Influence And Mechanisms On Offspring Neurodevelopment After Exposure To Two-dimensional Ultrathin MXene（Ti₃C₂） Nanosheets During Early Pregnancy

Background:In recent two decades,various nanomaterials have pervaded different aspects of human lives through cosmetics,paints,medical products,textiles,personal care,and other products,with the development of nanotechnology.The widespread use of these products increases people’s attention to their safety and biological effects.MXene（Ti₃C₂）nanosheets are the earliest discovered in the family of new two-dimensional MXene,like dozens of MXenes,which were widely discovered because of their unique characteristics,including large surface area,abundant oxygen-containing groups within their structure,and cost-effective scale-up production.They have garnered increasing attention in biomedical applications,such as wastewater treatment,photothermal therapy（PTT）in near-infrared bio-windows,antimicrobial formulations,antibacterial material,etc.One of the important key factors for whether nanomaterials can be widely used in human production and living life due to their toxic effect on organisms.Recently,few studies of biocompatibility and toxicity have been described for MXene（Ti₃C₂）nanosheets,of which only a few studies are based on cell experiments or short-term assays.In the present study,we established a mouse model exposed to different doses of MXene（Ti₃C₂）nanosheets by tail-vein injection during early pregnancy and assessed the effects of MXene（Ti₃C₂）nanosheets on the female reproductive function and long-term effects on offspring.To further elucidate the potential mechanisms,we used metabolomics and RNA-seq approaches to identify molecular changes in the placenta following maternal MXene（Ti₃C₂）nanosheets exposure.The results described herein should provide an important reference for the female reproductive toxicity of MXene（Ti₃C₂）nanosheets,and form an experimental basis for determining the safety of MXene（Ti₃C₂）nanosheets.This study can also provide a meaningful reference for the application of this nanomaterial in human life.Methods:1.Synthesis and characterization of MXene（Ti₃C₂）nanosheets（1）Synthesis of MXene（Ti₃C₂）nanosheets:The HCl/Li F mixture was prepared for etching,and MXene（Ti₃C₂）nanosheets were obtained by etching Ti₃Al C₂.Thereafter,the obtained compound was washed with distilled water and the MXene（Ti₃C₂）nanosheets dispersion was collected after sonication under an Ar flow.（2）Characterization of MXene（Ti₃C₂）nanosheets:The ultrastructure and morphology of MXene（Ti₃C₂）nanosheets were examined by atomic force microscopy,scanning electron microscopy,transmission electron microscopy,X-ray photoelectron spectroscopy,and X-ray diffraction.2.Mouse model exposed to different doses of MXene（Ti₃C₂）nanosheets establishedAdult,virgin female,and fertile male mice（KM-strain,30–33 g）were purchased.The appearance of the vaginal plug in the morning（8:00-8:30）was considered the gestational day（GD）1.Due to the high sensitivity to environmental factors during early pregnancy,we injected mice intravenously from GD5 when the blastocyst initially implants into the uterus for 3 consecutive days.The respective solvents and blank（no injection）groups as control groups.3.Measurement of the titanium（Ti）content in tissuesUsing ICP-MS to measure the titanium（Ti）content in the uterus on GD8,GD9,and GD10 and in the placenta on GD12;Inferring the accumulation of MXene（Ti₃C₂）nanosheets in various tissues.4.Analysis of the spatial learning and memory abilities in offspring mice（1）To clarify the effect of MXene（Ti₃C₂）nanosheets exposure on reproduction,we first determined the pregnancy outcome of mice.The average pregnancy days,the number of fetuses produced by each female mouse,the average neonatal weight,and the malformation of the fetus.（2）Morris water maze test was conducted to examine the spatial learning and memory of offspring;using the elevated plus-maze test to assess the behavior of the offspring.H&E staining was used to measure the morphological changes in the cerebral cortex and hippocampus.5.Effects of MXene（Ti₃C₂）nanosheets exposure on pregnancy statusThe maternal body weight was recorded from GD5 to GD12 to measure the whole pregnancy status.Calculating the vital organ coefficient（heart,liver,spleen,lung,kidney,and ovary）,and observing the micromorphology of vital organs by H&E staining.Recording the implantation sites on GD8,GD9,GD10,and GD12 at different doses and calculating the organ coefficient of the uterus.Using DBA stanning to detect the decidualization of the endometrium.6.Effects of MXene（Ti₃C₂）nanosheets exposure on placental development and functionUsing IHC stanning to detect the expression of BMP-2 on GD8 and the expression of CK8 on GD9;Using H&E staining to detect the morphological changes of the placenta;Using IHC stanning to perform the expression of