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Advances in gene editing are leading to new medical interventions where patients' own cells are used for stem cell therapies and immunotherapies. One of the key limitations to translating these treatments to the clinic is the need for scalable technologies for engineering cells efficiently and safely. Toward this goal, microfluidic strategies to induce membrane pores and permeability have emerged as promising techniques to deliver biomolecular cargo into cells. As these technologies continue to mature, there is a need to achieve efficient, safe, nontoxic, fast, and economical processing of clinically relevant cell types. We demonstrate an acoustofluidic sonoporation method to deliver plasmids to immortalized and primary human cell types, based on pore formation and permeabilization of cell membranes with acoustic waves. This acoustofluidic-mediated approach achieves fast and efficient intracellular delivery of an enhanced green fluorescent protein-expressing plasmid to cells at a scalable throughput of 200,000 cells/min in a single channel. Analyses of intracellular delivery and nuclear membrane rupture revealed mechanisms underlying acoustofluidic delivery and successful gene expression. Our studies show that acoustofluidic technologies are promising platforms for gene delivery and a useful tool for investigating membrane repair.
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Técnicas de Transferencia de Gen , Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Sistema Hematopoyético , Células Madre , Supervivencia Celular , Citoplasma , Expresión Génica , Técnicas de Transferencia de Gen/instrumentación , Terapia Genética/instrumentación , Proteínas Fluorescentes Verdes/genética , Humanos , Células Jurkat , Plásmidos , SonidoRESUMEN
Deficiencies in either lamin B1 or lamin B2 cause both defective migration of cortical neurons in the developing brain and reduced neuronal survival. The neuronal migration abnormality is explained by a weakened nuclear lamina that interferes with nucleokinesis, a nuclear translocation process required for neuronal migration. In contrast, the explanation for impaired neuronal survival is poorly understood. We hypothesized that the forces imparted on the nucleus during neuronal migration result in nuclear membrane (NM) ruptures, causing interspersion of nuclear and cytoplasmic contents-and ultimately cell death. To test this hypothesis, we bred Lmnb1-deficient mice that express a nuclear-localized fluorescent Cre reporter. Migrating neurons within the cortical plate of E18.5 Lmnb1-deficient embryos exhibited NM ruptures, evident by the escape of the nuclear-localized reporter into the cytoplasm and NM discontinuities by electron microscopy. The NM ruptures were accompanied by DNA damage and cell death. The NM ruptures were not observed in nonmigrating cells within the ventricular zone. NM ruptures, DNA damage, and cell death were also observed in cultured Lmnb1-/- and Lmnb2-/- neurons as they migrated away from neurospheres. To test whether mechanical forces on the cell nucleus are relevant to NM ruptures in migrating neurons, we examined cultured Lmnb1-/- neurons when exposed to external constrictive forces (migration into a field of tightly spaced silicon pillars). As the cells entered the field of pillars, there were frequent NM ruptures, accompanied by DNA damage and cell death.
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Muerte Celular/fisiología , Movimiento Celular/fisiología , Lamina Tipo B/metabolismo , Neuronas/metabolismo , Membrana Nuclear/metabolismo , Lámina Nuclear/metabolismo , Animales , Línea Celular , Supervivencia Celular , Citoplasma/metabolismo , Daño del ADN , Regulación de la Expresión Génica , Lamina Tipo B/genética , Ratones , Ratones Noqueados , Neuronas/citología , Lámina Nuclear/genéticaRESUMEN
Nanoribbon- and nanowire-based field-effect transistors (FETs) have attracted significant attention due to their high surface-to-volume ratios, which make them effective as chemical and biological sensors. However, the conventional nanofabrication of these devices is challenging and costly, posing a major barrier to widespread use. We report a high-throughput approach for producing arrays of ultrathin (â¼3 nm) In2O3 nanoribbon FETs at the wafer scale. Uniform films of semiconducting In2O3 were prepared on Si/SiO2 surfaces via a sol-gel process prior to depositing Au/Ti metal layers. Commercially available high-definition digital versatile discs were employed as low-cost, large-area templates to prepare polymeric stamps for chemical lift-off lithography, which selectively removed molecules from self-assembled monolayers functionalizing the outermost Au surfaces. Nanoscale chemical patterns, consisting of one-dimensional lines (200 nm wide and 400 nm pitch) extending over centimeter length scales, were etched into the metal layers using the remaining monolayer regions as resists. Subsequent etch processes transferred the patterns into the underlying In2O3 films before the removal of the protective organic and metal coatings, revealing large-area nanoribbon arrays. We employed nanoribbons in semiconducting FET channels, achieving current on-to-off ratios over 107 and carrier mobilities up to 13.7 cm2 V-1 s-1. Nanofabricated structures, such as In2O3 nanoribbons and others, will be useful in nanoelectronics and biosensors. The technique demonstrated here will enable these applications and expand low-cost, large-area patterning strategies to enable a variety of materials and design geometries in nanoelectronics.
