A comparative biodistribution and toxicity study of single and multi-component nanomaterials: NMs: TiO₂, SiC, and SiC@TiO₂

Abstract

Background

With the increasing integration of nanomaterials (NMs) into daily life, their technological advantages have become evident. However, their intricate interactions with biological systems introduce complexities that can lead to unpredictable toxicological outcomes. This study investigated the in vivo toxicokinetics and toxicodynamics of single- and multi-component NMs composed of silicon carbide (SiC), titanium dioxide (TiO₂), and a SiC@TiO₂ composite, along with a physical mixture of SiC and TiO₂ in the same ratio as the composite. Rats were exposed to these materials via single intratracheal instillation, and biological responses were assessed over time (1 h to 28 days) to identify the no-observed-adverse-effect level (NOAEL).

Results

All NMs induced minimal structural alterations in lung tissue and prompted varying degrees of inflammatory cell infiltration. Over time, translocation from the lungs to secondary organs (heart, spleen, liver, kidney) was observed, with distinct distribution patterns between Si- and Ti-containing NMs. Bronchoalveolar lavage fluid analysis revealed a minimal to mild inflammatory response that evolved in a time-dependent manner, even at NOAEL exposure levels, suggesting delayed-onset biological effects.

Conclusions

SiC@TiO₂ demonstrated a reduced pulmonary toxicological profile relative to its single-component counterparts, likely due to antagonistic effects between its constituents. These findings highlight the need to assess multicomponent nanomaterials as distinct entities and suggest that rational material design may help mitigate adverse biological effects, supporting safer nanotechnology development.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12989-026-00664-x.

Background

As industrialization continues to accelerate and the use of engineered nanomaterials increases [1, 2], there is need to understand, predict and manage potential health risks associated with human exposure, especially in the workplace where nanomaterials are produced [3–5]. For workers handling nanomaterials, inhalation of nanoparticles is the most concerning route of exposure [6], followed by skin exposure and oral ingestion [7, 8]. In many studies it has been shown that inhalation of man-made nanomaterials (NMs) such as titanium dioxide (TiO₂) and carbon nanotubes [9–11] can lead to lung inflammation and fibrosis. Evidence from epidemiological studies also confirms a significant association between inhalation of small particles, particularly nanomaterials, from ambient air and cardiovascular events such as angina pectoris, arrhythmias, ischemic heart failure and sudden death [12]. Because these particles are in the respirable size range, it is important to understand the potential pulmonary effects of NMs suspended in the air as an aerosol [13–15].

TiO₂ NMs are widely used in paint, plastics, cosmetics, photodynamic therapy and treatment of waste water [16–18]. TiO₂ may be suspended in the air during production, distribution and use. TiO₂ can be an integral part of environments and therefore may be present in the air we breathe. Conventional white TiO₂ particles exhibit low quantum yields as photocatalytic agents, primarily due to the rapid recombination of electrons (e-) and holes (h+) within their band structure [19], which limits their therapeutic effectiveness. Silicon carbide (SiC), known for its strong adhesion and chemical stability, presents excellent support for TiO₂. When the two are properly combined into SiC@TiO₂, SiC can make up for the shortage of TiO₂ and significantly improve the photocatalytic activity [20]. In light of the SUNSHINE project (https://www.h2020sunshine.eu) the intended application was to apply it on aluminium baking moulds as alternative to Teflon for hydrophobic and anti-sticking properties. TiO₂ was incorporated to improve the compatibility of SiC within the silicon-based paint matrix used in the coating.

Given the growing prevalence of multi-component nanomaterials (MCNMs) in industrial and environmental contexts, there is the need to understand their long-term health effects under realistic exposure conditions. Multi-component nanomaterials exhibit complex biological interactions due to differences in physicochemical properties among their constituents, potentially leading to synergistic or antagonistic effects on absorption, distribution, metabolism, and excretion. Moreover MCNMs may induce nonlinear toxicological responses, such as combined additive toxicity, synergism or antagonism, and even trigger systemic toxicity through intercomponent interference, challenging conventional risk assessment and impacting long-term safety. Therefore, the SUNSHINE project was aimed to provide safety information on MCNMs primarily based on in vitro toxicological data. However, data on how SiC@TiO₂ would behave in vivo in terms of local and systemic toxicity after translocation of NMs was needed for the purpose of ‘in vitro to in vivo extrapolation’ modelling and risk assessment. In addition, such data would also facilitate a safer-by-design strategy. Most published studies focus on short-term exposure to single-component NMs, but it remains unclear how this information can be used to assess the effects of multi-component NMs or mixtures of single NMs. Therefore, the aim of this study is to investigate the pulmonary toxicity of single and combined SiC and TiO₂ NMs after acute exposure of the lung, including the assessment of the biodistribution after several timepoints. We hypothesized that the MCNM would cause less pulmonary toxicity than its components separately or when mixed in the same ratio, and this would be, at least in part, a result of faster clearance from the lung. This work aims to bridge current knowledge gaps and provide a more comprehensive risk assessment approach for emerging multi-component nanomaterials.

Methods

Design

The study consisted of two parts. Part one was a range finding study to assess a dose level that would not cause clear toxicity up to 3 days as this may have a profound effect on translocation of NMs from the lung to other organs. For the main study, that was targeted to obtain information of the biodistribution of NMs over the main organs, we used not only the single and the multicomponent but also a mixture of the single components (see below) and 6 timepoints after a single dose.

Materials

SiC and TiO₂ and the MCNM SiC@TiO₂ (60 nm) were obtained from Laurentia Technologies SLL (Paterna, Valencia, Spain). Inductively Coupled Plasma Optical Emission Spectroscopy 85% SiC, 15% TiO₂ (by weight). Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) analysis was performed by an ICP-OES 5100 – vertical dual view apparatus coupled with OneNeb nebulizer (Agilent Technologies, Santa Clara, CA, USA). The quantification of elemental composition was performed by adding 10% vol/vol of ultrapure sulfuric acid, 1% vol/vol of hydrofluoric acid and 10% vol/vol of ultrapure nitric acid to ensure complete digestion of the sample. Calibration curves were obtained with 0.1, 1.0, 10.0 and 100.0 mg·L-1 standards prepared in MilliQ water, using the same procedure applied to samples.

