Mesoporous silica nanoparticles combining two-photon excited fluorescence and magnetic properties

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www.rsc.org/materials | Journal of Materials Chemistry

Mesoporous silica nanoparticles combining two-photon excited fluorescence and magnetic properties† Elena Chelebaeva,a Laurence Raehm,a Jean-Olivier Durand,*a Yannick Guari,*a Joulia Larionova,a Christian Guerin,a Alexandre Trifonov,b Marc Willinger,c Kalaivani Thangavel,de Alessandro Lascialfari,def Olivier Mongin,g Youssef Mirg and Mireille Blanchard-Desceg Received 21st October 2009, Accepted 10th December 2009 First published as an Advance Article on the web 26th January 2010 DOI: 10.1039/b922052f A new approach to the synthesis of multifunctional nanoparticles was developed by using covalent anchoring of cyano-bridged coordination polymer Ni2+/[Fe(CN)6]3 to the surface of two-photon dyedoped mesoporous silica nanoparticles. The obtained hybrid nanoparticles were studied by infrared (IR) spectroscopy, nitrogen adsorption (BET), X-ray diffraction, transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM), luminescence, and magnetic analysis. The synthesis leads to homogeneously dispersed uni-shaped nanoparticles of around 100 nm in length that are coated with cyano-bridged metallic coordination polymer nanoparticles. These hybrid nanoparticles combine effective two-photon excited fluorescence, porosity, high transverse nuclear relaxivity values (i.e. the magnetic resonance imaging efficiency) and superparamagnetic properties.

1. Introduction Multifunctional nanoparticles represent a class of nano-materials that combines several specific properties such as mechanical, electronic, optical, and magnetic in a single nano-object. Therefore, they are capable of exhibiting diverse physical responses when subjected to certain external stimuli. In the recent years, multifunctional nano-materials are at the forefront of research and technology due to their interesting properties and their potential applications in different fields.1 In particular, for biomedical applications, multifunctional nano-objects are capable of combining two or more functions, such as fluorescent markers or photothermal therapy agents with MRI contrast agents or hyperthermia therapy agents, drug delivery, clinical diagnosis, and others.2 Such hybrid nano-objects may either present simple coexistence of physical and chemical properties of components (which requires the various components to have no

a Institut Charles Gerhardt Montpellier, UMR 5253 CNRS-UM2-ENSCM-UM1, Chimie Mol eculaire et Organisation du Solide, Universit e Montpellier II, Place E. Bataillon, 34095 Montpellier cedex 5, France; Fax: +(33) 4 67 14 38 52 b G. A. Razuvaev Institute of Organometallic Chemistry of the Russian Academy of Science, Tropinina 49, GSP-44S, 603950 Nizhny Novgorod, Russia c Department of Chemistry, CICECO, University of Aveiro, 3810-193 Aveiro, Portugal d Dipartimento di Scienze Molecolari Applicate ai Biosistemi, Universit a degli studi di Milano, I-20134 Milano, Italy e CNR-INFM-S3 NRC, I-41100 Modena, Italy f Dipartimento di Fisica ‘‘A. Volta’’, Universit a degli studi di Pavia, I-27100 Pavia, Italy g Chimie et Photonique Mol eculaires, CNRS UMR 6510, Campus de Beaulieu, Universit e Rennes 1, 35042 Rennes Cedex, France † Electronic supplementary information (ESI) available: An EDS spectrum recorded from the nanocomposite material. See DOI: 10.1039/b922052f

