experiments 
 were carried out using polystyrene nps with either 
 an anionic , carboxylate - modified or cationic , amine - modified surface 
 ( table 1 ) .
dark , nonfluorescent nps 
 were used for experiments in which the np fluorescence would interfere 
 with the measurement .
we use the diameter supplied by the manufacturer , 
 which can differ from the hydrodynamic diameter depending on characterization 
 method , to denote the np throughout the text .
adsorption of proteins 
 onto both anionic and cationic nps has been observed previously by 
 our group and others .
np complexes are formed from both anionic and cationic 
 nps ( figures s1 , s2 , and s3 of the supporting 
 information ) .
importantly , bsa  np complexes formed with 
 anionic and cationic nps have identical corona compositions and surface 
 charges ( 18.5 mv and 19.0 mv , respectively , figure 
 s2 of the supporting information ) .
cells are commonly cultured in a buffered solution , such as minimum 
 essential medium ( mem ) , containing inorganic salts , amino acids , and 
 vitamins .
the medium is supplemented with serum proteins , typically 
 fetal bovine serum ( fbs ) .
since our goal was to observe molecular - level 
 effects , we used isolated bsa for the majority of experiments .
fluorescent 
 nps were incubated with monkey kidney epithelial ( bs - c-1 ) cells in 
 mem or in mem supplemented with 10 mg ml bsa . 

this concentration of bsa is approximately equal to the total protein 
 concentration used in typical cell culture ( materials 
 and methods ) .
cellular binding studies were carried out at 
 4 c , which allows np binding to the cell surface but blocks 
 cellular internalization of the nps . for the anionic , carboxylate - modified 
 nps , we observed np binding in mem ( figure 1a ) .
interestingly , 
 we observe the opposite trend for the cationic , amine - modified nps . 

minimal cellular binding was observed in mem , while bsa enhances cationic 
 np binding ( figure 1b ) .
similar results were 
 observed for chinese hamster ovary ( cho ) cells , indicating that this 
 trend is independent of cell type ( figure s4 of the supporting information ) .
fluorescence microscopy images show cellular 
 binding of nps ( green ) 
 in mem and mem supplemented with 10 mg ml bsa 
 ( mem + bsa ) to monkey kidney epithelial cells ( bs - c-1 ) at 4 c . 

cellular binding of the anionic , carboxylate - modified 
 nps was inhibited by bsa ( figure 1a ) , suggesting 
 that bsa 
np complexes formed from anionic nps compete with 
 bsa for binding to the native albumin receptor . to test this hypothesis
, 
 we carried out cellular binding competition studies with increasing 
 concentrations of bsa . using flow cytometry as a high - throughput , 
 quantitative measure of cellular binding , we observe decreased bsa 
np 
 binding in the presence of increasing concentrations of bsa ( figure 2a ) . at a bsa concentration of 10 mg ml , similar to the concentration used in cell culture , bsa 
np 
 binding was reduced to 32% relative to 100% in the absence of bsa . 

nps formed from anionic 
 nps bind to native albumin receptors on the cell surface .
previous 
 studies have shown that some nps and protein  np complexes bind 
 to scavenger receptors , specifically scavenger receptors with an affinity for modified albumins . to determine if the bsa  nps formed from cationic nps bind 
 to scavenger receptors , we carried out competition studies in the 
 presence of both fucoidan ( figure 2b ) and polyinosinic 
 acid ( figure 2c ) , known competitors for scavenger 
 receptors . increasing concentrations 
 of fucoidan and polyinosinic acid both led to decreased cellular binding 
 of the bsa  np complexes , although fucoidan was a stronger competitor 
 ( 11% binding at 2500 g ml compared to 25% ) . 
 as a control
, we measured cellular binding of bsa  nps formed 
 from cationic nps in the presence of polyadenylic acid ( figure 2d ) .
polyadenylic acid has a similar structure to 
 polyinosinic acid but is not a competitor for scavenger receptors .
we did not observe competition in the presence 
 of polyadenylic acid ( 88% binding compared to a control normalized 
 to 100% ) .
identification of cell surface receptors using cellular binding 
 competition assays measured with flow cytometry .
( a ) cellular binding 
 of 93 nm anionic , carboxylate - modified nps in mem with increasing 
 concentrations of bsa .
( b ) cellular binding of 87 nm cationic , amine - modified 
 nps in mem supplemented with 10 mg ml bsa with 
 increasing concentrations of fucoidan .
( c ) cellular binding of 87 
 nm cationic , amine - modified nps in mem supplemented with 10 mg ml bsa with increasing concentrations of polyinosinic 
 acid .
( d ) control experiments show cellular binding of 87 nm amine - modified 
 nps in the presence of polyadenylic acid ( polya ) and the autofluorescence 
 from cells in the absence of nps ( cells only , error bar is too small 
 to see ) .
the same protein binding to different cell surface receptors , 
 especially scavenger receptors , suggests a structural difference in 
 bsa following adsorption on the anionic and cationic nps .
we used 
 circular dichroism ( cd ) spectroscopy to probe the structure of bsa 
 following incubation with dark , nonfluorescent , polystyrene nps . for 

