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Published in final edited form as: Cell Biol Toxicol. 2021 Nov 11;39(1):259–275. doi: 10.1007/s10565-021-09678-x

The cytotoxicity of zinc oxide nanoparticles to 3D brain organoids results from excessive intracellular zinc ions and defective autophagy

Liangliang Liu 1, Junkang Wang 2, Jiaqi Zhang 2, Chaobo Huang 3, Zhaogang Yang 4, Yi Cao 5,6
PMCID: PMC12070400  NIHMSID: NIHMS2069844  PMID: 34766255

Abstract

Although the neurotoxicity of ZnO nanoparticles (NPs) has been evaluated in animal and nerve cell culture models, these models cannot accurately mimic human brains. Three-dimensional (3D) brain organoids based on human-induced pluripotent stem cells have been developed to study the human brains, but this model has rarely been used to evaluate NP neurotoxicity. We used 3D brain organoids that express cortical layer proteins to investigate the mechanisms of ZnO NP-induced neurotoxicity. Cytotoxicity caused by high levels of ZnO NPs (64 μg/mL) correlated with high intracellular Zn ion levels but not superoxide levels. Exposure to a non-cytotoxic concentration of ZnO NPs (16 μg/mL) increased the autophagy-marker proteins LC3B-II/I but decreased p62 accumulation, whereas a cytotoxic concentration of ZnO NPs (64 μg/mL) decreased LC3B-II/I proteins but did not affect p62 accumulation. Fluorescence micro-optical sectioning tomography revealed that 64 μg/mL ZnO NPs led to decreases in LC3B proteins that were more obvious at the outer layers of the organoids, which were directly exposed to the ZnO NPs. In addition to reducing LC3B proteins in the outer layers, ZnO NPs increased the number of micronuclei in the outer layers but not the inner layers (where LC3B proteins were still expressed). Adding the autophagy flux inhibitor bafilomycin A1 to ZnO NPs increased cytotoxicity and intracellular Zn ion levels, but adding the autophagy inducer rapamycin only slightly decreased cellular Zn ion levels. We conclude that high concentrations of ZnO NPs are cytotoxic to 3D brain organoids via defective autophagy and intracellular accumulation of Zn ions.

Introduction

Metal and metal oxide-based nanoparticles (NPs) have drawn much attention because of their use in perovskite solar cells (Siavash Moakhar et al. 2020), the food industry (Jafarzadeh and Jafari 2020), and biomedicine (Chen et al. 2018; Joseph et al. 2020). These NPs are readily available from commercial sources and are more commonly used than carbon and silica NPs (Yang and Westerhoff 2014; Vance et al. 2015). Zinc oxide (ZnO) NPs are important metal oxide-based NPs that are used in commercial products such as sunscreen (Vujovic and Kostic 2019), food packaging (Xia et al. 2020), and catalysts (Ponnamma et al. 2019). ZnO NPs may also be useful as antimicrobial agents (Gharpure and Ankamwar 2020), anti-cancer drugs (Yi et al. 2020), and bio-imaging tools (Jin and Jin 2019). The increasing use of ZnO NPs in a variety of applications in the future underscores the need to investigate their potential toxicity in reliable models.

One system that has been difficult to model accurately is the central nervous system (CNS), as it is surrounded by various physiological barriers like the blood–brain barrier (BBB), to maintain homeostasis, a process that is essential to human life. However, metal and metal oxide-based NPs including ZnO NPs are ultrasmall and may penetrate physiological barriers including the BBB (Chen et al. 2016; Cao et al. 2018), which raises public health concerns regarding their potential neurotoxicity. Moreover, the expanding uses of ZnO NPs in biomedicine, particularly as biomedical materials for CNS diseases, further emphasize the need to evaluate their potential neurotoxicity (Song et al. 2016; Mohammadipour et al. 2020). At present, the neurotoxicity of ZnO NPs has been studied in laboratory animal models such as mice, rats, and zebrafish (Chakraborty et al. 2016; Singh 2019). However, because human brains are much more complex than those of laboratory animals, the generalizability of results from such models to humans is unclear. The neurotoxicity of ZnO NPs has also been evaluated in conventional two-dimensional (2D) cell cultures derived from human neurons such as human glial cells (Engin and Engin 2019; Chang et al. 2021). However, conventional 2D cell cultures are also different from human organs (Cao et al. 2021), prompting the development of more complex models based on human neurons to evaluate the bio-effects of metal and metal oxide-based NPs in human brains. In 2019, Leite et al. developed human brain spheroids based on human dopaminergic neurons or induced pluripotent stem cells (iPSC) and showed that both spheroid types were good for studying the neurotoxicity of Au NPs (Leite et al. 2019). Sokolova and colleagues found that ultrasmall Au NPs conjugated with dyes could be internalized into 3D spheroids composed of human primary astrocytes, pericytes, and brain endothelial cells (Sokolova et al. 2020). These findings notwithstanding, better models are still needed to study the potential neurotoxicity of agents such as metal and metal oxide-based NPs and its underlying mechanisms (Cao et al. 2021).

