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Conjugation of biomolecules on the surface of nanoparticles (NPs) to achieve active targeting is widely investigated within the scientific community. However, while a basic framework of the physicochemical processes underpinning bionanoparticle recognition is now emerging, the precise evaluation of the interactions between engineered NPs and biological targets remains underdeveloped. Here, we show how the adaptation of a method currently used to evaluate molecular ligand-receptor interactions by quartz crystal microbalance (QCM) can be used to obtain concrete insights into interactions between different NP architectures and assemblies of receptors. Using a model bionanoparticle grafted with oriented apolipoprotein E (ApoE) fragments, we examine key aspects of bionanoparticle engineering for effective interactions with target receptors. We show that the QCM technique can be used to rapidly measure construct-receptor interactions across biologically relevant exchange times. We contrast random adsorption of the ligand at the surface of the NPs, resulting in no measurable interaction with target receptors, to grafted oriented constructs, which are strongly recognized even at lower graft densities. The effects of other basic parameters impacting the interaction such as ligand graft density, receptor immobilization density, and linker length were also efficiently evaluated with this technique. Dramatic changes in interaction outcomes with subtle alterations in these parameters highlight the general importance of measuring the interactions between engineered NPs and target receptors ex situ early on in the construct development process for the rational design of bionanoparticles.
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The progress achieved over the last three decades in the field of bioconjugation has enabled the preparation of sophisticated nanomaterial-biomolecule conjugates, referred to herein as bionanoconstructs, for a multitude of applications including biosensing, diagnostics, and therapeutics. However, the development of bionanoconstructs for the active targeting of cells and cellular compartments, both in vitro and in vivo, is challenged by the lack of understanding of the mechanisms governing nanoscale recognition. In this review, we highlight fundamental obstacles in designing a successful bionanoconstruct, considering findings in the field of bionanointeractions. We argue that the biological recognition of bionanoconstructs is modulated not only by their molecular composition but also by the collective architecture presented upon their surface, and we discuss fundamental aspects of this surface architecture that are central to successful recognition, such as the mode of biomolecule conjugation and nanomaterial passivation. We also emphasize the need for thorough characterization of engineered bionanoconstructs and highlight the significance of population heterogeneity, which too presents a significant challenge in the interpretation of in vitro and in vivo results. Consideration of such issues together will better define the arena in which bioconjugation, in the future, will deliver functional and clinically relevant bionanoconstructs.
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Productos Biológicos , NanoestructurasRESUMEN
Silica nanoparticles (SiNP) trigger a range of innate immune responses in relevant essential organs, such as the liver and the lungs. Inflammatory reactions, including NLRP3 inflammasome activation, have been linked to particulate materials; however, the molecular mechanisms and key actors remain elusive. Although many receptors, including several scavenger receptors, were suggested to participate in SiNP cellular uptake, mechanistic evidence of their role on innate immunity is lacking. Here we present an atomic force microscopy-based approach to physico-mechanically map the specific interaction occurring between nanoparticles and scavenger receptor A1 (SRA1) in vitro on living lung epithelial cells. We find that SiNP recognition by SRA1 on human macrophages plays a key role in mediating NLRP3 inflammasome activation, and we identify cellular mechanical changes as clear indicators of inflammasome activation in human macrophages, greatly advancing our knowledge on the interplay among nanomaterials and innate immunity.
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Inflamasomas , Nanopartículas , Humanos , Proteína con Dominio Pirina 3 de la Familia NLR , Macrófagos/metabolismo , Inmunidad Innata , Dióxido de Silicio/metabolismoRESUMEN
Despite the high level of interest in bio-nano interactions, detailed intracellular mechanisms that govern nanoscale recognition and signalling still need to be unravelled. Magnetic nanoparticles (NPs) are valuable tools for elucidating complex intracellular bio-nano interactions. Using magnetic NPs, it is possible to isolate cell compartments that the particles interact with during intracellular trafficking. Studies at the subcellular scale rely heavily on optical microscopy; therefore, combining the advantages of magnetic recovery with excellent imaging properties to allow intracellular NP tracking is of utmost interest for the nanoscience field. However, it is a challenge to prepare highly magnetic NPs with a suitable fluorescence for the fluorescence imaging techniques typically used for biological studies. Here we present the synthesis of biocompatible multifunctional superparamagnetic multicore NPs with a bright fluorescent silica shell. The incorporation of an organic fluorophore in the silica surrounding the magnetic multicore was optimised to enable the particles to be tracked with the most common imaging techniques. To prevent dye loss resulting from silica dissolution in biological environments, which would reduce the time that the particles could be tracked, we added a thin dense encapsulating silica layer to the NPs which is highly stable in biological media. The synthesised multifunctional nanoparticles were evaluated in cell uptake experiments in which their intracellular location could be clearly identified using fluorescence imaging microscopy, even after 3 days. The magnetic properties of the iron oxide core enabled both efficient recovery of the NPs from the intracellular environment and the extraction of cell compartments involved in their intracellular trafficking. Thus, the NPs reported here provide a promising tool for the study of the processes regulating bio-nano interactions.
