New approach methodologies for in vitro toxicity screening of nanomaterial using a pulmonary three-dimensional floating extracellular matrix model

Abstract

Background

Nanomaterials offer increasing applications across diverse sectors, including food science, medicine, and electronics. Environmental risk assessment is crucial for ensuring the safety and sustainability of nanomaterials. However, high-throughput screening (HTS) of their potential toxicity remains challenging owing to their unique physicochemical properties.

Results

This study introduces a novel pulmonary three-dimensional (3D) floating extracellular matrix (ECM) model utilizing a 384-pillar/well platform for HTS of nanotoxicity. Compared with conventional HTS models based on two-dimensional (2D) cells, the 3D model developed in this study successfully addressed the issues related to the aggregation and sedimentation of nanoparticles and their possible optical interference with the toxicity assays. Using 20 nm silica nanoparticles (SiNPs), we assessed cell viability and nanoparticle uptake in both serum-containing and serum-free culture media. While the 2D model showed high SiNPs toxicity regardless of the media composition, the pulmonary 3D floating ECM model demonstrated variable toxicities that depended on the SiNPs behaviors under different conditions.

Conclusions

By reducing the uncertainties associated with the sedimentation and optical interference of nanomaterials, our 3D model provided a more precise analysis of cytotoxicity. This study highlights the potential of using new approach methodologies and improved HTS approaches to enhance the efficiency and accuracy of risk assessment protocols for emerging nanomaterials.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13036-025-00532-w.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s13036-025-00532-w.

Background

Advanced materials are a rapidly evolving field of cutting-edge technology, offering unprecedented opportunities across diverse sectors, including food science, medicine, and electronics [1]. The unique properties of these materials, often at the nanoscale, also present significant challenges in terms of risk assessment and safety evaluation [2]. There is an urgent need to develop robust methodologies to assess the potential impacts of advanced materials on human health and the environment. The European Green Deal emphasizes the importance of balancing technological advancements with environmental sustainability and human safety [3]. In this context, developing effective risk assessment strategies for advanced materials is not only a scientific imperative but also a regulatory necessity. However, the complex physicochemical properties of these materials, particularly nanoparticles (NPs), pose significant challenges for conventional toxicity testing methods. Traditional in vitro assays used for assessing the safety of NPs often fail to yield reliable data due to the distinct physicochemical properties of NPs, including their small size, large surface area, high reactivity, and unique optical characteristics [4]. These properties can lead to unpredictable interactions with biological systems and experimental conditions, potentially biasing results and complicating data interpretation. Consequently, the development of precise and robust new approach methodologies (NAMs) is critical for accurately evaluating and mitigating the environmental and health risks associated with NPs. NAMs aim to provide reliable and physiologically relevant data by addressing the limitations of conventional testing methods related to the unique behaviors of NPs in biological systems.

Among the key challenges in applying traditional in vitro assays to nanomaterials are issues related to dispersion stability and optical interference, both of which can significantly impact assay reliability and result accuracy. Most in vitro assays assume that the NPs are dispersed uniformly during incubation. However, NPs often exhibit instability in culture media despite being well-dispersed in aqueous phases, such as deionized water (DIW). International Organization for Standards (ISO) have been developed to evaluate nanotoxicity based on cell viability assays or reactive oxygen species (ROS) levels. However, these standards are only applicable to a few classes of NPs that can be well dispersed in culture media [5, 6]. To address this limitation, NPs are pre-treated by sonication or bovine serum albumin to ensure their uniform dispersion. However, these processes can potentially alter the intrinsic physicochemical properties of the NPs [7]. In addition, due to their optical properties, NPs often interfere with the optical detection systems, including ultraviolet (UV) light, luminescence, and fluorescence-based assays, which can result in inconsistent and unreliable data [8].

High-throughput screening (HTS) plays a crucial role in the risk assessment of a wide variety of NPs and in dose-dependent screening. By employing HTS techniques, researchers can efficiently evaluate the potential risks associated with different types of NPs and provide valuable data for their safe development and applications [9]. HTS approaches, such as label-free intracellular identification, flow cytometry, impedance-based monitoring, and imaging-based micronucleus analysis, have been widely used for nanosafety research [10]. Previously, we developed a three-dimensional (3D) cell-based HTS method using various pillar/well platforms, such as 96-, 384-, or 532-well plates and validated its high performance by screening drug efficacy or toxicity [11–13]. Recent HTS technology development has shifted from 2D to 3D cells to recapitulate in vivo-like physiological responses [10] because many cells from normal and malignant origins lose their phenotypes during 2D cell culture [14]. 3D cell culture facilitates cell-cell or cell-extracellular matrix (ECM) interactions, leading to the expression of proteins/genes related to in vivo phenotype and the maintenance of more in vivo-like characteristics [15]. ECM-embedded organoid technology has been actively used in numerous studies, and organoid models can mimic key organs, such as the liver, kidneys, and intestine, for evaluating the toxicity of nanomaterials [16–18]. However, this field remains in its early stages, and further research and development are required to commercialize different organoid models and establish standardized methods for nanomaterial safety assessment. Recently, we developed a novel method for assessing nanomaterials’ toxicity using a 3D floating ECM model, which was published in an ISO technical report (ISO/TR22455). This assay allows the toxicity evaluation of nanomaterials in a 3D environment that closely mimics human physiology and provides a reliable approach with minimal interference from nanomaterials [19].