The 3D human brain organoids (also called organotypic cultures) developed to date are an intriguing research tool for understanding the pathophysiology of human brains (Chen et al. 2020; Zhang et al. 2020), but they have rarely been used in toxicological studies. Although one group reported the feasibility of using 3D brain organoids to investigate Au NP neurotoxicity, the model used was based on mouse stem cells (Ji et al. 2019). Even brain organoids derived from non-human primates differ significantly from human brain organoids (Pollen et al. 2019), and thus presumably brain organoids based on mouse stem cells probably also differ substantially from those based on human stem cells. We recently used 3D brain organoids based on human iPSC to test the effects of multi-walled carbon nanotubes (MWCNTs) on nitric oxide (NO) signaling pathways in brains (Jiang et al. 2020). Huang et al. recently utilized 3D brain organoids based on human embryonic stem cells to evaluate cadmium-induced neurodevelopmental toxicity (Huang et al. 2021). The results suggest that the potential of 3D human brain organoids for studying mechanism-based neurotoxicity should be further evaluated.

We report here findings from a novel 3D human brain organoid model based on human iPSC that was exposed to ZnO NPs, with changes in cellular viability as a surrogate for neurotoxicity. To further investigate mechanisms thought to underlie the neurotoxicity of Zn NPs in other models, we also measured changes in oxidative stress, intracellular Zn ion accumulation, and autophagy biomarkers (Liu et al. 2016; Mohammadinejad et al. 2019). We also imaged the spatial distribution of the autophagic protein LC3B and morphologic changes in nuclei throughout the organoids by using fluorescence micro-optical sectioning tomography (MOST). Our findings confirm that this model is useful for assessing the neurotoxicity of ZnO NPs.

Materials and methods

ZnO NP preparation

ZnO NPs (average size 20 nm) were obtained from Nanjing XFNANO Materials Tech Co., Ltd. NP morphology was evaluated by transmission electron microscopy (TEM, FEI Tecnai G20, USA). NP suspensions were created by sonicating 1280 μg/mL nanoparticles in 2% fetal bovine serum (FBS) 16 min at 4 °C by using an ultrasonic processor, and suspensions were used immediately thereafter. The hydrodynamic size, polydispersity index (PDI), and zeta potential of newly prepared NPs (at concentration of 64 μg/mL) were examined by using a Zetasizer Nano ZS90 (Malvern Panalytical, Westborough, MA). ZnO NP topography was visualized in both double-distilled water and cell culture medium by using atomic force microscopy (AFM) as previously described (Liang et al. 2018).

Three-dimensional brain organoid generation and characterization

The human iPSC cell line DYR0100 was provided by the Chinese Academy of Science Stem Cell Bank and differentiated into neural precursor cells (NPC) according to previously described protocols (Xu et al. 2016). The 3D brain organoids were obtained after 90 days of differentiation and maintained in human 3D cerebral organoid medium per manufacturer’s instructions (Hopstem Biotechnology Co. Ltd., China). As described previously (Jiang et al. 2020), we used fluorescent immunohistochemical staining to analyze the following markers of cortical layers: T-box brain protein 1 (TBR1, for cortical layers I, V, and VI), brain-2 (BRN2) and special AT-rich sequence-binding protein 2 (SATB2, for cortical layers II–IV), and chicken ovalbumin upstream promoter-transcription factor–interacting protein 2 (CTIP2; for layers V and VI). Marker staining in all samples was verified by light microscopy with an Axio Observer3 (Carl Zeiss).

