Thermally Robust Solvent-Free Liquid Polyplexes for Heat-Shock Protection and Long-Term Room Temperature Storage of Therapeutic Nucleic Acids.

Nucleic acid therapeutics have attracted recent attention as promising preventative solutions for a broad range of diseases. Nonviral delivery vectors, such as cationic polymers, improve the cellular uptake of nucleic acids without suffering the drawbacks of viral delivery vectors. However, these delivery systems are faced with a major challenge for worldwide deployment, as their poor thermal stability elicits the need for cold chain transportation. Here, we demonstrate a biomaterial strategy to drastically improve the thermal stability of DNA polyplexes. Importantly, we demonstrate long-term room temperature storage with a transfection efficiency maintained for at least 9 months. Additionally, extreme heat shock studies show retained luciferase expression after heat treatment at 70 °C. We therefore provide a proof of concept for a platform biotechnology that could provide long-term room temperature storage for temperature-sensitive nucleic acid therapeutics, eliminating the need for the cold chain, which in turn would reduce the cost of distributing life-saving therapeutics worldwide.

Diffusivelike Motions in a Solvent-Free Protein-Polymer Hybrid.

The interaction between proteins and hydration water stabilizes protein structure and promotes functional dynamics, with water translational motions enabling protein flexibility. Engineered solvent-free protein-polymer hybrids have been shown to preserve protein structure, function, and dynamics. Here, we used neutron scattering, protein and polymer perdeuteration, and molecular dynamics simulations to explore how a polymer dynamically replaces water. Even though relaxation rates and vibrational properties are strongly modified in polymer coated compared to hydrated proteins, liquidlike polymer dynamics appear to plasticize the conjugated protein in a qualitatively similar way as do hydration-water translational motions.

Solvent-free liquid avidin as a step toward cold chain elimination.

The temperature sensitivity of vaccines and therapeutic proteins forces the distribution of life-saving treatments to rely heavily on the temperature-controlled (usually 2-8°C) supply and distribution network known as the cold chain. Here, using avidin as a model, we demonstrate how surface engineering could significantly increase the thermal stability of therapeutic proteins. A combination of spectroscopic (Fourier transform infrared, circular dichroism, and ultraviolet-visible) and scattering techniques (dynamic light scattering, small-angle, and wide-angle X-ray scattering) were deployed to probe the activity, structure, and stability of the model protein. Temperature-dependent synchrotron radiation circular dichroism spectroscopy was used to demonstrate a significant increase in thermal stability, with a half denaturation temperature of 139.0°C and reversible unfolding with modified avidin returning to a 90% folded state when heated to temperatures below 100°C. Accelerated aging studies revealed that modified avidin retained its secondary structure after storage at 40°C for 56 days, equivalent to 160 days at 25°C. Furthermore, binding studies with multiple ligands revealed that the binding site remained functional after modification. As a result, this approach has potential as a storage technology for therapeutic proteins and the elimination of the cold chain, enabling the dissemination of life-saving vaccines worldwide.

Revealing the complexity of ionic liquid-protein interactions through a multi-technique investigation.

Ionic liquids offer exciting possibilities for biocatalysis as solvent properties provide rare opportunities for customizable, energy-efficient bioprocessing. Unfortunately, proteins and enzymes are generally unstable in ionic liquids and several attempts have been made to explain why; however, a comprehensive understanding of the ionic liquid-protein interactions remains elusive. Here, we present an analytical framework (circular dichroism (CD), fluorescence, ultraviolet-visible (UV/Vis) and nuclear magnetic resonance (NMR) spectroscopies, and small-angle X-ray scattering (SAXS)) to probe the interactions, structure, and stability of a model protein (green fluorescent protein (GFP)) in a range (acetate, chloride, triflate) of pyrrolidinium and imidazolium salts. We demonstrate that measuring protein stability requires a similar holistic analytical framework, as opposed to single-technique assessments that provide misleading conclusions. We reveal information on site-specific ionic liquid-protein interactions, revealing that triflate (the least interacting anion) induces a contraction in the protein size that reduces the barrier to unfolding. Robust frameworks such as this are critical to advancing non-aqueous biocatalysis and avoiding pitfalls associated with single-technique investigations.

Thermally robust solvent-free biofluids of M13 bacteriophage engineered for high compatibility with anhydrous ionic liquids.

Here, we demonstrate a chemical modification strategy to create biomaterials of the M13 bacteriophage with extraordinary thermal stability, and high compatibility with non-aqueous ionic liquids. The results provide a blueprint for developing soft materials with well-defined architectures that may find broad applicability in the next generation of flexible devices.

