Toxicological Aspects of Long‐Term Treatment of Keratinocytes with ZnO and TiO2 Nanoparticles

Sunscreens containing ZnO and TiO₂ nanoparticles (NPs) are increasingly applied to skin over long time periods to reduce the risk of skin cancer. However, long‐term toxicological studies of NPs are very sparse. The in vitro toxicity of ZnO and TiO₂ NPs on keratinocytes over short‐ and long‐term applications is reported. The effects studied are intracellular formation of radicals, alterations in cell morphology, mitochondrial activity, and cell‐cycle distribution. Cellular response depends on the type of NP, concentration, and exposure time. ZnO NPs have more pronounced adverse effects on keratinocytes than TiO₂. TiO₂ has no effect on cell viability up to 100 μg mL^?1, whereas ZnO reduces viability above 15 μg mL^?1 after short‐term exposure. Prolonged exposure to ZnO NPs at 10 μg mL^?1 results in decreased mitochondrial activity, loss of normal cell morphology, and disturbances in cell‐cycle distribution. From this point of view TiO₂ has no harmful effect. More nanotubular intercellular structures are observed in keratinocytes exposed to either type of NP than in untreated cells. This observation may indicate cellular transformation from normal to tumor cells due to NP treatment. Transmission electron microscopy images show NPs in vesicles within the cell cytoplasm, particularly in early and late endosomes and amphisomes. Contrary to insoluble TiO₂, partially soluble ZnO stimulates generation of reactive oxygen species to swamp the cell redox defense system thus initiating the death processes, seen also in cell‐cycle distribution and fluorescence imaging. Long‐term exposure to NPs has adverse effects on human keratinocytes in vitro, which indicates a potential health risk.     

Mechanistic Differences in Cell Death Responses to Metal‐Based Engineered Nanomaterials in Kupffer Cells and Hepatocytes

The mononuclear phagocyte system in the liver is a frequent target for nanoparticles (NPs). A toxicological profiling of metal‐based NPs is performed in Kupffer cell (KC) and hepatocyte cell lines. Sixteen NPs are provided by the Nanomaterial Health Implications Research Consortium of the National Institute of Environmental Health Sciences to study the toxicological effects in KUP5 (KC) and Hepa 1–6 cells. Five NPs (Ag, CuO, ZnO, SiO₂, and V₂O₅) exhibit cytotoxicity in both cell types, while SiO₂ and V₂O₅ induce IL‐1β production in KC. Ag, CuO, and ZnO induced caspase 3 generated apoptosis in both cell types is accompanied by ion shedding and generation of mitochondrial reactive oxygen species (ROS) in both cell types. However, the cell death response to SiO₂ in KC differs by inducing pyroptosis as a result of potassium efflux, caspase 1 activation, NLRP3 inflammasome assembly, IL‐1β release, and cleavage of gasdermin‐D. This releases pore‐performing peptide fragments responsible for pyroptotic cell swelling. Interestingly, although V₂O₅ induces IL‐1β release and delays caspase 1 activation by vanadium ion interference in membrane Na⁺/K⁺ adenosine triphosphate (ATP)ase activity, the major cell death mechanism in KC (and Hepa 1–6) is caspase 3 mediated apoptosis. These findings improve the understanding of the mechanisms of metal‐based engineered nanomaterial (ENM) toxicity in liver cells toward comprehensive safety evaluation.     

Growth of ZnO nanorods on TiO2 nanoparticles films and their application to the electrode of dye-sensitized solar cells

A ZnO nanorods (NRs)/TiO₂ nanoparticles (NPs) film has been prepared by electrochemical deposition of ZnO NRs growth on P25 TiO₂ NPs film surfaces. It was found that ZnO NRs/TiO₂ NPs could significantly improve the efficiency of dye-sensitized solar cells owing to its relatively enhanced light-scattering capability and efficient charge transport efficiency. The overall energy-conversion efficiency (η) of 3.48 % was achieved by the formation of ZnO NRs/TiO₂ NPs film, which is 33 % higher than that formed by TiO₂ NPs alone (η = 2.62 %). The charge recombination behavior of cells was investigated by electrochemical impedance spectra, and the results showed that ZnO NRs/TiO₂ NPs film has the longer electron lifetime than TiO₂ NPs alone, which could facilitate the reduction of recombination processes and thus would promote the photocatalysis and solar cell performance.     

