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Stable and Biocompatible Colloidal Dispersions of Superparamagnetic Iron Oxide Nanoparticles with Minimum Aggregation for Biomedical Applications Jiaqi Wan,*,† Ruiting Yuan,† Chongyu Zhang,† Ning Wu,‡ Fengying Yan,† Shoushan Yu,† and Kezheng Chen*,† †

School of Materials Science and Engineering, Qingdao University of Science and Technology, Qingdao 266042, China Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China

‡

S Supporting Information *

ABSTRACT: In this article, a simple and scalable method for preparing well-defined and highly stable colloidal dispersions of superparamagnetic iron oxide nanoparticles (IONPs) is reported. The IONPs with narrow size distribution were synthesized by polyol process. Nonhazardous sodium tripolyphosphate (STPP) was immobilized on the surface of IONPs via effective ligand exchange in aqueous phase. Then the STPP-capped IONPs were purified by tangential flow ultrafitration. The polyanionic nature of STPP and its strong coordination capability to iron oxide warrant the IONPs longterm colloidal stability even in phosphate-buffer saline. Because the ligand exchange and purification process did not involve repeated precipitation by organic solvents, the unwanted irreversible aggregation and organic impurities were avoided to the utmost extent. The absence of aggregation renders the IONPs well-defined magnetic behaviors and optimized relaxometric properties for T1-weighted magnetic resonance imaging. The in vitro cytotoxicity test suggests that the STPP-capped IONPs possess little toxicity. In vivo MRI experiment carried out with a mouse model demonstrates the excellent T1-weighted MR contrast enhancement capability of the IONPs. This new kind of IONPs is expected to be applicable in various biomedical applications.

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INTRODUCTION In the past two decades, colloidal dispersions of superparamagnetic iron oxide nanoparticles (IONPs) have attracted increasing attention as a platform for a variety of promising biomedical applications such as magnetic resonance imaging (MRI), hyperthermia for cancer treatment, targeted drug delivery, and magnetic separation.1−4 The usefulness of these magnetic colloidal dispersions is mainly determined by the particle size, surface coating, and dispersion state of IONPs in the solution. To date, a huge amount of publications exist on the synthesis and surface functionalization of IONPs.5−8 Nevertheless, the dispersion state of the IONPs in solution is often overlooked and poorly controlled by the majority of researchers. Actually, many sophisticated processes for preparation of IONPs-based colloids often result in irreversible particle aggregation during synthesis or postsynthetic procedure, which leads to unfavorable reproducibility and elusive outcome regarding MR signal enhancement, hyperthermia heating efficiency, and magnetic attractive force.9−12 Therefore, facile preparation of well-defined and highly stable colloidal dispersions of IONPs is still a significant challenge for both fundamental studies and practical applications in this field. Until now, two major synthetic methods have been commonly adopted to fabricate IONPs: coprecipitation reaction and thermal decomposition. However, each of them © 2016 American Chemical Society

has its inherent problems difficult to be addressed. Coprecipitation of iron salts in alkaline water solution seems to be a straightforward choice for synthesis of water-soluble IONPs.13,14 However, the as-synthesized IONPs have a rather broad size distribution, and further size sorting process is needed to reduce the particle polydispersity.15,16 IONPs with narrow size distribution can be developed by thermal decomposition of the metal−organic iron precursor in organic media.17,18 Unfortunately, the nanoparticles are only soluble in nonpolar solvents due to the capped hydrophobic surfactant ligands in situ which limited their biomedical applications. Further multistep phase transfer procedure not only involves various hazardous organic solvents difficult to be separated but also often causes irreversible particle aggregation. Recently, we have developed a synthetic method for water-soluble IONPs with a narrow size distribution by polyol process,19−21 which offers many economical and environmental advantages compared to traditional coprecipitation and thermal decomposition methods.22−24 The polyols in this method play a triple role as sustainable green solvent, reducing agent, as well as stabilizer to control particle growth, and rendering IONPs Received: July 8, 2016 Revised: September 18, 2016 Published: September 23, 2016 23799

DOI: 10.1021/acs.jpcc.6b06614 J. Phys. Chem. C 2016, 120, 23799−23806

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The Journal of Physical Chemistry C

