Self-enhanced mobility enables vortex pattern formation in living matter.

Ranging from subcellular organelle biogenesis to embryo development, the formation of self-organized structures is a hallmark of living systems. Whereas the emergence of ordered spatial patterns in biology is often driven by intricate chemical signalling that coordinates cellular behaviour and differentiation1-4, purely physical interactions can drive the formation of regular biological patterns such as crystalline vortex arrays in suspensions of spermatozoa5 and bacteria6. Here we discovered a new route to self-organized pattern formation driven by physical interactions, which creates large-scale regular spatial structures with multiscale ordering. Specifically we found that dense bacterial living matter spontaneously developed a lattice of mesoscale, fast-spinning vortices; these vortices each consisted of around 104-105 motile bacterial cells and were arranged in space at greater than centimetre scale and with apparent hexagonal order, whereas individual cells in the vortices moved in coordinated directions with strong polar and vortical order. Single-cell tracking and numerical simulations suggest that the phenomenon is enabled by self-enhanced mobility in the system-that is, the speed of individual cells increasing with cell-generated collective stresses at a given cell density. Stress-induced mobility enhancement and fluidization is prevalent in dense living matter at various scales of length7-9. Our findings demonstrate that self-enhanced mobility offers a simple physical mechanism for pattern formation in living systems and, more generally, in other active matter systems10 near the boundary of fluid- and solid-like behaviours11-17.

Viscoelastic control of spatiotemporal order in bacterial active matter.

Active matter consists of units that generate mechanical work by consuming energy1. Examples include living systems (such as assemblies of bacteria2-5 and biological tissues6,7), biopolymers driven by molecular motors8-11 and suspensions of synthetic self-propelled particles12-14. A central goal is to understand and control the self-organization of active assemblies in space and time. Most active systems exhibit either spatial order mediated by interactions that coordinate the spatial structure and the motion of active agents12,14,15 or the temporal synchronization of individual oscillatory dynamics2. The simultaneous control of spatial and temporal organization is more challenging and generally requires complex interactions, such as reaction-diffusion hierarchies16 or genetically engineered cellular circuits2. Here we report a simple technique to simultaneously control the spatial and temporal self-organization of bacterial active matter. We confine dense active suspensions of Escherichia coli cells and manipulate a single macroscopic parameter-namely, the viscoelasticity of the suspending fluid- through the addition of purified genomic DNA. This reveals self-driven spatial and temporal organization in the form of a millimetre-scale rotating vortex with periodically oscillating global chirality of tunable frequency, reminiscent of a torsional pendulum. By combining experiments with an active-matter model, we explain this behaviour in terms of the interplay between active forcing and viscoelastic stress relaxation. Our findings provide insight into the influence of bacterial motile behaviour in complex fluids, which may be of interest in health- and ecology-related research, and demonstrate experimentally that rheological properties can be harnessed to control active-matter flows17,18. We envisage that our millimetre-scale, tunable, self-oscillating bacterial vortex may be coupled to actuation systems to act a 'clock generator' capable of providing timing signals for rhythmic locomotion of soft robots and for programmed microfluidic pumping19, for example, by triggering the action of a shift register in soft-robotic logic devices20.

Precision size and refractive index analysis of weakly scattering nanoparticles in polydispersions.

Characterization of the size and material properties of particles in liquid suspensions is in very high demand, for example, in the analysis of colloidal samples or of bodily fluids such as urine or blood plasma. However, existing methods are limited in their ability to decipher the constituents of realistic samples. Here we introduce iNTA as a new method that combines interferometric detection of scattering with nanoparticle tracking analysis to reach unprecedented sensitivity and precision in determining the size and refractive index distributions of nanoparticles in suspensions. After benchmarking iNTA with samples of colloidal gold, we present its remarkable ability to resolve the constituents of various multicomponent and polydisperse samples of known origin. Furthermore, we showcase the method by elucidating the refractive index and size distributions of extracellular vesicles from Leishmania parasites and human urine. The current performance of iNTA already enables advances in several important applications, but we also discuss possible improvements.

Eulerian simulation of complex suspensions and biolocomotion in three dimensions.