Nano‐TiO2 Drives Epithelial–Mesenchymal Transition in Intestinal Epithelial Cancer Cells

The majority of cancer mortality is associated with cancer metastasis. Epithelial‐to‐mesenchymal transition (EMT) is a process by which cells attain migratory and invasive properties, eventually leading to cancer metastasis. Here, it is shown that titanium dioxide nanoparticles (nano‐TiO₂), a common food additive, can induce the EMT process in colorectal cancer cells. Nano‐TiO₂ exposure is observed to activate transforming growth factor‐β (TGF‐β)/mitogen‐activated protein kinase (MAPK) and wingless (Wnt) pathways, and drive the EMT process. Similarly, silica nanoparticles (nano‐SiO₂) and hydroxyapatite nanoparticles (nano‐HA), as food‐based additives, can be ingested and accumulated in the stomach, and are found to be able to induce the EMT progression. The implication of this work can be profound for colorectal cancer patients where these food additives may unknowingly and unnecessarily hasten the progression of their cancers.     

Foodborne Titanium Dioxide Nanoparticles Induce Stronger Adverse Effects in Obese Mice than Non‐Obese Mice: Gut Microbiota Dysbiosis,Colonic Inflammation,and Proteome Alterations

The recent ban of titanium dioxide (TiO₂) as a food additive (E171) in France intensified the controversy on safety of foodborne‐TiO₂ nanoparticles (NPs). This study determines the biological effects of TiO₂ NPs and TiO₂ (E171) in obese and non‐obese mice. Oral consumption (0.1 wt% in diet for 8 weeks) of TiO₂ (E171, 112 nm) and TiO₂ NPs (33 nm) does not cause severe toxicity in mice, but significantly alters composition of gut microbiota, for example, increased abundance of Firmicutes phylum and decreased abundance of Bacteroidetes phylum and Bifidobacterium and Lactobacillus genera, which are accompanied by decreased cecal levels of short‐chain fatty acids. Both TiO₂ (E171) and TiO₂ NPs increase abundance of pro‐inflammatory immune cells and cytokines in the colonic mucosa, indicating an inflammatory state. Importantly, TiO₂ NPs cause stronger colonic inflammation than TiO₂ (E171), and obese mice are more susceptible to the effects. A microbiota transplant study demonstrates that altered fecal microbiota by TiO₂ NPs directly mediate inflammatory responses in the mouse colon. Furthermore, proteomic analysis shows that TiO₂ NPs cause more alterations in multiple pathways in the liver and colon of obese mice than non‐obese mice. This study provides important information on the health effects of foodborne inorganic nanoparticles.     

Cytotoxicity of titanium dioxide nanoparticles differs in four liver cells from human and rat

Titanium dioxide (TiO₂) nanoparticles (NPs) are the important nanoscale components of composites. Although TiO₂ NPs and their related nanocomposites have been widely used in industrial and medical applications, the adverse effects of TiO₂ nanomaterials have not been well studied. Here, we investigated the cytotoxicity of TiO₂ NPs in vitro using four liver cell lines: human hepatocellular carcinoma cell line (SMMC-7721), human liver cell line (HL-7702), rat hepatocarcinoma cell line (CBRH-7919) and rat liver cell line (BRL-3A). We checked cell viability, cell morphology, and the levels of reactive oxygen species (ROS) and glutathione (GSH) after TiO₂ exposure at varying concentrations (0.1–100 μg/mL) and different exposure periods of time (12–48 h). Compared to the NP-free control, all four cell lines exposed to TiO₂ NPs showed cytotoxicity in a dosage-dependent and time-dependent manner, which was associated with the changes of cell viability and cell morphology, increased intercellular ROS levels, and decreased intracellular GSH levels. Further, we observed that carcinomatous liver cells and human liver cells exhibited more tolerance to TiO₂ NPs exposure for 24 h, compared to normal liver cells and rat liver cells, respectively. The results indicate that the in vitro cytotoxicity induced by NPs should be assessed with great caution before the use of nanocomposites and that there is a need to standardize the cytotoxicity testing procedure of nanoscale components in composites when using different cell lines.     

