superparamagnetic iron oxide nanoparticles 
 ( nps ) , with core diameters 
 of 315 nm , are used in a rapidly expanding number of applications 
 in the biomedical field ; the most common include cell labeling , hyperthermia , drug 
 delivery , and as contrast agents for magnetic 
 resonance imaging . for these applications , 
 the iron oxide cores are coated with polymer , lipid , or other dispersants 
 to enable dispersion of nps in aqueous solutions containing biomolecules . 

the most common method to stabilize iron oxide nps for biomedical 
 applications has been to enwrap them in a weakly adsorbed shell of 
 high molecular weight polymer ( often dextran ) or amphiphiles such as block copolymers or lipids .
however , weakly adsorbed shells lead to low polymer densities 
 on the particle surface that are further reduced with time under dilute 
 conditions , resulting in low colloidal stability .
chemically grafted 
 polymer shells are physically and chemically stable ; they can also 
 be made denser than physisorbed shells , and therefore they have received 
 increasing attention . with recent 
 improvements in the synthesis of nps there 
 has been a push toward more well - defined , core 
spherical iron oxide nps can now be synthesized 
 with monodisperse size ( sd < 5% ) , but they are as - synthesized capped 
 with a strongly adsorbed , hydrophobic shell of oleate that is difficult 
 to replace ; these nps require stabilization 
 with a shell of linear , end - grafted polymer dispersants of sufficient 
 thickness and grafting density to ensure that the particles remain 
 monodisperse during application .
defined core  shell architectures 
 enable the prediction of all colloidal properties and can be used 
 to define biological interactions through the attachment of organic 
 ligands .
the main advantage of grafting dispersants to the core is 
 that the hydrodynamic size of the nps , the stability of the shell , 
 and the presentation of ligands can be precisely controlled , in contrast 
 to for nps with shells consisting of physisorbed high molecular weight 
 dispersants ; this critically determines np performance in a biological 
 fluid .
precise control over monodisperse 
 size , purity , and colloidal interactions is crucial for nps in biomedical 
 applications to control biodistribution and self - assembly of drug 
 delivery structures . achieving 
 high dispersant grafting density on monodisperse iron 
 oxide nanoparticles is challenging since the dispersant has to densely 
 replace the ( oleic acid ) organic shell carried by the as - synthesized 
 particles .
in addition , a major problem for the synthesis and characterization 
 of core  shell nanoparticles is the efficient separation of 
 excess dispersants .
the peg - dispersant density on 
 nanoparticles is difficult to determine since continuous and complete 
 removal of excess dispersants from nanoparticle dispersions can lead 
 to particle aggregation and precipitation ; alternatively , not all 
 free ligands are removed , which leads to overestimation of the dispersant 
 density on the nanoparticles .
the actual stability and correct core  shell 
 structure can not be determined if excess dispersants are not removed . 

efficient separation methods can also contribute to nanoparticle degradation 
 and should be identified and avoided .
an irreversibly grafted 
 shell of > 2 chains / nm of peg(5 
 kda ) on polydisperse and irregularly shaped single nps was shown to 
 be required for colloidal stability under application conditions .
np curvature facilitates polymer grafting by 
 grafting - to of presynthesized polymer coils and chain grafting densities 
 up to 2.5 chains / nm have been claimed on iron oxide nps . until recently , only 0.5 chains / nm of peg(5 kda )
was reported grafted on spherical fe3o4 nps after ligand replacement by nitro - dihydrohyphenylalanine 
 ( nitro - dopa)-peg(5 kda ) on monodisperse nanoparticles synthesized 
 using oleylamine as capping agent .
lak 
 and colleagues recently synthesized large , 25 nm in diameter , fe3o4 ferrimagnetic nanoparticles that were stabilized 
 through ligand replacement with nitrodopamine ( nda)-peg . due to their size
, these multidomain particles 
 are optimized for applications where brownian relaxation mechanisms 
 are desired , while particles with weaker magnetic interactions and 
 nel relaxation as the dominant relaxation mechanism require 
 iron oxide nps smaller than 15 nm . these large particles form 
 small aggregates in cell culture and pbs , which might be due to the 
 stronger long - range core 
quantitatively addressed the problem of replacing oleic acid by ligand 
 exchange on superparamagnetic iron oxide nanoparticles .
full ligand replacement was not shown , although 
 the superiority of dopa and nitro - dopa anchor groups to displace oleic 
 acid from the surface was demonstrated .
these and other previous studies 
 did not address the effect of purification from excess ligands on 
 the results .
shell 
 nanoparticles under relevant conditions such as biological buffers 
 and elevated temperature was also not investigated .
we present 
 protocols to replace oleic acid for a dense spherical 
 brush peg - shell on monodisperse , monocrystalline , and spherical iron 
 oxide nanoparticles in the superparamagnetic size range of 310 
 nm diameter at physiological temperatures . the colloidal stability 
 in both phosphate buffered saline ( pbs ) and biofluids of these densely 
 pegylated core 
shell particles significantly exceeds what has 
 previously been described for particles synthesized by ligand exchange . 

importantly , we also present an evaluation of different purification 
 methods to separate pegylated particles from free peg , and we suggest 
 a protocol for iterated solvent precipitation and magnetic extraction 
 as most suitable to completely purify these nanoparticles at high 
 yield . only through this advancement
is the high grafting density 
 and colloidal stability conclusively demonstrated , with important 
 implications for correct comparison of previous and future results 
 on np ligand density .
all chemicals ( see supporting information for details ) were purchased from sigma - aldrich , 
 all solvents were from roth , and peg was from jenkem and used as received 
 without further purification .
all nps and nitrocatechol ligands originated 
 from the same respective batch to ensure maximum reproducibility .
tem studies were performed on a fei tecnai 
 g2 20 transmission electron 
 microscope operating at 120 or 200 kv for high resolution imaging . 

samples were prepared by dropping toluene dispersions ( as - synthesized 
 nps ) or aqueous dispersions ( pegylated nps ) onto a 300-mesh carbon - coated 
 copper grid and subsequently evaporating the solvent in air .
size 
 distributions were evaluated using the pebbles software package with a local intensity - model fitting algorithm . 