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Nanopartículas Multifuncionales , Nanopartículas , Colorantes Fluorescentes , Nanopartículas Magnéticas de Óxido de Hierro , Dióxido de SilicioRESUMEN
Fabricating future materials by self-assembly of nano-building blocks programmed to generate specific lattices is among the most challenging goals of nanotechnology and has led to the recent concept of patchy particles. We report here a simple strategy to fabricate polystyrene nanoparticles with several silica patches based on the solvent-induced self-assembly of silica/polystyrene monopods. The latter are obtained with morphological yields as high as 99% by seed-growth emulsion polymerization of styrene in the presence of 100 nm silica seeds previously modified with an optimal surface density of methacryloxymethyl groups. In addition, we fabricate "magnetic" silica seeds by silica encapsulation of preformed maghemite supraparticles. The polystyrene pod, i.e., surface nodule, serves as a sticky point when the monopods are incubated in a bad/good solvent mixture for polystyrene, e.g., ethanol/tetrahydrofuran mixtures. After self-assembly, mixtures of particles with two, three, four silica or magnetic silica patches are mainly obtained. The influence of experimental parameters such as the ethanol/tetrahydrofuran volume ratio, monopod concentration and incubation time is studied. Further developments would consist of obtaining pure batches by centrifugal sorting and optimizing the relative position of the patches in conventional repulsion figures.
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The field of nanomedicine has the potential to be a game-changer in global health, with possible applications in prevention, diagnostics, and therapeutics. However, despite extensive research focus and funding, the forecasted explosion of novel nanomedicines is yet to materialize. We believe that clinical translation is ultimately hampered by a lack of understanding of how nanoparticles really interact with biological systems. When placed in a biological environment, nanoparticles adsorb a biomolecular layer that defines their biological identity. The challenge for bionanoscience is therefore to understand the evolution of the interactions of the nanoparticle-biomolecules complex as the nanoparticle is trafficked through the intracellular environment. However, to progress on this route, scientists face major challenges associated with isolation of specific intracellular compartments for analysis, complicated by the diversity of trafficking events happening simultaneously and the lack of synchronization between individual events. In this perspective article, we reflect on how magnetic nanoparticles can help to tackle some of these challenges as part of an overall workflow and act as a useful platform to investigate the bionano interactions within the cell that contribute to this nanoscale decision making. We discuss both established and emerging techniques for the magnetic extraction of nanoparticles and how they can potentially be used as tools to study the intracellular journey of nanomaterials inside the cell, and their potential to probe nanoscale decision-making events. We outline the inherent limitations of these techniques when investigating particular bio-nano interactions along with proposed strategies to improve both specificity and resolution. We conclude by describing how the integration of magnetic nanoparticle recovery with sophisticated analysis at the single-particle level could be applied to resolve key questions for this field in the future.
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Here we present a blood-brain barrier (BBB) model that enables high-resolution imaging of nanoparticle (NP) interactions with endothelial cells and the capture of rare NP translocation events. The enabling technology is an ultrathin silicon nitride (SiN) membrane (0.5 µm pore size, 20% porosity, 400 nm thickness) integrated into a dual-chamber platform that facilitates imaging at low working distances (â¼50 µm). The platform, the µSiM-BBB (microfluidic silicon membrane-BBB), features human brain endothelial cells and primary astrocytes grown on opposite sides of the membrane. The human brain endothelial cells form tight junctions on the ultrathin membranes and exhibit a significantly higher resistance to FITC-dextran diffusion than commercial membranes. The enhanced optical properties of the SiN membrane allow high-resolution live-cell imaging of three types of NPs, namely, 40 nm PS-COOH, 100 nm PS-COOH, and apolipoprotein E-conjugated 100 nm SiO2, interacting with the BBB. Despite the excellent barrier properties of the endothelial layer, we are able to document rare NP translocation events of NPs localized to lysosomal compartments of astrocytes on the "brain side" of the device. Although the translocation is always low, our data suggest that size and targeting ligand are important parameters for NP translocation across the BBB. As a platform that enables the detection of rare transmission across tight BBB layers, the µSiM-BBB is an important tool for the design of nanoparticle-based delivery of drugs to the central nervous system.
