Biocatalytic PEI-PSS membranes through aqueous phase separation: influence of casting solution pH and operational temperature.

Biocatalytic membranes combine the separation properties of membranes and the catalytic abilities of enzymes, holding great promise for industries where both purification and conversion are required. In this work, polyelectrolyte complex membranes incorporated with lysozyme were prepared using polyethyleneimine (PEI) and poly(sodium 4-styrenesulfonate) (PSS) through a one-step and mild pH shift aqueous phase separation (APS) approach. The effects of lysozyme addition and casting solution pH on the membrane properties were studied. All the membranes, both with and without added lysozyme, exhibited asymmetric structures with relatively dense top surfaces and porous cross-sections with finger-like macrovoids. The incorporation of lysozyme did not significantly influence the structure and permeability of the formed membranes. The PEI-PSS biocatalytic membranes exhibited temperature dependent enzymatic activity. The activity strongly increased with increased operational temperature, with the highest activity of 4.30 ± 0.15 U cm-2 at 45 °C. This indicates a responsive effect, where a higher temperature leads to some swelling of the polyelectrolyte complex membrane, making the enzyme more accessible to the used substrate. Moreover, the biocatalytic membranes demonstrate desirable enzymatic stability, maintaining 60% activity even after 60 days of storage. This study validates the potential of the water-based APS process as a straightforward approach for integrating enzymes into responsive biocatalytic membranes.

Distribution of polyelectrolytes and counterions upon polyelectrolyte complexation.

HYPOTHESIS: Understanding polyelectrolyte complexation remains limited due to the absence of a systematic methodology for analyzing the distribution of components between the polyelectrolyte complex (PEC) and the dilute phases. EXPERIMENTS: We developed a methodology based on NMR to quantify all components of solid-like PECs and their supernatant phases formed by mixing different ratios of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid)-sodium salt (PAA). This approach allowed for determining relative and absolute concentrations of polyelectrolytes in both phases by 1H NMR studies. Using 23Na and 35Cl NMR spectroscopy we measured the concentration of counterions in both phases. FINDINGS: Regardless of the mixing ratio of the polyelectrolytes the PEC is charge-stoichiometric, and any excess polyelectrolytes to achieve charge stoichiometry remains in the supernatant phase. The majority of counterions were found in the supernatant phase, confirming counterion release being a major thermodynamic driving force for PEC formation. The counterion concentrations in the PEC phase were approximately twice as high as in the supernatant phase. The complete mass balance of PEC formation could be determined and translated into a molecular picture. It appears that PAH is fully charged, while PAA is more protonated, so less charged, and some 10% extrinsic PAH-Cl- pairs are present in the complex.

Sequestration of Small Ions and Weak Acids and Bases by a Polyelectrolyte Complex Studied by Simulation and Experiment.

Mixing of oppositely charged polyelectrolytes can result in phase separation into a polymer-poor supernatant and a polymer-rich polyelectrolyte complex (PEC). We present a new coarse-grained model for the Grand-reaction method that enables us to determine the composition of the coexisting phases in a broad range of pH and salt concentrations. We validate the model by comparing it to recent simulations and experimental studies, as well as our own experiments on poly(acrylic acid)/poly(allylamine hydrochloride) complexes. The simulations using our model predict that monovalent ions partition approximately equally between both phases, whereas divalent ones accumulate in the PEC phase. On a semiquantitative level, these results agree with our own experiments, as well as with other experiments and simulations in the literature. In the sequel, we use the model to study the partitioning of a weak diprotic acid at various pH values of the supernatant. Our results show that the ionization of the acid is enhanced in the PEC phase, resulting in its preferential accumulation in this phase, which monotonically increases with the pH. Currently, this effect is still waiting to be confirmed experimentally. We explore how the model parameters (particle size, charge density, permittivity, and solvent quality) affect the measured partition coefficients, showing that fine-tuning of these parameters can make the agreement with the experiments almost quantitative. Nevertheless, our results show that charge regulation in multivalent solutes can potentially be exploited in engineering the partitioning of charged molecules in PEC-based systems at various pH values.