In this study, a 3D cell-based assay using a cell-embedded floating ECM model was developed to evaluate NPs cytotoxicity. This model allows the easy transfer of NPs-exposed cells to new wells containing wash buffer or cell-staining dyes. Due to its unique design, this system enables NPs cytotoxicity evaluation without the limitations associated with dispersion stability, such as sedimentation issues, or optical interference. To investigate the influence of these factors, silica nanoparticles (SiNPs) and silver nanoparticles (AgNPs) were selected as representative NPs, and the performance of the proposed pulmonary 3D floating ECM model was systematically assessed. Monodisperse and well-controlled 20 nm silica nanoparticles (SiNPs) were employed to confirm the applicability of the system under different experimental conditions. There are many studies on the toxicity of SiNPs; however, the results are controversial and depend on the cell type and culture medium [20]. Smaller SiNPs cause higher cytotoxicity, and agglomeration or sedimentation occurs in the presence of serum, increasing the size of the SiNPs [21]. Although serum provides a more in vivo-like physiological environment, most in vitro assays are performed under serum-free conditions, considering the significant increase in SiNP size and the difficulty in controlling the behavior of SiNPs in serum. The toxicity data of SiNPs reported by different laboratories can vary significantly based on the sonication methods employed. Here, we characterized the size and stability of SiNPs in different culture media and compared the cell viabilities obtained from conventional and pulmonary 3D floating ECM models. AgNPs, on the other hand, are widely known to interfere with in vitro assays due to their optical properties such as light absorption and scattering, which can lead to inaccuracies in absorbance, luminescence, and fluorescence-based assays [22]. AgNPs were utilized to assess the feasibility of the 3D floating ECM model in evaluating NPs that may cause optical interference. This study aims to advance NAMs by overcoming the challenges posed by the unique properties of NPs and providing a more accurate and reliable framework for assessing their potential risks to human health and the environment.

Methods

Materials

Human lung normal bronchial epithelium (BEAS-2B) and lung carcinoma (A549) cell lines were obtained from ATCC (USA; CRL-9609 and CCL-185, respectively). The bronchial epithelial cell growth medium bullet kit (BEGM), RPMI 1640 (RPMI), penicillin-streptomycin, and trypsin-EDTA were purchased from Lonza (USA). Fetal bovine serum (FBS) was purchased from Gibco (USA). Dulbecco’s phosphate-buffered saline (DPBS) was purchased from Welgene (South Korea). CellTiter 96^® aqueous one solution cell proliferation assay (MTS) and CellTiter Glo^® luminescent cell viability assay (CellTiter-Glo) were supplied by Promega (USA), Calcein AM-green or -Red-Orange, and Hoechst 33342 were purchased from Invitrogen (USA). Polyvinylpyrrolidone-capped silver nanoparticles (AgNPs) with particle sizes of 10 nm were purchased from NanoComposix (USA). Fluorescein 5(6)-isothiocyanate (FITC, > 90%, HPLC grade), tetraethyl orthosilicate (TEOS, > 98%), 3-aminopropyl triethoxysilane (APTES, > 99%), butanol (> 99%), L-arginine (> 98%), and cyclohexane (anhydrous, > 99.5%) were purchased from Sigma-Aldrich (USA) and all of the chemicals were analytical reagent grade. Ultrapure Milli-Q water (18.2 MΩ cm ^− 1) was used for all the procedures.

NP synthesis, preparation, and characterization

The 20 nm SiNPs were supplied by Korea Research Institute of Standards and Science (KRISS) as certified reference material (301-01-002). The synthesis method of the SiNPs is described in the following procedure. Briefly, 350 mL of 8.2 mM aqueous L-arginine solution was heated to 50°C in a 500 mL round bottom flask, and a mixture of TEOS and cyclohexane was added under vigorous stirring at 900 rpm. The temperature and stirring speed were maintained overnight, and approximately 300 mL of the resulting SiNP dispersion in the aqueous phase was extracted using a separating funnel [23]. FITC-labeled SiNPs (FITC-SiNPs) were prepared using the same procedure with the addition of a silane-dye conjugate. Fluorescein isothiocyanate (FITC) was pre-reacted with (3-aminopropyl) triethoxysilane (APTES) in a butanol solution containing L-arginine prior to the addition of TEOS, enabling incorporation of the fluorescent dye into the silica matrix via sol-gel condensation. The reaction was conducted at elevated temperature under high space velocity to promote uniform nucleation and monodisperse particle growth. The aqueous phase-containing FITC-SiNPs was separated from the organic phase and purified by centrifugation.

The diameter and size distribution of the SiNPs were measured using transmission electron microscopy (TEM, JEOL, JEM-ARM200F at an accelerating voltage of 200 kV), scanning electron microscopy (SEM, ZEISS, GeminiSEM 500 at an accelerating voltage of 10 kV), dynamic light scattering (DLS, Brookhaven Instruments using a BI9000AT digital correlator), and a scanning mobility particle sizer (SMPS, consisting of a DMA and CPC module, TSI Inc., 3081). The crystallographic structure and surface composition of the SiNPs were analyzed using X-ray diffraction (XRD, SmartLab, Rigaku) and time-of flight secondary ion mass spectrometry (ToF-SIMS V, IONTOF GmbH), respectively. The uncertainty was calculated according to the Guide to Expression of Uncertainty in Measurement (GUM) [24]. The UV-vis spectra of the AgNPs were measured using a Spectramax M3 spectrophotometer (Molecular Devices, USA).