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deleterious effect on the properties conveyed by other components) or, more rarely, exhibit novel properties due to the mutual interaction between the individual components. One of the promising multifunctional nano-objects presents a combination of magnetic and optical properties within a single nano-system. In order to achieve such multifunctional hybrid nanoparticles several approaches have been used.3 In particular, the development of multifunctional nanoparticles combining both magnetic properties and two-photon excited fluorescence (TPEF) is highly desired. Indeed, the two-photon excitation technique provides many advantages for optical imaging or phototherapy4 such as intrinsic three dimensional resolution, increased excitation selectivity4i and penetration depth, and lower scattering losses.4a,e However the literature dealing with the synthesis of such nanoparticles is scarce. We can cite bifunctional Fe3O4–Ag heterodimer nanoparticles synthesized for two-photon fluorescence imaging and magnetic manipulation of macrophage cells5 as well as quantum dots and Fe3O4 nanoparticles co-encapsulated in ormosil (organically modified silica) in which the fluorescence of the nanoparticles was monitored by two-photon excitation.6 Concerning organic dyes, two publications from Prasad and coworkers 7 deal with Fe2O3 nanoparticles with a two-photon fluorophore covalently attached to the surface. The nanoparticles were entrapped inside silica in order to attach a bio-targeting group to the surface. These nanoparticles were shown to be internalized in cancer cells as monitored by two-photon fluorescence. In all cases, superparamagnetic iron oxide nanoparticles (SPIONs) or magnetic metallic nanoparticles are usually used to confer the magnetic properties to the hybrid nano-objects. Recently another type of magnetic inorganic nanoparticles called coordination polymer nanoparticles (CPNs) has been developed.8 These nanoparticles were discovered a decade ago and the number of articles devoted to their synthesis, and their study is in constant expansion. The reason for the present interest J. Mater. Chem., 2010, 20, 1877–1884 | 1877

in CPNs is due to their specific nature, which is different in comparison to other inorganic nanoparticles and their interesting properties. For instance, these objects present all the advantages of bulk coordination polymers such as controlled and flexible molecular structures, tunable physical and chemical properties, porosity, low density, and the possibility to combine several properties in the same multifunctional nano-objects. Such CPNs may be obtained from molecular precursors by combining ‘‘soft’’ chemistry self-assembling reactions with conventional nano-chemistry approaches, and thus, enable the design of nanoobjects a way that allows the control of size, shape and organization at the nano-scale level, as well as a combination of desired physical and chemical properties. Indeed, numerous cyanobridged coordination or metal–organic frameworks nanoparticles presenting magnetic or photo-magnetic properties have been synthesized and studied.9 Furthermore, very recently it was shown that some CPNs may present high magnetic resonance relaxivities, hence they may be considered as a new family of efficient contrast agents for magnetic resonance imaging (MRI).10 The present work is devoted to explore a new approach to achieve multifunctional hybrid nano-objects based on dye-doped mesostructured silica nanoparticles (MSNs) with two-photon excited fluorescence associated with magnetic cyano-bridged CPNs. Recently, some of us reported on the synthesis and studies of MSNs containing dye molecules based on the quadrupolar derivative bearing pyridinium end-group conjugated to a fluorenyl core moiety (see Scheme 1) incorporated into the walls of silica presenting bright fluorescence and high two-photon absorption cross-section.11 These results encourage us to further investigate these dye-doped MSNs and anchor magnetic CPNs on their surface in order to form original hybrid magneto-TPEF nanoparticles.

2. Results and discussion Synthesis and structural characteristics of the hybrid nanoparticles Scheme 1 shows the synthetic method used for the fabrication of hybrid multifunctional nanoparticles. MSNs containing dye molecules based on the quadrupolar derivative bearing pyridinium end-groups conjugated to

a fluorenyl core moiety (Scheme 1) incorporated into the walls of silica were synthesized following our previously described procedure.11 The surface of the MSN was then functionalized with NH2 groups by grafting aminopropyltriethoxysilane which is able to coordinate metal ions by NH2 groups.12 Then, the sequential growth of cyano-bridged networks on the surface of the silica particles occurs at specific sites by consecutive coordination of Ni2+ and [Fe(CN)6]3 ions. The growth of the coordination polymer nanoparticles onto the silica nanoparticles was performed by consecutive treatment of silica nanoparticles first with a methanolic solution of Ni(H2O)6](BF4)2 and second with a methanolic solution of [N(C4H9)4]3[Fe(CN)6]. At each step of the treatment, the silica particles were thoroughly washed with methanol and dried in vacuo. The procedure was repeated twice. The M/M i.e. Ni2+/Fe3+ atomic ratio extracted from elemental and energy dispersive spectroscopy analysis (EDS) analysis is equal to 1. The IR spectroscopy was performed on the hybrid nanoparticles especially in the 2000–2300 cm1 spectral window, i.e. in the vicinity of the CN stretching mode, which is a fingerprint of structural and electronic changes occurring in Prussian Blue analogues. The CN stretching frequency of a free CN ion is 2080 cm1, whereas upon coordination to a metal ion, it shifts to higher frequencies.13 The IR spectrum of the obtained hybrid nanoparticles shows two characteristic absorption bands in the CN stretching region at 2101 and 2165 cm1. Similar CN stretching frequencies can also be found in the spectra of the respective bulk cyano-bridged coordination polymer. The high frequency bands can be attributed to the stretching of the CN ligand bridged between Ni2+ and Fe3+ in Ni2+-CN–Fe3+ mode and the low frequency bands can be assigned to the presence of the linkage isomer with Ni2+-NC–Fe3+ coordination mode, as it was reported for the bulk cyano-bridged coordination polymers.14 As expected, the IR spectra of the particles also present SiO2 vibration bands at 1080, 958, 801 and 460 cm1.15 Due to the low concentration of the fluorophore incorporated inside the nanoparticles it cannot be detected by IR spectroscopy. The electronic spectra of these nanoparticles show an absorption band at 420 nm corresponding to inter-metal charge-transfer bands from Ni2+ to Fe3+, which can also be found in the UV-Vis spectra of the bulk counterpart. This band is superimposed on the fluorophore’s absorption band.14

