animals : sixteen pregnant icr mice at gestation day 16 ( gd16 ) ( 1517 weeks 
 old , 5575 g ) were obtained from clea japan inc . commercial diet ce-2 ( clea 
 japan inc . ) and tap water were given ad libitum throughout the experiment . the mice were housed at approximately 25c and 5570% relative humidity under a 12-hr 
 light / dark cycle . the experiments were approved by the institutional animal care and use 
 committee , and all procedures were conducted according to the tottori university guidlines 
 for animal welfare . particles morphology of gold nanoparticles : the experiment employed 20- 
 and 50-nm colloidal gold solutions ( mean diameters of 19.6 nm and 49.3 nm respectively , 
 according to the manufacturer s information ; bb international , cardiff , u.k . ) . the 
 morphology of the gold nanoparticles was examined by transmission electron microscopy 
 ( tem-100cx ; japan electron optical laboratory , tokyo , japan ) . the 20-nm and 50-nm colloidal 
 gold solutions contained electron - dense , spherical , uniform and individual or slightly 
 agglomerated particles ( fig . 1.electron micrographs of 20-nm ( a ) and 50-nm ( b ) gold nanoparticles show 
 electron - dense , spherical , uniform and individual or slightly agglomerated appearance . electron micrographs of 20-nm ( a ) and 50-nm ( b ) gold nanoparticles show 
 electron - dense , spherical , uniform and individual or slightly agglomerated appearance . 
 bar=100 nm . experimental protocol : at gd16 , pregnant icr mice were intravenously 
 injected with 0.5 ml of saline solution ( control , n=4 ) , 20-nm or 50-nm 
 colloidal gold solutions through the tail vein ( n=6 per treatment ) . at 24 hr after the first 
 injection , the pregnant mice ( gd17 ) were intravenously injected with the same solutions as 
 the previous injections . at 24 hr after the second injection , the pregnant mice ( gd18 ) were 
 sacrificed by performing exsanguination under deep anesthesia induced by the intraperitoneal 
 injection of sodium pentobarbital . then , tissue samples were collected . sampling for maternal organs , plancentas and fetal organs : maternal 
 livers , placentas and fetal livers the samples were used for histopathological , immunohistochemical , 
 autometallography ( amg ) and transmission electron microscopy examinations . inductively coupled plasma - mass spectroscopy ( icp - ms ) : maternal livers , 
 placentas and fetal livers ( 0.1 g of each tissue ; n=2 from the control or n=3 from mice that 
 were injected with gold nanoparticles ) were digested with 2 ml of aqua 
 regia . after digestion , the inorganic residues were dissolved in 5 ml of 
 0.05 n hcl and ultrasonicated for 20 min , and the samples were then analyzed by performing 
 inductively coupled plasma - mass spectroscopy ( icp - ms ) ( hp 4500 ; agilent technologies , santa 
 clara , ca , u.s.a . ) . detection and quantification limits for gold were 0.5 
 ng / ml and 1 ng / ml , 
 respectively . histopathology : maternal livers , placentas and fetal livers were fixed in 
 10% neutral - buffered formalin . the formalin - fixed tissues were processed using routine 
 pathological methods and embedded in paraffin blocks . paraffin - embedded sections 
 ( 4-m - thick ) were cut for hematoxylin and eosin staining , 
 immunohistochemistry and amg . histopathological examination was performed by 2 pathologists 
 in a blind manner under a light microscope . autometallography ( amg ) : paraffin - embedded sections of placenta , maternal 
 liver and fetal liver were used for amg staining [ 7 , 
 8 ] . the sections were developed in a physical 
 developer consisting of 50% gum arabic , 50% citrate buffer , 5.6% hydroquinone and 17% silver 
 nitrate . the reaction was conducted in a water bath at 26c for 1 hr in the dark . excessive 
 silver residue was removed with a 5% sodium thiosulfate solution for 10 min . a positive result was demonstrated as small 
 black silver grains inside the cells of interest . immunohistochemistry : paraffin - embedded sections of the placentas were 
 used for the immunohistochemical detection of clathrin , caveolin , interleukin ( il)-6 , tumor 
 necrotic factor ( tnf)- , dimeric copper- and zinc - containing superoxide dismutase ( cu / zn 
 sod ) , inducible nitric oxide synthase ( inos ) and caspase-3 . for antigen retrieval , the 
