Dose-dependent cell necrosis induced by silica nanoparticles

In recent years, much attention has been given to nanoparticles (NPs) due to their many possible applications, and as research has progressed, these NPs have become valuable tools for medical purposes. Among many different types of NPs, silica nanoparticles (SiO₂NPs) have been specifically evaluated for medical purposes and have also been used in many different types of products. Although SiO₂NPs have already been applied and are believed to be nontoxic, there is still a concern regarding possible adverse effects that may be triggered after SiO₂NP exposure. Therefore, in the present study, we employed a recommended cell line (BALB/c 3T3) for the toxicity evaluation to investigate the cytotoxic effects of SiO₂NPs produced by chemical synthesis at a laboratory scale. First, we employed OECD guideline 129 in order to evaluate cytotoxicity effects and also estimate the starting doses for acute oral systemic toxicity tests. We evaluated the cytotoxic effects of two types of SiO₂NPs (nonfluorescent and fluorescent) and found that they were not significantly different (IC₅₀ = 1986.39 ± 237 μg/mL and IC₅₀ = 1861.13 ± 186.72 μg/mL, respectively). Then, we used the predicted LD₅₀ of both types of SiO₂NPs to suggest that they could be categorized as GHS category 4 substances. By ultrastructural evaluation, we found that SiO₂NPs are internalized by 3 T3 cells and are located in vacuole-like structures with no other significant changes in cell structure. We also found that SiO₂NPs lead to cell necrosis in a dose-dependent manner.     

Cytotoxicity of Au,ZnO and SiO2 NPs using in vitro assays with mussel hemocytes and gill cells: Relevance of size,shape and additives

Metal-bearing nanoparticles (NPs) possess unique physico-chemical characteristics that make them useful for an increasing number of industrial products and applications, but could also confer them a higher toxicity due to their higher reactivity compared to bulk forms of the same materials. There is a considerable interest in the use of in vitro techniques in environmentally relevant species, such as marine mussels, to evaluate NPs toxicity. In the present work, mussel hemocytes and gill cells were used to assess the potential toxic effects of Au, ZnO and SiO₂ NPs with different sizes and shapes in parallel with their respective ionic and bulk forms and additives used in the NPs preparations. Cytotoxicity (neutral red and MTT assays) was screened at a wide range of concentrations, and LC50 values were calculated. Uptake of fluorescently labeled SIO₂ NPs of 27?nm by hemocytes was also investigated. Au, ZnO and SiO₂ NPs were less toxic than the corresponding ionic forms but more toxic than the bulk forms. ZnO NPs were the most toxic NPs tested which could be related with their capacity to release free ions. SiO₂ NPs were not taken up by hemocytes and were not toxic to either hemocytes or gill cells. Size-dependent toxicity was found for Au NPs. Shape influenced the cytotoxicity of ZnO NPs. Finally, the presence of the additives Na-citrate and Ecodis P90 contributed to the toxicity of Au and ZnO NPs, respectively. As a general conclusion, solubility appears to play a key role in NPs toxicity to mussel cells.     

In vitro intestinal toxicity of copper oxide nanoparticles in rat and human cell models

Abstract

Human oral exposure to copper oxide nanoparticles (NPs) may occur following ingestion, hand-to-mouth activity, or mucociliary transport following inhalation. This study assessed the cytotoxicity of Cupric (II) oxide (CuO) and Cu₂O-polyvinylpyrrolidone (PVP) coated NPs and copper ions in rat (intestine epithelial cells; IEC-6) and human intestinal cells, two- and three-dimensional models, respectively. The effect of pretreatment of CuO NPs with simulated gastrointestinal (GI) fluids on IEC-6 cell cytotoxicity was also investigated. Both dose- and time-dependent decreases in viability of rat and human cells with CuO and Cu₂O-PVP NPs and Cu²⁺ ions was observed. In the rat cells, CuO NPs had greater cytotoxicity. The rat cells were also more sensitive to CuO NPs than the human cells. Concentrations of H₂O₂ and glutathione increased and decreased, respectively, in IEC-6 cells after a 4-h exposure to CuO NPs, suggesting the formation of reactive oxygen species (ROS). These ROS may have damaged the mitochondrial membrane of the IEC-6 cells causing a depolarization, as a dose-related loss of a fluorescent mitochondrial marker was observed following a 4-h exposure to CuO NPs. Dissolution studies showed that Cu₂O-PVP NPs formed soluble Cu whereas CuO NPs essentially remained intact. For GI fluid-treated CuO NPs, there was a slight increase in cytotoxicity at low doses relative to non-treated NPs. In summary, copper oxide NPs were cytotoxic to rat and human intestinal cells in a dose- and time-dependent manner. The data suggests Cu₂O-PVP NPs are toxic due to their dissolution to Cu ions, whereas CuO NPs have inherent cytotoxicity, without dissolving to form Cu ions.     