We present a numerical method specifically designed for simulating three-dimensional fluid-structure interaction (FSI) problems based on the reference map technique (RMT). The RMT is a fully Eulerian FSI numerical method that allows fluids and large-deformation elastic solids to be represented on a single fixed computational grid. This eliminates the need for meshing complex geometries typical in other FSI approaches and greatly simplifies the coupling between fluid and solids. We develop a three-dimensional implementation of the RMT, parallelized using the distributed memory paradigm, to simulate incompressible FSI with neo-Hookean solids. As part of our method, we develop a field extrapolation scheme that works efficiently in parallel. Through representative examples, we demonstrate the method's suitability in investigating many-body and active systems, as well as its accuracy and convergence. The examples include settling of a mixture of heavy and buoyant soft ellipsoids, lid-driven cavity flow containing a soft sphere, and swimmers actuated via active stress.

Rheology of debris flow materials is controlled by the distance from jamming.

Debris flows are dense and fast-moving complex suspensions of soil and water that threaten lives and infrastructure. Assessing the hazard potential of debris flows requires predicting yield and flow behavior. Reported measurements of rheology for debris flow slurries are highly variable and sometimes contradictory due to heterogeneity in particle composition and volume fraction ([Formula: see text]) and also inconsistent measurement methods. Here we examine the composition and flow behavior of source materials that formed the postwildfire debris flows in Montecito, CA, in 2018, for a wide range of [Formula: see text] that encapsulates debris flow formation by overland flow. We find that shear viscosity and yield stress are controlled by the distance from jamming, [Formula: see text], where the jamming fraction [Formula: see text] is a material parameter that depends on grain size polydispersity and friction. By rescaling shear and viscous stresses to account for these effects, the data collapse onto a simple nondimensional flow curve indicative of a Bingham plastic (viscoplastic) fluid. Given the highly nonlinear dependence of rheology on [Formula: see text], our findings suggest that determining the jamming fraction for natural materials will significantly improve flow models for geophysical suspensions such as hyperconcentrated flows and debris flows.

Assessing suspension and infectivity times of virus-loaded aerosols involved in airborne transmission.

Airborne transmission occurs through droplet-mediated transport of viruses following the expulsion of an aerosol by an infected host. Transmission efficiency results from the interplay between virus survival in the drying droplet and droplet suspension time in the air, controlled by the coupling between water evaporation and droplet sedimentation. Furthermore, droplets are made of a respiratory fluid and thus, display a complex composition consisting of water and nonvolatile solutes. Here, we quantify the impact of this complex composition on the different phenomena underlying transmission. Solutes lead to a nonideal thermodynamic behavior, which sets an equilibrium droplet size that is independent of relative humidity. In contrast, solutes do not significantly hinder transport due to their low initial concentration. Realistic suspension times are computed and increase with increasing relative humidity or decreasing temperature. By uncoupling drying and suspended stages, we observe that enveloped viruses may remain infectious for hours in dried droplets. However, their infectivity decreases with increasing relative humidity or temperature after dozens of minutes. Examining expelled droplet size distributions in the light of these results leads to distinguishing two aerosols. Most droplets measure between 0 and 40 µm and compose an aerosol that remains suspended for hours. Its transmission efficiency is controlled by infectivity, which decreases with increasing humidity and temperature. Larger droplets form an aerosol that only remains suspended for minutes but corresponds to a much larger volume and thus, viral load. Its transmission efficiency is controlled by droplet suspension time, which decreases with increasing humidity and decreasing temperature.

Tracking colloidal silica particles to evaluate their dispersion and interactions in concentrated suspensions under shear force applications.

This study aimed to characterize interactions within colloidal silica particles in their concentrated suspensions, using rheo-confocal measurements and imaging, followed by image analysis. We studied the effect of shear rate (0-500 s-1) and solution pH (6, 10) on the dispersion degree of colloidal silica particles via the determination and comparison of interparticle distances and their modeling. Images corresponding to different shear rates were analyzed to identify the coordinates of the particles. These coordinates were further analyzed to calculate the distance among the particles and then their surface-to-surface distance normalized by the particle diameter (H/D). It was found that the population of the particles per unit area of the image and H/D varied with increasing shear rate. The comparison between experimentally measured and theoretically calculated H/D identified that for some particles, the former was shorter than the latter, indicating the unexpected attractions among them against the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Then, the modification of previously reported equations for H/D was suggested and confirmed its validity. Assuming pair potential interaction and hydrodynamic interaction were the main non-DLVO interactions, their magnitudes were calculated and confirmed the significance of pH and shear application strength on particle dispersion/coagulation.

