A Gauzzi

Flavio P Milton

Pascotto Gastaldo

M Verseils

A Gualdi

D Von Dreifus

Y Klein

David Garcia

A J A De Oliveira

P Bordet

Ferroelectricity in the 1 µC cm -2 range induced by canted antiferromagnetism in (LaMn3)Mn4O12

Ferroelectricity in the 1 µC cm -2 range induced by canted antiferromagnetism in (LaMn 3 )Mn 4 O 12 A. Gauzzi, 1, a) F. Milton, 2 V. Pascotto Gastaldo, 2 M. Verseils, 1 A. Gualdi, 2 D. von Dreifus, 2 Y. Klein, 1 D. Garcia, 2 A.J.A de Oliveira, 2 P. Bordet, 3 and E. Gilioli 4 1) IMPMC, Sorbonne Université, CNRS, MNHN, 4, place Jussieu, 75005 Paris, France 2) Departamento de Física, Universidade Federal de São Carlos, Rodovia Washington Luís, km 235, São Carlos -São Paulo, 13565-905 Brazil 3) Institut Neél, CNRS and Université Grenoble Alpes, 38042 Grenoble, France 4) Istituto dei Materiali per Elettronica e Magnetismo-CNR, Area delle Scienze, 43100 Parma, Italy (Dated: 6 November 2019) Pyroelectric current and field-dependent specific heat measurements on polycrystalline samples of the quadruple perovskite (LaMn 3 )Mn 4 O 12 give evidence of ferroelectricity driven by the canted antiferromagnetic ordering of the B-site Mn 3+ ions at T N,B =78 K with record large remnant electric polarization up to 0.56 µC cm -2 . X-ray diffraction measurements indicate an anomalous behavior of the monoclinic β angle at T N,B , which suggests that the polarization lies in the ac-plane, where the moments are collinear, and that symmetric exchange striction is the mechanism of spindriven ferroelectricity. Polarization values up to ∼3-6 µC cm -2 are expected in single crystals or epitaxial films, which would enable the development of practical multiferroic applications.

In the search for multiferroic materials for applications, magnetic ferroelectrics, where ferroelectricity is induced by magnetic order, are promising for their inherently strong magnetoelectric couplings 1 . The challenge is to enhance the remnant polarization, which is typically small in these materials, P ∼ 0.1µC cm -2 . This has been attributed to the weakness of the spin-orbit interaction in non-collinear spin structures 2 , where P arises from the antisymmetric exchange striction (inverse Dzyaloshinskii-Moriya interaction) between neighbouring spins. More promising are collinear spin structures, where values up to ∼10 µC cm -2 are predicted due to the comparatively strong symmetric exchange striction 3 . This prediction is supported by the large value P ∼ 1µC cm -2 reached under pressure in the perovskitelike compound TbMnO 3 , characterized by a collinear E-structure 4 . The symmetric exchange mechanism has been invoked to account for a remarkable Penhancement up to 0.27 µC cm -2 in another manganese oxide, (CaMn 3 )Mn 4 O 12 (CMO) 5,6 , with quadruple perovskite (QP) AA 3 B 4 O 12 structure, characterized by two distinct A and B Mn sites 7 . Though, the role of this mechanism in CMO remains controversial 8,9 possibly because of a complex interplay of incommensurate helicoidal spin structure and charge and orbital ordering of the Mn 3+ and Mn 4+ ions 10 .

Here we show that (LaMn 3 )Mn 4 O 12 11,12 (LMO) is a model system to investigate the role of symmetric exchange striction on magnetic ferroelectricity. LMO shares with CMO a similar QP structure, but exhibits simpler structural and electronic properties. LMO is a single-valent Mn 3+ system with neither charge orderings nor incommensurate structural modulations 11 . The Im3 cubic structure stable at high temperatures undergoes a monoclinic I2/m distortion at 653 K 12 . The B-site Mn 3+ ions antiferromagnetically (AFM) order forming a canted C-type structure at T N,B =78 K; the A -sites Mn 3+ ions also order AFM at lower temperatures T N,A =21 a) Corresponding Author. E-mail: andrea.gauzzi@sorbonne-universite.fr K 11 . Here we report on record high electric polarizations ∼ 0.5µC cm -2 in polycrystalline LMO samples induced by the magnetic order at T N,B , so even higher values promising for applications are expected in single-crystalline samples.

