Nanoscale coordination polymers exhibiting luminescence properties and NMR relaxivity

Abstract

This article presents the first example of ultra-small (3–4 nm) magneto-luminescent cyano-bridged coordination polymer nanoparticles Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻ (Ln = Eu (x = 0.34), Tb (x = 0.35)) enwrapped by a natural biocompatible polymer chitosan. The aqueous colloidal solutions of these nanoparticles present a luminescence characteristic of the corresponding lanthanides (⁵D₀ → ⁷F_0–4 (Eu³⁺) or the ⁵D₄ → ⁷F_6–2 (Tb³⁺)) under UV excitation and a green luminescence of the chitosan shell under excitation in the visible region. Magnetic Resonance Imaging (MRI) efficiency, i.e. the nuclear relaxivity, measurements performed for Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻nanoparticles show r_1p and r_2p relaxivities slightly higher than or comparable to the ones of the commercial paramagnetic compounds Gd-DTPA® or Omniscan® indicating that our samples may potentially be considered as a positive contrast agent for MRI. The in vitro studies performed on these nanoparticles show that they maybe internalized into human cancer and normal cells and well detected by fluorescence at the single cell level. They present high stability even at low pH and lack of cytotoxicity both in human cancer and normal cells.

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I. 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 which is capable of exhibiting diverse physical responses when subjected to certain external stimuli. In 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 able to combine two or more functions, such as different types of imaging or imaging with drug delivery, targeting, or various therapies.² One of the most promising multifunctional nano-objects should present a combination of magnetic and optical properties within a single hybrid nano-system in order to combine luminescence biolabelling and Magnetic Resonance Imaging (MRI). Generally, the approaches used for the synthesis of those multifunctional nanomaterials consist in design complex hybrid nano-objects containing two or more components with luminescence and magnetic relaxivity. We can cite numerous works on magnetic metallic or metal-oxide core–shell nanoparticles where luminescent organic dyes, Au nanoparticles or metal complexes are incorporated into silica or polymer shells or attached on their surface.^2g,3 Other works concern silica or polymer nanoparticles used as a platform for incorporation or covalent surface anchoring of magnetic nanoparticles, luminescent complexes, organic dyes or paramagnetic Gd³⁺ complexes.⁴ Note that in most cases, superparamagnetic iron oxide nanoparticles or more rarely Gd–macrocycles are used to confer the magnetic properties to these hybrid nano-objects.

Very recently, nanoparticles of molecule-based materials as a new type of magnetic inorganic nanoparticles were explored.⁵ These nanoparticles present an increasing interest due to their specific nature, which is different in comparison to other inorganic nano-objects. They present all advantages of bulk molecular-based materials, such as determined and flexible molecular structures, adjustable physical and chemical properties, porosity, low density, and the possibility to combine several properties in the same multifunctional nano-objects. Furthermore, as their bulk analogous, the nanoparticles may be obtained from molecular precursors by using “soft” chemistry methods by self-assembling reactions. On the other hand, it is possible to design the nano-objects in such a way that they possess controlled size, shape and organization at the nano-scale level, and thus their physical and chemical properties may be controlled. Indeed, the synthesis of nano-objects based on cyanometallates, carboxylates or phosphate ligands presenting exciting magnetic or optical properties was performed.⁶ These nanoparticles present an increasing interest for biomedical applications, and few works on the investigation of lanthanides-⁷iron-based⁸ Metal–Organic Frameworks (MOFs) nanoparticles, and cyano-bridged coordination polymer nanoparticles⁹ as new systems of contrast agents for Magnetic Resonance Imaging (MRI), optical imaging and drug delivery have been reported recently. Among these, a small number of examples have been devoted to designing multifunctional nanoparticles. We can cite hybrid polymer or silica coated nanoparticles of Gd-¹⁰ and Mn-based¹¹ MOFs with organic fluorophores on their surface, supramolecular coordination polymer networks based on lanthanides ions and phosphate-modified nucleotides with encapsulated organic fluorophore¹² or silica nanoparticles containing fluorophore coated with magnetic cyano-bridged coordination poymer.¹³ Among those, only one recently published work concerns the synthesis of nanorods with a bi-functional magneto-luminescent core by a combination of paramagnetic gadolinium with luminescent europium or terbium ions in the same MOF in order to achieve a bi-modal MRI-optical imaging probe.¹⁴ However, these nano-objects present a relatively large size with large size distribution that makes difficult their internalization into the cells and more importantly their elimination from the body. In addition, it was shown that due to their relatively low stability, MOFs nanoparticles release the metal ions.^10,14

In the present work, we demonstrate a new approach to the synthesis of ultra small magneto-luminescent coordination polymer nanoparticles designed from octacyanomolybdate building block and lanthanides ions which can be considered as a new family of bi-functional probe for MRI and optical imaging. This approach is based on an association of luminescent Ln³⁺ and paramagnetic Gd³⁺ ions with paramagnetic octacyanomolybdate building blocks in order to obtain magneto-luminescent cyano-bridged core of nanoparticles stabilized by the biopolymer chitosan. These small nanoparticles of 3–4 nm are biocompatible, they present high stability event at low pH and may potentially be used for imaging in specific conditions (for instance a gastro-intestinal tract). Here, we present the synthesis, the evaluation of the Nuclear Magnetic Resonance (NMR) relaxivity and the luminescence properties of ultra-small, bi-functional magneto-luminescent coordination polymer nanoparticles Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻ (Ln = Eu (x = 0.34), Tb (x = 0.35)) enwrapped by the natural biopolymer chitosan used as a stabilizing agent. A first characterization of the cells uptake of these nanoparticles indicates their efficient internalization and fluorescence detection into human cancer and normal cell line and the lack of cytotoxicity related to their high stability.

