Universität zu Köln

Horn, Mareike (2017). Model membrane systems, membrane-active peptides and application possibilities. PhD thesis, Universität zu Köln.

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Abstract

The ability of cell-penetrating peptides (CPPs) to translocate a variety of covalently or non-covalently linked cargoes, particularly therapeutics and imaging agents, across the plasma membrane of living cells renders them of broad interest in the medical field as well as in biotechnological development. Thus, the use of CPPs, e.g. in therapeutics, increases the necessity of understanding their mechanism of action, a subject still under debate. For the first step of peptide internalization, the interaction between the positively charged amino acid side chains within the peptide sequence and the anionic constituents of the membrane bilayer is assumed. However, owing to the complexity of biological plasma membranes, studying the mode of action of CPPs on a molecular level is very difficult. With this in mind, model membrane systems, especially large and giant unilamellar vesicles and giant plasma membranes vesicles derived from live cells plasma membranes, were prepared and several techniques were established in order to monitor the initial step of cell entry – binding to and translocation through the cell membrane as well as identification of key components of the plasma membrane required for efficient uptake. The first chapter focuses on the membrane interactions and perturbation mechanism of two structural modified variants of the well-described CPP sC18, a cyclized and a dimeric branched version, with artificial model membranes. Fluorescence analysis demonstrated the importance of negatively charged lipids for an efficient uptake of Arg-rich CPPs, supporting the assumption that electrostatic interactions are required for the initial stage of cell entry. In case of dimeric variants, the strong affinity was further accompanied by intense membrane destabilizing effects, presumably responsible for their lytic behavior as anticancer peptide. Moreover, the cyclization of the peptide backbone turned out to be a promising strategy as well to improve their cell-penetrating capability. Using CD spectroscopy, structural arrangements when in contact with neutral and anionic vesicles further revealed that a certain flexibility and thus the static arrangement of the guanidinium groups within the cycle seem to positively affect peptide-membrane interaction. In summary, these evidences suggest that the internalization of the peptide variants depends highly on the membrane composition of the target cell. In the second chapter, the focus lay on the design and characterization of novel peptide conjugates, composing of a wound healing promoting peptide and sC18 as cell penetrating peptide, with the final goal to further enhance the wound healing activity. Besides, the new conjugate Tylotoin-sC18* exhibited further promising antimicrobial activity and moreover a certain selectivity towards cancer cells, suggesting that Tylotoin-sC18* is a good candidate for further studies and the possible development as a therapeutic agent for infected wounds.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Horn, Mareike mareikehorn@gmx.de UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-80005
Date:	23 October 2017
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Medicine > Biochemie > Institut I für Biochemie
Subjects:	Chemistry and allied sciences Life sciences
Uncontrolled Keywords:	Keywords Language membrane-active peptides English peptide-lipid interaction English cell-penetrating peptide English wound healing English
Date of oral exam:	19 December 2017
Referee:	Name Academic Title Neundorf, Ines Prof. Dr. Baumann, Ulrich Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/8000