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. 2022 Jan 7;7(2):2031–2040. doi: 10.1021/acsomega.1c05486

Fabricating Dual-Functional Plasmonic–Magnetic Au@MgFe2O4 Nanohybrids for Photothermal Therapy and Magnetic Resonance Imaging

Enhui Qiu †, Xiaofang Chen †, Da-Peng Yang †,‡,*, Michelle D Regulacio §,*, Rufus Mart Ceasar R Ramos §,∥, Zheng Luo ⊥, Yun-Long Wu ⊥, Ming Lin #, Zibiao Li #, Xian Jun Loh #, Enyi Ye #,*
PMCID: PMC8771950  PMID: 35071891

Abstract

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Bifunctional nanohybrids possessing both plasmonic and magnetic functionalities are of great interest for biomedical applications owing to their capability for simultaneous therapy and diagnostics. Herein, we fabricate a core–shell structured plasmonic–magnetic nanocomposite system that can serve as a dual-functional agent due to its combined photothermal therapeutic and magnetic resonance imaging (MRI) functions. The photothermal activity of the hybrid is attributed to its plasmonic Au core, which is capable of absorbing near-infrared (NIR) light and converting it into heat. Meanwhile, the magnetic MgFe2O4 shell exerts its ability to act as a MRI contrast agent. Our in vivo studies using tumor-bearing mice demonstrated the nanohybrids’ excellent photothermal and MRI properties. As a photothermal therapeutic agent, the nanohybrids were able to dramatically shrink solid tumors in mice through NIR-induced hyperthermia. As T2-weighted MRI contrast agents, the nanohybrids were found capable of substantially reducing the MRI signal intensity of the tumor region at 10 min postinjection. With their dual plasmonic–magnetic functionality, these Au@MgFe2O4 nanohybrids hold great promise not only in the biomedical field but also in the areas of catalysis and optical sensing.

1. Introduction

Hybrid nanomaterials have been the focus of intense research in the past two decades as they provide a means of combining different functionalities into a single nanoplatform.1−3 Due to their integrated properties, they have gained ample significance in a multitude of applications that span across diverse areas of science and technology. In biomedicine, an emerging trend is the development of nanostructured theranostic agents that can perform therapeutic and diagnostic functions simultaneously.4−8 Such a kind of agent has enormous potential in cancer treatment and diagnosis. In several cases, the therapeutic action is based on the light-absorbing ability of nanostructures to eradicate cancer cells through the photothermal effect.9−12 This minimally invasive treatment approach strongly relies on an optically active material that can efficiently absorb light and transform it into heat. The generated heat is then used to ablate diseased cells through the hyperthermia effect. The wavelength of light that is used for this purpose is within the near-infrared (NIR) spectral window, which is also known as the therapeutic window or the biological transparency window.13,14 This is because NIR light (λ = 700–1400 nm) is weakly absorbed by tissue components; hence, it can allow for deeper tissue penetration and can reach a high volume of deep-seated tumors. It is therefore highly desirable to develop photothermally active nanomaterials that are capable of intense NIR absorption.

Gold (Au) nanostructures are regarded as ideal photothermal therapeutic agents because they are photothermally responsive, biocompatible, chemically stable, and easily modifiable.13−17 Their optical behavior is governed by localized surface plasmon resonances (LSPRs) that arise from the collective oscillation of free electrons in the conduction band.18 While early reports on Au nanostructures have centered mainly on Au nanospheres that absorb visible light, more recent studies have focused on extending their plasmonic absorption to the NIR window.19 For instance, increasing both the size and the shape anisotropy of Au nanostructures has been found to be an effective way to shift the plasmon absorption band from the visible region to the NIR region.17−24 The surrounding environment is another factor that can be manipulated to tune the plasmon-induced optical response to achieve strong NIR absorption.25 Coupling Au nanostructures with a high-refractive-index material has been successful in this regard. An example of such a material is Fe3O4, a spinel-structured ferrite that has a refractive index of 2.42.26 Over the past decade, hybrid nanostructures that consist of Au and Fe3O4 have been prepared in a variety of configurations, including Janus (or dumbbell), core–shell (both Au@Fe3O4 and Fe3O4@Au), and flower-like structures.27−33 Apart from enhancing the NIR absorption, a major advantage of hybridizing Au with Fe3O4 is the incorporation of a magnetic functionality. The presence of magnetic Fe3O4 enables these hybrids to realize magnetic resonance imaging (MRI), a noninvasive medical diagnostic technique that can be used to obtain high-resolution images of organs and tissues in the body.34,35 Consequently, a plasmonic–magnetic dual functional system with both therapeutic and diagnostic functions can be achieved.