Calcipotriol Nanosuspension-Loaded Trilayer Dissolving Microneedle Patches for the Treatment of Psoriasis: In Vitro Delivery and In Vivo Antipsoriatic Activity Studies.

Psoriasis, affecting 2-3% of the global population, is a chronic inflammatory skin condition without a definitive cure. Current treatments focus on managing symptoms. Recognizing the need for innovative drug delivery methods to enhance patient adherence, this study explores a new approach using calcipotriol monohydrate (CPM), a primary topical treatment for psoriasis. Despite its effectiveness, CPM's therapeutic potential is often limited by factors like the greasiness of topical applications, poor skin permeability, low skin retention, and lack of controlled delivery. To overcome these challenges, the study introduces CPM in the form of nanosuspensions (NSs), characterized by an average particle size of 211 ± 2 nm. These CPM NSs are then incorporated into a trilayer dissolving microneedle patch (MAP) made from poly(vinylpyrrolidone) and w poly(vinyl alcohol) as needle arrays and prefrom 3D printed polylactic acid backing layer. This MAP features rapidly dissolving tips and exhibits good mechanical properties and insertion capability with delivery efficiency compared to the conventional Daivonex ointment. The effectiveness of this novel MAP was tested on Sprague-Dawley rats with imiquimod-induced psoriasis, demonstrating efficacy comparable to the marketed ointment. This innovative trilayer dissolving MAP represents a promising new local delivery system for calcipotriol, potentially revolutionizing psoriasis treatment by enhancing drug delivery and patient compliance.

Multi-population dissolution in confined active fluids.

Autonomous out-of-equilibrium agents or cells in suspension are ubiquitous in biology and engineering. Turning chemical energy into mechanical stress, they generate activity in their environment, which may trigger spontaneous large-scale dynamics. Often, these systems are composed of multiple populations that may reflect the coexistence of multiple species, differing phenotypes, or chemically varying agents in engineered settings. Here, we present a new method for modeling such multi-population active fluids subject to confinement. We use a continuum multi-scale mean-field approach to represent each phase by its first three orientational moments and couple their evolution with those of the suspending fluid. The resulting coupled system is solved using a parallel adaptive level-set-based solver for high computational efficiency and maximal flexibility in the confinement geometry. Motivated by recent experimental work, we employ our method to study the spatiotemporal dynamics of confined bacterial suspensions and swarms dominated by fluid hydrodynamic effects. Our in silico explorations reproduce observed emergent collective patterns, including features of active dissolution in two-population active-passive swarms, with results clearly suggesting that hydrodynamic effects dominate dissolution dynamics. Our work lays the foundation for a systematic characterization and study of collective phenomena in natural or synthetic multi-population systems such as bacteria colonies, bird flocks, fish schools, colloid swimmers, or programmable active matter.

Differentiating Microplastics from Natural Particles in Aqueous Suspensions Using Flow Cytometry with Machine Learning.

Microplastics (MPs) in natural waters are heterogeneously mixed with other natural particles including algal cells and suspended sediments. An easy-to-use and rapid method for directly measuring and distinguishing MPs from other naturally present colloids in the environment would expedite analytical workflows. Here, we established a database of MP scattering and fluorescence properties, either alone or in mixtures with natural particles, by stain-free flow cytometry. The resulting high-dimensional data were analyzed using machine learning approaches, either unsupervised (e.g., viSNE) or supervised (e.g., random forest algorithms). We assessed our approach in identifying and quantifying model MPs of diverse sizes, morphologies, and polymer compositions in various suspensions including phototrophic microorganisms, suspended biofilms, mineral particles, and sediment. We could precisely quantify MPs in microbial phototrophs and natural sediments with high organic carbon by both machine learning models (identification accuracies over 93%), although it was not possible to distinguish between different MP sizes or polymer compositions. By testing the resulting method in environmental samples through spiking MPs into freshwater samples, we further highlight the applicability of the method to be used as a rapid screening tool for MPs. Collectively, this workflow can be easily applied to a diverse set of samples to assess the presence of MPs in a time-efficient manner.