High-resolution TEM on SiC@TiO₂ 60 nm confirmed a core-shell structure, where larger SiC cores are coated with smaller TiO₂ particles hetero-nucleated on their surface, forming a flower-like morphology. Electron diffraction analysis showed the presence of β-SiC in the core and anatase-phase TiO₂ in the shell. The TiO₂ used as a reference for the single component and the physical mixture (SiC + TiO₂) was the commercial Aeroxide TiO₂ P25. Further analysis revealed that the TiO₂ particles on the SiC surface have a mean diameter of 6–7 nm. The SiC nanoparticles used in both the single-component and multicomponent systems are identical, with a size range of 40–60 nm. XRD analysis confirmed these nanoparticles to be in the β-SiC phase according to JCPDS card no. 01-089-5012. Dissolution tests in H₂O, Roswell Park Memorial Institute (RPMI) cell culture medium with or without 10% Fetal Bovine Serum and phagolysosomal simulant fluid. TiO₂ was not detected by ICP-OES, it was under the LOD (about 0.02 ppm).

Animal

Specific-pathogen-free (SPF) Sprague-Dawley rats, weighing with a range of 190–210 g, were purchased from Beijing Vital River Laboratory Animal Technology. Rats were maintained in standard housing conditions (temperature: 25 ± 1 °C, relative humidity: 50 ± 5%; light and dark cycle: 12 h:12 h) and had free access to standard rat chow and water. Food (sterile feed) and water (through automatic watering system) were provided ad libitum. This study was approved by the Ethical Committee of the CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety & CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, 100,190 Beijing, China as documented in PONY-2022-FL-12. All procedures used in this experiment were compliant with the local ethics committee.

Sample preparation and animal exposure

The NMs were suspended in water and then dispersed using a vortex shaker before administration to the animals. Briefly, rats were deeply anesthetized with isoflurane until fully unconscious. To ensure precise and uniform delivery of the nanomaterial suspension directly into the trachea, an exposed intratracheal instillation procedure was performed. The neck area was shaved, the skin was incised, and the trachea was gently exposed for accurate administration. A plastic syringe was inserted into the trachea (endotracheal instillation) and a suspension of NMs was instilled at a volume of 0.1 mL irrespective of the bodyweight. The control group received 0.1 mL of phosphate buffered saline via intratracheal administration. Following administration, the trachea and the skin were closed.

For the range finding study, groups of rats were exposed to 0.05–0.5–5 mg per rat (Table 1) and sacrificed on day 3. The heart, liver, spleen, lungs, and kidneys were subsequently removed and rinsed three times with normal saline to ensure complete blood removal. These organs were used for ICP-OES testing and histopathology.

Table 1.

Summary of administered doses in the DRF and main studies

Study	Concentration (mg/mL)	Instillation volume (mL/rat)	Dose (mg/rat)	Number of animals (N)	Observation duration
DRF study	0.5 / 5 / 50	0.1	0.05 / 0.5 / 5	3 / group	3 days
Main study	1.5	0.1	0.15	4 / group	1 h, 1, 3, 7, 14, 28 days

For the main study to assess the biodistribution, the 1.5 mg/mL was derived from the range finding study as a level that was expected not to induce acute effects that would affect the clearance of and retention from the lung. Rats were examined at various time points (1 h, 1d, 3d, 7d, 14d and 28d) after having received a single, one-time, dose of 0.15 mg of SiC and TiO₂ pre rat, the dose (0.15 mg/rat; ; see Table 1) that was derived from the range finding study as expected not to cause clear toxicity or pathology in main study. To investigate whether the combination and their binding influence their biological effect, we included an SiC+TiO₂ experimental group as a physical mixture at an 85:15 (w/w) ratio, consistent with the elemental composition of the multicomponent SiC@TiO₂ material, as well as the multi-component SiC@TiO₂ group itself.

Lungs from rats sacrificed 1 h after dosing were taken for ultrastructure analysis via electron microscopy to asses the size and shape of the retained NMs in the lung. Only at day 3 and 28 post-exposure bronchoalveolar lavage fluid (BALF) was collected by performing lavages with 2 mL of sterile saline, followed by centrifugation at 1000 ×g for 10 min at 4 °C. BALF was used for the assessment of cytotoxicity and the supernatant was then stored at − 80 °C, in order to detect total protein and cytokines concentrations. For the organ burdens, the lungs, heart, liver, spleen and kidneys were subsequently removed and blood vessels were rinsed three times with normal saline to ensure complete blood removal and weighed. These organs were used for assessment of Si and Ti content by Inductively Coupled Plasma Optical Emission spectroscopy (ICP-OES) and pathological examination. The limits of detection (LOD) were 0.01 mg/L for Si and 0.003 mg/L for Ti, and the limits of quantification (LOQ) were 0.05 mg/L for Si and 0.012 mg/L for Ti.

Transmission electron microscopy (TEM)

Lungs from rats sacrificed 1 h after dosing were taken for ultrastructure analysis by Transmission Electron Microscope (Hitachi TEM HT7700, Japan). The lung tissues were fixed using glutaraldehyde and osmium tetroxide solution, dehydrated in ethanol, and embedded in epoxy resin. At room temperature, ultrathin sections of lung tissue were stained with 2% uranyl acetate solution and 0.5% lead citrate solution and examined for particle deposition.

Characterization of NMs

All NMs were obtained from partners of the SUNSHINE project, that was funded from the European Union’s Horizon 2020 research and innovation program under grant agreement No 952,924. To ensure morphology and size distribution, high-resolution imaging measurements were conducted using a G2 20 S-TWIN TEM (Tecnai, Oregon, USA) operating at an accelerating voltage of 200 kV. TEM grids were prepared by drop-casting 10 µL of NMs dispersion in water at a final concentration of 0.01 mg/mL and allowed to dry overnight before TEM analysis. The hydrodynamic sizes and Zeta potentials of the NMs suspensions used to dose the animals were determined by dynamic light scattering (DLS) using a Malvern Zeta sizer Nano series Nano-ZS instrument (Malvern Panalytical Ltd, Malvern, UK). The dissolution tests were performed on SiC and SiC@TiO₂ dispersed at 100 mg/L in different test media: MilliQ water, RPMI, RPMI with 10% FBS, and phagolysosomal simulant fluid. The suspensions were incubated at 37 °C under shaking conditions for 1, 24, and 48 h. After the incubation time, samples were ultracentrifuged at 50,000 rpm for 15 min (Ultracentrifuge OptimaTM MAX-XP, Beckman Coulter). 3 mL of the media were collected from the top of the centrifuge tube, acid digested and analyzed by ICP-OES for elemental analysis (ICP-OES 5100—vertical dual view apparatus - Agilent Technologies, Santa Clara, CA, USA). The instrument was calibrated with standards ranging from 0.1 to 100.0 mg/L for silicon and titanium.