Acridine orange/4′,6-diamidino-2-phenylindole (AO/DAPI) staining

The 3D brain organoids were treated with ZnO NPs at three concentrations: 0 (control), 16, or 64 μg/mL for 24 h. We also treated the organoids with 64 μg/mL ZnO NPs plus 100 nM bafilomycin A1 (Baf A1, an inhibitor of autophagic flux, CST Cell Signaling Technologies, USA) or 200 nM rapamycin (RAPA, an autophagy activator; Vetec™, Sigma-Aldrich, USA). After the 24-h treatment, the organoids were washed and dissociated in Accutase-Enzyme Cell Detachment Medium (Thermo Fisher, USA). After centrifugation, the resulting cell pellets were incubated in an AO/DAPI solution (ChemoMetec A/S, Allerod, Denmark), resulting in live cells being stained with green fluorescence by AO and dead cells with blue fluorescence by DAPI. The stained cells were then counted, and their viability was automatically evaluated by using a NucleoCounter NC-3000 advanced image cytometer (ChemoMetec A/S).

Superoxide determination

To determine superoxide levels, the 3D brain organoids were incubated with various concentrations of ZnO NPs for 24 h, after which superoxide levels were measured in each cell by using DHE staining (Sigma-Aldrich, USA) as described elsewhere (Jiang et al. 2020). All samples were counted and evaluated by using a NucleoCounter NC-3000.

Inductively coupled plasma mass spectroscopy (ICP-MS)

To measure levels of Zn element, the 3D brain organoids were treated with various concentrations of ZnO NPs for 24 h, thoroughly washed with Hanks solution, and measured by using a NexIon 350X ICP-MS instrument (PerkinElmer, Waltham, MA, USA), as described (Li et al. 2019).

Intracellular Zn Ion measurement

To investigate whether inhibiting or activating autophagy affects intracellular Zn ion levels, the 3D brain organoids were incubated with 64 μg/mL ZnO NPs, ZnO NPs plus 100 nM Baf A1, or ZnO NPs plus 200 nM RAPA. After 24-h incubation, the organoids were washed and dissociated in Accutase. After centrifugation, the resulting cell pellets were incubated for 30 min with 24 μM Zinquin ethyl ester to stain intracellular Zn ions with blue fluorescence (Sigma-Aldrich). The results were counted and evaluated by using a NucleoCounter NC-3000.

Western blotting

After 3D brain organoids were incubated with various concentrations of ZnO NPs for 24 h, LC3B and p62 proteins were measured by western blotting as described elsewhere (Wang et al. 2018). The primary antibodies used in these experiments were LC3B (1:1000), P62 (1:3000), and β-actin (1:5000). All antibodies were purchased from Proteintech, USA.

Fluorescence micro-optical sectioning tomography

The localization of LC3B proteins and morphologic changes in the nuclei in the 3D brain organoids, treated or not treated with 64 μg/mL ZnO NPs, was visualized by fluorescence MOST. After incubation for 24 h, the cells were fixed and incubated with primary antibody (1:200 LC3B antibody, Sigma-Aldrich) and then secondary antibody (1:1000 Alexa Flour 488 Donkey anti-mouse IgG, Invitrogen), as described (Jiang et al. 2020). Finally, the cells were sealed in HM20 resin and imaged by using fluorescence MOST system as described elsewhere (Gong et al. 2016). Samples were simultaneously stained by propidium iodide (PI) during imaging to visualize morphologic changes in the nuclei. Fluorescence MOST and image analysis were done by Wuhan OE-Bio Co., Ltd.

Statistical analysis

All western blotting experiments were repeated in triplicate, and the results are reported as means ± standard deviations (SD). One-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) tests were used to compare differences among treatment conditions by using R 3.3.3. A p-value of < 0.05 by Student’s t test was accepted as indicating statistical difference.

Results

Characterization of ZnO NPs

Our previous characterization of ZnO NPs is reported elsewhere (Zhou et al. 2017). Briefly, X-ray diffractograms indicated that ZnO NPs are hexagonal, about 20 nm in size, with a surface area of 19.07 m2/g. These NPs are partially soluble in water and medium (dissolution rate of 40–50% in 24 h), but the Zn ions released from extracellular dissolution of these NPs are much lower than the threshold to induce cytotoxicity, which suggests that NPs mediate the cytotoxicity (He et al. 2017; Wang et al. 2018). In the current study, the primary size of the ZnO NPs (mean ± SD) was 30.73 ± 9.76 nm (n = 20) (Fig. 1A). The height of the ZnO NPs measured by AFM was 33.68 ± 9.71 nm in water (Fig. 1B; n = 30) and was slightly greater at 45.78 ± 15.74 nm in cell culture medium (Fig. 1C; n = 20). In double-distilled water, the average hydrodynamic size was 271.77 ± 3.86 nm; the PDI was 0.172 ± 0.033; and the zeta potential was −20.9 ± 0.1 mV. Suspension in cell culture medium led to a hydrodynamic size of 301.27 ± 11.53 nm, a PDI of 0.253 ± 0.016, and a zeta potential of −0.38 ± 1.69 mV.

