The mechanical properties of a cell determine many aspects of its behavior, and these mechanics are largely determined by the cytoskeleton. Although the contribution of actin filaments and microtubules to the mechanics of cells has been investigated in great detail, relatively little is known about the contribution of the third major cytoskeletal component, intermediate filaments (IFs). To determine the role of vimentin IF (VIF) in modulating intracellular and cortical mechanics, we carried out studies using mouse embryonic fibroblasts (mEFs) derived from wild-type or vimentin −/− mice. The VIFs contribute little to cortical stiffness but are critical for regulating intracellular mechanics. Active microrheology measurements using optical tweezers in living cells reveal that the presence of VIFs doubles the value of the cytoplasmic shear modulus to ∼10 Pa. The higher levels of cytoplasmic stiffness appear to stabilize organelles in the cell, as measured by tracking endogenous vesicle movement. These studies show that VIFs both increase the mechanical integrity of cells and localize intracellular components.
Simultaneous encapsulation of multiple active substances in a single carrier is essential for therapeutic applications of synergistic combinations of drugs. However, traditional carrier systems often lack efficient encapsulation and release of incorporated substances, particularly when combinations of drugs must be released in concentrations of a prescribed ratio. We present a novel biodegradable core shell carrier system fabricated in a one-step, solvent-free process on a microfluidic chip; a hydrophilic active (doxorubicin hydrochloride) is encapsulated in the aqueous core, while a hydrophobic active (paclitaxel) is encapsulated in the solid shell. Particle size and composition can be precisely controlled, and core and shell can be individually loaded with very high efficiency. Drug-loaded particles can be dried and stored as a powder. We demonstrate the efficacy of this system through the simultaneous encapsulation and controlled release of two synergistic anticancer drugs using two cancer-derived value for encapsulation of other active cell lines. This solvent-free platform technology is also of high potential ingredients and chemical reagents.
We introduce a novel type of droplet generator that produces droplets of a volume set by the geometry of the droplet generator and not by the flow rates of the liquids. The generator consists of a classic T-junction with a bypass channel. This bypass directs the continuous fluid around the forming droplets, so that they can fill the space between the inlet of the dispersed phase and the exit of the bypass without breaking. Once filled, the dispersed phase blocks the exit of the bypass and is squeezed by the continuous fluid and broken off from the junction. We demonstrate the fixed-volume droplet generator for (i) the formation of monodisperse droplets from a source of varying flow rates, (ii) the formation of monodisperse droplets containing a gradation of solute concentration, and (iii) the parallel production of monodisperse droplets. (C) 2013 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4801637]
Colloidal particles are microscopic solid particles suspended in a fluid. Colloids are small enough that thermal energy drives their dynamics and ensures equilibration with the suspending fluid; they are also large enough that their positions and motions can be measured precisely using optical methods, such as light scattering and laser-scanning confocal fluorescence microscopy. Colloidal suspensions are a powerful model system for the study of other phenomena in condensed matter physics, where the collective phase behavior of the solid particles mimics that of other condensed systems. We review three classes of interacting colloidal particles, crystals, glasses, and gels, each of which represents fascinating properties of colloidal particles as well as a model for more general types of materials and their behavior.
We report a microfluidic approach to produce monodisperse pH-responsive microcapsules with precisely controlled release behavior. The solid microcapsule shells are composed of a biocompatible pH-responsive polymer and robustly encapsulate an active material; however, when exposed to a trigger pH, the shells degrade and ultimately release the microcapsule contents. We control the trigger pH by using polymers that dissolve at different pH values. We independently control the time at which the microcapsule contents are released by carefully controlling the shell thickness. Moreover, we independently control the rate at which the encapsulated contents are released by making hybrid shells composed of a mixture of a pH-responsive polymer and varying proportions of another, solid, pH-unresponsive polymer. This enables us to achieve monodisperse microcapsules that robustly encapsulate an active material, only releasing it when exposed to a desired pH, after a prescribed time delay, and at a prescribed rate.
Droplet-based microfluidic techniques can form and process micrometer scale droplets at thousands per second. Each droplet can house an individual biochemical reaction, allowing millions of reactions to be performed in minutes with small amounts of total reagent. This versatile approach has been used for engineering enzymes, quantifying concentrations of DNA in solution, and screening protein crystallization conditions. Here, we use it to read the sequences of DNA molecules with a FRET-based assay. Using probes of different sequences, we interrogate a target DNA molecule for polymorphisms. With a larger probe set, additional polymorphisms can be interrogated as well as targets of arbitrary sequence.