volumes, an optically clear black-brown suspension containing purified STPP-capped IONPs was obtained. For comparative analysis, citrate-capped IONPs were also fabricated via the same procedure by using sodium citrate as dispersants instead of STPP. TREG-capped IONPs were obtained by diluting the reaction solution containing TREG-protected IONPs by ultrapure water without dispersants of STPP or sodium citrate and then treated by TFU to remove the excess TREG and other small molecules. It should be noted that, in laboratories where TUF equipment is not available, the mixed solution can be purified by dialyzing against ultrapure water in a cellulose acetate dialysis bag (Biosharp, MW: 8000−14 400) for 72 h to remove the free ligands and other small molecules as well. Materials Characterization. Transmission electron microscope (TEM) and high-resolution TEM (HRTEM) images were obtained on a JEOL JEM 2010 electron microscope at an accelerating voltage of 200 kV. X-ray powder diffraction (XRD) patterns of the products were recorded with a Rigaku D/maxγB diffractometer equipped with a rotating anode and a Cu Kα source (λ = 0.154 056 nm). Fourier transform infrared (FT-IR) spectra were recorded on a PerkinElmer Spectrum One spectrometer. Magnetic measurements were carried out using a superconducting quantum interference device (SQUID) magnetometer (Quantum Design MPMS-5XL). Dynamic light scattering (DLS) and ξ potential measurements were performed with a Zetasizer Nano ZS (Malvern Instruments), provided with a He/Ne laser of 633 nm wavelength. To determine the isoelectric point (IEP), the ξ potential of the NPs was measured as a function of the pH by using an MPT-2 accessory. Elemental analysis was carried out by using inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Perkin−Elmer Optima 5300 DV). Relaxometric Measruements. Longitudinal relaxation times (T1) and transverse relaxation times (T2) of the aqueous solution of the as-prepared IONPs were measured on a 1.41 T (60 MHz) Bruker Minispec mq60 MR Analyzer operating at 37 °C. T1 values were obtained using inversion−recovery pulse sequences. T2 values were obtained using CPMG pulse sequence. Relaxivity values (r1, r2) were calculated by fitting relaxation rates R1 (=1/T1) and R2 (=1/T2) as a function of different Fe concentrations.21 Additional T1 and T2 measurements at 0.55 T (23.3 MHz) were performed on a MesoMR23 system (Niumag Corporation, China) at 32 °C. Cell Viability Study. Hela cells were routinely cultured in DMEM supplemented with 10% fetal bovine serum, 100 kU/L penicillin, and 100 mg/L streptomycin at 37 °C in a humidified CO2 atmosphere. Cell counts were carried out using a hemocytometer. To evaluate cytotoxicity of the IONPs, Hela cells were seeded into 96-well plates at densities of 5 × 103 cells per well for 24 h. Then the cells were incubated with the STPPcapped IONPs at various concentrations of 12.5, 25, 50, 100, and 200 mg Fe/L for 12, 24, and 48 h. The control well was a culture medium with no particles. After incubation, the culture media were removed and the cells were washed with PBS. Subsequently, 100 μL of DMEM with 0.5 mg/mL of MTT reagent was added into each well and incubated for another 3 h. After that, the medium in each well was discarded. Then, 100 μL of DMSO was added to each well. Finally, plates were shaken for 15 min, and ODs were monitored at 550 nm using a microplate reader (Bio-Rad 680, U.S.A.) immediately. Cell viabilities were calculated with the ODs of cells treated with normal culture medium as a blank group.33

water soluble. However, the polyol ligands are liable to detach from the surface of the IONPs due to their weak coordination nature to iron oxide, which would be detrimental to the longterm colloidal stability the IONPs. Therefore, an efficient surface modification strategy to improve the colloidal stability of the IONPs is highly desired. So far, various surface modification approaches have been developed to enhance particle stability of IONPs, including attachment of surfactants or polymeric stabilizers and synthesis of core−shell structured composites via silica or Au encapsulation.2,7 Although widely applied, many existing methods have limitations for low affinity, significant increase in overall particle size, and issues of biocompatibility and immunogenicity. Polyphosphate, such as sodium tripolyphosphate (STPP) and sodium hexametaphosphate (SHMP), is a kind of nonhazardous inorganic polyelectrolyte, which has attracted much attention as an effective buffer, emulsifier, water softener, sequestrant, and dispersion agent of various nanoparticles such as Au, Ag, ZnO, and ZnS.25−29 Some polyphosphates even have FDA approval and are used as food additives.30 However, research work on polyphosphate as a robust and biocompatible capping agent for IONPs synthesized by polyol process has rarely been reported.31,32 In this work, we report a simple and scalable approach for preparing well-defined and stable colloidal dispersions of IONPs with narrow hydrodynamic size, based on posttreatment of the polyol synthesized IONPs with STPP. STPP can serve as convenient and robust ligands for the colloidal stabilization of IONPs. Compared to the hydrophobic IONPs obtained by thermal decomposition method, the current protocol does not involve repeated precipitation process and can be performed without leaving aqueous media, which is favorable to minimize irreversible particle aggregation and avoid organic impurities. The as-prepared STPP-capped IONPs exhibit long-term colloidal stability and excellent biocompatibility and enhanced T1-weighted MR contrast capability. The use of these IONPs as novel T1-weighted MRI contrast agents for in vivo imaging is also demonstrated.

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EXPERIMENTAL SECTION Synthesis of IONPs. IONPs were synthesized by polyol process based on decomposition of Fe(acac)3 in triethylene glycol (TREG) at elevated temperature according to our previously reported procedure.19−21 Briefly, 4 mmol Fe(acac)3 was dissolved in 50 mL of TREG in a three-neck roundbottomed flask under magnetic stirring and N2 protection. Then the solution was slowly heated to reflux (∼280 °C) and kept at reflux for 30 min. After cooling down to room temperature by removal of the heating source, a homogeneous black polyol solution containing free-standing polyol-protected IONPs was obtained. Surface Modification of IONPs by STPP. Twenty-five milliliter reaction solution containing TREG-protected IONPs was directly mixed with 200 mL of aqueous solution of sodium tripolyphosphate (STPP, 2.5 mM) under violent stirring for 1 h to ensure the ligand exchange between TREG and STTP. Then the mixture solution was transferred to a retentive reservoir of a tangential flow ultrafiltration (TFU) system (UF-20, HuaTai Purification Technology Co. Ltd., China) and pumped through 5 kDa hollow fiber ultrafiltration membranes and eluted by excess water to remove the excess STPP, TREG, and other small molecules. While IONPs were retained, small molecules passed through the membranes. After five ultrafiltration 23800


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The Journal of Physical Chemistry C In Vivo MRI Experiment with a Mouse. The in vivo MR imaging efficiency of our colloidal dispersion of STPP-capped IONPs was further studied on live mice to demonstrate the potential in T1-positive contrast agent. All animal experiments were conducted in accordance with IACUC approved protocols. A Kunming mouse was anesthetized by 2% isoflurane. Then 200 μL water dispersion of STPP-protected IONPs (2 mM) was administrated from the tail vein. MR imaging procedure was performed on a 0.55 T MR scanner (MesoMR23 system, Niumag Corporation, China) before and after the injection of the IONPs. T1-weighted images were acquired using MSE imaging sequence (TR = 400 ms, TE = 18.2 ms, slice width = 3 mm, slices = 6, average 12).

