Mesoporous silica-coated luminescent Eu³⁺ doped GdVO₄ nanoparticles for multimodal imaging and drug delivery

Abstract

We describe a simple method for synthesizing mesoporous silica-coated luminescent europium-doped gadolinium vanadate (GdVO₄:Eu³⁺@mSiO₂) nanoparticles. Their biomedical applications as a potential imaging nanoprobe for both fluorescence imaging and magnetic resonance imaging (MRI) and as a simultaneous anti-cancer drug delivery vehicle are also discussed. Eu³⁺ doped GdVO₄ nanoparticles exhibit strong red photoluminescence and the Gd³⁺ in GdVO₄ can be used as a T₁ contrast agent for MRI. T₁-weighted MR contrast enhancement as well as sustained intracellular drug delivery can be achieved by the mesoporous silica layer coating onto a single nanoparticle. Enhanced T₁ MR contrast was proven by relaxometric studies on water access to the paramagnetic core. GdVO₄:Eu³⁺@mSiO₂ nanoparticles as a new type of theragnostic (imaging and treatment) agent can provide new opportunities in a cancer treatment.

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1. Introduction

The development of nanostructured materials for multimodal imaging is a rapidly growing research field. Particularly, nanostructured materials that possess both fluorescence and magnetic resonance imaging (MRI) properties have received wide recognition since they have both the high sensitivity of fluorescence phenomenon and the high spatial resolution of magnetic resonance imaging.^1–5 Hybrid nanoparticles comprising a silica matrix which incorporates fluorescent dyes or QDs and magnetic nanoparticles, are one of the approaches in achieving multimodal capability.^6–8 Another is to use nanoparticles having attributes of both fluorescence and magnetic resonance imaging.

Metal oxides containing alkaline, transition, or lanthanide metals, exhibit strong photoluminescence when they are doped with other lanthanide metal ions.^9,10 The red-emitting europium(III) ion (Eu³⁺) serves as a highly efficient luminescent center,^11,12 while gadolinium vanadate (GdVO₄) has been reported as an excellent host matrix for photoluminescence applications.¹³ Therefore, Eu³⁺ doped GdVO₄ nanoparticles (denoted by GdVO₄:Eu³⁺ NPs) can be an efficient photoluminescent nanoprobe for fluorescence imaging. In addition, unpaired electrons of Gd³⁺ on the surface of GdVO₄ NPs can be exploited to enhance contrast in magnetic resonance imaging. Using the photoluminescence of Eu³⁺ dopants and the T₁-shortening effect of Gd³⁺ in the host matrix, GdVO₄:Eu³⁺ NPs can be a possible imaging nanoprobe for both fluorescence and magnetic resonance imaging.^14,15

The MRI technique mostly relies on water proton relaxation, which depends on magnetic fields, MRI pulse sequence, and the heterogeneous distribution of water in the organism.¹⁶ MRI signals can be enhanced by using two types of contrast agents, negative and positive.^17,18 Superparamagnetic iron oxide nanoparticles which are widely used as negative T₂ contrast agents provide a long-range disturbance in the magnetic field homogeneity and activate the dephasing of protons. Therefore, iron oxide nanoparticles accelerate spin–spin relaxation and shorten T₂ relaxation times of their environments, and they produce a decreased signal intensity in T₂-weighted MR imaging.^19,20 On the other hand, Gd³⁺, one of the paramagnetic ions, enhances the spin–lattice relaxation and reduces the T₁ relaxation times,²¹ which can generate positive enhancement of T₁-weighted MR imaging without any distortion in the environment. This positive enhancement maximizes signal intensity with higher spatial resolution. For the T₁ contrast mechanism, the accessibility of water molecules to the magnetic center is a key of designing highly efficient MRI contrast agents.^22,23 Therefore, a mesoporous silica coating allows the easier water diffusion across mesoporous silica shell, leading to efficient relaxation of water in the vicinity of the nanoparticles.^24–26

Furthermore, their structural and surface properties have made mesoporous silica nanostructure materials applicable in biomedicine, as controlled drug release vehicles in an anti-cancer chemotherapy. The large surface area and pore volume of mesoporous silica nanoparticles can ensure facile adsorption as well as high loading of various therapeutics.^27–29 In several studies, treatment using drug-loaded mesoporous silica nanoparticles suppressed tumor growth in cancer xenograft mouse models, demonstrating the anti-cancer drug delivery capability of mesoporous silica nanoparticles.^30,31

We synthesized a very uniform and dispersive mesoporous silica-coated NPs with a single GdVO₄:Eu³⁺ core in their bodies (denoted by GdVO₄:Eu³⁺@mSiO₂ NPs) and demonstrated their capabilities to perform simultaneous T₁ MR imaging, fluorescence imaging, and drug delivery. This study is to prove these newly developed GdVO₄:Eu³⁺@mSiO₂ NPs as one of the ideal candidates of multifunctional nanoparticles for cancer treatment.

