Cell penetrating peptides (CPPs) have received considerable attention as cellular delivery vectors for small-molecule and macromolecular therapeutics. Hundreds of CPP sequences have now been described including bioportides that serve as single entity modifiers of cell physiology. Translation of promising CPP pre-clinical studies have however been disappointing as few identified delivery-systems have progressed to clinical trials. This project aimed to extend on studies using CPPs for delivering peptides influencing NF-B signalling and to search for novel membrane active peptides that could be classed as CPPs or bioportides. In-house methods measuring IB-α degradation and luciferase expression assays were developed to study the capacity of penetratin to influence NF-B activity via delivery of the NBD peptide (Pen-NBD: DRQIKIWFQNRRMKWKKTALDWSWLQTE). Results from these studies highlighted how two methods investigating the same signalling pathway could give very different results. Pen-NBD did not reduce TNF-α-induced IB-α degradation but induced reduction in TNF-α-induced luciferase activity. This effect was also observed in cells treated with penetratin alone or DMSO- diluent. EJP-18 (LFMRRRHIVRKRTLRRLL) represents a novel peptide from the EGFR juxtamembrane region and was found to be membrane active and internalised into cells. This thesis further characterised this peptide including two other sequences from this region of EGFR (EJP-21- ii LFMRRRHIVRKRTLRRLLQER and E-64562- RRRHIVRKRTLRRLLQER). In studied cancer cell lines, EJP-18 was toxic in an EGFR-dependant manner and aside from being a potential bioportide, also effectively delivered protein (BSA) and siRNA into cells. EJP-18/BSA was delivered to the lysosome and it remains to be determined whether it could be utilised to deliver siRNA to the cytosol. Cytotoxicity and protein delivery studies with EJP-21 and E-64562 highlighted the critical importance of terminal hydrophobicity in the EJP-18 sequence; this may extend to other studies involving CPPs. This thesis provides new information on in vitro activity of CPPs and CPP-chimeras. Additionally, new peptides were discovered that could potentially be classed as bioportides and CPPs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:681262 |
| Date | January 2015 |
| Creators | Eissa, Noura Gamal |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/86622/ |
Page generated in 0.0129 seconds