ZnO nanowire/TiO2 nanoparticle photoanodes prepared by the ultrasonic irradiation assisted dip-coating method

Hybrid ZnO/TiO₂ photoanodes for dye-sensitized solar cells were prepared by combining ZnO nanowire (NW) arrays and TiO₂ nanoparticles (NPs) with the assistance of the ultrasonic irradiation assisted dip-coating method. Results show that the ultrasonic irradiation was an efficient way to promote the gap filling of TiO₂ NPs in the interstices of ZnO NWs. Hybrid ZnO NW/TiO₂ NP electrodes prepared with ultrasonic treatment exhibited better gap filling efficiency and higher visible absorptance. The overall conversion efficiency of the hybrid electrode was 0.79%, representing 35% improvement compared with that of the traditional one (0.58%). The enlarged surface area and improved attachments of TiO₂ NPs onto the walls of ZnO NWs induced by the application of ultrasonic irradiation may be the underlying reason. Electrochemical impedance spectroscopy measurements indicated that hybrid electrodes combined the advantages of improved electron transport along the ZnO NWs and increased surface area provided by infiltrated TiO₂ NPs, both of which are responsible for the improved cell efficiency.     

Quantitative Proteomics Study Reveals Changes in the Molecular Landscape of Human Embryonic Stem Cells with Impaired Stem Cell Differentiation upon Exposure to Titanium Dioxide Nanoparticles

The increasing number of nanoparticles (NPs) being used in various industries has led to growing concerns of potential hazards that NP exposure can incur on human health. However, its global effects on humans and the underlying mechanisms are not systemically studied. Human embryonic stem cells (hESCs), with the ability to differentiate to any cell types, provide a unique system to assess cellular, developmental, and functional toxicity in vitro within a single system highly relevant to human physiology. Here, the quantitative proteomics approach is adopted to evaluate the molecular consequences of titanium dioxide NPs (TiO₂ NPs) exposure in hESCs. The study identifies ≈328 unique proteins significantly affected by TiO₂ NPs exposure. Proteomics analysis highlights that TiO₂ NPs can induce DNA damage, elevated oxidative stress, apoptotic responses, and cellular differentiation. Furthermore, in vivo analysis demonstrates remarkable reduction in the ability of hESCs in teratoma formation after TiO₂ NPs exposure, suggesting impaired pluripotency. Subsequently, it is found that TiO₂ NPs can disrupt hESC mesoderm differentiation into cardiomyocytes. The study unveils comprehensive changes in the molecular landscape of hESCs by TiO₂ NPs and identifies the impact which TiO₂ NPs can have on the pluripotency and differentiation properties of human stem cells.     

Low doses of TiO2-polyethylene glycol nanoparticles stimulate proliferation of hepatocyte cells

This paper describes the effect of low concentrations of 100 nm polyethylene glycol-modified TiO₂ nanoparticles (TiO₂-PEG NPs) on HepG2 hepatocellular carcinoma cells. Proliferation of HepG2 cells increased significantly when the cells were exposed to low doses (<100 μg ml^–1) of TiO₂-PEG NPs. These results were further confirmed by cell counting experiments and cell cycle assays. Cellular uptake assays were performed to determine why HepG2 cells proliferate with low-dose exposure to TiO₂-PEG NPs. The results showed that exposure to lower doses of NPs led to less cellular uptake, which in turn decreased cytotoxicity. We therefore hypothesized that TiO₂-PEG NPs could affect the activity of hepatocyte growth factor receptors (HGFRs), which bind to hepatocyte growth factor and stimulate cell proliferation. The localization of HGFRs on the surface of the cell membrane was detected via immunofluorescence staining and confocal microscopy. The results showed that HGFRs aggregate after exposure to TiO₂-PEG NPs. In conclusion, our results indicate that TiO₂-PEG NPs have the potential to promote proliferation of HepG2 cells through HGFR aggregation and suggest that NPs not only exhibit cytotoxicity but also affect cellular responses.     

Role of toll-like receptors 3, 4 and 7 in cellular uptake and response to titanium dioxide nanoparticles

Innate immune response is believed to be among the earliest provisional cellular responses, and mediates the interactions between microbes and cells. Toll-like receptors (TLRs) are critical to these interactions. We hypothesize that TLRs also play an important role in interactions between nanoparticles (NPs) and cells, although little information has been reported concerning such an interaction. In this study, we investigated the role of TLR3, TLR4 and TLR7 in cellular uptake of titanium dioxide NP (TiO₂ NP) agglomerates and the resulting inflammatory responses to these NPs. Our data indicate that TLR4 is involved in the uptake of TiO₂ NPs and promotes the associated inflammatory responses. The data also suggest that TLR3, which has a subcellular location distinct from that of TLR4, inhibits the denaturation of cellular protein caused by TiO₂ NPs. In contrast, the unique cellular localization of TLR7 has middle-ground functional roles in cellular response after TiO₂ NP exposure. These findings are important for understanding the molecular interaction mechanisms between NPs and cells.     

