
==== Front
J Phys Chem B
J Phys Chem B
jp
jpcbfk
The Journal of Physical Chemistry. B
1520-6106
1520-5207
American Chemical Society

39231525
10.1021/acs.jpcb.4c02804
Article
Cellular and In Vivo Response to Industrial, Food Grade, and Photocatalytic TiO2 Nanoparticles
https://orcid.org/0009-0009-6346-8739
Heckman Morgan M. †
Albright Michaela C. ‡
Poulsen Karsten M. †§
Tighe Robert M. *‡
https://orcid.org/0000-0002-2370-0101
Payne Christine K. *†
† Thomas Lord Department of Mechanical Engineering and Materials Science, Duke University, Durham, North Carolina 27708, United States
‡ Department of Medicine, Duke University School of Medicine, Durham, North Carolina 27710, United States
* Email: robert.tighe@duke.edu.
* Email: christine.payne@duke.edu.
04 09 2024
19 09 2024
128 37 88788885
29 04 2024
01 08 2024
15 07 2024
© 2024 The Authors. Published by American Chemical Society
2024
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

We encounter titanium dioxide nanoparticles (TiO2 NPs) throughout our daily lives in the form of food coloring, cosmetics, and industrial materials. They are used on a massive industrial scale, with over 1 million metric tons in the global market. For the workers who process these materials, inhalation is a major concern. The goal of our current research is to provide a direct comparison of the three major types of TiO2 NPs (P25, E171, R101) in terms of surface characterization, cellular response, and in vivo response following introduction into the lungs of mice. In both cellular and in vivo experiments, we observe a pro-inflammatory response to the P25 TiO2 NPs that is not observed in the E171 or R101 TiO2 NPs at mass-matched concentrations. Cellular experiments measured a cytokine, TNF-α, as a marker of a pro-inflammatory response. In vivo experiments in mice measured the number of immune cells and four pro-inflammatory cytokines (IL-6, MIP-2, IP-10, and MCP-1) present in bronchoalveolar lavage fluid. A detailed physical and chemical characterization of the TiO2 NPs shows that the P25 TiO2 NPs are distinguished by smaller primary particles suggesting that samples matched by mass contain a larger number of P25 TiO2 NPs. Cellular dose–response measurements with the P25, E171, and R101 TiO2 NPs support this hypothesis showing increased TNF-α release by macrophages as a function of TiO2 NP dose. Overall, this direct comparison of the three major types of TiO2 NPs shows that the number of particles in a dose, which is dependent on the particle diameter, is a key parameter in TiO2 NP-induced inflammation.

National Institute of Environmental Health Sciences 10.13039/100000066 5R21-ES031191 Simons Foundation 10.13039/100000893 SFARI 594594 Division of Mathematical Sciences 10.13039/100000121 NSF-DMS1764406 document-id-old-9jp4c02804
document-id-new-14jp4c02804
ccc-price
Special Issue

Published as part of The Journal of Physical Chemistry Bspecial issue “Advances in Cellular Biophysics”.
==== Body
pmcIntroduction

The use of nanomaterials in industry ranges from construction to electronics to agriculture to personal care products.1−7 The production of nanomaterials for these industries is rapidly growing, with a projected doubling of the global market between 2020 and 2031 to nearly 3.5 million tons of nanomaterials produced per year.1 This high level of use is associated with manufacturing and environmental exposures. It is projected that 21,000 tons of nanomaterials will be released into the atmosphere by 2031, with more in the water, soil, and landfills.1,7 Titanium dioxide nanoparticles (TiO2 NPs) are the second most highly used nanomaterial, following silicon dioxide NPs.1

The diversity of TiO2 NP properties has made them of special interest to physical chemists.8 Different types of TiO2 NPs are produced for use in paint, plastics, rubber, adhesives, coatings, cosmetics, and food products.1−4,7,9−11 Photocatalytic TiO2 NPs are mixed phase (80% anatase and 20% rutile) and are well studied due to their ability to generate reactive oxygen species when exposed to ultraviolet light.12−14 These are often referred to by their original supplier and number, Degussa P25. Food grade TiO2 NPs provide a bright white coloring in candy, gum, and frostings.1,10,11 These are anatase TiO2 and are often described by the European Union food additive designation of E171. Industrial rutile TiO2 NPs are produced by Chemours with the product name R101.15

In the United States, exposure to aerosolized TiO2 particles in the work environment is regulated by the Occupational Safety and Health Administration (OSHA) based on recommendations from the National Institute of Occupational Safety and Health (NIOSH).11,16 NIOSH recommends that exposure to TiO2 particles should be limited to concentrations of 2.4 mg m–3 (fine; primary particle diameter = 100 – 10 mm) and 0.3 mg m–3 (ultrafine; <100 nm) in the air of the work environment.11 The United States Food and Drug Administration (FDA) limits the concentration of TiO2 in food products to 1 wt/wt %.17 In comparison, as of January 2020, the use of TiO2 in food products was banned in France, followed by the rest of the European Union in 2022.18,19

At the most fundamental level, the possible toxicity of TiO2 NPs is dependent on the physical and chemical properties of the TiO2 NPs. Physical properties include the diameter, aggregation properties, effective surface charge, and surface defects. Chemical properties include the chemical composition and the effective surface charge. While OSHA classifies TiO2 NPs only based on diameter,11,16 many of these other surface properties could affect toxicity, but are less explored. Particle diameter is a known concern for inhalation exposures, reflected in the lower exposure limits for ultrafine (<100 nm) TiO2 particles compared to fine (100 – 10 mm) TiO2 particles set by OSHA.11,16 Previous studies of TiO2 NPs examined the impact of surface properties on inflammation and cytotoxicity showing that primary particle diameter, surface area, and aggregation behavior are driving factors in cytotoxicity and pulmonary inflammation.20−26

