Neurotoxicity of four frequently used nanoparticles: a systematic review to reveal the missing data

Abstract

Systemic exposure to nanoparticles (NPs) adversely affects different organs, including the nervous system. We systematically extracted data from publication on PubMed and Embase database up to the year 2020, and analyzed in vitro and in vivo neurotoxicity of 4 of the most well studied NPs (silver NPs, carbon-based NPs, iron NPs and silica NPs). A relatively good correlation was observed between in vitro and in vivo effects, including genotoxicity, oxidative stress, apoptosis and pro-inflammatory effects. However, crucial knowledge gap exists in current understanding of the underlying mechanisms. Some of the critical knowledge gaps and research needs identified in relation to neurotoxicity of nanoparticles include (1) lack of physio-chemical characteristics of NPs used, (2) cellular/tissue uptake of NP, (3) NP translocation across the blood-brain barrier (BBB), (4) Effect of exposure routes.

Appendices

Appendix

In vitro result

Cellular uptake

AgNPs

Pristine AgNPs (6.5 nm, 10 µg/ml) were taken up by rat primary BME cells, astrocytes and pericytes in triple culture (Xu et al. 2015a). PVP-coated AgNPs of different sizes (23–102 nm) at 1–50 µg/ml were internalized by rat primary BME cells and rat astrocytes (Sun et al. 2016; Trickler et al. 2010). The internalization of citrate-coated AgNPs (49.7 nm, 50 µg/ml) was observed in N9 cells, a mouse microglia-derived cell line (Gonzalez-Carter et al. 2017).

Carbon-based NPs

Graphene oxide (50 µg/ml) was taken up by PC12 cells time-dependently (1–24 h) (Feng et al. 2018). Different types of MWCNTs at 10–30 µg/ml were internalized by neurons and mixed glia cells (Bussy et al. 2015; Visalli et al. 2017; Meng et al. 2013; Phillips et al. 2013). The internalized MWCNTs were found in cytoplasm, vacuoles, endosome, lysosome and nucleus.

IONPs

Fe₃O₄ NPs were internalized and found in cytoplasm and the perinuclear area of PC12 cells (Liu et al. 2018). O-IONPs were internalized by human astrocytes (Fernández-Bertólez et al. 2019). Cellular uptake of O-IONPs in human neurons was dose- (10–200 µg/ml) and time- (3–24 h) dependent (Fernández-Bertólez et al. 2018). Monocultures of D384 and SH-SY5Y cells and co-cultures/3D cultures of SH-SY5Y and D384 cells treated with PVP-coated Fe₃O₄ NPs showed dose- (1–100 µg/ml) and time- (24–48 h) dependent cellular uptake (Simone et al. 2018). SPIONs at 214 µg/ml were internalized by PC12 cells (SCoEaNIHR ES 2010). Uptake of MIRB by primary mouse neurons was also detected by FITC microscopy (Badman et al. 2020).

SiNPs

SiO₂ NPs (12.1–25 nm, 10–200 µg/ml) were internalized by SK–N–SH cells and PC12 cells and found in cytoplasm or vacuoles (Yang et al. 2014; Xie and Wu 2016). Amorphous SiO₂ NPs (100 µg/ml) were internalized via macropinocytosis and found in cytoplasm of primary BME cells (Liu et al. 2017). Internalization of FITC-labeled Stöber SiO₂ NPs of different sizes (46–306 nm) by microglia cells and BME cells was size- and dose- (100–400 µg/ml) dependent. Neurons barely took up these NPs (Du et al. 2019; Sun et al. 2017). RuBpy-doped SiO₂ NPs at 0.1 µg/ml were taken up by rat primary hippocampal neurons and found in endolysosomes (Ye et al. 2019).

Oxidative stress

AgNPs

Oxidative stress was induced by AgNPs (18.3 nm, 10 µg/ml), AgNPs (20 nm, 1 µg/ml), GSH-coated AgNPs (7.5 nm, 1 µg/ml) and green tea-coated AgNPs (12.5 nm, 1 µg/ml) in rat PC12 cells and NF-κB reporter gene-transfected rat N27 cells (Wang et al. 2009; Baruwati et al. 2013). Exposure to PVP-coated AgNPs of different sizes (15–23.18 nm) resulted in dose- (0.01–20 µg/ml) and time- (0.5–24 h) dependent oxidative stress responses in human ESCs, rat NSCs and rat primary astrocytes (Sun et al. 2016; Begum et al. 2016). PVP-coated (15 nm) AgNPs at 75 µg/ml also induced oxidative stress in rat primary cerebellar granule cells (Ziemińska et al. 2014). Citrate-coated AgNPs of different sizes (7.9–20.3 nm) induced a dose-dependent (0.1–50 µg/ml) oxidative stress response in human ESCs (Oh et al. 2016; Sun et al. 2016; Begum et al. 2016), with no oxidative stress observed when citrate-coated AgNPs of different sizes (34.08–49.7 nm, 1–50 µg/ml) were applied to mouse ESCs and mouse microglia cells (Yin et al. 2018; Gonzalez-Carter et al. 2017).

