peptide toxins are used in the defense and offense systems of all organisms, including plants and humans. A major group includes antimicrobial peptides, which serve as a nonspecific defense system that complements the highly specific cell-mediated immune response. The increasing resistance of bacteria to conventional antibiotics stimulated the isolation and characterization of many antimicrobial peptides for potential use as new target antibiotics. The finding of thousands of antimicrobial peptides with variable lengths and sequences, all of which are active at similar concentrations, suggests a general mechanism for killing bacteria rather than a specific mechanism that requires preferred active structures. Such a mechanism is in agreement with the "carpet model" that does not require any specific structure or sequence. It seems that when there is an appropriate balance between hydrophobicity and a net positive charge the peptides are active on bacteria. However, selective activity depends also on other parameters, such as the volume of the molecule, its structure, and its oligomeric state in solution and membranes. Further, although many studies support that bacterial membrane damage is a lethal event for bacteria, other studies point to a multihit mechanism in which the peptide binds to several targets in the cytoplasmic region of the bacteria.
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              Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake.

              Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48-60 and (Arg)(9). We demonstrate here that cell fixation, even in mild conditions, leads to the artifactual uptake of these peptides. Moreover, we show that flow cytometry analysis cannot be used validly to evaluate cellular uptake unless a step of trypsin digestion of the cell membrane-adsorbed peptide is included in the protocol. Fluorescence microscopy on live unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake are similar to the kinetics of endocytosis. Peptide uptake is inhibited by incubation at low temperature and cellular ATP pool depletion. Similar data were obtained for Tat-conjugated peptide nucleic acids. These data are consistent with the involvement of endocytosis in the cellular internalization of cell-penetrating peptides and their conjugates to peptide nucleic acids.
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                Author and article information

                Journal
                J Biophys
                JBP
                Journal of Biophysics
                Hindawi Publishing Corporation
                1687-8000
                1687-8019
                2011
                7 April 2011
                : 2011
                : 414729
                Affiliations
                1Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, 10691 Stockholm, Sweden
                2Department of Neurochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, 10691 Stockholm, Sweden
                3Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
                Author notes
                *Astrid Gräslund: astrid@ 123456dbb.su.se

                Academic Editor: Eaton Edward Lattman

                Article
                10.1155/2011/414729
                3103903
                21687343
                074a83e8-58e9-4951-b5ea-128461dcfd02
                Copyright © 2011 Fatemeh Madani et al.

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 24 November 2010
                : 24 January 2011
                Categories
                Review Article

                Biophysics
                Biophysics

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