Elemental analysis

ICP-OES samples were prepared by weighing approximately 0.4–0.5 g of tissue samples, followed by the addition of 6 mL of nitric acid for overnight digestion. On the next day, 2 mL of H₂O₂ and 100 µL of HF were added to the digestion vessel. The digestion process was conducted using a microwave digester, the temperature was gradually increased from room temperature to 150 °C, over 20 min, followed by continuous heating at 150 °C for 30 min. After cooling to room temperature, the digested sample solution was transferred to a Teflon-lined vessel for acid extraction, where it was heated until the volume was reduced to approximately 0.3–0.5 mL. The remaining volume was washed with a 2% (v/v) HNO₃, adjusting the final volume to 3 mL. The prepared samples were measured in duplicate using a PerkinElmer Optima 8000 ICP-OES (USA).

Toxicity assessment

To assess cytotoxicity as a measure of lung damage, Bronchoalveolar lavage (BALF) in the main study was performed using three sequential lavages: the first lavage with 2 mL PBS, and two subsequent lavages with 5 mL PBS each. Only the first 2-mL lavage was used for cytokine and biochemical analyses, as this fraction contains the cells and soluble mediators representative of the airway lumen. Total protein (TP) concentration in BALF was measured at 3 and 28 days post-exposure as an indicator of alveolar-capillary barrier integrity and cell damage, reflecting increased permeability and potential tissue injury. TP concentration was determined with a Pierce BCA protein assay kit (Thermo Scientific, USA). Additionally, levels of pro-inflammatory and anti-inflammatory cytokines in the BALF, including TNF-α, IL-1β, IFN-γ, and IL-10 were measured using enzyme-linked immunosorbent assay (ELISA) kits (Thermo Scientific, USA), following the manufacturer’s instructions. Any unused supernatant was stored at − 80 °C for future analysis.

Histology

Two rats from each group, at each designated time point post-exposure, were used for lung histopathological assessment. The range of the lung weight of adult male Sprague-Dawley rats used in this study was 2.2–2.5 mg. The selected lung tissue sections were placed in embedding cassettes and immersed in 10% paraformaldehyde for fixation. After fixation, the tissues were routinely processed and embedded in paraffin for histopathological examination. Tissue sections, 4 μm thick, were cut and stained with haematoxylin and eosin (H&E) to visualize pulmonary inflammatory infiltration. Additionally, the embedded lung tissues were sectioned to a thickness of 3 μm and stained with Masson’s trichrome to evaluate lung fibrosis.

Statistical analysis

Data are presented as mean ± SD. Differences between the two groups were analysed using one-way and two-way ANOVA with GraphPad Prism 8.0 (GraphPad Software). Each group included at least three replicates, and P-values < 0.05 were considered statistically significant. Significance levels were denoted as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Results

NMs characterization

TEM images showed that the suspended NMs were spherical, most of them were in an agglomeration state, and the average size was 50–100 nm (Figs. 1 and S1a). DLS results showed that the hydrodynamic particle sizes of SiC, TiO₂, SiC@TiO_2, and SiC+TiO₂ were 204.3 nm (PDI = 0.41), 260.2 nm (PDI = 0.45), 194.3 nm (PDI = 0.33) and 586.3 nm (PDI = 0.31) (Figs. 1d and S1b), and the Zeta potentials were − 13.76 ± 0.22 mV, 11.47 ± 1.07 mV, -21.78 ± 0.66 mV and − 7.85 ± 1.33 mV (Figs. 1e and S1c) respectively. The MCNM (SiC@TiO₂) exhibit a narrow hydrodynamic size distribution, indicating better colloidal stability in suspension compared to their single-component counterparts. This agglomeration state can significantly influence their biodistribution in tissues. Additional characterization data are presented the supplement (Fig. S2).

Fig. 1.

Physicochemical characterization of NMs. a–c TEM images of SiC, TiO_2, and SiC@TiO₂, respectively, with a scale bar of 200 nm. d TEM images with high magnification of SiC@TiO2 indicating a flower morphology. e Hydrodynamic diameter and (f) Zeta potential in water at room temperature as measured by dynamic light scattering. A summary of the the main physicochemical characterization results is in press in form of proceedings that can be cited by the present paper. Blosi at al “SSbD Industrial Case Study: Sustainable Alternatives to Anti-Stick Coatings for Aluminum Molds in Bakery Application” Safe and Sustainable Value Creation by Design - Proceedings of the 21st Global Conference on Sustainable Manufacturing (GCSM 2025) September 10–12, 2025, Bologna, Italy - Volume 1, Lecture Notes in Mechanical Engineering

SiC (band gap 3.0 eV) forms a heterojunction when it comes into contact with TiO₂ (band gap 3.2 eV). Because the conduction band position of TiO₂ is higher than that of SiC, electrons migrate from TiO₂ to SiC, resulting in increased electron density on the SiC surface, enhancing electronegativity. The positive charge on the TiO₂ surface decreases or even reverses due to electron loss.

Figure 1d shows the flower-like morphology observed for the SiC@TiO₂ sample throughout a dedicated campaign analysis addressed to explore the TiO₂ morphology. The particle size distribution was calculated on more than 50 TiO₂ nanoparticles surrounding the SiC core and highlighting a mean diameter of 6–7 nm. Figure 1b highlights the lattice parameters derived from the electron diffraction analysis and consistent with β-SiC in the core and anatase TiO₂ in the shell

Range finding

Incorporation into a multicomponent nanomaterial (SiC@TiO₂) May reduce organ tropism of pure TiO₂ NM

In the dose range-finding study, the biodistribution of Ti and Si following intratracheal instillation was quantified in major organs 3 days post-exposure using three widely separated dose levels: 5 mg/rat, 0.5 mg/rat, and the lowest dose, 0.05 mg/rat (Fig. 2; Tables 2 and S1). The objective was to investigate the dose-dependent effects of single- and multi-component nanomaterials on biological distribution and inflammatory response.

Fig. 2.

The biodistribution of NMs after a single dose (0.5, 5, and 50 mg/mL) applied to rats by endotracheal instillation (a–d). Si and Ti contents in organs were measured by inductively coupled plasma optical emission spectrometry (ICP-OES). The Si content is similar between SiC and SiC@TiO₂. The Ti content is similar between TiO₂ and SiC@TiO₂. Error bar denotes standard deviation (n = 3)

Table 2.