Carbon-based NPs

Different types of MWCNTs induced dose-dependent (5–50 µg/ml) nitric oxide (NO) production in primary mixed glia cells and increased reactive oxygen species (ROS) levels in retinoid acid (RA)-differentiated SH-SY5Y cells (Bussy et al. 2015; Visalli et al. 2017). Long (20 µm) SWCNTs (5–400 µg/ml) increase ROS production, lipid peroxidation, glutathione (GSH) depletion and decreased superoxide dismutase, catalase and glutathione peroxidase (GSH-Px) levels in PC12 cells (Wang et al. 2011).

IONPs

Fe₃O₄ NPs (30 nm) dose-dependently (25–200 µg/ml) induced oxidative stress in PC12 cells (Wu et al. 2013), but 45 nm of Fe₃O₄ NPs at 250–500 µg/ml did not produce oxidative stress in rat primary microglia cells (Xue et al. 2012). IONPs (10.8, 30.1 nm) in SH-SY5Y cells and rat primary BME cells dose-dependently (0.1–10 µg/ml) induced oxidative stress (Imam et al. 2015). PMA-coated IONPs dose-dependently (7–140 nM) induced oxidative stress in mNSCs and ReNcell VM, LAN-N-2 and Neuron-2a cells and dose-dependently (7–140 nM) decreased oxidative stress in hNSCs and C17.2 cells (Joris et al. 2017). DMSA-coated IONPs dose-dependently (7–140 nM) increased oxidative stress in hNSCs and ReNcell VM, LAN-N-2 and Neuron-2a cells and dose-dependently (7–140 nM) decreased oxidative stress in mNSCs and C17.2 cells (Joris et al. 2017). No obvious oxidative stress was observed after exposure of primary rat neurons to MIRB (Badman et al. 2020).

SiNPs

SiO₂ NPs (12.1–15 nm) dose-dependently (2.5–200 µg/ml) increased the oxidative stress response in rat, mouse and human neurons (Wu et al. 2011; Yang et al. 2014), but 20 nm of SiO₂ NPs at 250–500 µg/ml did not induce oxidative stress in rat primary microglia cells (Xue et al. 2012). Exposure to all sizes of Stöber SiO₂ NPs (46–306 nm) at 50 µg/ml induced oxidative stress in microglia cells (Du et al. 2019). Amorphous SiO₂ NPs (100 µg/ml) induced oxidative stress in human primary BME cells (Liu et al. 2017). Different types of mesoporous SiO₂ NPs induced oxidative stress in NGF-treated PC12 cells (Zhou et al. 2016). LUDOX^® TM SiO₂ NPs at 100 µg/ml did not induce oxidative stress in SH-SY5Y or RA-induced SH-SY5Y cells (Kim and Yang 2011).

Pro-inflammatory responses

AgNPs

Exposure to different-sized PVP-coated AgNPs (1–50 µg/ml) induced secretion of cytokines, including CINC-2a/b, CINC-3, fratalkine, L-selectin, thymus chemokine, prostaglandin E2 (PGE2), IL-10, IL-1β, IP-10 and TNF-α, in rat primary astrocytes and rat primary BME cells (Sun et al. 2016; Trickler et al. 2010). Citrate-coated AgNPs at 50 µg/ml reduced lipopolysaccharide-induced TNF-α release in mouse microglia cells (Gonzalez-Carter et al. 2017).

Carbon-based NPs

Levels of TNF-α, IL-6 and IL-8 were increased in RA-treated SH-SY5Y cells after exposure to 12.5 µg/ml pristine and carboxyl-functionalized MWCNTs (Visalli et al. 2017).

IONPs

Fe₃O₄ NPs at 500 µg/ml induced TNF-α, IL-6 and IL-1β secretion in rat primary microglia cells (Xue et al. 2012).

SiNPs

SiO₂ NPs (20 nm) at 500 µg/ml induced TNF-α, IL-6 and IL-1β release in rat primary microglia cells (Xue et al. 2012). Stöber SiO₂ NPs (50 nm) at 100 µg/ml induced IL-1β secretion from mouse primary microglia cells and a macrophage cell line (J744A.1) (Du et al. 2019).

Morphology

Pristine AgNPs at 1–10 µg/ml commonly induced mitochondrial shrinkage in triple cultures of rat primary BME cells, astrocytes and pericytes (Xu et al. 2015b). PVP-coated AgNPs of different sizes induced cell perforation and dose-dependently (0.1–25 µg/ml) inhibited neurite outgrowth in rat primary BME cells and human ESCs (Trickler et al. 2010; Begum et al. 2016). Human ESCs treated with citrate-coated AgNPs showed a similar dose-dependent (0.1–50 µg/ml) reduction in neurite outgrowth and cell detachment and a round/irregular cell shape (Oh et al. 2016; Begum et al. 2016).

Carbon-based NPs

Different types of MWCNTs at 30 µg/ml induced organelle damage in PC12 and NGF-induced PC12 neurons (Meng et al. 2013). The PC12 neurons also tended to adhere to and grew on the MWCNTs (Phillips et al. 2013). Apoptotic markers were observed after PC12 neurons were exposed to SWCNTs (20 µm) at 100–400 µg/ml (SCoEaNIHR ES 2010).

