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Multicellular tumour cell spheroids embedded within three-dimensional (3D) hydrogels or extracellular matrices (ECM) are widely used as models to study cancer growth and invasion. Standard methods to embed spheroids in 3D matrices result in random placement in space which limits the use of inverted fluorescence microscopy techniques, and thus the resolution that can be achieved to image molecular detail within the intact spheroid. Here, we leverage UV photolithography to microfabricate PDMS (polydimethylsiloxane) stamps that allow for generation of high-content, reproducible well-like structures in multiple different imaging chambers. Addition of multicellular tumour spheroids into stamped collagen structures allows for precise positioning of spheroids in 3D space for reproducible high-/super-resolution imaging. Embedded spheroids can be imaged live or fixed and are amenable to immunostaining, allowing for greater flexibility of experimental approaches. We describe the use of these spheroid imaging chambers to analyse cell invasion, cell-ECM interaction, ECM alignment, force-dependent intracellular protein dynamics and extension of fine actin-based protrusions with a variety of commonly used inverted microscope platforms. This method enables reproducible, high-/super-resolution live imaging of multiple tumour spheroids, that can be potentially extended to visualise organoids and other more complex 3D in vitro systems.
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Neoplasias , Humanos , Neoplasias/diagnóstico por imagem , Esferoides Celulares/patologia , Colágeno , Matriz ExtracelularRESUMO
Delivery of molecules into intracellular compartments is one of the fundamental requirements in molecular biology. However, the possibility of delivering a precise number of nano-objects with single-particle resolution is still an open challenge. Here we present an electrophoretic platform based on 3D hollow nanoelectrodes to enable delivery of single nanoparticles into single selected cells and monitoring of the single-particle delivery by surface-enhanced Raman scattering (SERS). The gold-coated hollow nanoelectrode capable of confinement and enhancement of electromagnetic fields upon laser illumination can distinguish the SERS signals of a single nanoparticle flowing through the nanoelectrode. Tight wrapping of cell membranes around the nanoelectrodes allows effective membrane electroporation such that single gold nanorods are delivered on demand into a living cell by electrophoresis. The capability of the 3D hollow nanoelectrodes to porate cells and reveal single emitters from the background in continuous flow is promising for the analysis of both intracellular delivery and sampling.
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The dynamic interface between the cellular membrane and 3D nanostructures determines biological processes and guides the design of novel biomedical devices. Despite the fact that recent advancements in the fabrication of artificial biointerfaces have yielded an enhanced understanding of this interface, there remain open questions on how the cellular membrane reacts and behaves in the presence of sharp objects on the nanoscale. Here we provide a multifaceted characterization of the cellular membrane's mechanical stability when closely interacting with high-aspect-ratio 3D vertical nanostructures, providing strong evidence that vertical nanostructures spontaneously penetrate the cellular membrane to form a steady intracellular coupling only in rare cases and under specific conditions. The cell membrane is able to conform tightly over the majority of structures with various shapes while maintaining its integrity.
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Adesão Celular , Nanoestruturas/ultraestrutura , Fenômenos Biomecânicos , Linhagem Celular , Membrana Celular/ultraestrutura , Forma Celular , Eletroporação , Células HEK293 , Humanos , Miócitos Cardíacos/citologia , Nanoestruturas/química , Nanotecnologia , Propriedades de SuperfícieRESUMO
Three-dimensional vertical micro- and nanostructures can enhance the signal quality of multielectrode arrays and promise to become the prime methodology for the investigation of large networks of electrogenic cells. So far, access to the intracellular environment has been obtained via spontaneous poration, electroporation, or by surface functionalization of the micro/nanostructures; however, these methods still suffer from some limitations due to their intrinsic characteristics that limit their widespread use. Here, we demonstrate the ability to continuously record both extracellular and intracellular-like action potentials at each electrode site in spontaneously active mammalian neurons and HL-1 cardiac-derived cells via the combination of vertical nanoelectrodes with plasmonic optoporation. We demonstrate long-term and stable recordings with a very good signal-to-noise ratio. Additionally, plasmonic optoporation does not perturb the spontaneous electrical activity; it permits continuous recording even during the poration process and can regulate extracellular and intracellular contributions by means of partial cellular poration.
