The Translocation And Its Potential Mechanisms Of ZrO₂ NPs Crossing Placental And Fetal Blood-brain Barriers

BackgroundNanomaterials(NMs)are defined as the diameter smaller than 100 nm at least one dimension.Nanomaterials can be divided into zero dimension,one-dimensional and two-dimension.Due to their special physicochemical characteristics(size effect,surface effect,macroscopic quantum tunneling effect,high surface reactivity,surface adsorption and so on),nanomaterials have been widely used in many fields,such as electronics,cosmetics,auto industry,medical industry and so on.So far,NMs have been applied in 1833 commercial products including 764 health-related products.With the wide applications of NMs increase,the potential risk for human attracted more and more attention.Furthermore,embryonic development is sensitive to the harmful xenobiotics and prenatal exposure might induce irreversible damage of offspring.In pregnancy,the placental barrier and blood-brain barrier(BBB)are important for fetal development to prevent the harmful xenobiotics entering into fetus.The placental barrier was consisted of the junctional and labyrinthine zones,and protected the fetus from potentially noxious xenobiotics.As nanoproducts increasing in environmental and daily life,the current studies mainly payed attention on whether nanoparticles could cross the biological barriers.In addition to the various administration route and exposure dose,the previous studies of nanoparticle crossing-barrier problem mainly focused on the characteristics of nanoparticles,such as particle size,surface charge and surface coating.However,the contribution of barrier-development on nanoparticle crossing-barriers is still unclear.Similarly,as mainly composing of capillary endothelial cells,astrocytes and pericytes,BBB is a dynamic structure that regulates the entry of endogenous and exogenous substrates into the brain.However,whether fetal BBB development affected the translocation of nanoparticles to fetus is also still unclear.Due to their toxicity,nanoparticles crossed the biological barriers with inducing the oxidative stress and inflammation and then affected the permeability of barriers and the quantity of nanoparticle translocation.Therefore,to exclude the various nanotoxicity inducing different permeability of biological barriers,the lower toxicity and high biocompatible nanoparticles were good for investigating the barrier-development and nanoparticle crossing-barrier problem.Due to their properties of heat-resistant,corrosion resistance,high refractive index and good stability,zirconia nanoparticles(ZrO2 NPs)have been widely used in sensor,catalyst,ceramics,thermal insulation and other fields.Especially,polymer-ZrO2 NPs has a promising prospect in wastewater treatment and drinking water purification.Here,in order to exclude this confounding factor,we used ZrO2 NPs to investigate the role of development of biological barriers on nanoparticle crossing barriers problem.Research objectiveTo confirm the better biocompatibility of ZrO2 NPs,we first observed the reproductive and developmental toxicity of ZrO2 NPs.Then,we investigated the role of barrier-development on nanoparticle translocation.We further determined the translocation mechanisms of nanoparticle crossing the mature barriers.Method1.Experimental designThe previous studies showed that the placental barrier in pregnant mice matured at gestational day of 12.5(GD 12.5)and the fetal blood-brain barrier matured at GD 15.5.Based on the development of placental and fetal blood-brain barriers,we designed two part experiments.Part 1,we established three barrier-development models:Model 1(GD9-11)both two barriers are immature,Model 2(GD13-15)the BPB is mature but the fetal BBB is immature,Model 3(GD16-18)both two barriers are mature.According to the three models,we investigated the biocompatibility of ZrO2 NPs and the role of barrier-development on nanoparticles translocation.Part 2,we investigated the translocation mechanisms of nanoparticle crossing barriers based on the mature barrier model.Moreover,we observed the characteristics of ZrO2 NPs in vitro study.2.Animal grouping and exposure doseBased on the released ZrO2 NPs in drinking water,we designed three exposure groups,including 2.5,25 and 50 mg/kg.After ultrasonic processing for 30-60 min,the dissolution of ZrO2 NPs(0.25,2.5 and 5 mg/ml)were orally administrated to pregnant mice.Model 1 were exposing at GD 9-11 and then were sacrificed at GD 12.Similarly,Model 2 were exposing at GD 13-15 and Model 3 were at GD 16-18.In Part 1,ICR pregnant mice were randomly divided into four groups in per model(12 groups and 12 mice in per group).In Part 2,the pregnant mice were randomly divided into four groups(12 mice in per group).3.Research contents3.1 The characteristics of ZrO2 NPsTEM observed the morphology of nanoparticles.DLS measured the hydrate particle size of ZrO2 NPs.The Zeta potential was also determined.3.2 The stable and biocompatible properties of ZrO2 NPsAfter oral administration of ZrO2 NPs in three models,we observed the differences of maternal body weight,little size and fetal weight.We also observed the fetal growth.fetal body length and tail length,and fetal malformations.Moreover,we examined the histopathological changes of placenta as analyzed by hematoxylin-eosin(HE)staining and TUNEL assay.After exposures to pregnant mice at various doses,we measured the oxidative stress in molecular levels by determining the activity of GSH and GSSG and inflammation by measuring the expression levels of TNF-? and IL-1? mRNA.To further determine fetal neurotoxicity,we observed the neuron,microglia and astrocyte by immunofluorescence in fetal brains at three models.3.3 The development of BPB and fetal BBB accounts for ZrO2 NPs crossing barriersWe used ICP-MS to determine the biodistribution of ZrO2 NPs and further confirmed the particle form by TEM and EDX.Furthermore,we observed the normal physiologic morphology of tight junctions of