Scheme 1 A schematic representation of CPNs growth on dye-doped silica nanoparticles.

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Textural characteristics of the nanocomposites In order to determine the effect of the cyano-bridged coordination polymer anchoring at the silica nanoparticles surface, the hybrid particles were studied by scanning electronic microscopy (SEM), transmission electronic microscopy (TEM) and high resolution transmission electronic microscopy (HRTEM). A typical SEM image of the hybrid nanoparticles is shown in Fig. 1a. It reveals that the ovoid shape of the MSNs was preserved during the anchoring of CPNs on their surface. A distinctive signal for Ni and Fe was detected by EDS with the atomic ratio Ni/Fe ¼ 1/1 (Fig. 1S, ESI†). The HRTEM image recorded along the long axis of a particle shows that the crosssection resembles the shape of a hexagon. Indeed, the hexagonal arrangement of channels in the host material is still retained, as revealed by the arrangement of spots in the fast-Fourier transformation of the image (Fig. 1b and inset). SEM and TEM analysis did not reveal the presence of a secondary phase or aggregates of coordination polymer nanoparticles. This is indicative of a homogeneous dispersion of the cyano-bridged polymer network on the silica matrix. In order to locate the metallic coordination polymer, dark field scanning TEM images were recorded using an annular dark field detector (Fig. 1c). At small camera lengths, the signal is highly sensitive to variations in atomic number. Above that, the intensity is modulated by the thickness of the particles, which varies across the diameter and is further modulated due to the presence of the pores. Nevertheless, slight intensity variations can be attributed to the presence of the CPNs. From this, it can be estimated that their size is only in the

Fig. 1 (a) A SEM image demonstrating the homogeneity of the sample. (b) A HRTEM image recorded along the long axis of a particle. It reveals the hexagonal arrangement of the pores. The power spectrum as well as a small square region containing a Fourier filtered image are shown as insets. (c) A high angle annular dark field STEM image recorded from the hybrid dye-doped MSN with Ni2+/[Fe(CN)6]3 shell. Intensity variations are due to thickness variations and differences in atomic number. (d) A TEM image of a hybrid nanoparticle after calcination.