Our previous work has examined the cellular response to noncytotoxic concentrations of P25 and E171 TiO2 NPs and found a range of potentially detrimental effects including protein oxidation,27−29 lipid peroxidation,30 and changes to epigenetic modifying enzymes.27−29,31 Specific to inhalation, we have found that a “corona” of lung fluid proteins on the surface of P25 TiO2 NPs leads to an enhanced pro-inflammatory response compared to bare TiO2 NPs or albumin-coated TiO2 NPs.32 This suggests that specific lung fluid proteins and lipids are responsible for an increase in cytokine expression through protein-dependent responses, an increased uptake of NPs, or a combination of both.32

This current work determines the relationship between TiO2 NP properties and the pro-inflammatory response in macrophages and mice through a comparison of the in vitro and in vivo responses to P25, E171, and R101 TiO2 NPs. Macrophages were chosen based on their previous use in evaluating the cellular response to nanomaterials, as well as their presence in an inflammatory response in the lungs.33,34 The production of cytokines, including tumor necrosis factor-α (TNF-α), by macrophages has been associated with lung inflammation,35−37 and is the focus of our current study. Our prior work has shown an increase in this cytokine when macrophages were incubated with TiO2 NPs. In addition, previous work with murine macrophages (RAW 264.7 and J774) has also shown that TiO2 NPs increased pro-inflammatory cytokines associated with lung inflammation such as TNF-α, IL-4, IL-6, IL-10, IL-1β, and MIP-2.38−40 We also measure the pro-inflammatory response in mice to determine the in vivo relevance. To the best of our knowledge, this is the first study to provide a direct comparison of the three major types of TiO2 NPs.

We find that P25 TiO2 NPs have an increased pro-inflammatory response both in vitro and in vivo that is not observed in the E171 or R101 TiO2 NPs at the same concentration (250 μg mL–1). Our experiments suggest that this increased pro-inflammatory response may be attributed to a dose effect that results from the smaller diameter of the P25 TiO2 NPs. P25 TiO2 NPs are composed of a greater number of smaller diameter NPs than the E171 or R101 TiO2 NPs, resulting in a larger number of NPs for equal masses of samples. Increasing doses of both the E171 and R101 TiO2 NPs show an increase in pro-inflammatory response. We hope our work will help address the underlying relationship between TiO2 NPs and the observed lung toxicity and inflammation associated with the inhalation of TiO2 NPs with the goal of designing safer nanomaterials.41

Materials and Methods

TiO2 NPs and Characterization

Three different types of TiO2 NPs (P25, no. 718467, Sigma-Aldrich, Burlington, MA; E171, no. 100805, Millipore Sigma, Billerica, MA; R101, no. 816719, Chemours, Fayetteville, NC) were used for experiments. Surface area was determined using Brunauer–Emmett–Teller (BET) theory reported by the Micromeritics Particle Testing Authority (Norcross, GA). Dynamic light scattering (DLS; Zetasizer, Malvern Instruments, Worcestershire, England) was used to measure the hydrodynamic diameter and polydispersity index (10 μg mL–1 in Opti-MEM Reduced Serum Media; #31985070, Thermo Fisher Scientific, Waltham, MA) following sonication (5 min; #Q700, Qsonica, Newtown, CT) and ζ-potential of the NPs (10 μg mL–1 in 0.01× phosphate-buffered saline (PBS) in water). Measurements were carried out in triplicate with three distinct samples. Average and standard deviations are reported for all measurements. Electrophoretic mobility was converted to ζ-potential using the Smoluchowski approximation. Primary particle and aggregate diameters were measured from transmission electron microscopy (TEM; Tecnai G2 Twin; FEI; Hillsboro, Oregon; 25× to 700,000× magnitude, 20 to 200 kV). TiO2 NPs (1 mg mL–1 in Opti-MEM, 5 min sonication) were drop-cast onto carbon grids and allowed to sit for 30 s, then excess liquid was wicked away and samples were washed with deionized (DI) water to remove salt crystals. TEM was carried out at Duke University’s Shared Materials Instrumentation Facility. Primary particle diameters were measured for isolated particles that were not part of larger aggregates or from clearly visible boundaries within aggregates. Aggregate diameters were also measured from bright-field images taken on an inverted light microscope (Olympus 1X71; 60× objective, water immersion, Olympus, Tokyo, Japan) of TiO2 NPs suspended in Opti-MEM (0.5 mg mL–1, 5 min sonication). For both TEM and bright-field images, aggregate diameters were measured along the long axis. Image analysis was carried out with ImageJ.42 Elemental composition of the TiO2 NPs was determined with X-ray photoelectron spectroscopy (XPS; Axis Ultra XPS, Kratos, Manchester, U.K.) at Duke University’s Shared Materials Instrumentation Facility.

Cell Culture and TiO2 NP Incubations

RAW 264.7 mouse macrophages (TIB-71, ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, #12100046, Thermo Fisher Scientific) supplemented with 10% FBS (#F4135, Sigma-Aldrich) at 37 °C and 5% CO2. Cells were passaged by scraping (no. 08100240, Thermo Fisher Scientific) every 2–3 days.

For TiO2 NP incubation studies, cells were seeded at 700,000 cells mL–1 in 6-well plates (#353046, Corning, Corning, NY). Cells were grown for 24 h in DMEM with 10% FBS. Prior to the addition of TiO2 NPs, this media was removed and replaced with Opti-MEM to prevent the formation of an in situ protein corona. TiO2 NPs were sonicated (5 min, room temperature (RT); #Q700, Qsonica, Newtown, CT) in Opti-MEM and then incubated with cells for 24 h. The concentration of TiO2 NPs added to cells is noted in the figures and figure captions.

