although it is a relatively new modality , 
 magnetic resonance imaging ( mri ) has emerged as a prominent noninvasive 
 and nonradioactive imaging tool in diagnostic medicine and biomedical 
 research . in general , the mri signal intensity ( si ) is a function of the water proton density 
 of tissues under examination and the relative longitudinal and transverse 
 relaxation times , t1 and t2 , of the protons in the tissues . in some cases ,
image contrast can be directly produced by the inherent 
 variation of proton density in different tissues by altering the various 
 image acquisition parameters .
in other cases , it is necessary to administer 
 a contrast agent ( ca ) to improve the quality of the image and aid 
 in diagnosis .
the use of a contrast agent 
 also makes it possible to visualize certain pathological regions externally , 
 such as organs or cells of the human body via target - specific delivery 
 of the agent .
the majority of existing mri cas can be divided into two different 
 types based on which relaxation time they alter to a greater extent , t1-weighted and t2-weighted agents .
the most widely used 
 cas in clinical imaging are the paramagnetic gd - chelates 
 that can produce t1-weighted or positive 
 contrast enhancement .
the principal drawback of such 
 cas is their limited relaxivity because the observed relaxivity values 
 are usually a few percent of the theoretically attainable values predicted 
 by the solomon  bloembergen 
rather high tissue concentrations 
 ( 0.10.3 mm ) are required for these cas to be effective when 
 used with mr scanners of low magnetic fields from 0.3 to 3.0 t. the 
 paramagnetic relaxation enhancement has two separate components ( i.e. , 
 inner - sphere and outer - sphere relaxation contributions ) .
the mechanism 
 of inner - sphere relaxation in small molecular gd - chelates 
 requires that at least one water molecule be directly coordinated 
 to the paramagnetic metal center and be able to undergo a rapid exchange 
 with the water molecules in the outer sphere .
although increasing the number 
 of coordination water molecules can lead to a significant increase 
 of the inner - sphere relaxivity , the thermodynamic stability and/or 
 kinetic inertness of the gd - chelates will decrease as 
 a result .
the consequence is to make the complexes more susceptible 
 to displacement by proteins , biological ligands , or endogenous metal 
 ions , often resulting in the toxicity of the cas .
therefore , all of 
 the current clinical gd - chelates contain strictly one 
 coordinated water molecule .
furthermore , because 
 of nuclear magnetic relaxation dispersion with increasing larmor frequency , 
 the relaxivity of such cas decreases drastically with increasing magnetic 
 field strength to render them less effective on high - field scanners .
recently , the use of high - field scanners ( 4.7 
 to 9.0 t ) has become increasingly prevalent because they provide greater 
 spatial resolution , a shortened data acquisition time , and an improved 
 signal - to - noise ratio .
another drawback 
 of these cas is that as small molecules , the gd - chelates 
 act largely as extracellular agents because they are unable to penetrate 
 cells without attaching to a cell - targeting vector .
however , high - relaxivity and cell - penetrating superparamagnetic 
 iron oxides ( spios ) have been developed as effective t2-weighted contrast agents for both clinical diagnosis 
 and cellular imaging .
however , the negative contrast enhancement produced 
 by spios is far less desirable than that produced by the small - molecule t1 agents .
for example , 
 it is often difficult to differentiate between the darkened spots 
 produced by the accumulation of a t2 ca 
 and signals caused by bleeding , calcification , metal deposits , or 
 other artifacts from the background . in 
 addition , the high magnetic susceptibility of the t2 cas 
 often induces a distortion of the magnetic field on neighboring normal 
 tissues , which complicates the interpretation of mr radiograms .
it should be noted that because of the strong magnetic coupling 
 in spios , water coordination to the metal centers in such cas is not 
 necessary .
instead , the field inhomogeneity induced by the presence 
 of the tiny magnetic particles can cause a rapid dephasing in the 
 transverse plane , which in turn accelerates the t2 relaxation as well as the t1 relaxation , albeit to a much smaller extent for the latter .
the use of nanoparticulate coordination polymers as contrast 
 agents in different imaging modalities represents an important new 
 direction in the development of next - generation contrast agents .
of particular relevance to the design of t1-weighted mr cas is the ability to tailor the porosity , water 
 coordination , and thermodynamic / kinetic stability in such crystalline 
 polymers through the proper selection of the metal centers and ligands . 
 in this article
, we describe the synthesis , characterization , and 
 mr imaging studies of biocompatible nanoparticles ( nps ) of a coordination 
 polymer , kgd(h2o)2[fe(cn)6]h2o .
we have demonstrated that these nps exhibit extremely large t1 relaxivity because of the presence of two 
 water molecules coordinated to the gd(iii ) center .
furthermore , such 
 nps also possesses unprecedentedly high thermodynamic stability and 
 kinetic inertness due to the maximum ligand field stabilization energy 
 ( lfse ) found in the [ fe(cn)6 ] ligand , 
 coupled with the considerable lattice energy of the extended 3d solid - state 
 structure .
we have also shown that such 
 nps have no cytotoxicity and can be readily internalized by cells 
 to act as effective cellular mr probes .
equimolar aqueous solutions of gd(no3)3 ( 0.25 
 mmol , 20 ml ) and k4[fe(cn)6 ] ( 0.25 mmol , 20 
 ml ) were mixed at room temperature under vigorous stirring .
the solution 
 turned pale yellow and cloudy after continuous stirring at room temperature 
 for ca .
the reaction product was dialyzed using a regenerated 
 cellulose tubular membrane ( mwco = 12  00014  000 ) 
 against distilled water for 8 h , followed by lyophilization .
the solid 
 material obtained from this process was redispersed in water by sonication 
 to form a slurry and lyophilized one more time to increase its crystallinity . 

