RESUMEN
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.
Asunto(s)
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
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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Using glycopolymer surfaces, we have stimulated Shewanella oneidensis bacterial colonization and induced where the bacteria attach on a molecular pattern. When adherent bacteria were rinsed with methyl α-d-mannopyranoside, the glycopolymer-functionalized surfaces retained more cells than self-assembled monolayers terminated by a single mannose unit. These results suggest that the three-dimensional multivalency of the glycopolymers both promotes and retains bacterial attachment. When the methyl α-d-mannopyranoside competitor was codeposited with the cell culture, however, the mannose-based polymer was not significantly different from bare gold surfaces. The necessity for equilibration between methyl α-d-mannopyranoside and the cell culture to remove the enhancement suggests that the retention of cells on glycopolymer surfaces is kinetically controlled and is not a thermodynamic result of the cluster glycoside effect. The MshA lectin appears to facilitate the improved adhesion observed. Our findings that the surfaces studied here can induce stable initial attachment and influence the ratio of bacterial strains on the surface may be applied to harness useful microbial communities.
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Materiales Biocompatibles Revestidos/química , Manosa/química , Polímeros/química , Shewanella/metabolismo , Resinas Acrílicas/química , Biopelículas , Adhesión Celular , Células Cultivadas , Galactanos/química , Glicosilación , Oro/química , Cinética , Lectinas/química , Mananos/química , Polimerizacion , Propiedades de Superficie , TermodinámicaRESUMEN
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
Periodontitis is a common chronic inflammatory disease that affects tooth-supporting tissues. We engineer a multifunctional periodontal membrane for the guided tissue regeneration of lost periodontal tissues. The major drawback of current periodontal membranes is the lack of tissue regeneration properties. Here, a series of nanofibrous membranes based on poly(ε-caprolactone) with tunable biochemical and biophysical properties were developed for periodontal tissue regeneration. The engineered membranes were surface coated using biomimetic polydopamine to promote the adhesion of therapeutic proteins and cells. We demonstrate successful cellular localization on the surface of the engineered membrane by morphological patterning. Polydopamine accelerates osteogenic differentiation of dental-derived stem cells by promoting hydroxyapatite mineralization. Such multiscale designs can mimic the complex extracellular environment of periodontal tissue and serve as functional tissue constructs for periodontal regeneration. In a periodontal defect model in rats, our engineered periodontal membrane successfully promoted the regeneration of periodontal tissue and bone repair. Altogether, our data demonstrate that our biomimetic membranes have potential as protein/cell delivery platforms for periodontal tissue engineering.
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Indoles/química , Células Madre Mesenquimatosas/citología , Nanofibras/química , Periodoncio/citología , Polímeros/química , Ingeniería de Tejidos/métodos , Animales , Materiales Biocompatibles/química , Regeneración Ósea , Células Cultivadas , Humanos , Masculino , Membranas Artificiales , Nanofibras/ultraestructura , Osteogénesis , Poliésteres/química , Ratas , Andamios del Tejido/químicaRESUMEN
Serotonin-based nanoparticles represent a class of previously unexplored multifunctional nanoplatforms with potential biomedical applications. Serotonin, under basic conditions, self-assembles into monodisperse nanoparticles via autoxidation of serotonin monomers. To demonstrate potential applications of polyserotonin nanoparticles for cancer therapeutics, we show that these particles are biocompatible, exhibit photothermal effects when exposed to near-infrared radiation, and load the chemotherapeutic drug doxorubicin, releasing it contextually and responsively in specific microenvironments. Quantum mechanical and molecular dynamics simulations were performed to interrogate the interactions between surface-adsorbed drug molecules and polyserotonin nanoparticles. To investigate the potential of polyserotonin nanoparticles for in vivo targeting, we explored their nano-bio interfaces by conducting protein corona experiments. Polyserotonin nanoparticles had reduced surface-protein interactions under biological conditions compared to polydopamine nanoparticles, a similar polymer material widely investigated for related applications. These findings suggest that serotonin-based nanoparticles have advantages as drug-delivery platforms for synergistic chemo- and photothermal therapy associated with limited nonspecific interactions.
