most synthetic routes to high - quality 
 nanoparticles ( nps ) , with 
 respect to uniformity , size control , and crystallinity , generally 
 make use of organic solutions and predominantly employ long - chain 
 hydrocarbons that contain a coordinating headgroup such as oleic acid 
 ( oa ) as the ligand to sterically stabilize nps in nonpolar , hydrophobic 
 solvents .
however , biological applications 
 require nps to be fully dispersible in hydrophilic or aqueous media 
 without degradation of their properties . therefore , organically - synthesized 
 hydrophobic nps need to go through a surface modification process 
 before use in bioapplications .
the first general approach to such surface modification entails 
 replacement of the original ligands by a specially designed hydrophilic 
 species through coordinate bonding in a process known as ligand exchange . while this approach is very practical 
 and widely used , one major limitation of this method is that most 
 of the ligand - exchange reactions employ stronger surface binding molecules 
 that replace the original ligand , often yielding an irreversible ligand 
 exchange , thus making it difficult for further functionalization of 
 the nps .
another popular approach , known as ligand attraction , uses 
 hydrophobic van der waals interactions , through which the hydrophobic 
 tail of an amphiphilic ligand interacts with , but does not replace , 
 the hydrophobic ligand on nanocrystals and leads to the complete encapsulation 
 of the np core through a bilayer formation ; the hydrophilic part renders 
 the nps fully dispersible in aqueous media . in the ligand attraction method , the original ligand layers do not 
 get replaced 
the particle dispersity of the newly functionalized 
 nps is nearly as good as the original nps solution with almost no 
 aggregation .
the nature of the surface ligands on nps is an important consideration 
 in determining their stability as well as toxic effects in biological 
 systems .
depending on the nanoparticle type and the solvent in which 
 the particles are dispersed , the choice of appropriate ligand may 
 not only provide the stability of the nps but also prevent any aggregation 
 in solution .
therefore , the ligand molecules have to be bound to the 
 nps surface by some kind of attractive interaction , for example , by 
 chemisorption , electrostatic attraction , or hydrophobic interaction . though different types of ligands and attachment 
 strategies have been developed for efficient use of nps ,
ligands are 
 highly sensitive to conditions such as solution ph , ionic concentration , 
 and the nature of the interactions , which can lead to dissociation 
 from the nps .
we have chosen to study an up - converting 
 phosphor np ( nayf4:yb , er ) as representative of a subset 
 of the class of particles 
 with high potential for future biological applications . during up - conversion
, long wavelength , low energy photons are absorbed , 
 leading to the emission of shorter wavelength , higher energy photons . 

these nps offer unique optical properties for biolabeling , including 
 narrow emission band - widths , large anti - stokes shifts , long luminescence 
 lifetimes , and excellent photostability , making them more attractive 
 for use as biomarkers than conventional organic dyes or quantum dots .
similarly to other controlled synthesis methods of nps , the up - converting 
 phosphors are commonly synthesized in organic media followed by postsynthesis 
 modification of their surfaces , especially for bioapplications .
importantly , 
 this class of nps is known to be less toxic than its counterparts 
 such as quantum dots .
although the cytotoxicity of nps generally depends on a number 
 of critical parameters such as particle size , shape , surface charge , 
 hydrophobicity and the nature of the surface ligands , up - converting 
 nps have only been tested for a limited range of functionalization 
 chemistries .
therefore , there is a clear need to test this class of materials 
 at different conditions , especially under different surface - ligand 
 binding conditions , which are critical for maintaining biocompatibility . to assess the fate of the nps under physiological conditions , we 
 have engineered nps with three types of surface conditions : ( i ) particles 
 with no ligands ( i.e. , bare surface nps ) ; ( ii ) particle with an additional 
 biocompatible ligand layer attached by hydrophobic ligand attraction ; 
 and ( iii ) particles with a biocompatible ligand
poly(ethylene glycol ) ( peg ) was chosen as the 
 hydrophilic ligand for its extreme good biocompatibility , which renders 
 stability to nanoparticles and prevent undesired protein adhesion . 

