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. 2023 Nov 8;2(2):76–84. doi: 10.1021/envhealth.3c00119

Silica Nanoparticle Exposure Caused Brain Lesion and Underlying Toxicological Mechanism: Route-Dependent Bio-Corona Formation and GSK3β Phosphorylation Status

Wei Wei 1,2, Bingwei Yang 1,2, Xiangyu Zhu 1, Xuting Liu 1, Erqun Song 2, Yang Song 1,2,*
PMCID: PMC11503913  PMID: 39474100

Abstract

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When nanoparticles (NPs) enter a physiological environment, they tend to adsorb proteins to form a so-called corona. A comprehensive understanding of the effect of protein corona on NPs’ toxicity is required. Our previous study indicated that silica nanoparticles (SiO2 NPs) exposure with different routes resulted in distinct brain damage; however, an exact molecular mechanism of protein corona on the regulation of SiO2 NPs-induced damages needs further investigation. SiO2 NPs exposure via intravenous injection may encounter a protein-rich bio-matrix, which drives the adsorption of serum protein on their surface to form a stable SiO2 NPs@serum complex. On the contrary, SiO2 NPs exposure via intranasal instillation remained their original feature, due to a protein infertile environment of cerebrospinal fluid. Apparently, surface adsorption of proteinaceous substances altered inherent toxic behavior of SiO2 NPs. In addition, glycogen synthase kinase 3 beta (GSK3β) phosphorylate was found at different residues, which play an essential role in orchestrating apoptosis and autophagy threshold. Route-dependent corona formation determined GSK3β phosphorylation status and ultimately the toxic behavior of SiO2 NPs. This work presented the evidence of bio-corona on the regulation of SiO2 NPs-induced toxicity, which can be used to guide risk assessment of environmental NPs.

Keywords: silica nanoparticle, brain damage, bio-corona, serum, GSK3β

1. Introduction

The rapid development of nanoparticles (NPs) industrial production and application raised the global concern of their safety and potential detrimental effect to human beings. Due to their small size, NPs are able to translocate across the blood-brain barrier (BBB);1 meanwhile, inhaled NPs are able to migrate to the brain via the olfactory region in the nasal cavity.2−4 Therefore, their neurotoxicity is warranted.

Silica nanoparticles (SiO2 NPs), one of the most commonly produced classes of nanomaterials, exhibit high biocompatibility and stability, making them promising candidates as food additives and cosmetic products as well as in various biomedical applications.5 In 2017, the estimation of SiO2 NPs-based products consumption reached four million tons globally.6 However, the toxic potential of SiO2 NPs was proposed.6,7 Occupational exposure to SiO2 NPs is commonly associated with pulmonary diseases.8 Our recent work demonstrated SiO2 NPs inhalation resulted in cardiovascular toxic effects.9 However, relatively few studies have been conducted on the neurotoxicity of SiO2 NPs.

Here, we use intravenous injection (IVI) and intranasal instillation (INI) exposure routes to mimic biomedical applications and occupational exposure of SiO2 NPs, respectively. We were surprised to discover that SiO2 NP exposures via IVI and INI possess different toxic potential to the brain. To understand why IVI SiO2 NPs did not bring about the same toxicity outcomes, a mechanism study at the molecular level was conducted. In this study, we presented that serum constituted the IVI SiO2 NPs (but not INI SiO2 NPs) protein corona, inactivated glycogen synthase kinase 3 beta (GSK3β) through the manipulation of its phosphorylation status, and ultimately alleviated brain lesions.

This study not only provided a further neurotoxic mechanism of SiO2 NPs but also calls attention to the assessment of the current knowledge about toxicity of SiO2 NPs. In addition, we encourage researchers to consider the effect of bio-corona in the risk assessment of NPs-induced toxicities, especially in the translation of results from different experimental models, e.g., in vitro to in vivo, and vice versa.