the bulk material was collected , washed with water and acetone , and 
 dried in vacuum .
an aqueous solution of gd(no3)3 ( 1 mm , 10 
 ml ) containing 50 mg of sodium citrate was added dropwise to an aqueous 
 mixture of k4[fe(cn)6 ] ( 1 mm , 10 ml ) and pvp 
 ( 111 mg , mw = 8000 ) under vigorous stirring for ca . 3 h. the reaction 
 product was dialyzed using a regenerated cellulose tubular membrane 
 ( mwco is 12  00014  000 ) against distilled water 
 for 2 days .
metal analysis was carried out using inductively 
 coupled plasma optical emission spectroscopy ( icp - oes ) with a perkinelmer 
 optima 3200 system .
a sample of 60 mg of kgd(h2o)2[fe(cn)6]h2o bulk material was first 
 decomposed at 620 c for ca .
6 h to obtain an amorphous oxide 
 powder that was then extracted with 5 ml of concentrated hno3 .
after dilution in a volumetric flask , the solution was analyzed 
 by icp - oes for potassium , gadolinium , and iron .
homogenous aqueous dispersions of nps with measured concentrations 
 of the gd ions in the range of 5.0  10 to 3.5  10 mm were used for t1 and t2 relaxivity measurements 
 at 25 c using a 60 mhz bruker minispec relaxometer and a 7.0 
 t bruker biospec small animal mri system ( bruker , inc . ,
billerica , 
 ma ) . for relaxivity measurements at 1.4 t , t1 and t2 relaxiviy values were 
 obtained from the minispec relaxometer using conventional inversion 
 recovery ( t1 ) and multiecho spin echo 
 ( t2 ) acquisitions . for relaxivity measurements 
 at 7.0 t , an inversion recovery gradient echo sequence with te = 4 
 ms was used for t1 relaxiviy measurements . 

t2 measurements were performed using a spin - echo sequence 
 of tr of 10  000 ms and a te of 10.6340 ms .
the t1-weighted mr images were acquired using the 
 7.0 t scanner with a matrix size of 128  128 , a field of view 
 of 3.0  3.0 cm , a slice thickness of 0.5 mm , a te 
 of 9.4 ms , and a tr of 13.91500 ms .
data analysis was performed 
 by fitting to relaxivity curves with self - written programs .
leaching experiments were performed 
 to verify the stability of nps under physiologically relevant conditions . 

about 200 mg nps were sealed in a membrane dialysis bag ( mwco = 3000 ) . 

