Chinese Journal of Chemical Physics  2019, Vol. 32 Issue (3): 391-398

The article information

Bei-chen Duan, Peng-ping Xu, Zheng Guo, Qian-wang Chen
段北晨, 徐鹏平, 郭振, 陈乾旺
Mesoporous MnSiO$_3 $@Fe$_3 $O$_4 $@C Nanoparticle as pH-responsive T1-T2 Dual-modal Magnetic Resonance Imaging Contrast Agent for Tumor Diagnosis
具有介孔结构的MnSiO3@Fe3O4@C纳米粒子的制备以及作为pH响应的T1-T2双模MRI造影剂的研究应用
Chinese Journal of Chemical Physics, 2019, 32(3): 391-398
化学物理学报, 2019, 32(3): 391-398
http://dx.doi.org/10.1063/1674-0068/cjcp1805105

Article history

Received on: May 14, 2018
Accepted on: June 2, 2018
Mesoporous MnSiO$_3 $@Fe$_3 $O$_4 $@C Nanoparticle as pH-responsive T1-T2 Dual-modal Magnetic Resonance Imaging Contrast Agent for Tumor Diagnosis
Bei-chen Duana , Peng-ping Xua , Zheng Guob , Qian-wang Chena     
Dated: Received on May 14, 2018; Accepted on June 2, 2018
a. Hefei National Laboratory for Physical Sciences at the Microscale, Collaborative Innovation Center of Suzhou Nano Science and Technology, Department of Materials Science & Engineering, CAS High Magnetic Field Laboratory, University of Science and Technology of China, Hefei 230026, China;
b. Anhui Key Laboratory for Cellular Dynamics and Chemical Biology and School of Life Sciences, University of Science and Technology of China, Hefei 230026, China
Abstract: Mesoporous structured MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C nanoparticles (NPs) were prepared via a facile and efficient strategy, with negligible cytotoxicity and minor side efforts. The as-prepared MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs hold great potential in serving as pH-responsive $ T_1 $-$ T_2 $$ ^* $ dual-modal magnetic resonance (MR) imaging contrast agents. The released Mn$ ^{2+} $ shortened $ T_1 $ relaxation time, meanwhile the superparamagnetic Fe$ _3 $O$ _4 $ enhanced $ T_2 $ contrast imaging. The release rate of Mn ions reaches 31.66% under the condition of pH$ = $5.0, which is similar to tumor microenvironment and organelles. Cytotoxicity assays show that MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs have minor toxicity, even at high concentrations. After intravenous injection of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs, a rapid contrast enhancement in tumors was achieved with a significant enhancement of 132% after 24 h of the administration. Moreover, a significant decreasement of 53.8% was witnessed in $ T_2 $ MR imaging signal. It demonstrated that MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs can act as both positive and negative MR imaging contrast agents. Besides, owing to the pH-responsive degradation of mesoporous MnSiO$ _3 $, MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs can also be used as potential drug systems for cancer theranostics.
Key words: Magnetic resonance imaging    MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C    Dual-modal    Contrast agent    
Ⅰ. INTRODUCTION