exhibit a typical magnetite diffractogram pattern (JCPDS No. 65-3107) with spinel structure. Preparation of STTP-Capped IONPs. Because of being capped with hydrophilic polyol ligands in situ, the TREGcapped IONPs are intrinsically water-soluble without phase transformation procedure.20 However, the polyol ligands are not covalently linked to IONPs. They tend to agglomerate due to desorption of polyol ligands when these nanoparticles are diluted in physiological conditions. To substitute the weak polyol ligands with stronger binding entities, we treated the IONPs with a nonhazardous inorganic polyanion, sodium tripolyphosphate (STPP). We choose STPP for ligand exchange because STPP can chemically anchor to the oxide surface via the formation of stable P−O−M binds through the phosphate groups.34,35 The study by Riffle et al. suggested that the phosphonate end group remains stably bound to the magnetite even in phosphate-buffered saline (PBS), whereas the carboxylate and ammonium end groups are prone to desorption in PBS in the presence of phosphate salts.36 In addition, STPP might provide colloidal stabilization while enabling excellent biocompatibility. Thanks to the intermiscibility between TREG and water, the raw reaction solution containing TREG-capped IONPs can be directly stirred with STPP aqueous solution for ligand exchange without separation by nonpolar solvent-induced precipitation. After the ligand exchange reaction was completed, a membrane-based tangential flow ultrafitration (TFU) method was used to remove the unbinding ligands and other small molecules rapidly, resulting in a purified optically clear colloidal solution containing freestanding STPP-capped IONPs in liter-scale (Figure S2). Compared to conventional separation techniques such as precipitation, centrifugation, chromatography, extraction, dialysis, or lyophilization, TFU method offers considerable potential for the efficient purification of water-soluble NPs with low polydispersity in less time on a large scale. The whole procedure avoids the unwanted irreversible aggregation and organic impurity to the utmost.37−39 Nevertheless, if TFU equipment is not available in particle synthesis laboratories, dialysis method can be employed to purify the STPP-capped IONPs as well. Characterization of STTP-Capped IONPs. TEM analysis (Figure 1b) suggests that the size, morphology, and the nonaggregation nature of the IONPs were unaltered after surface modification by STPP. SEAD and XRD analysis (Figure 1b inset and Figure 2a) confirmed that the ligand exchange reaction also did not modify the magnetite structure of the IONPs. Figure 2b displays the Fourier transform infrared (FTIR) spectra of the IONPs before and after ligand exchange. The characteristic bands at 2937−2862 cm−1 of C−H stretching and 1115−1075 cm−1 of C−O stretching revealed the presence of TREG molecules on the surface of the IONPs before surface modification. The broad band centered at 3421 cm−1 comes from hydrogen-bonded surface hydroxyl groups and adsorbed TREG and water on the IONPs. The strong Fe− O absorption band around 578 cm−1 validates that the main phase of the as-prepared IONPs is magnetite. After surface modification by STPP, the characteristic bonds of TREG disappeared, while a broad band P−O and P = O stretching between 900 and 1200 cm−1, which correspond to STPP, appeared with main feature at 1076 cm−1 and two shoulders at 1186 and 915 cm−1. These results demonstrate that the TREG molecules adsorbed on IONPs have been successfully substituted by STPP during the ligand exchange process.

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RESULTS AND DISCUSSION IONPs Prepared by Polyol Process. Superparamagnetic iron oxide nanoparticles (IONPs) with narrow size distribution and high crystallinity were synthesized by decomposition of iron(III) acetylacetonate in triethylene glycol (TREG) at elevated temperature as reported previously.19−21 Because TRGE can act as a stabilizer to control particle growth and prevent their aggregation and precipitation, no other surfactant is needed in the reaction. Transmission electron microscope (TEM) image of the as-prepared IONPs dispersed in ethanol (Figure 1a) suggests that the nanoparticles are uniform in size

Figure 1. TEM images of (a) TREG-capped IONPs dispersed in ethanol and (b) STTP-capped IONPs dispersed in water. The insets are the corresponding HRTEM images and SEAD patterns.

and well isolated due to capping with TREG ligands in situ. The particle size is found to be 8.5 ± 1.2 nm by analyzing the TEM micrographs of the sample (Figure S1). In the highresolution TEM (HRTEM) image presented in Figure 1a inset, lattice fringes of a group of atomic planes are clearly visible, indicating the single crystalline nature of the IONPs with good crystallinity. We employed selected area electron diffraction (SAED) (Figure 1a inset) and X-ray diffraction (XRD) (Figure 2a) to identify the crystal structure of the product, which 23801


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Figure 2. (a) XRE patterns, (b) FTIR spectra, and (c) zeta-potential curves of the IONPs before and after ligand exchange.

