iron oxide nanoparticles have been used in various fields of biomedical research and clinical applications , including magnetic resonance imaging , cell labeling , cancer therapy and drug delivery.14 in particular , resovist , a preparation of carboxydextran - coated superparamagnetic iron oxide nanoparticles , has been approved as a diagnostic contrast agent in many countries . previous studies have shown that iron oxide nanoparticles can be taken up by the reticuloendothelial system upon systemic administration , with the liver and spleen being the major organs of drug deposition.57 in addition , immune organs have been shown to be the main sites for the deposition of other nanoparticles following systemic exposure.8,9 hence , the interaction between nanoparticles and immune cells and the consequences of such interactions are relevant issues in addressing the potential health impact of iron oxide and other nanoparticles . for example , iron oxide nanoparticles have been shown to suppress the phagocytic activity of raw 264.7 cells , a murine macrophage line.10 exposure of primary macrophages to iron oxide nanoparticles in culture resulted in a marked induction of oxidative stress and apoptosis.11 furthermore , animal studies have shown that intratracheal instillation of mice with iron oxide nanoparticles induced a marked infiltration of inflammatory cells in the lungs and elevated levels of proinflammatory cytokines , including interleukin ( il)-1 , il-6 and tumor necrosis factor- in the bronchoalveolar lavage fluid.12 these results clearly demonstrated that the functionality of macrophages was modulated by iron oxide nanoparticles upon in vitro and in vivo exposure . in addition to macrophages , a recent study has shown that t cells were another target in the immune system sensitive to iron oxide nanoparticles . a single intravenous administration of iron oxide nanoparticles to normal mice increased the number of both cd4 and cd8 cells in the peripheral blood , and the serum levels of il-2 and interferon ( ifn)- , two critical cytokines predominantly released by t cells.13 together , these reports demonstrated that exposure to iron oxide nanoparticles affected some aspects of immune reactions mediated by macrophages and t cells . however , it is currently unclear if antigen - specific immunity is influenced by iron oxide nanoparticles . in light of the available evidence showing the impacts of iron oxide nanoparticles on the functionality of macrophages and t cells , the objective of the present study was to investigate the effect of iron oxide nanoparticles on antigen - specific immune responses in a murine model sensitized with ovalbumin ( ova ) , a t cell - dependent antigen.14,15 all reagents were purchased from sigma ( st louis , mo ) unless otherwise stated . enzyme - linked immunosorbent assay ( elisa ) sets for cytokine measurement were purchased from bd biosciences ( san diego , ca ) . fetal bovine serum ( fbs ) and cell culture supplies resovist ( schering ag , berlin , germany ) , a commercial preparation of carboxydextran - coated iron oxide nanoparticles containing 28 mg fe / ml , was used in the present study . male balb / c mice ( 5 weeks old ) were obtained from biolasco ( ilan , taiwan ) . on arrival , mice were randomly transferred to plastic cages containing aspen bedding and quarantined for at least 1 week . mice were housed in a temperature ( 22c  2c ) , humidity ( 50%  20% ) and light ( 12-hour light / dark cycle)-controlled environment . food and water were supplied ad libitum . a previously described sensitization protocol was employed with minor modifications.16,17 briefly , the mice were randomly divided into the following groups ( 35 animals / group ) : nave ( na ) , ovalbumin - sensitized ( ova ) , vehicle - treated and ova - sensitized ( vh ) , and iron oxide nanoparticle - treated and ova - sensitized ( figure 1 ) . except for na and ova groups , the mice were administered intravenously with a single dose of iron oxide nanoparticles ( 10 , 30 and 60 mg fe / kg ; 0.2 ml / mouse ) and/or vehicle ( vh ; saline ; 0.2 ml / mouse ) . except for the na group , the mice were sensitized 1 hour after drug administration with ova by intraperitoneal injection using a sensitization solution containing 100 g ova plus 1 mg alum ( as adjuvant ) in saline ( 250 l / mouse ) . blood samples from individual mice were