Abstract:During studies on the absorption and interactions between silver nanoparticles and mammalian cells grown in vitro it was observed that large extracellular rings of silver nanoparticles were deposited on the microscope slide, many located near post-mitotic cells. Silver nanoparticles (AgNP, 80nm), coated with citrate, were incubated at concentrations of 0.3 to 30 μg/ml with a human-derived culture of retinal pigment epithelial cells (ARPE-19) and observed using darkfield and fluorescent microscopy, 24 h after t… Show more
“…Li et al reported that 20 nm PVP-AgNPs (slightly positively charged) could trigger cytotoxicity by targeting mitochondria and disturbing mitochondrial dynamics, which led to a series of mitochondrial effects in HepG2 cells . Using the same visualization system as ours, Zucker et al reported that positively charged bPEI-AgNPs showed higher particle accumulation in the mitochondrial region as well as greater mitochondrial toxicity than PVP-coated AgNPs, perhaps because mitochondria are the most electronegative organelles in cells . In fact, positively charged coatings were associated with higher cellular uptake in cells as a whole, compared to their negative-charged counterparts, possibly because the cell membrane is also slightly negatively charged, potentially facilitating electrostatic attraction to the cell membrane followed by endocytotic uptake .…”
Section: Discussionmentioning
confidence: 63%
“…15 Using the same visualization system as ours, Zucker et al reported that positively charged bPEI-AgNPs showed higher particle accumulation in the mitochondrial region as well as greater mitochondrial toxicity than PVPcoated AgNPs, perhaps because mitochondria are the most electronegative organelles in cells. 27 In fact, positively charged coatings were associated with higher cellular uptake in cells as a whole, compared to their negative-charged counterparts, possibly because the cell membrane is also slightly negatively charged, potentially facilitating electrostatic attraction to the cell membrane followed by endocytotic uptake. 42 A key point, however, is that surface charge is likely to be modified in cell culture media and after interaction with cell and their biomolecules.…”
Section: Discussionmentioning
confidence: 99%
“…We adapted our previous methods to examine subcellular colocalization of AgNPs and mitochondria. − Images were obtained from a combination of dark-field microscopy and fluorescence microscopy with a 60× or 100× Plan Fluor objective and an iris diaphragm (Nikon Ti2 inverted microscope, Melville, NY). Methods for imaging AgNPs and mitochondria were described previously …”
Silver nanoparticles (AgNPs) are
well-proven antimicrobial nanomaterials,
yet little is elucidated regarding the mechanism underlying cytotoxicity
induced by these nanoparticles. Here, we tested the hypothesis that
mitochondria are primary intracellular targets of two AgNPs and silver
ions in mouse hepatocytes (AML12) cultured in glucose- and galactose-based
media. AML12 cells were more sensitive to mitochondrial uncoupling
when grown with galactose rather than glucose. However, 24 h treatments
with 15 nm AgNPs and 6 nm GA-AgNPs (5 and 10 μg/mL) and AgNO3 (1 and 3 μg/mL), concentrations that resulted in either
10 or 30% cytotoxicity, failed to cause more toxicity to AML12 cells
grown on galactose than glucose. Furthermore, colocalization analysis
and subcellular Ag quantification did not show any enrichment of silver
content in mitochondria in either medium. Finally, the effects of
the same exposures on mitochondrial respiration were mild or undetectable,
a result inconsistent with mitochondrial toxicity causing cell death.
Our results suggest that neither ionic Ag nor the AgNPs that we tested
specifically target mitochondria and are inconsistent with mitochondrial
dysfunction being the primary cause of cell death after Ag exposure
under these conditions.
“…Li et al reported that 20 nm PVP-AgNPs (slightly positively charged) could trigger cytotoxicity by targeting mitochondria and disturbing mitochondrial dynamics, which led to a series of mitochondrial effects in HepG2 cells . Using the same visualization system as ours, Zucker et al reported that positively charged bPEI-AgNPs showed higher particle accumulation in the mitochondrial region as well as greater mitochondrial toxicity than PVP-coated AgNPs, perhaps because mitochondria are the most electronegative organelles in cells . In fact, positively charged coatings were associated with higher cellular uptake in cells as a whole, compared to their negative-charged counterparts, possibly because the cell membrane is also slightly negatively charged, potentially facilitating electrostatic attraction to the cell membrane followed by endocytotic uptake .…”
Section: Discussionmentioning
confidence: 63%
“…15 Using the same visualization system as ours, Zucker et al reported that positively charged bPEI-AgNPs showed higher particle accumulation in the mitochondrial region as well as greater mitochondrial toxicity than PVPcoated AgNPs, perhaps because mitochondria are the most electronegative organelles in cells. 27 In fact, positively charged coatings were associated with higher cellular uptake in cells as a whole, compared to their negative-charged counterparts, possibly because the cell membrane is also slightly negatively charged, potentially facilitating electrostatic attraction to the cell membrane followed by endocytotic uptake. 42 A key point, however, is that surface charge is likely to be modified in cell culture media and after interaction with cell and their biomolecules.…”
Section: Discussionmentioning
confidence: 99%
“…We adapted our previous methods to examine subcellular colocalization of AgNPs and mitochondria. − Images were obtained from a combination of dark-field microscopy and fluorescence microscopy with a 60× or 100× Plan Fluor objective and an iris diaphragm (Nikon Ti2 inverted microscope, Melville, NY). Methods for imaging AgNPs and mitochondria were described previously …”
Silver nanoparticles (AgNPs) are
well-proven antimicrobial nanomaterials,
yet little is elucidated regarding the mechanism underlying cytotoxicity
induced by these nanoparticles. Here, we tested the hypothesis that
mitochondria are primary intracellular targets of two AgNPs and silver
ions in mouse hepatocytes (AML12) cultured in glucose- and galactose-based
media. AML12 cells were more sensitive to mitochondrial uncoupling
when grown with galactose rather than glucose. However, 24 h treatments
with 15 nm AgNPs and 6 nm GA-AgNPs (5 and 10 μg/mL) and AgNO3 (1 and 3 μg/mL), concentrations that resulted in either
10 or 30% cytotoxicity, failed to cause more toxicity to AML12 cells
grown on galactose than glucose. Furthermore, colocalization analysis
and subcellular Ag quantification did not show any enrichment of silver
content in mitochondria in either medium. Finally, the effects of
the same exposures on mitochondrial respiration were mild or undetectable,
a result inconsistent with mitochondrial toxicity causing cell death.
Our results suggest that neither ionic Ag nor the AgNPs that we tested
specifically target mitochondria and are inconsistent with mitochondrial
dysfunction being the primary cause of cell death after Ag exposure
under these conditions.
“…The rings were most likely the result of NP binding to adhesion membrane proteins released during mitotic cytokinesis contraction that remained on the slide ( Figure 4 ). 62 Thus, DFM could also serve as a tool for studying normal and abnormal mitosis. Figure 4 Retinal pigment epithelial cells (ARPE-19) incubated with 3 µg/mL AgNP.…”
Section: Imaging Techniquesmentioning
confidence: 99%
“…2020;15(12):e0240268. The work is made available under the Creative Commons CC0 public domain dedication 62 . …”
Label-free detection of nanoparticles is essential for a thorough evaluation of their cellular effects. In particular, nanoparticles intended for medical applications must be carefully analyzed in terms of their interactions with cells, tissues, and organs. Since the labeling causes a strong change in the physicochemical properties and thus also alters the interactions of the particles with the surrounding tissue, the use of fluorescently labeled particles is inadequate to characterize the effects of unlabeled particles. Further, labeling may affect cellular uptake and biocompatibility of nanoparticles. Thus, label-free techniques have been recently developed and implemented to ensure a reliable characterization of nanoparticles.
This review provides an overview of frequently used label-free visualization techniques and highlights recent studies on the development and usage of microscopy systems based on reflectance, darkfield, differential interference contrast, optical coherence, photothermal, holographic, photoacoustic, total internal reflection, surface plasmon resonance, Rayleigh light scattering, hyperspectral and reflectance structured illumination imaging. Using these imaging modalities, there is a strong enhancement in the reliability of experiments concerning cellular uptake and biocompatibility of nanoparticles, which is crucial for preclinical evaluations and future medical applications.
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