Cell penetrating peptides (CPPs) have been used in many areas of drug delivery for mediating the delivery of peptides, protein, DNA, siRNA and liposomes. Additionally they have shown an ability to overcome drug resistance in cells enhancing chemotherapeutic activity. Our group has recently designed a promising multifunctional peptide, Tat-LK15, originating from the fusion of Tat peptide (49-57) with the synthetic amphipathic peptide, LK15. TAT-LK15 was found to be efficient at mediating DNA and siRNA delivery to cells especially contributing to the silencing of bcr-abl oncoprotein over a long period of time. To date, Tat-LK15 peptide’s cells uptake mechanism, which is expected to be influenced by the presence of the amphipathic sequence LK15, which is known to be lytic, has not been assessed. The aim of this project is to study the cellular uptake of the Tat-LK15 peptide and any influence it exerts on the uptake of p-glycoproteins substrates. Also we aimed to explore whether the peptide characteristics could be exploited to improve the delivery of the cytotoxic agent, doxorubicin.Flow cytometry and confocal microscopy experiments revealed that Tat-LK15 uptake was dependent on two mechanisms depending on the concentration of peptide used. At low concentrations (2.5µM and below) the uptake of TAMRA-Tat-LK15 peptide appeared to be temperature-dependent and was inhibited by sodium azide suggesting endocytosis as the main route of entry in the studied cell lines. At 5µM and above, the peptide’s uptake was less reliant on temperature and not be inhibited by sodium azide, but relied on membrane potential. Interestingly in K562 cells, the peptide accumulated on the cell membrane.The Tat-LK15 peptide membrane activity was also characterised: Tat-LK15 lytic activity was concentration-dependent. At 5µM the Tat-LK15 peptide led to approximately 60% LDH release in HT29 cells which is likely to be related to the amphipathic sequence, LK15 renowned for its membrane lytic activity. Further to this, the uptake of a membrane impermeant dye (SYTOX® blue) incubated with the peptide was followed as a function of time in K562 cells with a high content screening. The results appeared to substantiate the above findings that Tat-LK15 peptide has a concentration- and time-dependent membrane lytic activity. An MTT assay, after four hours of incubation, indicated very limited variation in cytotoxicity (EC50 ~ 5µM) over six different cell lines. More importantly, an Annexin V assay suggested a possible induction of apoptosis in the MCF-7 cell line, in contrast a Tat-LK15 concentration-dependent necrosis was observed in a K562 cell line. Finally attempts were made to exploit the properties of Tat-LK15 to overcome drug resistance of doxorubicin. Firstly Tat-LK15 peptide co-incubation with a model P-gp substrate (calcein AM) revealed a significant reduction of calcein fluorescence in K562 and HT29 cells and their doxorubicin resistant sublines (p<0.05). Attempts to conjugate doxorubicin to TAT-LK15 proved difficult. Consequently doxorubicin/Tat-LK15 admixtures were used to study whether the P-gp efflux of doxorubicin in doxorubicin resistance sublines could be circumvented. A significant increase of the toxicity relative to doxorubicin alone for long incubation times on several cell lines (p<0.05) indicated that using TAT-LK15 as an additive could potentially be considered as a drug delivery strategy. Thus the concept of conjugating a well known cell penetrating peptide to an amphipathic sequence is worthwhile however the findings in this study are not sufficient to highlight the full potential and major improvements in efficacy needs to be achieved.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:566572 |
| Date | January 2012 |
| Creators | Al-Kotaji, Myasar |
| Contributors | Aojula, Harmesh; Berk, David; Pluen, Alain |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/the-multifunctional-peptide-tatlk15a-study-of-its-cellular-uptake-and-its-potential-use-in-drug-delivery(92514766-2626-4949-8e02-ca77dc88ff7b).html |
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