IONPs

Fe₃O₄ NPs (17.9 nm) and n-octyltriethoxysilane-coated Fe₃O₄ NPs (18.7 nm) at 6–98 µg/ml both induced shape changes and inhibited neurite outgrowth in ReNcell VM cells (Ma et al. 2019). However, Fe₃O₄ NPs or n-octyltriethoxysilane-coated Fe₃O₄ NPs at 6–98 µg/ml induced no significant morphological changes in PC12 cells. PVP-coated Fe₃O₄ NPs at 1–100 µg/ml induced changes in cell shape and reduced cell density when applied to D384 cells in monoculture, in 3D culture or when co-cultured with SH-SY5Y cells (Simone et al. 2017; Simone et al. 2018). SH-SY5Y cells in 3D culture disaggregated in the presence of PVP-coated Fe₃O₄ NPs at 1–100 µg/ml. PMA-coated IONPs (3.5–70 nM) induced contractions in cell size and morphological changes in hNSCs, mNSCs and ReNcell VM, C17.2, Neuro-2a and LA-N-2 cells (Joris et al. 2017). DMSA-coated IONPs (3.5–70 nM) produced similar results in hNSCs, mNSCs and ReNcell VM, C17.2, Neuro-2a, and LA-N-2 cells (Joris et al. 2017). In contrast, DMSA-coated IONPs (2 mM) induced no observable morphological changes in rat primary cerebellar granule neurons (Petters and Dringen 2015).

SiNPs

Different types of pristine SiO₂ NPs (12.1–15 nm) at 2.5–200 µg/ml induced morphological changes, inhibition of neurite outgrowth and cell detachment in cultures of mouse neuro-2 rat PC12 cells (Wu et al. 2011; Yang et al. 2014). Stöber SiO₂ NPs (200 µg/ml) of different sizes (46–306 nm) induced a pyroptotic cell morphology in a microglia cell line, a primary microglia cell line and a macrophage cell line (Du et al. 2019). SH-SY5Y cells detached and became more irregularly shaped in the presence of LUDOX^® TM SiO₂ NPs (1.5 nm, 100 µg/ml) (Kim and Yang 2011). However, neurite outgrowth in RA-treated SH-SY5Y cells was not altered by exposure to LUDOX^® TM SiO₂ NPs (Kim and Yang 2011). Exposure to 100–400 µg/ml FITC-Stöber SiO₂ NPs induced disruption of the plasma membrane, vacuolated cytoplasm, damaged organelles and apoptotic cell morphology in C17.2 neurons (Sun et al. 2017).

Cytotoxicity and cell death

AgNPs

PVP-coated AgNPs of different sizes dose-dependently (0.01–80 µg/ml) induced cytotoxicity in human ESCs, human NSCs and rat NSCs and primary astrocytes (Liu et al. 2015; Begum et al. 2016). In human BME cells, cytotoxicity was induced time-dependently (24–48 h) with NPs applied at 10 µg/ml, whereas in rat primary BME cells size-dependent cytotoxicity was induced with NPs applied at 50 µg/ml (Trickler et al. 2010; Khan et al. 2019). Cytotoxicity was also induced by PVP-coated AgNPs (15 nm, 10 µg/ml) in rat primary cerebellar granule cells (Ziemińska et al. 2014). Citrate-coated AgNPs of different sizes (8–34 nm) dose- (0.1–200 µg/ml) and time-dependently (6–24 h) induced cytotoxicity in human ESCs (Begum et al. 2016). In contrast, no cytotoxicity was observed in mouse ESCs and mouse microglia cells with citrate-coated AgNPs (34–50 nm) at 1–50 µg/ml (Yin et al. 2018; Gonzalez-Carter et al. 2017). Citrate-coated (20 and 110 nm) and PVP-coated (110 nm) gold core AgNPs at 50 µg/ml induced cytotoxicity in mouse primary midbrain cells (Weldon et al. 2018).

PVP-coated AgNPs of different sizes (23–23.18 nm, 5–10 µg/ml) induced apoptosis in rat primary astrocytes and NSCs (Sun et al. 2016; Liu et al. 2015). Exposure to citrate-coated AgNPs (7.9 nm, 25–50 µg/ml) time-dependently (6–24 h) induced apoptosis in human ESCs (Oh et al. 2016).

Carbon-based NPs

Graphene dose-dependently induced cytotoxicity (10–100 µg/ml) in rat BME cells (Rosas-Hernandez et al. 2019). Graphene oxide dose- (5–60 µg/ml) and time-dependently (6–24 h) induced cytotoxicity in rat neurons. Carbon black NPs induced cytotoxicity in umbilical vein endothelial cells (14 nm, 100 µg/ml) but had no cytotoxic effect on primary astrocytes (19 nm, 128 µg/ml) (Zhang et al. 2019). PC12 cells exposed to different types of SWCNTs exhibited dose-dependent cytotoxicity (0.01–600 µg/ml) (Wang et al. 2011; Zeinabad et al. 2016). Different types of MWCNTs (pristine, high iron impunities, carboxyl functionalized) dose- (0.01–200 µg/ml) or time-dependently (6–72 h) induced cytotoxicity in PC12 and SH-SY5Y cells (Zeinabad et al. 2016; Phillips et al. 2013). MWCNTS (pristine or functionalized) induced cytotoxicity in rat primary mixed glia cells, related to the brain region from where cells were isolated (Bussy et al. 2015).

