Magnetic hybrid TiO2/Alg/FeNPs triads for the efficient removal of methylene blue from water

A new adsorbent material with combined adsorption, photocatalytic, and magnetic properties has been successfully synthesized and tested for the efficient dye removal from methylene blue (MB) contaminated water. A facile non-thermal method was applied to synthesize a hybrid nanocomposite consisting of TiO₂/calcium alginate (TiO/Alg) and magnetite (Fe₃O₄) nanoparticles (FeNPs). The potential of the adsorbent Alg as a barrier to prevent direct contact between the magnetic core and TiO₂ was experimented by varying the synthesis conditions. The performance of four differently synthesized TiO₂/Alg/FeNPs samples (TiO₂/Alg/FeNPs-1, TiO₂/Alg/FeNPs-2, TiO₂/Alg/FeNPs-3, and TiO₂/Alg/FeNPs-4) was found to be fairly comparable and stable based on their efficiency in removing MB from aqueous solution due to the physico-chemical characterization (surface morphology, functional groups and elemental analysis) which supports the performance of TiO₂/Alg/FeNPs. For the optimization study using the response surface methodology (RSM) with three factorial Box-Behnken experimental designs, TiO₂/Alg/FeNPs-2 was selected as it exhibited the highest MB removal of 97.6% after 120 min under ultra violet irradiation (254 nm wavelength). Among the three independent variables studied (i.e., pH, contact time and initial MB concentration), the initial concentration of MB had significant effect towards the MB removal performance. A recycling study was performed, thus confirming the stability of TiO₂/Alg/FeNPs-2 up to three cycles, with only a slight drop in the removal efficiency from 93.1% to 88.5%. The fabricated TiO₂/Alg/FeNPs nanocomposites could be a potential functional material for treating artificial dye laden wastewater such as in textile, cosmetic, and paper industries.     

Diclofenac degradation properties of a La-doped visible light-responsive TiO2 photocatalyst

Diclofenac (DCF), which is detected in the aquatic environment, can act as a harmful factor to the ecosystem and human health, but it is difficult to remove it with existing water treatment methods. DCF can be removed using the TiO₂ photocatalysis process, and in this study, a method for removing it using visible light was sought. A visible-light reactive TiO₂ photocatalyst doped with lanthanum (La) was synthesized using an LPP (Liquid Phase Plasma) process, and the photocatalytic activity was investigated by decomposing diclofenac. La was doped uniformly into the TiO₂ powder, and the content of doped La increased with increasing concentration of the initial precursor. The La dopant was in the form of La₂O₃, and the bandgap and photoluminescence intensity decreased with increasing amount of doped La. In the photolysis experiment using a UV light source, the La-doped TiO₂ photocatalysts (LDTPs) showed slightly higher activity than the bare TiO₂, but the photocatalyst activity of the LDTPs under the visible light irradiation was superior. In the degradation experiment of DCF by LDTP, four major by-product intermediates were detected. Moreover, it was estimated that degradation proceeded through two pathways: hydroxylation and ring closure.     

Assessment of cellular toxicity of TiO2 nanoparticles for cardiac tissue engineering applications

Abstract

Because of the increased use of titanium dioxide (TiO₂) nanoparticles (NPs) in tissue engineering (TE), and in new constructs for cardiac TE, their effect was studied on three relevant cell types: Adult rat ventricular cardiomyocytes, human embryonic stem cell-derived cardiomyocytes (hESC-CM) and fibroblasts. For adult rat myocytes, 10 μg/mL TiO₂ NPs showed no significant effect on myocyte survival over 24 h or acute myocyte contractility. Increasing the concentration to 100 μg/mL was seen to reduce contraction amplitude (p < 0.05). For hESC-CM, 10 μg/mL TiO₂ reduced the beating rate significantly by 24 h. No arrhythmias or cessation of beating were observed in either cell type. Culturing fibroblasts in 5–150 μg/mL TiO₂ significantly reduced cell proliferation at day 4 and increased cell death. We conclude that there may be modest but potentially adverse effects of TiO₂ NPs if used in fast degrading polymers for myocardial tissue engineering (MTE) applications.     