The biodistribution of NMs after a single dose (0.05, 0.5, and 5 mg/rat) 3 days after administration

Groups	Dose		Heart (µg/g)		Liver (µg/g)		Spleen (µg/g)		Lung (µg/g)		Kidney (µg/g)
SiC	0.05 mg		1.01 ± 0.04		0.47 ± 0.09		0.73 ± 0.18		0.57 ± 0.16		0.73 ± 0.11
	0.5 mg		1.58 ± 0.26		0.48 ± 0.06		0.65 ± 0.08		1.82 ± 0.25		2.54 ± 0.27
	5 mg		2.78 ± 0.01		0.69 ± 0.07		1.16 ± 0.06		45.3 ± 13.24		4.22 ± 0.11
TiO2	0.05 mg		0.16 ± 0.01		0.04 ± 0.01		0.4 ± 0.16		6.20 ± 1.66		2.55 ± 0.46
	0.5 mg		1.06 ± 0.77		0.17 ± 0.04		1.25 ± 0.36		48.13 ± 21.01		12.04 ± 2.19
	5 mg		0.37 ± 0.06		0.49 ± 0.11		1.56 ± 0.59		111.57 ± 23.93		27.49 ± 8.05

			Si	Ti	Si	Ti	Si	Ti	Si	Ti	Si	Ti
	0.05 mg		1.11 ± 0.26	0.11 ± 0.04	0.35 ± 0.06	0.03 ± 0.02	0.33 ± 0.14	0.22 ± 0.12	0.46 ± 0.07	4.08 ± 0.38	0.77 ± 0.15	0.36 ± 0.05
SiC@TiO2	0.5 mg		2.37 ± 1.15	0.15 ± 0.04	0.62 ± 0.16	0.03 ± 0.01	0.72 ± 0.12	0.22 ± 0.06	1.78 ± 0.18	5.87 ± 5.56	1.27 ± 0.27	12.27 ± 2.63
	5 mg		2.50 ± 0.68	0.28 ± 0.04	0.58 ± 0.14	0.15 ± 0.01	1.21 ± 0.59	0.28 ± 0.09	40.39 ± 13.88	100.45 ± 13.08	3.37 ± 1.87	16.22 ± 2.89

Overall, for the Si-containing nanomaterials (SiC and SiC@TiO₂), Si predominantly accumulated within the lung tissue. However, low levels of Si were detectable in the kidney at the higher doses (0.5 mg/rat and 5 mg/rat) at 3 days post-exposure. Considering the relatively large size of the Si-NMs, this observation suggests that the nanoparticles may undergo biotransformation in vivo, leading to the formation of non-particulate, more readily metabolized forms of the element that are subsequently excreted via the kidney. Interestingly, small amounts of Si were also detected in the heart at the highest dose level.

Regarding the Ti-containing nanomaterials (TiO₂ and SiC@TiO₂), significant tissue burdens were detected in both the lungs and kidneys. Ti concentrations in the lungs exhibited a dose-dependent increase and were significantly higher than controls at doses of 0.5 mg/rat and 5 mg/rat. Renal translocation was also evident, with Ti levels in the kidney significantly elevated at the two highest doses for both pure TiO₂ and the SiC@TiO₂ composite. Conversely, Ti levels in the liver, spleen, and heart did not differ significantly from baseline control values across the tested dose range.

Collectively, the range-finding study identified the lung as the primary site of deposition for all materials, with measurable but limited kidney accumulation observed for the Ti-containing materials.

Toxicity assessment

H&E staining was used to reveal clear changes in alveolar structure and infiltration of inflammatory cells (such as neutrophils, macrophages), serving as an important method for evaluating acute lung inflammatory response to NMs (Figs. 3a and S3). These images were used to assess the pathological effects of these nanomaterials on lung tissue after single-dose exposure.

Fig. 3.

Histology assessment of lungs exposure to NMs at highest dosages (5 mg/rat). a Hematoxylin and eosin (H&E) staining and (b) Masson’s trichrome staining images. Hyperplasia of alveolar epithelial cells and thickening of the alveolar wall are indicated by the red arrows. Detached bronchial epithelial cells are indicated by the blue arrows. Inflammatory cell infiltration are indicated by the yellow arrows. Scale bar: 50 μm. c Statistics are based on the percentage of fibrosis measured in Masson’s trichrome-stained images of each group. Data were subjected to one-way ANOVA to assess significance

At the highest dose (5 mg/rat), the lung tissue structure was severely disrupted in both the SiC and SiC@TiO₂ groups. Extensive areas of lung parenchymal damage were observed, with the absence of clear alveolar structures (yellow arrows). A large number of infiltrating inflammatory cells were present throughout the tissue (red arrows), and substantial nanoparticle deposition was visible within the bronchial lumen (blue arrows). While SiC@TiO₂ appeared more uniformly distributed, it induced a similar degree of pulmonary damage as SiC at high doses. In contrast, the TiO₂ group showed moderate structural abnormalities, characterized by alveolar wall thickening and partial loss of alveolar integrity.

Quantitative analysis of pulmonary fibrosis was based on the percentage of fibrotic area measured in Masson’s trichrome-stained images to visualize collagen deposition (Fig. 3b-c). At the highest dosage of 5 mg, both SiC and TiO₂ nanoparticles induced significant early fibrotic responses in the lungs. Fibrosis was characterized by thickening of the alveolar walls, a sign of cellular damage and collagen deposition, particularly in the alveolar epithelial cells. The increased severity of fibrosis was more pronounced in the lung tissue exposed to 5 mg of SiC and TiO₂. The average fibrotic area at the high dose (5 mg) was lower in the SiC@TiO₂ group (12.04%) compared with SiC (17.2%) and TiO₂ (22.05%), indicating a trend toward reduced fibrotic response in the multicomponent material.

The pathological findings from this range-finding study (Fig. 3) were essential for selecting the final dose for the long-term biodistribution study. Specifically, the acute, severe disruption of lung structure and significant fibrotic response observed at the 0.5 mg and 5 mg dose levels indicated that these concentrations would likely lead to “lung overload” and altered physiological clearance. Therefore, the intermediate dose of 0.15 mg was chosen as it minimized acute toxicity, allowing us to assess the intrinsic biodistribution and clearance kinetics over the 28-day period without the confounding factor of massive pathological damage.