isolated bsa , a primarily -helix protein , the far - uv cd spectrum 
 contains a positive band at 195 nm and two negative bands at 208 and 
 222 nm ( figure 3a ) , in good agreement with 
 previous cd measurements .
incubation with 60 
 nm anionic , carboxylate - modified nps shows little change in the cd 
 spectrum ( figure 3a ) . in comparison ,
incubation 
 with 58 nm cationic , amine - modified nps results in more positive mean 
 residue ellipticity ( mre ) values , indicating a loss in protein secondary 
 structure ( figure 3a ) .
cd difference spectra 
 were calculated by subtracting the spectrum of bsa alone from the 
 spectrum of bsa in the presence of nps ( figure 3b ) .
the difference spectra reveal a larger change in bsa secondary 
 structure in the presence of cationic , compared to anionic , nps .
we 
 observe similar results for bsa in the presence of 200 nm nps ( figure 
 s5 of the supporting information ) .
percent 
 -helicity was calculated using the cd peak at 208 nm ( table 2 , materials and methods ) . 

for bsa in the presence of the 58 and 200 
 nm cationic , amine - modified nps , a loss in -helicity was observed 
 ( 48% and 37% , respectively ) . for bsa in the presence of 60 nm anionic , 
 carboxylate - modified nps , a slight increase in -helicity to 
 71% was observed . for the 200 nm carboxylate - modified nps ,
the equilibrium association constant , 
 enthalpy , and binding stoichiometry of bsa adsorbed on nps was determined 
 using itc ( figure 4 and figures s6 and s7 of 
 the supporting information ) .
cd spectra 
 of bsa in the presence of 60 nm carboxylate - modified 
 nps ( red ) , 58 nm amine - modified nps ( blue ) , and in the absence of 
 nps ( black ) .
spectra , in units of mean residue ellipticity ( mre ) , 
 are the average of 10 consecutive scans .
( b ) cd 
 difference spectra were calculated by subtracting the spectrum of 
 bsa from bsa in the presence of 60 nm carboxylate - modified ( red ) or 
 58 nm amine - modified nps ( blue ) .
black dashed lines correspond to 
 spectral peaks at 195 , 208 , and 222 nm .
similar results were obtained 
 for 200 nm nps ( figure s5 of the supporting information ) .
an independent 
 site model was used to fit the integrated titration 
 curves and extract thermodynamic parameters ( table 3 , materials and methods ) .
the association 
 constant was nearly an order of magnitude higher for bsa adsorbed 
 on anionic nps with ka values of 2.4  
 0.9  10 m and 4.0  0.5 
  10 m for 60 nm anionic , carboxylate - modified 
 and 58 nm cationic , amine - modified nps , respectively .
the enthalpy 
 of bsa binding to the 60 nm carboxylate - modified ( 1.4  
 0.4  10 kj mol ) and 58 nm amine - modified 
 nps ( 1.4  0.7  10 kj mol ) was almost identical .
a significantly greater number of bsa molecules 
 adsorbed onto the surface of the 60 nm carboxylate - modified nps ( 871 
  21 proteins per np ) compared to the 58 nm amine - modified nps 
 ( 27  8 proteins per np ) , although this is likely an underestimate 
 for the 58 nm nps due to dimerization of these nps under these conditions 
 ( figure s8 of the supporting information ) .
the percent coverage , relative to a monolayer of 100% , was calculated 
 assuming bsa binds end - on , maximizing the number of bsa molecules 
 in a monolayer .
the coverage of bsa on the 60 
 nm carboxylate - modified nps was 230  6% , compared with 8  
 2% on the 58 nm amine - modified nps .
fluorescence spectroscopy was used as a 
 complementary technique to measure equilibrium association constants 
 by monitoring the quenching of tryptophan at 340 nm ( figure 5a ) .
the ratio of fluorescence intensities in the absence 
 and presence of nps were plotted versus np concentration in a stern 
as the plots were nonlinear 
 at higher np concentrations , the first four points were used for a 
 linear fit to extract an effective ksv , equivalent to an effective ka ( materials and methods ) . for bsa in the presence 
 of 60 nm carboxylate - modified nps ,
