Cell-penetrating peptides: A comparative membrane toxicity study

Saar, Külliki; Lindgren, Maria; Hansen, Mats; Eiríksdóttir, Emelía; Jiang, Yang; Rosenthal-Aizman, Katri; Sassian, Meeri; Langel, Ülo

doi:10.1016/j.ab.2005.07.033

Cited by 256 publications

(217 citation statements)

References 73 publications

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“…peptides, siRNA, ODNs, proteins), and results of this assay can be predictive of performance as an intracellular drug delivery vehicle. 3,4,8,18,19 Thus, this assay represents an effective screen to gauge the ability of polymeric drug carriers to mediate intracellular drug delivery based on their pH-dependent membrane disruption.…”

Section: Discussionmentioning

confidence: 99%

“…6,7 One useful model system for screening endosomolytic behavior is the ex vivo pH-dependent hemolysis assay. 8 In this model system, the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. This generalizable model has been used by others to evaluate the endosomolytic behavior of cell-penetrating peptides and other polymeric gene delivery systems.…”

Section: Introductionmentioning

confidence: 99%

“…This generalizable model has been used by others to evaluate the endosomolytic behavior of cell-penetrating peptides and other polymeric gene delivery systems. [8][9][10][11] In this experiment, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular (7.4), early endosomal (6.8), and late endo-lysosomal (< 6.8) environments. The amount of hemoglobin released during the incubation period is quantified as a measure of red blood cell lysis, which is normalized to the amount of hemoglobin released in positive control samples lysed with a detergent.…”

Section: Introductionmentioning

confidence: 99%

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<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Evans

Nelson

et al. 2013

JoVE

271

245

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Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems.In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Evans

Nelson

et al. 2013

JoVE

271

245

View full text Add to dashboard Cite

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“…At this concentration, the TAT peptide has no cellular toxicity as tested by cell leakage of cell lines and primary erythrocytes (32). PNAs have also been tested for toxicity after injection into mice and, under maximal dosage (100 mg∕Kg) with repeated administration, were considered nontoxic as major organ functions were normal and no irreversible damage was detected (33).…”

Section: Discussionmentioning

confidence: 99%

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo

Chen

Peterson²,

Iancu‐Rubin³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Pharmacological treatments designed to reactivate fetal γ-globin can lead to an effective and successful clinical outcome in patients with hemoglobinopathies. However, new approaches remain highly desired because such treatments are not equally effective for all patients, and toxicity issues remain. We have taken a systematic approach to develop an embedded chimeric peptide nucleic acid (PNA) that effectively enters the cell and the nucleus, binds to its target site at the human fetal γ-globin promoter, and reactivates this transcript in adult transgenic mouse bone marrow and human primary peripheral blood cells. In vitro and in vivo DNAbinding assays in conjunction with live-cell imaging have been used to establish and optimize chimeric PNA design parameters that lead to successful gene activation. Our final molecule contains a specific γ-promoter-binding PNA sequence embedded within two amino acid motifs: one leads to efficient cell/nuclear entry, and the other generates transcriptional reactivation of the target. These embedded PNAs overcome previous limitations and are generally applicable to the design of in vivo transcriptional activation reagents that can be directed to any promoter region of interest and are of direct relevance to clinical applications that would benefit from such a need.γ-globin gene reactivation | primary human CD34+ cells

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“…Cell-penetrating peptides (CPPs), also known as peptide transduction domains (PTDs), make a new class of transmembrane delivery vectors with high pharmaceutical potential [1]. CPPs can be taken up by living cells and can deliver different covalently coupled cargoes into cells both in vitro [2] or in vivo [3,4].…”

Section: Introductionmentioning

confidence: 99%

Protein and siRNA delivery by transportan and transportan 10 into colorectal cancer cell lines

Wierzbicki

Kogut-Wierzbicka

Ruczyński

et al. 2015

Folia Histochem Cytobiol.

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Introduction. Cell penetrating peptides (CPPs) have the ability to translocate through cell membranes with high efficiency and therefore can introduce biological agents with pharmaceutical properties into the cell. Transportan (TP) and its shorter analog transportan 10 (TP10) are among the best studied CPPs, however, their effects on viability of and cargo introduction into colorectal cancer (CRC) cells have yet not been investigated. The aim of our study was to evaluate the cytotoxic effects of TP and TP10 on representative CRC lines and the efficiency of protein (streptavidin) and siRNA cargo delivery by TP-biotinylated derivatives (TP-biot). Material and methods. HT29 (early stage CRC model) and HCT116 (metastatic CRC model) cell lines were incubated with TP, TP10, TP-biot1, TP-biot13 and TP10-biot1. The effects of studied CPPs on cell viability and cell cycle were assessed by MTT and annexin V assays. The uptake of streptavidin-FITC complex into cells was determined by flow cytometry and fluorescence microscopy, with the inhibition of cellular vesicle trafficking by brefeldin A. The efficiency of siRNA for SASH1 gene delivery was measured by quantitative PCR (qPCR). Results. Since up to 10 µM concentrations of each CPP showed no significant cytotoxic effect, the concentrations of 0.5-5 µM were used for further analyses. Within this concentration range none of the studied CPPs affected cell viability and cell cycle. The efficient and endocytosis-independent introduction of streptavidin-FITC complex into cells was observed for TP10-biot1 and TP-biot1 with the cytoplasmic location of the fluorescent cargo; decreased SASH1 mRNA level was noticed with the use of siRNA and analyzed CPPs. Conclusions. We conclude that TP, TP10 and their biotinylated derivatives can be used as efficient delivery vehicles of small and large cargoes into CRC cells.

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Cell-penetrating peptides: A comparative membrane toxicity study

Cited by 256 publications

References 73 publications

<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo

Protein and siRNA delivery by transportan and transportan 10 into colorectal cancer cell lines

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