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Barrera Hematoencefálica/metabolismo , Modelos Biológicos , Nanopartículas/metabolismo , Imagen Óptica , Silicio/metabolismo , Transporte Biológico , Barrera Hematoencefálica/química , Línea Celular , Técnicas de Cocultivo , Humanos , Nanopartículas/química , Tamaño de la Partícula , Silicio/química , Propiedades de SuperficieRESUMEN
We know surprisingly little about the long-term outcomes for nanomaterials interacting with organisms. To date, most of what we know is derived from in vivo studies that limit the range of materials studied and the scope of advanced molecular biology tools applied. Long-term in vitro nanoparticle studies are hampered by a lack of suitable models, as standard cell culture techniques present several drawbacks, while technical limitations render current three-dimensional (3D) cellular spheroid models less suited. Now, by controlling the kinetic processes of cell assembly and division in a non-Newtonian culture medium, we engineer reproducible cell clusters of controlled size and phenotype, leading to a convenient and flexible long-term 3D culture that allows nanoparticle studies over many weeks in an in vitro setting. We present applications of this model for the assessment of intracellular polymeric and silica nanoparticle persistence and found that hydrocarbon-based polymeric nanoparticles undergo no apparent degradation over long time periods with no obvious biological impact, while amorphous silica nanoparticles degrade at different rates over several weeks, depending on their synthesis method.
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Técnicas de Cultivo de Célula , Nanopartículas/química , Polímeros/química , Dióxido de Silicio/química , Células A549 , Proliferación Celular , Humanos , Cinética , Tamaño de la Partícula , Fenotipo , Factores de TiempoRESUMEN
Due to the wealth of actors involved in the development of atherosclerosis, molecular imaging based on the targeting of specific markers would substantiate the diagnosis of life-threatening atheroma plaques. To this end, TEG4 antibody is a promising candidate targeting the activated platelets (integrin αIIbß3) highly represented within the plaque. In this study, scFv antibody fragments were used to functionalize multimodal imaging nanoparticles. This grafting was performed in a regio-selective way to preserve TEG4 activity and the avidity of the nanoparticles was studied with respect to the number of grafted antibodies. Subsequently, taking advantage of the nanoparticle bimodality, both near infrared fluorescence and magnetic resonance imaging of the atheroma plaque were performed in the ApoE-/- mouse model. Here we describe the design of the targeted nanoparticles, and a quantification method for their detection in mice, both ex vivo and in vivo, highlighting their value as a potential diagnosis agent.
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Aterosclerosis/diagnóstico , Imagen Molecular , Imagen Multimodal , Nanopartículas/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Aterosclerosis/patología , Fluorescencia , Imagen por Resonancia Magnética , Masculino , Ratones , Conejos , Distribución TisularRESUMEN
We report a new route to synthesize clusters, or so-called colloidal molecules (CMs), which mimic the symmetry of molecular structures made of one central atom. We couple site-specifically functionalized patchy nanoparticles, i.e., valence-endowed colloidal atoms (CAs), with complementary nanospheres through amide bonds. By analogy with the Gillespie formalism, we show that AX4, AX3E1 and AX2E2 CMs can be obtained from tetravalent sp3-like CAs when the relative amount of both building units is varied in a controlled manner. We obtain AX2 CMs from divalent sp-like CAs. We also show that it is possible to covalently attach two different types of satellites to the same central patchy nanoparticle to create more complex CMs, opening the way to the fabrication of new multifunctional nanostructures with well-controlled shape and composition.
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Key practical challenges such as understanding the immunological processes at the nanoscale and controlling the targeting and accumulation of nano-objects in vivo now further stimulate efforts to underpin phenomenological knowledge of the nanoscale with more mechanistic and molecular insight. Thus, the question as to what constitutes nanoscale biological identity continues to evolve. Certainly nanoparticles in contact with a complex biological milieu develop a biological identity, differing from the original nanomaterial, now referred to as the "biomolecular corona". However, this surface-adsorbed layer of biomolecules may in some circumstance lead to different forms of receptor-particle interactions not evident only from the identity of the surface-adsorbed biomolecules and hard to predict or detect by current physicochemical methods. Here we show that scavenger receptors may recognize complex as yet unidentified biomolecular surface layer motifs, even when no current physicochemical analysis is capable of doing so. For instance, fluorescently labeled SiO2 nanoparticles in a biological milieu are strongly recognized by the macrophage receptor with collagenous structure (MARCO) in even dense biological media (human serum) apparently using a form of binding with which most of the MARCO's known ligands ( e. g., LPS, modified LDL) fail to compete. Such observations may suggest the need for a much stronger emphasis on nanoscale receptor-corona and other biomolecular interaction studies if one wishes to unravel how biomolecular recognition drives outcomes in the nanoscale biological domain.
