Thermoreversible gels of hollow silica nanorod dispersions.

Colloidal suspensions of anisotropic particles are ubiquitous in particle-based industries. Consequently, there is a need to quantify the effects of particle shape on equilibrium phases and kinetic state transitions, particularly at lower aspect ratios (L/D ≈ 1-10). We present a new, colloidal system comprised of hollow, octadecyl-coated silica rods with 40 nm diameter with controlled aspect ratio and thermoreversible short-range attractions. Rheology and dynamic light scattering measurements on suspensions of these hollow adhesive hard rods with nominal aspect ratio ≈3 suspended in tetradecane exhibit thermoreversible gelation without complicating effects of gravitational settling. Small angle neutron scattering measurements of the microstructure are analyzed to determine the effective strength of attraction in the form of Baxter sticky parameter. Quantitative agreement is found with simulation predictions of the thermoreversible gel transition as a function of volume fraction, further validating a universal state diagram and providing guidance for the effects of aspect ratio on gelation.

Surface-mediated spontaneous emulsification of the acylated peptide, semaglutide.

Acylated peptides composed of glucagon-like peptide-1 receptor agonists modified with a fatty acid side chain are an important class of therapeutics for type 2 diabetes and obesity but are susceptible to an unusual physical instability in the presence of hydrophobic surfaces, i.e., spontaneous emulsification, also known as ouzo formation in practice. In this work, light scattering, small-angle X-ray scattering, and circular dichroism measurements are used to characterize the physical properties of the semaglutide colloidal phase, including size distribution, shape, secondary structure, internal structure, and internal composition, as a function of solution physico-chemical conditions. The existence and size of the colloids formed are successfully predicted by a classical Rayleigh model, which identifies the parameters controlling their size and formation. Colloid formation is found to be catalyzed by hydrophobic surfaces, and formation rates are modeled as an autocatalytic reaction, enabling the formation of a master curve for various surfaces that elucidates the mechanism. Surfaces differ due to differences in surface wettability, which can be correlated with Hansen solubility parameters. This work provides insights into this unusual colloidal phenomenon and guides the peptide synthesis process and drug product formulation in the pharmaceutical industry.

Combined Effects of Pressure and Ionic Strength on Protein-Protein Interactions: An Empirical Approach.

Proteins are exposed to hydrostatic pressure (HP) in a variety of ecosystems as well as in processing steps such as freeze-thaw, cell disruption, sterilization, and homogenization, yet pressure effects on protein-protein interactions (PPIs) remain underexplored. With the goal of contributing toward the expanded use of HP as a fundamental control parameter in protein research, processing, and engineering, small-angle X-ray scattering was used to examine the effects of HP and ionic strength on ovalbumin, a model protein. Based on an extensive data set, we develop an empirical method for scaling PPIs to a master curve by combining HP and osmotic effects. We define an effective pressure parameter that has been shown to successfully apply to other model protein data available in the literature, with deviations evident for proteins that do not follow the apparent Hofmeister series. The limitations of the empirical scaling are discussed in the context of the hypothesized underlying mechanisms.

Hydrogen-Deuterium Exchange Dynamics of NISTmAb Measured by Small Angle Neutron Scattering.

