Materials identification
This study aimed to prepare a new form of T2-weighted MRI contrast agent with good biocompatibility and high saturation magnetization. The layer structure can limit the space of the magnetic material and optimize magnetic efficiency. For this reason, coating layered ceramic materials, such as MTT with magnetic nanoparticles (such as FePt or iron oxide NPs) can increase the saturation magnetization and reduce the coercive force. The primary reason is that the NPs are subjected to uniaxial compression stress at a vertical angle to the MMT layer. The resulting mechanical stress causes the magnetic dipoles of the NPs to rearrange and makes them parallel to the direction of the compressive stress. Therefore, the saturation magnetization follows the magnetization direction and enhances the magnetic flux, as shown in Figure S1a. However, as a candidate for the T2 contrast agent, iron oxide has the drawback of being prone to corrosion and degradation in unpredictable biochemical environments. FePt NPs have good biocompatibility, high chemical and physical stability, and a low cost to increase the saturation magnetization via co-synthetic nanocomposites. They can be integrated with MMT to achieve a high adsorption performance of the drug. In this study, bulk MMT material was first processed via CTAB and ultrasound to produce a layered structure. To observe the surface morphology of CTAB-modified MMT, scanning electron microscopy (SEM) was used to evaluate the form of the particles (Figure S1b). It was determined from the low-magnification images that the MMT material is evenly distributed in a layered structure after modification. Since the CTAB polymer needs to be removed from MMT, the calcining process converts CTAB to CO2. After an evaluation with thermogravimetric analysis (TGA), it was confirmed that CTAB was dismissed at 500 degrees (Figure S1c). An X-ray powder diffractometer (XRD) was used to analyze the crystal structure of the MMT and CTAB-modified MMT, as shown in Figure S1d.
The measured data were then compared with the MMT standard map (JCPDS 29-1498). The comparison showed that the MMT modified by CTAB has higher characteristic diffraction peaks than the pure MMT at 8.9, 19.6, 34.9, and 61.7 degrees. This indicated that the MMT modified by CTAB is superior in crystallinity to pure MMT. In addition to the diffraction peaks of MMT, various other diffraction peaks can be seen, caused by impurities (quartz, muscovite, and feldspar). Functional groups on the surface of MMT and CTAB-modified MMT were analyzed via Fourier transform infrared spectrometry (FTIR). As seen in Figure S1e, after the surface modification of CTAB, the stretching of the Si-O bond results in a change in the absorption peak at 1033 cm− 1, and the absorption band is stretched at 2820 − 2960 cm− 1, corresponding to the C-H bond. The peak at 1440 cm− 1 on the MMR corresponds to the bending of the C-H bond, which indicates that long-chain alkyl groups are present in the CTAB of the CTAB-modified MMT.
This research uses a simple, easy-to-implement, safe and effective method to produce a FePt nanocomposite material with magnetic and adsorption properties. Since more than half of the biological components in the body are water, this work is also intended to improve the dispersibility and biocompatibility in a hydrophilic environment. The FePt nanocomposites for the experiment were prepared using a one-pot synthesis. The one-pot synthesis method is chosen because it is simple to use, has a large output, and is hoped to produce crystalline materials at low temperatures. It is thus expected that a multifunctional nanocomposite material with high adsorption properties and magnetic properties can be prepared (Fig. 1a and 1b). First, Figs. 1c and 1d show the structural analysis of FePt@MMT using SEM and Transmission Electron Microscopy (TEM). It can be seen that FePt NPs are sandwiched between the layered structures of the MMT. To optimize the concentration gradient of MMT modified for the FePt NPs, we chose 3.5, 7, 10.5, 14, and 17.5% for further evaluation. The crystal structure of the FePt NPs was analyzed using X-ray powder diffraction (XRD), as shown in Fig. 1e, compared to its standard map (JCPDS No. 43-1359). The prominent diffraction peaks of FePt NPs are located at 41.05, 47, 71, and 84 degrees with orientations of (111), (200), (202), and (311), respectively. Due to the lack of external energy, synthesized FePt NPs cannot overcome the activation energy of the ordered phase transition. This indicates that a chemically disordered fcc Fe-Pt structure with a (111) orientation is formed. With varying amounts of MMT in the FePt@MMT nanocomposite, the diffraction peak of MMT was observed at 8.9, 19.6, and 23.9 degrees at MMT concentrations greater than 7%, and it