Jean-Baptiste Lugagne

Gwennhaël Brackx

Emek Seyrek

Sophie Nowak

Yann Sivry

Leticia Vitorazi

Jean-François Berret

Pascal Hersen

Gaëlle Charron
email: gaelle.charron@univ-paris-diderot.fr

Assembly and Characterizations of Bifunctional Fluorescent and Magnetic Microneedles Displaying Length Tunability over One Decade

Keywords: microneedles, magnetic nanoparticles, quantum dots, nanoparticle assembly, confocal fluorescence microscopy

We report on the fabrication of bifunctional magnetic and fluorescent microneedles (µNDs) made of a ternary mixture of magnetic nanoparticles (NPs), quantum dots (QDs) and polyelectrolyte. The assembly relies on the electrostatic complexation of negatively charged NPs with positively charged polymer strands and is controlled by the charge ratio between the nanoparticulate building blocks and the polymer mortar. We show that the resulting µNDs retain the fluorescent and superparamagnetic ú

Introduction

1D-magnetic microstructures made of single magnetic microparticles or assemblies of magnetic nanoparticles can find applications in a wide variety of fields that includes composite materials, microfluidics and micro-mechanics. Micro-rods of non-magnetic material coated with iron oxide nanoparticles have been used to mechanically reinforce polymer matrices through applying small magnetic fields [START_REF] Erb | Composites Reinforced in Three Dimensions by Using Low Magnetic Fields[END_REF]. Micro-machined pieces of magnetic materials or chains of magnetic colloids have been applied as micro-stir bars to increase the e ciency of mixing in microfluidic channels and micro-droplets [START_REF] Ward | Mixing in microfluidic devices and enhancement methods[END_REF][START_REF] Han | Stirring in Suspension: Nanometer-Sized Magnetic Stir Bars[END_REF][START_REF] Bruyker | Rapid mixing of sub-microlitre drops by magnetic microstirring[END_REF][START_REF] Suk Ryu | Micro magnetic stir-bar mixer integrated with parylene microfluidic channels[END_REF][START_REF] Sibani | Micromixing with Linked Chains of Paramagnetic Particles[END_REF]. Mixing at the microscale can also be enhanced using micropillars or artificial cilia consisting of magnetic micro or nanoparticle assemblies that can be actuated through application of a rotating or a swinging magnetic field [START_REF] Yu | Mixing control by frequency variable magnetic micropillar[END_REF]. These magnetic cilia can form the basis of flow or vibration sensing when coupled to a giant magnetoresistive sensing element that monitors their deflection [START_REF] Alfadhel | Magnetic Nanocomposite Cilia Tactile Sensor[END_REF][START_REF] Alfadhel | A magnetic nanocomposite for biomimetic flow sensing[END_REF]. Alternatively, the bending of these cilia under a static external field can serve as a mean to apply controlled stresses to soft materials deposited on the substrate such as tissues, films or cells in order to study their mechanical responses [START_REF] Gao | Three Dimensional and Homogenous Single Cell Cyclic Stretch within a Magnetic Micropillar Array (mMPA) for a Cell Proliferation Study[END_REF][START_REF] Le Digabel | Magnetic micropillars as a tool to govern substrate deformations[END_REF][START_REF] Sniadecki | Magnetic microposts for mechanical stimulation of biological cells: Fabrication, characterization, and analysis[END_REF]. In the same line, free standing magnetic micro-rods can be used as active micro-rheometers to probe the viscoelastic properties of complex fluids, including living cells, with microscale resolution [START_REF] Berret | Local viscoelasticity of living cells measured by rotational magnetic spectroscopy[END_REF][START_REF] Chevry | Magnetic wire-based sensors for the microrheology of complex fluids[END_REF].

Many of these applications would benefit from the addition of a fluorescent functionality to the magnetic one. Incorporating fluorophores into the 1D-microstructures enables easier imaging in scattering media. It could allow for optical bar-coding of the magnetic micro-2 actuators. In cell mechanics studies, it could give insights into the environment of the magnetic probe through colocalisation experiments involving organelle-specific fluorophores.

Moreover, most of these applications simultaneously require the length of the 1D-magnetic microstructures to be adjusted to the problem at play. Micro-stirrers need to be attuned to the dimensions of the fluid compartment and to its viscosity. The range of forces to which magnetic microposts are sensitive depends on their length and thickness. To be able to resolve inhomogeneities in viscoelastic properties at the microscale, magnetic microrheometers need to be of dimensions comparable to the typical length scale of the structural inhomogeneities [START_REF] Wirtz | Particle-Tracking Microrheology of Living Cells: Principles and Applications[END_REF][START_REF] Waigh | Microrheology of complex fluids[END_REF].

Bifunctional magnetic and fluorescent nano or microspheres have been reported in the past. They were developed mostly towards applications in bimodal magnetic resonance and fluorescence imaging, drug delivery or bioassays. These materials have been synthesised according to various strategies: core-shell particles, where a core of magnetic material (e.g. iron oxide nanoparticles) is encased in a shell of fluorescent one (e.g. dye-labelled silica or quantum dots) or the other way round [START_REF] Labiadh | A facile method for the preparation of bifunctional Mn:ZnS/ZnS/Fe 3 O 4 magnetic and fluorescent nanocrystals[END_REF][START_REF] Jing | Controlled fabrication of bi-functional Fe3o4@SiO2@Gd2o3:Yb,Er nanoparticles and their magnetic, up-conversion luminescent properties[END_REF][START_REF] Lu | Bifunctional Magnetic-Fluorescent Nanoparticles: Synthesis, Characterization, and Cell Imaging[END_REF]; raspberry-like particles, where a large particles of one material are decorated on their surface with smaller particles of the other material [START_REF] Hassan | Continuous Multistep Microfluidic Assisted Assembly of Fluorescent, Plasmonic, and Magnetic Nanostructures[END_REF][START_REF] Peng | Multifunctional Manganese Carbonate Microspheres with Superparamagnetic and Flu-22 orescent Properties: Synthesis and Biological Application[END_REF]; colloidosomes self-assembled from fluorescent and magnetic colloids [START_REF] Bollhorst | Bifunctional Submicron Colloidosomes Coassembled from Fluorescent and Superparamagnetic Nanoparticles[END_REF]; beads in which particles of both materials have been coprecipitated or co-embedded in a silica or polymer matrix [START_REF] Sun | Bifunctional Superparticles Achieved by Assembling Fluorescent CuInS2@ZnS Quantum Dots and Amphibious Fe3o4 Nanocrystals[END_REF][START_REF] Di Corato | Multifunctional Nanobeads Based on Quantum Dots and Magnetic Nanoparticles: Synthesis and Cancer Cell Targeting and Sorting[END_REF][START_REF] Wang | Magnetic/NIR-thermally responsive hybrid nanogels for optical temperature sensing, tumor cell imaging and triggered drug release[END_REF]; and particles made of oxides of rare earth ions [START_REF] Kumar Gupta | Bifunctional Luminomagnetic Rare-Earth Nanorods for High-Contrast Bioimaging Nanoprobes[END_REF]. Most of these strategies lead to bifunctional spheres in the 0.1 to 1 µm range whose size cannot be easily tuned. In general, they are not amenable to synthesize 1D structures. Moreover, in many cases the synthetic cost is high, either because of the minute scale of production or to the time or number of steps required. We report here on the fabrication of bifunctional magnetic and fluorescent 1D-structures of micrometric length assembled by controlled aggregation of a ternary mixture of polyelectrolyte, superparamagnetic nanoparticles and quantum dots. The building blocks are simple and easily sourced.

Assembly proceeds in water, in less than half an hour, to give rise to wire or needle-like aggregates of millions of nanoparticles that are responsive under an external magnetic field and can be imaged by conventional epifluorescence microscopy. Interestingly, by playing on the ternary composition, their length can be tuned in the 5-50 µm range to match the needs of many microfluidic or biophysical applications. As an illustration of the potential of the microneedles (µNDs) for cell mechanics studies, we show that they are readily internalised by HeLa cells and that their fluorescence can be used to assess their localisation with regards to cellular compartments.

Results and discussion

2.1 General strategy for assembling fluorescent magnetic microneedles

Assembly of 1st generation µNDs

The bifunctional magnetic µNDs derive from a first generation of magnetic-only 1D-microstructures reported previously. This first generation of magnetic µNDs was obtained by electrostatic complexation of negatively charged magnetic nanoparticles (mNPs) with polycationic polymers in a kinetically controlled fashion [START_REF] Fresnais | Electrostatic Co-Assembly of Iron Oxide Nanoparticles and Polymers: Towards the Generation of Highly Persistent Superparamagnetic Nanorods[END_REF][START_REF] Yan | Growth mechanism of nanostructured superparamagnetic rods obtained by electrostatic co-assembly[END_REF][START_REF] Safi | Interactions between Magnetic Nanowires and Living Cells: Uptake, Toxicity, and Degradation[END_REF]. The magnetic building blocks consisted of 6-8 nm superparamagnetic Fe 2 O 3 maghemite NPs coated in a 3 nm-thick layer of sodium polyacrylate that imparted them a persistent colloidal stability, even at high ionic strength (Ø 1M). Various polyelectrolytes bearing positive charges through ammoniums (either quaternary or proton-exchanging) were investigated and all lead to successful assembly [START_REF] Yan | Magnetic Nanowires Generated via the Waterborne Desalting Transition Pathway[END_REF].

Due to the strong driving force for electrostatic assembly, direct mixing of the NP building blocks and polyelectrolyte glue resulted in macroscopic precipitation of large aggregates.

The assembly protocol was therefore designed to slow down the interaction. To this end, the constituents were mixed in the presence of a screening electrolyte at high ionic strength (typically in the M range), at which stage they remained individually dispersed. Assembly was then driven by slow dialysis of the screening electrolyte to give rise to spherical clusters. When performing the assembly under a static magnetic field (typically of 0.3 T), 1D structures grew and lead to microneedles between 150 and 400 nm in width and 1 to 500 µm in length that displayed superparamagnetism inherited from their iron oxide constituents.