collected by cardiac puncture , and the serum was obtained by centrifugation of the coagulated blood at 3000  g for 15 min . all animal experiments were approved by the institutional animal care and use committee of the national taiwan university . spleens from the mice of the same group were aseptically isolated , pooled and made into single cell suspensions as described previously.18 splenocytes were cultured in rpmi 1640 medium supplemented with 100 u / ml penicillin , 100 g / ml streptomycin , and 10% heat inactivated fbs . in all cases , ova - specific igg1 and igg2a were measured as previously described.19 in brief , elisa plates were coated with 0.05% ova in coating buffer ( 0.1 m nahco3 ) , and blocked with 1% bovine serum albumin in phosphate - buffered saline ( pbs ) containing 0.05% tween20 ( pbst ) . after washing with pbst , serum samples were placed into wells ( 50 l / well ) and incubated for 1 hour . after another washing , horse radish peroxidase - conjugated anti - mouse igg1 and igg2a was added ( 50 l / well ) and incubated for 1 hour . lastly , wells were washed and a tetramethylbenzidine solution ( 50 l / well ) was added for colorimetric detection of bound peroxidase conjugate . the reaction was stopped by adding 150 l per well of 3n h2so4 . the optical density ( od ) was measured at 450 nm using a microplate reader ( molecular devices , inc . , sunnyvale , ca ) . as the assay does not include a standard curve , the results are expressed as od values . the viability of splenocytes was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl - tetrazolium bromide ( mtt ) assay as previously described.20 briefly , splenocytes ( 5  10 cells / ml ) were cultured in triplicate ( 100 l / well ) in 96-well plates in the absence ( no stimulation ) or presence of ova ( 50 g / ml ) for 72 hours or concanavalina ( cona ; 5 g / ml ) for 48 hours . an mtt stock solution ( 5 mg / ml in pbs ) was added to each well ( 10 l / well ) and incubated for 4 hours . the formed formazan was then dissolved by adding 200 l 0.1 n acid - isopropanol per well and shaken gently . od was measured at 570 nm , and at 630 nm as a background reference , using a microplate reader . the splenocytes ( 5  10 cells / ml ) were cultured in quadruplicate in 48-well plates ( 0.25 ml / well ) , and stimulated with ova ( 50 g / ml ) for 72 hours or cona ( 5 g / ml ) for 48 hours . the supernatants were collected and quantified for il-4 and ifn- by elisa as previously described.19 splenocytes obtained from each group were stained with rat anti - mouse cd4 conjugated with fluorescein isothiocyanate ( fitc ) and rat anti - mouse cd8 and b220 conjugated with pe - cy5 antibodies ( biolegend , san diego , ca ) in pbs containing 2% fetal bovine serum ( fbs ) . after washing , the single cell fluorescence of 10,000 cells for each sample was measured by a flow cytometer ( bd facscalibur , san jose , ca ) . data were analyzed using the software flowjo 5.7 ( tree star inc , ashland , or ) . the mean  standard error ( se ) was determined for each treatment group in the individual experiments . homogeneous data were evaluated by a parametric analysis of variance , and dunnett s two - tailed t - test was used to compare treatment groups to the control group . all reagents were purchased from sigma ( st louis , mo ) unless otherwise stated . enzyme - linked immunosorbent assay ( elisa ) sets for cytokine measurement were purchased from bd biosciences ( san diego , ca ) . fetal bovine serum ( fbs ) and cell culture supplies resovist ( schering ag , berlin , germany ) , a commercial preparation of carboxydextran - coated iron oxide nanoparticles containing 28 mg fe / ml , was used in the present study . male balb / c mice ( 5 weeks old ) were obtained from biolasco ( ilan , taiwan ) . on arrival , mice were randomly transferred to plastic cages containing aspen bedding and quarantined for at least 1 week . mice were housed in a temperature ( 22c  2c ) , humidity ( 50%  20% ) and light ( 12-hour light / dark cycle)-controlled environment . a previously described sensitization protocol was employed with minor modifications.16,17 briefly , the mice were randomly divided into the following groups ( 35 animals / group ) : nave ( na ) , ovalbumin - sensitized ( ova ) , vehicle - treated and ova - sensitized ( vh ) , and iron oxide nanoparticle - treated and ova - sensitized ( figure 1 ) . except for na and ova groups , the mice were administered intravenously with a single dose of iron oxide nanoparticles ( 10 , 30 and 60 mg fe / kg ; 0.2 ml / mouse ) and/or vehicle ( vh ; saline ; 0.2 ml / mouse ) . except for the na group , the mice were sensitized 1 hour after drug administration with ova by intraperitoneal injection using a sensitization solution containing 100 g ova plus 1 mg alum ( as adjuvant ) in saline ( 250 l / mouse ) . blood samples from individual mice were collected by cardiac puncture , and the serum was obtained by centrifugation of the coagulated blood at 3000  g for 15 min . all animal experiments were approved by the institutional animal care and use committee of the national taiwan university . spleens from the mice of the same group were aseptically isolated , pooled and made into single cell suspensions as described previously.18 splenocytes were cultured in rpmi 1640 medium supplemented with 100 u / ml penicillin , 100 g / ml streptomycin , and 10% heat inactivated fbs . in all cases , splenocytes were cultured at 37c in 5% co2 . ova - specific igg1 and igg2a were measured as previously described.19 in brief , elisa plates were coated with 0.05% ova in coating buffer ( 0.1 m nahco3 ) , and blocked with 1% bovine serum albumin in phosphate - buffered saline ( pbs ) containing 0.05% tween20 ( pbst ) . after washing with pbst , serum samples were placed into wells ( 50 l / well ) and incubated for 1 hour . after another washing , horse radish peroxidase - conjugated anti - mouse igg1 and igg2a was added ( 50 l / well ) and incubated for 1 hour . lastly , wells were washed and a tetramethylbenzidine solution ( 50 l / well ) was added for colorimetric detection of bound peroxidase conjugate . the reaction was stopped by adding 150 l per well of 3n h2so4 . the optical density ( od ) was measured at 450 nm using a microplate reader ( molecular devices , inc . , as the assay does not include a standard curve , the results are expressed as od values . the viability of splenocytes was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl - tetrazolium bromide ( mtt ) assay as previously described.20 briefly , splenocytes ( 5  10 cells / ml ) were cultured in triplicate ( 100 l / well ) in 96-well plates in the absence ( no stimulation ) or presence of ova ( 50 g / ml ) for 72 hours or concanavalina ( cona ; 5 g / ml ) for 48 hours . an mtt stock solution ( 5 mg / ml in pbs ) was added to each well ( 10 l / well ) and incubated for 4 hours . the formed formazan was then dissolved by adding 200 l 0.1 n acid - isopropanol per well and shaken gently . od was measured at 570 nm , and at 630 nm as a background reference , using a microplate reader . the splenocytes ( 5  10 cells / ml ) were cultured in quadruplicate in 48-well plates ( 0.25 ml / well ) , and stimulated with ova ( 50 g / ml ) for 72 hours or cona ( 5 g / ml ) for 48 hours . splenocytes obtained from each group were stained with rat anti - mouse cd4 conjugated with fluorescein isothiocyanate ( fitc ) and rat anti - mouse cd8 and b220 conjugated with pe - cy5 antibodies ( biolegend , san diego , ca ) in pbs containing 2% fetal bovine serum ( fbs ) . after washing , the single cell fluorescence of 10,000 cells for each sample was measured by a flow cytometer ( bd facscalibur , san jose , ca ) . data were analyzed using the software flowjo 5.7 ( tree star inc , ashland , or ) . the mean  standard error ( se ) was determined for each treatment group in the individual experiments . homogeneous data were evaluated by a parametric analysis of variance , and dunnett s two - tailed t - test was used to compare treatment groups to the control group . mice were intravenously administered with a single dose of iron oxide nanoparticles ( 1060 mg fe / kg of body weight ) and then sensitized with ova ( figure 1 ) . the doses were chosen on the basis of previous studies using a similar range for magnetic resonance imaging of the liver and musculoskeletal infections in rats.21,22 to measure humoral responses , serum samples from individual mice were collected 7 days after the ova sensitization and ova - specific antibodies were examined as previously described.19 a marked increase in the serum levels of ova - specific igg1 and igg2a was observed in ova - sensitized mice , as compared to non - sensitized nave mice ( figure 2a and b ; ova versus