The emerging threat of antibiotic-resistant bacteria has become a global challenge in the last decades, leading to a rising demand for alternative treatments for bacterial infections. One approach is to target the bacterial cell envelope, making understanding its biophysical properties crucial. Specifically, bacteriophages use the bacterial envelope as an entry point to initiate infection, and they are considered important building blocks of new antibiotic strategies against drug-resistant bacteria.. Depending on the structure of the cell wall, bacteria are classified as Gram-negative and Gram-positive. Gram-negative bacteria are equipped with a complex cell envelope composed of two lipid membranes enclosing a rigid peptidoglycan layer. The synthesis machinery of the Gram-negative cell envelope is the target of antimicrobial agents, including new physical sanitizing procedures addressing the outer membrane (OM). It is therefore very important to study the biophysical properties of the Gram-negative bacterial cell envelope. The high complexity of the Gram-negative OM sets the demand for a model system in which the contribution of individual components can be evaluated separately. In this respect, giant unilamellar vesicles (GUVs) are promising membrane systems to study membrane properties while controlling parameters such as membrane composition and surrounding medium conditions.
The aim of this work was to develop methods and approaches for the preparation and characterization of a GUV-based membrane model that mimics the OM of the Gram-negative cell envelope. A major component of the OM is the lipopolysaccharide (LPS) on the outside of the OM heterobilayer. The vesicle model was designed to contain LPS in the outer leaflet and lipids in the inner leaflet. Furthermore, the interaction of the prepared LPS-GUVs with bacteriophages was tested. LPS containing GUVs were prepared by adapting the inverted emulsion technique to meet the challenging properties of LPS, namely their high self-aggregation rate in aqueous solutions. Notably, an additional emulsification step together with the adaption of solution conditions was employed to asymmetrically incorporate LPS containing long polysaccharide chains into the artificial membranes. GUV membrane asymmetry was verified with a fluorescence quenching assay. Since the necessary precautions for handling the quenching agent sodium dithionite are often underestimated and poorly described, important parameters were tested and identified to obtain a stable and reproducible assay. In the context of varied LPS incorporation, a microscopy-based technique was introduced to determine the LPS content on individual GUVs and to directly compare vesicle properties and LPS coverage. Diffusion coefficient measurements in the obtained GUVs showed that increasing LPS concentrations in the membranes resulted in decreased diffusivity.
Employing LPS-GUVs we could demonstrate that a Salmonella bacteriophage bound with high specificity to its LPS receptor when presented at the GUV surface, and that the number of bound bacteriophages scaled with the amount of presented LPS receptor. In addition to binding, the bacteriophages were able to eject their DNA into the vesicle lumen. LPS-GUVs thus provide a starting platform for bottom-up approaches for the generation of more complex membranes, in which the effects of individual components on the membrane properties and the interaction with antimicrobial agents such as bacteriophages could be explored.
Influenza A virus (IAV) is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. During the viral assembly process in the infected cells, the plasma membrane (PM) has to bend in localized regions into a vesicle towards the extracellular side. Studies in cellular models have proposed that different viral proteins might be responsible for inducing membrane curvature in this context (including M1), but a clear consensus has not been reached. M1 is the most abundant protein in IAV particles. It plays an important role in virus assembly and budding at the PM. M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. However, the details of M1 interactions with the cellular PM, as well as M1-mediated membrane bending at the budozone, have not been clarified.
In this work, we used several experimental approaches to analyze M1-lipids and M1-M1 interactions. By performing SPR analysis, we quantified membrane association for full-length M1 and different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region). This allowed us to obtain novel information on the protein regions mediating M1 binding to membranes. By using fluorescence microscopy, cryogenic transmission electron microscopy (cryo-TEM), and three-dimensional (3D) tomography (cryo-ET), we showed that M1 is indeed able to cause membrane deformation on vesicles containing negatively-charged lipids, in the absence of other viral components. Further, sFCS analysis proved that simple protein binding is not sufficient to induce membrane restructuring. Rather, it appears that stable M1-M1 interactions and multimer formation are required to alter the bilayer three-dimensional structure through the formation of a protein scaffold.
Finally, to mimic the budding mechanism in cells that arise by the lateral organization of the virus membrane components on lipid raft domains, we created vesicles with lipid domains. Our results showed that local binding of M1 to spatial confined acidic lipids within membrane domains of vesicles led to local M1 inward curvature.