Cell viability was determined by measuring the mitochondrial activity. Mitochondrial activity was quantified using a Vybrant 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Cell Proliferation Assay Kit (No. V13154, Invitrogen, Carlsbad, CA). Cells were seeded on a 24-well plate (#25–107MP, Genesee Scientific, Morrisville, NC) at 200,000 cells mL–1 and grown for 24 h in DMEM with 10% FBS. Cells were then washed with Opti-MEM to remove FBS and incubated in Opti-MEM with TiO2 NPs (250 μg mL–1, 24 h). A detergent, Triton X-100 (10% v/v in PBS, 1 min incubation; #X100–100 ML, Sigma-Aldrich), was used as a positive control to damage cells. The MTT assay was performed following the manufacturer’s instructions.

Cytokine Assay

Following NP incubation, the cell media was harvested and centrifuged at 14,000 rpm for 10 min to remove cell debris and NPs. The supernatant was collected and stored at −20 °C. TNF-α release was measured using ELISA according to the manufacturer’s instructions (Mouse TNF-α DuoSet ELISA, #DY410, R&D Systems, Minneapolis, MN) and normalized against the untreated control. Data was analyzed in Prism (v. 9.5.1, GraphPad Software, San Diego, CA).

Mouse Exposures and Inflammation Analysis

C57BL/6 male mice (8–12 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME). All procedures were approved by the Duke University Institutional Animal Care and Use Committee (IACUC) and were performed under an IACUC-approved animal protocol (A053-21–03). Prior to in vivo TiO2 NP administration, mice were anesthetized using aerosolized isoflurane in a plexiglass chamber (VIP3000 MDX Matrix). Following anesthesia, TiO2 NPs were introduced to the lungs via oropharyngeal aspiration (40 μg of TiO2 in 50 μL PBS) and then the mice were monitored until returning to recumbency. 24 h following administration of the TiO2 NPs, the mice were deeply anesthetized with an intraperitoneal injection of a ketamine/xylazine mixture (ketamine (100 mg kg–1), xylazine (100 mg kg–1), dosed by weight). The chest wall was opened and the trachea cannulated for bronchoalveolar lavage (BAL), as described previously.37 In brief, BAL was performed by passive instillation and drainage to 20 cm of H2O. BAL cells were assessed for total cells and differentials to identify macrophages and neutrophils. BAL fluid was used to assess total protein by BCA assay and to assess cytokine responses by BAL fluid multiplex. Cytokines were analyzed using a ProcartaPlex Multiplex Immunoassay kit (Affymetrix, Bioscience) per the manufacturer’s protocol and analyzed on a MagPlex instrument (Luminex, TX). Before running the cytokine assay, BAL samples were concentrated 5 times by centrifugation in Amicon Ultra-4 Centrifugal Filter Units (EMD Millipore, MA) at 3000g (18,000 rcf), 4 °C.

Results and Discussion

TiO2 NPs and Characterization

Three different types of TiO2 NPs were used in experiments, P25 (Sigma-Aldrich), E171 (Millipore Sigma), and R101 (Chemours) (Table 1 and Figures 1 and 2). Surface area measurements (BET) show that the P25 TiO2 NPs have a much larger surface area (56.6 m2 g–1) than those of the E171 and R101 TiO2 NPs. These BET values are in agreement with supplier-reported values (P25 = 35–65 m2 g–1)43 and previously published data (P25 = 49.3–54.03 m2 g–1;44,45 E171 = 6.137–8.05 m2 g–1).45,46 No published BET values for the R101 TiO2 NPs were found. Particle diameters were measured following suspension of TiO2 NPs in Opti-MEM. Opti-MEM was chosen to mimic the state of the TiO2 NPs in the cellular experiments. All three types of TiO2 NPs show high aggregation in Opti-MEM, even after sonication, with PDI > 0.5 (Table 1). The level of aggregation is much greater than TiO2 NPs (P25 and E171) characterized in ultrapure water.27−29,31,32 It is possible that a component of the Opti-MEM solution leads to increased aggregation. ζ-Potential was measured following suspension of TiO2 NPs in 0.01× PBS diluted in water. Primary particle and aggregate diameters are reported for TEM measurements from a solution prepared in Opti-MEM and then dried (Figure 1). Aggregate diameters of these irregularly shaped NPs are measured along the long axis. Primary particle diameters are comparable to previously reported values and supplier-reported values (P25 = 21 nm;43 E171 = 148 nm;28 R101 = 290 nm),15 with the P25 TiO2 NPs having a much smaller primary particle diameter (28 ± 10 nm). Aggregate diameters were also measured with bright-field microscopy following suspension in Opti-MEM (Figure 2). The larger field of view provided by bright-field microscopy results in the observation of larger aggregates and a larger average aggregate diameter. Both TEM and bright-field microscopy images show that the P25 TiO2 NPs have the largest range in aggregate diameter.

Figure 1 Representative TEM images of TiO2 NPs: (A) P25, (B) E171, (C) R101. (D) Distribution of aggregate TiO2 NP diameters measured along the long axis from TEM images. n = 40. (E) Values from 0–1 μm are replotted from (D) to highlight the smaller diameters. Average values are shown with a blue bar, which is not within the range of (E).

Figure 2 Representative bright-field images of TiO2 NPs: (A) P25, (B) E171, (C) R101. (D) Distribution of aggregate TiO2 NP diameters measured along the long axis from bright-field microscopy images. n = 100. (E) Values from 0–5 μm are replotted from (D) to highlight the smaller diameters. Average values are shown with a blue bar, which is not within the range of (E).