The LMO samples studied in the present work are 4 mm diameter cylinders synthesized under high-pressure as described elsewhere 11 . Depending on synthesis conditions, either powders or small, 0.5 mm-sized, single crystals are obtained. Powders are typically 95 % pure and contain minor LaMnO 3 and Mn 3 O 4 impurities. One powder sample was studied by x-ray diffraction as a function of temperature in a D8 Bruker diffractometer equipped with a focusing primary Ge(111) monochromator for Cu K α1 radiation and a closecycle He cryostat. Pyrocurrent, I p , measurements were carried out on three 0.2 mm thin disks of sintered powders of the same batch. The size of the available single crystals was too small for these measurements. Electrodes were realized by depositing a 0.1µm thick Au layer on both sides of the disks by magnetron sputtering. The disks were cooled in a close-cycle cryogenic system down to T p =100 K, at which a poling field E p = 3-36 kV cm -1 was applied for 30 min. At this temperature, the dc resistance of the samples exceeds 1 GΩ, which ensures their full polarization. We subsequently cooled the samples down to 15 K, removed the poling field and applied a short-circuit for 10 min to remove space charges. I p was then measured upon warming-up the samples up to 150 K at rates of 2 -5 K min -1 . On the same polycrystalline samples, we carried out complementary measurements of impedance, Z(T, f ), at frequencies, f , in the 1 kHz -1 MHz range, using a commercial LCR-meter combined with a PPMS system, and of field-dependent specific-heat, C(T, H), using a 2τ relaxation method in zero-field cooling by ramping the field up to H=9 T at constant temperature in the 2-60 K range.

Fig. 1 shows the behavior of the unit cell parameters down to 9 K. No anomalous behavior is found for any of these parameters, except for the monoclinic angle, β, which displays a kink at T N,B , consistent with previous powder neu-tron diffraction data 11 . This indicates a sizable magnetostriction along the diagonal of the ac-plane. In Fig. 2a, the I p (T ) results at different heating rates for both positive and negative E p show that this distortion is concomitant to the appearance of ferroelectricity. The I p (T ) curves reproducibly exhibit a pronounced peak near T N,B , which changes sign according to the polarity of E p , a signature of ferroelectricity induced by the ordering of the B-site Mn 3+ ions. The shift of the I p peak towards high temperatures with increasing heating rate is due to a delay in sample thermalization, which explains the difference between the onset temperature of the peak and T N,B . Above 120 K, one observes a sudden increase of I p , a characteristic signature of the leak current of thermally activated charges since the sign of this current does not change with polarity of E p . This explanation is consistent with the rapid drop of sample resistance above this temperature, seen in the impedance curve, Z(T ), of Fig. 2b. This drop reflects the previously reported semiconducting behavior of LMO 11 . The dependence of the phase, φ(T ), of Z confirms that, below T p =100 K, the response of the sample is predominantly capacitive, i.e. φ ≈ 90 • , thus supporting the reliability of the I p measurements. The Z data are therefore described by a parallel RC equivalent circuit, which enables us to extract the behavior of the sample capacitance, C(T ), shown in Fig. 2b, where one notes a kink at T N,B . This anomaly corroborates the scenario of ferroelectricity induced by magnetic order. 13 Fig. 3 shows the remnant polarization curve, P (T ), obtained by integrating the I p (T ) curve of samples nr. 1 and 2 for different poling fields, E p . In the inset, a plot of the lowtemperature (15 K) values of P with E p shows the characteristic dependence expected for ferroelectrics: P first rapidly increases with E p and then tends to level off at 0.45 µC cm -2 for the maximum value of poling field used, E p =36 kV cm -1 . At this value, saturation is not yet achieved. To give an estimate of the saturation value, P s , we fit the data using a Langevin function and obtain P s = 0.56µC cm -2 . This value -which would not change significantly using alternative fitting functions -is a record for polycrystalline samples of magnetic ferroelectrics, twice as large as that previously reported on CMO single crystals 6 . About 6-10 times larger values are expected in single crystalline film or bulk samples, as previously found in other manganese oxides displaying magnetic ferroelectricity 15 . In CMO, for example, for poling fields of 4 kV cm -1 , P -values increase from ≈ 0.03µC cm -2 in polycrystalline samples 5 up to 0.28 µC cm -2 in single crystals 6 . To explain the observation of magnetic ferroelectricity, we investigated the existence of a magnetoelastic coupling by measuring the effect of a magnetic field H on the specific heat, C(T, H), at low-temperature. This coupling is expected to alter the phonon frequencies and thus the lattice contribution to C. This is confirmed in Fig. 4, where we report the H-dependence of C(T, H) at constant temperature in fieldcooling mode measured on the same batch of LMO powder samples. Note a dramatic reduction of C with field, the relative reduction at 2 K being as large as 40% at 9 T. The reduction progressively decreases with temperature. Above 20 K, the contribution of the AFM transition T N,A =21 K dominates over the magnetoelastic contribution.