With the growing interest in the use of plasmonic–magnetic nanohybrids for cancer therapy, other members of the spinel ferrite family have been actively explored as the magnetic component. For instance, Ravichandran et al. prepared a hybrid that consists of a cobalt ferrite core and a multilayered Au shell (i.e., CoFe2O4@Au) and demonstrated its use as a multifunctional nanoplatform that was not only capable of MRI- and hyperthermia-based therapy but also allowed for targeted drug delivery.36 Lee and co-workers constructed a hybrid based on manganese ferrite with a MnFe2O4@SiO2@Au configuration and reported its potential for the simultaneous imaging and treatment of human epithelial cancer.37 Magnesium ferrite (MgFe2O4) is another promising spinel ferrite compound that can form a hybrid with Au, but its utility in cancer therapy has not yet been thoroughly examined. Because Mg is a biocompatible element that has been known to play vital roles in many important processes in the human body, the use of MgFe2O4 is favorable in biomedical applications. Nonkumwong et al. fabricated a core–shell hybrid where magnetic MgFe2O4 was coated with a plasmonic Au shell (i.e., MgFe2O4@Au).38 They observed the noncytotoxicity of the hybrid to mouse areola fibroblast (L-929) cells, indicating that it was compatible with mammalian cells. However, the saturation magnetization of the hybrid was far lower than the value obtained for uncoated MgFe2O4, which was attributed to the diamagnetism exhibited by the thick Au shell. The group did not probe on the therapeutic and MRI capabilities of MgFe2O4@Au, thus warranting further studies of the potential applications of this hybrid combination.

In this work, we present a promising dual functional nanohybrid system that is composed of a plasmonic Au core and a magnetic MgFe2O4 shell. In vivo studies showed that the as-prepared Au@MgFe2O4 nanohybrids exhibited photothermal therapeutic effects as they were able to annihilate cancer cells in tumor-bearing mice under NIR illumination. In addition, they possess a saturation magnetization value that is sufficient for effective T2-based MR imaging as evidenced by the substantial darkening of the tumor region after the administration of the hybrid. Suffice to say that these bifunctional Au@MgFe2O4 nanohybrids could potentially enable the MRI-guided photothermal therapy of cancer cells.

