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Abstract
Superparamagnetic nanoparticles have been widely used as contrast agents in magnetic resonance imaging (MRI). The combined use of multiple imaging modes can provide more accurate information for clinical diagnosis. In this paper, a MRI/fluorescence dual-mode imaging contrast agent was developed by a simple method. The method is to make the fluorescent carbon quantum dots (CDs) adsorbed on the surface of the magnetic composite with pore structure by ultrasonic dispersion. Replacing the traditional methods such as chemical bonding, the fluorescent material is coated on the surface of the composite material. The synthesized composite materials were characterized by the transmission electron microscopy method (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and vibration sample magnetometer (VSM). The results of TEM, FTIR and XPS showed that CDs were successfully coated on the surface of C60@Fe3O4 magnetic composite. The VSM results show that the composite material still maintains superparamagnetism. The cytotoxicity of the material on SMMC-7721 liver cancer cells was detected by the MTT method, and the biocompatibility of the material was verified. By observing the fluorescence distribution in the cell, it is proved that the composite material successfully enters the cell and produces fluorescence. Finally, through the analysis of T2-weighted imaging, it is found that the addition of materials results in an enhanced dark contrast compared to control cells. Therefore, the composite nanomaterials synthesized in this paper can be used as MRI/fluorescence dual-mode imaging contrast agents.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Medical imaging technology plays an important role in clinical medical practice. In terms of imaging methods, medical imaging technologies include optical imaging, thermal imaging, X-ray imaging, and magnetic resonance imaging [1–7]. In order to enhance the details of the image as much as possible, new methods and technologies need to be continuously explored.
The use of contrast agent in the image acquisition process is an effective method to enhance the image [8–10]. Contrast agents based on nanomaterials are widely used and very promising medical diagnostic tools. For example, magnetic nanoparticles are often used in nuclear magnetic resonance imaging [11]. N. Vilaca et al.developed a magnetic zeolite nanocomposite material (MZNC) [12]. The ability of MZNC as an MRI imaging probe has been confirmed and verified in 3T's MRI in vitro clinical experiments. Z. Mohammadi et al. [13] study synthesized and investigated the potential use of CoFe2O4 as a contrast agent in magnetic resonance imaging (MRI) by using a conventional MRI system. The synthesized CoFe2O4 has a high r2/r1 value and can be used as a T2-weighted contrast agent.Where r1 is the longitudinal relaxation efficiency and r2 is the transverse relaxation efficiency.
Various imaging technologies have their own advantages and disadvantages, and it is difficult to obtain comprehensive information of the human body by only one imaging method. High-resolution imaging technologies tend to have low sensitivity, while high-sensitivity imaging technologies have relatively low resolution [14]. For example, MRI has high resolution, but low sensitivity. The optical imaging mode cannot obtain quantitative information of deep tissues [15]. The combined use of MRI/fluorescence dual-mode imaging can solve the above problems and provide more accurate information for clinical diagnosis. Therefore, multi-mode imaging technology has received more and more attention [16].
Fluorescent quantum dot nanomaterials have the advantages of narrow spectrum, stable luminescence, and high yield [17]. The development of new high-resolution and highly sensitive nanoprobes has been rapid, MRI imaging is sensitive but resolution needs to be improved, and fluorescence imaging is limited in depth, so combining the two to develop dual-mode probes can combine the advantages.
The current preparation methods of MRI/fluorescence dual-mode imaging probes are mostly complicated. Su et al. [18] reported a nanoprobe Fe3O4@SiO2@GQD-FA/DOX with graphene quantum dots as the luminescent group, which was used for tumor targeted drug delivery and OI/MRI dual-mode imaging. GQD (C-GQD) with a carboxyl group on the surface is first combined with Fe3O4@SiO2; then the tumor targeting recognition molecule folic acid (FA) is coating on the surface of the nanoparticle; finally, the chemotherapeutic drug DOX is loaded onto the nanoprobe by π-π accumulation. Dai et al. [19] covalently bonded Eu(III) luminescent complex and tumor targeting recognition molecule folic acid (FA) to the surface of FeCoO magnetic nanoparticles, and prepared a multifunctional nano-organism with long-lived fluorescence and superparamagnetism. The probe PTTA-Eu3+-CoFeO-FA successfully achieved fluorescence imaging and T2 weighted magnetic resonance imaging detection of cancer cells in vitro and in vivo.