We show that when a non-wetting fluid drains a stratified porous medium at sufficiently small capillary numbers Ca, it flows only through the coarsest stratum of the medium; by contrast, above a threshold Ca, the non-wetting fluid is also forced laterally, into part of the adjacent, finer strata. The spatial extent of this partial invasion increases with Ca. We quantitatively understand this behavior by balancing the stratum-scale viscous pressure driving the flow with the capillary pressure required to invade individual pores. Because geological formations are frequently stratified, we anticipate that our results will be relevant to a number of important applications, including understanding oil migration, preventing groundwater contamination, and sub-surface CO2 storage. Copyright (C) EPLA, 2013
We report a parallelized capillary microfluidic device for enhanced production rate of monodisperse polymersomes. This device consists of four independent capillary microfluidic devices, operated in parallel; each device produces monodisperse water-in-oil-in-water (W/O/W) double-emulsion drops through a single-step emulsification. During generation of the double-emulsion drops, the innermost water drop is formed first and it triggers a breakup of the middle oil phase over wide range of flow rates; this enables robust and stable formation of the double-emulsion drops in all drop makers of the parallelized device. Double-emulsion drops are transformed to polymersomes through a dewetting of the amphiphile-laden middle oil phase on the surface of the innermost water drop, followed by the subsequent separation of the oil drop. Therefore, we can make polymersomes with a production rate enhanced by a factor given by the number of drop makers in the parallelized device.
Polymeric magnetic microparticles have been created using a microfluidic device via ultraviolet (UV) polymerization of double emulsions, resulting in cores of magnetorheological (MR) fluids surrounded by polymeric shells. We demonstrate that the resultant particles can be manipulated magnetically to achieve triggered rupture of the capsules. This illustrates the great potential of our capsules for triggered release of active ingredients encapsulated in the polymeric magnetic microparticles. (C) 2012 Elsevier BY. All rights reserved.
The fast dynamics generated by the Brownian motion of particles in colloidal drops, and the related relaxation during drying, which play key roles in suspension systems, were investigated incorporating multispeckle diffusing wave spectroscopy (MSDWS). MSDWS equipment was implemented to analyze the relaxation properties of suspensions under a nonergodic and nonstationary drying process, which cannot be elucidated by conventional light scattering methods, such as dynamic light scattering and diffusing wave spectroscopy. Rapid particle movement can be identified by the characteristic relaxation time, which is closely related to the Brownian motion due to thermal fluctuations of the particles. In the compacting stage of the drying process, the characteristic relaxation time increased gradually with the drying time because the particles in the colloidal drop were constrained by themselves. Moreover, variations of the initial concentration and particle size considerably affected the complete drying time and characteristic relaxation time, producing a shorter relaxation time for a low concentrated suspension with small particles.
Polymersomes, vesicles composed of bilayer membranes of amphiphilic block-copolymers, are promising delivery vehicles for long-term storage and controlled release of bioactives; enhanced stability of the membrane makes polymersomes potentially useful in a wide range of biological delivery applications by comparison with liposomes. However, unilamellar structure is intrinsically fragile when subjected to external stress. Here, we report a microfluidic approach to produce polymersomes with double bilayers, providing higher stability and lower permeability than unilamellar polymersomes. To achieve this, we developed a new design of a capillary microfluidic device to produce quadruple-emulsion drops which serve as a template for the polymersomes-in-polymersomes. When two bilayers are attracted by depletion in polymersomes-in-polymersomes, the inner polymersomes protrude and bud, forming double bilayers. We confirm these structures are indeed double bilayers using microaspiration and selective doping of the leaflets with nanoparticles. The resultant polymersomes have great potential as highly stable and biocompatible microcarriers for robust encapsulation and storage of bioactives such as drugs, cosmetics and nutrients.
Gas-in-oil-in-water-in-oil triple emulsions are fabricated with a microfluidic method. The encapsulating layers can be triggered for release by ultrasound, owing to the gas core. Due to the stability in the atmosphere, the emulsions are polymerized by using UV light outside the device to fabricate compound particles with a gas-in-liquid-in-solid structure.
Hole in one! Hole–shell microparticles (blue, see picture) with controllable structures and flexible internal surfaces have been fabricated from W/O/W emulsions. These microparticles could be used as microcontainers for the controlled capture/release of molecules, microsphere classification/separation, confined cell culture, or as microreactors for catalysis.
Many bacteria on earth exist in surface-attached communities known as biofilms. These films are responsible for manifold problems, including hospital-acquired infections and biofouling, but they can also be beneficial. Biofilm growth depends on the transport of nutrients and waste, for which diffusion is thought to be the main source of transport. However, diffusion is ineffective for transport over large distances and thus should limit growth. Nevertheless, biofilms can grow to be very large. Here we report the presence of a remarkable network of well-defined channels that form in wildtype Bacillus subtilis biofilms and provide a system for enhanced transport. We observe that these channels have high permeability to liquid flow and facilitate the transport of liquid through the biofilm. In addition, we find that spatial variations in evaporative flux from the surface of these biofilms provide a driving force for the flow of liquid in the channels. These channels offer a remarkably simple system for liquid transport, and their discovery provides insight into the physiology and growth of biofilms.
We introduce a facile approach for the production of gas-filled microcapsules designed to withstand high pressures. We exploit microfluidics to fabricate water-filled microcapsules that are then externally triggered to become gas-filled, thus making them more echogenic. In addition, the gas-filled microcapsules have a solid polymer shell making them resistant to pressure-induced buckling, which makes them more mechanically robust than traditional prestabilized microbubbles; this should increase the potential of their utility for acoustic imaging of porous media with high hydrostatic pressures such as oil reservoirs.