this dialysis bag was gently stirred in 25 ml solutions of distilled 
 water at ph 7 or 1 for 24 h to reach equilibrium .
the leaching experiment 
 was also conducted in saline solution or in a solution containing 
 a biologically relevant divalent metal ion ( i.e. , zn , 
 ca , or mg ) at a concentration of 100 ppm 
 for each ion with the ph value adjusted to 7.4 .
the resulting solutions 
 were analyzed for free cn ions by a fluorometric 
 method using the cyanide test kit from lamotte co. ( chestertown , md ; 
 code 7387 - 01 ) .
the calibration curve was established using the standard 
 kcn solutions with concentrations at the ppm level .
the quantitative 
 determination of the leached gd ions was carried out 
 by the icp - oes method .
hela 
 cells were seeded in a 96-well plate at a density of 1  10 cells per well with the dmem low - glucose medium and incubated 
 for 12 h at 37 c in an atmosphere of 5% co2 and 95% 
 air to allow cells to attach to the surface .
cells in each well were 
 then treated with 100 l of fresh medium containing varying 
 concentrations of the nps and then incubated for 24 or 48 h. control 
 wells contained the same medium without nps .
the cells were incubated 
 in media containing 0.1 mg / ml 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium 
 bromide ( i.e. , the mtt dye ) for 3 h. after the mtt solution was removed , 
 the precipitated violet crystals were dissolved in 200 l of 
 dmso .
the assay was run in triplicate , and the results were presented 
 as percent viable cells . to conjugate the fluorescent 
 dye molecules to the surfaces of nps
, we used a slightly modified 
 synthetic procedure derived from the above - mentioned process for preparing 
 pvp - citrate - coated kgd(h2o)2[fe(cn)6]h2o nps .
specifically , 200 mg of pvp ( mw = 40000 ) 
 was added to an aqueous solution of k4[fe(cn)6 ] ( 1 mm , 50 ml ) under stirring , followed by the addition of a 50 
 ml gd(no3)3 solution ( 1 mm ) containing 100 mg 
 of citric acid .
the product was resuspended in 2 
 ml of aqueous pvp solution ( 100 mg pvp / ml ) .
ethylenediamine ( 1.5 equiv ) 
 and 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide ( edc , 1.2 equiv ) 
 reagents were then added to it under vigorous stirring .
the resultant 
 mixture was stirred for 24 h before the excess ethylenediamine was 
 removed by dialysis against distilled water for 2 days .
next , 5 ml 
 of carboxyfluorescein ( 0.35 mg / ml ) was allowed to react with 1.2 equiv 
 of edc ( 1.5 mg ) for 24 h. the ethylenediamine - coated nps were 
 then added to 2 ml of the above reaction mixture and stirred for ca . 

24 h. finally , the product was dialyzed for 2 days to remove unconjugated 
 dye molecules .
the fluorescence spectrum of the dye - conjugated nps 
 clearly showed the presence of dye molecules that are covalently bound 
 to the surfaces of nps .
confocal 
 scanning microscopy was used to visualize the cellular uptake of the 
 fluorescence dye - labeled nps in hela cells .
the cells were first seeded 
 in an 8-well chamber slide at a density of approximately 1.5  
 10 cells per well and incubated at 37 c for 
 24 h in a complete medium without antibiotics .
the cells were then 
 incubated with the fluorescent dye - labeled nps for 3 h at the same 
 temperature .
after the cells were washed with the pbs buffer solution 
 three times to remove free nps , the cellular uptake of nps was directly 
 imaged on the living cells under the confocal microscope with the 
 488 nm excitation wavelength .
approximately 1  10 pc3 cells were placed in t25 
 flasks and incubated for 48 h. the cells were then rinsed with serum - free 
 medium and incubated with varying concentrations of nps for 6 h. the 
 treated cells were washed with pbs three times and harvested by trypsinisation . 

mr imaging 
 of cell pellets was performed on a bruker 9.4-t mri scanner at 37 
 c using a conventional gradient echo acquisition with an inversion 
 recovery preparation .
the other acquisition parameters were field 
 of view = 3.0  3.0 cm , matrix = 128  128 , 
 and slice thickness = 3.0 mm .
intracellular gd and fe 
 concentrations were determined from the cell lysate using icp - ms . 

after incubation with nps , cells were washed three times with pbs 
 and centrifuged .
cells were then lysed with concentrated nitric acid 
 and diluted to determine the gd concentration using icp - ms .
the direct combination 
 of a soluble gd(iii ) salt with k4[fe(cn)6 ] in 
 aqueous solution resulted in the formation of a pale - yellow precipitate 
 with the metallic ratio k / gd / fe from the elemental analysis being 
 close to unity and a singlet ir stretching band at 2070 cm , suggesting the formation of a single - phase coordination polymer . 