Culture media-dependent stability analysis of SiNPs and AgNPs

The size distributions of SiNPs and AgNPs were measured in each culture medium by DLS for up to 72 h. The dispersion behavior of the NPs was evaluated in real time using a Turbiscan Tower analyzer (Microtrac, France), as previously described [25, 26]. Briefly, SiNPs and AgNPs were diluted with DIW, serum-free BEGM (BEGM-SF), serum-free RPMI (RPMI-SF), complete BEGM medium with supplements recommended by the manufacturer (BEGM-CM), and complete RPMI medium with 10% FBS (RPMI-CM). SiNPs (250 µg/mL) and AgNPs (100 µg/mL) suspensions were prepared in a 4 mL glass vial with a height of 48 mm, and the dispersion stability was monitored in real time at 37°C for 72 h. The transmitted signal intensity was measured every 1 h. The Turbiscan stability index (TSI) was calculated based on the cumulative sum of the transmitted intensities (T) using the following formula:

where H represents the sample height from the bottom of the cell to the meniscus; scan_i(h) represents the transmitted intensity at a specific height h and time i; and scan_i−1(h) represents the transmitted intensity at the same height h at the previous time point _i−1. The index i ranges from 1 to k (k = total time/scanning speed).

Cell culture and cell viability assay in a 2D culture model

BEAS-2B cells were cultured in a BEGM medium supplemented with bovine serum albumin/fibronectin/collagen solution-coated culture vessels according to ATCC instructions. A549 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were cultured in 75 cm² T flasks and incubated in a 5% CO₂ incubator at 37℃, and cells were sub-cultured every 2 days. For the cell viability assay, BEAS-2B and A549 cells were seeded in a 96-well plate at 1.5 × 10⁴ cells/well and 1 × 10⁴ cells/well, respectively, and cultured for 24 h. The serially diluted NPs were well-dispersed in culture media with or without serum by vigorous vortexing for 1 min and added to BEAS-2B and A549 cells in a two-fold serial manner. After 24 h of incubation, cell viability was measured using MTS assays by colorimetric detection, CellTiter-Glo assays by luminescence detection, and Calcein AM assays by fluorescence detection. The NP-treated cells were incubated with 20 µL of MTS reagent for 1 h at 37°C, and the optical density (OD) was determined at 490 nm using a GloMax Discover microplate reader (Promega, USA). For luminescence detection using the CellTiter-Glo assay, the cells were lysed in the same amount of assay reagent for 2 min and allowed to react for 10 min at room temperature. The luminescence was measured using a microplate reader. For the Calcein AM assay, cells were stained with Calcein Red-Orange AM and Hoechst 33342 for 30 min, and fluorescence intensity was measured using the ImageXpress Micro XLS system (Molecular Devices, USA). Dose-response analysis was performed based on a nonlinear regression algorithm with four-parameter equations, and the 50% inhibitory concentration (IC₅₀) was calculated using the GraphPad Prism software (GraphPad, USA).

Cell viability assay using pulmonary 3D floating ECM model

The pulmonary 3D floating ECM models were fabricated by plastic injection molding, and 3D cell culture was performed by previously reported methods with some modifications [13, 27]. Briefly, a 1 µL alginate spot containing approximately 500 cells of BEAS-2B and 0.5 w/w % alginate was automatically dispensed onto a 384-pillar plate using ASFA™ Spotter ST (Medical & Bio Device, South Korea) with a solenoid valve (The Lee Company, USA). Cells were cultured in the 384-well plate containing culture media for 24 h at 37˚C. The 3D cells were then transferred to a new 384-well plate filled with serially diluted NP suspensions. The combined plates were incubated with NPs for 24 h. The cell viability was measured using Calcein AM, where viable cells emitted red fluorescence. The staining dye solution was prepared by adding 1.0 µL of 4 mM Calcein AM to 8 mL of staining buffer (MBD-STA50, Medical & Bio Device). After staining for 1 h, the alginate spots on the 384-pillar plate were dried. Images of dried alginate spots were captured using an automated optical fluorescence scanner (ASFA™ Scanner ST, Medical & Bio Device). A red fluorescence filter (EM 570–600, EX 612–640) was used for cell viability analysis, and a green fluorescence filter (EM 485–510, EX 515–540) was used for detecting FITC-SiNPs in the cellular uptake analysis.

Bio-TEM analysis of cellular uptake of NPs

The control culture media and 12.5 µg/mL of 20 nm FITC-SiNPs were used to treat 2D and 3D BEAS-2B cells for 24 h in 96- and 384-well plates, respectively. The treated cells were harvested and fixed with 2.5% glutaraldehyde in 0.1 M phosphate (pH 7.3) for 2 h at room temperature. After fixation, the cells were stained with 1% OsO₄ and 1.5% potassium ferrocyanide in 0.1 M phosphate buffer (pH 7.3) for 1 h at 4°C in the dark and embedded in Epon 812 (Electron Microscopy Sciences, USA) after dehydration in an ethanol and propylene oxide series. Polymerization was performed using pure resin at 70°C for 2 days. Ultrathin Sect. (70 nm) were cut using an ultramicrotome (UltraCut-UCT, Leica, Austria) and collected on 150 mesh copper grids. After staining with 2% uranyl acetate (10 min) and lead citrate (5 min), the sections were examined under a TEM at 120 kV (Tecnai G2 Spirit Twin, FEI, USA).

Statistical analysis

All data are presented as the mean ± standard deviation (SD) for NP size and cell viability measurements. Statistically significant differences between groups were analyzed using one-way analysis of variance (ANOVA) with GraphPad Prism software (GraphPad, USA). A P-value of < 0.05 indicates a statistically significant difference between groups.

