Registered charity number: 207890 Showcasing research from teams at the Laboratory for Vascular Translational Science, INSERM U1148, Laboratoire de Toxicologie, Pharmacologie et Signalisation Cellulaire, INSERM U1124, and at CEA: the Unité d’Imagerie par Résonance Magnétique et de Spectroscopie NeuroSpin and the Laboratoire d’Etude du Métabolisme des Médicaments. Optimization of pegylated iron oxide nanoplatforms for antibody coupling and bio-targeting Covalently immobilized fluorescent antibodies onto iron oxide nanoparticles, via the control of the PEG polymer architecture surface, allows a bimodal, fluorescent and Magnetic Resonance Imaging contrast agent for cancer diagnosis to be obtained. rsc.li/materials-b As featured in: See L. Motte et al., J. Mater. Chem. B, 2017, 5, 2896.
Transcript
Registered charity number: 207890
Showcasing research from teams at the Laboratory for
Optimization of pegylated iron oxidenanoplatforms for antibody couplingand bio-targeting†
S. Richard,a M. Boucher,b A. Saric,c A. Herbet,d Y. Lalatonne,ef P. X. Petit,c
S. Meriaux,b D. Boquetd and L. Motte *e
PEGylation has been established as a valuable strategy to minimize nanoparticle clearance by the
reticulo-endothelial system due to hydrophilicity and steric repulsion of PEG chains. In this study we
functionalized superparamagnetic iron oxide nanoparticle surface with two PEG differing in their length
(n = 23 and 44) and terminal functionality, COOH and CH3. By varying the ratio of the two different
PEG, we optimized the molecular architecture of the nanoplatform to obtain maximum stability and low
toxicity under physiological conditions. The best nanoplatform was evaluated as MRI contrast for mouse
brain vascularization imaging at 7 T. The carboxylic acid functions of the nanoplatform were used to
covalently bind an antibody, Ab. This antibody, labeled with a fluorophore, targets the ETA receptor,
a G-protein-coupled receptor involved in the endothelin axis and overexpressed in various solid tumours,
including ovarian, prostate, colon, breast, bladder and lung cancers. In vitro studies, performed by flow
cytometry and magnetic quantification, showed the targeting efficiency of the Ab-nanoplatforms. Clearly,
an imaging tracer for cancer diagnosis from a bimodal contrast agent (fluorescence and MRI) was
thus obtained.
Introduction
Surface chemistry plays a critical role in the behavior of ironoxide nanoparticles (NPs) for applications such as in diagnosisand drug delivery. Usual methods for NP tumoral targeting aremainly based on passive and active mechanisms. By passivetargeting, NPs are accumulated and retained in the tumor
interstitial space mainly through the enhanced permeabilityand retention (EPR) effect. The active mechanism needs mole-cular ligands such as antibodies, peptides, or small moleculesrecognizing specific receptors on the tumor cell surface, oftenfollowed by receptor-mediated endocytosis and nanoparticleinternalization. In the last case, functional groups, such as–COOH, are introduced for molecular conjugation with the NPsurface. One of the most popular strategies involves amide bondformation through carbodiimide coupling between carboxylic andamine moieties. For both passive and active targeting strategies,the reticulo-endothelial system has the ability to clear intra-venously administered NPs from the blood within hours.1,2
Therefore, NPs developed for systemic application need to beengineered to reduce the opsonization process. To impart stealthproperties, the most common approach consists of coating theNP surface with the polyethylene glycol (PEG) polymer. The PEGinduces steric hindrance limiting protein adsorption and improv-ing the colloidal stability and stealth in biological medium.3,4 Theligand density and molecular weight of PEG on the NP surface arekey factors that control PEG structure and consequently theparticle stealth properties.
The theory originally proposed by Alexander and de Genneset al. suggests the existence of a variety of conformations that canbe predicted by comparing the distance (D) between two PEGchains on the surface and the radius of gyration or Flory radius
a Laboratoire Matiere et Systemes Complexes (MSC), UMR 7057,
CNRS and Universite Paris Diderot, 75205 Paris Cedex 05, Franceb Unite d’Imagerie par Resonance Magnetique et de Spectroscopie,
CEA/DRF/I2BM/NeuroSpin, F-91191 Gif-sur-Yvette, Francec Laboratoire de Toxicologie, Pharmacologie et Signalisation Cellulaire,
INSERM U1124, Universite Paris-Descartes, Centre Universitaire des Saints-Peres,
F-75270 Paris Cedex 06, Franced Laboratoire d’Etude du Metabolisme des Medicaments, CEA/DRF/iBiTec-S/SPI,
Universite Paris 13, Sorbonne Paris Cite, F-93017 Bobigny, France.
E-mail: [email protected] Service de Medecine Nucleaire Hopital Avicenne Assistance Publique-Hopitaux de
Paris F-93000 Bobigny, France
† Electronic supplementary information (ESI) available: Hydrodynamic diameter(DH) and zeta potential as a function of pH for bare NPs (Fig. S1), absorptionspectrum (Fig. S2), fluorescence emission spectra (Fig. S3), variation of the averagenumber of PO-PEG per nanoparticle considering a preferential interaction of 1200-PO-PEG-COOH (Table S1), average number of Ab/NP deduced with fluorescenceand OPA titration (Table S2). See DOI: 10.1039/c6tb03080g
Received 28th November 2016,Accepted 22nd March 2017
(RF) of the polymer.1,5–8 Considering the number n and the lengtha (a = 0.35 nm) of EG monomers, the Flory radius is expressed asRF (nm) = a�n3/5. Thus it is expected that if D 4 RF, correspondingto low grafting density, the chains do not interact laterally andadopt a ‘‘mushroom’’ conformation (Scheme 1). In contrast, forhigh grafting density, this distance decreases (D o RF) and chainsinteract strongly. The overlap between chains forces them tostretch away from the surface resulting in the so-called ‘‘brush’’conformation (Scheme 1). Generally, it is recognized that the‘‘brush’’ conformation is the most efficient in limiting proteinadsorption.5,6 This conformation is achieved with high molecularweight polymers (M 4 44 000 g mol�1), or EG with more than1000 monomers. However, the behavior towards opsonization,but also NP internalization in cells, will also depend on thenumber of PEG grafted onto the surface of the NPs. Thus, it hasbeen shown that the grafting of low molecular weight PEG(2000 g mol�1) with a density of 1 PEG per nm2 effectivelyreduced protein adsorption, while promoting NP internalization incells. On the other hand, a PEG with a molecular weight of5000 g mol�1 limits both protein adsorption and NP internaliza-tion.9 Another study also showed that PEG of 2000 g mol�1
molecular weight appears to be the best compromise in order tominimize opsonization compared to PEG chains from 1000 to5000 g mol�1.10
In this study the surface of iron oxide NPs has been function-alized with two phosphonate polyethylene glycol molecules,differing in their length (n E 23 units, 1200 g mol�1 andn E 44 units, 2100 g mol�1) and terminal functionality, COOHor CH3, respectively. The phosphonate group presents highaffinity for metallic surfaces whereas the PEG chain improvesthe in vivo nanoparticle biodistribution and COOH groups allowbiomolecular conjugation for active targeting. The aim was tooptimize the molecular architecture of the nanoplatform byvarying the ratio of the two phosphonate polyethylene glycolmolecules on the NP surface in order to improve stability andfurtivity.
Several complementary methods are used to evaluate theeffect of PEGylation on relevant aspects, including surface liganddensity, hydrodynamic size, stability, with important repercus-sions on stealth properties and cellular toxicity.
The best nanoplatform was evaluated as MRI contrast formouse brain vascularization imaging at 7 T. To validate andelaborate multifunctional nanoplatforms, the carboxylic acid
functions of the nanoplatform were used to covalently bind,through carbodiimide coupling, an antibody, Ab, labeled with afluorescent dye Alexa Fluor 488.11,12 This Ab targets a membraneprotein: the endothelin A receptors (ETAR) overexpressed ondifferent cancer cells.13–15 We demonstrated the full functionalityof our Ab-NPs by flow cytometry and magnetic measurementsusing CHO-ETAR cells, overexpressing ETAR on the external cellsurface, and CHO-WT as a negative control. This new generationof nanoplatform opens the way to promising early diagnosticcancer approaches combining the high MRI contrasting effici-ency and the active targeting for tumor pathologies.
Results and discussionPhysico-chemical characterization of the variousnanoplatforms cFe2O3@PO-PEGx
The gFe2O3 NPs with an average diameter of 9.6 nm (Fig. 1a and b)were synthesized according to a procedure already described.11,12
Phosphonate (PO) groups are effective chelating agents for variousmetallic oxide surfaces (Al2O3, Ta2O5, TiO2, ZrO2, Fe2O3, Fe3O4
ZnGa2O4:Cr3+).16–24 The NPs were surface passivated withvarious mixtures of 1200-PO-PEG-COOH and 2100-PO-PEG-CH3,defined by the ratio x = 100� n1200-PO-PEG-COOH/(n1200-PO-PEG-COOH +n2100-PO-PEG-CH3
), by simply mixing the NPs and PO-PEG in anacidic medium. Excess coating molecules were eliminated byultrafiltration.
Table 1 reports the zeta potential and the hydrodynamicdiameter at physiological pH of the various nanoplatforms. Thezeta potential and the hydrodynamic diameter are stable for eachx ratio except for x = 100%.
Compared to the bare NPs that were stable only in acidic orbasic media and agglomerated at physiological pH owing to anisoelectric point around 6–7 (Fig. S1, ESI†), the negative zetapotential clearly shows efficient binding of the PO-PEG to theNP surface. The phosphonate (PO3H2) group displays twopKa values, 3.1 and 5.4, and the pKa of the carboxylic acid/carboxylate couple is about 4.4 and thus the two PO-PEG-R(R = CH3 or COOH) molecules are negatively charged in a largepH range. Various metal coordinations are reported for ironoxide NPs from monodentate to tridendate binding.25 Thenegative charge observed for x = 0% at physiological pH seemsto be in favor of monocomplexation of iron compared to abidendate or tridendate binding. Such assumption is corro-borated by considering the isoelectric point (IP) value equal to4.1 for x = 0 (Fig. S1, ESI†) corresponding to the main species
Scheme 1 Schematic representation of ‘‘mushroom’’ and ‘‘brush’’ PEGconformation.
R-PO(OH)O�� � �Fe (with R = PEG-CH3). For the other x ratioalso, the IP values are around 4.
Fig. 2a shows the IR spectra of the bare NPs (pH = 2) and ofthe two PO-PEG (pH = 7). Iron oxide NPs are characterizedby the Fe–O vibration band at around 600 cm�1 (red curve) andthe two PO-PEG by the their PO asymmetric vibration bands ataround 1061 cm�1 and the characteristic band at 1100 cm�1
due to the stretching vibration of aliphatic ether C–O. The twoPO-PEG can be distinguished by the terminal COOH functionfor 1200-PO-PEG-COOH. The antisymmetric and symmetricvibration bands due to carboxylate are observed at 1400 cm�1
and 1557 cm�1 and the band at 1670 cm�1 is due to the carboxylicfunction (red curve).
The analysis of the band position indicates the conforma-tions of polymer chains.26–32 A preferential helical conformationis the result of glycol moieties with a trans (T) conformationaround the C–O bonds and a gauche (G) conformation aroundthe C–C bond (TGT). In the amorphous phase, the predominantconformation around the C–C bond is still gauche, but the C–Obond can be trans or gauche (TGT, TGG, GGT). For the twopolymers, the CH2 scissoring at 1470 cm�1, the wagging band at
around 1360 cm�1, the twisting (1240 cm�1) and CH2 rocking(950 and 845 cm�1) as well as CH2 twisting (trans conformationof C–O) at 1300 cm�1 are characteristics for the crystalline phasewith TGT conformations. The band at 1450 cm�1 is due to theCH2 asymmetric deformation mode. Nevertheless, it should benoted that the presence of a band around 1340 cm�1 assigned tothe stretching vibration of the CH2 in the trans conformationalso indicates co-existence of mushroom orientation for thefree molecule.
Fig. 2b shows the IR spectra of the various nanoplatformsafter surface functionalization with various ratios x (at pH = 7.4).All spectra are normalized with the Fe–O vibration band. Allspectra show the characteristic band around 1100 cm�1 due toC–O of PEG indicating efficient complexation of PO-PEG chainsonto the NP surface. The CH2 vibration bands localized at950, 1240, 1300 and 1470 cm�1 are also clearly observed forthe various nanoplatforms, indicating PEG chain more or lesscompressed in brush conformation. Nevertheless, the bandscorresponding to the P–O (1061 cm�1) and the CH2 wag forgauche (1360 cm�1) and trans (1340 cm�1) conformation arebroadened on the NP surface. Therefore their identifications areno more possible. Finally, it has to be noticed that carboxylatebands at 1400, 1550 and 1670 cm�1 induced by the grafting ofPO-PEG-COOH are not observed. This last result could beexplained based on the fact that the PEG length of 2100PO-PEG-CH3 is larger than that of 1200 PO-PEG-COOH, andconsequently that the carboxylate groups are masked by thelong PEG chains.
Hence, with IR analysis, it was not possible to confirm theproportional grafting of the terminated end functionality ofPO-PEG-CH3 and PO-PEG-COOH onto the NP surface with theinitial ratio x, and there was no clear evidence of the conforma-tion of PEG grafted onto the NP surface.
The number of PO-PEG molecules per NP was quantitativelydetermined by EDX analysis measuring the ratio of iron tophosphorus signals (Fig. S2, ESI†) as already reported.11,33 Theresults obtained for the various nanoplatforms differing by theinitial ratio x are presented in Fig. 3 and Table 1.
The average number (Nb) of PO-PEG grafted per NP appearsto be dependent on the initial ratio x (black point in Fig. 3, andTable 1). Hence the loading is more important considering thesurface coating with 100% of 2100-PO-PEG-CH3 compared to100% of 1200-PO-PEG-COOH. Considering the surface area ofthe phosphonate group (0.24 nm2)34 and the molecular weight ofeach PO-PEG (1200 g mol�1 and 2100 g mol�1), the Flory radiusis found to be equal to 2.3 nm for x = 100% (PO-PEG-COOH)and 3.4 nm for x = 0% (PO-PEG-CH3). Hence the averagedistance between two PO-PEG chains on the NP surface couldbe evaluated as D = (SPO-PEG)1/2 = (SNP/NbPO-PEG)1/2 with S beingthe surface area. Thus, the value of D is between 0.43 nm (2100-PO-PEG-CH3) and 0.56 nm (1200-PO-PEG-COOH) (Table 1). Thisdistance is lower than RF indicating a brush conformation forthe two chain lengths. The value of D for 1200-PO-PEG-COOH isgreater than that for 2100-PO-PEG-CH3, suggesting a reducedbrush conformation. The higher loading with PO-PEG-CH3 couldbe correlated to the chain length of PO-PEG-CH3 (n = 44),
Table 1 Hydrodynamic diameter (in volume), polydispersity index (PDI),zeta potential at physiological pH, number of PO-PEG per NP or per nm2
for the various nanoplatforms, and average distance (D) between twoPO-PEG chains on the NP surface
which is larger compared to that of PO-PEG-COOH (n = 23),suggesting a more rigid ‘‘brush’’ orientation for high molecularweight PEG in accordance with the literature.35 Similar resultswere observed considering the grafting of thiolated Self-AssembledMonolayers (SAMs) onto the gold titanium oxide substrate: thelonger alkyl chains pack more densely than the shorter ones inthese SAMs.36,37 Moreover, as observed in the case of COOH-terminated SAMs,38,39 it can be expected that hydrogen bondingbetween the terminating COOH functions occurs, considering ourNP surface functionalisation process performed in acid solution.Such hydrogen interaction induces the formation of gauche defectsand decreases the van der Waals interactions between PEG chainsand hence reduces the PO-PEG-COOH loading compared toPO-PEG-CH3 loading. Nevertheless, when increasing the initialratio x, i.e. increasing the initial ratio of PO-PEG-COOH com-pared to that of PO-PEG-CH3 (black point in Fig. 3), EDX resultsshow a decrease of PO-PEG density per NP up to x = 50%whereupon it reaches a plateau. Compared to the proportionaldistribution of the initial ratio x (red point in Fig. 3), thissuggests a preferential interaction of PO-PEG-COOH comparedto PO-PEG-CH3 with the NP surface for x o 50%.
If we assume that all the PO-PEG-COOH are grafted onto theNP surface for various initial x ratios, the average number ofPO-PEG-CH3 grafted onto the NP surface can be deduced andtherefore an effective experimental x ratio can be calculated(Table S1, ESI†). Thus, with this assumption, the evolution ofthe average experimental number of PO-PEG grafted per NP canbe correlated to this effective experimental x ratio. The resultsare reported as green points in Fig. 3 and are in rather goodaccordance with the experimental results. This indicates a pre-ferential interaction of 1200 PO-PEG-COOH with the NP surfacecompared with 2100 PO-PEG-CH3.
The better affinity of PO-PEG-COOH for NP surface could bedue to two factors:
– firstly, phosphonate as well as carboxylic functions areefficient complexing functions for iron oxide NPs. Indeed, themain functions reported in the literature as anchoring groups forthe surface of the iron oxide NPs are the carboxylic acid (throughthe use of the citrate ions or oleic acid), catechol, phosphonate and
hydroxyl methyl bis phosphonate functions.11,19,40–42 One assump-tion could be a cooperative effect of the affinity of phosphonate(PO) and carboxylic (COOH) functions for iron oxide complexa-tion surface.
– secondly, competitive adsorption related to the chainlength of the two polymers could occur.43,44 Indeed Tsai et al.have reported that the presence of mercaptopropionic acidmolecules inhibits the adsorption of thiolated polyethylene glycol(1 and 5 kDa) in the case of gold NP surface functionalization.40,41
This behavior was related to steric hindrance between longpolymer chains in solution which reduces the access to bareNPs. A similar behavior could be expected in our experimentalconditions.
Considering the preferential interaction of PO-PEG-COOHwith the NP surface and the presence of gauche defects, thissuggests phase segregation of the two components onto the NPsurface, as observed for mixed SAMs with short-chain-CH2OHand long chain CH3-terminated alkanethiol.45 This effect wasattributed to the prevalence of van der Waals interactions amongthe long CH3-terminated alkanethiol SAMs. Besides, in the caseof a random mixture of the two components, it was expected thatsteric effects inhibit hydrogen bonding.39
Iron oxide NPs are characterized by their absorption spectrabetween 350 and 550 nm (Fig. S3, ESI†). Hence, a decrease of theabsorption versus time reflects NP instability owing to their pre-cipitation. Fig. 4 shows the stability of the various nanoplatforms inphysiological medium (NaCl = 150 mM, pH = 7.4). It can beobserved that when increasing the initial ratio x, the absorp-tion, measured at 480 nm, decreases with time. In particular thegFe2O3@PO-PEG100 nanoplatform presents lower stability inaccordance with DLS measurements. At pH = 7.4, the terminatingfunctions are carboxylate (COO–) groups and the stability of thenanoplatform is mainly related to electrostatic repulsion due tocarboxylate groups. However, in the presence of high salt concen-trations (NaCl = 150 mM), the electric field is shielded, and thenanoparticles agglomerate. For intermediate x, and when increasingthe PO-PEG-CH3 loading, steric repulsion due to the largest PEGchain length predominates over charge shielding and increases thestability of the other nanoplatforms in physiological medium.
Hence in our conditions, by modulating the experimentalinitial x ratio, we have demonstrated that we are able to modulate
Fig. 3 Variation of the average number of PO-PEG per nanoparticle: deducedby EDX (black point), considering proportional distribution (red point), and witha preferential interaction of 1200-PO-PEG-COOH (green point).
Fig. 4 Stability of the various nanoplatforms in physiological medium(NaCl = 150 mM, pH = 7.4).
the proportion of PO-PEG differing in their chain length andtheir terminal function (COOH and CH3). Considering theore-tical and experimental results, brush orientation was deducedwhatever the PEG chain length investigated in this work (n = 23or 44) for the iron oxide NP surface (9.6 nm as the averagecrystalline diameter). Moreover, a preferential interaction ofPO-PEG-COOH with the NP surface occurs certainly due to thecooperative effect of the affinity of PO and COOH groups for NPsurface and due to the steric hindrance in solution of 2100-PO-PEG-CH3, due to the largest PEG chain length compared to that of1200 PO-PEG-COOH. These results have motivated us to investi-gate the potential of these various nanoplatforms as candidatesfor MRI diagnostic and/or therapeutic agents.
Nanoparticle toxicity
Determination of the ROS production associated withcFe2O3@PO-PEGx NP incubation. In order to detect any potentialeffect of gFe2O3@PO-PEGx NPs, the cellular ROS homeostasis(i.e., superoxide anions) was assessed by measuring the changesin MitoSOX fluorescence between control cells and after NPtreatment. The NPs were incubated with U87-MG cell lines for24 h at two extracellular concentrations ([NP] = 50 nM and 100 nM).As control NPs, we used gFe2O3 nanoparticles surface function-alized with caffeic acid (CA), a well-known anti-oxidant.12,46
Compared to control cells, the fluorescence intensity decreasesafter incubation with the various pegylated nanoplatforms (Fig. 5).This result indicates a decrease of the superoxide anion level. Thiseffect seems to be related to the ratio x. Hence, the oxidative stressdecreases when the amount of PO-PEG-COOH per NP decreases.The same phenomenon is observed for higher extracellularNP concentrations (100 nM). Finally, gFe2O3@PO-PEG0 andgFe2O3@PO-PEG15 present similar anti-oxidant properties tothose of gFe2O3@AC NPs used as control NPs.
Impedance-based label-free test of toxicity (xCELLigence oncellular proliferation)
The experiments needed prior calibration of the cellular curveat different cell concentrations. For U87-MG cells, 20 000 cellssuspended in a volume of 200 mL were used for calibration. Thecontrol cells (Fig. 6, black line) adhered to the substrate and the
cell index (CI) increased until reaching a plateau at around20 hours at which point the cells started to proliferate. That wasthe time point chosen to inject the NPs (50 nM), correspondingto an early proliferation state. After incubation with the variouspegylated nanoplatforms, cell proliferation remained similar tothe control cells.
Hence the various platforms present antioxidative properties,in particular for low x ratios, but have no effect on cell growth.
Multimodal nanoplatforms
For targeting or therapeutic application the NPs have to befunctionalized with specific molecules. This implies the covalentcoupling of these molecules and in our case using carboxylicfunctions present on the outer surface of NPs via carbodiimidechemistry. So far the nanoplatforms gFe2O3@PO-PEG0 can beeliminated due to the absence of carboxylic acid functions.In this work, we have chosen to specifically functionalize thenanoplatform surface with an antibody, Ab. This Ab has beenelaborated in order to target endothelin A receptors (ETAR)overexpressed on some cancer cells. Moreover labeling Ab witha fluorescent dye, Alexa Fluor 488 (AF-488), allows one to obtaina dual (MRI and fluorescence) imaging agent.
Ab covalent coupling, stability study and selection of the bestnanoplatform
In the first step, the Ab covalent coupling was evaluated on thevarious nanoplatforms with x = 15% to 100%, using the con-ventional carbodiimide coupling procedure. This reaction wasperformed at a molar ratio R = nAb-AF488/nNP corresponding toR = 4. To evaluate the efficient Ab grafting, two procedures wereused. The first one consisted to measure the Fluorescence emis-sion Intensity (FI) of the supernatant after the coupling proce-dure. The second one was the titration of amino groups usingOPA reactant, after chemical decomposition of the amidecoupling onto the various nanoplatforms, were quantitativelymeasured. Below x = 50%, an efficient Ab grafting yield couldnot be evaluated using our analytical techniques. In contrast forx = 85% and 100%, analytical results indicate the grafting of2 Ab-AF488 per NP. These results are in accordance with thefact that on increasing the initial ratio x, the number of COOH
Fig. 5 Flow cytometry analysis of the cell behavior treated with mitoSOX(superoxide anion detection) and TOPRO-3 (assessment of plasma membranepermeability linked to cellular viability) in the presence of gFe2O3@AC (50 nM)and gFe2O3@PEGx (50 or 100 nM). Control experiment corresponds toU87-MG cells incubated without NPs.
Fig. 6 RTCA profiles (xCELLigence measurements) generated by U87-MGcells with NP nanoplatforms gFe2O3@PEGx (50 nM) during 120 hours.
groups and thus their accessibility for amide coupling increase.Below x = 50%, the longer PO-PEG-CH3 chains mask the COOHfunctions of PO-PEG-COOH, due to the difference of polymerlength. It has to be noticed that the fluorescence emissionintensity is related to the quenching effect. Hence, the quenchingfactor for gFe2O3@PO-PEG85-Ab-AF488 is found to be equal to 3(Fig. S4, ESI†). Compared to previous results with the iron oxideNP surface functionalized with a smaller bifunctional molecule(caffeic acid, CA) and conjugated with Ab-AF488, the fluores-cence quenching factor (equal to 14) is reduced.12,46 This effecthas to be related to the increased distance induced by the PEGchain between AF488 and NP surface. Finally considering aswell the physiological stability of the various nanoplatforms(Fig. 4), toxicity evaluation (Fig. 6) and the coupling efficiencyof Ab, gFe2O3@PO-PEG85 seems to be the best candidate fortargeting evaluation.
cFe2O3@PO-PEG85 nanoplatform
Control of the average number of Ab-AF488 grafted per NP.In order to evaluate the potential avidity effect, we first checkedfor the control of the average number of Ab-AF488 grafted perNP. For such an aim, we first evaluated the covalent couplingof Ab-AF488 onto gFe2O3@PO-PEG85, using two initial ratiosR = nAb-AF488/nNP = 2 or 4. Fluorescence analytical results showthat a grafting yield of about 50% is achieved whatever theinitial ratio with a fluorescence quenching factor equal to 3 inboth cases (Fig. S4, ESI†).
Fig. 7 and Table 2 report the hydrodynamic diameter and zetapotential at physiological pH of the various nanoplatforms beforeand after coupling of Ab-AF488 onto gFe2O3@PO-PEG85. A morenegative zeta potential and a slight increase of the hydrodynamicdiameter with the number of grafted Ab-AF488 are observed.
These results confirm the efficient coupling of Ab-AF488 ontothe NP surface. The increase of the zeta potential of the nano-platforms has to be correlated to the zeta potential of Ab-AF488(�15 mV) at physiological pH.
The antibody-NP stability was studied after dilution in 10%serum with or without antiproteases. The release of Ab-AF488was monitored by fluorescence spectroscopy through the kineticdisplacement of the quenched and bound Ab-AF488 on the NPsurface (Fig. 8).
After 48 hours, less than 15% release was reached for the tworatios R with or without antiproteases, indicating the release ofPO-PEG-Ab-AF488 rather than the cleavage of the amide bondbetween PO-PEG and Ab-AF488 entities. This evidences the bio-stability of the nanoplatform through the high PO-PEG anchoringon the iron oxide surface and the covalent coupling throughamide binding. These results underline NP compatibility forin vivo imaging applications.
MRI properties. Diluted samples of the different NPs werehomogenously suspended in an agar matrix, with iron concen-trations ranging from 0 to 0.25 mM. Longitudinal r1 and trans-verse r2 relaxivities were measured with dedicated T1 and T2
mapping sequences using a 7 T MRI preclinical scanner (Table 2).The high r2/r1 ratios (Table 2) confirmed the great potential ofthese NPs as T2-shortening contrast agents for contrast-enhancedMRI applications. The increase in transverse relaxivity r2 observedwith the antibody-NPs compared to the NPs alone can be relatedto the increase in hydrodynamic size.2,47,48 The addition offunctionalization, such as antibodies, results in an increase ofthe hydrodynamic size of the contrast agent (Fig. 7), and thus in aslower Brownian NP rotation. Consequently, the interaction timebetween the electronic spins of the iron-oxide based contrastagent and the nuclear spins of the surrounding water protonsincreases, resulting in an increase of transverse relaxivity r2. Thenantibody-NPs exhibit high values (r2 around 220 mM�1 s�1 at 7 T)greater than the reported relaxivities of commercially available T2
contrast agents such as Resovist (r2 = 177 mM�1 s�1 at 7 T) andEndorem (r2 = 160 mM�1 s�1 at 7 T).49
To assess the high MRI contrasting efficiency of pegylated NPs,an in vivo study was performed at 7 T. Male Swiss mice (25 g)
Fig. 7 (a) Hydrodynamic diameter distribution (in volume) and (b) zetapotential measured in water (pH 7 and [Fe] = 0.25 mM) for the nanoplat-forms gFe2O3@PO-PEG85 without (green) or with 1 (blue) or 2 (red) anti-bodies mAb-AF488.
Table 2 Hydrodynamic diameter (in volume), polydispersity index (PDI),zeta potential at physiological pH, and MRI longitudinal (r1) and transverse(r2) relaxivity values of the nanoplatforms gFe2O3@PO-PEG85 without orwith 1 or 2 antibodies Ab-AF488 per nanoparticle
Fig. 8 Release of Ab-AF488 in 10% FBS without (dashed line, &) or withantiproteases (solid line,B) for 1 (blue) or 2 (red) antibodies mAb-AF488per nanoparticle gFe2O3@PO-PEG85.
were injected via the caudal vein with a bolus of 100 mL ofpegylated NPs alone. The tested dose was 200 mmolFe kg�1,which is usually injected in preclinical studies. The ability ofthese NPs to be used as in vivo MRI nanoprobes is illustrated inFig. 9. An increase in the number of detected hypo-intense voxelswas observed in FLASH images when NPs were circulating in thebloodstream, leading to better defined angiograms of the mousebrain compared to the pre-injection images, in which case onlyendogenous iron is circulating (hemoglobin protein complexesbinding iron ions in red blood cells). This brain vasculaturehighlighting confirms the efficiency of pegylated NPs as aT2-shortening MRI contrast agent.
Targeting properties and toxicity. The targeting properties ofantibody-NPs were established first by flow cytometry at 4 1C.The different NPs, differing in their number of grafted Ab, wereincubated with CHO-ETAR cells, overexpressing ETAR on theexternal cell surface, and CHO-WT cells used as a negative control.Under these conditions, the endocytosis pathway is inhibited andthe targeting effect is expected to be highlighted.50,51
In the second experimental sets, Ab-NPs were incubated at37 1C. In this case, NP internalization occurs via the endo-cytosis pathway.52 Thus for CHO-ETAR cells, a cumulative effectis expected due both to the targeting by the Ab grafted on theNP surface and to the NP cell uptake, whereas for CHO-WT,only nonspecific internalization will occur. In addition to flowcytometry evaluation, magnetic measurements were performedin order to deduce the average number of Ab-NP internalizedinto one cell.
NPs were incubated at 4 1C with cells in suspension for3 hours, firstly with a non-saturating extracellular concentrationof antibody equal to 25 nM and corresponding to [NP] = 25 nMfor R = 1 and 12.5 nM for R = 2. The cell autofluorescence(negative control denoted Cells) was measured and a positivecontrol was performed with the labeled Ab at a concentration of100 nM, allowing one, in this condition, to obtain the highestfluorescence signal (saturating conditions). The results are pre-sented in Fig. 10.
It is clearly observed that the cell autofluorescence is verylow (lower than 3), and that the free AF488-Ab (positive control)shows the highest MFI with the CHO-ETAR (142) compared to
CHO-WT (5) demonstrating the efficiency of the Ab to targetspecifically the receptor ETAR. The two Ab-NPs also presenthigh fluorescence signals after incubation with CHO-ETAR com-pared to CHO-WT cells. These results demonstrate that activetargeting occurs and that the functionality of the antibody isconserved even after grafting onto the NP surface. It has to benoticed that the NP functionalized with 2 Ab (extracellular con-centration [NP] = 12.5 nM) induced a higher signal (around 1.5�)compared with NP functionalized with only one Ab (extra-cellular concentration [NP] = 50 nM). Considering that NPtargeting is enhanced with the average number of targetingmolecules on the NP surface and with the extracellular NPconcentration, these results suggest a cooperative effect or anavidity phenomenon.11
Such results are confirmed by evaluating the influence ofextracellular NP concentration versus cell targeting (Fig. 11).
As expected, low MFI signals are observed with the CHO-WTcells compared with CHO-ETAR cells. Increasing the extracellularAb-NP concentration induces an increase of the MFI signaland the response is higher with CHO-ETAR than with CHO-WT.Nevertheless, nonspecific adhesion is observed (less than 10%)(Fig. 11a). For the same extracellular NP concentration, thenanoplatform with two antibodies induces a 2.3 times greatersignal than the nanoplatform with one antibody (Fig. 11b). Inaddition, better results are observed with 50 nM NP concentration(R = 2), close to the value obtained with 100 nM Ab. Considering that
Fig. 9 3D angiograms of the mouse brain acquired (a) before and(b) after intravenous injection of the nanoplatforms gFe2O3@PO-PEG85 at200 mmolFe kg�1. The contrast enhancement (in %) of mouse brain vascu-lature observed between pre- and post-injection of gFe2O3@PO-PEG85
corresponds to an increase of 78.9%.
Fig. 10 Flow cytometry analysis for cells CHO-WT (hatched bars) andCHO-ETAR (full bars) without treatment (green), incubated with the anti-body (100 nM, black) or with the nanoplatforms with 1 ([NP] = 25 nM, blue)or 2 antibodies ([NP] = 12.5 nM, red) per NP at 4 1C.
Fig. 11 Flow cytometry analysis for cells CHO-WT (hatched bars) andCHO-ETAR (full bars) incubated with various extracellular concentrations ofnanoplatforms with 1 (blue) or 2 antibodies (red) per nanoparticle at 4 1C.
100 nM concentration for Ab corresponds to saturating condi-tions ([NP] = 100 nM for R = 1 and 50 nM for R = 2) and takinginto account the fluorescence quenching factor equal to 3,these results confirm the efficiency of our Ab-NP to targetthe ETAR.
To evaluate the NP internalization in the two cell lines,the magnetic signal of the cells was measured at 37 1C usinga magnetic reader.53,54 Experiments were performed using asaturating Ab concentration equal to 100 nM (correspondingto NP with 1 and 2 Ab grafted per NP concentration and thusequal to 100 nM and 50 nM NP respectively). The cytometryresults and the magnetic measurements are shown in Fig. 12.
At 37 1C, as expected the MFI signal for CHO-ETAR cellsincreases markedly for both the nanoplatforms in comparisonwith the CHO-WT cells (Fig. 12a) as already observed at 4 1C.The magnetic quantification (Fig. 12b) shows a good corre-lation between the two techniques (magnetic quantificationand flow cytometry) as already demonstrated with the othertype of NPs (surface functionalized with oligonucleotides)and validating our magnetic innovative quantification.53,54
The average number of internalized NPs per cell is found tobe higher for the CHO-ETAR cells (between 1.4 million and1.7 million NPs per cell) than for the CHO-WT cells (between140 000 and 240 000). Once again these results demonstrate thetargeting efficiency of the nanoplatforms. Hence compared toprevious results with iron oxide Ab-NPs surface functionalizedwith caffeic acid (CA), a smaller linker than PEG, the amountof magnetic NPs per cell is found to be lower for the CHO-WTcells (4.4 � 105 and 1.3 � 106 for the CA nanoplatforms versus1.4 � 105 and 2.4 � 105 for the pegylated nanoplatforms,respectively with 1 or 2 antibodies grafted per NP).12,46 Theseresults suggest lower internalization of the pegylated nano-platforms compared to CA ones. This behavior could be dueto the difference in NP potential charge and hydrodynamic size
induced by the nature of the coating layer and modifying thequantity of NPs inside the cells. In the first approximation, thetargeting specificity can be evaluated at 4 1C by the factorbetween cells overexpressing the EtA receptor and the controlcells. Factors are estimated to be 1.36 and 2 for the Ab-CA-nanoplatforms and 11 and 5.8 for the Ab-PEG-nanoplatforms,respectively grafted with 1 and 2 antibodies.12,46 Thus, thespecificity seems to be more important with the pegylatedNPs than with the CA NPs. However, such difference could bealso due to various effects: (i) the specificity of the Ab-AC NPsfor the ETBR receptor compared to targeting of Ab-PEG NPs forETAR due to the specificity of each Ab; (ii) the difference in thecharge and hydrodynamic diameter of the various nanoplat-forms, as well as (iii) the fact that Ab-AC NP targeting evaluationwas performed with adherent cells whereas with Ab-PEG NPswe used cells in suspension.
To check the toxicity of Ab-PEG nanoplatforms, MTT testswere performed at 37 1C during 3 and 24 hours on CHO-WTand CHO-ETAR cells using increasing extracellular NP concen-trations (Fig. 13).
After 3 hours (Fig. 13a and b), a slight toxicity occurs even forthe highest NP concentrations, although it remains very lowwith the CHO-ETAR cells (cell viability greater than 90%) andslightly higher with the CHO-WT cells (cell viability greater than60%). In addition, during a long incubation (24 hours), addi-tional toxicity is not observed. The cell viability is always greaterthan 80%. Indeed, various studies show that the presence ofPEG chains on the surface of the nanoparticles could induce adecrease of the toxicity due to the lowest internalization of thepegylated nanoparticles in comparison with the non-pegylatednanoparticles.55,56
Hence we demonstrated that fine-tuning and control ofchemical–physical properties of the iron oxide nanoparticledesign is a key point for the stability and the targeting of suchnanomaterial. This proof-of-concept study will be completed inthe future by an in vivo investigation for confirming the targetingeffect and functional diagnostic imaging of tumor.
Fig. 12 (a) Flow cytometry analysis and (b) magnetic quantification forCHO-WT (hatched bars) and CHO-ETAR (full bars) cells incubated withnanoplatforms with 1 ([NP] = 100 nM, blue) or 2 antibodies ([NP] = 50 nM,red) per nanoparticle at 37 1C.
Fig. 13 MTT viability assay after incubation, 3 hours (a and b) or 24 hours(c and d), of the nanoplatforms with 1 (blue) or 2 antibodies (red) withCHO-WT (a and c) and CHO-ETAR (b and d) cells.
The reagents were obtained from the following sources: all thefluorescent probes, MitoSOX and TO-PRO-3 iodide, YO-PRO-1,are from Molecular Probes (Invitrogen) except propidium iodide(PI, Sigma Aldrich). Reagents for particle synthesis were fromSigma-Aldrich (Saint Louis, MO, USA). Phosphonate-poly(ethyleneglycol) PO-PEG-COOH (MW 1200 g mol�1; ref. SP-1P-10-001) andPO-PEG-CH3 (MW 2100 g mol�1; ref. SP-1P-1-001) were purchasedfrom Specific Polymers (Castries, France).
Cell lines
The first cell line used in the study was the human U-87 MG cellline from ATCCr (also named HTB-14s, issued from the brainand that is defined as originating from glioblastoma, astrocytoma,classified as grade IV as of 2007). The cells were maintained inDMEM containing 4.5 g of glucose per L supplemented with 10%decomplemented fetal calf serum, 1% penicillin, 1% streptomycin,1% glutamine (Gibco) supplemented with 1 mM Na-pyruvate and1 mM HEPES (NaOH). All cultures were grown at 37 1C in ahumidified atmosphere of 5% CO2.
The second cell line used in the study was CHO-WT (wild-type)and CHO-ETAR cells which overexpress the endothelin A receptor.CHO cells (European Collection of Cell Cultures (ECACC),Salisbury, UK) were cultured in Ham-F12 medium supplementedwith 10% fetal calf serum, 1% pyruvate, 1% nonessential aminoacids, 1% glutamine, 1% penicillin and streptomycin, and weremaintained at 37 1C in a humidified atmosphere of 5% CO2.All media and cell culture supplements were from Invitrogen(Life Technologies, Carlsbad, USA).
Non-coated gFe2O3 NPs were synthesized by the reaction of ferrousdodecyl sulfate with dimethylamine as previously described.19 After2 hours at 28 1C, the solution was rinsed under acidic conditions.The magnetic NPs were then separated from the supernatant usinga permanent magnet and dispersed in water at pH 2.
Two PEG molecules were used, differing in their molecularweight, 2100 g mol�1 (n E 44 units) and 1200 g mol�1 (n E23 units), and in their terminal function, CH3 and COOH,respectively (called 2100-PO-PEG-CH3 and 1200-PO-PEG-COOH).The NPs were coated with various mixtures of 2100-PO-PEG-CH3
and 1200-PO-PEG-COOH, defined by the ratio x = n1200-PO-PEG-COOH/(n1200-PO-PEG-COOH + n2100-PO-PEG-CH3
) � 100. To coat the NPs, the twoPO-PEG were mixed at pH 2 with a mass (PO-PEG/NP) ratioequal to 10. After the washing procedure to remove excessmolecules using an ultrafiltration process (Amicon 30 kDa,Merck Millipore), the magnetic gFe2O3@PO-PEGx particles weredispersed in distilled water at pH 7.
The grafting of the molecules to the surface of the variousplatforms was studied by Fourier transform infrared (FTIR)analysis. The FTIR spectra were recorded as thin films on KBrpellets on a Thermo Scientific Nicolet 380 FTIR and are reportedas frequency of absorption (cm�1).
Quantification of PO-PEG coating and grafting per particle wasevaluated by EDX. EDX microanalyses were performed using a TM3000 tabletop microscope equipped with a Swift EDX-ray 3000microanalysis system (Oxford Instruments). Samples were depositedas powder on a copper surface, and data were collected using a15 kV accelerating voltage, studying the ratio of iron versus phos-phorus and knowing the average number of iron atoms/particles.
The hydrodynamic size and zeta potential of the variousnanoplatforms ([Fe] = 0.25 mM) were investigated by dynamiclaser light scattering measurements using a Nano-ZS (Red Badge)ZEN 3600 device (Malvern Instruments, Malvern, UK). The stabi-lity in physiological medium (150 mM NaCl, [Fe] = 1.5 mM) wasstudied by measuring over time the evolution of the absorption at480 nm. UV-vis spectra were recorded on a Varian Cary 50 ScanUV-vis spectrophotometer.
TEM images were obtained using a FEI CM10 microscope(Philips), and samples were prepared by depositing a drop of NPsuspension on carbon-coated copper grids placed on a filter paper.The median diameter was deduced from TEM data measurements,simulating the diameter distribution with a log-normal function.
Antibody Ab coupling procedure onto cFe2O3@PO-PEGx NPs
First, the carboxylic acid functions at the outer surface of thenanocrystals were activated using 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC, nEDC = 4nCOOH) in grafting buffer(0.3% Pluronic F127, 100 mM H3PO4, pH 4) for 10 minutesat 37 1C. The second step was the linkage of the amine functionof Ab-AF488 with the activated carboxylic acid functions on thenanocrystals. The procedure was carried out with molar ratiosR = nAF488-Ab/nNP equal to 2 and 4. AF488-mAb was dissolved ingrafting buffer at pH 4 and added to the ferrofluid for 60 minutesat 37 1C. The activated carboxylic functions that did not react withthe amine function of the antibody were saturated in graftingethanolamine (EA) buffer (nEA = 5nCOOH) for 30 minutes at 37 1C.The modified particles were isolated with a magnet in blockingbuffer (50 mM Tris, 0.2% Tween, pH 8.6) 2 times and washed2 times with deionized water by centrifugation (14 600 rpm, 3 min).The particles were dispersed in water at physiological pH forphysicochemical characterization.
Evaluation of Ab grafting yield with OPA titration
The average number of grafted Ab per nanocrystal was deducedusing the OPA (o-phthalaldehyde and 2-mercaptoethanol) reagent.The OPA reacts with amines to form blue fluorescent isoindolesin the presence of reduced thiols. The amount of grafted Ab pernanoparticle was deduced after chemical decomposition (NaOH2 M, 60 1C, overnight) of the coupling and titration of aminogroups in the supernatant. To do this quantification, 50 mL of thesample was diluted in 50 mL of 2 M NaOH and left overnight at60 1C. Then, 900 mL of OPA reagent was added to the mixture andfluorescence was measured at 595 nm (lex = 330 nm).
Nanoparticle toxicity
Flow cytometry measurements. MitoSOX (1 mM stock solution,Molecular Probes) was used at 5 mM final concentration for super-oxide anion determination (controls are realized by incubation
with 5 mM paraquat). The incubation time is 15 min at 37 1C.A double staining was realized in order to simultaneously assaythe cellular viability with TO-PRO-3 iodide. Only TOPRO-3 nega-tive cells that are viable are considered for MitoSOX analysis. Allthe various NPs were evaluated at 50 nM and 100 nM and wereanalyzed by flow cytometry as previously described on a FACSCalibur 4C and using the Cell quest software.57,58 The FL2 channelis a 585 � 20 nm Bandpass used for mitoSOX fluorescence andthe FL4 channel is a 662 � 10 nm Bandpass used for TO-PRO-3fluorescence. Dead cells if any are TOPRO-3 positive. Iron oxideNPs surface functionalized with caffeic acid were used ascontrol NPs.12,46
Impedance-based label-free assays. The Xcelligence real-time cellanalyzer (RTCA, ACEA Biosciences) has been widely described as apowerful and reliable tool that can be used in drug discovery fortoxicity and pharmacology studies. This device measures the relativecellular impedance changes over time (here, 120 h or 5 days) thatare related to cellular binding to the substrate and cellular prolifera-tion. Potential changes in cellular binding and proliferation may arerelated to cellular toxicity of the added compounds. All the variousNPs were evaluated at 50 nM and were tested in two independentexperiments in quadruplicate. The principles of data assessments aswell as the formula for the normalized cell index (NCI) calculationhave been described previously.
MTT assays. Cells were seeded at a density of 1 � 104 cells perwell in 96-well flat-bottom plates (Falcon, Strasbourg, France) andincubated in complete culture medium for 24 hours. Then themedium was removed and replaced by complete culture mediumcontaining increasing concentrations of gFe2O3@PEG-Ab*AF488and gFe2O3@PEG-(Ab*AF488)2 NPs. After 3 or 24 hours, themedium was removed and PBS with MTT (0.5 mg mL�1) wasadded for 2 hours at 37 1C. The medium was removed againand insoluble formazan crystals were dissolved by the additionof DMSO. The optical density was measured at 570 nm using aspectramax microplate reader (Molecular Devices, Sunnyvale,CA, USA).
Evaluation of Ab-NP targeting. Flow cytometric analysis wasperformed on CHO-WT (wild-type) and CHO-ETAR cells at 4 1Cand 37 1C. Cells were washed twice and resuspended in 500 mLof DPBS. Fluorescence was assayed using a FACS-Calibur (BDBioscience, Franklin Lakes, USA) flow cytometer and the meanfluorescence intensity (MFI) was measured (30 000 cells per count).For the experiments performed at 37 1C, the cell number wasincreased (5 times) keeping the same concentrations by adjust-ment of the volume, in order to measure sufficient magneticdetection (see below).
Evaluation of Ab-NP uptake. The magnetic properties of thenanoparticles were recorded at room temperature using a mag-netic sensor which measures a signal that is proportional to thethird derivative of magnetization at a zero magnetic field.53,54
First, a calibration curve is established with the gFe2O3@PEG,gFe2O3@PEG-Ab*AF488 and gFe2O3@PEG-(Ab*AF488)2 NPs atvarious concentrations. After the flow cytometry measurements,the MIAtek signal is measured for the different samples. From theprevious calibration curve, the amount of internalized nano-particles in the sample was deduced.
In vitro MRI relaxivity measurements. Longitudinal r1 andtransverse r2 relaxivities of gFe2O3@PO-PEG85 NPs were measuredin vitro at 37 1C and 7 T (Bruker PharmaScan preclinical scanner,Ettlingen, Germany) using dedicated phantoms consisting of11 tubes filled with different concentrations of the contrastagent (from 0.001 to 0.25 mM) suspended in 0.3% w/w agarmatrix. T1 and T2 maps were acquired respectively with anInversion Recovery Fast Gradient Echo Sequence (TE = 2.5 ms,90 inversion times with DTI = 100 ms, 6 segments, TR = 15 000 ms,field of view = 3 � 3 cm2 in-plane, acquisition matrix = 120 � 120,6 slices of 1.25 mm thickness, total acquisition time = 54 min) anda Multi-Slice Multi-Echo sequence (TE = 7.7 ms, 64 echoes withDTE = 7.7 ms, TR = 10 000 ms, field of view = 3 � 3 cm2 in-plane,acquisition matrix = 120 � 120, 6 slices of 1.25 mm thickness,total acquisition time = 1 h 20 min). In the first post-processingstep, T1 (resp. T2) maps were reconstructed with a homemadepipeline of Matlab routines (MathWorks, Natick, USA), by fittingthe signal intensity versus inversion time (resp. echo time)using Bloch equations. Then, the mean proton relaxation rateR1 (resp. R2), equal to the inverse of relaxation time T1 (resp. T2),was computed for each tube, and a linear fit of R1 (resp. R2)values versus iron concentrations was performed to estimatethe longitudinal relaxivity r1 (resp. transverse relaxivity r2) asthe slope of the fitted curve.
In vivo MRI experiments. All in vivo experiments wereconducted in strict accordance with the recommendations ofthe European Community (86/609/EEC) and the French legisla-tion (decree no. 87/848) for use and care of laboratory animals.The protocol for contrast agent injection was approved by theComite d’Ethique en Experimentation Animale du Commissariata l’Energie Atomique et aux Energies Alternatives – Direction desSciences du Vivant Ile-de-France (CETEA/CEA/DSV IdF) underprotocol ID 12-058. MRI acquisitions were performed on a 7 Tpreclinical scanner equipped with a volume radiofrequencycoil dedicated to mouse brain (Bruker, Ettlingen, Germany),in order to maximize the detection of the T2* effect induced bygFe2O3@PO-PEG85 NPs. Male Swiss mice of 25 g were anesthetizedusing an air/O2 mixture (50 : 50) and isoflurane (1–3%), beforebeing positioned into dedicated cradles. The respiration ratewas continuously monitored and body temperature was keptconstant at 37 1C owing to a warm air blowing system. Injectionof 200 mmolFe kg�1 of gFe2O3@PO-PEG85 NP suspension (100 mL,71 mMFe) was performed via the caudal vein with a 29 G needlewhile keeping the mouse positioned in the cradle.
To visualize the biodistribution of injected NPs, a T2*-weightedFLASH (Fast Low Angle SHot) sequence was used with the follow-ing parameters: TE/TR = 16/485 ms, in-plane field-of-view = 1.92 �1.68 cm2, in-plane resolution = 160� 160 mm2, 22 slices of 320 mmthickness, 48 averages, total acquisition time = 40 min. MR imageswere reconstructed from raw data using a homemade pipelineconsisting of Matlab routines.
Segmentation of the mouse brain was done manually, follow-ing the edges of the brain, slice by slice in all FLASH images.A mask was generated from these manual segmentations, whichenables one to consider the MRI signal only in this region ofinterest. A filter based on the multiscale vessel enhancing method
proposed by Frangi was applied to the segmented FLASH image.This filter computes the likelihood that a vessel is present in eachvoxel, considering the second order gradient values in the sur-rounding voxels to enhance vasculature and reduce the noise inthe image.59 3D visualization of vessels enhanced by the Frangifilter was obtained owing to the volume rendering and MaximumIntensity Projection tools provided by Anatomist software (CEANeuroSpin, Gif-sur-Yvette, France, http://brainvisa.info). The com-parison between the 3D pre- and post-injection angiograms pro-vides an insight into the efficiency of the tested contrast agent.
Conclusions
This article presented the improvements of the stability ofsuperparamagnetic iron oxide nanoparticles in physiologicalconditions owing to their surface functionalization using poly-ethylene glycol chains differing in their terminal functionality,COOH and CH3, as well as PEO length. Various ratios of the twophosphonate polyethylene glycols have been evaluated to deter-mine the most relevant nanoplatform for further studies.Pegylated nanoplatforms were successfully functionalized bycarbodiimide chemistry with an antibody labeled with a fluoro-phore AF488. In vitro studies, performed by flow cytometry andmagnetic quantification, demonstrated the efficiency of the ETA
receptor targeting. In addition, a low toxicity was observed. Thus,an imaging tracer from a bimodal contrast agent (fluorescenceand MRI) was obtained and opens the way to the detection ofsmall tumor masses.
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