thermograms were recorded on a mettler - toledo 
 tga / dsc 1 star system in the temperature range 25650 c 
 with a ramp of 10 k / min in synthetic air stream of 80 ml / s in order 
 to ensure complete combustion of ligands as nda was found to polymerize 
 by pyrolization under n2 .
70 l aluminum oxide crucibles 
 were filled with 0.51.5 mg of sample , and the total organic 
 content ( toc ) was evaluated as the mass loss fraction at 500 c 
 by horizontal setting .
h and c solution 
 spectra were collected on a bruker dpx operating at 300 mhz in cdcl3 and d6-dmso as solvents using 
 tms as an internal standard .
mid - ir powder spectra of the lyophilized 
 samples were collected on a single reflection bruker tensor 37 ftir 
 spectrometer with bruker platinum diamond single - reflection atr equipment 
 at a resolution of 4 cm by averaging 32 scans .
samples were measured 
 in type 0500 glass capillaries ( hilgenberg , germany ) with nominal 
 diameter of 1 mm and wall thickness of 10 m .
measurements were 
 carried out using a rigaku s - max 3000 saxs system equipped with a 
 copper - target microfocus x - ray tube micromax-002 + ( 45 kv , 0.88 ma ) 
 with an energy of 8.05 kev , collimated through three pinholes ( 400 , 
 200 , and 700 m ) to achieve a beam diameter at the sample position 
 of 210 m ( fwhm ) and a triton 200 2d multi wire gas - filled x - ray 
 detector ( 200 mm diameter of active area , spatial resolution 200 m ) . 
 data were acquired in the q - range from 0.01 to 0.26 
  with a measurement time of 28.800 s for 
 each scattering pattern at vacuum conditions better than 10 mbar .
subsequent data treatment included background correction based 
 on the measured transmission and radial integration with the saxsgui 
 2.8.03 software package .
superparamagnetic 
 oleic acid ( oa)-stabilized magnetite nanoparticles were synthesized 
 by thermal decomposition of an iron precursor according to a slightly 
 modified heat - up procedure described by hyeon et al .
briefly , for 9.6 nm nps a mixture of 50 ml of dioctyl ether 
 ( oct2o ) and 7.04 ml of oa was heated to 100 c under 
 n2 .
1 ml of iron pentacarbonyl ( fe(co)5 ) was 
 injected rapidly , and the reaction mixture was heated to 290 c 
 with a temperature ramp of 3 c / min .
after aging for 1 h the 
 np dispersion was allowed to cool to room temperature and precipitated 
 thrice with ethanol ( etoh ) from toluene in order to remove excess 
 oa .
the size was controlled by the fe(co)5:oa ratio ; details 
 can be found in the supporting information ( table s1 ) .
6-nitrodopamine 
 hemisulfate ( nda - hso4 ) was synthesized according to literature 
 with slight modifications .
nda - peg(5 
 kda ) was synthesized by ( 1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylaminomorpholinocarbenium 
 hexafluorophosphate ( comu)-mediated peptide - coupling reactions ( see supporting information for experimental 
 details ) . typically , 1.3 
 g of wet ( from etoh 
 washing ) nps ( containing 0.11 g cores )
were dispersed in 30 
 ml of dmf ; the desired amount of nda - peg(5 kda ) , usually a 10-fold 
 excess ( with respect to the grafting density of 1 nda - peg / nm expected from preliminary measurements of the maximum achievable 
 grafting density by ligand replacement , e.g. , 1.9 g nda - peg(5 kda ) 
 for 9.6 nm nps ) , was dissolved in dmf and mixed with the nps .
note 
 that only a low excess is needed for the ligand exchange , but to allow 
 for fast ligand exchange and easier determination of the efficiency 
 of the purification methods , a large excess was used .
the dispersion 
 was sonicated for 26 h at slightly elevated temperature ( 35 c ) . 

preliminary tests showed that 26 h was sufficient to replace oa , and 
 longer sonication did not improve the exchange .
subsequently , the 
 mixture was extracted thrice with n - hexane ( 30 ml 
 each ) in order to remove released oleic acid .
afterward , the solvent 
 was evaporated ; the pegylated nps were lyophilized for 24 h and stored 
 as dry powder for further analysis and are referred to as 
nps were obtained as a light brown powder 
 in 97% yield with respect to the amount of cores .
all purification 
 methods were applied after the extraction of the nps with n - hexane .
all nps were characterized and analyzed before 
 and after purification by tem , tga , ir , and dls .
additionally , nps 
 purified by precipitation and magnetic decantation were analyzed by 
 saxs .
0.3 g of the 
 nps after ligand replacement 
 ( nps raw ) was dissolved in 2 ml of h2o and sonicated for 
 15 s. sephadex was swollen overnight in milli - q water at 40 c 
 and manually packed into a 25  2 cm column .
the length 
 of the column was sufficient to split the np sample in six to seven 
 identifiable , differently colored fractions ( figure s1 ) .
the eluates were collected and freeze - dried for further 
 analysis . for all np sizes fraction ii could be identified as the 
 main fraction . for two np sizes ( 9.6 and 7.1 nm )
this main fraction 
 was reapplied onto a fresh column , and the eluate was again characterized . the 1000 kda molecular weight 
 cutoff ( mwco ) membranes
were equilibrated for 10 min in 10% v / v etoh 
 and 30 min in h2o .
0.3 g of the raw nps were dissolved 
 in 5 ml of milli - q and dialyzed against 5 l of milli - q . to monitor 
 the dialysis progress ,
equivalents of 0.5 ml were taken after 1 , 2 , 
 4 , 6.5 , and 24 h and freeze - dried .
the 9.6 and 7.1 nm nps were further 
 dialyzed for 48 h and the small nps ( 4.9 and 3.5 nm ) for 70 h. the 
 dispersions were lyophilized for further analysis by tem , tga , ir , 
 and dls .
typically , 0.15 g of raw nps 
 were dispersed in 2 ml of milli - q and loaded into a 50 kda amicon - ultra-15 
 membrane centrifugation filter unit and centrifuged at 4500 g for 15 min .
the centrifuge effluent was collected , and 
 the nps were redispersed in 1 ml of milli - q .
this procedure was repeated 
 up to 15 times ; equivalents were taken after 5 , 7 , and 11 centrifugation 
 steps to track the removal of excess nda - peg .
all dispersions and 
 the collected effluents were freeze - dried for further analysis by 
 tem , tga , and dls .
typically 0.3 
 g of the raw nps ( containing 0.019 g cores for 9.6 nm nps ) were dissolved 
 in 10 ml of etoh by sonication and slight heating with a hot gun to 
 60 c .
the clear dispersion was poured into a small beaker and 
 mixed with 10 ml of ice - cold petrolether whereupon it got slightly 
 turbid .
the beaker was placed on a 5  5 cm 1 t magnet in the 
 fridge at 4 c , and the nps were soaked from the cloudy mixture 
 to the bottom of the beaker within a few minutes , dependent on the 
 np size .
the supernatant was decanted by holding back the nps with 
 the magnet , collected , combined , and freeze - dried for tga measurements . 

