Elucidating siRNA Cellular Delivery Mechanism Mediated by Quaternized Starch Nanoparticles.

Starch-based nanoparticles are highly utilized in the realm of drug delivery taking advantage of their biocompatibility and biodegradability. Studies have utilized Quaternized starch (Q-starch) for small interfering RNA (siRNA) delivery, in which quaternary amines enable interaction with negatively charged siRNA, resulting in self-assembly complexation. Although reports present numerous applications, the demonstrated efficacy is nonetheless limited due to undiscovered cellular mechanistic delivery. In this study, a deep dive into Q-starch/siRNA complexes' cellular mechanism and kinetics at the cellular level is revealed using single-particle tracking and cell population level using imaging flow cytometry. Uptake studies depict the efficient cellular internalization via endocytosis while a significant fraction of complexes' intracellular fate is lysosome. Utilizing single-particle tracking, it is found that an average of 15% of cellular detected complexes escape the endosome which holds the potential for the integration in the cytoplasmatic gene silencing mechanism. Additional experimental manipulations (overcoming endosomal escape) demonstrate that the complex's disassembly is the rate-limiting step, correlating Q-starch's structure-function properties as siRNA carrier. Structure-function properties accentuating the high affinity of the interaction between Q-starch's quaternary groups and siRNA's phosphate groups that results in low release efficiency. However, low-frequency ultrasound (20 kHz) application may have induced siRNA release resulting in faster gene silencing kinetics.

Resonant oscillation of droplets under an alternating electric field to enhance solute diffusion.

This study investigates a novel microfluidic mixing technique that uses the resonant oscillation of coalescent droplets. During the vertical contact-separation process, solutes are initially separated as a result of the combined effects of diffusion and gravity. We show that the application of alternating current (AC) voltage to microelectrodes below the droplets causes a resonant oscillation, which enhances the even distribution of the solute. The difference in concentration between the top and bottom droplets exhibits frequency dependence and indicates the existence of a particular AC frequency that results in a homogeneous concentration. This frequency corresponds to the resonance frequency of the droplet oscillation that is determined using particle tracking velocimetry. To understand the mixing process, a phenomenological model based on the equilibrium between surface tension, viscosity, and electrostatic force was developed. This model accurately predicted the resonance frequency of droplet flow and was consistent with the experimental results. These results suggest that the resonant oscillation of droplets driven by AC voltage significantly enhances the diffusion of solutes, which is an effective approach to microfluid mixing.

Highly sensitive volumetric single-molecule imaging.

Volumetric subcellular imaging has long been essential for studying structures and dynamics in cells and tissues. However, due to limited imaging speed and depth of field, it has been challenging to perform live-cell imaging and single-particle tracking. Here we report a 2.5D fluorescence microscopy combined with highly inclined illumination beams, which significantly reduce not only the image acquisition time but also the out-of-focus background by ∼2-fold compared to epi-illumination. Instead of sequential z-scanning, our method projects a certain depth of volumetric information onto a 2D plane in a single shot using multi-layered glass for incoherent wavefront splitting, enabling high photon detection efficiency. We apply our method to multi-color immunofluorescence imaging and volumetric super-resolution imaging, covering ∼3-4 µm thickness of samples without z-scanning. Additionally, we demonstrate that our approach can substantially extend the observation time of single-particle tracking in living cells.

Probing Twist-induced Endocytotic Membrane Fission using Anisotropic Gold Homodimers.

Membrane fission involves a crucial step of lipid remodeling, in which the dynamin collar constricts and severs the tubulated lipid membrane at the neck of budding vesicles. Nevertheless, the difficulty in accurately determining the rotational dynamics of live endocytotic vesicles poses a limit on the elucidation of dynamin-induced membrane remodeling for endocytotic vesicle scission. Herein, we designed a DNA-modified gold homodimer (AuHD)-based anisotropic plasmonic probe with uniform surface chemistry, minimizing orientational fluctuation within vesicle encapsulation. Using AuHDs as cargos to image the dynamics of cargo-containing vesicles during endocytosis, we showed that, prior to detachment from plasma membrane, the cargo-containing vesicles underwent multiple intermittent twists of ~4° angular orientation relative to plasma membrane with a ~0.2 s dwell time. These findings suggest that the membrane torques resulting from dynamin actions in vivo constitute the pathway to membrane fission, potentially shedding light on how dynamin-mediated lipid remodeling orchestrates membrane fission.