Control over Charge Density by Tuning the Polyelectrolyte Type and Monomer Ratio in Saloplastic-Based Ion-Exchange Membranes.

Membranes based on polyelectrolyte complexes (PECs) can now be prepared through several sustainable, organic solvent-free approaches. A recently developed approach allows PECs made by stoichiometric mixing of polyelectrolytes to be hot-pressed into dense saloplastics, which then function as ion-exchange membranes. An important advantage of PECs is that tuning their properties can provide significant control over the properties of the fabricated materials, and thus over their separation properties. This work studies the effects of two key parameters─(a) ratio of mixing and (b) choice of polyelectrolytes─on the mechanical, material, and separation properties of their corresponding hot-pressed saloplastic-based ion-exchange membranes. By varying these two main parameters, charge density─the key property of any IEM─was found to be controllable. While studying several systems, including strong/strong, strong/weak, and weak/weak combinations of polyelectrolytes, it was observed that not all systems could be processed into saloplastic membranes. For the processable systems, expected trends were observed where a higher excess of one polyelectrolyte would lead to a more charged system, resulting in higher water uptake and better permselectivities. An anomaly was the polystyrenesulfonate-polyvinylamine system, which showed an opposite trend with a higher polycation ratio, leading to a more negative charge. Overall, we have found that it is possible to successfully fabricate saloplastic-based anion- and cation-exchange membranes with tunable charge densities through careful choice of polyelectrolyte combination and ratio of mixing.

Biomolecular condensates can both accelerate and suppress aggregation of α-synuclein.

Biomolecular condensates present in cells can fundamentally affect the aggregation of amyloidogenic proteins and play a role in the regulation of this process. While liquid-liquid phase separation of amyloidogenic proteins by themselves can act as an alternative nucleation pathway, interaction of partly disordered aggregation-prone proteins with preexisting condensates that act as localization centers could be a far more general mechanism of altering their aggregation behavior. Here, we show that so-called host biomolecular condensates can both accelerate and slow down amyloid formation. We study the amyloidogenic protein α-synuclein and two truncated α-synuclein variants in the presence of three types of condensates composed of nonaggregating peptides, RNA, or ATP. Our results demonstrate that condensates can markedly speed up amyloid formation when proteins localize to their interface. However, condensates can also significantly suppress aggregation by sequestering and stabilizing amyloidogenic proteins, thereby providing living cells with a possible protection mechanism against amyloid formation.

Biocatalytic membranes through aqueous phase separation.

HYPOTHESIS: 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.

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.

Hot-pressed polyelectrolyte complexes as novel alkaline stable monovalent-ion selective anion exchange membranes.

Polymeric ion-exchange membranes have come a long way since their invention, benefiting a wide range of processes, ranging from desalination to fuel cells. However, challenges such as alkaline stability, monovalent ion selectivity, cost-effectivity, and process sustainability largely persist. This work showcases alkaline stable anion-exchange membranes made by hot-pressing of a polyelectrolyte complex of poly(styrenesulfonate) (PSS) and poly(diallyldimethylammonium) (PDADMA). This completely aqueous production approach leads to especially dense (non-porous) saloplastic films with an excess of cationic groups, demonstrating good stability even at high salinities (up to 2 M NaCl). On key performance indicators for anion exchange membranes, such as water uptake (~40%), permselectivity (up to 97%), ion exchange capacity (1.01 mmol g-1), and resistance (2.3 O·cm2) the membranes show comparable values to commercial membranes. A drop in permselectivity at high salinities, however, indicates that the charge density of the membranes could be further improved. Still, what really sets these membranes apart is their natural long term (up to 60 days) stability at extreme acidic (pH 0) and alkaline conditions (pH 14) and a relevant monovalent selectivity of up to 6.3 for Cl- over SO42-. Overall this work showcases PDADMA/PSS based saloplastics as highly promising and stable anion-exchange membranes, that can be produced by a simple, scalable, and sustainable approach.