Figure 2c presents the zeta-potential curve as a function of pH value before and after ligand exchange. Before ligand exchange, the isoelectric point (IEP) of the TREG-capped IONPs is located at pH = 6.56, closed to the values reported in literature for magnetite nanoparticles.40 When the pH is increased, the surface charge of IONPs varies from positive to negative, due to deprotonation of surface hydroxyls. After treatment with STPP, the surface of IONPs becomes negatively charged as observed on the zeta potential curve. At pH value above 3, the IONPs exhibit highly negative charge (< −30 mV) sufficient to warrant colloidal stability. The significantly reduction of surface change is in agreement with the specific bonding of the tripolyphosphate ions on the surface of IONPs, which are negatively charged at this pH range.41 For comparative analysis, the IONPs were also treated with sodium citrate, a commonly used stabilizer of IONPs, via the same ligand exchange process. As shown in Figure 2c, the citratecapped IONPs also exhibit negative charge according the zeta potential curve, indicating that the IONPs were grafted with citrate. However, the surface charge density of the citratecapped IONPs is less than that of STTP-capped IONPs as indicated by the zeta potential values at the same pH range, indicating the lesser efficiency of citrate as a capped ligand. Dispersion State. The dispersion state of STPP-capped IONPs in aqueous solution was evaluated by dynamic light scattering (DLS) analyses through hydrodynamic diameter measurement. Figure 3 shows the number-weighted, volumeweighted, and intensity-weighted hydrodynamic diameter distribution of the IONPs capped with STPP and citrate. The STPP-capped IONPs show unimodal and log-normal size distribution in aqueous solution with mean particle diameters of 11.87 ± 3.32 nm (number-weighted), 15.21 ± 5.83 nm (volume-weighted), and 23.49 ± 10.12 nm (intensityweighted), respectively. The value of the number-average diameter is comparable to the mean size obtained from TEM measurements, indicating that the STPP-capped IONPs are almost individually distributed in the solution. The increase in the values of volume-weighted and intensity-weighted diameter is because these data are sensitive to the larger particles that present in the distribution.42 The citrate-capped IONPs also exhibit unimodal particle size distribution in number-weighted and volume-weighted distribution, but the values (dn = 14.54 ± 4.47 nm, dv = 20.80 ± 11.73 nm) are slightly larger than those of STPP-capped IONPs. In contrast, the intensity-weighted distribution of citrate-capped IONPs is much broader than that of STPP-capped IONPs, and a shoulder at about 400 nm is clearly observed as shown in Figure 3c. Because intensity

Figure 3. (a) Number-weighted, (b) volume-weighted, and (c) intensity-weighted hydrodynamic size distribution of the IONPscapped with STPP and citrate.

average diameter is a function of the sixth power of the physical diameter,42 it is very sensitive to the presence of particle aggregation and reflects more detailed information about the dispersion state of the IONPs in aqueous solution. The result indicates that more particle aggregation presents in the dispersion of the citrate-capped IONPs than in the dispersion of the STPP-capped IONPs. Irreversible aggregation of IONPs formed during the ligand exchange process possibly owing to the less electrostatic repulsion of the citrate-capped IONPs. Colloidal Stability. The colloidal stability of the IONP dispersions is a key factor in their biomedical applications. Replacing polyol ligands with STPP increased the electrostatic and spatial repulsion significantly, which warrants the IONPs long-term colloidal stabilization. The STPP-capped IONPs can remain stable in aqueous solution for years. In contrast, the citrate-capped IONPs began to precipitate after several months. The separation of the STPP-capped IONPs from the aqueous media was not even possible by centrifugation at 12 000 rpm 23802


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addition, the ZFC curve of the citrated-capped IONPs is relatively broader than that of the STPP-capped IONPs. According to literature,43,44 the increase of TB and relatively broadening of the ZFC curve are clearly correlated to aggregation and increase of dipolar magnetic interaction between magnetic NPs, which confirms the DLS measurements. These results show the important effect of the dispersion state in the magnetic behaviors of IONP suspensions. MRI Contrast Enhancement Effects. Up to now, iron oxide nanoparticles have been predominantly used as negative T2-weighted MRI contrast agents due to their strong magnetic moment.2 Their performances as contrast agents mainly depend on particle size, dispersion state, and coating material used.45,46 To evaluate the MRI contrast enhancement effects, longitudinal relaxation time (T1) and transverse relaxation time (T2) of aqueous solutions containing STTP-capped IONPs and citrate -capped IONPs were measured at clinical field strength (1.41 T). The inverse relaxation times as a function of the Fe concentrations are shown Figure 6. Then the longitudinal and transverse relaxivities (r1 and r2), which represent the efficiency of the IONPs as a contrast agent, were determined by fitting the inverse relaxation time against the Fe concentration. Although dispersions of STTP-capped IONPs and citratecapped IONPs are derived from the same “parent” particles, they bring about substantially different outcomes for the proton relaxation. For the STPP-capped IONPs, the r1 and r2 values are 18.878 and 73.024 mM−1 s−1, respectively. Compare to commercial iron oxide MRI contrast agents, such as Endorem or Sinerem,2,43 the STTP-capped IONPs have a large r1 value and relatively small r2 value. For the citrate-capped IONPs, the r1 and r2 values are 18.691 and 122.431 mM−1 s−1, respectively. While the longitudinal relaxivity r1 value is comparable with that of STPP-capped IONPs, the transverse relaxivity r2 value increases by nearly twofold. In general, the T1 contrast effect is induced by the interactions between water molecules and electron spins of the contrast agents.45,47 Because the vast majority of IONPs are individually dispersed in the solution as revealed by DLS measurements, a small fraction of aggregation has ignorable impact on the T1 contrast enhancement. In contrast, the T2 contrast effect reflects the ability of the nanoparticles to produce local magnetic inhomogeneity in the applied magnetic field.45,47 Therefore, a small fraction of aggregation should drastically shorten the transverse relaxation time of the IONPs because of increased dipolar magnetic interactions between IONPs. Indeed, the results highlight that the dispersion state of IONPs in colloidal solution must be taken into account in order to understand relaxometric properties. The r2/r1 ratio is an important parameter to estimate the efficiency of T1 or T2 contrast agents. In literature, great efforts have been spent to suppress the r2/r1 ratio down to at least 5 in order to enable IONPs as T1 contrast agent,48,49 because T1 contrast enhancement by IONPs may offer great potential applications of clinical diagnosis, especially in patients suffering from nephrogenic systemic fibrosis (NSF) with impaired kidney function.46,50 The r2/r1 ratios of STTP-capped IONPs and citrated-capped IONPs were found to be 3.868 and 6.550, respectively. The results suggest that the STPP-capped IONPs are more suitable for T1 imaging. In addition, the STPP-capped IONPs were also measured using an animal MRI scanner (0.55 T, 23.3 MHz). The r1 and r2 values of the STPP-capped IONPs were measured to be 36.901 and 87.355 mM−1 s−1, and the r2/