This journal is ª The Royal Society of Chemistry 2010

range of a few nm and that they are equally distributed across the surface of the particles, including the channels. All attempts to perform extractive replicas which consists of removing the silica part of the nanocomposite material using an HF treatment in order to visualize the CPNs were unsuccessful.15 We previously published the formation of metallic alloys nanoparticles from cyano-bridged CPNs under heating up to 700  C.16 Thus, to prove that nanoparticles are present on the MSNs, we performed an additional calcination step of the hybrid nanoparticles under air up to 700  C which induces the formation of metallic nanoparticles, hence proving the presence of CPNs decorating the MSNs. A TEM image of the heated hybrid nanoparticles showing the metallic nanoparticles decorating the silica nanoparticles is presented in Fig. 1d. N2 adsorption-desorption was used to characterize the nanocomposite material. The starting MSN with APTS grafted on the surface possesses a specific surface area of 505 m2 g1 with a pore diameter of 1.7 nm. After formation of the cyano-bridged network, the specific surface area dropped to 35 m2g1, which is indicative for an anchorage of the CPNs blocking the pores of the MSNs towards N2 adsorption. Powder X-ray diffraction measurements recorded in the range of 2q ¼ 10–50 is depicted Fig. 2. The attempted NaCl-type structure with a lattice parameter of 10.2 nm corresponding to the profile pattern of the bulk analogous Ni1.5[Fe(CN)6] should display three main diffraction peaks at 17.8 , 25,3 and 36.1 , corresponding to the (200), (220) and (400) reflections, respectively. The (200) and (400) reflections are clearly visible as rather broad peaks, as expected for nanosized materials.9 The peaks corresponding to the (200) and (400) reflections have been deconvoluted to Lorentzian curves for the determination of the full-width at half maximum (fwhm) value. The crystalline domain size have been calculated from the Debye-Scherrer formula, using the fwhm value of respective index peaks giving values in the range of 7–10 nm. The (220) reflection could not be observed due to the presence of a very broad peak at 22.1 which is attributed to amorphous silica. Four additional peaks are observed at 18.9 , 31.1 , 38.3 and 44.4 which are attributed to the (N(C4H9)4)BF4 compound formed during the sequential growth of the CPNs which is visibly trapped in the nanomaterial. It should be noted that (N(C4H9)4)BF4 couldn’t be removed even after several washing cycles.

Fig. 2 Powder X-ray diffraction measurements of the hybrid nanomaterial. * indicates impurity attributed to (N(C4H9)4)BF4 and # is attributed to SiO2.

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Magnetic measurements The magnetic measurements of the hybrid nanoparticles were performed by using dc and ac modes on the SQUID magnetometer working in the temperature range between 1.8 and 350 K. The zero field-cooled (ZFC)/field-cooled (FC) magnetization curves performed for the nanoparticles in the 2–30 K range are shown in Fig. 3. The ZFC curve is obtained by recording the magnetization when the sample is heated under a field of 100 Oe after being cooled in zero field. The FC data were obtained by cooling the sample under the same magnetic field after the ZFC experiment and recording the change in sample magnetization with temperature. The ZFC/FC thermal irreversibility can be attributed to the blocking-unblocking process of the particle magnetic moment when thermal energy is varied17 or, for strongly interacting particle assemblies, inducing a spin-glasslike transition.18 In both kind of systems, the ZFC curve exhibit a maximum at a temperature Tmax, which corresponds to the blocking temperature (TB) of the nanoparticles with mean volume for a superparamagnetic system or to the freezing temperature (Tf) for a spin-glass-like regime.18 For the sample, the ZFC curve shows a narrow peak with a maximum at 12 K and the FC curve increases as the temperature decreases and tends to saturation at low temperature. The FC and ZFC curves coincide at high temperatures and start to separate below 14.2 K. Similar curves were previously observed for Ni2+/[Fe(CN)6]3 CPNs.8a, 9d, 9l–n, 15 The irreversibility of the ZFC/FC curves was investigated in details by using alternating current (ac) susceptibility measurements. The temperature dependence of the in-phase, c0 (absorptive), and out-of-phase, c00 (dispersive), components of the ac susceptibility measured with no static field applied for frequencies from 1 Hz to 1488 Hz, are shown in Fig. 4. At 1 Hz, both c0 and c00 responses present a peak at 13.0 K and 11.87 K, respectively, which shifted toward higher temperatures as the frequency increases. The frequency-dependent behavior of the present system may be either attributed to (i) the blocking process of not or weakly interacting superparamagnetic nanoparticles,19 (ii) spin-glass like transition, which may be caused by strong dipolar interparticle interactions and by randomness18,20 or (iii) to intraparticle spin glass-like regime due to the particle

Fig. 3 Zero field-cooled and field-cooled magnetization (ZFC/FC) versus temperature curves for the hybrid obtained by applying an external magnetic field of 100 Oe.