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Materiales Biocompatibles/química , Portadores de Fármacos/química , Nanopartículas/química , Serotonina/química , Antineoplásicos/química , Terapia Combinada , Doxorrubicina/química , Humanos , Hipertermia Inducida , Indoles/química , Rayos Infrarrojos , Simulación de Dinámica Molecular , Nanopartículas/efectos de la radiación , Fototerapia/métodos , Polímeros/química , Corona de Proteínas/química , Células Madre/citología , Microambiente TumoralRESUMEN
The supported monolayer of Au that accompanies alkanethiolate molecules removed by polymer stamps during chemical lift-off lithography is a scarcely studied hybrid material. We show that these Au-alkanethiolate layers on poly(dimethylsiloxane) (PDMS) are transparent, functional, hybrid interfaces that can be patterned over nanometer, micrometer, and millimeter length scales. Unlike other ultrathin Au films and nanoparticles, lifted-off Au-alkanethiolate thin films lack a measurable optical signature. We therefore devised fabrication, characterization, and simulation strategies by which to interrogate the nanoscale structure, chemical functionality, stoichiometry, and spectral signature of the supported Au-thiolate layers. The patterning of these layers laterally encodes their functionality, as demonstrated by a fluorescence-based approach that relies on dye-labeled complementary DNA hybridization. Supported thin Au films can be patterned via features on PDMS stamps (controlled contact), using patterned Au substrates prior to lift-off (e.g., selective wet etching), or by patterning alkanethiols on Au substrates to be reactive in selected regions but not others (controlled reactivity). In all cases, the regions containing Au-alkanethiolate layers have a sub-nanometer apparent height, which was found to be consistent with molecular dynamics simulations that predicted the removal of no more than 1.5 Au atoms per thiol, thus presenting a monolayer-like structure.
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The electrical conductivity measured in Shewanella and Geobacter spp. is an intriguing physical property that is the fundamental basis for possible extracellular electron transport (EET) pathways. There is considerable debate regarding the origins of the electrical conductivity reported in these microbial cellular structures, which is essential for deciphering the EET mechanism. Here, we report systematic on-chip nanoelectronic investigations of both Shewanella and Geobacter spp. under physiological conditions to elucidate the complex basis of electrical conductivity of both individual microbial cells and biofilms. Concurrent electrical and electrochemical measurements of living Shewanella at both few-cell and the biofilm levels indicate that the apparent electrical conductivity can be traced to electrochemical-based electron transfer at the cell/electrode interface. We further show that similar results and conclusions apply to the Geobacter spp. Taken together, our study offers important insights into previously proposed physical models regarding microbial conductivities as well as EET pathways for Shewanella and Geobacter spp.
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Conductividad Eléctrica , Geobacter , Nanotecnología , Shewanella , Fuentes de Energía Bioeléctrica , Biopelículas , Electrodos , Transporte de ElectrónRESUMEN
Cell size control and homeostasis are fundamental features of bacterial metabolism. Recent work suggests that cells add a constant size between birth and division ("adder" model). However, it is not known how cell size homeostasis is influenced by the existence of heterogeneous microenvironments, such as those during biofilm formation. Shewanella oneidensis MR-1 can use diverse energy sources on a range of surfaces via extracellular electron transport (EET), which can impact growth, metabolism, and size diversity. Here, we track bacterial surface communities at single-cell resolution to show that not only do bacterial motility appendages influence the transition from two- to three-dimensional biofilm growth and control postdivisional cell fates, they strongly impact cell size homeostasis. For every generation, we find that the average growth rate for cells that stay on the surface and continue to divide (nondetaching population) and that for cells that detach before their next division (detaching population) are roughly constant. However, the growth rate distribution is narrow for the nondetaching population, but broad for the detaching population in each generation. Interestingly, the appendage deletion mutants (ΔpilA, ΔmshA-D, Δflg) have significantly broader growth rate distributions than that of the wild type for both detaching and nondetaching populations, which suggests that Shewanella appendages are important for sensing and integrating environmental inputs that contribute to size homeostasis. Moreover, our results suggest multiplexing of appendages for sensing and motility functions contributes to cell size dysregulation. These results can potentially provide a framework for generating metabolic diversity in S. oneidensis populations to optimize EET in heterogeneous environments.
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The microbiome presents great opportunities for understanding and improving the world around us and elucidating the interactions that compose it. The microbiome also poses tremendous challenges for mapping and manipulating the entangled networks of interactions among myriad diverse organisms. Here, we describe the opportunities, technical needs, and potential approaches to address these challenges, based on recent and upcoming advances in measurement and control at the nanoscale and beyond. These technical needs will provide the basis for advancing the largely descriptive studies of the microbiome to the theoretical and mechanistic understandings that will underpin the discipline of microbiome engineering. We anticipate that the new tools and methods developed will also be more broadly useful in environmental monitoring, medicine, forensics, and other areas.