the use of nanoparticles for cellular imaging is among the most important clinical breakthrough of the past decade . in particular , superparamagnetic iron oxide ( spio ) nanoparticles enhance contrast in magnetic resonance imaging ( mri ) , which allows clinicians to monitor anatomical , physiological , and molecular changes during the evolution of a disease or treatment .
following intravenous injection , these nanoparticles accumulate in macrophages residing in the liver , bone marrow , and spleen , as well as tumors and sites of inflammation .
accordingly , applications include the detection of inflammatory diseases , in vivo stem cell tracking,1 hyperthermia therapy,2 lymph node detection,3 and anticancer drug delivery.4 the current applications for spio nanoparticles are limited because these relatively large particles , with an average diameter of 80 nm , are rapidly internalized by the mononuclear phagocytic system of the liver and the spleen .
this problem is currently addressed by the development of ultrasmall superparamagnetic iron oxide ( uspio ) nanoparticles ( < 50 nm).5 in vivo studies recently showed that uspio nanoparticles of 35 nm in diameter have a longer half - life in the circulation system , allowing the labeling of macrophages migrating to remote areas.6,7 these studies opened the door to a number of new applications for molecular imaging because macrophages migrate and accumulate at sites of inflammation,8 autoimmune neuritis,9 renal ischemia,10 and solid organ transplant rejection.11 the use of uspio agents has allowed for the direct visualization of macrophage infiltration of carotid atheroma in clinical study.12,13 the surface coating of uspio nanoparticles also influences their stability and cellular uptake by macrophages.6 dextran - based coatings are preferred because of their low toxicity and they are biodegradable.14 for instance , dextran - coated spio nanoparticles and ferucarbotran are approved for liver mri . moreover , carboxydextran - coated spio nanoparticles have undergone clinical trials for mri evaluation of lymph node metastasis;15,16 however , the impact of uspio nanoparticles size and dextran coating composition on the uptake by macrophages has not been determined .
the aim of the present study is to determine the impact of particle size and surface coating on the cellular uptake and relaxing time of spio nanoparticles in mouse macrophages by mri and microscopy .
the uspio ( 28 nm ) and spio ( 72 and 74 nm ) nanoparticles were coated with alkali - treated dextran ( atdm ) or carboxymethyl dextran ( cmdm ) .
atdm ( surface voltage potential : 15 mv ) was tested with particle sizes of 28 and 74 nm .
cmdm ( surface voltage potential : 24 mv ) was tested with particle sizes of 28 and 72 nm .
all spio and uspio compounds were purchased from the meito sangyo company , ltd ( aichi , japan).17 the mouse macrophage cell line raw264 was provided by the riken bioresource center ( riken tsukuba institute , tsukuba , ibaraki , japan ) .
raw264 cells ( 1  10 ) were cultured in minimum essential medium ( sigma - aldrich , tokyo , japan ) , 10% fetal calf serum ( nichirei corporation , tokyo , japan ) , and 0.1 mm nonessential amino acids ( sigma - aldrich ) in 93 mm  21 mm petri dishes ( iwaki , tokyo , japan ) with 18 mm square cover glasses ( matusnami glass ind , ltd , osaka , japan ) .
the contrast of proton mri images depends on the relaxation times of the nuclear magnetization ( t1 , longitudinal ; t2 , transverse ) .
iron oxide nanoparticles primarily affect t2 and work as negative contrast agents . a high intracellular concentration of spio and uspio nanoparticles results in the reduction of t2 relaxation time . prior to the mri experiment ,
the concentrations of uspio particles were adjusted to 0.005 mg fe / ml and prepared in five sample tubes with water samples serving as controls .
mri experiments for the three times measurements of the t2 relaxation times were performed on an 11.7-t mri scanner ( bruker biospin , ettlingen , germany ) and used a volume rf coil with a 25 mm inner diameter for transmission and reception ( m2 m imaging corp , cleveland , oh ) .
t2 mapping was performed using a multislice , multiecho spin - echo sequence ( repetition time = 3000 ms ; slice thickness = 1 mm ; field of view = 25.6  25.6 mm ; matrix = 128  128 ; slice orientation = transaxial ; number of repetitions = 1 ) with echo times ranging from 10 to 100 ms in steps of 10 ms .