 sections were treated with citrate buffer ( ph 5.4 ) and then microwaved . endogenous 
 peroxidase activity was quenched with 3% h2o2 at room temperature for 
 30 min . the slides were then blocked with 10% normal goat serum ( for the detection of 
 clathrin , caveolin-1 , il-6 , tnf- , cu / zn sod , inos and caspase-3 ) or 10% bovine serum 
 albumin ( for the detection of il-6 ) for 5 min in a microwave . next , the sections were 
 incubated with primary antibodies at 4c overnight ( anti - clathrin heavy chain : abcam , 
 cambridge , ma , u.s.a . 1:7,000 dilution ; anti - caveolin-1 : santa cruz biotechnology , santa 
 cruz , ca , u.s.a . 1:1,500 dilution ; anti - il-6 : santa cruz biotechnology 1:200 dilution ; 
 tnf- : monosan , uden , the netherlands , 1:15 dilution ; anti - cu / zn sod : stressgen bioreagents , 
 victoria , canada , 1:200 dilution ; anti - inos : bd transduction laboratories , lexington , ky , 
 u.s.a . 1:125 dilution ; and anti - caspase-3 : promega , madison , wi , u.s.a 1:250 ) or an 
 equivalent amount of phosphate - buffered saline as a negative control . a labeled streptavidin 
 biotin kit ( dako , glostrup , denmark ) was used to detect immunoreaction complexes in the 
 avidin  biotin complex assay . the sections were counterstained with 
 hematoxylin and observed by light microscopy in 4 randomly selected lesions at 100 
 magnification . positive immunolabeling areas were analyzed in 5 randomly selected areas from 
 each group by using image - pro plus 6.1 software ( mediacybernetics , inc . , silver spring , md , 
 u.s.a . ) . double immunofluorescence : the sections were placed in citrated buffer 
 solution ( ph=5.4 ) , microwaved and then treated with proteinase k for 15 min for antigen 
 retrieval . next , the sections were incubated with normal goat serum for 5 min in a 
 microwave . after incubation with normal goat serum , the sections were applied to the primary 
 antibodies against clathrin ( 1:500 dilution ) or caveolin-1 ( 1:350 dilution ) overnight at 4c 
 and then incubated with alexa fluor 488-conjugated secondary antibody ( 1:200 dilution ) for 1 
 hr at room temperature . then , the sections were reacted with primary antibody against 
 cytokeratin ( dako , 1:100 dilution ) overnight at 4c and then incubated with alexa fluor 
 555-conjugated secondary antibody ( 1:300 dilution ) for 30 min at room temperature . analyses were 
 performed with a confocal imaging system ( ax-70 , olympus laboratory , tokyo , japan ) . electron microscopy : half of the longitudinal sections from the placenta 
 were cut into cubes measuring 12 mm . next , tissues were fixed in glutaraldehyde 
 for 3 hr at 4c , rinsed in 0.1 m phosphate buffer ( ph 7.4 ) , postfixed with 1% osmium 
 tetroxide for 1 hr , dehydrated in alcohol and embedded in epoxy resin . the areas of interest 
 were selected for electron microscopy examination from 1% toluidine - stained semi - thin 
 ( 1-m - thick ) sections and subsequently cut into ultra - thin ( 70-nm - thick ) 
 sections . after staining with uranyl acetate and lead citrate , ultra - thin sections were 
 examined using a transmission electron microscope ( tem-100cx ; japan electron optical 
 laboratory , tokyo , japan ) . western blot analysis : approximately 100 mg of placental tissue ( n=4 per 
 group ) was harvested and processed in lysis buffer ( roche , basel , switzerland ) to extract 
 protein . tissue lysates were clarified by centrifugation at 14,000 rpm for 10 min , and the 
 protein content of the supernatant was determined . lysate supernatants were diluted 1:1 with 
 2 electrophoresis sample buffer ( 1 sample buffer=125 mm tris  hcl , ph 6.8 , 2% sodium 
 dodecyl sulfate [ sds ] , 5% glycerol , 0.003% bromophenol blue and 1% -mercaptoethanol ) , and 
 aliquots equivalent to 20 g of total protein per sample were resolved in a 
 15% sds / polyacrylamide gel . the gels were electroblotted onto a polyvinylidene difluoride 
 membrane ( biorad , hertfordshire , u.k . ) , and the membrane was probed with the primary 
 antibodies , rabbit anti - clathrin heavy chain ( 1:1,000 dilution ) or rabbit anti - caveolin-1 
 ( 1:1,000 dilution ) . after overnight incubation at 4c , the membrane was washed and probed 
 with the secondary antibody , which was an anti - rabbit horse radish - peroxidase - conjugated 
 igg . the immunoblot procedure was 
