Cao, T. ; Wang, Y. ; Tao, Y. ; Zhang, L. ; Zhou, Y. - L. ; Zhang, X. - X. ; Heyman, J. A. ; Weitz, D. A. DNAzyme-powered nucleic acid release from solid supports. Chemical Communications 2020, 56, 647–650. Publisher's VersionAbstract
Here, we demonstrate use of a Mg 2+ -dependent, site-specific DNA enzyme (DNAzyme) to cleave oligos from polyacrylamide gel beads, which is suitable for use in drop-based assays. , Here, we demonstrate use of a Mg 2+ -dependent, site-specific DNA enzyme (DNAzyme) to cleave oligos from polyacrylamide gel beads, which is suitable for use in drop-based assays. We show that cleavage efficiency is improved by use of a tandem-repeat cleavage site. We further demonstrate that DNAzyme-released oligos function as primers in reverse transcription of cell-released mRNA.
Lu, H. ; Mutafopulos, K. ; Heyman, J. A. ; Spink, P. ; Shen, L. ; Wang, C. ; Franke, T. ; Weitz, D. A. Rapid additive-free bacteria lysis using traveling surface acoustic waves in microfluidic channels. Lab on a Chip 2019, 19, 4064-4070. Publisher's VersionAbstract

We report an additive-free method to lyse bacteria and extract nucleic acids and protein using a traveling surface acoustic wave (TSAW) coupled to a microfluidic device. We characterize the effects of the TSAW on E. coli by measuring the viability of cells exposed to the sound waves and find that about 90% are dead. In addition, we measure the protein and nucleic acids released from the cells and show that we recover about 20% of the total material. The lysis method should work for all types of bacteria. These results demonstrate the feasibility of using TSAW to lyse bacteria in a manner that is independent of the type of bacteria.

Kanai, T. ; Nakai, H. ; Yamada, A. ; Fukuyama, M. ; Weitz, D. A. Preparation of monodisperse hybrid gel particles with various morphologies via flow rate and temperature control. Soft Matter 2019, 15, 6934-6937. Publisher's VersionAbstract

We report a facile method for preparing monodisperse hybrid smart gel particles with various morphologies by using microfluidic techniques and the swelling–shrinking phenomenon of thermosensitive poly(N-isopropylacrylamide) (PNIPAM) gel particles. We demonstrate that PNIPAM–polyacrylamide snowman-like, raspberry-like, and dumbbell-like hybrid gel particles can be prepared by controlling the flow rate and temperature.

Mutafopulos, K. ; Spink, P. ; Lofstrom, C. D. ; Lu, P. J. ; Lu, H. ; Sharpe, J. C. ; Franke, T. ; Weitz, D. A. Traveling surface acoustic wave (TSAW) microfluidic fluorescence activated cell sorter (μFACS). Lab on a Chip 2019, 19, 2435-2443. Publisher's VersionAbstract

We report a microfluidic fluorescence activated cell-sorting (μFACS) device that employs traveling surface acoustic waves (TSAW) to sort cells at rates comparable to conventional jet-in-air FACS machines, with high purity and viability. The device combines inertial flow focusing and sheath flow to align and evenly space cells, improving the sorting accuracy and screening rate. We sort with an interdigital transducer (IDT) whose tapered geometry allows precise positioning of the TSAW for optimal cell sorting. We sort three different cell lines at several kHz, at cell velocities exceeding one meter per second, while maintaining both sorting purity and cell viability at around 90% simultaneously.

Chowdhury, M. S. ; Zheng, W. ; Kumari, S. ; Heyman, J. ; Zhang, X. ; Dey, P. ; Weitz, D. A. ; Haag, R. Dendronized fluorosurfactant for highly stable water-in-fluorinated oil emulsions with minimal inter-droplet transfer of small molecules. Nature Communications 2019, 10, 1-10. Publisher's VersionAbstract