Understanding protein dynamics and conformational stability holds great significance in biopharmaceutical research. Hydrogen-deuterium exchange (HDX) is a quantitative methodology used to examine these fundamental properties of proteins. HDX involves measuring the exchange of solvent-accessible hydrogens with deuterium, which yields valuable insights into conformational fluctuations and conformational stability. While mass spectrometry is commonly used to measure HDX on the peptide level, we explore a different approach using small-angle neutron scattering (SANS). In this work, SANS is demonstrated as a complementary and noninvasive HDX method (HDX-SANS). By assessing subtle changes in the tertiary and quaternary structure during the exchange process in deuterated buffer, along with the influence of added electrolytes on protein stability, SANS is validated as a complementary HDX technique. The HDX of a model therapeutic antibody, NISTmAb, an IgG1κ, is monitored by HDX-SANS over many hours using several different formulations, including salts from the Hofmeister series of anions, such as sodium perchlorate, sodium thiocyanate, and sodium sulfate. The impact of these formulation conditions on the thermal stability of NISTmAb is probed by differential scanning calorimetry. The more destabilizing salts led to heightened conformational dynamics in mAb solutions even at temperatures significantly below the denaturation point. HDX-SANS is demonstrated as a sensitive and noninvasive technique for quantifying HDX kinetics directly in mAb solution, providing novel information about mAb conformational fluctuations. Therefore, HDX-SANS holds promise as a potential tool for assessing protein stability in formulation.

Interfacial Pressure and Viscoelasticity of Antibodies and Their Correlation to Long-Term Stability in Formulation.

Monoclonal antibodies (mAbs) form viscoelastic gel-like layers at the air-water interface due to their amphiphilic nature, and this same protein characteristic can lead to undesired aggregation of proteins in therapeutic formulations. We hypothesize that the interfacial viscoelasticity and surface pressure of mAbs at the air-water interface will correlate with their long-term stability. To test this hypothesis, the interfacial viscoelastic rheology and surface pressure of five different antibodies with varying visible particle counts from a three-year stability study were measured. We find that both the surface pressures and interfacial elastic moduli correlate well with the long-time mAb solution stability within a class of mAbs with the interfacial elastic moduli being particularly sensitive to discriminate between stable and unstable mAbs across a range of formulations. Furthermore, X-ray reflectivity was used to gain insight into the interfacial structure of mAbs at the air-water interface, providing a possible molecular mechanism to explain the relationship between interfacial elastic moduli and the long-term stability.

Micelle Formation and Phase Separation of Poloxamer 188 and Preservative Molecules in Aqueous Solutions Studied by Small Angle X-ray Scattering.

Multi-injection pharmaceutical products such as insulin must be formulated to prevent aggregation and microbial contamination. Small-molecule preservatives and nonionic surfactants such as poloxamer 188 (P188) are thus often employed in protein drug formulations. However, mixtures of preservatives and surfactants can induce aggregation and even phase separation over time, despite the fact that all components are well dissolvable when used alone in aqueous solution. A systematic study is conducted here to understand the phase behavior and morphological causes of aggregation of P188 in the presence of the preservatives phenol and benzyl alcohol, primarily using small-angle x-ray scattering (SAXS). Based on SAXS results, P188 remains as unimers in solution when below a certain phenol concentration. Upon increasing the phenol concentration, a regime of micelle formation is observed due to the interaction between P188 and phenol. Further increasing the phenol concentration causes mixtures to become turbid and phase-separate over time. The effect of benzyl alcohol on the phase behavior is also investigated.

Instrument for measurement of interfacial structure-property relationships with decoupled interfacial shear and dilatational flow: "Quadrotrough".

Understanding the interfacial structure-property relationship of complex fluid-fluid interfaces is increasingly important for guiding the formulation of systems with targeted interfacial properties, such as those found in multiphase complex fluids, biological systems, biopharmaceuticals formulations, and many consumer products. Mixed interfacial flow fields, typical of classical Langmuir trough experiments, introduce a complex interfacial flow history that complicates the study of interfacial properties of complex fluid interfaces. In this article, we describe the design, implementation, and validation of a new instrument capable of independent application of controlled interfacial dilation and shear kinematics on fluid interfaces. Combining the Quadrotrough with both in situ Brewster angle microscopy and neutron reflectometry provides detailed structural measurements of the interface at the mesoscale and nanoscale in relationship to interfacial material properties under controlled interfacial deformation histories.

Anomalous rheological aging of a model thermoreversible colloidal gel following a thermal quench.

