Diaminated Cellulose Beads as a Sustainable Support for Industrially Relevant Lipases.

Environmentally persistent polystyrene or polyacrylic beads are used as supports in enzyme large-scale bioprocesses, including conversion glucose isomerization for high-fructose corn syrup production, hydrolysis of lactose, and synthesis of active pharmaceutical ingredients. In this paper, we report the development of a novel sustainable and scalable method to produce diaminated cellulose beads (DAB) as highly efficient alternative supports for industrially relevant lipases. Regenerated cellulose beads were grafted with diaminated aliphatic hydrocarbons via periodate oxidation and reductive amination. The oxidation step indicated that aldehyde content can be easily tuned through the reaction time and concentration of reactants. Reductive amination of dialdehyde cellulose was more efficient as the length of the diaminated hydrocarbon compound increased. Morphological analysis of DAB showed that cellulose chemical grafting enabled the preservation of the bead shape and internal structure upon freeze-drying. Enzymatic degradability studies demonstrated that chemical functionalization did not undermine enzyme cellulose hydrolysis. The addition of aminated moieties on cellulose dramatically increased absorption efficiency for all industrially relevant lipases used, reaching 100% for Thermomyces lanuginosus lipase (TLL). Storage and recyclability experiments demonstrated that enzymes were retained and recyclable for at least nine cycles, although the activity gradually declined after each cycle. Medium chain triacylglycerol hydrolysis in a SpinChem reactor using TLL immobilized on 1,6 DAB exhibited higher activity compared to acrylic beads (588 vs 459 U/g) suggesting that biodegradable cellulose-based materials could be a valid and attractive alternative to plastics carriers.

Nanostructure in Amphiphile-Based Deep Eutectic Solvents.

Deep eutectic solvents (DESs) are an emerging class of modern, often "green" solvents with unique properties. Recently, a deep eutectic system based on amphiphilic surfactant N-alkyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C12 & C14 sulfobetaine) and (1S)-(+)-10-camphor-sulfonic acid in the molar ratio 1:1.5 has been reported. Nanostructuring can be expected in this DES due to the nature of the components. In this work, we have investigated the native nanostructure in the DES comprising C12-C18 alkyl chain sulfobetaines with camphor sulfonic acid and how it interacts with polar and nonpolar species, water and dodecane, respectively, using small angle neutron scattering. By using contrast variation to highlight the relative position of the solvent components and additives, we can resolve the structure of the solvent and how it changes upon interaction with water and dodecane. Scattering from the neat DES shows structures corresponding to the self-assembly of sulfobetaines; the size of the structure increases as the alkyl chain length of the sulfobetaines increases. Water and dodecane interact, respectively, with the hydrophilic and hydrophobic moieties in the DES structure, primarily the sulfobetaine, thereby swelling and solvating the entire structure. The extent of the shift of the peak position, and the swelling, depend on concentration of the additive. The solution phase organization and the interaction of polar and nonpolar species as observed here, have the potential to affect the ordering of inorganic or polymeric materials grown in such solvents, paving new avenues for templating applications.

Evidence for an L3 phase in ternary deep eutectics: composition-induced L3-to-Lα transition of AOT.

Pure and hydrated deep eutectic solvents (DES) are proposed to form self-assembled nanostructures within the fluid bulk, similar to the bicontinuous L3 phase common for ionic liquids (ILs). Labelled choline chloride : urea : water DES were measured using small-angle neutron scattering (SANS), showing no long-range nanostructure. However, solutions of the surfactant AOT in this DES yielded scattering consistent with the L3 "sponge" phase, which was fitted using the Teubner-Strey model. A disclike model gave local structural information, namely, a linear increase in radius versus solvent water content (w = molar ratio of DES : water), eventually forming large, turbid lamellar phases at 10w; an L3-to-Lα transition was observed. Simultaneous multi-contrast SANS fits show the surfactant headgroup region is dominated by interactions with poorly-soluble Na+ at low water contents, and numerically-superior [cholinium]+ as water content increases. The modified interfacial Gaussian curvature from cation : anion volume matching stabilizes the lamellar morphology, allowing the bilayer aggregation number to increase.