this procedure was repeated 79 times ; after each step a small 
 sample of nps was kept and freeze - dried for tga analysis to monitor 
 the removal of excess ligands and impurities .
the final products after 
 the seventh step were freeze - dried and further analyzed .
the nps were 
 obtained as dark brown powder in 96% yield with respect to the cores .
freeze - dried samples 
 of all purification methods were stored at room temperature without 
 protection from light .
after 6 months dry storage , suspensions of 
 nps in milli - q water and pbs of the nps purified by dialysis ( 4.9 
 nm cores ) and magnetic decantation ( 7.1 nm cores ) were prepared ( 4 
 mg / ml ) and characterized by tem and dls .
the np 
 dispersions were stored for 4 months at 4 c to prevent microbe 
 growth and analyzed .
additionally a sample of 7.1 nm cores purified 
 by magnetic decantation was dissolved in pbs and water at 3 mg / ml ; 
 10% bovine calf serum was added to investigate if the nps aggregate 
 upon nonspecific protein adsorption .
dls was measured over a temperature 
 cycle from 207020 c in 5k steps with an equilibration 
 time of 5 min at each temperature .
spherical , monodisperse , and single - crystalline 
 superparamagnetic iron oxide nanoparticles were synthesized using 
 a modified heat - up method introduced by hyeon and co - workers . the np size could be precisely tuned in the 
 range of 310 nm without any further size selection process 
 by varying the fe(co)5 to oleic acid molar ratio ( see table 
 s1 in the supporting information for details ) . 

the obtained nps were characterized by low- and high - resolution tem , 
 tga , and atr - ftir .
figure 1 shows tem micrographs of monodisperse 3.5  0.3 , 4.9 
  0.4 , 7.1  0.4 , and 9.6  0.4 nm magnetite cores , 
 calculated with pebbles by evaluation of 900 nps from multiple 
 hr and lr - tem micrographs of the respective batches .
the hr - tem ( inset 
 in figure 1c ) shows 
 the single - crystallinity of the nps with lattice spacing in the ( 311 ) 
 direction of 0.26 nm .
the ring diffraction pattern ( inset figure 1d ) reveals the highly 
 crystalline structure of the nps .
the ratio of d - spacings 
 in the obtained pattern show good agreement with the jcpds database 
 numbers for maghemite or magnetite .
transmission 
 electron micrographs of as - synthesized monodisperse 
 oa - capped fe3o4 nps : ( a ) 3.5 nm .
( c ) 7.1 nm ; inset : high resolution tem image showing that the particles 
 are single - crystalline .
( d ) 9.6 nm ; inset : electron diffraction pattern 
 of 9.6 nm nps reveal the highly crystalline nature of the nps .
the grafting of nda - peg to the nanoparticle 
 cores was performed in large excess of peg in order to ensure fast 
 and full ligand replacement and to provide an easily distinguished 
 baseline for evaluation of different methods to remove unreacted dispersant . 

the separation of all excess free peg - dispersant after grafting is 
 challenging , as indicated by previous work ; we performed a thorough comparison and analysis 
 of different methods that have been applied for core 
shell 
 nanoparticle preparation : cross - linked dextran size - exclusion column 
 separation , dialysis , membrane centrifugation and precipitation 
 with magnetic decantation .
the different methods were compared on 
 their ability to remove excess peg to a stable value determined by 
 tga and their effect on nanoparticle stability .
the tga results with 
 corresponding nda - peg(5 kda ) grafting density are summarized for all 
 purification methods and core sizes in table 1 .
the total organic content ( toc ) was determined 
 as the mass loss fraction up to 650 c and converted to nda - peg(5 
 kda ) dispersant grafting density using the known molecular weight 
 of nda - peg , the average iron oxide core area determined by tem , and 
 a core density of 5.17 g / cm .
as described in the materials 
 and methods section , the nps were after column purification 
 collected in 67 dark brown , red , orange , and yellow fractions . 

the same colors and amounts were observed for all np core sizes ( see figure 2b ) .
all samples passed 
 the column without visible sticking to the dextran matrix , which demonstrates 
 a high and homogeneous peg shell density on the particles .
fraction 2 could be identified as the main fraction for all sizes ; 
 it had the largest amount of nps with the smallest amount of nda - peg(5 
 kda ) .
figure 2 shows 
 the results of the column purification in terms of grafting density 
 based on measurement of organic content by tga . for two np sizes ( 9.6 
 and 7.1 nm )
the main fraction was dispersed in mq and reapplied onto 
 a fresh sephadex column .
for the 3.5 and 4.9 nm nps the amounts contained 
 in fraction 2 were too small for a second pass through the column . 

the reapplied samples passed the column as a single band with a very 
 small fraction of particles that passed the column faster .
the organic 
 content of the reapplied fractions could be decreased from 66% to 
 49% ( 9.6 nm nps ) and 91% to 73% ( 7.1 nm nps ) by the second pass . for 
 the 9.6 nm nps
this translates into a grafting density of 1.0 nda - peg(5 
 kda)/nm , while for 7.1 nm nps a grafting density of 2 
 nda - peg(5 kda)/nm is calculated .
( a ) total organic content 
 of the column purification , measured 
 by tga and converted into nda - peg(5 kda)/nm using the 
 known molecular weight of nda - peg(5 kda ) and average core surface 
 area .
toc includes large excesses of free peg and sephadex and was 
 converted into dispersants / nm for comparison with the 
 other methods but does therefore not reflect the actual grafting density 
 of nda - peg .
( b ) fractions 
 of nps after column purification dispersed in water ( 9.6 nm , upper ) 
 and freeze - dried ( 7.1 nm , lower ) .
dialysis has to be performed with membranes 
 having a pore size sufficiently large to allow diffusion of the dispersant 
 through the pores ; the mwco of membranes is however typically given 
 for equivalent protein or dextran sizes and can not be used directly 
 for comparison to highly solvated polymer molecular weights for which 
 the radius of gyration is much bigger than for e.g. protein of the 
 same molecular weight .
a rule of thumb of choosing a pore size > 5 
 that of the dialyzed molecule is often invoked .
this was confirmed 
 by attempting dialysis of dissolved peg(5 kda ) ( 0.2 g / ml against 5 
 l of milli - q ) in a membrane of 1214 kda mwco , i.e. , nominally 
 23 times the molecular weight of the nda - peg , which was used 
 in previous publications studying grafting - to and ligand replacement 
 of pegylated nps .
after 24 h only 50% of the free 
 peg was removed , and only 60% had been removed after 5 days despite 
 exchanging milli - q in the reservoir five times .
thus , choosing a too 
 low mwco results in remaining free dispersant and subsequent overestimation 
 of the dispersant grafting density by tga .
we therefore changed to cellulose - based 
 membranes with a mwco of 1000 kda , which nominally corresponds to 
 a cutoff hydrodynamic size of 37 nm ( assuming a scaling related 
 to equivalent sizes of dextran ) .
nps with a hydrodynamic radius rh between 15 and 23 nm ( for 7.1 nm cores ) were 
 still retained while free nda - peg(5 kda ) with an rh of 4 nm was presumably 
 removed from the dispersion .
figure 3a shows the removal of excess ligands by 
 dialysis for all sizes of nps .
large - core nps ( 7.1 and 9.6 nm ) dialyzed 
 for 24 h yielded an average grafting density of 1 nda - peg(5 
 kda)/nm determined by tga .
however , nps also started to 
 stick to the dialysis membrane after 24 h. tem of large nps ( 7.1 and 
 9.6 nm ) extracted from the dialyzed dispersion after 24 h showed aggregated 
 morphology ( figure 3b ) .
the 24 h dialyzed samples could be redispersed in water but showed 
 poor long - term stability .
visible aggregates were obtained after 1 
 week . continued dialysis of the large cores led to an even lower grafting 
 density ( 0.5 nda - peg(5 kda)/nm ) and increased 
 aggregation .
( a ) removal of excess ligands by dialysis with a 1000 
 kda mwco 
 membrane .
total organic content was measured by tga and converted 
 into nda - peg(5 kda)/nm using the known molecular weight 
 of nda - peg(5 kda ) and average core surface area determined by tem . 