however , because of poor crystallinity , the powder x - ray diffraction 
 ( xrd ) patterns of this product could not be indexed .
refluxing of 
 the reaction in water or an organic solvent ( e.g. , ethanol and dmf ) 
 to increase crystallinity caused the product to decompose into a mixture 
 that invariably contained prussian blue ( pb ) and other unidentified 
 components . although the single - crystal structure of a coordination 
 polymer formed from these two starting materials was reported in 1996 , there are few synthesis details except for the 
 mention that single crystals were obtained from the diffusion method 
 in a u tube for 6 months given in the structural report .
we were able 
 to shorten the crystal - growth time to 45 days using 1 m nitric 
 acid as the crystallization medium with a similar diffusion method , 
 but the yield we obtained for the intended compound was less than 
 1% whereas the major product formed was pb powder . to develop a suitable 
 synthesis method that could later be adopted for preparing nps of 
 this coordination polymer
, we explored a variety of preparative techniques 
 including hydro(solvo)thermal synthesis and mechanical milling .
the 
 best method we have found is an unusual low - temperature solid - state 
 crystallization process .
this method entails the mixing of an equimolar 
 quantity of aqueous gd(no3)3 and k4[fe(cn)6 ] solutions at millimolar concentrations , followed 
 by stirring the solution at room temperature for 8 h. the 
 product was dialyzed in water to remove potassium and nitrate ions . 

the solution was then frozen while water was removed under vacuum 
 to afford a light - yellow powder .
this synthesis process resembles the hydrothermal reaction for crystal 
 growth in that the supersaturating condition suitable for crystallization 
 is slowly induced at low temperatures .
the advantage of such an approach 
 is that the heating of the reaction is avoided to prevent the reaction 
 product from decomposing into prussian blue .
the x - ray powder 
 diffraction ( xrd ) patterns of this bulk sample were well indexed on 
 the basis of an orthorhombic unit cell with the same lattice parameters 
 as those obtained from the single - crystal structure analysis ( supporting information ) .
therefore , atomic parameters 
 of the latter structure were used as the starting model in the rietveld 
 refinement . the final refinement in space 

group pnma gave a = 12.6098(4 )  , b = 13.6161(4 )  , c = 7.2490(3 )  , v = 1244.63(7 )  , and calc = 2.4351(1 ) g / cm ( figure s1 ) .
the structure of kgd(h2o)2[fe(cn)6]h2o consists of fe octahedra and gd biface - capped trigonal prisms joined in a 3d framework by 
 cn groups ( figure 1 ) . the 
 fe atom
is coordinated by six c atoms of the cn groups whereas the gd atom is coordinated by six n atoms and additionally 
 by two o atoms of water molecules o1 and o2 ( table 1 ) .
the cavities in the framework are filled up by the k ion and water of crystallization ( o3 ) , showing site - occupancy 
 disorder in the same cavity . both k and o3
are slightly shifted from 
 each other by 0.4  so that k distances to nearby o and n atoms 
 are in the range of 2.85 to 2.99  , whereas the o3 water molecule 
 forms two h bonds with o1 and o2 ( table 1 ) . 