 performed using the chemiluminescence detection reagent ( luminata forte western 
 hrp substrate , millipore , billerica , ma , u.s.a . ) the bands on the clarified blots were measured with an image analysis system ( image - pro plus 
 6.1 , mediacybernetics , inc . , silver spring , md , u.s.a . ) , and the digital numbers that were 
 obtained were integrated density values of the intensity of each band . in situ apoptosis detection ( tunel assay ) : paraffin - embedded sections of 
 placenta were treated with proteinase k at room temperature . then , the sections were 
 inactivated for endogenous peroxidase by applying 3% h2o2 for 5 min . after washing , the labeling reaction mixture ( consisting of tdt enzyme and labeling safe 
 buffer , in situ apoptosis detection kit : takara bio inc . , otsu , japan ) was 
 applied to the sections , and they were incubated in a 37c humidified chamber for 70 min . following this , the sections were treated with anti - fitc hrp conjugate ( takara bio inc . ) and 
 incubated at 37c for 30 min . after coloring with dab , the sections were counterstained with 
 3% methyl green and observed under a light microscope . statistical analysis : all of the data were expressed as the mean  
 standard error . statistical significance was determined by performing the mann - whitney s 
 u - test or the student s t - test for 2-group comparisons . for all 
 comparisons , p values less than 5% ( p<0.05 ) were 
 considered statistically significant . icp  ms : gold was detected in the maternal livers and placentas from mice 
 injected with 20- and 50-nm gold nanoparticles ( table 
 1table 1.gold contents in the tissues measured by icp - msgroupgold content ( g / g dry tissue)maternal liverplacentafetal livercontrol(n=2)0.2  0.07ndnd20-nm gold nps(n=3)640.3  171.801.3  0.61nd50-nm gold nps(n=3)99.2  2.751.0  0.78ndvalues are the mean  standard error . a ) significantly different between the control 
 and 20 nm gold nps injected mice , p<0.05 . b ) significantly 
 different between 20-nm gold nps injected mice and 50-nm gold nps injected mice , 
 p<0.05 . the level of gold in both maternal livers and placentas from mice injected 
 with 20-nm gold nanoparticles was significantly higher than the level of gold from mice 
 injected with 50-nm gold nanoparticles . gold was not detected in maternal livers and 
 placentas from control mice and fetal livers from the control mice and the mice injected 
 with 20- and 50-nm gold nanoparticles . a ) significantly different between the control 
 and 20 nm gold nps injected mice , p<0.05 . b ) significantly 
 different between 20-nm gold nps injected mice and 50-nm gold nps injected mice , 
 p<0.05 . histopathology : placental tissues from mice injected with 20- and 50-nm 
 gold nanoparticles demonstrated no significant pathological changes at the maternal fetal 
 barrier in the labyrinthine zone of the placentas compared to the control mice ( fig . 2fig . 2.maternalfetal barrier in the labyrinthine zone of the placentas from control mice 
 ( a ) and from mice injected with 20-nm ( b ) and 50-nm gold nanoparticles ( c ) . mild swelling of the cytoplasm ( asterisks ) of 
 syncytiotrophoblastic cells ( t ) and endothelial cells ( arrows ) are observed at the 
 maternal  fetal barrier from mice injected with 20- and 50-nm gold nanoparticles . swelling of the cytoplasm of syncytiotrophoblastic cells and fetal 
 endothelial cells in the maternal  fetal barrier from mice injected with gold nanoparticles 
 was occasionally observed ( fig . 2b and 2c ) . maternal  fetal barrier in the labyrinthine zone of the placentas from control mice 
 ( a ) and from mice injected with 20-nm ( b ) and 50-nm gold nanoparticles ( c ) . mild swelling of the cytoplasm ( asterisks ) of 
 syncytiotrophoblastic cells ( t ) and endothelial cells ( arrows ) are observed at the 
 maternal  fetal barrier from mice injected with 20- and 50-nm gold nanoparticles . no pathological lesions were found in other maternal and fetal organs from the control mice 
 and the mice injected with 20- and 50-nm gold nanoparticles . autometallography : silver - enhanced gold nanoparticles were detected in 
 kupffer cells in the maternal livers of mice injected with 20- and 50-nm gold nanoparticles 
 ( fig . 3b and 3cfig . 3.autometallographic-enhanced gold nanoparticles in maternal livers ( a  c ) , placentas 