The effect of polymer end-group on the formation of styrene - maleic acid lipid particles (SMALPs).

A series of block copolymers comprising styrene and maleic acid (SMA) has been prepared using RAFT polymerisation. RAFT often results in a large hydrophobic alkylthiocarbonylthio end group and this work examines its effect on the solution behaviour of the copolymers. SMA variants with, and without, this end group were synthesised and their behaviour compared with a commercially-available random copolymer of similar molecular weight. Dynamic light scattering and surface tension measurements found the RAFT-copolymers preferentially self-assembled into higher-order aggregates in aqueous solution. Small angle neutron scattering using deuterated styrene varients add support to the accepted model that these agreggates comprise a solvent-protected styrenic core with an acid-rich shell. Replacing the hydrophobic RAFT end group with a more hydrophilic nitrile caused differences in the resulting surface activity, attributed to the ability of the adjoining styrene homoblock to drive aggregation. Each of the copolymers formed SMALP nanodiscs with DMPC lipids, which were found to encapsulate a model membrane protein, gramicidin. However, end group variation affected solubilisition of DPPC, a lipid with a higher phase transition temperature. When using RAFT-copolymers terminated with a hydrophobic group, swelling of the bilayer and greater penetration of the homoblock into the nanodisc core occurred with increasing homoblock length. Conversely, commercial and nitrile-terminated RAFT-copolymers produced nanodisc sizes that stayed constant, instead indicating interaction at the edge of the lipid patch. The results highlight how even minor changes to the copolymer can modify the amphiphilic balance between regions, knowledge useful towards optimising copolymer structure to enhance and control nanodisc formation.

Trace Water Changes Metal Ion Speciation in Deep Eutectic Solvents: Ce3+ Solvation and Nanoscale Water Clustering in Choline Chloride-Urea-Water Mixtures.

Eutectic mixtures of choline chloride, urea, and water in deep eutectic solvent (DES)/water molar hydration ratios (w) of 2, 5, and 10, with dissolved cerium salt, were measured using neutron diffraction with isotopic substitution. Structures were modeled using empirical potential structure refinement (EPSR). Ce3+ was found to form highly charged complexes with a mean coordination number between 7 and 8, with the shell containing mostly chloride, followed by water. The shell composition is strongly affected by the molar ratio of dilution, as opposed to the mass or volume fraction, due to the high affinity of Cl- and H2O ligands that displace less favorable interactions with ligands such as urea and choline. The presence of Ce3+ salt disrupted the bulk DES structure slightly, making it more electrolyte-like. The measured coordination shell of choline showed significant discrepancies from the statistical noninteracting distribution, highlighting the nonideality of the blend. Cluster analysis revealed the trace presence of percolating water clusters (25 ≥ n ≥ 2) in solvent compositions of 5 and 10w for the first time.

Role of the Deep Eutectic Solvent Reline in the Synthesis of Gold Nanoparticles.

This work presents a mechanistic understanding of the synthesis of small (<3 nm) gold nanoparticles in a nontoxic, eco-friendly, and biodegradable eutectic mixture of choline chloride and urea (reline) without the addition of external reducing or stabilization agents. Reline acts as a reducing agent by releasing ammonia (via urea hydrolysis), forming gold nanoparticles even at trace ammonia concentration levels. Reline also affects the speciation of the gold precursor forming gold chloro-complexes, stabilizing Au+ species, leading to an easier reduction and avoiding the otherwise fast disproportionation reaction. Such a capability is however lost in the presence of large amounts of water, where water replaces the chloride ligands in the precursor speciation. In addition, reline acts as a weak stabilizing agent, leading to small particles (<3 nm) and narrow distributions although agglomerates quickly form. Such properties are maintained in the presence of water, indicating that it is linked to the urea stabilization rather than the hydrogen-bonding network. This work has important implications in the field of green synthesis of nanoparticles with small sizes, especially for biomedical and health care applications, due to the nontoxic nature of the components of deep eutectic solvents in contrast to the conventional routes.

Cellulose Acetate Microbeads for Controlled Delivery of Essential Micronutrients.