Figure 1.

Figure 1

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Physicochemical characterization of ZnO NPs. A ZnO NP morphology visualized by TEM. B and C ZnO NP topography in water (B) and in cell culture medium (C) visualized by using AFM

Characteristics of 3D brain organoids

The 3D brain organoids were generally spheroidal, with a diameter of about 1.5 mm (Fig. 2A). Immunohistochemical staining confirmed that the organoids expressed all four tested cortical layer markers, namely TBR1, BRN2, SATB2, and CTIP2 (Fig. 2B). Besides these cortical layer markers, the 3D brain organoids also contained a high level of glial cells (glial fibrillary acidic protein or oligodendrocyte transcription factor 2-positive cells; information available from http://www.hopstem.com/index.php?language=2), but we did not further attempt to stain these cells.

Figure 2.

Figure 2

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Characteristics of 3D brain organoids. The 3D human brain organoids were developed from the sPSC cell line DYR0100 as described in the Methods. A 3D brain organoid morphology shown by light microscopy. B Expression of cortical layer proteins including BRN1, CTIP (green fluorescence), TBR1, and SATB2 (red fluorescence). The nuclei were stained by DAPI (blue fluorescence)

Cellular viability of the 3D brain organoids

The viability of cells within the organoids incubated with 0 or 16 μg/mL ZnO NPs was greater than 90%, but the viability of the cells in the organoids exposed to 64 μg/mL ZnO NPs decreased slightly to 83.6% (Fig. 3).

Figure 3.

Figure 3

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Cytotoxicity of ZnO NPs to the 3D brain organoids. Organoids were incubated with ZnO NPs at 0 (A), 16 (B), or 64 μg/mL ZnO NPs (C), after which they were dissociated into single cells and counterstained with AO (green fluorescence, stains the entire population) and DAPI (blue fluorescence, stains non-viable cells) for assessing cellular viability. Data are representative of two independent experiments

Intracellular superoxide levels

Only a small portion of cells showed orange fluorescence staining by DHE (i.e., were positive for intracellular superoxide) in control or ZnO NP-treated organoids (Fig. 4A–C, upper panel). No obvious increases in DHE intensity were observed after ZnO NP exposure (Fig. 4A–C, lower panel).

Figure 4.

Figure 4

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Intracellular superoxide. The 3D brain organoids were incubated with ZnO NPs at 0 (A), 16 (B), or 64 μg/mL (C), dissociated into single cells, and stained with DHE to visualize intracellular superoxide (red) and with Hoechst 33,342 to visualize nuclei (blue). Upper panel shows representative fluorescence images; lower panel shows changes in DHE intensity versus Hoechst 33,342 intensity

ZnO NP internalization

The concentration of Zn elements in control organoids was too low to be detected by ICP-MS. Organoids exposed to 16 or 64 μg/mL ZnO NPs had high levels of Zn elements, with the concentration higher in the 16 μg/mL condition than in the 64 μg/mL condition (Fig. 5A). However, concentrations of Zn ions showed a dose-dependent increase upon exposure to ZnO NPs (Fig. 5B–D). Interestingly, even in organoids exposed to 64 μg/mL ZnO NPs, only some cells were stained by Zinquin, although the intensity of the blue fluorescence was high (Fig. 5D, upper panel).

Figure 5.

Figure 5

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Accumulation of intracellular Zn elements (A) and Zn ions (B–D). The 3D brain organoids were incubated with ZnO NPs at 0, 16, or 64 μg/mL, after which the concentrations of intracellular Zn elements were determined by ICP-MS (A). B–D The 3D brain organoids were treated with ZnO NP at 0 (control, B), 16 μg/mL (C), or 64 μg/mL (D), dissociated and stained with Zinquin to measure intracellular Zn ions (blue fluorescence). Cellular events were identified by dark field microscopy. Middle panel, representative fluorescence images; lower panel, changes in Zinquin intensity versus dark-field intensity