 ( d  f ) and fetal livers ( g  i ) of control mice ( a , d and g ) , mice intravenously injected 
 with 20-nm gold nanoparticles ( b , e and h ) and 50-nm gold nanoparticles ( c , f and i ) . gold nanoparticles are demonstrated exclusively in the maternal livers from mice 
 injected with 20- and 50-nm gold nanoparticles . the deposition of gold nanoparticles 
 is shown in kupffer cells ( arrowheads ) ( b and c ) . autometallography shows negative 
 staining in all tissues from the control mice ( a , d and g ) , placentas ( e and f ) and 
 fetal livers ( h and i ) from mice injected with 20- and 50-nm gold nanoparticles . gold nanoparticles were not detected by using the amg method in the placentas , 
 maternal livers and fetal livers from the control mice and placentas and fetal livers from 
 the mice injected with 20- and 50-nm gold nanoparticles ( fig . autometallographic - enhanced gold nanoparticles in maternal livers ( a  c ) , placentas 
 ( d  f ) and fetal livers ( g  i ) of control mice ( a , d and g ) , mice intravenously injected 
 with 20-nm gold nanoparticles ( b , e and h ) and 50-nm gold nanoparticles ( c , f and i ) . gold nanoparticles are demonstrated exclusively in the maternal livers from mice 
 injected with 20- and 50-nm gold nanoparticles . the deposition of gold nanoparticles 
 is shown in kupffer cells ( arrowheads ) ( b and c ) . autometallography shows negative 
 staining in all tissues from the control mice ( a , d and g ) , placentas ( e and f ) and 
 fetal livers ( h and i ) from mice injected with 20- and 50-nm gold nanoparticles . c ; 
 bar=10 m ; d  i ; bar=25 m . immunohistochemistry : clathrin immunohistochemistry demonstrated positive 
 reaction in syncytiotrophoblastic cells and fetal endothelial cells ( fig . 4.clathrin immunohistochemistry showing intense positivity in the cytoplasm of 
 trophoblastic cells ( arrows ) and endothelial cells ( white arrowheads ) in the 
 maternal  fetal barrier in the labyrinthine zone of placentas from mice injected with 
 20-nm ( b ) and 50-nm ( c ) gold nanoparticles . weak positivity is shown in the cytoplasm 
 of trophoblastic cells ( arrows ) and fetal endothelial cells ( white arrowheads ) in the 
 maternal  fetal barrier from a control mouse ( a ) . weak positivity was observed in syncytiotrophoblastic cells and fetal endothelial 
 cells at the maternal  fetal barrier from the control mice ( fig . 4a ) compared to mice injected with gold nanoparticles ( fig . 4b and 4c ) . the image analysis of clathrin 
 immunopositivity showed higher intensity in the placental tissues from mice injected with 
 gold nanoparticles . no significant differences were observed between clathrin 
 immunoreactivity in the placental tissues from mice injected with 20- and 50-nm gold 
 nanoparticles as seen by using image analysis ( table 
 2table 2.positive immunolabeling area in the labyrinthine zone of placentasgroup% positive areaanti - caveolin-1anti - clathrincontrol0.28  0.1792.73  0.43020-nm gold nps3.57  0.71812.04  1.49550-nm gold nps2.57  0.9269.10  1.618values are the mean  standard error . a ) positive area ( % ) of 94,850 
 m of 4-m - thick paraffin sections . clathrin immunohistochemistry showing intense positivity in the cytoplasm of 
 trophoblastic cells ( arrows ) and endothelial cells ( white arrowheads ) in the 
 maternal  fetal barrier in the labyrinthine zone of placentas from mice injected with 
 20-nm ( b ) and 50-nm ( c ) gold nanoparticles . weak positivity is shown in the cytoplasm 
 of trophoblastic cells ( arrows ) and fetal endothelial cells ( white arrowheads ) in the 
 maternal  fetal barrier from a control mouse ( a ) . a ) positive area ( % ) of 94,850 
 m of 4-m - thick paraffin sections . caveolin-1 immunoreactivity was detected exclusively in the cytoplasm of fetal endothelial 
 cells at the maternal  fetal barrier in the labyrinthine zone of the placentas ( fig . 5fig . 5.caveolin-1 immunohistochemistry showing immunopositivity in the cytoplasm of fetal 
 endothelial cells at the maternal  fetal barrier in the labyrinthine zone of the 
 placentas ( white arrowheads ) from mice injected with 20-nm ( b ) and 50-nm ( c ) gold 
 nanoparticles . weak positivity is shown in the cytoplasm of fetal endothelial cells in 
 the maternal  fetal barrier from a control mouse ( a ) . the positivity of caveolin-1 immunolabeling was lesser in the placental tissues 