2. Materials and Methods

2.1. Chemical and Reagents

A Cell Counting Kit-8 (CCK8) assay kit was purchased from Bimake Inc. (Houston, TX, USA). 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) and Hifair TM II 1st Strand cDNA Synthesis SuperMix were purchased from YESEN Inc. (Shanghai, China). A total protein quantitative assay kit was provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). BeyoECL Star and LY294002 were acquired from Beyotime Institute of Biotechnology (Nanjing, China). Lithium chloride (LiCl) was purchased from Aladdin Reagent Database, Inc (Shanghai, China). A total RNA purification kit was purchased from BioTeke Inc (Beijing, China). Acridine orange (AO) was obtained from Biosharp Inc (Hefei, China). Okadaic acid (OA) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Detailed information for antibodies used in this study was listed in Table S1. Other chemicals used were of the highest commercial grade.

2.2. Preparation of Fluorescent Silica Nanoparticles (SiO2 NPs)

Fluorescent SiO2 NPs were synthesized according to a previous publication.10 Triton X-100, cyclohexane, n-hexanol, and ultrapure water were mixed to form a transparent microemulsion. Methylene blue and tris (2,2′-bipyridine) dichlororuthenium(II) hexahydrate dyes were synchronously doped in the silica matrix by using hydrolysis of tetraethyl orthosilicate in a microemulsion.

2.3. Animal Experiment

Male C57BL/6 mice (8-10 weeks old, 22-25 g) were purchased from Mode Animal Centre of Nanjing University (Nanjing, China). Animals were maintained in our laboratory animal facility (22 ± 1 °C and 60 ± 10% relative humidity, 12 h light/dark cycle) with free access to food and water. All experimental procedures were approved by the Southwest University Animal Care and Use Committee (IACUC-20190310-01). Experiments were carried out in accordance with the National Institutes of Health’s guidelines for the care and use of laboratory animals. For INI, mice were treated with 20 mg/kg SiO2 NPs [suspended in phosphate buffer saline (PBS) at a concentration of 20 mg/mL] once (for silicon content assay) or daily for 28 days (damage examination). For IVI, mice were treated with 40 mg/kg SiO2 NPs at the same interval. The mice in the control group received no treatment. After 24 h of the last SiO2 NPs administration, animals were anesthetized with isoflurane. Then, mice were heart perfused with normal saline, and organs were removed for further investigation.

2.4. Cell Culture and Treatment

Mouse microglial BV2 cell line was obtained from American Type Culture Collection (ATCC, USA). Cells were maintained in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2 humidified environment. Cells were treated with 50 μg/mL SiO2 NPs or SiO2 NPs@serum for an additional 12 h. Cells in the control group received serum-free medium treatment for 12 h. For pharmaceutical inhibition assay, cells were treated with corresponding inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (30 μmol/L) or serine phosphatases inhibitor OA (10 nmol/L) or glycogen synthase kinase 3 beta (GSK3β) activity inhibitor LiCl (10 mmol/L) for 2 h prior to NPs treatment for 12 h.

2.5. Immunofluorescence Staining

For animal study, brain tissues were fixed in 4% paraformaldehyde and then cut into 3 μm-thick serial coronal sections. For cultured cells, BV2 cells were washed three times with PBS, then fixed with 4% paraformaldehyde containing 0.1% Triton X-100 for 20 min. The sections and cells were then blocked with 10% skimmed milk for 2 h at room temperature and then incubated overnight at 4 °C with primary antibodies. After rinsing with PBS, a secondary antibody Cy3 (1:300) or Alexa Fluor 488 (1:250) was applied for 2 h at room temperature. Nuclei were stained with DAPI. After being washed five times with PBS, sections and cells were observed under a confocal microscope (Nikon, N-SIME). Image analysis was performed in ImageJ software.

2.6. Tissue Silicon Content Determination

Organs were collected and immersed in a concentrated nitric acid and perchloric acid mixture (v/v = 5:1). The samples were then heated on a hot plate in a fume hood at 90 °C until the tissue was dissolved. Samples were digested with hydrofluoric acid and analyzed by using colorimetric analysis of blue molybdosilicate anion.11

2.7. Preparation of Mouse Serum

Mouse serum was collected from C57BL/6 mice freshly. Blood samples were drawn from orbit and placed in a 5 mL Eppendorf tube at 4 °C for 8 h. The upper layer of serum was centrifuged at 4000g, 4 °C for 10 min to obtain the supernatants.