Assessment of the in vitro genotoxicity of TiO2 nanoparticles in a regulatory context

A review of in vitro genotoxicity studies on titanium dioxide nanoparticles (TiO₂-NPs) published between 2010 and 2016 was performed by France in the framework of the CLP Regulation 1272/2008/EC. Neither the few in vivo studies of low quality nor the larger number of acceptable in vitro studies available for genotoxicity allowed France to conclude on the genotoxicity of TiO₂-NPs. Based on this work, it was decided to compare the acceptable in vitro studies to understand the reasons for the diverging results observed, such as the materials tested or of the protocols used and their inherent interferences. The systematic review performed on in vitro genotoxicity data for TiO₂-NPs was then restricted to studies with the highest level of confidence among studies following OECD guidelines and the largely applied comet assay. Indeed, the aim of this article is to understand why, even if judged of good quality, the 36 publications selected and analyzed did not lead to a clear picture. Some recommendations to be taken into account before performing new in vitro genotoxicity assays for insoluble particles such as TiO₂-NPs are proposed. Although secondary genotoxic effects consequent to oxidative stress seem to be the major mechanism responsible for the genotoxicity of TiO₂-NPs reported in some studies, primary genotoxic effects cannot be excluded. Further studies are needed to clarify the exact mode of action of TiO₂-NPs and to highlight which physicochemical properties lead to their genotoxicity in vitro to ultimately identify a specific combination of parameters that could represent a risk in vivo.     

Contribution of oxidative stress to TiO2 nanoparticle-induced toxicity

With the rapid development of nanotechnology, titanium dioxide nanoparticles (TNPs) are widely used in many fields. People in such workplaces or researchers in laboratories are at a higher risk of being exposed to TNPs, so are the consumers. Moreover, increasing evidence revealed that the concentrations of TNPs are elevated in animal organs after systematic exposure and such accumulated TNPs could induce organ dysfunction. Although cellular responses such as oxidative stress, inflammatory response, apoptosis, autophagy, signaling pathways, and genotoxic effects contribute to the toxicity of TNPs, the interrelationship among them remains obscure. Given the pivotal role of oxidative stress, we summarized relevant articles covering the involvement of oxidative stress in TNPs’ toxicity and found that TNP-induced oxidative stress might play a central role in toxic mechanisms. However, available data are far from being conclusive and more investigations should be performed to further confirm whether the toxicity of TNPs might be attributed in part to the cascades of oxidative stress. Tackling this uncertain issue may help us to comprehensively understand the interrelationship among toxic cellular responses induced by TNPs and might shed some light on methods to alleviate toxicity of TNPs.     

TiO2 nanoparticles and bulk material stimulate human peripheral blood mononuclear cells

Nanomaterials are increasingly produced and used throughout recent years. Consequently the probability of exposure to nanoparticles has risen. Because of their small 1–100 nm size, the physicochemical properties of nanomaterials may differ from standard bulk materials and may pose a threat to human health. Only little is known about the effects of nanoparticles on the human immune system. In this study, we investigated the effects of TiO₂ nanoparticles and bulk material in the in vitro model of human peripheral blood mononuclear cells (PBMC) and cytokine-induced neopterin formation and tryptophan breakdown was monitored. Both biochemical processes are closely related to the course of diseases like infections, atherogenesis and neurodegeneration. OCTi60 (25 nm diameter) TiO₂ nanoparticles and bulk material increased neopterin production in unstimulated PBMC and stimulated cells significantly, the effects were stronger for OCTi60 compared to bulk material, while P25 TiO₂ (25 nm diameter) nanoparticles had only little influence. No effect of TiO₂ nanoparticles on tryptophan breakdown was detected in unstimulated cells, whereas in stimulated cells, IDO activity and IFN-γ production were suppressed but only at the highest concentrations tested. Because neopterin was stimulated and tryptophan breakdown was suppressed in parallel, data suggests that the total effect of particles would be strongly pro-inflammatory.     

