A deep eutectic solvent is an effective cryoprotective agent for platelets.

The most widely used method of platelet cryopreservation requires the addition of dimethyl sulfoxide (DMSO; Me2SO) as a cryoprotective agent (CPA) and pre-freeze removal of Me2SO before freezing to mitigate toxicity. However, alternative CPAs such as deep eutectic solvents (DES), which are less toxic could simplify this process. The aim of this study was to determine the effectiveness of a Proline-Glycerol (Prol-Gly 1:3) DES as a platelet CPA. Platelets were cryopreserved at -80 °C using 10 % Prol-Gly 1:3 (DES; n = 6), or in the absence of a cryoprotectant (no CPA; n = 6). Platelets were also cryopreserved according to the gold-standard blood-banking method using 5.5 % Me2SO (n = 6), with centrifugation and pre-freeze removal of the excess Me2SO. Platelet quality was assessed by flow cytometry and thromboelastography (TEG). Post-thaw recovery was similar between the three groups. The abundance of labile platelet glycoproteins GPIbα and GPVI were highest in the DES group, however, markers of activation (CD62P and annexin-V) were also higher in this group. In terms of function, the strength of the clot (maximum amplitude; TEG) and extent of clot retraction was better with DES platelets compared to no CPA, but lower than Me2SO platelets. DES provides a cryoprotective advantage to platelets when compared to no CPA. Importantly, when compared to Me2SO platelets, most quality parameters were similar in DES platelets. The major advantage with using a DES is biocompatibility, therefore it does not need to be removed prior to transfusion. This greatly simplifies the freezing and thawing process, avoiding the toxic effects of Me2SO.

Amino Acid-Coated Zeolitic Imidazolate Framework for Delivery of Genetic Material in Prostate Cancer Cell.

Metal-organic frameworks (MOFs) are currently under progressive development as a tool for non-viral biomolecule delivery. Biomolecules such as proteins, lipids, carbohydrates, and nucleic acids can be encapsulated in MOFs for therapeutic purposes. The favorable physicochemical properties of MOFs make them an attractive choice for delivering a wide range of biomolecules including nucleic acids. Herein, a green fluorescence protein (GFP)-expressing plasmid DNA (pDNA) is used as a representative of a biomolecule to encapsulate within a Zn-based metal-organic framework (MOF) called a zeolitic imidazolate framework (ZIF). The synthesized biocomposites are coated with positively charged amino acids (AA) to understand the effect of surface functionalization on the delivery of pDNA to prostate cancer (PC-3) cells. FTIR and zeta potential confirm the successful preparation of positively charged amino acid-functionalized derivatives of pDNA@ZIF (i.e., pDNA@ZIFAA). Moreover, XRD and SEM data show that the functionalized derivates retain the pristine crystallinity and morphology of pDNA@ZIF. The coated biocomposites provide enhanced uptake of genetic material by PC-3 human prostate cancer cells. The AA-modulated fine-tuning of the surface charge of biocomposites results in better interaction with the cell membrane and enhances cellular uptake. These results suggest that pDNA@ZIFAA can be a promising alternative tool for non-viral gene delivery.

Photoinitiated Energy Transfer in Porous-Cage-Stabilised Silver Nanoparticles.

We report a new composite material consisting of silver nanoparticles decorated with three-dimensional molecular organic cages based on light-absorbing porphyrins. The porphyrin cages serve to both stabilize the particles and allow diffusion and trapping of small molecules close to the metallic surface. Combining these two photoactive components results in a Fano-resonant interaction between the porphyrin Soret band and the nanoparticle-localised surface-plasmon resonance. Time-resolved spectroscopy revealed the silver nanoparticles transfer up to 37 % of their excited-state energy to the stabilising layer of porphyrin cages. These unusual photophysics cause a 2-fold current increase in photoelectrochemical water-splitting measurements. The composite structure provides a compelling proof of concept for advanced photosensitiser systems with intrinsic porosity for photocatalytic and sensing applications.

Probing the dynamics of turbid colloidal suspensions using differential dynamic microscopy.