Results

Schematic of the NPs exposure system to the pulmonary 3D floating ECM model

Figure 1 shows a schematic of the exposure system of NPs to the conventional 2D cell-based model and the proposed pulmonary 3D floating ECM model. In conventional 2D models, NPs are deposited onto 2D monolayer cells via gravity. The cells are then exposed to the aggregated and sedimented NPs. In contrast, the inverted 3D floating ECM model ensures that the cells are only exposed to well-distributed NPs, thereby reducing the interference caused by NPs sedimentation. Previously, the performance of 3D floating ECM models in cytotoxicity assays was validated in various cell lines, including A549 lung cells, THLE-2 hepatocytes, and primary neuronal cells, using 96-, 384-, or 532-well platforms [11, 13, 28, 29]. In this study, the culture conditions were optimized for BEAS-2B lung cells, and the morphological characteristics of the 3D BEAS-2B cells are shown in Fig. 1B.

Fig. 1.

Schematic of the NPs exposure systems to cells. (A) 2D and 3D NPs exposure systems to cells. The left panel shows the conventional 2D model, and the right panel illustrates the newly proposed pulmonary 3D floating ECM model to eliminate the interference of nanomaterials. (B) Schematic of the experimental procedure used to evaluate the cell viability of NPs using a pulmonary 3D floating ECM model. Cells and media are dispensed into columns and well plates, respectively. Cells are then immobilized on alginate at the top of the column and incubated in well plates containing growth medium for 24 h to form 3D cell structures. Then, the well plate filled with NPs is used to replace the original one for NPs treatment. 3D cells are exposed to the live cell staining dyes by transferring them to a new well plate containing the Calcein AM staining dye. The fluorescence images are scanned for data analysis. The representative image of 3D BEAS-2B cells is shown in the right panel; the live 3D BEAS-2B cells are stained with Calcein AM. This schematic illustrates how the floating ECM model improves uniform NPs exposure by minimizing gravitational sedimentation and enhancing compatibility with live-cell fluorescence-based assay. The image was created with Biorender.com

Physicochemical properties of SiNPs depending on the culture media

The particle sizes of the synthesized SiNPs were characterized using TEM, SEM, DLS, and SMPS (Table 1). The average sizes of SiNPs determined by TEM and SEM were 19.6 nm and 21.1 nm, respectively, consistent with those determined by DLS and SMPS (19.5 nm and 21.5 nm, respectively). The DLS analysis across concentrations ranging from 0.015 to 1 mg/mL showed no significant variation in the size of SiNPs (Table S1). Through XRD and ToF-SIMS analyses, the 20 nm SiNPs were confirmed to be amorphous silica nanoparticles with no detectable functional groups (Fig. S1 and S2). Morphological observation and hydrodynamic size distribution indicated that the 20 nm SiNPs were highly monodispersed in DIW without agglomeration or aggregation. (Fig. 2A and B). DLS was used to characterize the size of 20 nm SiNPs in different types of culture media, such as BEGM and RPMI, at 37°C for 72 h (Fig. 2C and Table S2). In the absence of serum, 20 nm SiNPs maintained their original sizes in both media for 72 h. However, the size of 20 nm SiNPs increased significantly with the addition of serum. The size of SiNPs in RPMI-CM was initially 71.1 ± 3.8 nm and gradually increased to 382.2 ± 7.1 nm during 72 h of incubation. Meanwhile, the size of SiNPs increased to 3000 ± 445.8 nm in BEGM-CM and remained stable for up to 72 h. These results indicate that the SiNPs agglomerate in serum-containing media.

Table 1.

Analysis of the particle size of SiNPs in DIW

Measurement methods	Diameter	Measurement uncertainty (k = 2)
TEM	Areal: 19.6 nm Feret: 21.8 nm	0.5 nm 0.6 nm
SEM	Areal: 21.1 nm Feret: 22.5 nm	0.5 nm 0.5 nm
DLS	19.5 nm	2.4 nm
SMPS	21.5 nm	1.1 nm

Fig. 2.

Physicochemical characterization of 20 nm SiNPs. (A) TEM image, scale bar = 50 nm. (B) hydrodynamic size distribution measured by DLS. (C, D) Size and stability of 20 nm SiNPs in DIW and four different culture media. These characterizations were performed to monitor dispersion stability and sedimentation kinetics of SiNPs in physiologically relevant media over 72 h; (C) Hydrodynamic size measured by DLS, (D) TSI profiles showing the stability kinetics for 72 h using Turbiscan analysis. DIW, distilled water; BEGM-SF, BEGM serum-free medium; BEGM-CM, BEGM complete medium with supplemental serum; RPMI-SF, RPMI 1640 serum-free medium; RPMI-CM, RPMI 1640 complete medium with 10% FBS. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 compared to the controls (Dunnett test with a one-way ANOVA)

Next, the stability of the 20 nm SiNPs was monitored in real time for 72 h using a Thurbiscan tower analyzer (Fig. 2D). The degree of NPs agglomeration can be evaluated by measuring the differences between the transmission and backscatter signals of near-infrared pulsed light (λ = 880 nm) [30]. The SiNP suspensions were prepared in sterile glass vials containing each culture medium, and transmission changes were observed from the bottom to the neck of the vial at 37°C for 72 h. The transmission profile showed that the stability of the SiNPs depended on the culture media, and agglomeration and sedimentation were observed in BEGM-SF and BEGM-CM, respectively (Fig. S3). Fig. 2D also shows that the TSI, a kinetic index of destabilization, of SiNPs depends on the culture medium. The TSI value increased with NP size, indicating lower stability [31]. For BEGM-SF and RPMI-SF, the TSI of the SiNPs remained low for 72 h, indicating their stability in the SF medium. The TSI of SiNPs was high in RPMI-CM, indicative of agglomeration. A significant increase in TSI was observed in BEGM-CM from the start of the incubation, suggesting that SiNPs were highly unstable, leading to agglomeration and sedimentation. These results are consistent with the DLS data.