VEGFR2 and ki67 on the placenta,and to detect the development of labyrinth and proliferation of trophoblast.Calculating the organ coefficient of the placenta,placental efficiency,and the entire labyrinth surface area quantified on histological sections.Using TEM to detect the ultrastructure of placental cells.Using ELISA to detect the level ofβ-CG,Pl GF,and s Flt-1on serum which were collected on GD10 and GD12.7.Mechanism of MXene（Ti₃C₂）nanosheets exposure on placental and neurodevelopment of offspring（1）Using GC-MS analysis to detect the metabolite of serum and placental tissue.Using AMDI software to identify the metabolites.KEGG pathway was performed to predict the pathways linked to the metabolites and to explore the molecular mechanism of the impairment caused by MXene（Ti₃C₂）nanosheets.（2）The placental tissues in 2.5 mg/kg group and control group on GD12were collected for RNA-Seq.Differential genes on sequencing results were performed to draw a volcano diagram,heatmap,and circle diagram.Besides,GO analysis of differential genes and KEGG pathway analysis were used to identify mainly biological processes,and further investigate the differential genes related to amino acid metabolism and lipid metabolism.Results:1.Successful synthesis of MXene（Ti₃C₂）ultrathin nanosheetsThe lateral size of the ultrathin nanosheet structure was about 1-2μm.The thickness of well-dispersed nanosheets was about 2-3 nm.Various concentrations of MXene（Ti₃C₂）nanosheet solution showed good dispersibility.2.Accumulation of MXene（Ti₃C₂）nanosheets in uterus and placentaICP-MS analysis showed that Ti was rapidly accumulated in the uterus on GD8,GD9,and GD10 and in the placenta on GD12 after injection of different doses into pregnant mice.The Ti content was significantly increased in the uterus in the 2.5 mg/kg group.3.Exposure to MXene（Ti₃C₂）nanosheets did not affect the reproductive ability of miceMXene（Ti₃C₂）nanosheet did not affect the pregnancy outcome of mice after accumulation in the uterus and placenta.The average pregnancy days,the number of fetuses produced by each female mouse,and the average neonatal weight were not significantly different from those in the vehicle control group.Moreover,there was no significant malformation of the fetus at a dose of 2.5 mg/kg.4.Exposure to MXene（Ti₃C₂）nanosheets induced neurobehavioral abnormalities in offspringThe results of Morris water maze and elevated plus maze tests showed that maternal exposure to MXene（Ti₃C₂）nanosheets impaired cognitive function and had enduring effects on the emotional behavior of offspring（postnatal day 90）.H&E staining showed that MXene（Ti₃C₂）nanosheets caused morphological changes in the cerebral cortex and hippocampal,including neuronal cell loss,nuclei shrinkage,and dark staining of neurons.5.Effects of MXene（Ti₃C₂）nanosheets exposure on pregnancy status（1）The maternal body weight was recorded from GD5 to GD12.The maternal body weight showed a slow gain at 2.5 mg/kg dose compared with the gain in other dose groups.（2）The vital organ coefficient（heart,liver,spleen,lung,kidney,and ovary）,and observed the micromorphology of vital organs by H&E staining.No abnormalities were seen at the selected doses.However,compared with the organ coefficient of the uterus in the vehicle control group,an obvious decrease was observed in the 2.5 mg/kg group on GD12.（3）The number of implantation sites had no significant difference on GD8,GD9,GD10,and GD12 at different doses.While uneven embryo spacing and uterine bleeding were the observed in 2.5 mg/kg group,especially on GD12.（4）The number of u NK cells in endometrial decidua on GD8,GD9,and GD10 was no significant difference.6.Exposure to MXene（Ti₃C₂）nanosheets impaired the development and function of the placenta in mice（1）After exposure to 2.5 mg/kg,BMP-2 did not express in the ectoplacental cone（EPC）on GD8.As the result of the IHC staining of CK8,the thickness of chorion on GD9 was less than that in the vehicle control group.（2）H&E staining showed that histopathological changes in placenta tissues showed loose structure and hemorrhagic spots in the 2.5 mg/kg group.Compared with the vehicle control group,the organ coefficient of the placenta,placental efficiency,and the entire labyrinth surface area quantified on histological sections were significantly decreased.（3）IHC staining showed wedges of spongiotrophoblast invading the labyrinth in placentae treated with MXene（Ti₃C₂）nanosheets on GD12.Besides,Immunohistochemistry for ki67 showed that more proliferating trophoblasts were presented in placentae exposed to MXene（Ti₃C₂）nanosheets than those of control.（4）The level of serumβ-CG was significantly increased in MXene（Ti₃C₂）nanosheets exposure groups on GD10 and GD12.The level of PIGF was dose-dependently increased,whereas that of s Flt-1 was dose-dependently decreased in groups exposed to MXene（Ti₃C₂）nanosheets on GD10 and GD12.