t2 maps were calculated from a single exponential fitting of mri signal intensities at each echo time point .
the experiments were conducted to measure the t2 relaxation times of the macrophages labeled with spio or uspio nanoparticles .
all uspio particles were dissolved in the cell growth medium at a concentration of 0.3 mg fe / ml , which is similar to the concentration used in another published report.18 cells ( 2  10 ) were incubated with medium containing uspio particles for 1 , 2 , or 4 h ( same time points noted in a previous report)19 at 37c under 5% co2 .
after incubation , the medium was removed by two washes with phosphate - buffered saline ( pbs ) followed by fixation of the cells for 30 min with 7.5% formalin .
the presence of iron oxides in the raw264 cells was detected by staining with berlin blue ( kfe(3)fe(2)(cn)6 ) ( wako pure chemical industries , ltd , osaka , japan)20 for 20 min . after washing away the berlin blue with pbs , the cell nuclei were counter - stained with nuclear fast red for 5 min .
the nuclear fast red was removed by washing with pbs three times , and the cell specimens were fixed to the cover glass . for each experiment ,
the labeling efficiency of each type of uspio nanoparticle was assessed visually by three observers using a bz-9000 microscope ( keyence corporation , osaka , japan ) . the labeling efficiencies ( percentages of spio / uspio uptake )
were qualitatively assessed from the microscope images in three independent experiments by estimating the number of iron - positive cells within five randomly selected 200 m fields .
viable cells are not stained by trypan blue , while dead and lysed cells are stained .
all uspio particles were dissolved in cell growth medium at a concentration of 0.3 mg fe / ml , which is a lower concentration when compared to a sample test because the concentrations of spio and uspio in macrophage cells were lower than sample spio and uspio concentrations .
cells ( 2  10 ) were incubated in a medium containing uspio particles for 1 , 2 , or 4 h at 37c under 5% co2 .
after incubation , the medium was removed with two washes of pbs and the cells were fixed with formalin .
cells were stained with trypan blue for 10 min and the viable and dead cells were counted under a microscope .
the percentages of viable cells were qualitatively assessed by counting the number of trypan blue - positive cells within five randomly selected 200 m fields .
all uspio nanoparticles were dissolved in cell growth medium at a concentration of 0.3 mg / ml . raw264 cells ( 2  10 ) were incubated with medium containing uspio particles for 30 min at 37c under 5% co2 .
after incubation , the medium was removed by two washes with pbs and the cells were fixed with formalin .
the concentration of raw264 cells was adjusted to 4  10 cells / ml and the cells ( 0.5 ml ) were dissolved in 0.1% agarose gel ( 0.5 ml ) to give a final concentration of 2.0  10 cells / ml . the sample temperature was maintained at approximately 23c and the cells were imaged after settling into a pellet by gravity . for measurement of t2 relaxation time , t2 mapping was performed using a multislice multiecho spin - echo sequence ( repetition time = 3000 ms ; slice thickness = 1 mm ; field of view = 25.6  25.6 mm ; matrix = 128  128 ; slice orientation = transaxial ; number of repetitions = 1 ) with echo times ranging from 10 to 100 ms in steps of 10 ms .
t2 maps were calculated from a single exponential fitting of mri signal intensities at each echo time point .
all statistical analysis was performed using prism ( version 5 ; graphpad software inc , la jolla , ca ) .
one - way analysis of variance with the bonferroni correction was applied in order to compare changes in percentages of spio and uspio uptake , cell viability , and t2 relaxation times for all spio / uspio nanoparticles and labeled cells .
atdm ( surface voltage potential : 15 mv ) was tested with particle sizes of 28 and 74 nm .
cmdm ( surface voltage potential : 24 mv ) was tested with particle sizes of 28 and 72 nm .
all spio and uspio compounds were purchased from the meito sangyo company , ltd ( aichi , japan).17
the mouse macrophage cell line raw264 was provided by the riken bioresource center ( riken tsukuba institute , tsukuba , ibaraki , japan ) .