Few techniques can reliably measure the dynamics of colloidal suspensions or other soft materials over a wide range of turbidities. Here we systematically investigate the capability of Differential Dynamic Microscopy (DDM) to characterise particle dynamics in turbid colloidal suspensions based on brightfield optical microscopy. We measure the Intermediate Scattering Function (ISF) of polystyrene microspheres suspended in water over a range of concentrations, turbidities, and up to 4 orders of magnitude in time-scales. These DDM results are compared to data obtained from both Dynamic Light Scattering (DLS) and Two-colour Dynamic Light Scattering (TCDLS). The latter allows for suppression of multiple scattering for moderately turbid suspensions. We find that DDM can obtain reliable diffusion coefficients at up to 10 and 1000 times higher particle concentrations than TCDLS and standard DLS, respectively. Additionally, we investigate the roles of the four length-scales relevant when imaging a suspension: the sample thickness L, the imaging depth z, the imaging depth of field DoF, and the photon mean free path . More detailed experiments and analysis reveal the appearance of a short-time process as turbidity is increased, which we associate with multiple scattering events within the imaging depth of the field. The long-time process corresponds to the particle dynamics from which particle-size can be estimated in the case of non-interacting particles. Finally, we provide a simple theoretical framework, ms-DDM, for turbid samples, which accounts for multiple scattering.

Differential dynamic microscopy for the characterisation of motility in biological systems.

Differential Dynamic Microscopy (DDM) is a relatively new technique which measures the dynamics of suspended particles using a dynamic light scattering formalism. Videos are recorded using standard light microscopy at moderate frame rates, and fluctuations in pixel intensity are measured as a function of time. As only pixel intensity is analysed, it is not necessary to resolve individual particles. This allows for low magnifications and wide fields of view, and therefore dynamics can be measured on tens of thousands of scattering objects, providing robust statistics. A decade ago the technique was successfully applied to measure bacterial motility. Since then, it has been applied to a range of motile systems, but has not yet reached the wider biological community. This perspective reviews the work done so far, and provides the basic background to enable the broader application of this promising technique.

Translational and rotational diffusion coefficients of gold nanorods functionalized with a high molecular weight, thermoresponsive ligand: a depolarized dynamic light scattering study.

Probing the rotational and translational diffusion and colloidal stability of nanorods is of significant fundamental interest with implications for many different applications. Recently R. Nixon-Luke and G. Bryant presented a method to analyze angle-dependent depolarized dynamic light scattering data allowing for the clear separation of the translational and rotational diffusion coefficients of gold nanorods in dilute suspension (R. Nixon-Luke and G. Bryant, Part. Part. Syst. Charact., 2018, 36, 1800388). In the present work we applied this analysis to gold nanorods decorated with high molecular weight, thermoresponsive poly-N-isopropylacrylamide ligands, which results in particles with lower effective aspect ratios. The temperature response of the ligand shell is studied. We precisely determine the translational and rotational diffusion coefficients over a broad range of temperatures and the results are compared to theoretical predictions. The results show that as temperature increases the ligands collapse, and the effective aspect ratio increases as the particle shape transitions from prolate spheroid at low temperatures to more cylindrical at high temperatures.

Analysis of Pathogenic Bacterial and Yeast Biofilms Using the Combination of Synchrotron ATR-FTIR Microspectroscopy and Chemometric Approaches.

Biofilms are assemblages of microbial cells, extracellular polymeric substances (EPS), and other components extracted from the environment in which they develop. Within biofilms, the spatial distribution of these components can vary. Here we present a fundamental characterization study to show differences between biofilms formed by Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), Gram-negative Pseudomonas aeruginosa, and the yeast-type Candida albicans using synchrotron macro attenuated total reflectance-Fourier transform infrared (ATR-FTIR) microspectroscopy. We were able to characterise the pathogenic biofilms' heterogeneous distribution, which is challenging to do using traditional techniques. Multivariate analyses revealed that the polysaccharides area (1200-950 cm-1) accounted for the most significant variance between biofilm samples, and other spectral regions corresponding to amides, lipids, and polysaccharides all contributed to sample variation. In general, this study will advance our understanding of microbial biofilms and serve as a model for future research on how to use synchrotron source ATR-FTIR microspectroscopy to analyse their variations and spatial arrangements.

A Hitchhiker's Guide to Particle Sizing Techniques.