Non-aqueous homogenous biocatalytic conversion of polysaccharides in ionic liquids using chemically modified glucosidase.

The increasing requirement to produce platform chemicals and fuels from renewable sources means advances in biocatalysis are rapidly becoming a necessity. Biomass is widely used in nature as a source of energy and as chemical building blocks. However, recalcitrance towards traditional chemical processes and solvents provides a significant barrier to widespread utility. Here, by optimizing enzyme solubility in ionic liquids, we have discovered solvent-induced substrate promiscuity of glucosidase, demonstrating an unprecedented example of homogeneous enzyme bioprocessing of cellulose. Specifically, chemical modification of glucosidase for solubilization in ionic liquids can increase thermal stability to up to 137 °C, allowing for enzymatic activity 30 times greater than is possible in aqueous media. These results establish that through a synergistic combination of chemical biology (enzyme modification) and reaction engineering (solvent choice), the biocatalytic capability of enzymes can be intensified: a key step towards the full-scale deployment of industrial biocatalysis.

Solubilizing and Stabilizing Proteins in Anhydrous Ionic Liquids through Formation of Protein-Polymer Surfactant Nanoconstructs.

Nonaqueous biocatalysis is rapidly becoming a desirable tool for chemical and fuel synthesis in both the laboratory and industry. Similarly, ionic liquids are increasingly popular anhydrous reaction media for a number of industrial processes. Consequently, the use of enzymes in ionic liquids as efficient, environment-friendly, commercial biocatalysts is highly attractive. However, issues surrounding the poor solubility and low stability of enzymes in truly anhydrous media remain a significant challenge. Here, we demonstrate for the first time that engineering the surface of a protein to yield protein-polymer surfactant nanoconstructs allows for dissolution of dry protein into dry ionic liquids. Using myoglobin as a model protein, we show that this method can deliver protein molecules with near native structure into both hydrophilic and hydrophobic anhydrous ionic liquids. Remarkably, using temperature-dependent synchrotron radiation circular dichroism spectroscopy to measure half-denaturation temperatures, our results show that protein stability increases by 55 °C in the ionic liquid as compared to aqueous solution, pushing the solution thermal denaturation beyond the boiling point of water. Therefore, the work presented herein could provide a platform for the realization of biocatalysis at high temperatures or in anhydrous solvent systems.

Molecular dynamics simulations reveal a dielectric-responsive coronal structure in protein-polymer surfactant hybrid nanoconstructs.

Solvent-free liquid proteins are a new class of thermally stable hybrid bionanomaterials that are produced by extensive lyophilization of aqueous solutions of protein-polymer surfactant nanoconjugates followed by thermal annealing. The hybrid constructs, which consist of a globular protein core surrounded by a monolayer of electrostatically coupled polymer surfactant molecules, exhibit nativelike structure, function, and backbone dynamics over a large temperature range. Despite the key importance of the polymer surfactant shell, very little is known about the atomistic structure of the corona and how it influences the phase behavior and properties of these novel nanoscale objects. Here we present molecular dynamics simulations of protein-polymer surfactant nanoconjugates consisting of globular cores of myoglobin or lysozyme and demonstrate that the derived structural parameters are highly consistent with experimental values. We show that the coronal layer structure is responsive to the dielectric constant of the medium and that the mobility of the polymer surfactant molecules is significantly hindered in the solvent-free state, providing a basis for the origins of retained protein dynamics in these novel biofluids. Taken together, our results suggest that the extension of molecular dynamics simulations to hybrid nanoscale objects could be of generic value in diverse areas of soft matter chemistry, bioinspired engineering, and biomolecular nanotechnology.

Enzyme activity in liquid lipase melts as a step towards solvent-free biology at 150 °C.

Water molecules play a number of critical roles in enzyme catalysis, including mass transfer of substrates and products, nucleophilicity and proton transfer at the active site, and solvent shell-mediated dynamics for accessing catalytically competent conformations. The pervasiveness of water in enzymolysis therefore raises the question concerning whether biocatalysis can be undertaken in the absence of a protein hydration shell. Lipase-mediated catalysis has been undertaken with reagent-based solvents and lyophilized powders, but there are no examples of molecularly dispersed enzymes that catalyse reactions at sub-solvation levels within solvent-free melts. Here we describe the synthesis, properties and enzyme activity of self-contained reactive biofluids based on solvent-free melts of lipase-polymer surfactant nanoconjugates. Desiccated substrates in liquid (p-nitrophenyl butyrate) or solid (p-nitrophenyl palmitate) form can be mixed or solubilized, respectively, into the enzyme biofluids, and hydrolysed in the solvent-free state. Significantly, the efficiency of product formation increases as the temperature is raised to 150 °C.