II. Experimental part

Synthesis

All of the chemical reagents used in these experiments are analytical grade: Ln(NO₃)₃·6H₂O (Eu from Alfa Aesar, Tb and Gd from Rhone-Poulenc). (N(C₄H₉)₄)₃[Mo(CN)₈] was prepared according to the literature procedure.¹⁵ The pristine porous chitosan beads were synthesized as previously described.¹⁶ The degree of acetylation (DA) which is the percent of remaining acetyl groups was 10% as measured by IR spectroscopy.

Synthesis of Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻/chitosan (Ln = Eu (x = 0.34), Tb (x = 0.35)) nanoparticles. The synthesis of Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻/chitosan nanoparticles (Ln = Eu, Tb) was performed in two steps, consisting first in the synthesis of chitosan nanocomposites containing the Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻nanoparticles (1a, 2a) incorporated into the chitosan beads and second in the solubilization of the most part of the chitosan matrix in acidic water in order to obtain aqueous colloidal solutions of the nanoparticles (1b, 2b). The nanocomposite beads Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻/chitosan (Ln = Eu 1a, Tb 2a) were prepared by subsequent incorporation of Gd(NO₃)₃·6H₂O, Ln(NO₃)₃·6H₂O and (N(C₄H₉)₄)₃[Mo(CN)₈] into the pristine porous chitosan beads. The typical synthesis for 1a or 2a was performed as follows: the pristine chitosan beads (90 mg) were added to a mixture of methanolic solutions of Ln(NO₃)₃·6H₂O (Ln = Eu or Tb) (5 g L⁻¹) and of Gd(NO₃)₃·6H₂O (5 g L⁻¹). The mixture was stirred overnight at room temperature and then the solution was filtered off. The beads were washed copiously with methanol and then dried in vacuo. Then, the composite Ln_0.33³⁺Gd_x³⁺/chitosan (Ln = Eu (x = 0.34), Tb (x = 0.35)) (90 mg) was added to a 10⁻² M methanolic solution of the (N(C₄H₉)₄)₃[Mo(CN)₈]. The mixture was stirred 48 h, the beads were filtered, thoroughly washed with methanol and dried in vacuo. The consecutive treatment with a mixture of methanolic solutions of Gd(NO₃)₃·6H₂O and Ln(NO₃)₃·6H₂O and then with a methanolic solution of (N(C₄H₉)₄)₃[Mo(CN)₈] was repeated twice. In the second step, in order to obtain colloidal solutions of the coordination polymer nanoparticles (1b, 2b), the respective nanocomposite beads (100 mg) were immersed in a buffer solution (20 mL, pH 4.25) overnight with stirring. The resulting suspension was centrifuged (15 min, 20 000 trs min⁻¹) and the supernatant containing the nanoparticles recovered. As a result, colloidal solutions containing nanoparticles Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻/chitosan (Ln = Eu 1b, or Tb 2b) were obtained. These colloidal solutions are very stable and no any precipitate was observed even after four months. The solutions may be concentrated or diluted in water. For biological assays, the pH of solutions was adjusted to 7.2 by using 0.1 M aqueous KOH solution. The stability tests of the nanoparticles were performed both in buffered aqueous solution with pH = 4.25 (acetic acid buffered solution) and in a physiological media McCoy's 5A, supplemented with 10% fetal bovine serum, penicillin 100 U mL⁻¹and streptomycin 100 µg mL⁻¹ used for the maintenance of HCT116 colon cancer cells. The TEM image of the nanoparticles 1b dispersed into these physiological media shown that the nanoparticles are well dispersed and not aggregated (ESI, Fig. S1†). The nanoparticles were then precipitated from both solutions with ethanol and the ICP-MS analysis of the mother solutions shows no detectable presence of Gd³⁺.