Table 1 Characterization of TiO2 NP Surface Area (BET), Hydrodynamic Diameter (dh), Polydispersity Index (PDI), ζ-Potential (ZP), and Primary Particle and Aggregate Diameters Measured by TEM (dTEM) or Bright-Field (dBF) Microscopy

TiO2 NP	application (supplier)	BET (m2 g–1)	dh (nm)	PDI	ZP (mV)	dTEM primary (nm)	dTEM aggregate (μm)	dBF aggregate (μm)	
P25	photocatalysis (Sigma-Aldrich)	56.6	3010 ± 630	0.6 ± 0.5	–35 ± 1	28 ± 10	1.02 ± 7.70	4.66 ± 25.56	
E171	food coloring (Millipore Sigma)	10.1	5800 ± 3600	0.98 ± 0.06	–49 ± 5	131 ± 41	1.58 ± 2.25	4.82 ± 13.53	
R101	plastics, paint (Chemours)	7.2	1710 ± 450	0.7 ± 0.3	–29 ± 13	230 ± 84	0.57 ± 0.97	5.66 ± 17.97	

XPS showed that all three TiO2 NPs are primarily composed of titanium, oxygen, and carbon (Table 2). P25 TiO2 NPs showed no other elements. E171 TiO2 NPs showed a small amount of phosphorus (0.59%). R101 TiO2 NPs showed the inclusion of aluminum in addition to titanium, oxygen, and carbon. A previous XPS study of P25 and E171 TiO2 NPs also found that P25 TiO2 NPs consist solely of titanium (26.40%), oxygen (55.84%), and carbon (17.75%), while E171 TiO2 NPs were composed of titanium (22.52%), oxygen (61.86%), carbon (11.76%), phosphorus (2.27%), and potassium (1.59%).47 A separate study of E171 TiO2 NPs found a composition of titanium (19.8%), oxygen (62.4%), carbon (2.84%), phosphorus (2.84%), potassium (8.07%), aluminum (2.8%), and silicon (0.54%).48 We did not identify previous XPS studies of the R101 TiO2 NPs.

Table 2 XPS Analysis of the TiO2 NP Elemental Percentages

TiO2 NP	%Ti	%O	%C	%Al	%P	
P25	14.66	65.68	19.67	n.d.	n.d.	
E171	16.52	75.82	7.07	n.d.	0.59	
R101	13.52	53.22	27.70	5.56	n.d.	

P25 TiO2 NPs Result in Higher Levels of TNF-α Release from Macrophages

Based on previous work that observed a pro-inflammatory response in human lung cells (A549), human cervical cancer cells (HeLa), and murine macrophages (RAW 264.7) treated with P25 TiO2 NPs,27,29,32 we measured the production of the pro-inflammatory cytokine TNF-α using an ELISA (Figure 3). Murine macrophages (RAW 264.7) were incubated with TiO2 NPs (250 μg mL–1, 24 h). This concentration of TiO2 NPs was selected based on our previous studies on E171 TiO2 NPs that showed these TiO2 NPs were not cytotoxic at concentrations <3200 μg mL–1 in human lung cells (A549).28 Cell viability of the macrophages used in these experiments, for all three types of TiO2 NPs, was confirmed by the MTT assay (Figure S1). Experiments were conducted in serum-free media to avoid formation of a protein corona. Incubation of macrophages with P25 TiO2 NPs showed a significant increase in the expression of TNF-α compared to that of an untreated control. The E171 and R101 TiO2 NPs did not cause a significant increase in the level of TNF-α release.

Figure 3 TNF-α cytokine release by macrophages incubated with P25, E171, and R101 TiO2 NPs (250 μg mL–1, 24 h). Control cells had no NPs added. n = 6 wells of a 6-well plate. Significance was determined using one-way ANOVA. * p < 0.05, *** p < 0.001, nonsignificant comparisons are not shown.

Increasing Concentrations of E171 and R101 TiO2 NPs Result in Higher Levels of TNF-α Release from Macrophages

The cellular experiments described above used TiO2 NPs concentration matched by mass (250 μg mL–1), which results in a greater number of NPs in samples with smaller particles. An alternative approach is to compare the cellular response as a function of the number of particles. A particle number-dependent dose–response has been observed in previous in vitro and in vivo studies of TiO2 NPs.11,21 Based on the primary particle diameter measured by TEM (dTEM) (Table 1), we expect 81× more P25 TiO2 NPs than E171 TiO2 NPs and 450× more P25 TiO2 NPs than R101 TiO2 NPs in the same mass. Increasing the concentration of E171 and R101 TiO2 NPs to provide a particle number-matched sample would result in cell death.28 Decreasing the concentration of P25 TiO2 NPs to a matched level would result in a lack of cellular response. Instead, we compared a range of concentrations for each type of TiO2 NP to examine the impact of increasing particle number and determine the limits of the cytokine response (Figure 4). For P25 TiO2 NPs, we examined lower concentrations, which show a rapidly increasing response with a plateau at 100 μg mL–1 (Figure 4A). E171 TiO2 NPs also show a rapidly increasing response, but at higher concentrations than the P25 TiO2 NPs (Figure 4B). R101 TiO2 NPs show a generally low TNF-α response, but with increased levels of TNF-α release at very high concentrations (≥4000 μg mL–1) (Figure 4C). Cell viability at these higher TiO2 NPs concentrations was confirmed by the MTT assay (Figure S2).

Figure 4 TNF-α cytokine release by macrophages incubated with increasing concentrations of (A) P25, (B) E171, and (C) R101 TiO2 NPs (24 h). Control cells had no NPs added. Control values n = 12, treatment values n = 6 wells of a 6-well plate. Significance was determined using one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, nonsignificant comparisons are not shown. The control and 250 μg mL–1 data are replotted from Figure 3.