The controlled delivery of micronutrients to soil and plants is essential to increase agricultural yields. However, this is today achieved using fossil fuel-derived plastic carriers, posing environmental risks and contributing to global carbon emissions. In this work, a novel and efficient way to prepare biodegradable zinc-impregnated cellulose acetate beads for use as controlled release fertilizers is presented. Cellulose acetate solutions in DMSO were dropped into aqueous antisolvent solutions of different zinc salts. The droplets underwent phase inversion, forming solid cellulose acetate beads containing zinc, as a function of zinc salt type and concentration. Even higher values of zinc uptake (up to 15.5%) were obtained when zinc acetate was added to the cellulose acetate-DMSO solution, prior to dropping in aqueous zinc salt antisolvent solutions. The release profile in water of the beads prepared using the different solvents was linked to the properties of the counter-ions via the Hofmeister series. Studies in soil showed the potential for longer release times, up to 130 days for zinc sulfate beads. These results, together with the efficient bead production method, demonstrate the potential of zinc-impregnated cellulose acetate beads to replace the plastic-based controlled delivery products used today, contributing to the reduction of carbon emissions and potential environmental impacts due to the uptake of plastic in plants and animals.

Lignin recovery from cocoa bean shell using microwave-assisted extraction and deep eutectic solvents.

Lignin is the second most abundant natural polymer after cellulose, and valorisation of lignin-rich streams has attracted increasing attention recently. This paper presents a novel and sustainable method to recover lignin from Cocoa Bean Shells (CBS) using Deep Eutectic Solvents (DES) and microwaves. A DES containing p-toluenesulfonic acid, choline chloride and glycerol (2:1:1 M ratio) was selected based on its dielectric properties. Under 200 W microwave power, the optimum yield of 95.5 % lignin was achieved at 130 °C and 30 min. DES-extracted lignin exhibited unique structural characteristics including larger particle sizes (242.5 µm D50 size), structural diversity (410.4 µm D90-D10 size) and H/G sub-unit ratio (71.9 %) compared with commercial Kraft lignin (77.2 µm, 157.9 µm and 0.1 % respectively), indicating the potential of DES in the modification and upgrading of lignin for novel value-added products.

Surfactant induced gelation of TEMPO-oxidized cellulose nanofibril dispersions probed using small angle neutron scattering.

In this work, we studied TEMPO-oxidized cellulose nanofibril (OCNF) suspensions in the presence of diverse surfactants. Using a combination of small angle neutron scattering (SANS) and rheology, we compared the physical properties of the suspensions with their structural behavior. Four surfactants were studied, all with the same hydrophobic tail length but different headgroups: hexaethylene glycol mono-n-dodecyl ether (C12EO6, nonionic), sodium dodecyl sulfate (SDS, anionic), cocamidopropyl betaine (CapB, zwitterionic), and dodecyltrimethylammonium bromide (DTAB, cationic). Contrast variation SANS studies using deuterated version of C12EO6 or SDS, or by varying the D2O/H2O ratio of the suspensions (with CapB), allowed focusing only on the structural properties of OCNFs or surfactant micelles. We showed that, in the concentration range studied, for C12EO6, although the nanofibrils are concentrated thanks to an excluded volume effect observed in SANS, the rheological properties of the suspensions are not affected. Addition of SDS or CapB induces gelation for surfactant concentrations superior to the critical micellar concentration (CMC). SANS results show that attractive interactions between OCNFs arise in the presence of these anionic or zwitterionic surfactants, hinting at depletion attraction as the main mechanism of gelation. Finally, addition of small amounts of DTAB (below the CMC) allows formation of a tough gel by adsorbing onto the OCNF surface.

Structural investigation of sulfobetaines and phospholipid monolayers at the air-water interface.