2. Experimental

2.1. Materials

Gadolinium(III) acetate hydrate (99%), vanadyl sulfate hydrate (97%), oleic acid (90%), 3-aminopropyltriethoxysilane (APTES), xylene (98.5+%), gelatin, 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), polyoxyethylene(5) nonylphenyl ether (Igepal CO-520, containing 50 mol% hydrophilic group), doxorubicin, and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich. Europium(III) acetate hydrate (99.9%), oleylamine (approximate C₁₈-content 80–90%), cetyltrimethylammonium bromide (CTAB), and tetraethylorthosilicate (TEOS) were purchased from Acros Organics. Methoxy poly(ethylene glycol) succinimidyl glutarate (mPEG-SG, MW = 5000) was purchased from Sunbio company. Ammonium hydroxide (28.0–30.0%), ethylacetate (99%), hydrochloric acid (35–37%), n-hexane (99%), ethanol (99%), chloroform (99%), and acetone (99%) were purchased from Samchun Chemicals. Dulbecco's modified eagle medium (DMEM), penicillin/streptomycin, fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were purchased from GIBCO.

2.2. Preparation of GdVO₄:Eu³⁺@mSiO₂ NPs

2.2.1. Synthesis of GdVO₄:Eu³⁺ NPs. A mixture of gadolinium(III) acetate and europium(III) acetate (total 1 mmol), 6 mmol of oleic acid, and 6 mmol of oleylamine were dissolved in 15 mL of xylene. After stirring for 2 h at room temperature, the solution was slowly heated to 90 °C. 1 mL of aqueous vanadyl sulfate solution (1 mM) was then added under vigorous stirring, and the resulting solution was aged at 90 °C for 3 h. 100 mL of ethanol was added to precipitate the nanoparticles that were retrieved by centrifugation. The resulting GdVO₄:Eu³⁺ NPs were well dispersed in organic solvents such as n-hexane and chloroform.

2.2.2. Synthesis of GdVO₄:Eu³⁺@mSiO₂ NPs. GdVO₄:Eu³⁺@mSiO₂ NPs were synthesized by sol–gel reaction using tetraethylorthosilicate (TEOS) as a silica precursor in an aqueous solution containing CTAB and GdVO₄:Eu³⁺ NPs. The GdVO₄:Eu³⁺ NPs stabilized with oleic acid and oleylamine were dispersed in chloroform with a concentration of 4.8 mg Gd mL⁻¹ (the concentration of Gd in the solution was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES)). GdVO₄:Eu³⁺ NPs in chloroform was added to 5 mL of 0.55 M aqueous CTAB solution and the resulting mixture was stirred vigorously for 30 min. After the formation of oil-in-water micro-emulsions, the solution was heated to 60 °C and aged at the same temperature for 10 min under stirring to evaporate the chloroform. The aqueous solution of GdVO₄:Eu³⁺/CTAB was added to a mixture of 45 mL of water and 0.3 mL of 2 M NaOH solution, and the mixture was heated to 70 °C under stirring, followed by the addition of 0.5 mL of TEOS and 3 mL of ethylacetate. During the reaction, 50 μL of APTES was added to introduce amine groups (–NH₂) and the solution was stirred for 3 h. To extract CTAB from the nanoparticles, 20 μL of HCl was added to the dispersion (pH ∼ 2.3) and stirred for 1 h at 60 °C. Finally, CTAB extracted GdVO₄:Eu³⁺@mSiO₂ NPs were redispersed in 10 mL ethanol after washing with ethanol twice.