We explain this result by Callen and Callen's theory 16 of magnetoelastic coupling for cubic crystals, suitable for the present pseudocubic LMO system. The theory describes the magnetoelastic (ME) energy as an expansion of orthogonal modes for the strain field: H

M E = - µ j,l B µ j,l i ε µ,j i K µ,l i (1)
where B µ j,l (T ) are temperature-dependent magnetoelastic coefficients, j numbers the modes of a given irreducible representation Γ µ (IR) of the strain coordinates ε µ,j i , i numbers the dimensions of the given IR, K µ,l i are tensor cubic operators (TKOs) of degree l forming an orthogonal basis. The Hamiltonian contains only even-degree TKOs, since the Hamiltonian is time-invariant. At low temperatures, the theory predicts that the B µ j,l (T ) coefficients scale with the magnetization of the sublattice, consistent with the l(l + 1)/2 power law originally established by Kittel and van Vleck 17 :

B µ j,l (T )/ Bµ j,l (0) = [M (T )/M (0)] l(l+1)/2 (2)
By knowing the elastic constants of the compound, eqs. (1-2) enable to calculate all thermodynamic quantities like the specific heat of interest here. These constants are not available for LMO, so we limit ourselves to analyze the power-law behavior of the C(H) curve expected from eq. 2. To do so, we consider the external strain caused by an external field, H, which corresponds to the j = 0 strain coordinates for a given IR. The leading contribution is given by the lowest-order (l=2) mode present in the quadrupolar (E g ) Γ γ IR, which is expected to give a characteristic ∼ M (T ) 3 dependence 16 . Since the free energy depends quadratically on the ME coefficients, one finds that the corresponding ME contribution to the specific heat is described by a sum of two power-laws:

C M E T ∼ - ∂ 2 Bγ 0,2 2 ∂T 2 ∼ 3M 5 ∂ 2 M ∂T 2 + 7M 4 ∂M ∂T 2 (3)
Therefore, at a given temperature, the ME contribution C M E grows rapidly with M following a combination of two power laws of index 4 and 5, the weight of each depending on the slope and curvature of the M (T ) curve, respectively.

We apply the above considerations to LMO whose M vs. H dependence exhibits a pronounced kink at H * ∼ 0.1 T (see Fig. 5a and11 ). Consistent with the hysteretic behavior of the magnetization in Fig. 3a, this dependence is characteristic of canted AFM structures 18 like the AFM C-type structure of LMO 11 . Namely, with increasing field, the canting angle is gradually reduced until saturation of the canted component is achieved at H * corresponding to M * ≈ 52 emu/g below 10 K. At higher fields, the response then becomes AFM-like, i.e. M increases linearly with H, as indeed observed in Fig. 5. The experimental M vs H dependence enables us to plot C(H) as a function of M , which we did in Fig. 5b for the 2 K data. Interestingly, note a threshold behavior exactly at M * above which the specific heat decreases rapidly, which gives evidence that the specific heat reduction scales with the magnetization. In addition, a simple data fit indicates that the M vs C behavior above M * is well described by the sum of power laws of eq. 3. An analysis of the magnitude of the Creduction with M would require a measurement of the elastic constants, which goes beyond the scope of the present work. The above finding of large ferroelectric polarization induced by a canted AFM structure and the evidence of a large magnetoelastic coupling raise a number of questions.

(i) As in previously reported magnetic ferroelectrics like TbMnO 3 19 , we have no indication of noncentrosymmetric distortions in LMO, as confirmed by recent high-resolution synchrotron x-ray diffraction study below T N,B 20 . This discrepancy may be due to the difficulty in detecting these distortions by means of diffraction techniques. In alternative, we envisage the existence of polar domains in the nm-scale detected by microscopic and spectroscopic techniques, as reported re-cently on (Ca,Sr) 3 Ti 2 O 7 21 . Therefore, a similar experimental study may be suitable to verify this scenario in LMO.

(ii) As mentioned before, the magnetoelastic response of the β angle indicates that P should lie along the ac-diagonal, thus lowering the I2/m symmetry to Im. Hence, considering that the magnetic structure displays AFM-coupled collinear moments in the ac-plane, the ferroelectricity should be driven by the symmetric exchange coupling. In the scenario of magnetic ferroelectricity proposed for collinear E-type magnetic structures like that of TbMnO 3 3 , a polar distortion is driven by a competition between AFM and ferromagnetic (FM) superexchange interaction between neighboring Mn 3+ ions. A similar scenario may apply here, for in LMO as well the strong tilt of the MnO 6 octahedra characteristic of the QP structure, corresponding to a Mn-O-Mn bond angle ≈136 • does indeed lead to a strong competition, evident from the fact that, in the C-type structure of LMO, the interaction is AFM along the aor c-direction and FM along the b-direction 11 . In presence of this competition, the Im symmetry would therefore trigger the formation of non-equivalent pairs of neighboring Mn 3+ ions within the ac-plane, thus leading to polar distortions.