LC3B-p62 protein levels

Because the above data suggest that 24-h exposure is enough for ZnO NPs to induce neurotoxicity in 3D brain organoids, we then used this time point to investigate the changes of autophagic proteins by Western blotting analysis. The LC3B-II/I ratio was significantly elevated after exposing cells to 16 μg/mL ZnO NPs relative to the control condition, but the decline in LC3B-II/I ratio after 64 μg/mL by 40.56% relative to the control condition was not significantly different (95% confidence interval (CI): –68.71 to 149.8%; p > 0.05). The LC3B-II/I ratio was significantly higher in organoids exposed to 16 μg/mL ZnO NPs than in organoids exposed to 64 μg/mL ZnO NPs (p < 0.01; Fig. 6A). Accumulation of p62 was drastically decreased after treatment with 16 μg/mL ZnO NPs (p < 0.01), but not after 64 μg/mL ZnO NPs (p > 0.05; all compared with control conditions). Finally, p62 protein expression was significantly lower in the 16 μg/mL ZnO NPs condition than in the 64 μg/mL ZnO NPs condition (p < 0.01; Fig. 6B).

Figure 6.

Figure 6

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Changes in LC3B-p62 protein levels. The 3D brain organoids were incubated with 0, 16, or 64 μg/mL ZnO NPs, and levels of LC3B proteins (A) and p62 proteins (B) in the treated organoids were detected by western blotting. * p < 0.05, ** p < 0.01, VS control. Mean ± SD, n = 4

Localization of LC3B proteins and nuclear morphology

In representative images, LC3B protein localization (indicated by green fluorescence staining) showed that relative to the control condition (Fig. 7A), the intensity of LC3B staining at the outer layers of ZnO NP-exposed organoids was reduced, but little difference in intensity was apparent at the inner layers (Fig. 7B). To investigate the changes in LC3B protein localization further, we reconstructed areas with intensive LC3B protein staining to compare the volume of LC3B staining under the different treatment conditions. If we assume that the outer layer of PI-stained nuclei (red fluorescence) indicated the marginal surface of 3D brain organoids, then we could calculate the ratio of LC3B protein volume to the entire organoid volume. We found that the volume of LC3B proteins decreased from 32.03 in the control condition (Fig. 8A and B) to 28.62% in the ZnO NP-exposed condition (Fig. 8C and D).

Figure 7.

Figure 7

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Representative fluorescent MOST images. The 3D brain organoids were treated with ZnO NPs at 0 μg/mL (control, A) or 64 μg/mL (B) and stained by LC3B antibodies (green) and PI (red). At left, from top to bottom: overview of stained organoids; green fluorescence (LC3B localization) and red fluorescence (PI-stained nuclei). At right: the merged images

Figure 8.

Figure 8

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Changes in LC3B protein volume. Assuming that the outer layer of red fluorescence (from PI-stained nuclei) is the marginal surface of 3D brain organoids that allows the LC3B protein volume (green fluorescence) to be calculated. The 3D brain organoids were treated with Zn NPs at 0 μg/mL (control) (A and B) or 64 μg/mL (C and D)

We also reconstructed high-resolution PI-stained areas in the outer layers and inner layers of the organoids to detect changes in nuclear morphology. In the control group, only a few micronuclei were present, both in the outer layers (Fig. 9A, arrow) and inner layers (Fig. 9B, arrow). However, the numbers of micronuclei in the outer layers of organoids exposed to ZnO NPs were clearly increased (Fig. 9C, arrows), although the exact numbers could not be quantified. No increase in micronuclei was apparent in the inner layers of organoids exposed to ZnO NPs (Fig. 9D, arrow).

Figure 9.

Figure 9

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Reconstructed high-resolution images of PI-stained areas. A total volume of 70 μm3 was reconstructed. The 3D brain organoids were treated with Zn NPs at 0 μg/mL (control, A and B) or 64 μg/mL (C and D). A The outer layers of the control, B the inner layers of the control, C the outer layers of the brain organoid treated with 64 μg/mL ZnO NPs, and D the inner layers of the brain organoid treated with 64 μg/mL ZnO NPs. Arrows indicate micronuclei

Effects of autophagic inhibition or activation

The presence of the autophagic inhibitor Baf A1 markedly enhanced the cytotoxicity of ZnO NPs (p < 0.01 VS control), but the autophagic inducer RAPA had no significant effect (p > 0.05 VS control). Nevertheless, cellular viability after treatment with ZnO NPs plus RAPA was greater than that after treatment with ZnO NPs plus Baf A1 (p < 0.01; Fig. 10A). In contrast to the organoids exposed to ZnO NPs only (Fig. 10B), intracellular Zn ion levels in ZnO NP-exposed organoids were markedly increased by Baf A1 (Fig. 10C) but only slightly decreased by RAPA (Fig. 10D).