na ; p < 0.05 ) , indicating a successful induction of humoral responses . no significant difference between the vh and ova groups was observed ( figure 2a and b ; vh versus ova ) , demonstrating that the vh treatment per se has no effect on the antibody responses . the production of ova - specific igg1 was attenuated by the doses of 30 and 60 mg fe / kg ( figure 2a ; p < 0.05 ) , whereas the low dose ( 10 mg fe / kg ) was ineffective . these results showed a trend of dose - dependency by iron oxide nanoparticles on igg1 production . iron oxide nanoparticles ( 1060 mg fe / kg ) also demonstrated a suppressive effect on the serum production of ova - specific igg2a ( figure 2b ; p < 0.05 ) , in which the magnitude of suppression by all three doses was comparable , and no dose - dependency was observed . as t cells play a pivotal role in antigen - specific humoral responses , we examined the effect of iron oxide nanoparticles on the functionality of t cells . splenocytes isolated from nave and ova - sensitized mice were stimulated with ova ( 50 g / ml ) in culture for 72 hours to induce antigen - specific cytokine production . as expected , the amount of cytokines produced by splenocytes of the non - sensitized mice ( na ) was very low , and the ova stimulation markedly increased the production of il-4 and ifn- by splenocytes of ova - sensitized mice ( figure 3a and b ; ova versus na ; p < 0.05 ) . the production of ifn- was significantly suppressed in all iron oxide nanoparticle - treated groups with a comparable magnitude of inhibition between the 3 doses ( figure 3a ; p < 0.05 ) . the production of il-4 was also markedly attenuated by the doses of 30 and 60 mg fe / kg ( figure 3b ; p < 0.05 ) , whereas the low dose ( 10 mg fe / kg ) was ineffective . as the expression of antigen - induced cytokines was suppressed by iron oxide nanoparticle treatment , the viability of splenocytes was further examined . as shown in figure 4a , the viability of the ova - stimulated splenocytes was attenuated in all three iron oxide nanoparticle - treated groups ( p < 0.05 ) . the cellularity of splenocytes was also examined by flow cytometry , and no significant difference was observed in the percentage of splenic cd4 , cd8 and b220 cells among na , vh- and iron oxide nanoparticle - treated groups ( table 1 ) . to further investigate the potential influence of iron oxide nanoparticles on t cell reactivity , splenocytes were stimulated with the t - cell mitogen cona ( 5 g / ml ) for 48 hours and their viability was measured . interestingly , the viability of cona - stimulated splenocytes was not affected by treatment with iron oxide nanoparticles ( figure 4b ) . mice were intravenously administered with a single dose of iron oxide nanoparticles ( 1060 mg fe / kg of body weight ) and then sensitized with ova ( figure 1 ) . the doses were chosen on the basis of previous studies using a similar range for magnetic resonance imaging of the liver and musculoskeletal infections in rats.21,22 to measure humoral responses , serum samples from individual mice were collected 7 days after the ova sensitization and ova - specific antibodies were examined as previously described.19 a marked increase in the serum levels of ova - specific igg1 and igg2a was observed in ova - sensitized mice , as compared to non - sensitized nave mice ( figure 2a and b ; ova versus na ; p < 0.05 ) , indicating a successful induction of humoral responses . no significant difference between the vh and ova groups was observed ( figure 2a and b ; vh versus ova ) , demonstrating that the vh treatment per se has no effect on the antibody responses . the production of ova - specific igg1 was attenuated by the doses of 30 and 60 mg fe / kg ( figure 2a ; p < 0.05 ) , whereas the low dose ( 10 mg fe / kg ) was ineffective . these results showed a trend of dose - dependency by iron oxide nanoparticles on igg1 production . iron oxide nanoparticles ( 1060 mg fe / kg ) also demonstrated a suppressive effect on the serum production of ova - specific igg2a ( figure 2b ; p < 0.05 ) , in which the magnitude of suppression by all three doses was comparable , and no dose - dependency was observed . as t cells play a pivotal role in antigen - specific humoral responses , we examined the effect of iron oxide nanoparticles on the functionality of t cells . splenocytes isolated from nave and ova - sensitized