Photocatalysts synthesized via plant mediated extracts for degradation of organic compounds: A review of formation mechanisms and application in wastewater treatment

Photocatalysis is a promising technology to remove several persistent and recalcitrant pollutants in water and wastewater. Most of the conventional chemical and physical synthesis routes of photocatalysts result in the release of toxic pollutants to the environment and hence green synthesis is a better alternative. Green synthesis of photocatalyst using plants, fungi, bacteria, yeast, and other biological sources is widely explored in recent years. The presence of various biomolecules used in green synthesis replaces conventional reducing and stabilizing agents such as hydrazine, sodium borohydride, ascorbic acids, and other polymer substitutes. This review focuses on the synthesis of photocatalysts using plant extracts through different synthesis routes and explores the efficacy of photocatalyst synthesized via plant extract mediated synthesis routes in the photodegradation of organic compounds present in water. The pathway as well as the factors affecting the formation of photocatalysts, the role of plant extract in the photocatalytic properties are explored in detail. The action of plant-mediated photocatalyst on the pollutant and the underlying predominant degradation mechanism are also discussed. Besides, this study gives an insight into the recyclability of the photocatalyst synthesized via plant extract mediated synthesis routes and addresses future perspectives and the challenges of green photocatalysts.     

光敏剂TiO2诱导下米诺环素降解的研究

目的 探究米诺环素光催化降解条件,有利于水环境中米诺环素的防治。方法 米诺环素是一种新型污染物。自然光条件下米诺环素与可见光没有明显的响应,在体系中加入TiO2光敏剂,米诺环素可发生显著的可见光降解。通过研究TiO2的用量,米诺环素的浓度,溶液中的金属离子对TiO2可见光降解米诺环素的影响。结果 TiO2诱导下的光催化降解可有效的去除水环境中的米诺环素,pH3,温度为75℃,米诺环素的初始浓度为20mg/L,TiO2的浓度为1.0g/L时米诺环素降解率最高,最高降解率为78%。结论 TiO2能够有效的降解废水中的米诺环素,为此类废水的净化提供方法。     

The effect of TiO(2) and Ag nanoparticles on reproduction and development of Drosophila melanogaster and CD-1 mice

In the last two decades, nanoparticles (NPs) have found applications in a wide variety of consumer goods. Titanium dioxide (TiO₂) and silver (Ag) NPs are both found in cosmetics and foods, but their increasing use is of concern due to their ability to be taken up by biological systems. While there are some reports of TiO₂ and Ag NPs affecting complex organisms, their effects on reproduction and development have been largely understudied. Here, the effects of orally administered TiO₂ or Ag NPs on reproduction and development in two different model organisms were investigated. TiO₂ NPs reduced the developmental success of CD-1 mice after a single oral dose of 100 or 1000 mg/kg to dams, resulting in a statistically significant increase in fetal deformities and mortality. Similarly, TiO₂ NP addition to food led to a significant progeny loss in the fruit fly, Drosophila, as shown by a decline in female fecundity. Ag NP administration resulted in an increase in the mortality of fetal mice. Similarly in Drosophila, Ag NP feeding led to a significant decrease in developmental success, but unlike TiO₂ NP treatment, there was no decline in fecundity. The distinct response associated with each type of NP likely reflects differences in NP administration as well as the biology of the particular model. Taken together, however, this study warns that these common NPs could be detrimental to the reproductive and developmental health of both invertebrates and vertebrates.     