2.2.3. Surface modification of GdVO₄:Eu³⁺@mSiO₂ NPs with PEG. During the sol–gel reaction, amine groups were introduced to the surface of GdVO₄:Eu³⁺@mSiO₂ NPs by adding APTES to the solution. Surface modification of the GdVO₄:Eu³⁺@mSiO₂ NPs with PEG was carried out by the formation of covalent bonds between amine groups on the surface of GdVO₄:Eu³⁺@mSiO₂ NPs and the succinimidyl groups of mPEG-SG. 10 mL of an ethanol solution of the GdVO₄:Eu³⁺@mSiO₂ NPs was added to 20 mL of ethanol solution containing 50 mg of mPEG-SG. The mixture was stirred at 40 °C for 3 h. The GdVO₄:Eu³⁺@mSiO₂-PEG NPs were collected after removing unreacted mPEG-SG. For in vitro applications, the GdVO₄:Eu³⁺@mSiO₂-PEG NPs were dispersed in 0.7 mL of phosphate buffer solution (PBS, 10 mM, pH 7.4) and filtered using a 0.8 μm cellulose acetate filter.

2.3. Synthesis of GdVO₄:Eu³⁺@dSiO₂ NPs

Dense silica-coated GdVO₄:Eu³⁺ NPs (GdVO₄:Eu³⁺@dSiO₂ NPs) were prepared as previously described along with some modifications.⁷ 0.544 mmol of polyoxyethylene(5) nonylphenyl ether (0.23 g, Igepal CO-520) was dispersed in a scintillation tube containing 4.5 mL of cyclohexane by sonication. Next, 300 μL of GdVO₄:Eu³⁺ NPs, dispersed in cyclohexane (15.6 mg Gd mL⁻¹), was added to the tube. The mixture was vortexed until that became transparent. 50 μL of ammonium hydroxide was then added to form a reverse microemulsion. After 1 h, 50 μL of TEOS was added and the reaction continued for 24 h. The resulting GdVO₄:Eu³⁺@dSiO₂ NPs were precipitated by adding ethanol, and collected by centrifugation. Washing steps were repeated three times, and finally, the nanoparticles were redispersed in distilled water.

2.4. Characterizations

The transmission electron microscopy (TEM) analysis was conducted by JEOL JEM-2010 (JEOL Ltd. Japan). N₂ adsorption/desorption isotherm was measured at 77 K using a Micromeritics ASAP 2000 gas adsorption analyzer (Norcross, USA) after the samples were degassed at 293 K under 10 μTorr for 5 h. The surface area and the total pore volume were evaluated using the BET (Brunauer–Emmett–Teller) model and the pore size was evaluated using the BJH (Barrett–Joyner–Halenda) model. DLS measurements were carried out using a zeta-potential & particle size analyzer (ELS-Z, Otsuka Electronics, JAPAN). Inductively coupled plasma atomic emission spectrometer (ICP-AES, Shimadzu ICPS-1000IV, JAPAN) was used for a quantitative analysis. Absorption and fluorescence spectra were obtained using the JASCO V-550 UV-Vis spectrophotometer, and JASCO FP-6500 spectrofluorometer (JASCO Inc., JAPAN), respectively. The confocal laser scanning microscopy data were obtained using LSM 510 (Carl Zeiss, Germany). In vitro fluorescence images were obtained using the fluorescence imaging system (Kodak IS4000MM pro, USA) and MR images were obtained using a 1.5 T MR scanner (GE Sigma Excite, USA).

2.5. Relaxivity measurements

Relaxivity measurements were performed on 1.5 T MR scanner (GE Sigma Excite, USA). The samples were diluted to concentrations of 0.52, 0.26, 0.13, 0.06, 0 mM Gd by adding deionized water and the resulting solutions were transferred to a 200 μL-well plastic microtiter plate. T₁ values were obtained from the IR-FSE sequence with 30 multiple values of TIs (TR/TE/TI = 4400 ms/13 ms/24–4000 ms). T₁ and T₂ relaxation times were calculated by fitting the signal intensities with increasing TEs or TIs to a mono-exponential function using a non-linear least squares fit of the Levenberg–Marquardt algorithm. T₁ and T₂ values were averaged over the region of interest and plotted as 1/T₁ or 1/T₂ vs. the concentration of Gd. The slopes of these lines provide the molar relaxivities, r₁ and r₂, respectively.