Fluorosurfactant-stabilized microfluidic droplets are widely used as pico- to nanoliter volume reactors in chemistry and biology. However, current surfactants cannot completely prevent inter-droplet transfer of small organic molecules encapsulated or produced inside the droplets. In addition, the microdroplets typically coalesce at temperatures higher than 80 °C. Therefore, the use of droplet-based platforms for ultrahigh-throughput combination drug screening and polymerase chain reaction (PCR)-based rare mutation detection has been limited. Here, we provide insights into designing surfactants that form robust microdroplets with improved stability and resistance to inter-droplet transfer. We used a panel of dendritic oligo-glycerol-based surfactants to demonstrate that a high degree of inter- and intramolecular hydrogen bonding, as well as the dendritic architecture, contribute to high droplet stability in PCR thermal cycling and minimize inter-droplet transfer of the water-soluble fluorescent dye sodium fluorescein salt and the drug doxycycline.

Gerber, G. ; Bensouda, M. ; Weitz, D.  A. ; Coussot, P. Self-Limited Accumulation of Colloids in Porous Media. Physical Review Letters 2019, 123, 158005. Publisher's VersionAbstract

We present local direct imaging of the progressive adsorption of colloidal particles inside a 3D model porous medium. By varying the interparticle electrostatic interactions, we observe a large range of particle deposition regimes, from a single layer of particles at the surface of the medium to multiple layers and eventually clogging of the system. We derive the complete deposition dynamics and show that colloid accumulation is a self-limited mechanism towards a deposited fraction associated with a balance between the particle interactions and the imposed flow rate. These trends are explained and predicted using a simple probability model considering the particle adsorption energy and the variation of the drag energy with evolving porosity. This constitutes a direct validation of speculated particle transport mechanisms, and a further understanding of accumulation mechanisms.

    Zhang, W. ; Qu, L. ; Pei, H. ; Qin, Z. ; Didier, J. ; Wu, Z. ; Bobe, F. ; Ingber, D. E. ; Weitz, D. A. Controllable Fabrication of Inhomogeneous Microcapsules for Triggered Release by Osmotic Pressure. Small 2019, 15, 1903087. Publisher's VersionAbstract
    Inhomogeneous microcapsules that can encapsulate various cargo for controlled release triggered by osmotic shock are designed and reported. The microcapsules are fabricated using a microfluidic approach and the inhomogeneity of shell thickness in the microcapsules can be controlled by tuning the flow rate ratio of the middle phase to the inner phase. This study demonstrates the swelling of these inhomogeneous microcapsules begins at the thinnest part of shell and eventually leads to rupture at the weak spot with a low osmotic pressure. Systematic studies indicate the rupture fraction of these microcapsules increases with increasing inhomogeneity, while the rupture osmotic pressure decreases linearly with increasing inhomogeneity. The inhomogeneous microcapsules are demonstrated to be impermeable to small probe molecules, which enables long‐term storage. Thus, these microcapsules can be used for long‐term storage of enzymes, which can be controllably released through osmotic shock without impairing their biological activity. The study provides a new approach to design effective carriers to encapsulate biomolecules and release them on‐demand upon applying osmotic shock.
    Mao, A. S. ; Ozkale, B. ; Shah, N. J. ; Vining, K. H. ; Descombes, T. ; Zhang, L. ; Tringides, C. M. ; Wong, S. - W. ; Shin, J. - W. ; et al. Scadden, D. T. Programmable microencapsulation for enhanced mesenchymal stem cell persistence and immunomodulation. Proceedings of the National Academy of Sciences 2019, 116, 15392–15397. Publisher's VersionAbstract

    Mesenchymal stem cell (MSC) therapies demonstrate particular promise in ameliorating diseases of immune dysregulation but are hampered by short in vivo cell persistence and inconsistencies in phenotype. Here, we demonstrate that biomaterial encapsulation into alginate using a microfluidic device could substantially increase in vivo MSC persistence after intravenous (i.v.) injection. A combination of cell cluster formation and subsequent cross-linking with polylysine led to an increase in injected MSC half-life by more than an order of magnitude. These modifications extended persistence even in the presence of innate and adaptive immunity-mediated clearance. Licensing of encapsulated MSCs with inflammatory cytokine pretransplantation increased expression of immunomodulatory-associated genes, and licensed encapsulates promoted repopulation of recipient blood and bone marrow with allogeneic donor cells after sublethal irradiation by a ∼2-fold increase. The ability of microgel encapsulation to sustain MSC survival and increase overall immunomodulatory capacity may be applicable for improving MSC therapies in general.