for 30 min. The STPP-capped IONPs can also be stable in PBS buffer solution for at least 2 weeks, which has the same pH value and ionic strength as physiological conditions. Figure 4

Figure 4. Time-dependence hydrodynamic diameter evolution of the STPP-capped IONPs and citrate-capped IONPs in PBS.

shows the time-dependence evolution of the STPP-capped IONPs and citrate-capped IONPs in PBS buffer solution during the 2 week measurement period. It can be seen that the hydrodynamic diameters of STPP-capped IONPs did not increase. In control experiment, the citrate-capped IONPs began to aggregate after 1 week, mainly owning to the replacement of citrate ligands with phosphate salts.36 Magnetic Behaviors. Magnetic behaviors of the colloidal dispersions of as-prepared STPP-capped IONPs and citratecapped IONPs, for comparison purpose, were investigated with a SQUID magnetometer. Figure 5 shows the zero-field cooling

Figure 5. Zero-field cooling (ZFC) and field cooling (FC) curves of the aqueous dispersions of the IONPs capped with STPP and citrate measured in a probe field of 100 Oe.

and field cooling (ZFC−FC) curves carried out in an applied field of 100 Oe. Both STPP-capped IONPs and citrate-capped IONPs exhibit a characteristic feature of superparamagnetism, an important property required for biomedical applications. In the ZFC measurement, magnetization curves show a cusp defined as blocking temperature (TB). Above TB the sample is superparamagnetic and below TB it is ferromagnetic. The STPP-capped IONPs have a low blocking temperature (26.2 K) as compared with that of the citrate-capped IONPs (38.9 K). In 23803


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Figure 6. T1 and T2 relaxation rates (1/T1, 1/T2) plotted against the Fe concentration for the aqueous solutions containing STTP-capped IONPs and citrate-capped IONPs.

images, the T1-weighted signal intensity of kidneys drastically increased by 170% 10 min after injection and remained brighter until 120 min later at the end of the experiment. In the case of liver, the T1-weighted signal intensity increased 118% at the maximum at 10 min after injection and then gradually became negative enhancement due to the uptake of IONPs by Kupffer cells.51,52 Liver and kidney are highly vascularized organs, and the bright T1 contrast in kidney and liver is attributed to the STPP-capped IONPs highly dispersed in the blood pool and having prolonged blood circulation time as well as their small size. It should be noted that, during in vivo experiment, no visible sign of toxicity or side effects is observed. After testing, the model mouse recovered from anesthesia spontaneously and lived normally for weeks, which demonstrates biocompatibility and little toxicity of the STPP-capped IONPs. These results make the STPP-capped IONPs an ideal candidate for highlyefficient T1 positive MR imaging.

r1 ratio is 2.37. This indicates that the STPP-capped IONPs can be a good T1 contrast agent at this field strength, as described below. Cytotoxicity. Before in vivo MRI experiments, a standard MTT viability test was carried out on Hela cells after 12, 24, and 48 h incubation with the STPP-capped IONPs at various incubation concentrations (12.5−200 Fe mg L−1) to evaluate cytotoxicity of the STPP-capped IONPs. The viability profiles of the cells treated at various particle concentrations and incubation times are shown in Figure 7. It can be seen that the

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CONCLUSIONS

In summary, we reported a facile and scalable protocol for preparation of well-defined, highly stable, and biocompatible IONPs. Monodisperse water-soluble IONPs were prepared by polyol process. By capping with high-affinity STPP ligands and keeping the whole grafting process within aqueous media, highly stable colloidal dispersion of IONPs could be obtained in large scale with minimum aggregation. Magnetization and relaxivity measurements demonstrated that the dispersion state of IONPs in colloidal solution greatly impacts the magnetic and relaxometric behaviors. The minimized aggregation of the STPP-capped IONPs successfully suppresses the T2 contrast effect by decreasing inhomogeneity of the local magnetic field, leading to an optimized T1-weighted MR contrast enhancement of the colloidal dispersion. In vitro cytotoxicity test suggests that the as-prepared IONPs possess a good safety profile. In vivo T1-weighted MR imaging carried out on a mouse model shows the prolonged cycling time and excellent T1-weighted MR contrast enhancement capability of the IONPs. This new kind of IONPs hold great promise as a novel MRI contrast agent for better diagnosis as well as desired start materials for various biomedical applications.53,54

Figure 7. Cytotoxicity profile of the STPP-capped IONPs via MTT assay using Hela cells at different Fe concentrations and incubation time.

cell viability is not statistically less than that of the corresponding cells incubated with normal culture medium, even up to 200 [Fe] mg L−1 iron concentrations for 48 h. This result suggests that the as-prepared STPP-capped IONPs possess a good safety profile. In Vivo MRI Experiments. On the basis of above in vitro results, we performed in vivo MRI experiments on a live mouse using STPP-capped IONPs as contrast agents by an animal MRI scanner (0.55 T, 23.3 MHz). Figure 8a shows the typical T1-weighted images of mouse liver and kidney collected at different intervals after injection of the STPP-capped IONPs with a dosage of 0.013 mmol [Fe] kg−1 (200 μL, 2 mM [Fe]) through tail vein. The signal intensity of labeled areas was plotted in Figure 8b. In comparison with the precontrast 23804


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Figure 8. (a) T1-weighted in vivo MRI images of a mouse collected at different time points before and after intravenous injection of the STPPcapped IONPs. (b) T1-weighted MR signal intensity of the selected liver and kidney areas at different time points before and after administration of the STPP-capped IONPs.

