RESUMEN
High throughput screening of small molecules and natural products is costly, requiring significant amounts of time, reagents, and operating space. Although microarrays have proven effective in the miniaturization of screening for certain biochemical assays, such as nucleic acid hybridization or antibody binding, they are not widely used for drug discovery in cell culture due to the need for cells to internalize lipophilic drug candidates. Lipid droplet microarrays are a promising solution to this problem as they are capable of delivering lipophilic drugs to cells at dosages comparable to solution delivery. However, the scalablility of the array fabrication, assay validation, and screening steps has limited the utility of this approach. Here we take several new steps to scale up the process for lipid droplet array fabrication, assay validation in cell culture, and drug screening. A nanointaglio printing process has been adapted for use with a printing press. The arrays are stabilized for immersion into aqueous solution using a vapor coating process. In addition to delivery of lipophilic compounds, we found that we are also able to encapsulate and deliver a water-soluble compound in this way. The arrays can be functionalized by extracellular matrix proteins such as collagen prior to cell culture as the mechanism for uptake is based on direct contact with the lipid delivery vehicles rather than diffusion of the drug out of the microarray spots. We demonstrate this method for delivery to 3 different cell types and the screening of 92 natural product extracts on a microarray covering an area of less than 0.1 cm2. The arrays are suitable for miniaturized screening, for instance in high biosafety level facilities where space is limited and for applications where cell numbers are limited, such as in functional precision medicine.
Asunto(s)
Gotas Lipídicas , Humanos , Gotas Lipídicas/metabolismo , Análisis por Micromatrices/métodos , Animales , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodosRESUMEN
Lipids are known to play a vital role in the molecular organization of all cellular life. Molecular recognition is another fundamental biological process that is generally attributed to biological polymers, such as proteins and nucleic acids. However, there is evidence that aggregates of lipids and lipid-like molecules are also capable of selectively binding to or regulating the partitioning of other molecules. We previously demonstrated that a model two-phase octanol/water system can selectively partition Red 40 and Blue 1 dyes added to an aqueous phase, with the selectivity depending on the surfactant (e.g., cetyltrimethylammonium bromide) dissolved in the organic phase. Here, we elucidate the mechanism of molecular recognition in this system by using quantitative partitioning experiments and molecular dynamics (MD) simulations. Our results indicate that the selectivity for the red dye is thermodynamically favored at all surfactant concentrations, while selectivity for the blue dye is kinetically favored at high surfactant concentrations. The kinetic selectivity for the blue dye correlates with the presence of molecular aggregation at the oil-water interface. Coarse-grained MD simulations elucidate nanoscale supramolecular structures that can preferentially bind one small molecule rather than another at an interface, providing a selectively permeable barrier in the absence of proteins. The results suggest a new supramolecular mechanism for molecular recognition with potential applications in drug delivery, drug discovery, and biosensing.
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Surfactantes Pulmonares , Tensoactivos , Tensoactivos/química , Agua/química , Simulación de Dinámica Molecular , Lipoproteínas , Colorantes/químicaRESUMEN
Biological systems routinely extract and organize ions in complex yet highly ordered and active systems. Much of this function is attributed to proteins, although recent evidence indicates aggregates of lipids are also capable of molecular recognition. Here we tested the hypothesis that combinatorial mixtures of organic solutes might lead to enhanced liquid/liquid extraction. We started with liquid oleic acid as an organic phase extracting copper ions from water and added a library of additives. By using Bayesian optimization to autonomously direct the combinatorial formulation, we discovered mixtures that enhanced the extraction performance. The main additive that improved the system was octylphosphonic acid. Interestingly, the optimal mixture has a significant improvement compared to this additive alone. This suggests that the combinations of organic solutes are better than using pure components in liquid/liquid extraction. Furthermore, we found that precipitation occurs in the samples showing better extraction efficiency, which has interesting material properties and potential for new types of supramolecular biosensors.