(iii) The large polarizations in CMO and LMO, suggest that the (AMn 3 )Mn 4 O 12 system is favorable for hosting magnetic ferroelectricity. This may be explained by the fact that the aforementioned AFM-FM competition is maximized by the above tilt angle -remarkably constant in all quadruple perovskites -for this angle is intermediate between 90 • and 180 • . Further favorable conditions may be given by the following features of this structure 22 : 1) the absence of oxygen defects; 2) the high 2/m symmetry of the oxygen sites (higher than in simple perovskites) hinders the distortions of the oxygen sublattice that would tend to screen the electric dipole.

In conclusion, we reported on ferroelectricity with a record large remnant polarization, P s =0.56 µC cm -2 , in polycrystalline samples of the quadruple perovskite LMO, concomitant to a canted AFM ordering of the B-site Mn 3+ ions at T N,B =78 K, accompanied by sizable magnetostriction and magnetoelastic effects. The present finding is striking in two aspects: (i) The above values of polarization, twice as large as those found in CMO single crystals, are obtained on polycrystalline samples, so one expects values as high as ∼ 3 -6 µC cm -2 -comparable to those obtained in proper ferroelectrics -in single crystalline LMO samples, which would be very promising for applications. (ii) If the picture of polarization in the ac-plane will be confirmed, the collinear properties of the AFM order in the ac-plane would support a scenario of ferroelectricity driven by a symmetric exchange coupling between Mn 3+ ions, thus providing a clear guideline for the optimization of the multiferroic properties of magnetic ferroelectrics.

FIG. 1 .

 1 FIG. 1. Temperature dependence of the I2/m unit cell parameters and volume, V , of (LaMn3)Mn4O12 powders. Note the sudden increase of the β angle below the AFM ordering temperature of the B-sublattice, TN,B, which indicates a large magnetoelastic coupling.

FIG. 2 .

 2 FIG. 2. (a):Pyrocurrent, Ip, measured on polycrystalline sample nr. 1 at different heating rates (see legend) for positive and negative poling field, Ep = ±10 kV cm -1 . Tp denotes the poling temperature. Note the peak near the AFM ordering at TN,B=78 K. Inset: the measured disk of LMO sintered powders covered by a Au layer. (b): temperature-dependence of the sample capacitance, C, extracted from the complex-impedance, Z and φ, data of the inset using a parallel RC equivalent circuit (see inset). f denotes the frequency of the measurements. The C-values are not converted into dielectric constant owing to the uncertain contribution of the Schottky barrier created by the Au layer, as discussed in Ref.[START_REF] Cabassi | [END_REF] .

FIG. 3 .

 3 FIG. 3. (a): Magnetic susceptibility of a representative LMO single crystal measured at 100 Oe. Vertical broken lines indicate the AFM ordering temperatures, T N,A and TN,B of the A -and B-sublattices, respectively. Inset: detail of the hysteresis behavior showing the weakly ferromagnetic response of the canted magnetic structure of LMO. (b): Remnant polarization, P , for different poling fields indicated in the legend and for a heating rate of 4 K/min. The curves are obtained by integrating the Ip curves of polycrystalline samples nr. 1 (3 kV/cm data) and 2 (remaining data). Inset: dependence of P upon Ep. A fit of the experimental points using a Langevin function (red solid line) yields a saturation value Ps = 0.56 µC cm -2 .

FIG. 4 .

 4 FIG.4. Field-dependence of the specific heat, C, of a polycrystalline LMO sample at various temperatures. Inset: temperature dependence measured at different fields. Note the jumps at the AFM ordering temperatures, T N,A and TN,B, smeared by the field.

FIG. 5 .

 5 FIG. 5. (a): Magnetization, M (H), curves measured at various temperatures on the same LMO single crystal of Fig. 3a. (b): Mdependence of the 2 K C(H) data of Fig. 4. The experimental points (black dots) are obtained using the 5 K M (H) data of panel (a), unchanged below 10 K. The solid blue line is a best fit using eq. (3). Note the same threshold behavior at M * for both curves.