Edaravone Oral Suspension: A Neuroprotective Agent to Treat Amyotrophic Lateral Sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is characterized by loss of motor neurons due to degeneration of nerve cells within the brain and spinal cord. Early symptoms include limb weakness, twitching or muscle cramping, and slurred speech. As the disease progresses, difficulty breathing, swallowing, and paralysis can lead to death. Currently, there are no medications that cure ALS, and guidelines recommend treatments focused on symptom management. Intravenous (IV) edaravone was approved by the US Food and Drug Administration (FDA) in 2017 as a treatment to slow the progression of ALS. In May 2022, the FDA approved an oral suspension (ORS) formulation of edaravone. MECHANISM OF ACTION: The mechanism of action of edaravone is not well defined. However, its neuroprotective effects are thought to result from antioxidant properties occurring through elimination of free radicals. PHARMACOKINETICS: Edaravone ORS (105 mg) has a bioavailability of 57% when compared with edaravone IV (60 mg). The ORS should be taken on an empty stomach in the morning, with water and no food or beverages, for 1 hour. Edaravone is bound to albumin (92%), has a mean volume of distribution of 63.1 L, a half-life of 4.5-9 hours, and a total clearance of 35.9 L/h after intravenous administration. Edaravone is metabolized into nonactive sulfate and glucuronide conjugates. CLINICAL TRIALS: The FDA approval was based on studies of the pharmacokinetics, safety, tolerability, and bioavailability of edaravone ORS. A phase III, global, multicenter, open-label safety study was conducted on edaravone ORS in 185 patients with ALS over 48 weeks. The most reported treatment-emergent adverse events were falls, muscular weakness, and constipation. Serious treatment-emergent adverse events included disease worsening, dysphagia, dyspnea, and respiratory failure. THERAPEUTIC ADVANCE: Oral edaravone is an ALS treatment that can be self-administered or administered by a caregiver, precluding the need for administration by a health care professional in an institutional setting.

Viscosity of Suspensions of Strongly Bonded Spherical Particles of Nickel-Manganese-Cobalt Mixed Oxides (NMC) in Molten Poly(Ethylene Carbonate) for Batteries.

Ionically conductive polymers highly filled with active materials, such as metal oxides are increasingly studied for their potential use in all solid-state batteries. They offer the desirable processing ease of polymers for mass production despite interfacial issues that remain to be solved. In this study, it is shown that spherical particles of transition metal oxides can be introduced in co-polymers of alkene carbonate and ethylene oxide at loading close to the maximum packing fraction, without imparting the processability in the melt of the material. In particular, the viscosity does not show any yield stress and the increase of viscosity shows that the intrinsic viscosity of the filler does not match with the usual 2.5 value in the limit of the Einstein's equation. Conversely, rheological data show that the value is rather close to unity consistently with theoretical arguments that predicted that this scaling factor should be unity when particle rotation is precluded. In the present case, this behavior is attributed to strong bonding between polymer and filler that is proved by electronic microscopy and by dynamical mechanical spectroscopy showing a relaxation due to bound polymer.

Exploring operational boundaries for acoustic concentration of cell suspensions.

The development of a standardized, generic method for concentrating suspensions in continuous flow is challenging. In this study, we developed and tested a device capable of concentrating suspensions with an already high cell concentration to meet diverse industrial requirements. To address typical multitasking needs, we concentrated suspensions with high solid content under a variety of conditions. Cells from Saccharomyces cerevisiae, Escherichia coli, and Chinese hamster ovary cells were effectively focused in the center of the main channel of a microfluidic device using acoustophoresis. The main channel bifurcates into three outlets, allowing cells to exit through the central outlet, while the liquid evenly exits through all outlets. Consequently, the treatment separates cells from two-thirds of the surrounding liquid. We investigated the complex interactions between parameters. Increasing the channel depth results in a decrease in process efficiency, attributed to a decline in acoustic energy density. The study also revealed that different cell strains exhibit distinct acoustic contrast factors, originating from differences in dimensions, compressibility, and density values. Finally, a combination of high solid content and flow rate leads to an increase in diffusion through a phenomenon known as shear-induced diffusion. KEY POINTS: • Acoustic focusing in a microchannel was used to concentrate cell suspensions • The parameters influencing focusing at high concentrations were studied • Three different cell strains were successfully concentrated.

Enhancing Corneal Drug Penetration Using Penetratin for Ophthalmic Suspensions.