2.8. Preparation of Cerebrospinal Fluid (CSF)

The mice were anesthetized by isoflurane and placed horizontally. The occipital bone to the atlas muscles were cut with iris scissors to expose the white dura mater. CSF samples were collected using a capillary tube with a tapered tip that was inserted through the exposed meninges.

2.9. Preparation of SiO2 NPs@serum and SiO2 NPs@CSF Complexes

SiO2 NPs (3 mg) were incubated with 100 μL of mice serum or CSF at 37 °C for 12 h. At the end of incubation, the suspensions were centrifuged at 10,000g for 10 min and pellets were collected. The samples were washed three times using 1 mL of PBS and re-dispersed in PBS.

2.10. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis of Protein Corona

SiO2 NPs@serum and SiO2 NPs@CSF complexes were re-dispersed in loading buffer and boiled at 95 °C for 10 min to elute and denature the coronal proteins. Isolated proteins were loaded on a 12.5% SDS-PAGE and separated by electrophoresis under reducing conditions. The gels were stained using 0.25% w/w Coomassie Brilliant Blue dispersed in 45% methanol and 10% acetic acid and destained overnight in 45% ethanol/10% acetic acid/45% H2O solution. Finally, the membranes were analyzed with Infinity Cap software.

2.11. Cell Viability Assay

A volume of 100 μL of DMEM medium and 10 μL of CCK8 reagent were added to each well and incubated at 37 °C for 2 h. The sample absorbance was measured at 450 nm using a microplate reader. Experiments were repeated 3 times independently.

2.12. Flow Cytometry Analysis for SiO2 NPs Uptake

Approximately 1 × 106 cells/well were seeded in 6-well plates overnight followed by the treatment of SiO2 NPs or SiO2 NPs@serum at 50 μg/mL for 12 h. Then, cells were washed with PBS and analyzed using a BD FACS MelodyTM flow cytometry. A total of 1 × 104 events was acquired for each sample from three independent experiments.

2.13. RNA Extraction and Real-Time Quantitative PCR (RT-qPCR)

Total RNA was extracted from brain tissue homogenates or BV2 cells using a total RNA purification kit (BioTeke, Beijing, China). The concentration of RNA was measured using a NanoDrop (Thermo Fisher Scientific, USA). Total RNA was reverse-transcribed into cDNA using Hifair TM II 1st Strand cDNA Synthesis SuperMix (Yeasen, shanghai, China). The comparative threshold cycle (Ct) value for housekeeping gene β-actin was used to normalize the loading variations. Relative gene expression was analyzed using the 2–ΔΔCt method. The primers with their sequences were listed in Table S2.

2.14. Western Blot

Total protein was extracted from mouse brain tissue or BV2 cells. Protein was separated by SDS-PAGE and transferred to poly(vinylidene fluoride) (PVDF) membranes. After they were blocked with 5% skimmed milk for 2 h at room temperature, samples were incubated with the primary antibodies at 4 °C overnight and secondary antibody for 1 h. Proteins were visualized with an electrogenerated chemiluminescence system. Protein levels were standardized by comparison with β-actin or total protein.

2.15. Acidic Vesicular Organelles (AVOs) Detection

The formation of AVOs (a morphological characteristic of autophagy) was detected by AO staining.12,13 After SiO2 NPs exposure, BV2 cells were stained with 1 μg/mL of AO at 37 °C for 20 min and washed three times with PBS. The cells were observed immediately under a confocal microscope (Nikon, N-SIME). The absorbance/emission wavelength for AVOs was 512 nm/526 nm.

2.16. Statistical Analysis

Statistical analysis was performed with SPSS 20.0 (SPSS Inc., Chicago) software and GraphPad Prism version 7.04 (GraphPad Software, La Jolla). All data were expressed as the mean and standard deviation (mean ± SD). One-way analysis of variance (ANOVA) was used followed by multiple comparisons between groups using least significant difference (LSD) post hoc test or Tukey’s multiple comparison test. A P value less than 0.05 was considered statistically significant.

