Robert Rennert

Quantum Dot−Carrier Peptide Conjugates Suitable for Imaging and Delivery Applications

We developed multifunctional fluorescent nanoparticles suitable for the nonviral delivery of negatively charged molecules like RNA. Therefore, we incorporated the recently developed branched hCT-derived carrier peptide hCT(18-32)-k7 on the surface of luminescent quantum dots (QDs). Besides detailed characterization of our QD-peptide conjugates concerning stability, toxicity, and uptake mechanism. we used them for efficient RNA delivery into different cell lines. The results of our studies indicate the involvement of more than one endocytotic uptake pathway in the internalization process. Furthermore, we could show that the QD-peptide bioconjugates exhibit no effect on cell viability and possess high stability inside living cells. The efficacy of our newly designed constructs for oligonucleotide drug delivery is highlighted by the successful intracellular transport of Cy-3 labeled RNA. Moreover, by using the chemotherapeutic chloroquine the efficient release of the assemblies out of endosomes was demonstrated. These results prove that our multifunctional platforms are versatile tools for diagnostic and therapeutic imaging purposes applicable for biologically active siRNA or aptamer sequences.

Fusion of a Short HA2-Derived Peptide Sequence to Cell-Penetrating Peptides Improves Cytosolic Uptake, but Enhances Cytotoxic Activity

Cell-penetrating peptides (CPP) have become a widely used tool for efficient cargo delivery into cells. However, one limiting fact is their uptake by endocytosis causing the enclosure of the CPP-cargo construct within endosomes. One often used method to enhance the outflow into the cytosol is the fusion of endosome-disruptive peptide or protein sequences to CPP. But, until now, no studies exist investigating the effects of the fusion peptide to the cellular distribution, structural arrangements and cytotoxic behaviour of the CPP. In this study, we attached a short modified sequence of hemagglutinin subunit HA2 to different CPP and analysed the biologic activity of the new designed peptides. Interestingly, we observed an increased cytosolic distribution but also highly toxic activities in the micromolar range against several cell lines. Structural analysis revealed that attachment of the fusion peptide had profound implications on the whole conformation of the peptide, which might be responsible for membrane interaction and endosome disruption.

Generation of carrier peptides for the delivery of nucleic acid drugs in primary cells.

Now that the human genome has been decoded, the demand for novel therapeutic concepts, such as gene and stem cell therapy, is higher than ever before. Although new and better pharmaceutical agents are available, their efficient delivery to the intracellular site of action is still a serious challenge. A possible solution to this problem is the use of cell‐penetrating peptides as delivery vectors, including derivatives of human calcitonin (hCT). The aim of this study was to synthesise novel branched hCT‐derived peptides for the noncovalent delivery of nucleic acids. The uptake of the resulting oligocationic peptides into various cell lines as well as primary cells was monitored by fluorescence microscopy. To determine the appropriate peptide–plasmid charge ratios for efficient cell transfection, electromobility shift assays were carried out. Finally, flow cytometric and fluorescence microscopic studies of gene expression highlighted two novel hCT‐derived peptides as highly effective in the delivery of noncovalently complexed plasmid DNA. Thus, the absence of cytotoxicity paired with highly efficient cell internalisation and transfection rates, in primary cells as well, make both peptides powerful candidates as drug delivery vectors, especially for plasmid DNA, for both inu2005vivo and exu2005vivo therapeutic applications.

Calcitonin-derived carrier peptide plays a major role in the membrane localization of a peptide–cargo complex

Bilayers made of dioleoylphosphatidylcholine (DOPC)/dipalmitoylphosphatidylcholine (DPPC) mixture containing or not cholesterol (Chl) were used to investigate the interaction of a carrier peptide with membranes. Atomic force microscopy revealed that the C‐terminal 9‐32 fragment of human calcitonin (hCT (9‐32)), free or coupled to enhanced green fluorescent protein (hCT‐eGFP) cargo forms aggregates in the DOPC fluid phase in absence of Chl and in the DPPC enriched liquid‐ordered phase when Chl is present. The data show that hCT (9‐32) plays a determinant role in the membrane localization of the peptide–cargo complex. They suggest that carpet‐like mechanism for membrane destabilization may be involved in the carrier function of hCT (9‐32).