2. Results and Discussion

2.1. Synthesis and Characterization of the Au@MgFe2O4 Nanohybrids

Hybrid nanostructures of Au@MgFe2O4 were colloidally prepared following a two-step seeded-growth approach, which began with the synthesis of Au seeds. Using our previously established protocol for Au nanoparticle synthesis, Au seeds were produced at 150 °C in oleylamine, which acted as both the reducing agent and the capping ligand.39 The change in color of the reaction mixture to wine red indicated the formation of spherical Au nanoparticles. The transmission electron microscopy (TEM) image of the product (Figure 1a) showed that the oleylamine-capped Au seeds exhibited a quasi-spherical morphology with an average diameter of 12 ± 2 nm. To prepare the Au@MgFe2O4 nanocrystals, MgO and Fe(acac)3 were decomposed in the presence of the preformed Au seeds at 300 °C in a solvent mixture that contained oleylamine, octadecene, and oleic acid. The X-ray diffraction (XRD) pattern of the resulting hybrid (Figure S1 in the Supporting Information) showed diffraction peaks that could be indexed to Au (JCPDS no. 04-0784) and MgFe2O4 (JCPDS no. 73-1720), confirming the presence of the two components. TEM imaging revealed a core–shell structure with an average size of 42 ± 7 nm (Figure 1b and c). The high-resolution TEM (HRTEM) image in Figure 1d showed distinct lattice fringes of the shell with a d-spacing that matched the (311) planes of MgFe2O4. For the core, lattice fringes characteristic of the (111) planes of Au were evident. The Au core appeared darker in the TEM images than the MgFe2O4 shell because of its higher electron density, which allowed fewer electrons to be transmitted. To verify the core–shell configuration of the Au@MgFe2O4 hybrid, additional imaging techniques were performed. Figure 2a displays the high-angle annular dark-field scanning TEM (HAADF-STEM) image of the Au@MgFe2O4 nanohybrids, which clearly shows that Au is at the center and is covered by MgFe2O4. Au is much brighter in the image due to its higher atomic number as compared to the elements present in MgFe2O4. This is a consequence of the Z-contrast in HAADF-STEM. The corresponding energy-dispersive X-ray (EDX) elemental maps (Figure 2b–e) further confirmed the elemental distribution within the hybrid. Au is present only at the center, whereas Mg, Fe, and O enclose the Au core.

Figure 1.

Figure 1

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(a) TEM image of the starting Au seeds. (b and c) TEM images of the Au@MgFe2O4 nanohybrids. (d) HRTEM image of the red square region in panel c, showing distinct lattice fringes of the Au core and the MgFe2O4 shell.

Figure 2.

Figure 2

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(a) HAADF-STEM image of the Au@MgFe2O4 nanohybrids and the corresponding EDX elemental maps for (b) Au, (c) Mg, (d) Fe, and (e) O.

Compared to the starting 12 nm quasi-spherical Au seeds, the Au cores in the nanohybrids have irregular anisotropic shapes with significantly larger dimensions (length × width = 27 × 20 nm), indicating that a change in both size and morphology took place during the synthesis. It is likely that the Au cores were formed through coalescence of the Au seeds and that the high temperature at which the synthesis was performed (300 °C) played a crucial role in this process. Since elevated temperatures can induce the desorption of capping ligands, the surface of the starting Au seeds becomes less densely capped at 300 °C due to the desorption of oleylamine; therefore, coalescence becomes favorable. The merging of the Au seeds appears to have happened in a random nonoriented manner judging from the irregular shape, polydispersity, and polycrystallinity of the Au cores. From the TEM images, it can be seen that MgFe2O4 was able to coat the entire Au core. However, the thickness of the shell is uneven, implying that the deposition of MgFe2O4 did not occur uniformly. Considering the polycrystalline nature of the Au core, where multiple crystal domains are exposed, it is possible for MgFe2O4 to nucleate and grow randomly on multiple sites of the Au core surface. This explains the flower-like arrangement of the core–shell hybrid, where MgFe2O4 forms multiple petal-like structures that surround the Au core. Flower-like core–shell architectures have also been reported for analogous composites of Au with other ferrites, MFe2O4 (where M = Fe, Co, Mn).40,41

Next, the plasmon-induced optical absorptions of the Au seeds and the Au@MgFe2O4 nanohybrids were examined through UV–vis spectroscopy. For the Au seeds, a well-defined plasmon absorption band is present in the visible region with a maximum at around 520 nm (Figure S2 in the Supporting Information). This is typical for small-sized Au nanoparticles with a spherical morphology.19 On the other hand, the Au@MgFe2O4 nanohybrids exhibit a broader plasmon absorption band centered at around 600 nm (Figure 3a). It is worth noting that although the absorption maximum is located within the visible region, strong absorption is present even at the NIR spectral range. The shift toward longer wavelengths with respect to the starting Au seeds can be ascribed to a number of factors. It has been well established that the size, morphology, composition, and local environment of Au nanostructures have a profound influence on their plasmonic behavior.25 In the case of the Au@MgFe2O4 nanohybrids, the larger size and the increased shape anisotropy of the Au core as compared to the quasi-spherical Au seeds contributed to the broadening and the red-shifting of the plasmon absorption band. In addition, these observed changes in optical properties were induced by the change in the surrounding environment when the Au seeds were coated with MgFe2O4, which has a high refractive index of 2.39. This is consistent with earlier studies that reported that the plasmon resonances of Au nanostructures are highly sensitive to the refractive index of the surrounding medium, whereby an increase in the local refractive index leads to a red-shift in the plasmon absorption band.42,43 Similar plasmonic behavior has been observed for Au@Fe3O4 nanocrystals.31,41