The ultra-sensitive imaging is crucial for early diagnosis of cancer. Magnetic resonance (MR) imaging is a vital three-dimensional imaging technology means based on the relaxivity of protons within the tissues among common molecular imaging techniques, containing computed tomography (CT), positron emission tomography (PET), fluorescence optical imaging (FOI), ultrasonic imaging (US), and so on [1-3]. It can offer the three-dimensional reconstruction images of tissues at a high spatial/temporal resolution without penetration depth limitation as a non-invasive diagnostic tool [3-5]. It also has no radiation damage to normal tissues. Especially, MR imaging demands contrast agents (CAs) to improve the sensitivity of diagnostic imaging, due to the slight difference in relaxation time between pathological tissue and normal tissue. Generally, CAs make a diminished impact on relaxation time of protons to modulate the MR signal [6]. Two categories of materials were applied to enhance the signal-noise ratio of MR signals at present. Paramagnetic gadolinium (Gd)-based compounds acted as $ T_1 $ CAs to drop the longitudinal relaxation time of protons, which provided a positive contrast to enhance the resolution and sensitivity [7, 8]. Superparamagnetic iron oxide NPs with strong saturation magnetization were served as $ T_2 $ CAs to achieve histopathologic diagnosis [9]. Nevertheless, Gd-based MR imaging CAs pervasively have the drawback of inevitably intrinsic toxicity and short blood circulation time [10]. It caused great side effects on kidneys and other organs [11]. Superparamagnetic $ T_2 $ CAs take a negative contrast owing to lowered MR signal. Evident defacts of calcification, bleeding, and the susceptibility artifacts restricted the clinical application to certain extent [7, 12]. Taken together, all CAs now available have ineluctable defact and restrictions. Some dual-modal CAs were designed to improve the resolution and sensitivity of MR imaging through offering integrated imaging information simultaneously [13, 14]. It contributed to concurrently achieving accurate diagnosis ($ {T_1}^* $-weighted signal) and detection of lesions ($ {T_2}^* $-weighted signal) [15]. Since the MRI signal increases with the aggregation of CAs in abnormal tissues, there is a major trend for nano-sized contrast agents because of the enhanced permeability and retention effect. The pH-responsive NPs are beneficial to achieve specific imaging of tumor tissue owing to the common phenomenon of acidic tumor microenvironment [16]. With consideration of these, Mn$ ^{2+} $ with strong magnetic moment may be a great alternative. Since the relaxivity can be largerly increased in response to the interaction between the released Mn$ ^{2+} $ and binding proteins, Mn-based systems possess tremendous advantages on contrast amplification among all paramagnetic and magnetic contrast agents [17-19]. And paramagnetic Mn$ ^{2+} $ can play a role in shortening relaxation time of $ T_1 $ and $ T_2 $ at the same time. It is a valuable contrast agent for MR imaging [20-22].

In this work, we synthesized a superparamagnetic mesoporous structured MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs via a facile hydrothermal method, which can be served as a pH-responsive $ T_1 $-$ T_2 $$ ^* $ dual-modal CA to rapidly amplify MR signals for the precise imaging of tumors. The MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs are stable in the neutral normal tissue and blood circulatory system. And Mn$ ^{2+} $ ions released from the MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs under acidic conditions (lysosome/organelle) can effectively shorten the longitudinal relaxation of surrounding protons to increase the $ T_1 $-weighted MR imaging sensitivity. Superparamagnetic Fe$ _3 $O$ _4 $ acts on $ T_2 $$ ^* $-weighted imaging to enhance contrast, distinguishing the lesion sites from normal tissues. Besides, all the elements (Mn, Si, O, Fe, C) contained in this nanoplatform belong to the common components of human tissues, which greatly depress its potential risks to the human body. Our work demonstrates that MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs are a pH-responsive $ T_1 $-$ T_2 $$ ^* $ dual-modal MR imaging contrast agent and a potential drug carrier.

Ⅱ. Results and discussion A. Synthesis and characterization of MnSiO$_3 $@Fe$_3 $O$_4 $@C NPs

The preparation of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs was carried out through a safe and efficient route (see supplementary materials). Firstly, uniform and monodisperse SiO$ _2 $ was used to prepare superparamagnetic and carboxyl-functionalized SiO$ _2 $@Fe$ _3 $O$ _4 $@C nanospheres by a facile hydrothermal method. Through chemical etching, the solid state SiO$ _2 $ core of SiO$ _2 $@Fe$ _3 $O$ _4 $@C (20 mg) precursor was etched into mesoporous MnSiO$ _3 $ by adding 29.4 mg of Na℃$ _6 $H$ _5 $O$ _7 $$ \cdot $2H$ _2 $O and 34 mg of MnSO$ _4 $$ \cdot $H$ _2 $O [23]. Here the superparamagnetic Fe$ _3 $O$ _4 $@C can act on $ T_2 $$ ^* $-weighted MR contrast imaging to detect diseased tissue and Mn$ ^{2+} $ ions released from mesoporous MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs can shorten the longitudinal relaxation of water protons to increase MR imaging sensitivity. Besides, mesoporous MnSiO$ _3 $ can be used as a potential drug carrier for cancer theranostics. The morphology and particle size of the SiO$ _2 $ are not only related to the feed ratio of ammonia, TEOS, and deionized water, but also related to the reaction temperature and time. Uniform and monodispersed SiO$ _2 $ nanosphere is controlled by continuously adjusting the above experimental factors to keep a mean size of 12 nm. In order to ensure that the solid state SiO$ _2 $ core was completely converted to mesoporous MnSiO$ _3 $ after chemical etching, we have set up a series of control groups to determine the optimal experimental conditions. We first perform 6-group experiments to determine reaction time (4 h), then another 3-group experiments were performed to determine the reaction temperature (180 ℃).