2.6. In vitro cellular experiments

2.6.1. Cell viability. Human breast cancer cell lines, MCF-7 cells and SKBR-3 cells were cultivated in monolayer in DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 3.7 g L⁻¹ sodium carbonate, and 5 mM glucose in 5% CO₂/95% air at 37 °C. For cell viability, MCF-7 cells and SKBR-3 cells were initially seeded in 96-well plates at ∼1 × 10⁴ cells per well. The cell viability after labeling was assessed by a standard MTT assay and was compared with the viability of unlabeled cells.

2.6.2. Doxorubicin loading in GdVO₄:Eu³⁺@mSiO₂-PEG NPs and drug release profile. GdVO₄:Eu³⁺@mSiO₂-PEG NPs dispersed in PBS were centrifuged and redispersed with 1 mL of doxorubicin (DOX) solution in PBS (0.6 mg mL⁻¹). After stirring overnight, DOX-loaded NPs were centrifuged and washed with DI water to remove the unadsorbed free DOX. The supernatant containing unadsorbed DOX was collected for UV measurement to calculate the adsorbed amount of DOX in NPs. To evaluate time-dependent drug release, drug loaded nanoparticles (0.3 mg) was dispersed in 1 mL of buffer at different pH conditions, 1 mM phosphate buffer (pH 7.4) and 1 mM acetate buffer (pH 5.0). DOX release was monitored by measurement of the absorption intensity at 480 nm of supernatant solution after centrifugation at 2 h, 4 h, 7 h, 10 h, 14 h, 18 h, and 24 h incubations.

2.6.3. In vitro cytotoxicity against MCF-7 cells with free DOX and DOX-loaded NPs. MCF-7 cells were seeded in a 96-well plate at a density of ∼1 × 10⁴ cells per well and cultivated in DMEM/F12 medium supplemented with 10% FBS for 24 h at 37 °C. Then, free DOX and DOX-loaded GdVO₄:Eu³⁺@mSiO₂-PEG NPs were added to the medium, and the cells were incubated for 24 h at 37 °C. The concentrations of DOX were 15, 1.5, 0.15, and 0.015 μM, respectively. Finally, the cell viability was evaluated by the MTT assay.

2.6.4. T₁-weighted MR and fluorescence imaging. For in vitro T₁-weighted MR and fluorescence images, unlabeled cells and labeled cells (1 × 10⁶) with GdVO₄:Eu³⁺@mSiO₂-PEG NPs were counted and transferred to 1.5 mL Eppendorf tubes at a concentration of ∼1 × 10⁶ cells per tube and pelleted by centrifugation. 0.5 mL of 4% gelatin was dropped to each tube. MRI of the cell pellets was conducted on 1.5 T MR scanner. Fluorescence imaging of the pellets was obtained using the fluorescence imaging system.

2.6.5. In vitro drug delivery and confocal laser scanning microscopy (CLSM). MCF-7 cells were plated in chamber slides (Labtek chamber slide 4-well, Nunc) at a density of 2 × 10⁴ cells per well for 24 h before cellular uptake. GdVO₄:Eu³⁺@mSiO₂-PEG NPs loaded with DOX were incubated in MCF-7 cells for 2 h at 37 °C in serum free medium. Then, the cells were washed with PBS three times and fixed in 1% paraformaldehyde solution. The slides were covered with a mounting medium (SuperMount, InnoGenex, USA). The cells were imaged by a confocal laser scanning microscope using a helium–neon laser with an excitation wavelength of 543 nm and 560–615 nm emission band-pass filter.

3. Results and discussion

3.1. Synthesis of GdVO₄:Eu³⁺@mSiO₂ NPs

GdVO₄:Eu³⁺ NPs were prepared using a modified reverse micelle method,³² which enabled the synthesis of uniform GdVO₄:Eu³⁺ NPs under facile and mild reaction conditions. Specifically, they were synthesized from the reaction of gadolinium(III) acetate and europium(III) acetate (9 mol% doped of Eu³⁺) with vanadyl sulfate solution in xylene at 90 °C in the presence of oleylamine and oleic acid under air atmosphere. Transmission electron microscopy (TEM) images of the GdVO₄:Eu³⁺ NPs reveal the formation of square-shaped nanoplates with average side dimension of 15 nm and thickness of around 3.5 nm (Fig. 1A and B). Fig. 1C shows an X-ray diffraction (XRD) pattern of the synthesized GdVO₄:Eu³⁺ NPs, indicating that it corresponds well with the typical XRD pattern of zircon-type (ZrSiO₄) GdVO₄ crystal structure (a = 7.212 Å and c = 6.348 Å, Joint Committee on Powder Diffraction Standards (JCPDS) file number 17-0260).