Figure 10.

Figure 10

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Influence of autophagic modulators on ZnO NP cytotoxicity (A) and ZnO NP-induced intracellular Zn ions (B to D). The 3D brain organoids were treated with 64 μg/mL ZnO NPs, 64 μg/mL ZnO NPs plus the autophagic inhibitor Baf A1, or 64 μg/mL ZnO NPs plus the autophagic activator RAPA for 24 h. A Changes in organoid viability, measured by the AO/DAPI assay. B to D Changes in intracellular Zn ions in organoids treated with 64 μg/mL ZnO NPs (B), 64 μg/mL ZnO NPs plus Baf A1 (C), or 64 μg/mL ZnO NPs with RAPA (D) as detected by Zinquin staining. ** p < 0.01 vs control

Discussion

We applied a novel 3D brain organoid model to study the mechanisms of ZnO NP cytotoxicity in human brains, with a focus on the possible roles of autophagy and intracellular Zn ions. First, our counterstaining cells with AO/DAPI revealed reduced cellular viability after exposure to 64 μg/mL ZnO NPs (Fig. 3C). In our previous studies, exposure to the same type of ZnO NPs at concentrations of ≥ 32 μg/mL reduced cellular viability to nearly zero in different types of conventional 2D cell cultures including macrophages, vascular smooth muscle cells, Caco-2, HepG2, THP-1 monocytes, and endothelial cells (Wang et al. 2018; Luo et al. 2018; Chen et al. 2019). However, lung epithelial tissue models (He et al. 2017; Liu et al. 2019) and 3D Caco-2 spheroids (Cao et al. 2022) apparently were more resistant to ZnO NP exposure. In the current study, ZnO NPs at concentration as high as 64 μg/mL seemed to have only modest cytotoxic effects (cellular viability > 80%, Fig. 3C), which could reflect the presence of multiple layers of tissue-based cells in our 3D brain organoid model (Fig. 2), which seems to confer more resistance to NP exposure. A previous study also found that 3D spheroids were more resistant to NP exposure than regular 2D cell cultures because of the tissue-like morphology and phenotypic changes in 3D cultures (Lee et al. 2009). Moreover, Elje et al. reported that 3D HepG2 spheroids were less sensitive to NPs than a 2D model, but this effect depended on the type of NP, with differences noted for Ag NPs but not ZnO NPs (Elje et al. 2020).

Although the mechanisms by which ZnO NPs induce cytotoxicity are not fully known, ZnO NP-induced oxidative stress, excessive intracellular Zn ions, or both are thought to be involved (Liu et al. 2016; Król et al. 2017). Our findings showed only minimal changes in intracellular superoxide (Fig. 4), indicating that ZnO NPs had negligible influence on oxidative stress. However, we did note a dose-dependent increase in intracellular Zn ions (Fig. 5B–D). We previously found that the same type of ZnO NPs also induced excessive intracellular Zn ions but not oxidative stress (Luo et al. 2018; Chen et al. 2019; Cao et al. 2022). Others have shown that stabilizing ZnO NPs with oleate reduced the intracellular concentrations of Zn ions and consequently alleviated NP-induced cytotoxicity to HepG2 and Caco-2 cells (Fang et al. 2017). Chelation of Zn ions reduced the cytotoxicity of ZnO NPs to HEK293T (Yan et al. 2020) and immune cells (Johnson et al. 2015), whereas increasing the concentration of intracellular Zn ions by vitamin C (Wang et al. 2014) or 3-hydroxyflavone (Luo et al. 2018) enhanced this cytotoxicity. Previous reports as well as the findings reported here suggest that the cytotoxicity of NPs in 3D brain organoids resulted mainly from excessive Zn ions but not oxidative stress. We further found that concentrations of Zn as an element were not dose-dependent on ZnO NPs (Fig. 5A). This may be a protective response to internalize smaller amounts of ZnO NPs. Nevertheless incubation with 16 or 64 μg/mL ZnO NPs still markedly increased the concentration of Zn elements, whereas its concentration in controls was below detectable limits.