In this paper, a method of adsorbing carbon quantum dots on porous composite materials was designed. Through this simple approach, fluorescent carbon quantum dots were coated on the surface of C60@Fe3O4 magnetic particles to realize dual-mode imaging of fluorescence and magnetic resonance. It’s effects on liver cancer cells were studied. The characterization results show that the material has excellent magnetic properties, and the cell viability test shows that the synthesized material is non-toxic to cells. Most of these nanoparticles are endocytosed by cells. On the other hand, the material's ability to act as a T2-MRI probe and generate dark contrast enhancement has been verified in vitro. This paper investigates the preparation of C60@Fe3O4&CDs dual-mode probes and verifies the imaging properties of the nanomaterials by in vitro MRI and fluorescence imaging experiments on liver cancer cells.
2. Materials and methods
2.1 Materials
Human hepatocellular carcinoma cells (SMMC-7721) were purchased from Shanghai Institutes for Life Sciences, Chinese Academy of Sciences. Dimethyl sulfoxide (DMSO), m-phenylenediamine (m-PD) and cysteine were purchased from China Pharmaceutical Group Ltd. Fetal bovine serum (FBS) was purchased from Hangzhou Shiziqing Biological Company. Penicillin-streptomycin solution Nanjing Mike Ward Biological Co. DMEM high sugar medium was purchased from Nanjing Maclean Biotechnology Co.
2.2 Preparation of C60@Fe3O4&CDs
C60@Fe3O4 is synthesized by co-precipitation method. Adding hydroxylated C60 to the solution can cause C60 adsorb on the surface of Fe3O4. The specific preparation steps and material characterization results have been described in detail in our previous work [20].Add 50 mg of hydroxylated C60, 158 mg of FeCl2·4H2O, and 108 mg of FeCl3·6H2O in 100 ml of deionized water and add NaOH solution with a concentration of 0. 4 mol/L until the pH of the reaction solution is 12. Then put the solution in a water bath at a constant temperature of 50°C for 1 h to complete the crystallization. Finally, the generated magnetic particles were adsorbed with a magnet and washed repeatedly with ethanol and deionized water until the washing solution became neutral to obtain pure C60@Fe3O4. C60@Fe3O4&CDs are obtained by ultrasonically dispersing C60@Fe3O4 and CDs for 30 minutes. The CDs solution was prepared by the other member of our research group [21]. The work done in reference 18 was done to improve the dispersion of C60@Fe3O4, when the prepared particles do not have fluorescent properties. The carbon dots made in reference 19 do not possess magnetic properties and cannot be MRI imaging agents. The traditional method regarding MRI dual-mode imaging is achieved by chemical bonding, while in this paper the dual-mode probes are prepared by direct adsorption and used for fluorescence imaging and MRI.
2.3 Characterization of C60@Fe3O4&CDs
The automatic surface area analyzer (AutoPore Iv 9500) is used to test the pore structure of C60@Fe3O4. The microstructure of the sample is measured by field emission transmission electron microscope (TEM, JEOL2100F); Fourier transform infrared spectroscopy (FTIR, Thermo Scientific Nicolet iS5) is used to measure the functional groups and molecular structure of the samples. A vibrating sample magnetometer (VSM, LakeShore7404) was used to test the hysteresis loop of the sample at room temperature within ±1.5T. The X-ray photoelectron spectroscopy (XPS) equipment model is Thermo ESCALAB 250XI, the fine spectrum of Fe, C and O and the full spectrum of the sample are tested.
2.4 Characterization of C60@Fe3O4&CDs
Liver cancer cells (SMMC-7721) were purchased from Youze Biological Company. DMEM high-sugar medium (500 mL/bottle) and MTT (analytical pure) were purchased from Nanjing Macleans Biotechnology Co., Ltd. Microplate reader (Multiskan FC) were purchased from Thermo Fisher Scientific.
In order to evaluate the biocompatibility of C60@Fe3O4&CDs, the MTT method was used to detect its effect on the activity of liver cancer cells. SMMC-7721 liver cancer cells were seeded on a 96-well plate at a density of 2×104 cells per well. Then the cells were placed in the incubator and incubated for 24 h. Then the cells were treated with culture medium containing different mass concentrations of C60@Fe3O4&CDs (1.25-20 µg/mL) for an additional 24 h. The untreated cells were used as control. 10 µL of MTT solutions (5 mg/mL) were added to each well and continue to incubate for 4 h. Finally, 100 µL of DMSO was added to dissolve the blue-violet crystals, and a microplate reader was used to measure the absorbance of each well at a wavelength of 450 nm.
