PhD defence, Zsófia Szathmáry

Water is the foundation of life on planet Earth, however it is not an inexhaustible resource. Water scarcity and global thirst are becoming some of the most pressing matters of the century as clean water and adequate sanitation are inaccessible for almost two billion people globally. Advances in membrane filtration technologies provide a low-cost and easily scalable way of water desalination and purification, enabling the reuse and recycling of different water types such as wastewater or seawater. In recent years, a new subclass of membranes called biomimetic membranes, have been gaining significant interest due to their unique architecture and promising wide range of use cases. Biomimetic membranes combine the fundamentals of synthetic membranes with a biological element, to create an improved product. This was the principle that the Danish water-tech company, Aquaporin A/S was founded upon, creating membranes that incorporate nature’s water channel proteins, aquaporins. Aquaporin proteins are present in the cell membrane of most organisms and are responsible for the bi-directional transport of water molecules across it, while rejecting charged solutes. While they function elegantly on a cellular level, once the protein is extracted from its in vivo environment, care needs to be taken to ensure that its structure and functionality remain the same. Maintaining the activity of the aquaporin protein within a synthetic membrane is a complex task, which was tackled in this work by the formation of nanoassemblies that have the potential of stabilizing and orienting these delicate biomolecules. Several systems built up either by natural lipids or synthetic block copolymers were tested for their self-assembly and reconstitution ability, enabling the formation of structures that can host the protein and sustain its water filtration ability.  The first nanocarrier system tested was the liposome. Liposomes resemble the natural cellular environment of the aquaporin protein the most, as they provide a closed compartment-based system surrounded by a lipid bilayer, which is ideal for protein reconstitution. Aquaporin containing proteoliposomes were subjected to different analytical techniques to verify protein localization within the lipid assemblies; to study changes within the overall vesicle size, polydispersity and bilayer characteristics; to test the protein’s activity within the liposomes as well as to attempt the quantification of the reconstitution process. While liposomes were proven to host the aquaporin protein in a structurally stable manner and serve as the ideal model system to study the bilayer-anchored protein on a nanoscale, they generally lack long-term stability which limits their applications. Therefore, it was of importance to investigate alternative systems with more industrial relevance.

Complex lipid assemblies were evaluated next, as they combine the in vivo-like protein environment with an enhanced stability profile compared to liposomes, while allowing for increased protein loading due to the presence of a high lipid bilayer area within a given volume. These latter two properties hold a particularly high industrial relevance for the production of better performing biomimetic membranes with an increased shelf-life. Complex lipid structures have unique architectures as they can form the so-called sponge or cubic phases, that can be further dispersed in excess water as their corresponding nanoparticles. Their characteristics can be tailored to the needs of the protein by changing their composition and formation parameters. In this study, a gradual increase of phase parameters was demonstrated via optimizations within the used lipid mixtures and a decrease in the salt content of the sample buffer. These changes favored the incorporation of structurally intact aquaporin proteins, proven by different analytical approaches. Furthermore, findings of this work showcased the protein’s preference towards a flatter bilayer with less curvature, which was a valuable result for the design of optimized reconstitution assemblies.

Lastly, polymersomes built up by different copolymers were tested, as they provide highly stable reconstitution assemblies that are attractive for industrial applications. As their commercial availability is limited, all polymers used in this work were custom synthesized during the project of Aquaporin A/S’ industrial PhD student, Karolis Norinkevicius. The evaluation of three different systems was carried out, built up by blocks of poly(ethylene glycol) (PEG), poly(caprolactone) (PCL), poly(dimethylsiloxane) (PDMS) and poly(2-methyl-2-oxazoline) (PMOXA). The triblock PEG-b-PCL-b-PEG, diblock PEG-g-PDMS and triblock PMOXA-b-PDMS-b-PMOXA copolymers were tested for the formation of polymer vesicles and the reconstitution of the aquaporin protein via electron and light microscopic methods. The last mentioned copolymer was also subjected to photoinitiated crosslinking via its specific methacrylate end groups, which was expected to further enhance the mechanical stability of the vesicles.

                             
Overall, this PhD project focused on the evaluation of different membrane protein carrier systems regarding the aspects of protein loading and stabilization on a structural and functional level. Emphasis was placed on learning more about the behavior and characteristics of the aquaporin protein within a given bilayer system, to aid the design of the next generation of nanocarriers that can host large quantities of the protein. Findings of this work are expected to ensure that the protein remains structurally and functionally intact both during the steps of membrane manufacturing as well as during the long-term storage of the finished membrane product.