Eye drops, including solutions and suspensions, are essential dosage forms to treat ophthalmic diseases, with poorly water-soluble drugs typically formulated as ophthalmic suspensions. In addition to low bioavailability, suspensions exhibit limited efficacy, safety, and usability due to the presence of drug particles. Improving bioavailability can reduce the drug concentrations and the risk of problems associated with suspended drug particles. However, practical penetration enhancers capable of improving bioavailability remain elusive. Herein, we focused on penetratin (PNT), a cell-penetrating peptide (CPP) that promotes active cellular transport related to macromolecule uptake, such as micropinocytosis. According to the in vitro corneal uptake study using a reconstructed human corneal epithelial tissue model, LabCyte CORNEA-MODEL24, PNT enhanced the uptake of Fluoresbrite® YG carboxylate polystyrene microspheres without covalent binding. In an ex vivo porcine eye model, the addition of 10 µM PNT to rebamipide ophthalmic suspension markedly improved the corneal uptake of rebamipide; however, the addition of 100 µM PNT was ineffective due to potentially increased particle size by aggregation. This article provides basic information on the application of PNT as a penetration enhancer in ophthalmic suspensions, including the in vitro and ex vivo studies mentioned above, as well as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay and storage stability at different pH values.

An effective and efficient model of the near-field hydrodynamic interactions for active suspensions of bacteria.

Near-field hydrodynamic interactions in active fluids are essential to determine many important emergent behaviors observed, but have not been successfully modeled so far. In this work, we propose an effective model capturing the essence of the near-field hydrodynamic interactions through a tensorial coefficient of resistance, validated numerically by a pedagogic model system consisting of an Escherichia coli bacterium and a passive sphere. In a critical test case that studies the scattering angle of the bacterium-sphere pair dynamics, we prove that the near-field hydrodynamics can make a qualitative difference even for this simple two-body system: Calculations based on the proposed model reveal a region in parameter space where the bacterium is trapped by the passive sphere, a phenomenon that is regularly observed in experiments but cannot be explained by any existing model. In the end, we demonstrate that our model also leads to efficient simulation of active fluids with tens of thousands of bacteria, sufficiently large for investigations of many emergent behaviors.

Bacteria hinder large-scale transport and enhance small-scale mixing in time-periodic flows.

Understanding mixing and transport of passive scalars in active fluids is important to many natural (e.g., algal blooms) and industrial (e.g., biofuel, vaccine production) processes. Here, we study the mixing of a passive scalar (dye) in dilute suspensions of swimming Escherichia coli in experiments using a two-dimensional (2D) time-periodic flow and in a simple simulation. Results show that the presence of bacteria hinders large-scale transport and reduces overall mixing rate. Stretching fields, calculated from experimentally measured velocity fields, show that bacterial activity attenuates fluid stretching and lowers flow chaoticity. Simulations suggest that this attenuation may be attributed to a transient accumulation of bacteria along regions of high stretching. Spatial power spectra and correlation functions of dye-concentration fields show that the transport of scalar variance across scales is also hindered by bacterial activity, resulting in an increase in average size and lifetime of structures. On the other hand, at small scales, activity seems to enhance local mixing. One piece of evidence is that the probability distribution of the spatial concentration gradients is nearly symmetric with a vanishing skewness. Overall, our results show that the coupling between activity and flow can lead to nontrivial effects on mixing and transport.

Edge current and pairing order transition in chiral bacterial vortices.

Bacterial suspensions show turbulence-like spatiotemporal dynamics and vortices moving irregularly inside the suspensions. Understanding these ordered vortices is an ongoing challenge in active matter physics, and their application to the control of autonomous material transport will provide significant development in microfluidics. Despite the extensive studies, one of the key aspects of bacterial propulsion has remained elusive: The motion of bacteria is chiral, i.e., it breaks mirror symmetry. Therefore, the mechanism of control of macroscopic active turbulence by microscopic chirality is still poorly understood. Here, we report the selective stabilization of chiral rotational direction of bacterial vortices in achiral circular microwells sealed by an oil/water interface. The intrinsic chirality of bacterial swimming near the top and bottom interfaces generates chiral collective motions of bacteria at the lateral boundary of the microwell that are opposite in directions. These edge currents grow stronger as bacterial density increases, and, within different top and bottom interfaces, their competition leads to a global rotation of the bacterial suspension in a favored direction, breaking the mirror symmetry of the system. We further demonstrate that chiral edge current favors corotational configurations of interacting vortices, enhancing their ordering. The intrinsic chirality of bacteria is a key feature of the pairing order transition from active turbulence, and the geometric rule of pairing order transition may shed light on the strategy for designing chiral active matter.