Fig. 1 Characterizations of the GdVO₄:Eu³⁺ NPs. (A and B) The bright field TEM image (A) and tilted TEM image (B) of the 15 nm-sized GdVO₄:Eu³⁺ NPs. (C) XRD pattern of the GdVO₄:Eu³⁺ NPs (XRD pattern for zircon-type structure GdVO₄ is shown as blue lines for reference).

In a previous report on the synthesis of Eu³⁺ doped metal oxide nanoparticles, it was demonstrated that Eu³⁺ doped nanoparticles exhibited strong red emission with wavelength of around 618 nm.^11–13 The concentration of Eu³⁺ in the NPs is easily controlled by initial ratio of Eu³⁺/Gd³⁺. The inductively coupled plasma (ICP) data of GdVO₄:Eu³⁺ NPs shows that the concentration of Eu³⁺ in the as-prepared NPs was well matched with the concentration of it in the starting material, indicating that Eu³⁺ was successfully incorporated in the GdVO₄ NPs (see the ESI Table S1†). Fig. 2 displays the room temperature PL spectra of GdVO₄:Eu³⁺ NPs excited at 330 nm, showing that the emission was considerably enhanced by increasing the Eu³⁺ concentration up to 9 mol%. This phenomenon was primarily attributed to enhanced population of energy transfer from host to the dopants. On the other hand, under the higher Eu³⁺ concentration than 9 mol%, intensity of emission spectrum was quickly decreased possibly due to the Eu³⁺–Eu³⁺ cross relaxation.³³

Fig. 2 Room temperature PL spectra of GdVO₄:Eu³⁺ NPs excited at 330 nm, showing GdVO₄:Eu³⁺ NPs exhibited the strongest PL emission when the concentration of Eu³⁺ was 9 mol%.

In this synthesis, CTAB serves as not only a capping agent to transfer hydrophobic GdVO₄:Eu³⁺ NPs to the aqueous phase³⁴ but also an organic template to form mesoporous silica structures on the surface of NPs.^35,36 For biomedical applications, the surface of the NPs was modified with PEG to render them biocompatible after removing the CTAB templates from the as-synthesized materials by refluxing in acidic ethanol solution (pH 2.3). TEM (Fig. 3A) and high-resolution TEM (HRTEM) (Fig. 3B) images reveal that a single GdVO₄:Eu³⁺ core was encapsulated with a porous silica shell without any interparticular aggregation and its overall size is about 75 nm. The mesoporous structures of GdVO₄:Eu³⁺@mSiO₂ NPs were also confirmed using N₂ adsorption/desorption isotherms. The average diameter of pores calculated by the Barrett–Joiner–Halenda (BJH) method was 3.3 nm. The Brunauer–Emmett–Teller (BET) surface area and pore volume of GdVO₄:Eu³⁺@mSiO₂ NPs were measured to be 158.57 m² g⁻¹ and 0.36 cm³ g⁻¹ (Fig. 3C), respectively, indicating the large surface area of GdVO₄:Eu³⁺@mSiO₂ NPs. The terminal succinimidyl group of PEG was conjugated with the amine groups in the amine-modified GdVO₄:Eu³⁺@mSiO₂ NPs to improve the dispersibility in an aqueous environment, which is essential to be theragnostic agents. GdVO₄:Eu³⁺@mSiO₂-PEG NPs showed excellent colloidal stability without agglomerations in phosphate-buffered saline (PBS). Their hydrodynamic diameter measured by dynamic light scattering (DLS) according to the number distribution was obtained to be 124 nm, and maintained at 110–125 nm for over 7 days (see the ESI Fig. S1†). We should note that our design strategy was to produce highly dispersible mesoporous nanoparticles with a single contrasting core in their bodies. In the previous studies, GdVO₄:Eu³⁺/mesoporous silica hybrids usually have a structure of decorated GdVO₄:Eu³⁺ phosphors outside of mesoporous silica and large overall size (>100 nm).^37,38 Our GdVO₄:Eu³⁺@mSiO₂ NPs is more uniform and dispersive with a very robust and single core–shell nanostructure. The colloidal stability and biocompatibility is also improved by pegylation on the surface of nanoparticles.³⁹ Therefore, as-synthesized GdVO₄:Eu³⁺@mSiO₂-PEG NPs ended up being very stable in biological media and desirable as an effective bio-imaging nanoprobe and an intracellular drug carrier system. In particular, the aqueous dispersibility is a key factor to affect magnetic relaxivity of contrast agents, particularly, T₁ relaxation, and a GdVO₄:Eu³⁺ single core with a manipulated shell structure (mesoporous/dense silica) could offer the relaxometric study on the water accessibility to a magnetic core.