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Técnicas Eletroquímicas/métodos , Miócitos Cardíacos/fisiologia , Nanoestruturas/química , Neurônios/fisiologia , Potenciais de Ação , Animais , Citoplasma/metabolismo , Espaço Extracelular/fisiologia , Espaço Intracelular/fisiologia , Microeletrodos , Fenômenos Físicos , Ratos Sprague-Dawley , Razão Sinal-RuídoRESUMO
Periodontal disease is a significant burden for oral health, causing progressive and irreversible damage to the support structure of the tooth. This complex structure, the periodontium, is composed of interconnected soft and mineralised tissues, posing a challenge for regenerative approaches. Materials combining silicon and lithium are widely studied in periodontal regeneration, as they stimulate bone repair via silicic acid release while providing regenerative stimuli through lithium activation of the Wnt/ß-catenin pathway. Yet, existing materials for combined lithium and silicon release have limited control over ion release amounts and kinetics. Porous silicon can provide controlled silicic acid release, inducing osteogenesis to support bone regeneration. Prelithiation, a strategy developed for battery technology, can introduce large, controllable amounts of lithium within porous silicon, but yields a highly reactive material, unsuitable for biomedicine. This work debuts a strategy to lithiate porous silicon nanowires (LipSiNs) which generates a biocompatible and bioresorbable material. LipSiNs incorporate lithium to between 1% and 40% of silicon content, releasing lithium and silicic acid in a tailorable fashion from days to weeks. LipSiNs combine osteogenic, cementogenic and Wnt/ß-catenin stimuli to regenerate bone, cementum and periodontal ligament fibres in a murine periodontal defect.
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Nanofios , beta Catenina , Animais , Camundongos , Silício/farmacologia , Porosidade , Lítio/farmacologia , Ácido Silícico/farmacologia , Cemento DentárioRESUMO
The epicardium has recently gained interest in the cardiovascular field due to its capacity to support heart regeneration after ischemic injury. Models to study the epicardium of large animals in vitro are limited and mainly based on epicardial cell isolation/differentiation from stem cells, followed by 2D cells culture. In this method paper, we describe the procedure to obtain and culture 3D organotypic heart slices presenting an intact epicardium, as a novel model to study the epicardial physiology and activation. Epicardial slices are obtained from porcine hearts using a high-precision vibratome and retain a healthy epicardial layer embedded in its native extracellular environment and connected with other cardiac cells (cardiomyocytes, fibroblasts, vascular cells etc.). Epicardial slices can be cultured for 72 h, providing an ideal model for studying the epicardium physiology or perform pharmacological interventions/gene therapy approaches. We also report on methods to assesses the viability and composition of the epicardial slices, and evaluate their architecture in 3D through tissue decoloration. Finally, we present a potential application for a nanomaterial-based gene transfer method for tracking of epicardial cells within the slice. Crucially, given the similarity in morphology and physiology of porcine heart with its human counterpart, our system provides a platform for translational research while providing a clinically relevant and ethical alternative to the use of small animals in this type of research.
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Understanding, reproducing, and regulating the cellular and molecular processes underlying human embryogenesis is critical to improve our ability to recapitulate tissues with proper architecture and function, and to address the dysregulation of embryonic programs that underlies birth defects and cancer. The rapid emergence of stem cell technologies is enabling enormous progress in understanding embryogenesis using simple, powerful, and accessible in vitro models. Biomaterials are playing a central role in providing the spatiotemporal organisation of biophysical and biochemical signalling necessary to mimic, regulate and dissect the evolving embryonic niche in vitro. This contribution is rapidly improving our understanding of the mechanisms underlying embryonic patterning, in turn enabling the development of more effective clinical interventions for regenerative medicine and oncology. Here we highlight how key biomaterial approaches contribute to organise signalling in human embryogenesis models, and we summarise the biological insights gained from these contributions. Importantly, we highlight how nanotechnology approaches have remained largely untapped in this space, and we identify their key potential contributions.