Physical measurements

IR spectra were recorded on a Perkin Elmer 1600 spectrometer with a 4 cm⁻¹ resolution. TEM measurements were carried out with a microscope JEOL 1200 EXII operated at 100 kV, HRTEM observations were performed on a JEOL JEM2010 operated at 200 kV. In the latter, coupled EDS analysis was performed on single nanoparticles. Samples for TEM measurements were prepared using ultramicrotomy techniques and then deposited on copper grids for the solids. In the case of the colloids, samples were prepared simply by depositing a drop of the nanoparticles' solutions on carbon coated copper grids. The nanoparticles' size distribution histogram was determined using enlarged TEM micrographs taken at magnification of 100 K on a statistical sample of ca. 300 nanoparticles. An evaluation of the Ln/Gd/Mo ratio on assemblies of nanoparticles was performed by using an Environmental Secondary Electron Microscope FEI Quanta 200 FEG coupled with an Electrons Dispersive Spectroscope Oxford INCA detector. Elemental analyses (ICP-MS) were performed by the Service Central d'Analyse (CNRS, Vernaison, France). Magnetic susceptibility data were collected with a Quantum Design MPMS-XL SQUID magnetometer. The photoluminescence spectra were recorded at room temperature on a Fluorolog-3 Model FL3-2T with double excitation spectrometer and a single emission spectrometer (TRIAX 320) coupled to a R928 photomultiplier, using a front face acquisition mode. The excitation source was a 450 W Xenon lamp. Emission was corrected for the spectral response of the monochromators and the detector using a typical correction spectrum provided by the manufacturer and the excitation spectra were corrected for the spectral distribution of the lamp intensity using a photodiode reference detector. Emission decay curve measurements were carried out at room temperature with the setup described for the luminescence spectra using a pulsed Xe–Hg lamp (6 µs pulse at half width and 20–30 µs tail). 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 ≤ ν ≤ 10 MHz,¹⁷ and of Stelar Spinmaster and Apollo-Tecmag spectrometers for ν> 10 MHz. Standard radio frequency excitation sequences CPMG-like (T₂) and saturation-recovery (T₁) were used. From the measured T₁ and T₂ values, we calculated the longitudinal and transverse relaxivities with the usual formula. To perform the confocal analysis, the nanoparticles solution was added in serum free culture medium of each cell type at the dose of 100 ng mL⁻¹. The day prior to the experiment, HCT116 and Capan-1 cells were seeded onto bottom glass dishes (World Precision Instrument, Stevenage, UK) at a density of 30 000 cells cm⁻². On the day of the experiment, cells were washed once and incubated in 1 mL red-free medium containing Eu_0.33³⁺Gd_0.35³⁺/[Mo(CN)₈]³⁻/chitosan nanoparticles 1b at a concentration of 100 ng mL⁻¹ for 6 h. 30 min before the end of incubation, cells were loaded with Hoechst 33342 (Invitrogen, Cergy Pontoise, France) for nuclear staining at a final concentration of 5 µg mL⁻¹. For the lysosome labelling, 3 h before the end of the experiment, 50 nM of lysotracker red DND-99 (Invitrogen) were added to phenol red-free medium. Before visualization, cells were washed gently with phenol red-free medium. Cells were then scanned with a LSM 5 LIVE confocal laser scanning microscope (Carl Zeiss, Le Pecq, France), with a slice depth (Z stack) of 0.67 µm and at 489 nm excitation wavelength. The distributions of fluorescent nanoparticles were analyzed by CLSM using nucleus and lysosome markers. Merged images of fluorescent nanoparticles and lysosome marker allow establishment of the level of nanoparticles into lysosomes.

Cell culture

Cancer cell types were purchased from ATCC (American Type Culture Collection, Manassas, VA), HUVECs were purchased from PromoCell and normal fibroblasts were obtained from Prof. Dr Kurt von Figura (Göttingen). All cell types were allowed to grow in humidified atmosphere at 37 °C under 5% CO₂. Capan-1 human pancreatic cancer cells were routinely maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 50 µg mL⁻¹gentamycin. HCT116 colon cancer cells were routinely maintained in McCoy's 5A medium, supplemented with 10% fetal bovine serum, penicillin 100 U mL⁻¹and streptomycin 100 µg mL⁻¹. HUVECs (human umbilical vein endothelial cells) were routinely cultured in Endothelial Cell Growth Medium 2 (ECGM 2) supplemented with fetal calf serum (2%), epidermal growth factor (5 ng mL⁻¹), basic fibroblast growth factor (10 ng mL⁻¹), insulin-like growth factor (20 ng mL⁻¹), vascular endothelial growth factor (0.5 ng mL⁻¹), ascorbic acid (1 µg mL⁻¹), heparin (22.5 µg mL⁻¹), hydrocortisone (0.2 µg mL⁻¹), penicillin (100 U mL⁻¹) and streptomycin (100 µg mL⁻¹). Human fibroblasts were routinely maintained in DMEM supplemented with 7.5% fetal bovine serum, penicillin 100 U mL⁻¹ and streptomycin 100 µg mL⁻¹.

MTT cell viability assay

For cell viability experiments, Capan-1 and HCT-116 cells were seeded into 96-well plates at 10 000 cells per well in 100 µL of their respective culture medium. HUVECs and fibroblasts were seeded into 96-well plates at 5000 cells per well in 100 µL of their culture medium. One day after seeding, cells were then incubated 4 days with different doses of Eu_0.33³⁺Gd_0.35³⁺/[Mo(CN)₈]³⁻/chitosan nanoparticles 1b (0, 25, 50, 100 ng mL⁻¹). Following this incubation, cells were incubated for 4 h with 0.5 mg mL⁻¹ of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Promega) in media. The MTT/media solution was then removed and the precipitated crystals were dissolved in ethanol/DMSO (1/1). The solution absorbance was read at 540 nm.