The goal of this work was to study the binding of ions to polymers and lipid bilayer membranes in aqueous solutions. In the first part of this work, the influence of various inorganic salts and polyelectrolytes on the structure of water was studied using Isothermal Titration Calorimetry (ITC). The heat of dilution of the salts was used as a scale of water structure making and breaking of the ions. The heats of dilution could be attributed to the Hofmeister Series. Following this, the binding of Ca2+ to poly(sodium acrylate) (NaPAA) was studied. ITC and a Ca2+ Ion Selective Electrode were used to measure the reaction enthalpy and binding isotherm. Binding of Ca2+ ions to PAA, was found to be highly endothermic and therefore solely driven by entropy. We then compared the binding of ions to the one-dimensional PAA polymer chain to the binding to lipid vesicles with the same functional groups. As for the polymer, Ca2+ binding was found to be endothermic. Binding of calcium to the lipid bilayer was found to be weaker than to the polymer. In the context of these experiments, it was shown that Ca2+ not only binds to charged but also to zwitterionic lipid vesicles. Finally, we studied the interaction of two salts, KCl and NaCl, to a neutral polymer gel, PNIPAAM, and to the ionic polymer PAA. Combining calorimetry and a potassium selective electrode we observed that the ions interact with both polymers, whether containing charges or not.
Die vorliegende Arbeit beschäftigt sich mit der Synthese und den Eigenschaften von linearen und verzweigten amphiphilen Polypeptid-Blockcopolymeren. Die Frage nach dem Einfluss der Topologie und Konformation der Blockcopolymere auf die supramolekularen und kolloidalen Eigenschaften bildete einen wichtigen Aspekt bei den Untersuchungen. Die Blockcopolymere wurden nach einem mehrstufigen Reaktionsschema durch Kombination von anionischer und ringöffnender Polymerisation von Aminosäuren-N-Carboxyanhydriden (NCA) synthetisiert. Die Untersuchung der Polypeptid-Blockcopolymere hinsichtlich ihres Aggregationsverhaltens in fester Phase sowie in verdünnter wässriger Lösung erfolgte mittels Streumethoden (SAXS, WAXS, DLS) sowie abbildender Methoden (TEM). Durch Einsatz der Blockcopolymere als polymere Stabilisatoren in der Emulsionspolymerisation wurden Oberflächen funktionalisierte Latizes erhalten. Als Beispiel für eine pharmazeutische Anwendung wurden bioverträgliche Polypeptid-Blockcopolymere als Wirkstoff-Trägersysteme in der Krebstherapie eingesetzt.
The present thesis focuses on the synthesis of nanostructured iron-based compounds by using β-FeOOH nanospindles and poly(ionic liquid)s (PILs) vesicles as hard and soft templates, respectively, to suppress the shuttle effect of lithium polysulfides (LiPSs) in Li-S batteries. Three types of composites with different nanostructures (mesoporous nanospindle, yolk-shell nanospindle, and nanocapsule) have been synthesized and applied as sulfur host material for Li-S batteries. Their interactions with LiPSs and effects on the electrochemical performance of Li-S batteries have been systematically studied.
In the first part of the thesis, carbon-coated mesoporous Fe3O4 (C@M-Fe3O4) nanospindles have been synthesized to suppress the shuttle effect of LiPSs. First, β-FeOOH nanospindles have been synthesized via the hydrolysis of iron (III) chloride in aqueous solution and after silica coating and subsequent calcination, mesoporous Fe2O3 (M-Fe2O3) have been obtained inside the confined silica layer through pyrolysis of β-FeOOH. After the removal of the silica layer, electron tomography (ET) has been applied to rebuild the 3D structure of the M-Fe2O3 nanospindles. After coating a thin layer of polydopamine (PDA) as carbon source, the PDA-coated M-Fe2O3 particles have been calcinated to synthesize C@M-Fe3O4 nanospindles. With the chemisorption of Fe3O4 and confinement of mesoporous structure to anchor LiPSs, the composite C@M-Fe3O4/S electrode delivers a remaining capacity of 507.7 mAh g-1 at 1 C after 600 cycles.