ACKNOWLEDGMENTS

The authors acknowledge fruitful discussions with L. Chapon, M. Marezio, I. Mirebeau and M. Mostovoy and financial support provided by the FAPESP project 2012/21171-5, CAPES-COFECUB project Ph 878-17 and the Leonardo da Vinci program of the Université Franco-Italienne.


Andrea Pucci

Erica Locatelli

Jessica Ponti

Chiara Uboldi

Valerio Molinari

Mauro Comes

Á E Locatelli

Á V Molinari

Á M Comes

Á C Uboldi

Click chemistry on the surface of PLGA-b-PEG polymeric nanoparticles: a novel targetable fluorescent imaging nanocarrier

Keywords: Click, Fluorescein, Functionalized polymers, Nanochemistry, Glioblastoma

In the quest for biocompatible nanocarriers for biomedical applications, a great deal of effort is put on engineering the nanocomposites surface in order to render them specific to the particular purpose. We developed biocompatible PLGA-b-PEG-based nanoparticles carrying a double functionality (i.e., carboxylic and acetylenic) able to serve as flexible highly selective grafting centers for cancer diagnosis and treatment. As a proof of concept, the nanocarrier was successfully functionalized with a tailored fluorescent molecule by means of click chemistry and with a targeting agent specific for glioblastoma multiforme via amidic bond formation.

Introduction

The addition of functional groups to the surface of nanoparticles (NPs) is usually highly required to enable the bioconjugation, and therefore, to obtain multifunctional nanobiomaterials. Generally, linkers with terminal amino, thiol, or carboxylic groups are preferred [START_REF] Hermanson | Bioconjugate techniques[END_REF]). However, these functional groups are quite common and abundant in the biomolecules; thus, even though often used, in some cases, they could compromise selective conjugation. Moreover, charged species (NH 2 or COOH) give rise to electrostatic (unspecific) interactions that could lead to particles aggregation.

Nevertheless, the multiple presence of functional groups on nanocarriers would certainly be an important item for the synthesis of biohybrid polymeric nanoparticles (PNPs) [START_REF] Metallo | Using bifunctional polymers presenting vancomycin and fluorescein groups to direct anti-fluorescein antibodies to self-assembled monolayers presenting D-alanine-D-alanine groups[END_REF] and the combination of tracking agents, synthetic polymers, and biomolecules into new classes of nanomaterials has indeed gained much interest in recent years and has now become an important field in diagnostics.

Among the most important synthetic polymers, the poly(lactic-co-glycolic acid)-block-poly(ethyleneglycol) (PLGA-b-PEG) deserves a special mention [START_REF] Gu | Biodegradable PLGA-b-PEG polymeric nanoparticles: synthesis, properties and nanomedical applications as drug delivery system[END_REF]. Our interest is (Locatelli and Comes Franchini 2012;[START_REF] Gentili | Double phase transfer of gold nanorods for surface functionalization and entrapment into PEG-based nanocarriers[END_REF] in the PLGA-b-PEG-COOH due to special properties, such as amphiphilicity and its simple synthesis. It is indeed a unique biomaterial: it possess both the biodegradability and biocompatibility of PLGA polymer and the stealth behavior of PEG. Due to its well-distinct lipophilic (PLGA) and hydrophilic (PEG) portions, it can form micelles displaying internally the hydrophobic PLGA part and externally the hydrophilic PEG, which forms a stabilizing external shell. These unique physicochemical characteristics and behavior make the micelles extremely versatile and able to easy entrap, in the inner core constituted of PLGA chains, lipophilic molecules such as drugs or smaller nanostructures. The residual functional groups on the outer shell linked to the PEG can be further be elaborated from the synthetic point of view.

Taking into consideration, the above mentioned problem related to the common functional groups, there is a well-established combination of functional groups that emerged in the last years. Huisgen 1,3dipolar cycloaddition (1,3-DC), involves the reaction of an azido group to an alkyne group and provide fast access to an enormous variety of robust and stable heterocycles called triazoles [START_REF] Huisgen | 1,3-Dipolar cycloadditions[END_REF]. This so called ''click-chemistry'' is therefore an attractive reaction because these two organic functional groups are hardly present in proteins and oligomers. The application of click-chemistry in bioconjugation reactions works for molecularly dissolved species, but can also be used to functionalize the nanostructured materials, giving thus an easy entry to hybrid multifunctional nanobiomaterials [START_REF] Opsteen | Click chemistry in (bio) materials sciences[END_REF]. Moreover, the catalytic effect of Cu(I) ions of this cycloaddition, discovered by Meldal [START_REF] Tornøe | Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides[END_REF] and Sharpless [START_REF] Rostovtsev | Stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective ''ligation'' of azides and terminal alkynes[END_REF], gave a great stimulus to the application of this reaction [START_REF] Moses | The growing applications of click chemistry[END_REF]. Notably, in terms of biological applications, as the bioconjugation, the metal-catalyzed click 1,3-DC proceeds in high yields and occurs at room temperature in aqueous solution at neutral pH.