Evaluation of cellular effects of silicon dioxide nanoparticles

Silica nanoparticles (nSiO₂s) are an important type of manufactured nanoparticles. Although there are some reports about the cytotoxicity of nSiO₂, the association between physical and chemical properties of nSiO₂s and their cellular effects is still unclear. In this study, we examined the correlation between the physiochemical properties and cellular effects of three kinds of amorphous nSiO₂s; sub-micro-scale amorphous SiO₂, and micro-scale amorphous and crystalline SiO₂ particles. The SiO₂ particles were dispersed in culture medium and applied to HaCaT human keratinocytes and A549 human lung carcinoma cells. nSiO₂s showed stronger protein adsorption than larger SiO₂ particles. Moreover, the cellular effects of SiO₂ particles were independent of the particle size and crystalline phase. The extent of cell membrane damage and intracellular ROS levels were different among nSiO₂s. Upon exposure to nSiO₂s, some cells released lactate dehydrogenase (LDH), whereas another nSiO₂ did not induce LDH release. nSiO₂s caused a slight increase in intracellular ROS levels. These cellular effects were independent of the specific surface area and primary particle size of the nSiO₂s. Additionally, association of solubility and protein adsorption ability of nSiO₂ to its cellular effects seemed to be small. Taken together, our data suggest that nSiO₂s do not exert potent cytotoxic effects on cells in culture, especially compared to the effects of micro-scale SiO₂ particles. Further studies are needed to address the role of surface properties of nSiO₂s on cellular processes and cytotoxicity.     

Varying the morphology of silver nanoparticles results in differential toxicity against micro-organisms,HaCaT keratinocytes and affects skin deposition

The use of silver nanoparticles (Ag NPs) within the healthcare sector and consumer products is rapidly increasing. There are now a range of diverse-shaped Ag NPs that are commercially available and many of the products containing nanosilver are topically applied to human skin. Currently, there is limited data on the extent to which the antimicrobial efficacy and cytotoxicity of Ag NPs is related to their shape and how the shape of the Ag NPs affects their distribution in both intact and burn wounded human skin after topical application. In this study, we related the relative Ag NP cytotoxicity to potential skin pathogens and HaCaT keratinocytes in vitro with the shape of the Ag NPs. We employed multiphoton fluorescence lifetime imaging to map the distribution of the native and unlabeled Ag NPs after topical application to both intact and burn wounded human skin using the localized surface plasmon resonance signal of the Ag NPs. Truncated plate shaped Ag NPs led to the highest cytotoxicity against both bacteria (IC₅₀ ranges from 31.25 to 125?μg/mL depending on the bacterial species) and HaCaT keratinocytes (IC₅₀ 78.65?μg/mL [95%CI 63.88, 96.83]) thus both with similar orders of magnitude. All Ag NPs were less cytotoxic than solutions of silver nitrate (IC₅₀ of 7.85?μg/mL [95%CI 1.49, 14.69]). Plate-shaped Ag NPs displayed the highest substantivity within the superficial layers of the stratum corneum when topically applied to intact skin and the highest deposition into the wound bed when applied to burned ex vivo human skin relative to other Ag NP shapes.     

Effects of SiO2 nanoparticles on HFL‐I activating ROS‐mediated apoptosis via p53 pathway

Silicon dioxide nanoparticles (SiO₂ NPs) have attracted increasing interest as nanovehicles for delivering drugs, genes and bio‐active molecules into cells. However, it is still unknown whether SiO₂ NPs could cause side‐effects to normal cells. In the present study, human lung fibroblasts (HFL‐Is) were directly exposed to two different sizes of SiO₂ NPs. The effect of size and concentration on cell response was studied by analyzing the cell viability, the ratio of apoptosis and the pathway of cell injury. The results demonstrated that a size‐associated and a dose‐dependent toxicity of HFL‐Is was induced by SiO₂ NPs. Meanwhile, the expression of reactive oxygen species in HFL‐I was significantly increased. This activation effect was accompanied by upregulation of p53 expression, release of cytochrome C from chondriosomes, inhibition of Bcl2, and activation of Bax and caspase 9. These findings implied that SiO₂ NPs might induce apoptosis of HFL‐Is by stimulating reactive oxygen species release and subsequently causing the activation of p53 pathway in vitro. Copyright © 2011 John Wiley & Sons, Ltd.     