In the second part of the thesis, a series of iron-based compounds (Fe3O4, FeS2, and FeS) with the same yolk-shell nanospindle morphology have been synthesized, which allows for the direct comparison of the effects of compositions on the electrochemical performance of Li-S batteries. The Fe3O4-carbon yolk-shell nanospindles have been synthesized by using the β-FeOOH nanospindles as hard template. Afterwards, Fe3O4-carbon yolk-shell nanospindles have been used as precursors to obtain iron sulfides (FeS and FeS2)-carbon yolk-shell nanospindles through sulfidation at different temperatures. Using the three types of yolk-shell nanospindles as sulfur host, the effects of compositions on interactions with LiPSs and electrochemical performance in Li-S batteries have been systematically investigated and compared. Benefiting from the chemisorption and catalytic effect of FeS2 particles and the physical confinement of the carbon shell, the FeS2-C/S electrode exhibits the best electrochemical performance with an initial specific discharge capacity of 877.6 mAh g-1 at 0.5 C and a retention ratio of 86.7% after 350 cycles.
In the third part, PILs vesicles have been used as soft template to synthesize carbon nanocapsules embedded with iron nitride particles to immobilize and catalyze LiPSs in Li-S batteries. First, 3-n-decyl-1-vinylimidazolium bromide has been used as monomer to synthesize PILs nanovesicles by free radical polymerization. Assisted by PDA coating route and ion exchange, PIL nanovesicles have been successfully applied as soft template in morphology-maintaining carbonization to prepare carbon nanocapsules embedded with iron nitride nanoparticles (FexN@C). The well-dispersed iron nitride nanoparticles effectively catalyze the conversion of LiPSs to Li2S, owing to their high electrical conductivity and strong chemical binding to LiPSs. The constructed FexN@C/S cathode demonstrates a high initial discharge capacity of 1085.0 mAh g-1 at 0.5 C with a remaining value of 930.0 mAh g-1 after 200 cycles.
The results in the present thesis demonstrate the facile synthetic routes of nanostructured iron-based compounds with controllable morphologies and compositions using soft and hard colloidal templates, which can be applied as sulfur host to suppress the shuttle behavior of LiPSs. The synthesis approaches developed in this thesis are also applicable to fabricating other transition metal-based compounds with porous nanostructures for other applications.
This dissertation contains theoretical investigations on the morphology and statistical mechanics of vesicles. The shapes of homogeneous fluid vesicles and inhomogeneous vesicles with fluid and solid membrane domains are calculated. The influence of thermal fluctuations is investigated. The obtained results are valid on mesoscopic length scales and are based on a geometrical membrane model, where the vesicle membrane is described as either a static or a thermal fluctuating surface. The thesis consists of three parts. In the first part, homogeneous vesicles are considered. The focus in this part is on the thermally induced morphological transition between vesicles with prolate and oblate shape. With the help of Monte Carlo simulations, the free energy profile of these vesicles is determined. It can be shown that the shape transformation between prolate and oblate vesicles proceeds continuously and is not hampered by a free energy barrier. The second and third part deal with inhomogeneous vesicles which contain intramembrane domains. These investigations are motivated by experimental results on domain formation in single or multicomponent vesicles, where phase separation occurs and different membrane phases coexist. The resulting domains differ with regard to their membrane structure (solid, fluid). The membrane structure has a distinct effect on the form of the domain and the morphology of the vesicle. In the second part, vesicles with coexisting solid and fluid membrane domains are studied, while the third part addresses vesicles with coexisting fluid domains. The equilibrium morphology of vesicles with simple and complex domain forms, derived through minimisation of the membrane energy, is determined as a function of material parameters. The results are summarised in morphology diagrams. These diagrams show previously unknown morphological transitions between vesicles with different domain shapes. The impact of thermal fluctuations on the vesicle and the form of the domains is investigated by means of Monte Carlo simulations.