P25 TiO2 NPs Lead to an Increased Immune Response in Mice

To determine if a pro-inflammatory response was also observed in vivo, the individual TiO2 NPs (40 μg TiO2 NPs in 50 μL of PBS) versus PBS control were introduced into the lungs of mice via oropharyngeal aspiration. Dosing and time point (24 h postexposure) of lung inflammatory assessments were based on results from the NIEHS Nano GO Consortium.49 BAL total cells and neutrophils were measured as markers of lung airspace inflammation (Figure 5). When compared to the PBS control, we observed that P25 TiO2 NPs increased BAL total cells and neutrophils, consistent with the Nano GO Consortium observations. Interestingly, the E171 and R101 TiO2 NPs, when compared to the PBS control, did not demonstrate an increase in BAL total cells or neutrophils. We observed significant differences between the P25 TiO2 NPs and the E171 and R101 TiO2 NP where the P25 NP values were elevated over these two other TiO2 NPs. This suggests that airspace inflammation is different among the different types of TiO2 NPs.

Figure 5 Immune response in mice following a 24 h exposure to TiO2 NPs via oropharyngeal aspiration. PBS was used as a vehicle control. n = 5 male mice per group. A. Total cell count. B. Neutrophil cells. Significance was determined by one-way ANOVA. ** p < 0.01, *** p < 0.001, **** p < 0.0001, nonsignificant comparisons are not shown.

Given the difference in airspace inflammation, we wanted to correlate airspace inflammation with pro-inflammatory cytokine/chemokine production. We assessed IL-6, MIP-2, IP-10, and MCP-1 from the BAL fluid via multiplex ELISA (Figure 6). These pro-inflammatory cytokines and chemokines were selected based on our prior in vivo lung injury studies.37,50,51 We observed an increase in BAL fluid IL-6, MIP-2, and IP-10 when comparing the P25 TiO2 NP versus PBS control exposures (Figure 6A–C). However, there was no increase in cytokine/chemokine production in the E171 or R101 TiO2 NP exposures compared to the PBS control. The expression of MCP-1 was high enough for detection in only the mice exposed to the P25 TiO2 NPs (Figure 6D). The mice exposed to E171 and R101 TiO2 NPs had no detectable levels of MCP-1. This suggests that, similar to the in vitro experiments (Figure 3), P25 TiO2 NPs lead to an increased pro-inflammatory response compared to other types of TiO2 NPs at mass-matched concentrations.

Figure 6 Immune response in mice measured from BAL fluid after a 24 h exposure to TiO2 NPs via oropharyngeal aspiration. PBS was used as a vehicle control. n = 5 male mice per group. A. IL-6. B. MIP-2. C. IP-10. D. MCP-1. Significance was determined by one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, nonsignificant comparisons are not shown.

Conclusions

Overall, we find that in vitro and in vivo exposures to P25 TiO2 NPs result in an increased pro-inflammatory response that is not present in response to the same mass of E171 and R101 TiO2 NPs (Figures 3, 5, and 6). One unique aspect of the P25 TiO2 NPs is the range of particle diameters (Table 1 and Figures 1 and 2). The surface elemental compositions of all types of TiO2 NP are similar (Table 2). This suggests that the increased pro-inflammatory response to the P25 TiO2 NPs is likely due to the greater number of particles in these mass-matched experiments (Figure 3). In vitro experiments measuring cytokine release over a range of TiO2 NP concentrations showed that all TiO2 NPs lead to an increase in the pro-inflammatory response at higher concentrations (Figure 4).

Although these results could suggest that E171 and R101 TiO2 NPs are safer than P25 TiO2 NPs, it is important to note that all cellular exposure studies will depend on the dose (Figure 4) and cell type. Other studies have shown potential negative health effects from the E171 TiO2 NPs. For example, E171 TiO2 NPs incubated with colon cells (24 h) lead to nuclei enlargement, DNA damage, and tubulin depolymerization.52 Oral exposure of rats to E171 TiO2 NPs has led to cardiac damage.53 It is possible that different cell types or exposure routes will lead to different TiO2 NP responses.54 Recent reviews and metanalyses have highlighted gaps in the current understanding of the risk of E171 TiO2 NPs and the need for further research to correctly evaluate potential health impacts.55,56

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcb.4c02804.MTT assays of RAW 264.7 cells incubated for 24 h with P25; E171; and R101 TiO2 NPs (PDF)

Supplementary Material

jp4c02804_si_001.pdf

Author Present Address

§ Donaldson Company, Bloomington, Minnesota 55431, United States

The authors declare no competing financial interest.

Acknowledgments

The authors thank Chemours for the R101 TiO2 NPs and Faisal Anees for assistance with cell culture. This research was supported by the NSF-Simons Southeast Center for Mathematics and Biology (SCMB) through NSF-DMS1764406 and Simons Foundation-SFARI 594594, the National Institutes of Health (NIH 5R21-ES031191), and Duke MEDx.
==== Refs
References

