Unveiling the Rational Development of Stimuli-Responsive Silk Fibroin-Based Ionogel Formulations.

We present an approach for the rational development of stimuli-responsive ionogels which can be formulated for precise control of multiple unique ionogel features and fill niche pharmaceutical applications. Ionogels are captivating materials, exhibiting self-healing characteristics, tunable mechanical and structural properties, high thermal stability, and electroconductivity. However, the majority of ionogels developed require complex chemistry, exhibit high viscosity, poor biocompatibility, and low biodegradability. In our work, we overcome these limitations. We employ a facile production process and strategically integrate silk fibroin, the biocompatible ionic liquids (ILs) choline acetate ([Cho][OAc]), choline dihydrogen phosphate ([Cho][DHP]), and choline chloride ([Cho][Cl]), traditional pharmaceutical excipients, and the model antiepileptic drug phenobarbital. In the absence of ILs, we failed to observe gel formation; yet in the presence of ILs, thermoresponsive ionogels formed. Systems were assessed via visual tests, transmission electron microscopy, confocal reflection microscopy, dynamic light scattering, zeta potential and rheology measurements. We formed diverse ionogels of strengths ranging between 18 and 642 Pa. Under 25 °C storage, formulations containing polyvinylpyrrolidone (PVP) showed an ionogel formation period ranging over 14 days, increasing in the order of [Cho][DHP], [Cho][OAc], and [Cho][Cl]. Formulations lacking PVP showed an ionogel formation period ranging over 32 days, increasing in the order of [Cho][OAc], [Cho][DHP] and [Cho][Cl]. By heating from 25 to 60 °C, immediately following preparation, thermoresponsive ionogels formed below 41 °C in the absence of PVP. Based on our experimental results and density functional theory calculations, we attribute ionogel formation to macromolecular crowding and confinement effects, further enhanced upon PVP inclusion. Holistically, applying our rational development strategy enables the production of ionogels of tunable physicochemical and rheological properties, enhanced drug solubility, and structural and energetic stability. We believe our rational development approach will advance the design of biomaterials and smart platforms for diverse drug delivery applications.

Decreased Water Mobility Contributes To Increased α-Synuclein Aggregation.

The solvation shell is essential for the folding and function of proteins, but how it contributes to protein misfolding and aggregation has still to be elucidated. We show that the mobility of solvation shell H2 O molecules influences the aggregation rate of the amyloid protein α-synuclein (αSyn), a protein associated with Parkinson's disease. When the mobility of H2 O within the solvation shell is reduced by the presence of NaCl, αSyn aggregation rate increases. Conversely, in the presence CsI the mobility of the solvation shell is increased and αSyn aggregation is reduced. Changing the solvent from H2 O to D2 O leads to increased aggregation rates, indicating a solvent driven effect. We show the increased aggregation rate is not directly due to a change in the structural conformations of αSyn, it is also influenced by a reduction in both the H2 O mobility and αSyn mobility. We propose that reduced mobility of αSyn contributes to increased aggregation by promoting intermolecular interactions.

Decreased Water Mobility Contributes To Increased α-Synuclein Aggregation.

The solvation shell is essential for the folding and function of proteins, but how it contributes to protein misfolding and aggregation has still to be elucidated. We show that the mobility of solvation shell H2O molecules influences the aggregation rate of the amyloid protein α-synuclein (αSyn), a protein associated with Parkinson's disease. When the mobility of H2O within the solvation shell is reduced by the presence of NaCl, αSyn aggregation rate increases. Conversely, in the presence CsI the mobility of the solvation shell is increased and αSyn aggregation is reduced. Changing the solvent from H2O to D2O leads to increased aggregation rates, indicating a solvent driven effect. We show the increased aggregation rate is not directly due to a change in the structural conformations of αSyn, it is also influenced by a reduction in both the H2O mobility and αSyn mobility. We propose that reduced mobility of αSyn contributes to increased aggregation by promoting intermolecular interactions.

Ionic Liquid-Based Strategy for Predicting Protein Aggregation Propensity and Thermodynamic Stability.