III.Results and discussion

III.1. Synthesis, structural and textural characterizations of Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻/chitosan (Ln = Eu (x = 0.34), Tb (x = 0.35)) nanoparticles

Our approach consists in an association of luminescent Ln³⁺ (Ln = Eu, Tb) and paramagnetic Gd³⁺ ions with the paramagnetic octacyanomolybdate building block in order to obtain a bi-functional magneto-luminescent core of the nanoparticles enwrapped by a biopolymer chitosan. The choice of these components was performed from the reasons given hereafter. The paramagnetic [Mo(CN)₈]³⁻ do not efficiently absorb visible light and prevents the quenching of the Ln³⁺ luminescence by energy transfer processes contrarily to what is observed for the [Fe(CN)₆]³⁻ building block. In association with lanthanide ions at the macroscopic level [Mo(CN)₈]³⁻ forms very stable bulk two-dimensional cyano-bridged coordination polymer network presenting magnetic properties and the lanthanides luminescence.¹⁸ Further, the choice of the chitosan beads for the formation and stabilization of nano-sized cyano-bridged coordination polymers has been done due to its interesting physical and chemical properties. The chitosan is a biocompatible, biodegradable, bioactive, and non-toxic polymer derived from the partial deacetylation of chitin and thus possessing free amino groups allowing coordination of metal ions (Scheme 1a). It presents high water solubility and can be produced in the form of porous beads obtained by drying gel beads of the polymer chitosan under supercritical CO₂ conditions in order to provide easy circulation of precursors into the pores and increasing the access to the functional amino groups.¹⁶

Scheme 1 Schematic representation of (a) the chemical structure of chitosan; (b) the synthesis of coordination polymer nanoparticles Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻/chitosan (Ln = Eu (x = 0.34), Tb (x = 0.35)).

The synthesis of Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻/chitosan (Ln = Eu (x = 0.34), Tb (x = 0.35)) nanoparticles was performed by using a two-step procedure. The first step consists in the intrapore growth of the cyano-bridged network of the nanoparticles into the chitosan matrix by step-by-step coordination of lanthanides ions and octacyanomolybdenum in order to obtain the nanocomposite beads containing the coordination polymer nanoparticles (1a, 2a). The second step concerns the solubilization of the chitosan matrix in acidic water to obtain the corresponding aqueous colloidal solutions containing Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻/chitosan (Ln = Eu (x = 0.34), Tb (x = 0.35)) nanoparticles (1b, 2b) (Scheme 1).

As the first step of our approach, two subsequent treatments of the pristine porous chitosan beads with methanolic solutions of Gd(NO₃)₃·6H₂O, Ln(NO₃)₃·6H₂O (Ln = Eu, Tb) and (N(C₄H₉)₄)₃[Mo(CN)₈] allow us to obtain two types of nanocomposite beads Eu_0.33³⁺Gd_0.34³⁺/[Mo(CN)₈]³⁻/chitosan 1a and Tb_0.33³⁺Gd_0.35³⁺/[Mo(CN)₈]³⁻/chitosan 2a. At each step of the treatment, the chitosan beads were thoroughly washed with methanol and dried in vacuo. The atomic ratio Ln and Gdvs.Mo as inferred from elemental and Energy Dispersive Spectroscopy (EDS) analyses is given in Table 1. Compared to the stoichiometry found for the corresponding bulk compounds¹⁸i.e. 1/1, the nanoparticles present a lack in lanthanide ion vs.molybdenum (ca. 0.65/1). A similar ratio was observed in the case of coordination polymer nanoparticles containing lanthanide and hexacyanometallates,^19a and may be attributed to the specific step-by-step growth of the cyano-bridged framework onto the chitosan matrix instead of the co-precipitation method employed for the preparation of the bulk materials.

Table 1 Some relevant characteristics for the samples 1a,b and 2a,b

Composition	Sample	Ln/Gd/Mo ratio^a	IR, ν(CN)/cm^−1	NPs size in aqueous solution/nm
a Calculated from elemental analysis.
Eu0.33^3+Gd0.34^3+/[Mo(CN)8]^3^−/chitosan	1a,b	0.33/0.34/1	2113	4.5(2.4)
Tb0.33^3+Gd0.35^3+/[Mo(CN)8]^3^−/chitosan	2a,b	0.33/0.35/1	2113	2.8(0.8)

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In order to obtain information about the homogeneity of the cyano-bridged network dispersion into the chitosan beads, EDS analysis was performed on the total internal surface of a cleaved nanocomposite bead. As an example, Fig. 1a shows an internal view of the cleaved nanocomposite bead 1a with the line along which the EDS analysis was performed. Eu, Gd and Mo contents are shown to be homogenous on the entire surface of the cleaved bead (Fig. 1b).

Fig. 1 (a) Internal view of a cleaved bead of 1a. The yellow line represents the line (total length = 600 µm) along which the profile curves were obtained; (b) EDS profile curves of 1a for Eu, Gd and Mo.

As the second step, the straw-colored nanocomposite beads were solubilized in slightly acidic water (acetate buffer, pH 4.25) giving rise to aqueous solutions of the same color of Eu_0.33³⁺Gd_0.34³⁺/[Mo(CN)₈]³⁻/chitosan 1b or Tb_0.33³⁺Gd_0.35³⁺/[Mo(CN)₈]³⁻/chitosan 2b (Scheme 1b). It is important to note that these colloidal solutions are extremely stable over time and no any precipitation or aggregation of nanoparticles was observed during few months. Such stability in time was attributed to the presence of a residual corona of chitosan surrounding the nanoparticles.^9a,19 The dry residues of these solutions present the same Ln/Gd/Mo ratio as for the nanocomposite beads (Table 1).