A role for surface reactivity in TiO2 and quartz-related nanoparticle pulmonary toxicity

A variety of pulmonary hazard studies in rats have demonstrated that exposures to ultrafine or nanoparticles (generally defined as particles in the size range < 100 nm) produce more intensive inflammatory responses when compared with bulk-sized particle-types of similar chemical composition. However, this common perception of greater nanoparticle toxicity is based on a limited number of studies, conducted primarily with titanium dioxide and carbon black particle-types. Apart from variables such as particle size and surface area, it is conceivable that several additional physicochemical particle characteristics could play more significant roles in facilitating the development of nanoparticle-related toxicity; particularly when considering particle surface-cell interactions. These include but are not limited to: (i) Surface reactivity of particle-types; (ii) surface coatings; (iii) aggregation/disaggregation potential; and (iv) the method of nanoparticle synthesis. We present results of pulmonary bioassay hazard/safety studies with quartz particles of varying sizes/surface areas. These demonstrated that intratracheal instillation exposures to fine-sized, Min-U-Sil quartz particles (0.5 µm [particle size] – 5 m²/g [surface area]) produced (persistent) enhanced pulmonary toxicity (inflammation, cytotoxicity, cell proliferation and/or histopathology) in rats when compared to nanoscale quartz particles (50 nm–31 m²/g), but not when compared to smaller nanoscale quartz sizes (e.g., 12 nm–91 m²/g). The toxicity results correlated with red blood cell hemolytic potency as a measure of particle surface reactivity. In a second pulmonary bioassay study in rats, pulmonary hazard effects were measured following exposures to three different ultrafine (nano) TiO₂ particle-types, each with similar particle size distributions. The various TiO₂ particles differed in their crystal structures and surface reactivity endpoints as measured by the Vitamin C yellowing assay. Moreover, the surface activity characteristics correlated with potency of hazard biomarkers as described above, in these dose/response, time-course studies. It is concluded that particle surface reactivity, rather than particle size/surface area endpoints correlated best with lung inflammatory potency following exposures to particles.     

Application of TiO2-ceramic/UVA photocatalyst for the photodegradation of sulfamethoxazole

Sulfamethoxazole (SMX) is one of the most antibiotic compounds detected in the aquatic environment. The potential concentration of SMX could raise the ecotoxicological effects on microbial. The photocatalyst based on TiO₂ has been demonstrated as the effective material for the elimination of SMX in water. However, due to the non-selected attack of hydroxyl radical derived from photocatalysis to S–N, C–N bond, –NH₂ group, or isoxazole ring, therefore SMX could be transferred to different intermediate products. In this study, SMX at 5 mg L^?1 has been removed up to 84.7% after 7 h of irradiation by TiO2-ceramic photocatalyst. The highest removal efficiency could maintain at pH (5–6) where SMX existed in neutral form and the TiO₂ reached to point of zero charge (PZC). On the other hand, environmental parameters such as bicarbonate ions and the dissolved organic matter (DOM), which are generally existing in ambient water, have negative effects on the elimination of SMX. Therefore, we investigated the effect of these factors on SMX photocatalyst removal. The efficiency of TiO₂-ceramic was stable for 15 cycles of the experiment. In addition, the intermediate products were identified by UPLC-MS/MS and the degradation pathways were proposed. There were 11 intermediates detected with a molecular mass higher than 254. The result suggested that there was the recombination of free radicals to form new compounds. Therefore, although TiO₂-ceramic is a promising photocatalyst for SMX removal an ecotoxicity test still needs to be evaluated for a deeper understanding of transformation products.     