Novel drug candidates are continuously being developed to combat the most life-threatening diseases; however, many promising protein therapeutics are dropped from the pipeline. During biological and industrial processes, protein therapeutics are exposed to various stresses such as fluctuations in temperature, solvent pH, and ionic strength. These can lead to enhanced protein aggregation propensity, one of the greatest challenges in drug development. Recently, ionic liquids (ILs), in particular, biocompatible choline chloride ([Cho]Cl)-based ILs, have been used to hinder stress-induced protein conformational changes. Herein, we develop an IL-based strategy to predict protein aggregation propensity and thermodynamic stability. We examine three key variables influencing protein misfolding: pH, ionic strength, and temperature. Using dynamic light scattering, zeta potential, and variable temperature circular dichroism measurements, we systematically evaluate the structural, thermal, and thermodynamic stability of fresh immunoglobin G4 (IgG4) antibody in water and 10, 30, and 50 wt % [Cho]Cl. Additionally, we conduct molecular dynamics simulations to examine IgG4 aggregation propensity in each system and the relative favorability of different [Cho]Cl-IgG4 packing interactions. We re-evaluate each system following 365 days of storage at 4 °C and demonstrate how to predict the thermodynamic properties and protein aggregation propensity over extended storage, even under stress conditions. We find that increasing [Cho]Cl concentration reduced IgG4 aggregation propensity both fresh and following 365 days of storage and demonstrate the potential of using our predictive IL-based strategy and formulations to radically increase protein stability and storage.

An experimental approach probing the conformational transitions and energy landscape of antibodies: a glimmer of hope for reviving lost therapeutic candidates using ionic liquid.

Understanding protein folding in different environmental conditions is fundamentally important for predicting protein structures and developing innovative antibody formulations. While the thermodynamics and kinetics of folding and unfolding have been extensively studied by computational methods, experimental methods for determining antibody conformational transition pathways are lacking. Motivated to fill this gap, we prepared a series of unique formulations containing a high concentration of a chimeric immunoglobin G4 (IgG4) antibody with different excipients in the presence and absence of the ionic liquid (IL) choline dihydrogen phosphate. We determined the effects of different excipients and IL on protein thermal and structural stability by performing variable temperature circular dichroism and bio-layer interferometry analyses. To further rationalise the observations of conformational changes with temperature, we carried out molecular dynamics simulations on a single antibody binding fragment from IgG4 in the different formulations, at low and high temperatures. We developed a methodology to study the conformational transitions and associated thermodynamics of biomolecules, and we showed IL-induced conformational transitions. We showed that the increased propensity for conformational change was driven by preferential binding of the dihydrogen phosphate anion to the antibody fragment. Finally, we found that a formulation containing IL with sugar, amino acids and surfactant is a promising candidate for stabilising proteins against conformational destabilisation and aggregation. We hope that ultimately, we can help in the quest to understand the molecular basis of the stability of antibodies and protein misfolding phenomena and offer new candidate formulations with the potential to revive lost therapeutic candidates.

Advancing predictions of protein stability in the solid state.

The ß-relaxation associated with the sub-glass transition temperature (Tg,ß) is attributed to fast, localised molecular motions which can occur below the primary glass transition temperature (Tg,α). Consistent with Tg,ß being observed well-below storage temperatures, the ß-relaxation associated motions have been hypothesised to influence protein stability in the solid state and could thus impact the quality of e.g. protein powders for inhalation or reconstitution and injection. Why then do distinct solid state protein formulations with similar aggregation profiles after drying and immediate reconstitution, display different profiles when reconstituted following prolonged storage? Is the value of Tg,ß, associated with the ß-relaxation process of the system, a reliable parameter for characterising the behaviour of proteins in the solid state? Bearing this in mind, in this work we further explore the different relaxation dynamics of glassy solid state monoclonal antibody formulations using terahertz time-domain spectroscopy and dynamical mechanical analysis. By conducting a 52 week stability study on a series of multi-component spray-dried formulations, an approach for characterising and analysing the solid state dynamics and how these relate to protein stability is outlined.

Exploring conformational preferences of proteins: ionic liquid effects on the energy landscape of avidin.