a hydrophobic oleate - coupled peg was chosen as the amphiphilic ligand 
 for the bilayer formation as oleic acid is one of the extensively 
 used hydrophobic ligand used for nanoparticle synthesis .
a number 
 of peg - coupled amphiphilic ligands have been reported in the literature 
 for hydrophobic ligand attraction mediated phase transfer such as 
 octylamine 
poly(acrylic acid)poly(ethylene glycol ) 
 ( oa  paa  peg ) , poly(ethylene 
 glycol)-block - poly(carprolactone ) ( peg - b - pcl ) , poly((ethylene glycol)-block - lactic acid ) ( peg - b - pla ) , and poly(ethylene glycol ) monooleate ( peg - oa ) .
oleate 
 hydrophobic bilayer with the synthesized oleate - capped up - converting 
 nps and minimally affects the original surface conditions of the nps . 
 for covalent
binding , peg - phosphate was used as the ligand for its 
 widespread use in similar usage .
the interaction of the cells with 
 these types of nps can provide general insight into the potential 
 for toxic effects due to the different surface conditions for any 
 organically synthesized nanomaterial .
to understand the interaction , the as - synthesized nayf4:yb , er ( i.e. , oleate - capped nayf4:yb , er ) nps were specifically 
 modified as follows : ( i ) nayf4:yb , er with no ligands ; ( ii ) 
 nayf4:yb , er nps coated with a peg - oleate bilayer ; and ( iii ) 
 covalently bound peg - nayf4:yb , er nps ( figure 1 ) .
cytotoxicity was assessed with an mtt enzymatic activity 
 assay that was confirmed by a calcein 
schematic of surface modification of the oleate - capped 
 nayf4:yb , er nps as ( i ) ligand - free ( top ) , ( ii ) peg - oleate 
 bilayered 
 ( middle ) , and ( iii ) covalently bound peg nps ( bottom ) .
yttrium chloride hexahydrate ( > 99.9% ) , 
 erbium 
 chloride hexahydrate ( > 99.995% ) , ytterbium chloride hexahydrate 
 ( > 
 99.9% ) , oleic acid ( tech . , 90% ) , 1-octadecene ( tech . , 90% )
, ammonium 
 fluoride ( > 99.99% ) , sodium hydroxide ( pellets , reagent grade > 98% ) , 
 poly(ethylene glycol ) ( mn  2000 ) , 
 peg monooleate ( mn  860 ) were 
 purchased from sigma - aldrich .
cyclohexane and hydrochloric acid were 
 purchased from emd , hexane ( acs ,  98.5% ) , methanol ( acs , 99% ) , 
 and ethanol were purchased from fisher scientific , and chloroform 
 ( acs , 99.8% ) was purchased from alfa aesar .
oleate - capped nayf4:yb , er nps were synthesized 
 using a high - temperature synthesis 
 method developed by li and zhang .
typically , 
 0.8 mmol of ycl36h2o , 0.18 mmol of ybcl36h2o , and 0.02 
 mmol of ercl36h2o were dissolved in a mixture of 6 ml oleic 
 acid and 15 ml 1-octadecene and heated to 120 c under vacuum 
 for 2 h. the mixture was cooled to room temperature and a solution 
 of 2.5 mmol naoh and 2.5 mmol nh4f dissolved in 10 ml methanol was 
 added .
the resulting mixture was further stirred for 30 min at room 
 temperature and then slowly heated to 70 c to remove methanol . 

once methanol was removed , the resulting solution was heated to 300 
 c under an argon atmosphere and kept for 1 h. subsequently , 
 the solution was cooled down to room temperature and precipitated 
 with ethanol .
the washed nps were dispersed in 5 ml chloroform 
 for further surface modification and characterization .
the synthesis approach is based on removing the 
 oleate ligands from the surface of as - synthesized nps using an acid 
 treatment process as developed by bogdan et al .
typically , the as - synthesized oleate - capped nps ( 50 mg ) 
 were dispersed in 10 ml cyclohexane , then 20 ml of 0.1 m hcl ( ph = 
 3 ) was introduced . after vigorous stirring for 3 h , the nps were transferred 
 from the upper cyclohexane to the water below . during the reaction 
 the carboxylate groups of the oleate ligands were protonated to yield 
 oleic acid . after separating the aqueous layer ,
the nps in the water dispersible 
 fraction were collected by centrifugation and were rinsed by acetone . 

