Facile one-pot synthesis of different surfactant-functionalized water-soluble Fe₃O₄ nanoparticles as magnetic resonance imaging contrast agents for melanoma tumors

Abstract

A facile and efficient solvothermal strategy is described to synthesize modified Fe₃O₄ nanoparticles (NPs) as magnetic resonance (MR) imaging contrast agents. In this study, glycerine was used not only as a solvent but also as a reducing agent due to its nontoxicity and high viscosity, which result in the advantages of good dispersibility and narrow size distribution of the Fe₃O₄ NPs. In addition, sodium citrate, L-asparagine and polyvinylpyrrolidone were employed as surfactants for the surface modification of Fe₃O₄ NPs on account of their non/low-toxicity, water-solubility and biocompatibility. The modification process was completed by one step rather than complicated grafting approaches. Moreover, the resultant aqueous solution of the modified Fe₃O₄ NPs turned out to be stable for more than 6 months with no flocculates or precipitates appearing in the stock fluids. Then, T₂ weighted images and T₂ relaxation times of the modified Fe₃O₄ NPs were examined to investigate their biological applications for magnetic resonance (MR) imaging as T₂ contrast agents. As a consequence, the sodium citrate-modified Fe₃O₄ NPs exhibit a higher r₂ relaxivity than that of sodium citrate combined with PVP and L-asparagine-modified Fe₃O₄ NPs. Furthermore, in order to investigate their contrast effect for melanoma tumors, we also carried out a study of in vitro cytotoxicity and an observation of the tissue sections. The results suggest that these modified Fe₃O₄ NPs may be potential contrast agents for detecting melanoma tumors.

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1. Introduction

In recent years, biological imaging has been a rapidly growing field, not only in fundamental biology but also in medical science, and it has attracted much attention due to its analytic and diagnostic ability at the molecular or cellular level.¹ As a result, a new discipline, known as “molecular imaging” has emerged, which combines molecular biology and in vivo imaging.¹ Molecular imaging techniques including computed tomography (CT) imaging,^2,3 position emission tomography (PET) imaging,^4,5 single photon emission computed tomography imaging,^6–8 optical imaging^9,10 and magnetic resonance (MR) imaging^11–14 have been illustrated to have promising potential in disease diagnosis. Among these different molecular imaging techniques, MR imaging is a powerful noninvasive imaging technique because of its high image contrast, good temporal and spatial resolution, arbitrary fault orientation, various image types, non-ionizing radiation source, and long effective imaging windows.^12,15,16 It has been considered as the preferred tool for imaging the brain and the central nervous system, examining soft tissue, assessing cardiac function and detecting tumors.¹ Based on the extensive application of MR imaging, the MR imaging contrast agents, which can make images more clear and allow better interpretation, have also been of great interest to worldwide researchers. Recent advances in nanotechnology show that various nanoparticles (NPs) can be used as contrast agents for molecular imaging applications.^2–14,17 Among the various nanoparticles, magnetic iron oxide (IO) nanostructured materials are currently one of the most promising contrast agents for MR imaging.^18–21 IONPs have been extensively developed as negative contrast agents that can shorten the T₂ relaxation time of water protons, resulting in enhanced imaging contrast and sensitivity.^1,22 Until now, IONPs are the only commercial T₂ contrast agents due to their biocompatibility and superior magnetic properties. Besides, they are relatively easy to synthesize and have a very active area of research.^23–26

As is known to all, for the applications of MR imaging, IONPs must have good dispersibility, water solubility, good colloidal stability, high magnetization values and good biocompatibility under biological conditions. So as to meet the above requirements, tremendous efforts have been made in fabricating the required IONPs. Fortunately, by means of recent studies, the surface modification of IONPs by non-toxicity and hydrophilic polymers has been proven to be an effective strategy to make particles that have good colloidal stability and biocompatibility, such as dextran, dendrimers, polyethylene glycol (PEG), or polyethylene oxide (PEO) and polyethyleneimine (PEI).²⁷ With in-depth research, scientists’ focus has turned away from general organs and tissues to tumors (or cancer). In this respect, many remarkable results have been achieved. For example, Hu et al.²⁸ prepared PEG-coated Fe₃O₄ (denoted Fe₃O₄@PEG-COOH) nanocrystals, which can potentially be used as effective MR imaging contrast agents for cancer diagnosis when coupled with a specific cancer-targeting antibody. Based on the combination of an amino-functionalized poly(isoprene) preligand (PI-N3) and a polyisoprene-block-poly(ethylene oxide) diblock copolymer (PI-b-PEO), IO nanocrystals of excellent water solubility and biocompatibility have been successfully obtained for in vitro and in vivo tumor imaging by Pöselt et al.²⁹ Shi and coworkers³⁰ synthesized dendrimer-functionalized shell-crosslinked IONPs for in vivo magnetic resonance imaging of tumors.