A major challenge for a drug-delivery system is to engineer stable drug carriers with excellent biocompatibility, monodisperse size, and controllable release profiles. In this study, we used a microfluidic technique to encapsulate thermally hydrocarbonized porous silicon (THCPSi) microparticles within solid lipid microparticles (SLMs) to overcome the drawbacks accompanied by THCPSi microparticles. Formulation and process factors, such as lipid matrixes, organic solvents, emulsifiers, and methods to evaporate the organic solvents, were all evaluated and optimized to prepare monodisperse stable SLMs. FTIR analysis together with confocal images showed the clear deposition of THCPSi microparticles inside the monodisperse SLM matrix. The formation of monodisperse THCPSi solid lipid microcomposites (THCPSi SLMCs) not only altered the surface hydrophobicity and morphology of THCPSi microparticles but also remarkably enhanced their cytocompatibility with intestinal (Caco-2 and HT-29) cancer cells. Regardless of the solubility of the loaded therapeutics (aqueous insoluble, fenofibrate and furosemide; aqueous soluble, methotrexate and ranitidine) and the pH values of the release media (1.2, 5.0, and 7.4), the time for the release of 50% of the payloads from THCPSi SLMC was at least 1.3 times longer than that from the THCPSi microparticles. The sustained release of both water-soluble and -insoluble drugs together with a reduced burst-release effect from monodisperse THCPSi SLMC was achieved, indicating the successful encapsulation of THCPSi microparticles into the SLM matrix. The fabricated THCPSi SLMCs exhibited monodisperse spherical morphology, enhanced cytocompatibility, and prolonged both water-soluble and -insoluble drug release, which makes it an attractive controllable drug-delivery platform.
Chromatin structure and dynamics control all aspects of DNA biology yet are poorly understood, especially at large length scales. We developed an approach, displacement correlation spectroscopy based on time-resolved image correlation analysis, to map chromatin dynamics simultaneously across the whole nucleus in cultured human cells. This method revealed that chromatin movement was coherent across large regions (4-5 mu m) for several seconds. Regions of coherent motion extended beyond the boundaries of single-chromosome territories, suggesting elastic coupling of motion over length scales much larger than those of genes. These large-scale, coupled motions were ATP dependent and unidirectional for several seconds, perhaps accounting for ATP-dependent directed movement of single genes. Perturbation of major nuclear ATPases such as DNA polymerase, RNA polymerase II, and topoisomerase II eliminated micron-scale coherence, while causing rapid, local movement to increase; i.e., local motions accelerated but became uncoupled from their neighbors. We observe similar trends in chromatin dynamics upon inducing a direct DNA damage; thus we hypothesize that this may be due to DNA damage responses that physically relax chromatin and block long-distance communication of forces.
We describe a microfluidic device with an integrated microwave heater specifically designed to dielectrically heat non-aqueous droplets using time-varying electrical fields with the frequency range between 700 and 900 MHz. The precise control of frequency, power, temperature and duration of the applied field opens up new vistas for experiments not attainable by conventional microwave heating. We use a non-contact temperature measurement system based on fluorescence to directly determine the temperature inside a single droplet. The maximum temperature achieved of the droplets is 50 degrees C in 15 ms which represents an increase of about 25 degrees C above the base temperature of the continuous phase. In addition we use an infrared camera to monitor the thermal characteristics of the device allowing us to ensure that heating is exclusively due to the dielectric heating and not due to other effects like non-dielectric losses due to electrode or contact imperfection. This is crucial for illustrating the potential of dielectric heating of benzyl alcohol droplets for the synthesis of metal oxides. We demonstrate the utility of this technology for metal oxide nanoparticle synthesis, achieving crystallization of tungsten oxide nanoparticles and remarkable microstructure, with a reaction time of 64 ms, a substantial improvement over conventional heating methods.
Modern confocal microscopes enable high-precision measurement in three dimensions by collecting stacks of 2D (x-y) images that can be assembled digitally into a 3D image. It is difficult, however, to ensure position accuracy, particularly along the optical (z) axis where scanning is performed by a different physical mechanism than in x-y. We describe a simple device to calibrate simultaneously the x, y, and z pixel-to-micrometer conversion factors for a confocal microscope. By taking a known 2D pattern and positioning it at a precise angle with respect to the microscope axes, we created a 3D reference standard. The device is straightforward to construct and easy to use. (C) 2013 American Institute of Physics. [http://dx.doi.org/10.1063/1.4776672]
Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deformability. We probed neutrophil-differentiated HL-60 cells lacking expression of lamin B receptor, which fail to develop lobulated nuclei during granulopoiesis and present an in vitro model for Pelger-Huet anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. We then investigated the unique nuclear envelope composition of neutrophil-differentiated HL-60 cells, which may also impact their deformability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constrictions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron-scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential.