Ensilicated tetanus antigen retains immunogenicity: in vivo study and time-resolved SAXS characterization.

Our recently developed ensilication approach can physically stabilize proteins in silica without use of a pre-formed particle matrix. Stabilisation is done by tailor fitting individual proteins with a silica coat using a modified sol-gel process. Biopharmaceuticals, e.g. liquid-formulated vaccines with adjuvants, frequently have poor thermal stability; heating and/or freezing impairs their potency. As a result, there is an increase in the prevalence of vaccine-preventable diseases in low-income countries even when there are means to combat them. One of the root causes lies in the problematic vaccine 'cold chain' distribution. We believe that ensilication can improve vaccine availability by enabling transportation without refrigeration. Here, we show that ensilication stabilizes tetanus toxin C fragment (TTCF), a component of the tetanus toxoid present in the diphtheria, tetanus and pertussis (DTP) vaccine. Experimental in vivo immunization data show that the ensilicated material can be stored, transported at ambient temperatures, and even heat-treated without compromising the immunogenic properties of TTCF. To further our understanding of the ensilication process and its protective effect on proteins, we have also studied the formation of TTCF-silica nanoparticles via time-resolved Small Angle X-ray Scattering (SAXS). Our results reveal ensilication to be a staged diffusion-limited cluster aggregation (DLCA) type reaction. An early stage (tens of seconds) in which individual proteins are coated with silica is followed by a subsequent stage (several minutes) in which the protein-containing silica nanoparticles aggregate into larger clusters. Our results suggest that we could utilize this technology for vaccines, therapeutics or other biopharmaceuticals that are not compatible with lyophilization.

Thermal resilience of ensilicated lysozyme via calorimetric and in vivo analysis.

Ensilication is a novel method of protein thermal stabilisation using silica. It uses a modified sol-gel process which tailor fits a protective silica shell around the solvent accessible protein surface. This, electrostatically attached, shell has been found to protect the protein against thermal influences and retains its native structure and function after release. Here, we report the calorimetric analysis of an ensilicated model protein, hen egg-white lysozyme (HEWL) under several ensilication conditions. DSC, TGA-DTA-MS, CD, were used to determine unfolding temperatures of native, released and ensilicated lysozyme to verify the thermal resilience of the ensilicated material. Our findings indicate that ensilication protects against thermal fluctuations even at low concentrations of silica used for ensilication. Secondly, the thermal stabilisation is comparable to lyophilisation, and in some cases is even greater than lyophilisation. Additionally, we performed a mouse in vivo study using lysozyme to demonstrate the antigenic retention over long-term storage. The results suggest that protein is confined within the ensilicated material, and thus is unable to unfold and denature but is still functional after long-term storage.

Self-assembly and surface behaviour of pure and mixed zwitterionic amphiphiles in a deep eutectic solvent.

Recent investigations have shown that deep eutectic solvents provide a suitable environment for self-organisation of biomolecules, in particular phospholipids and proteins. However, the solvation of complex lyophilic moieties by deep eutectic solvents still remains unclear. Here we explore the behaviour of zwitterionic surfactants in choline chloride:glycerol eutectic mixture. Dodecyl-2-(trimethylammonio)ethylphosphate and N-alkyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (alkyl = dodecyl, tetradecyl) surfactants were investigated by means of surface tension, X-ray reflectivity and small-angle neutron scattering. These surfactants were found to remain surface active and form globular micelles in deep eutectic solvents. Still, the surface behaviour of these species was found to differ depending on the headgroup and tail structure. The morphology of the micelles also slightly varies between surfactants, demonstrating differences in the packing of individual monomers. The characteristics of mixtures of the dodecyl surfactants is also reported, showing a deviation from ideal mixing associated with attractive interactions between sulfobetaine and phosphocholine headgroups. Such non-ideality results in variation of the surface behaviour and self-assembly of these surfactant mixtures. The results presented here will potentially lead to the development of new alternatives for drug-delivery, protein solubilisation and biosensing through a better fundamental understanding of the behaviour of zwitterionic surfactants in deep eutectic solvents.

Counterion binding alters surfactant self-assembly in deep eutectic solvents.

Micellisation of surfactants in deep eutectic solvents has been recently demonstrated to provide a controllable way to modify micelle morphology. Ion-pair interactions between the solvent and the surfactant headgroup were identified as affecting the micellisation by modifying the charge density of the micelle. Here we explore the micellisation of dodecylsulfate surfactants with different counterions (Li+, Cs+, Mg2+, Bmim+, Emim+, cholinium+) dissolved in two deep eutectic solvents: choline chloride:urea and choline chloride:glycerol. Surface tension results show a solvent and counterion dependence of the CMC of the surfactants. Small-angle neutron scattering was subsequently used to investigate the morphology of the micelles formed. The results show that the elongation of the micelles is strongly dependent on the solvent, showing more elongated aggregates in choline chloride:urea than in choline chloride:glycerol. The morphology of micelles in DES was also found to depend on the counterion, where the affinity of binding showed similarities to that in water.