Overcharging and charge inversion: Finding the correct explanation(s).

Both overcharging and charge inversion denote a general observation that the sign of a surface charge can flip in the presence of interacting species such as surfactants, polyelectrolytes, proteins and multivalent ions. Moreover, charge inversion of proteins through charge regulation, is one explanation for protein adsorption to similarly charged surfaces. While overcharging and charge inversion have been long studied, the explanations for these phenomena are often still debated. Broadly these explanations can be categorized as "chemical" where specific attractive interactions are seen as the cause of charge inversion, and "physical" where purely electrostatic interactions and constraints of geometry are used as explanation. In this review, charge inversion is discussed from a very broad viewpoint, where we draw connections between the various explanations proposed for very different systems. Especially, we highlight the work of Johannes Lyklema, who always carefully balanced between the competing chemical and physical explanations, and demonstrated that only few experimental systems allow just a single explanation.

Charge-Based Separation of Proteins Using Polyelectrolyte Complexes as Models for Membraneless Organelles.

Membraneless organelles are liquid compartments within cells with different solvent properties than the surrounding environment. This difference in solvent properties is thought to result in function-related selective partitioning of proteins. Proteins have also been shown to accumulate in polyelectrolyte complexes, but whether the uptake in these complexes is selective has not been ascertained yet. Here, we show the selective partitioning of two structurally similar but oppositely charged proteins into polyelectrolyte complexes. We demonstrate that these proteins can be separated from a mixture by altering the polyelectrolyte complex composition and released from the complex by lowering the pH. Combined, we demonstrate that polyelectrolyte complexes can separate proteins from a mixture based on protein charge. Besides providing deeper insight into the selective partitioning in membraneless organelles, potential applications for selective biomolecule partitioning in polyelectrolyte complexes include drug delivery or extraction processes.

Biocompatible Single-Chain Polymer Nanoparticles for Drug Delivery-A Dual Approach.

Single-chain polymer nanoparticles (SCNPs) are protein-inspired materials based on intramolecularly cross-linked polymer chains. We report here the development of SCNPs as uniquely sized nanocarriers that are capable of drug encapsulation independent of the polarity of the employed medium. Synthetic routes are presented for SCNP preparation in both organic and aqueous environments. Importantly, the SCNPs in organic media were successfully rendered water soluble, resulting in two complementary pathways toward water-soluble SCNPs with comparable resultant physicochemical characteristics. The solvatochromic dye Nile red was successfully encapsulated inside the SCNPs following both pathways, enabling probing of the SCNP interior. Moreover, the antibiotic rifampicin was encapsulated in organic medium, the loaded nanocarriers were rendered water soluble, and a controlled release of rifampicin was evidenced. The absence of discernible cytotoxic effects and promising cellular uptake behavior bode well for the application of SCNPs in controlled therapeutics delivery.

TEMPO-oxidised cellulose nanofibrils; probing the mechanisms of gelation via small angle X-ray scattering.

The structure of dispersions of TEMPO-oxidised cellulose nanofibrils (OCNF), at various concentrations, in water and in NaCl aqueous solutions, was probed using small angle X-ray scattering (SAXS). OCNF are modelled as rod-like particles with an elliptical cross-section of 10 nm and a length greater than 100 nm. As OCNF concentration increases above 1.5 wt%, repulsive interactions between fibrils are evidenced, modelled by the interaction parameter νRPA > 0. This corresponds to gel-like behaviour, where G' > G'' and the storage modulus, G', shows weak frequency dependence. Hydrogels can also be formed at OCNF concentration of 1 wt% in 0.1 M NaCl(aq). SAXS patterns shows an increase of the intensity at low angle that is modelled by attractive interactions (νRPA < 0) between OCNF, arising from the screening of the surface charge of the fibrils. Results are supported by ζ potential and cryo-TEM measurements.