Figure 3.

Figure 3

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(a) Absorption spectrum of the Au@MgFe2O4 nanohybrids. (b) Cytotoxicity effects of Au@MgFe2O4 nanohybrids on HepG2 cells with (red bars) and without (green bars) NIR irradiation. (c) Photothermal effect of Au@MgFe2O4 nanohybrids. The temperature vs time plot was recorded for Au@MgFe2O4 nanohybrids (0.1 mg mL–1) upon irradiation by a 808 nm laser (power density of 1 W cm–2). The plot for the control PBS solution was also recorded. (d) Thermographic images of a 1 mL aqueous dispersion of Au@MgFe2O4 nanohybrids (0.1 mg mL–1) recorded at different time intervals.

2.2. Au@MgFe2O4 Nanohybrids as Photothermal Therapeutic Agents

The strong NIR light-absorbing ability of the Au@MgFe2O4 nanohybrids makes them attractive materials for photothermal therapy, where the heat generated from the absorbed light can be used to obliterate cancer cells. Prior to use, the nanohybrids were made water-dispersible and biocompatible through a ligand exchange process that replaced oleylamine with citrate as the surface-capping ligand. In addition, an in vitro assay was performed to assess their cellular toxicity against HepG2, a human hepatic cancer cell line. Figure 3b shows that after incubation for 24 h, the nanohybrids did not induce cell loss at very low concentrations, and around 90% of the cells remained viable at the maximum testing concentration (i.e., 100 μg mL–1). To assess their photothermal activity, a parallel assay was done with 10 min of NIR irradiation performed after 22 h of incubation (i.e., 2 h before the end of the 24 h incubation period). A significant decrease in cell viability was observed even at a very low concentration of 0.1 μg mL–1, and the decrease became more pronounced as the concentration of the nanocrystals increased. The cell viability was reduced to only 12% at 100 μg mL–1, demonstrating dramatic cell loss with NIR irradiation. This suggests that the Au@MgFe2O4 nanohybrids are photothermally active and are able to annihilate cells through NIR-induced hyperthermia. In order to confirm this, we have evaluated the photothermal effect of the Au@MgFe2O4 nanohybrids. A 1 mL aqueous dispersion that contained 0.1 mg of the nanohybrids was irradiated using an 808 nm NIR laser with a power density of 1 W cm–2, then the temperature changes were monitored over time using an infrared (IR) thermal camera. As shown in Figure 3C, the temperature of the Au@MgFe2O4 solution increased from 25.7 to 45.7 °C after irradiation for 4 min (see Supporting Video 1), while there was no significant temperature change for the control solution that only contained PBS. The change in temperature of the nanohybrid solution was also visualized in the thermographic images captured by the IR camera (Figure 3d). The observed temperature increase is a clear indication that the Au@MgFe2O4 nanohybrids are photothermally responsive.