FIG. 1 shows the SEM images of Fe$ _3 $O$ _4 $@C nanospheres and MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs synthesized under different experimental conditions, including different reaction time and different reaction temperature, respectively. Through the SEM image, it can be observed that all particles are uniform monodisperse spheres with basically same mean size. When reaction time was 4 h, there was no obvious damage to the outer carbon layer of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs. And the experimental temperature had no obvious effect on the morphology. When the reaction experiment was increased to 6 h, the outer Fe$ _3 $O$ _4 $@C layer was damaged by etching. Under the condition of 8 h, almost entire particles were etched seriously. There were numerous broken particles scattered around MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs. The high temperature also promoted the damage of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs. By analyzing the morphology of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs under different conditions, it was found that the optimal reaction time was 4 h. In order to further determine the synthesis temperature of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs, we set up another three sets of experiments. FIG. 2(a–f) exhibited SEM and TEM images in sequence. In view of SEM images, it was found that the morphology of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs was well intact and carbon layer was not significantly damaged under the three experimental conditions. By further observing TEM image of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs, it can be clearly observed that the MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs remain a portion of solid material under condition of 140 ℃, which corresponds to the SiO$ _2 $ incompletely etched. The shallower peripheral areas refer to mesoporous MnSiO$ _3 $, and the outer layer is the thin Fe$ _3 $O$ _4 $@C layer. When temperature increased to 160 ℃, the solid SiO$ _2 $ core was obviously reduced, but there were still some solid SiO$ _2 $ in the core. When temperature increased to 180 ℃, the dark SiO$ _2 $ core completely disappeared, demonstrating that solid SiO$ _2 $ has been completely transformed into mesoporous MnSiO$ _3 $, and that the outer Fe$ _3 $O$ _4 $@C layer has not been significantly damaged. Combining with SEM and TEM results, the best temperature and reaction time for preparing MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs are 180 ℃ and 4 h, respectively. Through TEM images (FIG. 2(h)), we can observe that Fe$ _3 $O$ _4 $@C layer was etched away, and that bare MnSiO$ _3 $ scattered around. It indicated that MnSiO$ _3 $ possess a mesoporous structure [24].

FIG. 1 SEM of (a) Fe3O4@C NPs and (b–d) MnSiO3@Fe3O4@C NPs prepared at 180 oC for 4 h, 6 h, and 8 h, respectively. SEM of (e) Fe3O4@C NPs and (f–h) MnSiO3@Fe3O4@C NPs prepared at 140 oC for 4 h, 6 h, and 8 h, respectively
FIG. 2 (a–c) SEM and (e–g) TEM of MnSiO3@Fe3O4@C NPs prepared under three experimental conditions at 140 oC for 4 h, 160 oC for 4 h, and 180 oC for 4 h, respectively. (d) SEM and (h) TEM of MnSiO3@Fe3O4@C NPs prepared at 180 oC for 8 h

In the light of the published crystal structure data, XRD patterns shown in FIG. 3(a) have demonstrated the presence of Fe$ _3 $O$ _4 $ and MnSiO$ _3 $ phases of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs [25, 26]. It can be seen from FIG. 3(a) that the diffraction peaks centered at 30.1°, 35.4°, 56.9°, and 62.5° correspond to the standard PDF card (JCPDS 19-0629) of the Fe$ _3 $O$ _4 $. The peaks at 30.1°, 33.8°, and 37.1°correspond well to strong peaks of the standard PDF of the MnSiO$ _3 $ phase (JCPDS 24-0735), and diffraction peaks at 55.9° and 58.2° also correspond to the standard diffraction peaks of the MnSiO$ _3 $ phase. It can be discovered that the crystallinity of MnSiO$ _3 $ is poor according to the diffraction peak, which is due to the mesoporous structure of MnSiO$ _3 $ formed by chemical etching. FIG. 3(b) shows the FT-IR spectra of Fe$ _3 $O$ _4 $@C nanospheres and mesoporous MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs. Blue line represented the mesoporous MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C and the black line represented Fe$ _3 $O$ _4 $@C. The absorption peak in the range of 1360–1720 cm$ ^{-1} $ is caused by the stretching of the C=C and the vibration of the C$ = $O, which root in the carboxyl functional group on the carbon layer [27, 28]. MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs have a distinct absorption peak at 1019 cm$ ^{-1} $, which corresponds to the Si–O of MnSiO$ _3 $. Raman spectroscopy (FIG. 3(c)) was also used to demonstrate the presence of MnSiO$ _3 $. Compared with the Fe$ _3 $O$ _4 $@C NPs, the prepared MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs have a new absorption peak at 645 cm$ ^{-1} $ in Raman spectrum, corresponding to Mn–O from MnSiO$ _3 $. The absorption band at 1590 cm$ ^{-1} $ comes from the carboxyl group linked on the outer carbon layer. This conclusion is in accord with FT-IR spectrum, and further testifies the presence of MnSiO$ _3 $ and the presence of vast carboxyl groups on the surface of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs.