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Odor detection and discrimination in mammals is known to be initiated by membrane-bound G-protein-coupled receptors (GPCRs). The role that the lipid membrane may play in odor discrimination, however, is less well understood. Here, we used model membrane systems to test the hypothesis that phospholipid bilayer membranes may be capable of odor discrimination. The effect of S-carvone, R-carvone, and racemic lilial on the model membrane systems was investigated. The odorants were found to affect the fluidity of supported lipid bilayers as measured by fluorescence recovery after photobleaching (FRAP). The effect of odorants on surface-supported lipid multilayer microarrays of different dimensions was also investigated. The lipid multilayer micro- and nanostructure was highly sensitive to exposure to these odorants. Fluorescently-labeled lipid multilayer droplets of 5-micron diameter were more responsive to these odorants than ethanol controls. Arrays of lipid multilayer diffraction gratings distinguished S-carvone from R-carvone in an artificial nose assay. Our results suggest that lipid bilayer membranes may play a role in odorant discrimination and molecular recognition in general.
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Lipid multilayer gratings are promising optical biosensor elements that are capable of transducing analyte binding events into changes in an optical signal. Unlike solid state transducers, reagents related to molecular recognition and signal amplification can be incorporated into the lipid grating ink volume prior to fabrication. Here we describe a strategy for functionalizing lipid multilayer gratings with a DNA aptamer for the protein thrombin that allows label-free analyte detection. A double cholesterol-tagged, double-stranded DNA linker was used to attach the aptamer to the lipid gratings. This approach was found to be sufficient for binding fluorescently labeled thrombin to lipid multilayers with micrometer-scale thickness. In order to achieve label-free detection with the sub-100 nm-thick lipid multilayer grating lines, the binding affinity was improved by varying the lipid composition. A colorimetric image analysis of the light diffracted from the gratings using a color camera was then used to identify the grating nanostructures that lead to an optimal signal. Lipid composition and multilayer thickness were found to be critical parameters for the signal transduction from the aptamer functionalized lipid multilayer gratings.
RESUMEN
Information contained in the sequences of biological polymers such as DNA and protein is crucial to determining their function. Lipids are not generally thought of as information-containing molecules. However, from a supramolecular perspective, the number of possible combinations of lipids in a mixture is comparable to the complexity of DNA or proteins. Here, we test the idea that an organic composome can exhibit molecular recognition. We use water/octanol as a model two-phase system and investigate the effect of organic solutes in different combinations in the organic phase on selective partitioning of two water-soluble dyes (Brilliant Blue FCF and Allura Red AC) from the aqueous phase into the organic phase. We found that variation in the concentration of the surfactant cetyltrimethylamonium bromide (CTAB) in the octanol phase alone was sufficient to cause a switch in selectivity, with low CTAB concentrations being selective for the red dye and high CTAB concentrations being selective for the blue dye. Other organic components were added to the organic phase to introduce molecular diversity into the composome and directed evolution was used to optimize the relative concentrations of the solutes. An improvement of selective partitioning in the heterogeneous system over the pure CTAB solution was observed. The results indicate that supramolecular composomes are sufficient for molecular recognition processes in a way analogous to nucleic acid aptamers.
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Existing clinical cell therapies, which rely on the use of biological functionalities of living cells, can be further enhanced by conjugating functional particles to the cells to form cell-particle complexes. Disk-shaped microparticles produced by the top-down microfabrication approach possess unique advantages for this application. However, none of the current mechanisms for conjugating the microfabricated microparticles to the cells are principally applicable to all types of cells with therapeutic potentials. On the other hand, membrane intercalation is a well-established mechanism for attaching fluorescent molecules to living cells or for immobilizing cells on a solid surface. This paper reports a study on conjugating disk-shaped microparticles, referred to as micropatches, to living cells through membrane intercalation for the first time. The procedure for producing the cell-micropatch complexes features an unprecedented integration of microcontact printing of micropatches, end-grafting of linear molecules of octadecyl chain and poly(ethylene glycol) to the printed micropatches, and use of gelatin as a temperature-sensitive sacrificial layer to allow the formation and subsequent release of the cell-micropatch complexes. Complexes composed of mouse neuroblastoma cells were found to be stable in vitro, and the micropatch-bound cells were viable, proliferative, and differentiable. Moreover, complexes composed of four other types of cells were produced. The membrane-intercalation mechanism and the corresponding fabrication technique developed in this study are potentially applicable to a wide range of therapeutic cells and thus promise to be useful for developing new cell therapies enhanced by the disk-shaped microparticles.