Herein, we present a facile one-pot hydrothermal strategy to synthesize three kinds of Fe₃O₄ NPs modified with sodium citrate, L-asparagine, and sodium citrate combined with polyvinylpyrrolidone (PVP), respectively. It is known that sodium citrate is nontoxic, stable and water-soluble. PVP as a kind of polymer compound also has the advantages of excellent biocompatibility, low-toxicity and good water solubility. In particular, L-asparagine intrinsically exists in organisms as one of the amino acids that make up human proteins, and is completely nontoxic. Therefore, these three kinds of modifier are employed for the surface modification of Fe₃O₄ NPs. Furthermore, these three kinds of modified Fe₃O₄ NPs, used as contrast agents for melanoma tumors, were separately investigated.

2. Experimental section

2.1. Materials

Hexahydrated ferric chloride (FeCl₃·6H₂O, 99.0%), sodium hydroxide (NaOH > 96.0%), sodium citrate (>99.0%), PVP (K-30), L-asparagine (>99.0%), and glycerine (C₃H₈O₃ > 99.0%) were all of analytical grade and used as received. Deionized water was used in all experiments.

2.2. Characterization

An Agilent Cary 50 UV-vis spectrophotometer was used to collect the UV-vis spectra for the determination of [Fe] concentration in solutions. The crystalline structures were identified using an X-ray diffractometer (XRD, Rigaku D/max 2500pc) with copper-K alpha (Cu Kα) radiation (λ = 1.5406 Å) at a scan rate of 0.02°/1(s). Electron micrographs of the samples were taken using a transmission electron microscopy (TEM) at 200 kV (Philips Tecnai 20). A drop of the sample was placed on a Cu mesh coated with an amorphous carbon film and then air-dried before measurements. A Fourier Transform Infrared Spectrometer (FT-IR, NEXUS, 670) was used for FT-IR characterization. The M–H magnetization curves at 300 K were measured using a vibrating sample magnetometer (VSM-100, Lakeshore, USA). Transverse relaxivity measurements were conducted on a 1.5 T scanner (GE, HDx), equipped with a wrist solenoid coil.

2.3. Synthesis of modified Fe₃O₄ NPs

Three kinds of modified Fe₃O₄ NPs were obtained through a facile one-pot hydrothermal strategy. In detail, to synthesize sodium citrate modified Fe₃O₄ NPs, FeCl₃·6H₂O (1 mmol) and sodium citrate (0.5 g) were first dissolved in 5 mL of deionized water to form a homogeneous solution with the assistance of ultrasonication. Afterward, 16 mL of glycerine was added to the solution, and then a certain amount of aqueous solution of NaOH (2 mM) was added under vigorous stirring. Subsequently, the mixed solution was transferred into a 50 mL Teflon lined stainless steel autoclave. The autoclave was sealed and maintained at 200 °C for 4 h and then cooled to room temperature naturally. The resulting products were collected by centrifugation, washed several times with distilled water and absolute ethanol, and finally redispersed into deionized water.

In order to obtain sodium citrate combined with PVP modified IONPs, FeCl₃·6H₂O (1 mmol), sodium citrate (0.1 g) and PVP (0.5 g) were first dissolved in 5 mL of deionized water to form a homogeneous solution with the assistance of ultrasonication. The remaining steps were the same as for the synthesis of the sodium citrate modified IONPs.

For the preparation of L-asparagine modified Fe₃O₄ NPs, FeCl₃·6H₂O (1 mmol) and L-asparagine (0.3 g) were first dissolved in 7 mL of deionized water to form a homogeneous solution with the assistance of ultrasonication. Afterward, 14 mL of glycerine was added to the solution, and then a certain amount of aqueous solution of NaOH (2 mM) was added under vigorous stirring. Subsequently, the mixed solution was transferred into a 50 mL Teflon lined stainless steel autoclave. The autoclave was sealed and maintained at 200 °C for 3 h and then cooled to room temperature naturally. The remaining steps were the same as for the synthesis of the sodium citrate modified Fe₃O₄ NPs.