FIG. 3 (a) X-ray diffraction pattern of MnSiO3@Fe3O4@C NPs, (b) FT-IR spectra, and (c) Raman spectra of MnSiO3@Fe3O4@C NPs and Fe3O4@C NPs

XPS spectra of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs are given in FIG. 4 to further determine the atomic valence of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C. The Mn 2p spectrum (FIG. 4(b)) shows two symmetrical peaks with binding energies of 642.2 and 654.3 eV, which correspond to Mn 2p$ _{3/2} $ and Mn 2p$ _{1/2} $ [29], respectively. The characteristic peaks with binding energies of 641.9 and 653.4 eV are attributed to Mn$ ^{2+} $ [30], while Mn$ ^{2+} $ in MnSiO$ _3 $ is easily oxidized to Mn$ ^{4+} $ in aqueous solution, corresponding to the characteristic peaks at the binding energies of 642.6 and 653.9 eV [29, 31]. The characteristic peaks with binding energy of 102.4 eV corresponds to Si 2p (FIG. 4(c)). By analysis of the Fe 2p spectrum (FIG. 4(d)), binding energies of 710 eV (Fe 2p$ _{3/2} $) and 724 eV (Fe 2p$ _{1/2} $) are attributed to the Fe$ _3 $O$ _4 $ connected to the carbon layer [34, 35]. Binding energies of 711.3 and 724.6 eV correspond to Fe$ ^{2+} $ of Fe$ _3 $O$ _4 $ [32, 33]. The C 1s spectrum (FIG. 4(e)) has three different components at binding energies of 284.5, 286.3, and 288.5 eV, corresponding to C–C, C–O, and C$ = $O, respectively [34]. The C$ = $O bond demonstrates the presence of carboxyl groups attached on carbon layer of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs. FIG. 4(f) shows the O 1s spectrum containing four components. The characteristic peaks of the binding energies at 532.6 and 530 eV are assigned to the O–Si bond and M–O, derived from MnSiO$ _3 $. Binding energy at 531.6 eV corresponds to the HO–Fe bond, derived from the interaction of iron ions with adsorbed water. The Fe–O bond at the binding energy of 530.8 eV originates from Fe$ _3 $O$ _4 $ [35].

FIG. 4 (a) XPS spectrum of MnSiO3@Fe3O4@C NPs. (b) XPS spectra of the Mn 2p, (c) Si 2p, (d) Fe 2p, (e) C 1s, and (f) O 1s
B. pH-responsive MR imaging

We performed the release experiment of Mn ions from Fe$ _3 $O$ _4 $@C@MnO$ _2 $ with different pH values of PBS by means of ICP-AES to demonstrate the acid-responsiveness of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs. Taking into account the blood environment and the tumor microenvironment, the pH of PBS was set to 7.4 and 5.0, respectively. After mechanical stirring at 37 ℃ for 24 h, ICP-AES was used to determine the concentration of Mn ions in the supernatant. We can find only a few Mn ions (4.54%) were released by MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs under the condition of pH$ = $7.4, and the Mn ions content reached 31.66% (FIG. 5(a)) under the same environment of pH$ = $5.0. It demonstrates that monodisperse MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs possess great acid sensitivity. The magnetic properties of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs were measured by an SQUID magnetometer at 27 ℃. The hysteresis loop (FIG. 5(b)) shows that the MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs and Fe$ _3 $O$ _4 $@C nanospheres all maintain typical super-paramagnetism. However, the saturation magnetization value ($ M_\rm{s} $) of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs is only 12.69 emu/g, which is significantly lower than the saturation magnetic induction (39.15 emu/g) of Fe$ _3 $O$ _4 $@C nanospheres. This is because inner mesoporous structure of MnSiO$ _3 $ performs non-magnetism. The $ M_\rm{s} $$ = $ 12.69 emu/g ensures that MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs enhance the effect of $ T_2 $ contrast imaging. And reduced $ M_\rm{s} $ is beneficial to the release of Mn ions in an acidic condition, which performs an enhancement contrast for $ T_1 $ imaging.