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Materiais Biocompatíveis , Medicina Regenerativa , Desenvolvimento Embrionário , Humanos , Nanotecnologia , Células-TroncoRESUMO
BACKGROUND: Proteins are efficient supramolecular scaffolds to drive self-assembly of nanomaterials into regular colloidal structures suitable for several purposes, including cell imaging and drug delivery. Proteins, in particular, can bind to gold nanoparticles (AuNPs) through van der Waals and electrostatic forces as well as coordination and hydrogen bonds leading their assembly into responsive nanostructures. METHODS: Bioconjugation of alkyne Raman tag-labeled 20 nm AuNPs with the ring-shaped protein Peroxiredoxin (Prx), characterized by a symmetric homo-oligomeric circular arrangement, has been investigated by absorption spectroscopy, transmission and scanning electron microscopy. The plasmonic behavior of the resulting hybrid assemblies has been assessed by Surface Enhanced Raman Scattering (SERS). RESULTS: The ring-shaped Prx molecules are demonstrated to adsorb onto the gold surface acting as "sticky" bio-linkers between adjacent nanoparticles to drive self-assembly into small colloidal AuNPs arrays. The arrays show nanometric interparticle gaps tailored by the protein ring thickness. The arrays exhibit improved optical activity due to SERS allowing detection of the Raman signals from both the protein and alkyne molecules. CONCLUSIONS: This method can be used to build up SERS-active nanostructures using Prx as both a bio-linker and platform for attaching dyes, two-dimensional materials, such as graphene, and other biomolecules including DNA and enzymes. GENERAL SIGNIFICANCE: The development of colloidal SERS nanostructures is considered a significant step forward in spectroscopic bioanalysis. Though protein-tailored nanofabrication is in a childhood stage, these results demonstrate the versatility of supramolecular proteins as tools to build-up nanostructures which are still impractical to obtain through top-down techniques.
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Ouro/química , Nanopartículas Metálicas/química , Peroxirredoxinas/química , Adsorção , Alcinos/química , Animais , Modelos Moleculares , Tamanho da Partícula , Peroxirredoxinas/metabolismo , Schistosoma mansoni/enzimologia , Análise Espectral Raman , Propriedades de SuperfícieRESUMO
3D vertical nanostructures have become one of the most significant methods for interfacing cells and the nanoscale and for accessing significant intracellular functionalities such as membrane potential. As this intracellular access can be induced by means of diverse cellular membrane poration mechanisms, it is important to investigate in detail the cell condition after membrane rupture for assessing the real effects of the poration techniques on the biological environment. Indeed, differences of the membrane dynamics and reshaping have not been observed yet when the membrane-nanostructure system is locally perturbed by, for instance, diverse membrane breakage events. In this work, new insights are provided into the membrane dynamics in case of two different poration approaches, optoacoustic- and electro-poration, both mediated by the same 3D nanostructures. The experimental results offer a detailed overview on the different poration processes in terms of electrical recordings and membrane conformation.
