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Table 2 In vitro studies on nanosilica particles (SNPs) toxicity

From: The nanosilica hazard: another variable entity

Silica form Size (primary) Material characterization Cells used Test Biological endpoints and findings Ref
Amorphous 40 nm- 5 μm Not specified A549
HEp-2
RPMI 2650
RLE-6TN
N2a
• Replication and transcription assays
• Cell proliferation and cell viability assay
• Proteasome activity assay
• Immunofluorescence and microscopy
• Uptake of all particles into the cytoplasm and nuclear localization of nanoparticles between 40 and 70 nm
• The uptake of NSPs in the nucleus induced aberrant clusters of topoisomerase I and protein aggregates in the nucleoplasm
[100]
Amorphous
(luminescent)
50 nm • Synthesis (ref. to literature) A549
rat alveolar macrophages
• laser scanning confocal microscope
• Comet Assay
• Pulse Field Gel Electrophoresis (PFGE)
• Western Blot Analysis of DNA Adducts/DNA Agarose Gel
• DNA Repair Enzyme Activity Assay
• Cell Proliferation Assay
• Vybrant Apoptosis Assay
• Uptake not detected in the nuclear region
• As compared to the A549 cells, the nanoparticle penetration rate was much faster in the rat alveolar macrophages
• No significant toxic effects observed at the molecular and cellular levels below a concentration of 0.1 mg/ml
[101]
Amorphous (colloidal) 15 and 46 nm • Particle sizes and distribution
• Surface area (268 and 52.5 m2/g for 15 and 46 nm particle, respectively), crystalline structure, major trace metal impurities
• Hydrodynamic particle size in water suspension
A549 • SRB (sulforhodamine B) and LDH assays
• Reduced glutathione (GSH) level
• DCFH assay (ROS generation)
• Malondialdehyde (MDA) assay
• Cytotoxicity was dose- and time-dependent
• Reduced glutathione (GSH) levels and elevated MDA production after exposure to 15 nm SNPs
[102]
Amorphous 60 and
100 nm
• Size distribution analysis
• Endotoxin concentration
A549
THP-1
Mono Mac 6;
co-cultures
• LDH assay
• Cytokine expression (TNF, IL-6, IL-8)
• Light and transmission electron microscopy (TEM)
• Cytotoxicity differed among the cell lines and was dose- and size-dependent (smaller particles were more toxic)
• co-cultures showed an increased sensitivity to particles concerning the cytokine release in comparison to the mono-cultures of each cell type
[103]
Amorphous ~14 nm • Size distribution A549
L-132
HeLa
MNNG/
HOS
• MTT and WST-1 assays
• Trypan blue exclusion and LDH assay
• Annexin V-PI assay (fluorescence microscopy)
• DCFH assay
• IL-8 expression (ELISA)
• Little cytotoxic effects in 4 cell lines tested at the concentration below 250 μg/ml within 48 h
• Exposing cancer cells to high concentrations (250-500 μg/ml) for 72 h resulted in an inflammatory response with oxidative stress and membrane damage, which varied with cell type (A549>HOS > HeLa)
• SNPs triggered an inflammation response without causing considerable cell death for both cancer cells and normal cells
[104]
Amorphous 10 and 80 nm o Provided by producer for the primary particles (surface area: 640 and 440 m2/g for 10 and 80 nm particle, respectively)
o Hydrodynamic particle size (in cell culture medium)
A549 • MTT and LDH assays
• DCFH assay
• Intracellular glutathione (GSH) concentration
• Membrane lipid peroxidation (LPO)
• Assay of glutathione reductase and glutathione peoxidase
• Cytotoxicity was dose-dependent
• SNPs induced reactive oxygen species and membrane lipid peroxidation in dose-dependent manner
• Both sizes of SNPs had little effect on GSH level and the activities of glutathione metabolizing enzymes
[105]
Amorphous 7 and 5-15 nm o Surface area (350 and 644 m2/g for 7 and 5-15 nm particle, respectively)
o Size distribution (in the test medium)
Beas-2B • Incorporation of SNPs into the cells (confocal LSM)
• MTT assay
• PI staining (flow cytometry)
• Apoptosis
• DCFH assay
• Oxidative stress responding transcription factors (Western blotting)
• SNPs were incorporated into the cells and distributed around the nucleus area
• SNPs induced oxidative stress via ROS formation and induction of of antioxidant enzymes (SOD and HO-1)
• Induction of Nrf-2-ERK MAP kinase signaling pathway was observed
• Overall, cells exposed to 5-15 nm SNPs (porous) showed a more sensitive response than those exposed to 7 nm SNPs (fumed)
[106]
Amorphous 10-20 nm o Provided by manufacturer (surface area: 140-180 m2/g)
o Primary particle size
o Endotoxin content (LPS)
A549 • MTT and LDH assays
• DCFH assay
• SOD activity determination
• Nitrate/nitrite determination
• DNA oxidative damage assay
• Cytotoxicity was dose- and time-dependent
• SNPs stimulated the ROS generation, GSH depletion and lower expression of SOD activity in a dose-dependent manner
• No NO production and significant DNA oxidative damage was observed after treatment of cells with SNPs
• Co-treatment of LPS with SNPs enhanced observed cytoxicity and generation of oxidative stress
[107]
Amorphous 30, 48, 118 and 535 nm • Synthesis method
• Hydrodynamic particle size (in water and cell culture medium)
HEL-30 • MTT and LDH assays
• Reduced glutathione (GSH) and DCFH assay
• Transmission electron microscopy (TEM)
• Cytotoxicity was dose- and size-dependent (smaller particles were more toxic)
• Uptake of all particles into the cytoplasm (nuclear uptake not studied)
• GSH level reduced significantly of after exposure to 30 nm nanoparticles
• No significant Reactive Oxygen Species (ROS) formation
[108]
Amorphous 70, 300 and 1000 nm Not specified XS52 • TEM analysis of cells
• LDH assay
• Proliferation ([3H]-Thymidine incorporation assay)
• SNPs of 300 and 1000 nm were incorporated into the cells and located in cytoplasm only; nanoparticles of 70 nm were located in nucleus as well as cytoplasm
• Cell proliferation was inhibited by treatment with SNPs of all sizes in dose-dependent manner
• The growth of the cells was more strongly inhibited by smaller-sized SNPs
[109]
Amorphous 15, 30 and 365 nm • Size distribution
• Zeta potential
• Amorphous structure
HaCaT • CCK assay
• Cell cycle assay
• Annexin V-PI assay (Flow cytometry)
• 2D-DIGE and, IEF and SDS_PAGE (protein expression)
• Western blot
• Cytotoxicity was dose- and size-dependent (smaller particles were more toxic)
• Apoptosis was dose- and size-dependent (smaller particles induced higher apoptosis frequency)
• Up-regulated proteins were classified as oxidative stress-associated proteins; cytoskeleton-associated proteins; molecular chaperones; energy metabolism-associated proteins; apoptosis and tumor-associated proteins
[110]
Amorphous 15 nm • Size distribution
• Zeta potential
• Amorphous structure
HaCaT • Flow cytometric analysis of methylated DNA
• Real-time PCR
• Western blot
• Treatment with SNPs induced Global DNA hypomethylation [111]
Amorphous 21 and 80 nm • Particle preparation and dispersion
• Size, morphology and chemical states of elements
• Hydrodynamic particle size (dispersed in water)
WS1
CCD-966sk
MRC-5
A549
MKN-28
HT-29
• MTT and LDH assays • Toxicity was seen at concentrations exceeding 138 μg/ml
• Susceptibility to NSPs differed among tested cell lines
[113]
Amorphous 20 nm Only provided by producer (surface area: 640 ± 50 m2/g) RAW264.7 • Membrane fluidity measurements (FRAP technique by LSCM)
• DCFH assay
• Intracellular free calcium content
• Exposure to SNPs increased ROS generation and decrease of the membrane fluidity
• Perturbation of Intracellular free calcium homeostasis was responsible for observed cytotoxicity
[114]
Amorphous 14 nm Only provided by producer (surface area: 200 m2/g) Caco-2 • LDH and WST-1 assay
• Fpg-modified comet assay
• Total GSH content
• Cytotoxicity observed
• Oxidative DNA damage
• Significant depletion of intracellular GSH
[115]
Amorphous 21, 48 and 86 nm • Size distribution analysis
• Surface area (225, 106 and 39 m2/g for 21, 48 and 86 nm particle, respectively)
• structure
L-02 • MTT and LDH assays
• TEM assay
• DCFH, MDA and GSH assay
• Annexin V-PI assay (flow cytometry)
• DNA ladder assay
• Western blot
• Cytotoxicity was dose- time - and size-dependent (smaller particles were more toxic)
• 21 nm SNPs induced ROS generation, lipid peroxidation and GSH depletion in a dose-dependent manner
• 21 nm SNPs induced apoptosis in a dose-dependent manner
[116]
Amorphous 4-40 nm (mean size: 14) Not specified HDMEC • MTS assay
• transmission electron microscopy (TEM)
• Ki67 expression and IL-8 release
• The particles were internalized but they did not exert cytotoxic effects
• Reduction of the proliferative activity and a pro-inflammatory stimulation were observed
[117]
Amorphous (monodisperse) 14, 15, 16, 19, 60, 104, 335 nm • Particle preparation and stability
• shape and size distribution
• surface area (196, 179, 183, 145, 33, 28 and 7.7 m2/g for 14, 15, 16, 19, 60, 104 and 335 nm particle, respectively)
• micropore volume
• Hydrodynamic particle size (in water and cell culture medium)
EAHY926 • MTT and LDH assays
• Annexin V-PI assay
• Cytotoxicity was dose- and size-dependent (smaller particles were more toxic and affected the exposed cells faster)
• Cell death predominantly caused by necrosis
[118]
Amorphous 21 and 48 nm • Size distribution analysis
• Surface area (225 and 106 m2/g for 21 and 48 nm particle, respectively)
• structure
H9c2(2-1) • MTT and LDH assays
• Hematoxylin and eosin staining
• DCFH, intracellular MDA and GSH assays
• Flow cytometry (cell cycle)
• Western blot
• Cytotoxicity was dose- time - and size-dependent (smaller particles were more toxic)
• ROS generation in a dose-dependent manner; increased level of MDA and decreased concentration of GSH indicated oxidative stress
• Cell cycle arrest in G1 phase
• Dose-dependent expression of p53 and p21 for 21 nm SNP
[119]
Amorphous From 20 nm to below 400 nm • the dispersion characteristics (size, size distribution, size evolution)
• zeta potential
3T3-L1 • comet assay • No detectable genotoxicity (the results were independently validated in two separate laboratories) [120]
Amorphous (monodisperse) 16, 60 and 104 nm • Particle preparation and stability
• shape and size distribution
• surface area (183, 33 and 28 m2/g for 16, 60 and 104 nm particle, respectively)
• micropore volume
• Hydrodynamic particle size (in water and cell culture medium)
A549 • MTT assay
• cytochalasin-B micronucleus assay (CBMN) alone or in combination with FISH-centromeric staining
• Alkaline Comet assay
• Measurements of cell-associated silica (ICP-MS)
• Results suggest that non-cytotoxic doses of SNPs may be capable of inducing slight chromosome breakage, loss and mitotic slippage, and at higher concentration possibly mitotic arrest. [122]
Amorphous (monodisperse) from 2 up to 335 nm • Particle preparation and stability
• shape and size distribution
• surface area (from 232 to 7.7 m2/g)
• micropore volume
• Hydrodynamic particle size (in water and cell culture medium)
• Zeta potential
J774
EAHY926
3T3
Human erythrocytes
• MTT and WST-1 assays
• RBC hemolysis
• in murine macrophages, the cytotoxic response, after treatment with SNPs of 17 different sizes, increased with external surface area and decreased with micopore volume
• in human endothelial cells and mouse embryo fibroblast the cytotoxicity increased with surface roughness and decrease in diameter
• the hemolytic activity of SNPs in human erythrocytes increased with the diameter of SNPs
[141]
Amorphous 30 nm • Provided by producer for primary partilcles (surface area: 165 m2/g)
• Hydrodynamic particle size (in PBS and cell culture medium)
• Adsorption of proteins from the test media in the absence of cells
3T3
hT
RAW264.7
• MTS assay
• Uptake (flow cytometry)
• DCFH assay
• Lysosomal membrane integrity
• Mitochodrial membrane potential
• Apoptosis (caspase-3, and caspase-7 activation; Annexin V-PI assay)
• SNPs depleted serum proteins from cell culture media
• SNPs cytotoxicity was dose-, time- and cell line dependent-dependent
• SNPs induced significant ROS generation in all cell lines
• No detectable destabilization of lysosomal membranes was observed
• Incubation with SNPs decreased mitochodrial membrane potential in hT and RAW cells
• SNPs triggered different extent of cell apoptosis depending on the cell line tested
[140]
Amorphous (mesoporous) 110 nm (pore diameter of ~2.5 nm) • Structure
• surface area (910 m2/g)
• pore volume
• stability in aqueous solution
3T3-L1
MCF-7
K562
• Confocal microscope
• TEM
• Flow cytometry
• Particles were internalized into cells and accumulated in cytoplasm
• No apparent cytotoxicity
[123]
Amorphous (mesoporous) Not specified (MCM-41 particle type) • Synthesis and functionalization of particles
• Zeta potential
• Cylindrical pores with a diameter around 5 nm
HeLa • MTT, WST-1 and LDH assays
• Flow cytometry for PI
• TEM observations
• No cytotoxicity was observed up to 50 μg/ml
• Particles interfered with MTT assay
[126]
Amorphous (mesoporous) 108, 110, 111 and 115 nm • Synthesis (ref to the previous study) and surface modification
• Zeta potential
• Surface area (780, 980, 930 and 1050 m2/g for 108, 110, 111 and 115 nm particle, respectively)
• pore volume and pore size distribution (2.6-2.0 nm)
hMSCs
3T3-L1
• MTT assay
• Flow cytometry for the uptake
• Cellular differentiation and cytochemical assay
• The modulation of surface charge and its threshold affects the uptake and is specific to cell type
• Positive correlation of positive surface charge and the uptake by the cells
• Uptake was through clathrin and actin-dependent endocytosis
• Uptake did not affect cells viability, proliferation and differentiation
[125]
Amorphous (mesoporous silica nanorods capped with iron oxide NPs) 200 × 80 nm (pore diameter of ~3 nm) • Preparation and functionalization HeLa • Confocal fluorescence microscopy • Particles were endocytosed by the cells and biocompatible (concentration used: 0.2 mg/mL) [127]
Amorphous (mesoporous) 30, 50, 110, 170 and 280 nm • Synthesis, suspension stability (no interparticle aggregation), hydrodynamic diamaters, zeta potential HeLa • MTT
• onfocal laser scanning microscopy
• ICP-MS
• Cellular uptake is highly particle size-dependent (with the optimum size of 50 nm); little cytotoxicity up to 100 mg/ml [128]
Amorphous (mesoporous) loaded with anticancer drugs) <130 nm (pore diameter of ~2 nm) • Preparation, shape, aggregation/stability in aqueous solution PANC-1
AsPC-1
Capan-1
MKN45
SW480
• Fluorescence and confocal microscopy • The particles offer the possibility of controlled release of anticancer drugs (non-loaded particles did not caused cytotoxicity) [129]
Amorphous (mesoporous) 150 nm (pore diameter of ~2.4 nm) • Synthesis, functionalization, surface area (850 m2/g), zeta potential HeLa • Flow cytometry
• Fluorescence microscopy
• Uptake of particles can be regulated by different surface functionalization
• More negatively charged particles were able to escape from endosomes
[130]
Amorphous (mesoporous)
Commercially available amorphous silica material
100 - 300 nm (pore diameter of ~3 nm) - • Synthesis (ref. to the previous study), funcionalization, surface area (1138 m2/g), pore volumes, number of silanol group
• Funcionalization
Rabbit RBCs • Hemolysis assay
• UV/Vis spectroscopy
• Flow cytometry
• The hemolytic activity of silica nanoparticles depends only on the concentration of negatively charged silanol groups
• Mesoporous particles exhibit a high compatibility towards RBCs as most of the silanols are located in the interior of the particles that are not accessible by the RBCs membranes
[131]
Amorphous (mesoporous) 300-650 nm (pore diameter of 31Å) and SBA-15 type
(>hundreds of nm, pore diameter of 55 Å)
• Synthesis,
• Order of mesostructures, surface area (821 and 506 m2/g), wall thickness, composition
HL-60
Jurkat
• Oxygen consumption assay
• ATP formation assay
• Cellular GSH assay
• Particles with larger size and larger pores caused concentration- and time dependent inhibition of cellular respiration