 from the control mice ( fig . 5a ) compared to mice 
 injected with gold nanoparticles ( fig . 5b and 5c ) . the image analysis of caveolin-1 immunolabeling showed higher intensity in the placental 
 tissues from mice injected with gold nanoparticles . no significant differences were observed 
 between caveolin-1 immunoreactivity in the placental tissues from mice injected with 20- and 
 50-nm gold nanoparticles as seen by using image analysis ( table 2 ) . caveolin-1 immunohistochemistry showing immunopositivity in the cytoplasm of fetal 
 endothelial cells at the maternal  fetal barrier in the labyrinthine zone of the 
 placentas ( white arrowheads ) from mice injected with 20-nm ( b ) and 50-nm ( c ) gold 
 nanoparticles . weak positivity is shown in the cytoplasm of fetal endothelial cells in 
 the maternal  fetal barrier from a control mouse ( a ) . there were occasional positive findings in il-6 , tnf- , cu / zn sod and inos immunolabeling 
 in the cytoplasm of syncytiotrophoblasts and fetal endothelial cells in the maternal  fetal 
 barrier . no difference in the intensity and distribution of il-6 , tnf- , cu / zn sod and inos 
 immunolabeling between the control mice and the mice injected with 20- and 50-nm gold 
 nanoparticles was observed ( data not shown ) . no positivity in caspase-3 immunolabeling was 
 detected in the maternal  fetal barrier from the control mice and the mice injected with 20- 
 and 50-nm gold nanoparticles ( data not shown ) . double immunofluorescence : positive clathrin immunoreactivity was 
 demonstrated in the syncytiotrophoblast layer and fetal vascular endothelium ( fig . 6.double immunofluorescence of clathrin and cytokeratin proteins in the labyrinthine 
 zone of mice placenta . double immunolabeling by using antibodies against clathrin as 
 shown by alexa 488 ( green ) ( a ) and cytokeratin as shown by alexa 555 ( red ) ( b ) . merged 
 image with the 2 fluorophores ( c ) . the syncytiotrophoblast layer ( white arrows ) and 
 fetal vascular wall are strongly labeled with clathrin ( white arrowheads ) ( a ) . the 
 syncytiotrophoblast layer is exclusively labeled with cytokeratin ( white arrows ) ( b ) . co - expression of clathrin and cytokeratin ( yellowish ) is shown in the 
 syncytiotrophoblast layer ( white arrows ) ( c ) . bars=100 m . ) . the co - localization of clathrin and cytokeratin was observed in the 
 syncytiotrophoblast layer ( fig . in contrast , 
 caveolin-1 immunoreactivity revealed strong immunoreactivity exclusively in fetal vascular 
 endothelial cells ( fig . 7afig . 7.double immunofluorescence of caveolin-1 and cytokeratin proteins in the labyrinthine 
 zone of mice placenta . double immunolabeling by using antibodies against caveolin-1 as 
 shown by alexa 488 ( green ) ( a ) and cytokeratin as shown by alexa 555 ( red ) ( b ) . the fetal vascular wall is exclusively labeled with 
 caveolin-1 ( white arrows ) ( a ) . the syncytiotrophoblast layer is labeled with 
 cytokeratin ( white asterisks ) ( b ) . co - expression of caveolin-1 and cytokeratin is not 
 shown in the labyrinthine zone of the placenta . bars=100 m . ) . the co - localization of caveolin-1 and cytokeratin was not observed in the 
 labyrinthine zone of the placentas ( fig . double immunofluorescence of clathrin and cytokeratin proteins in the labyrinthine 
 zone of mice placenta . double immunolabeling by using antibodies against clathrin as 
 shown by alexa 488 ( green ) ( a ) and cytokeratin as shown by alexa 555 ( red ) ( b ) . merged 
 image with the 2 fluorophores ( c ) . the syncytiotrophoblast layer ( white arrows ) and 
 fetal vascular wall are strongly labeled with clathrin ( white arrowheads ) ( a ) . the 
 syncytiotrophoblast layer is exclusively labeled with cytokeratin ( white arrows ) ( b ) . co - expression of clathrin and cytokeratin ( yellowish ) is shown in the 
 syncytiotrophoblast layer ( white arrows ) ( c ) . bars=100 m . double immunofluorescence of caveolin-1 and cytokeratin proteins in the labyrinthine 
 zone of mice placenta . double immunolabeling by using antibodies against caveolin-1 as 
 shown by alexa 488 ( green ) ( a ) and cytokeratin as shown by alexa 555 ( red ) ( b ) . merged 
 image with the 2 fluorophores ( c ) . the fetal vascular wall is exclusively labeled with 
 caveolin-1 ( white arrows ) ( a ) . the syncytiotrophoblast layer is labeled with 