mice were stimulated with ova ( 50 g / ml ) in culture for 72 hours to induce antigen - specific cytokine production . as expected , the amount of cytokines produced by splenocytes of the non - sensitized mice ( na ) was very low , and the ova stimulation markedly increased the production of il-4 and ifn- by splenocytes of ova - sensitized mice ( figure 3a and b ; ova versus na ; p < 0.05 ) . the production of ifn- was significantly suppressed in all iron oxide nanoparticle - treated groups with a comparable magnitude of inhibition between the 3 doses ( figure 3a ; p < 0.05 ) . the production of il-4 was also markedly attenuated by the doses of 30 and 60 mg fe / kg ( figure 3b ; p < 0.05 ) , whereas the low dose ( 10 mg fe / kg ) was ineffective . as the expression of antigen - induced cytokines was suppressed by iron oxide nanoparticle treatment , the viability of splenocytes was further examined . as shown in figure 4a , the viability of the ova - stimulated splenocytes was attenuated in all three iron oxide nanoparticle - treated groups ( p < 0.05 ) . the cellularity of splenocytes was also examined by flow cytometry , and no significant difference was observed in the percentage of splenic cd4 , cd8 and b220 cells among na , vh- and iron oxide nanoparticle - treated groups ( table 1 ) . to further investigate the potential influence of iron oxide nanoparticles on t cell reactivity , splenocytes were stimulated with the t - cell mitogen cona ( 5 g / ml ) for 48 hours and their viability was measured . interestingly , the viability of cona - stimulated splenocytes was not affected by treatment with iron oxide nanoparticles ( figure 4b ) . a common application of nanomedicine is the clinical use of superparamagnetic iron oxide nanoparticles as contrast agents to enhance magnetic resonance imaging . previous studies have shown that a considerable portion of systemically administered iron oxide nanoparticles is taken up by the reticuloendothelial system leading to a potential exposure of immune cells to high concentrations of the nanoparticles.57,23,24 although a few studies demonstrated that iron oxide nanoparticles affected the functionality of macrophages and lymphocytes,10,25,26 evidence pertaining to the influence of iron oxide nanoparticles on antigen - specific immunity is scarce . in the present study , we investigated the in vivo effect of iron oxide nanoparticles on systemic antibody production and t cell responses in mice sensitized with ova . our data demonstrated that a single intravenous administration of iron oxide nanoparticles significantly attenuated the subsequent production of antigen - specific antibodies , as evidenced by a marked reduction in the serum levels of ova - specific igg1 and igg2a ( figure 2 ) . the viability and cytokine production by splenocytes re - stimulated with the antigen ova were also suppressed by iron oxide nanoparticles ( figures 3 and 4 ) . in contrast , the viability of splenocytes stimulated with cona , a t cell mitogen , was unaltered . collectively , these results indicate that systemic exposure to a single dose of iron oxide nanoparticles differentially suppressed antigen - specific t cell responses , in which the suppression of antigen - specific cytokine production may be attributed to a reduced viability of ova - stimulated splenocytes . t helper ( th ) cell - derived cytokines play a critical role in the activation and differentiation of b cells.27 in particular , the class switching from igm to igg2a and igg1 is dictated by th1 ( ie , ifn- ) and th2 ( ie , il-4 ) cytokines , respectively.27 in line with the attenuation of igg2a and igg1 , both ifn- and il-4 were suppressed in response to iron oxide nanoparticle exposure , suggesting that the attenuated antibody production may be attributed to the changes in th cell functions . this notion is further substantiated by our data showing that the viability of splenocytes re - stimulated with the sensitized antigen ova was attenuated by iron oxide nanoparticles . antibody production is a complex process that requires interactions among various immune cells and the action of many mediators . thus , in addition to t cells , other targets and/or mediators may be affected by iron oxide nanoparticles and contribute to the impaired humoral immunity . for example , a recent study showed that the capability of dendritic cells to process