To further assess the utility of the Au@MgFe2O4 nanohybrids as a photothermal therapeutic agent, in vivo studies were conducted using 12 live nude mice, each bearing a HepG2 cell-transplanted solid tumor. The mice were randomly divided into four groups and were subjected to the following different treatment conditions: (1) injection of PBS solution (i.e., control), (2) injection of PBS followed by 10 min of NIR irradiation, (3) injection of a PBS dispersion of the Au@MgFe2O4 nanohybrids, and (4) injection of a PBS dispersion of the Au@MgFe2O4 nanohybrids followed by 10 min of NIR irradiation. Injections were done intratumorally, and the weight of the mice and the volume of the tumor were measured every day for seven days. Figure 4a shows the photographs of four tumor-bearing mice on the last day of measurement, with one representative mouse for each treatment. Each tumor is highlighted with a red circle. It is evident from the photographs that the smallest tumor size is for the mouse that was treated with Au@MgFe2O4 nanohybrids coupled with NIR irradiation (i.e., treatment 4). This observation is supported by Figure 4b and c, which show the photographs of the tumors after excision and the daily size measurements, respectively. All the tumors from the mice that were subjected to treatments 1–3 grew continuously for seven days from their initial size of around 100 mm3. On the other hand, a reduction in size can be seen for the tumors excised from the mice that received treatment 4. As treatments 2 (NIR only) and 3 (Au@MgFe2O4 only) did not lead to tumor regression, it can be inferred that the success of treatment 4 (Au@MgFe2O4 and NIR) is a consequence of the photothermal therapeutic property of Au@MgFe2O4 that was stimulated by NIR light. Figure 4d shows that the weight of mice in all groups remained practically stable throughout the study, implying that treatment 4 had no adverse effects on mice. However, we observed that a scab (dark spot seen on mouse 4) formed on the skin covering the tumor region, which indicated burns caused by the heat generated from the photothermal effect. Experimental parameters such as laser power, irradiation time, and nanocomposite dosage can be adjusted to prevent this from occurring.

Figure 4.

Figure 4

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In vivo evaluation of the photothermal therapeutic effects of the Au@MgFe2O4 nanohybrids under NIR irradiation. Tumor-bearing mice were subjected to four different treatment conditions: (1) control, (2) NIR irradiation, (3) Au@MgFe2O4 nanohybrids, and (4) Au@MgFe2O4 nanohybrids with NIR irradiation. (a) Representative photographs of mice on the seventh day after treatment. Tumor regions are highlighted with red circles. (b) Photograph of excised tumors from the four different groups (three trials each). (c) Average tumor volumes and (d) mice weight were measured every day for seven days.

Histological changes were examined by slicing the tumors into frozen sections and staining with hematoxylin and eosin (H&E). Displayed in Figure 5 are the stained histological images of tumor tissues from mice that were given different treatments. Consistent with the results discussed above, only treatment 4 was successful in massively destroying tumor cells, as evidenced by the large number of necrotic and apoptotic cells that are present in the histological image in Figure 5d. No significant necrosis and apoptosis were observed in the histological images obtained for the other three treatments. This further proves the excellent photothermal therapeutic efficacy of the Au@MgFe2O4 nanohybrids.

Figure 5.

Figure 5

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Hematoxylin and eosin (H&E) stained histological images of tumor tissues from mice that were exposed to four different treatment conditions: (a) control, (b) NIR irradiation, (c) Au@MgFe2O4 nanohybrids, and (d) Au@MgFe2O4 nanohybrids with NIR irradiation. The scale bar is 50 μm.

2.3. Au@MgFe2O4 Nanohybrids as MRI Contrast Agents

To investigate the magnetic properties of the Au@MgFe2O4 nanohybrids, the magnetic field dependence of magnetization was measured at room temperature (300 K) using a vibrating sample magnetometer (VSM). The resulting magnetization curve is displayed in Figure 6. An enlarged view of the center of the curve (inset of Figure 6) revealed a narrow hysteresis loop with a remanence of 4.32 emu g–1 and a coercivity of 77.6 Oe. Such a small hysteresis loop reflects the soft ferrimagnetic character of MgFe2O4.44,45 The saturation magnetization value was determined to be 36.8 emu g–1, which is comparable to literature values for pristine MgFe2O4 nanostructures.46,47 It is worth noting that the saturation magnetization value that was achieved for the previously reported MgFe2O4@Au nanohybrids (i.e., 2.52 emu g–1) is much lower than the value observed for our Au@MgFe2O4 nanohybrids.38 For the MgFe2O4@Au nanohybrids where MgFe2O4 is situated at the core, the decrease in the saturation magnetization value is believed to have been caused by the thick Au shell, which is diamagnetic. Our results indicate that this can be circumvented by reversing the core–shell arrangement such that MgFe2O4 is positioned as the outer component.