2.4. Cell culture

Mouse melanoma cell lines (B16 cells) came from the Department of Immunology of Bethune Basic Medical Sciences, Jilin University. B16 cells were cultured in RPMI Medium 1640 containing 10% (v/v) heat-inactivated Fetal Bovine Serum (FBS) supplemented with 100 U mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin. B16 cells were subcultured every two days and cells in the logarithmic growth phase were digested with 0.25% trypsin and 0.02% Ethylene Diamine Tetraacetic Acid (EDTA). The passaged ratio was approximately 1 : 3.

2.5. Animal models

Female C57 mouse inbred lines of 18–20 g body weight were supplied by the Department of Experimental Animals, Jilin University, and maintained under standard housing conditions. For the preparation of the B16 tumor model, mouse hair on the back was cut and the corresponding skin was disinfected with 75% alcohol. Then, 0.1 mL of a cell suspension containing 2 × 10⁶ B16 cells was injected intradermally into the bare back region. When the tumor diameter size was more than 0.5 cm, the tumor model animals were ready for the experiments.

2.6. In vitro cytotoxicity study and cell morphology observation

B16 cells were regularly cultured in 25 cm² plates in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, penicillin (100 U mL⁻¹) and streptomycin (100 U mL⁻¹) under 37 °C and 5% CO₂. The cytotoxicity of the as synthesized modified Fe₃O₄ NPs at different concentrations was assessed by using a MTT assay. Briefly, B16 cells were seeded into a 96-well plate at an initial density of 1 × 10⁴ cells per well in 200 mL RPMI 1640 medium. After overnight incubation to bring the cells to confluence, the medium was substituted with fresh medium (200 μL) containing pure PBS buffer (control), sodium citrate modified Fe₃O₄ NPs, sodium citrate combined with PVP modified Fe₃O₄ NPs and L-asparagine modified Fe₃O₄ NPs at different concentrations (10, 25, 50, 75 and 100 μg mL⁻¹, respectively). After 24 h incubation at 37 °C and 5% CO₂, 20 μL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 0.5 mg mL⁻¹ in PBS buffer) was added to each well to detect the metabolically active cells. After further incubation for 4 h in an incubator at 37 °C, 200 μL DMSO was added to each well to replace the culture medium and dissolve the insoluble formazan crystals. The plates were oscillated for one hour to make sure all the formazan was dissolved in DMSO. The assays were carried out according to the manufacturer’s instructions and the absorbance of each well was measured using a BIOTEK Epoch Microplate Spectrophotometer at 570 nm. All experiments were performed in triplicate.

After incubation with sodium citrate modified Fe₃O₄ NPs, sodium citrate combined with PVP modified Fe₃O₄ NPs and L-asparagine modified Fe₃O₄ NPs at different concentrations (10, 25, 50, 75 and 100 μg mL⁻¹) for 24 h, the cells were washed with PBS for 5 times and observed by phase contrast microscopy (IX71 inverted fluorescence microscope). The magnification was set at 200× for each sample.

2.7. In vitro MR imaging measurements for the Fe₃O₄ NPs

For T₂-weighted imaging, the samples were first diluted with water to different Fe concentrations (0.1–1.0 mM) in 4 mL tubes. Then measurements were performed on a 1.5 T scanner to achieve MR images. The instrument parameters were set at a section thickness of 0.5 mm, TR of 2000 ms, TE of 18 ms, and excitation number of 1. The T₂ relaxivity was calculated by a linear fit of the inverse T₂ (1/T₂) relaxation time as a function of the Fe concentration.

2.8. Observation of tissue sections

Animal experiments were performed according to protocols approved by the institutional ethical committee for animal care, and also in accordance with the policy of the National Ministry of Health. A mouse was randomly selected as the control group, which was not injected. For the three experimental groups, the aqueous solutions of sodium citrate, L-asparagine and sodium citrate combined with PVP-modified Fe₃O₄ NPs were injected into the tail vein of tumor-bearing mice (B16 tumor) at a dose of 10 mg Fe per mouse, respectively.

To observe the nanoparticle distribution in the tissues, the sections of tissues harvested from the mice 1 day after injection were processed with Prussian blue and hematoxylin and eosin (H&E) staining.

