This thesis focuses on the application of lipid-based nanomedicine in drug delivery, including small molecular antitumor drugs and biomacromolecules including mRNA, and evaluates their biological... Show moreThis thesis focuses on the application of lipid-based nanomedicine in drug delivery, including small molecular antitumor drugs and biomacromolecules including mRNA, and evaluates their biological performance. We have modified liposomes and LNPs with fusogenic coiled-coil peptides to enhance the drug/mRNA delivery efficiency (Chapter 2-4), and also investigated how the lipid composition of LNPs influences the immune response (Chapter 5). Show less
A structural investigation of coiled coil peptides used as membrane fusogens, mimicing naturally occuring coiled-coil fusion proteins. Synthetic modifications have been made to alter lipid... Show moreA structural investigation of coiled coil peptides used as membrane fusogens, mimicing naturally occuring coiled-coil fusion proteins. Synthetic modifications have been made to alter lipid attachment, secondary structure and to insert photoactive azobenzene moieties for active control over coiled coil structure. Finally, the underlying photocontrol mechanism investigated in coiled coil peptides is extended to beta-structured peptides, and was shown to be universally applicable. Show less
This dissertation contains four works during my PhD. Different biomaterials have been designed based on coiled-coil peptides. These biomaterials have a range of applications, inclusing drug... Show moreThis dissertation contains four works during my PhD. Different biomaterials have been designed based on coiled-coil peptides. These biomaterials have a range of applications, inclusing drug delivery, cell sorting to cell-cell fusion. By knowledge-guideddesign and modification, coiled-coil induced membrane fusion systems are expected to achieve drug delivery in vivo. Finally, L-forms obtained by coiled-coil induced fusion of different strains are expected to be an ideal model for studying questions related to the origin of life and novel antibiotics. Show less
O-Nitrobenzyl groepen worden veel gebruikt als moleculaire beschermgroepen die je met licht kunt verwijderen. Deze zogenaamde photocages worden vaak toegepast in zowel de organische chemie als in... Show moreO-Nitrobenzyl groepen worden veel gebruikt als moleculaire beschermgroepen die je met licht kunt verwijderen. Deze zogenaamde photocages worden vaak toegepast in zowel de organische chemie als in de biologie. Photocaging blokkeert de functionaliteit van een molecuul en deze blokkade kan verwijderd worden door middel van licht met een specifieke golflengte. Dit proefschrift laat zien dat licht gebruikt kan worden om precieze controle te verkrijgen over waar en wanneer geneesmiddelenafgifte plaatsvindt. Show less
Giant Unilamellar Vesicles (GUVs) are spherical assemblies of natural or synthetic lipids composed of a single lipid bilayer which separates the aqueous interior compartment from the... Show more Giant Unilamellar Vesicles (GUVs) are spherical assemblies of natural or synthetic lipids composed of a single lipid bilayer which separates the aqueous interior compartment from the exterior. Because of their “giant” size (5-100 µm) which allows the imaging of the lipid bilayer by optical microscopy, GUVs have been used as a minimal model system of the cellular membrane. Several protocols have been proposed to form GUVs at low salt concentrations; however, the study of biological interactions requires the use of biologically relevant salt concentrations. To tackle this drawback from traditional preparation methods, this thesis presents a new method, based on a chemically crosslinked hydrogel as a substrate for the growth of GUVs at relevant biological salt conditions. Furthermore, the physical chemical properties of this hydrogel network and the characterization of the final GUVs in terms of yield and size distribution are discussed in this work. Finally, GUVs are used as a biophysical platform for studying drug delivery and membrane fusion processes at biological salt concentrations. Overall, this work covers some biophysical aspects and applications of the GUV model applied at relevant biologically salt concentrations. Show less
For decades a large amount of research has dealt with membrane interactions of peptides and proteins as well as peptide-peptide interactions to understand the mechanisms of essential biological... Show moreFor decades a large amount of research has dealt with membrane interactions of peptides and proteins as well as peptide-peptide interactions to understand the mechanisms of essential biological processes such as protein-driven vesicle budding and fission, cell penetration and lysis by peptides, and of course protein-driven membrane fusion. The advance of these fields, in combination with recent progress in cell biology, has inspired chemists to mimic these biological processes with simple model systems. However, it becomes apparent that these model systems are more complex than initially thought and the lessons that were learned from natural systems can also be applied here. The work reported in this thesis applied and extended classical methods for the study of peptide-peptide and peptide-membrane interactions to study the properties of the fusogenic coiled-coil forming lipopeptides in different membrane model systems or in solution. Hypotheses were constructed and tested based on the current biochemical and biophysical models of natural membrane fusion. Taken together, the results reported here led to a new perspective on lipopeptide mediated vesicle fusion. Show less
Fusion of lipid bilayers in cells facilitates the active transport of chemicals. Non-viral membrane fusion is regulated by a cascade of proteins as the process is highly regulated both in space and... Show moreFusion of lipid bilayers in cells facilitates the active transport of chemicals. Non-viral membrane fusion is regulated by a cascade of proteins as the process is highly regulated both in space and time. In eukaryotic cells, the so-called SNARE protein complex is at the heart of fusion. However, little is known about the actual mechanism at the molecular level. Inspired by the SNARE protein complex, our group previously developed a model system composed of a pair of lipidated complementary coiled coil peptides enabling targeted liposome-liposome fusion. This model system possesses all the key characteristics of membrane fusion similar to SNARE mediated fusion. The tetrameric coiled-coil of SNAREs is mimicked by a complementary pair of coiled coil forming peptides composed of three heptad repeat units (denoted __coil-E__ and __coil-K__). A flexible poly(ethylene glycol) spacer is conjugated to the N-terminus ensuring rotational freedom of the peptides. Lipidation warrants the anchoring of the peptides in the membrane by means of a phospholipid anchor (DOPE), mimicking the transmembrane domain of SNAREs. In order to develop future applications of this model system, the mechanism of membrane fusion needs to be studied in more detail and this has been the goal of this thesis. Show less
In this thesis the behavior and functionality of peptide amphiphiles at the surface of bilayer vesicles is examined. By controlling the behavior of the surface bound peptides, I was able to... Show moreIn this thesis the behavior and functionality of peptide amphiphiles at the surface of bilayer vesicles is examined. By controlling the behavior of the surface bound peptides, I was able to construct assemblies which could: 1) release their content (triggered by pH), 2) fuse in a targeted and controlled manner or 3) dock to cells and zebrafish embryos Show less
Of the various biomolecular building blocks in use in nature, coiled-coil forming peptides are amongst those with the most potential as building blocks for the synthetic self-assembly of... Show moreOf the various biomolecular building blocks in use in nature, coiled-coil forming peptides are amongst those with the most potential as building blocks for the synthetic self-assembly of nanostructures. Native coiled coils have the ability to function in, and influence, complex systems composed of multiple building blocks. However, there have only been a limited number of synthetic coiled-coil assemblies that mimic native coiled coils by incorporating multiple assembling components. This thesis represents efforts at extending this aspect of coiled-coil self-assembly. In order to achieve this, a range of hybrid molecules were synthesized which combine coiled-coil peptides with a hydrophobic component. In this way the highly specific coiled-coil self-assembly is juxtaposed with the non-specific, but structure-inducing aggregation of the hydrophobic section. This thesis asked simple questions: can coiled coils function when covalently attached to large hydrophobic blocks? How large can the hydrophobic blocks be? Can coiled coils function when incorporated noncovalently with a supramolecular assembly? By answering these fundamental questions the possibilities of prescriptive self-assembly have been probed and expanded, novel preparative methods have been developed, and specific applications have arisen. Show less