Fig. 3 (A and B) TEM and HRTEM images of the 75 nm-sized GdVO₄:Eu³⁺@mSiO₂ NPs. (C) N₂ adsorption/desorption isotherms of the GdVO₄:Eu³⁺@mSiO₂ NPs (inset: pore size distribution; V: pore volume, D: pore size). (D) TEM image of the 65 nm-sized GdVO₄:Eu³⁺@dSiO₂ NPs.

3.2. Photoluminescence of GdVO₄:Eu³⁺@mSiO₂ NPs

In the previous photoluminescence studies of Eu³⁺ in GdVO₄ matrix,¹³ the emission spectrum in the wavelength of 540–620 nm could be described by the intrinsic transitions between ⁵D₀/⁵D₁ and ⁷F_j (j = 1, 2), which include the ⁵D₁ → ⁷F₁ transition at 540 nm, the ⁵D₀ → ⁷F₁ transition at 595 nm, and the ⁵D₀ → ⁷F₂ transition at 618 nm. Particularly, the ⁵D₀ → ⁷F₂ transition in the GdVO₄:Eu³⁺ NPs was greatly enhanced and produced a strong red emission upon UV irradiation. Even though the PL efficiency dropped 30% (Fig. S2†), the photoluminescent behavior of GdVO₄:Eu³⁺ NPs was successfully maintained after mesoporous silica coating and a bright red emission was observed (Fig. 4). Turbid gray colored aqueous solution of the GdVO₄:Eu³⁺@mSiO₂ NPs turned red under excitation of UV lamp at 307 nm (inset of Fig. 4). The emission peaks at 540 nm, 585 nm, and 618 nm appeared in the PL spectrum of GdVO₄:Eu³⁺@mSiO₂ NPs, and strong red emission was observed at 618 nm. These peaks were similar to the previously reported values.¹³

Fig. 4 Room-temperature PL spectrum of the GdVO₄:Eu³⁺@mSiO₂ NPs in water. Inset shows photographic images of GdVO₄:Eu³⁺@mSiO₂ NP dispersion under room light (left) and UV irradiation (right).

3.3. MRI contrasting effect of GdVO₄:Eu³⁺@mSiO₂ NPs

Fig. 5A shows the T₁-weighted MR images of the GdVO₄:Eu³⁺@mSiO₂ NPs with various concentrations of Gd ion (0, 0.06, 0.13, 0.26, and 0.52 mM). MR signal intensity increased as Gd ion concentration rose. The efficacy of a contrast agent is generally expressed by its relaxivity, which is defined as the gradient of the linear plot of relaxation rates (1/T_i, i = 1, 2) versus Gd concentration [Gd] (i.e., 1/T_i = 1/T_o + r_i·[Gd], where T_i is the relaxation time for a contrast agent solution concentration [Gd], and T_o is the relaxation time in the absence of a contrast agent).⁴⁰ Fig. 5B shows linear correlation between the relaxation rate (R₁ = 1/T₁) and the concentration of Gd. Particularly, the r₁ value of GdVO₄:Eu³⁺@mSiO₂ NPs (2.21 mM⁻¹ s⁻¹) was larger than that of dense silica-coated GdVO₄:Eu³⁺ NPs (GdVO₄:Eu³⁺@dSiO₂ NPs) (0.64 mM⁻¹ s⁻¹) (Fig. 3D, and see also ESI Fig. S3†), indicating that the mesoporous silica coating increase the T₁ MRI contrast. It is known that the change of the longitudinal relaxivity (r₁) is influenced by the local diffusion of water molecules in the vicinity of an MRI contrast agent.⁴¹ Therefore, the longitudinal relaxivity of gadolinium-based contrast agents was enhanced by the entrapment of Gd³⁺ ions in mesoporous structures.^42,43 In our GdVO₄:Eu³⁺@mSiO₂ NPs, the mesoporous silica coating facilitates the access of water molecules to the Gd core, thereby leading to the efficient relaxation of water, and subsequently enhanced T₁-weighted MRI contrast.^44,45 The performance of MRI contrast agents is also characterized by the r₂/r₁ ratio, which must be 1 to be the best positive contrast agent. For example, “Magnevist” (Gadopentetate dimeglumine; a commercial gadolinium complex) has an optimal r₂/r₁ ratio of 1 (r₁ = 4.6 mM⁻¹ s⁻¹, r₂ = 4.5 mM⁻¹ s⁻¹) at 1.5 T.^46,47 The relaxometric ratio (r₂/r₁ ratio) of GdVO₄:Eu³⁺@mSiO₂ NPs was calculated to be 3.39. When compared to the above-mentioned commercial Gd compound, it is understood that the relaxometric properties of GdVO₄:Eu³⁺@mSiO₂ NPs need to be substantially improved. However, considering that the GdVO₄:Eu³⁺@mSiO₂ NPs are specifically designed as a multifunctional theragnostic agent, they are excellent candidates to be NP–based positive MRI contrast agents in T₁-weighted MRI applications.