Cell viability of SiNPs in 2D and 3D BEAS-2B cells

The viability of 2D BEAS-2B cells was evaluated in different culture media with or without serum (Fig. 3A). SiNPs were used to treat 2D BEAS-2B cells for 24 h at concentrations up to 250 µg/mL, with serial 2-fold dilutions. Cell viability was evaluated using multiple assays, including MTS, CellTiter-Glo, and Calcein AM staining. The result showed that SiNPs were more cytotoxic in BEGM-SF (IC₅₀ = 69.2 µg/ml) than in BEGM-CM (IC₅₀ = 127.7 µg/ml) (Fig. 3B). However, the cytotoxicity of SiNPs was observed in RPMI-SF (IC₅₀ = 25.7 µg/ml) but not in RPMI-CM (Fig. S4A). In addition, the cytotoxic effects of SiNPs in SF medium on 2D A549 cells were similar to those on 2D BEAS-2B cells (Fig. S5). These results indicated that the cytotoxicity of the SiNPs strongly depends on their stabilities in different medium compositions. The well-dispersed 20 nm SiNPs were cytotoxic, and sedimentary SiNPs in BEGM-CM also induced cytotoxicity, whereas agglomerated SiNPs in RPMI-CM were not cytotoxic in 2D BEAS-2B cells.

Fig. 3.

Viability of BEAS-2B cells after being treated with 20 nm SiNPs in different culture media. (A) Cell viability of 20 nm SiNPs in 2D BEAS-2B cells using different detection methods. 20 nm SiNPs were used to treat 2D BEAS-2B cells in BEGM-SF and BEGM-CM media for 24 h. Cell viability was measured using MTS assays by colorimetric detection, CellTiter-Glo assays by luminescence detection, and Calcein AM assays by fluorescence detection. For fluorescence detection, total cells were stained with Hoechst 33342, and live cells were calculated by the intensity of the Calcein Red-Orange staining. (B) Cell viability of 20 nm SiNPs in 2D and 3D BEAS-2B cells in different culture media. 20 nm SiNPs were treated in 2D BEAS-2B in BEGM-SF and -CM media for 24 h and cell viability was measured using MTS assays. (C) 3D BEAS-2B cells were treated with 20 nm SiNPs for 24 h in BEGM-SF and BEGM-CM media according to the experimental procedure. Cell viability was calculated by the intensity of Calcein Red-Orange AM staining. Multiple detection platforms were employed to reveal potential interference caused by the optical properties of NPs. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 compared to the controls (Dunnett test with a one-way ANOVA)

A 3D floating ECM model was used to evaluate the viability of 3D BEAS-2B cells after they were treated with SiNPs dispersed in different culture media (Fig. 3C). SiNPs in BEGM and RPMI with or without serum were exposed to 3D BEAS-2B grown on the pillar plate, and the cell viability was analyzed by Calcein AM staining. The cytotoxicity of SiNPs in 3D BEAS-2B cells was confirmed in both types of SF media. Contrary to the results observed in 2D BEAS-2B cells, SiNPs in BEGM-CM did not exhibit cytotoxicity in 3D BEAS-2B cells. In RPMI medium with or without serum, the cytotoxic effect of SiNPs on 3D BEAS-2B cells was similar to that on 2D BEAS-2B cells (Fig. S4B). These results indicate that the sedimentation of SiNPs in BEGM-CM induces cytotoxicity in 2D BEAS-2B cells during static incubation, whereas the cytotoxic effects due to sedimentation are insignificant in 3D BEAS-2B cells using a 3D floating ECM model.

Cellular uptake of SiNPs in 3D BEAS-2B cells

To explore the correlation between the uptake of NPs into 3D cells and their toxicity in the 3D floating ECM model, cell viability and cellular uptake were determined by fluorescence microscopy using 20 nm FITC-SiNPs (Fig. 4). The particle sizes of the synthesized FITC-SiNPs were characterized using SEM and DLS (Fig. S6). The average size of FITC-SiNPs, as determined by SEM, was 25.3 ± 1.8 nm, which was consistent with DLS measurements. Furthermore, its size was comparable to that of the unlabeled 20 nm SiNPs used in this study. The intensity of green fluorescence indicates cellular uptake. For BEGM-SF, cell viability decreased after FITC-SiNPs treatment, and the fluorescence images showed that FITC-SiNPs was taken up by 3D BEAS-2B cells (Figs. 4 and S7). In contrast, the cellular uptake and cell viability of BEGM-CM remained unchanged before and after FITC-SiNPs treatment.

Fig. 4.

Cellular uptake of 20 nm FITC-SiNPs in 3D BEAS-2B cells via the pulmonary 3D floating ECM. Cell viability and cellular uptake were measured after the cells were treated with 20 nm FITC-SiNPs for 24 h in the presence and absence of serum. The green and red fluorescence indicate cellular uptake and cell viability, respectively. Increased uptake in SF conditions may reflect reduced viability-associated permeability. BEGM-SF, BEGM serum-free medium; BEGM-CM, BEGM complete medium with supplemental serum. The dose-response curve was analyzed based on the four-parameter equation. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 compared to the controls (Dunnett test with a one-way ANOVA)

The cellular uptake of SiNPs in both 2D and 3D culture models was confirmed using bio-TEM analysis (Fig. 5). BEAS-2B cells were treated with 12.5 µg/mL of 20 nm SiNPs for 24 h and showed approximately 90% cell viability. In 2D BEAS-2B cells, cellular uptake of SiNPs was observed in both SF and CM, with greater uptake in the SF medium. The TEM image showed that some sedimented SiNPs were internalized by the cells in BEGM-CM, while most SiNPs were observed outside the cell membrane. Meanwhile, SiNP uptake in 3D BEAS-2B cells was not observed in BEGM-CM, consistent with the cell viability and cellular uptake data obtained by fluorescence microscopy. Morphological changes, including membrane rupture or blebs in 3D BEAS-2B, were also observed, indicating cellular damage after SiNPs treatment in SF medium.