raw264 cells ( 1  10 ) were cultured in minimum essential medium ( sigma - aldrich , tokyo , japan ) , 10% fetal calf serum ( nichirei corporation , tokyo , japan ) , and 0.1 mm nonessential amino acids ( sigma - aldrich ) in 93 mm  21 mm petri dishes ( iwaki , tokyo , japan ) with 18 mm square cover glasses ( matusnami glass ind , ltd , osaka , japan ) .
the contrast of proton mri images depends on the relaxation times of the nuclear magnetization ( t1 , longitudinal ; t2 , transverse ) . iron oxide nanoparticles primarily affect t2 and work as negative contrast agents . a high intracellular concentration of spio and uspio nanoparticles results in the reduction of t2 relaxation time .
prior to the mri experiment , the concentrations of uspio particles were adjusted to 0.005 mg fe / ml and prepared in five sample tubes with water samples serving as controls .
mri experiments for the three times measurements of the t2 relaxation times were performed on an 11.7-t mri scanner ( bruker biospin , ettlingen , germany ) and used a volume rf coil with a 25 mm inner diameter for transmission and reception ( m2 m imaging corp , cleveland , oh ) .
t2 mapping was performed using a multislice , multiecho spin - echo sequence ( repetition time = 3000 ms ; slice thickness = 1 mm ; field of view = 25.6  25.6 mm ; matrix = 128  128 ; slice orientation = transaxial ; number of repetitions = 1 ) with echo times ranging from 10 to 100 ms in steps of 10 ms .
t2 maps were calculated from a single exponential fitting of mri signal intensities at each echo time point .
the experiments were conducted to measure the t2 relaxation times of the macrophages labeled with spio or uspio nanoparticles .
all uspio particles were dissolved in the cell growth medium at a concentration of 0.3 mg fe / ml , which is similar to the concentration used in another published report.18 cells ( 2  10 ) were incubated with medium containing uspio particles for 1 , 2 , or 4 h ( same time points noted in a previous report)19 at 37c under 5% co2 .
after incubation , the medium was removed by two washes with phosphate - buffered saline ( pbs ) followed by fixation of the cells for 30 min with 7.5% formalin .
the presence of iron oxides in the raw264 cells was detected by staining with berlin blue ( kfe(3)fe(2)(cn)6 ) ( wako pure chemical industries , ltd , osaka , japan)20 for 20 min .
after washing away the berlin blue with pbs , the cell nuclei were counter - stained with nuclear fast red for 5 min .
the nuclear fast red was removed by washing with pbs three times , and the cell specimens were fixed to the cover glass . for each experiment ,
the labeling efficiency of each type of uspio nanoparticle was assessed visually by three observers using a bz-9000 microscope ( keyence corporation , osaka , japan ) .
the labeling efficiencies ( percentages of spio / uspio uptake ) were qualitatively assessed from the microscope images in three independent experiments by estimating the number of iron - positive cells within five randomly selected 200 m fields .
viable cells are not stained by trypan blue , while dead and lysed cells are stained .
all uspio particles were dissolved in cell growth medium at a concentration of 0.3 mg fe / ml , which is a lower concentration when compared to a sample test because the concentrations of spio and uspio in macrophage cells were lower than sample spio and uspio concentrations .
cells ( 2  10 ) were incubated in a medium containing uspio particles for 1 , 2 , or 4 h at 37c under 5% co2 .
after incubation , the medium was removed with two washes of pbs and the cells were fixed with formalin .
cells were stained with trypan blue for 10 min and the viable and dead cells were counted under a microscope .
the percentages of viable cells were qualitatively assessed by counting the number of trypan blue - positive cells within five randomly selected 200 m fields .
all uspio nanoparticles were dissolved in cell growth medium at a concentration of 0.3 mg / ml . raw264 cells ( 2  10 ) were incubated with medium containing uspio particles for 30 min at 37c under 5% co2 .
after incubation , the medium was removed by two washes with pbs and the cells were fixed with formalin .
the concentration of raw264 cells was adjusted to 4  10 cells / ml and the cells ( 0.5 ml ) were dissolved in 0.1% agarose gel ( 0.5 ml ) to give a final concentration of 2.0  10 cells / ml . the sample temperature was maintained at approximately 23c and the cells were imaged after settling into a pellet by gravity . for measurement of t2 relaxation time , t2 mapping was performed using a multislice multiecho spin - echo sequence ( repetition time = 3000 ms ; slice thickness = 1 mm ; field of view = 25.6  25.6 mm ; matrix = 128  128 ; slice orientation = transaxial ; number of repetitions = 1 ) with echo times ranging from 10 to 100 ms in steps of 10 ms .