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(7) Lee, H.; Shin, T.-H.; Cheon, J.; Weissleder, R. Recent developments in magnetic diagnostic systems. Chem. Rev. 2015, 115 (19), 10690−10724. (8) Thorat, N. D.; Lemine, O. M.; Bohara, R. A.; Omri, K.; El Mir, L.; Tofail, S. A. M. Superparamagnetic iron oxide nanocargoes for combined cancer thermotherapy and MRI applications. Phys. Chem. Chem. Phys. 2016, 18 (31), 21331−21339. (9) Roca, A. G.; Veintemillas-Verdaguer, S.; Port, M.; Robic, C.; Serna, C. J.; Morales, M. P. Effect of nanoparticle and aggregate size on the relaxometric properties of MR contrast agents based on high quality magnetite nanoparticles. J. Phys. Chem. B 2009, 113 (19), 7033−7039. (10) Smolensky, E. D.; Park, H.-Y. E.; Berquó, T. S.; Pierre, V. C. Surface functionalization of magnetic iron oxide nanoparticles for MRI applicationsEffect of anchoring group and ligand exchange protocol. Contrast Media Mol. Imaging 2011, 6 (4), 189−199. (11) Coral, D. F.; Mendoza Zélis, P.; Marciello, M.; Morales, M. d. P.; Craievich, A.; Sánchez, F. H.; Fernández van Raap, M. B. Effect of nanoclustering and dipolar interactions in heat generation for magnetic hyperthermia. Langmuir 2016, 32 (5), 1201−1213. (12) Orozco-Henao, J. M.; Coral, D. F.; Muraca, D.; MoscosoLondoño, O.; Mendoza Zélis, P.; Fernandez van Raap, M. B.; Sharma, S. K.; Pirota, K. R.; Knobel, M. Effects of nanostructure and dipolar interactions on magnetohyperthermia in iron oxide nanoparticles. J. Phys. Chem. C 2016, 120 (23), 12796−12809. (13) Massart, R. Preparation of aqueous magnetic liquids in alkaline and acidic media. IEEE Trans. Magn. 1981, 17 (2), 1247−1248. (14) Baumgartner, J.; Dey, A.; Bomans, P. H.; Le Coadou, C.; Fratzl, P.; Sommerdijk, N. A.; Faivre, D. Nucleation and growth of magnetite from solution. Nat. Mater. 2013, 12 (4), 310−314. (15) Lefebure, S.; Dubois, E.; Cabuil, V.; Neveu, S.; Massart, R. Monodisperse magnetic nanoparticles: Preparation and dispersion in water and oils. J. Mater. Res. 1998, 13 (10), 2975−2981. (16) Guibert, C.; Dupuis, V.; Peyre, V.; Fresnais, J. Hyperthermia of magnetic nanoparticles: Experimental study of the role of aggregation. J. Phys. Chem. C 2015, 119 (50), 28148−28154. (17) Sun, S.; Zeng, H. Size-controlled synthesis of magnetite nanoparticles. J. Am. Chem. Soc. 2002, 124 (28), 8204−8205. (18) Park, J.; An, K. J.; Hwang, Y. S.; Park, J. G.; Noh, H. J.; Kim, J. Y.; Park, J. H.; Hwang, N. M.; Hyeon, T. Ultra-large-scale syntheses of monodisperse nanocrystals. Nat. Mater. 2004, 3 (12), 891−895. (19) Cai, W.; Wan, J. Q. Facile synthesis of superparamagnetic magnetite nanoparticles in liquid polyols. J. Colloid Interface Sci. 2007, 305 (2), 366−370. (20) Wan, J.; Cai, W.; Meng, X.; Liu, E. Monodisperse water-soluble magnetite nanoparticles prepared by polyol process for highperformance magnetic resonance imaging. Chem. Commun. 2007, 47, 5004−5006. (21) Wan, J.; Jiang, X.; Li, H.; Chen, K. Facile synthesis of zinc ferrite nanoparticles as non-lanthanide T1 MRI contrast agents. J. Mater. Chem. 2012, 22 (27), 13500−13505.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.6b06614. Size distribution of as-prepared IONPs by analyzing the TEM micrographs of the sample; photograph showing 3.5 L of the colloidal solution containing free-standing STPP-capped IONPs purified by TUF method one time (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +86 0532 84022509. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge support from the National Natural Science Foundation of China (No. 51472133), the Natural Science Foundation of Shandong Province (No. ZR2013EMM006), and the China Scholarship Council (201607890002).

