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 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] |