We further studied the potential effects of Zn NPs on autophagy signaling. Autophagy is a highly controlled process that degrades damaged biomolecules and internalized foreign materials; defective or excessive autophagy has been linked with NP toxicity (Mohammadinejad et al. 2019). Decreased LC3B-II/I levels and p62 accumulation are hallmarks of defective autophagy, which presumably would impair the ability of cells to degrade unwanted materials; in contrast, increased LC3B-II/I and p62 clearance can indicate autophagy induction (Kirkin and Rogov 2019). We found that ZnO NPs at 16 μg/mL led to increases in LC3B-II/I ratio but decreased p62 accumulation (Fig. 6A). In contrast, ZnO NPs at 64 μg/mL led to decreases in LC3B-II/I ratio but did not affect p62 accumulation (Fig. 6B), suggesting that ZnO NPs at different concentrations induce different autophagic responses. Arakha et al. found that exposure to ZnO NPs led to recovery of LC3B-II from NP-induced stress, whereas cells with unrecoverable damage underwent apoptosis (Arakha et al. 2017). Other studies showed that NP-induced defects in autophagy led to retention of NPs and consequently toxic effects (Mao et al. 2016; Rong et al. 2019; Azimee et al. 2020). Thus, 3D brain organoids that have lower LC3B-II/I ratios might be expected to have impaired ability to degrade NPs and damaged biomolecules, which could lead to NP cytotoxicity.

Findings from traditional western blotting indicated decreased LC3B-II/I levels, but did not reveal which areas were the most sensitive. For this reason, we used fluorescence MOST to show the localization of LC3B proteins. Interestingly, decreases in LC3B fluorescence were more obvious in the outer layers (which were directly exposed to ZnO NPs) than in the inner layers (which were indirectly exposed to NPs; Fig. 7). If this is true for human brains, use of traditional nerve cell cultures could lead to misleading results because of differences in nerve cell sensitivity to NPs. The difference in response between the outer and inner layers in the current study could be explained by NPs accumulating more efficiently in the outer layers, as recently shown by Sobańska et al. using MoS2 NP-exposed 3D HepG2 spheroids (Sobańska et al. 2020). Also possible is that signaling communications vary among the various types of cells that constitute the 3D organoids (Fig. 2) (Cao et al. 2021). We previously reported that MWCNTs caused endoplasmic reticulum stress-mediated cytotoxicity, but only in directly exposed lung epithelial cells and not in co-cultured endothelial cells (Chang et al. 2018). Cao et al. reported that exposing lung epithelial cells to MWCNTs had minimal effects on indirectly exposed endothelial cells (Cao et al. 2016). However, Hawkins et al. reported that Co and Cr NPs altered the autophagic flux across BeWo cell barriers and also induced DNA damage in indirectly exposed neuronal cell cultures (Hawkins et al. 2018). Bengalli et al. also observed that apical exposure of NCI-H441 cells to ZnO NPs led to endothelial activation in basal chambers (Bengalli et al. 2017). Another possibility is that the indirect effects of NPs depend on NP types, endpoints, and cell types. Our results showed that ZnO NPs decreased LC3B proteins in the directly exposed outer layers, but any such changes were minimal in the indirectly exposed inner layers. This finding is different from our recent report that MWCNTs decreased neuronal NO synthase proteins in both outer and inner layers of this 3D model (Jiang et al. 2020). This discrepancy may result from NO being able to directly diffuse into different layers of cells, whereas autophagic signaling depends on the activation of autophagic proteins, which may not be directly transferred among the different layers (Kirkin and Rogov 2019).

Another previous study proposed a model suggesting that “onion-like” outer layers of 3D cultures entrapped NPs to protect the inner layers (Chia et al. 2015). Interestingly, we found here that ZnO NPs decreased LC3B protein volume (Fig. 8) yet increased the appearance of micronuclei in the outer layers. However, these effects were less apparent in the inner layers, where LC3B protein levels were still relatively high (Fig. 9). Considering the importance of autophagy in degrading damaged proteins and organelles (Kirkin and Rogov 2019), it is possible that decreases in LC3B proteins in the outer layers lead to the cells being more sensitive to ZnO NP-induced damage, such as damage to nuclei.