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Membrana Celular , Nanoestruturas , Animais , Linhagem Celular , Membrana Celular/química , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Eletrofisiologia , Eletroporação , Desenho de Equipamento , Camundongos , Microeletrodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Técnicas FotoacústicasRESUMO
Gaining access to the cell interior is fundamental for many applications, such as electrical recording and drug and biomolecular delivery. A very promising technique consists of culturing cells on micro-/nanopillars. The tight adhesion and high local deformation of cells in contact with nanostructures can promote the permeabilization of lipids at the plasma membrane, providing access to the internal compartment. However, there is still much experimental controversy regarding when and how the intracellular environment is targeted and the role of the geometry and interactions with surfaces. Consequently, we investigated, by coarse-grained molecular dynamics simulations of the cell membrane, the mechanical properties of the lipid bilayer under high strain and bending conditions. We found out that a high curvature of the lipid bilayer dramatically lowers the traction force necessary to achieve membrane rupture. Afterward, we experimentally studied the permeabilization rate of the cell membrane by pillars with comparable aspect ratios but different sharpness values at the edges. The experimental data support the simulation results: even pillars with diameters in the micron range may cause local membrane disruption when their edges are sufficiently sharp. Therefore, the permeabilization likelihood is connected to the local geometric features of the pillars rather than diameter or aspect ratio. The present study can also provide significant contributions to the design of three-dimensional biointerfaces for tissue engineering and cellular growth.
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Membrana Celular , Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Nanoestruturas , TraçãoRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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Live intracellular imaging is a valuable tool in modern diagnostics and pharmacology. Surface Enhanced Raman Spectroscopy (SERS) stands out as a non-destructive and multiplexed technique, but intracellular SERS imaging still suffers from interfering background from endogenous components. Here we show the assembly of small colloidal SERS probes with Raman signal in the cell-silent window of 1800-2900 cm-1 for biorthogonal intracellular SERS imaging of dopamine that was undistinguishable from the endogenous cell background. By linking colloidal silver nanoparticles with alkyne-dopamine adducts, clusters are formed by 2-6 nanoparticles spaced by tight interparticle gaps that exhibited high electric field enhancement and strong SERS signals of alkyne and dopamines. Due to the cell-silent signals of the alkyne, intracellular in-vitro Raman imaging shows that the dopamines on the internalized clusters remain distinguishable across the cytoplasm with good spatial resolution. Our method can be a general-purpose method for real-time imaging of biomolecules, such as proteins, peptides, DNA and drugs.
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Dopamina/análise , Imagem Molecular/métodos , Análise Espectral Raman/métodos , Alcinos/química , Animais , Citoplasma/química , Nanopartículas Metálicas/química , Camundongos , Células NIH 3T3 , Prata/química , Propriedades de SuperfícieRESUMO
Biological studies on in vitro cell cultures are of fundamental importance to investigate cell response to external stimuli, such as new drugs for the treatment of specific pathologies, or to study communication between electrogenic cells. Although three-dimensional (3D) nanostructures brought tremendous improvements on biosensors used for various biological in vitro studies, including drug delivery and electrical recording, there is still a lack of multifunctional capabilities that could help gain deeper insights in several bio-related research fields. In this work, the electrical recording of large cell ensembles and the intracellular delivery of few selected cells are combined on the same device by integrating microfluidic channels on the bottom of a multi-electrode array decorated with 3D hollow nanostructures. The novel platform allows the recording of intracellular-like action potentials from large ensembles of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) and from the HL-1 line, while different molecules are selectively delivered into single/few targeted cells. The proposed approach shows high potential for enabling new comprehensive studies that can relate drug effects to network level cell communication processes.
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Técnicas Biossensoriais/instrumentação , Espaço Intracelular/metabolismo , Linhagem Celular , Fenômenos Eletrofisiológicos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Microeletrodos , Miócitos Cardíacos/citologiaRESUMO
3D nanostructures are widely exploited in cell cultures for many purposes such as controlled drug delivery, transfection, intracellular sampling, and electrical recording. However, little is known about the interaction of the cells with these substrates, and even less about the effects of electroporation on the cellular membrane and the nuclear envelope. This work exploits 3D plasmonic nanoelectrodes to study, by surface-enhanced Raman scattering (SERS), the cell membrane dynamics on the nanostructured substrate before, during, and after electroporation. In vitro cultured cells tightly adhere on 3D plasmonic nanoelectrodes precisely in the plasmonic hot spots, making this kind of investigation possible. After electroporation, the cell membrane dynamics are studied by recording the Raman time traces of biomolecules in contact or next to the 3D plasmonic nanoelectrode. During this process, the 3D plasmonic nanoelectrodes are intracellularly coupled, thus enabling the monitoring of different molecular species, including lipids, proteins, and nucleic acids. Scanning electron microscopy cross-section analysis evidences the possibility of nuclear membrane poration compatible with the reported Raman spectra. These findings may open a new route toward controlled intracellular sampling and intranuclear delivery of genic materials. They also show the possibility of nuclear envelope disruption which may lead to negative side effects.
