Gloria D. Elliott

Energy applications of ionic liquids

Ionic liquids offer a unique suite of properties that make them important candidates for a number of energy related applications. Cation–anion combinations that exhibit low volatility coupled with high electrochemical and thermal stability, as well as ionic conductivity, create the possibility of designing ideal electrolytes for batteries, super-capacitors, actuators, dye sensitised solar cells and thermo-electrochemical cells. In the field of water splitting to produce hydrogen they have been used to synthesize some of the best performing water oxidation catalysts and some members of the protic ionic liquid family co-catalyse an unusual, very high energy efficiency water oxidation process. As fuel cell electrolytes, the high proton conductivity of some of the protic ionic liquid family offers the potential of fuel cells operating in the optimum temperature region above 100 °C. Beyond electrochemical applications, the low vapour pressure of these liquids, along with their ability to offer tuneable functionality, also makes them ideal as CO2 absorbents for post-combustion CO2 capture. Similarly, the tuneable phase properties of the many members of this large family of salts are also allowing the creation of phase-change thermal energy storage materials having melting points tuned to the application. This perspective article provides an overview of these developing energy related applications of ionic liquids and offers some thoughts on the emerging challenges and opportunities.

Cyto-toxicity and biocompatibility of a family of choline phosphate ionic liquids designed for pharmaceutical applications

Recently, the ionic liquid (IL) choline dihydrogen phosphate was demonstrated to improve the thermostability and shelf life of several model proteins, thus exhibiting potential as a stabilizing excipient or solvent for protein therapeutics. Before novel ILs can be used for biomedical applications, comprehensive data is required to establish biocompatibility, including cytotoxicity effects and solution behavior. In this study five phosphate-based anion moieties were analyzed: H2PO4− (DHP), dibutyl phosphate (DBP), bis(2-ethylhexyl) phosphate (BEH), bis(2,4,4-trimethylpentyl) phosphinate (TMP), and O,O′-diethyl dithiophosphate (DEP), all paired with the cation choline (C). Toxicity levels for these ILs, and a common sugar and salts, were established using a J774 murine macrophage cell line. The sugar trehalose, and the simple salts sodium chloride and choline chloride yielded EC50 values of >100, 63 and 34 mM, respectively. The EC50 values (mM) of CDHP (20), CDBP (9.1), and CDEP (8.2) were lower than, but within the range of simple salts NaCl (62.8) and choline Cl (33.7). The EC50 values of CTMP and CBEH were considerably lower, 0.25 and 0.30 mM, respectively. CDHP and CBEH displayed a hormetic response. Osmolality measurements indicated that CDHP, CDBP, and CDEP exhibit nearly complete dissociation in aqueous solution, with osmotic coefficients of 1.0, 0.9, and 0.8, whereas CTMP and CBEH have coefficients of 0.5 and 0.3, and are more molecular in character. A high correlation between the EC50 value and the anion mass fraction indicated that anion size and the presence of moderately long and/or branched alkyl chains may affect viability.

Biocompatible Ionic Liquids: A New Approach for Stabilizing Proteins in Liquid Formulation

Ionic liquids (ILs) have shown excellent promise as both solutes and solvents for stabilizing proteins at room temperature. Because many modern drugs are protein-based, these stabilizing characteristics have great potential to provide advances in the field of liquid formulation of therapeutic proteins. However, before these developments can be translated into clinical solutions it is essential to establish data related to the biocompatibility of these ILs. The current work investigates the cytotoxicity of several ILs that were rationally synthesized from natural biomolecules and compounds that have already been approved as excipients for drug formulations. The effect of choline dihydrogen phosphate (choline dhp), choline saccharinate, and 1-butyl 3-methyl imidazolium lactate (bmim lactate) on the metabolic activity of a mouse macrophage cell line (J774) was assessed using the reduction in resazurin as an indicator of activity and, by extension, viability. Two formulations of lysozyme (10 mg/ml and 100 mg/ml) in 80 wt % choline dhp (aq) were prepared and the proteins were evaluated for structural stability immediately following formulation and again at 1 month. Equivalent formulations in 0.1 M Na acetate aqueous buffer were evaluated as controls. A differential scanning microcalorimeter (DSC) was used to evaluate the structural stability on the basis of the unfolding temperature and the enthalpy of unfolding, and a micrococcus lysodiekticus activity test was used to evaluate functional activity. All compounds were found to be relatively benign, with toxicity increasing in the order choline dhp<choline saccharinate<bmim lactate. At 1 month lysozyme that had been stored in choline dhp had a higher activity and folded fraction than lysozyme that had been stored in aqueous buffer. These results suggest that biocompatibility and protein stabilization characteristics can be rationally designed into ionic liquids.