was concluded that the higher the proportion of MMT, the higher the peak intensity. FTIR was then used to analyze the surface functional groups of the FePt@MMT-CTAB nanocomposites. As shown in Fig. 1f, the functional groups are not much different from pure FePt NPs or CTAB-modified MMT. The change in the absorption peak at 1033 cm− 1 is the stretching of the Si-O-Si bond. Thus, as the proportion of CTAB-modified MMT increases, the absorption peak intensity also becomes more vigorous. The thermal stability of the FePt@MMT-CTAB nanocomposite was analyzed via TGA. As shown in Fig. 1g, the weight loss of FePt@MMT-CTAB nanocomposites before 100°C is mainly due to the evaporation of physically adsorbed water, while the weight loss at 240 − 400℃ increases with an increase in the proportion of MMT-modified CTAB. From the results, it can be inferred that the cleavage of the surfactant CTAB mainly causes the difference in weight loss. According to previous experimental results, the weight loss at 240 − 400°C also includes the decomposition of surface functional groups modified by the surfactant oleic acid. To confirm that the magnetization ability of the FePt material will not be affected by the interlayer of MMT to produce a shadowing effect, the catalytic efficiency of Pt can act as a catalyst in the presence of methanol, which causes reduction and oxidation peaks in the cyclic voltammetry (CV) curve according to Eq. 3.
An oxidation peak presence of methanol at 0.6 ~ 0.75 V, and methanol can then generate a Pt-CO bond on the surface after catalysis via Pt. For the induced potential by reduction, the peak of CO reduction to CO2 desorption occurs at 0.35 ~ 0.5 V. As seen in Figure S2a–S2f, the current density of the sample after adding montmorillonite increases proportionally with the amount added. It is presumed that the relative Pt content after adding montmorillonite is less than the initial concentration, thus resulting in a relatively lower density of the products. A potentiostat electrochemical analysis confirmed that different concentrations of MMT did not affect the reaction ability of FePt.
Magnetic characteristics measurement of FePt NPs and FePt@MMT
The magnetic properties of FePt NPs and FePt@MMT were measured using a vibrating sample magnetometer. The measurement conditions were at room temperature with an external magnetic field of 16000 Oe to -16000 Oe. As shown in Figs. 2a and 2b, its magnetic behavior is paramagnetic, and the amount of magnetization shows a trend of increasing with the addition of MMT-modified CTAB. The saturation magnetization of the FePt NPs is 14.67 emu/g, which rises to 24.54 emu/g after a combination with a 10.5% MMT sheet (FePt@MMT/10.5%). However, when the proportion of MMT increased to 14%, the saturation magnetization began to decrease. It is speculated that the reason may be that the influence of nonferromagnetic MMT has a higher total weight ratio than FePt NPs, resulting in a decrease in the saturation magnetization of FePt@MMT/14.5% to 18.37 emu/g and FePt@MMT/17.5% to 15.32 emu/g. Analyzing the material with a magnetic resonance imager is suitable for use as an MRI imaging agent. In this experiment, the size of the nuclear magnetic resonance field was 7T, samples of different concentrations were prepared in a 0.5% agar peptizer, and the spin-spin relaxation time (T2) of the material was measured using a nuclear magnetic resonance instrument. The material's transverse relaxation rate (r2) was then obtained by plotting the relaxation rate (1/T2) against the ion concentration. The FePt NPs and FePt@MMT/10.5% were first measured at concentrations of 0.01, 0.02, 0.04 and 0.08 mg/mL. As seen in Fig. 2c, the T2-weighted image darkens as FePt NPs increase, which means that the higher the concentration of nanoparticles, the better the contrast imaging effect. Then, T2 was converted to 1/T2. As shown in Fig. 2d, linear regression is performed on 1/T2, and r2 is 41.835 mg− 1s− 1mL, which indicates that Feplatin nanoparticles can shorten the transverse relaxation time and reduce the effect of magnetic resonance. The signal intensity can then be used as a contrast agent for MRI. Next, the FePt@MMT/10.5% nanocomposite was measured. The T2-weighted image darkens as the concentration of FePt@MMT/10.5% increases. This means that the higher the concentration of the nanocomposite, the better the contrast effect. Then, T2 was converted to 1/T2, as shown in Fig. 2e. Linear regression was performed on 1/T2, and r2 was obtained as 40.32 mg− 1s− 1mL. The r2 value did not change significantly. The reason for this is that, although FePt@MMT/10.5% sacrifices the number of magnetic nanoparticles, it is due to the saturation magnetization. This amount is higher than that of FePt NPs, so it will ultimately enhance the T2 imaging effect of MRI.