2.5 In vitro fluorescence of liver cancer cells
A DN3000 confocal fluorescence microscope from Leica, Germany, was used to achieve fluorescence imaging. Under aseptic conditions, SMMC-7721 cells stored in liquid nitrogen were resuscitated and stably cultured in an incubator with 37 °C, 5% CO2 to third generation. Then the cells in the logarithmic growth phase were selected and were digested and collected by centrifugation afterwards. Then fresh culture medium was used to prepare cell suspension of appropriate concentration. Then the cell suspension was inoculated 1 mL per well in a 24-well plate containing sterile cell slides, and it was placed in incubator for 24 hours. When the cell growth status is good, aspirate the waste culture medium, rinse thoroughly with PBS three times to ensure that there are no dead cells in each well. Then each well was replaced with fresh culture medium (5 µg/mL) containing C60@Fe3O4&CDs, and cultured for 24 h, aspirated the waste culture medium. The cells were rinsed again with PBS three times, fixed by adding 4% paraformaldehyde, and prepared for fluorescence imaging with Cytation5 imaging reader to observe the fluorescence distribution of carbon quantum dots in the cells. The excitation wavelength of the fluorescence microscope is 488 nm.
2.6 In vitro MRI of liver cancer cells
The instrument used for magnetic resonance imaging (MRI) is BAP 70/20 (BRUKER BioSpin). For in vitro imaging, the sample at different concentrations was incubated with SMMC-7721 cells for 4 hours. The specific experimental conditions for incubation are as follows: lyse the frozen cells in a water bath at 37°C and place them in a centrifuge tube with 2-3mL of culture medium. Place in a centrifuge at 1000rps/min for three minutes, discard the supernatant, add new culture medium and place in a new culture flask. Place in a 37°C, 5% CO2 incubator for recovery. When the cells occupy about 80% of the bottom of the flask, discard the used culture medium, rinse the cells with PBS three times, then add 1mL 0.25% trypsin digestion solution to completely submerge the cells and put them into the incubator to rest. After the cells became round, the digestion was terminated using 4Ml medium. Blow the cells to form a suspension, remove the suspension into a centrifuge tube, and centrifuge at 1000rps/min for 3min in the centrifuge. discard the supernatant, add 1ml of new medium and blow well, inoculate into a new culture flask at a ratio of 1:3, and place in a constant temperature incubator at 37°C with 5% CO2 for passaging. After incubation, the cells were washed 3 times with PBS, trypsinized and resuspended in 300 µL warm 2% agar, and transferred into PCR microtubes with a pipette. These microtubes were immediately watered for 30 minutes to solidify the gel, and then stored in the refrigerator until imaging analysis. For T2-weighted imaging, the following parameters were used: TE = 68 ms and TR = 4800 ms. Image analysis adopts MicroDicom viewer software, and the signal intensity is calculated by Matlab.
3. Results and discussion
3.1 Synthesis and characterization of C60@Fe3O4&CDs
Magnetic nanoparticles have a high specific surface area and a large number of dangling bonds, and they also have the attraction of magnetic poles and have a tendency to aggregate. Therefore, when used for imaging, magnetic particles need good water solubility, dispersibility, stability and biocompatibility to avoid agglomeration in organisms. Therefore, surface coating of magnetic nanoparticles is required. The surface coating of Fe3O4 with hydroxylated C60 is of great significance for improving these properties of Fe3O4 [20].
At the same time, after coated with C60, the surface of Fe3O4 has a pore structure.To determine the specific surface area and pore size distribution curves of porous materials by nitrogen cryosorption, the samples are generally first degassed by heating and evacuation to remove the impurity gases adsorbed on the surface. Then it is weighed and then placed in liquid nitrogen. At the temperature of liquid nitrogen, the amount of atmospheric gas adsorbed by the sample is measured at different preset pressure points to obtain the adsorption isotherm. Then the data are then processed by computer to calculate the specific surface area, pore volume, average pore size and pore size distribution from the adsorption isotherm. Figure 1(b) shows the nitrogen adsorption isotherm test. The category is type IV adsorption isotherm. The line type is convex in the low-pressure zone, capillary condensation in the high-pressure zone, and adsorption and desorption are not overlapped. It is suitable to use the BET formula to calculate the pore distribution of the sample. The calculated results of the pore distribution are shown in Fig. 1(c). The size of the mesopores is approximately distributed between 5–25 nm. This indicates that the fluorescent carbon quantum dots can be easily adsorbed on the surface of C60@Fe3O4. Therefore, the ultrasonic dispersion method was used to prepare C60@Fe3O4& CDs composite nanomaterials.