Main study

Biodistribution

To better understand the mixed biological effects of composite nanomaterials, we introduced an additional experimental group in which the two individual component nanomaterials were directly mixed. Furthermore, the safety profile under low-dose exposure (no-adverse effect level, NOAEL) is likely more relevant to the everyday use of nanomaterials and holds greater practical significance for risk assessment. Therefore, we further investigated the inflammatory responses induced by each nanomaterial under low-dose exposure conditions. The 0.15 mg/rat was derived from the range finding study as a level that was expected not to induce acute effects that would affect the clearance of and retention from the lung.

The biological effects of SiC, TiO₂, the physical mixture of SiC and TiO₂ (SiC+TiO₂), and the multi-component SiC@TiO₂ NMs were evaluated at different post-exposure periods (1 h, 1 day, 3 days, 7 days, 14 days, and 28 days) to investigate possible retention, biodistribution and adverse outcomes on lung tissue. As shown in the lung TEM images (Fig. 4), SiC@TiO₂ exhibited superior dispersion within the pulmonary environment, while the SiC + TiO₂ mixture appeared to aggregate into larger structures, likely due to electrostatic interactions between the two particle types.

Fig. 4.

Representative TEM images of lung tissue sections at 1 h post-exposure, scale bar: 2 μm

The Si and Ti contents in the lungs at various time points were quantified using inductively coupled plasma optical emission spectrometry (ICP-OES), while histological analysis was conducted to evaluate corresponding tissue changes. The results (Fig. 5) resulted in an initial value (1 h after instillation) of approx. 5.5 µg Si/g lung tissue, corresponding to approx. 7.9 µg SiC/g lung tissue. The initial dose was 150 µg per rat lung, thus, the recovery of the analysis showed approx. 10%.

Fig. 5.

The biodistribution of NMs at no-adverse effect level (NOAEL) obtained from the range finding study assessed in tissue of rats after 1 h, 1d, 3d, 7d, 14d, and 28d of the single endotracheally applied dose of 0.15 mg/rat. a Si and b Ti contents in tissues were measured by inductively coupled plasma optical emission spectrometry (ICP-OES). The Si content is similar between SiC and SiC@TiO₂. The Ti content is similar between TiO₂ and SiC@TiO₂. Error bar denotes standard deviation (n = 4)

To enable clearer interpretation of organ-specific biodistribution patterns, lung data were analyzed separately from extrapulmonary organs (Fig. 5; Tables 3 and S2). For Si-containing materials, the highest Si concentration in lung occurred at 1 h post-instillation, followed by a pronounced decline at day 1, indicating rapid early-phase clearance from the respiratory tract. In extrapulmonary organs, Si exhibited distinct temporal behaviors: concentrations in the heart and spleen reached their maxima at day 1 and subsequently decreased, whereas kidney concentrations showed a delayed peak around day 7 before declining. In contrast, liver Si levels remained relatively stable across the 28-day period, suggesting persistent hepatic retention.

Table 3.

The biodistribution of NMs at no-adverse effect level (NOAEL) obtained from the range finding study assessed in tissue of rats after 1 h, 1d, 3d, 7d, 14d, and 28d of the single endotracheally applied dose of 0.15 mg/rat

NMs		Heart (µg/g)		Liver (µg/g)		Spleen (µg/g)		Lung (µg/g)		Kidney (µg/g)
SiC	1 h	0.30 ± 0.09		0.67 ± 0.18		0.30 ± 0.17		5.84 ± 1.24		0.40 ± 0.04
	1d	0.64 ± 0.11		0.81 ± 0.34		0.73 ± 0.06		2.68 ± 0.17		0.38 ± 0.05
	3d	0.58 ± 0.02		0.90 ± 0.12		0.30 ± 0.03		1.58 ± 0.72		0.43 ± 0.06
	7d	0.45 ± 0.10		0.85 ± 0.33		0.29 ± 0.04		1.30 ± 0.48		0.69 ± 0.33
	14d	0.51 ± 0.09		0.76 ± 0.17		0.39 ± 0.10		0.79 ± 0.19		0.87 ± 0.30
	28d	0.37 ± 0.06		0.61 ± 0.11		0.19 ± 0.03		0.19 ± 0.07		0.56 ± 0.08
TiO2	1 h	0.23 ± 0.06		0.38 ± 0.23		0.17 ± 0.06		4.34 ± 0.73		0.21 ± 0.02
	1d	0.37 ± 0.12		0.57 ± 0.14		0.53 ± 0.05		3.87 ± 0.38		0.34 ± 0.06
	3d	0.21 ± 0.03		0.52 ± 0.05		0.35 ± 0.02		2.48 ± 0.58		0.30 ± 0.07
	7d	0.59 ± 0.12		0.51 ± 0.03		0.32 ± 0.04		1.60 ± 0.21		0.27 ± 0.01
	14d	0.52 ± 0.20		0.42 ± 0.03		0.57 ± 0.22		0.83 ± 0.46		0.24 ± 0.05
	28d	0.37 ± 0.03		0.38 ± 0.02		0.21 ± 0.04		0.65 ± 0.21		0.20 ± 0.03