Magnetic hyperthermia analysis of FePt NPs and FePt@MMT
The high-frequency heater can be used to determine whether the FePt@MMT nanocomposite can be heated up in a short amount of time under the interaction of an external magnetic field to a temperature sufficient to kill cancer cells. The high-frequency heater used in this research has a magnetic field strength of 3.8 kA/m and an output frequency of 800 kHz. FePt@MMT was dispersed in deionized water at a ratio of 10 mg/mL. It can be observed that the heating rate of FePt NPs is the slowest. As the proportion of MMT increases, the heating rate of other FePt@MMT/10.5% nanocomposites also increases significantly, compared with other groups. However, since the detection limit of the thermal imager is only approximately 50 degrees, it cannot match the value obtained by the thermometer in Figs. 3a and 3b. Based on the thermal analysis of the magnetically processed FePt@MMT/10.5% through a thermal imager, and it can be seen that the temperature inside the cuvette rises sharply. Next, the increased temperature effect of the prepared magnetic FePt@MMT/10.5% is discussed. The slope of the change in the temperature recorded in a certain period is calculated, which is the degree of increased temperature per unit time in Figs. 3c and 3d. Then, Eq. 2 was used to calculate the magnetic nanocomposite material. The heating capacity (SAR) is shown in Table 1, where it can be seen that the greater the heating rate, the greater the slope of the sample, indicating that it can be heated to a higher temperature at the same time. We can determine that we have successfully prepared a magnetic FePt@MMT nanocomposite material that can quickly heat up to a temperature sufficient to kill cancer cells from the above results. It has a considerable biomedicine potential to kill cancer cells with magnetic hyperthermia.
FePt@MMT drug loading and evaluation experiments
An ultraviolet-visible light absorption spectrometer was used to measure the change in different dye concentrations after adding FePt@MMT to judge the ability of the sample to adsorb dyes (Figure S3a). The drug-carrying capacity of FePt@MMT was first determined through the dye adsorption test. We prepared 20 µg/mL of methylene blue (MB), 50 µg/mL of Congo red (CR) and 50 µg/mL of brilliant green (BG) and added 1 mg FePt NPs, FePt@MMT/10.5% and FePt@MMT/50%. After shaking and placing the samples at room temperature for 10 minutes, magnetic separation was used to remove the magnetic nanocomposite materials. It can be seen in the experimental results that the pure FePt NPs have almost no adsorption capacity for dyes (Figure S3b to S3d). In contrast, after adding the MMT layer to each dye, the adsorption percentage reached more than 50%, and the BG reached the highest value of 62.14%. Moreover, the adsorption percentage of FePt@MMT/10.5% reached as high as 65%. The highest is 76.07% of MB, as shown in Table S2. Compared with pure FePt NPs, MMT has more fragmented and smaller holes, contributing to an increase in the specific surface area and significantly increasing the efficiency and percentage of dye adsorption. However, suppose too much MMT is added to prepare the FePt@MMT/50% nanocomposites. In that case, the multilayer MMT may overlap to generate a thick clay layer without space interference from FePt particles and make it difficult for the system to carry dyes or other drug cargos (Figure S3b to S3d). The same experiment was also used to measure the chemotherapeutic drug MIT. MIT is an anthraquinone chemotherapy drug with a dark blue solution and anti-cancer effects on fast-growing and slow-growing malignant tumors. It can treat breast cancer, liver cancer, acute non-lymphatic leukemia, multiple sclerosis, etc. It is combined with prednisone for second-line treatment of hormone-insensitive prostate cancer with distant metastasis. In addition, MIT is a drug with fluorescent characteristics that can absorb light at 648 nm and has an emission wavelength of 695 nm (Figs. 4a and 4b). An ultraviolet-visible light absorption spectrometer can also obtain data that is consistent with the dye-loading analysis. The absorption efficiency of FePt@MMT/10.5% for MIT is close to 75% (Fig. 4c). In addition, it can be known from the confocal results that FePt@MMT-MIT is affected by magnetic force, and bringing drugs to the cells will cause cell apoptosis, so the number of cells seen under the field of view has decreased.
