Large-Scale, Facile Transfer of Oleic Acid-Stabilized Iron Oxide

Feb 1, 2017 - Large-Scale, Facile Transfer of Oleic Acid-Stabilized Iron Oxide Nanoparticles to the Aqueous Phase for Biological Applications. Jing Ca...
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Large-Scale, Facile Transfer of Oleic Acid-Stabilized Iron Oxide Nanoparticles to the Aqueous Phase for Biological Applications Jing Cai,†,§ Yu Qing Miao,† Bao Zhi Yu,‡ Pei Ma,† Li Li,*,§ and Hai Ming Fan*,† †

School of Chemical Engineering and ‡Institute of Photonics and Photon-Technology, Northwest University, Xi’an 710069, China § State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China S Supporting Information *

ABSTRACT: Fe3O4 nanoparticles synthesized via thermal decomposition in the organic phase have attracted tremendous research interest because of their unique morphology, size dispersion, and crystallinity. However, their poor water dispersibility strongly limited their development in biomedical applications. Therefore, a phase-transfer strategy through which hydrophobic nanoparticles with good performance in the aqueous phase can be obtained is an extremely critical issue. Herein, we present a large-scale, facile, highly efficient strategy for the phase transfer of oleic acid-coated Fe3O4 nanoparticles via a reverse-micelle-based oxidative reaction. The reverse micelle system improves the efficiency of the interface oxidative reaction and prevents the aggregation of nanoparticles during the reaction, facilitating the transfer of Fe3O4 nanoparticles from the organic phase to the aqueous phase. The transferred Fe3O4 nanoparticles are used as a T2 contrast agent to perform magnetic resonance imaging of CNE2 cells (nasopharyngeal carcinoma cell line). In addition, the free carboxyl groups on the surface of transferred nanoparticles can also be programmed to permit the conjugation of other molecules, in turn allowing nanoparticles to be extended in biological targeting or biological recognition applications. Therefore, this strategy offers a promising platform for the large-scale, highly efficient phase transfer of oleic acid-capped nanoparticles and may become a new paradigm to promote the development of diverse nanoparticles for widespread biomedical applications.

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INTRODUCTION Recently, in the field of biomedicine, functional inorganic nanoparticles exhibiting special magnetic, optical, or electric properties have been extensively studied.1−3 As FDA-approved materials, iron oxide nanoparticles have attracted significant attention for use as contrast or therapy agents in biomedical applications, including magnetic resonance imaging and magnetic hyperthermia therapy.4,5 Among the synthetic methods for preparing iron oxide nanoparticles, thermal decomposition is a common choice for obtaining nanoparticles with an advantageous size distribution, morphology, and crystallinity.6 However, nanoparticles synthesized by thermal decomposition are generally coated with oleic acid or oleylamine to facilitate their good dispersibility in a nonpolar organic solvent. These hydrophobic nanoparticles must be transferred to the aqueous phase for further biological applications. With the development of the surface chemistry of nanoparticles, there are mainly three methods that were used to transfer the hydrophobic nanoparticle to the water phase. They are the amphiphilic polymer coating,7−10 the ligand exchange,11 and the oxidation of surface fatty acid.12 Each method has its own advantages and disadvantages. For example, amphiphilic polymer coating will increase the hydrodynamic size of nanoparticles, and also it is not favorable for these application in which water molecules need to contact the © 2017 American Chemical Society

surface of the nanoparticles. Ligand exchange usually requires excess ligand molecules and elaborate reaction conditions. The oxidation of surface oleate acid seems to be a better method for industrial practice. However, early reported oxidation routes frequently lead to aggregations, which may be due to incomplete oxidation during the reactions.12−14 Hence, it is necessary to develop an efficient and simple oxidation method to transfer oleic acid-capped nanoparticles to aqueous solution and enable the nanoparticles to maintain good dispersity in water. Significant effort has been devoted to modifying the watertransfer oxidation process of oleic acid-capped nanoparticles to improve the reaction efficiency and water dispersibility. Efforts have included using strong oxidants, extending the reaction time, changing the polarity of reaction solvents, and grafting hydrophilic molecules onto the as-oxidized oleic acid molecules.13,15,16 Although these efforts have modified the process to some extent, improvements are still necessary to prepare nanoparticles with stable water dispersion on a large scale. Thus, there still exists a need for a strategy that is able to shorten the reaction time, increase the yield of the nanoReceived: September 12, 2016 Revised: February 1, 2017 Published: February 1, 2017 1662