Keller A. A. ; Ehrens A. ; Zheng Y. ; Nowack B. Developing Trends in Nanomaterials and Their Environmental Implications. Nat. Nanotechnol. 2023, 18 , 834–837. 10.1038/s41565-023-01409-z.37280284
Mohajerani A. ; Burnett L. ; Smith J. ; Kurmus H. ; Milas J. ; Arulrajah A. ; Horpibulsuk S. ; Kadir A. A. Nanoparticles in Construction Materials and Other Applications, and Implications of Nanoparticle Use. Materials 2019, 12 , 3052 10.3390/ma12193052.31547011
Hanus M. J. ; Harris A. T. Nanotechnology Innovations for the Construction Industry. Prog. Mater. Sci. 2013, 58 , 1056–1102. 10.1016/j.pmatsci.2013.04.001.
Lee J. ; Mahendra S. ; Alvarez P. J. J. Nanomaterials in the Construction Industry: A Review of Their Applications and Environmental Health and Safety Considerations. ACS Nano 2010, 4 , 3580–3590. 10.1021/nn100866w.20695513
Giraldo J. P. ; Kruss S. Nanosensors for Monitoring Plant Health. Nat. Nanotechnol. 2023, 18 , 107–108. 10.1038/s41565-022-01307-w.36609485
Newkirk G. M. ; De Allende P. ; Jinkerson R. E. ; Giraldo J. P. Nanotechnology Approaches for Chloroplast Biotechnology Advancements. Front. Plant Sci. 2021, 12 , 691295 10.3389/fpls.2021.691295.34381480
Zahra Z. ; Habib Z. ; Chung S. ; Badshah M. A. Exposure Route of TiO2 NPs from Industrial Applications to Wastewater Treatment and Their Impacts on the Agro-Environment. Nanomaterials 2020, 10 , 1469 10.3390/nano10081469.32727126
Kamat P. V. TiO 2 Nanostructures: Recent Physical Chemistry Advances. J. Phys. Chem. C 2012, 116 , 11849–11851. 10.1021/jp305026h.
Mineral Commodity Summaries; U.S. Department of the Interior, U.S. Geological Survey, 2023.
Weir A. ; Westerhoff P. ; Fabricius L. ; Hristovski K. ; Von Goetz N. Titanium Dioxide Nanoparticles in Food and Personal Care Products. Environ. Sci. Technol. 2012, 46 , 2242–2250. 10.1021/es204168d.22260395
Current Intelligence Bulletin 63: Occupational Exposure to Titanium Dioxide; U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, 2011.
Linsebigler A. L. ; Lu G. ; Yates J. T. Photocatalysis on TiO2 Surfaces: Principles, Mechanisms, and Selected Results. Chem. Rev. 1995, 95 , 735–758. 10.1021/cr00035a013.
Fujishima A. ; Zhang X. ; Tryk D. TiO2 Photocatalysis and Related Surface Phenomena. Surf. Sci. Rep. 2008, 63 (12 ), 515–582. 10.1016/j.surfrep.2008.10.001.
Diebold U. The Surface Science of Titanium Dioxide. Surf. Sci. Rep. 2003, 48 , 53–229. 10.1016/S0167-5729(02)00100-0.
R-101 Titanium Dioxide: Product Information; Chemours, 2020.
OSHA Fact Sheet: Working Safely with Nanomaterials, 2023.
Code of Federal Regulations Title 21, In Listing of Color Additives Exempt from Certification; U.S. Food and Drug Administration, 2019.
France Bans Titanium Dioxide in Food Products by January 2020; USDA Foreign Agriculture Service, 2019.
De Belder T. ; Fertig E. Titanium Dioxide Banned as a Food Additive in the EU; United States Department of Agriculture: Foreign Agricultural Service, 2022.
Andersson P. O. ; Lejon C. ; Ekstrand-Hammarström B. ; Akfur C. ; Ahlinder L. ; Bucht A. ; Österlund L. Polymorph- and Size-Dependent Uptake and Toxicity of TiO 2 Nanoparticles in Living Lung Epithelial Cells. Small 2011, 7 , 514–523. 10.1002/smll.201001832.21265017
Monteiller C. ; Tran L. ; MacNee W. ; Faux S. ; Jones A. ; Miller B. ; Donaldson K. The Pro-Inflammatory Effects of Low-Toxicity Low-Solubility Particles, Nanoparticles and Fine Particles, on Epithelial Cells in Vitro: The Role of Surface Area. Occup. Environ. Med. 2007, 64 , 609–615. 10.1136/oem.2005.024802.17409182
Brzicova T. ; Sikorova J. ; Milcova A. ; Vrbova K. ; Klema J. ; Pikal P. ; Lubovska Z. ; Philimonenko V. ; Franco F. ; Topinka J. ; Rossner P. Nano-TiO2 Stability in Medium and Size as Important Factors of Toxicity in Macrophage-like Cells. Toxicol. In Vitro 2019, 54 , 178–188. 10.1016/j.tiv.2018.09.019.30287216
Kose O. ; Tomatis M. ; Leclerc L. ; Belblidia N.-B. ; Hochepied J.-F. ; Turci F. ; Pourchez J. ; Forest V. Impact of the Physicochemical Features of TiO 2 Nanoparticles on Their In Vitro Toxicity. Chem. Res. Toxicol. 2020, 33 , 2324–2337. 10.1021/acs.chemrestox.0c00106.32786542
Kobayashi N. ; Naya M. ; Endoh S. ; Maru J. ; Yamamoto K. ; Nakanishi J. Comparative Pulmonary Toxicity Study of Nano-TiO2 Particles of Different Sizes and Agglomerations in Rats: Different Short- and Long-Term Post-Instillation Results. Toxicology 2009, 264 , 110–118. 10.1016/j.tox.2009.08.002.19666077