Coarse-grained empirical potential structure refinement: Application to a reverse aqueous micelle.

Conventional atomistic computer simulations, involving perhaps up to 106atoms, can achieve length-scales on the order of a few 10s of nm. Yet many heterogeneous systems, such as colloids, nano-structured materials, or biological systems, can involve correlations over distances up 100s of nm, perhaps even 1µm in some instances. For such systems it is necessary to invoke coarse-graining, where single atoms are replaced by agglomerations of atoms, usually represented as spheres, in order for the simulation to be performed within a practical computer memory and time scale. Small angle scattering and reflectivity measurements, both X-ray and neutron, are routinely used to investigate structure in these systems, and traditionally the data have been interpreted in terms of discrete objects, such as spheres, sheets, and cylinders, and combinations thereof. Here we combine the coarse-grained computer simulation approach with neutron small angle scattering to refine the structure of a heterogeneous system, in the present case a reverse aqueous micelle of sodium-dioctyl sulfosuccinate (AOT) and iso-octane. The method closely follows empirical potential structure refinement and involves deriving an empirical interaction potential from the scattering data. As in traditional coarse-grained methods, individual atoms are replaced by spherical density profiles, which, unlike real atoms, can inter-penetrate to a significant extent. The method works over an arbitrary range of length-scales, but is limited to around 2 orders of magnitude in distance above a specified dimension. The smallest value for this dimension is of order 1nm, but the largest dimension is arbitrary. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.

Protein conformation in pure and hydrated deep eutectic solvents.

Deep eutectic solvents (DES) have recently been postulated as possible environments where protein structure may be preserved in the absence of water. Here we present our results towards understanding protein conformation in choline chloride-based DES and mixtures with water. Lysozyme and bovine serum albumin have been investigated by means of circular dichroism and small-angle neutron scattering.

Micelle structure in a deep eutectic solvent: a small-angle scattering study.

In recent years many studies into green solvents have been undertaken and deep eutectic solvents (DES) have emerged as sustainable and green alternatives to conventional solvents since they may be formed from cheap non-toxic organic precursors. In this study we examine amphiphile behaviour in these novel media to test our understanding of amphiphile self-assembly within environments that have an intermediate polarity between polar and non-polar extremes. We have built on our recently published results to present a more detailed structural characterisation of micelles of sodium dodecylsulfate (SDS) within the eutectic mixture of choline chloride and urea. Here we show that SDS adopts an unusual cylindrical aggregate morphology, unlike that seen in water and other polar solvents. A new morphology transition to shorter aggregates was found with increasing concentration. The self-assembly of SDS was also investigated in the presence of water; which promotes the formation of shorter aggregates.

Surfactant Behavior of Sodium Dodecylsulfate in Deep Eutectic Solvent Choline Chloride/Urea.

Deep eutectic solvents (DES) resemble ionic liquids but are formed from an ionic mixture instead of being a single ionic compound. Here we present some results that demonstrate that surfactant sodium dodecyl sulfate (SDS) remains surface-active and shows self-assembly phenomena in the most commonly studied DES, choline chloride/urea. X-ray reflectivity (XRR) and small angle neutron scattering (SANS) suggest that the behavior is significantly different from that in water. Our SANS data supports our determination of the critical micelle concentration using surface-tension measurements and suggests that the micelles formed in DES do not have the same shape and size as those seen in water. Reflectivity measurements have also demonstrated that the surfactants remain surface-active below this concentration.

Multiple thin film formation from dilute mixtures of polyethyleneimine (PEI) and cetyltrimethylammonium bromide (CTAB).

Dilute mixtures of the water soluble polymer polyethyleneimine (PEI) and the cationic surfactant cetyltrimethylammonium bromide (CTAB) form mesostructured thin films at the air/solution interface. In this paper we show that these films form spontaneously, reaching an equilibrium composition. When the film is removed, a new solid film rapidly reforms, with a similar morphology when inspected by Brewster angle microscopy (BAM). Successive removal of films lead to a series of structurally similar films, until the amount of surfactant in solution approaches the lower limit of film forming concentration. The results obtained using surfactant-selective potentiometry suggest that the incipient polymer:surfactant aggregates are in a position to migrate to the surface rapidly after film removal, prior to mesostructure formation. X-ray reflectivity indicates that films formed at different PEI:CTAB compositions retain mesophase regular structures with the same d-spacing feature, equal to 52.2A. Grazing incidence diffraction measurements indicate that films are composed of small crystallites arranged in a random powder fashion, developing a rough surface morphology evidenced by BAM. The central finding is that PEI:CTAB films form when the amount of CTAB in solution is higher than a critical film formation concentration, very close to the critical aggregation concentration (CAC), allowing the formation of several equilibrated films from the same growing solution.