Sun, Z. ; Yang, C. ; Eggersdorfer, M. ; Cui, J. ; Li, Y. ; Hai, M. ; Chen, D. ; Weitz, D. A. A General Strategy for One-Step Fabrication of Biocompatible Microcapsules with Controlled Active Release. Chinese Chemical Letters 2020, 31, 249-252. Publisher's VersionAbstract
Fabrication of biocompatible core-shell microcapsules in a controllable and scalable manner remains an important but challenging task. Here, we develop a one-step microfluidic approach for the high-throughput production of biocompatible microcapsules, which utilizes single emulsions as templates and controls the precipitation of biocompatible polymer at the water/oil interface. The facile method enables the loading of various oils in the core and the enhancement of polymer shell strength by polyelectrolyte coating. The resulting microcapsules have the advantages of controllability, scalability, biocompatibility, high encapsulation efficiency and high loading capacity. The core-shell microcapsules are ideal delivery vehicles for programmable active release and various controlled release mechanisms are demonstrated, including burst release by vigorous shaking, pH-triggered release for targeted intestinal release and sustained release of perfume over a long period of time. The utility of our technique paves the way for practical applications of core-shell microcapsules.
Haney, B. ; Werner, J. G. ; Weitz, D. A. ; Ramakrishnan, S. Absorbent-Adsorbates: Large Amphiphilic Janus Microgels as Droplet Stabilizers. ACS Applied Materials & Interfaces 2020, 12, 33439-33446. Publisher's VersionAbstract
Microgel particles are cross-linked polymer networks that absorb certain liquids causing network expansion. The type of swelling-fluid and extent of volume change depends on the polymer-liquid interaction and the network’s cross-link density. These colloidal gels can be used to stabilize emulsion drops by adsorbing to the interface of two immiscible fluids. However, to enhance the adsorption abilities of these predominantly hydrophilic gel particles, some degree of hydrophobicity is needed. An amphiphilic Janus microgel with spatially distinct lipophilic and hydrophilic sides is desired. Here, we report the fabrication of polyethylene glycol diacrylate/ polypropylene glycol diacrylate Janus microgels (JM) using microfluidic drop making. The flow streams of the two separate and immiscible monomer solutions are brought into contact and intersected by a third immiscible fluid in a flow-focusing junction to form Janus droplets. The individual droplets are crosslinked via UV irradiation to form monodispersed microgel particles with opposing hydrophilic and hydrophobic 3D-networked polymer matrices. By combining two chemically different polymer gel networks, an amphiphilic emulsion stabilizer is formed that adsorbs to the oil/water interface while its faces absorb their respective water or hydrocarbon solvents. Both particle sides swell at the liquid/liquid interface as water in oil emulsions are stabilized and destabilized via thermal responsive hydrogel. Stimuli responsive droplets are demonstrated by adding a short chain oligo ethylene glycol acrylate molecule to the hydrogel formulation on the Janus microgel particle. Droplets stabilized by these particles experience a sudden increase in droplet diameter around 60˚C. This work with absorbent particles may prove useful for applications in bio catalysis, fuel production, and oil transportation.
Cohen, L. ; Cui, N. ; Cai, Y. ; Garden, P. M. ; Li, X. ; Weitz, D. A. ; Walt, D. R. Single Molecule Protein Detection with Attomolar Sensitivity Using Droplet Digital Enzyme-Linked Immunosorbent Assay. ACS Nano 2020, 14, 9491-9501. Publisher's VersionAbstract
Many proteins are present at low concentrations in biological samples, and therefore, techniques for ultrasensitive protein detection are necessary. To overcome challenges with sensitivity, the digital enzyme-linked immunosorbent assay (ELISA) was developed, which is 1000× more sensitive than conventional ELISA and allows sub-femtomolar protein detection. However, this sensitivity is still not sufficient to measure many proteins in various biological samples, thereby limiting our ability to detect and discover biomarkers. To overcome this limitation, we developed droplet digital ELISA (ddELISA), a simple approach for detecting low protein levels using digital ELISA and droplet microfluidics. ddELISA achieves maximal sensitivity by improving the sampling efficiency and counting more target molecules. ddELISA can detect proteins in the low attomolar range and is up to 25-fold more sensitive than digital ELISA using Single Molecule Arrays (Simoa), the current gold standard tool for ultrasensitive protein detection. Using ddELISA, we measured the LINE1/ORF1 protein, a potential cancer biomarker that has not been previously measured in serum. Additionally, due to the simplicity of our device design, ddELISA is promising for point-of-care applications. Thus, ddELISA will facilitate the discovery of biomarkers that have never been measured before for various clinical applications.