		Si	Ti	Si	Ti	Si	Ti	Si	Ti	Si	Ti
SiC+TiO2	1 h	0.16 ± 0.09	0.10 ± 0.05	0.46 ± 0.03	0.10 ± 0.02	0.22 ± 0.02	0.09 ± 0.04	3.31 ± 0.42	1.10 ± 0.25	0.21 ± 0.01	0.10 ± 0.02
	1d	0.44 ± 0.08	0.25 ± 0.10	0.51 ± 0.09	0.31 ± 0.05	0.25 ± 0.08	0.42 ± 0.05	0.86 ± 0.59	0.78 ± 0.36	0.20 ± 0.04	0.21 ± 0.02
	3d	0.26 ± 0.08	0.18 ± 0.02	0.46 ± 0.05	0.35 ± 0.08	0.16 ± 0.04	0.22 ± 0.09	1.13 ± 0.25	0.75 ± 0.24	0.27 ± 0.02	0.21 ± 0.05
	7d	0.17 ± 0.05	0.30 ± 0.06	0.48 ± 0.08	0.19 ± 0.01	0.22 ± 0.07	0.30 ± 0.11	1.03 ± 0.28	0.44 ± 0.13	0.55 ± 0.32	0.15 ± 0.03
	14d	0.17 ± 0.10	0.31 ± 0.02	0.43 ± 0.08	0.17 ± 0.01	0.27 ± 0.06	0.20 ± 0.06	0.47 ± 0.35	0.33 ± 0.04	0.49 ± 0.33	0.13 ± 0.01
	28d	0.23 ± 0.08	0.23 ± 0.07	0.54 ± 0.10	0.15 ± 0.05	0.14 ± 0.02	0.23 ± 0.03	0.17 ± 0.04	0.16 ± 0.04	0.26 ± 0.11	0.11 ± 0.03
SiC@TiO2	1 h	0.16 ± 0.08	0.10 ± 0.05	0.36 ± 0.25	0.06 ± 0.01	0.22 ± 0.01	0.09 ± 0.03	3.10 ± 0.29	1.18 ± 0.18	0.17 ± 0.03	0.10 ± 0.02
	1d	0.37 ± 0.10	0.16 ± 0.08	0.55 ± 0.15	0.30 ± 0.04	0.31 ± 0.10	0.26 ± 0.11	1.95 ± 0.54	1.07 ± 0.53	0.17 ± 0.02	0.23 ± 0.03
	3d	0.20 ± 0.06	0.22 ± 0.08	0.49 ± 0.14	0.27 ± 0.04	0.19 ± 0.04	0.30 ± 0.16	1.34 ± 0.31	0.86 ± 0.08	0.17 ± 0.06	0.22 ± 0.06
	7d	0.13 ± 0.06	0.29 ± 0.07	0.53 ± 0.45	0.19 ± 0.03	0.26 ± 0.08	0.29 ± 0.08	0.57 ± 0.11	0.56 ± 0.18	0.52 ± 0.26	0.16 ± 0.01
	14d	0.19 ± 0.05	0.19 ± 0.04	0.21 ± 0.06	0.17 ± 0.01	0.33 ± 0.13	0.24 ± 0.12	0.46 ± 0.08	0.32 ± 0.06	0.33 ± 0.23	0.16 ± 0.02
	28d	0.21 ± 0.06	0.14 ± 0.03	0.47 ± 0.10	0.15 ± 0.04	0.14 ± 0.02	0.19 ± 0.03	0.15 ± 0.07	0.25 ± 0.06	0.18 ± 0.02	0.08 ± 0.05

Ti-containing materials displayed a different kinetic profile. Ti concentrations in lung decreased progressively over time without a sharp early drop, indicating a longer pulmonary retention half-life compared with Si. In liver, spleen, and kidney, Ti concentrations peaked at day 1, consistent with their role in clearance, while the heart exhibited a delayed accumulation pattern, with concentrations reaching a maximum at day 7 before declining.

Notably, the biodistribution and clearance patterns of the physical mixture (SiC + TiO₂) and the multicomponent material (SiC@TiO₂) were highly similar for both Si and Ti, supporting the interpretation that the Si- and Ti-containing domains undergo partial dissociation or differential processing in vivo, resulting in behavior comparable to their corresponding single-component nanomaterials.

Further analysis of the Si/Ti ratio across different organs (Table 4) revealed discrepancies in the expected uniformity of Si and Ti distribution, suggesting a possible dissociation of SiC@TiO₂ in biological environments. The Si/Ti ratio was found to vary at different time points, particularly in heart and liver tissues, which implies that SiC and TiO₂ components may not remain entirely bound after inhalation exposure. The observed inconsistency in the ratio further supports the notion that SiC@TiO₂ undergoes partial dissociation or differential clearance, influencing its biodistribution profile.

Table 4.

The Ti/(Si + Ti) ratio of NMs at NOAEL (1 h, 1d, 3d, 7d, 14d, and 28d) after 1 h, 1d, 3d, 7d, 14d, and 28d of the single endotracheally applied dose of 0.15 mg/rat

		Heart (µg/g)	Liver (µg/g)	Spleen (µg/g)	Lung (µg/g)	Kidney (µg/g)
		Ti/(Si + Ti)	Ti/(Si + Ti)	Ti/(Si + Ti)	Ti/(Si + Ti)	Ti/(Si + Ti)
SiC+TiO2	1 h	38%	46%	54%	48%	51%
	1d	36%	51%	51%	49%	50%
	3d	41%	48%	52%	49%	50%
	7d	64%	43%	53%	49%	50%
	14d	65%	43%	53%	49%	50%
	28d	50%	46%	53%	49%	50%
SiC@TiO2	1 h	38%	46%	54%	48%	51%
	1d	30%	51%	52%	49%	51%
	3d	52%	45%	53%	49%	50%
	7d	69%	42%	53%	49%	50%
	14d	50%	45%	53%	49%	50%
	28d	40%	47%	53%	49%	51%

Toxicity assessment

Histological analysis of lung tissues was performed using HE staining (Figs. 6a and S4) and Masson’s trichrome staining (Figs. 6b and S5), revealing notable tissue alterations in response to nanomaterial (NM) exposure. By day 28 post-exposure, pulmonary fibrosis was evident in all treated groups. The severity of fibrosis, indicated by the thickening of the alveolar walls (red arrows), was notably higher in the SiC and TiO₂ groups compared to the control group. In addition, the inflammatory cell infiltration (yellow arrow) indicates significant pathological changes and sustained tissue injury induced by these nanomaterials.

Fig. 6.

The biological effects of NMs (1 h, 1d, 3d, 7d, 14d, and 28d). a and b HE and Masson’s trichrome staining images of lungs at 28 d. Hyperplasia of alveolar epithelial cells and thickening of the alveolar wall are indicated by the red arrows. Inflammatory cell infiltration are indicated by the yellow arrow. Scale bar: 50 μm. c The percentage of fibrosis in Masson staining images of each group. A statistical T-Test was performed on the fibrosis area data; P-values indicate the significance level between the SiC@TiO₂ and SiC+TiO₂ treatment groups

On day 28 following a single endotracheal instillation of NMs, potential early-stage fibrotic and inflammatory responses were evaluated. Although 28 days may not be sufficient to fully establish chronic fibrosis, histopathological changes such as alveolar wall thickening, epithelial hyperplasia, and collagen deposition observed in Masson’s trichrome staining are considered early markers associated with fibrotic progression. As shown in Fig. 3, the effects seem somewhat less for the multi-component NM compared to both single components based on hyperplasia of alveolar epithelial cells and atrophy of the alveoli with thickening of the alveolar wall, infiltration of inflammatory cells, suggesting that the multi-component formulation may elicit a comparatively attenuated pulmonary response.