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Scheme 1. (a) Photographs, (b) Schematic Models, and (c) Molecular Models of the Phase Transfer of Hydrophobic Fe3O4 Nanoparticles from the Organic Phase to the Aqueous Phase

particles with good water-dispersibility and high stability, and simplify the process for large-scale phase transfer. Since Pileni reported in 1993 that reverse micelles can be used as microreactors to perform an interface chemical reaction, scientific research into reverse micelles has increased rapidly.17 A reverse micelle consists of an aqueous microdomain and surrounding surfactants. This structure can reduce the interfacial tension and increase the reaction area; using reverse micelles as media in phase-transfer reactions can improve the efficiency of interface reactions.18 By exploiting these properties, the use of reverse micelles in chemical reactions has wide applications in the fields of nanocrystal preparation and enzyme biocatalysis.19,20 To the best of our knowledge, the use of reverse micelles to optimize the oxidative reaction of oleic acidcapped nanoparticles for phase transfer has not been previously reported. Herein, we propose an oxidative cleavage strategy that uses reverse micelles to transfer Fe3O4 nanoparticles from the organic phase to the water phase. Poly(vinylpyrrolidone) (PVP) has high biocompatibility and is used as an interface agent for forming reverse micelles in this reaction system.21 The main advantages of the phase-transfer strategy include the following: (1) The aqueous microdomain facing polar groups may protect water-soluble oxidants and transferred nanoparticles from organic adverse effects. (2) There is improved oxidation due to an increased interfacial contact area between reactants in two immiscible liquids. (3) The micelles possess fluidity, which is beneficial for reaction substance exchange. (4) The steric effects of amphiphilic polymer PVP stabilizes the water domain during the interfacial reaction process, avoiding nanoparticle aggregation. This strategy opens up the possibility to easily functionalize with other molecules bearing amino or hydroxyl groups for the biological targeting of molecular recognition applications and also provides a powerful platform

for transferring oleic acid-capped nanoparticles of variable composition, i.e., not only iron oxide. Furthermore, owing to the good performance, such as ideal colloidal stability, high water-dispersibility, biocompatibility, and surface functionalization, the nanoparticles prepared by this strategy are potential candidates for a widespread range of biomedical applications.

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EXPERIMENTAL SECTION

Synthesis of Magnetite Nanoparticles. All chemicals were purchased from Sigma-Aldrich and used as received. Superparamagnetic Fe3O4 nanoparticles were prepared by the thermal decomposition method, where an iron−oleate complex was used as the precursor.6 In a typical synthesis, 8 mmol of iron chloride (FeCl3· 6H2O, 98%) and 24 mmol of sodium oleate (95%) were dissolved in a mixture solvent consisting of distilled water (12 mL), ethanol (16 mL), and hexane (28 mL). The mixed solution was heated to 70 °C and kept for 4 h. After the reaction was finished, the product was washed with water and then dried in a vacuum oven at 60 °C for 4 h. The iron−oleate complex was obtained. The Fe3O4 nanoparticles were then synthesized by the thermal decomposition of the iron−oleate complex at high temperature. In brief, 8 mmol of the iron−oleate complex and 4 mmol of oleic acid (90%) were dissolved in 40 g of 1octadecene (90%). The mixture was heated to 320 °C and then kept at that temperature for 30 min. After that, the mixture was cooled to room temperature. The nanoparticles were obtained by centrifugation separation and then redispersed in hexane for further use. Phase-Transfer Synthesis. In a typical water-transfer process, asprepared hydrophobic Fe3O4 nanoparticles (1 g) were dispersed in 0.5 L of cyclohexane. Then, 0.35 L of tertiary butanol, 0.025 L of K2CO3 (5 wt %), 0.05 L of a PVP aqueous solution (40 wt %), and 0.2 L of an oxidizing agent solution (90 mg KMnO4 and 4.5 g NaIO4 aqueous solution) were added, and the resultant mixture was stirred at room temperature for 2 h. After oxidation, the products were centrifuged and washed three times with ethanol and water. Finally, the hydrophilic nanoparticles was redispersed in water, filtered through a 0.22 μm filter, and stored at 4 °C before use. 1663