（5）The result of TEM was obvious that lipid droplets gradually increased after being treated with MXene（Ti₃C₂）nanosheets.7.Analysis of the metabolome data after treatment with MXene（Ti₃C₂）nanosheets（1）The result of PCA showed that exposure to MXene（Ti₃C₂）nanosheets changed metabolites of the placenta in the 2.5 mg/kg group.Meanwhile,the metabolites of the serum in the 2.5 mg/kg group didn’t show a distinct disparity from the vehicle control group.（2）A total of 145 metabolites were identified in the placenta,including amino acids,saturated fatty acids,branched unsaturated fatty acids,long-chain unsaturated fatty acids,etc.Only 5 metabolites were found to be significantly increased in the 1.25 mg/kg group.32 metabolites were found to be significantly different between the 2.5 mg/kg group and the vehicle control group,including 5 were decreased and 27 were increased.A total of152 metabolites were identified in the serum.（3）Compared with the control group,seven amino acids were significantly increased,including threonine,lysine,glutamic acid,and histidine.Besides,six long-chain unsaturated fatty acids were significantly increased in the 2.5 mg/kg group,including DHA,linoleic acid,nervonic acid,etc.（4）The KEGG alignment revealed that 26 metabolic pathways were significantly altered in the 2.5 mg/kg group.Five amino acid metabolism pathways were upregulated（phenylalanine,tyrosine and tryptophan biosynthesis,arginine biosynthesis,valine,leucine and isoleucine biosynthesis,cysteine and methionine metabolism,glycine,serine and,threonine metabolism）while three lipid metabolisms were significantly downregulated（linoleic acid metabolism,glycerolipid metabolism,and sphingolipid metabolism）in the 2.5 mg/kg group.8.Exposure to MXene（Ti₃C₂）nanosheets changed the expression of genes（1）Genome sequencing of the placenta tissue in the 2.5 mg/kg group and control group revealed that there were 408 differential genes（|log₂Fold Change|>1）related to treatment with MXene（Ti₃C₂）nanosheets.306 genes were up-regulated and 102 genes were down-regulated.（2）The GO analysis showed that the differential genes are mainly involved in the small molecule metabolic process,lipid transporter activity,and transporter activity.（3）The KEGG analysis showed that 16 genes related to amino acid metabolism（tryptophan metabolism,histidine metabolism,arginine and proline metabolism,tyrosine metabolism,phenylalanine metabolism,and lysine degradation）.15 genes were up-regulated and 1 gene was down-regulated.There are 9 genes related to lipid metabolism（arachidonic acid metabolism and fatty acid degradation）,and all of those were up-regulated.Conclusion:（1）MXene（Ti₃C₂）nanosheets quickly accumulated in the uterus and placenta after injecting intravenously.Surprisingly,even though MXene（Ti₃C₂）nanosheets barely affected the reproductive ability of female mice,the neurotoxic effects on offspring were obvious.（2）The abnormal development and function of the placenta might be caused by dysregulation of proliferation and differentiation of trophoblasts after exposure to MXene（Ti₃C₂）nanosheets during early pregnancy,which might be the key contributing factor to neurobehavioral abnormalities in offspring.（3）The metabolites significantly accumulated in the placenta after exposure to MXene（Ti₃C₂）nanosheets.The high level of glutamic acid and histidine and upregulation of related transport genes improved the efficiency of amino acid transfer from mother to fetus through the placenta,which satisfied the nutrient needs of the fetus in the late pregnancy period,and ensure that the neonatal weight still maintained within the normal range.However,the high activity genes that involved in tryptophan metabolic pathway may induce the lack of neurotransmitter（5-HT）and increase the level of quinolinic acid,which impair the normal neurodevelopment of the fetus.（4）The upregulation of Cyp4a12a and Cyp4a12b suggests that the catabolism of long-chain fatty acids in the placenta was activated,while the downregulation of Mfsd2a indicates the transport efficiency of DHA was decreased.The insufficient transport of DHA might be one of the key contributing factors to the altered learning and spatial memory ability and emotional state of offspring.In summary,the research is the first time confined that maternal exposure MXene（Ti₃C₂）nanosheets did not affect the pregnancy outcome,while induced neurobehavioral abnormalities in offspring.These abnormalities were closely related to the development and function of the placenta.The alteration of amino acid metabolism and lipid metabolism and the insufficient transport of essential fatty acids in the placenta might be the key contributing factor to neurobehavioral abnormalities.These data provide a better understanding of MXene（Ti₃C₂）nanosheets’safety and provide an experimental basis for the prevention of its negative biological effects.