We investigate the aging behavior in a well-studied model system comprised of a colloidal suspension of thermoreversible adhesive hard spheres (AHS) but thermally quenched below the gel transition to much larger depths than previously studied. The aging behavior in the model AHS system is monitored by small amplitude oscillatory shear rheology measurements conducted while rapidly quenching from the liquid state at 40 °C to a temperature below the gel temperature, and new, anomalous aging behaviors are observed. Shallow quenches lead to monotonic development of the elastic modulus with time, consistent with prior reports for the development of a homogeneous gel [Gordon et al., J. Rheol. 61, 23-34 (2017)]. However, for deeper quenches, a unique and new phenomenon is reported, namely, after an initial rise in the modulus, a reproducible drop in the modulus is observed, followed by a plateau in the modulus value. This drop can be gradual or sudden and the extent of the drop depends on the quench depth. After this drop in the modulus, AHS gel evolves toward a quench-path independent state over the experimental timescale. These effects of the extent of quenching on aging behavior are hypothesized to be a consequence of quenching into different underlying thermodynamic states of colloidal gels and the possible influence of the adhesive glass dynamical arrest for the deepest quenches. The research connects homogeneous gelation with heterogeneous gel formation due to phase separation and shows that the extent of quench can be used as an independent parameter to govern the rheological response of the arrested gel.

A Thermodynamically Consistent, Microscopically-Based, Model of the Rheology of Aggregating Particles Suspensions.

In this work, we outline the development of a thermodynamically consistent microscopic model for a suspension of aggregating particles under arbitrary, inertia-less deformation. As a proof-of-concept, we show how the combination of a simplified population-balance-based description of the aggregating particle microstructure along with the use of the single-generator bracket description of nonequilibrium thermodynamics, which leads naturally to the formulation of the model equations. Notable elements of the model are a lognormal distribution for the aggregate size population, a population balance-based model of the aggregation and breakup processes and a conformation tensor-based viscoelastic description of the elastic network of the particle aggregates. The resulting example model is evaluated in steady and transient shear forces and elongational flows and shown to offer predictions that are consistent with observed rheological behavior of typical systems of aggregating particles. Additionally, an expression for the total entropy production is also provided that allows one to judge the thermodynamic consistency and to evaluate the importance of the various dissipative phenomena involved in given flow processes.

Microstructure of continuous shear thickening colloidal suspensions determined by rheo-VSANS and rheo-USANS.

Research on shear thickening colloidal suspensions demonstrates that measurements of the microstructure can elucidate the source of the rheological material properties in the shear thickened state as well as critically test simulations and theory based on a variety of mechanisms such as enhanced lubrication hydrodynamics, elastohydrodynamics, and contact friction. Prior work on continuous shear thickening dispersions with a well-defined shear thickened state identified the formation of hydroclusters as characteristic of this state, determined the anisotropy in the nearest neighbor distribution, and used this information to test prevailing theories and simulations. However, important questions remain about the mesoscale (i.e., particle cluster scale) microstructure of the shear thickened state. Here we employ neutron scattering methods applied to shearing colloidal dispersions of spherical particles with two extremes of friction and lubrication surface properties to resolve the longer-length scale microstructure in the shear thickened state. Hydroclusters are shown to be highly localized, in agreement with prior neutron scattering and direct optical measurements, but in disagreement with the most recent simulations that predict a longer-range structure formation. These results combined with prior measurements provide experimental evidence about the length scale of microstructure formation in continuous shear thickening suspensions necessary to improve our understanding of the phenomenon as well as guide theoretical investigations that quantitatively link nanoscale forces to macroscopic properties in the shear thickened state.

Aggregation Kinetics of Polysorbate 80/m-Cresol Solutions: A Small-Angle Neutron Scattering Study.