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Langmuir Scheme 2. (a) Models and (b) Reaction Equation of Oxidation Cleavage in the Reverse Micelles System

Characterization. The size and morphologies of nanoparticles before and after phase transfer were determined using transmission electron microscopy (TEM, JEOL JEM-1010) at 200 kV. Dynamic light scattering (DLS) and zeta potential measurements were performed using a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, U.K.). The crystal phase of nanoparticles was verified by X-ray diffractometry (XRD, Bruker D8 Advance diffractometer system). The structures of molecules on the surface of nanoparticles were characterized by Fourier transform infrared (FTIR) spectroscopy (Varian 3100 FT-IR spectrophotometer). The samples were pressed into potassium bromide pellets before measurement. The magnetic properties of the samples were measured using a LakeShore model 7407 vibrating sample magnetometer (VSM). Thermogravimetric analysis (TGA) curves were recorded on a DTG-60H (Shimadzu) at a heating rate of 10 °C/min from room temperature to 600 °C. The iron content of samples was determined by ion-coupled plasma−mass spectrometry using a Hewlett-Packard 4500 ICP−MS. In Vitro Cytotoxicity Assay. The toxicity of nanoparticles on nasopharyngeal carcinoma CNE2 cells (obtained from State Key Laboratory of Oncology in South China, Guangzhou, China) was assessed by an in vitro cytotoxicity assay. Cell viability was quantitatively measured using a cell counting kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). A CNE2 cell suspension (100 μL) was seeded into a 96-well plate (cell concentration, 1 × 104 cell/well) and incubated for 12 h at 37 °C in 5% CO2 in a humidified incubator. The cells were treated with various concentrations of Fe 3O4 nanoparticles for 12 h, which was sufficient time for the nanoparticles to be absorbed by cells. Then, 10 μL of a CCK-8 solution was added to each well of the plate and incubated for 2 h. The absorbance of each sample was measured at 450 nm with an iMark microplate reader (BioRad Laboratories, Hercules, CA, USA). The Fe concentration of Fe3O4 nanoparticles was measured by ICP-MS. MR Images and Relaxivity Measurements. The MRI measurements of as-transferred nanoparticles were performed using an MRI scanner at 3T field (Siemens Medical Solutions, Germany). The astransferred nanoparticles in different Fe concentrations were dispersed in 0.5% agarose solution before use. The spin−echo sequence (repetition time (TR) = 5000 ms, echo time (TE) = 13, 30, 92,

113, 141, 170, 198, and 210 ms) was used for T2 measurements. T2 relaxation times were obtained for each sample by fitting the decay curve with a nonlinear monoexponential algorithm

M TE = M 0e−TE/ T2 where TE is the echo time and M is the MRI signal intensity. The transverse relaxivity (r2) was determined from the slope of a 1/T2 − C (iron concentration) fitted line. Fast spin-echo T2-weighted MR images were acquired using the following parameters: flip angle = 120°, TR = 2000 ms, TE = 76 ms, slice thickness = 2.5 mm, FOV = 224 × 320 mm2, and NEX = 4. In Vitro T2-Weighted MR Images. For Fe3O4 nanoparticles that were absorbed by CNE2 cells, the MR contrast effects were tested using a 3T MRI scanner with a head coil. A detailed procedure follows. The cells were seeded onto culture dishes in 4 mL of media and grown for 12 h. After the cells reached 70−80% confluence and fully adhered to the bottom surface, different concentrations of hydrophilic Fe3O4 nanoparticles were added and incubated for 12 h. Subsequently, the cells were washed with PBS and centrifuged. Then, the cells were resuspended in a 1% agarose solution for MR images. The spin-echo sequence was used in the measurement of T2-weighted MR images.