Hydrophobic-Interaction-Induced Stiffening of α-Synuclein Fibril Networks.

In water, networks of semiflexible fibrils of the protein α-synuclein stiffen significantly with increasing temperature. We make plausible that this reversible stiffening is a result of hydrophobic contacts between the fibrils that become more prominent with increasing temperature. The good agreement of our experimentally observed temperature dependence of the storage modulus of the network with a scaling theory linking network elasticity with reversible cross-linking enables us to quantify the endothermic binding enthalpy and estimate the effective size of hydrophobic patches on the fibril surface. Our findings may not only shed light on the role of amyloid deposits in disease conditions, but can also inspire new approaches for the design of thermoresponsive materials.

Oligonucleotide Length-Dependent Formation of Virus-Like Particles.

Understanding the assembly pathway of viruses can contribute to creating monodisperse virus-based materials. In this study, the cowpea chlorotic mottle virus (CCMV) is used to determine the interactions between the capsid proteins of viruses and their cargo. The assembly of the capsid proteins in the presence of different lengths of short, single-stranded (ss) DNA is studied at neutral pH, at which the protein-protein interactions are weak. Chromatography, electrophoresis, microscopy, and light scattering data show that the assembly efficiency and speed of the particles increase with increasing length of oligonucleotides. The minimal length required for assembly under the conditions used herein is 14 nucleotides. Assembly of particles containing such short strands of ssDNA can take almost a month. This slow assembly process enabled the study of intermediate states, which confirmed a low cooperative assembly for CCMV and allowed for further expansion of current assembly theories.

Accumulation of small protein molecules in a macroscopic complex coacervate.

To obtain insight into the accumulation of proteins into macroscopic complex coacervate phases, the lysozyme concentration in complex coacervates containing the cationic polyelectrolyte poly-(N,N dimethylaminoethyl methacrylate) and the anionic polyelectrolyte polyacrylic acid was investigated as a function of the mixing ratio, protein concentration and ionic strength. Maximal protein enrichment of the complex coacervate phase was observed to require the presence of all three macromolecules. Under optimized conditions the protein concentrations in the complex coacervate were as high as 200 g L(-1). Such high concentrations are comparable to the protein concentration in the cytosol, suggesting that these interesting liquid phases may serve a suitable model system for the phase behavior of the cytosol and genesis and function of membrane-less organelles. The high stability of the complexes and the salt dependent uptake of protein suggest that complex coacervates may provide a way to store hydrated proteins at high concentrations and might therefore be of interest in the formulation of high protein foods.

Oligomers of Parkinson's Disease-Related α-Synuclein Mutants Have Similar Structures but Distinctive Membrane Permeabilization Properties.

Single-amino acid mutations in the human α-synuclein (αS) protein are related to early onset Parkinson's disease (PD). In addition to the well-known A30P, A53T, and E46K mutants, recently a number of new familial disease-related αS mutations have been discovered. How these mutations affect the putative physiological function of αS and the disease pathology is still unknown. Here we focus on the H50Q and G51D familial mutants and show that like wild-type αS, H50Q and G51D monomers bind to negatively charged membranes, form soluble partially folded oligomers with an aggregation number of ~30 monomers under specific conditions, and can aggregate into amyloid fibrils. We systematically studied the ability of these isolated oligomers to permeabilize membranes composed of anionic phospholipids (DOPG) and membranes mimicking the mitochondrial phospholipid composition (CL:POPE:POPC) using a calcein release assay. Small-angle X-ray scattering studies of isolated oligomers show that oligomers formed from wild-type αS and the A30P, E46K, H50Q, G51D, and A53T disease-related mutants are composed of a similar number of monomers. However, although the binding affinity of the monomeric protein and the aggregation number of the oligomers formed under our specific protocol are comparable for wild-type αS and H50Q and G51D αS, G51D oligomers cannot disrupt negatively charged and physiologically relevant model membranes. Replacement of the membrane-immersed glycine with a negatively charged aspartic acid at position 51 apparently abrogates membrane destabilization, whereas a mutation in the proximal but solvent-exposed part of the membrane-bound α-helix such as that found in the H50Q mutant has little effect on the bilayer disrupting properties of oligomers.