Mixtures of sulfobetaine based lipids with phosphocholine phospholipids are of interest in order to study the interactions between zwitterionic surfactants and the phospholipids present in cell membranes. In this study we have investigated the structure of mixed monolayers of sulfobetaines and phosphocholine phospholipids. The sulfobetaine used has a single 18-carbon tail, and is referred to as SB3-18, and the phospholipid used is DMPC. Surface pressure-area isotherms of the samples were used to determine whether any phase transitions were present during the compression of the monolayers. Neutron and X-ray reflectometry were then used to investigate the structure of these monolayers perpendicular to the interface. We found that the average headgroup and tail layer thickness was reasonably consistent across all mixtures, with a variation of less than 3 Å reported in the total thickness of the monolayers at each surface pressure. However, by selective deuteration of the two components of the monolayers, it was found that the two components have different tail layer thicknesses. For the mixture with equal compositions of DMPC and SB3-18 or with a higher composition of DMPC the tail tilts were found to be constant, resulting in a greater tail layer thickness for SB3-18 due to its longer tail. For the mixture higher in SB3-18 this was not the case, the tail tilt angle for the two components was found to be different and DMPC was found to have a greater tail layer thickness than SB3-18 as a result.

Continuous production of cellulose microbeads by rotary jet atomization.

The replacement of plastic microbeads with biodegradable alternatives is essential due to the environmental persistence of plastics and their accumulation within the human food chain. HYPOTHESIS: Cellulose microbeads could be such alternative, but their production is hindered by the high viscosity of cellulose solutions. It is expected that this viscosity can be harnessed to induce filament thinning of jets of cellulose solutions to create droplets with diameters within the micrometre range, which can then be converted to solid cellulose microbeads via phase inversion. EXPERIMENTS: A 3D printed rotating multi-nozzle system was used to generate jets of cellulose dissolved in solutions of [EMIm][OAc] and DMSO. The jets were subject to Rayleigh breakup to generate droplets which were captured in an ethanol anti-solvent bath, initiating phase-inversion, and resulting in regeneration of the cellulose into beads. FINDINGS: Control of both process (e.g. nozzle dimensions) and operational (e.g. rotational speed and pressure) parameters has allowed suppression of both satellite droplets generation and secondary droplet break-up, and tuning of the filament thinning process. This resulted in the continuous fabrication of cellulose microbeads in the size range 40-500 µm with narrow size distributions. This method can produce beads in size ranges not attainable by existing technologies.

The interaction of styrene maleic acid copolymers with phospholipids in Langmuir monolayers, vesicles and nanodiscs; a structural study.

HYPOTHESIS: Self-assembly of amphipathic styrene maleic acid copolymers with phospholipids in aqueous solution results in the formation of 'nanodiscs' containing a planar segment of phospholipid bilayer encapsulated by a polymer belt. Recently, studies have reported that lipids rapidly exchange between both nanodiscs in solution and external sources of lipids. Outstanding questions remain regarding details of polymer-lipid interactions, factors influencing lipid exchange and structural effects of such exchange processes. Here, the dynamic behaviour of nanodiscs is investigated, specifically the role of membrane charge and polymer chemistry. EXPERIMENTS: Two model systems are investigated: fluorescently labelled phospholipid vesicles, and Langmuir monolayers of phospholipids. Using fluorescence spectroscopy and time-resolved neutron reflectometry, the membrane potential, monolayer structure and composition are monitored with respect to time upon polymer and nanodisc interactions. FINDINGS: In the presence of external lipids, polymer chains embed throughout lipid membranes, the extent of which is governed by the net membrane charge. Nanodiscs stabilised by three different polymers will all exchange lipids and polymer with monolayers to differing extents, related to the properties of the stabilising polymer belt. These results demonstrate the dynamic nature of nanodiscs which interact with the local environment and are likely to deposit both lipids and polymer at all stages of use.

Neutron Diffraction Study of Indole Solvation in Deep Eutectic Systems of Choline Chloride, Malic Acid, and Water.

Deep eutectic systems are currently under intense investigation to replace traditional organic solvents in a range of syntheses. Here, indole in choline chloride-malic acid deep eutectic solvent (DES) was studied as a function of water content, to identify solute interactions with the DES which affect heterocycle reactivity and selectivity, and as a proxy for biomolecule solvation. Empirical Potential Structure Refinement models of neutron diffraction data showed [Cholinium]+ cations associate strongly with the indole π-system due to electrostatics, whereas malic acid is only weakly associated. Trace water is sequestered into the DES and does not interact strongly with indole. When water is added to the DES, it does not interact with the indole π-system but is exclusively in-plane with the heterocyclic rings, forming strong H-bonds with the -NH group, and also weak H-bonds and thus prominent hydrophobic hydration of the indole aromatic region, which could direct selectivity in reactions.