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RESULTS AND DISCUSSION Scheme 1 illustrates a versatile strategy for the large-scale transfer of Fe3O4 nanoparticles from an organic phase to the water phase. A photograph, schematic illustration, and nanoparticle models of the procedure are shown in a−c, respectively. First, Fe3O4 nanoparticles that were coated with oleic acid were prepared by thermal decomposition and dispersed in the upper organic phase. The carboxyl groups of the oleic acid were bound to the surfaces of nanoparticles, and the terminal alkyl chain provided the nanoparticles with hydrophobicity. Then, oxidation agents and PVP were added to the system to initiate the oxidation reactions. The contacted areas between oxidation agents and oleic acid were maximized because of the existence of PVP molecules; this enabled the 1664

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Figure 1. (a) Photographs and (b, d) TEM images of Fe3O4 nanoparticles before and after phase transfer. The inset of (d) is the size distribution histogram of Fe3O4 nanoparticles after phase transfer. (c) Size distribution histogram of Fe3O4 nanoparticles before phase transfer. (e) Hydrodynamic size profiles of Fe3O4 nanoparticles after phase transfer.

Figure 2. (a) FT-IR spectra, (b) X-ray diffraction patterns (XRD), (c) TGA, and (d) magnetic properties of Fe3O4 nanoparticles before and after phase transfer. The red lines in (b) represent the positions of peaks in XRD patterns of typical magnetite structures according to JCPDS file number 65-3107. Lines I and II in the graphs represent the Fe3O4 nanoparticles before and after phase transfer, respectively.

To further investigate the process of phase transfer in this work, a possible mechanism was discussed. Scheme 2 illustrates the mechanism model and reaction equation for the oxidation cleavage reaction in reverse micelles. The micelles size during the transfer process was approximately 1.23 ± 0.5 μm (Figure S1). It is speculated that hydrophobic Fe3O4 nanoparticles are

reaction to proceed to completion in a reasonable period of time. After oxidation, the double bonds of oleic acid were cleaved and transformed into hydrophilic carboxyl groups on the surface of the nanoparticles; consequently, the nanoparticles dispersed well in the water layer. 1665

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peaks at 1512 and 1412 cm−1 are attributed to the asymmetric and symmetric stretching vibrations of the carboxylic groups of oleic acid.23 In the spectra of samples after water transfer, the peaks corresponding to asymmetric and symmetric stretching vibrations of the carboxylic group are shifted to 1637 and 1560 cm−1.13 The peak at 1051 cm−1 of hydrophilic nanoparticles is attributed to the C−O single-bond stretching of the carboxylic group.24 In addition, the peaks at approximately 576 cm−1 in the spectra of both samples correspond to the vibrations of Fe− O bonds, indicating that the ligands anchored on the nanoparticles were stable during oxidation. To confirm the role of PVP in this system, IR spectra were obtained for pure PVP, the reactant mixture, and the products after washing (Figure S3). The spectrum of pure PVP has three characteristic peaks at 1664, 1421, and 1289 cm−1 that are attributed to the vibrations of the carbonyl, C−N bond, and C−H bond, respectively.22 Characteristic peaks of PVP were also observed in the IR spectrum of the reaction mixture. For the hydrophilic nanoparticles after phase transfer, a broad peak between 1000 and 1800 cm−1 appears, which is consistent with the previous reports.13 It is noted that if the hydrophilic nanoparticles were coated with PVP, then the characteristic peaks of samples in the FT-IR spectrum should show three typical peaks in the range between 1000 to 1800 cm−1, which is different from our observation.25 To further confirm the role of PVP, 1H NMR measurements for oleic acid-coated Fe3O4 nanoparticles, the astransferred nanoparticles, and pure PVP were carried out (Figure S4). The 1H NMR characteristic peaks of nanoparticles before phase transfer are consistent with previously reported data of oleic acid-capped nanoparticles. The 1H NMR spectrum of the as-transferred sample shows that the peak centered at 5.4 and 0.9 ppm disappeared after phase transfer, indicating the cleavage of the double bond in oleic acid. No characteristic peaks of PVP were observed in the 1H NMR spectrum of the as-transferred sample, showing that PVP is completely removed from the as-transferred sample. Hence, it is concluded that PVP acts as a surfactant to form a microemulsion, which can offer the stable reaction interface areas and improve the efficiency of the surface oxidation reaction. The X-ray diffraction (XRD) patterns of Fe3O4 nanoparticles before and after phase transfer are shown in Figure 2b. The XRD analysis of both samples of Fe3O4 nanoparticles shows peaks corresponding to (220), (311), (400), (422), (511), and (440) Bragg reflections based on the cubic structure of magnetite (JCPDS file number 65-3107). According to the calculation using the Scherrer equation, the crystalline sizes of the original hydrophobic and as-transferred nanoparticles are ∼13.49 and ∼14.07 nm, respectively. The organic content in hydrophobic and as-transferred Fe3O4 nanoparticles was evaluated by TGA analysis. In Figure 2c, TGA curves demonstrate the weight loss of samples. Below 200 °C, the weight loss may be attributed to water or organic solvents absorbed on samples.14 In this temperature range, the TGA curve of as-transferred nanoparticles was slowly decreasing, which may be due to the high water content in hydrophilic nanoparticles. When the temperature increased from 200 to 600 °C, the capping organic ligands on the nanoparticles decomposed. The weight percentage of the residue represents the amounts of inorganic materials in the samples. The results of TGA show that the content of oleic acid ligands in hydrophobic nanoparticles was approximately 21 wt % and the content of ligands after water transfer was approximately 19 wt %. The decrease in organic content