Fig. 5 MR property of the GdVO₄:Eu³⁺@mSiO₂ NPs. (A) T₁-weighted MR images of the GdVO₄:Eu³⁺@mSiO₂ NP dispersion in water. The contents of Gd in the dispersions are indicated above the images. (B) Linear plot of Gd concentration versus 1/T₁ with a relaxivity value (r₁) of 2.21 mM⁻¹ s⁻¹.

3.4. In vitro multimodal imaging and drug delivery

The cell viability was assessed by the MTT assay. The cell viability was not hindered by the presence of GdVO₄:Eu³⁺@mSiO₂-PEG NPs up to a concentration of 50 μg Gd mL⁻¹ for both SK-BR-3 cell lines and MCF-7 cell lines (Fig. S4†). Within this concentration of Gd ion, we investigated the potentials of GdVO₄:Eu³⁺@mSiO₂-PEG NPs for multimodal imaging and drug delivery. Fig. 6A shows the results of in vitro T₁-weighted MRI of the pelleted MCF-7 cells labeled with GdVO₄:Eu³⁺@mSiO₂-PEG NPs (concentration 20 μg Gd mL⁻¹). No hyperintense signal was detected when the unlabeled cell pellet was imaged. However, a hyperintense signal appeared on pelleted MCF-7 cells labeled with nanoparticles in both T₁-weighted MR and their color-coded maps. Like of the MR results, the fluorescence appeared on the pelleted MCF-7 cells labeled with GdVO₄:Eu³⁺@mSiO₂-PEG NPs (Fig. 6B). The tumor cell responses to chemotherapy can be monitored real-time due to the combination of MR and fluorescence imaging capabilities of GdVO₄:Eu³⁺@mSiO₂-PEG NPs, which provide important feedbacks in cancer treatment.⁴⁸

Fig. 6 In vitro T₁-weighted MR and fluorescence images of the pelleted MCF-7 cells labeled with GdVO₄:Eu³⁺@mSiO₂-PEG NPs. (A) T₁-Weighted MR images and their color-coded maps. (B) Fluorescence images.

Doxorubicin (DOX), a chemotherapeutic agent for cancer,⁴⁹ was used as a model drug and was incorporated into the nanoparticles. DOX can be easily attracted and adsorbed onto the large amount of open channels of mesoporous silica shell via electrostatic attraction or/and through forming hydrogen bonds with the surface silanols.⁵⁰ The loading efficiency was assessed by UV-Vis spectroscopy and the typical DOX loading was measured to be 14.6 wt% (0.54 mg DOX/mg NPs). After 2 h incubation, 15.5% of DOX was released from the NPs at pH 7.4, whereas 32.5% of DOX released at pH 5.0 (See the ESI Fig. S5†). The drug release profile depicts a faster initial release of the absorbed DOX in pores of silica, followed by a plateau (Fig. S5C†). In particular, loaded drugs were released from the NPs in a sustained manner at pH 7.4, resulting 50% of DOX was still loaded in the NPs even after 24 h. However, lowering of the pH results in an increase of drug release. The electrostatic interaction between payload and silica is decreased due to the protonation of surface silanol at low pH.⁵¹