3. Results and discussion

TEM was used to characterize the morphology and size distribution of the formed sodium citrate, sodium citrate combined with PVP and L-asparagine modified Fe₃O₄ NPs (Fig. 1a–c). It is clear that all the three kinds of NPs possess an irregular spherical shape and quite a uniform size distribution with a mean diameter of 10.9 nm for the sodium citrate-modified Fe₃O₄ NPs, 9.4 nm for the sodium citrate combined with PVP-modified Fe₃O₄ NPs and 5.9 nm for the L-asparagine-modified Fe₃O₄ NPs. In the case of the sodium citrate-modified NPs, from the well-resolved lattice fringes of the HRTEM image (Fig. 1d) taken from one nanoparticle, an interplanar spacing of 0.253 nm was obtained, being consistent with the distance of the (311) plane of Fe₃O₄ crystals with an inverse spinel structure. The corresponding SAED (selected area electron diffraction) pattern (inset of Fig. 1d) shows that the obtained Fe₃O₄ NPs are polycrystalline in nature. On the whole, all the modified Fe₃O₄ NPs have a narrow size distribution, good dispersibility and crystallinity, which is beneficial for the biological application as contrast agents.

Fig. 1 TEM micrographs and size distribution of the (a) sodium citrate, (b) sodium citrate combined with PVP, and (c) L-asparagine modified Fe₃O₄ NPs. (d) HRTEM image of the sodium citrate modified NPs. The upper right inset shows the corresponding SAED pattern.

The hydrodynamic size of the three kinds of modified Fe₃O₄ NPs was analyzed by DLS (dynamic light scattering) (Table 1). The sodium citrate, sodium citrate combined with PVP and L-asparagine modified Fe₃O₄ NPs dispersed in water were measured to be 34.57 nm, 26.09 nm and 260.4 nm, respectively. It is noteworthy that the obtained hydrodynamic sizes of the three kinds of samples were all larger than those obtained by TEM. According to the previous reports, this is attributed to the fact that DLS measures the size of aggregated clusters of particles in aqueous solution that may consist of many single Fe₃O₄ NPs, while TEM just measures the single Fe₃O₄ NPs.^18,20 Furthermore, it can be seen that all particles have a relatively small polydispersity index (PDI), indicating that all the three kinds of Fe₃O₄ NPs have a quite uniform size distribution.

Table 1 Zeta potential and hydrodynamic size of the sodium citrate-Fe₃O₄ NPs, the sodium citrate combined with PVP-Fe₃O₄ NPs and the L-asparagine-Fe₃O₄ NPs

Sample	Zeta potential value (mV)	Hydrodynamic size (nm)	PDI
Sodium citrate-Fe3O4 NPs	−22.8 ± 0.25	34.57 ± 4.13	0.290 ± 0.015
Sodium citrate combined with PVP-Fe3O4 NPs	−10.3 ± 0.18	26.02 ± 2.96	0.392 ± 0.021
L-Asparagine-Fe3O4 NPs	−12.2 ± 0.20	260.4 ± 6.87	0.366 ± 0.024

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Zeta potential measurements were also employed to confirm the particle surface potential. As shown in Table 1, the sodium citrate, sodium citrate combined with PVP and L-asparagine modified Fe₃O₄ NPs are negatively charged with surface potentials of −22.8 mV, −10.3 mV and −12.2 mV, respectively. This should be due to the fact that a carboxylate group (COO⁻) existed in both sodium citrate and L-asparagine. That is, sodium citrate and L-asparagine are negatively charged in an aqueous solution due to the existence of COO⁻, thus, after modification by sodium citrate, sodium citrate combined with PVP and L-asparagine, all the three kinds of Fe₃O₄ NPs are negatively charged. It also suggests that the NPs have been successfully modified.

XRD was performed on the three samples to characterize the phase and crystallization of the modified Fe₃O₄ NPs (Fig. 2). It can be seen that the three kinds of modified Fe₃O₄ NPs have the same diffraction peaks, and all the peaks can be indexed to the face-centered cubic structure of magnetite according to JCPDS Card no. 19-0629. The lattice spacing calculated from the diffraction peaks observed at 18.2, 30.1, 35.7, 43, 53.7, 57.1, and 62.5° match the (111), (220), (311), (400), (422), (511), and (440) planes of the Fe₃O₄ crystals, respectively. The sharpness of the X-ray diffraction peaks confirm that the as-prepared samples should be highly crystallized without any other impurities.