Wu, B. ; Yang, C. ; Li, B. ; Feng, L. ; Hai, M. ; Zhao, C. - X. ; Chen, D. ; Liu, K. ; Weitz, D. A. Active Encapsulation in Biocompatible Nanocapsules. Small 2020, 16, 2002716. Publisher's VersionAbstract
Co‐precipitation is generally refers to the co‐precipitation of two solids and is widely used to prepare active‐loaded nanoparticles. Here, it is demonstrated that liquid and solid can precipitate simultaneously to produce hierarchical core–shell nanocapsules that encapsulate an oil core in a polymer shell. During the co‐precipitation process, the polymer preferentially deposits at the oil/water interface, wetting both the oil and water phases; the behavior is determined by the spreading coefficients and driven by the energy minimization. The technique is applicable to directly encapsulate various oil actives and avoid the use of toxic solvent or surfactant during the preparation process. The obtained core–shell nanocapsules harness the advantage of biocompatibility, precise control over the shell thickness, high loading capacity, high encapsulation efficiency, good dispersity in water, and improved stability against oxidation. The applications of the nanocapsules as delivery vehicles are demonstrated by the excellent performances of natural colorant and anti‐cancer drug‐loaded nanocapsules. The core–shell nanocapsules with a controlled hierarchical structure are, therefore, ideal carriers for practical applications in food, cosmetics, and drug delivery.
Wu, H. ; Shen, Y. ; Wang, D. ; Herrmann, H. ; Goldman, R. D. ; Weitz, D. A. Effect of divalent cations on the structure and mechanics of vimentin intermediate filaments. Biophysical Journal 2020, 119, 55-64. Publisher's VersionAbstract
Divalent cations behave as effective cross-linkers of intermediate filaments (IFs) such as vimentin IF (VIF). These interactions have been mostly attributed to their multivalency. However, ion-protein interactions often depend on the ion species, and these effects have not been widely studied in IFs. Here, we investigate the effects of two biologically important divalent cations, Zn2+ and Ca2+, on VIF network structure and mechanics in vitro. We find that the network structure is unperturbed at micromolar Zn2+ concentrations, but strong bundle formation is observed at a concentration of 100 μM. Microrheological measurements show that network stiffness increases with cation concentration. However, bundling of filaments softens the network. This trend also holds for VIF networks formed in the presence of Ca2+, but remarkably, a concentration of Ca2+ that is two orders higher is needed to achieve the same effect as with Zn2+, which suggests the importance of salt-protein interactions as described by the Hofmeister effect. Furthermore, we find evidence of competitive binding between the two divalent ion species. Hence, specific interactions between VIFs and divalent cations are likely to be an important mechanism by which cells can control their cytoplasmic mechanics.
