188. Internalization of Novel Delivery Vector TAT-Streptavidin into Human Cells

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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“…This observation is in agreement with the reported location of other CPPs, such as TAT [53] or TP and its derivatives TP10 and TP14 [8].…”

Section: Discussionsupporting

confidence: 93%

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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“…Actually, smaller cargoes tend to be taken up by membrane transduction, while larger molecules are mainly trapped in endocytosis. [14][15][16] Our triple-helical cell-penetrating peptide probably could not form pores in the membranes. Some progresses in overcoming this drawback such as combination with membrane-disruptive liposomes, peptides or polymers have been made.…”

Section: )mentioning

confidence: 89%

Cellular Uptake of IgG Using Collagen-Like Cell-Penetrating Peptides

Masuda

Yamamoto

Koide

2016

Biological & Pharmaceutical Bulletin

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Cell-penetrating peptides (CPPs) are attractive tools for delivering macromolecules that have poor membrane permeability, such as antibodies, into cells. However, the major drawback of conventional CPPs is their instability in bodily fluids. We previously reported a novel CPP employing a collagen-like triple-helical structure that exhibited remarkable resistance against serum proteases. Herein, we report the delivery of full-length immunoglobulin G (IgG) antibody into cells using a triple-helical CPP. The CPP was conjugated to IgG via a one-pot reaction using 2-iminothiolane as a crosslinking reagent. The triple-helical CPP was less prone to being aggregated and neutralized by serum than was octaarginine, a conventional CPP. However, most of the conjugates were found to be entrapped in endosomes.

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“…Firstly, it allows the delivery of a non-modified cargo and avoids potential problems associated with the covalent linkage of a PTD, such as reduced expression and purification yields and impairment of the biophysical properties of the cargo (10). Secondly, in contrast to previously designed transmembrane delivery systems, such as Tat11-SA for biotinylated molecules (27,34) or Tat11-fused protein A for antibodies (35,36), the transporter employed here is less bulky since it only consists of low-MW components: a PTD and a small ligand. Thirdly, since the active site of the internalized cargo is reversibly occupied by a PTD-fused ligand, only molecules possessing a higher affinity to the cargo should be able to displace the ligand which increases specificity.…”

Section: Discussionmentioning

confidence: 99%

Binding proteins internalized by PTD-fused ligands allow the intracellular sequestration of selected targets by ligand exchange

Moosmeier

Bulkescher²,

Hoppe‐Seyler³

et al. 2010

Int J Mol Med

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Abstract. The targeted inactivation of intracellular molecules has important therapeutic potential. For this purpose, it could be envisioned to introduce specifically designed binding proteins into cells by covalent linkage to protein transduction domains (PTDs). However, stable linkage of a PTD to a cargo may affect its conformation and, hence, its binding property inside the cell. Here, we analyzed the ability of non-covalently linked PTDs to internalize the model binding proteins streptavidin (SA) and Strep-Tactin (ST). Notably, inside the cell, the PTD-Strep-tag II ligand used for internalization of SA was displaced by the model target biotin which exhibits a higher binding affinity for the same binding pocket. Thus, specifically designed binding proteins can be internalized into cells by non-covalent binding to a PTD and subsequently be used for capturing given intracellular target molecules by ligand exchange. Under therapeutic aspects, it could be envisioned to further develop such systems for the intracellular sequestration, and consequently, functional inactivation of pathologically relevant factors.

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188. Internalization of Novel Delivery Vector TAT-Streptavidin into Human Cells

Cited by 20 publications

References 0 publications

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

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

Cellular Uptake of IgG Using Collagen-Like Cell-Penetrating Peptides

Binding proteins internalized by PTD-fused ligands allow the intracellular sequestration of selected targets by ligand exchange

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