Fig. 2 XRD patterns of the Fe₃O₄ NPs modified by sodium citrate (a), sodium citrate combined with PVP (b), and L-asparagine (c).

Experimental results show that the synthesized Fe₃O₄ NPs have good water solubility, and they can remain stable for at least six months in ambient conditions (Fig. S1†). In order to explore the surface nature of the hydrophilic NPs, FT-IR spectra measurements were performed on the NPs. Fig. 3a depicts the FT-IR spectra of the (I) sodium citrate, (II) sodium citrate combined with PVP, and (III) L-asparagine modified Fe₃O₄ NPs. As shown in spectrum (I) in Fig. 3a, the IR peak at 575 cm⁻¹ corresponds to the stretching vibration of the Fe–O bond for the Fe₃O₄ NPs. The three adsorption peaks at 1616, 1385 and 1070 cm⁻¹ are attributed to the asymmetric and symmetric stretching modes of COO⁻ and C–O stretching.³¹ The two characteristic peaks at 2926 and 2854 cm⁻¹ are assignable to the asymmetric and symmetric stretching modes of –CH₂. In addition, the IR spectrum of the sodium citrate modified Fe₃O₄ NPs is distinct compared with single sodium citrate, which probably arises from the strong interaction between the NPs and COO⁻ in citrates.³² Thereby, it indicates that some citrates adsorb on the NP surface via the carboxyl group resulting in excellent water solubility. By comparison with spectrum (I), the adsorption peak at 1293 cm⁻¹ in spectrum (II) corresponds to the stretching vibration of the C–N bond in N-vinyl pyrrolidone molecules.³³ The peak at 1639 cm⁻¹ may be ascribed to the combined action of the COO⁻ in citrates and the C=C bond in PVP. In general, the data illustrates the successful conjugation of N-vinyl pyrrolidone molecules onto the surface of the Fe₃O₄ NPs. From spectrum (III) we can see that the adsorption peaks appear at 1625, 1385, 575 cm⁻¹and so on. The IR peak at 575 cm⁻¹ is attributed to the stretching vibration of the Fe–O bond. A certain extent of displacement appeared due to the interaction between groups, and the peaks at 1625, 1385 and 1081 cm⁻¹ are also assigned to the asymmetric and symmetric stretching modes of COO⁻ and C–O stretching, respectively. The absence of an absorption peak corresponding to acylamino (−CONH₂), can be observed, which may be covered by the COO⁻ bond. Based on the above analysis of the FT-IR spectra, we can conclude that the Fe₃O₄ NPs were successfully modified by sodium citrate, PVP and L-asparagine.

Fig. 3 (a) The FT-IR spectra of the (I) sodium citrate, (II) sodium citrate combined with PVP, and (III) L-asparagine modified Fe₃O₄ NPs. (b) The structural formulas of sodium citrate – C₆H₅Na₃O₇·2H₂O, PVP – (C₆H₉NO)_n and L-asparagine – C₄H₈N₂O₃.

Fig. 3b shows the structural formulas of sodium citrate, PVP and L-asparagine. It is observed that all the three kinds of surfactants contain polar groups, such as the –OH and –COO⁻ groups of sodium citrate, the lactam group (–CON–) of PVP, and the –COOH, –CONH₂ and –NH₂ groups of L-asparagine. The polar groups can interact with polar water molecules to form hydrogen bonds, which leads to a considerable solubility in water. Therefore, the good water solubility of the Fe₃O₄ NPs is induced by the modification of the polar groups containing surfactants. However, the Fe₃O₄ NPs have poor water solubility when PVP is used alone. Based on this point, sodium citrate combined with PVP is suggested to modify the IONPs.

These modified Fe₃O₄ NPs were also characterized using UV-vis spectroscopy, which further confirmed the modification process (Fig. 4a). After modifying the Fe₃O₄ NPs with sodium citrate, sodium citrate combined with PVP and L-asparagine, there was no significant change in the UV-vis spectra between the three types of Fe₃O₄ NPs. This indicates the formation of stable and monodispersed nanospheres. Furthermore, the minimal shift in the spectra can be associated with the change in Fe₃O₄ NPs surface modifications, and suggests a negligible increase in the nanoparticle size after modification.³⁴

Fig. 4 (a) Optical properties of the synthesized Fe₃O₄ NPs. (b) Magnetic hysteresis loops of the prepared Fe₃O₄ NPs modified by sodium citrate (I), sodium citrate combined with PVP (II), and L-asparagine (III). The inset shows the low-field region.