Giant vesicles may contain several spatial compartments formed by phase separation within their enclosed aqueous solution. This phenomenon might be related to molecular crowding, fractionation and protein sorting in cells. To elucidate this process we used two chemically dissimilar polymers, polyethylene glycol (PEG) and dextran, encapsulated in giant vesicles. The dynamics of the phase separation of this polymer solution enclosed in vesicles is studied by concentration quench, i.e. exposing the vesicles to hypertonic solutions. The excess membrane area, produced by dehydration, can either form tubular structures (also known as tethers) or be utilized to perform morphological changes of the vesicle, depending on the interfacial tension between the coexisting phases and those between the membrane and the two phases. Membrane tube formation is coupled to the phase separation process. Apparently, the energy released from the phase separation is utilized to overcome the energy barrier for tube formation. The tubes may be absorbed at the interface to form a 2-demensional structure. The membrane stored in the form of tubes can be retracted under small tension perturbation. Furthermore, a wetting transition, which has been reported only in a few experimental systems, was discovered in this system. By increasing the polymer concentration, the PEG-rich phase changed from complete wetting to partial wetting of the membrane. If sufficient excess membrane area is available in the vesicle where both phases wet the membrane, one of the phases will bud off from the vesicle body, which leads to the separation of the two phases. This wetting-induced budding is governed by the surface energy and modulated by the membrane tension. This was demonstrated by micropipette aspiration experiments on vesicles encapsulating two phases. The budding of one phase can significantly decrease the surface energy by decreasing the contact area between the coexisting phases. The elasticity of the membrane allows it to adjust its tension automatically to balance the pulling force exerted by the interfacial tension of the two liquid phases at the three-phase contact line. The budding of the phase enriched with one polymer may be relevant to the selective protein transportation among lumens by means of vesicle in cells.
In this work, the basic principles of self-organization of diblock copolymers having the in¬herent property of selective or specific non-covalent binding were examined. By the introduction of electrostatic, dipole–dipole, or hydrogen bonding interactions, it was hoped to add complexity to the self-assembled mesostructures and to extend the level of ordering from the nanometer to a larger length scale. This work may be seen in the framework of biomimetics, as it combines features of synthetic polymer and colloid chemistry with basic concepts of structure formation applying in supramolecular and biological systems. The copolymer systems under study were (i) block ionomers, (ii) block copolymers with acetoacetoxy chelating units, and (iii) polypeptide block copolymers.
Die vorliegende Arbeit beschäftigt sich mit der Synthese von Disulfiden, der Thiol-Disulfid Metathesereaktion als Möglichkeit, Polymere zu funktionalisieren, und der Synthese von Polydisulfiden. Im ersten Teil der Arbeit wird die Aminolyse von RAFT-Polymeren und die Abhängigkeit der Polymer-Polymer Disulfidbildung von der Molmasse untersucht. Dabei wurde durch die Aufnahme von Reaktionskinetiken mittels Gel-Permeations-Chromatographie (GPC) festgestellt, dass je länger die Polymerketten sind, desto weniger Disulfid Polymerkopplung tritt auf. RAFT-Polymere werden oft genutzt, um die RAFT-Polymer Endgruppe nach der Polymerisation zu modifizieren oder in einer chemischen Reaktion zu funktionalisieren. Hier kann die Aminolyse in Anwesenheit von kurzkettigen Disulfiden, wie zum Beispiel Cystin, durchgeführt werden, um die Bildung von Polymer-Polymer Disulfiden vollständig zu unterdrücken und ein endgruppenfunktionalisiertes Polymer zu erhalten. Bei dieser Reaktion greift das bei der Aminolyse entstehende Polymerthiolat die kurzkettigen Disulfide an, und es kommt zur Bildung von funktionalisierten Polymeren. Es wurde ein Polyethylenglykoldisulfid eingesetzt, um ein amphiphiles Blockcopolymer zu erhalten. Als RAFT-Polymer wurde Polystyrol (PS) verwendet, und es konnte die Bildung von Polystyrol-Polyethylenglykol Copolymeren nachgewiesen werden. Das amphiphile Polymer bildet im wässrigen Medium Vesikel. Die Oberfläche der Vesikel konnte mittels der Thiol-Disulfid Metathese umfunktionalisiert werden. Die Aminolyse von PS RAFT-Polymeren mit einem Polylaktiddisulfid oder einem Polybenzylglutamatdisulfid ergab Polystyrol-block-Polyester und Polystyrol-block-Polyaminosäuren Copolymere. Im zweiten Teil der Arbeit liegt der Fokus auf der Synthese von Polydisulfiden und ihren thermischen Eigenschaften. Es wurden verschiedene Alkyldithiole synthetisiert und mittels Wasserstoffperoxid und Triethylamin polymerisiert. Dabei konnte gezeigt werden, dass die Polymere teilkristallin sind und dass der Schmelzpunkt und die Kristallinität der Polymere mit steigender Alkylkettenlänge zwischen den Disulfidbindungen zunehmen. Die Möglichkeit einer Polymerkettenerweiterung nach der Polymerisation ist mit diesem System gegeben. Die Abbaubarkeit der Polydisulfide konnte durch den Einsatz von Thiolen im basischen Milieu gezeigt werden.