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Indio/química , Nanotecnología/métodos , Nanotubos de Carbono/química , Semiconductores , Técnicas Biosensibles/instrumentación , Diseño de Equipo , Oro/química , Nanotecnología/economía , Nanotecnología/instrumentación , Nanotubos de Carbono/ultraestructura , Dióxido de Silicio/química , Titanio/químicaRESUMEN
We designed and fabricated large arrays of polymer pens having sub-20 nm tips to perform chemical lift-off lithography (CLL). As such, we developed a hybrid patterning strategy called polymer-pen chemical lift-off lithography (PPCLL). We demonstrated PPCLL patterning using pyramidal and v-shaped polymer-pen arrays. Associated simulations revealed a nanometer-scale quadratic relationship between contact line widths of the polymer pens and two other variables: polymer-pen base line widths and vertical compression distances. We devised a stamp support system consisting of interspersed arrays of flat-tipped polymer pens that are taller than all other sharp-tipped polymer pens. These supports partially or fully offset stamp weights thereby also serving as a leveling system. We investigated a series of v-shaped polymer pens with known height differences to control relative vertical positions of each polymer pen precisely at the sub-20 nm scale mimicking a high-precision scanning stage. In doing so, we obtained linear-array patterns of alkanethiols with sub-50 nm to sub-500 nm line widths and minimum sub-20 nm line width tunable increments. The CLL pattern line widths were in agreement with those predicted by simulations. Our results suggest that through informed design of a stamp support system and tuning of polymer-pen base widths, throughput can be increased by eliminating the need for a scanning stage system in PPCLL without sacrificing precision. To demonstrate functional microarrays patterned by PPCLL, we inserted probe DNA into PPCLL patterns and observed hybridization by complementary target sequences.
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There is increasing interest in the study of chiral degrees of freedom occurring in matter and in electromagnetic fields. Opportunities in quantum sciences will likely exploit two main areas that are the focus of this Review: (1) recent observations of the chiral-induced spin selectivity (CISS) effect in chiral molecules and engineered nanomaterials and (2) rapidly evolving nanophotonic strategies designed to amplify chiral light-matter interactions. On the one hand, the CISS effect underpins the observation that charge transport through nanoscopic chiral structures favors a particular electronic spin orientation, resulting in large room-temperature spin polarizations. Observations of the CISS effect suggest opportunities for spin control and for the design and fabrication of room-temperature quantum devices from the bottom up, with atomic-scale precision and molecular modularity. On the other hand, chiral-optical effects that depend on both spin- and orbital-angular momentum of photons could offer key advantages in all-optical and quantum information technologies. In particular, amplification of these chiral light-matter interactions using rationally designed plasmonic and dielectric nanomaterials provide approaches to manipulate light intensity, polarization, and phase in confined nanoscale geometries. Any technology that relies on optimal charge transport, or optical control and readout, including quantum devices for logic, sensing, and storage, may benefit from chiral quantum properties. These properties can be theoretically and experimentally investigated from a quantum information perspective, which has not yet been fully developed. There are uncharted implications for the quantum sciences once chiral couplings can be engineered to control the storage, transduction, and manipulation of quantum information. This forward-looking Review provides a survey of the experimental and theoretical fundamentals of chiral-influenced quantum effects and presents a vision for their possible future roles in enabling room-temperature quantum technologies.