Asunto(s)
Células Madre Mesenquimatosas/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos , Micropartículas Derivadas de Células , Humanos , Microtecnología , Tamaño de la Partícula , Polietilenglicoles/química , TemperaturaRESUMEN
The burgeoning field of nanotechnology aims to create and deploy nanoscale structures, devices, and systems with novel, size-dependent properties and functions. The nanotechnology revolution has sparked radically new technologies and strategies across all scientific disciplines, with nanotechnology now applied to virtually every area of research and development in the US and globally. NanoFlorida was founded to create a forum for scientific exchange, promote networking among nanoscientists, encourage collaborative research efforts across institutions, forge strong industry-academia partnerships in nanoscience, and showcase the contributions of students and trainees in nanotechnology fields. The 2019 NanoFlorida International Conference expanded this vision to emphasize national and international participation, with a focus on advances made in translating nanotechnology. This review highlights notable research in the areas of engineering especially in optics, photonics and plasmonics and electronics; biomedical devices, nano-biotechnology, nanotherapeutics including both experimental nanotherapies and nanovaccines; nano-diagnostics and -theranostics; nano-enabled drug discovery platforms; tissue engineering, bioprinting, and environmental nanotechnology, as well as challenges and directions for future research.
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Gradient patterns comprising bioactive compounds over comparably (in regard to a cell size) large areas are key for many applications in the biomedical sector, in particular, for cell screening assays, guidance, and migration experiments. Polymer pen lithography (PPL) as an inherent highly parallel and large area technique has a great potential to serve in the fabrication of such patterns. We present strategies for the printing of functional phospholipid patterns via PPL that provide tunable feature size and feature density gradients over surface areas of several square millimeters. By controlling the printing parameters, two transfer modes can be achieved. Each of these modes leads to different feature morphologies. By increasing the force applied to the elastomeric pens, which increases the tip-surface contact area and boosts the ink delivery rate, a switch between a dip-pen nanolithography (DPN) and a microcontact printing (µCP) transfer mode can be induced. A careful inking procedure ensuring a homogeneous and not-too-high ink-load on the PPL stamp ensures a membrane-spreading dominated transfer mode, which, used in combination with smooth and hydrophilic substrates, generates features with constant height, independently of the applied force of the pens. Ultimately, this allows us to obtain a gradient of feature sizes over a mm2 substrate, all having the same height on the order of that of a biological cellular membrane. These strategies allow the construction of membrane structures by direct transfer of the lipid mixture to the substrate, without requiring previous substrate functionalization, in contrast to other molecular inks, where structure is directly determined by the printing process itself. The patterns are demonstrated to be viable for subsequent protein binding, therefore adding to a flexible feature library when gradients of protein presentation are desired.
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Polímeros/química , Tinta , Nanotecnología , Fosfolípidos , ImpresiónRESUMEN
Specific size, shape and surface chemistry influence the biological activity of nanoparticles. In the case of lipophilic nanoparticles, which are widely used in consumer products, there is evidence that particle size and formulation influences skin permeability and that lipophilic particles smaller than 6 nm can embed in lipid bilayers. Since most nanoparticle synthetic procedures result in mixtures of different particles, post-synthetic purification promises to provide insights into nanostructure-function relationships. Here we used size-selective precipitation to separate lipophilic allyl-benzyl-capped silicon nanoparticles into monodisperse fractions within the range of 1 nm to 5 nm. We measured liposomal encapsulation and cellular uptake of the monodisperse particles and found them to have generally low cytotoxicities in Hela cells. However, specific fractions showed reproducibly higher cytotoxicity than other fractions as well as the unseparated ensemble. Measurements indicate that the cytotoxicity mechanism involves oxidative stress and the differential cytotoxicity is due to enhanced cellular uptake by specific fractions. The results indicate that specific particles, with enhanced suitability for incorporation into lipophilic regions of liposomes and subsequent in vitro delivery to cells, are enriched in certain fractions.