Fig. 1. (a) The schematic diagram of the composite material successfully entering the cell and generating fluorescence; (b) Nitrogen adsorption desorption curve of C60@Fe3O4; (c) pore distribution of C60@Fe3O4; (d) histogram statistics of particle size of the C60@Fe3O4&CDs; (e) TEM morphology of CDs; (f) lattice of CDs; (g) TEM morphology of C60@Fe3O4&CDs; (h) lattice of CDs; (i) FTIR of C60@Fe3O4&CDs and C60@Fe3O4; (j) VSM of C60@Fe3O4&CDs and C60@Fe3O4.
The schematic diagram of the composite material successfully entering the cell and generating fluorescence (Fig. 1(a)). In order to verify the composition of the product, Figs. 1(e) and 1(f) characterize the morphology and crystal lattice of the carbon dots, respectively. Figures 1(g) and 1(h) show the TEM characterization of C60@Fe3O4 coated with carbon dots. In Fig. 1(h), there are both the lattice fringe characteristics of Fe3O4 and the carbon dots, indicating that the carbon dots are successfully coated on the material. The area where the interplanar distance is 0.29 nm is Fe3O4 [22], and the area where the interplanar distance is 0.36 nm is similar to the carbon quantum dots in Fig. 1(f), indicating that this area is carbon dot. Therefore, it can be seen that the carbon dots were successfully adsorbed into the pores of the C60@Fe3O4. Figure1d shows the histogram statistics of particle size of the C60@Fe3O4&CDs in Fig. 1(g). Using ImageJ software to determine the size and distribution of particles. The C60@Fe3O4&CDs are well dispersed with an average diameter of 15.65 nm, as shown in Fig. 1(d).
In order to further characterize the functional groups of the sample, Fig. 1(i) shows the FTIR spectra before and after C60@Fe3O4 coated carbon dots. The spectrum shows a broad vibration peak near 3400 cm−1, which is caused by the hydroxyl group in the complex. The vibration peaks at 1095, 1385 and 1622 cm−1 are attributed to the C-O, C-O-H and C = C structures present in the complex.The C-O, C-O-H and C = C in C60@Fe3O4 come from hydroxylated C60 and the peaks of the C = C double bonds are more pronounced after modification of the CDs. The similarity between the two contrast bond positions is due to the fact that both carbon dots and C60 are carbon nanomaterials. The comparison of the peak strengths shows that the target product was obtained on the carbon dot coating. A small Fe-O vibration peak is shown in the region of 500–600 cm−1. Comparison of the C60@Fe3O4 and C60@Fe3O4&CDs spectra shows that the latter has a more pronounced C = C double bond peak, indicating the presence of carbon dots.
In order to verify the magnetic properties of the sample, Fig. 1(j) shows the comparison of the hysteresis loop before and after C60@Fe3O4 coated carbon dots. This is because after the experiments were performed for adsorption, a cladding layer was formed on the surface of Fe3O4@C60, so the degree of magnetization was reduced. The hysteresis loops of the samples are all typical S-shaped curves, indicating that it has superparamagnetism at room temperature, and the saturation magnetization of C60@Fe3O4 is slightly reduced after the carbon dots are coated. Its saturation magnetization is 69.5 emu/g and 53.3 emu/g, respectively.
Figure 2 shows the XPS spectra before (Fig. 2(a), b, c, d) and after C60@Fe3O4 coated carbon dots (Fig. 2(e), f, g, h). The full spectrum and Fe 2p spectrum of the two are basically the same. The survey scan (Fig. 2(a), e) confirmed the presence of the elements Fe, O and C in the sample. The sub-XPS spectrum of Fe 2p (Fig. 2(b), f) shows two peaks at 724.5 eV and 711.6 eV, corresponding to the Fe 2p 1/2 and Fe 2p 3/2 spin-orbit peaks of Fe3O4, which are consistent with the standard XPS spectrum of Fe3O4 [23]. The fitting curve of the C 1s peak of C60@Fe3O4 (Fig. 2(c)) contains three peak types of 284.8, 286.6eV and 288.4eV, which can be assigned to unoxidized C (C-C and C = C), C-O bond and C = O bond, respectively [24,25,26]. The C = O bond may be produced by the incomplete reaction of C60 during the hydroxylation process. After modifying the carbon dots (Fig. 2(g)), the C-O bond is significantly strengthened which is attributed to the presence of carboxyl and hydroxyl groups in the carbon dots. The fitting curve of the O 1s peak of C60@Fe3O4 (Fig. 2(d)) shows three peaks at 529.9, 530.5 and 532.9eV, respectively, corresponding to the Fe-O bond in ferroferric oxide, the C-O bond and O-H bond in hydroxylated C60. After modifying the carbon dots (Fig. 2(h)), the C-O bond and O-H bond are also significantly strengthened, indicating that the carbon dots were successfully coated on the surface of C60@Fe3O4.