The infrared (IR) spectra of the nanocomposite beads 1a and 2a and their corresponding aqueous solutions 1b and 2b show the band at 2113 cm⁻¹ corresponding to the stretching vibrations of the bridging –Mo⁵⁺–CN–Ln³⁺– indicating the formation of the cyano-bridged network (Table 1, ESI, Fig. S2†). Similar band has also been observed in the case of the bulk analogous.¹⁸ As expected, the IR spectra also present chitosan vibration bands at 3237 (NH₂ and OH groups), 1660 (acetamide groups) and 1598 cm⁻¹ (NH₂ groups).¹⁹

Transmission Electronic Microscopy (TEM) observations were performed on the nanocomposite beads 1a, 2a and on the corresponding aqueous solutions 1b, 2b. As an example, Fig. 2a shows a TEM image of the nanocomposite beads 1a. The porosity of the chitosan matrix can be clearly seen as well as the presence of non-aggregated homogeneously dispersed nanoparticles appearing as black dots into the chitosan (inset on the left of Fig. 2a). A High Resolution Transmission Electronic Microscopy (HRTEM) image of a single nanoparticle displaying the atomic planes alignment is also given in the inset on the right of Fig. 2a (ESI, Fig. S3†). The EDS analysis coupled with HRTEM on isolated nanoparticles 1a confirmed the homogeneous dispersion of Mo, Eu and Gd ions in proportions identical to those obtained on assemblies of nanoparticles (i.e. a Eu/Gd/Mo ratio of ca. 0.3/0.3/1) (ESI, Fig. S4†). Fig. 2b and c present TEM and HRTEM images of nanoparticles 1b obtained after solubilization of the chitosan matrix in water. They show spherical in shape, non-aggregated and well dispersed nanoparticles with the size distribution centered at 4.5 nm. The size distribution histogram for nanoparticles 1b is given in Fig. 2d. The nanoparticles 2a,b present similar textural characteristics: narrow sized spherical coordination polymer nanoparticles were observed with the size distribution centered 2.8 nm (ESI, Fig. S5†).

Fig. 2 (a) TEM image of the nanocomposite beads 1a. Insets: magnification of this image and HRTEM image of a single nanoparticle; (b) TEM and (c) HRTEM images of an aqueous colloidal solution of 1b; (d) Size distribution histogram obtained for the nanoparticles 1b. White circles are given as guide for the eyes.

Consequently, the step-by-step coordination of the respective precursors allowed us to achieve cyano-bridged coordination polymer nanoparticles with a good control of the Ln/Mo stoichiometry in the nanoparticle core as well as of the nanoparticles size which cannot be achievable by using a co-precipitation method. Note also that these nanoparticles are perfectly soluble in aqueous solutions, which may be concentrated or diluted in water and their pH may be increased up to 7.2. These nanoparticles may also be dispersed into physiological media without any precipitation (see Experimental part concerning the composition of the physiological media and ESI Fig. S1† for the TEM images of this solution). These are important requirements for the use of the nanoparticles for potential biological applications.

III.2. Investigations on magneto-luminescent properties of Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻/chitosan nanoparticles

Photoluminescence properties. In order to prove the bi-functionality of the obtained nanoparticles, we investigate both the photoluminescence and the relaxivity studies of their aqueous colloidal solutions. The photoluminescent properties of the nanoparticles 1b, 2b were investigated at room temperature. Fig. 3 shows the emission spectra of Eu_0.33³⁺Gd_0.34³⁺/[Mo(CN)₈]³⁻/chitosan 1b and Tb_0.33³⁺Gd_0.35³⁺/[Mo(CN)₈]³⁻/chitosan 2bnanoparticles under distinct excitation wavelengths. For both, the emission is formed by a large broad band ascribed to the chitosan intrinsic emission²⁰ and by a series of intra-4f lines attributed to the ⁵D₀ → ⁷F₀₋₄ (Eu³⁺) and ⁵D₄ → ⁷F₆₋₀ (Tb³⁺) transitions. The relative intensity between the intra-4f lines and the chitosan-related emission depends on the excitation wavelength, in such a way that for lower excitation wavelengths (280–340 nm) the intra-4f lines dominates, whereas for higher excitation wavelength (350–464 nm), the opposite is observed, as illustrated in Fig. 3 for selected excitation wavelengths. The energy and full width at half maximum (fwhm) of the intra-4f lines are independent on the excitation wavelength suggesting that, in each material, the Ln³⁺ ions occupy the same average local environment. Moreover, for the particular case of the Eu³⁺-containing material 1b the presence of a single line for the non-degenerated ⁵D₀ → ⁷F₀ transition, the Stark splitting of the ⁷F_1,2 levels into 3 and 4 components and the high relative intensity of the ⁵D₀ → ⁷F₂ transition (ESI, Fig. S6†), indicates that the Eu³⁺ ions occupy a low symmetry local coordination site without an inversion centre.²¹ Nevertheless, the high fwhm of the intra-4f lines (e.g. 44 ± 1 cm⁻¹ for the ⁵D₀→⁷F₀ transition) indicates a high degree of disorder for the Eu³⁺ local coordination (site-to-site variations). Accordingly, it should be noted that Eu³⁺ local site disorder has been found in the case of the Eu(H₂O)₅[Mo(CN)₈] bulk coordination polymer material.¹⁸

Fig. 3 Room-temperature emission spectra of (a) 1a and (b) 2a excited at (1) 280, (2) 340, (3) 420, and (4) 460 nm. The inset shows a magnification of the ⁵D₄ → ⁷F_2-0 (Tb³⁺) transitions.