after 24 h the surface coverage was 1 nda - peg(5 kda)/nm independent of size , but core size - dependent aggregation 
 and precipitation were observed .
dialysis for 70 h lowered the grafting 
 density to 0.3 nda - peg(5 kda)/nm , resulting in colloidally 
 unstable dispersions .
( b ) typical tem of nanoparticles with 9.6 nm 
 core size that had visible aggregates ( inset ) grafted with nda - peg(5 
 kda ) after 24 h dialysis against water using a 1000 kda mwco cellulose 
 membrane .
nps with small core sizes ( 4.9 
 and 3.5 nm ) had grafting densities 
 of 0.8 and 1.7 nda - peg(5 kda)/nm , respectively , after 
 24 h dialysis and showed good colloidal stability .
the small nps were 
 dialyzed also for 70 h , which resulted in lowering of the average 
 grafting density to 0.3 nda - peg(5 kda)/nm . during 
 dialysis
the np suspension underwent visible color change 
 from light brown to almost black ( figure s2 in the supporting information ) , indicating that as dialysis proceeds , 
 surface - bound nda - peg is ripped off in addition to the initial removal 
 of free nda - peg .
in contrast 
 to diffusion - driven 
 dialysis , a mwco of 50 kda was sufficient to remove excess nda - peg . 

this can be ascribed to the convective flow and high forces induced 
 by the centrifugation that deform the free polymer to flow through 
 narrow pores .
figure 4 shows the results of the membrane centrifugation for the different 
 np sizes . ( a ) removal of excess ligands by repeated membrane centrifugation 
 with amicon 50 kda centrifugation filter units .
after 11 repetitions 
 all sizes show an average grafting density around 0.50.7 nda - peg(5 
 kda)/nm .
( b ) more centrifugation steps lead to a decrease 
 in grafting density and colloidal instability .
left to right : 4.6 
 nm nps after 5 , 4.6 nm nps after 15 , and 7.1 nm nps after 11 centrifugation 
 steps .
the average grafting density at this point was measured to 
 be 1 nda - peg(5 kda)/nm by tga , but the resuspended 
 nps were colloidally unstable , as can be seen by the visible aggregates 
 sticking to the vial in figure 4b . in order to study
if the grafting density stays constant , 
 the centrifugation 
 steps were repeated up to 15 times .
as shown in figure 4a , the grafting density did not stay constant 
 but could be reduced to 0.4 nda - peg(5 kda)/nm .
these nps 
 could be redispersed in milli - q but showed visible aggregation and 
 precipitation after 2 days ( figure 4b ) .
analysis of the combined centrifugates showed a 
 99.8% organic composition , meaning that only ligands were removed 
 from the dispersion and all cores were retained by the membrane .
all 
 nps purified by this method showed strong aggregation and were therefore 
 not analyzed further . both column 
 separation and filtration ( dialysis and membrane centrifugation )
are 
 established methods , but they failed to produce a high yield of colloidally 
 stable nanoparticles for all sizes when the peg - shell was grafted 
 through ligand replacement .
we therefore attempted a variation on 
 precipitation that exploits the possibility to magnetize the nanoparticle 
 cores . while hydrophilic , polymer - stabilized superparamagnetic nanoparticles 
 can not be pulled out of aqueous solution using a fixed magnet , such 
 extraction is possible in solvent / nonsolvent mixtures in which the 
 poor solubility leads to formation of small aggregates . by dispersing 
 the particles in a solvent mixture of etoh with an addition of petrolether , 
 a cloudy suspension of poorly dispersed core 
the poorly suspended nps could be pulled to the 
 container wall by application of a fixed magnet .
the decantation 
 step has to be repeated several times due to the poor solubility also 
 of the free peg dispersant in the etoh / petrolether .
tga was therefore 
 performed after each decantation step to evaluate the stepwise reduction 
 in dispersant concentration ( figure 5a ) . after the third precipitation and magnetic decantation 

step the grafting density stayed constant , interpreted as that no 
 further dispersant removal is possible and that all remaining dispersants 
 are strongly bound to the iron oxide core surface .
the grafting density 
 is within the measurement error independent of core size at 1 
 nda - peg(5 kda)/nm .
standard deviations were calculated 
 based on results measured on 67 different samples for each 
 core size .
the combined supernatants were analyzed with tga , and 1.5% 
 inorganic material was found , which means that this purification method 
 removes free nda - peg and no cores .
after reaching the constant grafting 
 density by precipitation and magnetic decantation the retained product 
 included 98% of the cores initially added for ligand replacement independent 
 of np size . ( a ) step - by - step removal of excess ligands by repeated precipitation / magnetic 
 decantation
total organic content was measured by tga and converted 
 into nda - peg(5 kda)/nm .
after three precipitations with 
 magnetic extraction the free dispersant is removed for all core sizes . 
 a stable grafting density of 1 nda - peg(5 kda)/nm independent of core size remains with a yield of 98% purified nps . 