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Nanopartículas , Silicio , Transporte Biológico , Supervivencia Celular , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas , Nanopartículas/química , Nanopartículas/ultraestructura , Tamaño de la Partícula , Silicio/químicaRESUMEN
The dynamic self-organization of lipids in biological systems is a highly regulated process that enables the compartmentalization of living systems at micro- and nanoscopic scales. Consequently, quantitative methods for assaying the kinetics of supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Here, a new nanotechnology-based method for quantitative measurements of lipid-protein interactions is presented and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1 is demonstrated. Lipid multilayer gratings are printed onto surfaces using nanointaglio and exposed to Sar1, resulting in the inflation of lipid multilayers into unilamellar structures, which can be observed in a label-free manner by monitoring the diffracted light. Local variations in lipid multilayer volume on the surface is used to vary substrate availability in a microarray format. A quantitative model is developed that allows quantification of binding affinity (K D ) and kinetics (kon and koff ). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1-induced inflation of single bilayers from surface supported multilayers, the semicylindrical grating lines are observed to remodel into semispherical buds when a critical radius of curvature is reached.
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Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/metabolismo , Fenómenos Ópticos , Cinética , Luz , Microscopía FluorescenteRESUMEN
Liquid microdroplet arrays on surfaces are a promising approach to the miniaturization of laboratory processes such as high-throughput screening. The fluid nature of these droplets poses unique challenges and opportunities in their fabrication and application, particularly for the scalable integration of multiple materials over large areas and immersion into cell culture solution. Here, we use pin spotting and nanointaglio printing to screen a library of lipids and their mixtures for their compatibility with these fabrication processes, as well as stability upon immersion into aqueous solution. More than 200 combinations of natural and synthetic oils composed of fatty acids, triglycerides, and hydrocarbons were tested for their pin-spotting and nanointaglio print quality and their ability to contain the fluorescent compound tetramethylrhodamine B isothiocyanate (TRITC) upon immersion in water. A combination of castor oil and hexanoic acid at the ratio of 1:1 (w/w) was found optimal for producing reproducible patterns that are stable upon immersion into water. This method is capable of large-scale nanomaterials integration.
RESUMEN
Lipid multilayer gratings are recently invented nanomechanical sensor elements that are capable of transducing molecular binding to fluid lipid multilayers into optical signals in a label free manner due to shape changes in the lipid nanostructures. Here, we show that nanointaglio is suitable for the integration of chemically different lipid multilayer gratings into a sensor array capable of distinguishing vapors by means of an optical nose. Sensor arrays composed of six different lipid formulations are integrated onto a surface and their optical response to three different vapors (water, ethanol and acetone) in air as well as pH under water is monitored as a function of time. Principal component analysis of the array response results in distinct clustering indicating the suitability of the arrays for distinguishing these analytes. Importantly, the nanointaglio process used here is capable of producing lipid gratings out of different materials with sufficiently uniform heights for the fabrication of an optical nose.
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Nariz Electrónica , Gases/análisis , Lípidos/química , Nanoestructuras/química , Nanotecnología/instrumentación , Fenómenos Ópticos , Humedad , Concentración de Iones de Hidrógeno , Análisis de Componente PrincipalRESUMEN
The structural organization of proteins in biological membranes can affect their function. Photosynthetic thylakoid membranes in chloroplasts have the remarkable ability to change their supramolecular organization between disordered and semicrystalline states. Although the change to the semicrystalline state is known to be triggered by abiotic factors, the functional significance of this protein organization has not yet been understood. Taking advantage of an Arabidopsis thaliana fatty acid desaturase mutant (fad5) that constitutively forms semicrystalline arrays, we systematically test the functional implications of protein crystals in photosynthetic membranes. Here, we show that the change into an ordered state facilitates molecular diffusion of photosynthetic components in crowded thylakoid membranes. The increased mobility of small lipophilic molecules like plastoquinone and xanthophylls has implications for diffusion-dependent electron transport and photoprotective energy-dependent quenching. The mobility of the large photosystem II supercomplexes, however, is impaired, leading to retarded repair of damaged proteins. Our results demonstrate that supramolecular changes into more ordered states have differing impacts on photosynthesis that favor either diffusion-dependent electron transport and photoprotection or protein repair processes, thus fine-tuning the photosynthetic energy conversion.
