Specific enzymatic cleavage and payload release from peptide-based hybrid nanocapsules

Within this thesis, new approaches for the concepts of peptide-polymer conjugates and peptide-based hybrid nanomaterials are investigated. In the first part, the synthesis of a triblock polymer-peptide-polymer is carried out following a typical peptide coupling reaction, both in solution and on solid-phase. The peptide sequence is chosen, so that it is cleaved by an enzyme preparation of trypsin. End-functionalized polystyrene is used as a model hydrophobic polymer and coupled to the peptide sequence. The results show successful coupling reactions in both methods, while the solid phase method produced a more defined product. Suspensions, consisting of peptide-polymer conjugates particles, are prepared in water by ultrasonication. In contact with the enzyme, the peptide constituting the conjugated particles is cleaved. This demonstrates the enzymatic cleavage in heterophase of enzymatic sequence bond to hydrophobic polymers, and is of great interest for the encapsulation and delivery of hydrophobic molecules.rnA second approach is the preparation of peptide-based hybrid nanocapsules. This is achieved by interfacial polyaddition in inverse miniemulsion with the peptide sequence functionalized with additional amino acids. A method suitable to the use of a peptide sequence for interfacial polyaddition was developed. It is shown that, the polarity of the dispersed phase influences the structures prepared, from particle-like to polymeric shell with a liquid core.rnThe peptide sequence is equipped with a FRET pair (more exactly, an internally-quenched fluorescent system) which allows the real-time monitoring of the enzymatic cleavage of the recognition site. This system shows the successful cleavage of the peptide-based nanocapsules when trypsin preparation is added to the suspensions. A water-soluble fluorescent polymer is efficiently entrapped and its possible use as marker for the capsules is highlighted. Furthermore, a small water-soluble fluorescent dye (SR-101) is successfully encapsulated and the encapsulation efficiency as a function of the functionality of the peptide and the amount of comonomer equivalent (toluene diisocyanate) is studied. The dye is encapsulated at such a high concentration, that self-quenching occurs. Thus, the release of the encapsulated dye triggered by the enzymatic cleavage of the peptide results in a fluorescence recovery of the dye. The fluorescence recovery of the FRET pair in the peptide and of the encapsulated dye correlate well.rnFinally, nanocapsules based on a hepsin-cleavable peptide sequence are prepared. Hepsin is an enzyme, which is highly upregulated in prostate cancer cells. The cleavage of the nanocapsules is investigated with healthy and “cancerous” (hepsin-expressing) cell cultures. The degradation, followed via fluorescence recovery of the FRET system, is faster for the suspensions introduced in the hepsin expressing cell cultures.rnIn summary, this work tackles the domain of responsive nanomaterials for drug delivery from a new perspective. It presents the adaptation of the miniemulsion process for hybrid peptide-based materials, and their successful use in preparing specific enzyme-responsive nanoparticles, with hydrophilic payload release properties.rn

Functional characterization of the placenta specific protein PLAC1 and its use for cancer immunotherapy

Summary Antibody-based cancer therapies have been successfully introduced into the clinic and have emerged as the most promising therapeutics in oncology. The limiting factor regarding the development of therapeutical antibody vaccines is the identification of tumor-associated antigens. PLAC1, the placenta-specific protein 1, was categorized for the first time by the group of Prof. Sahin as such a tumor-specific antigen. Within this work PLAC1 was characterized using a variety of biochemical methods. The protein expression profile, the cellular localization, the conformational state and especially the interacting partners of PLAC1 and its functionality in cancer were analyzed. Analysis of the protein expression profile of PLAC1 in normal human tissue confirms the published RT-PCR data. Except for placenta no PLAC1 expression was detectable in any other normal human tissue. Beyond, an increased PLAC1 expression was detected in several cancer cell lines derived of trophoblastic, breast and pancreatic lineage emphasizing its properties as tumor-specific antigen. rnThe cellular localization of PLAC1 revealed that PLAC1 contains a functional signal peptide which conducts the propeptide to the endoplasmic reticulum (ER) and results in the secretion of PLAC1 by the secretory pathway. Although PLAC1 did not exhibit a distinct transmembrane domain, no unbound protein was detectable in the cell culture supernatant of overexpressing cells. But by selective isolation of different cellular compartments PLAC1 was clearly enriched within the membrane fraction. Using size exclusion chromatography PLAC1 was characterized as a highly aggregating protein that forms a network of high molecular multimers, consisting of a mixture of non-covalent as well as covalent interactions. Those interactions were formed by PLAC1 with itself and probably other cellular components and proteins. Consequently, PLAC1 localize outside the cell, where it is associated to the membrane forming a stable extracellular coat-like structure.rnThe first mechanistic hint how PLAC1 promote cancer cell proliferation was achieved identifying the fibroblast growth factor FGF7 as a specific interacting partner of PLAC1. Moreover, it was clearly shown that PLAC1 as well as FGF7 bind to heparin, a glycosaminoglycan of the ECM that is also involved in FGF-signaling. The participation of PLAC1 within this pathway was approved after co-localizing PLAC1, FGF7 and the FGF7 specific receptor (FGFR2IIIb) and identifying the formation of a trimeric complex (PLAC1, FGF7 and the specific receptor FGFR2IIIb). Especially this trimeric complex revealed the role of PLAC1. Binding of PLAC1 together with FGF7 leads to the activation of the intracellular tyrosine kinase of the FGFR2IIIb-receptor and mediate the direct phosphorylation of the AKT-kinase. In the absence of PLAC1, no FGF7 mediated phosphorylation of AKT was observed. Consequently the function of PLAC1 was clarified: PLAC1 acts as a co-factor by stimulating proliferation by of the FGF7-FGFR2 signaling pathway.rnAll together, these novel biochemical findings underline that the placenta specific protein PLAC1 could be a new target for cancer immunotherapy, especially considering its potential applicability for antibody therapy in tumor patients.