( b ) representative tem micrograph of well - dispersed , nda - peg - grafted 
 nps with 9.6 nm core diameter . in comparison to oa - capped nps
it is 
 possible to dry the nps well dispersed and without order on the grid 
 due to the steric repulsion of the added peg shell ( cf .
tem micrographs 
 of smaller cores purified by this method can be found in the supporting information ( figure s7 ) .
compared 
 to the oa - capped nps ( figure 1d ) , the interparticle distance is increased due to the bulky 
 nda - peg shell , and importantly an inspection of thousands of particles 
 did not reveal any aggregated cores .
this strongly suggests that the 
 peg shell was formed around individual cores and remained homogeneous . 

the morphology of the np cores did not change after oa was replaced 
 by nda - peg . a color change from light brown to dark brown
the difficulty of achieving complete exchange of oa for 
 new dispersants previously reported by us and others demonstrates the need to 
 establish that the total organic content determined by tga is traced 
 exclusively to nda - peg .
significant amounts of other dispersants on 
 the core surface might alter the physicochemical properties of the 
 core  shell particles .
atr - ftir analysis was performed to verify 
 the composition of the dispersant shell after purification ( figure 6a ; insets can be 
 found in figure s4 ) .
ir spectra were also 
 recorded on the complete supernatant collected from the precipitation 
 and magnetic decantation method .
the presence of nda - peg on the np 
 surface could be confirmed by several marker bands below 1000 cm as well as bands in the region from 1345 to 1240 
 cm and from 1144 to 840 cm that can be mainly attributed to ethylene glycol ch2 and 
 c  o / c  c stretching vibrations , respectively .
( a ) atr - ftir spectra of 7.1 nm iron oxide nanoparticles : 
 ( a ) as - synthesized 
 cores with oa ligands , ( b ) nda - peg - nps after ligand exchange before 
 purification , ( c ) peg - nda - nps purified by precipitation and magnetic 
 decantation , ( d ) peg - nda - nps dialyzed 24 h , ( e ) peg - nda - nps purified 
 through sephadex g75 column ( fr ii ) , ( f ) sephadex g75 , ( g ) peg - nda , 
 and ( h ) combined supernatants of all magnetic decantation steps .
labeling : 
 (  ) 1960 cm oh - bending from peg - cooh , ( + ) 
 1740 cm coor or cooh , (  ) 1702 cm ( c = o ) free oa , (  ) 1640 cm amide 
 conh or nh2 , (  ) 1074974 cm sugar band , (  ) 965 cm ( ( c = o)oh ) 
 free oa ; 680400 cm np bands . ( b ) representative 
 tga ( solid lines ) and dsc ( dashed ) graphs of 4.9 nm nps measured in 
 synthetic air : oa - capped nps ( orange ) , nda - peg - nps  raw  
 after ligand exchange before purification ( black ) , precipitation and 
 magnetic decantation ( green ) , dialyzed ( light blue ) , membrane filtration 
 with amicon ( dark cyan ) , column purified ( dark yellow ) , and pure ligand 
 nda - peg ( navy ) .
the amide stretching 
 vibrations at 1640 ( co ) and 1550 cm ( nh ) ( figure 6a (  ) ) that can be found in all spectra 
 except for the oa - np are a good indication of successful coupling 
 of nda to peg and its presence at the np surface ; this peak is slightly 
 larger for nps purified by magnetic decantation compared to dialyzed 
 nps .
a weak peak ( figure s4b (  ) ) 
 at 1491 cm fits monodeprotonated nitrocatechol and is attributed to surface - bound nda - peg . 

the band at 1728 cm ( figure s4a (  ) ) arises from residual carboxylic carbonyl groups 
 of free peg(5 kda ) that has not been coupled to nda and is found only 
 in the nonpurified sample and the combined supernatants .
a similar 
 trend for the band at 1960 cm ( figure 6a (  ) ) suggests a common 
 origin ; we therefore attribute this vibration to an overtone or combination 
 vibration of the out - of - plane oh bending mode of peg carboxylic acid
. the bands at 1730 and 1740 cm ( figure 6 ( + ) ) that were found in all 
 samples except in the precipitation purified nps and oa - nps might 
 originate from peg - cooh or coor groups from unreacted peg or cross - reacted 
 nda - peg ( o - ph ) . however , these byproducts were removed by precipitation / magnetic 
 decantation .
an important observation is that the nps purified 
 by column show 
 deviations from the ir absorption spectrum for iron oxide nanoparticles 
 purified by other means .
the column material sephadex dextran shows 
 strong similarities ( bands at 1074974 and 790740 cm , figure 5a (  ) , oh vibrations at 36003000 cm ) with these additional peaks in the spectrum , indicating that nps 
 purified by column are additionally covered with small amounts of 
 dextran .
a final important question is the presence of significant 
 amounts 
 of weakly or strongly bound oleic acid after ligand replacement and 
 purification .
in particular , free or weakly bound oleic acid can greatly 
 influence the colloidal stability .
the presence of free oleic acid 
 can be determined from absorptions at 1702 cm in 
 the ftir spectra ( figure 6 (  ) ) assigned to the c = o stretch of h - bonded 
 ( dimeric ) alkylcarboxylic acids and from other bands characteristic 
 for free acid groups such as the oh in - plane and out - of - plane bending 
 at 1410 and 965 cm which is overlaid ( figure 6 (  ) ) .
the absence of the bands at 1702 cm in all samples 
 ( also the raw sample ) demonstrates that free oa was successfully removed 
 by the dmf  hexane extraction .
the presence of oleate can be evaluated at 1605 , 1520 , and 1410 
 cm corresponding to two asymmetric and one symmetric 
 stretching vibrations of the carboxylate headgroup . whereas the peaks at 1605 and 1520 cm are overlaid 
 by bands from the peg , a slight shoulder of the band at 1410 cm can be found in all samples .
this band , however , 
 is more prominent in the raw and the dialyzed samples than in the 
 precipitated samples . for the column purified nps this peak
multistep 
 tga profiles observed below 400 c have been attributed 
 alternatively to vaporization of physisorbed oleic acid , to chemisorbed 
 oleate species with different binding strengths , or to partial cleavage of the capping agent .
our data ( figure 6b ) show that a significant second step in tga only 
 is observed when free or weakly bound impurities such as physisorbed 
 oleate or dextran are present .
a one - step profile is only observed 
 for nps purified by magnetic decantation .
we conclude from the 
 ir measurements that the ligand replacement 
 is not complete in any sample .
this is also suggested by that the 
 measured high surface coverage of 1 nda - peg(5 kda)/nm is still lower than the theoretical maximum surface coverage 
 of > 2 nda - dispersants / nm .
however , 
 for samples purified by precipitation and magnetic extraction only 
 a minor amount of strongly complexed oleate remains on the particle 
 surface shielded by a dense peg brush .
if oleate with a negligible 
 desorption rate is distributed on the particle surface , its short 
 extension combined with the high peg - brush density still results in 
 excellent colloidal stability as shown in the next section .
the long - term stability of samples 
 that were purified by dialysis and magnetic decantation was investigated 
 with tem , dls , and saxs .
long - term stability of dialyzed samples was 
 only investigated for 4.9 nm core size as the dispersions of nps with 
 larger core diameters showed visible aggregation after a few days . 

