Tat-Conjugated Synthetic Macromolecules Facilitate Cytoplasmic Drug Delivery To Human Ovarian Carcinoma Cells

Nori, Aparna; Jensen, Keith D.; Tijerina, Monica; Kopečková, Pavla; Kopeček, Jindřich

doi:10.1021/bc0255900

Cited by 135 publications

(90 citation statements)

References 19 publications

(20 reference statements)

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“…The one most commonly employed is trypsin treatment, which removes the surface-bound fusion proteins and reduces the chances of nonspecific migration of proteins upon fixation of cells during immunostaining. In addition, some studies have shown successful transduction of PTD-fusion proteins into cells without fixation [47,98,[158][159][160][161]. Further, many of these studies have also shown biological effects upon treatment with PTD-fusion proteins, which could be due to the interactions with surface receptors [162] or due to the release of proteins from lysosomes into the cytoplasm and nucleus.…”

Section: Validation Of True Ptd-mediated Protein Transductionmentioning

confidence: 99%

The taming of the cell penetrating domain of the HIV Tat: Myths and realities

Chauhan

Tikoo²,

Kapur

et al. 2007

Journal of Controlled Release

155

136

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Protein transduction with cell penetrating peptides over the past several years has been shown to be an effective way of delivering proteins in vitro and now several reports have also shown valuable in vivo applications in correcting disease states. An impressive bioinspired phenomenon of crossing biological barriers came from HIV transactivator Tat protein. Specifically, the protein transduction domain of HIV-Tat has been shown to be a potent pleiotropic peptide in protein delivery. Various approaches such as molecular modeling, arginine guanidinium head group structural strategy, multimerization of PTD sequence and phage display system have been applied for taming of the PTD. This has resulted in identification of PTD variants which are efficient in cell membrane penetration and cytoplasmic delivery. Inspite of these state of the art technologies, the dilemma of low protein transduction efficiency and target specific delivery of PTD fusion proteins remains unsolved. Moreover, some misconceptions about PTD of Tat in the literature require considerations. We have assembled critical information on secretory, plasma membrane penetration and transcellular properties of Tat and PTD using molecular analysis and available experimental evidences.

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Section: Validation Of True Ptd-mediated Protein Transductionmentioning

confidence: 99%

The taming of the cell penetrating domain of the HIV Tat: Myths and realities

Chauhan

Tikoo²,

Kapur

et al. 2007

Journal of Controlled Release

155

136

View full text Add to dashboard Cite

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“…Most studies involving PTDmediated cellular uptake have been carried out with Tat peptide. Tat-modified nanoparticles, such as cross-linked iron oxide for cell tracking by magnetic resonance [18] or polymeric drug carriers [19] showed improved uptake properties when compared to unmodified particles. The Tat peptide was also used to investigate PTD-mediated liposome uptake.…”

mentioning

confidence: 99%

Enhanced heparan sulfate proteoglycan-mediated uptake of cell-penetrating peptide-modified liposomes

Marty

Meylan

Schott

et al. 2004

CMLS, Cell. Mol. Life Sci.

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Abstract. Protein transduction domains (PTDs) are used to enhance cellular uptake of drugs, proteins, polynucleotides or liposomes. In this study, functionalized Antennapedia (Antp, aa 43 -58) and HIV Tat (aa 47 -57) peptides were coupled to small unilamellar liposomes via thiol-maleimide linkage. Modified liposomes showed higher uptake into a panel of cell lines including tumor and dendritic cells than unmodified control liposomes. Liposome uptake was time and concentration dependent as analyzed by flow cytometry and live-cell microscopy. At least 100 PTD molecules per small unilamellar liposome (100 ± 30 nm) were necessary for efficient translocation into cells. Cellular uptake of PTD-modified liposomes was 15-to 25-fold increased compared to unmod-CMLS, Cell. Mol. Life Sci. 61 (2004) 1785 -1794 1420-682X/04/141785-10 DOI 10.1007/s00018-004-4166-0 © Birkhäuser Verlag, Basel, 2004 CMLS Cellular and Molecular Life Sciencesified liposomes and was inhibited by preincubation of liposomes with heparin. Glycosaminoglycan-deficient CHO cells showed dramatically reduced cell association of PTD-modified liposomes, confirming the important role of heparan sulfate proteoglycans in PTD-mediated uptake. Antp-liposomes used as carriers of the cytotoxic drug N 4 -octadecyl-1-b-D-arabinofuranosylcytosine-(5¢-5¢)-3¢-C-ethinylcytidine showed a reduction of the IC 50 by 70 % on B16F1 melanoma cells compared with unmodified liposomes. PTD-functionalized liposomes, particularly Antp-liposomes, represent an interesting novel carrier system for enhanced cell-specific delivery of a large variety of liposome-entrapped molecules.

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“…To understand if both intracellular phenotypes are two distinct intermediate stages of the same process or indicate different uptake routes, we monitored the cytoplasmic and nucleolar localization of RRPs upon inhibition of endocytosis. In addition to TAT, we used the peptide PTD 4 , which is an artificial, more amphipathic CPP with a reduced number of arginines and increased ␣-helical content compared with TAT (29). Most importantly, to suppress endocytic pathways, we restricted ourselves to the usage of genetically inducible systems, knock-out (KO) cell cultures, or physical methods, thus avoiding any potential side effects of chemical inhibitors of endocytosis.…”

mentioning

confidence: 99%

Cell Entry of Arginine-rich Peptides Is Independent of Endocytosis

Ter-Avetisyan

Tünnemann

Nowak

et al. 2009

Journal of Biological Chemistry

202

174

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Arginine-rich peptides are a subclass of cell-penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytic mode of uptake and a subsequent release from vesicles or to direct membrane penetration (transduction). To distinguish between both possibilities, we have blocked endocytic pathways suggested to be involved in uptake of cell-penetrating peptides. We have then monitored by confocal microscopy the uptake and distribution of the cell-penetrating transactivator of transcription (TAT) peptide into living mammalian cells over time. To prevent side effects of chemical inhibitors, we used genetically engineered cells as well as different temperature. We found that a knockdown of clathrin-mediated endocytosis and a knock-out of caveolin-mediated endocytosis did not affect the ability of TAT to enter cells. In addition, the TAT peptide showed the same intracellular distribution throughout the cytoplasm and nucleus as in control cells. Even incubation of cells at 4°C did not abrogate TAT uptake nor change its intracellular distribution. We therefore conclude that this distribution results from TAT peptide that directly penetrated (transduced) the plasma membrane. The formation of nonselective pores is unlikely, because simultaneously added fluorophores were not taken up together with the TAT peptide. In summary, although the frequency and kinetics of TAT transduction varied between cell types, it was independent of endocytosis.

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Tat-Conjugated Synthetic Macromolecules Facilitate Cytoplasmic Drug Delivery To Human Ovarian Carcinoma Cells

Cited by 135 publications

References 19 publications

The taming of the cell penetrating domain of the HIV Tat: Myths and realities

The taming of the cell penetrating domain of the HIV Tat: Myths and realities

Enhanced heparan sulfate proteoglycan-mediated uptake of cell-penetrating peptide-modified liposomes

Cell Entry of Arginine-rich Peptides Is Independent of Endocytosis

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