Wang J. ; Fan Y. Lung Injury Induced by TiO2 Nanoparticles Depends on Their Structural Features: Size, Shape, Crystal Phases, and Surface Coating. Int. J. Mol. Sci. 2014, 15 , 22258–22278. 10.3390/ijms151222258.25479073
Danielsen P. H. ; Knudsen K. B. ; Štrancar J. ; Umek P. ; Koklič T. ; Garvas M. ; Vanhala E. ; Savukoski S. ; Ding Y. ; Madsen A. M. ; et al. Effects of Physicochemical Properties of TiO2 Nanomaterials for Pulmonary Inflammation, Acute Phase Response and Alveolar Proteinosis in Intratracheally Exposed Mice. Toxicol. Appl. Pharmacol. 2020, 386 , 114830 10.1016/j.taap.2019.114830.31734322
Jayaram D. T. ; Runa S. ; Kemp M. L. ; Payne C. K. Nanoparticle-Induced Oxidation of Corona Proteins Initiates an Oxidative Stress Response in Cells. Nanoscale 2017, 9 , 7595–7601. 10.1039/C6NR09500C.28537609
Jayaram D. T. ; Payne C. K. Food-Grade TiO2 Particles Generate Intracellular Superoxide and Alter Epigenetic Modifiers in Human Lung Cells. Chem. Res. Toxicol. 2020, 33 , 2872–2879. 10.1021/acs.chemrestox.0c00331.33064449
Jayaram D. T. ; Kumar A. ; Kippner L. E. ; Ho P.-Y. ; Kemp M. L. ; Fan Y. ; Payne C. K. TiO2 Nanoparticles Generate Superoxide and Alter Gene Expression in Human Lung Cells. RSC Adv. 2019, 9 , 25039–25047. 10.1039/C9RA04037D.35321350
Runa S. ; Hussey M. ; Payne C. K. Nanoparticle–Cell Interactions: Relevance for Public Health. J. Phys. Chem. B 2018, 122 , 1009–1016. 10.1021/acs.jpcb.7b08650.29111728
Jayaram D. T. ; Payne C. K. Intracellular Generation of Superoxide by TiO2 Nanoparticles Decreases Histone Deacetylase 9 (HDAC9), an Epigenetic Modifier. Bioconjugate Chem. 2020, 31 , 1354–1361. 10.1021/acs.bioconjchem.0c00091.
Poulsen K. M. ; Albright M. ; Niemuth N. J. ; Tighe R. M. ; Payne C. K. Interaction of TiO2 Nanoparticles with Lung Fluid Proteins and the Resulting Macrophage Inflammatory Response. Environ. Sci.: Nano 2023, 10 , 2427–2436. 10.1039/D3EN00179B.38009084
Borgognoni C. F. ; Kim J. H. ; Zucolotto V. ; Fuchs H. ; Riehemann K. Human Macrophage Responses to Metal-Oxide Nanoparticles: A Review. Artif. Cells, Nanomed., Biotechnol. 2018, 46 , 694–703. 10.1080/21691401.2018.1468767.29726285
You D. J. ; Bonner J. C. Susceptibility Factors in Chronic Lung Inflammatory Responses to Engineered Nanomaterials. Int. J. Mol. Sci. 2020, 21 , 7310 10.3390/ijms21197310.33022979
Duque G. A. ; Descoteaux A. Macrophage Cytokines: Involvement in Immunity and Infectious Diseases. Front. Immunol. 2014, 5 , 491 10.3389/fimmu.2014.00491.25339958
Hussain S. ; Johnson C. G. ; Sciurba J. ; Meng X. ; Stober V. P. ; Liu C. ; Cyphert-Daly J. M. ; Bulek K. ; Qian W. ; Solis A. ; Sakamachi Y. ; Trempus C. S. ; Aloor J. J. ; Gowdy K. M. ; Foster W. M. ; Hollingsworth J. W. ; Tighe R. M. ; Li X. ; Fessler M. B. ; Garantziotis S. TLR5 Participates in the TLR4 Receptor Complex and Promotes MyD88-Dependent Signaling in Environmental Lung Injury. eLife 2020, 9 , e50458 10.7554/eLife.50458.31989925
Tighe R. M. ; Birukova A. ; Yaeger M. J. ; Reece S. W. ; Gowdy K. M. Euthanasia- and Lavage-Mediated Effects on Bronchoalveolar Measures of Lung Injury and Inflammation. Am. J. Respir. Cell Mol. Biol. 2018, 59 , 257–266. 10.1165/rcmb.2017-0357OC.29481287
Miller T. J. ; Knapton A. ; Adeyemo O. O. ; Noory L. S. ; Weaver J. L. ; Hanig J. P. ; Honchel R. ; Zhang J. ; Espandiari P. ; Benedick M. F. ; et al. Toxicology of Titanium Dioxide (TiO2) Nanoparticles: In Vitro and in Vivo Evaluation of Macrophage Uptake of TiO2. FASEB J. 2007, 21 , A812 10.1096/fasebj.21.6.A812.
Kang J. L. ; Moon C. ; Lee H. S. ; Lee H. W. ; Park E.-M. ; Kim H. S. ; Castranova V. Comparison of the Biological Activity between Ultrafine and Fine Titanium Dioxide Particles in RAW 264.7 Cells Associated with Oxidative Stress. J. Toxicol. Environ. Health, Part A 2008, 71 , 478–485. 10.1080/15287390801906675.
Shi H. ; Magaye R. ; Castranova V. ; Zhao J. Titanium Dioxide Nanoparticles: A Review of Current Toxicological Data. Part. Fibre Toxicol. 2013, 10 , 15 10.1186/1743-8977-10-15.23587290
Gakis G. P. ; Aviziotis I. G. ; Charitidis C. A. Metal and Metal Oxide Nanoparticle Toxicity: Moving towards a More Holistic Structure–Activity Approach. Environ. Sci.: Nano 2023, 10 , 761–780. 10.1039/D2EN00897A.