To further study the potential contributions of autophagy and excessive Zn ions to the cytotoxicity of ZnO NPs, we co-exposed the 3D brain organoids with both ZnO NPs and autophagic modulators. As expected, co-exposure with autophagic inhibitor Baf A1 led to marked increases in cytotoxicity (Fig. 10A) and in levels of intracellular Zn ions (Fig. 10C). We previously reported that cyanidin chloride modestly protected Caco-2 cells from ZnO NP treatment, whereas an autophagic blocker inhibited this protective effect (Jiang et al. 2019). Similarly, we found that exposure to carbon nanotubes induced defective autophagy in human endothelial cells, and the addition of autophagic modulators affected the sensitivity of endothelial cells to the carbon nanotubes (Zhao et al. 2019). Wu et al. reported that superparamagnetic iron oxide NPs induced autophagy and promoted cellular viability, whereas inhibiting autophagy reduced cellular viability upon NP challenge (Wu et al. 2017). Hence, defective autophagy could be related to ZnO NP-induced cytotoxicity. However, we also found here that adding the autophagy inducer RAPA did not drastically affect the cytotoxicity of ZnO NPs (Fig. 10A). In contrast, a previous study reported that stimulating autophagic flux promoted the release of carbon nanotubes and, thus, attenuated carbon nanotube cytotoxicity to endothelial cells (Orecna et al. 2014), which might be explained by our finding here that RAPA decreased intracellular Zn ions only slightly (Fig. 10D). However, we did not use higher levels of RAPA because we previously showed that excessive induction of autophagy could also increase the cytotoxicity of carbon nanotubes to endothelial cells (Zhao et al. 2019).

Our study has the following potential limitations. First, during environmental exposure, ZnO NPs need to penetrate physiological barriers before entering CNS, but we cannot mimic this process by using our in vitro model. Nevertheless, many types of NMs including ZnO NPs are capable to penetrate physiological barriers, although the translocation rate is relatively low (Cao et al. 2018; Cao 2021). Second, the human brains are protected by BBB, whereas 3D brain organoids used in this study lack BBB. Recent studies developed human brain organoids with BBB (Cakir et al. 2019; Pellegrini et al. 2020), and it would be interesting to use these models to further investigate the neurotoxicity of NMs and compare the differences. Third, brain development is a very complex and largely unknown process, and the organoids used in this study may probably only reflect the toxicity of NPs to brains at early developmental stages, such as fetal brains. Brain organoids with more advanced tissue architecture and maturation, which may mimic the brains at later developmental stages, should be used in the future to evaluate the neurotoxicity of NMs to developed brains (Chiaradia and Lancaster 2020). Nonetheless, 3D brain organoids are considered as advanced model compared with conventional 2D cell cultures and can be used as the in vitro tool to investigate the neurotoxicity and the mechanisms (Cao et al. 2021).

In summary, our experiments with novel 3D brain organoids based on human iPSC demonstrated that high levels of ZnO NPs were cytotoxic and that this cytotoxicity was regulated by defective autophagy and excessive accumulation of intracellular Zn ions. We further found by fluorescence MOST technology that the responses of the autophagic marker proteins LC3B were not equal throughout the entire organoids and that decreases in LC3B proteins were associated with more micronuclei in the outer layers of ZnO NP-exposed brain organoids. Our results may provide novel insights into ZnO NP neurotoxicity to human brains. Because 3D brain organoids resemble human brains, our results may be more easily extrapolated to humans than other models, especially animal models.

Acknowledgements

The authors thank the Wuhan OE-Bio Co., Ltd for performing the fluorescence micro-optical sectioning tomography experiments and data analysis. The authors also thank Dr. Damiana Chiavolini from the Department of Radiation Oncology at UT Southwestern Medical Center as well as Christine F. Wogan, MS, ELS, from MD Anderson’s Division of Radiation Oncology for editing the manuscript. The authors also thank Milton S. Wang from Oakville Trafalgar High School for editing the TOC image.

Funding

This study is supported in part by the Cancer Center Support (Core) Grant P30 CA01667 from the National Cancer Institute, National Institutes of Health, to The University of Texas MD Anderson Cancer Center.

Abbreviations

2D

Two-dimensional

3D

Three-dimensional

AFM

Atomic force microscopy

ANOVA

Analysis of variance

Baf A1

Bafilomycin A1

BBB

Blood brain barrier

BRN2

Brain-2

CI

Confidence interval

CTIP2

Chicken ovalbumin upstream promoter-transcription factor interacting protein 2

CNS

Central nervous system

DHE

Dihydroethidium

FBS

Fetal bovine serum

HSD

Honestly significant different

ICP-MS

Inductively coupled plasma mass spectroscopy

iPSC

Induced pluripotent stem cells

MOST

Micro-optical sectioning tomography

MWCNTs

Multi-walled carbon nanotubes

NO

Nitric oxide

NPs

Nanoparticles

NPC

Neural precursor cells

PDI

Polydispersity index

RAPA

Rapamycin

SATB2

Special AT-rich sequence-binding protein 2

TBR1

T-box brain protein 1

TEM

Transmission electron microscopy

Availability of data and material

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

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

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

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