Optimizing distal and proximal splenic artery embolization with patient-specific computational fluid dynamics.

Splenic artery embolization (SAE) has become a favored alternative to splenectomy, offering a less invasive intervention for injured spleens while preserving spleen function. However, our understanding of the role that hemodynamics plays during embolization remains limited. In this study, we utilized patient-specific computational fluid dynamics (CFD) simulations to study distal and proximal embolization strategies commonly used in SAE. Detailed 3D computer models were constructed considering the descending aorta, various major visceral arteries, and the iliac arteries. Subsequently, the blood flow and pressure associated with different coil placement locations in proximal embolization were studied considering the collateral vessels. Coil induced variations in pressure fields were quantified and compared to baseline. The coil induced flow stagnation was also quantified with particle residence time. Distal embolization was modeled with Lagrangian particle tracking and the effect of particle size, release location, and timing on embolization outcome was studied. Our findings highlight the crucial role of collateral vessels in maintaining blood supply to the spleen following proximal embolization. It was demonstrated that coil location can affect distal pressure and that strategic coil placement guided by patient-specific CFD simulations can further reduce this pressure as desired. Additionally, the results point to the critical roles that particle size, release timing, and location play in distal embolization. Our study provides an early attempt to use patient-specific computer modeling for optimizing embolization strategies and ultimately improving patient outcomes during SAE procedures.

Seasonal change in fate and transport of plastics from Red River to the coast of Vietnam.

A Lagrangian-particle tracking model, Delft3D-PART, combined with hydrodynamics models are used to investigate the fate and transport of buoyant plastics from Ba Lat river mouth in Red River Delta, northern Vietnam. It was found that during the dry season (Dec-Feb), 23 % (26.43 ton) of the plastics reached the shoreline while 76.1 % (68.3 ton) moved towards the coast further south of Red River Delta. During the wet season (Jun-Aug), 42 % (56.3 ton) were transported offshore away from the coast and 20 % (26.43 ton) distributed along the shore. The two bays adjacent to the river mouth are major hotspots with the intensity skewed towards the upwind side relative to the seasonal monsoon. This phenomenon is exacerbated by storm events which reverse the typical transport and lead to formation of hotspots at the upwind side of the plastic source. Guidance of model results for targeted cleanup operations is discussed.

Porcine ex-vivo intestinal mucus has age-dependent blocking activity against transmissible gastroenteritis virus.

Transmissible gastroenteritis virus (TGEV) causes high mortality in young piglets (< 3 days of age). With aging, the susceptibility/morbidity/mortality rates drop. We previously hypothesized that the age-related changes in the intestinal mucus could be responsible for this resistance. Hence, this study investigated the effect of porcine intestinal mucus from 3-day and 3-week-old pigs on the free mobility of the virulent TGEV Miller strain, and on the infection in swine testicle (ST) cells. Single particle tracking (SPT) revealed that TGEV had significantly higher diffusion coefficients in 3-day mucus compared to 3-week mucus. TGEV and charged and uncharged control nanoparticles diffused freely in 3-day mucus but were hindered by 3-week mucus in the diffusion model; TGEV mimicked the diffusion behavior of negatively charged carboxylated particles. Inoculation of ST cells with TGEV in the presence of 3-week mucus resulted in a significantly lower average number of infected cells (30.9 ± 11.9/5 fields) compared with 3-day mucus (84.6 ± 16.4/5 fields). These results show that 3-week mucus has a significant TGEV-blocking activity compared to 3-day mucus in free diffusion and infection of the underlying susceptible cells. Additionally, a label-free proteomics analysis revealed an increased expression of mucin 13, known for negatively regulating the tight junctions in intestinal epithelium, in 3-day-old pigs. In 3-week-old pigs, a higher expression of mucin 2, a type of secreted mucin which is known for inhibiting coronavirus infection, was observed. Concludingly, this study demonstrated a protective effect of 3-week mucus against viral infections.