In vitro toxicity analysis with drug tracking and cell viability
This experiment was mainly designed to prove whether FePt@MMT-MIT can be guided by external magnetic forces in a short amount of time, with FePt@MMT-MIT entering cells and rapidly accumulating in the cytosol to improve the therapeutic effect. FePt@MMT-MIT was initially added to the cells at a 50 µg/mL concentration in a 12-well dish and immediately guided with a magnetic force. After approximately 12 hours, the cells were removed for observation. As shown in Fig. 4d, we used extra magnetic guidance to help FePt@MMT-MIT accumulate in Mahlavu liver cancer cells, indicating that magnetic induction of MIT drug accumulation can be used as a cumulative drug in specific tissues or a targeted approach for target locations. Moreover, this result was also evaluated by flow cytometry. Based on the cell gating to circle the cell region, FePt@MMT-MIT can be guided by a magnetic field to accumulate the drug in the cells actively.
Here we used two different types of liver cancer cell lines to confirm the therapeutic effect of the FePt@MMT-MIT nanosystem, namely, Mahlavu as an HCC model (tends to be malignant) and SK-Hep1 (tends to be benign) as a hepatic adenocarcinoma cell line. The 293T cell line was chosen as the normal cell control group. We first compared FePt and MMT, which had very low toxicity towards those cell lines (Figure S4a, S4b, and S4e). After combining these two materials into FePt@MMT nanocomposites with different hybrid ratios, the cytotoxicity remained in a state not significantly increased in either cell line (Figure S4c and S4d). At the same time, we also separately measured the cytotoxicity of MIT to these three types of cell lines to infer the concentration of IC50. In Figs. 4f and 4g, where the cytotoxicity results of FePt@MMT-MIT on Mahlavu and SK-Hep1 cells were evaluated. Related IC50 of FePt@MMT-MIT was measured by serial dilution, 83–250 mg/mL for Mahlavu and 27–83 mg/mL for SK-Hep1, in which the IC90 results show 250 mg/mL with cell proliferation suppression on SK-Hep1 cells due to it was more benign than Mahlavu.
Moreover, the 293T as the normal control group was also evaluated for the cytotoxicity in Figures S4f. The normal cell demonstrates more sensitive results after treating with FePt@MMT-MIT. The IC50 effect can be measured at a concentration of approximately 9–27 mg/mL.