Starting binary compositions were referenced using the charge ratio Z, defined as the number of negative charges brought by the mNPs divided by the number of positive charges brought by the polymer [START_REF] Erb | Composites Reinforced in Three Dimensions by Using Low Magnetic Fields[END_REF].

Z = C °mN P V mN P C ü pol V pol (1)

Choice of constituents for fluorescent magnetic µNDs

One possible strategy to incorporate a fluorescent functionality into the magnetic µNDs described above consists in substituting fluorescent building blocks for a fraction of the magnetic ones. Fluorescent NPs then need to present the same chemical reactivity as mNPs with regards to the assembly mechanism. This implies that they must bear negative charges on their surface and, critically, display colloidal stability even at high ionic strength. With the aim of tracking the µNDs using fluorescence (video) microscopy, the µNDs should ideally be as bright as possible. Indeed, strong brightness not only increases contrast but also enables the use of short exposure times and therefore give access to shorter timescales of dynamic tracking. Moreover, the µNDs should display homogeneous fluorescence, , namely should be homogeneously doped with fluorescent blocks, so that the fluorescence profile tightly mirrors the shape of the object. In a solid state solution, doping is facilitated by a similarity of size between the dopant and the host. Based on the same principle, the fluorescent building blocks and the magnetic ones should have diameters of the same order of magnitude.

Taking into account these criteria, we selected the AgInS 2 ≠ZnS (ZAIS) quantum dots (QDs) described by Regulacio et al. [START_REF] Regulacio | Aqueous synthesis of highly luminescent AgInS2â ZnS quantum dots and their biological applications[END_REF] as fluorescent building blocks (Fig. 1). These hydrosoluble QDs were synthesised directly in water and were a few nm in diameter. Their emission was composition-dependent and could be tuned from 525 to 640 nm, with a maximum reported quantum yield of 20%. Interestingly, these QDs were synthesized in the presence of polyacrylic acid, which acted as a capping agent. Having a diameter within the same range as the mNPs and the exact same coating, we expected these QDs to be an ideal dopant for the µNDs. Table 1: Summary of QD and mNP characteristics

Characterisations of magnetic and fluorescent building blocks for NW assembly

The mNPs used for µND assembly were synthesised following the Massart method and coated in a post-synthetic fashion with PAA, as described earlier [START_REF] Massart | Preparation and Properties of Monodisperse Magnetic Fluids[END_REF][START_REF] Qi | Stability and Adsorption Properties of Electrostatic Complexes:â Design of Hybrid Nanostructures for Coating Applications[END_REF]. The detailed characterizations of the exact batch used in this study are presented in ESI. Hereafter, we present the characterisations of the QDs we synthesized using the procedure from Regulacio et al. with few modifications, and compare the QD characteristics to those of the mNPs used for µND assembly (Table 1) [START_REF] Regulacio | Aqueous synthesis of highly luminescent AgInS2â ZnS quantum dots and their biological applications[END_REF].

Optical properties. Iron oxide NPs present an intense and continuous absorption in

the UV-vis range that vanishes close to 610 nm. We therefore aimed at synthesizing orange to red-emitting QDs to prevent fluorescence quenching through energy transfer to or reabsorption by the host matrix of the µND. The synthesized QDs had a maximum emission peak centered at 600 nm and a moderate quantum yield of 7%, which was compensated by the emission sitting well outside the iron oxide absorption range (Figure 2). According to Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) analysis, the QDs had a non-stoechiometric Ag 0.23 In 0.58 Zn 1.54 S 3 formula, reflecting an excess chalcogen compared to the metal ions and a defect in silver compared to indium, which is consistent with orange emission (Table S1) [START_REF] Subramaniam | Generation of a Library of Non-Toxic Quantum Dots for Cellular Imaging and siRNA Delivery[END_REF].

Size properties. TEM images of QDs reveal a 5.8 ± 1.2 nm core diameter based on counting 175 NPs (Fig. S1). The hydrodynamic diameter as determined by DLS was 13 nm in pure water at pH 8.5 (Fig. S2). The QDs and mNPs therefore had dimensions of the same order of magnitude, both regarding their inorganic core and their size in solution. Importantly, the hydrodynamic diameter of QDs remained unchanged in the screening electrolyte solution used for µND assembly (1M NH 4 Cl/NH 4 OH) over a period of days, evidencing remarkable colloidal stability due to the PAA coating. Just as the mNPs, the QDs were resilient towards the NW assembly conditions.

Charge density and concentration. As evidenced by gel electrophoresis on agarose

and consistently with PAA coating, the QDs were negatively charged (Fig. S9). They also exhibited a higher number of surface carboxylates per core mass unit than the mNPs, a fact that was confirmed by analysis of the composition of both batches of QDs and mNPs, by ICP-OES for the inorganic core elemental concentrations and pH-titration for the surface carboxylates (see ESI) [START_REF] Charron | On the Use of pH Titration to Quantitatively Characterize Colloidal Nanoparticles[END_REF]. Detailed compositions of the stock solutions are provided in Tables S1 & S5.

Assembly of fluorescent magnetic microneedles

Assembly of fluorescent magnetic µNDs was performed following the same synthetic procedure as those developped previously for magnetic-only µNDs, but for the substitution of QDs for a fraction w of mNPs (Eq. ( 2)). For a given assembly procedure, the starting reagent stoechiometry is unambiguously defined by two synthetic parameters: w and the previously defined Z ratio between negative charges brought by the nanoparticles and positive charges brought by the polymer, which now takes into account the contribution of QDs (Eq. ( 3)). Alternatively, the ternary composition can be depicted using the molar fractions of the charges respectively brought by QDs, mNPs and polymer, according to Equations (4). A set of 15 {Z;w} compositions with Z oe [0.62 ≠ 7.01] and w oe [0 ≠ 0.2] was investigated (Table S6). The individually dispersed mNPs are too small to be collected using a table magnet but their aggregates have a much higher saturation magnetization. Successful decantation under an applied magnetic field (typically of few hundredth of Tesla) therefore evidences that assembly has taken place. According to this indicator, we observed that all investigated compositions successfully led to µND assembly. Figure 3b displays the same specimen area imaged by epifluorescence microscopy. The µND emit in the red range under green excitation and appear homogeneously bright, evidencing homogeneous incorporation of the QDs in the host µND matrix and retention of their optical properties. When depositing a soft permanent magnet on the microscope stage, the µND respond to the external magnetic actuation by aligning along the field lines (Fig. 3c &3d). Upon suppressing the external magnetic field, the µND relax towards randomized orientations, thereby evidencing superparamagnetism.

w = V QD V mN P + V QD (2) Z = C °QD V QD + C °mN P V mN P C ü pol V pol (3) x QD = C °QD C °QD + C °mN P + C ü pol (4)
x mN P = C °mN P C °QD + C °mN P + C ü pol x pol = C ü pol C °QD + C °mN P + C ü
Interestingly, the morphology and in particular the length of the µNDs vary with the starting composition (Fig. 4). While {Z = 4.20; w = 0.20}-µNDs have a mean length of 13 µm, {Z = 1.40; w = 0.20}-µNDs that have the same QDs substitution ratio are on average 3 times longer (39 µm). Both samples display straight persistent needle-like aggregates. In contrast, the {Z = 1.21; w = 0.15} sample contains large, soft, hair strand-like bundles of µNDs whose length cannot be systematically determined because of inter-µNDs aggregation, along with some individual µNDs. In addition, the longer the µNDs or bundles of µNDs, the brighter they are. As both the length of the µNDs and their brightness ultimately determine their potential application, a systematic investigation of these parameters as a function of ternary composition was conducted.

Systematic investigation of length and brightness as a function of ternary composition

Tools for image analysis. The distribution of length was analysed from phase contrast microscopy images acquired with 10, 20 and 40X objectives. Lengths were extracted using a custom-written, dedicated image analysis Matlab code The algorithm for µND recognition was based on the linear Hough transform, which is a feature extraction technique devoted to the identification of lines [START_REF] Duda | Use of the Hough Transformation to Detect Lines and Curves in Pictures[END_REF]. The software is successful in recognizing single objects in an entangled bunch of µNDs but not when the µNDs are bundled (Fig. S11). It is biased against smaller µNDs due to their comparatively lower contrast. The software features a graphical user interface (GUI) for quick and handy correction of the most obvious errors, such as µNDs identified shorter than they are due to uneven contrast along their length or wires aligned tip-to-tip mistaken for one long object. Using this automated image analysis tool, 750 µNDs on average were counted per sample comprising individual µNDs, with no less than 200 µNDs counted for that having the highest mean length. All length distributions were log-normal with relative standard deviations ranging from 15 to 23% (Fig. 4 & S12). Fluorescence analysis was performed based on epifluorescence microscopy images acquired with a single 20X objective. The brightness of each sample was taken as the mean background-corrected intensity of the identified bright pixels in the set of sample images. The dependence of brightness and mean length on ternary composition are depicted in Fig. 5 (see also Table S7 and Fig. S14) Tuning of brightness. For convenience, the QD-doping ratio w is mapped over the range of ternary compositions in Figure 5 (left). As a general trend, starting compositions richer in QDs display higher relative brightness. This observation evidences that the actual incorporation ratio parallels the starting doping ratio and supports the hypothesized similarity in reactivity between PAA-coated QDs and PAA-coated mNPs.

Tuning of length. Out of 15 compositions, the mean length of 11 samples could be extracted. Mapping the Z ratio over the ternary diagram reveals that the length decreases with increasing Z, with the geometric mean length ranging from 5.7 to 48.5 µm for Z = 6.03 to 1.21 respectively. Three compositions lead to large bundles of aggregated µNDs whose length could not be extracted; they corresponded to the weakest Z ratios across the whole range of molar fractions (Z=0.62, 0.82 & 1.01 respectively). The last remaining sample corresponded to the highest Z value (7.01). For this sample, the µNDs could not be resolved using the 40X objective, although assembly had indeed taken place since the product could be magnetically decanted with a table magnet, albeit admittedly very slowly.