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Arabidopsis/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Tilacoides/metabolismo , Cristalización , Transporte de Electrón , Electrones , Recuperación de Fluorescencia tras Fotoblanqueo , Microscopía Electrónica , Mutación , Oxígeno/metabolismo , Fotosíntesis , Espectrometría de Fluorescencia , EspectrofotometríaRESUMEN
Lipid multilayer microarrays are a promising approach to miniaturize laboratory procedures by taking advantage of the microscopic compartmentalization capabilities of lipids. Here, we demonstrate a new method to pattern lipid multilayers on surfaces based on solvent evaporation along the edge where a stencil contacts a surface called evaporative edge lithography (EEL). As an example of an application of this process, we use EEL to make microarrays suitable for a cell-based migration assay. Currently existing cell migration assays require a separate compartment for each drug which is dissolved at a single concentration in solution. An advantage of the lipid multilayer microarray assay is that multiple compounds can be tested on the same surface. We demonstrate this by testing the effect of two different lipophilic drugs, Taxol and Brefeldin A, on collective cell migration into an unpopulated area. This particular assay should be scalable to test of 2000 different lipophilic compounds or dosages on a standard microtiter plate area, or if adapted for individual cell migration, it would allow for high-throughput screening of more than 50,000 compounds per plate.
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A crucial component of protein homeostasis in cells is the repair of damaged proteins. The repair of oxygen-evolving photosystem II (PS II) supercomplexes in plant chloroplasts is a prime example of a very efficient repair process that evolved in response to the high vulnerability of PS II to photooxidative damage, exacerbated by high-light (HL) stress. Significant progress in recent years has unraveled individual components and steps that constitute the PS II repair machinery, which is embedded in the thylakoid membrane system inside chloroplasts. However, an open question is how a certain order of these repair steps is established and how unwanted back-reactions that jeopardize the repair efficiency are avoided. Here, we report that spatial separation of key enzymes involved in PS II repair is realized by subcompartmentalization of the thylakoid membrane, accomplished by the formation of stacked grana membranes. The spatial segregation of kinases, phosphatases, proteases, and ribosomes ensures a certain order of events with minimal mutual interference. The margins of the grana turn out to be the site of protein degradation, well separated from active PS II in grana core and de novo protein synthesis in unstacked stroma lamellae. Furthermore, HL induces a partial conversion of stacked grana core to grana margin, which leads to a controlled access of proteases to PS II. Our study suggests that the origin of grana in evolution ensures high repair efficiency, which is essential for PS II homeostasis.
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Arabidopsis/metabolismo , Evolución Molecular , Complejo de Proteína del Fotosistema II/metabolismo , Proteolisis , Tilacoides/metabolismo , Arabidopsis/genética , Complejo de Proteína del Fotosistema II/genética , Tilacoides/genéticaRESUMEN
We report the development of a low-cost method to generate a centimetre-scale periodic array of single plasmid DNA molecules of 11 kilobase pairs. The arrayed DNA molecules are amenable to enzymatic and physical manipulations.
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ADN/química , Análisis de Secuencia por Matrices de Oligonucleótidos/economía , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Plásmidos/genética , Emparejamiento Base , Costos y Análisis de Costo , Vidrio/química , Humanos , Nanotecnología/economía , Nanotecnología/instrumentación , Nanotecnología/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Impresión/economía , Propiedades de SuperficieRESUMEN
DNA fluorescence in situ hybridization (FISH) is a powerful cytogenetic assay, but conventional sample-preparation methods for FISH do not support large-scale high-throughput data acquisition and analysis, which are potentially useful for several biomedical applications. To address this limitation, we have developed a novel FISH sample-preparation method based on generating a centimetre-sized cell array, in which all cells are precisely positioned and separated from their neighbours. This method is simple and capable of patterning nonadherent human cells. We have successfully performed DNA FISH on the single-cell arrays, which facilitates analysis of the FISH results with the FISH-FINDER computer program.