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Lipoproteínas LDL/química , Nanopartículas/química , Nanopartículas/metabolismo , Corona de Proteínas/química , Receptores Inmunológicos/metabolismo , Dióxido de Silicio/química , Adsorción , Animales , Bovinos , Expresión Génica , Células HEK293 , Humanos , Dominios Proteicos , Desplegamiento Proteico , Receptores Inmunológicos/genética , Albúmina Sérica Bovina/química , Propiedades de Superficie , TransfecciónRESUMEN
The data presented in this article are related to the publication entitled "Iron oxide core oil-in-water nanoemulsion as tracer for atherosclerosis MPI and MRI imaging" (Prévot et al., 2017) [1]. Herein we describe the synthesis and the characteristics of the Superparamagnetic Iron Oxide Nanoparticles (SPION) loaded inside nanoemulsions (NEs). Focus was set on obtaining SPION with narrow size distribution and close to superparamagnetic limit (20 nm) in order to reach a reasonable magnetic signal. Nanoparticles (NPs) of three different sizes were obtained (7, 11 and 18 nm) and characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), diffuse reflectance infrared Fourier transform (DRIFT) and thermogravimetric analysis (TGA). SPION were coated with oleic acid (OA) in order to load them inside the oily core of NEs droplets. SPION loaded NEs were magnetically sorted using MACS® MS Column (Miltenyi Biotec) and iron quantification was performed by UV-spectrometry measurements.
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PURPOSE: For early atherosclerosis imaging, magnetic oil-in-water nanoemulsion (NE) decorated with atheroma specific monoclonal antibody was designed for Magnetic Particle Imaging (MPI) and Magnetic Resonance Imaging (MRI). MPI is an emerging technique based on direct mapping of superparamagnetic nanoparticles which may advantageously complement MRI. METHODS: NE oily droplets were loaded with superparamagnetic iron oxide nanoparticles of 7, 11 and 18nm and biofunctionalized with atheroma specific scFv-Fc TEG4-2C antibody. RESULTS: Inclusion of nanoparticles inside NE did not change the hydrodynamic diameter of the oil droplets, close to 180nm, nor the polydispersity. The droplets were negatively charged (ζ=-30mV). In vitro MPI signal was assessed by Magnetic Particle Spectroscopy (MPS). NE displayed MRI and MPS signals confirming its potential as new contrast agent. NE MPS signal increase with NPs size close to the gold standard (Resovist). In MRI, NE displayed R2* transversal relaxivity of 45.45, 96.04 and 218.81mM-1s-1 for 7, 11 and 18nm respectively. NE selectively bind atheroma plaque both in vitro and ex vivo in animal models of atherosclerosis. CONCLUSION: Magnetic NE showed reasonable MRI/MPS signals and a significant labelling of the atheroma plaque. These preliminary results support that NE platform could selectively image atherosclerosis.