the nps were then dispersed in water and collected by ultracentrifugation 
 at 100  000 g for 1 h. the pellets were redispersed 
 in 10 ml milli - q water ( 18 m ) for further use . the phase transfer protocol was adapted from 
 the method developed by colvin et al .
typically , 
 1 ml of nps ( 0.2 mmol ) dispersion in chloroform ( i.e. , stock solution 
 of the oleate - capped nps ) was added to 500 mg peg - monooleate dissolved 
 in 9 ml chloroform .
then , chloroform was removed slowly under vacuum at room temperature , 
 leaving a waxy layer at the bottom of the flask .
about 15 ml of milli - q 
 water ( 18 m ) was added to the waxy liquid and dispersed very 
 well by sonication ( 2510 branson ultrasonic cleaner ) for 30 min .
the flask 
 was put under vacuum and the remaining chloroform was removed slowly 
 at room temperature .
the nps were collected using a microcentrifuge 
 ( accuspin micro , fisher scientific ) at a speed of 13  000 rpm 
 for 10 min .
the collected pellet was redispersed in milli - q water 
 to a concentration of 7.8 mg / ml for cell study .
nps were further diluted 
 in dpbs to 2000 g / ml . this stock solution was used for all 
 subsequent experiments .
nanoparticle stock solutions were ultrasonicated 
 for 5 min immediately prior to final dilution in culture medium for 
 cell exposure .
covalently bound peg- nayf4:yb , er 
 nps were synthesized using a modified protocol developed by boyer 
 et al .
approximately , 50 mg of ligand 
 free nanoparticles dispersed in 2 ml of absolute ethanol were taken 
 into a 20 ml glass vial and 300 mg of peg ( 750)-phosphate ligand was 
 added to it .
the vial was capped tightly and the resulting solution 
 was stirred overnight at 60 c .
it was then cooled to room temperature , 
 and nps were collected via centrifugation at 10000 rpm .
transmission 
 electron microscopy ( tem ) images were recorded using a philips cm-12 
 tem equipped with a ccd camera operating at 120 kv .
the samples were 
 prepared by drop - casting the np dispersions on a formvar - coated 400-mesh 
 copper tem grid and air - dried .
powder x - ray diffraction ( xrd ) pattern 
 of the nps were collected using a scintag powder x - ray diffractometer 
 ( xrd ) with cu k radiation ( = 1.54056  , operated 
 at 45 kv and 40 ma ) with 4 mm divergence slit , 1 mm scattering slit , 
 and 0.2 mm receiving slit .
the scanning step size was 0.015 
 in 2 with a counting time of 1 s per step .
the hydrodynamic 
 size distribution and state of agglomeration / dispersion of the nps 
 was analyzed by dynamic light scattering ( dls ) technique using a brookhaven 
 zetaplus instrument ( brookhaven instrument inc .
the 
 particle size distributions were calculated by the 90plus software 
 ( brookhaven instruments ) . the zeta potential ( zp ) of the np samples 

the nps were excited by a continuous wave laser ( lasermate ) 
 operating 
 at 980 nm . a princeton instruments pi - max camera fitted with a charge - coupled 
 device sensor and an acton spectrapro 300i series spectrometer
fourier transformation infrared ( ftir ) spectra were collected 
 using 
 a thermo scientific nicolet ir100 spectrometer ( fisher scientific ) 
 to quantitatively compare ftir spectra before and after ligand exchange , 
 and at different surface conditions .
thermogravimetric analysis 
 ( tga ) was conducted using a setsys evolution 1600 tg / dsc ( setaram 
 instrumentation , caluire , france ) at a heating rate of 10 c / min 
 in air to determine the percent of organic content on the nps .
primary human aortic endothelial cells 
 ( haecs ) were procured from lifeline cell technologies ( fc-0014 ) and 
 cultured in vasculife vegf cell culture medium containing 2% fbs , 
 10 mm l - glutamine , 0.75 u / ml heparin sulfate , 1.0 g / ml 
 hydrocortisone hemisuccinate , 50 g / ml ascorbic acid , 15 ng / ml 
 rh igf-1 , 5 ng / ml rh fgf basic , and 5 ng / ml rh egf at 37 c in 
 a humidified 5% co2 incubator .
cells were proliferated 
 to 95% confluency for all experiments and were used up to passage 
 6 . for confocal imaging , cells were cultured in glass bottom culture 
 dishes ( mattek part number p50g-1.5 - 30-f ) that were treated with 5 
 ng / ml ti rat tail collagen ( bd biosciences part number 354236 ) in 
 0.01 mm ascorbic acid solution for 30 min prior to cell plating .
an mtt assay ( life technologies v13154 ) was used to determine how 
 the nps affected cellular metabolic activity .
per kit instructions , 
 cells were plated at 500010  000 cells per well and 
 allowed 48 to 72 h to reach confluency , at which point np incubation 
 experiments were begun .
after np incubation , cells were rinsed three 
 times in pbs to remove any loose particles before adding 100 l 
 of fresh media ; 10 ul of 12 mm mtt in pbs was then added to each well 
 and incubated at 37 c for 4 h. one hundred microliters of sds - hcl 
 solution was added to each well and incubated for an additional 418 
 h. samples were then mixed , and absorbance was read at 570 nm in a 
 molecular devices spectramax m2 plate reader ( sunnyvale , ca ) .
nayf4:yb , er was tested in the as - synthesized pegylated state as 
 well as one stripped of all surface ligands .
concentrations ranged 
 from 5 to 75 g / ml with 200 l of solution per well in 
 a 96 well culture plate .
two np incubation times were tested , 4 and 
 24 h. experiments were run in triplicate using a standard , clear , 
 flat bottomed 96 well culture plate ( corning part number 3596 ) .
calcein 
 and propidium iodide assays ( invitrogen part # c3100mp and p3566 , 
 respectively ) were used to determine cellular viability .
calcein am 
 is a nonfluorescent membrane permeant dye that is hydrolyzed by intracellular 
 acetoxylmethyl ester to a fluorescent calcein conjugate which then 
 accumulates inside the cell and can be used to indicate membrane integrity 
 and cellular viability .
propidium iodide is a membrane impermeant 
 nucleic acid stain that binds the dna of dead cells and is excluded 
 from viable cells .
passage 5 ( p5 ) cells were plated on 50 mm glass 
 bottom culture dishes ( mattek part # p50g-1.5 - 39-f ) and were grown 
 to 95% confluency .
pegylated and ligand - free nayf4:yb , er 
 nps were then incubated for 24 h at 5 g / ml and 75 g / ml 
 concentrations .
the np solution was then gently aspirated out and 
 replaced with 3 m calcein in dpbs and incubated for 15 min . 