Microplastics in ecosystems: Critical review of occurrence, distribution, toxicity, fate, transport, and advances in experimental and computational studies in surface and subsurface water.

Microplastics (MPs), particles under 5 mm, pervade water, soil, sediment, and air due to increased plastic production and improper disposal, posing global environmental and health risks. Examining their distribution, quantities, fate, and transport is crucial for effective management. Several studies have explored MPs' sources, distribution, transport, and biological impacts, primarily focusing on the marine environment. However, there is a need for a comprehensive review of all environmental systems together for enhanced pollution control. This review critically examines the occurrence, distribution, fate, and transport of MPs in the following environments: freshwater, marine, and terrestrial ecosystems. The concentration of MPs is highly variable in the environment, ranging from negligible to significant amounts (0.003-519.223 items/liter in water and 0-18,000 items/kg dry weight sediment, respectively). Predominantly, these MPs manifest as fibers and fragments, with primary polymer types including polypropylene, polystyrene, polyethylene, and polyethylene terephthalate. A complex interplay of natural and anthropogenic actions, including wastewater treatment plant discharges, precipitation, stormwater runoff, inadequate plastic waste management, and biosolid applications, influences MPs' presence and distribution. Our critical synthesis of existing literature underscores the significance of factors such as wind, water flow rates, settling velocities, wave characteristics, plastic morphology, density, and size in determining MPs' transport dynamics in surface and subsurface waters. Furthermore, this review identifies research gaps, both in experimental and simulation, and outlines pivotal avenues for future exploration in the realm of MPs.

Cell-Material Interactions in Covalent Adaptable Thioester Hydrogels.

Covalent adaptable networks (CANs) are polymeric networks with cross-links that can break and reform in response to external stimuli, including pH, shear, and temperature, making them potential materials for use as injectable cell delivery vehicles. In the native niche, cells rearrange the extracellular matrix (ECM) to undergo basic functions including migration, spreading, and proliferation. Bond rearrangement enables these hydrogels to mimic viscoelastic properties of the native ECM which promote migration and delivery from the material to the native tissue. In this work, we characterize thioester CANs to inform their design as effective cell delivery vehicles. Using bulk rheology, we characterize the rearrangement of these networks when they are subjected to strain, which mimics the strain applied by a syringe, and using multiple particle tracking microrheology (MPT) we measure cell-mediated remodeling of the pericellular region. Thioester networks are formed by photopolymerizing 8-arm poly(ethylene glycol) (PEG)-thiol and PEG-thioester norbornene. Bulk rheology measures scaffold properties during low and high strain and demonstrates that thioester scaffolds can recover rheological properties after high strain is applied. We then 3D encapsulated human mesenchymal stem cells (hMSCs) in thioester scaffolds. Using MPT, we characterize degradation in the pericellular region. Encapsulated hMSCs degrade these scaffolds within ≈4 days post-encapsulation. We hypothesize that this degradation is mainly due to cytoskeletal tension that cells apply to the matrix, causing adaptable thioester bonds to rearrange, leading to degradation. To verify this, we inhibited cytoskeletal tension using blebbistatin, a myosin-II inhibitor. Blebbistatin-treated cells can degrade these networks only by secreting enzymes including esterases. Esterases hydrolyze thioester bonds, which generate free thiols, leading to bond exchange. Around treated cells, we measure a decrease in the extent of pericellular degradation. We also compare cell area, eccentricity, and speed of untreated and treated cells. Inhibiting cytoskeletal tension results in significantly smaller cell area, more rounded cells, and lower cell speeds when compared to untreated cells. Overall, this work shows that cytoskeletal tension plays a major role in hMSC-mediated degradation of thioester networks. Cytoskeletal tension is also important for the spreading and motility of hMSCs in these networks. This work informs the design of thioester scaffolds for tissue regeneration and cell delivery.

Simultaneous Mapping of the Nanoscale Organization and Redox State of Extracellular Space in Living Brain Tissue.