Amorphous Silica Nanoparticles Promote Monocyte Adhesion to Human Endothelial Cells: Size‐Dependent Effect

There is evidence that nanoparticles can induce endothelial dysfunction. Here, the effect of monodisperse amorphous silica nanoparticles (SiO₂‐NPs) of different diameters on endothelial cells function is examined. Human endothelial cell line (EA.hy926) or primary human pulmonary artery endothelial cells (hPAEC) are seeded in inserts introduced or not above triple cell co‐cultures (pneumocytes, macrophages, and mast cells). Endothelial cells are incubated with SiO₂‐NPs at non‐cytotoxic concentrations for 12 h. A significant increase (up to 2‐fold) in human monocytes adhesion to endothelial cells is observed for 18 and 54 nm particles. Exposure to SiO₂‐NPs induces protein expression of adhesion molecules (ICAM‐1 and VCAM‐1) as well as significant up‐regulation in mRNA expression of ICAM‐1 in both endothelial cell types. Experiments performed with fluorescent‐labelled monodisperse amorphous SiO₂‐NPs of similar size evidence nanoparticle uptake into the cytoplasm of endothelial cells. It is concluded that exposure of human endothelial cells to amorphous silica nanoparticles enhances their adhesive properties. This process is modified by the size of the nanoparticle and the presence of other co‐cultured cells.     

Penetration,distribution and brain toxicity of titanium nanoparticles in rodents' body: a review

Titanium dioxide (TiO₂) has been vastly used commercially, especially as white pigment in paints, colorants, plastics, coatings, cosmetics. Certain industrial uses TiO₂ in diameter <100 nm. There are three common exposure routes for TiO₂ : (i) inhalation exposure, (ii) exposure via gastrointestinal tract, (iii) dermal exposure. Inhalation and gastrointestinal exposure appear to be the most probable ways of exposure, although nanoparticle (NP) penetration is limited. However, the penetration rate may increase substantially when the tissue is impaired. When TiO₂ NPs migrate into the circulatory system, they can be distributed into all tissues including brain. In brain, TiO₂ lead to oxidative stress mediated by the microglia phagocytic cells which respond to TiO₂ NPs by the production and release of superoxide radicals that convert to multiple reactive oxygen species (ROS). The ROS production may also cause the damage of blood–brain barrier which then becomes more permeable for NPs. Moreover, several studies have showed neuron degradation and the impairment of spatial recognition memory and learning abilities in laboratory rodent exposed to TiO₂ NPs.Inspec keywords: nanoparticles, permeability, health hazards, molecular biophysics, biochemistry, biological tissues, toxicology, oxidation, brain, neurophysiology, blood, reviews, cosmetics, cellular biophysics, titanium compoundsOther keywords: rodents, white pigment, gastrointestinal tract, dermal exposure, gastrointestinal exposure, nanoparticle penetration, penetration rate, blood–brain barrier, titanium nanoparticles, brain toxicity, size 100.0 nm, TiO₂      

Multiphoton Microscopy of Nonfluorescent Nanoparticles In Vitro and In Vivo

Nanotechnology holds great promise for a plethora of potential applications. The interaction of engineered nanomaterials with living cells, tissues, and organisms is, however, only partly understood. Microscopic investigations of nano‐bio interactions are mostly performed with a few model nanoparticles (NPs) which are easy to visualize, such as fluorescent quantum dots. Here the possibility to visualize nonfluorescent NPs with multiphoton excitation is investigated. Signals from silver (Ag), titanium dioxide (TiO₂), and silica (SiO₂) NPs in nonbiological environments are characterized to determine signal dependency on excitation wavelength and intensity as well as their signal stability over time. Ag NPs generate plasmon‐induced luminescence decaying over time. TiO₂ NPs induce photoluminescent signals of variable intensities and in addition strong third harmonic generation (THG). Optimal settings for microscopic detection are determined and then applied for visualization of these two particle types in living cells, in murine muscle tissue, and in the murine blood stream. Silica NPs produce a THG signal, but in living cells it cannot be discriminated sufficiently from endogenous cellular structures. It is concluded that multiphoton excitation is a viable option for studies of nano‐bio interactions not only for fluorescent but also for some types of nonfluorescent NPs.     

