Now showing 1 - 10 of 34
  • Publication
    Locating Reactive Groups on Nanomaterials with Gold Nanoclusters: Toward a Surface Reactive Site Map
    Nanoparticles (NPs) are often functionalized with reactive groups like amines or thiols for the subsequent conjugation of further molecules, e.g., stabilizing polymers, drugs and proteins or targeting cells or specific diseases, etc. In addition to the quantitative estimation of the reactive conjugation sites, their nanoscale and molecular positioning and local arrangement on single nanoparticles becomes more and more important for tailored engineering and design of functional nanomaterials. Here, we use maleimide or sulfo-succinimidyl ester modified 1.4 nm gold nanoclusters (AuNCs) to specifically label reactive thiol and amine groups with sub –2 nm precision on metal oxide and polymeric nanostructures. We confirm the binding of AuNCs by measuring and modelling sedimentation properties using analytical centrifugation, image their surface distribution and surface distances by transmission electron microscopy (TEM), and compare the results to ensemble measurements of numbers of reactive surface groups obtained by common photometric assays. We map thiol and amine groups introduced on silica NPs (SiNPs), titania stars (Ti), silica inverse opals (SiOps), and polystyrene NPs (PS NPs). We show that the method is suitable to map local, clustered inhomogeneities of the reactive sites on single SiNPs introduced by masking certain areas during surface functionalization. Mapping precise positions of reactive surface groups is essential for the design and the tailored ligation of multifunctional nanomaterials.
      463Scopus© Citations 2
  • Publication
    Mapping protein binding sites on the biomolecular corona of nanoparticles
    Nanoparticles in a biological milieu are known to form a sufficiently long-lived and well-organized 'corona' of biomolecules to confer a biological identity to the particle. Because this nanoparticle-biomolecule complex interacts with cells and biological barriers, potentially engaging with different biological pathways, it is important to clarify the presentation of functional biomolecular motifs at its interface. Here, we demonstrate that by using antibody-labelled gold nanoparticles, differential centrifugal sedimentation and various imaging techniques it is possible to identify the spatial location of proteins, their functional motifs and their binding sites. We show that for transferrin-coated polystyrene nanoparticles only a minority of adsorbed proteins exhibit functional motifs and the spatial organization appears random, which is consistent, overall, with a stochastic and irreversible adsorption process. Our methods are applicable to a wide array of nanoparticles and can offer a microscopic molecular description of the biological identity of nanoparticles.
      574Scopus© Citations 300
  • Publication
    Role of cell cycle on the cellular uptake and dilution of nanoparticles in a cell population
    Nanoparticles are considered a primary vehicle for targeted therapies because they can pass biological barriers, enter and distribute in cells by energy-dependent pathways1-3. Until now, most studies have shown that nanoparticle properties, such as size4-6 and surface7,8, can affect how cells internalise nanoparticles. Here we show that the different phases of cell growth, which constitute the cell cycle, can also influence nanoparticle uptake. Although cells in different cell cycle phases internalised nanoparticles with similar rates, after 24 hours of uptake the concentration of nanoparticles in the cells is ranked according to the different cell cycle phases: G2/M > S > G0/G1. Nanoparticles were not exported from cells but the internalised nanoparticle concentration is split when the cell divides. Our results suggest that future studies on nanoparticle uptake should consider the cell cycle because in a cell population, the internalised nanoparticle dose in each cell varies as the cell cycles.
      5929Scopus© Citations 514
  • Publication
    Regimes of Biomolecular Ultrasmall Nanoparticle Interactions
    Ultrasmall nanoparticles (USNPs), usually defined as NPs with core in the size range 1–3 nm, are a class of nanomaterials which show unique physicochemical properties, often different from larger NPs of the same material. Moreover, there are also indications that USNPs might have distinct properties in their biological interactions. For example, recent in vivo experiments suggest that some USNPs escape the liver, spleen, and kidney, in contrast to larger NPs that are strongly accumulated in the liver. Here, we present a simple approach to study the biomolecular interactions at the USNPs bio-nanointerface, opening up the possibility to systematically link these observations to microscopic molecular principles.
      509Scopus© Citations 81
  • Publication
    Ultrathin Silicon Membranes for in Situ Optical Analysis of Nanoparticle Translocation across a Human Blood-Brain Barrier Model
    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 (∼50 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.
