Table 2 In vitro studies on nanosilica particles (SNPs) toxicity
| 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-α, 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] |