thermal gravimetric analysis ( tga ) on the bulk sample showed a two - step 
 loss of water ( i.e. , the first step of weight loss was found in the 
 range from room temperature to about 100 c , corresponding 
 to the loss of one water molecule , and the second step of weight loss 
 was found in the range from 100 to 160 c , corresponding 
 to the loss of two water molecules ( figure s2 ) ) .
this observation is consistent with the existence of one zeolitic 
 and two coordinative water molecules per formula as revealed by the 
 powder x - ray structure determination .
furthermore , the fourier transform 
 infrared ( ft - ir ) spectra of the bulk sample of kgd(h2o)2[fe(cn)6]h2o showed a strong characteristic 
 cn stretching vibration at 2070 cm , attributable 
 to the fe(ii)-cn 
unit - cell packing diagram of kgd(h2o)2[fe(cn)6]h2o ( left ) with the k ion and zeolitic water molecule omitted for clarity .
the coordination 
 environment of the gd ion ( middle and right ) showing 
 two water molecules directly bound to the metal center .
the reaction of gd(no3)3 in 
 a sodium citrate solution with k4[fe(cn)6 ] in 
 an aqueous solution containing polyvinylpyrrolidone ( pvp ) under similar 
 conditions to the above preparation led to the formation of stable 
 pvp - citrate - coated kgd(h2o)2[fe(cn)6]h2o nps ( pvp - c - kgdfecn nps ) .
we found that the 
 concurrent use of sodium citrate as a capping agent and pvp as a coating 
 polymer is necessary for controlling the size and stabilizing the 
 nps in aqueous solution and in the solid state . when either sodium 
 citrate or pvp was used alone , the nps would aggregate and separate 
 from the solution after the aqueous dispersion was left to stand at 
 room temperature for a few days .
the ft - ir spectra of the nps after 
 24 h of dialysis to remove unbound citrate and pvp contained the characteristic 
 ir vibrations attributable to the citrate anion and pvp in addition 
 to the cn stretching vibration at 2070 cm , confirming the presence of both the capping agent and the coating 
 polymer on the surface ( figure s3 ) .
the 
 elemental analysis of the sample digested with nitric acid showed 
 the expected k / gd / fe to be 1:1:1 .
furthermore , the xrd patterns of 
 the pvp - c - kgdfecn nps match well with those of the bulk sample ( figure s4 ) .
the morphology and the size 
 of the nps were examined by transmission electronic microscopy ( tem ) . 
 as shown in figure 2 , the tem image of pvp - c - kgdfecn 
 nps
energy - dispersive 
 x - ray analysis ( edxa ) showed that the nanomaterial contains gadolinium , 
 potassium , and iron ( figure s5 of the si ) .
the efficiency of a ca is measured by its ability to 
 enhance the proton relaxation of water and is commonly expressed as 
 proton relaxivity .
the latter is defined 
 as the longitudinal or transverse relaxation rate increase of the 
 water protons by unity concentration of the agent in mm .
to evaluate 
 the efficacy of our nps as mri contrast agents , we performed a series 
 of proton t1 and t2 relaxation measurements in order to determine their longitudinal 
 and transverse relaxivity values , r1 and r2 , at both low ( 1.4 t ) and high ( 7.0 t ) magnetic 
 field strengths .
the 1.4 t relaxometry results were obtained on a 
 bruker minispec 60 mhz relaxometer , and the 7.0 t results were obtained 
 on a bruker biospec 7.0-t mri scanner .
experimentally , the change 
 in relaxation rate with increasing concentration of ca was measured , 
 and the numeric value of the relaxivity , r1 or r2 , was then extracted from the plot 
 of 1/t1 ( or 1/t2 ) vs the concentration of gd ions in nps using the following 
 equationwhere 1/ti , d ( i = 1 , 
 2 ) is the diamagnetic contribution to the relaxation rate and [ gd ] 
 is the concentration of gd ions in nps .
the relaxation 
 rate of pure water is taken as the diamagnetic contribution in all 
 of the experiments .
all of the data are reported on a per gd ion basis at two different magnetic fields in table 2 and compared to commercial contrast agent magnevist ( i.e. , 
 gd - dtpa ) and several known nanoparticle - based gd(iii ) mr contrast 
 agents . as shown in figure 3 ,
the resulting 
 values for these nps are r1 = 17 mms and r2 = 24 mm s at 7.0 
 t and r1 = 35 mm s and r2 = 38 mm s at 1.4 t , respectively . for comparison ,
the 
 relaxivity values of gd - dtpa are r1 = 
 3.4 mm s and r2 = 3.7 mm s at 
 1.4 t ( table 2 ) .
if the citrate - coating layer on the surfaces of the nps 
 would impede water exchange between the bulk and coordinated h2o molecules , we intentionally prepared the nps under the same 
 synthesis conditions with pvp as the only coating layer of the nps . 
 as shown in figure s6 , there is a slight 
 increase in the r1 value from 35 to 37 
 mm s for the pvp - coated nps , 
 whereas the simultaneous change in the r2 value for such nps from 38 to 47 mm s appears to be more significant .
these results suggest that the presence 
 of the citrate coating layer on the surfaces of the nps may block 
 the access of the bulk water to the sites of the coordinated h2o because the slight increase in the r1 relaxivity may well be due to the increased outer - sphere 
 contribution as a result of a higher r2 value in the pvp - coated nps .
however , the opposite may be said about 
 the tight , covalently bound citrate coating layer ; that is , its presence 
 on the surfaces of the nps can act as a magnetic shield to reduce 
 to the effectiveness of the localized tiny magnetic fields generated 
 by the nps , which in turn would induce the r2 relaxation of the bulk water protons .
it is conceivable that 
 the gd(iii ) ions on the surface of the nps have a higher ability to 
 induce the relaxation of the bulk water protons than the contribution 
 to gd(iii ) inside the nps .
we therefore prepared pvp - c - kgdfecn nps 
 with four different sizes by controlling the nucleation rate and coating . 