The uptake and internalization of the free DOX and DOX-loaded GdVO₄:Eu³⁺@mSiO₂-PEG NPs in MCF-7 cells were evaluated by the confocal laser scanning microscopy (CLSM). CLSM images of the nuclei of cancer cells were obtained from blue fluorescence by Hoechst 33342 dye, and red fluorescence from DOX indicated that nanoparticles can deliver the chemotherapeutic agent. As shown in Fig. 7A and B, the intracellular distribution of the DOX-loaded GdVO₄:Eu³⁺@mSiO₂-PEG NPs is different from that of free DOX. Free DOX was mostly found in the cell nuclei (Fig. 7A), since DOX is known to be transported to cells by a passive diffusion mechanism.⁵² In contrast, the DOX-loaded GdVO₄:Eu³⁺@mSiO₂-PEG NPs were accumulated mostly in the cytoplasm (Fig. 7B), which is the same result obtained from other nanostructured drug-delivery vehicles.^31,53 These results demonstrate that the GdVO₄:Eu³⁺@mSiO₂-PEG NP is an efficient delivery vehicle with sustained delivery behavior. NPs loaded with DOX were internalized to the cytoplasm via endocytosis, and DOX may be released to enter cell nuclei. The delivered DOX inside the cell nucleus intercalates into DNA and interacts with topoisomerase II to cause DNA cleavage, cell growth inhibition, and finally cell death.⁵⁴ Therefore, in vitro cytotoxicity effects of the free DOX and DOX-loaded GdVO₄:Eu³⁺@mSiO₂-PEG NPs were tested on the MCF-7 cells for assessment of drug delivery efficacies of nanoparticles. As shown in Fig. 7C, growth inhibition was observed when the cells were treated with either the free DOX or DOX-loaded GdVO₄:Eu³⁺@mSiO₂-PEG NPs. Furthermore, consistent with CLSM results, the entrapped DOX from DOX-loaded GdVO₄:Eu³⁺@mSiO₂-PEG NPs seems to exhibit sustained release behavior.^55,56 These results demonstrate that the GdVO₄:Eu³⁺@mSiO₂-PEG NPs can be an efficient drug-delivery vehicle.

Fig. 7 Uptake of the free DOX and DOX-loaded GdVO₄:Eu³⁺@mSiO₂-PEG NPs in cancer cells and cytotoxicity data. (A and B) Optical and CLSM images of the free DOX (A) and of the DOX-loaded GdVO₄:Eu³⁺@mSiO₂-PEG NPs (B) incubated in MCF-7 cells for 2 h (blue: the nuclei of cells stained by Hoechst 33342, red: DOX fluorescence). (C) In vitro cytotoxicity of the free DOX (red) and of the DOX-loaded GdVO₄:Eu³⁺@mSiO₂-PEG NPs (blue) against MCF-7 cells, demonstrating that the GdVO₄:Eu³⁺@mSiO₂-PEG NPs can be used as anti-cancer drug delivery vehicles.

4. Conclusion

We have successfully synthesized discrete and uniform mesoporous silica-coated GdVO₄:Eu³⁺ NPs. The GdVO₄:Eu³⁺ NPs have potential to be used as a multimodal imaging probe of fluorescence and magnetic resonance imaging. Eu³⁺ doped in GdVO₄ NPs exhibit strong red emission and Gd³⁺ in GdVO₄ NPs can be used as a T₁ contrast agent for MRI. Furthermore, when the nanoparticles were coated with mesoporous silica shell, water accessibility to the magnetic core is increased, providing more efficient T₁ MR imaging contrast. Mesoporous silica shell also enables drug delivery with sustained release. We have thus successfully demonstrated simultaneous T₁ MR and fluorescence imaging as well as drug delivery using the mesoporous silica-coated GdVO₄:Eu³⁺ NPs.

Acknowledgements

This work was supported by the Korean Health Technology R&D Project, Korean Ministry of Health & Welfare (A111014), the Research Center Program of the Institute for Basic Science (IBS) in Korea (IBS-R006-D1), the Engineering Research Center of Excellence Program of Korea (Grant NRF-2014R1A5A1009799) and the Basic Science Research Program (Grant NRF-2014R1A1A1003555) through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra06628f

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