also , samples purified by membrane filtration were not subjected to 
 long - term stability studies as the dispersions showed visible aggregates 
 after 2 days . for long - term studies of core 
shell nps 
 purified by precipitation and magnetic decantation , 7.1 nm nps were 
 dispersed at a high concentration of 4 mg / ml that speeds up aggregation 
 in both h2o and pbs .
pbs tests the stability at physiological 
 ionic strengths and is additionally known to cause aggregation and 
 precipitation of poorly stabilized iron oxide nps due to the strong 
 interaction of phosphates with iron ions .
tem , dls , and saxs were 
 measured after 1 week and 4 months .
for nps in water and pbs a rh of 16 and 23 nm ( intensity weighted ) , respectively , 
 was measured by dls ( table 2 ) , which is in good agreement with previous findings for individually 
 stabilized core 
shell np with nda - peg monolayer , considering the 
 length of hydrated nda - peg(5 kda ) to be 4 nm and the core 7.1 nm , resulting in a rh(theoretical ) of 15 nm .
these values are significantly 
 smaller than in other recent publications investigating direct ligand 
 replacement on oleic acid - capped nanoparticles with peg dispersants . 

the narrow size distributions strongly support the interpretation 
 of individually stabilized and colloidally stable core 
no precipitation and negligible 
 change in the hydrodynamic radius were found for nps in milli - q as 
 well as pbs after storage of the dispersions for 4 months at 4 c 
 ( table 2 ) .
the rh of dialyzed 4.9 nm cores was 
 measured to be 16 nm and increased slightly after 4 months to 18 nm . 
 tem imaging of the nanoparticles stored for 4 months in milli - q and 
 pbs showed no trace of core aggregation or aging of the particles 
 ( figure s5 ) .
the stability of the dialyzed 
 sample during storage , but not to further dialysis , indicates a degradation 
 mechanism related to the dialysis membranes .
scattering curves and fits for samples in milli - q and pbs 
 fresh and after 4 months are shown in figure 7b .
the high monodispersity of the cores is 
 evident from the scattering curves . a core size of 6.8  0.4 
 nm could be fitted to the data .
this 
 is in excellent agreement with the core size determined by image analysis 
 of the tem data of the same particles ( 7.1  0.4 nm ) .
after storage 
 for 4 months at 4 c there is no significant deviation in the 
 scattering attributable to aggregates , such as a structure factor 
 peak and/or a change in the low q range .
( a ) dls measurements 
 of 7.1 nm nps purified by magnetic decantation ; 
 3 mg / ml in pbs with 10% v / v fetal calf serum added .
temperature cycle 
 from 20 to 70 c ( circles ) and back ( triangles ) with 5 min equilibration 
 time at each temperature .
( b ) saxs curves of 7.1 nm nps in pbs and 
 h2o , freshly prepared and after storage for 4 months at 
 4 c .
inset : clear colloidal dispersion of nps from measurement 
 ( a ) stored in pbs / fcs 10% v / v for 2 months at 4 c after temperature 
 cycling .
the colloidal stability of the 
 nps was further challenged by heating 
 the nps ( 3 mg / ml ) up to 70 c in pbs with 10% v / v fetal calf 
 serum ( fcs ) .
this test is applied to distinguish between particles 
 that show stability in serum but interact with denatured protein aggregates 
 and those for which interactions between core and denatured proteins 
 also do not occur.figure 7a shows the evolution of the hydrodynamic 
 radius with temperature .
no increase in rh or precipitation of nanoparticles during the temperature cycle was 
 observed .
the inset in figure 7b shows a picture of the same np dispersion subjected to temperature 
 cycling after being stored for 2 months at 4 c .
aggregation 
 or precipitation also can not be observed after storage ; the dispersion 
 remains clear and colloidally stable .
tem micrographs ( figure s6 ) show that the nps in pbs / fcs remain 
 well - dispersed after storage for 2 months in serum .
dialysis has to be performed with membranes 
 having a pore size sufficiently large to allow diffusion of the dispersant 
 through the pores ; the mwco of membranes is however typically given 
 for equivalent protein or dextran sizes and can not be used directly 
 for comparison to highly solvated polymer molecular weights for which 
 the radius of gyration is much bigger than for e.g. protein of the 
 same molecular weight .
a rule of thumb of choosing a pore size > 5 
 that of the dialyzed molecule is often invoked .
this was confirmed 
 by attempting dialysis of dissolved peg(5 kda ) ( 0.2 g / ml against 5 
 l of milli - q ) in a membrane of 1214 kda mwco , i.e. , nominally 
 23 times the molecular weight of the nda - peg , which was used 
 in previous publications studying grafting - to and ligand replacement 
 of pegylated nps .
after 24 h only 50% of the free 
 peg was removed , and only 60% had been removed after 5 days despite 
 exchanging milli - q in the reservoir five times .
thus , choosing a too 
 low mwco results in remaining free dispersant and subsequent overestimation 
 of the dispersant grafting density by tga .
we therefore changed to cellulose - based 
 membranes with a mwco of 1000 kda , which nominally corresponds to 
 a cutoff hydrodynamic size of 37 nm ( assuming a scaling related 
 to equivalent sizes of dextran ) .
nps with a hydrodynamic radius rh between 15 and 23 nm ( for 7.1 nm cores ) were 
 still retained while free nda - peg(5 kda ) with an rh of 4 nm was presumably 
 removed from the dispersion .
figure 3a shows the removal of excess ligands by 
 dialysis for all sizes of nps .
large - core nps ( 7.1 and 9.6 nm ) dialyzed 
 for 24 h yielded an average grafting density of 1 nda - peg(5 
 kda)/nm determined by tga .
however , nps also started to 
 stick to the dialysis membrane after 24 h. tem of large nps ( 7.1 and 
 9.6 nm ) extracted from the dialyzed dispersion after 24 h showed aggregated 
 morphology ( figure 3b ) .
the 24 h dialyzed samples could be redispersed in water but showed 
 poor long - term stability .
visible aggregates were obtained after 1 
 week . continued dialysis of the large cores led to an even lower grafting 
 density ( 0.5 nda - peg(5 kda)/nm ) and increased 
 aggregation .
( a ) removal of excess ligands by dialysis with a 1000 
 kda mwco 
 membrane .
total organic content was measured by tga and converted 
 into nda - peg(5 kda)/nm using the known molecular weight 
 of nda - peg(5 kda ) and average core surface area determined by tem . 