Different types of MWCNT at 10–66.5 µg/ml induced apoptosis in primary mixed glia cells or necrosis in PC12 cells (Bussy et al. 2015; Zeinabad et al. 2016). Different types of SWCNTs at 22.7–100 µg/ml induced apoptosis in PC12 cells (Wang et al. 2011; Zeinabad et al. 2016). Graphene oxide at 40–60 µg/ml induced autophagy in PC12 cells (Feng et al. 2018).

IONPs

O-IONPs dose-dependently (5–200 µg/ml) induced cytotoxicity in human astrocytes and neurons (Fernández-Bertólez et al. 2018; Fernández-Bertólez et al. 2019). PVP-coated Fe₃O₄ NPs dose- (1–100 µg/ml) and time-dependently induced cytotoxicity in 3D cultures of D384 and SH-SY5Y cells, monocultures of D384 and SH-SY5Y cells and D384 and SH-SY5Y co-cultures (Simone et al. 2017, 2018). Repeated dosing of PVP-coated Fe₃O₄ NPs dose-dependently (0.1–25 µg/ml) induced cytotoxicity in both 3D cultures of D384 and SH-SY5Y cells. Lysosomal-targeted IONPs (200 µg/ml) but not mitochondria-targeted IONPs or pristine IONPs induced cytotoxicity in SH-SY5Y cells (Huang et al. 2019). PMA-coated IONPs dose-dependently (3–140 nM) induced cytotoxicity in hNSCs, mNSCs, and ReNcell VM, C17.2, LAN-N-2 and Neuron-2a cells (Joris et al. 2017). Exposure to DMSA-coated IONPs dose-dependently (3–140 nM) induced cytotoxicity in hNSCs, mNSCs and ReNcell VM and LAN-N-2 cells (Joris et al. 2017). DMSA (2 mM)-coated IONPs did not induce cytotoxicity but rather delayed cytotoxicity in rat primary cerebellar granule neurons (Petters and Dringen 2015).

Fe₃O₄ NPs of different sizes dose-dependently (25–200 µg/ml) induced apoptosis in PC12 cells (Fernández-Bertólez et al. 2018; Wu et al. 2013). Exposure to 10.8 nm IONPs at 2.5–40 µg/ml triggered apoptosis in SH-SY5Y cells (Imam et al. 2015). In contrast, exposure to 30.1 nm IONPs did not induce apoptosis in SH-SY5Y cells (Imam et al. 2015). MIRB (10 and 20 µg/ml) after 24-h exposure induced significant cytotoxicity in primary mouse neurons (Badman et al. 2020).

SPIONs (214 µg/ml) induced apoptosis in PC12 cells (Sun et al. 2015). O-IONPs dose-dependently (5–200 µg/ml) induced apoptosis in human astrocytes and human neurons (Fernández-Bertólez et al. 2018; Fernández-Bertólez et al. 2019).

SiNPs

Stöber SiO₂ NPs size- (46–306 nm) and dose- (25–200 µg/ml) dependently induced cytotoxicity in microglia cells and macrophages (Du et al. 2019). Stöber SiO₂ NPs (46 nm) at 100 µg/ml also induced cytotoxicity in primary microglia cells (Du et al. 2019). In monocultures of human primary BME cells, amorphous SiO₂ NPs dose-dependently (25–800 µg/ml) induced cytotoxicity (Liu et al. 2017). In co-cultures of human primary BME cells and human primary astrocytes, exposure to amorphous SiO₂ NPs dose-dependently (25–200 µg/ml) induced cytotoxicity in only BME cells. LUDOX^® TM SiO₂ NPs dose-dependently (10–1000 µg/ml) induced cytotoxicity in SH-SY5Y and RA-induced SH-SY5Y cells (Kim and Yang 2011). Different types of mesoporous SiO₂ NPs (266–498.4 nm) induced cytotoxicity dose-dependently (12.5–100 µg/ml) (Zhou et al. 2016). Different sizes of SiO₂ NPs (15–68 nm) all dose-dependently (0.01–200 µg/ml) induced cytotoxicity in rat primary hippocampal neurons, rat PC12 cells and mouse neuro-2a and human SK-N-SH cells (Wu et al. 2011; Ye et al. 2019).

Different sizes of SiO₂ NPs (12.1–15 nm) dose-dependently (2.5–200 µg/ml) induced apoptosis in rat, mouse and human neuronal cell lines (Wu et al. 2011; Yang et al. 2014). SiO₂ NPs (25 nm) at 25 to 200 µg/ml induced autophagy in rat neurons (SCoEaNIHR ES 2010). Stöber SiO₂ NPs size- (46–306 nm) and dose-dependently (25–200 µg/ml) induced pyroptosis in mouse microglia cells (Du et al. 2019). Exposure to LUDOX^® TM SiO₂ NPs induced apoptosis in both SH-SY5Y and RA-treated SH-SY5Y cells (Kim and Yang 2011). FITC-Stöber SiO₂ NPs dose-dependently (100–400 µg/ml) induced apoptosis in microglia.

Genotoxicity

AgNPs

PVP-coated (23 nm) AgNPs at 5 µg/ml induced DNA damage in rat ESCs (Liu et al. 2015).

Carbon-based NPs

Pristine and carboxyl-functionalized MWCNTs at 12.5–25 µg/ml induced DNA damage in RA-treated SH-SY5Y cells (Visalli et al. 2017). SWCNTs dose-dependently (50–100 µg/ml) induced cell cycle arrest (G2/M phase) in PC12 cells (Wang et al. 2011).