3.2 In vitro toxicity
Since only in the mitochondria of living cells, MTT can be reduced to blue-purple crystals by succinate dehydrogenase, whereas dead cells cannot produce crystals. Therefore, the in vitro killing effect of C60@Fe3O4&CDs on liver cancer cell SMMC-7721 can be accurately detected. Liver cancer cells were incubated with C60@Fe3O4&CDs of different concentrations (0-2 mg/ml) for 24h. After 24 hours of culture in the presence of 2 mg/mL C60@Fe3O4&CDs, the cell viability was still >83%, as shown in Fig. 3. The results show that C60@Fe3O4&CDs has good biocompatibility and does not show serious cytotoxicity to liver cancer cells.
3.3 In vitro fluorescence/MRI imaging
The nanomaterials were prepared into a solution of 1 mg/ml and immersed in the liver cancer cells. The fluorescence imaging of the cells is shown in Fig. 4(a). The cells were successfully lit, and the fluorescence effect was significant, indicating that C60@Fe3O4&CDs can be taken up by tumor cells. Figure 4(b) is the corresponding bright field photo. Through comparison, it is found that each cell is successfully labeled, which proves the excellent fluorescence imaging ability of the material.
Fig. 4. (a) Fluorescence imaging map of CDs-stained cells; (b) Bright field photographs of CDs stained cells accordingly.(c) Fluorescence imaging map of C60@Fe3O4&CDs-stained cells; (b) Bright field photographs of C60@Fe3O4&CDs stained cells accordingly.
We found that T2 localization was effective in distinguishing labeled cells from unlabeled cells. Figure 5(a) shows the T2-MRI imaging of cells after the cells incubated with different concentrations of C60@Fe3O4&CDs. The higher the concentration of the sample, the darker the color after imaging. When the sample concentration is 0.1 mg/ml, 0.3 mg/ml, 0.6 mg/ml, the signal intensity decreases by 30.1%, 52.6%, and 29.8%, respectively. As the concentration of the sample increases, the T2-weighted image signal decreases significantly. Figure 5(b) shows the T2 values of the samples. For the control group and the samples added with three different concentrations of materials, the values of T2 were 15.1 ms, 10.2 ms, 2.5 ms and 2.0 ms, respectively, indicating that the r2 value increased with the increase of concentration of C60@Fe3O4&CDs. MRI imaging results show that our C60@Fe3O4&CDs is a suitable T2-weighted MRI contrast agent, which can greatly enhance the diagnosis of clinical diseases, thereby reducing the incidence of false positive diagnosis.
4. Conclusion
In summary, the synthesized C60@Fe3O4&CDs composite nanomaterials successfully coated CDs on the surface of C60@Fe3O4 through the adsorption effect of pore structure. The material still maintains superparamagnetism, and the saturation magnetization reaches 53.3 emu/g. MTT assay showed that for C60@Fe3O4&CDs with a concentration of up to 0.02 g/ml, it did not have much impact on cell viability. In vitro fluorescence imaging showed that liver cancer cells have the ability to internalize C60@Fe3O4&CDs. The composite material is suitable for fluorescent labeling of cells. The MRI characteristics of C60@Fe3O4&CDs labeled cells were studied in agar gel, which proved that the material has the ability to enhance the contrast of T2-MRI. Therefore, the composite nanomaterial synthesized in this study can be used as MRI/fluorescence dual-mode imaging contrast agent for high-resolution and high-sensitivity tumor imaging in vitro.
Funding
National Natural Science Foundation of China (81971655); China Postdoctoral Science Foundation (2018M641677); ShanXi Science and Technology Department (201805D131010).
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not currently publicly available but may be obtained from the authors upon reasonable request.
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