Feni, Lucia (2019). Improving cargo delivery in cancer therapy with the help of cell-penetrating peptides. PhD thesis, Universität zu Köln.

Doctoral dissertation_publication_Lucia Feni.pdf

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A major obstacle of many active pharmaceutical compounds is their low ability to cross body barriers, especially cell membranes. Cell permeability of a drug is therefore considered as a key step for therapeutic efficacy. Over the last decades, different approaches to overcome this limitation have been studied intensively. Among these are so-called cell-penetrating peptides (CPPs). CPPs are able to autonomously internalize into cells without the need for auxiliary proteins. However, not only the cellular uptake is important but also cell selectivity has to be addressed. Over the past two decades, cancer research has dramatically evolved, particularly with the appearance of targeted molecular therapies and advances in antibody engineering that allowed the discovery and validation of innovative molecules, more effective and less harmful than conventional chemotherapy. Especially small molecule-drug conjugates, like peptide-drug conjugates, became of particular interest since they combine several advantages as deep tissue penetration, possibility of cell organelle targeting and relatively easy access by chemical synthesis. This work focuses on the design and synthesis of an array of tumor-targeting peptide-drug conjugates combining known tumor-homing peptides with a well-described CPP and potent cytotoxic drugs. The development of these hybrids was followed by a validation of the model via in vitro studies where their selectivity towards different cell lines was evaluated. Two targeting ligands (GnRH-III and c[DKPf3RGD]) were employed for the conjugation to the CPP sC18 and a very straightforward synthesis could be developed in both cases. The conjugates maintained a remarkable binding affinity in low nanomolar range towards GnRH and αvβ3 integrin receptors, respectively, and for further in vitro experiments, the expression of the receptors in different cell lines was explored. For the investigation of the final compounds, a new in vitro model based on a short contact time with the cells was established in order to emphasize the role of the fast CPP-mediated internalization after reversible binding to the receptors. While for the GnRH-III-conjugates a selectivity was difficult to detect, the c[DKPf3RGD] was identified as very effective targeting moiety for the synthesis of an efficient drug delivery system. Different drugs were attached to the CPP and daunorubicin turned out to be the most advantageous in terms of simple synthesis and stability. Fluorescence analysis demonstrated that the internalization was mainly mediated by the CPP but that the ligand had an important role in targeting the surface of the cells overexpressing the receptor. The selectivity could also be proved by anti-proliferative assays providing another demonstration that with this approach it would be possible to overcome the drawbacks of CPP-mediated drug transport leading to higher target selectivity and better bioavailability. In the second part of the thesis, cyclic CPPs with peculiar diketopiperazine scaffolds (trans DKP3 and cis DKP1) were synthesized starting from the sequence of a truncated variant of II sC18. An optimized cyclization strategy could be developed and the secondary structure of these peptides was analyzed and compared with the linear counterparts by different techniques. The biological activity of these compounds was also evaluated in cell systems where the ability to transport cytotoxic drugs inside the cells was explored by using both a non-covalent as well as covalent drug coupling approach. Notably, the cycle actually showed a higher ability to increase the activity of daunorubicin than the linear CPP, proving that cyclization via a diketopiperazine scaffold is a promising strategy to improve CPP-mediated drug delivery.

Item Type: Thesis (PhD thesis)
CreatorsEmailORCIDORCID Put Code
Feni, Lucialfeni@uni-koeln.deUNSPECIFIEDUNSPECIFIED
URN: urn:nbn:de:hbz:38-97584
Date: 4 February 2019
Language: English
Faculty: Faculty of Mathematics and Natural Sciences
Divisions: Faculty of Medicine > Biochemie > Institut I für Biochemie
Subjects: Natural sciences and mathematics
Chemistry and allied sciences
Life sciences
Uncontrolled Keywords:
Cell-penetrating peptidesUNSPECIFIED
Tumor-targeted therapyUNSPECIFIED
Small-molecule drug conjugatesUNSPECIFIED
Cargo deliveryUNSPECIFIED
Date of oral exam: 29 March 2019
NameAcademic Title
Neundorf, InesProf. Dr.
Sewald, NorbertProf. Dr.
Refereed: Yes
URI: http://kups.ub.uni-koeln.de/id/eprint/9758


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