it is interesting that both r1 and r2 relaxivities decrease 
 with increasing particle size at almost the same rate .
plots of 1/ti ( i = 1 , 2 ) vs the 
 gd concentration at magnetic field strengths of 1.4 t 
 ( left ) and 7.0 t ( right ) for pvp - c - kgdfecn nps .
these observed values of r1 relaxivity 
 would place the current nps among the best nanoparticle - based t1-weighted cas that contain the gd ion . besides the extremely 
 high r1 relaxivity , the small r2/r1 ratios of ca . 

1.4 at 7.0 t and 1.1 at 1.4 t indicate that these nps are suitable 
 as a t1-weighted agent to enhance the 
 longitudinal relaxation of the water protons .
in contrast , metal oxide - based 
 nps , particularly iron oxide nps , have a tendency to exhibit superparamagnetic 
 behavior with an exceedingly large r2/r1 ratio ( i.e. , r2/r1 > 10 ) . as a result ,
plots of r1 ( left ) and r2 ( right ) vs the hydrodynamic particle size 
 for pvp - c - kgdfecn nps . according to sbm theory , the mechanism of inner - sphere relaxation 
 in small molecular gd - chelates can be understood from 
 the following equationwhere q is the number of water molecules directly coordinated 
 to the gd center , [ c ] is the molar concentration 
 of the contrast agent in mm , t1 m is the 
 longitudinal relaxation time of the bound water , and m is the mean residence lifetime of the coordinated water molecule . 

if t1 m and m are fixed , 
 then increasing the number of water molecules bound to the gd center from one to two will result in a doubling of the 
 inner - sphere t1 relaxivity .
however , the 
 decreased thermodynamic stability and/or kinetic inertness will render 
 the complex highly susceptible to displacement by proteins , biological 
 ligands , or endogenous zn , ca , and mg ions .
it is tempting to conjecture that the presence of two water molecules 
 directly coordinated to the gd(iii ) center as well as the zeolitic 
 water in the structural cavities plays an important role in contributing 
 to the inner - sphere longitudinal relaxation of the water protons . 

furthermore , the structural rigidity resulting from the 3d extended 
 polymeric network and the reduced tumbling rate of such particulate 
 ca also favor high relaxivity .
recently , perrier and coworkers reported 
 the synthesis and nmr relaxivity studies of nps based on a similar 
 coordination polymer with the formula gd(h2o)2[fe(cn)6]h2o , where gd contains 
 two coordinated water molecules as well , although the cytotoxicity 
 and cellular penetrating ability of these nps remained to be seen .
as expected , the pegylated nps of the coordination 
 polymer also exhibit high t1-weighted 
 relaxivity with a low r2/r1 ratio ( table s1 ) .
however , 
 the [ fe(cn)6 ] anion in gd(h2o)2[fe(cn)6]h2o contains the 
 low - spin fe(iii ) center with s = /2 , which may be magnetically coupled with the gd(iii ) center 
 with s = = /2 to give rise 
 to a significant contribution of the outer - sphere relaxation to the 
 overall t1-weighted relaxivity , thus making 
 the distinction of the coordinated water molecules to the inner - sphere 
 longitudinal relaxation from the outer - sphere relaxation impossible . 

