**5. Circumvention of the blood-brain barrier by NPs**

Some of the ways by which NP can circumvent the blood brain barrier include the following (**Figure 1**):


#### **Figure 1.**

*Possible pathways through which nanoparticles cross the blood-brain barrier (BBB) and damage the neurons. Engineered nanomaterials with specific physicochemical properties can cross the BBB through various transport pathways such as (A) transcellular diffusion; (B) paracellular diffusion; (C) receptor-mediated transcytosis; (D) adsorptive-mediated transcytosis; and (E) cell mediated transcytosis. Nanoparticles interact directly with neuronal cells and cause neurotoxicity.*


#### **6. Translocation of nanoparticles through the placenta**

Exposure of pregnant mice to different NPs has been reported to induce pregnancy complications or damage to the fetus. Placenta is the maternal-fetal interface, which is formed of both maternal and fetal tissues that protects the embryo from harmful substances in the maternal blood. Placenta functions to exchange oxygen, nutrients, metabolic waste, and other molecules between the maternal and fetal bloodstream [49]. Factors that control the transfer of substances between maternal and fetal circulation include membrane surface area and thickness, blood flow, hydrostatic pressure in the intervillous chamber and the difference between fetal and maternal osmotic pressure [50]. Beside the placenta, amnion, chorion and parietal decidua also surround the fetus. These membranes are impervious to most of the xenobiotics in the maternal blood [51].

The brains from the fetuses of rats and mice have shown the presence of NP when the pregnant mothers were exposed to NP [52, 53]. Nano-silica and nano-TiO2 have been reported to accumulate in the placenta, fetal liver, and fetal brain when injected to pregnant mice [54]. The extent of transfer of nanoparticle across the placenta depends on the characteristics and functionalization of the particles [55, 56]. NPs with diameters 1–100 nm have been shown to transverse the placental barrier and were detected in the brain of the offspring [57, 58]. Gestational age is an important factor affecting the toxicity of NP on the fetus [50]. Fennell et al. [59] have demonstrated that AgNP administered through oral and IV route on gestational day 18 resulted in placental accumulation after 48 h. Campagnolo et al. [60] demonstrated that inhalation of Ag NP during the first gestational day until the fifteenth gestational day in female rats caused fetal resorption. This was accompanied with an increased expression of pregnancy-relevant inflammatory cytokines in the placentas. Zhang et al. [19] have shown that maternal exposure of mice to TiO2 NP decreased in angiogenesis in placental tissue and activated apoptotic pathways through caspase-3 in placental tissue.

Studies have demonstrated that various NPs can cross the BBB and placental barrier [61, 62]. Titanium dioxide nanomaterials (nTiO2) have been reported to cross the placental barrier in pregnant mice and cause neurotoxicity in their offspring. Toxicity to the brain cells was reported to be caused due to necrosis (**Figure 2**) [63].

#### **6.1 Mammalian embryonic model**

Rodents, primarily mice and rats have been commonly used for gestational translocation of NPs [15]. Mice have been commonly used for mammalian embryo toxicity studies [64–66]. Although rabbits have been used in fewer studies, rabbit placentae bear closer resemblance to human placentae than that of other rodents. Therefore, rabbits should be the preferable animal model to study gestational particle exposure [15]. Other nonmammalian species like drosophila and zebrafish have also been used in *in vivo* studies [67].

#### **Figure 2.**

*Maternal exposure of nanoparticles (NPs) results in neural fetotoxicity and developmental abnormalities. Direct translocation of NPs from maternal circulation across the placental barrier into growing fetus has been recognized as the major factor involved in NP-induced fetotoxicity. Accumulation of NPs in the fetus can cause structural and functional abnormalities in various fetal tissues, including the central nervous system (CNS) which is the main target of metallic NPs. Oxidative stress, induction of inflammatory responses, alterations in gene expression, DNA damage, necrosis, and apoptosis are the mechanisms associated with NP-induced neural fetotoxicity.*

### **6.2 Effects of nanoparticles on fetal brain**

The developing brain is highly vulnerable to nanomaterials [18] due to the incomplete development of BBB in the fetus [68]. The CNS shows considerable plasticity in the early stages of development and therefore highly susceptible to the toxic effects of NP [69]. The placenta is a multifunctional organ forming a barrier between maternal and fetal tissues. In utero exposure to NPs is one of main routes of exposure during the development of the nervous system [70]. Neurodevelopmental studies have shown that both male and female offspring show differential phenotypes after prenatal insults by NPs [18].