after 24 h the surface coverage was 1 nda - peg(5 kda)/nm independent of size , but core size - dependent aggregation 
 and precipitation were observed .
dialysis for 70 h lowered the grafting 
 density to 0.3 nda - peg(5 kda)/nm , resulting in colloidally 
 unstable dispersions .
( b ) typical tem of nanoparticles with 9.6 nm 
 core size that had visible aggregates ( inset ) grafted with nda - peg(5 
 kda ) after 24 h dialysis against water using a 1000 kda mwco cellulose 
 membrane .
nps with small core sizes ( 4.9 
 and 3.5 nm ) had grafting densities 
 of 0.8 and 1.7 nda - peg(5 kda)/nm , respectively , after 
 24 h dialysis and showed good colloidal stability .
the small nps were 
 dialyzed also for 70 h , which resulted in lowering of the average 
 grafting density to 0.3 nda - peg(5 kda)/nm . during 
 dialysis
the np suspension underwent visible color change 
 from light brown to almost black ( figure s2 in the supporting information ) , indicating that as dialysis proceeds , 
 surface - bound nda - peg is ripped off in addition to the initial removal 
 of free nda - peg .
first - generation , hydrophilic iron oxide nps were mainly stabilized 
 with dextran or carbohydrate derivatives physically adsorbed without using a separately 
 defined anchor such as nitrodopamine .
a second generation of polymer - coated 
 iron oxide nps could be said to have polydisperse cores but defined 
 polymer shells end - grafted with defined anchor groups .
shell architecture 
 allows for exact characterization and functionalization , which is 
 crucial to medical applications . a third generation with monodisperse 
 iron oxide cores to perfectly tailor uniform properties
several mutually contradicting conditions 
 during the ligand replacement reaction have to be fulfilled : ( 1 ) to 
 dissolve the capping agent ( oleic acid ) , ( 2 ) to solubilize the dispersant , 
 ( 3 ) to keep the dispersant at low coil size , quantitatively described 
 by e.g. rg , which determines the grafting 
 footprint , and ( 4 ) to provide the right conditions ( protonation ) of 
 the anchor group to irreversibly bind to the core .
we could 
 optimize the ligand exchange procedure within the boundaries 
 of the contradicting requirements and thus produce nps with grafting 
 densities consistently around 1 nda - peg(5 kda)/nm .
the 
 grafting densities of 1 nda - peg(5 kda)/nm for 
 monodisperse nps were shown to be sufficient to render the nps long - term 
 stable in biologically relevant media .
this is in contrast to what 
 was reported earlier for polydisperse systems . for polydisperse cores
a grafting density 
 of 2 nda - peg(5 kda)/nm was found as the threshold 
 for colloidal stability at temperatures above body temperature and 
 in biological fluids .
we hypothesize that the accurate exchange of 
 ligands into a homogeneously coated shell is what enables the improved 
 stability at a lower average grafting density for monodisperse , spherical 
 nps . on the other hand
, we were unable to achieve grafting densities 
 higher than 1 nda - peg(5 kda)/nm by direct ligand 
 replacement . that the grafting density was constant with respect to 
 core size indicates that surface curvature is not the determinant 
 of the ultimate grafting density by ligand replacement in the superparamagnetic 
 size range .
this is similar to previous observations for ligand replacement 
 of hydrophobic dispersants and nda - peg 
 grafting in the melt , for which within 
 the experimental error a difference in grafting density with respect 
 to curvature could not be demonstrated . in the present case
the constant 
 grafting density is lower and suggests that the size of the peg - coil 
 is the main determinant of the grafting density .
recent work 
 by e.g. mefford and co - workers reported very high ligand 
 densities from ligand replacement in their study of oleic acid displacement . in their study ,
one precipitation step followed by dialysis with 
 low mwco membranes was used to remove excess dispersant .
our results 
 show that this is insufficient to remove excess dispersants so their 
 unusually high dispersant density in comparison with literature can 
 likely be traced to this fact .
a higher polydispersity of the cores 
 can also have contributed to uncertainty in the grafting density .
, large monodisperse cores were grafted with nda - peg and purified 
 by repeated ultracentrifugation from aqueous solution , which is only 
 feasible with very large cores .
they report a grafting density of 
 1 nda - peg(5 kda)/nm , which is inconsistent with 
 the presented tga results that yield a grafting density within a large 
 interval of 528 nda - peg(5 kda)/nm .
this organic 
 content , which corresponds to an order of magnitude higher grafting 
 density than the theoretical maximum , demonstrates that also a single 
 centrifugation step and dialysis are insufficient to purify also large 
 core nps .
these samples even showed low colloidal stability , which 
 is typically associated with a low grafting density .
however , one 
 should keep in mind that stabilizing ferrimagnetic nanoparticles with 
 large cores using thin polymer brushes is more challenging than in 
 the superparamagnetic range . a different ligand replacement protocol 
 in this study
might also have left a higher fraction of destabilizing 
 or heterogeneously distributed oleate bound to the np surface . finally , it was recently shown that grafting of polymer shells 
 under solvent - free , polymer - melt conditions can lead to a near complete 
 coverage of end - grafted polymer dispersants , with unique shell structure 
 and colloidal properties as the result .
while this might seem superior we show here that optimized protocols 
 for grafting - to can yield the required colloidal properties for standard 
 medical applications .
our grafting - to by one - step ligand replacement 
 has the benefits of ( a ) providing near - total yields ( 98% ) under optimal 
 conditions , while melt - grafting requires considerable excess dispersant 
 and was reported with a maximum yield of 30% calculated on 
 nanoparticle cores ; ( b ) multifunctional particles can be constructed 
 by molar mixing of differently functionalized dispersants without 
 risking degradation of the functional group under the melt - grafting 
 conditions ; and ( c ) direct ligand replacement is easier to scale up 
 for synthesis of large batches .
purification of as - synthesized 
 core  shell nps is crucial 
 for proper determination of np properties , and we emphasize the thorough 
 analysis that we have done of the efficiency and effect of different 
 purification methods as a central result of the presented work . to 
 our knowledge , there has been no previous study on the colloidal stability 
 of core 
we 
 report a strong time - dependent degradation of the particles when dialyzed 
 through high mwco regenerated cellulose membranes .
cellulose ester 
 should be inert and not the cause of the observed particle degradation , 
 which from tem clearly included the particle core .
a similar trend 
 was found for nps purified by membrane filtration : np degradation 
 in terms of removal of surface - bound nda - peg was observed , and a colloidally 
 stable product could not be obtained .
degradation of the core can 
 occur by that a dispersant with a strongly binding anchor group leaves 
 the core surface and removes the surface iron ion with it .
since the other purification methods do not 
 lead to this degradation , i.e. , the nda anchor is stably bound to 
 the core , we hypothesize that the degradation during dialysis and 
 membrane filtration involves an osmotic / mechanical strain related 
 to dispersant interaction with the pores of the membranes where the 
 particles are also found to precipitate .
stretching of the peg chain 
 leads to a spring - like increase in energy ; it could effectively weaken 
 the bond to the surface and lead to polymer detachment and the observed 
 surface dissolution .
size - exclusion column chromatography with 
 cross - linked dextran 
 as column material is often used for purification of biotechnological 
 samples in the size range of nanoparticles ; it was previously advocated 
 by us and others as a suitable way to purify core 
shell nanoparticles 
 after grafting - to and simultaneously testing their grafting density 
 and colloidal stability .
dextran has high affinity to iron oxide and 
 has 
 been used in commercial contrast agent coatings of iron oxide nanoparticles . 

