34 research outputs found
High-Speed Imaging of Rab Family Small GTPases Reveals Rare Events in Nanoparticle Trafficking in Living Cells
Despite the increased application of nanomaterials in diagnostics and therapeutics, methods to study the interactions of nanoparticles with subcellular structures in living cells remain relatively undeveloped. Here we describe a robust and quantitative method that allows for the precise tracking of all cell-associated nanoparticles as they pass through endocytic compartments in a living cell. Using rapid multicolor 3D live cell confocal fluorescence microscopy, combined with transient overexpression of small GTPases marking various endocytic membranes, our studies reveal the kinetics of nanoparticle trafficking through early endosomes to late endosomes and lysosomes. We show that, following internalization, 40 nm polystyrene nanoparticles first pass through an early endosome intermediate decorated with Rab5, but that these nanoparticles rapidly transfer to late endosomes and ultimately lysosomes labeled with Rab9 and Rab7, respectively. Larger nanoparticles of 100 nm diameter also reach acidic Rab9- and Rab7-positive compartments although at a slower rate compared to the smaller 40 nm nanoparticles. Our work also reveals that relatively few nanoparticles are able to access endocytic recycling pathways, as judged by lack of significant colocalization with Rab11. Finally, we demonstrate that this quantitative approach is sufficiently sensitive to be able to detect rare events in nanoparticle trafficking, specifically the presence of nanoparticles in Rab1A-labeled structures, thereby revealing the wide range of intracellular interactions between nanoparticles and the intracellular environment
Ordered Surface Structuring of Spherical Colloids with Binary Nanoparticle Superlattices
Surface-patterning
colloidal matter in the sub-10 nm regime generates
exceptional functionality in biology and photonic and electronic materials.
Techniques of artificially generating functional patterns in the small
nanoscale advanced in a fascinating manner in the last several years.
However, they remain often restricted to planar and noncolloidal substrates.
Patterning colloidal matter in solution via bottom-up assembly of
smaller subunits on larger core particles is highly challenging because
it is necessary to force the subunits onto randomly moving objects.
Consequently, the non-equilibrium conditions present during nanoparticle
self-assembly are difficult to control to eventually achieve the desired
material structures. Here, we describe the formation of surface patterns
with intrinsic periodic repeats of 8.9 ± 0.9 nm and less on hard,
amorphous colloidal core particles by assembling binary nanoparticle
superlattices on the curved particle surface. The colloidal environment
is preserved during the entire bottom-up crystallization of variable
building blocks (here, monodispersed 5 nm Au and 2.4 nm Pd nanoparticles
(NPs) and 230 nm SiO<sub>2</sub> core particles) into AB<sub>13</sub>-like, binary, and isotropic superlattice domains on the amorphous
cores. The three-dimensional, bottom-up assembly technique is a new
tool for patterning colloidal matter in the sub-10 nm surface regime
for gaining access to multicomponent metamaterials for bionanoscience,
photonics, and electronics
Ordered Surface Structuring of Spherical Colloids with Binary Nanoparticle Superlattices
Surface-patterning
colloidal matter in the sub-10 nm regime generates
exceptional functionality in biology and photonic and electronic materials.
Techniques of artificially generating functional patterns in the small
nanoscale advanced in a fascinating manner in the last several years.
However, they remain often restricted to planar and noncolloidal substrates.
Patterning colloidal matter in solution via bottom-up assembly of
smaller subunits on larger core particles is highly challenging because
it is necessary to force the subunits onto randomly moving objects.
Consequently, the non-equilibrium conditions present during nanoparticle
self-assembly are difficult to control to eventually achieve the desired
material structures. Here, we describe the formation of surface patterns
with intrinsic periodic repeats of 8.9 ± 0.9 nm and less on hard,
amorphous colloidal core particles by assembling binary nanoparticle
superlattices on the curved particle surface. The colloidal environment
is preserved during the entire bottom-up crystallization of variable
building blocks (here, monodispersed 5 nm Au and 2.4 nm Pd nanoparticles
(NPs) and 230 nm SiO<sub>2</sub> core particles) into AB<sub>13</sub>-like, binary, and isotropic superlattice domains on the amorphous
cores. The three-dimensional, bottom-up assembly technique is a new
tool for patterning colloidal matter in the sub-10 nm surface regime
for gaining access to multicomponent metamaterials for bionanoscience,
photonics, and electronics
Ordered Surface Structuring of Spherical Colloids with Binary Nanoparticle Superlattices
Surface-patterning
colloidal matter in the sub-10 nm regime generates
exceptional functionality in biology and photonic and electronic materials.
Techniques of artificially generating functional patterns in the small
nanoscale advanced in a fascinating manner in the last several years.
However, they remain often restricted to planar and noncolloidal substrates.
Patterning colloidal matter in solution via bottom-up assembly of
smaller subunits on larger core particles is highly challenging because
it is necessary to force the subunits onto randomly moving objects.