FIG. 5 (a) Mn2+ released from MnSiO3@Fe3O4@C NPs buffer solutions at pH=7.4 and 5.0, respectively. (b) Magnetic hysteresis loops of Fe3O4@C nanospheres and MnSiO3@Fe3O4@C NPs

On account of the above characterization analysis, MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs have an excellent acid sensitivity. Mesoporous MnSiO$ _3 $ releases abundant Mn ions under acidic environment, which enhance $ T_1 $$ ^* $-weighted imaging and improve imaging resolution. In order to confirm the effect of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs as $ T_1 $-$ T_2 $$ ^* $ dual-modal MR imaging contrast agent, we further explored how the pH value modulated the MR imaging contrast performance of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs with pH values of 7.4, 6.5, and 5.0, respectively (FIG. 6). The molar ratio of Mn to iron of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs is approximately 1.58$ : $1 by AES-ICP. The experimental groups with the addition of iron molar concentration from 0.00625 mmol/L to 0.2 mmol/L were used to verify the enhancement effect of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs on MR imaging. The corresponding concentration gradient of Mn ions was 0.01, 0.02, 0.04, 0.081, 0.161, and 0.322 mmol/L. From FIG. 6, it can be clearly seen that as the solution ion concentration increases, $ T_1 $$ ^* $-weighted MR imaging becomes brighter and $ T_2 $$ ^* $-weighted MR imaging becomes darker. In the neutral solution of pH$ = $7.4, MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs play a minor role in $ T_1 $$ ^* $-weighted imaging. The longitudinal relaxation $ r_1 $ is only 0.9778 (mmol/L)$ ^{-1} $$ \cdot $s$ ^{-1} $, and transverse relaxation $ r_2 $$ = $199.0553 (mmol/L)$ ^{-1} $$ \cdot $s$ ^{-1} $. When the solution environment became weakly acidic at pH$ = $6.5, the contrast effects of $ T_1 $$ ^* $-weighted MR imaging increased, $ r_1 $$ = $1.4597 (mmol/L)$ ^{-1} $$ \cdot $s$ ^{-1} $, $ r_2 $$ = $218.0718 (mmol/L)$ ^{-1} $$ \cdot $s$ ^{-1} $, $ T_1 $ imaging became brighter and $ T_2 $ imaging became darker with the same ion molarity. It demonstrates that MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs possess excellent pH responsiveness which can be used as both positive contrast agent and negative contrast agent. Mesoporous MnSiO$ _3 $ is corroded and degraded into Mn$ ^{2+} $ under acidic condition, resulting in an increase in the number of water molecules within the core. This result increases the interaction between Fe$ _3 $O$ _4 $ and water molecules, favoring the contrast agents to modulate the MR signal, thereby enhancing $ r_2 $. When the MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs are dispersed in an acidic solution (pH$ = $5.0) similar to tumor microenvironment, $ r_1 $$ = $2.372 (mmol/L)$ ^{-1} $$ \cdot $s$ ^{-1} $, $ r_2 $$ = $258.3328 (mmol/L)$ ^{-1} $$ \cdot $s$ ^{-1} $. It demonstrates that the synthesized MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs have excellent acid sensitivity in MR contrast imaging, so it can be used as a great pH responsive $ T_1 $-$ T_2 $$ ^* $ dual-mode MR imaging contrast agent. Furthermore, $ T_1 $ imaging contrast effect of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs under acidic environment is superior to other Mn-based MR imaging contrast agents, for example, Mn-doped mesoporous silica $ r_1 $$ = $0.76 (mmol/L)$ ^{-1} $$ \cdot $s$ ^{-1} $ [24], longitudinal relaxation of FeMn(SiO$ _4 $) being only 1.92 (mmol/L)$ ^{-1} $$ \cdot $s$ ^{-1} $ [26]. The mesoporous MnSiO$ _3 $ has the potential to become an excellent drug carrier.