    Du, J. ; Fan, Y. ; Guo, Z. ; Wang, Y. ; Zheng, X. ; Huang, C. ; Liang, B. ; Gao, L. ; Cao, Y. ; et al. Chen, Y. Compression Generated by a 3D Supracellular Actomyosin Cortex Promotes Embryonic Stem Cell Colony Growth and Expression of Nanog and Oct4. Cell Systems 2019, 9 214-220.e5. Publisher's VersionAbstract
    Mechanical factors play critical roles in mammalian development. Here, we report that colony-growing mouse embryonic stem cells (mESCs) generate significant tension on the colony surface through the contraction of a three-dimensional supracellular actomyosin cortex (3D-SAC). Disruption of the 3D-SAC, whose organization is dependent on the Rho/Rho-associated kinase (ROCK) signals and E-cadherin, results in mESC colony destruction. Reciprocally, compression force, which is generated by the 3D-SAC, promotes colony growth and expression of Nanog and Oct4 in mESCs and blastocyst development of mouse embryos. These findings suggest that autonomous cell forces regulate embryonic stem cells fate determination and provide insight regarding the biomechanical regulation of embryonic development.
    Chen, R. ; Liu, J. ; Yang, C. ; Weitz, D. A. ; He, H. ; Li, D. ; Chen, D. ; Liu, K. ; Bai, H. Transparent Impact-Resistant Composite Films with Bioinspired Hierarchical Structure. ACS Applied Materials & Interfaces 2019, 11, 23616-23622. Publisher's VersionAbstract
    Inspired by the helicoidally organized microstructure of stomatopods’ smasher dactyl club, a type of impact-resistant composite film reinforced with periodic helicoidal nanofibers is designed and fabricated, which reproduces the structural complexity of the natural material. To periodically align nanofibers in a helicoidal structure, an electrospinning system is developed to better control the alignment of electrospun nanofibers. When the nanofiber scaffold is embedded in an epoxy matrix, the presence of a hierarchical structure allows the composite films to achieve properties well beyond their constituents. The composite film exhibits excellent optical transparency and mechanical properties, such as enhanced tensile strength, ductility, and defect tolerance. With elegant design mimicking nature’s hierarchical structure at multilength scales, the composite films could effectively release the impact energy and greatly increase the impact resistance, suggesting that the transparent composite films are promising protective layers suitable for various applications.
    Kong, L. ; Chen, R. ; Wang, X. ; Zhao, C. - X. ; Chen, Q. ; Hai, M. ; Chen, D. ; Yang, Z. ; Weitz, D. A. Controlled co-precipitation of biocompatible colorant-loaded nanoparticles by microfluidics for natural color drinks. Lab Chip 2019, 19, 2089-2095. Publisher's VersionAbstract
    Natural colorants, which impart a vivid color to food and add additional health benefits, are favored over synthetic colorants; however, their applications are limited by their low solubility in water and low stability. Here, we develop a versatile microfluidic strategy to incorporate natural colorants in shellac nanoparticles with controlled physicochemical properties. The rapid mixing in the microfluidic channels ensures that the mixing time is shorter than the aggregation time, thus providing control over the co-precipitation of the colorant and the polymer. By introducing molecular interactions, colorant nanoaggregates are efficiently embedded in the polymer matrix, forming hierarchical colorant-loaded nanoparticles. The colorant-loaded nanoparticles dispersed in water are transparent and stable over a wide pH range and their polymer matrix also provides a favorable microenvironment that greatly improves the shelf life of the colorants. The improved solubility, stability and bioavailability of the natural colorants suggest that shellac nanoparticles are ideal carriers and the stable, transparent dispersions of biocompatible colorant-loaded nanoparticles in water are well-suited for the development of functional foods, such as natural color drinks.
    Hwang, H. ; Weitz, D. A. ; Spaepen, F. Direct observation of crystallization and melting with colloids. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 1180–1184. Publisher's VersionAbstract