Dielectric investigations of pristine and modified ZnO nanoparticles for energy storage devices

Pristine ZnO, Al-doped ZnO, and TiO₂ coated ZnO nanoparticles (NPs) were synthesized by the wet chemical precipitation technique. All the synthesized NPs were characterized using X-ray diffraction (XRD), Field emission scanning electron microscopy (FESEM), Transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy. XRD analysis of pristine ZnO and Al-doped ZnO NPs revealed the hexagonal wurtzite structure with P6₃mc space group with no secondary phases and impurities. FESEM micrographs also depicted hexagonal grains with well-defined grain boundaries. TEM images showed hexagonal polyhedral shape for pure ZnO NPs and spherical shape dominating polyhedral particle for Al-doped ZnO NPs, and pseudospherical particles for TiO₂ coated ZnO NPs. Energy-dispersive X-ray spectroscopy of Al-doped ZnO indicates the eminent exchange of dopant in the lattice site of Zn. Dielectric Studies reveal the highest value of the dielectric constant and lowest value of dielectric loss for Al-doped ZnO as compared to pure and TiO₂-coated ZnO NPs. Suggesting Al-doped ZnO to be used as a dielectric material that can serve as a basic building block of the energy storage devices such as dielectric capacitor. TiO₂-coated ZnO NPs demonstrated higher AC conductivity in comparison to pure ZnO and Al-doped ZnO NPs suggesting their use as a conductive nanofiller materials in a polymer-based nanocomposite to achieve higher energy density.

     

PEGylated TiO2 nanoparticles mediated inhibition of cell migration via integrin beta 1

Nanoparticles (NPs) elicit various physiological responses in cellular environment, and the effect of NPs on cell migration is of high interest. In this work, the effects of NPs on cell migration and their possible mechanisms were studied. Here, we showed that after exposure to pegylated titanium dioxide nanoparticles (TiO₂-PEG NPs, where PEG stands for the polyethylene glycol), NCI-H292 cells exhibited slower migration than control cells. Furthermore, larger NPs inhibited cell migration much stronger than smaller NPs. Following NP exposure, the cells showed decreased expression of integrin beta 1 and phosphorylated focal adhesion kinase (pFAK), and disrupted F-actin structures. We demonstrated that a possible mechanism involved NP-mediated promotion of the lysosomal degradation of integrin beta 1, thus leading to reduced expression of pFAK and cytoskeletal disruption and inhibited cell migration. Therefore, our results showed that inhibition of NCI-H292 cell migration by NPs is mediated through integrin beta 1, which provides useful information for the application of NPs in cancer therapy and related fields.     

Construction of hierarchical TiO<Subscript>2</Subscript> nanorod array/graphene/ZnO nanocomposites for high-performance photocatalysis

The photocatalytic performance of heterostructure photocatalysts is limited in practical use due to the charge accumulation at the interface and its low efficiency in utilizing solar energy during photocatalytic process. In this work, a ternary hierarchical TiO₂ nanorod arrays/graphene/ZnO nanocomposite is prepared by using graphene sheets as bridge between TiO₂ nanorod arrays (NRAs) and ZnO nanoparticles (NPs) via a facile combination of spin-coating and chemical vapor deposition techniques. The experimental study reveals that the graphene sheets provide a barrier-free access to transport photo-excited electrons from rutile TiO₂ NRAs and ZnO NPs. In addition, there generates an interface scattering effect of visible light as the graphene sheets provide appreciable nucleation sites for ZnO NPs. This synergistic effect in the ternary nanocomposite gives rise to a largely enhanced photocurrent density and visible light-driven photocatalytic activity, which is 2.6 times higher than that of regular TiO₂ NRAs/ZnO NPs heterostructure. It is expected that this hierarchical nanocomposite will be a promising candidate for applications in environmental remediation and energy fields.     