Consequently, the non-equilibrium conditions present during nanoparticle
self-assembly are difficult to control to eventually achieve the desired
material structures. Here, we describe the formation of surface patterns
with intrinsic periodic repeats of 8.9 ± 0.9 nm and less on hard,
amorphous colloidal core particles by assembling binary nanoparticle
superlattices on the curved particle surface. The colloidal environment
is preserved during the entire bottom-up crystallization of variable
building blocks (here, monodispersed 5 nm Au and 2.4 nm Pd nanoparticles
(NPs) and 230 nm SiO<sub>2</sub> core particles) into AB<sub>13</sub>-like, binary, and isotropic superlattice domains on the amorphous
cores. The three-dimensional, bottom-up assembly technique is a new
tool for patterning colloidal matter in the sub-10 nm surface regime
for gaining access to multicomponent metamaterials for bionanoscience,
photonics, and electronics
High-Speed Imaging of Rab Family Small GTPases Reveals Rare Events in Nanoparticle Trafficking in Living Cells
Despite the increased application of nanomaterials in diagnostics and therapeutics, methods to study the interactions of nanoparticles with subcellular structures in living cells remain relatively undeveloped. Here we describe a robust and quantitative method that allows for the precise tracking of all cell-associated nanoparticles as they pass through endocytic compartments in a living cell. Using rapid multicolor 3D live cell confocal fluorescence microscopy, combined with transient overexpression of small GTPases marking various endocytic membranes, our studies reveal the kinetics of nanoparticle trafficking through early endosomes to late endosomes and lysosomes. We show that, following internalization, 40 nm polystyrene nanoparticles first pass through an early endosome intermediate decorated with Rab5, but that these nanoparticles rapidly transfer to late endosomes and ultimately lysosomes labeled with Rab9 and Rab7, respectively. Larger nanoparticles of 100 nm diameter also reach acidic Rab9- and Rab7-positive compartments although at a slower rate compared to the smaller 40 nm nanoparticles. Our work also reveals that relatively few nanoparticles are able to access endocytic recycling pathways, as judged by lack of significant colocalization with Rab11. Finally, we demonstrate that this quantitative approach is sufficiently sensitive to be able to detect rare events in nanoparticle trafficking, specifically the presence of nanoparticles in Rab1A-labeled structures, thereby revealing the wide range of intracellular interactions between nanoparticles and the intracellular environment
Ordered Surface Structuring of Spherical Colloids with Binary Nanoparticle Superlattices
Surface-patterning
colloidal matter in the sub-10 nm regime generates
exceptional functionality in biology and photonic and electronic materials.
Techniques of artificially generating functional patterns in the small
nanoscale advanced in a fascinating manner in the last several years.
However, they remain often restricted to planar and noncolloidal substrates.
Patterning colloidal matter in solution via bottom-up assembly of
smaller subunits on larger core particles is highly challenging because
it is necessary to force the subunits onto randomly moving objects.
Consequently, the non-equilibrium conditions present during nanoparticle
self-assembly are difficult to control to eventually achieve the desired
material structures. Here, we describe the formation of surface patterns
with intrinsic periodic repeats of 8.9 ± 0.9 nm and less on hard,
amorphous colloidal core particles by assembling binary nanoparticle
superlattices on the curved particle surface. The colloidal environment
is preserved during the entire bottom-up crystallization of variable
building blocks (here, monodispersed 5 nm Au and 2.4 nm Pd nanoparticles
(NPs) and 230 nm SiO<sub>2</sub> core particles) into AB<sub>13</sub>-like, binary, and isotropic superlattice domains on the amorphous
cores. The three-dimensional, bottom-up assembly technique is a new
tool for patterning colloidal matter in the sub-10 nm surface regime
for gaining access to multicomponent metamaterials for bionanoscience,
photonics, and electronics
High Content Analysis Provides Mechanistic Insights on the Pathways of Toxicity Induced by Amine-Modified Polystyrene Nanoparticles
<div><p>The fast-paced development of nanotechnology needs the support of effective safety testing. We have developed a screening platform measuring simultaneously several cellular parameters for exposure to various concentrations of nanoparticles (NPs). Cell lines representative of different organ cell types, including lung, endothelium, liver, kidney, macrophages, glia, and neuronal cells were exposed to 50 nm amine-modified polystyrene (PS-NH<sub>2</sub>) NPs previously reported to induce apoptosis and to 50 nm sulphonated and carboxyl-modified polystyrene NPs that were reported to be silent. All cell lines apart from Raw 264.7 executed apoptosis in response to PS-NH<sub>2</sub> NPs, showing specific sequences of EC50 thresholds; lysosomal acidification was the most sensitive parameter. Loss of mitochondrial membrane potential and plasma membrane integrity measured by High Content Analysis resulted comparably sensitive to the equivalent OECD-recommended assays, allowing increased output. Analysis of the acidic compartments revealed good cerrelation between size/fluorescence intensity and dose of PS-NH<sub>2</sub> NPs applied; moreover steatosis and phospholipidosis were observed, consistent with the lysosomal alterations revealed by Lysotracker green; similar responses were observed when comparing astrocytoma cells with primary astrocytes. We have established a platform providing mechanistic insights on the response to exposure to nanoparticles. Such platform holds great potential for <i>in vitro</i> screening of nanomaterials in highthroughput format.</p></div
Primary human astrocytes respond to PS-NH<sub>2</sub> and PS-COOH comparably to 1321N1 astrocytoma cells.