 cytokeratin ( white asterisks ) ( b ) . co - expression of caveolin-1 and cytokeratin is not 
 shown in the labyrinthine zone of the placenta . bars=100 m . electron microscopy : the maternal  fetal barrier in the labyrinthine zone 
 of the placentas from the control mice consisted of 3 layers of syncytiotrophoblastic cells 
 ( syncytiotrophoblast layers i , ii and iii ) with underlying fetal endothelial cells . electron 
 microscopy demonstrated no signs of cell / tissue damage in the placenta from the mice treated 
 with gold nanoparticles . numerous microplicae and infolding with a few vesicle - like 
 structures were observed in the cytoplasm of syncytiotrophoblast layers ii and iii in the 
 maternal  fetal barrier from the control mice ( fig . 8.transmission electron micrographs of maternal  fetal barrier in the labyrinthine zone 
 of the placentas . the placenta from a control mouse consists of 3 syncytiotrophoblast 
 layers with underlying fetal endothelial cells . numerous microplicae and infolding 
 with a few of vesicle - like structures ( arrowheads ) are shown in the cytoplasm of 
 syncytiotrophoblast layers ii and iii in the maternal  fetal barrier from a control 
 mouse ( a ) . enlargement of the cytoplasm of syncytiotrophoblast layer i , ii , iii and 
 fetal endothelial cells with an increase in the number of vesicle - like structures 
 ( arrowheads ) is shown in the maternal  fetal barrier from mice injected with 20-nm ( b ) 
 and 50-nm ( c ) gold nanoparticles . bar=0.5 m . ms = maternal blood sinus , 
 i = syncytiotrophoblast layer i , ii = syncytiotrophoblast layer ii , 
 iii = syncytiotrophoblast layer iii , fe = fetal endothelial cell , fc = fetal capillary . ) . moderate enlargement with an increase in the number of vesicle - like structures was 
 observed in the cytoplasm of syncytiotrophoblast layers ii and iii in the maternal  fetal 
 barrier from mice injected with 20- and 50-nm gold nanoparticles ( figs . 9.transmission electron micrograph of the maternal  fetal barrier in the labyrinthine 
 zone of the placenta from mouse injected with 20-nm gold nanoparticles demonstrating 
 an increase in the number of vesicle - like structures ( arrowheads ) in the cytoplasm of 
 syncytiotrophoblast layers i , ii , iii , and fetal endothelial cells . i = syncytiotrophoblast layer i , ii = syncytiotrophoblast layer ii , 
 iii = syncytiotrophoblast layer iii , fe = fetal endothelial cell , fc = fetal capillary . ) . an increase in the number of vesicle - like structures was also shown in the 
 endothelial cells in the maternal  fetal barrier from mice injected with 20- and 50-nm gold 
 nanoparticles compared to the control mice ( figs . transmission electron micrographs of maternal  fetal barrier in the labyrinthine zone 
 of the placentas . the placenta from a control mouse consists of 3 syncytiotrophoblast 
 layers with underlying fetal endothelial cells . numerous microplicae and infolding 
 with a few of vesicle - like structures ( arrowheads ) are shown in the cytoplasm of 
 syncytiotrophoblast layers ii and iii in the maternal  fetal barrier from a control 
 mouse ( a ) . enlargement of the cytoplasm of syncytiotrophoblast layer i , ii , iii and 
 fetal endothelial cells with an increase in the number of vesicle - like structures 
 ( arrowheads ) is shown in the maternal  fetal barrier from mice injected with 20-nm ( b ) 
 and 50-nm ( c ) gold nanoparticles . bar=0.5 m . ms = maternal blood sinus , 
 i = syncytiotrophoblast layer i , ii = syncytiotrophoblast layer ii , 
 iii = syncytiotrophoblast layer iii , fe = fetal endothelial cell , fc = fetal capillary . transmission electron micrograph of the maternal  fetal barrier in the labyrinthine 
 zone of the placenta from mouse injected with 20-nm gold nanoparticles demonstrating 
 an increase in the number of vesicle - like structures ( arrowheads ) in the cytoplasm of 
 syncytiotrophoblast layers i , ii , iii , and fetal endothelial cells . i = syncytiotrophoblast layer i , ii = syncytiotrophoblast layer ii , 
 iii = syncytiotrophoblast layer iii , fe = fetal endothelial cell , fc = fetal capillary . western blot analysis : immunoblotting of tissue lysates from placentas of 
 mice injected with 20- and 50-nm gold nanoparticle solutions demonstrated a 171-kda band of 