Fig. 4 displays the selective excitation spectra monitored within the large broad band (475 nm) and within the Eu³⁺ (1b) and Tb³⁺ (2b) transitions (612 and 544 nm, respectively). The excitation spectra monitored within the chitosan-related emission at 475 nm are similar for the Eu³⁺ and Tb³⁺-containing materials, being formed of a broad band with two main components at ca. 345 and 375 nm ascribed to the chitosan excited states.²² For intra-4f monitored wavelengths, the excitation spectra reveal clearly the presence of the chitosan-related excited states at ca. 345 nm and at ca. 375 nm superimposed on the ⁷F₀ → ⁵L₆, ⁵D₂ (1b) and ⁷F₆ → ⁵D₄ (2b) transitions. The very-low relative intensity of the intra-4f lines points out that the Ln³⁺ ions are mainly populated via the chitosan-related excited states, rather than by direct intra-4f excitation. Chitosan-to-Ln³⁺ energy transfer, already reported for lanthanide-containing chitosan–silica hybrids,²⁰ can occur through the dipole–dipole, dipole–2^λ pole (λ = 2, 4, and 6) and exchange mechanisms.²³

Fig. 4 Room-temperature excitation spectra of (a) 1b and (b) 2b monitored at (1) 475, (2) 544, and (3) 612 nm. The inset shows a magnification of the ⁷F₀ → ⁵D₂ (Eu³⁺) transition.

The decay curves of the ⁵D₀ (Eu³⁺) and ⁵D₄ (Tb³⁺) excited states, monitored at 612 and 544 nm, respectively, and under excitation at 350 nm, are well modeled by a single exponential function yielding ⁵D₀ (1b) and ⁵D₄ (2b) lifetime values of 0.235 ± 0.002 ms and 0.415 ± 0.046 ms, respectively. The single exponential behavior of the emission decay curves is in good agreement with the presence of a single average local-environment for the Ln³⁺ ions.

Thus, the obtained aqueous colloidal solutions of the nanoparticles exhibit at room temperature the typical fluorescence characteristic for Eu³⁺ (⁵D₀ → ⁷F₀₋₄ transitions) or Tb³⁺ (⁵D₄ → ⁷F₆₋₂ transitions) ions under excitation in UV region, while a green luminescence of chitosan shell is observed when the nanoparticles are excited in the visible region. As a result, the luminescence of our nanoparticles may allow for multiplexing because different excitation sources provide different color of luminescence, red for lanthanides and green for chitosan shell. When compared with other inorganic nanoparticles such as dye-doped nanoparticles and QDs,²⁴ the Ln₃₃³⁺Gd_x³⁺/[Mo(CN)₈]₃ (Ln = Eu (x = 0.34), Tb (x = 0.35)) cyano-bridged coordination polymer nanoparticles combine the advantages of high photobleaching threshold and good chemical stability with readily tunable spectral properties. In particular, the bimodal emission with distinct dynamics (the long-lived Ln³⁺ narrow red lines and the short-lived chitosan broad green band) may be easily attained by controlling physical (excitation wavelength) and chemical (Ln³⁺ ratio) parameters, rather than making use of the size-dependent excitation and emission wavelengths typical of colloidal semiconductor nanoparticles. Note that bimodal (or multicolored) emission can be applied in multiplexed detection and imaging of therapeutic cells both in vitro and in vivo,²⁵ multiplex fluorescent detection assays in which a specific fluorescence image could be selected using appropriate optical filters and innovative barcodes systems.²⁶

Relaxivity properties. The longitudinal and transverse relaxivities of the nanoparticles were also estimated at room temperature in aqueous solutions. The ¹H NMR relaxometry characterization of the aqueous solutions Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻/chitosan nanoparticles (Ln = Eu (x = 0.34), Tb (x = 0.35)) 1b, 2b was performed by measuring the longitudinal and the transverse nuclear relaxation times T₁ and T₂, in the frequency range 10 KHz ≤ ν ≤ 100 MHz, for T₁, and 8 MHz ≤ ν ≤ 60 MHz, for T₂. Relaxivity values, r, are simply defined as the inverse of the relaxation time normalized for the contrast agent concentration, once previously corrected by the host diamagnetic contribution. So, the efficiency of the MRI contrast agents may be determined by measuring the nuclear relaxivities r_1p,2p defined as:²⁷

r_ip = [(1/T_i)_meas − (1/T_i)_dia]/c, i = 1,2

where (1/T_i)_meas is the measured value on the sample with concentration c (mmol L⁻¹) of magnetic center (0.0267 mmol L⁻¹ in our case), and (1/T_i)_dia refers to the nuclear relaxation rate of the diamagnetic host solution (water in our case).