The spatial organization characteristics and redox status of the extracellular space (ECS) are crucial in the development of brain diseases. However, it remains a challenge to simultaneously capture dynamic changes in microstructural features and redox states at the submicron level within the ECS. Here, we developed a reversible glutathione (GSH)-responsive nanoprobe (RGN) for mapping the spatial organization features and redox status of the ECS in brain tissues with nanoscale resolution. The RGN is composed of polymer nanoparticles modified with GSH-responsive molecules and amino-functionalized methoxypoly(ethylene glycol), which exhibit exceptional single-particle brightness and excellent free diffusion capability in the ECS of brain tissues. Tracking single RGNs in acute brain slices allowed us to dynamically map spatial organizational features and redox levels within the ECS of brain tissues in disease models. This provides a powerful super-resolution imaging method that offers a potential opportunity to study the dynamic changes in the ECS microenvironment and to understand the physiological and pathological roles of the ECS in vivo.

Kinetic Landscape of Single Virus-like Particles Highlights the Efficacy of SARS-CoV-2 Internalization.

The efficiency of virus internalization into target cells is a major determinant of infectivity. SARS-CoV-2 internalization occurs via S-protein-mediated cell binding followed either by direct fusion with the plasma membrane or endocytosis and subsequent fusion with the endosomal membrane. Despite the crucial role of virus internalization, the precise kinetics of the processes involved remains elusive. We developed a pipeline, which combines live-cell microscopy and advanced image analysis, for measuring the rates of multiple internalization-associated molecular events of single SARS-CoV-2-virus-like particles (VLPs), including endosome ingression and pH change. Our live-cell imaging experiments demonstrate that only a few minutes after binding to the plasma membrane, VLPs ingress into RAP5-negative endosomes via dynamin-dependent scission. Less than two minutes later, VLP speed increases in parallel with a pH drop below 5, yet these two events are not interrelated. By co-imaging fluorescently labeled nucleocapsid proteins, we show that nucleocapsid release occurs with similar kinetics to VLP acidification. Neither Omicron mutations nor abrogation of the S protein polybasic cleavage site affected the rate of VLP internalization, indicating that they do not confer any significant advantages or disadvantages during this process. Finally, we observe that VLP internalization occurs two to three times faster in VeroE6 than in A549 cells, which may contribute to the greater susceptibility of the former cell line to SARS-CoV-2 infection. Taken together, our precise measurements of the kinetics of VLP internalization-associated processes shed light on their contribution to the effectiveness of SARS-CoV-2 propagation in cells.

Reduction scenarios of plastic waste emission guided by the probability distribution model to avoid additional ocean plastic pollution by 2050s.

Marine plastic pollution is progressing worldwide and will become increasingly serious if plastic waste emissions continue at the current rate or increase with economic growth. Here, we report a particle tracking-based probability distribution model for predicting the abundances of marine macroplastics and microplastics, which undergo generation, transport, and removal processes in the world's upper ocean, under various scenarios of future land-to-sea plastic waste emissions. To achieve the Osaka Blue Ocean Vision, which aims to reduce additional pollution by marine plastic litter to zero by 2050, plastic waste emission in ∼2035 should be reduced by at least 32 % relative to 2019. It is necessary to take stringent measures such as 'system change scenario' or 'improve waste management scenario' proposed in previous studies to reduce the marine plastic pollution by 2050.

Plasticity-induced actin polymerization in the dendritic shaft regulates intracellular AMPA receptor trafficking.

AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing plasticity to increase synaptic transmission, but it is not fully understood if and how AMPAR-containing vesicles are selectively trafficked to these synapses. Here, we developed a strategy to label AMPAR GluA1 subunits expressed from their endogenous loci in cultured rat hippocampal neurons and characterized the motion of GluA1-containing vesicles using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced structural plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of synaptic activity.

Multiple Particle Tracking: A Method for Probing Biologically Relevant Mobility of Bacterial Extracellular Vesicles.