Accurate characterization of particle size and particle size distributions is mandatory in nanotechnology and a broad range of colloidal sciences. The size of colloidal particles can be determined using various techniques in direct and reciprocal space, including electron microscopy and static and dynamic light scattering. Differential dynamic microscopy was introduced recently and offers a new alternative. In this paper we present a systematic study of particle size determination using various techniques. We compare the results and highlight advantages and disadvantages. Unexpectedly we find that differential dynamic microscopy offers the unique possibility to determine the particle size in highly turbid samples.

Encapsulation, Visualization and Expression of Genes with Biomimetically Mineralized Zeolitic Imidazolate Framework-8 (ZIF-8).

Recent work in biomolecule-metal-organic framework (MOF) composites has proven to be an effective strategy for the protection of proteins. However, for other biomacromolecules such as nucleic acids, the encapsulation into nano MOFs and the related characterizations are in their infancy. Herein, encapsulation of a complete gene-set in zeolitic imidazolate framework-8 (ZIF-8) MOFs and cellular expression of the gene delivered by the nano MOF composites are reported. Using a green fluorescent protein (GFP) plasmid (plGFP) as a proof-of-concept genetic macromolecule, successful transfection of mammalian cancer cells with plGFP for up to 4 days is shown. Cell transfection assays and soft X-ray cryo-tomography (cryo-SXT) demonstrate the feasibility of DNA@MOF biocomposites as intracellular gene delivery vehicles. Expression occurs over relatively prolonged time points where the cargo nucleic acid is released gradually in order to maintain sustained expression.

Interaction of Giant Unilamellar Vesicles with the Surface Nanostructures on Dragonfly Wings.

The waxy epicuticle of dragonfly wings contains a unique nanostructured pattern that exhibits bactericidal properties. In light of emerging concerns of antibiotic resistance, these mechano-bactericidal surfaces represent a particularly novel solution by which bacterial colonization and the formation of biofilms on biomedical devices can be prevented. Pathogenic bacterial biofilms on medical implant surfaces cause a significant number of human deaths every year. The proposed mechanism of bactericidal activity is through mechanical cell rupture; however, this is not yet well understood and has not been well characterized. In this study, we used giant unilamellar vesicles (GUVs) as a simplified cell membrane model to investigate the nature of their interaction with the surface of the wings of two dragonfly species, Austrothemis nigrescens and Trithemis annulata, sourced from Victoria, Australia, and the Baix Ebre and Terra Alta regions of Catalonia, Spain. Confocal laser scanning microscopy and cryo-scanning electron microscopy techniques were used to visualize the interactions between the GUVs and the wing surfaces. When exposed to both natural and gold-coated wing surfaces, the GUVs were adsorbed on the surface, exhibiting significant deformation, in the process of membrane rupture. Differences between the tensile rupture limit of GUVs composed of 1,2-dioleoyl- sn-glycero-3-phosphocholine and the isotropic tension generated from the internal osmotic pressure were used to indirectly determine the membrane tensions, generated by the nanostructures present on the wing surfaces. These were estimated as being in excess of 6.8 mN m-1, the first experimental estimate of such mechano-bactericidal surfaces. This simple model provides a convenient bottom-up approach toward understanding and characterizing the bactericidal properties of nanostructured surfaces.

Visible-Light-Driven "On"/"Off" Photochromism of a Polyoxometalate Diarylethene Coordination Complex.

Herein we report the first photochromic polyoxometalate (POM)-based diarylethene (DAE) coordination complex, prepared by ligation of two cobalt(III)-incorporated borotungstates [BIIIWVI11O39CoIII]6- with the ditopic pyridyl-containing diarylethene (C25H16N2F6S2). The solution-state composition, structure, and stability of the assembly were probed using 1H and 19F nuclear magnetic resonance spectroscopy (NMR), electrospray ionization quadrupolar time-of-flight mass spectrometry (ESI-QTOF-MS), ultraviolet-visible spectroscopy (UV-vis), and small-angle X-ray scattering (SAXS), revealing that the complex self-organizes to adopt a molecular dumbbell structure due to electrostatic and steric considerations. This conformation is a prerequisite for the photocyclization reaction. The assembly was found to be switchable between two states using visible light due to the perturbation of the DAE electronic structure on coordination to the POM. We present photophysical data, including the reaction quantum efficiency of the molecular switch in both directions measured using a custom-built quantum yield determination setup in addition to fatigue resistance on prolonged irradiation.