 clathrin protein . the positive band from the control mice was less intense compared to 
 positive bands from mice injected with gold nanoparticles ( fig . 10.western blotting demonstrates clathrin and caveolin-1 protein expression in the 
 placental tissues from pregnant mice . the intensity of clathrin protein band ( 171 kda ) 
 of placental tissue lysates from control mice is less than the bands of tissue lysates 
 from mice injected with 20- and 50-nm gold nanoparticles ( a ) . the intensity of 
 caveolin-1 protein bands is not different between the control mice and the mice 
 treated with 20- and 50-nm gold nanoparticles ( b ) . ) . the integrated density values of the intensity of bands of tissue lysates from mice 
 injected with gold nanoparticles were significantly greater than those of the control mice 
 ( table 3table 3.integrated density values of the immunoblot band intensity using the image 
 analysis systemproteindensity of immunoblot band intensity(optical density 
 unit)control ( n=3)20-nm gold nps ( n=3)50-nm gold nps ( n=3)clathrin0.20  0.0621.05  0.0291.06  0.010caveolin-11.46  0.1671.36  0.2241.55 western blotting demonstrates clathrin and caveolin-1 protein expression in the 
 placental tissues from pregnant mice . the intensity of clathrin protein band ( 171 kda ) 
 of placental tissue lysates from control mice is less than the bands of tissue lysates 
 from mice injected with 20- and 50-nm gold nanoparticles ( a ) . the intensity of 
 caveolin-1 protein bands is not different between the control mice and the mice 
 treated with 20- and 50-nm gold nanoparticles ( b ) . the 
 positive bands showed no difference in intensity in the placental lysates from the control 
 mice and the mice injected with gold nanoparticles ( fig . the integrated density values of the intensity of bands of the tissue lysates 
 were not significantly different between all groups ( table 3 ) . in situ apoptosis detection ( tunel assay ) : the tunel assay showed negative 
 results in the labyrinthine zone of the placentas of control , 20- and 50-nm gold 
 nanoparticle injected mice ( data not shown ) . at present , the risk of exposure to nanosized materials in pregnancy is increasing . however , there are only a few studies on the translocation pathway of nanoparticles across 
 the maternal  fetal barrier ( placental barrier ) [ 5 , 
 9 , 15 ] . the 
 present study aims to determine the possible translocation mechanism of gold nanoparticles 
 across the maternal  fetal barrier of mice . icp - ms analysis demonstrated a significant amount of gold deposited in the maternal livers 
 and placentas , but no detectable level of gold in the fetal organs . although amg staining 
 also showed the dominant accumulation of gold nanoparticles in kupffer cells in the livers 
 of the pregnant mice , no amg positive findings were observed in the fetuses after 
 intravenous administration . a previous study also reported 
 that gold nanoparticles are primarily phagocytized by kupffer cells and that almost of the 
 administered nanoparticles present in the liver after intravenous administration in rodents 
 [ 2 , 29 , 30 , 33 ] . no pathological changes were observed in the placentas , maternal organs and fetal organs 
 from mice treated with 20-nm and 50-nm gold nanoparticle solutions , suggesting that gold 
 nanoparticles would not be harmful to pregnant mice and their placentas at the doses that we 
 used . previous in vitro and in vivo studies also reported 
 that various sizes and doses of gold nanoparticles showed no obvious toxicity to mice and 
 rats [ 17 , 31 , 
 33 ] . previous studies on the translocation pathway of nanoparticles across the fetal  maternal 
 barrier of the human placenta suggest that endocytosis plays an important role in the 
 transportation of nanoparticles [ 12 , 15 , 32 , 38 ] . in the present study on the mouse placenta , an 
 increase in the number of endocytic vesicles was also observed in the cytoplasm of the 
 syncytiotrophoblasts and fetal endothelial cells by electron microscopy , suggesting that 
 endocytosis was upregulated in the maternal  fetal barrier after administration of gold 
 nanoparticle solutions . endocytosis plays an important role for the transport of nutrients 
 or biomolecules , such as albumin , folic acid , hormones , etc . at the placenta in the 
 physiological condition clathrin - mediated endocytosis was previously described in the cytoplasm of 