Polysorbate 80 (PS80), a nonionic surfactant used in pharmaceutical formulation, is known to be incompatible with m-cresol, an antimicrobial agent for multi-dose injectable formulations. This incompatibility results in increased turbidity caused by micelle aggregation progressing over weeks or longer, where storage temperature, ionic strength, and component concentration influence the aggregation kinetics. Small-angle neutron scattering (SANS) analysis of PS80/m-cresol solutions over a pharmaceutically relevant concentration range of each component reveals the cause of aggregation, the coalescence mechanism, and aggregate structure. PS80 solutions containing m-cresol concentrations below ≈2.0 mg/mL and above ≈4.5 mg/mL are kinetically stable and do not aggregate over a 50 h period. At 5 mg/mL of m-cresol, the mixture forms a kinetically stable microemulsion phase, despite being well below the aqueous solubility limit of m-cresol. Solutions containing intermediate m-cresol concentrations (2.0-4.5 mg/mL) are unstable, resulting in aggregation, coalescence, and eventual phase separation. In unstable solutions, two stages of aggregate growth (nucleation and power-law growth) are observed at m-cresol concentrations at or below ≈3.6 mg/mL. At higher m-cresol concentrations, aggregates experience a third stage of exponential growth. A single kinetic model is developed to explain the stages of aggregate growth observed in both kinetic mechanisms. This work establishes the phase diagram of PS80/m-cresol solution stability and identifies component concentrations necessary for producing stable formulations.

Direct Observation of COVID-19 Prevention Behaviors and Physical Activity in Public Open Spaces.

Mask wearing and physical distancing are effective at preventing COVID-19 transmission. Little is known about the practice of these behaviors during physical activity (PA). In this longitudinal study, direct observation was used to describe COVID-19 prevention behaviors among physically active individuals. The Viral Transmission Scan (VT-Scan) was used to assess COVID-19 prevention behaviors of people standing, sitting, walking, jogging, and cycling in educational, retail, and residential areas. The VT-Scan was performed once per week over 22 weeks between 11:00 a.m. and 2:30 p.m. Information was manually extracted from videos collected during VT-Scans. A total of 4153 people were described, of which 71.2% were physically active, 80.0% were 18-30 years of age, 14.0% were non-white, 61.0% were female, and were 19.6% obese. Individuals not engaged in PA were less compliant with COVID-19 prevention behaviors than physically active people. Compliance differed by PA type, with walkers less compliant with COVID-19 prevention behaviors than joggers and cyclists. Among those physically active, non-compliance with COVID-19 prevention behaviors was higher in 18-30-year-olds, whites, and men. Engagement in COVID-19 prevention behaviors varies as a function of PA. Efforts to promote compliance with recommendations may benefit from tailored messaging, taking into account PA participation, PA type, and characteristics of physically active individuals.

Design of PLGA-Based Drug Delivery Systems Using a Physically-Based Sustained Release Model.

An extensive data set has been developed and used to further the progress of a model-informed design of controlled drug release. An improved drug-release model with mechanistic modeling of hydrolytic polymer degradation is used and validated by comparing model predictions to in vitro experiments. Combining parameter estimates from the literature with model fits to the data set, this study can aid in achieving a priori design of controlled drug release from a model PLGA release system. A systematic series of model release systems were formulated with FITC-labeled dextran, as a surrogate for biopharmaceuticals, in PLGA rods over a broad range of compositions. While general comparisons between the model and experiments were favorable, important discrepancies were identified for several formulations with significant first-phase drug release. Supported by cross-sectional fluorescence microscopy images of the FITC-dextran distribution within the rods, this first-phase release was attributed to a combination of two main factors: (1) percolation of the drug particles and (2) swelling of and pore formation in the rods due to water uptake. These observations indicate the importance of careful selection of the PLGA polymer grade when designing drug release systems but also reflect a need for better understanding of phenomena such as pore formation. Adapting model parameters, without modifying the physical processes included in the model, enabled accurate fitting of the experimental data for all formulations, highlighting the applicability of the model.

Nanocrystalline protein domains via salting-out.