Stable Cellulose Nanofibril Microcapsules from Pickering Emulsion Templates.

Electrostatic attractions are essential in any complex formation between the nanofibrils of the opposite charge for a specific application, such as microcapsule production. Here, we used cationized cellulose nanofibril (CCNF)-stabilized Pickering emulsions (PEs) as templates, and the electrostatic interactions were induced by adding oxidized cellulose nanofibrils (OCNFs) at the oil-water interface to form microcapsules (MCs). The oppositely charged cellulose nanofibrils enhanced the solidity of interfaces, allowing the encapsulation of Nile red (NR) in sunflower oil droplets. Microcapsules exhibited a low and controlled release of NR at room temperature. Furthermore, membrane emulsification was employed to scale up the preparation of microcapsules with sunflower oil (SFO) encapsulated by CCNF/OCNF complex networks.

Membrane extraction with styrene-maleic acid copolymer results in insulin receptor autophosphorylation in the absence of ligand.

Extraction of integral membrane proteins with poly(styrene-co-maleic acid) provides a promising alternative to detergent extraction. A major advantage of extraction using copolymers rather than detergent is the retention of the lipid bilayer around the proteins. Here we report the first functional investigation of the mammalian insulin receptor which was extracted from cell membranes using poly(styrene-co-maleic acid). We found that the copolymer efficiently extracted the insulin receptor from 3T3L1 fibroblast membranes. Surprisingly, activation of the insulin receptor and proximal downstream signalling was detected upon copolymer extraction even in the absence of insulin stimulation. Insulin receptor and IRS1 phosphorylations were above levels measured in the control extracts made with detergents. However, more distal signalling events in the insulin signalling cascade, such as the phosphorylation of Akt were not observed. Following copolymer extraction, in vitro addition of insulin had no further effect on insulin receptor or IRS1 phosphorylation. Therefore, under our experimental conditions, the insulin receptor is not functionally responsive to insulin. This study is the first to investigate receptor tyrosine kinases extracted from mammalian cells using a styrene-maleic acid copolymer and highlights the importance of thorough functional characterisation when using this method of protein extraction.

Fluorescent styrene maleic acid copolymers to facilitate membrane protein studies in lipid nanodiscs.

Fluorescently-labelled variants of poly(styrene-co-maleic acid), SMA, have been synthesised by RAFT copolymerisation. We show that low ratios of vinyl fluorophores, analogous to styrene, can be successfully incorporated during polymerisation without detriment to nanodisc formation upon interaction with lipids. These novel copolymers are capable of encapuslating lipids and the model membrane protein, gramicidin, and hence have the potential to be applied in fluorescence-based biological studies. To demonstrate this, energy transfer is used to probe polymer-protein interactions in nanodiscs. The copolymers may also be used to monitor nanodisc self assembly by exploiting aggregation-caused-quenching (ACQ).

Styrene-Maleic Acid Copolymer Nanodiscs to Determine the Shape of Membrane Proteins.

Lipid nanodiscs can be used to solubilize functional membrane proteins (MPs) in nativelike environments. Thus, they are promising reagents that have been proven useful to characterize MPs. Both protein and non-protein molecular belts have shown promise to maintain the structural integrity of MPs in lipid nanodiscs. Small-angle neutron scattering (SANS) can be used to determine low-resolution structures of proteins in solution, which can be enhanced through the use of contrast variation methods. We present theoretical contrast variation SANS results for protein and styrene-maleic acid copolymer (SMA) belt 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) nanodiscs with and without additional bound or transmembrane proteins. The predicted scattering properties are derived from atomistic molecular dynamics simulations to account for conformational fluctuations, and we determine deuterium-labeling conditions such that SANS intensity profiles only include contributions from the scattering of the MP of interest. We propose strategies to tune the neutron scattering length densities (SLDs) of the SMA and DMPC using selective deuterium labeling such that the SLD of the nanodisc becomes homogeneous and its scattering can essentially be eliminated in solvents containing an appropriate amount of D2O. These finely tuned labeled polymer-based nanodiscs are expected to be useful to extract the size and molecular shape information of MPs using SANS-based contrast variation experiments, and they can be used with MPs of any molecular weight.