A Gallium-Based Magnetocaloric Liquid Metal Ferrofluid.

We demonstrate a magnetocaloric ferrofluid based on a gadolinium saturated liquid metal matrix, using a gallium-based liquid metal alloy as the solvent and suspension medium. The material is liquid at room temperature, while exhibiting spontaneous magnetization and a large magnetocaloric effect. The magnetic properties were attributed to the formation of gadolinium nanoparticles suspended within the liquid gallium alloy, which acts as a reaction solvent during the nanoparticle synthesis. High nanoparticle weight fractions exceeding 2% could be suspended within the liquid metal matrix. The liquid metal ferrofluid shows promise for magnetocaloric cooling due to its high thermal conductivity and its liquid nature. Magnetic and thermoanalytic characterizations reveal that the developed material remains liquid within the temperature window required for domestic refrigeration purposes, which enables future fluidic magnetocaloric devices. Additionally, the observed formation of nanometer-sized metallic particles within the supersaturated liquid metal solution has general implications for chemical synthesis and provides a new synthetic pathway toward metallic nanoparticles based on highly reactive rare earth metals.

Blood-Catalyzed RAFT Polymerization.

The use of hemoglobin (Hb) contained within red blood cells to drive a controlled radical polymerization via a reversible addition-fragmentation chain transfer (RAFT) process is reported for the first time. No pre-treatment of the Hb or cells was required prior to their use as polymerization catalysts, indicating the potential for synthetic engineering in complex biological microenvironments without the need for ex vivo techniques. Owing to the naturally occurring prevalence of the reagents employed in the catalytic system (Hb and hydrogen peroxide), this approach may facilitate the development of new strategies for in vivo cell engineering with synthetic macromolecules.

Intrinsically Disordered Stress Protein COR15A Resides at the Membrane Surface during Dehydration.

Plants from temperate climate zones are able to increase their freezing tolerance during exposure to low, above-zero temperatures in a process termed cold acclimation. During this process, several cold-regulated (COR) proteins are accumulated in the cells. One of them is COR15A, a small, intrinsically disordered protein that contributes to leaf freezing tolerance by stabilizing cellular membranes. The isolated protein folds into amphipathic α-helices in response to increased crowding conditions, such as high concentrations of glycerol. Although there is evidence for direct COR15A-membrane interactions, the orientation and depth of protein insertion were unknown. In addition, although folding due to high osmolyte concentrations had been established, the folding response of the protein under conditions of gradual dehydration had not been investigated. Here we show, using Fourier transform infrared spectroscopy, that COR15A starts to fold into α-helices already under mild dehydration conditions (97% relative humidity (RH), corresponding to freezing at -3°C) and that folding gradually increases with decreasing RH. Neutron diffraction experiments at 97 and 75% RH established that the presence of COR15A had no significant influence on the structure of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. However, using deuterated POPC we could clearly establish that COR15A interacts with the membranes and penetrates below the headgroup region into the upper part of the fatty acyl chain region. This localization is in agreement with our hypothesis that COR15A-membrane interaction is at least, in part, driven by a hydrophobic interaction between the lipids and the hydrophobic face of the amphipathic protein α-helix.

Easy Come, Easy Go: Capillary Forces Enable Rapid Refilling of Embolized Primary Xylem Vessels.