The quantitative analysis of fibrosis (Fig. 6c) supported these histological findings, showing a dose- and time-dependent increase in fibrosis in the lung tissues of SiC, TiO₂, SiC@TiO₂, and SiC+TiO₂ exposed groups. The enhanced fibrotic response could be attributed to the prolonged retention of the SiC and TiO₂ in lung tissue, leading to sustained inflammation and tissue remodelling over the 28 days. The progression of fibrosis and lung damage observed over the different time points indicates that both SiC and TiO₂ NMs have the potential to cause chronic lung injuries. The extent of fibrosis was less pronounced in the SiC@TiO₂ and SiC+TiO₂ groups at 28 days, suggesting that the combined material might induce a less severe fibrotic response compared to the individual SiC and TiO₂ nanoparticles. This is consistent with the higher accumulation of both Si and Ti in the lungs over time, which likely contributes to the prolonged inflammatory response and fibrosis. The combined nanoparticles exhibited both decreased bioaccumulation of Ti in the lungs and a less pronounced fibrotic response compared to TiO₂ or SiC alone. This suggests an antagonistic effect between the two materials, which may alter the bioavailability and toxicity of the individual components. The MCNM SiC@TiO₂ and combined SiC+TiO₂ formulations possibly delaying the fibrotic process. This highlights the importance of considering the long-term effects of these NMs, especially when used in applications that could result in prolonged exposure, such as in inhalation studies or occupational settings.

The results from ELISA (Fig. 7) further strengthen the previous findings regarding the inflammatory response elicited by the nanomaterials, particularly the multicomponent SiC@TiO₂. The total protein levels in bronchoalveolar lavage fluid (BALF) (Fig. 7a), measured as a marker of lung injury, were significantly elevated in the SiC and TiO₂ groups at the 3-day day 3 post-exposure, indicating acute epithelial damage. In addition, by the 28-day mark, the combined SiC@TiO₂ group demonstrated a less pronounced and persistent increase in protein levels, suggesting a shorter duration of tissue injury and inflammation. This observation aligns well with earlier findings of heightened cellular stress and morphological changes in lung tissue, further supporting a potential antagonistic or mitigating effect conferred by the composite nanomaterial.

Fig. 7.

Inflammatory response in lungs. After a single dose, rats were sacrificed to collect BALF at 3 or 28 d post-exposure. (a) Levels of total cell damage markers and (b-e) levels of pro-inflammatory cytokines in BALF supernatant (n = 3)

The cytokine analysis reveals a similar trend, exposure to SiC@TiO₂ induced lower levels of pro-inflammatory cytokines, including TNF-α (Fig. 7b), IL-1β (Fig. 7c), IFN-γ (Fig. 7d), and IL-6 (Fig. 7e), compared to single-component exposures. This suggests that the multicomponent nanomaterial induces a milder inflammatory response, reinforcing its comparatively favourable pulmonary biocompatibility.

Cytokines are critical mediators of inflammation and play pivotal roles in the recruitment and activation of immune cells, which can lead to tissue damage. For instance, TNF-α, observed to be significantly elevated at both time points in the SiC and TiO₂ group (Fig. 7b), is known to amplify inflammatory cascades and contribute to the chronic inflammatory state observed in the lungs. This increase in TNF-α correlates with oxidative stress reported by others [21] (REF), further reinforcing the hypothesis that the NMs exacerbates immune activation through reactive oxygen species (ROS)-mediated pathways but the MCNM SiC@TiO₂ results in a lower effect compared to the sum of the effects of the single components. Similarly, the sustained elevation of IL-1β (Fig. 7c) and IFN-γ (Fig. 7d) in the SiC@TiO₂ group at the 28-day mark indicates the progression from an acute to a chronic inflammatory response. IL-1β, a key cytokine induced by the activation of inflammasomes, suggests that the SiC and TiO₂ NMs may persist within the lung environment, leading to prolonged immune cell activation and inflammation. This persistent activation may also explain the structural remodelling observed in lung tissue, as IFN-γ is known to promote macrophage recruitment and fibrotic processes [22]. IL-6 (Fig. 7e), a cytokine with both pro- and anti-inflammatory roles, showed a significant increase in the SiC and TiO₂ group, particularly at the later time point, which is consistent with the findings of epithelial injury and prolonged immune activation. This prolonged elevation of IL-6 might reflect its role in bridging the acute-phase response with chronic inflammation, contributing to the pathological changes observed.

The temporal dynamics of these inflammatory markers further underline the potential for SiC@TiO₂ to induce a less severe and sustained inflammatory state compared to single-component materials. This finding is crucial, as it suggests that the interaction between SiC and TiO₂ nanoparticles may alter their biological impact, creating an antagonistic effect that reduces immune activation. Importantly, the enhanced levels of cytokines and protein damage markers observed here complement published findings of oxidative stress and cellular toxicity [23], providing a holistic understanding of how these NMs disrupt lung homeostasis. The sustained inflammatory response observed with SiC and TiO₂ also raises concerns about the long-term health implications of exposure to such multi-component NMs. The potential for chronic inflammation, as suggested by elevated cytokine levels at 28 days, underscores the need for careful evaluation of their safety profiles, particularly in occupational or environmental settings where prolonged exposure is possible. These results highlight the necessity of assessing not only individual NMs but also their combined forms, as the interaction between components can significantly alter their biological impact.