The noncellular reduction of MTT tetrazolium salt by TiO2 nanoparticles and its implications for cytotoxicity assays

We report results of noncellular tests, revealing the occurrence of photocatalytic interactions between titanium dioxide (TiO₂, titania) nanoparticles and the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide] cytotoxicity indicator. These interactions induce the reduction of MTT and formation of purple formazan under biologically relevant conditions. Classical MTT assays have been performed to evaluate the production of formazan in DMEM-F12 and RPMI-1640 cell culture media (containing 10% fetal bovine serum-FBS) treated with Degussa-P25 TiO₂ nanoparticles, in the absence of cells. The colorimetric determinations revealed the noncellular MTT to formazan transformation induced by TiO₂ nanoparticles, under conditions commonly used for in vitro cytotoxicity testing of nanomaterials. The formazan precipitation was found to be proportional to the TiO₂ concentration, being enhanced under laboratory daylight exposure. The photocatalytic nature of the studied effect was assessed under UV irradiation at 365 nm. The biological significance of the reported reaction was established with respect to cellular reference experiments performed on V79-4, HeLa and B16 cell lines. The results show false viability increases with up to 14% (for TiO₂ concentrations generally higher than 50 μg/ml), induced by the TiO₂–MTT reaction. This type of artifacts may lead to underestimated toxicity or false proliferation results.     

The potential health challenges of TiO2 nanomaterials

Titanium dioxide (TiO₂) nanomaterials (NMs) have found widespread applications owing to their attractive physical and chemical properties. As a result, the potential adverse impacts of nano‐TiO₂ exposure on humans have become a matter of concern. This review presents the state‐of‐the‐art advances on the investigations of the adverse effects of NMs, including the potential exposure routes of nano‐TiO₂ (e.g. respiratory system, skin absorption and digestive system), the physico‐chemical characterizations of nano‐TiO₂ (e.g. crystal structure, shape,size, zeta potential, treatment media, aggregation and agglomeration tendency, surface characteristics and coatings), risk evaluation of nanotoxicity (e.g. cytotoxicity, ecotoxicity, phototoxicity, and phytotoxicity) and potential mechanisms of adverse effects (e.g. generation of reactive oxygen species, oxidative stress and organelle dysfunction). The review aims to facilitate scientific assessments of health risks to nano‐TiO₂, which would guide the safe applications of NMs in our daily life. Copyright © 2015 John Wiley & Sons, Ltd.     

Nanotoxicity of TiO2 nanoparticles to erythrocyte in vitro

Titanium dioxide (TiO(2)) has been considered as non-toxic mineral particles widely used in the fields like cosmetics, food and drug. When the scale come to nanometer, TiO(2) nanoparticles (nano-TiO(2)) exhibits multiple specific characteristics coupled with unknown risks on health. The purpose of this study was to systematically research the influence of nano-TiO(2) on erythrocyte. The results indicated that the erythrocytes treated with nano-TiO(2) underwent abnormal sedimentation, hemagglutination and dose dependent hemolysis, totally differing from those treated with micro-TiO(2). The ghost cells were firstly investigated by using ultra-thin cell section in the case under nano-TiO(2). The mechanism of such adverse effects is (1) the attachment around erythrocyte change the surface native properties and ultimately lead to hemoagglutination; (2) the content leak to the outside of erythrocyte through the breakage induced by both the nano-TiO(2) trans-membrane and the oxidative stress under nano-TiO(2). Our findings imply that nano-TiO(2) may have potential toxicity to human being health.     

Pulmonary toxicity study in rats with three forms of ultrafine-TiO2 particles: differential responses related to surface properties