In vivo T2-weight MRI and tumor inhibition analysis
An in vivo mouse experiment was conducted at the Genomics Research Center, Academia Sinica. We followed the protocol guidelines with our manager institute, Institutional Animal Care and Use Committee, to apply the protocol with passing number 18-03-1202. We optimized the dose for all different material concentrations of 10 mg/kg in the animal experiments. Only in the T2-weighted MRI data did we use two concentrations to compare the difference in contrast. In the T2-weighted MRI diagnostic test, FePt@MMT was separately injected into mice at different concentrations of 2.5 mg/mL and 10 mg/mL and then induced by a magnetic field to accumulate in the tumor tissue to demonstrate their magnetic characteristics. Clinical 7T MRI imaged mice and different views to analyze the liver contrast image, as shown in Fig. 5a (coronal slice at y = 0) and 5b (horizontal piece at z = 0). Based on the different segments of the body parts, the results in fraction 7 and fraction 8 demonstrate that FePt@MMT can be easily guided into liver tissue and actively accumulate in the tumor site under magnetic guidance in in vivo whole-body images of NOD-SCID mice and can be marked with a yellow circle to indicate the tumor site. After applying FePt@MMT, the T2-weighted MRI signal of the orthotopic HCC tumor became darker. Before injection, the appearance of the tumor area could not be observed under an MRI analysis, which means that the solid magnetic decay was attributed to FePt@MMT, and the dark field imaging of FePt@MMT was more evident at a concentration of 10 mg/mL, indicating that FePt@MMT is a prospective comparative reagent for orthotopic liver MRI contrast.
First of all, we used the Mahlavu cell line to observe various treatments in the mouse model. Mahlavu and SKHep1 cell lines have been confirmed to establish observable animal models in the NOD scid gamma (NSG) mouse system. The mice were sacrificed in the fifth week to ensure enough mice for statistical calculations, and the liver tissues were excised, as shown in Figs. 5c and S5a. In the following animal experiment analysis, we divided all the experimental groups into four groups: the control group, the MIT treatment group (drug only), the MFH treatment group (FePt@MMT with external magnetic treatment but without MIT), and the MFH + MIT group (FePt@MMT under external magnetic treatment). The first is the analysis and tracking of mouse Mahlavu tumor images within five weeks. An excellent therapy via MFH and MIT was demonstrated with a reduction in tumor size and weight by an almost sevenfold decrease can be evaluated under IVIS investigation in Fig. 5d (the IVIS average radiance values decrease from 4×106 to 3×104 p/s/cm2/sr).
Moreover, we compiled the IVIS system in mice every week and compared the original data of all the data in Figure S5a. The mouse group using only MIT caused local tumor shrinkage, while FePt@MMT material can effectively rely on the growth of tumors with the effect of MFH. Unlike the previous two groups, the FePt@MMT-MIT group can simultaneously inhibit tumor growth and prevent its metastasis from entering other tissues. After the sacrifice, a significant photon count decrease in the removed livers was distributed in the FePt@MMT-MIT group compared with the control, MFH treatment only, and MIT treatment only groups (Fig. 5e). The MIT + MFH group is more effective in suppressing tumors than the single-drug therapy (MIT and MFH) (about 125 mm3), and there is no significant difference in mouse body weight, as shown in Fig. 5f. Compared to weight gain in other groups, only the MIT group showed a slight weight loss, which the I.V. injection may cause, and these results support that FePt@MMT-MIT has no significant side effects on mice. (Fig. 5g). Kaplan-Meier survival curves of mice were also evaluated to track the health of the mice in situ. After 10 weeks, the control group mice no longer survived without any treatment. Compared with the MIT group, the MFH group and FePt@MMT-MIT group effectively inhibited the growth of Mahlavu cells and reserved the live number of mice from 9 to 12 (Fig. 5g).
Furthermore, the liver tumor tissues were image through the H&E staining. It is noteworthy that a clear cavity structure can be observed in the MFH group, which indicates tumor cells may cause necrosis by the heating effect and may be using as an alternative treatment for cancer and make it easier for chemotherapy-related drugs to destroy the remaining cancer cells. The relative curative effect after two weeks of treatment was shown in Fig. 5i. In addition, we analyzed different kinds of orthotopic liver cancer models, where SK-Hep1 was another treatment target. We only compare three different groups: the control, MIT group, and MFH + MIT group, to evaluate this nanosystem on other cell lines. The liver tissues were also removed from the mouse body, and tumor growth was assessed via IVIS (Figure S5b) and H&E staining (Figure S5c). We only tested three different groups (control, MIT, and MFH + MIT treatment groups). Tumor suppression ability was evaluated by the size of the four groups of tumors. The results support that the FePt@MMT-MIT nano-targeted drug package can be used as an MFH and MIT-based chemotherapy platform for targeting HCC.