FIG. 6 (a) T1- and (b) T2*-weighted MR images of MnSiO3@Fe3O4@C NPs at pH=7.4, 6.5, and 5.0. (c) Relaxation rate r1* vs. Mn concentration or (d) relaxation rate r2* vs. Fe concentration for nanospheres (relaxivity values r1 or r2 were obtained from the slopes of linear fits of experimental data)
C. Cytotoxicity test

The contrast agents demand for great biocompatibility, therefore careful evaluation of the cytotoxicity of the prepared MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs is crucial. HeLa cells were used to evaluate the cytotoxicity of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs, which were cultured with different concentrations (0, 12.5, 25, 50, 100, and 200 μg/mL) of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs and then detected by MTT colorimetry. As shown in FIG. 7, after incubating HeLa cells with 200 μg/mL MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs for 24 h, HeLa cells still showed a high cell viability of 87.21%. It indicates MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs have no significantly cytotoxicity because Mn$ ^{2+} $ released from MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs enters acidic organelles through endocytosis [36], excreted through the kidneys. In addition, Mn element is a basic element of the organism, MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs are usually stable in the neutral blood environment and normal tissues [37].

FIG. 7 In vitro cytotoxicity of MnSiO3@Fe3O4@C NPs to HeLa cell with concentrations of 12.5, 25, 50, 100, and 200 µg/mL, respectively
D. MR imaging $in $ $ vivo$

Tumor-bearing mice were randomly selected to be intravenously injected certain dose of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs (3 mg/kg Mn) to evaluate the effects of $ T_1 $-$ T_2 $$ ^* $ dual-modal MR imaging by a 3T MR scanner. According to the data of pre-injection and after administration of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C at 24 h collected from kindey, MR imaging contrast effects of the corresponding $ T_1 $ and $ T_2 $ imaging were obtained (FIG. 8(a)). It can be clearly observed that after 24 h of injection of contrast agent, the $ T_1 $ imaging shows a significant contrast enhancement and is obviously brighter, while $ T_2 $$ ^* $-weighted image is significantly darker because of the excellent acid sensitivity of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs (FIG. 8(b)). The cause for the phenomenon is that the particle size of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs is much larger than 10 nm. These NPs are usually absorbed by the liver kupffer cells and cleared by RES to vitro [38-40]. The Mn ions released by MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs under acid conditions are excreted by the kidneys. In consideration of the $ T_1 $-$ T_2 $$ ^* $ dual-modal MR imaging contrast agents, MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs have capability to shorten the relaxation time of water protons, and we quantified the imaging effect of a defined area of the tumor. Quantitative analysis displayed that the $ T_1 $ signal intensity increased approximately by 32% after intravenous injection 24 h compared with the pre-injection (FIG. 8(c)). Meanwhile, there was a decrease of $ T_2 $ signal intensity to 53.8% (FIG. 8(d)). It proved that MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs have a unique dual-mode MR imaging effect to enhance $ T_1 $-$ T_2 $ contrast imaging simultaneously and it can distinguish the tumors from the normal tissues easily depending on the sensitivity to tumor acidic microenvironments and $ T_1 $-$ T_2 $$ ^* $ dual-modal imaging ability, thereby improving the accuracy of cancer diagnosis. Meanwhile, the minor toxic and side effects of MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs can be neglected, which is different from the harmful Gd-based MR imaging contrast agents. In addition, mesoporous MnSiO$ _3 $ can be used as a potential drug carrier for cancer theranostics.

FIG. 8 In vivo (a) T1 and (b) T2 MR images of mice after intravenous administration of MnSiO3@Fe3O4@C NPs. (c, d) Quantification of relative signal intensity collected before and after administration of MnSiO3@Fe3O4@C NPs
Ⅲ. Conclusion

