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

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]