3. Results

3.1. Effect of SiO2 NPs Exposure Via Different Routes on Brain Lesions

In order to achieve the same brain accumulation of brain-targeted NPs via two routes, we adjusted the treatment dosage to reach identical internal exposure dosage; we chose the dosages for INI and IVI at 20 and 40 mg/kg for the subsequent assays.14 SiNPs (50 nm in diameter) were synthesized according to a previous publication.14 A typical transmission electron microscopy (TEM) image was presented in Figure 1A. The hydrodynamic diameter and zeta potential of SiNPs were presented in Table S3. We have presented this study’s strategy in a flow chart diagram (Figure 1B). After 28 continuous days of administration, INI SiNPs and IVI SiNPs accumulated in mice hippocampus, striatum, cerebellum, and brainstem at comparable internal exposure dose (Figure 1C,D). Although comparable SiNPs biodistribution of INI and IVI SiNPs was found in the brain, the damages are different.14 As the primary immunological cells in the brain, the microglia were activated to engulf NPs when the latter first entered the brain.15 Here, we used an immunofluorescence assay to reveal the aggregation of microglia in the brain (Figure 1E,F). Compared with the control group, the number of ionized calcium binding adapter molecule 1 (IBA-1)-positive cells in the hippocampal region of the INI group and the IVI group was increased. Microglia of the INI group and the IVI group were both gathered at the damaged sites in the hippocampus; however, compared with the IVI group, the INI group showed more microglia aggregation at the damaged sites.

Figure 1.

Figure 1

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Effect of SiNPs transferred to the brain via the INI and IVI routes on microglia in brains. (A) The morphology of SiNPs was characterized by TEM. (B) Flow chart diagram of the present study. Male C57BL/6 mice received SiNPs INI (20 mg/kg) or SiNPs IVI (40 mg/kg) daily for 28 continuous days, respectively. The mice in the control group received no treatment. (C) Different brain regions distribution of SiNPs in mice. Scale bar = 50 μm. (D) Quantification of fluorescent intensity of SiNPs in the brain. (E) Brains were sectioned and double-stained with DAPI (blue) and Cy3 (red). DAPI labeled nucleus, IBA-1 labeled microglia, Cy3 was used to color microglia. Image analysis was performed in ImageJ software. Scale bar = 50 μm. (F) Quantification of fluorescent intensity of IBA-1.

3.2. SiO2 NPs Enriched Protein Corona Via IVI Route, but Not INI Route

Given that the same amount of SiO2 NPs was delivered to the brain and yet resulted in different degrees of brain damage, we hypothesized that the interfacial corona generated via different administration routes affected the toxicity of SiO2 NPs. Generally, NPs administered by IVI entered the bloodstream and encounter serum proteins before being distributed to the brain.16 NPs transferred from the bloodstream to the brain may be blocked by the BBB; however, as the hard corona was particularly stable, it was exceedingly plausible that the brain-targeting IVI SiO2 NPs carried typical serum proteins as their corona.17,18 The mechanisms by which NPs administered by the INI route entered the brain have also been extensively studied in the literature. After escaping nasal mucociliary clearance, INI-administered NPs mainly accessed the brain region through the olfactory nerve pathway,19 which started at the nasal olfactory epithelium, entered the olfactory bulb chamber, and then entered the CSF. Thus, INI-administered SiO2 NPs may contact CSF en route to the brain.

