title: Hybrid Multimeric Architectures for Enhanced Receptor Targeting and Intracellular Delivery of Bioactive Cargoes
Creators: name: Englert, Simon Peter
Publisher: name: UNSPECIFIED
Year: 2023

description: In recent decades, the spectrum of available therapeutics has been expanded by the ever-growing field of biopharmaceuticals such as peptides, proteins, and nucleic acids, which are characterized by advantageous properties including high specificity and potency as a consequence of their molecular structural and functional diversity. However, the application of biotherapeutics is mostly limited to the extracellular environment due to their intrinsic disability to traverse the cellular membrane. In order to overcome this obstacle and expand the range of potential targets for biotherapeutics into the cell´s interior, numerous strategies such as cell-penetrating peptides (CPPs) have been developed. While these cationic peptides facilitate cellular uptake by simple conjugation to the cargo molecule, more sophisticated architectures can be designed to further improve the transduction efficiency. Multimeric presentation of CPPs on scaffold molecules is one of the promising approaches to reach this goal. Analogously, receptor-targeting peptides could also benefit from multimerization on a suited platform. The presented doctoral thesis summarizes results of a comprehensive study aimed at the development of hybrid multimeric architectures for intracellular delivery of bioactive cargoes and enhancing the performance of receptor-targeting peptides. The first part of the work addressed the evaluation of various monomeric CPPs, non-peptidic architectures and the CPP L17E, multimerized on the polysaccharide dextran, in their ability to facilitate cellular uptake of bioactive 18mer peptide nucleic acid (PNA) as payload. Thereby, the eGFP654 mis-splicing correction assay was performed as functional assay that allowed for comparison of the delivery modules in a quantitative fashion. Due to the remarkable performance of dextran-L17E hybrids in the mis-splicing assay, the architecture was subjected to further validation in a second functional assay based on the cytosolic delivery of a 16mer peptide. The dextran-L17E approach was able to facilitate cellular uptake of the bioactive peptide, which induced restoration of fluorescence upon complementation to non-functional green fluorescent protein (GFP). Additionally, dextran-CPP chimeras were assessed in their capabilities to mediate intracellular transport of large payload. In a proof-of-concept study, dextran-CPP hybrids were equipped with biotin and employed as delivery modules for fluorescently labeled streptavidin (Sav) as model cargo. L17E-functionalized dextran was able to facilitate intracellular delivery of the core protein Sav and, furthermore, a biotinylated GFP as additional cargo. However, the impressive performance of efficient protein delivery was accompanied by intolerable cytotoxicity, at least for therapeutic applications. The first part of this work confirmed the capability of L17E-dextran hybrids as versatile platform for intracellular delivery of relevant payload. Beside PNA and peptides, these architectures could facilitate the cellular uptake of cargo proteins, albeit with significant cytotoxicity. Thus, future investigations could optimize the architecture of dextran-L17E hybrids or replace the peptide by novel, high-performing CPPs. The second part of this work assessed dextran-Sav hybrids as novel architectures for multimerization of receptor-binding peptides. Dextran equipped with multiple copies of death receptor 5 targeting peptide (DR5TP) was able to efficiently induce apoptosis in a cancer-relevant cell line at low nanomolar concentrations. The apoptotic potency of DR5TP-decorated dextran was distinctly increased upon further multimerization on Sav as centerpiece. For further evaluation of dextran-Sav architectures as multimerization platform for receptor-targeting peptides, integrin binding cyclic peptides harboring the RGD motif were chosen. While dextran bearing multiple copies of cyclic RGD peptides was able to bind to the isolated target integrin, these results did not translate into a cell-based assay using fluorescently labeled Sav as core protein. Using an antibody fragment (Fc) as dimerization platform for RGD-modified dextran, integrin overexpressing cancer cells could be efficiently targeted and, furthermore, the architecture was suited to deliver a cytotoxic agent into the target cells. The second part of this work presented dextran-Sav hybrids as promising platform for multimeric receptor-targeting peptides, whereby the scope of successfully-addressable receptors should be the subject of further research. As an alternative, Fc-dextran hybrids remain a highly efficient framework for multimerization of receptor-targeting peptides.