Preparation and in Vitro Characterization of Fatty-Acid Modified Pirarubicin Nanosuspensions Stabilized by Albumin.

Pirarubicin (THP) shows more rapid intracellular uptake, more effective antitumor activity, and less cardiac toxicity, compared to doxorubicin. However, THP is distributed to both tumor and normal tissues indiscriminately. This study aimed to develop a nanosuspension to deliver THP to tumor tissues more efficiently. Fatty-acid-modified THPs (FA-THPs; octanoic acid, dodecanoic acid, palmitic acid-THPs) were synthesized to increase the hydrophobicity of THP. Nanosuspensions of these FA-THPs were then prepared using an antisolvent precipitation technique. Among the FA-THPs, the most efficiently drug-loaded nanosuspension was obtained from palmitic acid-THP (pal-THP) using an aqueous antisolvent containing bovine serum albumin as a stabilizer. The pal-THP nanoparticles in the nanosuspension were confirmed to be of optimal size (100-125 nm) for delivery to tumor tissues using dynamic light scattering and transmission electron microscopy. The pal-THP nanosuspension showed cytotoxicity in colon 26 cells. The nanosuspension was shown to disintegrate in the presence of surfactants such as lecithin, liberating pal-THP, which was converted to free THP in acidic media. It is therefore proposed that pal-THP nanoparticles that reach tumor cells after intravenous administration would exert antitumor effect by liberating pal-THP (i.e., disintegration of nanoparticles by the interaction with cell membrane), followed by the release of free THP in the acidic milieu of tumor cells. These findings indicate that FA-THP nanosuspensions, particularly pal-THP nanosuspension, hold promise as a candidate for cancer treatment. However, further in vivo studies are necessary.

Rapid Bacterial Detection and Gram-Identification Using Bacterially Activated, Macrophage-Membrane-Coated Nanowired-Si Surfaces in a Microfluidic Device.

Bacterially induced sepsis requires rapid bacterial detection and identification. Hours count for critically ill septic patients, while current culture-based detection requires at least 10 h up to several days. Here, we apply a microfluidic device equipped with a bacterially activated, macrophage-membrane-coating on nanowired-Si adsorbent surfaces for rapid, bacterial detection and Gram-identification in bacterially contaminated blood. Perfusion of suspensions of Gram-negative or Gram-positive bacteria through a microfluidic device equipped with membrane-coated adsorbent surfaces detected low (<10 CFU/mL) bacterial levels. Subsequent, in situ fluorescence-staining yielded Gram-identification for guiding antibiotic selection. In mixed Escherichia coli and Staphylococcus aureus suspensions, Gram-negative and Gram-positive bacteria were detected in the same ratios as those fixed in suspension. Results were validated with a 100% correct score by blinded evaluation (two observers) of 15 human blood samples, spiked with widely different bacterial strains or combinations of strains, demonstrating the potential of the platform for rapid (1.5 h in total) diagnosis of bacterial sepsis.

The effects of formulation on the pharmacokinetics of itraconazole and amiodarone in dogs after oral administration of a combination product, commercial products, and compounded products.

This study evaluated four different formulations of itraconazole and amiodarone. Formulation 1 was Vida's combination tablet containing both active pharmaceutical ingredients (APIs). Formulation 2 was separate, commercially available human generic capsules and tablets of itraconazole and amiodarone, respectively. Formulation 3 was separate, compounded suspensions of itraconazole and amiodarone. Formulation 4 was a compounded chewable tablet of itraconazole. Eight female dogs were dosed with 5 mg/kg of itraconazole and 15 mg/kg amiodarone (except for formulation 4, which only received 5 mg/kg itraconazole) once weekly for 4 weeks using a modified Latin Square design, ensuring that all dogs received all formulations with a 7-day washout between treatments. Animals were fasted overnight prior to each dose administration, with food returned to all animals 4 h post-dose. Blood samples (3 mL) were collected pre-treatment (0) and at appropriate time points over 72 h after each dose for a total of 14 samples per dog per treatment. There was high variability in the serum concentration data within treatment groups for itraconazole. The compounded suspensions were difficult to dose due to the nature of the formulations. The volumes dosed were accurate and consistent, but the suspension was thin and settled immediately when shaking was stopped for both itraconazole and amiodarone. All serum samples following itraconazole chewable tablet administration were not detectable or just above itraconazole's LOQ and thus did not allow for pharmacokinetic determination.