Fig. 5.

Intracellular uptake of SiNPs in 2D and 3D BEAS-2B cells by Bio-TEM analysis. 2D and 3D BEAS-2B cells were exposed to 12.5 µg/mL of 20 nm SiNPs for 24 h in BEGM-SF and BEGM-CM media. Intracellular SiNPs are marked by arrows. Bio-TEM analysis visualized the spatial localization of SiNPs in cellular compartments to compare uptake patterns across culture models. Scale bar = 1 μm

Implications of the pulmonary 3D floating ECM model for cell viability assays using NPs exhibiting optical interference

Most NPs exhibit optical absorption over a wide range of absorbance wavelengths, which can interfere with the absorbance measured in cell viability assays [23, 32]. The 3D floating ECM model was applied to diminish the optical interference of NPs, enabling a more accurate evaluation of their cytotoxicity. The DLS analysis for AgNPs was conducted across concentrations ranging from 6.25 to 100 µg/mL, showing no significant variation (Table S3). Additionally, AgNPs exhibited optical absorption at a specific wavelength with increasing NP concentration (Fig. 6A). The stability of AgNPs was monitored in real time at 37°C for 72 h using a Thurbiscan tower analyzer (Fig. 6B). The TSI of AgNPs remained stable in DIW and showed significant increases in SF and CM from the initial incubation period. The average size of the AgNPs was approximately 1494 nm in SF and 1868 nm in CM medium (Table S4). Combined with the DLS data, these increased TSI values indicated that the AgNPs were highly unstable, exhibiting more agglomeration and sedimentation in all cell culture media. 2D and 3D BEAS-2B cells were treated with AgNPs in SF and CM for 24 h to evaluate cell viability. The AgNPs present in the culture medium tend to sediment and adsorb onto cells, potentially causing interference. Therefore, to eliminate these effects, the cells were washed with DPBS before performing the assay. In the 2D culture system, the cell viability of the AgNPs based on the MTS assay was remarkably different before and after the washing step (Fig. S8). When AgNPs were not washed, cell viability increased with increasing NP concentration, owing to the optical absorbance of the NPs. Although the washing step reduced the absorbance interference, the NPs could not be completely removed from the attached 2D cells, which led to cell loss and potentially resulted in false negative or positive data (Fig. 6C). Meanwhile, AgNPs were exposed to 3D BEAS-2B in SF and CM media using a 3D floating ECM model, and the cell viability was analyzed by Calcein AM staining (Fig. 6D). AgNPs were cytotoxic to 3D BEAS-2B cells in a dose-dependent manner and were more cytotoxic in SF than in CM. These results indicate that the 3D floating ECM model is more applicable than 2D model for assessing the cell viability of NPs, as it minimizes optical interference and reduces the likelihood of false negative or positive results.

Fig. 6.

Cell viability of AgNPs in 2D and 3D BEAS-2B cells. (A) UV-vis absorption of AgNPs. AgNPs of different concentrations were prepared by dilution in DIW, and their spectra were measured within the wavelength of 200–800 nm. (B) Stability of AgNPs in culture media for 72 h. TSI profiles measured by Turbiscan analysis show the stability kinetics. (C) Cell viability of AgNPs after exposure to 2D BEAS-2B cells in BEGM-SF and BEGM-CM media for 24 h. Cell viability was measured using the MTS assay. The results show an increase in cell viability due to AgNP attachment to the cells in the 2D conventional model; magnification of 10× (right). (D) Cell viability of AgNPs after exposure to 3D BEAS-2B cells in BEGM-SF and BEGM-CM media for 24 h. Cell viability was measured using Calcein AM staining. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 compared to the controls (Dunnett test with a one-way ANOVA)