Protein salting-out is a well established phenomenon that in many cases leads to amorphous structures and protein gels, which are usually not considered to be useful for protein structure determination. Here, microstructural measurements of several different salted-out protein dense phases are reported, including of lysozyme, ribonuclease A and an IgG1, showing that salted-out protein gels unexpectedly contain highly ordered protein nanostructures that assemble hierarchically to create the gel. The nanocrystalline domains are approximately 10-100 nm in size, are shown to have structures commensurate with those of bulk crystals and grow on time scales in the order of an hour to a day. Beyond revealing the rich, hierarchical nanoscale to mesoscale structure of protein gels, the nanocrystals that these phases contain are candidates for structural biology on next-generation X-ray free-electron lasers, which may enable the study of biological macromolecules that are difficult or impossible to crystallize in bulk.

Recent advances in blood rheology: a review.

Due to the potential impact on the diagnosis and treatment of various cardiovascular diseases, work on the rheology of blood has significantly expanded in the last decade, both experimentally and theoretically. Experimentally, blood has been confirmed to demonstrate a variety of non-Newtonian rheological characteristics, including pseudoplasticity, viscoelasticity, and thixotropy. New rheological experiments and the development of more controlled experimental protocols on more extensive, broadly physiologically characterized, human blood samples demonstrate the sensitivity of aspects of hemorheology to several physiological factors. For example, at high shear rates the red blood cells elastically deform, imparting viscoelasticity, while at low shear rates, they form "rouleaux" structures that impart additional, thixotropic behavior. In addition to the advances in experimental methods and validated data sets, significant advances have also been made in both microscopic simulations and macroscopic, continuum, modeling, as well as novel, multiscale approaches. We outline and evaluate the most promising of these recent developments. Although we primarily focus on human blood rheology, we also discuss recent observations on variations observed across some animal species that provide some indication on evolutionary effects.

A Direct Observation Video Method for Describing COVID-19 Transmission Factors on a Micro-Geographical Scale: Viral Transmission (VT)-Scan.

The COVID-19 pandemic severely affected many aspects of human life. While most health agencies agree mask wearing and physical distancing reduce viral transmission, efforts to improve the assessment of these behaviors are lacking. This study aimed to develop a direct observation video method [Viral Transmission (VT)-Scan] for assessing COVID-19 transmission behaviors and related factors (e.g., environmental setting). A wearable video device (WVD) was used to obtain videos of outdoor, public areas. The videos were examined to extract relevant information. All outcomes displayed good to excellent intra- and inter-reliability with intra-class correlation coefficients ranging from 0.836 to 0.997. The majority of people had a mask (60.8%) but 22.1% of them wore it improperly, 45.4% were not physical distancing, and 27.6% were simultaneously mask and physical distancing non-compliant. Transmission behaviors varied by demographics with white, obese males least likely to be mask-compliant and white, obese females least likely to physical distance. Certain environments (e.g., crosswalks) were identified as "hot spots" where higher rates of adverse transmission behaviors occurred. This study introduces a reliable method for obtaining objective data on COVID-19 transmission behaviors and related factors which may be useful for agent-based modeling and policy formation.

A comparative study of blood rheology across species.

Recent advances in hemorheology are extended to study blood rheology across species, which has important clinical implications particularly in intravenous drug scaleup as drugs undergoing clinical trials are first tested in animals. Some of the first hemorheological measurements from seven different species under both steady and transient shear conditions are presented and modeled using a rheological model developed and validated on human blood rheology fit to 20 different donors. Despite similar physiological properties across the blood samples from different species, significant differences are observed, particularly at low shear rates. Blood from species that form rouleaux exhibit a yield-like behavior and enhanced viscoelasticity at low shear rates, while blood from species without rouleaux exhibit nearly Newtonian behavior at similar shear rates. Viscoelasticity due to blood cell deformation is evident for all species at high shear rates. Novel, unidirectional large amplitude oscillatory shear measurements differentiate species. Using the newly acquired data in combination with previous literature data, a new allometric scaling relation is suggested for the low-shear blood viscosity for various mammalian evolutionary orders. Using an established model for arterial branching across species, it is conjectured that the observed hemorheological scaling across species is driven by maintaining a constant wall shear stress in arterial vessels.