however , because the [ fe(cn)6 ] anion 
 in our kgd(h2o)2[fe(cn)6]h2o contains the low - spin fe(ii ) center with s = 0 , the gd(iii ) ions can be viewed as isolated paramagnetic centers , 
 thus rendering the description of their t1-weighted relaxivity using the sbm model meaningful .
to further 
 confirm the efficacy and evaluate their performance as effective mr 
 contrast agents , we obtained both t1- 
 and t2-weighted mri phantom images of 
 pvp - c - kgdfecn nps in aqueous solution with various concentrations 
 on a 7.0 t scanner ( figure 5 ) .
the t1-weighted images rapidly become brighter with 
 increasing concentration of nps , and the t2-weighted images respond to the increase in the concentration by 
 slightly darkening the contrast .
these results suggest that the current 
 nps can act as an effective t1-weighted 
 ca at the high magnetic field .
t1-weighted 
 ( left ) and t2-weighted ( right ) mr phantom 
 images of pvp - c - kgdfecn nps with various gd concentrations 
 using a 7.0 t scanner .
the ability of nps to cross the cell membrane is a critical prerequisite 
 for cellular mr imaging applications to be realized .
we studied the 
 cellular uptake of our nps in hela cells using the fluorescent confocal 
 microscopic imaging technique .
for live cell imaging , cells were incubated 
 with the carboxyfluorescein ( cbf ) dye - labeled nps ( figure s7 in the si ) , washed with pbs , and directly imaged under 
 a laser scanning confocal microscope without fixation . the advantage 
 of using cbf attached to the nps as the fluorescent probe is that 
 because of its high anionic negative charge , the cbf dye molecule 
 itself is membrane - impermeable .
the images 
 of confocal microscopy showed the presence of bright - green fluorescent 
 signals inside the cells that were incubated with the dye - labeled 
 nps for 3 h. the untreated hela cells were used as the negative control . 

figure 6 shows the typical confocal fluorescent 
 images of hela cells treated with the dye - labeled nps and the control 
 cells .
the uniform fluorescence emission in the perinuclear region 
 of the cell indicates an untargeted cytoplasmic distribution of nps 
 ( i.e. , there is no specific binding of nps to any small organelle 
 in the region ) .
these observations are consistent with the notion 
 that the internalization of such nps is most likely to occur via endocytosis .
confocal 
 microscopic fluorescence image of hela cells incubated with dye - conjugated 
 nps for 3 h ( top left ) , the bright - field image of the cells from panel 
 a ( top right ) , the fluorescent image of untreated hela cells as the 
 negative control ( bottom left ) , and the bright - field image of the 
 cells from panel c ( bottom right ) .
a quantification of the cellular uptake of kgdfecn nps was 
 performed on the hela cell lysate using inductively coupled plasma 
 mass spectrometry ( icp - ms ) . as shown in figure 7 ,
there is clear correlation between the cellular fluorescence signals 
 and the increase in both intracellular gd and fe concentrations .
however , 
 the cellular uptake of gd - dtpa is greatly diminished ( i.e. , reduced 
 approximately 30-fold ) compared to that of kgdfecn nps as revealed 
 by the much lower gd concentration in the cell lysate treated with 
 gd - dtpa .
this is because as a small molecule , gd - dtpa penetrates the 
 cell membrane mainly by the process of passive diffusion .
gadolinium 
 and iron concentrations of np - treated ( 0.25 mm ) vs gd - dtpa - treated 
 hela ( 0.25 mm ) cells ( 5 h incubation ) along with untreated hela cells 
 as the control .
viability of hela cells 
 after incubation with pvp - c - kgdfecn nps for 24 and 48 h. the cytotoxicity of the nps in hela cells was evaluated 
 using the mtt assay with various concentrations of the nps and two 
 different incubation times to study both the dose response and time 
 effect . as shown in figure 8 ,
more than 90% 
 of the cells were still viable after 48 h of incubation with 1.00 
 mm pvp - c - kgdfecn nps , indicating the nontoxic nature of such nps . 