Zhang, W. ; Zhao, X. ; Yuan, Y. ; Miao, F. ; Li, W. ; Ji, S. ; Huang, X. ; Chen, X. ; Jiang, T. ; Weitz, D. A. ; et al. Microfluidic Synthesis of Multimode Au@CoFeB-Rg3 Nanomedicines and Their Cytotoxicity and Anti-Tumor Effects. Chemistry Materials 2020, 32, 5044-5056. Publisher's VersionAbstract
Nanomedicines (i.e., Au@CoFeB-Rg3) were developed by conjugating multimode nanohybrids with active ingredients of natural herbs using Au@CoFeB nanoparticles as one model of multimode nanohybrids and the ginsenoside Rg3 as one model of active ingredients of natural herbs. Au@CoFeB nanoparticles were first synthesized using a temperatureprogrammed microfluidics process. Then, the surface of Au@ CoFeB nanoparticles was modified via an amino-silane coupling agent of (3-aminopropyl) trimethoxysilane (APTMS) and then activated by the bifunctional amine-active cross-linker. They were thereafter conjugated to ginsenosides preactivated by APTMS by cross-linking the surface-activated nanohybrids, forming Au@ CoFeB-Rg3 nanomedicines. Their multimode imaging functions were evaluated with the characterization of their magnetic and optical properties and the response to X-ray radiation. They can be optically detected via dark-field microscopy and can be imaged through X-ray computed tomography. They can also be used as magnetic resonance imaging contrast agents with excellent T2-weighted spin−echo imaging effects. Au@CoFeB-Rg3 nanomedicines exhibited distinct cytotoxicity and inhibitory effects on the proliferation of human hepatocellular carcinoma cells (HepG2/C3) and human chronic myeloid leukemia cells (K562) but were less toxic to 3T3 cells than other cells at concentrations more than 200 μg/ mL. However, Au@CoFeB nanoparticles showed markedly lower cytotoxicity and inhibitory effects on the proliferation of these cell lines, particularly at concentrations <100 μg/mL, than Au@CoFeB-Rg3 nanomedicines. Clearly, there is a distinct synergistic effect between nanohybrids and Rg3. Additionally, Au@CoFeB nanohybrids showed almost no toxicity to Jurkat-CT cells at low concentrations (47 μg/mL), indicating that they may be used as multimode nanoprobes at a suitable concentration. These findings provide an efficient alternative for the synthesis of multifunctional antitumor nanomedicines based on multimode nanohybrids and active ingredients of natural resources.
Xia, J. ; Yuan, Y. ; Wu, H. ; Huang, Y. ; Weitz, D. A. Decoupling the effects of nanopore size and surface roughness on the attachment, spreading and differentiation of bone marrow-derived stem cells. Biomaterials 2020, 248, 120014. Publisher's VersionAbstract
The nanopore size and roughness of nanoporous surface are two critical variables in determining stem cell fate, but little is known about the contribution from each cue individually. To address this gap, we use two-dimensional nanoporous membranes with controlled nanopore size and roughness to culture bone marrow-derived mesenchymal stem cells (BMSCs), and study their behaviors such as attachment, spreading and differentiation. We find that increasing the roughness of nanoporous surface has no noticeable effect on cell attachment, and only slightly decreases cell spreading areas and inhibits osteogenic differentiation. However, BMSCs cultured on membranes with larger nanopores have significantly fewer attached cells and larger spreading areas. Moreover, these cells cultured on larger nanopores undergo enhanced osteogenic differentiation by expressing more alkaline phosphatase, osteocalcin, osteopontin, and secreting more collagen type I. These results suggest that although both nanopore size and roughness can affect BMSCs, nanopore size plays a more significant role than roughness in controlling BMSC behavior.
Adams, L. L. A. ; Lee, D. ; Mei, Y. F. ; Weitz, D. A. ; Solovev, A. A. Nanoparticle‐Shelled Catalytic Bubble Micromotor. Advanced Materials Interfaces 2020, 7 1901583. Publisher's VersionAbstract
Nanoparticle‐shelled bubbles, prepared with glass capillary microfluidics, are functionalized to produce catalytic micromotors that exhibit novel assembly and disassembly behaviors. Stable microbubble rafts are assembled at an air–solvent interface of nonaqueous propylene carbonate (PC) solvent by creating a meniscus using a glass capillary. Upon the addition of hydrogen peroxide fuel, catalytic microbubbles quickly break free from the bubble raft by repelling from each other and self‐propelling at the air–fuel interface (a mixture of PC and aqueous hydrogen peroxide). While most of micromotors generate oxygen bubbles on the outer catalytic shell, some micromotors contain cracks and eject bubbles from the hollow shells containing air. Nanoparticle‐shelled bubbles with a high buoyancy force are particularly attractive for studying novel propulsion modes and interactions between catalytic bubble micromotors at air–fuel interfaces.