Effects of silica nanoparticles on isolated rat uterine smooth muscle

In spite of their widespread use, toxicity of silica nanoparticles (SiO₂ NPs) to mammalian has not been extensively investigated. In the present study, it is aimed to investigate the effects and the mechanism of action of 20?nm sized SiO₂ NPs on isolated uterine smooth muscle. A total number of 84 preparations of uterine strips were used in the experiments. Study was designed as four groups: group I (control), group II (0.2?mM SiO₂ NPs), group III (0.4?mM SiO₂ NPs) and group IV (0.8?mM SiO₂ NPs). Spontaneous contractions were recorded using mechanical activity recording system. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) levels were measured using the spectrophotometric methods. Apoptosis of the cells was detected using immunofluorescence staining assay. SiO₂ NP distribution and ultrastructural changes were determined by transmission electron microscopy. In groups II–IV, the frequency of contraction was significantly lower than that of the group I, whereas the contraction energy significantly decreased only in group IV. SOD and GSH-Px activities were significantly lower in experimental groups compared to the control group. MDA level and apoptotic cells were significantly higher in all SiO₂ groups compared to the control group. Numerous SiO₂ NPs in cytoplasm and connective tissue were observed in all dose groups. These findings showed that 20?nm sized SiO₂ NPs enter the connective tissue and cytoplasm of uterine muscle cells and cause oxidative stress and apoptosis leading to impaired uterine contractile activity.     

Aluminium oxide nanoparticles induced morphological changes,cytotoxicity and oxidative stress in Chinook salmon (CHSE‐214) cells

Aluminium oxide nanoparticles (Al₂O₃ NPs) are increasingly used in diverse applications that has raised concern about their safety. Recent studies suggested that Al₂O₃ NPs induced oxidative stress may be the cause of toxicity in algae, Ceriodaphnia dubia, Caenorhabditis elegans and Danio rerio. However, there is paucity on the toxicity of Al₂O₃ NPs on fish cell lines. The current study was aimed to investigate Al₂O₃ NPs induced cytotoxicity, oxidative stress and morphological abnormality of Chinnok salmon cells (CHSE‐214). A dose‐dependent decline in cell viability was observed in CHSE‐214 cells exposed to Al₂O₃ NPs. Oxidative stress induced by Al₂O₃ NPs in CHSE‐214 cells has resulted in the significant reduction of superoxide dismutase, catalase and glutathione in a dose‐dependent manner. However, a significant increase in glutathione sulfo‐transferase and lipid peroxidation was observed in CHSE‐214 cells exposed to Al₂O₃ NPs in a dose‐dependent manner. Significant morphological changes in CHSE‐214 cells were observed when exposed to Al₂O₃ NPs at 6, 12 and 24 h. The cells started to detach and appear spherical at 6 h followed by loss of cellular contents resulting in the shrinking of the cells. At 24 h, the cells started to disintegrate and resulted in cell death. Our data demonstrate that Al₂O₃ NPs induce cytotoxicity and oxidative stress in a dose‐dependent manner in CHSE‐214 cells. Thus, our current work may serve as a base‐line study for future evaluation of toxicity studies using CHSE‐214 cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of serum proteins bound to industrial nanomaterials

Nanoparticles (NPs) are decorated with proteins and other biomolecules when they get into contact with biological systems. The presence of proteins in cell culture medium can therefore have effects on the biological outcome in cell-based tests. In this study, the manufactured nanomaterials silicon dioxide (SiO₂), titanium dioxide (TiO₂), iron-III-oxide (Fe₂O₃), and carbon black (CB) were used to study their interaction with single proteins from bovine and human plasma (albumin, fibrinogen and IgG) as well as with complete human serum. The protein binding capacity of the material was investigated and 1D gel electrophoresis was used to separate the bound proteins and to identify the bands by matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF) mass spectrometry.We found that the NP surface chemistry had a great impact on the amount of bound protein with distinct ligands for each of the tested particles. The hydrophobic CB NPs bound much more protein than the hydrophilic metal oxide NPs. Among the single proteins investigated, fibrinogen showed the strongest affinity for SiO₂, TiO₂ and CB NPs. The identified proteins from human serum adsorbed to these NPs were very different. Only apolipoprotein A1 was found to be adsorbed to all NPs.These studies will help to explain the different degree of biological responses observed after in vitro exposure of cells in the absence or presence of serum and might also support the interpretation of in vivo experiments were NPs come directly into contact with blood plasma.     