Surface properties are critical to assess effects of ultrafine-TiO(2) particles. The aim of this study was to assess lung toxicity in rats of newly developed, well characterized, ultrafine-TiO(2) particles and compare them to TiO(2) samples in two different size ranges and surface modifications. Groups of rats were intratracheally instilled with doses of 1 or 5mg/kg of either two ultrafine rutile TiO(2) particles (uf-1 or uf-2); rutile R-100 fine-TiO(2) (F-1); 80/20 anatase/rutile P25 ultrafine-TiO(2) (uf-3); or alpha-quartz particles. Phosphate-buffered saline (PBS) solution instilled rats served as vehicle controls. Following exposures, the lungs of PBS and particle-exposed rats were evaluated for bronchoalveolar lavage (BAL) fluid inflammatory markers, cell proliferation, and by histopathology at post-instillation time points of 24h, 1 week, 1 and 3 months. The ranking of lung inflammation/cytotoxicity/cell proliferation and histopathological responses was quartz>uf-3>F-1=uf-1=uf-2. Exposures to quartz and to a lesser degree, uf-3 anatase/rutile TiO(2) particles produced pulmonary inflammation, cytotoxicity and adverse lung tissue effects. In contrast, exposures to F-1 fine-TiO(2) particles or to uf-1/uf-2 ultrafine-TiO(2) particle-types produced transient inflammation. We conclude that differences in responses to anatase/rutile uf-3 TiO(2) particles versus the rutile uf-1 and uf-2 TiO(2) particles could be related to crystal structure, inherent pH of the particles, or surface chemical reactivity. Thus, based on these results, inhaled rutile ultrafine-TiO(2) particles are expected to have a low risk potential for producing adverse pulmonary health effects. Finally, the results demonstrate that exposures to ultrafine-TiO(2) particle-types can produce differential pulmonary effects, based upon their composition, and crystal structure. Thus, the lung toxicity of anatase/rutile uf-3 should not be viewed as representative for all ultrafine-TiO(2) particle-types.     

Endocytosis, oxidative stress and IL-8 expression in human lung epithelial cells upon treatment with fine and ultrafine TiO2: role of the specific surface area and of surface methylation of the particles

Inhaled ultrafine particles show considerably stronger pulmonary inflammatory effects when tested at equal mass dose with their fine counterparts. However, the responsible mechanisms are not yet fully understood. We investigated the role of particle size and surface chemistry in initiating pro-inflammatory effects in vitro in A549 human lung epithelial cells on treatment with different model TiO(2) particles. Two samples of TiO(2), i.e. fine (40-300 nm) and ultrafine (20-80 nm) were tested in their native forms as well as upon surface methylation, as was confirmed by Fourier transformed infrared spectroscopy. Radical generation during cell treatment was determined by electron paramagnetic resonance with 5,5-dimethyl-1-pyrroline-N-oxide or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl. Interleukin-8 mRNA expression/release was determined by RT-PCR and ELISA, whereas particle uptake was evaluated by transmission electron microscopy. TiO(2) particles were rapidly taken up by the cells, generally as membrane bound aggregates and large intracellular aggregates in vesicles, vacuoles and lamellar bodies. Aggregate size tended to be smaller for the ultrafine samples and was also smaller for methylated fine TiO(2) when compared to non-methylated fine TiO(2). No particles were observed inside nuclei or any other vital organelle. Both ultrafine TiO(2) samples but not their fine counterparts elicited significantly stronger oxidant generation and IL-8 release, despite their aggregation state and irrespective of their methylation. The present data indicate that ultrafine TiO(2), even as aggregates/agglomerates, can trigger inflammatory responses that appear to be driven by their large surface area. Furthermore, our results indicate that these effects result from oxidants generated during particle-cell interactions through a yet to be elucidated mechanism(s).     