surrounded by PVP because of a hydrophobic interaction between the alkyl chain of oleic acid and the long polyvinyl chain of PVP. Water-soluble oxidative agents are dissolved in the water pool core of the reverse micelles. The oxidative agents preferentially oxidize oleic acid near the aqueous phase. The double bond of oleic acid undergoes oxidative cleavage to form azelaic acid and nonanoic acid. Over time, all oleic acid molecules on the Fe3O4 nanoparticles are completely oxidized to hydrophilic molecules containing carboxyl groups; this facilitates the transfer of the Fe3O4 nanoparticles to inner water pools. Meanwhile, aggregation resulting from hydrophobic interactions of incompletely reacting nanoparticles is avoided, which is attributed to the steric effect and amphiphilic ability of PVP polymers. Finally, hydrophilic Fe3O4 nanoparticles were successfully obtained after removing the excess agents and organic solvent. Figure 1 shows the photographs, TEM images, and size distribution analysis of Fe3O4 nanoparticles before and after phase transfer. The hydrophobic nanoparticles produced by thermal decomposition were dispersible in the upper hexane layer; the nanoparticles after oxidation were dispersible in the bottom water layer. According to TEM images, the mean sizes of hydrophobic nanoparticles were approximately 14.3 ± 1.2 nm. According to the dynamic light scattering (DLS) measurement, the particle size in nonpolar solvent was around 18.1 ± 0.9 nm. After transfer, the size and shape of the nanoparticles in the water phase underwent no obvious changes (14.5 ± 0.8 nm). As determined by dynamic light scattering analysis, the hydrodynamic diameter of the as-transferred nanoparticles was approximately 16.6 ± 1.2 nm. This result indicates that the nanoparticles are well dispersed in water without aggregation. To further illustrate the importance of PVP in this system, two control experiments were performed under the same conditions: (1) a transfer process without PVP and (2) a transfer process without oxidative reagents. For the former control experiment, the reaction occurred at the interface of both bulk phases and the nanoparticles aggregated. According to DLS measurements, the hydrodynamic diameter of nanoparticles was above 200 nm (Figure S2). The result indicates that in the absence of PVP the contact areas between oxidative agents and oleic acid-coated nanoparticles were limited, resulting in incomplete oxidative cleavage. Nanoparticles that were only partially oxidized readily aggregated in aqueous solution because of the hydrophobic interactions. As for the latter control experiment, the phase transfer failed even after overnight stirring, indicating that the final nanoparticles could not disperse in water, which is in agreement with previous reports.22 Therefore, oxidants and PVP are both required for the successful transfer of nanoparticles to water with good dispersibility on a large scale. The characteristics of hydrophobic and as-transferred Fe3O4 nanoparticles are shown in Figure 2. In Figure 2a, FT-IR spectra illustrate the characteristics of different surface molecules capped on Fe3O4 nanoparticles before and after transfer. For the spectra of both samples, a broad band at around 3400 cm−1 is assigned to the OH stretching vibrations. The peaks at 2852 and 2921 cm−1 in both samples are associated with the symmetric and asymmetric vibrations of methylene (CH2) of the alkyl chain, respectively. Notably, the two characteristic peaks of CH stretching vibrations are significantly reduced after phase transfer, which is ascribed to the cleavage of the oleic acid alkyl chain during oxidation.13 According to the spectra of the hydrophobic nanoparticles, the 1666

