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.

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.

Bacterial wax synthesis.

Biological wax esters offer a sustainable, renewable and biodegradable alternative to many fossil fuel derived chemicals including plastics and paraffins. Many species of bacteria accumulate waxes with similar structure and properties to highly desirable animal and plant waxes such as Spermaceti and Jojoba oils, the use of which is limited by resource requirements, high cost and ethical concerns. While bacterial fermentations overcome these issues, a commercially viable bacterial wax production process would require high yields and renewable, affordable feedstock to make it economically competitive and environmentally beneficial. This review describes recent progress in wax ester generation in both wild type and genetically engineered bacteria, with a focus on comparing substrates and quantifying obtained waxes. The full breadth of wax accumulating species is discussed, with emphasis on species generating high yields and utilising renewable substrates. Key areas of the field that have, thus far, received limited attention are highlighted, such as waste stream valorisation, mixed microbial cultures and efficient wax extraction, as, until effectively addressed, these will slow progress in creating commercially viable wax production methods.

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.

Implementing a Pain Care Toolkit to Improve Patient Perception of Pain Care.

As a result of the Hospital Value-Based Purchasing (VBP) Program, hospitals are now faced with reimbursement for quality of care, including patient perception of care, as measured by the Hospital Compare Assessment of Health Plans Survey, which includes the patient experience with pain care. Through the implementation of a Pain Care Quality Toolkit, patient perception of pain care can be improved, thus increasing the possibility of maximum reimbursement through the VBP.