antigen and stimulate t cell cytokine expression was suppressed by exposure to iron oxide nanoparticles in vitro.28 since ova is a t cell - dependent antigen that requires to be processed and presented by antigen - presenting cells,29 we postulated that antigen presenting cells may be also involved in iron oxide nanoparticle - mediated attenuation of antibody production . this notion is indirectly supported by our data showing that the activation of splenocytes by cona , whose responses are not antigen - specific , was not influenced by iron oxide nanoparticles . further studies are required to determine whether the functionality of antigen - presenting cells is modulated by iron oxide nanoparticles in vivo . resovist has been used clinically as a liver - specific magnetic resonance ( mr ) contrast agent ; for this indication the recommended dose range is between 0.420.65 mg fe / kg in humans . notably , a wider range of doses ( 0.1422.4 mg fe / kg ) of resovist have been applied in rats for tracking mr signals in the liver.21 in addition to liver contrasting , resovist at greater doses has been employed for imaging other tissues . for example , doses between 0.5665 mg fe / kg have been used to assess musculoskeletal infections in rats.22 based on these lines of evidence , the dose range ( 1060 mg / kg ) used in our studies could be pharmacologically relevant for certain applications of iron oxide nanoparticles . the production of igg2a and ifn- , but not igg1 and il-4 , was suppressed by the low dose of iron oxide nanoparticles ( 10 mg fe / kg ) , whereas all the measured igg and cytokines were attenuated by the higher doses ( 30 and 60 mg fe / kg ) . these results suggest a differential sensitivity between th1 and th2 responses to iron oxide nanoparticles , in which the th1 immunity is slightly more sensitive to iron oxide nanoparticles . a previous report has shown that intratracheal administration of mice with iron oxide nanoparticles affected inflammation and t cell cytokine expression in the airways.25 given the pivotal role of th cells in antigen - specific immunity , systemic exposure to iron oxide nanoparticles may have a potential impact on the host immune responses to antigens . on one hand , iron oxide nanoparticle - mediated attenuation of t cell responses may be beneficial to people predisposed to allergy . on the other hand , the suppressed t cell immunity may be detrimental in situations where an appropriate immune response is necessary for the protection of the host . the present study demonstrates that a single intravenous administration of iron oxide nanoparticles markedly suppresses t cell - mediated immune reactions with a broad spectrum covering both th1- and th2-type responses . we speculate that systemic exposure to iron oxide nanoparticles may have an impact on host immunity in response to specific antigens . on the base of these results ,
background : superparamagnetic iron oxide nanoparticles have been used in clinical applications as a diagnostic contrasting agent . 
 previous studies showed that iron oxide nanoparticles deposited in the liver and spleen after systemic administration . 
 the present study investigated the effect of iron oxide nanoparticles on antigen - specific immune responses in mice sensitized with the t cell - dependent antigen ovalbumin ( ova).methods : balb / c mice were intravenously administered with a single dose of iron oxide nanoparticles ( 1060 mg fe / kg ) 1 hour prior to ova sensitization , and the serum antibody production and splenocyte reactivity were examined 7 days later.results:the serum levels of ova - specific igg1 and igg2a were significantly attenuated by treatment with iron oxide nanoparticles . 
 the production of interferon- and interleukin-4 by splenocytes re - stimulated with ova in culture was robustly suppressed in mice administered with iron oxide nanoparticles . 
 the viability of ova - stimulated splenocytes was also attenuated . in contrast , treatment with iron oxide nanoparticles did not affect the viability of splenocytes stimulated with concanavalin a , a t - cell mitogen.conclusion:collectively , these data indicate that systemic exposure to a single dose of iron oxide nanoparticles compromises subsequent antigen - specific immune reactions , including the serum production of antigen - specific antibodies , and the functionality of t cells .