Fig. 5 reports the frequency dependence of r_1p and r_2p for Eu_0.33³⁺Gd_0.34³⁺/[Mo(CN)₈]³⁻/chitosan 1b, together with the values for the commercial contrast agents Omniscan® and Gd-DTPA®. As can be seen, the r_1p values obtained for 1b are comparable to or slightly higher than the ones observed for Gd-DTPA®, while the values of r_2p relaxivities of our sample are higher in comparison to the measured values for Omniscan®. The same values were observed in the case of 2b. For this reason, it can be concluded that our nanoparticles behind their luminescent properties may also be used as a positive contrast agent.

Fig. 5 Longitudinal (r_1p) (top) and transversal (r_2p) (bottom) relaxivities of 1b (■), collected at T ≈ 25 °C, compared to relaxivities reported for the commercial compounds Gd-DTPA® and Omiscan® (▲).

III.3. Nanoparticles internalization, intracellular detection and cytotoxicity tests

Taking into account that our nanoparticles present magneto-luminescent properties, we estimate their internalization into the living cells and the possibility of their intracellular detection. Note that the uptake of cyano-bridged coordination polymer nanoparticles has never been reported previously. The cellular uptake of our nanoparticles was analysed by using a series of cancer cell lines such as colorectal HCT-116, pancreatic Capan-1 and also normal human fibroblasts and human umbilical endothelial HUVEC cells. For this experiment, cells were incubated during 6 h at 37 °C with 100 ng mL⁻¹ of the nanoparticles 1b in serum free culture medium and analyzed by confocal laser scanning microscopy (CLSM) of living cells in order to assess internalization and intracellular distribution of the nanoparticles. The confocal microscopy of living cells avoids the cell fixation step, a potential cause of artefacts on the entry and the cellular localization of the nanoparticles. Moreover, the 0.6 µm width of Z stacks ensures that the labelling is intracellular and not retains at the outside of the plasma membrane. For all investigated cells the green emission related to the chitosan shell of the nanoparticles is clearly seen under excitation in the visible region at 489 nm (Fig. 6b) demonstrating that the nanoparticles 1b were successfully internalized by all used cancer (HCT116 and Capan-1) and normal cells (HUVEC and fibroblasts). This result is not surprising regarding with their ultra-small size of 2.8–4.5 nm. More importantly is that the presence of the nanoparticles into these cells is highly detectable by fluorescence microscopy at an extremely low dose. Co-staining of cell nuclei and lysosome were also performed. For this reasons, nuclei were loaded with 5 µg mL⁻¹ Hoechst 33342 (blue) (Fig. 6a) and lysosomes were labelled by 50 nM lysotracker (red) (Fig. 6c). Merged images of nanoparticles fluorescence and lysosome marker show that in all cases the nanoparticles, which appear as orange spots, were mostly localized into lysosomes (Fig. 6d). Such cell localisation is typical for the most of the inorganic nanoparticles and is mainly due to the endocytosis pathway. Note that the effect of particle uptake and optical detection can be used to label cells and follow their pathway or fate.

Fig. 6 Localization of nanoparticles 1b in living cancer (HCT-116 and Capan-1), or normal (HUVECs and fibroblasts) cell lines, incubated 6 h with nanoparticles 1b. Merged pictures represent the co-localization of nanoparticles 1b (100 ng mL⁻¹) with the lysosomal marker. Images are representative of two independent experiments. Bars represent 4 µm.

In the in vivo imaging area, some challenges have been identified and the foremost obstacle is the difficulty to obtain new contrast agents without cytotoxic effects. To analyse the toxicity of these nanoparticles, normal human fibroblasts, umbilical vein endothelial cells (HUVECs) and human colorectal (HCT-116) and pancreatic (Capan-1) cancer cell lines were treated 4 days with increasing doses of Eu_0.33³⁺Gd_0.34³⁺/[Mo(CN)₈]³⁻/chitosan nanoparticles 1b (with concentrations of 0, 25, 50 and 100 ng mL⁻¹). Cell viability was monitored for 4 days using MTT assay to measure mitochondrial enzyme activity. Fig. 7 shows that these nanoparticles present no toxicity, neither in cancer cells analysed in this study (human colorectal and pancreatic cancer cells, Fig. 7a and b) nor in normal cells (human fibroblasts and umbilical endothelial HUVEC cells, Fig. 7c and d).

Fig. 7 Absence of toxicity of Eu_0.33³⁺Gd_0.34³⁺/[Mo(CN)₈]³⁻/chitosan nanoparticles 1b in human normal and cancer cells. (A) Human colorectal cancer cells (HCT116), (B) human pancreatic cancer cells (Capan-1), (C) human fibroblasts and (D) human umbilical endothelial HUVEC cells are incubated for 4 days in the absence (0 ng mL⁻¹) or in the presence of increasing doses of nanoparticles (25, 50 and 100 ng mL⁻¹). After treatment, cell proliferation was measured by MTT assay (see Experimental part). Values represent the mean ± standard deviations of triplicates from a typical experiment and were confirmed in two additive experiments.