In summary, we have developed a facile and mild process for mesoporous structured MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs, which possess negligible cytotoxicity with mean size of 120 nm. The monodisperse SiO$ _2 $@Fe$ _3 $O$ _4 $@C precursor were etched using Na℃$ _6 $H$ _5 $O$ _7 $$ \cdot $2H$ _2 $O and MnSO$ _4 $$ \cdot $H$ _2 $O to transform the solid SiO$ _2 $ core to mesoporous MnSiO$ _3 $. Superparamagnetic Fe$ _3 $O$ _4 $ acts on $ T_2 $$ ^* $-weighted imaging to detect the diseased tissue. MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs release abundant Mn ions at the acidic tumor site. The released Mn$ ^{2+} $ ions shorten the longitudinal relaxation of water protons aiming to improve MR imaging sensitivity. In the neutral environment (blood circulation system/normal tissue), MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs remain stable. MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs possess excellent acid sensitivity, and the release rate of Mn ions in acidic PBS (pH$ = $5.0) is about 10 times than that under neutral conditions (pH$ = $7.4). Cytotoxicity assays demonstrated MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs have no significant toxicity. Through synergistic $ T_1 $-$ T_2 $$ ^* $ signals, evident distinction can be observed between the normal tissues and tumors in vivo MR imaging tests. As a conclusion, the prepared MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs can act as pH-reponsive $ T_1 $-$ T_2 $$ ^* $ dual-modal contrast agents. Moreover, owing to super acid sensitivity, mesoporous MnSiO$ _3 $ has the potential to be used as a drug carrier to release drugs at the diseased tissue. Manganese, iron, carbon, and silicon are common elements of human tissues, therefore, MnSiO$ _3 $@Fe$ _3 $O$ _4 $@C NPs are a safe $ T_1 $-$ T_2 $$ ^* $ dual-modal MR imaging contrast agent that can be used for accurate diagnosis of tumors. It has a certain significance for the development of cancer theranostics.

Supplementary materials: Experiment details are described.

Ⅳ. Acknowledgments

This work was supported by the National Natural Science Foundation of China (No.21571168). We thank Core Facility Center for Life Sciences of University of Science and Technology of China for imaging support.

EXPERIMENTAL SECTION

Materials: Ferrocene (Fe(C5H5)2), ≥ 98%), acetone (C3H6O, ≥ 99%), hydrogen peroxide (H2O2, 30%), ammonium hydroxide (NH3·H2O), tetraethyl orthosilicate (Si(OC2H5)4), dihydrate trisodium citrate (Na3C6H5O7·2H2O), hydrated manganese sulfate (MnSO4·H2O) were of analytic grade and purchased from the Shanghai Chemical Factory, China. All chemicals were used as received without further purification.

Synthesis of SiO2@Fe3O4@C: According to relevant literature reports, 18 experiments were conducted to prepare monodisperse SiO2 nanospheres through a sol-gel process. Then the SiO2 nanospheres were served as template for preparation of SiO2@Fe3O4@C NPs. In a typical experiment, 25 mg SiO2 nanospheres were added into 30 mL acetone, followed by adding 100 mg ferrocene and 0.5 mL H2O2. After stiring for 30 minites, the transparent solution was then transfered to a 50 mL Teflon-lined stainless autoclave and heated to 210 ℃ for 48 h. Finally, the products were collected through a magnet and washed three times with ethanol.

Synthesis of MnSiO3@Fe3O4@C NPs: Through chemical etching, the solid state SiO2 core of SiO2@Fe3O4@C precursor is etched into mesoporous MnSiO3. 20 mg SiO2@Fe3O4@C NPs dispersed in deionized water, followed by adding into 29.4 mg of Na3C6H5O7·2H2O and 34 mg of MnSO4·H2O. The solution were mixed by ultrasonication for 30 minutes, then transfered to stainless steel autoclave, heated to a certain temperature and heat for a suitable time.23 In order to ensure that the solid SiO2 core can be completely transformed into mesoporous structure of MnSiO3, three parallel experiments with different reaction temperatures (140, 160 and 180 ℃) and reaction times (4, 6 and 8 h) were performed to determine the optimal reaction condition.

Mn ions release experiment: The release of Mn ions from MnSiO3@Fe3O4@C NPs was surveyed at 37 ℃. Separately, 100 μg of MnSiO3@Fe3O4@C NPs were dissolved in phosphate buffer (PBS) at different pH values to make a suspension. PBS of pH = 5.0 (group 1) and pH = 7.4 (group 2) were used as release media to simulate tumor environments and lysosomes, normal tissues and blood, respectively. After the whole day, the precipitated nanoparticles were removed by centrifugation, and the concentration of released Mn2+ ions in the supernatant was measured by inductively coupled plasma-atomic emission spectroscopy (ICP-AES).