The buccal mucosa as a route for TiO2 nanoparticle uptake

Abstract

The oral cavity, although part of the aero-digestive tract, is still neglected in terms of risk assessment with respect to nanoparticle uptake. If nanoparticles enter the oral cavity, either via oral products or inhaled materials, it is not clear whether they rapidly interact with the mucosae or are swallowed. In this study, interactions of three distinct titanium dioxide (TiO₂) particles (i.e. NM 100, NM 101 and NM 105) with oral tissues are presented. Physicochemical properties were addressed in relevant media, and particle penetration was investigated with an ex vivo model using porcine mucosa. To avoid modification of the particle surfaces via labeling, multiphoton microscopy was introduced as an accurate method to detect TiO₂ particles within the tissue. The spatiotemporal aspects of nanoparticle uptake, as well as the intracellular localization in human epithelial cells, were studied and potential toxic effects were evaluated. Although TiO₂ particles formed large aggregates once dispersed in media, 10–50% remained in the nanoscale range, rapidly interacting with the mucus layer and infecting the epithelium. However, differences in the penetration depth were observed depending on the particle characteristics. NM 100 and NM 105 were found in both the upper part and the lower part of the buccal mucosa, while NM 101 (smallest particle sizes) only penetrated the upper parts. Transport studies revealed that TiO₂ nanoparticles were found in vesicles, as well as freely distributed in the cytoplasm. Cell viability/integrity was not affected negatively; however, NM 105 triggered the production of reactive oxygen species. These data clearly suggest that the oral cavity should be considered in further risk assessment studies.     

Negligible respiratory irritation and inflammation potency of pigmentary TiO2 in mice

Abstract

Titanium dioxide (TiO₂) is manufactured in millions of tons yearly, and it is used widely as pigment in various applications. Until recently, TiO₂ was considered toxicologically harmless and without adverse health effects. In this study, respiratory irritation and inflammation potencies of commercially available pigmentary TiO₂ particles (<5?µm, rutile) were studied. Single head-only exposures (30?min) of male Crl:OF1 mice at mass concentrations 6, 11, 21, and 37?mg/m³, and repeated exposures (altogether 16?h, 1?h/day, 4 days/week for 4 weeks) of female BALB/c/Sca mice at mass concentration of 16?mg/m³ to pigmentary TiO₂ were conducted. Minor sensory irritation was observed during acute and repeated exposures seen as elongation of the break after the inhalation, which is typical in sensory irritation, and caused by closure of the glottis inhibiting airflow from the lungs after inspiration. No pulmonary irritation, airflow limitation, nasal or pulmonary inflammation was observed. In conclusion, the respiratory irritation and inflammation potencies of the studied pigmentary TiO₂ particles seemed to be low and thus can serve as an ideal control exposure agent in short-term studies in mice.     

Uptake of different crystal structures of TiO2 nanoparticles by Caco-2 intestinal cells

The gastrointestinal uptake of different crystal structures of TiO₂ was investigated using Caco-2 intestinal cells. Caco-2 monolayers exhibited time-dependent, saturable uptake of Ti from TiO₂ exposures of 1 mg l⁻¹ over 24 h, which was influenced by crystal type. Initial uptake rates were 5.3, 3.73, 3.58 and 4.48 nmol mg⁻¹ protein h⁻¹ for bulk, P25, anatase and rutile forms respectively. All exposures caused elevations of Ti in the cells relative to the control (ANOVA P < 0.05). Electron micrographs of the Caco-2 monolayer showed the presence of particles inside the cells, and energy dispersive spectroscopy (EDS) confirmed the composition as TiO₂. Incubating the cells with 120 IU nystatin (putative endocytosis inhibitor) or 100 μmol l⁻¹ vanadate (ATPase inhibitor) caused large increases in Ti accumulation for all crystal types relative to controls (ANOVA P < 0.05), except for the rutile form with vanadate. Incubating the cells with 90 μmol l⁻¹ genistein (tyrosine kinase inhibitor) or 27 μmol l⁻¹ chloropromazine (clathrin-mediated endocytosis inhibitor) caused a large decrease in Ti accumulation relative to the controls (ANOVA P < 0.05). Cell viability measures were generally good (low LDH leak, normal cell morphology), but there were some changes in the electrolyte composition (K⁺, Na⁺, Ca²⁺, Mg²⁺) of exposed cells relative to controls. A rise in total Ca²⁺ concentration in the cells was observed for all TiO₂ crystal type exposures. Overall, the data shows that Ti accumulation for TiO₂ NP exposure in Caco-2 cells is crystal structure-dependent, and that the mechanism(s) involves endocytosis of intact particles.