Bacterial extracellular vesicles (bEVs) are produced by both Gram-negative and Gram-positive bacteria. These biological nanoparticles transport small molecules, nucleic acids, and proteins, enabling communication with both bacterial and mammalian cells. bEVs can evade and disrupt biological barriers, and their lipid membranes protect their cargo from degradation, facilitating long-distance communication in vivo. Furthermore, bacteria are easily manipulated and easily cultured. These combined factors make bEVs an ideal candidate for drug delivery applications. Thus, the study of how bEVs interact with biological barriers is interesting from both a signaling and drug delivery perspective. Here we describe methods for tracking bEV motion in biological matrices ex vivo. We outline methods for growth, isolation, quantification, and labeling, as well as techniques for tracking bEV motion ex vivo and quantifying these data. The methods described here are relevant to bEV communication with host cells as well as drug delivery applications using bEVs.

Making Rift Valley Fever Viral Particles Fluorescent.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that represents a significant threat to both human and veterinary public health. Since its discovery in the Great Rift Valley of Kenya in the 1930s, the virus has spread across Africa and beyond, now posing a risk of introduction into Southern Europe and Asia. Despite recent progresses, early RVFV-host cell interactions remain largely uncharacterized. In this method chapter, we delineate the procedure for labeling RVFV particles with fluorescent organic dyes. This approach makes it feasible to visualize single viral particles in both fixed and living cells and study RVFV entry into host cells. We provide additional examples with two viruses closely related to RVFV, namely, Toscana virus and Uukuniemi virus. Furthermore, we illustrate how to utilize fluorescent viral particles to examine and quantify each step of the cell entry program of RVFV, which includes state-of-the-art fluorescence-based detection techniques such as fluorescence microscopy, flow cytometry, and fluorimetry.

Reciprocal effect on lateral diffusion of receptor for advanced glycation endproducts and toll-like receptor 4 in the HEK293 cell membrane.

Receptor for advanced glycation endproducts (RAGE) and toll-like receptor 4 (TLR4) are pattern-recognition receptors that bind to molecular patterns associated with pathogens, stress, and cellular damage. Diffusion plays an important role in receptor functionality in the cell membrane. However, there has been no prior investigation of the reciprocal effect of RAGE and TLR4 diffusion properties in the presence and absence of each receptor. This study reports how RAGE and TLR4 affect the mobility of each other in the human embryonic kidney (HEK) 293 cell membrane. Diffusion properties were measured using single-particle tracking (SPT) with quantum dots (QDs) that are selectively attached to RAGE or TLR4. The Brownian diffusion coefficients of RAGE and TLR4 are affected by the presence of the other receptor, leading to similar diffusion coefficients when both receptors coexist in the cell. When TLR4 is present, the average Brownian diffusion coefficient of RAGE increases by 40%, while the presence of RAGE decreases the average Brownian diffusion coefficient of TLR4 by 32%. Diffusion in confined membrane domains is not altered by the presence of the other receptor. The mobility of the cell membrane lipid remains constant whether one or both receptors are present. Overall, this work shows that the presence of each receptor can affect a subset of diffusion properties of the other receptor without affecting the mobility of the membrane.

Multi-laser nanoparticle tracking analysis (NTA): A unique method to visualize dynamic (shear) and dynamic (Brownian motion) light scattering and quantify nonliving natural organic matter (NNOM) in environmental water.

Application of simultaneous multi-laser nanoparticle tracking analysis (NTA) to environmental water samples to investigate nonliving natural organic matter (NNOM) is introduced as an innovative method for observing particles directly in their native media. Multi-laser NTA results of particle visualization, particle number concentration, and particle size distribution elucidated particle dynamics in low and high total dissolved solids (TDS) aqueous environmental samples. A pond water sample and concentrate from a reverse osmosis (RO) treatment process (Stage 1) had 1.3 × 108 and 5.62 × 1019 particles/mL, respectively, (at time = 0) after filtration at 0.45 µm. Beyond the traditional applications for this instrument, this research presents novel evidence-based investigations that probe the existence of supramolecular structures in environmental waters during turbulence or quiescence. The pond water sample exhibited time-dependent aggregation as the volume distribution shifted to greater diameter during quiescence, compared to turbulence. Disaggregation (increased numbers of particles over time) was noted in the >250 nm to <600 nm region, and aggregation of >450 nm particles was also noted in the quiescent RO concentrate sample, indicative of depletion of small particles to form larger ones. Multi-laser NTA and dynamic light scattering (DLS) capabilities were compared and contrasted. DLS and NTA are different (complementary) particle sizing techniques. DLS yielded more information about the physical hydrogel in the NNOM hierarchy whereas multi-laser NTA better characterized meta-chemical and chemical hydrogel characteristics. Operationalization of innovation-moving from fundamental investigations to application-is supported by implementing novel analytical instrumentation as we address issues involving climate change, drought, and the scarcity of potable water. Multi-laser NTA can be used as a tool to study and optimize complex water and wastewater treatment processes. Questions about water treatment efficiencies, membrane fouling, assistance of pollutant transport, and carbon capture cycles affected by NNOM will benefit from insights from multi-laser NTA.