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Chemical lift-off lithography (CLL) is a subtractive soft-lithographic technique that uses polydimethylsiloxane (PDMS) stamps to pattern self-assembled monolayers of functional molecules for applications ranging from biomolecule patterning to transistor fabrication. A hallmark of CLL is preferential cleavage of Au-Au bonds, as opposed to bonds connecting the molecular layer to the substrate, i.e., Au-S bonds. Herein, we show that CLL can be used more broadly as a technique to pattern a variety of substrates composed of coinage metals (Pt, Pd, Ag, Cu), transition and reactive metals (Ni, Ti, Al), and a semiconductor (Ge) using straightforward alkanethiolate self-assembly chemistry. We demonstrate high-fidelity patterning in terms of precise features over large areas on all surfaces investigated. We use patterned monolayers as chemical resists for wet etching to generate metal microstructures. Substrate atoms, along with alkanethiolates, were removed as a result of lift-off, as previously observed for Au. We demonstrate the formation of PDMS-stamp-supported bimetallic monolayers by performing CLL on two different metal surfaces using the same PDMS stamp. By expanding the scope of the surfaces compatible with CLL, we advance and generalize CLL as a method to pattern a wide range of substrates, as well as to produce supported metal monolayers, both with broad applications in surface and materials science.
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Silica-coated nanoparticles are widely used in biomedical applications such as theranostics, imaging, and drug delivery. While silica-coated nanoparticles are biocompatible, experimental evidence shows that they can trigger innate immune reactions, and a broader understanding of what types of reactions are caused and how to mitigate them is needed. Herein, we investigated how the noncovalent surface functionalization of silica nanoparticles with purified proteins can inhibit nanoparticle-induced complement activation and macrophage uptake, two of the most clinically relevant innate immune reactions related to nanomedicines. Silica nanoparticles were tested alone and after coating with bovine serum albumin, human serum albumin, fibrinogen, complement factor H (FH), or immunoglobulin G (IgG) proteins. Enzyme-linked immunosorbent assays measuring the generation of various complement activation products indicated that silica nanoparticles induce complement activation via the alternative pathway. All protein coatings other than IgG protected against complement activation to varying extents. Most proteins acted as steric blockers to inhibit complement protein deposition on the nanoparticle surface, while FH coatings were biologically active and inhibited a key step in the amplification loop of complement activation, as confirmed by Western blot analysis. Flow cytometry and fluorescence microscopy experiments further revealed that complement activation-inhibiting protein coatings blunted macrophage uptake as well. Taken together, our findings demonstrate a simple and effective way to coat silica nanoparticles with purified protein coatings in order to mitigate innate immune reactions. Such methods are readily scalable and might constitute a useful strategy for improving the immunological safety profile of silica and silica-coated nanoparticles as well as other types of inorganic nanoparticles.
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Nanopartículas , Dióxido de Silicio , Activación de Complemento , Proteínas del Sistema Complemento , HumanosRESUMEN
Supported lipid membranes are versatile biomimetic coatings for the chemical functionalization of inorganic surfaces. Developing simple and effective fabrication strategies to form supported lipid membranes with micropatterned geometries is a long-standing challenge. Herein, we demonstrate how the combination of chemical lift-off lithography (CLL) and easily prepared lipid bicelle nanostructures can yield micropatterned, supported lipid membranes on gold surfaces with high pattern resolution, conformal character, and biofunctionality. Using CLL, we functionalized gold surfaces with patterned arrays of hydrophilic and hydrophobic self-assembled monolayers (SAMs). Time-lapse fluorescence microscopy imaging revealed that lipid bicelles adsorbed preferentially onto the hydrophilic SAM regions, while there was negligible lipid adsorption onto the hydrophobic SAM regions. Functional receptors could be embedded within the lipid bicelles, which facilitated selective detection of receptor-ligand binding interactions in a model streptavidin-biotin system. Quartz crystal microbalance-dissipation measurements further identified that lipid bicelles adsorb irreversibly and remain intact on top of the hydrophilic SAM regions. Taken together, our findings indicate that lipid bicelles are useful lipid nanostructures for reproducibly assembling micropatterned, supported lipid membranes with precise pattern fidelity.