Preservative Induced Polysorbate 80 Micelle Aggregation.

Small angle neutron scattering (SANS) studies of a model pharmaceutical formulation reveal how formulation stability depends on the compatibility of individual components. Solutions of two common protein formulation excipients, polysorbate 80 (PS80), a nonionic surfactant that prevents aggregation, and m-cresol, an antimicrobial agent for multi-dose injectable formulations, are investigated. The addition of m-cresol to PS80 solutions leads to solution turbidity and irreversibly alters PS80 micelle morphology. This slow preservative-induced destabilization of PS80 micelles progresses over days or even weeks, which highlights the essential role that aggregation kinetics plays in preservative-surfactant interactions. The temperature-dependence of PS80 micelle growth kinetics is quantified by SANS in the presence of m-cresol. Aggregation is a two-step process, where initial formation of small aggregates is followed by a period of monotonic power-law growth, providing evidence for the mechanism. Total aggregate mass stays constant after initial aggregate formation, and addition of a pH-regulating citrate buffer dramatically accelerates aggregation kinetics.

Adsorption of non-ionic surfactant and monoclonal antibody on siliconized surface studied by neutron reflectometry.

The adsorption of monoclonal antibodies (mAbs) on hydrophobic surfaces is known to cause protein aggregation and degradation. Therefore, surfactants, such as Poloxamer 188, are widely used in therapeutic formulations to stabilize mAbs and protect mAbs from interacting with liquid-solid interfaces. Here, the adsorption of Poloxamer 188, one mAb and their competitive adsorption on a model hydrophobic siliconized surface is investigated with neutron scattering coupled with contrast variation to determine the molecular structure of adsorbed layers for each case. Small angle neutron scattering measurements of the affinity of Poloxamer 188 to this mAb indicate that there is negligible binding at these solution conditions. Neutron reflectometry measurements of the mAb show irreversible adsorption on the siliconized surface, which cannot be washed off with neat buffer. Poloxamer 188 can be adsorbed on the surface already occupied by mAb, which enables partial removal of some adsorbed mAb by washing with buffer. The adsorption of the surfactant introduces significant conformational changes for mAb molecules that remain on the surface. In contrast, if the siliconized surface is first saturated with the surfactant, no adsorption of mAb is observed. Competitive adsorption of mAb and Poloxamer 188 from solution leads to a surface dominantly occupied with surfactant molecules, whereas only a minor amount of mAb absorbs. These findings clearly indicate that Poloxamer 188 can protect against mAb adsorption as well as modify the adsorbed conformation of previously adsorbed mAb.

Structural and rheological aging in model attraction-driven glasses by Rheo-SANS.

Aging in a model colloidal suspension comprised of particles with a thermoreversible attraction is studied using Rheo-SANS techniques in the attractive-driven glass state. Multiple thermal pathways lead to a common rheological and microstructural aging trajectory, as was observed previously for a thermoreversible gel. SANS measurements of the colloidal glass microstructure as a function of temperature and time during various quench protocols are quantitatively characterized in terms of an effective interaction strength that becomes an order parameter defining the microstructural state of the glass. Using previously validated concepts of a fictive temperature, a semi-empirical, quantitative relationship similar to an Avrami relationship is established between the mechanical aging (elastic modulus) and microstructural aging (order parameter) that is independent of thermal history for the thermal profiles studied herein at long times. Furthermore, shear rejuvenation is studied, and while shear may only partially reduce the degree of structure in the glass, aging upon flow cessation is found to follow a common trajectory when viewed in terms of the microstructural order parameter.