Protoxylem plays an important role in the hydraulic function of vascular systems of both herbaceous and woody plants, but relatively little is known about the processes underlying the maintenance of protoxylem function in long-lived tissues. In this study, embolism repair was investigated in relation to xylem structure in two cushion plant species, Azorella macquariensis and Colobanthus muscoides, in which vascular water transport depends on protoxylem. Their protoxylem vessels consisted of a primary wall with helical thickenings that effectively formed a pit channel, with the primary wall being the pit channel membrane. Stem protoxylem was organized such that the pit channel membranes connected vessels with paratracheal parenchyma or other protoxylem vessels and were not exposed directly to air spaces. Embolism was experimentally induced in excised vascular tissue and detached shoots by exposing them briefly to air. When water was resupplied, embolized vessels refilled within tens of seconds (excised tissue) to a few minutes (detached shoots) with water sourced from either adjacent parenchyma or water-filled vessels. Refilling occurred in two phases: (1) water refilled xylem pit channels, simplifying bubble shape to a rod with two menisci; and (2) the bubble contracted as the resorption front advanced, dissolving air along the way. Physical properties of the protoxylem vessels (namely pit channel membrane porosity, hydrophilic walls, vessel dimensions, and helical thickenings) promoted rapid refilling of embolized conduits independent of root pressure. These results have implications for the maintenance of vascular function in both herbaceous and woody species, because protoxylem plays a major role in the hydraulic systems of leaves, elongating stems, and roots.

Mixed-Metal Hybrid Polyoxometalates with Amino Acid Ligands: Electronic Versatility and Solution Properties.

Eight new members of a family of mixed-metal (Mo,W) polyoxometalates (POMs) with amino acid ligands have been synthesized and investigated in the solid state and solution using multiple physical techniques. While the peripheral POM structural framework is conserved, the different analogues vary in nuclearity of the central metal-oxo core, overall redox state, metal composition, and identity of the zwitterionic α-amino acid ligands. Structural investigations reveal site-selective substitution of Mo for W, with a strong preference for Mo to occupy the central metal-oxo core. This core structural unit is a closed tetrametallic loop in the blue reduced species and an open trimetallic loop in the colorless oxidized analogues. Density functional theory calculations suggest the core as the favored site of reduction and reveal that the corresponding molecular orbital is much lower in energy for a tetra- versus trimetallic core. The reduced species are diamagnetic, each with a pair of strongly antiferromagnetically coupled MoV centers in the tetrametallic core, while in the oxidized complexes all Mo is hexavalent. Solution small-angle X-ray scattering and circular dichroism (CD) studies indicate that the hybrid POM is stable in aqueous solution on a time scale of days within defined concentration and pH ranges, with the stability enhanced by the presence of excess amino acid. The CD experiments also reveal that the amino acid ligands readily exchange with other α-amino acids, and it is possible to isolate the products of amino acid exchange, confirming retention of the POM framework. Cyclic voltammograms of the reduced species exhibit an irreversible oxidation process at relatively low potential, but an equivalent reductive process is not evident for the oxidized analogues. Despite their overall structural similarity, the oxidized and 2e-reduced hybrid POMs are not interconvertible because of the respective open- versus closed-loop arrangement in the central metal-oxo cores.

Disordered cold regulated15 proteins protect chloroplast membranes during freezing through binding and folding, but do not stabilize chloroplast enzymes in vivo.

Freezing can severely damage plants, limiting geographical distribution of natural populations and leading to major agronomical losses. Plants native to cold climates acquire increased freezing tolerance during exposure to low nonfreezing temperatures in a process termed cold acclimation. This involves many adaptative responses, including global changes in metabolite content and gene expression, and the accumulation of cold-regulated (COR) proteins, whose functions are largely unknown. Here we report that the chloroplast proteins COR15A and COR15B are necessary for full cold acclimation in Arabidopsis (Arabidopsis thaliana). They protect cell membranes, as indicated by electrolyte leakage and chlorophyll fluorescence measurements. Recombinant COR15 proteins stabilize lactate dehydrogenase during freezing in vitro. However, a transgenic approach shows that they have no influence on the stability of selected plastidic enzymes in vivo, although cold acclimation results in increased enzyme stability. This indicates that enzymes are stabilized by other mechanisms. Recombinant COR15 proteins are disordered in water, but fold into amphipathic α-helices at high osmolyte concentrations in the presence of membranes, a condition mimicking molecular crowding induced by dehydration during freezing. X-ray scattering experiments indicate protein-membrane interactions specifically under such crowding conditions. The COR15-membrane interactions lead to liposome stabilization during freezing. Collectively, our data demonstrate the requirement for COR15 accumulation for full cold acclimation of Arabidopsis. The function of these intrinsically disordered proteins is the stabilization of chloroplast membranes during freezing through a folding and binding mechanism, but not the stabilization of chloroplastic enzymes. This indicates a high functional specificity of these disordered plant proteins.