The dependence of the mean length on the composition was rationalised earlier for magnetic-only µNDs [START_REF] Yan | Growth mechanism of nanostructured superparamagnetic rods obtained by electrostatic co-assembly[END_REF]. The assembly of µNDs was shown to occur in two steps that are not separated in time (Fig. S10). In a first nucleation step, supra-colloidal clusters are formed due to the electrostatic complexation between negatively charged mNPs and positively charged polymer strands once the ionic strength falls below a certain threshold. In a second growth step, clusters align along the magnetic field lines, assemble and then fuse due to magnetic dipolar attraction (Fig. S10). The surface charge of the clusters was found to mirror the Z ratio of the starting mixture, with compositions featuring excess mNPs (Z > 1) leading to negatively charged clusters and those featuring an excess polycation (Z < 1) leading to positively-charged ones [START_REF] Yan | Magnetic Nanowires Generated via the Waterborne Desalting Transition Pathway[END_REF]. The kinetics of assembly therefore results from the balance between dipolar magnetic attraction and electrostatic repulsion between the supra-colloidal clusters. Accordingly, Z=1-magnetic-only µNDs, which stem from intermediate clusters having the weakest surface charge, were systematically found to be the largest [START_REF] Yan | Magnetic Nanowires Generated via the Waterborne Desalting Transition Pathway[END_REF].

Here, we attempted at modelling the dependency of the length on compositional parameters in the case of the bifunctional µNDs. The dependence of the length on the Z ratio in fluorescent magnetic µNDs appears fully consistent with the afore-mentioned mechanism.

There is strong anticorrelation at the 95% confidence level between length and Z ratio (see ESI for details of the t-test). The shortest µNDs correspond to compositions having the largest Z absolute values due to the increased electrostatic repulsion between clusters. As the QD building blocks have a higher surface charge density than the mNPs,these shortest µNDs are also the richest in QDs. Since ZAIS QDs are diamagnetic, their incorporation should lessen the saturation magnetisation of the clusters. For a given Z value, a higher QD doping ratio should therefore lead to less intense magnetic dipolar attraction between clusters. One might expect this phenomenon to slow down the assembly and lower the length of the resulting µNDs. However, a t-test conducted on the data indicated no significant correlation between the length and the doping ratio w (see ESI). Finally, the dependency of the length on the compositional parameters could be simply described as Equation ( 5) (Figure 6).

L ¥ 65 ◊ exp(≠0.33Z) ( 5
)
where L is the geometric mean length and Z varies in the 1.2 -6.0 range (Akaike information criterion was applied to choose the best model, see ESI). Because of their length tunability in the 5 -50 µm range and the ease with which they can be assembled, the bifunctional fluorescent magnetic µNDs could find applications in multiple domains. Microfluidic channels have a typical width of 50 -200 µm. At the upper end of the size window, 30-50 µm µNDs could be used as individual micro-stir bars as a cheaper alternative to micromachinned devices that have been reported earlier [START_REF] Suk Ryu | Micro magnetic stir-bar mixer integrated with parylene microfluidic channels[END_REF]. Shorter µNDs could be easily incorporated as multiple stir bars in arrays of microdroplets to e ciently churn up the whole solution compartments [START_REF] Han | Stirring in Suspension: Nanometer-Sized Magnetic Stir Bars[END_REF][START_REF] Bruyker | Rapid mixing of sub-microlitre drops by magnetic microstirring[END_REF]. The magnetic actuation and fluorescent features could be exploited in simultaneous magnetic and fluorescence-assisted cell sorting [START_REF] Di Corato | Magneticâ Fluorescent Colloidal Nanobeads: Preparation and Exploitation in Cell Separation Experiments[END_REF]. Alternatively, the bifunctional µNDs could act as probes for active viscosimetry-based label-free bioassays [START_REF] Sinn | Asynchronous Magnetic Bead Rotation Microviscometer for Rapid, Sensitive, and Label-Free Studies of Bacterial Growth and Drug Sensitivity[END_REF]. In both of these assaying applications, the synthetic control over the length would enable fine tuning of probe to the application through optimisation of the saturation magnetisation of the µNDs. Finally, µNDs could allow to probe the mechanical properties of biological materials such as tissues, films, individual cells or subcellular compartments using magnetic actuation-based techniques coupled to fluorescence imaging. Lately, the viscoelastic properties of cells and tissues have attracted much attention in the frame of medical research. For instance, the mechanical sti ness of cancer cells has been shown to correlate with their metastatic potential, with softer more deformable cells having the highest migratory potential and invasiveness [START_REF] Vinay Swaminathan | Mechanical sti ness grades metastatic potential in patient tumor cells and in cancer cell lines[END_REF]. Moreover, cell state or fate can be controlled by the viscoelastic properties of its environment [START_REF] Grevesse | Opposite rheological properties of neuronal microcompartments predict axonal vulnerability in brain injury[END_REF]. The phenomena at play are di cult to study by conventional macroscopic rheological methods since the samples typically come as tiny specimens and biological fluids are generally complex, ie. they behave di erently at di erent length scales. In this context, the size tunability over the 5 -50 µm range is especially appealing since it would enable the study of a given material from the scale of assemblies of cells down to the subcellular level with a single technique suited to microscopic specimens.

Such multiscale study would shine light on the respective contributions and interplay of the mechanics of cells, organites and surrounding matrix.

Intracellular imaging of fluorescent magnetic microneedles

Since many of these applications would rely on interaction of the bifunctional µNDs with cells and subsequent fluorescent imaging, we hereafter examine whether the present bifunctional fluorescent magnetic µNDs would be suitable to probe individual cells via confocal fluorescence microscopy. Magnetic-only µNDs were previously found to be internalised by mouse fibroblasts and human cancer cells, a phenomenon which was used to extract viscoelastic properties of these cells through rotational magnetic spectroscopy [START_REF] Berret | Local viscoelasticity of living cells measured by rotational magnetic spectroscopy[END_REF]. Here, HeLa were incubated with QD-doped µNDs, fixed and stained with DAPI in order to fluorescently label their nucleus.

Summary

In this work, bifunctional microneedles were readily assembled by electrostatic complexation of a ternary mixture of superparamagnetic iron oxide nanoparticles, red-emitting zinc-silverindium sulfide quantum dots and a cationic poylelectrolyte. The microneedles encompass the properties of both nanoparticulate components, namely superparamagnetism and fluorescence in the visible range. They can be actuated using small external magnetic fields and imaged using epifluorescence or confocal fluorescence microscopy. Interestingly, we found that by playing on the ternary composition, more specifically on the ratio of negative charges brought by the nanoparticles to positive charges brought by the polyelectrolyte, the length of the microneedles could be tuned in the 5-50 µm range. Because of their easy synthesis, their bifunctionality and their size tunability, these fluorescent and magnetic microneedles could find applications in multiple domains such as micromixing, bioassays or biomechanical studies and especially in the field of cell mechanics, since they were found to be readily internalised by model animal cells.

Methods

Synthesis and characterisation of nanoparticle building blocks

Maghemite mNPs were synthesized following the Massart procedure and surface-coated with sodium polyacrylate according to previously reported protocols [START_REF] Massart | Preparation and Properties of Monodisperse Magnetic Fluids[END_REF][START_REF] Qi | Stability and Adsorption Properties of Electrostatic Complexes:â Design of Hybrid Nanostructures for Coating Applications[END_REF]. PAA-coated ZAIS QDs were synthesized in one step following the method reported by Regulacio et al. with minor modifications [START_REF] Regulacio | Aqueous synthesis of highly luminescent AgInS2â ZnS quantum dots and their biological applications[END_REF]. Core material concentrations were determined by ICP-OES for QDs and X-ray fluorecence for mNPs. Concentrations of surface carboxylates were determined by pH-titration with HCl. The quantum yield of QDs was determined by comparison to those of fluorescein and rhodamine 6G [START_REF] Albert | Standards for photoluminescence quantum yield measurements in solution (IUPAC Technical Report)[END_REF][START_REF] Fischer | Fluorescence quantum yield of rhodamine 6g in ethanol as a function of concentration using thermal lens spectrometry[END_REF][START_REF] Porrès | Absolute Measurements of Photoluminescence Quantum Yields of Solutions Using an Integrating Sphere[END_REF]. Gel electrophoresis was performed on 1%wt agarose gel in TBE 1x bu er. A full description of the synthetic and characterisation procedures is provided in Supporting Information.

Assembly of fluorescent magnetic microneedles

The following gives the synthetic procedure for assembly of {Z = 4.20; w = 0.20}-µNDs as an example. First, solutions of building blocks in NH 4 Cl 1M pH 8 were prepared by mixing in a 1:1 ratio a NH 4 Cl 2M pH8 solution with the mNP (0.2% wt core, 2.02 mM carboxylate), the QD (0.4% wt core, 14.73 mM carboxylate) and polydiallyldimethylammonium chloride (0.2% wt, 13.4 mM ammonium) stock solutions respectively. Next, 400 µl, 100 µl and 40.5 µl of mNP, QD and polymer dilute solutions were mixed in an eppendorf tube and shaken for 10 s with the help of a vortex mixer. The ternary mixture was inserted in a 0.5 -3 ml Slide-a-Lyzer dialysis cassette with a 10 kDa cuto using a syringe. Air bubbles were removed. The cassette was nested into a custom made plexiglas sample holder sandwiched between two 5 ◊ 8 cm 2 0.3 T magnets. The sample holder was then immersed in a 2.5 L cylindrical flask filled with deionised water equipped with a stir bar and continuous monitoring of the conductivity was launched immediately. Dialysis was run for 2 hours after which the conductivity of the dialysis medium had reached a stable value. The content of the dialysis cassette had turned turbid, evidencing success of the assembly. The reaction media was carefully taken out using a syringe and inserted into a 2 ml glass vial. µNDs were sedimented using a soft table magnet and the supernatant was discarded. The µNDs were redispersed in 1 ml MilliQ water through gentle vortex mixing, sedimented again and the supernatant discarded. Two more washing steps using 2 ◊ 1 mL MilliQ water were performed. Finally, the µNDs were redispersed in 1 mL MilliQ water and stored in the dark at 4 ¶ C. The 14 other investigated compositions were prepared using the same protocol, with varying mNP, QD and polymer amounts, as given in Supporting Information.

Imaging of µNDs using phase contrast and epifluorescence microscopy

A square well was built on a glass slide using double-sided tape. The NW suspension was homogenized and diluted 4 times with MilliQ water. 8 µl of this diluted suspension were deposited in the well, which was then covered with a coverslip. The sample was imaged in phase contrast mode and in epifluorescence mode using excitation and emission filters suited for Rhodamine 6G, with 10X, 20X and 40X magnifications. Length and brightness distributions were extracted using custom-written Matlab codes described in Supporting Information.

Imaging of fluorescent magnetic µNDs in interaction with P32

HeLa cells using confocal fluorescence microscopy

Preparation of NW sample. For incubation of cells, we selected {Z=3.62, w=0.15}-µNDs. This sample had a mean length amongst the shortest in all investigated compositions (15 µm). However, it was shown previously on a similar sized sample of magnetic-only µNDs that cells operate a size sorting of µNDs upon internalization, with an enrichment in µNDs having a length around 4 µm [START_REF] Safi | Interactions between Magnetic Nanowires and Living Cells: Uptake, Toxicity, and Degradation[END_REF]. We therefore shortened the µNDs by ultrasonication before cell incubation. To this end, 400 µl were inserted in a 1 mm optical path quartz cuvette and sonicated for 2 X 1 min in an ultrasonic bath in horizontal position, with intermediate cooling for 5 min. The µNDs were sedimented in the cuvette using a table magnet and the supernatant was discarded. Next, they were washed 3 times with 500 µl MilliQ water through sedimentation and discarding of the supernatant. Finally, they were dispersed in 500 µl MilliQ water before autoclaving.

Cell culture. P32 HeLa cells were grown in T25-flasks as a monolayer in DMEM with high glucose (4.5 g/L) and stable glutamine (2 mM, PAA Laboratories GmbH, Austria). This medium was supplemented with 10%vt fetal bovine serum (FBS) and 1% penicillin/streptomycin (PAA Laboratories GmbH, Austria), referred to as cell culture medium.

Exponentially growing cultures were maintained in a humidified atmosphere of 5% CO 2 and 95% air at 37 ¶ C. In these conditions the plating e ciency was 70-90% and the cell duplication time was 12-14 h. Cell cultures were passaged twice weekly using trypsin-EDTA to detach the cells from their culture flasks and wells. For counting, the cells were pelleted by centrifugation at 1200 rpm for 5 min, supernatants were removed and cell pellets were resuspended in assay medium in Malassez counting chambers.

Seeding of P32 HeLa cells onto microscope glas coverslips. Sterilized microscope coverslips were nested in the wells of a 6-well plate, washed with 1 mL of PBS bu er and then with 1 mL of culture medium. Cells were detached from their culture flask and transferred to the wells; the cell concentration was 75 000 cells/ml and each well contained 2 mL of cell suspension. The cells were incubated for 24 hours to promote cell adhesion onto the coverslips.

of shortened µNDs were mixed with 1 mL of culture medium and the resulting suspension was added dropwise in each well. The cells were incubated with the µNDs for 20 hours after which they were washed by 2 rounds of medium exchange against PBS bu er.

Cell fixation. Next, the cells were fixed through incubation in 4% PFA solution (J61899, Alfa Aesar) in PBS for 20 min at room temperature. After copious rinsing of PFA using PBS, the nuclei were fluorescently stained with DAPI dilactate dye (Ø 98%, Aldrich) by incubation for 20 min at room temperature in a 2.2 µM dye solution in PBS. The medium was exchanged for PBS three times to remove excess dye. Finally, microscope slides were equipped with Gene Frame stickers. The coverslips were carefully taken out of the wells and mounted onto the Gene Frames after filling the wells with PBS bu er. Samples were examined with an inverted wide-field microscope (Olympus IX81) equipped with spinning disc module (Yokogawa CSU-X1), with an oil immersion objective (60X, NA 1.42) and an EMCCD camera (Andor iXon 897). Z-stacks of wide-field fluorescent images were acquired using a piezo at 0.5 µm increments. For the DAPI channel, 405 nm laser-line excitation and a 465 nm-centered emission filter were used. For the NW chanel, the excitation line was 488 nm and the emission filter was centered at 607 nm.

Figure 1 :

 1 Figure 1: Synthetic scheme for assembly of bifunctional fluorescent magnetic µNDs

Figure 2 :

 2 Figure 2: Absorption spectra of mNPs and QDs (plain) and emission spectrum of QDs under 490 nm irradiation (dots).

Figure 3 :

 3 Figure 3: Phase contrast and epifluorescence microscopy images of {Z = 3.62; w = 0.15} µNDs, without (a,b) and under (c,d) static magnetic field.

2. 3

 3 Figure 3a depicts a phase 20 X contrast image of the aggregates obtained starting from the {Z = 3.62; w = 0.15} composition. Straight µND are obtained with lengths ranging from about 5 to 50 µm and diameters of about 0.3-1.0 µm (see ESI for TEM characterisation of width). Their morphology is identical to the magnetic-only µND reported previously.

Figure 4 :

 4 Figure 4: Phase contrast (left), epifluorescence (middle) microscopy images and associated length distributions (right) of bifunctional µNDs with various compositions.

Figure 5 :

 5 Figure 5: Relative fluorescence brightness (left) and mean length (right) as a function of ternary molar composition.

Figure 6 :

 6 Figure 6: Geometric mean length of µNDs as a function of Z (blue dots) and best fit line according to Eq. (5) (black line).

Figure 7

 7 displays bright field (first row) and fluorescence (middle row) confocal images acquired in two focal plans lying 6 µm apart. The blue and red fluorescence channels correspond to DAPI and QD emissions, respectively. In the left panel, the nuclei are out of focus while they come into focus in the right panel. On bright field images, a few µND can be spotted, mostly in the right panel, but their localisation with regards to the cellular compartments is di cult to assess. On fluorescence images (middle row), µNDs can be more easily outlined as red dashes. On cell C1 in left panel, a few µNDs appear on top of the out-o-focus nucleus. These µNDs disappear when the C1-nucleus comes into focus in the right panel, while many more µNDs with sharp contours appear around it. Reconstruction of the Z-profile of cell C1(lower row) conveniently shows that µNDs are internalised within

Figure 7 :

 7 Figure 7: Bright field (upper row) and fluorescent (middle row) confocal images of HeLa cells incubated with bifunctional fluorescent magnetic µNDs (60X objective). Reconstructed Zprofiles of cells C1 and C2 in the planes perpendicular to the highlighted regions of interest (lower row).


G Ramniceanu

B.-T Doan

C Vezignol

A Graillot

C Loubat

Nathalie Mignet

Jean-Francois Berret
email: jean-francois.berret@univ-paris-diderot.fr

J.-F Berret

Delayed hepatic uptake of multi-phosphonic acid poly(ethylene glycol) coated iron oxide measured by real-time Magnetic Resonance Imaging

Keywords:

published or not. The documents may come

Delayed hepatic uptake of multi-phosphonic acid poly(ethylene glycol) coated iron oxide measured by real-time magnetic resonance imaging

iron oxide, magnetic particles provide several advantages among which a substantial biodegradability over time and an absence of cytotoxicity. In addition, depending on their size the particles can be used as positive or negative contrast agents, leading to an enhancement of longitudinal and transverse proton relaxation rates. [4][5][6] For applications, the pharmacokinetic profile of the MRI probes has to be known. Absorption, in vivo biodistribution, metabolic transformation and elimination from the tissues and organs are the primary mechanisms involved in pharmacokinetics. In the blood pool, nanoparticles administered intravenously are recognized from plasma proteins, which adsorb at the particle surface spontaneously. [7][8][9][10][11][12] The protein binding process, known as the protein corona formation prevents the particles to interact specifically with potential targets. Simultaneously, protein adsorption activates the particle uptake by the mononuclear phagocytic system through the circulating macrophages and monocytes. The two-step opsonization mechanism described here is responsible for the particle elimination from the blood stream and for their accumulation in unrelated organs, typically the liver and the spleen. 13 As a result, commercial contrast agents using iron oxide cores (e.g. Endorem® from Guerbet, Resovist® and Cliavist® from Bayer) were first designed for imaging hepatic lesions and tumors. 1 Ever since, iron oxide particles have been surveyed in the context of other clinical applications, including heart transplantation, brain lesions imaging and tumor targeting. 2,6,[14][15][16] To prevent opsonization, studies have shown that neutral hydrosoluble polymers such as poly(ethylene glycol) (PEG) or polysaccharides are efficient. 13,[17][18][19][20] Tethered at the chain extremity or from several monomeric units, the polymers in aqueous environment form a swollen brush that acts as a protective layer against protein adsorption and particle aggregation. In this context, efforts were mainly directed towards the molecular weight effect on the in vitro stability and in vivo stealthiness. 15,[21][22][23][24][25][26][27] From in vivo pharmacokinetics studies, Leal et al. have found an optimum PEG value of 3 kDa, 25 whereas particle accumulation in tumor was found to be efficient with 10 kDa polymers. 15 Recently, Ruiz et al. reported a doubling of the residence time in blood for PEG conjugated iron oxide particles, and a reduction in the liver and spleen uptake. 21,22 In some of the previous studies, the PEGylated contrast agents tested were prepared from 10 nm particles; however it was found that during synthesis or functionalization the particles aggregated and formed large clusters containing tens to hundreds of particles. 15,21,22,26,28 For aggregates, additional parameters such as size and morphology were found to affect the MRI contrast properties and the biodistribution in vivo. These properties were also different from those of single nanoparticles.

Another crucial issue in particle functionalization is the nature and strength of the link between the coat and the inorganic surface. A wide variety of techniques based on covalent or non-covalent binding are now achievable, including ligand adsorption, layer-by-layer deposition or surface-initiated polymerization. 29 Non-covalent strategies based on the assembly of separately synthesized components are known to exhibit enhanced yields in terms of quantity of particles produced. 30 Novel macromolecular architectures obtained by radical polymerization were also considered. 31 Na et al. designed multi-dentate catechol and PEG derivatized oligomers that provide colloidal stability over a broad range of pH and electrolyte concentrations. 32 Single or multiple phosphonic acid based polymers were also synthesized. 31,[33][34][35] Phosphonate groups are known to have a higher affinity towards metallic atoms or metal oxides compared to sulfonates and carboxylates, and are hence able to build stronger links with surfaces. Sandiford et al. exploited PEG polymer conjugates containing a terminal bisphosphonic acid group for binding to magnetic nanoparticles. 34 We have recently shown that multi-phosphonic acid PEG-copolymers with up to 4 anchoring groups per chain conferred considerable stabilization to 10 nm iron oxide cores. The coated particles were found to be stable for months in extreme salt and pH conditions, as well as in cell culture media. In a previous work, multi-phosphonic acid PEG coated particles were tested against fibroblast and macrophage cultures and exhibited exceptional low cellular uptake. 12 Here, these newly coated particles are assessed in vivo using a live mouse model. MRI is particularly suited for this study as it provides spatial and temporal resolved images with high contrast. The present survey reveals that the phosphonic acid PEG copolymers with multiple anchoring groups are able to prolong the particle lifetime in the blood up to 3 hours, i.e. 50 times greater than that of anionic particles or Cliavist® benchmark, and 10 times greater than that of recently published PEGylated probes. [21][22][23] A qualitative model taking into account the uptake and clearance is proposed and is found to be appropriate for all contrast agents studied. By comparing identical core particles with different coating, we are able to determine the optimum conditions for stealth MRI probes.

-Materials & Methods

-Iron oxide nanoparticles and Coating

-Iron oxide nanoparticles

Iron oxide nanoparticles were synthesized by alkaline co-precipitation of iron(II) and iron(III) salts. 36,37 The salts were dissolved in a hydrochloric solution and co-precipitated by a concentrated base (ammonia, NH 3 ). The particles were then concentrated magnetically and the supernatant was removed. The concentrated phase was washed with water using magnetic sedimentation followed again by supernatant removal. Nitric acid (HNO 3 ) was then added to the particles down to pH 1.5. Addition of a large excess of ferric nitrate at water boiling temperature led to the oxidation of magnetite into stable maghemite nanocrystals. The nanoparticles were sorted according to their size by successive phase separation steps. 38 At pH 1.5, the particles are positively charged and have nitrate counterions adsorbed on their surfaces. The resulting interparticle interactions are repulsive and impart an excellent stability to the dispersion. 37,39 Here, two -Fe 2 O 3 nanoparticles batches (diameter 6.8 nm and 13.2 nm) were synthesized (Figs. 1a-d). The magnetic and geometric size distributions were obtained from vibrating sample magnetometry (VSM) and from transmission electron microscopy (TEM), respectively. Table I provides a list of the diameters and dispersities (ratio between standard deviation and average diameter) obtained from these two techniques. The nanoparticle magnetization was also measured using VSM and its value was found slightly lower than that of bulk maghemite 41,42 (2.910 5 A m -1 instead of 3.910 5 A m -1 ). The particle crystallinity was studied by electron microdiffraction scattering. Five diffraction rings characteristic of the maghemite structure were observed (S1-S4). In the dispersed state, light scattering techniques allowed the measurements of the weight-averaged molecular weight and of the hydrodynamic diameter for the particles. 40 Throughout the paper, the concentrations are expressed in terms of the iron molar concentration, noted [Fe]. For instance, the [Fe] = 5 mM dispersion used for the intravenous injection corresponds then to a weight concentration of 400 µg mL -1 . Assuming a blood pool of 1 mL for wild-type BALB/c mice (age 8 weeks), the concentration of circulating particles is estimated at 40 µg mL -1 . The contrast agent used as a benchmark, Cliavist® was commercialized by the company Bayer (Germany). According to supplier specifications, the Cliavist® dispersion has an iron concentration of 500 mM and contains 4 nm magnetic core particles aggregated into 55 to 65 nm clusters. 1 The 1400 Da carboxydextran molecules used in the formulation serve both as a linker for the maghemite nanocrystals, the polymer role being indeed to prevent the dispersion sedimentation. showing the chemical formula, charge and molecular weight of the organic coatings used in this study. For the phosphonic acid PEG copolymer, only the PEG molecular weight is indicated.

-Coating

In this section, we describe the macromolecules and protocols used to coat the iron oxide particles (Fig. 1e). Poly(ethylene glycol methacrylate-co-dimethyl(methacryoyloxy) methyl phosphonic acid), abbreviated in this work phosphonic acid PEG copolymer was synthesized by Specific Polymers ® , France (http://www.specificpolymers.fr/). The synthesis was carried out by free radical polymerization of PEGmethacrylate (PEGMA, SP-43-3-002, CAS: 26915-72-0) and dimethyl(methacryoyloxy)methyl phosphonate (MAPC1, SP-41-003, CAS: 86242-61-7) monomers. Synthesis details can be found in previous reports. 12,43 The phosphonic acid PEG copolymers were characterized from 1 H NMR and 31 P NMR using a Bruker Avance 300 spectrometer operating at 300 MHz. For the copolymers, a molar-mass dispersity of 1.8 was obtained by size exclusion chromatography on PolyPore column using THF as eluent and polystyrene standards. From the molar equivalent of acid groups obtained from NMR and molecular weight determination, the number of phosphonic acids and PEG segments was estimated. This number was found to be 3.1 ± 0.2 and 3.7 ± 0.2 for copolymers with PEG 2K and PEG 5K , respectively (Table II). Here and below, the error bars are defined as the standard deviations (SD). These determinations confirm the existence of multiple functional groups on the same polymer backbone. H NMR. The numbers of the functional groups are determined from the molecular weight and from the molar equivalent of acid groups. 12 The error bars are here defined as the standard deviations (SD).

For the coating, we used a protocol derived in 2008. 44,45 Dispersions of oppositely charged iron oxides and phosphonic acid PEG copolymers at the same concentration ( = 0.1 wt. %) and same ( ) were mixed at increasing volume ratios between 10 -3 and 10 3 . The of the mixed solution was then raised to 8 by ammonium hydroxide addition, leading to well-dispersed coated particles. The mixed polymer/particle dispersion stability diagram was investigated by dynamic light scattering (Fig. S5). By plotting the hydrodynamic diameter against the mixing ratio , a transition between individual coated particles and micron-sized aggregates occurs at the critical value, . The existence of a critical mixing ratio suggests that adsorption occurred via a non-stoichiometric electrostatic binding process. 44,45 The model assumes that for each value of , the polymers are equally distributed among the particles in the dispersion. 45 Below , the iron oxide surfaces are saturated with polymers, the functional end-groups exceeding the number of binding sites. Above, the coverage is incomplete and the particles precipitate upon increase (as uncoated particles do). Here, we exploit this feature to derive the number of adsorbed chains per particle : 12 where and are the particle and polymer molecular weights, respectively. For PEG 2K on 6.8 nm and 13.2 nm particles, = 1.3 and 5, leading to a number of adsorbed polymers = 97 and 207, respectively. For PEG 5K , = 0.5 and 2, resulting in = 43 and 88 (Table S5). These -values correspond to a PEG density of 1 -2 nm -2 . With zeta potentials of -6 mV, electrokinetic measurements confirmed that the multi-phosphonic acid PEG coated particles were globally neutral (S4). Before use, the dispersions were finally dialyzed against deionized water using a 50 kD cut-off membrane to remove the excess polymer, and further concentrated by ultrafiltration. They were then autoclaved (Tuttnauer Steam Sterilizer 2340M) at 120 °C and atmospheric pressure during 20 min to prevent bacterial contamination, and stored at 4 °C in a secure environment.

Citric acid is a weak triacid of molecular weight = 192 Da, with acidity constants pK A1 = 3.1, pK A2 = 4.8 and pK A3 = 6.4. Surface charge complexation with citric acid was performed during the synthesis through simple mixing. At pH 8, citric acid molecules are ionized, and particles are coated with citrate ions. As ligands, citrates are characterized by adsorption isotherms and adsorbed species are in equilibrium with free citrates in the bulk. The free citrate concentration was kept at the value of 8 mM, 39 both in DI-water and in culture medium. The hydrodynamic citrate coated particle diameter was identical to that of bare particles, indicating a layer thickness under 1 nm.

Poly(sodium acrylate), the salt form of poly(acrylic acid) with a weight-average molecular weight = 2.1 kDa and a dispersity of 1.7 was purchased from Sigma Aldrich and used without purification. To adsorb polyelectrolytes on the particles, the precipitation-redispersion protocol was applied. 41 The precipitation of the iron oxide dispersion by PAA 2K was performed in acidic conditions (

). The precipitate was then separated by magnetic sedimentation and its pH was increased by ammonium hydroxide addition. The precipitate redispersed spontaneously at pH8. PAA 2K coated γ-Fe 2 O 3 hydrodynamic sizes were around 4 -6 nm larger than the hydrodynamic diameter of the uncoated particles, indicating a corona thickness of 2 -3 nm. Electrophoretic mobility and zeta-potential values are provided in the Supporting Information section (S4). As for the multi-phosphonic acid PEG coated particles, the dispersions were dialyzed and autoclaved to prevent bacterial contamination.

-Methods

-Dynamic light scattering (DLS)

Light scattering experiments were performed on a NanoZS spectrometer (Malvern) at the wavelength of 633 nm and in backscattering configuration (scattering angle 173°). From the scattered intensity evolution, the second-order autocorrelation function of the light is calculated and analyzed using different procedures provided by the instrument software. These procedures are the cumulant method and the CONTIN algorithm. Both gave consistent values for the hydrodynamic diameter . As mentioned previously, the nanoparticle hydrodynamic diameters are found to be systematically larger than those obtained by other techniques, in particular by electron microscopy and magnetometry (Table I). The reason for this difference is related to the particle size distribution, along with the fact that light scattering is sensitive to the largest objects of the distribution.

-Cell Culture

Adherent cells from mouse hepatocyte cells BWTG3 were studied. Hepatocyte cells BWTG3 were grown in T25-flasks as a monolayer in Dulbecco's Modified Eagle's Medium (DMEM) with high glucose (4.5 g L - 1) and stable glutamine (PAA Laboratories GmbH, Austria). The medium was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PAA Laboratories GmbH, Austria). Exponentially growing cultures were maintained in a humidified atmosphere of 5% CO 2 and 95% air at 37°C, and in these conditions the plating efficiency was 70 -90% and the cell duplication time was 12 -14 h. Cell cultures were passaged twice weekly using trypsin-EDTA (PAA Laboratories GmbH, Austria) to detach the cells (6 millions in average for T25 flasks) from their culture flasks and wells. The cells were pelleted by centrifugation at 1200 rpm for 5 min. Supernatants were removed and cell pellets were resuspended in assay medium and counted using a Malassez counting chamber.

-Toxicity assay

The method measured the cellular mitochondrial activity. Subconfluent cell cultures (60% confluency at treatment time) on 96 well plates were treated with 100 µL/well of nanoparticles at different concentrations for 24 h, culture medium was removed, cells were rinsed with culture media without phenol red and incubated with 100 µL/well of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4disulphophenyl)-2H-tetrazolium (WST-1, Roche Diagnostics), diluted 1/10 in culture medium without phenol red for 1 to 4 h. The assay is based upon the reduction of the tetrazolium salt WST-1 to formazan by cellular dehydrogenase. The generation of the dark yellow colored formazan was measured at 450 nm in a multiwell-plate reader against a blank containing culture media and WST-1 and it was corrected from the absorbance at 630 nm. The supernatant optical density is directly correlated to cell number.

-Relaxometry

Images were acquired with a 7 Tesla spectrometer equipped with a 1 H radiofrequency linear coil of inner diameter 40 mm (Bruker, Karlsruhe Germany). and relaxation times were measured on 200 µL solutions with increasing iron concentrations, [Fe] = 0.02, 0.05, 0.1, 0.2 and 0.5 mM to determine the longitudinal and transverse relaxivities and . For -measurement, a saturation sequence with 8 different repetition times from 60 ms to 15 s was used to evaluate the proton spin-lattice recovery. The field of view was of a 3 3 0.15 cm 3 with a pixel resolution of 0.234 0.234 1.5 mm 3 in all protocols. For -measurement, a sequence with 32 sequential echo times from 11 ms to 385 ms was applied to follow the decay of the proton spin-spin relaxation. The total scan time was 30 min. The relaxivities rates were obtained by adjusting the signal intensity of each agent at different concentrations using: 46 [Fe] . These measurements were made in physiological saline solution and in DMEM with serum.

-In vivo MRI (including data analysis)

All animal work was performed in accordance with the institutional animal protocol guidelines in place at the University Paris-Descartes (Saisine CEEA34.JS.142.1) and approved by the Institute's Animal Research Committee. Wild-type female 8 weeks BALB/c mice were anaesthetized by isoflurane inhalation (1.5% isoflurane in a 1:1 mixture of air and oxygen at the rate of 0.25 L min -1 ) and placed in a dedicated contention cradle in a 7 Tesla MR-scanner. 100 µL of iron oxide nanoparticles in saline 0.9 wt. % with an optimized concentration of [Fe] = 5 mM were intravenously injected via the tail vein thanks to a catheter specifically elaborated for non-magnetic use. The injected iron concentration was adjusted so that the hepatic uptake and clearance processes take a maximum of one week. For reproducibility studies, 2 to 4 mice were injected with the contrast agents tested. 13.2 nm citrate and PEG 5K coated particles were injected on one mouse only. For imaging purposes, two types of time resolution were used sequentially: a high temporal resolution with low spatial resolution for the first 180 minutes, and a lower time resolution with high spatial resolution for time range between 20 min and 7 days. With this protocol, it was ensured that the early stage of the hepatic uptake could be evaluated accurately. The low spatial resolution scans were compared and normalized to the higher resolution images using a zero filling technique for signal optimization. All images were recorded with a field of view of 3 3 cm 2 , corresponding to an in-plane resolution of 125 125 µm 2 in both sequences. The scanning protocol was developed using Paravision 5.1 software. To minimize motion artifacts, two methods of respiratory synchronization were used. The high spatial, low temporal resolution sequence synchronized the acquisition with the mouse breathing cycles. The low spatial, high temporal resolution sequence used an internal navigator that detects the mouse respiratory and cardiac cycles and reconstructs the images in a post-processing operation. This second method is independent of the breathing cycle changes. The time frame was set to one minute for the fast scans and three minutes for the slower scan. To recapitulate the chronological imaging experiments, images were recorded before injection and during the first 20 min at a rate of one image per minute, then at 30 min, 40 min, 1 h, 1h40, 2h40, 4h, 7h20 and at 1, 2, 3, 4 5, and 7 days after injection. 3 mice per days were investigated with the addition of 3 to 9 mice in the follow-up process between one day and one week. To study the probe biodistribution, several organs including liver, spleen, kidney and arteries (Supporting Information S6) were monitored. Comparison with commercial Cliavist® was also performed.

-Results and Discussion

-Iron oxide based contrast agents and coating

Iron oxide nanoparticles of diameters 6.8 nm and 13.2 nm were synthesized by alkaline co-precipitation of iron salts and subsequent oxidation into maghemite (-Fe 2 O 3 ) and coated with different macromolecules. 36,37 As coating agent, we used a statistical copolymer that combines multiple phosphonic acid groups and poly(ethylene glycol) chains on the same backbone. The full name of the polymer is poly(ethylene glycol methacrylate-co-dimethyl(methacryoyloxy) methyl phosphonic acid), abbreviated phosphonic acid PEG copolymer in the sequel of the paper. Synthesized by free radical polymerization (Specific Polymers ® , France), the polymers were designed to strongly adsorb onto metal and metal oxide surfaces via the multiphosphonic acid groups. 12 Copolymers with 2 kDa and 5 kDa PEG chains were synthesized and studied.

The aforementioned PEGylated copolymers were compared to other coating types, including citric acid and poly(acrylic acid) polymer (PAA). Citric acid is a well-known and widely used coating for inorganic particles. In the molecular imaging context, citrate coated particles were tested in a clinical phase I trial to evaluate their pharmacokinetics and safety. 47 Poly(acrylic acid) in contrast was less studied and to our knowledge the biodistribution of PAA coated contrast agents was not reported yet. In vitro, it was shown that PAA coat is biocompatible and non-toxic, and that the charged polymer layer brings significant protection against protein adsorption and particle agglomeration. 9,48-50 A recent in vivo study has revealed however acute effects on cardiovascular function. 49 The chemical formulae, charge and molecular weights of the coating agents probed are summarized in Fig. 1b. More details on the coating and characterization methods are given in the M&M section.

Important parameters for the particles are the total hydrodynamic diameter and the coating thickness , which were determined by light scattering (Table III). The polymer layers were found to have the same thickness for the 6.8 and for the 13.2 nm particles, and to agree with the brush structure. 51,52 In good solvent conditions, PEG 2K is a chain of gyration radius 1.3 nm, 53 whereas its fully stretched length is about 10 nm. -values of 5 ± 1 nm (Table III) are intermediate and indicate that the chains at the surface are slightly stretched, typically by 40 -50 %. The PEG 5K layer on the 6.8 nm iron oxide has a thickness of 16 ± 2 nm, which corresponds to a higher stretched configuration. 52 PAA layers have a thickness of 2 -3 nm, as mentioned in earlier work. the coating thickness as determined from dynamic light scattering measurements. Data for Cliavist® are from ref. 1 The error bars denote here the standard deviations.

-Stability in physiological and culture media

As for the evaluation of protein corona and particle agglomeration, the following protocol was applied. 9,44 A few microliters of a concentrated dispersion are poured and homogenized rapidly in 1 mL of the solvent to be studied, and simultaneously the scattered intensity and diameter are measured by light scattering. After mixing, the measurements are monitored over a 2-hour period, and subsequent measurements are made after 24 hours and 1 week. Nanoparticles are considered to be stable if their hydrodynamic diameter in a given solvent remains constant as a function of the time and equal to its initial value (given in Table III). Solvents surveyed here are DI-water (pH 7.4), phosphate buffer saline (PBS) and cell culture medium (Dulbecco's Modified Eagle's Medium, DMEM) with or without calf bovine serum.

Stability assays were performed on bare and coated iron oxide nanoparticles (Fig. 2). The commercial contrast agent Cliavist® was used as a benchmark. Bare particles with diameters 6.8 nm and 13.2 nm are stable at pH 1.8 (the pH at which they are synthesized) and stabilized by surface charges. Under rapid dilution, the pH variation induces a modification of the electrostatic charges at their surfaces. At physiological pH, the dispersions are close to their isoelectric point 54 (IP 6.7) and the particles are slightly positive. Bare particles hence aggregate due to van der Waals interactions. Citrate and carboxydextran (Cliavist®) coated particles are stable in PBS buffer. In culture media however, citrate coated particle precipitation occurs (Fig. 2) and it is attributed to the displacement of the ligands from the surfaces, as they are preferentially complexed by Ca 2+ and Mg 2+ divalent counterions. 9 For Cliavist®, the destabilization also takes place and come from the loosely tethered carboxydextran chains at the iron oxide surface. The particles coated with polymers, either poly(acrylic acid) or phosphonic acid PEG copolymers exhibit in contrast an outstanding stability over time. The dispersions are found to remain stable for months. For particles with polymer coats, protein corona was also not detected. 12,55 The stability results obtained in DMEM after one week are summarized in Fig. 2.

Figure 2: a) Hydrodynamic diameter versus time for contrast agents after they have been diluted in Dulbecco's Modified Eagle's Medium (DMEM). For uncoated particles, and for particles coated with citrates, aggregation and

precipitation occur rapidly, as indicated by the initial size increase. For cliavist®, the aggregation is slower, but is still visible on the time scale of a week. Particles coated with poly(acrylic acid) and phosphonic acid PEG copolymers were found to remain stable for months. b-e) Images of some selected iron oxide dispersions in DMEM after one week. Stability assays were performed at experiments at the weight concentration of 0.1 wt. %.

-Relaxometry

The longitudinal and transverse relaxivities and of the coated particles dispersed in DI-water and in cell culture medium were measured using -and -weighted MR images on a 7.0 T on a 300 MHz micro-imaging Bruker spectrometer. Dispersion weighted phantom images at molar concentration [Fe] = 0, 0.02, 0.05, 0.1, 0.2 and 0.5 mM were acquired, and their greyscale intensity was found to decrease with concentration, a result that is explained by the dipolar coupling between the magnetic moments and the water protons. In this range, the inverse of the relaxation times was found to vary linearly with [Fe], according to:

[Fe] where denotes the water longitudinal and transverse relaxation time. 46,56 Fig. 3a and 3b display the transverse relaxivity histograms for the two particle sizes and for the various coatings. Comparison with Cliavist® is also shown (Fig. 3c). The relaxivities and relaxivity ratios are listed in the Supporting Information Section (S7).

Data in Fig. 3 show that the particle core size has a strong impact on the contrast, as passes from 62 mM -1 s -1 to 171 mM -1 s -1 for uncoated particles, and from 74 mM -1 s -1 to 226 mM -1 s -1 for the PEGylated ones. This increase is in agreement with that reported in Vuong et al. 42 who suggested that the iron based contrast agent transverse relaxivity grows quadratically with particle size. As for the coating, the data also show that the presence of a layer around particles leaves rather unchanged and increases by 20% in average. With respect to the solvent, the effect is the strongest with uncoated particles. From DI-water to cell culture medium, the transverse relaxivity of 6.8 nm -Fe 2 O 3 is multiplied by a factor of 4 (from 62 mM -1 s -1 to 284 mM -1 s -1 ), whereas it is increased by a factor of 1.5 for the 13.2 nm particles (from 171 to 272 mM -1 s -1 ). This increase is consistent with particles being incorporated into large micron-sized aggregates, as shown in Fig. 2. 42,57 In conclusion, we have found that for maghemite the coating has a relatively weak impact on and . These findings are important for the later in vivo MR imaging results, as they suggest that the role of the coating can be determined independently of other parameters.

-Biocompatibility and toxicity

Murine hepatocyte cells BWTG3 viability was studied using the colorimetric assay WST-1. WST-1 was performed at iron dose levels between 10 -3 mM to 10 mM, to cover a broad concentration range and specifically the concentration used in vivo ([Fe] = 0.5 mM in the blood pool). Fig. S8 a-f display the percentage of viable BWTG3 cells treated during 24 h with Cliavist® and with the 6.8 nm and 13.2 nm iron oxides. For the 11 particles studied, the viability exhibits a slight to moderate decrease around [Fe] = 0.5 -5 mM, the effect being the strongest for bare and citrate coated particles. This latter result could be related to the particle agglomeration and sedimentation, which increase the amount of materials adsorbed at the plasma membrane. 55 Observations of cells in situ using optical microscopy indicate that the cellular morphology was modified at high dose levels. Except for bare particles, the BWTG3 hepatoma cells exhibit a viability above 50% for all coating. These findings corroborate results obtained with other cell lines, including NIH/3T3 mouse fibroblasts, 50 2139 Human lymphoblasts 9 and RAW264.7 macrophages. 12 Viability experiments with the coating agents alone, including phosphonic acid PEG copolymers, citrate and PAA polymers, were also done and reveal no cytotocity at relevant concentrations. 9,12,50

--weighted MR Imaging of the liver: particle size and coating effects

The uptake of intravenously injected contrast agents was monitored by measuring the signal enhancement on T 2 weighted images of different organs. The iron oxide dispersion injected to the mouse was 100 µL and the iron concentration 5 mM (corresponding to 16.7 µmol kg -1 or 0.93 mg kg -1 ). Previous reports have shown that iron oxide based contrast agents exhibit uptake times of the order of minutes and clearance times in the range of days. [21][22][23][58][59][60] A logarithmic temporal scale was hence used for imaging the mouse organs at times ranging from one minute to one week. Fig. 4 compares MR scans obtained in the first three hours for Cliavist® (Fig. 4a to d) and for the 6.8 nm PEG 2K particles (Fig. 4e to h). With the commercial agent, the mouse liver section exhibits a negative contrast enhancement shortly after injection (e.g. in Fig. 4b at 30 min). The darkening in the liver arises from the iron accumulation in the organ. 1,21,22,58,[60][61][62][63] Multi-phosphonic acid PEG coated particles display a different behavior (Fig. 4e-g). For this sample the darkening is delayed by about 2 -3 hours as compared to that of Cliavist®. During the period, the MR images show no contrast change of the liver and of other organs (spleen, kidneys), indicating a prolonged circulation in the bloodstream, as well as a delayed hepatic uptake (S6). At 3 hours post-injection (Fig. 4d and4h), MR images exhibit a negative contrast enhancement as with cliavist®. A movie comparing the liver MRI intensity after injection of PAA 2K and multi-phophonic acid PEG 2K coated particles has been added as Supporting Information. To quantify the MR contrast, the liver mapping was performed by defining manually regions of interest (ROI), and by integrating the grey scale intensity as an 8-bit integer (between 0 and 255). Spatially average intensities were obtained and normalized with respect to the pre-injection level. With these conventions, the MRI signal intensity varies between 1 and 0, and the liver darkening is associated with a decrease as a function of the time (Fig. 5). The procedure was carried out at each time point for 8 particles: cliavist® ( = 3), 6.8 nm particles coated with PAA 2K ( = 3), PEG 2K ( = 4) and PEG 5K ( = 2), and with 13.2 nm coated with citrate ( = 1), PAA 2K ( = 2), PEG 2K ( = 2) and PEG 5K ( = 1). Here, designates the number of mice tested. Intravenous injection into the tail vein was performed as a single bolus in the conditions of Fig. 4, i.e. 100 µL of dispersion at 5 mM. Fig. 5 illustrates the correspondence between the liver MR images and the greyscale intensity. In the pre-injection period, the intensity remains at the level of 1. The MR signal then decreases rapidly after injection and passes through a minimum.

hepatic uptake is progressive and slightly more rapid for PEG 5K than for PEG 2K . The difference may come from the hydrodynamic diameter that is about twice larger with the longer polymer, or from a lower PEG density (S5). For the 13.2 nm particles, the time dependent contrast enhancements remain similar in terms of hyposignal kinetics and amplitude. Combined together, these results show that the optimum stealthiness in vivo is obtained for the 6.8 nm particles and a 2 kDa PEG coating.

-Quantitative analysis of iron uptake and release by the liver

To retrieve the contrast agent pharmacokinetics parameters, the MRI signal intensity was adjusted using an expression of the form: (2) where is the intensity minimum, and the characteristic times for uptake and clearance. In the above model, it is assumed that the two mechanisms are occurring one after the other. In a first attempt to adjust the MRI intensities, single exponentials were used but were found to be inappropriate. The single exponentials were replaced by stretched exponentials characterized by exponents for the uptake and for the clearance. Stretched exponential functions have been found to describe many phenomena in Nature, in particular in complex systems with broad relaxation time distribution. 64,65 In Eq. 2, and are related respectively to the half-life times and that characterize the initial and terminal processes, via the expressions and . In the following, the data will be discussed in terms of the half-life times to ease comparison with earlier work. 23,58 Fig. 8a-h show the MRI signal intensities obtained for the different systems and the least-square calculations results using Eq. 2 (continuous lines). For the eight samples considered, Eq. 2 provides a satisfactory outcome over the entire time range. The adjustable parameters , the half-life times and , and the stretched exponents and are listed in Table IV. IV).

As anticipated, the half-life time for the uptake mechanism depends strongly on the coating. For PAA 2K 6.8 nm particles, the uptake time is estimated at 4.5 ± 0.3 min, whereas it is 55 times longer for PEG 2K particles, at 249 ± 18 min. The stretch exponent for this mechanism varies from for the agents that are rapidly uptaken, to for the stealthiest ones. In Table IV, the size effect can be clearly seen as 6.8 nm particles have systematically prolonged circulation times as compared to the 13.2 nm particles. 1 The results suggest that the uptake mechanism depends primarily on two parameters, the probe size and the coating type. In the present study, the effects of each of these parameters are separately identified.

The clearance phenomenon is associated with the particle degradation and with the iron incorporation into the main intracellular iron storage proteins (ferritin, hemosiderin). 58,59 The MRI signal intensity returns to its pre-injection level as iron oxide particles are metabolized and their degradation products cleared (Fig. 8). As can be seen from Table IV, a rather uniform kinetics is observed for the clearance, with half-life times between 2 to 4 days. Indeed, does not depend on the magnetic core sizes, nor on the coating. The benchmark agent Cliavist® exhibits also a comparable clearance time ( = 2.6 ± 0.1 day).

-Fe2O3 coating (min) (day)

-Conclusion

In this work, novel iron oxide based contrast agents dedicated to in vivo magnetic resonance imaging were synthesized, with the aim of understanding the relationship between the particle coating properties and their pharmacokinetics. Maghemite (-Fe 2 O 3 ) particles with diameters 6.8 nm and 13.2 nm were prepared and coated following bottom-up assembly processes. Different organic macromolecules were considered and a total of 10 different probes were evaluated both in vitro and in vivo. Statistical copolymers (Specific Polymers ® , France) combining multiple phosphonic acid groups and PEG chains on the same backbone were synthesized and used as coats for iron oxide particles. These polymers were characterized by 2 kDa and 5 kDa PEGylated side-chains.

The particles were first evaluated regarding their physico-chemical properties and characterized in terms of size, charge and coating thickness. The colloidal stability, protein adsorption in culture media, contrast agent relaxometry and probe toxicity were investigated. The first result that emerges from the in vitro assay is that polymers, either poly(acrylic acid) or poly(ethylene glycol) are efficient coatings, as they prevent particles to agglomerate in culture media or to be covered by plasma proteins. The particles and the PEG based polymers were found to be non-toxic and biocompatible at the in vivo dose level.

The dispersions were injected intravenously into the mouse tail vein at a relatively low dose (16.7 µmol of iron per kilogram of mouse) compared to earlier reports. Liver, spleen and kidneys -MR images were acquired prior and after injection, at time points between one minute and seven days. As nanoparticles are eliminated by the reticulo-endothelial system and filtered by the liver, the change in the liver MRI contrast was investigated and provided a direct indication of the nanoparticle pharmacokinetics. With this protocol, the particle uptake and clearance time evolution in and out of the liver could be retrieved. The main outcome from the in vivo MRI mouse assays is that coating appears as the primary parameter that affects the liver uptake kinetics and elimination from the blood pool. Phosphonic acid PEG 2K -copolymers provides the most efficient contrast agent in terms of stealthiness, as they are able to prolong the blood particle lifetime up to 250 minutes. In similar conditions, the life times for citrate and PAA 2K coated particles are typically 50 times shorter. Although poly(acrylic acid) is a robust coating ensuring colloidal stability in vitro, its in vivo performance is comparable to that of citrate or dextran. The time at which the MRI signal intensity returns to its pre-injection level was found to follow a more universal behavior. The clearance time was comprised between 2 to 4 days for all samples studied, including the commercial benchmark Cliavist®. In conclusion, we have shown that a 5 nm layer made of multi-phosphonic acid poly(ethylene glycol) at the iron oxide surface significantly improve the colloidal stability, protein adsorption properties and stealthiness in vivo of MRI probes. Our work also demonstrates that the phosphonic acid coupled to innovative polymer synthesis may open up new horizons regarding the surface functionalization.

Supporting Information

The Supporting Information includes sections on the iron oxide nanoparticle characterization in S1 (transmission electron microscopy), S2 (electron beam microdiffraction), S3 (vibrating sample magnetometry) and S4 (zeta potential). The PEG density determination at the particle surface is described in S5, the nanoparticle distribution kinetics in abdominal aorta in S6, the longitudinal and transverse relaxivity measurements in S7 and the cytotoxicity studies using murine hepatocytes in S8.

Figure 1 :

 1 Figure 1: a, b) Transmission electron microscopy (TEM) of the 6.8 and 13.2 nm iron oxide nanoparticles. The bars are 20 nm and 50 nm respectively. c, d) Particle size distribution determined by TEM. e) Table showing the chemical formula, charge and molecular weight of the organic coatings used in this study. For the phosphonic acid PEG copolymer, only the PEG molecular weight is indicated.

Figure 3 :

 3 Figure 3: Transverse relaxivity measured at 7 Teslas for 6.8 nm (a) and 13.2 nm (b) iron oxide nanoparticles dispersed in aqueous media. The data for Cliavist® (c) are shown for comparison. The error bars are defined as the standard deviations.

Figure 4 :

 4 Figure 4: Magnetic resonance imaging scans for wild-type female 8 weeks BALB/c mouse livers at different time points (30 min, 1 and 3 hours) after injection for Cliavist® (a to d) and for the 6.8 nm PEG 2K coated particles (e to h). In the assays, 100 µL of a [Fe] = 5 mM dispersion were injected to the mouse. The corresponding dose is 16.7 µmoles, or equivalently 0.93 mg of iron per kilogram of mouse. A comparison of the two time sequences shows a delay in the negative contrast enhancement at about 3 hours for the PEG particles, compared to that of the commercial contrast agent.

Figure 6 : 1 )

 61 Figure 6: Time dependences of the MRI signal intensity for a) 6.8 nm and b) 13.2 nm PAA 2K and PEG 2K coated nanoparticles. Cliavist® is shown for comparison. In the in vivo experiments, the iron oxide dispersion injected to the mouse was 100 µL and the iron concentration 5 mM (corresponding to 16.7 µmol kg -1 or 0.93 mg kg -1). With the conventions used, the darkening of the liver is associated to a decrease of the MRI signal as a function of the time. The error bars indicate the standard deviations.

Figure 7 :

 7 Figure 7: Time dependences of the MRI signal intensity for a) 6.8 nm and b) 13.2 nm nanoparticles coated with PEG 2K and PEG 5K . Cliavist® is shown for comparison. The experimental conditions are similar to those of Fig. 6. The error bars indicate the standard deviations.

Figure 8 :

 8 Figure 8: MRI signal intensity for the iron based contrast agents studied in this work, together with least-square calculations using Eq. 2 (continuous lines). The fitting of the uptake and clearance kinetics allows to determine the following parameters: the intensity minimum , the half-life times and and the stretched exponents and (TableIV).

Table I :

 I Characteristics

	dispersity TEM		0.18	0.23
	molecular weight	(Da)	1.3×10 6	12×10 6
	hydrodynamic diameter	14	27
		(nm)
	averaged molecular weight	was obtained from static light scattering.	40	is the bare particle hydrodynamic
	diameter in water.
				6.8 nm -Fe2O3	13.2 nm -Fe2O3
				nanoparticle	nanoparticle
	diameter VSM (nm)		6.7	10.7
	dispersity VSM		0.21	0.33
	diameter TEM (nm)		6.8	13.2

of the iron oxide particles used in this work. The bare particle size and size dispersity were determined by vibrating sample magnetometry (VSM) and by transmission electron microscopy (TEM). The weight-

polymer name acid group meq/g functional group per chain PEG density 6.8 nm PEG density 13.2 nm copolymer

	with PEG 2000 Da	0.873	3.1 ± 0.2	1.9	1.1
	copolymer with PEG 5000 Da	0.349	7.4± 0.4	2.0	1.1
	Table II: Structural parameters of the PEG copolymers used in this work. The first column denotes the molar
	equivalent of acid groups per gram (		) of polymer as determined by	1

Table III :

 III Characteristics of the iron oxide particles used in this work.

			9,40,44,50
	nanoparticles	coating	hydrodynamic	Layer thickness
			diameter
			nm	nm
	-Fe2O3	bare	14 ± 1	0
	6.8 nm	citrate	15 ± 1	0.5 ± 1
		PAA2K	18 ± 1	2 ± 1
		PEG2K	22 ± 1	4 ± 1
		PEG5K	46 ±3	16 ± 2
	-Fe2O3	bare	27 ± 1	0
	13.2 nm	citrate	28 ± 1	0.5 ± 1
		PAA2K	33 ± 1	3 ± 1
		PEG2K	37 ± 2	5 ± 1
		PEG5K	44 ± 3	8.5 ± 2
	Cliavist©	carboxy	66 ± 4	aggregates
	4 nm	dextran
				denotes the particle hydrodynamic
	diameter and

Table IV :

 IV Parameters obtained from the adjustment of the MRI signal intensity with Eq. 2. denotes the intensity minimum,

	6.8 nm	PAA2K	0.45	4.5 ± 1.3	0.46	2.8 ± 0.2	1.6
		PEG2K	0.62 249 ± 18	3.0	4.0 ± 0.3	1.4
		PEG5K	0.65 103 ± 15	1.2	1.5 ± 0.3	1.6
	13.2 nm	citrate	0.40	2.5 ± 0.2	0.42	3.9 ± 0.2	1.0
		PAA2K	0.30	1.4 ± 0.3	0.60	2.8 ± 0.3	0.4
		PEG2K	0.50	82 ± 13	0.80	2.8 ± 0.6	1.8
		PEG5K	0.54	59 ± 15	1.2	n.d.	n.d.
	Cliavist ®	Carboxy	0.42	6.0 ± 1.2	0.40	2.6 ± 0.1	1.3
		dextran

and the half-life times associated to the uptake and clearance mechanisms and and the stretched exponential exponents. The error bars are defined as the standard deviations (SD).

Acknowledgements

We thank Virginie Berthat, Nicoletta Giamblanco, Isabelle Margaill, Giovani Marletta, Fanny Mousseau, Olivier Sandre and Leticia Vitorazi for fruitful discussions. The Laboratoire Physico-chimie des Electrolytes, Colloïdes et Sciences Analytiques (UMR Université Pierre et Marie Curie-CNRS n° 7612) is acknowledged for providing us with the magnetic nanoparticles. ANR (Agence Nationale de la Recherche) and CGI (Commissariat à l'Investissement d'Avenir) are gratefully acknowledged for their financial support of this work through Labex SEAM (Science and Engineering for Advanced Materials and devices) ANR 11 LABX 086, ANR 11 IDEX 05 02.

In Fig. 6a and6b, data for 6.8 nm and 13.2 nm particles coated with PAA 2K and PEG 2K are compared. For the poly(acrylic acid) coated nanoparticles, as well as for Cliavist®, the MRI signal intensity exhibits a broad minimum down to 0.35. For these samples, the minimum extends over a period of tens to hundreds of minutes. At longer timescale, typically from 1 to 7 days after injection the MRI signal intensity increases again and reaches unity, indicating that the liver returns to its pre-injection level. This recovery is associated with the iron clearance out of the liver. 21,[58][59][60] On the opposite, PEGylated 6.8 nm and 13.2 nm particles exhibit prolonged circulations in the blood pool, the contrast enhancement appearing approximately after 2 -3 hours. Later, the MRI signal passes through a minimum as in the previous case, and recovers its pre-injection limit after a few days. Interestingly, the MRI level at the minimum (around 0.6) remains higher than for anionic particles, indicating a lower iron oxide concentration and a reduced uptake Concerning the polymer molecular weight, earlier studies have shown that the coating efficiency increases with the layer thickness. [1][2][3]6,15,23,25,26 To test this assumption, the pharmacokinetics of identical core particles with 2 kDa and 5 kDa PEG coating was investigated. From light scattering experiments, the layer thickness associated to PEG 2K and PEG 5K polymers was estimated at 5 nm and 8 nm, respectively (Table III). Fig. 7a and 7b compare the MRI signal intensity for the two particles. For 6.8 nm particles, the