the effective ka is 1.8  0.1  10 m , compared to the 58 nm amine - modified nps with an effective ka of 7.7  0.1  10 m. isothermal titration calorimetry ( itc ) plots of differential 
 power 
 throughout the titration ( top ) and integrated heat as a function of 
 the mole ratio of bsa adsorbed on nps ( bottom ) .
the heat of dilution 
 of bsa into buffer ( figure s6 of the supporting 
 information ) was subtracted for each injection from the heat 
 of bsa titrated into a np solution .
( a ) bsa titrated into a solution 
 of 60 nm carboxylate - modified nps .
( b ) bsa titrated into a solution 
 of 58 nm amine - modified nps .
raw data plotted in the ta instruments 
 software , nanoanalyze , is shown in figure s7 of the supporting information . ( a ) raw fluorescence spectra of bsa in 
 the presence of 60 nm carboxylate - modified 
 nps ( red ) , 58 nm amine - modified nps ( blue ) , and in the absence of 
 nps ( black ) .
the black dashed line at 340 nm corresponds to the emission 
 from tryptophan residues in bsa .
( b ) stern  volmer plot of bsa 
 quenching in the presence of increasing concentrations of nps .
the 
 contribution from nps alone and buffer were subtracted , and initial 
 emission at 250 nm was set to zero .
dashed lines are the initial slope 
 used to calculate an effective equilibrium constant ( eq 3 ) .
nps 
 used in any biological application are exposed to a complex 
 mixture of extracellular proteins that form a protein corona on the 
 np surface .
we have characterized the changes in protein secondary 
 structure that result from adsorption of bsa on np surfaces and then 
 relate these structural changes to the cell surface receptor used 
 by the protein  np complex .
although bsa  np complexes 
 formed with anionic and cationic 
 nps have identical corona compositions and surface charges ( figures 
 s1 and s2 of the supporting information ) , the cellular binding trends are drastically different .
cellular 
 binding of bsa  np complexes formed with anionic nps is inhibited 
 by the presence of excess bsa ( figure 1a ) . 
 in comparison ,
the cellular binding of complexes formed with cationic 
 nps is strongly enhanced by bsa ( figure 1b ) . 

this difference in binding is independent of cell type , as similar 
 results were obtained for cho cells ( figure s4 of the supporting information ) .
the differences in cellular 
 binding suggest that the bsa  np complexes formed with anionic 
 and cationic nps bind to different receptors . in a series of cellular 
 binding competition studies
np complexes 
 formed from anionic nps bind to native albumin receptors ( figure 2a ) .
albumin has a dedicated receptor on the cell 
 surface as it is an essential blood serum protein . in comparison , bsa  np complexes formed with initially cationic 
 nps bind to scavenger receptors ( figure 2 , 
 panels b and c ) .
scavenger receptors are a broad class of receptors 
 that bind modified proteins , polysaccharides , and polyribonucleotides , as well as nps and protein  np complexes .
schnitzer et al . determined 
 that chemically or structurally modified albumins , including albumin 
gold 
 np complexes , bind preferentially to the glycoprotein scavenger receptors 
 gp30 and gp18 rather than the general class of modified protein receptors .
mirkin et al . have observed that oligonucleotide - conjugated gold 
 nps bind to scavenger receptors .
previous results 
 using a complete mixture of fbs showed identical binding trends , demonstrating that this result is not specific 
 to isolated bsa .
interestingly , for the bsa  np complexes 
 formed from cationic 
 nps , we observed that fucoidan is a much stronger competitor than 
 polyinosinic acid ( figure 2 , panels b and c ) . 
 at the highest competitor concentration ( 2500 g ml ) , bsa  np binding was 11% in the presence of fucoidan compared 
 to 25% in the presence of polyinosinic acid .
it is important to note 
 that this concentration of polyinosinic acid is relatively high compared 
 to previously reported concentrations for cellular binding competition , further confirming that polyinosinic acid is a less efficient competitor . 