Therefore, a polymeric nanocarrier based on PLGA-b-PEG carrying a double functionality of such kinds at its hydrophilic surface, and so enabling easy docking of diverse organic bioactive species and tracking agents, would represents a perfect support for theranostic purposes or drug delivery.

Concerning the tracking, fluorescence imaging is one of the most powerful technique currently available for continuous observation of dynamic intracellular processes in living cells [START_REF] Johnson | Review: fluorescent probes for living cells[END_REF][START_REF] Agarwal | Fluorescence imaging: applications in drug delivery research[END_REF][START_REF] Urano | Evolution of fluorescein as a platform for finely tunable fluorescence probes[END_REF]. Fluorescein was first developed in the nineteenth century [START_REF] Weber | Fluorescence excitation spectrum of organic compounds in solution, part 1. Systems with quantum yield independent of the exciting wavelength[END_REF], and has become widely known as a highly fluorescent molecule that emits longer wavelength light upon excitation at around 500 nm in aqueous media. Bioconjugation on polymeric micelles with fluorescent moieties is still an important challenge [START_REF] Wang | Bioconjugation of biotin to the interfaces of polymeric micelles via in situ click chemistry[END_REF], therefore, the functionalization of the commercially available fluorescein isothiocyanate (FITC) in order to link the fluorescent part onto the surface of PNPs would be an important synthetic goal.

We report an innovative biohybrid nanomaterial based on the copolymer PLGA-b-PEG, having multiple outer shell decoration for selective double functionalization. In details, one functional group is an acetylenic group to develop a click-reaction with a synthetic azidofluorescein. The second functional group, the carboxylic group, is used to conjugate the Chlorotoxin (Cltx), a specific peptide for in vitro targeting of the human primary brain tumor glioblastoma U87MG cell line.

Results and discussion

While the PLAG-b-PEG-COOH has been previously fabricated [START_REF] Cheng | Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery[END_REF]) and it was reproduced in this work following a similar methodology, the copolymer ending with an acetylenic (CCH) group PLGA-b-PEG-CCH was synthesized for the first time by readapting the same procedure and replacing the NH 2 -PEG-COOH reagent by its acetylenic commercial counterpart NH 2 -PEG-CCH. The twosteps protocol is straightforward and leads to a clean and stable co-polymer in 85 % yield (Scheme 1). A derivative of fluorescein, which was synthesized for the first time by a common nucleophilic addition reaction between the FITC and the amino group of a p-azidoaniline, was chosen as a fluorescent dye. As reported in Scheme 1, the reaction was carried out in N,N-dimethylformamide (DMF) with triethylamine (TEA) giving 1-(4-azidophenyl)-3-(3 0 ,6 0 -dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9 0 -xanthen]-5-yl)thiourea 1 in 90 % yield. A similar approach to obtain azido-terminal fluorescein was already pursued exploiting more expensive and hazardous linkers [START_REF] Lin | Site-specific protein modification through CuI-catalyzed 1,2,3-triazole formation and its implementation in protein microarray fabrication[END_REF]). The substituted fluorescein 1 carries now a pendant azido group which can react with an acetylenic moiety via the copper-catalyzed cycloaddition (CuAAC) reaction.

The PNPs fabrication was achieved through the nanoprecipitation technique [START_REF] Franchini | Design and synthesis of novel 3,4-disubstituted pyrazoles for nanomedicine applications against malignant gliomas[END_REF]) by solubilizing in DMSO a 50:50 blend of the two copolymers PLGA-b-PEG-COOH and PLGA-b-PEG-CCH, and subsequently mixing it with water under vigorous stirring and maintaining the water/ organic ratio 10:1 with constant removal of the solution (Scheme 2). The as-fabricated nanocarrier PNPs-C/CCH presents a hydrodynamic size of 136.4 ± 0.9 nm with a rather small polydispersity (PDI = 0.067 ± 0.018). The bifunctionalized surface of the PNPs leads to a multimodal distribution of the fpotential with peaks falling in a voltage range lower than -50 mV, indicating very high stability of the NPs (see DLS results in the supporting information). The DLS analyses were repeated over time up to 4 months showing no major changes. Thus, we can assume that the NPs are stable for several months if stored at 4 °C. Next, we focused our attention on the tailoring of the surface functionalities in order to exploit the fertile docking centers. Indeed, the polymeric nanocarriers PNPs-C/CCH represent a perfect support enabling easy docking of diverse organic bioactive species and tracking agents. In order to prove the applicability, the nanocarrier was functionalized with, on one hand, the as-synthesized fluorescent dye 1 and, on the other hand, the peptide Cltx.