biological barriers by using TEM and immunofluorescence.We also examined the molecular levels of tight junction proteins(Claudin-5,Occludin and ZO-1)by Western blot.3.4 Translocation mechanisms of ZrO2 NPs to fetal brainsAfter dissolving of ZrO2 NPs in water,AGF and concentrated hydrochloric acid(HCL),we determined the quantity of Zr ions by ICP-MS.Subsequently,after oral administration of NPs at various dose,we determined the biodistribution of ZrO2 NPs in maternal organs and fetal organs by ICP-MS.In transcellular pathway,we used Qrt-PCR to determine the mRNA levels of Caveolae,clathrin and Arf6.We further confirmed the results by measuring these protein expressions by Western blot.In paracellular pathway,the morphological structure of tight junctions in biological barriers was measured by TEM and immunofluorescence.Western blot was used to quantify the protein expressions of Claudin-5,Occludin and ZO-1.4.Statistical analysisThe data were inputted by Excel and analyzed by SPSS26.0.The P value<0.05 was deemed as statistical significance.The experimental data were in accordance with normal distribution and the results were shown as mean±standard deviation.The data in three model groups were first analyzed by the homogeneity test for variance.Subsequently,if the variances were homogeneous,the comparisons of four groups were analyzed by One-way ANOVA and then the pairwise comparison was determined by Bonferroni test.If the variances were heterogeneous,the data in four groups were analyzed by nonparametric analysis(Mann-Whitney U test and Kruskal-Wall is H test)Results1.The characteristics of ZrO2 NPsIn our study,the mean size of ZrO2 NPs by TEM were 15.8± 3.9 nm.Next,the hydrodynamic diameters measured by DLS were 54.2 ± 18.4 nm in aqueous solution and 105.2± 27.3 nm in AGF.The zeta potentials of ZrO2 NPs were 25.0± 6.1 mV in DI water and-10.2 ± 3.1 mV in AGF.These results indicated that ZrO2 NPs were stable and not easy to accumulate2.The stable and biocompatible properties of ZrO2 NPsAfter oral administration of ZrO2 NPs at doses of 2.5,25 and 50 mg/kg in three models,the maternal body weight,little size and fetal weight were no significant change compared to the vehicle control group.During the experiment,animals in each group had good activities and no death occurred.Moreover,the placental weight and fetal malformations have no significant change compared to the control group.Similarly,the fetal length and tail length were also no significant change in different groups.These results indicated that ZrO2 NPs induced no observed reproductive and developmental toxicity.The HE and TUNEL results showed that after crossing the maternal BPB in three models,ZrO2 NPs did not affect the structure and function of placentas.We further examined the development of fetal brains and found that after ZrO2 NP crossing fetal BBB,no damage of neuron,microglia and astrocyte was observed in three models.The above results were accordance with the findings showing that ZrO2 NPs did not trigger oxidative stress and damaged the balance of GSH and GSSG.Moreover,we also found that the expression levels of TNF-? and IL-1? mRNA had no significant change at three models.These results indicated that the biocompatible ZrO2 NPs could be used for investigating the relationship of barrier-development and nanoparticle translocation.3.Development of barriers contributing to ZrO2 NP crossingAfter oral exposure of ZrO2 NPs to pregnant mice,the content of Zr in fetal liver increased 6.1 and 9.8 fold at dose of 25 and 50 mg/kg at Model 1 as well as increase of 3.7 or 5.6 fold at Model 2 and 2.5 or 3.5 fold at Model 3.Similarly,the quantity of Zr in fetal brain increased 3.4 or 5.0 fold at Model 1,2.3 or 3.5 fold at Model 2 and 1.8 or 2.6 fold at Model 3,respectively(P<0.05).We also found that after oral administration of ZrO2 NPs at doses of 25 and 50 mg/kg,Zr accumulations in placenta,fetal liver and brain were increased by 1.3,1.6 and 1.3 fold in Model 1 compared to these in Model 2.Similarly,the Zr content in fetal brain was 1.5 times higher than that in Model 3.Furthermore,64%more ZrO2 NPs were translocated into fetal liver in Model 1 compared to Model 2.After oral exposures at Model 2,ZrO2 NPs crossing fetal BBB increased by 55%compared to Model 3.We also found that translocation of ZrO2 NPs to fetal brains increased by 96%in Model 1 compared to both maturation times of BPB and fetal BBB.More importantly,we found the undeveloped tight junctions in immature barriers.The protein expressions of tight junctions(Claudin-5,Occludin and ZO-1)were time-dependent increase with the development of barriers.4.Translocation mechanisms of NPs to fetal barinsIn transcellular pathway,ZrO2 NPs induced a dose-dependent increase in protein expressions of Caveolae,Clathrin and Arf6 in placentas and fetal brains(P<0.05).In paracellular translocation,ZrO2 NPs broken the strands of cell-cell tight junctions as measured by immunofluorescence labeling tight junction proteins.We further confirmed the decreased protein expressions of Claudin-5,Occludin and ZO-1 by western blot(P<0.05).Conclusion1.The good biocompatibility of ZrO2 NPs indicated that ZrO2 NPs was better for investigating the relationship between the barrier development and nanoparticle translocation2.ZrO2 NPs crossed the immature and mature BPB and fetal BBB indicating the potential application of nano-drug carrier.3.The development of placental barrier and fetal blood-brain barrier affected the quantity of ZrO2 NPs cross barriers.More importantly,ZrO2 NPs cross the immature placental and fetal brain barriers more easily than the mature barriers.4.The underdeveloped tight junction in immature placental and fetal brain barriers might be an important reason why ZrO2 NPs cross the immature barriers more easily than the mature.5.Not only tight junction but also receptor-mediated endocytosis(caveolae-,clathrin-and arf6-mediated endocytosis)contributed to the transporting pathways of ZrO2 NPs across the barriers.