polyinosinic acid is a competitor for both gp30 and gp18 , as well as the general class of modified protein receptors . in comparison , fucoidan is only a competitor 
 for the gp30 and gp18 receptors .
np 
 complexes formed from the cationic nps only bind to the receptors 
 for modified albumins , gp30 and gp18 , and not the general receptors 
 for modified proteins .
this highlights the subtle differences in molecular - level 
 interactions of nps with cells .
the opposite binding trends 
 observed for bsa  np complexes 
 formed from anionic and cationic nps provided an ideal model system 
 to study the effect of protein structure on the cellular receptors 
 used by bsa 
we hypothesized that bsa adsorbed 
 on anionic nps retained its native structure , allowing the bsa 
bsa adsorbed 
 on cationic nps was disrupted , likely partially denatured , such that 
 it is no longer recognizable by the native protein receptor and is 
 instead redirected to a scavenger receptor .
cd spectra of bsa in the 
 presence of 60 nm anionic nps show that the secondary structure of 
 bsa is retained ( figure 3 ) . in comparison , 

there is a loss in -helicity for bsa in the presence of 58 
 nm cationic nps ( 65% to 48% , table 2 ) .
similar 
 results were obtained for bsa in the presence of 200 nm nps ( figure 
 s5 of the supporting information , table 2 ) .
the greater loss in -helicity in the presence 
 of the 200 nm nps is attributed to greater bsa adsorption on the larger 
 np . although the decrease in -helicity appears modest , it should 
 be noted that boiled bsa has a similar percent -helicity as 
 bsa incubated with carboxylate - modified nps ( 71% -helix , data 
 not shown ) .
this indicates both a significant change in -helicity 
 for bsa following exposure to cationic nps , as well as a difference 
 in secondary structure for bsa disrupted by heat denaturation compared 
 to np exposure .
changes in protein secondary structure can be accompanied 
 by the exposure of new peptide sequences .
while epitope exposure and 
 structural changes are difficult to separate , changes in secondary 
 structure are the driving force for both and ultimately determine 
 the cellular receptors used by the protein . a change in protein 
 secondary structure
should be associated with 
 changes in adsorption of the protein on the np surface .
using itc 
 ( figure 4 ) , we observe that fewer proteins 
 adsorbed on the cationic , amine - modified nps ( 27  8 proteins 
 per np , compared to 871  21 proteins per np for anionic nps ) . 