To determine the magnetic properties of the three kinds of modified Fe₃O₄ NPs, their powders are evaluated by a VSM at room temperature. Fig. 4b presents a typical plot of the magnetization versus the applied magnetic field. It can be observed that the saturation magnetization values (M_s) of the sodium citrate, sodium citrate combined with PVP and L-asparagine-modified NPs are 52.38, 50.19 and 44.36 emu g⁻¹, respectively. These M_s values are lower than the bulk Fe₃O₄ due to size-dependent magnetization and the presence of organic ligands on the surface of the Fe₃O₄ NPs.³⁵

The potential cytotoxicity of the three kinds of modified Fe₃O₄ NPs was explored. A cytotoxicity test of modified Fe₃O₄ NPs in vitro was performed on B16 cells. Fig. 5 shows the cell viability after incubation of the B16 cells with sodium citrate modified Fe₃O₄ NPs, sodium citrate combined with PVP modified Fe₃O₄ NPs and L-asparagine modified Fe₃O₄ NPs at different iron concentrations of 10, 25, 50, 75 and 100 μg mL⁻¹ for 24 h. From the statistical analysis, it can be seen that all the three types of modified Fe₃O₄ NPs showed very low cytotoxicity in the studied concentration range (0–100 μg mL⁻¹) when compared with the PBS control. This result indicates that the sodium citrate modified Fe₃O₄ NPs, sodium citrate combined with PVP modified Fe₃O₄ NPs and L-asparagine modified Fe₃O₄ NPs are non-cytotoxic even at a concentration of 100 μg mL⁻¹. Furthermore, it is worth noting that the cell viability of the L-asparagine modified Fe₃O₄ NPs increases with increasing iron concentration. This interesting phenomenon may be explained by the fact that L-asparagine can be more easily degraded and absorbed by the cells for further proliferation. In short, these results demonstrate that all the modified Fe₃O₄ NPs employed in our work have excellent biocompatibility and non-toxicity is observed for the NPs as contrast agents.

Fig. 5 MTT assay of B16 cell viability after incubation with sodium citrate modified Fe₃O₄ NPs, sodium citrate combined with PVP modified Fe₃O₄ NPs and L-asparagine modified Fe₃O₄ NPs at concentrations of 0–100 μg mL⁻¹ for 24 h. B16 cells treated with PBS were used as a control.

The morphology of B16 cells treated with sodium citrate modified Fe₃O₄ NPs, sodium citrate combined with PVP modified Fe₃O₄ NPs and L-asparagine modified Fe₃O₄ NPs at different concentrations for 24 h was also observed by phase contrast microscopy to further assess their cytotoxicity (Fig. 6 and S2 of ESI†). Compared with the control cells treated with PBS (Fig. 6a, S2a and g†), it is clearly observed that the cells do not display any appreciable morphological changes at concentrations up to 100 μg mL⁻¹. These results corroborate the above MTT assay data, suggesting that all the modified Fe₃O₄ NPs have a good cytocompatibility in the given concentration range.

Fig. 6 Phase contrast microscopy images of B16 cells treated with PBS buffer (a), sodium citrate modified Fe₃O₄ NPs at concentrations of 10 μg mL⁻¹ (b), 25 μg mL⁻¹ (c), 50 μg mL⁻¹ (d), 75 μg mL⁻¹ (e), 100 μg mL⁻¹ (f) for 24 h.