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Innovative technologies for intracellular delivery are ushering in a new era for gene editing, enabling the utilization of a patient's own cells for stem cell and immunotherapies. In particular, cell-squeezing platforms provide unconventional forms of intracellular delivery, deforming cells through microfluidic constrictions to generate transient pores and to enable effective diffusion of biomolecular cargo. While these devices are promising gene-editing platforms, they require frequent maintenance due to the accumulation of cellular debris, limiting their potential for reaching the throughputs necessary for scalable cellular therapies. As these cell-squeezing technologies are improved, there is a need to develop next-generation platforms with higher throughput and longer lifespan, importantly, avoiding the buildup of cell debris and thus channel clogging. Here, we report a versatile strategy to coat the channels of microfluidic devices with lipid bilayers based on noncovalent lipid bicelle technology, which led to substantial improvements in reducing cell adhesion and protein adsorption. The antifouling properties of the lipid bilayer coating were evaluated, including membrane uniformity, passivation against nonspecific protein adsorption, and inhibition of cell attachment against multiple cell types. This surface functionalization approach was applied to coat constricted microfluidic channels for the intracellular delivery of fluorescently labeled dextran and plasmid DNA, demonstrating significant reductions in the accumulation of cell debris. Taken together, our work demonstrates that lipid bicelles are a useful tool to fabricate antifouling lipid bilayer coatings in cell-squeezing devices, resulting in reduced nonspecific fouling and cell clogging to improve performance.
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Incrustaciones Biológicas/prevención & control , Dispositivos Laboratorio en un Chip , Membrana Dobles de Lípidos/química , Adhesión Celular , Células Cultivadas , Humanos , Células Jurkat , Estructura Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
We introduce a graphene-based nanofluidic cell that facilitates in situ imaging of liquid samples via transmission electron microscopy. The cell combines the benefits of graphene liquid cells-namely, high resolution, reduced charging effects, and excellent sample stability-with the ability to introduce reactants and control fluid concentrations as provided by conventional silicon-nitride-windowed flow cells. The graphene flow cell offers significantly less window bowing compared to existing commercial holders. We demonstrate the performance of the flow cell by imaging gold nanoparticle dynamics and uranyl acetate crystallization. Our results confirm the utility of graphene flow cells in obtaining the high spatial and temporal resolution required for probing the complex dynamics of nanoparticles and nucleation pathways in aqueous solutions.
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With mounting evidence that nanomaterials can trigger adverse innate immune responses such as complement activation, there is increasing attention to the development of strategies that mask the complement-activating properties of nanomaterials. The current gold standard to reduce complement activation of nanomaterials is the covalent attachment of polymer coatings on nanomaterial surfaces, even though this strategy provides only moderate protection against complement activation. Akin to protein coronas that form on nanomaterial surfaces in physiological fluids, noncovalent strategies based on protein adsorption would offer a simplified, biomimetic approach to mitigate complement activation. Herein, we demonstrate that precoating graphene-based nanomaterials with purified, natural proteins enables regulatory control of nanomaterial-triggered complement activation. When the graphene-based nanomaterials were coated with complement factor H, nearly complete protection (>90% reduction) against complement activation (a "stealth effect") was achieved. By contrast, coating the nanomaterials with a passivating layer of bovine or human serum albumins achieved moderate protection (â¼40% reduction), whereas immunoglobulin G amplified complement activation by several-fold. Taken together, our results demonstrate that surface-bound factor H, as well as serum albumins, can prevent graphene oxide-triggered complement activation, thereby offering a facile approach to inhibit complement activation completely down to naturally occurring levels.