IONPs

O-IONPs dose-dependently (5–100 µg/ml) induced DNA damage and cell cycle arrest (S phase) in human astrocytes (Fernández-Bertólez et al. 2019). O-IONPs induced DNA damage and dose-dependent (10–200 µg/ml) cell cycle arrest (G0/G1 phase) in human neurons (Fernández-Bertólez et al. 2018). Fe₃O₄ NPs (30 nm) at 25–200 µg/ml induced cell cycle arrest (G2/M phase) in PC12 cells (Wu et al. 2013).

SiNPs

Exposure to SiO₂ NPs (15 nm, 25–200 µg/ml) induced cell cycle arrest (G2/M phase) in NGF-treated PC12 cells (Wu et al. 2011). FITC-Stöber SiO₂ NP exposure induced cell cycle arrest (G2/M phase) in mouse microglia as shown by a dose-dependent decrease in proliferation rate (Sun et al. 2017). Exposure to LUDOX^® TM SiO2 NPs at 100 µg/ml induced DNA damage and cell cycle arrest in SH-SY5Y cells (G0/G1 phase) and RA-treated SH-SY5Y cells (G2/M phase) (Kim and Yang 2011).

In vivo results

Distribution of NPs into the nerve system

AgNPs

Sprague–Dawley (SD) rats (male/female) orally administered pristine AgNPs (6 nm; 1 and 10 mg/kg body weight [bw]t/day for 14 days) showed elevated Ag level in blood and a dose-dependent accumulation of Ag in the brain (Xu et al. 2015a). Ag level was not significantly elevated in the forebrain of male Wistar rats administered citrate-coated AgNPs (10 nm) via oral gavage (0.2 mg/kg bw/day for 14 days). TEM revealed elevated Ag level in the blood and nanosized dark granules in the hippocampus, which could be AgNPs (Skalska et al. 2015). IP-administered PVP-coated AgNPs in Male Wistar rats (14.3 nm; 200 µg/per rat/day, daily for 5 days) were found in cytoplasm and nuclei of hippocampal neurons (Ghooshchian et al. 2017). Ag level was elevated in the cerebellum of SD rats (male/female) after 14 weeks daily of NI of citrate-coated AgNPs (20 nm; 0.1, 0.2, 0.5, 1 mg/kg bw) (Yin et al. 2015). Fatemi et al. investigated possible prenatal neurotoxicity by exposing pregnant Wistar rats to citrate-coated AgNPs (20 nm; 25 mg/kg bw/day) starting from day 9 of gestation until the day of delivery. Ag level was elevated in the brain of pups (Fatemi et al. 2013).

Carbon-based NPs

No biodistribution of carbon-based NPS to the CNS or peripheral nervous system (PNS) was assessed in the studies reviewed.

IONPs

Fe level was increased dose-dependently in serum, frontal cortex, hippocampus and cerebellum in male albino (Mus musculus) mice orally administered Fe₂O₃ NPs (45 nm; 25, 50 mg/kg bw/day for 30 days) (Manickam et al. 2019). Fe level was also increased in serum, frontal cortex, hippocampus and cerebellum of Swiss albino mice (male/female) orally administered Fe₂O₃ NPs (50 nm) for 30 days (25, 50 mg/kg bw/day) (Manickam et al. 2018). Male CD ICR mice with NI of Fe₂O₃ NPs (21 nm) for 30 days (130 µg/per animal/every 2 days) had detectable levels of NPs in neurons of axons in the olfactory bulb and hippocampal mitochondria and lysosome (Wang et al. 2009). With NI of ¹²⁵I-radiolabeled Fe₃O₄ NPs (36 nm; 20 µg/per male SD rat/day for 1 and 7 days), NPs were observed in the olfactory bulb, striatum, hippocampus, brain stem and cerebellum; half of the NPs still remained in striatum and hippocampus after 14 days of administration. Higher Fe content was related to longer exposure (Wu et al. 2013). In male SD rats, IV-injected IONPs (10.8 nm, 50 mg/kg bw) translocated to the brain after 1 h (Imam et al. 2015). Wistar rats sacrificed 1, 7 or 21 days after IP administration of IONPs (62.5 nm; 2, 14 mg/kg bw) showed a dose-dependent increase in Fe level in the cerebral cortex. Prussian blue staining was positive at blood vessel intima and in macrophages of the soft meninges in the high-dose groups at both 1 day and 7 days’ post-injection (Sheida et al. 2017). Kim et al. investigated neurotoxicity in the PNS by injecting (iron concentration at 0.5, 15 mM) DMSA-coated Fe₂O₃ NPs (9 nm) into the sciatic nerve of female SD rats. NPs were accumulated at 48 h post-administration (Kim et al. 2013).

SiNPs

After NI of ¹²⁵I-radiolabeled SiO₂ NPs (20 µg/per rat/day for 1 and 7 days), accumulation of particles was time-dependently increased in the olfactory bulb, striatum, hippocampus, brain stem, cerebellum and frontal cortex of SD rats (male/female) (Wu et al. 2011). Yamashita et al. IV-injected cyanine dye (DY-676)-labeled SiO₂ NPs (70, 300, 1000 nm), carboxyl-functionalized SiO₂ NPs (70 nm) and amine-functionalized SiO₂ NPs (70 nm) in pregnant BALB/c mice (800 µg/per mouse); all types of SiO₂ NPs accumulated in liver, whereas only 70 nm-SiO₂ NPs were also found in placenta (regardless of whether they were functionalized or not) (Yamashita et al. 2011).

