Extraction of Lysozyme from Chicken Albumen Using Polyelectrolyte Complexes

Cells use droplet-like membrane-less organelles (MLOs) to compartmentalize and selectively take-up molecules, such as proteins, from their internal environment. These membraneless organelles can be mimicked by polyelectrolyte complexes (PECs) consisting of oppositely charged polyelectrolytes. Previous research has demonstrated that protein uptake strongly depends on the PEC composition. This suggests that PECs can be used to selectively extract proteins from a multi-protein mixture. With this in mind, the partitioning of the protein lysozyme in four PEC systems consisting of different weak and strong polyelectrolyte combinations is investigated. All systems show similar trends in lysozyme partitioning as a function of the complex composition. The release of lysozyme from complexes at their optimal lysozyme uptake composition is investigated by increasing the salt concentration to 500 mm NaCl or lowering the pH from 7 to 4. Complexes of poly(allylamine hydrochloride) and poly(acrylic acid) have the best uptake and release properties. These are used for selective extraction of lysozyme from a hen-egg white protein matrix. The (back)-extracted lysozyme retains its enzymatic activity, showing the capability of PECs to function as extraction media for proteins

Complex coacervates as extraction media

Various solvents such as ionic liquids, deep eutectic solvents, and aqueous two phase systems have been suggested as greener alternatives to existing extraction processes. We propose to add macroscopic complex coacervates to this list. Complex coacervates are liquid-like forms of polyion condensates and consist of a complex of oppositely charged polyions and water. Previous research focussing on the biological significance of these polyion-rich phases has shown that polyion condensates have the ability to extract certain solutes from water and back-extract them by changing parameters such as ionic strength and pH. In this study, we present the distribution coefficients of five commonly used industrial chemicals, namely lactic acid, butanol, and three types of lipase enzymes in poly(ethylenimine)/poly(acrylic acid) complex coacervates. It was found that the distribution coefficients can vary strongly upon variation of tunable parameters such as polyion ratio, ionic strength, polyion and compound concentrations, and temperature. Distribution coefficients ranged from approximately 2 to 50 depending on the tuning of the system parameters. It was also demonstrated that a temperature-swing extraction is possible, with back-extraction of butanol from complex coacervates with a recovery of 21.1%, demonstrating their potential as extraction media

Biocatalytic membranes through aqueous phase separation

Hypothesis: Test Polymer membranes play a critical role in water treatment, chemical industry, and medicine. Unfortunately, the current standard for polymer membrane production requires unsustainable and harmful organic solvents. Aqueous phase separation (APS) has recently been proposed as a method to produce membranes in a more sustainable manner through induced polyelectrolyte complexation in aqueous solutions. Experiments: We demonstrate that APS has another natural advantage that goes beyond sustainability: the easy incorporation of enzymes in the membrane structure. Biocatalytic membranes hold great promise in for example biorefinery, but the most common current post-production processes to immobilize enzymes on the membrane surface are complicated and expensive. Findings: In this study we demonstrated the first biocatalytic membrane produced via APS. We demonstrate an easy procedure to incorporate lysozyme in polyelectrolyte complex membranes made via APS. Our functionalized membranes have the same structure, water permeability (in the range of high nanofiltration, low ultrafiltration), and retention as membranes without lysozyme. Lysozyme is antibacterial by catalysing the hydrolysis of specific peptidoglycan bonds in bacteria walls. We demonstrate that the functionalized membranes are also capable of catalysing this reaction. The membranes remain enzymatically active for a period of at least one week. This opens new routes to produce polymer membranes with added biological function

Pancreatic islet macroencapsulation using microwell porous membranes

Allogeneic islet transplantation into the liver in combination with immune suppressive drug therapy is widely regarded as a potential cure for type 1 diabetes. However, the intrahepatic system is suboptimal as the concentration of drugs and nutrients there is higher compared to pancreas, which negatively affects islet function. Islet encapsulation within semipermeable membranes is a promising strategy that allows for the islet transplantation outside the suboptimal liver portal system and provides environment, where islets can perform their endocrine function. In this study, we develop a macroencapsulation device based on thin microwell membranes. The islets are seeded in separate microwells to avoid aggregation, whereas the membrane porosity is tailored to achieve sufficient transport of nutrients, glucose and insulin. The non-degradable, microwell membranes are composed of poly (ether sulfone)/polyvinylpyrrolidone and manufactured via phase separation micro molding. Our results show that the device prevents aggregation and preserves the islet's native morphology. Moreover, the encapsulated islets maintain their glucose responsiveness and function after 7 days of culture (stimulation index above 2 for high glucose stimulation), demonstrating the potential of this novel device for islet transplantation

An important step towards a prevascularized islet microencapsulation device: in vivo prevascularization by combination of mesenchymal stem cells on micropatterned membranes

Extrahepatic transplantation of islets of Langerhans could aid in better survival of islets after transplantation. When islets are transfused into the liver 60-70% of them are lost immediately after transplantation. An important factor for a successful extrahepatic transplantation is a well-vascularized tissue surrounding the implant. There are many strategies known for enhancing vessel formation such as adding cells with endothelial potential, the combination with angiogenic factors and / or applying surface topography at the exposed surface of the device. Previously we developed porous, micropatterned membranes which can be applied as a lid for an islet encapsulation device and we showed that the surface topography induces human umbilical vein endothelial cell (HUVEC) alignment and interconnection. This was achieved without the addition of hydrogels, often used in angiogenesis assays. In this work, we went one step further towards clinical implementation of the device by combining this micropatterned lid with Mesenchymal Stem Cells (MSCs) to facilitate prevascularization in vivo. As for HUVECs, the micropatterned membranes induced MSC alignment and organization in vitro, an important contributor to vessel formation, whereas in vivo (subcutaneous rat model) they contributed to improved implant prevascularization. In fact, the combination of MSCs seeded on the micropatterned membrane induced the highest vessel formation score in 80% of the sections. [Figure not available: see fulltext.]