The click-reaction ensures high specificity as well as processability in water [START_REF] Rostovtsev | Stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective ''ligation'' of azides and terminal alkynes[END_REF]. The water solution of PNPs-C/CCH was mixed with 1 in the presence of a catalytic amount of CuSO 4 and sodium ascorbate (Scheme 2). Upon reacting overnight at room temperature, the fluorescent moiety was linked, creating a triazole ring, onto the surface giving PNPs-C/Fluo, and leaving the COOH group still available. Using the typical 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) chemistry, we then chemically linked the commercially available Cltx to the outer surface (COOH, ratio Cltx:COOH 8.5:1) of the PNPs-C/Fluo. The reaction was carried out overnight at room temperature, and after purification we obtained PNPs-Fluo/Cltx. It is worth mentioning that the two organic molecules attached on the surface sparingly decrease the stability of the particle leading to a hydrodynamic size of 180.3 ± 3.1 nm with a PDI of 0.146 ± 0.014 and a f-potential of -38.1 mV at nearly neutral pH.

The sequence of the reaction proved not to be indifferent. Trials were also carried out conjugating the Cltx onto the nanocarrier surface as a first step, prior to the cycloaddition reaction. The as-fabricated nanocarriers presented bigger size as well as higher polydispersity at the DLS (data not shown). Evidently, once the 36-amino acids peptide is linked on the surface, it constitutes an obstacle to the coppercatalyzed click reaction.

The luminescence properties of the PNPs-Fluo/Cltx together with the one of azidofluorescein and of the bare PNPs were monitored using a spectrofluorometer. The excitation and emission spectra are depicted in Fig. 1a, b, respectively. The excitation of the azidoterminal fluorescein occurs at 460 nm and it undergoes a red shift to 491 upon linkage onto the particle surface. The green intense emission falls at 522 and at 516 nm for the p-azidofluorescein and the PNPs-Fluo/ Cltx, respectively. As expected, the polymeric NPs absorb in the near UV (309 nm), and consequently do not emit when excited in the blue spectral range. We decided to link the Cltx, a peptide that specifically binds to metalloproteinase 2 (MMP-2), a receptor over-expressed by brain cancer (glioblastoma cells) due to our interest in this research [START_REF] Franchini | Design and synthesis of novel 3,4-disubstituted pyrazoles for nanomedicine applications against malignant gliomas[END_REF]Locatelli et al. 2012), therefore, U87MG cells (a stabilized cell line derived from human glioma) were exposed to 0.88 lg/mL of PNPs-Fluo/Cltx. This testing concentration is no toxic, as previously observed (Locatelli et al. 2012). In fact, U87MG cells were exposed to 50 lg/mL of PNPs-Cltx for 72 h, and by trypan blue exclusion dye test, we observed cell viability of 84 % of the unexposed cells (negative control) (Locatelli et al. 2012).

As shown in Fig. 2, PNPs-Fluo/Cltx (in green, in Fig. 2b,e) were internalized by U87MG glioblastoma-astrocytoma cells after both 48 (Fig. 2a-c) and 72 h (Fig. 2d-f) of exposure. Micelles were mainly localized in the cytoplasm and we excluded their internalization in the nuclei analysing each image of the slices obtained by Z-stack (ApoTome slide module, Carl Zeiss, Germany). At the concentrations tested, they did not alter the cell morphology (Fig. 2c-f) showing, also considering the toxicological results, biocompatibility of the system. Our results suggest that the PNPs-Fluo/Cltx, thanks to the presence of both a fluorescent dye and to the specificity of Cltx for cancer cells, could be potentially used as targeting agent for diagnostic and therapy.

Conclusion

In conclusion, we have developed the synthesis of a simple polymeric NPs based on PLGA-b-PEG having two functional groups on the outer shell. This nanocarrier bears an acetylenic group on which we have linked a fluorescent tag using a click reaction and, moreover, a carboxylic group, that is able to conjugate peptides and proteins through simple amidation chemistry. As already mentioned, the click chemistry is becoming a major pathway among the biological conjugations and many dyes or bioactive molecules carrying an azido pendant are already commercially available. On the other hand, engineering the nanocarrier surface by modifying the pristine polymers instead of venturing on later modification seems to be a preferable approach which can be further expanded to a large variety of terminal groups. Accordingly, this novel targetable nanocarrier has been tested in the in vitro tracking of a glioma cell line, and is therefore currently being investigated in our laboratories for active drug delivery both in vitro and in vivo against glioblastoma.