In Vitro MR imaging testing: The relaxation properties of MnSiO3@Fe3O4@C NPs in different pH values of PBS buffers (pH = 7.4, 6.5, 5.0) were measured at 25 ℃ by using the clinical magnetic resonance (MR) scanner (GE HDxt, 3.0 T). MnSiO3@Fe3O4@C NPs suspension with different iron concentrations of 0.00625, 0.0125, 0.025, 0.05, 0.1 and 0.2 mM were preliminarily used as the detection solutions. T1*-weighted MR images were obtained by using a saturation recovery spin−echo sequence (repetition time (TR): 100-4000 ms; echo times (TE): 10 ms). The T2* relaxation times were determined from amultislice gradient echo sequence (repetition time (TR): 120 ms; echo times (TE): 2-22 ms).

Cytotoxicity assays: The vitro cytotoxicity of MnSiO3@Fe3O4@C NPs was determined by a tetrazolium dye (MTT) assay on HeLa cells obtained from the American Type Culture Collection (Rockville, MD). Cells were inoculated in a Duelbecco's Modified Eagle Medium containing 96-well plates at 37 ℃ and under 5 % CO2. The wells were covered with a single cells and 10% fetal bovine serum was added at 100 g/ml streptomycin and 100 units/ml penicillin (Invitrogen). After cultured for 24 h, different concentrations of MnSiO3@Fe3O4@C NPs (12.5, 25, 50, 100, 200 μg/ml) were sequentially added to the culture medium. After incubation for 24 h, MTT solution was put into each sieve well for another 4 h incubation. The yellow MTT was oxidized to dark blue formazan crystals by the viable cells, and the absorbance of each well is measured using an ELISA to determine the viability of the cells.

In vivo MR imaging: The animal experiments were conducted in compliance with the Ethical Committee of the Experimental Animal Center of Medical University of Anhui, China and the Animal Care Committee of University of Science and Technology of China guidelines. Intracellular MR imaging experiments were conducted on a clinical magnetic resonance (MR) scanner (GE HDxt; 3.0 T) at 37℃. HeLa cell suspension (0.1 ml, 5*107 cells/ml) was injected subcutaneously into healthy female mice. After 15 days, MR imaging was performed on the tumor mice. T1*-weighted MR images were obtained by using a saturation recovery spin−echo sequence (repetition time (TR): 100-4000 ms; echo times (TE): 10 ms). The T2* relaxation times were determined from amultislice gradient echo sequence (repetition time (TR): 120 ms; echo times (TE): 2-22 ms).

Characterization: The powder X-ray diffraction (XRD) patterns were collected on a X-ray diffractometer (Japan Rigaku D/MAX-γA) equipped with Cu-Kα radiation (λ=1.54178 ). The morphology of the as-synthesized products was observed using a transmission electron microscope (TEM, Hitachi model H-800) and a scanning electron microscope (SEM, JEOL JSM-6700M). The FT-IR spectrum was obtained using a Magna-IR 750 spectrometer in the range of 400−4000 cm-1 with a resolution of 4 cm-1. The Raman spectrumwas taken on a LABRAM-HR Confocal Laser Micro-Raman spectrometer. XPS measurements were performed on a VGESCALAB MKIIX-ray photoelectron spectrometer with an Mg excitation source (1253.6 eV). The magnetic property of the product was evaluated by a superconducting quantum interference device (SQUID) magnetometer (Quantum Design MPMS XL-7) at room temperature.

Reference
[1]
W. Schima, A. Mukerjee, and S. Saini, Clin. Radiol. 51, 235(1996). DOI:10.1016/S0009-9260(96)80339-4
[2]
P. Caravan, J.J. Ellison, T.J. Mc Murry, and R.B. Lauffer, Chem. Rev. 99, 2293(1999). DOI:10.1021/cr980440x
[3]
Y. Chen, M. Li, Y. Hong, J.W. Lam, Q. Zheng, and B.Z. Tang, ACS Appl. Mater. Interf. 6, 10783(2014).
[4]
Y. Huang, L. Hu, T. Zhang, H. Zhong, J. Zhou, Z. Liu, H. Wang, Z. Guo, and Q. Chen, Sci. Reports 3, 2647(2013). DOI:10.1038/srep02647
[5]
M. Mahmoudi, V. Serpooshan, and S. Laurent, Nanoscale 3, 3007(2011). DOI:10.1039/c1nr10326a
[6]
Z. Zhen, and J. Xie, Theranostics 2, 45(2012). DOI:10.7150/thno.3448
[7]
H.B. Na, I.C. Song, and T. Hyeon, Adv. Mater. 21, 2133(2009). DOI:10.1002/adma.v21:21
[8]
Y.C. Lee, D.Y. Chen, S.J. