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