Figure 6.

Figure 6

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Magnetization curve of the Au@MgFe2O4 nanohybrids at 300 K. Inset shows a magnified view of the hysteresis loop.

In the design magnetic nanostructures for use as a MRI contrast agent, a high saturation magnetization is desired to achieve an enhanced MRI sensitivity.48 MRI contrast agents can be classified into two types depending on their mechanism of operation. A T1 (or positive) contrast agent shortens the longitudinal relaxation time of nearby water protons and brightens the target tissue or organ, whereas a T2 (negative) contrast agent darkens the target region by shortening the transverse relaxation time.49 A high saturation magnetization translates to a high transverse relaxivity coefficient r2, which determines the T2 contrast ability of the MRI probe.50 The high saturation magnetization of our Au@MgFe2O4 nanohybrids prompted us to examine their use as a T2 contrast agent for in vivo MR imaging of transplanted tumors in mice. Figure 7 shows the T2-weighted MR images that were taken at two different periods: (a) preinjection and (b) postinjection. The nanohybrids were injected directly into the tumor site, and the postinjection MR image was taken after 10 min. The tumor region is considerably darker in the postinjection image, indicating that the tumor site can be clearly detected 10 min after injection of the hybrid. This demonstrates that the Au@MgFe2O4 nanohybrids are capable of pronounced and rapid contrast-enhanced effects and are therefore excellent candidates as MRI contrast agents.

Figure 7.

Figure 7

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T2-Weighted MR images of a tumor-bearing mouse that were taken (a) before injection and (b) 10 min after an intratumoral injection of Au@MgFe2O4 nanohybrids. The tumor is highlighted with a red arrow, and the darkened region after injection is highlighted with a yellow arrow.

3. Conclusions

In summary, a dual functional theranostics nanoplatform based on plasmonic–magnetic nanohybrids was successfully constructed. The synthesis was carried out in solution through a seeded-growth approach, where presynthesized quasi-spherical Au seeds were used as starting materials. TEM imaging coupled with elemental mapping revealed that the hybrid has a core–shell flower-like architecture, where a polycrystalline anisotropic Au core is surrounded by a cluster of MgFe2O4 petals. The plasmonic Au core endowed the hybrid with a photothermal therapeutic ability, while the magnetic MgFe2O4 shell provided the potential for noninvasive diagnosis through MRI. In vivo studies indicated that the intratumoral administration of Au@MgFe2O4 nanohybrids followed by NIR irradiation is effective to regress tumor growth through photothermal ablation. Furthermore, in vivo MRI scanning showed large signal attenuation at the tumor location after injecting the hybrids. This work affords us a facile strategy for fabricating effective nanoscale theranostic agents, which can lead to the realization of the simultaneous imaging and therapy of tumors.

4. Experimental Section

4.1. Chemicals and Reagents

Auric chloride (AuCl3), oleylamine (70%), magnesium oxide (MgO), iron(III) acetylacetonate (Fe(acac)3, 99.9%), oleic acid (90%), octadecene (90%), sodium citrate, ethanol, and hexane were purchased from Sigma-Aldrich and were used as received. The synthesis process was carried out under an argon flow by using a Schlenk line.

4.2. Synthesis of the Au seeds

Gold seeds were prepared according to our previously published protocol.39 In a typical experiment, 0.1 mmol AuCl3 was dissolved in 10 mL of oleylamine with magnetic stirring at room temperature. The resulting transparent solution was degassed under vacuum for 15 min to remove water and oxygen and then flushed with argon. The solution was then heated to 150 °C and maintained at this temperature for 30 min. The color of the mixture changed to wine red, which denoted the formation of Au nanoparticles. The reaction was quenched by removing the heating mantle and allowing the mixture to cool. The as-synthesized oleylamine-coated Au nanoparticles were isolated through centrifugation and purified using a mixed hexane/ethanol (1:2 v/v) solvent system. The nanoparticles were redispersed in 4 mL of octadecene/oleylamine (3:1 v/v) for further use.

4.3. Synthesis of Au@MgFe2O4 Nanohybrids

In a three-neck round-bottom flask, 0.5 mmol MgO and 1 mmol Fe(acac)3 were dissolved in a mixture of 4.5 mmol (1.43 mL) oleic acid and 5 mL of octadecene at 150 °C. To the mixture were then added the presynthesized Au seeds that were dispersed in octadecene/oleylamine (3:1 v/v), and the resulting mixture was degassed under vacuum for 10 min before being loaded with argon. The mixture was rapidly heated to 300 °C, and the reaction was allowed to proceed for 1 h. After cooling to room temperature, the black mixture was diluted with 10 mL of hexane and precipitated using 25 mL of ethanol. This was followed by centrifugation at 10 000 rpm for 10 min. Purification using hexane and ethanol was repeated twice, followed by isolation with a magnet. Those particles attracted to the magnet were collected and redispersed in 20 mL of toluene for further use. A dispersion of Au@MgFe2O4 nanohybrids in toluene was mixed with an aqueous solution of sodium citrate. The mixture was subjected to mild heating at 90 °C until all particles were transferred to the aqueous phase.

4.4. Characterization

The size and morphology of the Au seeds and the Au@MgFe2O4 nanohybrids were examined through transmission electron microscopy (TEM). An FEI Titan 80-300 electron microscope was used to collect bright-field images at an accelerating voltage of 200 kV. High-angle annular dark-field scanning TEM (HAADF-STEM) images were also obtained for the nanohybrids using the same instrument, which was equipped with a HAADF detector. Energy-dispersive X-ray (EDX) analysis of the nanohybrids was performed in the STEM mode at an equal acquisition time, with a nominal electron beam diameter of ∼1 nm for the measurement. No beam damage or contamination was observed during the experiments. The elemental maps of Au, Mg, Fe, and O were all obtained. Room-temperature absorption spectra of the Au seeds and the Au@MgFe2O4 nanohybrids were recorded on a SHIMADZU UV-1800 spectrophotometer. The X-ray diffraction (XRD) pattern of the nanocrystals was collected using a Bruker D8 general area detector diffraction system (GADDS) under Cu Kα radiation (1.5418 Å). Samples were prepared by dropping a dispersion of the nanocrystals onto a silicon (100) wafer and allowing it to dry in air. The magnetization curve of the nanohybrids was measured using a vibrating sample magnetometer (VSM 880) at room temperature (300 K) with 10.5 mg of the dry sample. Sample magnetization was recorded as a function of the applied magnetic field. The field strength was varied from −3 to 3 T.

4.5. Cell Viability Test

The cytotoxicity of (1) Au@MgFe2O4 and (2) Au@MgFe2O4 with NIR irradiation was examined through the MTT (1-methyltetrazole-5-thiol) assay. The HepG2 cells were seeded onto 96-well culture plates at a density of 104 cells per well in Dulbecco’s modified Eagle’s medium (DMEM, 100 μL) containing 10% fetal bovine serum (FBS). After 24 h of incubation at 37 °C, Au@MgFe2O4 in the DMEM buffer was added at different concentrations. After culturing for 22 h, a group of cells was irradiated with NIR light for 10 min and then continuously cultured for 2 h. To each well was then added 10 μL of the MTT solution (5 mg mL–1) to evaluate the cell viability. After 4 h at 37 °C, the solution was removed, and 100 μL of DMSO was added to dissolve formazan crystals. To quantify the cell viability, the absorbance at 490 nm was measured using a microplate reader. The percent cell viability was calculated by normalizing with the results obtained from the control (i.e., no nanohybrids).