Cytotoxicity of vanadium oxide nanoparticles and titanium dioxide-coated vanadium oxide nanoparticles to human lung cells

Due to excellent metal–insulator transition property, vanadium dioxide nanoparticles (VO₂ NPs)-based nanomaterials are extensively studied and applied in various fields, and thus draw safety concerns of VO₂ NPs exposure through various routes. Herein, the cytotoxicity of VO₂ NPs (N-VO₂) and titanium dioxide-coated VO₂ NPs (T-VO₂) to typical human lung cell lines (A549 and BEAS-2B) was studied by using a series of biological assays. It was found that both VO₂ NPs induced a dose-dependent cytotoxicity, and the two cell lines displayed similar sensitivity to VO₂ NPs. Under the same conditions, T-VO₂ NPs showed slightly lower cytotoxicity than N-VO₂ in both cells, indicating the surface coating of titanium dioxide mitigated the toxicity of VO₂ NPs. Titanium dioxide coating changed the surface property of VO₂ NPs and reduced the vanadium release of particles, and thus helped lowing the toxicity of VO₂ NPs. The induced cell viability loss was attributed to apoptosis and proliferation inhibition, which were supported by the assays of apoptosis, mitochondrial membrane damage, caspase-3 level, and cell cycle arrest. The oxidative stress, i.e., enhanced reactive oxygen species generation and suppressed reduced glutathione , in A549 and BEAS-2B cells was one of the major mechanisms of the cytotoxicity of VO₂ NPs. These findings provide safety guidance for the practical applications of vanadium dioxide-based materials.     

Cell cooperation and role of the P2X7 receptor in pulmonary inflammation induced by nanoparticles

Abstract

Macrophages and alveolar epithelial cells are the first targets of inhaled nanoparticles (NPs) reaching the alveoli. Mono- or co-cultures of lung epithelial (A549 or NCI-H441) and macrophage (THP-1) cell lines were used to study the cell cooperation and the involvement of the P2X₇ cell death receptor during the inflammation caused by SiO₂ and TiO₂ NPs. Here we show that, secretion of pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) in response to NPs exposure was higher in co-cultures than in mono-cultures. A functional P2X₇ receptor was found in all the cell lines studied. Its involvement in IL-1β secretion in co-cultures was demonstrated using a specific antagonist, the brilliant blue G. Furthermore, mono and co-cultures exhibited distinct secretion patterns of pro-inflammatory cytokines in response to NPs exposure, and we provide the first evidence that the P2X₇ receptor is involved in the inflammation triggered by SiO₂ and TiO₂ NPs, by increasing IL-1β secretion, and likely through the inflammasome pathway. Altogether, our data indicate that cell co-cultures used in this study represent valid models to study the inflammatory mechanisms of NPs within the alveoli.     

Cytotoxicity of TiO2 nanoparticles to mussel hemocytes and gill cells in vitro: Influence of synthesis method,crystalline structure,size and additive

Abstract

Increasing the production and applications of TiO₂ nanoparticles (NPs) has led to grow concerns about the consequences for the environment. In this study, we investigated the effects of a set of TiO₂ NPs on the viability of mussel hemocytes and gill cells using neutral red and thiazolyl tetrazolium bromide assays. For this, we compared the cytotoxicity of TiO₂ NPs (0.1–100?mg Ti/L) produced by different techniques: rutile NPs (60?nm) produced by milling and containing disodium laureth sulfosuccinate (DSLS), rutile NPs (10, 40 and 60?nm) produced by wet chemistry and anatase/rutile NPs (～100?nm) produced by plasma synthesis. The commercially available P25 anatase/rutile NPs (10–20?nm) were also tested. Exposures were performed in parallel with their respective bulk forms and the cytotoxicity of the additive DSLS was also tested. Z potential values in distilled water indicated different stabilities depending on the NP type and all NPs tested formed agglomerates/aggregates in cell culture media. In general, TiO₂ NPs showed a relatively low and dose-dependent toxicity for both cell models with the two assays tested. NPs produced by milling showed the highest effects, probably due to the toxicity of DSLS. Size-dependent toxicity was found for NPs produced by wet chemistry (10?nm?>?40?nm and 60?nm). All TiO₂ NPs tested were more toxic than bulk forms excepting for plasma produced ones, which were the least toxic TiO₂ tested. The mixture bulk anatase/rutile TiO₂ was more toxic than bulk rutile TiO₂. In conclusion, the toxicity of TiO₂ NPs varied with the mode of synthesis, crystalline structure and size of NPs and can also be influenced by the presence of additives in the suspensions.     