propidium iodide nucleic acid stain was added to a final concentration 
 of 4 m for the last 5 min of incubation prior to imaging .
the culture dishes were placed on an inverted olympus ix81 confocal 
 microscope and imaged using fluoview 1000 .
calcein was excited using 
 a 488 nm argon ion laser and emission collected using the fitc filter 
 set .
pi was excited with a 524.5 nm argon ion laser and emission collected 
 using the tritc filter set . an olympus ix81 confocal microscope fitted with
an 
 optoenergy 975 nm 330 mw laser driven by an arroyo instruments 6310 
 combosource was used to obtain images of up - converting nanophosphors . 

calcein fluorescence via a 488 nm argon ion laser source was used 
 with a fitc filter set in order to find the focus for nir imaging . 
 for up - converting phosphor imaging , an in - line rdm690 short pass dichroic 
 mirror ( dm )
was used to filter out reflected nir wavelengths and a 
 long - pass 570 nm dm was used to further separate the two emission 
 peaks of the up - converting nps .
images were taken at 150 mw source 
 power using a lucplfln 40x objective ( na 0.6 ) .
healthy 
 p5 or p6 haecs were incubated at 37 c in a 5% co2 humidified incubator in fresh media for a period of 24 h. 
 the supernatant was collected via gentle aspiration for further experimentation . 

the supernatant was further divided into 1 ml microcentrifuge tubes , 
 half of which were denatured at 100 c for 4 h. peg - oleate bilayered 
 and ligand - free nps were added to final concentrations of 5 g / ml 
 and 75 g / ml to both the supernatant and denatured supernatant . 

all microcentrifuge tubes were incubated for 24 h. a nonconditioned 
 media control was also run for each np at a concentration of 75 g / ml .
the 
 size of the as - synthesized 
 oleate - capped nps was 25.3  3.5 nm , determined by transmission 
 electron microscopy ( tem ) ( figure 2a ) .
the 
 peg - oleate bilayer capped nps , the oleate - free nps , and the covalent 
 coated peg nps exhibited similar uniformity and dispersity as can 
 be seen in the tem images of figure 2 and digital 
 photographs of np dispersion in figure s1 .
it 
 is insoluble in water ; however , in its bilayer form with the peg - oleate 
 ligands ( figure 1 ) , the nps contain a polar 
 interface that yields good dispersity and stability in aqueous solution . 
 the ligand - free ( i.e. , bare surface ) and
covalently conjugated peg - nps 
 also provided a very stable dispersion in aqueous solution .
tem images 
 of ( a ) as - synthesized oleate - capped nayf4:yb , er nps drop - cast 
 from chloroform dispersion ; phase transferred 
 in water via ( b ) peg - oleate bilayer formation ; ( c ) ligand free ( bare 
 nps ) , and ( d ) covalently attached peg - nps .
dynamic light scattering data 
 of the nanoparticles showed an increase 
 in overall hydrodynamic size of the nps compared to the size measured 
 by tem .
peg - oleate bilayer - coated nps showed the largest hydrodynamic 
 size ( 44.9  6.0 nm ) compared to the bare surface nps ( 31.2 
 2.1 nm ) and covalent peg - nps ( 37.6  3.2 nm ) as expected ( inset 
 of figure 2 ) .
peg - functionalization induced 
 a negative charge ( 19  1 mv ) on the nps . stripping off 
 the ligands ( i.e. , reducing them to bare nps ) transformed them to 
 positively charged nps ( 30  1.7 mv ) under the reaction condition 
 ( i.e. , at ph  4.0 ) . however , for the nps dispersion at ph 
 7.0 ( used for cell study ) the zeta potential changes to 23 
 3.7 mv .
the change is zeta potential with different surface condition 
 is shown in figure s2 . at acidic ph of 
 reaction condition
the zeta potential is positive ( 30  1.7 mv ) 
 suggesting that in acidic conditions the oleates are protonated and 
 detach from the nps as oleic acid ( oah ) , leaving a positively charged 
 ln - np .
however , at ph 7.0 the zeta potential is negative ( - 
 23 3.7 mv ) , which suggests a deprotonated [ lno]h3o and thus a negative surface charge .
ftir spectra were taken to qualitatively determine 
 the surface 
 functional groups of the nps .
the spectra ( figure 
 s3 ) confirmed the presence of different functional groups at 
 every functionalization step .
in addition 
 to surface characterization , powder xrd patterns of the synthesized 
 nps were obtained ; the results are presented in figure s4 .
figure 3 shows a 
 comparison of the up - conversion 
 emission spectra of the nayf4:yb , er nps with different 
 surface modifications , all excited at 980 nm .
the spectra exhibit 
 distinct er emission bands centered at 540 ( green ) and 
 650 nm ( red ) , corresponding to h11/2/s3/2  i and f9/2 i transitions 
 of er , respectively .
we note that the up - conversion luminescence 
 of the surface modified nps was decreased by a few fold in every case . 

peg - oleate bilayered nps exhibited higher luminescence compared to 
 the other two functionalization methods .
on the basis of the integrated 
 area of the luminescence spectra , it was found that the peg - oleate 
 biyaler coated nps , bare surface nps , and the covalent - peg nps yielded 
 luminescence that was reduced by 3.4 , 7 , and 10.5 times compared to 
 the oleate - capped nps in chloroform .
the quenching of up - conversion 
 luminescence has been extensively reported in the literature ; it is 
 mainly attributed to the large vibrational modes of the water molecules 
 ( 3600 cm ) and an increase in surface defects 
 due to the ligand modification processes .
however , the luminescence of the phase - transferred 
 nps was sufficient to be detected in a confocal microscope .
( a ) comparison 
 of up - conversion luminescence spectra of nayf4:yb , er with 
 different surface conditions at 980 nm excitation : 
 ( i ) oleate - capped nps in chloroform , ( ii ) peg - oleate bilayered nps 
 in mq water , ( iii ) bare surface ( oleate free ) nps in mq water , and 
 ( iv ) covalently bound peg in mq water .
all spectra were taken at 3 
 mg / ml concentration . to examine the effect 
 of ligand coordination on the cellular response ,
we first incubated 
 haec cells with the nps containing no ligands and with particles containing 
 peg - oleate bilayer ligands for 24 h. first , enzymatic activity of 
 the cells upon exposure to nps for 24 h was tested though an mtt assay . 

the result ( figure 4 ) showed a decrease in 
 metabolic activity for both types of particles tested .
the bare particles 
 were the least toxic yet showed some concentration dependent toxicity . 

compared to control groups , the bare particles showed minimal toxicity 
 at the lowest concentration tested ( i.e. , 5 g / ml ) and 30% 
 toxicity at the highest concentration ( 75 g / ml ) .
the mtt toxicity 
 scaled linearly with r = 0.911 for concentrations 
 between minimum and maximum dose .
however , the peg - oelate nps had 
 a surprisingly significant effect on the metabolic activity of the 
 cells .
the nps decreased the metabolic activity of the cells to nearly 
 50% of control levels at all concentrations with only a slight concentration 
 dependence .
the results were repeatable for different batches of nps . 
 therefore , we pursued further studies to understand the cause and 
 possible mechanism of higher toxicity of the peg - oleate bilayer nps .
mtt assay 
 comparing metabolic activity of haec s incubated 
 with peg - oleate capped and with bare nayf4:yb , er for 24 
 h. experiments were run in triplicate ; error bars are  1 sd . 
 linear best fit line r = 0.911 .
stars 
 indicate a significant difference via p - tests between 
 toxicity levels at each concentration with p - values 
 < 0.01 . to confirm
the result obtained 
 from the mtt assay , we ran live / dead 
 cell calcein and propidium iodide viability assays .
though calcein am itself is not a fluorescent 
 molecule , the calcein generated by esterase in a viable cell emits 
 strong green fluorescence .
on the other hand , propidium iodide ( pi ) , a nucleus - staining dye , 
 can not pass through a viable cell membrane , thus pi stains only dead 
 cells .
it reaches the nucleus by passing through disordered areas 
 of dead cell membrane and intercalates with the dna double helix of 
 the cell to emit red fluorescence .
as shown in figure 5 , the cell counts via calcein staining were significantly 
 reduced following 24 h incubation with the two types of nps . in agreement 
 with the mtt assay cell viability result
, we found that the peg - oleate 
 bilayer nps resulted in less viable cells at the 75 g / ml np 
 concentration ( figure 5e ) . to analyze and quantify 
 the result , we used image processing software imagej to quantify the 
 cell viability .
the analysis showed that peg - oleate bilayer nps decreased 
 the cell viability by 50% compared to the control .
the bare surface 
 nps reduced the cell count as well , less significantly , to 67% of 
 control .
the pi control cells were released from the surface of the 
 plate upon death , and hence only calcein counts were taken into account . 

we conclude that the peg - oleate bilayer nps induced 
 significant toxic response to the cells , even at low concentration .
fluorescent 
 images of cells from calcein assays ( a , c , e ) , and quantification 
 analysis of the confocal fluorescent images using imagej analysis 
 ( b , d , f ) .
all cells were incubated with 75 g / ml nps for 24 h. 
 surface area and cell count normalized by calcein control .
analysis 
 show that ligand - free nayf4:yb , er nps ( c , d ) resulted in 
 67% viable cells , while peg - oleate bilayered nps ( e , f ) had 50% viable 
 cells compared to control . to verify these previously unreported findings of toxicity 
 from 
 bilayer pegylated nps
, we examined covalently bound peg - nps at the 
 same condition for 24 h in a separate experiment .
it was found that the haec 
 control covered 28.6% of the imaged area , haecs incubated with covalently 
 bound peg - nps covered 32.2% , and peg - oleate bilayer nps covered 11.0% . 

this result conclusively showed that the peg - oleate nps induced the 
 observed toxicity .
the impact of ligands on toxicity is not altogether 
 unknown  an earlier study reported a ligand - based toxicity of 
 iron oxide nps . in that case ,
the iron 
 oxide was nontoxic , but different types of surface ligands gave rise 
 to toxicity in hepg2 cells .
we examined 
 the role of the ligands in our case . to determine the 
 amount of ligands present on the nps , tga was performed ( figure s7 ) .
as seen in figure 
 s7 , about 9% of the nanoparticle mass was attributable to the 
 additional peg - oleate layer .
we calculated the equivalent peg present 
 in the 9% peg - oleate ligand then used equivalent amounts ( 3.97 g / ml 
 or 4.61 m ) of both peg and peg - oleate to assess their cytotoxicity 
 independently of the nanoparticles .
the concentration used in the 
 control experiments ( i.e. , 4.61 m ) is below the critical micelle 
 concentration ( cmc ) reported for similar peg - fatty acids ( i.e. , 9.91 
 m ) in the literature .
it is also worth noting 
 that only about 2% weight loss was observed for the bare nps in a 
 tga analysis ; the weight loss is attributed to the residual ligands 
 on the bare nps .
the ligand content on all three nps is in good agreement 
 with the literature data .
as shown by 
 the calcein assay in figure 6 , no signs of 
 toxicity were observed after 24 h incubation with either type of ligand .
fluorescent 
 images of cells obtained from calcein assays ( top panel ) , 
 and quantification analysis of the confocal fluorescent images using 
 imagej analysis ( bottom panel ) of ( a ) control cells , and cells treated 
 with ( b ) peg ( mw = 2000 ) , and ( c ) peg - oleate 
 ( mw = 860 ) .
all showed similar cell viability 
 and cell counts . generally , peg is known 
 to be nontoxic , hence the result was expected
. 
 however , the relatively benign behavior of the peg - oleate ligand was 
 most likely due to formation of micelles , thus secluding the hydrophobic 
 oleate molecules in the micelle and exposing peg chains to the culture 
 media .
therefore , cells were only exposed to peg , which resulted in 
 no toxicity .
based on this observation , we ruled out the possibility 
 that our observed np toxicity is due to excess peg - oleate ligands 
 present in the np dispersion .
we hypothesized that the toxicity might 
 arise from the partial or complete dissociation of the oleate - capping 
 on the nps themselves .
the peg - oleate nps exhibited a unique behavior upon incubation with 
 a confluent monolayer of haec s .
after a 24 h incubation with 
 the peg - oleate bilayer nayf4:yb , er , a white precipitate 
 was observed to settle on the bottom of the culture dish .
this precipitation 
 only occurred with the peg - oleate nayf4:yb , er nps  not 
 with the other two types of nps .
this observation suggested that bilayered peg - oleates may dissociate 
 during their interaction with cells . to investigate this possibility , 

an aliquot of the conditioned medium from the peg - oleate bilayer nayf4:yb , er incubation was taken for analysis .
it was first mixed 
 well with an equal volume of hexane , followed by the separation of 
 organic layers .
it was expected that the oleate molecules would move 
 to the organic layer due to their higher affinity for hexane .
the 
 organic layer was then dried in a vacuum oven and the remaining waxy 
 liquid was analyzed with ftir .
as seen in figure 
 s3c , the waxy layer showed all the peaks that matched peg and 
 oleic acid .
hence , we identified the waxy material as the peg - oleate 
 ligands used for np phase transfer .
this experiment provided confirmation 
 of our hypothesis that ligand dissociation could result in a particle 
 surface that was capped with oleates only .
observation of white 
 precipitate / cloudy solution after a 24 h incubation 
 with the peg - oleate bilayered nayf4:yb , er nps ( c : far right 
 panel ) while the covalent peg - nps ( b : middle panel ) and control ( a : 
 far left panel ) resulted in clear supernatant .
( top panels ) mattek 
 cell culture dishes after a 24 h incubation of the respective nps 
 with cells .
to investigate the mechanism 
 of ligand dissociation , we suspended 
 the peg - oleate nayf4:yb , er nps in ( i ) fresh medium and 
 ( ii ) medium conditioned by growing cells . in fresh medium ( i.e. , never 
 used in cell culture ) , no precipitation was observed at the 75 g / ml 
 concentration after 24 h. however , when nps were suspended for 24 
 h in conditioned medium ( i.e. , medium gathered from a 95% confluent 
 haec culture flask grown for 24 h ) , we observed the formation of white 
 precipitate .
we hypothesized that changes in ph and ionic concentration 
 of the media caused by the cell culture may have destabilized the 
 peg - oleate nanoparticles .
however , ph and ionic conductivity measurements 
 taken over a 24 h incubation showed minimal changes , all of which 
 were within a range to preserve particle stability . to further understand 
 the observations ,
the collected conditioned medium was denatured at 
 100 c for 4 h before running the experiment again . upon completion
, 
 we observed that in the unmodified conditioned medium , the sample 
 became cloudy and a white precipitate had begun to settle out  on 
 the other hand , the denatured conditioned medium had only the slightest 
 hint of precipitate formation . from this result , we concluded that 
 the nanoparticles that transferred from one phase to another via a 
 bilayer formation may not be stable and useful in biological applications . 

we propose that the peg - oleate nayf4:yb , er nps undergo 
 a deintercalation process that is initiated by an extra - cellular protein 
 or enzyme produced by the cell .
this reaction is largely eliminated 
 by the denaturing of the extra - cellular proteins .
confocal microscopy 
 was employed to examine the cellular localization 
 of the nps . due to their unique up - converting optical properties
, 
 we were able to excite the nps within the cells using a low energy 
 near - infrared ( nir ) laser ( 980 nm ) .
nir excitation also minimizes 
 cellular autofluorescence . as shown in figure 8 , fluorescence from the nps
confocal 
 microscope images of the haec cells ( a ) bright field image 
 of the cells , ( b ) peg - oleate bilayered nps internalized by the cells 
 excited with a 980 nm excitation laser ( nps are false colored as red ) , 
 ( c ) fluorescent signal from calcein , and ( d ) overlay of a
the internalization of the nps 
 provided insight into the interaction 
 of the peg - oleate bilayered np with the cells .
based on np internalization , 
 observed through confocal microscopy , we propose the mechanism depicted 
 in figure 9 .
we suggest that the oleate bound 
 to the nps is most likely the cause of cytotoxicity .
investigated the impact of oleic acid on cytotoxicity 
 using nickel ferrite particles in neuro-2a cells .
their study revealed 
 that cell membranes have the capability to accumulate some oleic acid 
 molecules within the lipid bilayer due to their hydrophobic nature . 

they also noted that at high concentrations , cellular viability dropped 
 drastically , to as low as 10% for the highest concentration they used . 
 with regard to the peg - oleate bilayer nps
, we propose that as ligand 
 dissociation takes place , initiated by cell - secreted enzymes , the 
 separated peg - oleate layer formed micelles and remained suspended 
 in the cell culture medium as characterized by ftir . however , the 
 resultant nps capped with oleates passed through the lipid bilayer 
 and resulted in the observed drop in cellular viability . due to the 
 oleate induced toxicity , low cell viability
was observed even at very 
 low np concentrations ( e.g. , 510 g / ml ) .
we believe 
 that the lowest concentration of the oleate - capped nps had sufficient 
 oleates to saturate the metabolic response of the mitochondria , reflected 
 in the lack of a dose dependent response .
although oleate - induced 
 cytotoxicity has been reported in previous studies , ligand dissociation 
 from a phase - transferred np , leading to cytotoxicity , is a unique 
 observation .
proposed schematic of dissociation mechanism of peg - oleate 
 bilayered 
 nps and internalization in cells .
finally , we confirmed the result with another subset of lanthanide 
 nps , viz .
, nagdf4:yb , er ( figure s8 , avg . size 13 nm ) which have been widely studied for mri 
 and optical imaging applications .
we synthesized the nps and functionalized 
 the surface in exactly the same way as the nayf4:yb , er . 

an mtt assay shows ( figure 10 ) the same trend 
 that was observed for nayf4:yb , er for both the bare and 
 peg - oleate bilayer nps .
we conclude that there is a general potential 
 for ligand dissociation in bilayered peg - oleate ligands which can 
 lead to toxicity by exposing cells to oleates .
mtt assay comparing 
 metabolic activity of haec s incubated 
 with peg - oleate capped and with bare nagdf4:yb , er for 24 
 h. experiments were run in triplicate , error bars are  1 sd . 
 linear best fit line r = 0.916 .
stars 
 indicate a significant difference via p - tests between 
 toxicity levels at each concentration with p - values 
 < 0.01 .
it 
 has been found that it is crucial to consider not only the intrinsic 
 potential toxicity of nanomaterials that are applied to living systems , 
 but also to consider the type of ligands that are used to functionalize 
 or passivate particles and how they are attached .
the particular material 
 in this case was an up - converting phosphor np that by itself appeared 
 to pose a limited dose - dependent potential for toxicity . however , 
 capping the particles with oleates introduced a significant risk of 
 toxicity to cells .
because this class of nps is mostly synthesized 
 in a high temperature reaction with a hydrophobically coordinated 
 ligand such as oleic acid , they can be potentially toxic if weakly 
 coordinated surface groups are used to render them biocompatible . 

this study has shown that intercalated ligands can result in oleate - capped 
 nps upon interaction with cells , rendering them cytotoxic .
an improved 
 understanding of the interaction of this common capping agent with 
 cells can be used to design and construct improved nanoparticles for 
 a range of bioapplications where particle stability and biocompatibility 
 are extremely important .
 the 
 cellular toxicity of nanoparticles that were capped with a 
 bilayered ligand was studied using an up - converting ( uc ) phosphor 
 material as a representative nanoparticle ( np ) . 
 the results indicate 
 that although uc nps are known to be nontoxic , the toxicity of the 
 nps depends strongly on ligand coordination conditions , in addition 
 to the other commonly known parameters such as size , structure , surface 
 charge etc . 
 oleate - capped hydrophobic nayf4:yb , er nps were 
 surface modified to yield three extreme conditions : bare particles 
 that were stripped of the oleate ligands ; particles with covalently 
 bound poly(ethylene glycol ) ( peg ) ligands ; and particles with an bilayer 
 of peg - oleate ligands using the oleate surface group that was remained 
 after synthesis . 
 it was found that the bare particles and the covalent 
 peg nps induced little toxicity . 
 however , particles that were rendered 
 biocompatible by forming a bilayer with an amphiphilic ligand ( i.e. , 
 peg - oleate ) resulted in significant cell toxicity . 
 these findings 
 strongly suggest that the peg - oleate group dissociated from the bilayered 
 oleate - capped nps , resulting in significant toxicity by exposing the 
 hydrophobic oleate - capped nps to the cell . based on results with bare 
 particles , the nalnf4:yb , 
 er ( ln = y , gd ) up - converting 
 phosphors are essentially less - toxic . capping and functionalizing 
 these particles with ligand intercalation 
 may , however , not be a suitable 
 method for rendering the nps suitable for bioapplication as the ligand 
 can potentially dissociate upon cellular interaction , leading to significant 
 toxicity . 
Introduction
Materials and Methods
Results and Discussion
Conclusion