Discussion

This study is to our knowledge the first in which toxicity and biodistribution of a MCNM, SiC@TiO₂, in comparison with its single-component nanomaterial (SiC and TiO₂) and their physical mixture has been systematically evaluated the pulmonary. The results demonstrated that all used nanomaterials are predominantly dose-dependently (range finding study) retained in the lungs following intratracheal instillation, with time-dependent clearance observed over 28 days (main study). It seems plausible that the nanoparticles used in this study entering the bloodstream after crossing the lung-blood barrier [24, 25] and eventually can be primarily cleared through the kidneys as the initial increase burden in the kidney is decreased after 14–28 days post exposure. There is no evidence for accumulation in secondary organs such as heart, spleen, liver and kidney during the 4-week observation period and the data suggest that the levels returned to baseline values. While the size of SiC@TiO₂ is not significantly different from SiC, the MCNM seem to exhibit reduced pulmonary retention, attenuated histopathological alterations, and milder fibrotic and inflammatory responses relative to single-component exposures. This can be interpretated as once inhaled the MCNM might be slightly better cleared from the lungs compared to the mixture of SiC and TiO₂. This may result in a reduced risk of developing for example fibrosis after prolonged exposure to sufficiently high exposure levels. Albeit that these finding are in line with for example a study by Leinardi and colleagues [26], who focuses on silica and longer post-observation periods. Their findings revealed that accumulation in secondary organs may occur at a much later stage, i.e. 120 days post-exposure in lymph nodes, spleen, skin, liver and kidney. However the Min-U-Sil 5 material used in that study is significantly larger (1.3 μm ± 0.6(SD)) which will have a profound effect on the kinetics. The fact that the Ti content in SiC@TiO₂ was lower than that in pure TiO₂ across most organs (Fig. 2d) might be the result of the composite structure limiting TiO₂ exposure or altering its kinetics in vivo. In vitro dissolution assays, including the use of including phagolysosomal simulant fluid, confirmed the high stability of the TiO₂ component (Ti < LOD), suggesting that the Ti detected in secondary organs is present in particulate form, likely due to the dissociation of the 6–7 nm satellites from the SiC carrier, rather than ionic release (data not shown). The incorporation of TiO₂ into a SiC-based composite appears to suppress Ti biodistribution, which may help reduce systemic exposure risk. It is important to recognize that biodistribution is highly dependent on exposure route. For instance, previous studies have shown that intravenously injected TiO₂ preferentially accumulates in the liver, followed by the spleen, lungs, and kidneys [27, 28]. This pattern contrasts with our findings after pulmonary exposure, where the lung remained the primary depot, followed by the kidneys, with minimal hepatic accumulation. This divergence underscores the critical influence of the initial exposure portal on nanoparticle fate, as also highlighted in biodistribution studies comparing different administration routes [28]. Factors such as particle size, hydrophilicity, and biological barriers further modulate biodistribution patterns [29–31]. In particular, our observation that the multicomponent SiC@TiO₂, despite similar primary particle size, showed different agglomeration states and clearance kinetics aligns with the principle that effective dose and biological response are better correlated with deposited surface area than mass alone [31]. The slightly better colloidal stability (lower PDI) of SiC@TiO₂ observed in our DLS data (Fig. 1e) might contribute to its altered interaction with biological barriers and clearance pathways compared to the more agglomerated single components or physical mixture.

The composite structure may influence nanoparticle-cell interactions and uptake dynamics, potentially altering the toxicological profile. And although we aimed to avoid clear toxicity in the main study, our data revealed that some minor effects were observed such as the release of pro-inflammatory cytokines, that also maintained elevated throughout the 28-day study period. The cytokine data nicely fit the pathology data. As the biodistribution of SiC@TiO₂ is similar to that of SiC and TiO₂, differences in toxicity/pathology can be explained by differences in intrinsic toxicity of the (MC)NM. As suggested, this particle toxicity may also be a main driver for particle clearance [21] However, this also showed that the MCNM elicited lower levels of pro-inflammatory cytokines and protein leakage, suggesting a potential antagonistic interaction between its components that mitigates toxicity. Our central finding—that the designed multicomponent nanomaterial (SiC@TiO₂) exhibits a distinct and often reduced toxicity profile compared to its individual constituents or their simple mixture—strongly supports the argument put forth by Stone et al. [32]. They emphasized that multicomponent nanomaterials should be assessed as new, distinct entities rather than through the simple summation of their parts, due to potential emergent properties and complex interactions. The “antagonistic” effect we observed between SiC and TiO₂ in the composite form, leading to attenuated inflammation and fibrosis, is a direct example of such non-additive behavior. This underscores the necessity of a dedicated safety assessment framework for MCNMs, as their risks cannot be reliably extrapolated from data on single components. A relevant difference between the physical mixing (SiC+TiO₂) and the MCNM (SiC@TiO₂) stems from the different interaction between the components. Albeit, no X-ray Photoelectron Spectroscopy analysis was performed, there will be a chemical covalent bond for the between core shell structure SiC and TiO₂ in the MCNM, as it was prepared from the precursors by a sol gel method and typically this implies a strong chemical bond between components. In the physical mixing, Van der Waals Forces, (weaker interactions) are expected. To draw definite conclusions, a more extensive dose-effect relationship comparison has to be established as in the present study. These findings underscore the importance of evaluating MCNM as distinct entities rather than simple additive combinations of their constituents [32]. The reduced toxicity profile of SiC@TiO₂ highlights the potential for engineered nanocomposites to achieve functional performance while minimizing adverse biological effects.

This work contributes to the growing field of nanosafety by emphasizing the need for comprehensive toxicological assessments under realistic exposure scenarios, especially for hybrid or composite nanostructures used in industrial and biomedical applications. Our study, focused on acute to sub-acute pulmonary exposure, adds to the existing body of knowledge that includes investigations into translocation to secondary organs over longer periods [26] and via different routes [24, 25, 27, 28]. The relevance of findings from acute high-dose models to real-world chronic low-dose exposure remains limited. While such studies provide mechanistic insights, caution is required when extrapolating them for long-term human health risk assessment.

Conclusions

In the present study, the toxicity and biodistribution of a MCNM, SiC@TiO₂, were evaluated in comparison to its single-component counterparts (SiC and TiO₂) as well as a physical mixture (SiC + TiO₂), focusing on acute and subacute responses following respiratory exposure. The SiC@TiO₂ composite exhibited a faster lung clearance and lower pulmonary toxicity compared to its individual components as well as the mixture of the single components suggesting that SiC can be made less harmful, in part due to faster clearance by adding TiO₂ to make it a MCNM. These results underscore the importance of evaluating multicomponent nanomaterials as unique entities and suggest that thoughtful material design could reduce harmful biological effects, paving the way for safer nanotechnology advancements.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

WZ: performed experiments, data curation, statistical analysis, writing (original draft); MD: performed experiments, writing (original draft); YL: Funding acquisition, writing (review and editing); CC, FRC, MB, RC: supervision, conceptualization, funding acquisition, writing (review and editing), writing (review and editing). All authors reviewed the manuscript.

Funding

This study was funded by the National Natural Science Foundation of China (22388101), the National Key R&D Program of China (2021YFE0112600, 2021YFA1200900), Beijing Nova Program (20220484060), European Union’s Horizon 2020 research and innovation programme (SUNSHINE, 952924).

Data availability

All the experimental data collected during SUNSHINE project is in “SUNSHINE database”, which is part of SUNSHINE e-Infrastructure (https://www.sunshine.greendecision.eu/) hosted by Green Decisions. Users are required to be registered before they can access the database and other services within the system.

Declarations

Ethics approval and consent to participate

All procedures used in this experiment were compliant with the local ethics committee. This study was approved by the institute’s Ethical Committee at the National Center for Nanoscience and Technology as documented in PONY-2022-FL-12.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.