t2 maps were calculated from a single exponential fitting of mri signal intensities at each echo time point .
all statistical analysis was performed using prism ( version 5 ; graphpad software inc , la jolla , ca ) .
one - way analysis of variance with the bonferroni correction was applied in order to compare changes in percentages of spio and uspio uptake , cell viability , and t2 relaxation times for all spio / uspio nanoparticles and labeled cells .
the mri contrast provided by each type of nanoparticle formulation was compared by measuring the t2 relaxation time . at 0.005 mg fe / ml , t2 relaxation time of
the 74 nm atdm - coated nanoparticles was lower than for the other three formulations ( figure 1a ) .
since a low t2 relaxation time translates into a high mri negative contrast , these data suggest that atdm - coated spio nanoparticles would provide the best mri images among these formulations ( figure 1b ) .
microscopy analysis was used to compare the labeling efficiency of each nanoparticle formulation in terms of the percentage of macrophages labeled within 200-mm fields , and the rate of culture saturation over time .
overall , the atdm - coated nanoparticles provided a more efficient labeling of the macrophages than the cmdm - coated nanoparticles ( figure 2 ) .
quantitative analysis revealed that more than 60% of the macrophages were labeled by atdm - coated nanoparticles after 1 h , compared to < 10% with cmdm - coated nanoparticles , regardless of the particle size ( figure 3a ) .
this profile was maintained after 2 h. however , the efficiency of atdm - coated nanoparticle labeling had reached 100% , and cmdm - coated nanoparticles were detected only in 20%30% of macrophages .
after 4 h , all nanoparticle formulations had a labeling efficiency superior to 95% , except for 20%30% for cmdm - coated 28 and 72 nm nanoparticles .
figure 3b shows that none of the nanoparticle formulations affected the viability of the macrophages .
altogether , these data suggest that the use of uspio nanoparticles ( 28 nm ) , instead of spio nanoparticles ( 72 and 74 nm ) , does not improve the labeling efficiency of macrophages . in contrast , atdm coating provides a more rapid and efficient labeling of most cells for at least 4 h , which is an asset for mri imaging .
the t2 relaxation time of each nanoparticle formulation was measured following a 30-min labeling period of the macrophages to determine the impact of cellular uptake .
figure 4a shows the different mri signal intensities obtained for the four types of nanoparticles .
quantitative analysis revealed significantly higher t2 values for the cmdm - coated particles than the atdm - coated particles ( figure 4b ) . among the atdm - coated particles , uspio particles generated an even brighter signal change than the spio particles .
these data suggest that atdm - coated uspio particles would offer the best mri image contrast than all four formulations .
finally , the impact of cell density on the contrast quality of mri images was tested by measuring the t2 relaxation time of macrophages labeled with atdm - coated 74 nm nanoparticles for 30 min .
figure 5a shows that the t2 relaxation time decreases with increasing cell density . a linear relationship between relaxation time and cell density
these data suggest that the ability of mri negative contrast agent is increased at high cell density .
the mri contrast provided by each type of nanoparticle formulation was compared by measuring the t2 relaxation time . at 0.005 mg fe / ml , t2 relaxation time of
the 74 nm atdm - coated nanoparticles was lower than for the other three formulations ( figure 1a ) .
since a low t2 relaxation time translates into a high mri negative contrast , these data suggest that atdm - coated spio nanoparticles would provide the best mri images among these formulations ( figure 1b ) .
microscopy analysis was used to compare the labeling efficiency of each nanoparticle formulation in terms of the percentage of macrophages labeled within 200-mm fields , and the rate of culture saturation over time .
overall , the atdm - coated nanoparticles provided a more efficient labeling of the macrophages than the cmdm - coated nanoparticles ( figure 2 ) .
quantitative analysis revealed that more than 60% of the macrophages were labeled by atdm - coated nanoparticles after 1 h , compared to < 10% with cmdm - coated nanoparticles , regardless of the particle size ( figure 3a ) .