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In the version of this Article originally published, the affiliation for the author Francesca Santoro was incorrectly given; it should have been 'Center for Advanced Biomaterials for Healthcare, Istituto Italiano di Tecnologia, Napoli, Italy'. This has now been corrected in all versions of the Article.
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The ability to monitor electrogenic cells accurately plays a pivotal role in neuroscience, cardiology and cell biology. Despite pioneering research and long-lasting efforts, the existing methods for intracellular recording of action potentials on the large network scale suffer limitations that prevent their widespread use. Here, we introduce the concept of a meta-electrode, a planar porous electrode that mimics the optical and biological behaviour of three-dimensional plasmonic antennas but also preserves the ability to work as an electrode. Its synergistic combination with plasmonic optoacoustic poration allows commercial complementary metal-oxide semiconductor multi-electrode arrays to record intracellular action potentials in large cellular networks. We apply this approach to measure signals from human-induced pluripotent stem cell-derived cardiac cells, rodent primary cardiomyocytes and immortalized cell types and demonstrate the possibility of non-invasively testing a variety of relevant drugs. Due to its robustness and easiness of use, we expect the method will be rapidly adopted by the scientific community and by pharmaceutical companies.
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Potenciais de Ação , Miócitos Cardíacos/citologia , Técnicas Fotoacústicas/instrumentação , Semicondutores , Animais , Células Cultivadas , Eletrodos , Desenho de Equipamento , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Lasers , Miócitos Cardíacos/metabolismo , Ratos Sprague-DawleyRESUMO
Electroporation of in-vitro cultured cells is widely used in biological and medical areas to deliver molecules of interest inside cells. Since very high electric fields are required to electroporate the plasma membrane, depending on the geometry of the electrodes the required voltages can be very high and often critical to cell viability. Furthermore, in traditional electroporation configuration based on planar electrodes there is no a priori certain feedback about which cell has been targeted and delivered and the addition of fluorophores may be needed to gain this information. In this study we present a nanofabricated platform able to perform intracellular delivery of membrane-impermeable molecules by opening transient nanopores into the lipid membrane of adherent cells with high spatial precision and with the application of low voltages (1.5-2 V). This result is obtained by exploiting the tight seal that the cells present with 3D fluidic hollow gold-coated nanostructures that act as nanochannels and nanoelectrodes at the same time. The final soft-electroporation platform provides an accessible approach for controlled and selective drug delivery on ordered arrangements of cells.
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Técnicas Citológicas/métodos , Sistemas de Liberação de Medicamentos/métodos , Eletroquimioterapia/métodos , Eletrodos , Eletroporação/métodos , Animais , Adesão Celular , Linhagem Celular , Membrana Celular/metabolismo , Sobrevivência Celular , Camundongos , NanoporosRESUMO
A Universal plasmonic/microfluidic platform for spatial and temporal controlled intracellular delivery is described. The system can inject/transfect the desired amount of molecules with an efficacy close to 100%. Moreover, it is highly scalable from single cells to large ensembles without administering the molecules to an extracellular bath. The latter enables quantitative control over the amount of injected molecules.
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Sistemas de Liberação de Medicamentos/métodos , Nanotubos , Animais , Sistemas de Liberação de Medicamentos/instrumentação , Espaço Intracelular/metabolismo , Dispositivos Lab-On-A-Chip , Lasers , Camundongos , Células NIH 3T3 , Imagem Óptica , Análise Espaço-TemporalRESUMO
In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here - providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures - opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes.