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^{Dendronized iron oxide nanoparticles as contrast agent for MRI}

Brice Basly, ^a Delphine Felder-Flesch,*^a Pascal Perriat,^b Claire Billotey,^c Jacqueline Taleb,^c Geneviève Pourroy,^a and Sylvie Begin-Colin*^a

Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X

First published on the web Xth XXXXXXXXX 200X

DOI: 10.1039/b000000x

Covalent attachment, through a phosphonate anchor, of hydrophilic pegylated dendrons on iron oxide nanoparticles results in versatile, robust, and highly relaxing MRI contrast agents.

Some of the significant and most promising applications of inorganic nanoparticles (NPs) lie in the fields of biology and biomedicine.¹ A major issue with the development of inorganic nanoparticles for biological applications pertains to the stability and size of bio-functionalized NPs in biological media.^2,3 Due to their magnetic properties and mainly their very high transverse relaxivity,⁴ superparamagnetic iron oxide nanoparticles (SPIO) with appropriate surface chemistry can be used in numerous in vivo applications such as MRI contrast enhancement,⁵ hyperthermia treatment,⁶ cell sorting,⁷ drug delivery, immunoassay, tissue repair.⁸ In all these applications, it is necessary (mandatory) to engineer the surface of SPIO NPs not only to improve biocompatibility, solubility and stability in physiological media but also to ensure a small particle size distribution (below 100 nm) after decoration and to preserve good magnetic properties, e.g. a high saturation magnetization. In addition, the small size particles will allow a favourable biodistribution of nanoparticles after intra-venous injection.

Several natural and synthetic polymers have been employed to coat the surface of SPIO NPs: these polymers include Dextran,⁹ lipids,¹⁰ polyethylene glycol (PEG)¹¹ or polyethylene oxide (PEO)¹² and polyvinylpyrrolidone (PVP).¹³ All the polymers used are known to be biocompatible and able to promote dispersion in aqueous media. However, these polymer coatings are not robust and can easily be detached from particle surfaces under in vivo conditions¹⁴ inducing NP’s aggregation. Moreover, they induce a large organic shell around the NPs. Both these facts lead to a lower impact of the superparamagnetic core on the water proton relaxivity and hence a lower contrast.

With the double objective to increase the efficiency of the grafting and to ensure the possibility of tuning the characteristics (morphology, functionalities, physico-chemical properties…) of the organic coating, we propose in this paper a strategy based on a combination of a dendritic and phosphonate approach. (i) the choice of the dendritic molecules is motivated by the fact that they are discrete and monodisperse entities whose relevant characteristics (size, hydrophilicity, molecular weight…) can be tuned as a function of their generation.¹⁵ Additionaly, they allow different and reproducible polyfunctionalization at their periphery. (ii) the choice of phosphonate as coupling agent is justified by previous studies that have evidenced that they allow a significantly higher grafting rate and a stronger binding than carboxylate anchors.^16,17,18,19 They were also demonstrated to stabilize suspensions in water at physiological pH and to preserve magnetic properties.Error: Reference source not found^,20

This strategy illustrated in Scheme 1 is validated in this paper by comparing the highly relaxing colloidal water suspensions so elaborated to polymer-decorated commercial SPIO-based contrast agents (Fig. 1). From T1 and T2 values measured as a function of concentration by relaxometry at 1.5T (37°C) (Fig. 1), relaxivity values as high as 349 mM^-1.s^-1 for r₂ and 10 mM^-1.s^-1 for r₁, with a 44.8 R2/R1 ratio were obtained for our nanoparticles with the smallest hydrodynamic diameter. These values are significantly higher (1.5 times superior) than those relative to commercial polymer-decorated nanoparticles.


Scheme 1 Schematic representation of dendronized iron oxide nanoparticles through phosphonate anchor.

aggre



Fig.1 In vitro relaxivity studies (1.5T (room temperature) and 7T (37°C) of dendronized NPs, compared to commercially available polymer-coated NPs.
In vitro relaxivity measurements were performed on one millicube samples containing increased iron concentration of dendronized materials dispersed in water at 7T and 37°C. Enhancement Contrast ratio (EHC) were calculated for our dendronized NPs in reference to water [EHC (%)_w= [(((Signal value of dendronized NPs) – (signal value of water))/signal value of water)x 100]. In addition, EHC values vs Endorem™ at equivalent iron concentration were also calculated [EHC_En= [(((Signal value of dendronized NPs) – (signal value of Endorem™))/signal value of Endorem™) x 100] (Fig. 2).

Fig. 2 T₂-weighted MR images (conditions: 37°C, 7T, T2w sequence: MSME acquisition – TR/TEE = 2000/70 ms – 2mm thickness slice) of ghost constituted of increased and equivalent iron concentration of Endorem™ (Guerbet) and dendronized NPs diluted in water, with corresponding EHC (EHC_w and EHC_En) values calculated as defined in text.

The very high EHC values obtained for our NPs confirmed their very high contrast power even at high magnetic field (7T). For instance, on MR T_2w image at 7T, EHC values are from 15% to 75% higher than those obtained for Endorem™.

These interesting properties may be related to the design and properties of these nano-objects consisting of 12 nm SPIO NPs coated with generation 0 dendritic molecules via a phosphonate anchor. Indeed, this design permits both (i) to preserve the magnetic properties until the surface of the particles and (ii) to minimize the thickness of the organic shell necessary for a good colloidal stabilization. Precisely, the iron oxide nanoparticles and the dendritic phosphonate were prepared following the method described elsewhere.Error: Reference source not found^,21 The SPIO NPs were obtained by co-precipitation of Fe²⁺ and Fe³⁺ ions in the presence of tetramethyl ammonium hydroxide N(CH₃)₄OH (ESI). X-ray and electron diffraction showed that the nanoparticles display the typical spinel structure (ESI). The value of the lattice parameter a = 0,8383 nm obtained from X-ray diffraction is intermediate between those of magnetite (0.8396 nm, JCPDS file 19-629) and maghemite (0.8346 nm, JCPDS file 39-1346) meaning that the NPs are oxidized at their surface as already noticed for such small sizes.Error: Reference source not found^{,Error: Reference source not found,22} The mean size estimated from X-ray pattern according to the Scherrer formula is about 12±2 nm. Since it is consistent with the size of the particles imaged by TEM, the NPs are monocrystalline. The magnetization curve of bare NPs is characteristic of a superparamagnetic sample and the saturation magnetization is about 65 emu/g in agreement with earlier reported value.Error: Reference source not found^{,Error: Reference source not found,23}

As grafting was demonstrated to be favoured by electrostatic interactionsError: Reference source not found^{,Error: Reference source not found,24} it must be performed in a pH range determined by the pKa values of the dendritic phosphonate (3 and 5-6): pH must be superior to 3 and the highest as possible. Since it must also be significantly lower than the isoelectric point (IEP) value of bare NPs which is 6.8, Error: Reference source not found the chosen compromise was pH 5 (Fig. 3). To achieve this, 50 mg of nanoparticles were added to 40 mg of dendron dissolved in 50 ml of degassed water (pH = 3). Then N(CH₃)₄OH was added in order to reach a pH value of 5 . The suspension was sonicated for 90 min at 35°C and the grafted NPs were separated from the ungrafted molecules by ultrafiltration. After such purification step, the pH of the NPs suspension is around 6.



Fig. 3 Ideal pH grafting condition according to isoelectric point (IEP) of the bare nanoparticles and pKas of the dendron.
Assuming a surface area of 72 Ų for the dendron, a grafting rate of 1.3 molecules/nm² (150 mg of molecules per gramme of nanoparticle) leading to a surface coverage of about 95% was determined directly by elemental analyses and undirectly from the UV-Visible spectroscopy analyses of the washing solutions. It was also confirmed by thermogravimetric measurements (Supporting Informations), IR spectroscopy through the disappearance of the P-OH and P=O bands of the dendron (Supporting Informations) and finally by TEM micrographs (Fig. 4). Indeed, a surrounding organic layer of 2 nm is visible on particles at focus, confirming that a monolayer surrounds effectively the SPIO NP.

The zeta potential value of suspensions of grafted NP’s at pH = 7 is around -15.5 mV. Again, this confirms the efficiency of grafting since the IEP has been shifted to lower pH values compared to bare NPs. The electrostatic stability of the iron oxide NPs has thus been improved at physiological pH and such zeta potential value is in the range and sometimes slightly higher than those previously reported for pegylated NPs obtained by other methods.Error: Reference source not found^,25,26,27

Fig. 4 TEM micrograph (left) of the decorated NPs and particle size distribution in volume of NP’s water suspension at physiological pH (right).
The average particle size of the decorated NPs in suspensions after magnetic decantation is in the [40 to 60 nm] range (Fig. 1&4). From magnetic measurements performed on the grafted nanoparticles (ESI), the saturation magnetization of the iron oxide itself, e.g. after subtraction of the organic contribution, is found to be 70 emu/g, a value slightly higher than that obtained for bare NPs. This confirms that grafting through a phosphonate allows preserving the saturation magnetization of the grafted NPs.

All these analyses confirm the great interest in grafting small dendritic molecules through a phosphonate anchor in order to stabilize iron oxide suspensions by electrostatic and steric repulsions. Such hybrid and biocompatible nano-objects open a new route for the development of highly relaxing contrast agents displaying a quite satisfactory R2/R1 ratio even at high field. In addition, we expect that further grafting of biological effectors through the dendrimer platform will allow efficient targeted imaging and therapy.

We would like to thank Didier Burger (TG analysis), Corinne Ulhacq (TEM), Emilie Voirin, Emilie Couzigné (technical assistance). We thank the French Ministry of Research for a fellowship to B. Basly, CNRS, UDS and ECPM for financial support. This work was also supported by the european community through the NANOMAGDYE project CP-FP 214032-2.

Notes and references

^a IPCMS, UMR CNRS-UdS-ECPM 7504 23 rue du loess BP 43, 67034 Strasbourg, France. Fax: + 33 388 10 72 47; Tel:+ 33 388 10 71 92; E-mail: [e-mail], [e-mail]

^b Groupe d'Etudes de Métallurgie Physique et de Physique des Matériaux, UMR 5510 CNRS-INSA de Lyon, F-69621 Villeurbanne Cedex, France. Fax: + 33 472 43 85 28 ; Tel: + 33 472 43 82 53; E-mail: [e-mail]

^c Université Claude Bernard Lyon 1, Laboratoire CREATIS-LRMN, CNRS UMR 5220, Inserm U 630, INSA-Lyon, 7 avenue J Capelle, bat. Blaise Pascal F-69621 Villeurbanne cedex, France. Fax: + 33 472 11 69 57 ; Tel: + 33 472 68 46 17; E-mail: [e-mail]
† Electronic Supplementary Information (ESI) available: [Materials and methods, synthesis of the dendronized IO NPs, Xray pattern, IR spectra, thermogravimetric curves, magnetizaton curves, in vitro relaxivity graphes]. See DOI: 10.1039/b000000x/

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