Schneider C. A. ; Rasband W. S. ; Eliceiri K. W. NIH Image to ImageJ: 25 Years of Image Analysis. Nat. Methods 2012, 9 , 671–675. 10.1038/nmeth.2089.22930834
Product Specification: Titanium(IV) Oxide; Sigma-Aldrich, 2023.
Yu J. ; Yu H. ; Cheng B. ; Zhou M. ; Zhao X. Enhanced Photocatalytic Activity of TiO2 Powder (P25) by Hydrothermal Treatment. J. Mol. Catal. A: Chem. 2006, 253 , 112–118. 10.1016/j.molcata.2006.03.021.
Yusoff R. ; Kathawala M. H. ; Nguyen L. T. H. ; Setyawati M. I. ; Chiew P. ; Wu Y. ; Ch’ng A. L. ; Wang Z. M. ; Ng K. W. Biomolecular Interaction and Kinematics Differences between P25 and E171 TiO2 Nanoparticles. NanoImpact 2018, 12 , 51–57. 10.1016/j.impact.2018.10.001.
Jovanović B. ; Jovanović N. ; Cvetković V. J. ; Matić S. ; Stanić S. ; Whitley E. M. ; Mitrović T. L. The Effects of a Human Food Additive, Titanium Dioxide Nanoparticles E171, on Drosophila Melanogaster - a 20 Generation Dietary Exposure Experiment. Sci. Rep. 2018, 8 , 17922 10.1038/s41598-018-36174-w.30560898
Ortelli S. ; Costa A. L. ; Zanoni I. ; Blosi M. ; Geiss O. ; Bianchi I. ; Mehn D. ; Fumagalli F. ; Ceccone G. ; Guerrini G. ; Calzolai L. TiO2@BSA Nano-Composites Investigated through Orthogonal Multi-Techniques Characterization Platform. Colloids Surf., B 2021, 207 , 112037 10.1016/j.colsurfb.2021.112037.
Yang Y. ; Doudrick K. ; Bi X. ; Hristovski K. ; Herckes P. ; Westerhoff P. ; Kaegi R. Characterization of Food-Grade Titanium Dioxide: The Presence of Nanosized Particles. Environ. Sci. Technol. 2014, 48 , 6391–6400. 10.1021/es500436x.24754874
Bonner J. C. ; Silva R. M. ; Taylor A. J. ; Brown J. M. ; Hilderbrand S. C. ; Castranova V. ; Porter D. ; Elder A. ; Oberdorster G. ; Harkema J. R. ; et al. Interlaboratory Evaluation of Rodent Pulmonary Responses to Engineered Nanomaterials: The NIEHS Nano GO Consortium. Environ. Health Perspect. 2013, 121 , 676–682. 10.1289/ehp.1205693.23649427
Birukova A. ; Cyphert-Daly J. ; Cumming R. I. ; Yu Y.-R. ; Gowdy K. M. ; Que L. G. ; Tighe R. M. Sex Modifies Acute Ozone-Mediated Airway Physiologic Responses. Toxicol. Sci. 2019, 169 , 499–510. 10.1093/toxsci/kfz056.30825310
Dunigan-Russell K. ; Yaeger M. J. ; Hodge M. X. ; Kilburg-Basnyat B. ; Reece S. W. ; Birukova A. ; Guttenberg M. A. ; Novak C. ; Chung S. ; Ehrmann B. M. ; et al. Scavenger Receptor BI Attenuates Oxidized Phospholipid-Induced Pulmonary Inflammation. Toxicol. Appl. Pharmacol. 2023, 462 , 116381 10.1016/j.taap.2023.116381.36681128
Rodríguez-Ibarra C. ; Medina-Reyes E. I. ; Déciga-Alcaraz A. ; Delgado-Buenrostro N. L. ; Quezada-Maldonado E. M. ; Ispanixtlahuatl-Meráz O. ; Ganem-Rondero A. ; Flores-Flores J. O. ; Vázquez-Zapién G. J. ; Mata-Miranda M. M. ; et al. Food Grade Titanium Dioxide Accumulation Leads to Cellular Alterations in Colon Cells after Removal of a 24-h Exposure. Toxicology 2022, 478 , 153280 10.1016/j.tox.2022.153280.35973603
Herrera-Rodríguez M. A. ; del Pilar Ramos-Godinez M. ; Cano-Martínez A. ; Segura F. C. ; Ruiz-Ramírez A. ; Pavón N. ; Lira-Silva E. ; Bautista-Pérez R. ; Thomas R. S. ; Delgado-Buenrostro N. L. ; et al. Food-Grade Titanium Dioxide and Zinc Oxide Nanoparticles Induce Toxicity and Cardiac Damage after Oral Exposure in Rats. Part. Fibre Toxicol. 2023, 20 , 43 10.1186/s12989-023-00553-7.37978398
Evans S. J. ; Lawrence R. L. ; Ilett M. ; Burgum M. J. ; Meldrum K. ; Hondow N. ; Jenkins G. J. ; Clift M. J. D. ; Doak S. H. Industrial-Relevant TiO2 Types Do Not Promote Cytotoxicity in the A549 or TK6 Cell Lines Regardless of Cell Specific Interaction. Toxicol. In Vitro 2022, 83 , 105415 10.1016/j.tiv.2022.105415.35752104
Younes ; Younes M. ; Aquilina G. ; Castle L. ; Engel K. ; Fowler P. ; Fernandez M. J. F. ; Fürst P. ; Gundert-Remy U. ; Gürtler R. ; Husøy T. ; et al. Safety Assessment of Titanium Dioxide (E171) as a Food Additive. EFSA J. 2021, 19 , e06585 10.2903/j.efsa.2021.6585.33976718
Bischoff N. S. ; De Kok T. M. ; Sijm D. T. H. M. ; Van Breda S. G. ; Briedé J. J. ; Castenmiller J. J. M. ; Opperhuizen A. ; Chirino Y. I. ; Dirven H. ; Gott D. ; et al. Possible Adverse Effects of Food Additive E171 (Titanium Dioxide) Related to Particle Specific Human Toxicity, Including the Immune System. Int. J. Mol. Sci. 2021, 22 , 207 10.3390/ijms22010207.