<p>1321N1 and NHA cells were exposed to vehicle (ctrl) or increasing concentrations of PS-COOH or PS-NH<sub>2</sub> NPs for 24 hours and analysed using HCA. Comparison of results obtained between the two cell lines in DMEM supplemented with 10% FBS (A) or with 3% FBS (B). In the graphs 1321n1 cells are represented by the continuous line (âȘ) while HNA cells are represented by the dashed line (âĄ) C. EC50/IC50 measurements for both cell lines exposed to NPs in presence of 10% or 3% FBS. NHA primary astrocytes showed slightly higher sensitivity than 1321N1 astrocytoma cells, with EC50/IC50 values in the same order of magnitude. Data are shown as average +/â SD of 30 acquired images from a representative experiment. Curve fitting and EC50/IC50 were obtained as described in the Methods. Fluo-4 and Lysotracker green dose response plots were fitted with a gaussian curve for visual purposes only.â</p
Surfactant Titration of NanoparticleâProtein Corona
Nanoparticles
(NP), when exposed to biological fluids, are coated
by specific proteins that form the so-called protein corona. While
some adsorbing proteins exchange with the surroundings on a short
time scale, described as a âdynamicâ corona, others
with higher affinity and long-lived interaction with the NP surface
form a âhardâ corona (HC), which is believed to mediate
NP interaction with cellular machineries. In-depth NP protein corona
characterization is therefore a necessary step in understanding the
relationship between surface layer structure and biological outcomes.
In the present work, we evaluate the protein composition and stability
over time and we systematically challenge the formed complexes with
surfactants. Each challenge is characterized through different physicochemical
measurements (dynamic light scattering, ζ-potential, and differential
centrifugal sedimentation) alongside proteomic evaluation in titration
type experiments (surfactant titration). 100 nm silicon oxide (Si)
and 100 nm carboxylated polystyrene (PS-COOH) NPs cloaked by human
plasma HC were titrated with 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate
(CHAPS, zwitterionic), Triton X-100 (nonionic), sodium dodecyl sulfate
(SDS, anionic), and dodecyltrimethylammonium bromide (DTAB, cationic)
surfactants. Composition and density of HC together with size and
ζ-potential of NPâHC complexes were tracked at each step
after surfactant titration. Results on Si NPâHC complexes showed
that SDS removes most of the HC, while DTAB induces NP agglomeration.
Analogous results were obtained for PS NPâHC complexes. Interestingly,
CHAPS and Triton X-100, thanks to similar surface binding preferences,
enable selective extraction of apolipoprotein AI (ApoAI) from Si NP
hard coronas, leaving unaltered the dispersion physicochemical properties.
These findings indicate that surfactant titration can enable the study
of NPâHC stability through surfactant variation and also selective
separation of certain proteins from the HC. This approach thus has
an immediate analytical value as well as potential applications in
HC engineering
PS-NH<sub>2</sub> NPs cause dose-dependent changes in lysosomal properties.
<p>I321N1 and HepG2 cells were exposed to vehicle (ctrl) or increasing concentrations of PS-COOH or PS-NH<sub>2</sub> NPs for 24 hours and analysed by HCA. The Spot Detection Bioapplication was used to analyse the Lysotracker green positive vesicles inside the exposed cells. A. Representative images of cells exposed to vehicle or 25 ”g/ml PS-NH<sub>2</sub> NPs showing the increased Lysotracker green fluorescence and the analysis performed using the Spot Detector Bioapplication. Scale barâ=â20 ”m. B. Dose-dependent changes parameters associated with Lysotracker green positive vesicles: counts, area and intensity of Lysotracker green positive vescicles for each cell were calculated; dose dependent changes in individual spots were also recorded. C. Comparison among spot counts, spot total area and spot total intensity/cell within each cell line. The results suggest that increasing concentrations of PS-NH<sub>2</sub> NPs cause swelling of lysosomes, as indicated by increased Lysotracker green intensity, followed by a reduction in spot counts and reduciton in the spot area/cell and their intensity, indicative of lysosomal rupture. Data are shown as average +/â SD of object numbers, fluorescence intensity, area per cell, or average fluorescence intensity per identified object respectively from a representative experiment. Data were fitted with a gaussian curve for visual purposes only.</p