Discussion

Reducing variations in the assessment of the in vitro toxicity of NPs remains challenging. The physicochemical properties of NPs depend on the experimental design, including cell type and assay conditions, which can result in inconsistent in vitro data [33, 34]. In general, smaller NPs have a larger surface area, which increases their reactivity with cellular components, consequently interfering with cellular biological activities [35]. Studies on SiNPs of different sizes have shown that SiNPs smaller than 50 nm cause more significant cytotoxicity in A549 cells in SF media [20, 36]. In contrast, in BEAS-2B cells, the size of SiNPs significantly increased to over 500 nm in culture media containing serum, and cytotoxicity was observed when SiNPs underwent strong sonication [37, 38]. In this study, the original size of SiNPs was maintained in SF media, and the well-dispersed SiNPs were cytotoxic to both BEAS-2B and A549 cells. However, the SiNPs agglomerated/sedimented in the culture medium containing serum, resulting in different cytotoxicities between the two cell lines. The stability of NPs is a key factor in determining the state of agglomeration/sedimentation. DLS can be used to measure agglomeration based on the hydrodynamic size of NPs [39]; however, it is difficult to determine the NP stability during cell culture treatment. In this study, the stability profiles of 20 nm SiNPs by Turbiscan analysis showed different states of agglomeration/sedimentation in the culture media containing serum (Fig. S3B). Interestingly, agglomerated SiNPs were non-cytotoxic, whereas sedimented SiNPs were cytotoxic to both A549 and BEAS-2B cells. These results are related to the protein corona formed on the surface of NPs in serum-containing media, which inhibits the interaction with the cellular target and reduces the cytotoxicity [40]. The effects of protein corona on cellular uptake are complicated and depend on the nature of the NP and protein, the environment, and the cell type [41]. It is generally believed that protein corona can suppress cellular uptake of NPs [40, 42]. In this study, cellular uptake of SiNPs was enhanced in the SF medium, and little uptake was observed in the CM medium where SiNPs were sedimented. The cellular uptake of NPs depends on several factors, including size and cell type, and NPs that are too small or too large for endocytosis cannot be effectively internalized [43]. The increased size of agglomerated SiNPs in RPMI-CM might not induce cytotoxicity in 2D BEAS-2B cells because of the formation of protein corona; however, SiNPs sedimented in BEGM-CM had high cytotoxicity, even though their size was larger than that in RPMI-CM. NPs gravitationally settled on 2D monolayer cells during cell culture, and the sedimented SiNPs should be considered as the effective dose delivered to the cells in vivo. Unlike well-dispersed NPs, the sedimented NPs are transferred into the cell by passive uptake [40]. In a physiological state with an ECM barrier or dynamic blood circulation, the in vivo response to sedimented NPs is different from the toxic effects derived from in vitro assays. In an in vivo study, SiNPs smaller than 50 nm induced an inflammatory response in the lungs by inhalation exposure, and SiNPs were distributed in the liver, lungs, spleen, and kidneys by ingestion or intravenous injection. In most in vivo studies, SiNPs were administered at very high doses compared with the actual level of human exposure. Therefore, it is difficult to estimate the toxic effects in humans accurately based on in vivo toxicity studies of SiNPs [20]. In this study, experiments with a 3D floating ECM model showed that well-dispersed 20 nm SiNPs were cytotoxic in the SF medium but not in the CM medium owing to the sedimentation, which contrasts with the results observed in 2D culture. Quantifying the exposure levels of SiNPs in 2D and 3D models provides valuable insights for in vitro toxicity results. However, due to inherent differences in cell number, exposure methods such as insert pillars, and ECM presence, direct comparisons between these models remain challenging. Instead, we further estimated total SiNPs exposure amounts and density in both 2D and 3D floating ECM model based on assumed sedimentation levels (Table S5). The results indicate that at approximately 25% sedimentation, exposure densities in both models were comparable. However, at 50% sedimentation, exposure in 2D models was calculated to be four times higher than in 3D models, suggesting that sedimentation could lead to overexposure of NPs to cells in 2D models. Despite sedimentation, SiNPs remained adequately exposed within the 3D floating ECM model. This estimation enhances the reliability of experimental results, as it aligns with FITC-SiNPs fluorescence and TEM findings, which confirm SiNP uptake. The IC₅₀ levels for 3D cells and 2D cells were 164.5 µg/mL and 65.2 µg/ml, respectively, in BEGM-SF medium, suggesting that 2D cells were more vulnerable to cytotoxicity screening. Wiemann et al. demonstrated that an in vitro assay could generate comparable results to those of a short-term inhalation study conducted under serum-free conditions [44]. It has been suggested that 20 nm SiNPs may be potentially toxic to lung cells by inhalation exposure, but the toxic effects in the lung may be reduced by systemic exposure. This higher IC₅₀ level for 3D cells is consistent with their drug efficacy results, which show a better correlation with their in vivo responses [29]. 3D lung cells also show increased levels of structural and functional markers, including tight junctions, epithelial proteins, and mucin-specific proteins [45]. Previous studies have indicated that the toxic effects of NPs in 3D cellular models correlate closely with the data from animal studies [46].

In the current 3D cell culture model, alginate, a polysaccharide hydrogel, is used to encapsulate cells in a 3D structure [47]. Compared with Matrigel, alginate was more efficient in forming a 3D structure of BEAS-2B lung cells (Fig. S7A). At a neutral pH, alginate tends to form ionic hydrogels because it is polyanionic and can chelate with divalent cations. Alginate hydrogels enable cellular access to nutrients and prompt the removal of waste products [48]. The alginates showed an average pore diameter of approximately 20 μm (Fig. S7B). Cellular uptake of FITC-SiNPs was confirmed by fluorescence and TEM analyses, demonstrating that nano-sized SiNPs could penetrate the 3D alginate-encapsulated BEAS-2B cells (Fig. S7C and Fig. 5).