An ex vivo experiment was performed to predict the coronal compositions of the SiO2 NPs. SiO2 NPs were incubated with mouse serum and CSF, which mimicked the biological matrices that SiO2 NPs may encounter via the IVI (serum) and INI (CSF) routes, respectively. The ex vivo corona has a similar composition compared to its in vivo corona counterpart, although some fingerprint information may become untraced.20 Following centrifugation and washing to remove unbound proteins, the bound proteins were denatured and separated from SiO2 NPs using SDS-PAGE. Figure 2 clearly revealed the different patterns in coronal proteins from the two incubation fluids. Specifically, the SiO2 NPs@serum corona was characterized by a protein pattern consisting of numerous protein bands ranging from 20 to 130 kDa. The protein profile of the SiO2 NPs@serum corona resembled the serum, except that the percentage of the most predominant component in serum (∼66 kDa, albumin) was lower in the SiO2 NPs@serum corona. However, although a recent study characterized CSF-driven coronal protein components,21 our SDS-PAGE data indicated that SiO2 NPs@CSF did not adsorb an apparent amount of proteins. In addition, SiO2 NPs@serum showed higher values of hydrodynamic diameter and zeta potential compared with that of pristine SiO2 NPs. SiO2 NPs@CSF exhibited slight changes in either hydrodynamic diameter or zeta potential, compared to pristine SiO2 NPs (Table S3). This result suggested that the brain-targeting SiO2 NPs administered by the INI route may not possess a protein corona per se. In summary, these results implicated a key role of the protein corona in mitigating SiO2 NPs-induced toxicity.

Figure 2.

Figure 2

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SDS-PAGE analysis of protein corona on the surface of SiO2 NPs. SiO2 NPs were incubated with mice (A) serum or (B) CSF. SiO2 NPs@serum and SiO2 NPs@CSF were handled as described in the Methods Section. Serum, CSF, and isolated corona proteins were eluted for SDS-PAGE analysis.

3.3. Effect of Serum Constructed Bio-Corona on SiO2 NPs-Induced BV2 Cells Apoptosis and Autophagy

Next, we conducted an in vitro experiment in BV2 mouse microglial cells to explore the effect of serum constructed bio-corona on SiO2 NPs-induced cytotoxicity. As the major immunological cells in the brain, the microglia were activated to engulf NPs when the latter first entered the brain.15 Because microglia function as professional phagocytes for the central nervous system, they are widely recognized as an ideal model for in vitro neurobiological research.22 Our data also suggested microglia response to SiO2 NPs challenge in both delivery routes (Figure 1).

There was no significant difference in the cellular uptake of bare SiO2 NPs and SiO2 NPs@serum (Figure S1), which mimicked brain-targeting INI and IVI SiO2 NPs, respectively. Bare SiO2 NPs, but not SiO2 NPs@serum, caused a significant change in cell viability, compared with the control group (Figure 3A). DAPI staining showed atrophic nuclei, apoptotic bodies formation in bare SiO2 NPs-treated BV2 cells, while no apparent morphological changes for cells exposed to SiO2 NPs@serum (Figure 3B). In general, apoptotic signaling is divided into three branches, i.e., mitochondrial, death receptor, and endoplasmic reticulum-driven apoptosis, which involves several biomolecules, including Bax, Bcl-XL, caspase 3, caspase 8, caspase 9, Cytochrome c, Fas/FasL, Fadd, glucose regulatory protein 78 (GRP78), and C/EBP homologous protein (CHOP).23−25 The marker protein for autophagy is microtubule-associated protein 1A/1B-light chain 3 (LC3).26 Correspondingly, the protein and mRNA expressions of these biomolecules were confirmed. Interestingly, apoptosis was activated in bare SiO2 NPs- but not SiO2 NPs@serum-treated BV2 cells (Figure 3C–F and Figure S2); both bare SiO2 NPs and SiO2 NPs@serum induced autophagy in BV2 cells (Figure 3C,G and Figure S3).

Figure 3.

Figure 3

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Effects of naked SiO2 NPs and SiO2 NPs@serum on cytotoxicity, apoptosis, and autophagy of BV2 cells. BV2 cells were treated with 50 μg/mL of SiO2 NPs or SiO2 NPs@serum for 12 h. Cells in the control group received serum-free medium. (A) Cell viability. (B) DAPI staining (blue). The white arrows point to the apoptotic cell. (C) Western blot analysis. (D–G) RT-qPCR analysis. Data were expressed as means ± SD (n = 3).

3.4. Effect of Pristine SiO2 NPs and SiO2 NPs@serum on GSK3β Phosphorylation Status

NPs may acquire a personalized protein corona, which provided a new biological identity.27 We therefore hypothesized that a specific protein governed both apoptotic and autophagic responses to SiO2 NPs exposure. GSK3β was a protein kinase that participated in diverse physiological processes, encompassing a wide range of roles from cell survival to cognition.28 It was surprising to note the multifaceted effects of GSK3β in increasing or decreasing the cellular apoptotic threshold28 as well as its important role in autophagy modulation.29 GSK3β was highly abundant in the brain, while dysregulation of GSK3β activity was involved in various brain diseases.30−32 For this matter, the effects of bare SiO2 NPs and SiO2 NPs@serum on the activities of PI3K/AKT and GSK3β were investigated. However, the total protein (Figure 4A) and mRNA (Figure 4B–D) expression levels of PI3K, AKT, and GSK3β were not regulated by bare SiO2 NPs nor SiO2 NPs@serum.

Figure 4.

Figure 4

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Effect of naked SiO2 NPs and SiO2 NPs@serum on GSK3β phosphorylation in BV2 cells. BV2 cells were treated with 50 μg/mL of SiO2 NPs or SiO2 NPs@serum for 12 h. Cells in the control group received serum-free medium treatment. (A) Western blot analysis. (B–D) RT-qPCR analysis. Data were expressed as means ± SD (n = 3).

The GSK3β activity is highly dependent on the phosphorylation status of its amino acid residues. When cells received abnormal external stimuli, the upstream kinases in cells orchestrated GSK3β phosphorylation. Specifically, the phosphorylation of Ser9 inhibited GSK3β activity, while the phosphorylation of Tyr216 stimulated GSK3β activity.33 In addition, PI3K/AKT inactivated GSK3β by phosphorylating the Ser9 residue of GSK3β.28,34 To our surprise, the results showed decreased p-PI3K, p-AKT, and p-Ser9 GSK3β levels and increased p-Tyr216 GSK3β levels in the bare SiO2 NPs group; in contrast, this effect was absent in the SiO2 NPs@serum group (Figure 4A). The higher caspase 3 cleavage in the bare SiO2 NPs group (shown in Figure 3C) correlated with its p-Ser9/p-Tyr216 GSK3β ratio.28,34

Autophagy can function to promote apoptosis, which can lead to cell death in a cooperative manner; however, autophagy is also able to block apoptotic cell death by promoting cell survival.35 A previous study demonstrated that inactivation of GSK3β led to enhanced autophagy.36 To this end, we regulated the active state of GSK3β through different inhibitors: the PI3K inhibitor LY294002, serine inhibitor OA, and GSK3β activity inhibitor LiCl (Figure 5 and Table S4). Clearly, these inhibitors showed no effect on total GSK3β expression (both protein and mRNA levels), but GSK3β phosphorylation status was largely regulated. In general, these inhibitors abrogated pristine SiO2 NPs, and SiO2 NPs@serum caused irregular GSK3β phosphorylation as well as imbalanced apoptosis and autophagy. Together, through the manipulation of p-Ser9 and p-Tyr216 GSK3β levels, we concluded that GSK3β was an essential component in the regulation of SiO2 NPs-induced apoptosis and autophagy.

Figure 5.

Figure 5

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Effects of three inhibitors on GSK3β phosphorylation, apoptosis, and autophagy in SiO2 NPs and SiO2 NPs@serum-challenged BV2 cells. BV2 cells were treated with corresponding inhibitors of LY294002 (30 μM), OA (10 nM), or LiCl (10 mM) for 2 h, then cells were treated SiO2 NPs and SiO2 NPs@serum for 12 h. Cells were harvested for (A–C) Western blot analysis and (D–F) RT-qPCR analysis. Data are expressed as means ± SD (n = 3).

Of note, microglia can be dramatically different based on their location in the brain. Thus, it is unfeasible to directly translate in vitro cell culture results to an in vivo animal study. Thus, parallel in vivo and in vitro studies were conducted to investigate the role of coronal proteins. The effect of GSK3β on SiO2 NPs-mediated apoptosis was reinforced in an animal study, where markedly decreased levels of p-Ser9 and increased levels of p-Tyr216 GSK3β occurred in the INI group without affecting the total protein and mRNA expression (Figure 6). The effects of SiO2 NPs and SiO2 NPs@serum on the GSK3β activity of BV2 cells were the same as those of INI SiO2 NPs and IVI SiO2 NPs on the GSK3β activity in vivo. Moreover, the IVI groups showed an insignificant effect on the regulation of GSK3β phosphorylation.

Figure 6.

Figure 6

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Effect of naked SiO2 NPs and SiO2 NPs@serum on GSK3β phosphorylation in mice. Male C57BL/6 mice received INI of SiO2 NPs (20 mg/kg) or IVI (40 mg/kg) daily for continuous 28 days. The mice in the control group received no treatment. Brain tissues were collected. (A) Western blot analysis. (B–D) RT-qPCR analysis. Data were expressed as means ± SD (n = 3).

4. Discussion

Curiously, in vitro nanotoxicity results appear to be more severe when compared to their corresponding in vivo results.37 While there are many possible reasons for this divergence, the protein corona hypothesis affords one accepted explanation that surface adsorption of proteinaceous substances masks the underlying toxicity and dictates the biological fate of NPs.38−44 However, the lack of non-coronal, toxic NPs as an essential in vivo control makes this, a solution without a well-constructed problem.

Here, we introduced SiO2 NPs via the IVI and the INI routes, where the latter entailed a less protein-rich in vivo environment. We found that the SiO2 NPs administered via the INI route resulted in more activation and aggregation of microglia in brain compared to the IVI route. In parallel, the total protein content in SiO2 NPs incubated in mouse CSF was negligible compared to that of SiO2 NPs incubated in mouse serum, suggested that the INI-introduced SiO2 NPs were essentially naked, compared to the IVI-introduced SiO2 NPs.

To understand why IVI SiO2 NPs did not cause apoptosis, we found SiO2 NPs enriched protein corona in blood which inactivated GSK3β and ultimately alleviated microglia apoptosis. In contrast, the bare SiO2 NPs activated GSK3β and caused microglia apoptosis. The whole protein corona thus masked the toxicity of the underlying SiO2 NPs, and the corona further protected against any potential apoptosis-driven brain toxicities of the NPs. This result revealed brain toxicity elicited by non-coronal INI SiO2 NPs and its mitigation by the protein corona associated with IVI SiO2 NPs.

5. Conclusions

The implications of this study are multifold. First of all, the findings suggested that the route of administration/exposure had a remarkable impact on the biological identity of engineered NPs. This study highlighted opportunities in exploiting the bio-circulation and hence the protein corona of NPs for enabling their biological applications. Secondly, as far as we know, this study delineated the NP-brain bio-corona, demonstrating that the presence of a brain bio-corona yielded new biological properties on NPs at the molecular level, while weakening brain toxicity and brain damage induced by NPs. Thirdly, this study provided a control of toxic NPs in the body that has been missing in protein corona-nanotoxicity models, which helped to elucidate the role of specific corona components on the risk assessment of nano-neurotoxicity.

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its Supplementary Information files.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/envhealth.3c00119.

  • Figure S1. Cellular uptake of SiO2 NPs and SiO2 NPs@serum; Effects of naked SiO2 NPs and SiO2; Figure S2. NPs@serum on three branches of apoptotic signaling; Figure S3. Effects of naked SiO2 NPs and SiO2 NPs@serum on autophagic signaling; Table S1. Details of primary and secondary antibodies; Table S2. Sequence of all primers used in the RT-qPCR experiment; Table S3. Physical characterization of hydrodynamic diameter and zeta potential by DLS; Table S4. Effects of p-Ser9/p-Tyr216 GSK3β on 50 nm SiO2 NPs or SiO2 NPs@serum induced autophagy and apoptosis (PDF)

This work is supported by National Natural Science Foundation of China (22176206, 21976145, 22174116, and 21974110).

The authors declare no competing financial interest.

Supplementary Material

eh3c00119_si_001.pdf (484.1KB, pdf)

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Supplementary Materials

eh3c00119_si_001.pdf (484.1KB, pdf)

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All data generated or analyzed during this study are included in this published article and its Supplementary Information files.

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