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Aterosclerosis/diagnóstico por imagen , Medios de Contraste/administración & dosificación , Compuestos Férricos/administración & dosificación , Placa Aterosclerótica/diagnóstico por imagen , Anticuerpos de Cadena Única/administración & dosificación , Animales , Apolipoproteínas E/genética , Aterosclerosis/inmunología , Medios de Contraste/química , Diglicéridos/administración & dosificación , Diglicéridos/química , Emulsiones , Femenino , Compuestos Férricos/química , Humanos , Fenómenos Magnéticos , Imagen por Resonancia Magnética , Ratones Noqueados , Nanoestructuras/administración & dosificación , Nanoestructuras/química , Placa Aterosclerótica/inmunología , Conejos , Anticuerpos de Cadena Única/química , Agua/administración & dosificación , Agua/químicaRESUMEN
BACKGROUND: In the context of systematically administered nanomedicines, the physicochemistry of NP surfaces must be controlled as a prerequisite to improve blood circulation time, and passive and active targeting. In particular, there is a real need to develop NP stealth and labelling for both in vivo and microscopic fluorescence imaging in a mice model. METHODS: We have synthesized NIR/red dually fluorescent silica nanoparticles of 19nm covalently covered by a PEG layer of different grafting density in the brush conformational regime by using a reductive amination reaction. These particles were characterized by TEM, DRIFT, DLS, TGA, ζ potential measurements, UV-vis and fluorescence spectroscopy. Prostate tumors were generated in mice by subcutaneous injection of RM1-CMV-Fluc cells. Tumor growth was monitored by BLI after a D-luciferin injection. Four samples of PEGylated fluorescent NPs were individually intravenously injected into 6 mice (N=6, total 24 mice). Nanoparticle distribution was investigated using in vivo fluorescence reflectance imaging (FRI) over 48h and microscopy imaging was employed to localize the NPs within tumors in vitro. RESULTS: Fluorescent NP accumulation, due to the enhanced permeability and retention (EPR) effect, increases gradually as a function of increased PEG surface grafting density with a huge difference observed for the highest density grafting. For the highest grafting density, a blood circulation time of up to 24h was observed with a strong reduction in uptake by the liver. In vivo experimental results suggest that the biodistribution of NPs is very sensitive to slight variations in surface grafting density when the NPs present a high curvature radius. CONCLUSION: This study underlines the need to compensate a high curvature radius with a PEG-saturated NP surface to improve blood circulation and accumulation within tumors through the EPR effect. Dually fluorescent NPs PEGylated to saturation display physical properties useful for assessing the susceptibility of tumors to the EPR effect. GENERAL SIGNIFICANCE: Control of the physicochemical features of nanoparticle surfaces to improve blood circulation times and monitoring of the EPR effect. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.
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Colorantes Fluorescentes/administración & dosificación , Imagen Molecular/métodos , Nanomedicina/métodos , Nanopartículas/administración & dosificación , Polietilenglicoles/química , Neoplasias de la Próstata/diagnóstico por imagen , Dióxido de Silicio/administración & dosificación , Animales , Línea Celular Tumoral , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Inyecciones Intravenosas , Mediciones Luminiscentes , Masculino , Ratones Transgénicos , Nanopartículas/química , Nanopartículas/metabolismo , Tamaño de la Partícula , Permeabilidad , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Dióxido de Silicio/química , Dióxido de Silicio/metabolismo , Propiedades de Superficie , Factores de Tiempo , Distribución TisularRESUMEN
The present work aims to demonstrate that colloidal dispersions of magnetic iron oxide nanoparticles stabilized with dextran macromolecules placed in an alternating magnetic field can not only produce heat, but also that these particles could be used in vivo for local and noninvasive deposition of a thermal dose sufficient to trigger thermo-induced gene expression. Iron oxide nanoparticles were first characterized in vitro on a bio-inspired setup, and then they were assayed in vivo using a transgenic mouse strain expressing the luciferase reporter gene under transcriptional control of a thermosensitive promoter. Iron oxide nanoparticles dispersions were applied topically on the mouse skin or injected subcutaneously with Matrigel™ to generate so-called pseudotumors. Temperature was monitored continuously with a feedback loop to control the power of the magnetic field generator and to avoid overheating. Thermo-induced luciferase expression was followed by bioluminescence imaging 6 h after heating. We showed that dextran-coated magnetic iron oxide nanoparticle dispersions were able to induce in vivo mild hyperthermia compatible with thermo-induced gene expression in surrounding tissues and without impairing cell viability. These data open new therapeutic perspectives for using mild magnetic hyperthermia as noninvasive modulation of tumor microenvironment by local thermo-induced gene expression or drug release.
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The production of silica nanoparticles (NPs) exposing quaternary ammonium groups (NPQ(+)) has been achieved using an optimized chemical surface functionalization protocol. The procedures of surface modification and quaternization of amino groups were validated by diffuse reflectance infrared Fourier transform (DRIFT) and (1)H NMR spectroscopies. Compared to nonquaternized aminated NP, the colloidal stability of NPQ(+) was improved for various pH and salt conditions as assessed by ζ potential and light scattering measurements. In the context of their use for nucleic acid delivery, DNA efficiently bound to NPQ(+) analyzed by cosedimentation assays for a large pH range and various NaCl concentrations and exhibited a better efficacy at basic pH than nonquaternized NP. The study of NPQ(+)/DNA/cationic lipids ternary complexes was carried out with 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and analyzed by cryo-electron microscopy (cryo-EM). Cryo-EM images showed ternary assemblies where condensed DNA strands are sandwiched between the NPQ(+) surface and the cationic lipid bilayer. Because of an unusual electrostatic colloidal stability of NPQ(+) and a high propensity to bind DNA molecules particularly at high salt concentrations, a novel type of ternary assembly has been formed that might impact the delivery properties of these complexes including their stability in biological environment.