The effect of Fe2O3 and ZnO nanoparticles on cytotoxicity and glucose metabolism in lung epithelial cells

Metallic nanoparticles (NPs) have potential applications in industry and medicine, but they also have the potential to cause many chronic pulmonary diseases. Mechanisms for their cytotoxicity, glucose and energy metabolism responses need to be fully explained in lung epithelial cells after treatment with metallic nanoparticles. In our study, two different metallic nanoparticles (Fe₂O₃ and ZnO) and two cell‐based assays (BEAS‐2B and A549 cell lines) were used. Our findings demonstrate that ZnO nanoparticles, but not Fe₂O₃ nanoparticles, induce cell cycle arrest, cell apoptosis, reactive oxygen species (ROS) production, mitochondrial dysfunction and glucose metabolism perturbation, which are responsible for cytotoxicity. These results also suggest that the glucose metabolism and bioenergetics had a great potential in evaluating the cytotoxicity and thus were very helpful in understanding their underlying molecular mechanisms. Copyright © 2015 John Wiley & Sons, Ltd.     

The role of quercetin in the formation of titanium dioxide nanoparticles for nanomedical applications

The current work aimed to synthesize and characterize titanium dioxide nanoparticles (TiO₂NPs) using quercetin (QE) and evaluate their biological activities, i.e., anti-hemolytic, anti-inflammatory, and cytotoxicity effects. The crystallographic phase and morphology of biosynthesized QE-TiO₂NPs were characterized by XRD (X-Ray Diffraction) and TEM/FE-SEM (Transmission/Field-Emission Scanning Electron Microscopy) micrographs. Functional groups involved in the synthesis process were determined by FTIR spectroscopy (Fourier Transform-Infrared Spectroscopy). Based on the characterization results, selected QE-TiO₂NPs showed a rutile phase, spherical shape, and a size range of 7.3–39 nm. The QE-TiO₂NPs did not show a hemolytic effect. They indicated 95.3% red blood cells (RBCs) membrane stabilization activity and 82.6% inhibition of bovine serum albumin (BSA) denaturation, similar to a standard drug, which proved their anti-inflammatory effects. The attained results from cytotoxicity studies revealed the toxic effects of QE-TiO₂NPs with IC50 values below 100 and 50 μg/mL for human breast cancer cells of MCF-7 and melanoma cancer cells of A375, respectively. These NPs did not significantly affect normal skin fibroblast cells up to 50 μg/mL and only showed a 16% inhibition rate on the cell viability at 100 μg/mL. These NPs also induced excessive ROS generation. This work established the blood/biocompatibility and excellent nanomedical applications of biosynthesized QE-TiO₂NPs.     

Iron oxide nanoparticles mediated cytotoxicity via PI3K/AKT pathway: Role of quercetin

Recently Fe₂O₃ NPs (iron oxide nanoparticles) have been extensively used in medical imaging and in industry also. As a result, people are increasingly exposed day by day to those nanoparticles. The adverse effect of Fe₂O₃ NPs is not so significant at lower doses but at higher doses Fe₂O₃ NPs causes significant damage to cells. The present study investigates the cell signaling mechanism of Fe₂O₃ NPs induced oxidative stress and cytotoxicity in vitro using murine hepatocytes as the working model. In addition, the cytoprotective action of quercetin in this pathophysiology has also been investigated. Dose-dependent studies suggest that incubation of hepatocytes with 250 μg/ml Fe₂O₃ NPs for 4 h significantly decreased the cell viability and intra-cellular antioxidant ability. This study also showed that exposure to Fe₂O₃ NPs caused hepatocytes death via apoptotic pathway. Incubation of hepatocytes with quercetin (50 μmol/L) prior to 1 h of Fe₂O₃ NPs exposure protects the cells from the altering activities of antioxidant indices, cytotoxicity and apoptotic death. Results suggest that Fe₂O₃ NPs induced cellular damage and quercetin plays a protective role in Fe₂O₃ NPs induced cytotoxicity and apoptotic death.     