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REFERENCES

(1) Weissleder, R.; Bogdanov, A.; Neuwelt, E. A.; Papisov, M. Longcirculating iron oxides for MR imaging. Adv. Drug Delivery Rev. 1995, 16 (2−3), 321−334. (2) Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Elst, L. V.; Muller, R. N. Magnetic iron oxide nanoparticles: Synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. Chem. Rev. 2008, 108 (6), 2064−2110. (3) Lee, N.; Yoo, D.; Ling, D.; Cho, M. H.; Hyeon, T.; Cheon, J. Iron oxide based nanoparticles for multimodal imaging and magnetoresponsive therapy. Chem. Rev. 2015, 115 (19), 10637−10689. (4) Thorat, N. D.; Bohara, R. A.; Malgras, V.; Tofail, S. A. M.; Ahamad, T.; Alshehri, S. M.; Wu, K. C. W.; Yamauchi, Y. Multimodal superparamagnetic nanoparticles with unusually enhanced specific absorption rate for synergetic cancer therapeutics and magnetic resonance imaging. ACS Appl. Mater. Interfaces 2016, 8 (23), 14656− 14664. (5) Amstad, E.; Textor, M.; Reimhult, E. Stabilization and functionalization of iron oxide nanoparticles for biomedical applications. Nanoscale 2011, 3 (7), 2819−2843. (6) Erathodiyil, N.; Ying, J. Y. Functionalization of inorganic nanoparticles for bioimaging applications. Acc. Chem. Res. 2011, 44 (10), 925−935. 23805


Article


colloidal silver nanoparticles: A “green” laboratory experiment for chemistry and engineering students. J. Chem. Educ. 2014, 91 (7), 1044−1049. (40) Lamanna, G.; Kueny-Stotz, M.; Mamlouk-Chaouachi, H.; Ghobril, C.; Basly, B.; Bertin, A.; Miladi, I.; Billotey, C.; Pourroy, G.; Begin-Colin, S.; et al. Dendronized iron oxide nanoparticles for multimodal imaging. Biomaterials 2011, 32 (33), 8562−8573. (41) Johnson, S. B.; Brown, G. E.; Healy, T. W.; Scales, P. J. Adsorption of organic matter at mineral/water interfaces. 6. Effect of inner-sphere versus outer-sphere adsorption on colloidal stability. Langmuir 2005, 21 (14), 6356−6365. (42) Jung, C. W.; Jacobs, P. Physical and chemical properties of superparamagnetic iron oxide MR contrast agents: Ferumoxides, ferumoxtran, ferumoxsil. Magn. Reson. Imaging 1995, 13 (5), 661−674. (43) Casula, M. F.; Floris, P.; Innocenti, C.; Lascialfari, A.; Marinone, M.; Corti, M.; Sperling, R. A.; Parak, W. J.; Sangregorio, C. Magnetic resonance imaging contrast agents based on iron oxide superparamagnetic ferrofluids. Chem. Mater. 2010, 22 (5), 1739−1748. (44) De Sousa, M. E.; Fernández van Raap, M. B.; Rivas, P. C.; Mendoza Zélis, P.; Girardin, P.; Pasquevich, G. A.; Alessandrini, J. L.; Muraca, D.; Sánchez, F. H. Stability and relaxation mechanisms of citric acid coated magnetite nanoparticles for magnetic hyperthermia. J. Phys. Chem. C 2013, 117 (10), 5436−5445. (45) Roch, A.; Gossuin, Y.; Muller, R. N.; Gillis, P. Superparamagnetic colloid suspensions: Water magnetic relaxation and clustering. J. Magn. Magn. Mater. 2005, 293 (1), 532−539. (46) Taboada, E.; Rodriguez, E.; Roig, A.; Oro, J.; Roch, A.; Muller, R. N. Relaxometric and magnetic characterization of ultrasmall iron oxide nanoparticles with high magnetization. Evaluation as potential T1 magnetic resonance imaging contrast agents for molecular imaging. Langmuir 2007, 23 (8), 4583−4588. (47) Roch, A.; Muller, R. N.; Gillis, P. Theory of proton relaxation induced by superparamagnetic particles. J. Chem. Phys. 1999, 110 (11), 5403−5411. (48) Tromsdorf, U. I.; Bruns, O. T.; Salmen, S. C.; Beisiegel, U.; Weller, H. A highly effective, nontoxic T1 MR contrast agent based on ultrasmall PEGylated iron oxide nanoparticles. Nano Lett. 2009, 9 (12), 4434−4440. (49) Kim, B. H.; Lee, N.; Kim, H.; An, K.; Park, Y. I.; Choi, Y.; Shin, K.; Lee, Y.; Kwon, S. G.; Na, H. B.; Park, J.-G.; Ahn, T.-Y.; Kim, Y.-W.; Moon, W. K.; Choi, S. H.; Hyeon, T.; et al. Large-scale synthesis of uniform and extremely small-sized iron oxide nanoparticles for highresolution T1 magnetic resonance imaging contrast agents. J. Am. Chem. Soc. 2011, 133 (32), 12624−12631. (50) Penfield, J. G.; Reilly, R. F. What nephrologists need to know about gadolinium. Nat. Clin. Pract. Nephrol. 2007, 3 (12), 654−668. (51) Huang, J.; Wang, L.; Zhong, X.; Li, Y.; Yang, L.; Mao, H. Facile non-hydrothermal synthesis of oligosaccharide coated sub-5 nm magnetic iron oxide nanoparticles with dual MRI contrast enhancement effects. J. Mater. Chem. B 2014, 2 (33), 5344−5351. (52) Li, Z.; Wang, S. X.; Sun, Q.; Zhao, H. L.; Lei, H.; Lan, M. B.; Cheng, Z. X.; Wang, X. L.; Dou, S. X.; Lu, G. Q. Ultrasmall manganese ferrite nanoparticles as positive contrast agent for magnetic resonance imaging. Adv. Healthcare Mater. 2013, 2 (7), 958−964. (53) Schellenberger, E.; Schnorr, J.; Reutelingsperger, C.; Ungethüm, L.; Meyer, W.; Taupitz, M.; Hamm, B. Linking proteins with anionic nanoparticles via protamine: Ultrasmall protein-coupled probes for magnetic resonance imaging of apoptosis. Small 2008, 4 (2), 225−30. (54) Wang, S.; Tang, J.; Zhao, H.; Wan, J.; Chen, K. Synthesis of magnetite−silica core−shell nanoparticles via direct silicon oxidation. J. Colloid Interface Sci. 2014, 432, 43−46.