this profile was maintained after 2 h. however , the efficiency of atdm - coated nanoparticle labeling had reached 100% , and cmdm - coated nanoparticles were detected only in 20%30% of macrophages .
after 4 h , all nanoparticle formulations had a labeling efficiency superior to 95% , except for 20%30% for cmdm - coated 28 and 72 nm nanoparticles .
figure 3b shows that none of the nanoparticle formulations affected the viability of the macrophages .
altogether , these data suggest that the use of uspio nanoparticles ( 28 nm ) , instead of spio nanoparticles ( 72 and 74 nm ) , does not improve the labeling efficiency of macrophages . in contrast , atdm coating provides a more rapid and efficient labeling of most cells for at least 4 h , which is an asset for mri imaging .
the t2 relaxation time of each nanoparticle formulation was measured following a 30-min labeling period of the macrophages to determine the impact of cellular uptake .
figure 4a shows the different mri signal intensities obtained for the four types of nanoparticles .
quantitative analysis revealed significantly higher t2 values for the cmdm - coated particles than the atdm - coated particles ( figure 4b ) . among the atdm - coated particles , uspio particles generated an even brighter signal change than the spio particles .
these data suggest that atdm - coated uspio particles would offer the best mri image contrast than all four formulations .
finally , the impact of cell density on the contrast quality of mri images was tested by measuring the t2 relaxation time of macrophages labeled with atdm - coated 74 nm nanoparticles for 30 min .
figure 5a shows that the t2 relaxation time decreases with increasing cell density . a linear relationship between relaxation time and cell density
these data suggest that the ability of mri negative contrast agent is increased at high cell density .
the present study suggests that spio / uspio labeling efficiency in cultured mouse macrophages was influenced by the particle size and surface coating .
the labeling efficiencies of four spio / uspios in mouse macrophage cells could be visualized by microscopy and 11.7-t mri .
oude engberink et al reported that spio particles with a diameter of 150 nm were more efficiently incorporated into mononuclear cells than uspio particles with a diameter of 30 nm.19 particles with larger diameters were more efficiently incorporated into macrophages.21,22 after 1 to 4 h of administration , there were no differences in labeling efficiencies between atdm at 28 nm and at 74 nm ( figure 3a ) .
in addition , there was no difference in labeling efficiencies by cmdm 1 h after administration .
the diameter of spio used in previous studies was over 150 nm.21,22 the diameter of uspio used as a control was below 30 nm.23,24 the culture time varied from 30 min21 to 6 h.8 in our experiment , the diameters of uspio and spio particles that were compared were only about 30 and 70 nm and we show that particle size is not an important factor in the uptake of 28 and 74 nm iron nanoparticles irons . using an efficient labeling contrast agent is critical for cell imaging .
the most commonly used cell - labeling method is to culture cells with a contrast agent .
this method employs surface modification with cations,25 virus transfection,26 lipofectamine,27 and poly - l - lysine28 to increase the labeling efficiency .
these positively - charged reagents are cocultured with negatively - charged uspio to alter the uspio into a positively - charged compound .
such a charge alteration increases the affinity of uspio to negatively - charged cell membranes , thereby facilitating the intracellular uptake of the uspio contrast agent .
the mechanism through which the positively - charged contrast agent passes through the cell membrane remains unclear , however , it may include cell wall destruction , adhesion , and internalization .
the atdm - coated particles are more positively - charged compared to the cmdm - coated particles .
our experiment demonstrated a more efficient uptake of iron particles during approximately 0.54 h culture for more positively - charged atdm ( 15 mv ) than for cmdm ( 24 mv ) .
furthermore , in vitro 11.7-t mri of labeled cells also demonstrated a shorter t2 relaxation time for atdm - labeled than for cmdm - labeled cells , although cmdm and atdm had the same diameter ( figure 4b ) .
the different charge and coating states of the surface potential may have influenced the uptake by cultured macrophages .
, it has been shown that aminosilane - and dextran - coated nanoparticles did not affect the cell viability of mouse macrophage cells.29 however , silane - coated spio nanoparticles negatively affected mouse macrophage cell viability in a dose - dependent manner.29 moreover , silica - coated spio nanoparticles , rather than dextran - coated nanoparticles , displayed dose - dependent cytotoxicity.30 in our study , none of the nanoparticle formulations affected the viability of the macrophages ( figure 3b ) .
our dextran - coated nanoparticles are safer than nanoparticles coated with aminosiline and silica.29,30 the different charge and coating states of the surface potential did not influence the cell viability of cultured macrophages in the four types of spio and uspio contrast agents that we used . in the in vivo imaging of labeled macrophages ,
a longer half - life of blood is critical for increasing accessibility to target organs and tissues.7,8,28,29 spio particles with a diameter of about 50 nm are rapidly incorporated into kupffer cells in the liver and reticuloendothelial system in the spleen .
hence , the half - life of spio in the blood is as short as approximately 48 min.13,28 on the other hand , uspio particles with a diameter below 30 nm are barely recognized by the reticuloendothelial system .
the half - life of uspio in blood is as long as approximately 2436 h in humans and a few hours in rats.6,7,31 the current study shows that cmdm - coated nanoparticles label macrophages at a significantly lower rate than atdm - coated nanoparticles , suggesting that they should remain soluble in blood circulation for a longer period of time .
there are no differences in t2 relaxation time of labeled cells between atdm at 28 and 74 nm ( figure 4b ) ; however , the t2 relaxation time of contrast agent differs between atdm at 28 and 74 nm ( figure 1 ) .
hence , the shortened t2 relaxation time of labeled cells can not simply be explained by the difference in the labeling efficiency of cells .
also , no difference was noted in the t2 relaxation time of contrast agent between atdm at 28 and cmdm 28 nm ( figure 1 ) , even though the t2 relaxation times of atdm labeled cells were shorter than that of cmdm labeled cells ( figure 4b ) . thus , atdm uspio with a smaller diameter was more efficiently incorporated into cells than cmdm uspio , thereby providing a more effective in vivo contrast agent for macrophages .
we conclude that the best contrast agent would be atdm - coated 74 nm spio nanoparticles . despite recent interest in smaller particles ( uspio ) , this study shows that coating plays a more important role than size to optimize mri contrast , while increasing the nanoparticle labeling time .
 purposemagnetic resonance imaging ( mri ) using contrast agents like superparamagnetic iron oxide ( spio ) is an extremely versatile technique to diagnose diseases and to monitor treatment . 
 this study tested the relative importance of particle size and surface coating for the optimization of mri contrast and labeling efficiency of macrophages migrating to remote inflammation sites.materials and methodswe tested four spio and ultrasmall superparamagnetic iron oxide ( uspio ) , alkali - treated dextran magnetite ( atdm ) with particle sizes of 28 and 74 nm , and carboxymethyl dextran magnetite ( cmdm ) with particle sizes of 28 and 72 nm . 
 mouse macrophage raw264 cells were incubated with spios and uspios , and the labeling efficiency of the cells was determined by the percentage of berlin blue - stained cells and by measuring t2 relaxation times with 11.7-t mri . 
 we used trypan blue staining to measure cell viability.resultsanalysis of the properties of the nanoparticles revealed that atdm - coated 74 nm particles have a lower t2 relaxation time than the others , translating into a higher ability of mri negative contrast agent . among the other three candidates , cmdm - coated particles showed the highest t2 relaxation time once internalized by macrophages . regarding labeling efficiency , 
 atdm coating resulted in a cellular uptake higher than cmdm coating , independent of nanoparticle size . 
 none of these particle formulations affected macrophage viability.conclusionthis study suggests that coating is more critical than size to optimize the spio labeling of macrophages . among the formulations tested in this study , 
 the best mri contrast and labeling efficiency are expected with atdm - coated 74 nm nanoparticles . 
Introduction
Material and methods
Ultrasmall superparamagnetic iron oxides
Cell culture of mouse macrophage RAW264 cells
Measurement of SPIO and USPIO T2 relaxation times
Measurement of USPIO-labeling efficiency
Viability of USPIO-labeled cells
Measurement of labeled cells using 11.7-T MRI
Statistical analysis
Results
T
Evaluation of in vitro USPIO labeling efficiency by microscopy
Magnetic resonance imaging of labeled mouse macrophage cells
Discussion
Conclusion