    We study the kinetics of crystal growth and melting of two types of colloidal crystals: body-centered cubic (BCC) crystals and face-centered cubic (FCC) crystals. A dielectrophoretic “electric bottle” confines colloids, enabling precise control of the motion of the interface. We track the particle motion, and by introducing a structural order parameter, we measure the jump frequencies of particles to and from the crystal and determine from these the free-energy difference between the phases and the interface mobility. We find that the interface is rough in both BCC and FCC cases. Moreover, the jump frequencies correspond to those expected from the random walk of the particles, which translates to collision-limited growth in metallic systems. The mobility of the BCC interface is greater than that of the FCC interface. In addition, contrary to the prediction of some early computer simulations, we show that there is no significant asymmetry between the mobilities for crystallization and melting.
    Zhu, H. ; Nawar, S. ; Werner, J. G. ; Liu, J. ; Huang, G. S. ; Mei, Y. F. ; Weitz, D. A. ; Solovev, A. A. Hydrogel micromotors with catalyst-containing liquid core and shell. J. Phys.: Condens. Matter 2019, 31, 214004. Publisher's VersionAbstract
    Methacrylic anhydride-derived hydrogel microcapsules have unique properties, including reversibly tunable permeation, purification, and separation of dissolved molecular species. Endowing these dynamic encapsulant systems with autonomous motion will significantly enhance their efficiency and applicability. Here, hydrogel micromotors are realized using complex water-in-oil-in-water double emulsion drops and oil-in-water emulsion drops from glass capillary microfluidics and subsequent photopolymerization. Three hydrogel micromotor strategies are explored: microcapsules with thin shells and liquid cores with dispersed catalytic Pt nanoparticles, as well as water-cored microcapsules and homogeneous microparticles selectively coated with Ti/Pt catalytic layers. Autonomous motion of hydrogel particles and capsules is realized in hydrogen peroxide solutions, where generated oxygen microbubbles propel the dynamically responsive micromotors. The micromotors are balanced by weight, buoyancy, lateral capillary forces and show specific autonomous behaviours that significantly extend short range dynamic responses of hydrogels. Drop-based microfluidics represent a paradigm shift in the integration of multifunctional subsystems and high-throughput design of chemical micromachines in reasonable quantities towards their desired biomedical, environmental and flow/diffusion microreactor applications.
    Montessori, A. ; Lauricella, M. ; Stolovicki, E. ; Weitz, D. A. ; Succi, S. Jetting to dripping transition: Critical aspect ratio in step emulsifiers. Physics of Fluids 2019, 31, 021703. Publisher's VersionAbstract
    Fully three-dimensional, time-dependent, direct simulations of the non-ideal Navier-Stokes equations for a two-component fluid shed light into the mechanism which inhibits droplet breakup in step emulsifiers below a critical threshold of the width-to-height (w/h) ratio of the microfluidic nozzle. Below w/h ∼ 2.6, the simulations provide evidence of a smooth topological transition of the fluid from the confined rectangular channel geometry to an isotropic (spherical) expansion of the fluid downstream the nozzle step. Above such threshold, the transition from the inner to the outer space involves a series of dynamical rearrangements which keep the free surface in mechanical balance. Such rearrangements also induce a backflow of the ambient fluid which, in turn, leads to jet pinching and ultimately to its rupture, namely, droplet formation. The simulations show remarkable agreement with the experimental value of the threshold, which is found around w/h ∼ 2.56.
    Haney, B. ; Chen, D. ; Cai, L. - H. ; Weitz, D. ; Ramakrishnan, S. Millimeter-Size Pickering Emulsions Stabilized with Janus Microparticles. Langmuir 2019, 35, 4693–4701. Publisher's VersionAbstract