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Fig. 4 (Top) Temperature dependence of in-phase, c0 , component of the ac susceptibility of the hybrid nanoparticles; (Bottom) temperature dependence of out-of-phase, c00 , component of the ac susceptibility of the hybrid nanoparticles; frequencies: 1 Hz (B), 125 Hz (C), 499 Hz (,); 998 Hz (-) and 1498 Hz (:).

surface spin disorder.17 Note that the temperature dependence of the ac susceptibility measured for the analogous bulk Ni3[Fe(CN)6]2$13H2O shows no frequency dependence, as expected for compounds presenting long range magnetic ordering.14 In order to understand the nature of the low temperature magnetic regime in this sample, the temperature dependence of the relaxation time extracted from the maximum of the c00 component of the ac susceptibility was fitted to an Arrhenius law, s ¼ s0exp(Ea/kBT). Here, where Ea is the average energy barrier for the reversal of the magnetization, s0 is the attempt time and kB is the Boltzmann constant. According to the Neel model, this law governs the temperature dependence of the relaxation of the magnetization of non-interacting superparamagnetic systems.19 The values of the energy barrier, Ea/kB, and of the pre-exponential factor s0 are equal to 1400 K and 2.35  1052 s, respectively. The obtained values of s0 are much smaller than the ones generally observed for pure superparamagnetic systems (109–1012 s)20 and have no physical meaning. However, similar small values of s0 were found for systems containing interacting metal, metal oxide or CPNs.9,20 They are normally interpreted as the signature of a magnetic moments correlation introduced by considerable dipole–dipole interparticle interactions. In order to investigate the possible presence of these interparticle interactions we further verified if the dynamics of these samples would exhibit critical slowing down, as observed in canonical spin-glass systems. If an equilibrium ordered phase occurs at a finite critical temperature, Tg s 0 K, the relaxation time dependence on the frequency can be fitted by the This journal is ª The Royal Society of Chemistry 2010

In the present work, the 1H NMR relaxometry characterization of the sample was performed at room temperature by measuring the longitudinal and the transverse nuclear relaxation times T1 and T2, in the frequency range 10 KHz # n # 100 MHz for T1 and 8 MHz # n # 60 MHz for T2. Relaxivity values, r, are simply defined as the inverse of the relaxation time with respect to the contrast agent concentration, once corrected by the host diamagnetic contribution. So, the efficiency of the MRI contrast agents is determined by measuring the nuclear relaxivities r1p,2p defined as:21 rip ¼ [(1/Ti)meas  (1/Ti)dia]/c i ¼ 1,2

Fig. 5 Thermal variation of the relaxation time according to the critical slowing down law (power law) for the hybrid nanoparticles.

conventional critical scaling law of the spin dynamics, s ¼ s0[Tg/ (Tmax  Tg)]zv, where Tg is the glass temperature, f is the frequency and zv is a critical exponent.20 The best fit gives Tg ¼ 10.9 K, s0 ¼ 1.74  1013 s and zn ¼ 11.83 (Fig. 5). The obtained zn value is in the 4–12 range and the s0 value is in the 107–1013 s range as expected for classical spin glass systems.18,20 In summary, contrarily to what was observed for the corresponding bulk coordination polymers, our samples exhibit a low temperature slow dynamics corresponding to a spin-glass-like regime. This cooperative spin-glass-like dynamics, usually observed in dense magnetic nanoparticles systems, results from the presence of strong interparticle interactions.9 All these results are in agreement with densely packed CPNs rather than a coordination polymer shell surrounding the MSNs. Further, the presence of low temperature magnetic properties suggests that the hybrid nanoparticles may have the ability to shorten the longitudinal and/or transversal relaxation times of protons from water and thus be considered as potential contrast agents for MRI. Relaxivity measurements Currently, magnetic resonance imaging (MRI) techniques generally employ two family of contrast agents (CAs): gadolinium macrocycles or superparamagnetic iron oxide nanoparticles (SPIONs).21 Gd-based CAs produce a large shortening of the nuclear longitudinal relaxation time (T1) thus giving high longitudinal relaxivity (r1) and are called positive CAs. On the contrary, SPIONs typically induce a large shortening of the nuclear transverse relaxation time (T2) with corresponding high transverse relaxivity (r2), leading to a darkening effect. They are thus called negative CAs. As there is a preference for the use of positive CAs at the clinical level, due to their wider dynamic range, CAs based on Gd3+ macrocycles are the most widely used. With the aim of increasing the relaxivity and stability of CAs, inorganic gadolinium oxides,22a gadolinium phosphate22b and gadolinium fluoride22c nanoparticles have been investigated. Along this line of thought, more recently, Gd and Mn based metal–organic frameworks (MOFs)10a,b and cyano-bridged coordination polymer nanoparticles10c have been reported. These are the first works concerning the employment of CPNs as contrast agent for MRI. This journal is ª The Royal Society of Chemistry 2010