In this work we experimentally investigate solvent and temperature induced conformational transitions of proteins and examine the role of ion-protein interactions in determining the conformational preferences of avidin, a homotetrameric glycoprotein, in choline-based ionic liquid (IL) solutions. Avidin was modified by surface cationisation and the addition of anionic surfactants, and the structural, thermal, and conformational stabilities of native and modified avidin were examined using dynamic light scattering, differential scanning calorimetry, and thermogravimetric analysis experiments. The protein-surfactant nanoconjugates showed higher thermostability behaviour compared to unmodified avidin, demonstrating distinct conformational ensembles. Small-angle X-ray scattering data showed that with increasing IL concentration, avidin became more compact, interpreted in the context of molecular confinement. To experimentally determine the detailed effects of IL on the energy landscape of avidin, differential scanning fluorimetry and variable temperature circular dichroism spectroscopy were performed. We show that different IL solutions can influence avidin conformation and thermal stability, and we provide insight into the effects of ILs on the folding pathways and thermodynamics of proteins. To further study the effects of ILs on avidin binding and correlate thermostability with conformational heterogeneity, we conducted a binding study. We found the ILs examined inhibited ligand binding in native avidin while enhancing binding in the modified protein, indicating ILs can influence the conformational stability of the distinct proteins differently. Significantly, this work presents a systematic strategy to explore protein conformational space and experimentally detect and characterise 'invisible' rare conformations using ILs.

Observation of high-temperature macromolecular confinement in lyophilised protein formulations using terahertz spectroscopy.

Characterising the structural dynamics of proteins and the effects of excipients are critical for optimising the design of formulations. In this work we investigated four lyophilised formulations containing bovine serum albumin (BSA) and three formulations containing a monoclonal antibody (mAb, here mAb1), and explored the role of the excipients polysorbate 80, sucrose, trehalose, and arginine on stabilising proteins. By performing temperature variable terahertz time-domain spectroscopy (THz-TDS) experiments it is possible to study the vibrational dynamics of these formulations. The THz-TDS measurements reveal two distinct glass transition processes in all tested formulations. The lower temperature transition, T g , ß , is associated with the onset of local motion due to the secondary relaxation whilst the higher temperature transition, T g , α , marks the onset of the α -relaxation. For some of the formulations, containing globular BSA as well as mAb1, the absorption at terahertz frequencies does not increase further at temperatures above T g , α . Such behaviour is in contrast to our previous observations for small organic molecules as well as linear polymers where absorption is always observed to steadily increase with temperature due to the stronger absorption of terahertz radiation by more mobile dipoles. The absence of such further increase in absorption with higher temperatures therefore suggests a localised confinement of the protein/excipient matrix at high temperatures that hinders any further increase in mobility. We found that subtle changes in excipient composition had an effect on the transition temperatures T g , α and T g , ß as well as the vibrational confinement in the solid state. Further work is required to establish the potential significance of the vibrational confinement in the solid state on formulation stability and chemical degradation as well as what role the excipients play in achieving such confinement.

Terahertz Spectroscopy: An Investigation of the Structural Dynamics of Freeze-Dried Poly Lactic-co-glycolic Acid Microspheres.

Biodegradable poly lactic-co-glycolic acid (PLGA) microspheres can be used to encapsulate peptide and offer a promising drug-delivery vehicle. In this work we investigate the dynamics of PLGA microspheres prepared by freeze-drying and the molecular mobility at lower temperatures leading to the glass transition temperature, using temperature-variable terahertz time-domain spectroscopy (THz-TDS) experiments. The microspheres were prepared using a water-in-oil-in-water (w/o/w) double-emulsion technique and subsequent freeze-drying of the samples. Physical characterization was performed by morphology measurements, scanning electron microscopy, and helium pycnometry. The THz-TDS data show two distinct transition processes, T g , ß in the range of 167-219 K, associated with local motions, and T g , α in the range of 313-330 K, associated with large-scale motions, for the microspheres examined. Using Fourier transform infrared spectroscopy measurements in the mid-infrared, we were able to characterize the interactions between a model polypeptide, exendin-4, and the PLGA copolymer. We observe a relationship between the experimentally determined T g , ß and T g , α and free volume and microsphere dynamics.