Experimental section

Synthesis of PNPs-C/CCH

PLGA-b-PEG-COOH (10 kDa, 25 mg, 0.0025 mmol) and PLGA-b-PEG-CCH (10 kDa, 25 mg, 0.0025 mmol) were solubilized in 5 mL of DMSO. The organic phase was mixed to 50 mL of ultrapure water under vigorous stirring, maintaining water/organic ratio 10/1 with a constant removal of the solution. The mixture was kept for 30 min under vigorous stirring. The solution was concentrated to a volume of 10 mL using a tangential flow filter (Pellicon XL filter device, Biomax membrane with 500.000 NMWL, Millipore, USA) following by filtration using a syringe filters Sterivex TM -GP of polyethersulfone (0.22 lm, Millipore, USA). Hydrodynamic size = 136.4 ± 0.9 nm, PDI = 0.067 ± 0.018, and f-potential = trimodal, far above the stability threshold.

Synthesis of PNPs-C/Fluo and PNPs-Fluo/Cltx

A water solution of PNPs-C/CCH (2 mL, -COOH and -CCH 1 lmol) was mixed with the p-azidofluorescein 1 (40 mg, 0.076 mmol) together with copper sulfate hydrate (0.7 mg, 0.0028 mmol) and (?)-sodium L- ascorbate (0.3 mg, 0.0015 mmol). The mixture was allowed to stir overnight. The resulting particles were washed with ultrapure water (up. water) using a centrifugal (3000 rpm, 15 min) filter devices (Amicon Ultra, Ultracel membrane with 100.000 NMWL, Millipore, USA) till the washing water resulted in a colorless fluid. Finally, the particles were redispersed in 5 mL of up. water.

To this dispersion, a water solution containing Nhydroxysulfosuccinimide (0.1 mg, 0.46 lmol) was added together with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 2 mg, 0.012 mmol) that was pre-acidified with 0.1 M HCl solution to neutral pH (*7). The solution was shacked for 30 min to permit the activation. Next, the Cltx (0.5 mL of a 0.017 M solution, 0.0085 mmol) was added to the reaction mixture and it was allowed to shake overnight. The resulting particles were washed with up. water using a centrifugal (3000 rpm, 15 min) filter devices (Amicon Ultra, Ultracel membrane with 100.000 NMWL, Millipore, USA). Finally, the particles were filtered through a Sterivex TM -GP of polyethersulfone (0.22 lm, Millipore, USA). Hydrodynamic size = 180.3 ± 3.1 nm, PDI = 0.146 ± 0.014, f-potential = -38.1 mV, and pH = 7.04. Polymer concentration obtained by weighing a dried aliquot of the final solution = 1.226 mg/mL.

Intracellular localization of PNPs-Fluo/Cltx

To perform microscopy studies, cells were seeded onto 4-chamber polystyrene vessel tissue culturetreated glass slides (BD Falcon, Italy) in 1 mL of complete cell culture medium and cultured under standard cells culture conditions (37 °C, 5 % CO 2 , 95 % humidity). After 24 h, cells were exposed to 0.88 lg/mL of PNPs-Fluo/Cltx for 48 h and 72 h; then fixed with 4 % (v/v) formaldehyde (Sigma Aldrich, Italy) solution in PBS and stained using Hoechst-33342 (Invitrogen, USA) for nuclei. Images were acquired using an Axiovert 200 M inverted microscope (Carl Zeiss, Germany) equipped with ApoTome slide module and AxioVision 4.8 software (Carl Zeiss, Germany), using 409/1.0 objective lens. Images were acquired using a black and white AxioCam MRm (Carl Zeiss, Germany) and pseudo-colors were applied after image acquisition.

  Scheme 1 Structures of PLGA-b-PEG-based polymers and synthesis scheme of 1-(4azidophenyl)-3-(3 0 ,6 0dihydroxy-3-oxo-3Hspiro[isobenzofuran-1,9 0xanthen]-5-yl)thiourea 1

Fig. 1

 1 Scheme 2 Schematic of the PNPs-Fluo/Cltx fabrication

Fig. 2

 2 Fig. 2 Intracellular localization of PNPs-Fluo/Cltx in U87MG cells. After 48 h (a-c) and 72 h (d-f) exposure, PNPs-Fluo/Cltx micelles were observed located in the cytoplasm of U87MG

J Nanopart Res (2013) 15:1818

Acknowledgments

The work was partly supported with the funds of SaveMe EU-FP7 project no. CP-IP 263307-2.