The promise of organ and tissue preservation to transform medicine

The ability to replace organs and tissues on demand could save or improve millions of lives each year globally and create public health benefits on par with curing cancer. Unmet needs for organ and tissue preservation place enormous logistical limitations on transplantation, regenerative medicine, drug discovery, and a variety of rapidly advancing areas spanning biomedicine. A growing coalition of researchers, clinicians, advocacy organizations, academic institutions, and other stakeholders has assembled to address the unmet need for preservation advances, outlining remaining challenges and identifying areas of underinvestment and untapped opportunities. Meanwhile, recent discoveries provide proofs of principle for breakthroughs in a family of research areas surrounding biopreservation. These developments indicate that a new paradigm, integrating multiple existing preservation approaches and new technologies that have flourished in the past 10 years, could transform preservation research. Capitalizing on these opportunities will require engagement across many research areas and stakeholder groups. A coordinated effort is needed to expedite preservation advances that can transform several areas of medicine and medical science.

A Role for Microwave Processing in the Dry Preservation of Mammalian Cells

Dry preservation involves removing water from samples so that degradative biochemical processes are slowed and extended storage is possible. Recently this approach has been explored as a method for preserving living mammalian cells. The current work explores the use of microwave processing to enhance evaporation rates and to improve drying uniformity, thereby overcoming some of the challenges in this field. Mouse macrophage cells (J774) were pre‐incubated in full complement media containing 50 mM trehalose, for 18‐h, to allow for endocytosis of trehalose. Droplets of experimental and control (no intracellular trehalose) cell suspensions were placed on coverslips in a microwave cavity. Water was evaporated using intermittent microwave heating (600 W, 30 s intervals). Samples were dried to various moisture levels, rehydrated, and then survival was assessed after a 45‐min recovery period using Calcein‐AM/PI fluorescence and Trypan Blue exclusion assays. The metabolic activity of dried cells (4.3 gH2O/gdw) was assessed after rehydration using a resazurin reduction assay. Apoptosis levels were also measured. Post‐ rehydration survival correlated with the final moisture content achieved, consistent with other drying methods. Intracellular trehalose provided protection against injury associated with moisture loss. Metabolic assays revealed normal growth in surviving cells, and these survival levels were consistent with results from apoptosis assays (P > 0.05). Brightfield and fluorescence images of microwave‐dried samples revealed a uniform distribution of cells within the dried matrix and profilometry analysis demonstrated that solids were uniformly distributed throughout the sample. Microwave‐processing successfully facilitated rapid and uniform dehydration of cell‐based samples. Biotechnol. Bioeng. 2008;100: 782–796.

Progressive elimination of microinjected trehalose during mouse embryonic development

Recently, sugars such as trehalose have been introduced into mammalian cells by overcoming the permeability barrier of cell membranes, and have provided improved tolerance against stresses associated with freezing and drying. However, the fate of the intracellular sugars has remained an open question. To address this issue, mouse oocytes were microinjected with 0.1 mol/l trehalose, and intracellular trehalose and glucose concentrations were determined during embryonic development using a high performance liquid chromatography and pulsed amperometric detection protocol. Trehalose was not detected in non-injected controls at any stage of development. In the microinjection group, the amount of intracellular trehalose progressively decreased as embryos developed. There was a corresponding increase in intracellular glucose concentration at the two-cell stage, suggesting cleavage of trehalose to two glucose molecules. In summary, this study presents a simple, highly sensitive protocol to determine intracellular sugars. The data reveal rapid elimination of microinjected trehalose during embryonic development. These findings have implications for designing osmolarity-optimized culture media for sugar-injected oocytes.

Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures

Cryopreservation has become a central technology in many areas of clinical medicine, biotechnology, and species conservation within both plant and animal biology. Cryoprotective agents (CPAs) invariably play key roles in allowing cells to be processed for storage at deep cryogenic temperatures and to be recovered with high levels of appropriate functionality. As such, these CPA solutes possess a wide range of metabolic and biophysical effects that are both necessary for their modes of action, and potentially complicating for cell biological function. Early successes with cryopreservation were achieved by empirical methodology for choosing and applying CPAs. In recent decades, it has been possible to assemble objective information about CPA modes of action and to optimize their application to living systems, but there still remain significant gaps in our understanding. This review sets out the current status on the biological and chemical knowledge surrounding CPAs, and the conflicting effects of protection versus toxicity resulting from the use of these solutes, which are often required in molar concentrations, far exceeding levels found in normal metabolism. The biophysical properties of CPAs that allow them to facilitate different approaches to cryogenic storage, including vitrification, are highlighted. The topics are discussed with reference to the historical background of applying CPAs, and the relevance of cryoprotective solutes in natural freeze tolerant organisms. Improved cryopreservation success will be an essential step in many future areas such as regenerative medicine, seed banking, or stem cell technology. To achieve this, we will need to further improve our understanding of cryobiology, where better and safer CPAs will be key requirements.

Two-stage fungal biopulping for improved enzymatic hydrolysis of wood

A novel two-stage, whole organism fungal biopulping method was examined for increasing the yield of enzymatic hydrolysis of wood into soluble glucose. Liriodendron tulipifera wood chips (1g) were exposed to liquid culture suspensions of white rot (Ceriporiopsis subvermispora) or brown rot (Postia placenta) fungi and incubated at 28°C, either alone in single-stage 30 day (one fungal species applied) or two-stage 60 day (both fungal species applied in alternative succession) treatments. Fungi grew in all treatments, but did not significantly decrease the percent carbohydrate content of the wood. Two-stage treatments differed significantly in mass loss depending on order of exposure, suggesting additive or inhibitory fungal interactions occurred. Treatments consisting of C. subvermispora followed by P. placenta exhibited 6 ± 0.5% mass loss and increased the yield of enzymatic hydrolysis by 67-119%. This significant hydrolysis improvement suggests that fungal biopulping technologies could support commercial lignocellulosic ethanol production efforts if further developed.

Dynamic and thermodynamic characteristics associated with the glass transition of amorphous trehalose–water mixtures

The glass transition temperature Tg of biopreservative formulations is important for predicting the long-term storage of biological specimens. As a complementary tool to thermal analysis techniques, which are the mainstay for determining Tg, molecular dynamics simulations have been successfully applied to predict the Tg of several protectants and their mixtures with water. These molecular analyses, however, rarely focused on the glass transition behavior of aqueous trehalose solutions, a subject that has attracted wide scientific attention via experimental approaches. Important behavior, such as hydrogen-bonding dynamics and self-aggregation has yet to be explored in detail, particularly below, or in the vicinity of, Tg. Using molecular dynamics simulations of several dynamic and thermodynamic properties, this study reproduced the supplemented phase diagram of trehalose-water mixtures (i.e., Tg as a function of the solution composition) based on experimental data. The structure and dynamics of the hydrogen-bonding network in the trehalose-water systems were also analyzed. The hydrogen-bonding lifetime was determined to be an order of magnitude higher in the glassy state than in the liquid state, while the constitution of the hydrogen-bonding network exhibited no noticeable change through the glass transition. It was also found that trehalose molecules preferred to form small, scattered clusters above Tg, but self-aggregation was substantially increased below Tg. The average cluster size in the glassy state was observed to be dependent on the trehalose concentration. Our findings provided insights into the glass transition characteristics of aqueous trehalose solutions as they relate to biopreservation.

Effect of choline carboxylate ionic liquids on biological membranes

Choline carboxylates, ChCm, with m=2-10 and choline oleate are known as biocompatible substances, yet their influence on biological membranes is not well-known, and the effect on human skin has not previously been investigated. The short chain choline carboxylates ChCm with m=2, 4, 6 act as hydrotropes, solubilizing hydrophobic compounds in aqueous solution, while the longer chain choline carboxylates ChCm with m=8, 10 and oleate are able to form micelles. In the present study, the cytotoxicity of choline carboxylates was tested using HeLa and SK-MEL-28 cells. The influence of these substances on liposomes prepared from dipalmitoylphosphatidylcholine (DPPC) was also evaluated to provide insights on membrane interactions. It was observed that the choline carboxylates with a chain length of m>8 distinctly influence the bilayer, while the shorter ones had minimal interaction with the liposomes.