Effects of polyelectrolyte complex micelles and their components on the enzymatic activity of lipase.

The enzymatic activity of Hl-lipase embedded in complexes of poly-2-methylvinylpyridinium-co-poly(ethylene oxide) (P2MVP(41)-PEO(205)) and poly(acrylic acid)(PAA(139)) is studied as a function of the PAA(139) + P2MVP(41)-PEO(205) complex composition. The measurements revealed that there are several factors that influence the enzymatic activity. When incorporated in micelles, the activity of lipase is increased, which suggests that the micelles favor the active state. The activity may further increase because the substrate tends to accumulate to the micelles. It is found that the presence of PAA(139) alone also increases the enzymatic activity somewhat. Increasing of the ionic strength decreases the enzymatic activity in all systems. However, at ionic strengths where the micelles are disintegrated (>0.5 M), the activity of lipase in the presence of both polyelectrolytes is still higher than the activity of free lipase. At 0.7 M NaCl it was found that lipase in the presence of (just) P2MVP(41)-PEO(205) is more active than lipase without this additive.

Salt-induced disintegration of lysozyme-containing polyelectrolyte complex micelles.

The salt-induced disintegration of lysozyme-filled polyelectrolyte complex micelles, consisting of positively charged homopolymers (PDMAEMA150), negatively charged diblock copolymers (PAA42-PAAm417), and lysozyme, has been studied with dynamic light scattering (DLS) and small-angle neutron scattering (SANS). These measurements show that, from 0 to 0.2 M NaCl, both the hydrodynamic radius (Rh) and the core radius (Rcore) decrease with increasing salt concentration. This suggests that the micellar structures rearrange. Moreover, from approximately 0.2 to 0.4 M NaCl the light-scattering intensity is constant. In this salt interval, the hydrodynamic radius increases, has a maximum at 0.3 M NaCl, and subsequently decreases. This behavior is observed in both a lysozyme-containing system and a system without lysozyme. The SANS measurements on the lysozyme-filled micelles do not show increased intensity or a larger core radius at 0.3 M NaCl. This indicates that from 0.2 to 0.4 M NaCl another structure is formed, consisting of just the diblock copolymer and the homopolymer, because at 0.12 M NaCl the lysozyme-PAA42-PAAm417 complex has disintegrated. One may expect that the driving force for the formation of the complex in this salt range is other than electrostatic.

Reversibility and relaxation behavior of polyelectrolyte complex micelle formation.

In this study, the formation and disintegration of polyelectrolyte complex micelles is studied by dynamic light scattering titrations with the aim to assess the extent to which these complexes equilibrate. Also, the time evolution of samples at fixed (electroneutral) composition was followed to obtain information about the relaxation time of the complex formation. We find that, in 3.5 mM phosphate buffer with pH 7, polyelectrolyte complex micelles consisting of the positively charged homopolymer PDMAEMA(150), the negatively charged diblock copolymer PAA(42)-PAAm(417) (both having a pH-dependent charge), as well as the positively charged protein lysozyme slowly equilibrate with a relaxation time of about 2 days. The same structures were obtained, independent of the way the polymers and proteins had been mixed. In contrast, polyelectrolyte complex micelles (at the same pH) consisting of (pH-dependent) negatively charged homopolymer PAA(139), the pH-independent positively charged diblock copolymer P2MVP(41)-PEO(205), and the negatively charged protein alpha-lactalbumin did not equilibrate. The way in which solutions containing these macromolecules were mixed yielded different results that did not change over the period of at least a week.