    The ability to make stable water-in-oil and oil-in-water millimeter-size Pickering emulsions is demonstrated using Janus particles—particles with distinct surface chemistries. The use of a highly cross-linked hydrophobic polymer network and the excellent water-wetting nature of a hydrogel as the hydrophobic and hydrophilic sides, respectively, permit distinct wettability on the Janus particle. Glass capillary microfluidics allows the synthesis of Janus particles with controlled sizes between 128 and 440 μm and control over the hydrophilic-to-hydrophobic domain volume ratio of the particle from 0.36 to 12.77 for a given size. It is shown that the Janus particle size controls the size of the emulsion drops, thus providing the ability to tune the structure and stability of the resulting emulsions. Stability investigations using centrifugation reveal that particles with the smallest size and a balanced hydrophilic-to-hydrophobic volume ratio (Janus ratio) form emulsions with the greatest stability against coalescence. Particles eventually jam at the interface to form nonspherical droplets. This effect is more pronounced as the hydrogel volume is increased. The large Janus particles permit facile visualization of particle-stabilized emulsions, which result in a better understanding of particle stabilization mechanisms of formed emulsions.

    Zhang, H. ; Cui, W. ; Qu, X. ; Wu, H. ; Qu, L. ; Zhang, X. ; Makila, E. ; Salonen, J. ; Zhu, Y. ; Yang, Z. ; et al. Photothermal-responsive nanosized hybrid polymersome as versatile therapeutics codelivery nanovehicle for effective tumor suppression. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 7744–7749. Publisher's VersionAbstract

    Effective cancer therapies often demand delivery of combinations of drugs to inhibit multidrug resistance through synergism, and the development of multifunctional nanovehicles with enhanced drug loading and delivery efficiency for combination therapy is currently a major challenge in nanotechnology. However, such combinations are more challenging to administer than single drugs and can require multipronged approaches to delivery. In addition to being stable and biodegradable, vehicles for such therapies must be compatible with both hydrophobic and hydrophilic drugs, and release drugs at sustained therapeutic levels. Here, we report synthesis of porous silicon nanoparticles conjugated with gold nanorods [composite nanoparticles (cNPs)] and encapsulate them within a hybrid polymersome using double-emulsion templates on a microfluidic chip to create a versatile nanovehicle. This nanovehicle has high loading capacities for both hydrophobic and hydrophilic drugs, and improves drug delivery efficiency by accumulating at the tumor after i.v. injection in mice. Importantly, a triple-drug combination suppresses breast tumors by 94% and 87% at total dosages of 5 and 2.5 mg/kg, respectively, through synergy. Moreover, the cNPs retain their photothermal properties, which can be used to significantly inhibit multidrug resistance upon near-infrared laser irradiation. Overall, this work shows that our nanovehicle has great potential as a drug codelivery nanoplatform for effective combination therapy that is adaptable to other cancer types and to molecular targets associated with disease progression.

    Vahabikashi, A. ; Park, C. Y. ; Perkumas, K. ; Zhang, Z. ; Deurloo, E. K. ; Wu, H. ; Weitz, D. A. ; Stamer, W. D. ; Goldman, R. D. ; Fredberg, J. J. ; et al. Probe Sensitivity to Cortical versus Intracellular Cytoskeletal Network Stiffness. Biophys. J. 2019, 116, 518–529. Publisher's VersionAbstract

    In development, wound healing, and pathology, cell biomechanical properties are increasingly recognized as being of central importance. To measure these properties, experimental probes of various types have been developed, but how each probe reflects the properties of heterogeneous cell regions has remained obscure. To better understand differences attributable to the probe technology, as well as to define the relative sensitivity of each probe to different cellular structures, here we took a comprehensive approach. We studied two cell types—Schlemm’s canal endothelial cells and mouse embryonic fibroblasts (MEFs)—using four different probe technologies: 1) atomic force microscopy (AFM) with sharp tip, 2) AFM with round tip, 3) optical magnetic twisting cytometry (OMTC), and 4) traction microscopy (TM). Perturbation of Schlemm’s canal cells with dexamethasone treatment, α-actinin overexpression, or RhoA overexpression caused increases in traction reported by TM and stiffness reported by sharp-tip AFM as compared to corresponding controls. By contrast, under these same experimental conditions, stiffness reported by round-tip AFM and by OMTC indicated little change. Knockout (KO) of vimentin in MEFs caused a diminution of traction reported by TM, as well as stiffness reported by sharp-tip and round-tip AFM. However, stiffness reported by OMTC in vimentin-KO MEFs was greater than in wild type. Finite-element analysis demonstrated that this paradoxical OMTC result in vimentin-KO MEFs could be attributed to reduced cell thickness. Our results also suggest that vimentin contributes not only to intracellular network stiffness but also cortex stiffness. Taken together, this evidence suggests that AFM sharp tip and TM emphasize properties of the actin-rich shell of the cell, whereas round-tip AFM and OMTC emphasize those of the noncortical intracellular network.

    Cao, T. ; Wang, Y. ; Zhao, L. ; Wang, Y. ; Tao, Y. ; Heyman, J. A. ; Weitz, D. A. ; Zhou, Y. ; Zhang, X. A simple mix-and-read bacteria detection system based on DNAzyme and molecular beacon. Chem. Commun. 2019, 55, 7358-7361. Publisher's VersionAbstract

    Here, we describe a simple mix-and-read method for the detection of specific bacterial strains that uses a DNAzyme and a molecular beacon to generate a signal. We have greatly improved upon the previously described DNAzyme-based bacteria detection method by eliminating a tedious preparation step while maintaining detection sensitivity.

    Arriaga, L. R. ; Huang, Y. ; Kim, S. - H. ; Aragones, J. L. ; Ziblat, R. ; Koehler, S. A. ; Weitz, D. A. Single-step assembly of asymmetric vesicles. Lab Chip 2019, 19, 749–756. Publisher's VersionAbstract

    Asymmetric vesicles are membranes in which amphiphiles are asymmetrically distributed between each membrane leaflet. This asymmetry dictates chemical and physical properties of these vesicles, enabling their use as more realistic models of biological cell membranes, which also are asymmetric, and improves their potential for drug delivery and cosmetic applications. However, their fabrication is difficult as the self-assembly of amphiphiles always leads to symmetric vesicles. Here, we report the use of water-in-oil-in-oil-in-water triple emulsion drops to direct the assembly of the two leaflets to form asymmetric vesicles. Different compositions of amphiphiles are dissolved in each of the two oil shells of the triple emulsion; the amphiphiles diffuse to the interfaces and adsorb differentially at each of the two oil/water interfaces of the triple emulsion. These middle oil phases dewet from the innermost water cores of the triple emulsion drops, leading to the formation of membranes with degrees of asymmetry up to 70%. The triple emulsion drops are fabricated using capillary microfluidics, enabling production of highly monodisperse drops at rates as high as 300 Hz. Vesicles produced by this method can very efficiently encapsulate many different ingredients; this further enhances the utility of asymmetric vesicles as artificial cells, bioreactors and delivery vehicles.

    Michelon, M. ; Huang, Y. ; de la Torre, L. G. ; Weitz, D. A. ; Cunha, R. L. Single-step microfluidic production of W/O/W double emulsions as templates for β-carotene-loaded giant liposomes formation. Chemical Engineering Journal 2019, 366, 27–32. Publisher's VersionAbstract

    We demonstrated the microfluidic production of W/O/W double emulsion droplets aiming formation of β-carotene-incorporated giant liposomes for food and/or pharmaceutical applications. For this purpose, glass-capillary microfluidic devices were fabricated to create a truly three-dimensional flow aiming production of giant unilamellar liposomes by solvent evaporation process after W/O/W double emulsion droplet templates formation. A great challenge of microfluidic production of monodisperse and stable W/O/W double emulsion templates for this proposal is the replacement of organic solvents potentially toxic for phospholipids dissolution. Besides, the high cost of several semi-synthetic phospholipids commonly used for giant liposome formation remains as a major technological challenge to be overcome. Thus, β-carotene-incorporated giant liposomes were generated using biocompatible solvents with low toxic potential (ethyl acetate and pentane) and non-purified soybean lecithin - a food-grade phospholipid mixture with low cost - by dewetting and evaporation of the solvents forming the oily intermediate phase of W/O/W double emulsion droplet templates. Our results showed monodisperse β-carotene-loaded giant liposomes with diameter ranging between 100 μm and 180 μm and a stability of approximately 7 days. In this way, a single-step microfluidic process with highly accurate control of size distribution was developed. This microfluidic process proposed is potentially useful for a broad range of applications in protection and delivery of active compounds.