similar results were obtained for sulfate - modified polystyrene nps , 
 which showed less than monolayer adsorption of bsa and disruption 
 of the bsa secondary structure .
the lower 
 binding stoichiometry is likely due to the loss of the bsa secondary 
 structure .
when spread out on the np surface , fewer bsa molecules 
 can access the surface without an energy cost .
the enthalpy of bsa binding 
 to anionic and cationic nps is identical ( 1.4  0.4  
 10 kj mol and 1.4  0.7 
  10 kj mol , respectively ) , which 
 is surprising given that the maximum bsa adsorption to the surface 
 of the 60 nm carboxylate - modified nps is approximately 30-fold greater 
 than on the 58 nm amine - modified nps .
however , the association of 
 bsa is much stronger on the anionic np surface ( 2.4  0.9  
 10 m compared with 4.0  0.5 
  10 m ) , which we attribute to 
 more energetically favorable packing of native bsa as well as some 
 degree of binding cooperativity .
previous itc experiments have examined 
 protein adsorption on nps as a function of protein species and hydrophobicity .
equilibrium association 
 constants and enthalpies are similar to our results , taking into consideration 
 the variability in itc measurements . however
, 27% coverage was observed 
 for carboxylate - modified nps and 63% coverage for amine - modified nps , compared to 230% and 8% , respectively , in our 
 experiments ( table 3 ) .
this difference could 
 be due to multiple factors , including differences in np diameter , 
 ph , buffer , and surface modification ligand .
previous fluorescence 
 correlation spectroscopy measurements showed that the number of human 
 serum albumin proteins adsorbed onto the surface of 1015 nm 
 cationic and anionic gold nps were similar .
while the difference between the values we obtained with itc and 
 the fluorescence correlation spectroscopy measurements could be due 
 to np diameter or surface modification , it should also be noted that 
 fluorescence correlation spectroscopy is insensitive to protein conformation 
 and can only be used to measure the hydrodynamic diameter of nps with 
 and without a protein corona .
these results highlight the difficulty 
 of comparing results obtained with different experimental methods 
 as well as itc experiments under even slightly different conditions . as a complementary method to itc
, fluorescence quenching was used 
 to measure equilibrium association constants ( figure 5 ) . the nonlinear stern  volmer plot ( figure 5b ) is indicative of a selective quenching mechanism . at low np concentrations ,
readily accessible tryptophan residues 
 on bsa are quenched uniformly . at higher np concentrations ,
the stronger association of 
 bsa on 60 nm anionic compared to 58 nm cationic nps was observed in 
 both the itc and fluorescence spectroscopy data , although the absolute 
 values differ by several orders of magnitude ( e.g. , ka = 2.4  0.9  10 m with itc versus 1.8  0.1  10 m from fluorescence spectroscopy for 60 nm anionic nps ) .
thermodynamic 
 parameters are highly sensitive to experimental conditions and , for 
 our experiments , the buffers required to optimize itc and fluorescence 
 spectroscopy experiments were not identical . while itc provides a 
 better quantitative measure of the association constant as it is a 
 direct , label - free measurement , the use 
 of multiple methods to confirm thermodynamic parameters is essential . fluorescence quenching has been used previously 
 to study the adsorption of bsa on gold , silver , and silver  titanium dioxide nps .
equilibrium binding constants of 10 to 10 were reported for gold , 10 for silver 
titanium 
 dioxide , and from 10 to 10 m for silver nps , depending on temperature .
the high variability in 
 equilibrium binding constants in the literature could be due to the 
 fluorescence spectroscopy method itself , in addition to the fact that 
 binding strength is highly sensitive to np material , size , and surface 
 modification .
our experiments provide 
 a molecular link between the structure 
 of the proteins that comprise the corona and the specific cellular 
 receptors used by the protein  np complex .
we show that changes 
 in protein structure upon adsorption to nps determine the specific 
 cell surface receptor used by the protein 
protein secondary structure is retained upon adsorption 
 to anionic nps , allowing the bsa  np complex to bind to native 
 albumin receptors .
the denaturation of bsa following adsorption to 
 cationic nps directs the bsa  np complexes to bind to scavenger 
 receptors .
these results have important implications for the in vivo 
 targeted delivery of nps . beyond merely confirming the presence of 
 a protein corona , it is critical to characterize the structure of 
 the corona proteins .
the diameter provided 
 by the supplier is used to identify the np : 93 nm carboxylate - modified 
 ( bang s laboratories , fc02f ) , 87 nm amine - modified ( invitrogen , 
 c29029 ) , 200 nm carboxylate - modified ( invitrogen , f8811 ) , 200 nm amine - modified 
 ( invitrogen , f8764 ) , 60 nm carboxylate - modified ( bang s laboratories , 
 pc02n ) , and 58 nm amine - modified ( bang s laboratories , pa02n ) .
the hydrodynamic diameter and zeta potential of the nps was measured 
 with a malvern zetasizer ( malvern instruments , nano - zs , table 1 ) .
nps were measured at the following concentrations : 
 93 nm carboxylate - modified ( 37 pm ) , 87 nm amine - modified ( 173 pm ) , 
 200 nm carboxylate - modified ( 13 pm ) , 200 nm amine - modified ( 15 pm ) , 
 60 nm carboxylate - modified ( 1.4 nm ) , and 58 nm amine - modified ( 1.4 
 nm ) .
the 60 nm carboxylate - modified nps and 58 nm amine - modified nps were 
 also measured in colorless mem ( invitrogen , 51200038 ) and mem supplemented 
 with either 10 mg ml bovine serum albumin ( bsa , 
 fisher , bp1600 ) or 10% ( v / v ) fetal bovine serum ( fbs , invitrogen , 
 10437028 ) . from the uv  vis spectrum of fbs ,
using the extinction 
 coefficient of bsa ( 43,824 m cm ) , the total protein concentration in 10% ( v / v ) fbs is approximately 
 10 mg ml .
solutions prepared with mem were diluted 
 by 10% ( v / v ) for zeta potential measurements to reduce the conductivity . 
 for the 58 nm amine - modified nps , solutions in mem supplemented with 
 10% ( v / v )
fbs were filtered with a 0.2 m syringe filter to 
 remove aggregates formed in the presence of protein .
all experiments were carried out with 
 three samples , and the mean and standard deviation are reported .
60 nm carboxylate - modified ( 14 
 nm ) and 58 nm amine - modified ( 0.4 nm ) nps were incubated in mem supplemented 
 with 10 mg ml bsa or 10% ( v / v ) fbs at 4 c 
 for 10 min and washed four times via centrifugation ( 16000 g , 4 c , 10 min ) .
np concentrations were optimized 
 based on the solubility and stability of the nps in solution .
previous 
 studies have demonstrated that four wash steps with centrifugation 
 followed by resuspension in water removes all observable unbound protein 
 from solution .
np complex was resuspended in either 
 buffer containing 6% ( w / v ) sds ( new england biolabs , # b7703s ) or water . 