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Figure 3. (a) Stability of as-transferred Fe3O4 nanoparticles. (b) Hydrodynamic size and zeta potential of as-transferred Fe3O4 nanoparticles in water at different pH values.

indirectly proves that the oleic acid molecules are cleaved and lose part of their components during oxidation. The magnetization hysteresis loops of two kinds of Fe3O4 nanoparticles were measured by sweeping the external magnetic field between −20 and 20 kOe at room temperature. As shown in Figure 2d, no coercivity or remanence was observed, indicating the superparamagnetic behaviors of both samples. The saturation magnetization of samples increased approximately from 34.7 to 39.3 emu/g after phase transfer. The slight increase in saturation magnetization is ascribed to the increased effective weight fraction of the magnetic nanoparticles and the possible increased particle size during the oxidation reaction.15,26 It is well known that there are positive correlations between the value of saturation magnetization and the efficiency of T2 contrast.27 Compared to nanoparticles transferred by encapsulating amphiphilic polymers or the ligand exchange of functional polymers, the nanoparticles transferred by this strategy have a lower organic content per unit of mass and may maintain a high saturation magnetization and result in high-quality images in T2-weighted magnetic resonance imaging. The colloid stability and zeta potentials of Fe3O4 nanoparticles after phase transfer are shown in Figure 3. Figure 3a shows that the hydrodynamic size was approximately 16−20 nm for at least 20 days, which illustrates that the as-transferred nanoparticles exhibit good stability over a long time. As shown in Figure 3b and Table S1, the hydrodynamic size of waterdispersible nanoparticles remained stable at approximately 17 nm when the pH was adjusted from 7 to 13. Simultaneously, the zeta potentials of nanoparticles were approximately −40 mV over the same pH range. When the pH was below 5, the hydrodynamic size sharply increased and the zeta potential decreased dramatically, illustrating that nanoparticles are unstable under acidic conditions. This instability may be due to the weakened repulsion of negative charges from surface carboxyl groups because the carboxylate groups are neutralized under acidic conditions. The nanoparticles also have good water-dispersibility and exhibit no aggregation in a biological PBS buffer (pH 7.4). Hence, the as-transferred nanoparticles have good colloid stability and suitable hydrodynamic sizes; therefore, they are ideal nanoprobes in bioapplications. To evaluate the cytotoxicity of water-transferred Fe3O4 nanoparticles, a standard CCK viability test was performed. The CNE2 cancer cells were incubated with various concentrations of Fe3O4 nanoparticles (from 6.25 to 100 Fe μg/mL). After 12 h, the viability of cells was evaluated by measuring the absorbance at 450 nm. Figure 4 shows that the

Figure 4. Cell viability of as-transferred Fe3O4 nanoparticles. Error bars = SEM, *p < 0.05, **p < 0.01, and ***p < 0.001.