Investigating local trawl fishing as a source of plastic beach litter.

This paper uses a particle tracking model to simulate the distribution of fishing-related marine-sourced plastic litter from demersal trawling activities along the Atlantic coast of Scotland. The modelled fishing litter dispersed widely across the region, with ∼50% of the particles beaching along the northwestern Scottish coast after a year-long simulation. The model was tuned using observations of beached litter loadings along the same coastline to estimate the annual input, by mass, of small (<1 kg) plastic litter. Model results suggest that between 107 g and 280 g of small fishing-related litter enters the ocean per hour of fishing, resulting in an estimated 234 t to 614 t of small fishing-related litter entering the ocean annually on the Scottish west coast. These results suggest that fishing on the Atlantic coast of Scotland may be a significant source of marine plastic. However, more modelled and observational data are required to reduce uncertainty.

Understanding stratification and turnover dynamics of a tropical lake using extensive field observations and 3D hydrodynamic simulations.

The stratification and turnover dynamics of a tropical lake were evaluated using field observations and 3D hydrodynamic simulations. Located in the Philippines, Sampaloc Lake is a 104-ha and 27-m deep volcanic crater lake with enclosed watershed, which is at risk of the impacts of intensive aquaculture, rapid urbanization and climate change. Temperature, dissolved oxygen (DO) and chlorophyll-a (Chl-a) were measured at seven sampling stations using a multiprobe. Kruskal-Wallis test revealed that the three parameters are not significantly different among stations, indicating that one sampling station can represent the water quality of the whole lake. Schmidt's Stability Index (SSI) and thermocline strength, together with DO and Chl-a gradients decreased from October 2022 (stratified) to January 2023 (turnover). After successfully verifying the 3D numerical model, sensitivity analyses of water temperature to varying weather, together with particle tracking simulations, were implemented to determine the timing of isothermal state, upwelling, partial mixing, and full turnover. Compared to air temperature, variations in wind speed have more pronounced effects on the delay or progression of isothermal conditions in the lake based on SSI, Lake Number and Wedderburn Number. Isothermal conditions do not necessarily coincide with the timing of full turnover, with the latter being delayed by two days than the former, on average. Results revealed that full turnover can occur several weeks earlier with the decrease in AT and increase in WS. This study can advance the understanding of thermal and turnover dynamics of stratified tropical lakes, leading to better management of the water quality of these water bodies.

A live cell imaging-based assay for tracking particle uptake by clathrin-mediated endocytosis.

A popular strategy for therapeutic delivery to cells and tissues is to encapsulate therapeutics inside particles that cells internalize via endocytosis. The efficacy of particle uptake by endocytosis is often studied in bulk using flow cytometry and Western blot analysis and confirmed using confocal microscopy. However, these techniques do not reveal the detailed dynamics of particle internalization and how the inherent heterogeneity of many types of particles may impact their endocytic uptake. Toward addressing these gaps, here we present a live-cell imaging-based method that utilizes total internal reflection fluorescence microscopy to track the uptake of a large ensemble of individual particles in parallel, as they interact with the cellular endocytic machinery. To analyze the resulting data, we employ an open-source tracking algorithm in combination with custom data filters. This analysis reveals the dynamic interactions between particles and endocytic structures, which determine the probability of particle uptake. In particular, our approach can be used to examine how variations in the physical properties of particles (size, targeting, rigidity), as well as heterogeneity within the particle population, impact endocytic uptake. These data impact the design of particles toward more selective and efficient delivery of therapeutics to cells.