samples were diluted with a 4x laemmli buffer ( boston bioproducts , 
 bp-110r ) and boiled for 5 min before loading onto the gel .
mini - protean 
 gradient gels ( bio - rad , 4561094 , 420% ) were used to 
 separate proteins at 40 ma and 100 v along with a 5225 kda 
 molecular weight marker ( lonza , 50547 ) .
proteins were stained for 
 1 h with simply blue safe stain ( invitrogen , lc6060 ) and imaged using 
 a li - cor odyssey imaging system .
african 
 green monkey kidney epithelial 
 cells ( bs - c-1 , atcc ) and chinese hamster ovary cells ( cho , atcc ) were 
 maintained in a 37 c , 5% carbon dioxide environment and passaged 
 every 3 days .
bs - c-1 cells were cultured in minimum essential medium 
 ( mem , invitrogen , 61100061 ) and cho cells in ham s f-12 ( f-12 , 
 invitrogen , 21700075 ) . both were supplemented with 10% ( v / v ) fbs . 

for all cell experiments , cells were grown in 35 mm glass - bottom dishes 
 ( mattek ) . for fluorescence imaging experiments , nuclei were stained 
 with 27 m 4,6-diamidino-2-phenylindole dilactate ( dapi , 
 invitrogen , d35671 ) at 37 c for 30 min .
cellular binding of nps was 
 imaged with an epifluorescence microscope ( olympus ix71 ) using a 1.20 
 n.a . , 60 water immersion objective ( olympus ) .
fucoidan 
 ( sigma - aldrich , f5631 ) and polyinosinic acid ( sigma - aldrich , p4154 ) 
 were used as competitors for scavenger receptors .
polyadenylic acid 
 ( sigma - aldrich , p9403 ) was used as a control .
the competitor or control 
 molecule was incubated with cells at 4 c for 20 min in mem for 
 93 nm carboxylate - modified nps or in mem supplemented with 10 mg ml bsa for 87 nm amine - modified nps .
the nps were then 
 incubated with cells in the presence of the competitor molecule for 
 10 min .
cells were rinsed twice with phosphate - buffered saline with 
 calcium and magnesium ( pbs , invitrogen , 14040182 ) to remove unbound 
 nps , twice with pbs without calcium and magnesium ( invitrogen , 14190250 ) , 
 and incubated in 10 mm ethylenediaminetetraacetic acid ( mallinckrodt , 
 49310 - 04 ) at 37 c for 30 min to remove the adherent cells from 
 the mattek dishes .
cells were rinsed twice with leibovitz s 
 l-15 buffer ( invitrogen , 21083027 ) via centrifugation at 10000 g for 10 min , filtered with a 40 m cell strainer 
 ( bd falcon , 352340 ) , and kept on ice for 1 h prior to analysis with 
 flow cytometry ( bd biosciences , lsr - ii ) .
a 488 nm laser was used to 
 excite the np fluorescence , and fluorescence emission was collected 
 on a 530/30 nm bandpass filter .
scatter plots and fluorescence histograms 
 were analyzed with weasel 3.0.1 ( walter and eliza hall institute of 
 medical research , victoria , australia ) .
cd spectra were 
 acquired on an olis cd spectrophotometer with the sample chamber maintained 
 at 20 c .
measurements were made using a 0.5 mm path length quartz 
 cell ( starna , 20/o - q-0.5 ) .
the bandwidth was set to 2 nm , and the 
 integration time was a function of the photomultiplier tube voltage . 

samples were measured in 10% ( v / v ) pbs without calcium and magnesium . 