4.6. Evaluation of the Photothermal Effect

The nanohybrids were first dispersed in water (0.1 mg/mL), then 1 mL of the dispersion was exposed to 808 nm laser light at a power density of 1 W cm–2. The temperature change at specified time intervals was monitored using an infrared (IR) thermal imaging system. The photothermal effect of PBS solution was also measured for comparison.

4.7. In Vivo Tumor Regression Study

All the experiments were in accordance with the Animal Care Guidelines of Xiamen University. Twelve nude mice weighing 19–21 g were fed in an IVC system and supplied with filtered air, sterile food, and water. For tumor transplantation, HepG2 cells were dispersed in sterile phosphate buffered saline (PBS) at a concentration of 1 × 107 cells per milliliter and injected into the flank of nude mice at a volume of 100 μL per mouse. When the solid tumor volume reached around 100 mm3, the mice were randomly divided into four groups (each having three mice), and each group received a different treatment as follows: (1) PBS, (2) PBS with NIR irradiation, (3) Au@MgFe2O4, and (4) Au@MgFe2O4 with NIR irradiation. PBS and the Au@MgFe2O4 dispersion in PBS (0.25 mg mL–1) were injected intratumorally at a volume of 100 μL. For treatments with NIR irradiation, the irradiation was performed using an 808 nm laser at a power density of 0.5 W cm–2 for 10 min. The weight of the mice and the tumor volume were monitored and measured every day for seven days starting from the day of treatment. On the last day, the mice were euthanized, and the transplanted tumors were removed for further study. The tumor tissues were then fixed sequentially in a 15% sucrose solution and a 30% sucrose solution for dehydration, embedded in an embedding medium for frozen tissue specimens, cut into 4 μm sections, and stained using hematoxylin and eosin (H&E) for the histological analysis.

4.8. In Vivo MRI of Tumors

Tumors were transplanted in nude mice using the protocol described above. The tumor-bearing mice were used when the solid tumor volume reached around 120 mm3. The mice were first anesthetized, and MR images were obtained (a) before injection and (b) 10 min after the injection of the hybrids. For the injection protocol, 50 μL of a PBS solution of Au@MgFe2O4 (0.1 mg mL–1) was directly injected into the tumor site. After 10 min, the mice were placed inside a custom-built rodent receiver coil, and MRI was performed using a 9.4T BioSpec MRI system. T2-Weighted MR images were obtained using a conventional spin–echo sequence, which was made up of a series of events as follows: 90° pulse, 180° rephasing pulse at TE/2, and signal reading at TE (echo time). This series was repeated at each time interval TR (repetition time). The following parameters were adopted: point resolution of 156 × 156 μm, TE = 60 ms, TR = 4000 ms, and number of acquisitions = 1.

Acknowledgments

This work was supported by the Orientative project funded by the Fujian Provincial Science and Technology Department (2019H0023) and the Quanzhou City Science and Technology Program of China (2017G023). M.D.R. and R.M.C.R.R. acknowledge the Natural Sciences Research Institute (NSRI, Project CHE-20-1-02) of UP Diliman for financial support. Z.B.L., X.J.L., and E.Y. thank the Institute of Materials Research and Engineering, A*STAR, for financial support.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c05486.

  • XRD pattern of the Au@MgFe2O4 nanohybrids and absorption spectrum of the Au seeds (PDF)

  • Temperature of the Au@MgFe2O4 solution after 4 min of irradiation (MP4)

Author Contributions

∇ Equal author contribution. D.P.Y., M.D.R., and E.Y. designed the experiments. E.Q., X.C., Z.L., Y.L.W., and M.L. performed the experiments. E.Q., X.C., D.P.Y., M.D.R., R.M.C.R.R., Z.L., X.L., and E.Y. analyzed the data. D.P.Y., M.D.R., R.M.C.R.R., and E.Y. wrote the paper. All authors read and approved the final manuscript.

The authors declare no competing financial interest.

Supplementary Material

ao1c05486_si_001.pdf (183.3KB, pdf)
ao1c05486_si_002.mp4 (4.6MB, mp4)

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