Tiribocchi, A. ; Montessori, A. ; Aime, S. ; Milani, M. ; Lauricella, M. ; Succi, S. ; Weitz, D. Novel nonequilibrium steady states in multiple emulsions. Physics of Fluids 2020, 32, 017102. Publisher's VersionAbstract
We numerically investigate the rheological response of a noncoalescing multiple emulsion under a symmetric shear flow. We find that the dynamics significantly depends on the magnitude of the shear rate and on the number of the encapsulated droplets, two key parameters whose control is fundamental to accurately select the resulting nonequilibrium steady states. The double emulsion, for instance, attains a static steady state in which the external droplet stretches under flow and achieves an elliptical shape (closely resembling the one observed in a sheared isolated fluid droplet), while the internal one remains essentially unaffected. Novel nonequilibrium steady states arise in a multiple emulsion. Under low/moderate shear rates, for instance, the encapsulated droplets display a nontrivial planetarylike motion that considerably affects the shape of the external droplet. Some features of this dynamic behavior are partially captured by the Taylor deformation parameter and the stress tensor. Besides a theoretical interest on its own, our results can potentially stimulate further experiments, as most of the predictions could be tested in the lab by monitoring droplets’ shapes and position over time.
Nawar, S. ; Stolaroff, J. K. ; Ye, C. ; Wu, H. ; Nguyen, D. T. ; Xin, F. ; Weitz, D. A. Parallelizable microfluidic dropmakers with multilayer geometry for the generation of double emulsions. Lab on a Chip 2020, 20, 147–154. Publisher's VersionAbstract
We present a multilayer dropmaker geometry that enables the modular fabrication of microfluidic devices containing precisely patterned channel surface wettability. The platform is used for the scalable production of uniform double emulsion drops. , Microfluidic devices enable the production of uniform double emulsions with control over droplet size and shell thickness. However, the limited production rate of microfluidic devices precludes the use of monodisperse double emulsions for industrial-scale applications, which require large quantities of droplets. To increase throughput, devices can be parallelized to contain many dropmakers operating simultaneously in one chip, but this is challenging to do for double emulsion dropmakers. Production of double emulsions requires dropmakers to have both hydrophobic and hydrophilic channels, requiring spatially precise patterning of channel surface wettability. Precise wettability patterning is difficult for devices containing multiple dropmakers, posing a significant challenge for parallelization. In this paper, we present a multilayer dropmaker geometry that greatly simplifies the process of producing microfluidic devices with excellent spatial control over channel wettability. Wettability patterning is achieved through the independent functionalization of channels in each layer prior to device assembly, rendering the dropmaker with a precise step between hydrophobic and hydrophilic channels. This device geometry enables uniform wettability patterning of parallelized dropmakers, providing a scalable approach for the production of double emulsions.
Shi, W. ; Chen, X. ; Li, B. ; Weitz, D. A. Spontaneous Creation of Anisotropic Polymer Crystals with Orientation-Sensitive Birefringence in Liquid Drops. ACS Applied Materials & Interfaces 2020, 12, 3912-3918. Publisher's VersionAbstract
It remains a grand challenge to prepare anisotropic crystal superstructures with sensitive optical properties in polymer science and materials field. This study demonstrates that semicrystalline polymers develop into anisotropic hollow spherulitic crystals spontaneously at interfaces of liquid drops. In contrast to conventional spherulites with centrosymmetric optics and grain boundaries, these anisotropic spherulitic crystals have vanished boundary defects, tunable aspect ratios, and noncentrosymmetric, orientationsensitive birefringence. The experimental finding is elaborated in poly(L-lactic acid) crystals and is further verified in a broad class of semicrystalline polymers, irrespective of molecular chirality, chemical constitution, or interfacial modification. The facile methods and general mechanism revealed in this study shed light on developing new types of optical microdevices and synthesis of anisotropic semicrystalline particles from liquid emulsions.
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.