 syncytiotrophoblasts of the mouse placenta . clathrin - mediated endocytosis is involved in the recycling of albumin in the term placenta , 
 neurotransmitter transport and the internalization of several antigens [ 16 , 21 , 23 ] . in this study , clathrin immunohistochemistry showed 
 an increase of intense positivity in the endocytic vesicles of both syncytiotrophoblasts and 
 fetal endothelial cells . the localization of clathrin expression was confirmed by performing 
 double immunofluorescence by using antibodies to clathrin and cytokeratin . immunoblot analysis 
 showed an increase in clathrin protein expression in the placental tissues from mice treated 
 with gold nanoparticles . these results suggested that gold nanoparticle administration 
 upregulates clathrin expression in the placenta and that clathrin - mediated endocytosis may 
 be one of the pathways of gold nanoparticle translocation in the maternal  fetal barrier . caveolae - mediated endocytosis is also considered to be one of the pathways for the 
 translocation of nanoparticles in the air caveolin-1 protein plays a role in the regulation of caveolar invagination 
 and the formation of caveolae , which is one form of the endocytic vesicle [ 19 , 25 , 27 , 28 ] . in murine 
 placental tissues , the localization of caveolin-1 has been demonstrated in the vasculatures , 
 especially in the fetal vascular endothelium [ 3 , 20 , 22 ] . in this 
 study , increased intensity of caveolin-1 immunohistochemistry was observed exclusively in 
 the fetal endothelium after the administration of gold nanoparticle solutions . in contrast , 
 immunoblotting showed no difference in the amount of caveolin-1 expression between the 
 control mice and the mice treated with gold nanoparticles , suggesting the possible 
 re - assembly of caveolin-1 protein from the cytosol to the caveolae structure . in this study with the experimental design , applied techniques including electron 
 microscopy failed to detect signs of actual translocation of the exposed particles as well 
 as signs of their toxicity . however , an increase of endocytic vesicles in the cytoplasm of 
 syncytiotrophoblasts and fetal endothelial cells in the maternal  fetal barrier of mice 
 treated with gold nanoparticles was demonstrated . a further study with prolonged duration , 
 different kinds of nanoparticles and sensitive techniques for the detection of nanoparticles 
 is required to ascertain whether exposed nanoparticles may have a chance to be translocated 
 through the maternal  fetal barrier by both clathrin - mediated and caveolae - mediated 
 endocytosis demonstrated in this study .
abstractexposure to nanoparticles during pregnancy is a public concern , because 
 nanoparticles may pass from the mother to the fetus across the placenta . 
 the purpose of 
 this study was to determine the possible translocation pathway of gold nanoparticles 
 across the maternal  fetal barrier as well as the toxicity of intravenously administered 
 gold nanoparticles to the placenta and fetus . 
 pregnant icr mice were intravenously 
 injected with 0.01% of 20- and 50-nm gold nanoparticle solutions on the 16th and 17th days 
 of gestation . 
 there was no sign of toxic damage to the placentas as well as maternal and 
 fetal organs of the mice treated with 20- and 50-nm gold nanoparticles . 
 icp - ms analysis 
 demonstrated significant amounts of gold deposited in the maternal livers and placentas , 
 but no detectable level of gold in the fetal organs . 
 however , electron microscopy 
 demonstrated an increase of endocytic vesicles in the cytoplasm of syncytiotrophoblasts 
 and fetal endothelial cells in the maternal  fetal barrier of mice treated with gold 
 nanoparticles . 
 clathrin immunohistochemistry and immunoblotting showed increased 
 immunoreactivity of clathrin protein in the placental tissues of mice treated with 20- and 
 50-nm gold nanoparticles ; clathrin immunopositivity was observed in syncytiotrophoblasts 
 and fetal endothelial cells . 
 in contrast , caveolin-1 immunopositivity was observed 
 exclusively in the fetal endothelium . 
 these findings suggested that intravenous 
 administration of gold nanoparticles may upregulate clathrin- and caveolin - mediated 
 endocytosis at the maternal  fetal barrier in mouse placenta .