Resistive Switching of Individual,Chemically Synthesized TiO2 Nanoparticles

Resistively switching devices are considered promising for next‐generation nonvolatile random‐access memories. Today, such memories are fabricated by means of “top–down approaches” applying thin films sandwiched between nanoscaled electrodes. In contrast, this work presents a “bottom–up approach” disclosing for the first time the resistive switching (RS) of individual TiO₂ nanoparticles (NPs). The NPs, which have sizes of 80 and 350 nm, respectively, are obtained by wet chemical synthesis and thermally treated under oxidizing or vacuum conditions for crystallization, respectively. These NPs are deposited on a Pt/Ir bottom electrode and individual NPs are electrically characterized by means of a nanomanipulator system in situ, in a scanning electron microscope. While amorphous NPs and calcined NPs reveal no switching hysteresis, a very interesting behavior is found for the vacuum‐annealed, crystalline TiO_2–x NPs. These NPs reveal forming‐free RS behavior, dominantly complementary switching (CS) and, to a small degree, bipolar switching (BS) characteristics. In contrast, similarly vacuum‐annealed TiO₂ thin films grown by atomic layer deposition show standard BS behavior under the same conditions. The interesting CS behavior of the TiO_2–x NPs is attributed to the formation of a core–shell‐like structure by re‐oxidation of the reduced NPs as a unique feature.     

Studies on interfacial interactions of TiO2 nanoparticles with bacterial cells under light and dark conditions

The probable underlying mechanism(s) of bacterial cell–TiO₂ nanoparticles (TiO₂ NPs) interaction in the absence of photo-irradiation has been less studied since most of the prior cytotoxicity studies focused on irradiated TiO₂. The present study draws attention to the possible role of cell surface–TiO₂ NP interactions under dark conditions, through an array of spectroscopic and microscopic investigations. A dominant freshwater bacterial isolate, Bacillus licheniformis, which interacted with environmentally relevant concentrations of TiO₂ NPs (1 μg/mL), was analysed and compared under both light and dark conditions. Aggregation of cells upon NP interaction and adsorption of NPs onto the cell membrane was evident from the scanning electron micrographs under both light and dark conditions. The FT–IR and FT–Raman spectra suggested stress response of bacterial cells by elevated protein and polysaccharide content in the cell–NP interaction. The Xray photoelectron spectroscopic data substantiated the reduction of titanium from Ti(IV) to Ti(III) species which might have contributed to the redox interactions on the cell surface under light as well as dark conditions. The internalization of NPs in the cytoplasm were obvious from the transmission electron micrographs. The consequent cell death/damage was confirmed through fluorescence spectroscopy and microscopy. To conclude, the current study established the substantial role of interfacial interactions in cytotoxicity of the TiO₂ NPs irrespective of the irradiation conditions.     

Extension of optical properties of ZnO/SiO2 materials induced by incorporation of Au or NiO nanoparticles

Incorporating noble metal nanoparticles (NPs) and oxides has been proved to be an effective method to tune the optical properties of silica based materials. In this paper the optical and photocatalytic properties have been studied for ZnO/SiO₂ modified with Au or NiO nanoparticles. Changes in the optical properties of semiconductor ZnO particles have been observed due to the deposition of coloured Au and NiO nanoparticles by reducing the band gap energy and thus extending light absorption to visible domain. The excellent surface characteristics of NiO/ZnO/SiO₂ and Au/ZnO/SiO₂ favour the adsorption behaviour of these materials and limit the recombination of electron–holes pairs. Crystal Violet degradation under VIS light proved to have higher efficiency in the presence of Au/ZnO/SiO₂ (97%) than for NiO/ZnO/SiO₂ (60%).     

Analysis of the cytotoxicity of differentially sized titanium dioxide nanoparticles in murine MC3T3-E1 preosteoblasts

There is an increased use of nanophase titanium dioxide (TiO₂) in bone implants and scaffolds. However, nano-debris is generated at the bone-biomaterial interface. Therefore, TiO₂ nanoparticles (NPs) of many sizes were investigated for cytotoxic effects on murine MC3T3-E1 preosteoblasts. These TiO₂ NPs induced a time- and dose-dependent decrease in cell viability. There was a significant increase in lactate dehydrogenase (LDH) release, apoptosis and mitochondrial membrane permeability following short-term exposure of the cells to TiO₂ NPs. These NPs also increased granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) gene expression. Compared with the 32 nm TiO₂ NPs, 5 nm TiO₂ NPs were more toxic, induced more apoptosis, increased mitochondrial membrane permeability and stimulated more GM-CSF expression at a high concentration (≥100 μg/ml). The results implied that the differential toxicity was associated with variations in size, so more attention should be given to the toxicity of small NPs for the design of future materials for implantation.