Direct Comparison of Disaccharide Interaction with Lipid Membranes at Reduced Hydrations.

Understanding sugar-lipid interactions during desiccation and freezing is an important step in the elucidation of cryo- and anhydro-protection mechanisms. We determine sucrose, trehalose, and water concentration distributions in intra-bilayer volumes between opposing dioleoylphosphatidylcholine bilayers over a range of reduced hydrations and sugar concentrations. Stacked lipid bilayers at reduced hydration provide a suitable system to mimic environmental dehydration effects, as well as a suitable system for direct probing of sugar locations by neutron membrane diffraction. Sugar distributions show that sucrose and trehalose both behave as typical uncharged solutes, largely excluded from the lipid bilayers regardless of sugar identity, and with no correlation between sugar distribution and the lipid headgroup position as the hydration is changed. These results are discussed in terms of current opinions about cryo- and anhydro-protection mechanisms.

Characterization of the phase behaviour of a novel polymerizable lyotropic ionic liquid crystal.

The development of new polymerizable lyotropic liquid crystals (LLCs) utilizing charged amphiphilic molecules such as those based on long chain imidazolium compounds, is a relatively new design direction for producing robust membranes with controllable nano-structures. Here we have developed a novel polymerizable ionic liquid based LLC, 1-hexadecyl-3-methylimidazolium acrylate (C16mimAcr), where the acrylate anion acts as the polymerizable moiety. The phase behaviour of the C16mimAcr upon the addition of water was characterized using small and wide angle X-ray scatterings, differential scanning calorimetry and polarized optical microscopy. We compare the phase behaviour of this new polymerizable LLC to that of the well known LLC chloride analogue, 1-hexadecyl-3-methylimidazolium chloride (C16mimCl). We find that the C16mimAcr system has a more complex phase behaviour compared to the C16mimCl system. Additional lyotropic liquid crystalline mesophases such as hexagonal phase (H1) and discontinuous cubic phase (I1) are observed at 20 °C for the acrylate system at 50 and 65 wt% water respectively. The appearance of the hexagonal phase (H1) and discontinuous cubic phase (I1) for the acrylate system is likely due to the strong hydrating nature of the acrylate anion, which increases the head group area. The formation of these additional mesophases seen for the acrylate system, especially the hexagonal phase (H1), coupled with the polymerization functionality offers great potential in the design of advanced membrane materials with selective and anisotropic transport properties.

Modular molecules: site-selective metal substitution, photoreduction, and chirality in polyoxometalate hybrids.

The first members of a promising new family of hybrid amino acid-polyoxometalates have emerged from a search for modular functional molecules. Incorporation of glycine (Gly) or norleucine (Nle) ligands into an yttrium-tungstoarsenate structural backbone, followed by crystallization with p-methylbenzylammonium (p-MeBzNH3(+)) cations, affords (p-MeBzNH3)6K2(GlyH)[As(III)4(Y(III)W(VI)3)W(VI)44Y(III)4O159(Gly)8(H2O)14]⋅47 H2O (1) and enantiomorphs (p-MeBzNH3)15(NleH)3[As(III)4(Mo(V)2Mo(VI)2)W(VI)44Y(III)4O160(Nle)9(H2O)11][As(III)4(Mo(VI)2W(VI)2)W(VI)44Y(III)4O160(Nle)9(H2O)11] (generically designated 2: L-Nle, 2 a; D-Nle, 2 b). An intensive structural, spectroscopic, electrochemical, magnetochemical and theoretical investigation has allowed the elucidation of site-selective metal substitution and photoreduction of the tetranuclear core of the hybrid polyanions. In the solid state, markedly different crystal packing is evident for the compounds, which indicates the role of noncovalent interactions involving the amino acid ligands. In solution, mass spectrometric and small-angle X-ray scattering studies confirm maintenance of the structure of the polyanions of 2, while circular dichroism demonstrates that the chirality is also maintained. The combination of all of these features in a single modular family emphasizes the potential of such hybrid polyoxometalates to provide nanoscale molecular materials with tunable properties.