Ab initio reconstruction of small angle scattering data for membrane proteins in copolymer nanodiscs.

Background: Small angle scattering techniques are beginning to be more widely utilised for structural analysis of biological systems. However, applying these techniques to study membrane proteins still remains problematic, due to sample preparation requirements and analysis of the resulting data. The development of styrene-maleic acid co-polymers (SMA) to extract membrane proteins into nanodiscs for further study provides a suitable environment for structural analysis. Methods: We use small angle neutron scattering (SANS) with three different contrasts to determine structural information for two different polymer nanodisc-incorporated proteins, Outer membrane protein F (OmpF) and gramicidin. Ab initio modelling was applied to generate protein/lipid structures from the SANS data. Other complementary structural methodologies, such as DLS, CD and TEM were compared alongside this data with known protein crystal structures. Results: A single-phase model was constructed for gramicidin-containing nanodiscs, which showed dimer formation in the centre of the nanodisc. For OmpF-nanodiscs we were able to construct a multi-phase model, providing structural information on the protein/lipid and polymer components of the sample. Conclusions: Polymer-nanodiscs can provide a suitable platform to investigate certain membrane proteins using SANS, alongside other structural methodologies. However, differences between the published crystal structure and OmpF-nanodiscs were observed, suggesting the nanodisc structure could be altering the folding of the protein. General significance: Small angle scattering techniques can provide structural information on the protein and polymer nanodisc without requiring crystallisation of the protein. Additional complementary techniques, such as ab initio modelling, can generate alternative models both the protein and nanodisc system.

Disposable Coverslip for Rapid Throughput Screening of Malaria Using Attenuated Total Reflection Spectroscopy.

Malaria is considered to be one of the most catastrophic health issues in the whole world. Vibrational spectroscopy is a rapid, robust, label-free, inexpensive, highly sensitive, nonperturbative, and nondestructive technique with high diagnostic potential for the early detection of disease agents. In particular, the fingerprinting capability of attenuated total reflection spectroscopy is promising as a point-of-care diagnostic tool in resource-limited areas. However, improvements are required to expedite the measurements of biofluids, including the drying procedure and subsequent cleaning of the internal reflection element to enable high throughput successive measurements. As an alternative, we propose using an inexpensive coverslip to reduce the sample preparation time by enabling multiple samples to be collectively dried together under the same temperature and conditions. In conjunction with partial least squares regression, attenuated total reflection spectroscopy was able to detect and quantify the parasitemia with root mean square error of cross-validation and R2 values of 0.177 and 0.985, respectively. Here, we characterize an inexpensive, disposable coverslip for the high throughput screening of malaria parasitic infections and thus demonstrate an alternative approach to direct deposition of the sample onto the internal reflection element.

Comparison of Cyclic and Linear Poly(lactide)s Using Small-Angle Neutron Scattering.

Small-angle neutron scattering (SANS) experiments were conducted on cyclic and linear polymers of racemic and l-lactides (PLA) with the goal of comparing chain configurations, scaling, and effective polymer-solvent interactions of the two topologies in acetone-d 6 and THF-d 8. There are limited reports of SANS results on cyclic polymers due to the lack of substantial development in the field until recently. Now that pure, well-defined cyclic polymers are accessible, unanswered questions about their rheology and physical conformations can be better investigated. Previously reported SANS experiments have used cyclic and linear polystyrene samples; therefore, our work allowed for direct comparison using a contrasting (structurally and sterically) polymer. We compared SANS results of cyclic and linear PLA samples with various microstructures and molecular weights at two different temperatures, allowing for comparison with a wide range of variables. The results followed the trends of previous experiments, but much greater differences in the effective polymer-solvent interaction parameters between cyclic and linear forms of PLA were observed, implying that the small form factor and hydrogen bonding in PLA allowed for much more compact conformations in the cyclic form only. Also, the polymer microstructure was found to influence polymer-solvent interaction parameters substantially. These results illustrate how the difference in polymer-solvent interactions between cyclic and linear polymers can vary greatly depending on the polymer in question and the potential of neutron scattering as a tool for identification and characterization of the cyclic topology.