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

Abstract

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.

The principles and applications of avidin-based nanoparticles in drug delivery and diagnosis

“…Cell-penetrating sequences such as transportan-10 (TP-10) and transportan (TP) have recently been evaluated by Wierzbicki et al Biotinylated analogues of TP and TP-10 have demonstrated an endocytosis-independent and highly efficient delivery of siRNA when complexed with streptavidin in HCT116 (metastatic CRC model) and HT29 (early stage CRC model) cell lines and resulted in a high silencing activity of SASH-1 mRNA. [76] It can clearly be inferred that streptavidin provides a substantial advantage for cellular and tissue internalization in addition to its use as a dynamic stoichiometric support (back-bone) for nanocomplexes.…”

Three Valuable Peptides from Bee and Wasp Venoms for Therapeutic and Biotechnological Use: Melittin, Apamin and Mastoparan

“…On the other hand, there is a chimeric galanin-mastoparan peptide called transportan, which contains the first 13 amino acids from the highly conserved amino-terminal part of galanin and the 14 amino acids sequence of mastoparan in the carboxyl terminus [ 179 ]. Transportan and some derivatives have the capacity to carry macromolecules [ 180 , 181 , 182 ].…”

Cell-penetrating peptides as a promising tool for delivery of various molecules into the cells

“…The role of the linker is often attributed to side chains of the residues present in CPP amino acid sequence, such as the thiol group of cysteine or the e-amino group of lysine. Non-covalent interactions, such as between streptavidin and biotin attached to CPP, are also possible [45].…”

“…Hence, cell biology techniques were used to determine the type of endocytosis contribution or the transport without energy contribution in the process of CPP absorption: -incubation of cells with CPPs at low temperatures (< 4°C) or under energy deficiency; -application of trypsin to remove surface receptor proteins; -use of confocal or freeze-fracture electron microscopy [43,44]; -incubation with compounds facilitating selective transport pathway, e.g. brefeldin A [45]; -binding to a cargo molecule determining a specific type of transport (e.g. transferrin, cholera toxin, dextran, or G protein) or to the target location in the cell allowing the determination of internalization type (e.g.…”