Fe₃O₄ NPs are known to be good T₂ contrast agents in MR imaging. To investigate the efficacy of the as-synthesized modified Fe₃O₄ NPs as enhanced MRI contrast agents, the T₂-weighted images of the modified Fe₃O₄ NPs for different Fe concentrations (0.1–1.0 mM) were acquired on a clinical 1.5 T MR imaging instrument and the transverse relaxivity (r₂, the transverse relaxation rate per mM of iron) was calculated (Fig. 7). From the T₂-weighted MR images (Fig. 7a), it can be seen that all the three kinds of Fe₃O₄ NPs are able to decline the MR signal intensity with increasing Fe concentration, due to the dipolar interaction of the magnetic moments of the particles and protons in the water, making the images darker. This result indicates that all the modified Fe₃O₄ NPs generate MR contrast on T₂-weighted sequences, and are promising T₂ MR imaging contrast agents. However, by comparison with the other two kinds of NPs, the sodium citrate-modified Fe₃O₄ NPs exhibit a more noticeable difference in the contrast compared with the control. Fig. 7b shows the relaxation rates 1/T₂ as a function of the Fe concentration for the Fe₃O₄ NPs. It was found that the plots were well-fitted by linear functions within the analyzed range of Fe concentration. The r₂ of the sodium citrate-modified Fe₃O₄ NPs, sodium citrate combined with PVP-modified Fe₃O₄ NPs and L-asparagine-modified Fe₃O₄ NPs were separately calculated to be 72.80, 55.10 and 46.79 mM⁻¹ s⁻¹, respectively, as shown in Table 2. In comparison, the sodium citrate-modified Fe₃O₄ NPs offered a stronger T₂ shortening effect with a better transverse relaxivity due to their larger particle size and higher magnetization.^36,37

Fig. 7 T₂-weighted images (a) and linear fitting of 1/T₂ (b) of the sodium citrate-modified Fe₃O₄ NPs, sodium citrate combined with PVP-modified Fe₃O₄ NPs and L-asparagine-modified Fe₃O₄ NPs with Fe concentrations of 0 (control), 0.1, 0.25, 0.5, 0.75, 1.0 mM.

Table 2 Relaxivity studies of the modified Fe₃O₄ NPs with different sizes and magnetization values

Fe3O4 NPs	Size (nm)	Ms (emu g^−1)	r2 (mM^−1 s^−1)
Sodium citrate modified	10.9	52.38	72.80
Sodium citrate combined with PVP modified	9.4	50.19	55.10
L-Asparagine modified	5.9	44.36	46.79

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Histology samples were also examined by Prussian blue staining (iron) to observe the NPs accumulated in the tumor, liver, heart and kidney (Fig. 8, S3 and S4†). We can see that the iron staining (the blue area) mainly appeared in the liver and tumor (especially in liver), and minimal staining was observed in the heart and kidney. Besides, it can be seen that the iron staining of sodium citrate modified Fe₃O₄ NPs in the tissues appeared stronger than that of the other two kinds of Fe₃O₄ NPs. This result is in accordance with the in vitro experiments. Thus, we can conclude that the sodium citrate, sodium citrate combined with PVP and L-asparagine modified Fe₃O₄ NPs can be successfully delivered into the tumors, and these modified Fe₃O₄ NPs should be effective MR imaging agents for tumors.

Fig. 8 Histological sections showing the distribution of the Fe₃O₄ NPs in the tumor and liver. Monodisperse Fe₃O₄ NPs, modified with sodium citrate, sodium citrate combined with PVP and L-asparagine were intravenously injected into mice at a dose of 10 mg Fe per kg, and the sections of tumor (a) and liver (b) tissues harvested from the mice 1 day after injection were processed with Prussian blue and H&E staining. The control samples were tissues from animals that were not injected with NPs. The scale bar indicates 50 μm.

4. Conclusions

In summary, we have successfully developed a facile one-pot hydrothermal approach to generating three types of Fe₃O₄ NPs functionalized with sodium citrate, sodium citrate combined with PVP and L-asparagine, and investigated their potential use as contrast agents for MR imaging. The fabricated modified Fe₃O₄ NPs displayed good dispersibility and were water-soluble, stable, nontoxic and biocompatible. The in vitro applications have demonstrated that all the as-prepared modified Fe₃O₄ NPs can potentially be used as effective MR imaging contrast agents. Furthermore, among the three kinds of modified Fe₃O₄ NPs, the sodium citrate-modified Fe₃O₄ NPs were demonstrated to exhibit a relatively high contrast effect. This finding suggests the potential use of the synthesized modified Fe₃O₄ NPs as excellent MRI contrast agents. Further investigations in terms of in vivo experiments of these modified Fe₃O₄ NPs are currently in progress.

Acknowledgements

This study was supported by grants provided by the Natural Science Foundation of China (Grant no. 81270315) and the Project of Science and Technology of Jilin Province (Grant no. 201115094).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Additional experimental results. See DOI: 10.1039/c4ra05786d

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