The toxicity tests also demonstrate a high stability of these nanoparticles in aqueous solutions. During 4 days at 37 °C no toxic Gd³⁺ or Ln³⁺ leaching has been detected. The stability of the nanoparticles in aqueous and physiological solutions has also been confirmed by the elemental analysis of the mother solution after precipitation of the nanoparticles in which no presence of free Gd³⁺ ion has been detected. Note that the high stability of the cyano-bridged coordination polymer nanoparticles is not surprising because Prussian Blue aggregated nanoparticles are well known as an effective per os treatment for human radioactive Cs⁺ and Tl⁺ decontamination.²⁸

An interesting point to note is that these ultra-small inorganic nanoparticles maybe interesting for imaging because they may be rapidly eliminated from the body. Recently three criteria for distinguishing inorganic nanoparticles that can present potential clinical utility were proposed: (i) a low hydrodynamic diameter permitting complete elimination from the body; or (ii) a formulation with completely nontoxic components; or (iii) biodegradability to clearable components. It was shown that the inorganic nanoparticles presenting the hydrodynamic diameter lower than 5.5 nm may be rapidly (during around 4 h) and efficiently excreted from the body by renal filtration while the larger nanoparticles cannot be evacuated and will be kept in the body for several days.²⁹ Taking into account the small size (2.8–4.5 nm) of the nanoparticles described here they may be completely eliminated by a renal clearance that satisfy the first criteria of potential clinical utility. In addition, high stability of these nanoparticles at low pH make them interesting in imaging or labeling of some cell compartments such as lysosomes, large acidic vesicles also called phagolysosomes or certain tissue environment such as the gastrointestinal tract where the physiological pH is less than 5.

IV. Conclusion

The development of multifunctional nanoparticles for imaging is one of the main objectives in the field of biology and nanomedicine. The main requirements for such nanoparticles include good image contrast at low dosage, high stability under physiological conditions which may be varying depending on the imaging objects, organs or body region, low toxicity and rapid elimination from the body. The research results described in this article are to propose a novel approach toward designing a new family of efficient imaging probes based on cyano-bridged coordination polymer nanoparticles presenting magneto-luminescent bi-functionality, ultra small size and high stability. This approach presents an alternative way to achieve multifunctional nanoparticles in comparison with designing of large sized complex hybrid nanoparticles where different components are incorporated together or attached onto the nanoparticles surface.

The approach that we adopted in order to obtain aqueous colloidal solutions of cyano-bridged coordination polymer nanoparticles Ln_0.33³⁺Gd_x³⁺/[Mo(CN)₈]³⁻/chitosan (Ln = Eu (x = 0.34), Tb (x = 0.35)) consists in the consecutive growing of the cyano-bridged metal network at the functional amino groups into the pores of the chitosan matrix and then in solubilization of the most part of the chitosan in water. Thus, the as-obtained nanoparticles are spherical in shape, non-aggregated and well dispersed in water or alcohols with mean sizes values in the range 2.8–4.5 nm, presenting a narrow size distribution according to the TEM and HRTEM measurements. The nanoparticles are perfectly soluble in aqueous solutions which may be concentrated or diluted in water and their pH may be increase up to 7.2. These colloidal solutions are also stable and no aggregation or nanoparticles degradation has been observed after few months. These properties are the first requirements for the potential use of these nanoparticles for biological applications.

In order to prove the bi-functionality of our nanoparticles, we evaluate their potential for MR contrast enhancement and their optical properties. The r_1p relaxivity of aqueous colloidal solutions of the nanoparticles is slightly higher than or comparable to the ones of the commercial contrast agents Omniscan® and Gd-DTPA® indicating that our nanoparticles may be considered as an efficient positive contrast agent for MRI. In addition to these properties in terms of relaxivity, the nanoparticles exhibit the typical fluorescence for Eu³⁺ (⁵D₀ → ⁷F_0–4 transitions) or Tb³⁺(⁵D₄ → ⁷F_6–2 transitions) under excitation in UV region, while a green luminescence of chitosan shell is observed when the nanoparticles are excited in the visible region. The magneto-luminescent property of the nanoparticles is suggestive of their use both for the luminescence labeling of cells and as new contrast agents for MRI imaging.

We also evaluate the nanoparticle uptake by living cells and their fluorescence detectionin vitro. The results clearly show that these nanoparticles not only were rapidly internalized by both healthy (HUVECs and fibroblasts) and living cancer (HCT-116 and Capan-1) cells, and localised into the lysosomes, but they can be highly detectable by fluorescence microscopy into these cells at an extremely low dose. Cell viabilities of human cancer or normal cells such as colorectal (HCT-116) and pancreatic cancer (Capan-1) cell lines, human fibroblasts and umbilical vein endothelial cells (HUVEC) were unaffected by these nanoparticles in the concentration range allowing their cellular imaging. The toxicity tests also demonstrate a high stability of these nanoparticles in aqueous solutions and no leaching of toxic Gd³⁺ or Ln³⁺ ions was detected.

These results present the first step toward a design of new multi-modal imaging probes of interest based on cyano-bridged metallic nanoparticles. Further works on functionalization of the nanoparticle surface with biological molecules in view of particular cells targeting are actually under run.

Acknowledgements

The authors thank Mme Corine Reibel (ICGM, Montpellier, France) for magnetic measurements. E.C. thanks the Russian Academy of Science and the French Ministry of Foreign Affairs. The authors also thank the Network of Excellence MAGMANet for funds support. M.G. and M.G.-B. thank Prof. Dr Kurt von Figura (Göttingen) for cell cultures.

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Footnote

† Electronic supplementary information (ESI) available: TEM images and size distribution histograms, IR and emission spectra, diffraction pattern and HRTEM coupled EDX analysis. See DOI: 10.1039/c0nr00709a

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