bsa ( 0.098 mg ml ) was measured alone and in the presence of 60 nm 
 carboxylate - modified nps ( 0.4 nm ) , 58 nm amine - modified nps ( 0.4 nm ) , 
 200 nm carboxylate - modified nps ( 13 pm ) , and 200 nm amine - modified 
 nps ( 13 pm ) .
spectra were smoothed with 
 a savitzy  golay least - squares fitting ( digital filter = 13 ) , 
 and the value at 260 nm was set to zero to account for spectral drift . 

spectra were acquired in millidegrees and converted to mean residue 
 ellipticity using eq 1.1 the 
 mean residue ellipticity in units 
 of degrees cm dmol ( [  ] ) is 
 a function of the observed signal in millidegrees , [ ]obs , the average molecular weight of the protein ( mw ) , path length in 
 cm ( l ) , protein concentration in g l ( c ) , and the total number of amino acids ( n ) .
percent -helicity was calculated from eq 2.2the percent -helix of a protein is 
 a function of the mean residue ellipticity at 208 nm ( [ ]mre ) , minus the contribution from the -form and random 
 coil conformations at 208 nm ( 4000 ) . the observed value is compared 
 to the mean residue ellipticity of a pure -helix protein ( 33000 ) .
itc measurements 
 were made on a nano itc ( ta instruments , low volume ) at 25 c 
 with a constant stirring speed of 250 rpm .
for all titrations , 16 
 3 l injections were made with a 300 s equilibration time before 
 and after each injection .
all solutions were prepared in a 20 mm hepes 
 buffer ( ph 7.4 ) .
titration of bsa ( 75 m ) alone into hepes buffer 
 was subtracted injection - by - injection from bsa titrations into np 
 solutions .
the 60 nm carboxylate - modified nps ( 5.0 pm ) or 58 nm amine - modified 
 nps ( 1.4 nm ) were loaded into the sample cell .
integration of differential power plots 
 as a function of time gave binding curves , and the raw data was fit 
 with the one independent site model using nanoanalyze ( ta instruments ) .
the theoretical 
 monolayer coverage of bsa molecules per np was calculated using the 
 assumption that bsa binds end - on to the np surface with a footprint 
 of 3.3  10 bsa molecules per cm .
all solutions 
 were prepared in 10% ( v / v ) pbs without calcium and magnesium .
samples 
 were excited at 280 nm , and emission was collected between 250 and 
 500 nm using 5 nm slit widths for both excitation and emission .
bsa 
 ( 9.8 g ml ) was measured in the presence 
 of nonfluorescent 60 nm carboxylate - modified and 58 nm amine - modified 
 nps . working np concentration ranged from 67 to 533 pm .
experiments 
 were repeated in triplicate , and the mean and standard deviation are 
 plotted for each data point .
corrected spectra are the raw spectra 
 with the np scatter peak and buffer contribution subtracted out and 
 the initial emission value at 250 nm set to zero . the stern 
volmer 
 equation was used to calculate the equilibrium constant ( eq 3).3 the fluorescence 
 intensity ratio of 
 bsa at max in the absence ( fo ) and presence ( f ) of a 
 quencher is calculated and plotted versus the np quencher concentration 
 ( [ np ] ) .
 nanoparticles used for biological 
 and biomedical applications encounter 
 a host of extracellular proteins . 
 these proteins rapidly adsorb onto 
 the nanoparticle surface , creating a protein corona . 
 poly(ethylene 
 glycol ) can reduce , but not eliminate , the nonspecific adsorption 
 of proteins . as a result , 
 the adsorbed proteins , rather than the nanoparticle 
 itself , determine the cellular receptors used for binding , the internalization 
 mechanism , the intracellular transport pathway , and the subsequent 
 immune response . 
 using fluorescence microscopy and flow cytometry , 
 we first characterize a set of polystyrene nanoparticles in which 
 the same adsorbed protein , bovine serum albumin , leads to binding 
 to two different cell surface receptors : native albumin receptors 
 and scavenger receptors . using a combination of circular dichroism 
 spectroscopy , isothermal titration calorimetry , and fluorescence spectroscopy 
 , 
 we demonstrate that the secondary structure of the adsorbed bovine 
 serum albumin protein controls the cellular receptors used by the 
 protein  nanoparticle complexes . 
 these results show that protein 
 secondary structure is a key parameter in determining the cell surface 
 receptor used by a protein  nanoparticle complex . 
 we expect 
 this link between protein structure and cellular outcomes will provide 
 a molecular basis for the design of nanoparticles for use in biological 
 and biomedical applications . 
Results
Discussion
Conclusions
Materials and Methods