Histopathology and ultrastructural change

AgNPs

Female SD rats showed shrinkage of neurons and swelling of both astrocytes and extravascular lymphocytes after oral administration of pristine AgNPs (6 nm; 1 mg/kw bw/day for 7 days) (Xu et al. 2015a). The forebrain of male Wistar rats showed myelin damage after oral administration of citrate-coated AgNPs (10 nm) (Dąbrowska-Bouta et al. 2016). Orally administered citrate-coated AgNPs (10 nm; 0.2 mg/kg bw/daily for 14 days) induced synaptic vesicles, meylin-like bodies, swollen synapses and blurred synaptic cleft in the forebrain and hippocampus of male Wistar rats (Skalska et al. 2015).

NI of citrate-coated AgNPs (20 nm) induced a loosen and separated granular layer with Purkinje cell deficiency in the cerebellum of SD rats (male/female) (Yin et al. 2015). The cerebrum of C57BL/6 mice (male/female) showed no significant histopathological change after inhalation administration (1.919 × 10⁷ particle number/m³, 6 h/day for 5 days/week for 2 weeks) of pristine AgNPs (22.18 nm) (Lee et al. 2010). Demyelination and BBB leakage were induced after IP, IV, ICA and IB injection of pristine AgNPs (55 nm). (Sharma et al. 2009).

Carbon-based NPs

The cerebral cortex of C57BL/6J mice (male/female) with NI of Printex 90 carbon black NPs (14 nm; 21, 103, 515 µg/per mouse/1 h/day for 10 days) showed decreased neuronal numbers (indicated as a reduction in microtubule-associated protein 2 staining), demyelination and fewer and shallower Nissl bodies (Zhang et al. 2019). IP-injected MWCNTs (diameter 15 nm, length 2 µm; 2.5 mg/kg/bw/day for 14 days) induced loosen and disordered hippocampal CA1 neurons in the brain of male Wistar rats (Gao et al. 2015). Prenatal neurotoxicity was measured by exposing pregnant ICR mice to NI of Printex 90 carbon black NPs (2.9, 15 or 73 μg/kg bw/day, on days 5 and 9 of gestation). The cerebral cortex of pups showed a dose-dependent increase in aquaporin-4 level (Onoda et al. 2017).

IONPs

The frontal cortex, hippocampus and cerebellum of male albino mice (M. musculus) showed demyelination and mitochondrial damage after oral administration of Fe₂O₃ NPs (45 nm) (Manickam et al. 2019). NI of Fe₂O₃ NPs (21 nm) induced dilated rough endoplasmic reticulum (ER)-degenerated dendrite and disrupted membrane structure in the olfactory bulb and hippocampus of male CD ICR mice (Wang et al. 2009). No brain lesions were observed in male SD rats after NI of Fe₃O₄ NPs (30 nm) (Wu et al. 2011). Striatum of male SD rats showed damage to the brain vasculature after IV injection of IONPs (10.8 nm) (Imam et al. 2015). The cerebral cortex of Wistar rats (male/female) showed neuronal damage, dose-dependent edema, cell dissociation, and glia and BME cell proliferation after IP injection of IONPs (62.5 nm) (Sheida et al. 2017). C57BL/6J mice IB injected with Fe₃O₄ NPs (49 nm; 10 μg/μl, daily for 7 and 14 days) showed a decrease in tyrosine hydroxylase-positive fibers in the dorsal striatum and in hematoxylin-labeled nuclei in hippocampus. However, no significant brain lesions were observed in either region (Liu et al. 2018). PNS neurotoxicity was reported in the sciatic nerve of female SD rats injected with DMSA-coated Fe₂O₃ NPs (9 nm); NP-laden phagocytes and edema were observed in the sub-perineurial area alongside severe edema, blood–nerve barrier disruption, loss of spindle-like cells and fiber in the perivascular area (Kim et al. 2013).

SiNPs

Male SD rats with NI of ¹²⁵I-radiolabeled SiO₂ NPs (20 nm) showed no significant histopathology changes (Wu et al. 2011). IP-administered amorphous SiO₂ NPs (20 nm; 50 mg/kg bw/day for 28 days) induced neuronophagia, perivascular edema and satellitosis in the cerebral cortex of male SD rats (Liu et al. 2017).

DNA damage

AgNPs and SiNPs

DNA damage was not studied in the studies reviewed.

Carbon-based NPs

No significant DNA damage was observed in the olfactory bulb, cerebellum or remaining brain areas of wild-type and p47phox-/- C57BL/6J mice (male/female) after inhalation delivery of pristine carbon NPs (58.8 nm; 154 µg/m3, 4 h) (Berlo et al. 2014).

IONPs

Male albino mice (M. musculus) orally exposed to Fe₂O₃ NPs (50 nm) showed G1/S cell-cycle checkpoint activation in the frontal cortex, hippocampus and cerebellum (Manickam et al. 2018).