Synthesis and cytotoxicity of silicon nanoparticles with covalently attached organic monolayers

Abstract

A series of highly monodisperse silicon nanoparticles (Si NPs) with either positively (amine), neutral (azide) or negatively (carboxylic acid) charged covalently attached organic monolayers were synthesized and investigated for their cytotoxicity. Infrared data confirmed the presence of these covalently attached surface groups. The Si NPs were characterized by absorption and fluorescence spectroscopy. The cytotoxicity was investigated in Caco-2 cells by determining the cell viability and proliferation. The IC50 values for the Si NPs ranged from 20 μg/l for the amine-terminated Si NPs, via 550–850 μg/l for the azide-terminated Si NPs to non-toxic (no measureable IC50) for the carboxylic acid-terminated Si NPs. These results indicate a trend in cytotoxicity, depending on surface charge, i.e., that positively charged Si NPs are more cytotoxic than negatively charged Si NPs. Interestingly, it appeared that the cytotoxicity of the Si NP-NH₂ depends strongly on the presence of fetal calf serum in the medium.     

Acute exposure to ZnO nanoparticles induces autophagic immune cell death

Abstract

The increasing risk of incidental exposure to nanomaterials has led to mounting concerns regarding nanotoxicity. Zinc oxide nanoparticles (ZnO NPs) are produced in large quantities and have come under scrutiny due to their capacity to cause cytotoxicity in vitro and potential to cause harm in vivo. Recent evidence has indicated that ZnO NPs promote autophagy in cells; however, the signaling pathways and the role of ion release inducing toxicity remain unclear. In this study, we report that ZnO NPs are immunotoxic to primary and immortalized immune cells. Importantly, such immunotoxicity is observed in mice in vivo, since death of splenocytes is seen after intranasal exposure to ZnO NPs. We determined that ZnO NPs release free Zn²⁺ that can be taken up by immune cells, resulting in cell death. Inhibiting free Zn²⁺ ions in solution with EDTA or their uptake with CaCl₂ abrogates ZnO NP-induced cell death. ZnO NP-mediated immune cell death was associated with increased levels of intracellular reactive oxygen species (ROS). ZnO NP death was not due to apoptosis, necroptosis or pyroptosis. Exposure of immune cells to ZnO NPs resulted in autophagic death and increased levels of LC3A, an essential component of autophagic vacuoles. Accordingly, ZnO NP-mediated upregulation of LC3A and induction of immune cell death were inhibited by blocking autophagy and ROS production. We conclude that release of Zn²⁺ from ZnO NPs triggers the production of excessive intracellular ROS, resulting in autophagic death of immune cells. Our findings suggest that exposure to ZnO NPs has the potential to impact host immunity.     

Cytotoxic and inflammatory responses of TiO2 nanoparticles on human peripheral blood mononuclear cells

Titanium dioxide nanoparticles (TiO₂‐NPs) have been widely used in many applications. Owing to their nanoscale size, interactions between cells and NPs have been expansively investigated. With the health concerns raised regarding the adverse effects of these interactions, closer examination of whether TiO₂‐NPs can induce toxicity towards human cells is greatly needed. Therefore, in this study, we investigated the cytotoxicity of TiO₂‐NPs towards human blood cells (peripheral blood mononuclear cells [PBMCs]) in serum‐free medium, for which there is little information regarding the cytotoxic effects of TiO₂‐NPs. Our results provide evidence that PBMCs treated with TiO₂‐NPs (at concentrations ≥25 μg ml^?1) for 24 h significantly reduced cell viability and significantly increased production of toxic mediators such as reactive oxygen species and inflammatory response cytokines such as interleukin‐6 and tumor necrosis factor‐α (P < 0.05). Cell apoptosis induction also occurred at these concentrations. Significant expressions of cyclooxygenase‐2 and interleukin‐1β were also observed in PBMCs treated with TiO₂‐NPs at concentrations ≥125 μg ml^?1. Our data presented here clearly indicate that the concentration of TiO₂‐NPs (at size ~26.4 ± 1.2 nm) applied to human blood cells has a strong impact on cytotoxic induction. Copyright © 2016 John Wiley & Sons, Ltd.