• Both nanoparticles were toxic to the isolated mitochondria
• No significant changes in cellular glutathione level was observed
[132]
Amorphous (mesoporous and silica nanospheres) 250 nm; 166x320 nm (pore diameter = 3.5 nm) • Synthesis and functionalization
• Number of particles per gram, surface area (4.1 and 0.2 m2/particle for mesoporous and spherical particle, respectively)
SK-N-SH • Staining with trypan blue and determination of viable cells using a hemacytometer • The cytotoxicity of particles was related to the adsorptive surface area of the particle (the most toxic malodorous silica are those with the largest BET surface areas)
• Dependency of cytotoxicity on the nature of the attached functional groups cannot be ruled out
[133]
Amorphous (mesoporous) 270 ± 50 nm (pore diameter of 3.9 nm) and 2.5 μm ± 500 nm (pore diameter of 2.8 nm) • Synthesis
• The structural and textural characterizations
• Surface area(520 and 547 m2/g for 270 nm and 2.5 μm particle, respectively)
• LPS concentration analysis
Human monocyte-derived
dendritic cells
• Apoptosis/necrosis (Annexin V/PI assay)
• production of cytokinesIL-10 and IL-12p70,IL-12, IL-10
• confocal microscopy, TEM
• Viability, uptake and immune regulatory markers were affected with increasing size and dose [134]
Amorphous (mesoporous) 190, 420 and 1220 nm • Synthesis and functionalization
• Size distribution
• Dispersity and porosity
• Surface area (220-650 m2/g)
• Zeta potential
MDA-MB-468 COS-7 • MTT assay
• The biodegradation experiments
• Intracellular localization of particles
• The cytotoxicity of particles was highly correlated with particle sizes ((smaller particles were more toxic)
• The biodegradation products of spherical E-MS particles showed no toxicity
• The residual surfactant bound to the particles has a much smaller contribution to the cytotoxicity than the free one
• The smaller particles were more easily endocytosed and
consequently located within lysosomes
[135]
Amorphous 100 and 200 nm • rod-shaped and spherical particles (Stöber), not-coated and coated with fibronectin or polyethylene glycol (PEG),
• Primary and aggregate size, surface area (9.2 and 4.6 m2/g for silica rods and 27.3 and 14.2 m2/g for silica spheres), crystallinity, impurities, zeta potential
MET-5A • LDH assay
• Expression of IL-8
• Simulated stretch imposed on the cells
• Dosimetric comparison of acicular and isotropic particulate materials is not straightforward
• In the absence of simulated lung function (stretch), cells showed no significant enhancement of cytotoxicity or inflammation release
• PEG surface treatment tended to reduce the cytotoxicity and IL-8 release from particle exposures suggesting the significance of adhesive interactions e.g. for membrane binding/signal transduction
[136]
Amorphous 130 nm and 155 nm; iron oxide particle with silica shell (80 nm) • Size distribution
• Reference given for the description in detail
Hmy2
Jurkat
U937
PC3;
human peripheral blood cells
• MTT assay and Trypan Blue exclusion
• Scanning electron microscopy
• DCFH assay
• The cytotoxicity of particles depended on the cell type tested
• No direct correlation between ROS production and cell toxicity.
• PEG-ylation of SNP protected the particles from protein adsorption on the external surface of the NPs and consequently no agglomeration in culture medium was observed.
• The availability of the particles to be internalized by the cells depended on the size and morphology of the aggregates.
[137]
Crystalline Particle sizes not uniform (7.21, 9.08 and 123.21 nm) • Size and concentration WIL2-NS • MTT assay
• Population Growth Assay
• Apoptosis Assay by Flow Cytometry
• Cytokinesis Block Micronucleus Assay
• Comet Assay
• HPRT Mutation Assay
• Significant dose-dependent decrease in viability
• with increasing dose of particles
• Fourfold increase in micronucleated binucleated cells frequency was detected, while no significant difference was measured by the Comet assay
[99]