Synthesis and application of peptides as drug carriers.

An efficient cellular drug delivery is a severe problem due to the charge, the hydrophilic character or the size of many therapeutic agents. High-drug doses, necessary to compensate the reduced bioavailability, often cause strong adverse effects. Synthetic drug delivery vectors will solve this problem, if limitations like low-cellular uptake efficiency or cytotoxicity can be overcome. Among these synthetic vectors, so-called cell-penetrating peptides (CPP) have proven their applicability as drug carriers. The ability to penetrate cellular membranes without the help of any receptor or transporter molecule was also found for derivatives of the native peptide hormone human calcitonin (hCT). We have shown that truncated hCT analogs with a branched peptide design and oligocationic side chain sequences - hCT(18-32)-k 7 and hCT(9-32)-2 br - are very interesting candidates as carrier peptides for drug delivery. Both peptides were found to efficiently shuttle covalently linked small molecules and non-covalently complexed DNA and RNA inside human embryonic kidney cells (HEK 293).

A cleavable cytolysin-neuropeptide Y bioconjugate enables specific drug delivery and demonstrates intracellular mode of action

Myxobacterial tubulysins are promising chemotherapeutics inhibiting microtubule polymerization, however, high unspecific toxicity so far prevents their application in therapy. For selective cancer cell targeting, here the coupling of a synthetic cytolysin to the hY1-receptor preferring peptide [F(7),P(34)]-neuropeptide Y (NPY) using a labile disulfide linker is described. Since hY1-receptors are overexpressed in breast tumors and internalize rapidly, this system has high potential as peptide-drug shuttle system. Molecular characterization of the cytolysin-[F(7),P(34)]-NPY bioconjugate revealed potent receptor activation and receptor-selective internalization, while viability studies verified toxicity. Triple SILAC studies comparing free cytolysin with the bioconjugate demonstrated an intracellular mechanism of action regardless of the delivery pathway. Treatments resulted in a regulation of proteins implemented in cell cycle arrest confirming the tubulysin-like effect of the cytolysin. Thus, the cytolysin-peptide bioconjugate fused by a cleavable linker enables a receptor-specific delivery as well as a potent intracellular drug-release with high cytotoxic activity.

Abstract 1745: NPY1 receptor specific peptide-drug-conjugates as novel treatment for metastatic breast cancer

The NPY1 receptor is a known marker for breast cancer that was characterized first at the university hospital Bern by the group of Reubi. In healthy breast tissue the NPY2 receptor (NPY2R) can be found, expression of which is switched to the NPY1R in tumor tissues (Reubi et al., Cancer Res. 2001, 61, 4636-4641). Particularly high NPY1R densities were also found in tumor biopsies of patients suffering from Ewing9s sarcoma, which is a rare, difficult to treat juvenile bone cancer (Korner et al., Clin. Cancer Res. 2008, 14, 5043-5049.). Our own results verify these literature reports, shown on a panel of patient biopsies where nearly 50% of the tumors strongly overexpress the NPY1R, whereas NPY2R could not be detected. OntoChem has developed peptide-drug-conjugates (“CytoPep”) that are selective agonists of the NPY1 receptor. As drugs we have used tubulin binding cytolysins and maytansines. CytoPep shall be developed as novel approach to treat Ewing9s sarcoma and NPY1R expressing breast cancer. We will present data on in vitro and in vivo pharmacology of these PDCs. IN particular efficacy is directly correlated with NPY1R expression levels in cells. We will also demonstrate internalization studies, showing a fast and selective internalization into NPY1R expressing cells. These in vitro data will be compared with corresponding data from ADCs such as for example trastuzumab emtansine, that show a considerably slower internalization and efficacy. Citation Format: Lutz Weber, Robert Rennert. NPY1 receptor specific peptide-drug-conjugates as novel treatment for metastatic breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1745. doi:10.1158/1538-7445.AM2015-1745