Self-organization of glucose oxidase-polymer surfactant nanoconstructs in solvent-free soft solids and liquids.

An anisotropic glucose oxidase-polymer surfactant nanoconjugate is synthesized and shown to exhibit complex temperature-dependent phase behavior in the solvent-free state. At close to room temperature, the nanoconjugate crystallizes as a mesolamellar soft solid with an expanded interlayer spacing of ca. 12 nm and interchain correlation lengths consistent with alkyl tail-tail and PEO-PEO ordering. The soft solid displays a birefringent spherulitic texture and melts at 40 °C to produce a solvent-free liquid protein without loss of enzyme secondary structure. The nanoconjugate melt exhibits a birefringent dendritic texture below the conformation transition temperature (Tc) of glucose oxidase (58 °C) and retains interchain PEO-PEO ordering. Our results indicate that the shape anisotropy of the protein-polymer surfactant globular building block plays a key role in directing mesolamellar formation in the solvent-free solid and suggests that the microstructure observed in the solvent-free liquid protein below Tc is associated with restrictions in the intramolecular motions of the protein core of the nanoconjugate.

Redox transitions in an electrolyte-free myoglobin fluid.

Redox responses associated with the heme prosthetic group in a myoglobin-polymer surfactant solvent-free liquid are investigated for the first time in the absence of an electrolyte solution. Cyclic voltammograms from the biofluid exhibit responses that are consistent with planar diffusion of mobile charges in the melt. Temperature-dependent dynamic electrochemical and rheological responses are rationalized in terms of the effective electron hopping rate between heme centers and the transport of intrinsic ionic species in the viscous protein liquid.

Isolation of a highly reactive ß-sheet-rich intermediate of lysozyme in a solvent-free liquid phase.

The thermal denaturation of solvent-free liquid lysozyme at temperatures in excess of 200 °C was studied by synchrotron radiation circular dichroism spectroscopy. Temperature-dependent changes in the secondary structure were used to map the equilibrium denaturation pathway and characterize a reactive ß-sheet-rich unfolding intermediate that was stable in the solvent-free liquid phase under anhydrous conditions but which underwent irreversible aggregation in the presence of water. The unfolding intermediate had a transition temperature of 78 °C and was extremely stable to temperature, eventually reaching the fully denatured state at 178 °C. We propose that the three-stage denaturation pathway arises from the decreased stability of the native state due to the absence of any appreciable hydrophobic effect, along with an entropically derived stabilization of the reactive intermediate associated with molecular crowding in the solvent-free liquid.

A polymer surfactant corona dynamically replaces water in solvent-free protein liquids and ensures macromolecular flexibility and activity.

The observation of biological activity in solvent-free protein-polymer surfactant hybrids challenges the view of aqueous and nonaqueous solvents being unique promoters of protein dynamics linked to function. Here, we combine elastic incoherent neutron scattering and specific deuterium labeling to separately study protein and polymer motions in solvent-free hybrids. Myoglobin motions within the hybrid are found to closely resemble those of a hydrated protein, and motions of the polymer surfactant coating are similar to those of the hydration water, leading to the conclusion that the polymer surfactant coating plasticizes protein structures in a way similar to hydration water.

Reversible dioxygen binding in solvent-free liquid myoglobin.

The ensemble of forces that stabilize protein structure and facilitate biological function are intimately linked with the ubiquitous aqueous environment of living systems. As a consequence, biomolecular activity is highly sensitive to the interplay of solvent-protein interactions, and deviation from the native conditions, for example by exposure to increased thermal energy or severe dehydration, results in denaturation and subsequent loss of function. Although certain enzymes can be extracted into non-aqueous solvents without significant loss of activity, there are no known examples of solvent-less (molten) liquids of functional metalloproteins. Here we describe the synthesis and properties of room-temperature solvent-free myoglobin liquids with near-native structure and reversible dioxygen binding ability equivalent to the haem protein under physiological conditions. The realization of room-temperature solvent-free myoglobin liquids with retained function presents novel challenges to existing theories on the role of solvent molecules in structural biology, and should offer new opportunities in protein-based nanoscience and bionanotechnology.