In addition to the agglomeration and sedimentation of NPs in culture media, optical interference is another obstacle to obtaining reliable cytotoxicity data using HTS approaches [4, 49]. Cell viability is typically measured using various spectroscopic methods, such as colorimetric, luminescent, and fluorescent detections. Many colorimetric detection methods, including MTS, water-soluble tetrazolium (WST), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays, and luminescence-based ATP detection assays, are affected by the optical properties of NPs [50]. AgNPs exhibit specific optical absorption at a wavelength of 405 nm, which overlaps with the absorption wavelengths of the detection reagents, including WST and MTS [22]. Although the residual NPs are removed by further washing steps or the experimental signal is subtracted by the background, the measured optical data still depend on the optical absorption of the NPs. The luminescence detection method is vulnerable to optical interference from NPs, and the intensity of luminescence is decreased by the NPs, possibly leading to false-positive cytotoxicity [49]. To address this issue, label-free detection methods have been proposed as alternatives, and impedance-based detection has been developed as an ISO protocol (TS 21633) [10]. However, the impedance approach is not suitable for precipitating NPs. We proposed a 3D floating ECM model in an ISO technical report (ISO/TR22455) [19] as an alternative HTS method for nanosafety research. It can address issues arising from the interference effect and the sedimentation of NPs. Further research may focus on understanding the biological differences between 2D and 3D cells and exploring various toxic mechanisms. In this study, we demonstrated the feasibility of a 3D floating ECM model for testing the cell viability of NPs with sedimentation or optical interference, which can be applied to HTS approaches for various NPs, including metal oxides and nanotubes. The crystallinity and functional groups of NPs are critical factors influencing their aggregation and dispersion stability. High-crystallinity nanomaterials generally exhibit lower surface energy, making aggregation more likely [51]. Additionally, surface modifications can impact dispersion behavior, highlighting the need to evaluate both crystallinity and functional groups in nanomaterial toxicity assessments [52]. In this study, the SiNPs were confirmed to be amorphous, suggesting they may exhibit enhanced dispersion stability [53]. Similarly, PVP-AgNPs play a crucial role in improving NPs dispersion [54]. However, despite being synthesized for stable dispersion, their dispersion behavior can vary depending on culture medium composition. Therefore, this study provides valuable insights for in vitro toxicity evaluations, offering a more in vivo-like environment through the floating ECM model, which helps mitigate artifacts caused by dispersion instability.

In this study, we present a 3D floating ECM model utilizing pulmonary cells for in vitro toxicity screening. This model provides a well-regulated microenvironment through ECM interactions, ensuring structural integrity and biologically relevant drug responsiveness. While organoids and other 3D models are increasingly recognized as promising tools for toxicity screening [16], their widespread adoption remains limited due to challenges such as reproducibility, technical complexity, and high costs. Efforts toward standardization, including those led by the ISO Biotechnology Committee (TC276), are still in the early stages. Unlike conventional 3D models, our system offers enhanced reproducibility and throughput, making it compatible with automated high-throughput screening platforms. Additionally, its recognition as a technical reference in ISO/TR 22455 highlights its potential for application to various NPs, including Cu₂O, SWCNTs, and quantum dots. Prior research has also demonstrated the adaptability of the floating ECM system to various organ-derived cells, including applications in organoid-based models [55]. This versatility suggests that the nanomaterial toxicity assessment technology developed in this study could be extended to multiple target organs, enabling broader toxicity evaluations. Further experimental validation is necessary to confirm its applicability across diverse cell models. While our floating ECM model offers significant advantages, it also has certain limitations that should be considered. Although it provides a biologically relevant environment, it does not fully replicate the intricate multicellular architecture of organoids or in vivo tissues. Additionally, its applicability to long-term studies and its ability to capture complex intercellular interactions require further validation. The effects of NPs aggregation and dynamic interactions with extracellular components must also be optimized to ensure accurate toxicity assessments. Future research should focus on refining these aspects to enhance the reliability and broader applicability of the platform.

Conclusions

We propose a novel pulmonary 3D floating ECM model as NAMs to evaluate the in vitro toxicity of NPs. This model allows a more reliable evaluation of the cytotoxic effects of NPs that may experience sedimentation or cause optical interference. Moreover, the model can be developed into an HTS approach to screening the potential cytotoxic effects of various NPs, supporting their risk assessment for human health and the environment.

Electronic supplementary material

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Abbreviations

2D

Two-Dimensional

3D

Three-Dimensional

AgNPs

Silver Nanoparticles

APTES

(3-aminopropyl) Triethoxysilane

BEGM

Bronchial Epithelial Cell Growth Medium Bullet Kit

DIW

Deionized Water

DLS

Dynamic Light Scattering

DPBS

Dulbecco’s Phosphate-Buffered Saline

ECM

Extracellular Matrix

FBS

Fetal Bovine Serum

FITC

Fluorescein Isothiocyanate

GUM

Guide to Expression of Uncertainty in Measurement

HTS

High-Throughput Screening

ISO

International Organization for Standards

MTS

CellTiter 96^® aqueous one solution cell proliferation assay

NAM

New Approach Methodologies

NPs

Nanoparticles

ROS

Reactive Oxygen Species

SEM

Scanning Electron Microscopy

SiNPs

Silica Nanoparticles

SMPS

Scanning Mobility Particle Sizer

TEM

Transmission Electron Microscopy

TEOS

Tetraethyl Orthsilicate

ToF-SIMS

Time-of Flight Secondary Ion Mass Spectrometry

TSI

Turbiscan Stability Index

UV

Ultraviolet

XRD

X-ray Diffraction

Author contributions

SK designed the study, performed the experiments, analyzed the data, and drafted the manuscript. MSC and JH performed the cytotoxicity and stability analyses. MBH and MJK performed SiNP synthesis and physicochemical analysis. HKS and SS performed SiNP surface and structural characterization. DWL performed the cytotoxicity and cellular uptake experiments and drafted the manuscript. JHP, TGL, and SY designed the study and drafted the manuscript. JHO drafted and revised the manuscript critically. All authors reviewed the manuscript.

Funding

This work was supported by the Research Program of the Ministry of Science and ICT [RS-2024-00452934, RS-2018-NR056535, RS-2023-NR076935], National Institute of Agricultural Sciences, Rural Development Administration (RS-2024-00400007) and the general research grant [No. 2710008763] funded by the Korea Institute of Toxicology. The funders had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.