we attribute the low cytotoxicity of the nps to the proper surface 
 coating and the high structural integrity of the coordination polymer 
 itself . to further confirm the stability of such nps against the leaching 
 of toxic cn and gd ions
, we carried 
 out a series of leaching experiments under physiologically relevant 
 conditions .
specifically , the solution concentrations of the cn and gd ions leached out of the nps were 
 determined using a static method in which the nps that were sealed 
 in a membrane dialysis bag were allowed to equilibrate with different 
 media that are physiologically relevant .
the resulting solutions were 
 then analyzed for free cn and gd ions 
 by a fluorometric method based on the konig reaction and icp - ms analysis , respectively .
figure 9 shows the concentrations of the cn ions 
 released from the nps under different conditions .
the highest cyanide 
 concentration was found in an extremely acidic , albeit physiologically 
 less relevant , solution of ph 1 to be approximately 3.5 ppm , and the 
 cyanide concentration released from the nps was found to be 0.8 
 ppm in a saline solution and 0.5 ppm in distilled water and 
 other media containing a biologically relevant divalent metal ion 
 at ph 7.4 . in comparison ,
the maximum allowable level of cyanide in 
 drinking water is 0.2 ppm as set by the environmental protection agency 
 ( epa ) .
it should be noted that in cigarette 
 smokers , the cyanide concentration in blood can be as high as 3565 
 ppm right after a cigarette is smoked . however , under all of the tested conditions , the concentrations 
 of the gd ions released from the nps were all below 1 
 ppm ( or < 6 m ) , except in the acidic solution of ph 1 where 
 the concentration of the gd ions was found to be approximately 
 3 ppm ( i.e. , 20 m , figure 10 ) .
these results suggest that the pvp - c - kgdfecn nps are stable against 
 dissociation to release both gd and cn ions under thermodynamic equilibrium conditions and kinetically 
 inert to metal substitution reactions .
this remarkable stability of 
 the coordination polymer stems from the fact that as a ligand , the 
 hexacyanoferrate(ii ) anion , [ fe(cn)6 ] , possesses the highest - possible ligand field stabilization energy 
 ( lfse ) for any complex containing the low - spin fe(ii ) center and that 
 the formation of an extended 3d coordination network structure results 
 in high lattice energy for the coordination polymer .
consequently , 
 both the cn group and the gd ion are 
 completely locked in their corresponding lattice positions and can not 
 be released from the structure by self - dissociation or ion exchange , 
 confirming that the nps are extremely stable and resistant to the 
 displacement of gadolinium in the presence of the biologically relevant 
 divalent metal ions . in sharp contrast , some small - molecule - based 
 cas are susceptible to in vivo transmetalation reactions with endogenous 
 zn and ca to release gd ions . 

the latter was linked to the development of nephrogenic systemic fibrosis 
 ( nsf ) in some renally impaired patients , which prompted the u.s .
food and drug administration ( fda ) in 2007 
 to issue a public health advisory regarding the use of gadolinium - containing 
 cas .
currently , the manufacturers of such cas are required by the 
 fda to include new boxed warnings and new warning sections in the 
 labels to describe the possible link between the use of such cas and 
 the development of nsf
gadolinium - releasing test for different conditions . to determine whether the internalized pvp - c - kgdfecn nps could enhance 
 the t1-weighted mri contrast of cells , 
 we incubated pc3 cells with various concentrations of pvp - c - kgdfecn 
 nps and examined the t1-weighted image 
 for each sample at 37 c using a spin - echo saturation recovery 
 sequence on a bruker 9.4-t mri scanner .
as shown in figure 11 , there is a considerable change in image brightness 
 in the pellets of the pc3 cells incubated with the nps . in particular , 
 the cells treated with the nps at a concentration equivalent to 0.25 
 mm gd ions prior to imaging exhibited a strong mri signal 
 brightening effect .
these results demonstrate that pvp - c - kgdfecn nps 
 have the potential to be used as an effective t1-weighted ca for cellular imaging at a high magnetic field
. t1-weighted mri phantoms of pc3 cells incubated 
 in pbs buffer ( left ) , 0.13 mm nps ( central ) , and 0.25 mm nps ( right ) 
 for 6 h. the images were collected using a bruker 9.4-t scanner .
we 
 have developed a simple one - step method for preparing extremely stable 
 and biocompatible nps of the gadolinium ferrocyanide coordination 
 polymer .
furthermore , we have demonstrated that such nps exhibit 
 extremely high t1-weighted relaxivity , 
 suggesting the potential of this coordination - polymer structural platform 
 in the development of new - generation t1-weighted cellular mr probes for biological receptors or markers 
 within the cell to study molecular events as well as for in vivo mr 
 imaging in biomedical research and clinical applications .
 a simple one - step method for preparing 
 biocompatible nanoparticles of gadolinium ferrocyanide coordination 
 polymer kgd(h2o)2[fe(cn)6]h2o is reported . 
 the crystal structure of this coordination 
 polymer is determined by x - ray powder diffraction using the bulk materials . 
 
 the stability , cytotoxicity , cellular uptake , and mr phantom and cellular 
 imaging studies suggest that this coordination - polymer structural 
 platform offers a unique opportunity for developing the next generation 
 of t1-weighted contrast agents with high 
 relaxivity as cellular mr probes for biological receptors or markers . 
 
 such high - relaxivity mr probes may hold potential in the study of 
 molecular events and may be used for in vivo mr imaging in biomedical 
 research and clinical applications . 
Introduction
Methods
and Materials
Results
and Discussion
Conclusions