Among various NPs, many studies have been reported on the neurotoxicity of TiO2 NP. Injection of TiO2 NP into pregnant mice resulted in altered expression of genes associated with brain development and function of the central nervous system in embryos [71]. The effects of TiO2 seem to continue on the developing brain even during lactation [72]. The effects of titanium dioxide nanomaterials in pregnant mice include reduced size of the placenta and disrupted anatomical structure of the fetal brain and liver. Toxicity to the brain cells was reported to be caused due to necrosis [63]. One study showed that TiO2 NPs administered subcutaneously to pregnant mice resulted in an increased number of apoptotic cells in the olfactory bulb of the brain and damage to cranial nerves [58]. A subsequent study showed that the mice fetuses that were exposed to TiO2 NPs prenatally exhibited an increased level of dopamine and its metabolites in the prefrontal cortex and neostriatum. This demonstrates that prenatal exposure to TiO2 NPs might affect the development of the central dopaminergic system in mouse offspring [73]. In utero exposure of mice to TiO2, NP has been shown to cause changes in the genes associated with the brain development and functions of central nervous system in the embryo [71]. Accumulation of TiO2 NP in the placenta may interfere with the development of nervous system of the fetus by impairing the transport of nutrients to the fetus [74].

Injection of silica (Si) NPs to pregnant mice resulted in their accumulation in the brain of the embryo [54]. Other studies have reported that ZnO and TiO2 NPs causes neurotoxic effects in fetus after passing through the placenta [71, 75]. Injection of cobalt-chrome (CoCr) NPs into pregnant mice has been reported to cause neurodevelopmental abnormalities, like reactive astrogliosis and increased DNA damage in the fetal hippocampus [76].

#### **6.3 Effects of prenatal exposure to NP on the offspring**

Here, we briefly enumerate some of the effects of NPs in offspring associated with prenatal exposure. The effects of prenatal exposure to nanoparticles include neurobehavioral alterations in the offspring [77]. Other effects of prenatal exposure include accumulation of NP in the hippocampus [58, 78, 79]. These NPs in the fetal brain cause disturbances in the CNS homeostasis. The accumulated NP has been reported to cause psychiatric disorders such as autism, schizophrenia, and depression in offspring [80]. Exposure of pregnant mice to aluminum NP has been shown to induce neurodevelopmental changes which persisted during adulthood. This was accompanied by an anxiety-like behavior and impairment of cognitive function in offspring exposed to aluminum nanoparticles during in utero life [20]. Prenatal exposure to TiO2 NPs has been shown to impair the antioxidant status, cause oxidative damage to nucleic acids and lipids in the brain of newborn pups and enhanced the depressivelike behaviors during adulthood. Prenatal exposure to TiO2 NP has been associated with depressive behavior in adults [81]. In the case of ZnO NP, the depressive behavior has been attributed to their neurotoxic effects on neural development [82].

Pups from mice exposed to Al2O3 before and during pregnancy have been shown to have higher levels of Al accumulation in the hippocampus [20]. Similarly, in the case of Sprague Dawley rats the pups of dams exposed to silver NP showed the accumulation of silver in the brain, lung, liver, and kidneys [78]. Subcutaneous injection of TiO2 NP to CD-1 pregnant mice caused the accumulation of TiO2 NPs in the brain and testis of offspring [58]. However, exposure of Sprague Dawley rats to Zn NPs before mating and during lactation caused no accumulation of these NPs in the brain of offspring [83]. Prenatal exposure of mice to TiO2 NPs causes anatomical alterations in cerebral cortex, olfactory bulb and regions associated with the dopamine systems in the offspring [84].

Studies of Mohammadipour et al. [85] and Gao et al. [72] showed that in pregnant rats treated with TiO2 NPs significantly decreased hippocampal cell proliferation, impaired learning, and memory, and affected synaptic plasticity in the hippocampal dentate gyrus area in newborn rats. Similarly, the study of Zhou and his collogues [86] showed that maternal exposure to TiO2 NP results in inhibition of hippocampal and dysfunction of the rho/NMDAR signaling pathway in offspring. Maternal CB-NP exposure induced the long-term activation of astrocytes resulting in reactive astrogliosis in the brains of young mice [87]. TiO2 NP injection to pregnant mice has been reported to cause symptoms akin to autism spectrum disorder (ASD) and neurodevelopment disorders in neonates, without the detectable

presence of NP in the placenta [88]. Another study indicated that nano-TiO2 can cross the blood-fetal barrier and placental barrier, thereby delaying the development of fetal mice and inducing skeletal malformation [89].