      852Scopus© Citations 35
  • Publication
    Time and Space Resolved Uptake Study of Silica Nanoparticles by Human Cells
    A spatio-temporal mapping of the uptake of silica (SiO2) nanoparticles of different sizes by lung epithelial cells has been obtained. Based on high control of nanoparticle dispersion in cell media and cell exposure, one obtains reproducible and quantitative time-resolved data using a combination of flow cytometry, fluorescence and electron microscopies. We are thereby able to give a rather detailed account of the journey of SiO2 nanoparticles from the early events of uptake to their final sub-cellular localization.
      1538Scopus© Citations 205
  • Publication
    Effects of the Presence or Absence of a Protein Corona on Silica Nanoparticle Uptake and Impact on Cells
    Nanoparticles enter cells through active processes, thanks to their capability of interacting with the cellular machinery. The protein layer (corona) that forms on their surface once nanoparticles are in contact with biological fluids, such as the cell serum, mediates the interactions with cells in situ. As a consequence of this, here we show that the same nanomaterial can lead to very different biological outcomes, when exposed to cells in the presence or absence of a preformed corona. In particular, silica nanoparticles exposed to cells in the absence of serum have a stronger adhesion to the cell membrane and higher internalization efficiency, in comparison to what is observed in medium containing serum, when a preformed corona is present on their surface. The different exposure conditions not only affect the uptake levels but also result in differences in the intracellular nanoparticle location and impact on cells. Interestingly, we also show that after only one hour of exposure, a corona of very different nature forms on the nanoparticles exposed to cells in the absence of serum. Evidence suggests that these different outcomes can all be connected to the different adhesion and surface properties in the two conditions.
      2759Scopus© Citations 890
  • Publication
    Nanoparticle Adhesion to the Cell Membrane and Its effect on Nanoparticle Uptake Efficiency
    The interactions between nanosized particles and living systems are commonly mediated by what adsorbs to the nanoparticle in the biological environment, its biomolecular corona, rather than the pristine surface. Here, we characterize the adhesion toward the cell membrane of nanoparticles of different material and size and study how this is modulated by the presence or absence of a corona on the nanoparticle surface. The results are corroborated with adsorption to simple model supported lipid bilayers using a quartz crystal microbalance. We conclude that the adsorption of proteins on the nanoparticle surface strongly reduces nanoparticle adhesion in comparison to what is observed for the bare material. Nanoparticle uptake is described as a two-step process, where the nanoparticles initially adhere to the cell membrane and subsequently are internalized by the cells via energy-dependent pathways. The lowered adhesion in the presence of proteins thereby causes a concomitant decrease in nanoparticle uptake efficiency. The presence of a biomolecular corona may confer specific interactions between the nanoparticle-corona complex and the cell surface including triggering of regulated cell uptake. An important effect of the corona is, however, a reduction in the purely unspecific interactions between the bare material and the cell membrane, which in itself disregarding specific interactions, causes a decrease in cellular uptake. We suggest that future nanoparticle-cell studies include, together with characterization of size, charge, and dispersion stability, an evaluation of the adhesion properties of the material to relevant membranes.
      2196Scopus© Citations 657
  • Publication
    Using single nanoparticle tracking obtained by nanophotonic force microscopy to simultaneously characterize nanoparticle size distribution and nanoparticle-surface interactions
    Comprehensive characterization of nanomaterials for medical applications is a challenging and complex task due to the multitude of parameters which need to be taken into consideration in a broad range of conditions. Routine methods such as dynamic light scattering or nanoparticle tracking analysis provide some insight into the physicochemical properties of particle dispersions. For nanomedicine applications the information they supply can be of limited use. For this reason, there is a need for new methodologies and instruments that can provide additional data on nanoparticle properties such as their interactions with surfaces. Nanophotonic force microscopy has been shown as a viable method for measuring the force between surfaces and individual particles in the nano-size range. Here we outline a further application of this technique to measure the size of single particles and based on these measurement build the distribution of a sample. We demonstrate its efficacy by comparing the size distribution obtained with nanophotonic force microscopy to established instruments, such as dynamic light scattering and differential centrifugal sedimentation. Our results were in good agreement to those observed with all other instruments. Furthermore, we demonstrate that the methodology developed in this work can be used to study complex particle mixtures and the surface alteration of materials. For all cases studied, we were able to obtain both the size and the interaction potential of the particles with a surface in a single measurement.
      359Scopus© Citations 8