our new results show that for highly but not extremely densely grafted 
 nps dextran can transfer from the column to the nps .
the cross - linked 
 dextran employed in a size - exclusion column should not transfer to 
 the nps , but the ir results unambiguously show its presence in the 
 eluted np fractions even after passing the same fraction through fresh 
 columns twice . although dextran was not found by ir for the same type 
 of np cores with grafting densities > 2 nda - peg(5 kda)/nm , a lower grafting density seems to 
 result in transfer of column material to the free core area .
the high 
 peg grafting density of 1 nda - peg(5 kda)/nm obviously 
 prevents further np aggregation or sticking to the column .
we interpret 
 this result as that transferred dextran also can stabilize nanoparticles 
 that are not extremely densely grafted .
this additional physisorbed 
 dextran coating can explain the high organic content in other particle 
 fractions that passed the column .
high - molecular - weight polymer physisorbed 
 to nps induce bridging interactions that cluster multiple cores ; this 
 leads to effective differences in size and column retention time , 
 which spreads the nps over several fractions and pushes down the yield 
 of the main fraction . thus ,
despite the stability of the nps purified 
 by column chromatography , the application of cross - linked dextran 
 as column material can not be generally recommended for purification 
 that should yield particles with controlled shell properties .
the 
 choice of another column material with less strong interaction with 
 iron oxide could remedy this drawback .
however , the yield and throughput 
 would based on our study remain low compared to magnetic decantation . among
the many investigated purification methods , our study suggests 
 a combination of precipitation and magnetic decantation repeated in 
 multiple cycles as the preferred choice .
this method reproducibly 
 yields nps with exceptional long - term colloidal stability , at high 
 yield and without free dispersant .
this very high yield 
 is far superior to the inherent losses of nps when column purification 
 or dialysis is used and can be attributed to the efficient magnetic 
 extraction .
similar approaches have previously been applied in combination 
 with centrifugation , which is sensitive to the choice 
 of solvent ratio and polymer shell molecular weight for their success 
 and do not make use of the superparamagnetic core . the crucial step 
 for successful separation by magnetic decantation was to discard the 
 supernatant prior to sedimentation of precipitated excess peg , since 
 peg and densely peg - grafted nps have similar solubility .
aggregation 
 of the superparamagnetic cores enables rapid collection of the nps 
 within 25 min depending on their size ; the spontaneous precipitation 
 and sedimentation of the free peg takes 10 min .
the petrolether 
 used in the magnetic decantation also aided removal of remaining oa 
 from the nanoparticle sample that can not be removed by dialysis or 
 centrifugation , since oa was found in the removed supernatant .
a final major advantage of the magnetic decantation method is that 
 in addition to its high yield and purity it can be scaled up and automated . 

large volumes and samples can be handled in a short time ( < 1 h 
 for > 3 cycles necessary for free dispersant removal ) , while dialysis 
 is inherently time - consuming ( relying on diffusion ) , membrane centrifugation 
 is limited by the volume of the filtration units , and columns are 
 difficult to scale .
we show magnetic decantation for synthesis of 
 gram quantities and further scaling up is possible .
we conclude that direct ligand replacement of oleic acid - coated 
 monodisperse iron oxide nps can be optimized to consistently yield 
 grafting densities of 1 nda - peg(5 kda)/nm regardless 
 of core size in the investigated superparamagnetic range ( diameter 
 310 nm ) .
shell nps colloidally stable over many 
 months in biologically relevant media as well as withstanding demanding 
 tests such as temperature cycling in serum .
the choice of purification 
 method is as important as the synthesis protocol for reproducible 
 results and greatly affects yield , purity , speed , and amount that 
 can be produced .
the best purification method that we found was magnetic 
 decantation , which compared to dialysis , centrifugation filtration , 
 and sephadex size - exclusion chromatography provides the highest quality 
 product and a means to verify purity ; it also provides the highest 
 yield and sample volumes that can be extracted per time unit .
the 
 methods described in this work form the fundament for development 
 of a whole set of diversely functionalized nps that are suitable for 
 biomedical applications such as targeted mri , multifunctional superparamagnetic 
 nps , and drug delivery .
 fundamental research 
 on nanoparticle ( np ) interactions and development 
 of next - generation biomedical np applications relies on synthesis 
 of monodisperse , functional , core  shell nanoparticles free 
 of residual dispersants with truly homogeneous and controlled physical 
 properties . 
 still , synthesis and purification of e.g. such superparamagnetic 
 iron oxide nps remain a challenge . comparing the success of different 
 methods 
 is marred by the sensitivity of analysis methods to the purity 
 of the product . 
 we synthesize monodisperse , oleic acid ( oa)-capped , 
 fe3o4 nps in the superparamagnetic size range 
 ( 310 nm ) . 
 ligand exchange of oa for poly(ethylene glycol ) 
 ( peg ) was performed with the peg irreversibly grafted to the np surface 
 by a nitrodopamine ( nda ) anchor . 
 four different methods were investigated 
 to remove excess ligands and residual oa : membrane centrifugation , 
 dialysis , size exclusion chromatography , and precipitation combined 
 with magnetic decantation . 
 infrared spectroscopy and thermogravimetric 
 analysis were used to determine the purity of samples after each purification 
 step . 
 importantly , only magnetic decantation yielded pure nps at high 
 yields with sufficient grafting density for biomedical applications 
 ( 1 nda - peg(5 kda)/nm2 , irrespective of size ) . 
 the 
 purified nps withstand challenging tests such as temperature cycling 
 in serum and long - term storage in biological buffers . 
 dynamic light 
 scattering , transmission electron microscopy , and small - angle x - ray 
 scattering show stability over at least 4 months also in serum . the 
 successful synthesis and purification route 
 is compatible with any 
 conceivable functionalization for biomedical or biomaterial applications 
 of pegylated fe3o4 nps . 