where (1/Ti)meas is the measured value of the sample with concentration c (mmol L1) of magnetic center (0.174 mmol L1 in total), and (1/Ti)dia refers to the nuclear relaxation rate of the diamagnetic host solution (water in our case). Fig. 6 reports the frequency dependence of r1p and r2p for our sample, together with the values for Endorem. As can be seen, the r1p values obtained for our sample are lower than the ones observed for Endorem, while the values of r2p relaxivities of our sample up to 2.5 times higher in comparison to the measured values for Endorem. The ratio of r2p/r1p is useful to establish the character of the CA, said negative if (as in our case) r2p/r1p > 2. The r2p/r1p relaxivities ratio of our sample is higher than the one of Endorem indicating that our nanoparticles may be used as an efficient contrast agent especially in the low frequency range. Fluorescence measurements The fluorescence properties of the nanoparticles were studied under one- and two-photon excitation. Data shown in Fig. 7 are in agreement with our previous work concerning dye-doped MSNs but without CPNs.11 Monophotonic fluorescence spectrum in EtOH confirmed the presence of the fluorophore moieties encapsulated inside the nanoparticles. A broad emission band was observed with a maximum intensity at 510 nm (lex ¼ 418 nm). Indeed, the mild conditions of the step-by-step growth of cyano-bridged metallic CPNs Ni2+/[Fe(CN)6]3 didn’t damage the fluorophore moiety. Two-photon excitation with near-infrared femtosecond light pulse was performed at 740 nm. Three different powers were used. The one-photon emission spectrum is superimposed for comparison. The TPEF emission spectra are identical to their monophotonic counterpart (OPEF). The fluorescence intensity depends quadratically on the excitation power, indicative of pure

Fig. 6 Longitudinal (r1p) (left) and transversal (r2p) (right) relaxivities of the hybrid nanoparticles (C), collected at T z 25  C, compared to the same quantity reported for the commercial compounds Endorem (B).

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Fig. 7 Two-photon excited fluorescence (TPEF) of the multifunctional nanoparticles (in suspension in ethanol, excited at 740 nm). The solid lines are the TPEF emission spectra at three different excitation powers, the dotted line is the one-photon excited fluorescence (OPEF, excited at 418 nm).

Fig. 8 Quadratic dependence of the emission intensity of the multifunctional nanoparticles on laser excitation power.

two-photon excitation without saturation or photobleaching as shown in Fig. 8. The TPEF properties of the fluorophore inside the nanoparticles are in agreement with the previous published results, i.e. two-photon excitation action cross section s2f z 400 GM per fluorophore at 740 nm. Since 8900 biphotonic fluorophores are encapsulated per particle, each multifunctional nanoparticle retains a TPEF cross-section of about 3.6  106 GM.

3. Conclusion The development of nanodevices for diagnostic and/or treatment of cancer is a major objective in the general field of nanomedicine. Despite the fact that the potential of such objects is clearly recognized, their clinical application is far from being achieved, partly because of the limited number of synthetic methods available. The research described in this article proposes a novel approach for the preparation of multifunctional nanoobjects combining both two-photon excited fluorescence and MRI relaxivity properties. To do this, we associate an organic dye and superparamagnetic coordination polymer nanoparticles on a common platform that is the mesostructured silica nanoparticles. The methodology is based on the synthesis of mesostructured silica nanoparticles encapsulating an organic 1882 | J. Mater. Chem., 2010, 20, 1877–1884

fluorophore with amine functions able to coordinate metal ions. Then, coordination polymers nanoparticles are formed and anchored on these amine sites by a step-by-step growth mechanism. The relaxivity properties of these nanoparticles are evaluated by measuring the longitudinal and the transverse nuclear relaxation times T1 and T2. The r2p relaxivity of our sample is up to 2.5 times higher than the one of Endorem indicating that our nanoparticles may be considered as an efficient negative contrast agent for MRI especially in the low frequency range. In addition to these interesting properties in terms of relaxivity, these nanoparticles exhibit two-photon excited fluorescence properties related to the presence of an organic fluorophore. These hybrid nanoparticles constitutes the first step towards new probes of interest in multimodal imaging but also as potential probes for theragnostics.

4. Experimental part Synthesis All of the chemical reagents used in these experiments were analytical grade. Amino-functionalized,12 dye-doped MSNs were obtained according to previously described procedures.11 The growth of cyano-bridged coordination polymer nanoparticles Ni2+/[Fe(CN)6]3 on the surface of dye-dopped MSN was performed by using the following procedure. Firstly, silica nanoparticles (75 mg) was added to a 102 M solution of [Ni(H2O)6](BF4)2 in methanol. The mixture was stirred overnight at room temperature. After filtration, the powder was thoroughly washed several times with methanol and dried at room temperature for 24 h in vacuo. Secondly, the so-obtained powder was added to a 102 M methanolic solution of the [N(C4H9)4]3[Fe(CN)6] complex.23 The mixture was stirred 48 h, the powder was filtered, thoroughly washed with methanol and dried in vacuo. Such consecutive treatments with metal salts and cyanometallate precursors were repeated again. The elemental analyses of the nanocomposite gives the ratio Ni/Fe/Si ¼ 1/1/40. Physical measurements IR spectra were recorded on a Perkin Elmer 1600 spectrometer with a 4 cm1 resolution. UV-Vis spectra were recorded in KBr disks on a Cary 5E spectrometer. Elemental analyses were performed by the Service Central d’Analyse (CNRS, Vernaison, France). The samples were heated at 3000  C under He. Oxygen was transformed in CO and detected by using an IR detector. Metals were determined with a High resolution ICP-MS using a ThermoFischer element. UV–vis spectra were recorded in KBr disks on a Cary5E spectrometer. Powder X-ray diffraction patterns were measured on a PanAnalytical diffractometer equipped with an ultra-fast X0 celerator detector X0 pert Pro with  The measurement Nickel-filtered copper radiation (1.5405 A). parameters are: stepsize, 0.01671; counting time, 60 s. Magnetic susceptibility data were collected with a Quantum Design MPMS-XL SQUID magnetometer working in the temperature range of 1.8–300 K and the magnetic field range of 0–50 kOe. The data were corrected for the sample holder. Samples for Transmission Electron Microscopy (TEM) measurements were prepared simply by depositing the nanoparticles on carbon coated copper grids. TEM measurements were carried out with This journal is ª The Royal Society of Chemistry 2010

a microscope JEOL 1200 EXII operated at 100 kV, HRTEM and STEM observations were performed on a JEOL 2200FS operated at 200 kV. Two-photon excited fluorescence measurements were performed as previously described11 using a mode-locked Ti:sapphire laser generating 150 fs wide pulses at a 76 MHz rate (Coherent Mira 900 pumped by a 5 W Verdi). Briefly, the setup allows for the recording of corrected fluorescence emission spectra under multiphoton excitation at variable excitation power and wavelength. Absolute values for the two-photon excitation action cross sections s2F were obtained according to the method described by Xu and Webb, using 1  104 M fluorescein in 0.01 M NaOH(aq) as a reference.24 We collected the NMR data by means of a Smartracer Stelar relaxometer (with the use of Fast-Field-Cycling technique) for frequencies in the range 10 kHz # n # 10 MHz, and of Stelar Spinmaster and Apollo-Tecmag spectrometers for n > 10 MHz. Standard radio frequency excitation sequences CPMG-like (T2) and saturationrecovery (T1) were used. From the measured T1 and T2 values, we have calculated the longitudinal and transverse relaxivities with the usual formula. 4 mg of the powder nanocomposite was suspended in 5 mL of water. The elemental analysis of the solution gave [Ni] ¼ 0.085 mmol L1 and [Fe] ¼ 0.089 mmol L1. Thus, the concentration of magnetic centers is equal to (0.085 + 0.089) ¼ 0.174 mmol L1.

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Acknowledgements The authors thank Mme Corine Rebeil (UM2, Institute Charles Gerhardt Montpellier, France) for magnetic measurements. The authors also thank the CNRS, the Universite Montpellier II and the network of excellence MAGMANet (FP6-NMP3-CT-2005515767) for financial support as well as the Portuguese network of electron microscopy, the RNME, FCT Project: REDE/1509/ RME/2005.

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