Oxidative stress

AgNPs

Male Wistar rats orally administered a repeating dose of citrate-coated AgNPs (10 nm; 0.2 mg/kg bw/day for 14 days) showed elevated ROS levels in brain and liver (Skalska et al. 2016). GSH and GSH-Px in the brains of pups were depleted with citrate-coated AgNPs (20 nm) prenatally administered by oral gavage (25 mg/kg bw/day, from day 9 of gestation to the last day of pregnancy) (Fatemi et al. 2013).

Carbon-based NPs

The levels of oxidative biomarkers (mRNA expression of 8-oxoguanine DNA glycosylase and heme oxygenase 1 [HO-1]) were not altered in the brains of wild type or p47 phox−/− C57BL/6J mice after inhalation (154 μg/m³, for 4 h) of pristine carbon-based NPs (58.8 nm) (Berlo et al. 2014).

IONPs

Levels of ROS in the frontal cortex, hippocampus and cerebellum were dose-dependently increased in male albino mice (M. musculus) orally administered Fe₂O₃ NPs (50 nm; 25 and 50 mg/kg bw/day for 30 days) (Manickam et al. 2018). Levels of oxidative stress biomarkers were changed in striatum (GSH level decreased, H₂O₂ level increased) and hippocampus (H₂O₂ level increased) after NI of ¹²⁵I-radiolabeled Fe₃O₄ NPs (30 nm; 25 and 50 mg/kg bw/day, for 1 and 7 days). Superoxide dismutase and malondialdehyde (MDA) levels were not changed in either brain region in this study (Wu et al. 2013). NI of Fe₂O₃ NPs (21 nm; 130 μg/animal, every other day for 12 h, 72 h and 14 days) induced various levels of oxidative stress in hippocampus, cortex, cerebellum and brain stem of male albino mice (M. musculus) (Wang et al. 2009). HO-1 protein expression was increased in female SD rats injected with DMSA-coated Fe₂O₃ NPs (9 nm) in the sciatic nerve (Kim et al. 2013).

SiNPs

MDA level was significantly increased in male SD rats IP injected with amorphous SiO₂ NPs (50 mg/kg bw, daily for 28 days).

Significant oxidative stress biomarkers were found in striatum (H₂O₂ and MDA levels increased and GSH level decreased) and hippocampus (GSH level decreased) in male SD rats after NI of SiO₂ NPs (15 nm; 20 µg/per rat, daily for 1 and 7 days).

Cell death mechanism

AgNPs

Three studies reported apoptotic cell death induced by AgNPs; no studies reported autophagy induced by AgNPs. Male Wistar rats IP-injected with PVP-coated AgNPs (14.3 nm; 100–400 µg/per rat) showed a dose-dependent increase in apoptosis (downregulation of Bcl-2 and overexpression of Bax) in hippocampus (Ghooshchian et al. 2017). Ganjuri et al. studied the prenatal neurotoxicity of pristine AgNPs (30 nm) by orally exposing pregnant Wistar rats to NPs (1–10 µg/ml NPs in drinking water over the entire gestational period). Pro-caspase 3 mRNA level was dose-dependently increased in the brains of pups (Ganjuri et al. 2015). Fatemi et al. also observed a significant increase in level of the apoptotic marker caspase 9 in the brains of pups exposed to citrate-coated AgNPs (20 nm) in utero (Fatemi et al. 2013).

Carbon-based NPs

Apoptosis was not studied in the reports covered by this review. One study reported induction of autophagy with the administration of carbon-based NPs.

Repeated IP injection of MWCNTs (diameter 10–20 nm, length 2 μm; 2.5 mg/kg bw/day for 14 days) induced autophagy biomarkers (ratio of LC II/LC I mRNAs and increased Beclin-1 mRNA level) in brains of male Wistar rats (Gao et al. 2015).

IONPs

Two studies reported induction of apoptosis by IONP administration; no studies investigated the possible link between IONPs and autophagy. Apoptotic biomarkers (upregulation of Bax and cleaved caspase 3, downregulation of total Bcl-2) were changed in the frontal cortex, hippocampus and cerebellum of albino mice orally administered Fe₂O₃ NPs (50 nm; 25 and 50 mg/kg bw, daily for 30 days) (Manickam et al. 2018). Apoptotic cells were increased in number in the dorsal striatum and hippocampus of C57BL/6J mice injected with Fe₃O₄ NPs (49 nm; 2 μg per animal for 7 and 14 days) (Liu et al. 2018). The sciatic nerve of female SD rats injected with DMSA-coated Fe₂O₃ NPs (9 nm) showed elevated levels of the apoptotic biomarkers cleaved and uncleaved caspase 3 in both S100⁺ Schwann cells and TCR⁺ lymphocytes (Kim et al. 2013).

SiNPs

SiNP-induced apoptosis and autophagy were not studied.

Pro-inflammatory responses

AgNPs

Female SD rats orally administered pristine AgNPs (6 nm; 1, 10 mg/kg bw, daily for 14 days) showed increased IL-4 level in the brain (Xu et al. 2015a).

Carbon-based NPs

No significant pro-inflammatory responses were observed in the brain of wild type or p47phox−/− C57BL/6J mice (male/female) exposed to pristine carbon NPs (58.8 nm) via inhalation (154 µg/m3, 4 h) (Berlo et al. 2014).

IONPs

The pro-inflammatory response was evaluated in only one study looking at the effects of peripheral NP administration. Pro-inflammatory biomarker levels were increased (IL-1β and phosphorylated p38; gelatinolytic matrix metalloproteinase 9) in S100⁺ Schwann cells, CD68⁺ macrophages and endothelial cells of the sciatic nerve in treated female SD rats (Kim et al. 2013).

SiNPs

NI of pristine SiO₂ NPs (15 nm; 20 µg/per animal, daily for 1 or 7 days) increased TNF-R, IL-1β, and IL-8 levels in the striatum of male SD rats, with no significant effects in the hippocampus (Wu et al. 2011). mRNA levels of NF-κB, P65 and TNF-α were increased in hippocampus dentate gyrus of C57BL/6J mice exposed to FITC-labeled SiO₂ NPs (91 nm) via inhalation (Fu et al. 2018).

Astrocyte activation

AgNPs

Astrocytes were activated in SD rats (male/female) with NI of citrate-coated AgNPs (20 nm; 0.1, 0.2, 0.5, 1 mg/kg bw, daily for 98 days) (Yin et al. 2015). Injected (IP, IV, ICA, IB) pristine AgNPs (55 nm) activated astrocytes in male C57Balb mice and male SD rats (Sharma et al. 2009).

Carbon-based NPs

No astrocyte activation was found in C57BL/6J mice (male/female) after inhalation of UFCP (37.5 nm) (Morris-Schaffer et al. 2019). Astrocytes were dose-dependently activated at the perivascular area of the cerebral cortex in 6-week-old ICR mouse offspring with prenatal NI of Printex 90 carbon black (14 nm; 2.9, 15, 73 µg/kg bw/day, on days 5 and 9 of gestation) (Onoda et al. 2017).

IONPs

Oral administration of Fe₂O₃ NPs activated astrocytes in the frontal cortex, hippocampus and cerebellum of male albino mice (M. musculus) (Manickam et al. 2019).

SiNPs

Astrocytes were activated in the cerebral cortex and hippocampus of male SD rats intraperitoneally administered amorphous SiO₂ NPs (20 nm; 50 mg/kg bw, daily for 28 days) (Liu et al. 2017).

Microglia activation

AgNPs, carbon-based NPs, IONPs

No study investigated microglia activation.

SiNPs

Fu et al. investigated microglia activation by exposing pregnant C57BL/6J mice to FITC-labeled SiO₂ NPs (91 nm) via inhalation. Microglia activation was observed in the dentate gyrus of pups (Fu et al. 2018).

Function assessment (animal behavior)

AgNPs

Male Wistar rats orally administered citrate-coated AgNPs (10 nm) showed slight anxiety-like behaviors and changes in nociception reaction but no significant differences in motor function (locomotor activity and motor coordination) or memory performance (Dąbrowska-Bouta et al. 2016) Motor activity (locomotor activity and motor coordination) was dose-dependently impaired in SD rats (male/female) after NI of citrate-coated AgNPs (20 nm) (Yin et al. 2015). Antsiferova et al. orally administered male C57BL/6 mice with PVP-coated AgNPs in distilled water (31 nm; 50 µg/day for 30, 60, 120, 180 days); cautious and anxiety-related behavior were observed after 30–60 days but disappeared after 120 days. Exploratory behavior was increased at 120 days post-administration, with impaired contextual memory and loss of cautious behavior reported after 180 days (Antsiferova et al. 2018). Tabatabaei et al. investigated prenatal functional impairment of pregnant NMRI mice by subcutaneous administration of citrate-coated AgNPs (10 nm; 0.2–2 mg/kg bw/day, every 3 days until delivery); sex-specific depressive-like behaviors were observed in offspring (Zhang et al. 2019)

Carbon-based NPs

Newborn C57BL/6J mice (male/female) administered UCFP (37.5 nm) via inhalation (50 µg/m³, postnatal days 4–7 and 10–13, 4 h/day) showed altered locomotor activity only in females. The authors detected no significant alterations in memory, learning, anxiety-like behavior, social preference and impulsivity (Morris-Schaffer et al. 2019). Gao et al. found spatial learning and functional memory impairments in male Wistar rats after repeated IP injection of MWCNTs (diameter 10–20 nm, length 2 μm; 2.5 mg/kg bw/day for 14 days) (Gao et al. 2015).

IONPs

Dose-dependent impairment of motor activity, significant alteration in gait pattern and memory defects were noted in male albino mice (M. musculus) orally administered Fe₃O₄ NPs (45 nm; 25 or 50 mg/kg bw/day for 30 days) (Liu et al. 2017). Wistar rats (male/female) with IP injection of IONPs (62.5 nm; 2 or 14 mg/kw bw, for 1, 7 or 21 days) showed dose-dependent anxiety and inhibited locomotory activity (Sheida et al. 2017). Manickam et al. injected Fe₂O₃ NPs (49 nm; 10 µg/µl/day for 7 or 14 days) in the dorsal striatum or hippocampus of C57BL/6J mice (male/female); Both spatial learning and memory were impaired. Motor coordination was decreased in mice injected in the striatum, with impairment appearing at 7 days post-injection (Zhang et al. 2019).

SiNPs

C57BL/6J mouse (male/female) offspring prenatally exposed to FITC-labeled SiO₂ NPs (91 nm) via inhalation (55.56, 111.11, 277.78 mg/m², 30 min/day for 7 days) showed decreased social novelty preference and increased anxiety-like behavior (Fu et al. 2018).