viability of CNE2 cells was above 70% even when the cells were incubated with hydrophilic Fe3O4 nanoparticles for 12 h at a concentration of 100 Fe μg/mL. Thus, the good cell viability of water-transferred Fe3O4 nanoparticles indicates that they could be a good candidate for biomedical applications. The potential biomedical applications of as-transferred nanoparticles were explored by a 3T clinical MR scanner. It is known that Fe3O4 nanoparticles can be developed as negative contrast agents in MRI because of their shortening transverse relaxation time of water protons. As shown in Figure 5a, the images illustrate that the intensity of the darkening signals was strongly dependent on the Fe concentrations. With increasing Fe concentration, the signal intensity of T2-weighted images decreased significantly. The plot of a sample containing 2 mM Fe was darker than for other samples. Figure 5b shows the transverse relaxation rate (1/T2) of hydrophilic Fe3O4 nanoparticles as a function of Fe concentration. According to the calculation, the transverse relaxivity (r2) value, which is represented by the slope, is 181.43 mM−1 S−1. Subsequently, in vitro MR imaging was performed with CNE2 cells, as shown in Figure 5c. After 12 h of incubation with Fe3O4 nanoparticles, the CNE2 cells absorbed the negative contrast agents with different concentrations. With increasing Fe concentration, the plots gradually go dark and the signal intensity exponentially declines (Figure 5d). These results demonstrate that hydrophilic Fe3O4 nanoparticles that are transferred via an oxidative reaction in a reverse micelle system are promising T2 contrast agents for biomedical MR imaging applications. 1667

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Figure 5. (a) T2-weighted MR images and (b) plot of 1/T2 vs Fe concentration of as-transferred Fe3O4 nanoparticles. (c) T2-weighted MR images from a 3T MR scanner of CNE2 cells after 12 h of incubation with as-transferred Fe3O4 nanoparticles. (d) Plot of normalized signal intensity of (c) as a function of Fe concentration.

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CONCLUSIONS

A versatile and efficient method for the large-scale transfer of hydrophobic Fe3O4 nanoparticles to the aqueous phase is proposed. The mechanism of this water phase transfer comprises the oxidative cleavage of oleic acid in the reverse micelle system with the assistance of PVP. According to characterization data, as-transferred Fe3O4 nanoparticles exhibit good water dispersibility, colloid stability, low cell cytotoxicity, and relatively good magnetic properties. The transverse relaxivity value (r2) of the as-transferred Fe3O4 nanoparticles was calculated to be 181.43 mM−1 S−1, which indicates that the nanoparticles are promising T2 contrast agents. T2-weighted MR imaging of CNE2 cells shows that the as-transferred Fe3O4 nanoparticles can retain their magnetic characteristics in biological systems, suggesting that they are potential candidates for MRI biomedical applications. In addition, this versatile phase-transfer strategy is suitable for hydrophobic Fe3O4 nanoparticles and also provides a platform for other oleic acid-capped nanoparticles (such as quantum dots and rare earth nanomaterials). Most importantly, the terminal carboxyl groups on the as-transferred nanoparticles may be further functionalized with drug molecules or active biomolecules. Given these superior features, we believe that our strategy for transferring various oleic acid-capped nanoparticles from the organic phase to the aqueous phase could be applied to a widespread range of applications in nanobiotechnology and nanomedicine.

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Additional hydrodynamic size, zeta potential, optical image, FT-IR spectra, and 1H NMR spectrum profiles of related samples (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Li Li: 0000-0002-7518-7426 Author Contributions

J.C. and Y.Q.M. contributed equally to this article. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (grant nos. 21376192, 81571809, 81271622, and 81471711), the Natural Science Foundation of Guangdong, China (no. 2014A030311036), and the State Key Laboratory of Optoelectronic Materials and Technologies (Sun Yat-sen Unversity) (no. OEMT-2015-KF-03).

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REFERENCES

(1) Wu, L.; Mendoza-Garcia, A.; Li, Q.; Sun, S. Organic Phase Syntheses of Magnetic Nanoparticles and Their Applications. Chem. Rev. 2016, 116, 10473. (2) Dreaden, E. C.; Alkilany, A. M.; Huang, X.; Murphy, C. J.; ElSayed, M. A. The Golden Age: Gold Nanoparticles for Biomedicine. Chem. Soc. Rev. 2012, 41 (7), 2740−79. (3) Fan, W.; Bu, W.; Shi, J. On the Latest Three-Stage Development of Nanomedicines Based on Upconversion Nanoparticles. Adv. Mater. 2016, 28 (21), 3987−4011.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b03360. 1668

DOI: 10.1021/acs.langmuir.6b03360 Langmuir 2017, 33, 1662−1669

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DOI: 10.1021/acs.langmuir.6b03360 Langmuir 2017, 33, 1662−1669