(22) Dong, H.; Chen, Y. C.; Feldmann, C. Polyol synthesis of nanoparticles: Status and options regarding metals, oxides, chalcogenides, and non-metal elements. Green Chem. 2015, 17 (8), 4107− 4132. (23) Hachani, R.; Lowdell, M.; Birchall, M.; Hervault, A.; Mertz, D.; Begin-Colin, S.; Thanh, N. T. K. Polyol synthesis, functionalisation, and biocompatibility studies of superparamagnetic iron oxide nanoparticles as potential MRI contrast agents. Nanoscale 2016, 8 (6), 3278−3287. (24) Bohara, R. A.; Yadav, H. M.; Thorat, N. D.; Mali, S. S.; Hong, C. K.; Nanaware, S. G.; Pawar, S. H. Synthesis of functionalized Co0.5Zn0.5Fe2O4 nanoparticles for biomedical applications. J. Magn. Magn. Mater. 2015, 378, 397−401. (25) Cini, N.; Ball, V. Polyphosphates as inorganic polyelectrolytes interacting with oppositely charged ions, polymers and deposited on surfaces: Fundamentals and applications. Adv. Colloid Interface Sci. 2014, 209, 84−97. (26) Liu, J.; Wang, L.-Q.; Samuels, W. D.; Exarhos, G. J. Aggregation and dispersion of colloidal suspensions by inorganic surfactants: Effect of chemical speciation and molecular conformation. J. Phys. Chem. B 1997, 101 (41), 8264−8269. (27) Song, S.; Lopez-Valdivieso, A.; Espinoza-Ortega, O. Role of sodium hexametaphosphate in the flotation of acanthite fines from finely disseminated ores. Sep. Sci. Technol. 2009, 44 (12), 2971−2982. (28) Tiwari, A.; Khan, S. A.; Kher, R. S. Study of size dependent photoluminescence properties of copper doped sodium hexametaphosphate capped ZnS nanoparticles. J. Lumin. 2012, 132 (6), 1564− 1567. (29) Omelon, S. J.; Grynpas, M. D. Relationships between polyphosphate chemistry, biochemistry, and apatite biomineralization. Chem. Rev. 2008, 108 (11), 4694−4715. (30) Parab, H. J.; Huang, J.-H.; Lai, T.-C.; Jan, Y.-H.; Liu, R.-S.; Wang, J.-L.; Hsiao, M.; Chen, C.-H.; Hwu, Y.-K. Biocompatible transferrin-conjugated sodium hexametaphosphate-stabilized Au NPs: Synthesis, characterization, cytotoxicity, and cellular uptake. Nanotechnology 2011, 22, 395706. (31) Majeed, J.; Barick, K. C.; Shetake, N. G.; Pandey, B. N.; Hassan, P. A.; Tyagi, A. K. Water-dispersible polyphosphate-grafted Fe3O4 nanomagnets for cancer therapy. RSC Adv. 2015, 5 (105), 86754− 86762. (32) Sharma, P.; Rana, S.; Barick, K. C.; Kumar, C.; Salunke, H. G.; Hassan, P. A. Biocompatible phosphate anchored Fe3O4 nanocarriers for drug delivery and hyperthermia. New J. Chem. 2014, 38 (11), 5500−5508. (33) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65 (1−2), 55−63. (34) Daou, T. J.; Begin-Colin, S.; Grenèche, J. M.; Thomas, F.; Derory, A.; Bernhardt, P.; Legaré, P.; Pourroy, G. Phosphate adsorption properties of magnetite-based nanoparticles. Chem. Mater. 2007, 19 (18), 4494−4505. (35) Queffélec, C.; Petit, M.; Janvier, P.; Knight, D. A.; Bujoli, B. Surface modification using phosphonic acids and esters. Chem. Rev. 2012, 112 (7), 3777−3807. (36) Goff, J. D.; Huffstetler, P. P.; Miles, W. C.; Pothayee, N.; Reinholz, C. M.; Ball, S.; Davis, R. M.; Riffle, J. S. Novel phosphonatefunctional poly(ethylene oxide)−magnetite nanoparticles form stable colloidal dispersions in phosphate-buffered saline. Chem. Mater. 2009, 21 (20), 4784−4795. (37) Sweeney, S. F.; Woehrle, G. H.; Hutchison, J. E. Rapid purification and size separation of gold nanoparticles via diafiltration. J. Am. Chem. Soc. 2006, 128 (10), 3190−3197. (38) Trefry, J. C.; Monahan, J. L.; Weaver, K. M.; Meyerhoefer, A. J.; Markopolous, M. M.; Arnold, Z. S.; Wooley, D. P.; Pavel, I. E. Size selection and concentration of silver nanoparticles by tangential flow ultrafiltration for SERS-based biosensors. J. Am. Chem. Soc. 2010, 132 (32), 10970−10972. (39) Dorney, K. M.; Baker, J. D.; Edwards, M. L.; Kanel, S. R.; O’Malley, M.; Pavel Sizemore, I. E. Tangential flow filtration of 23806


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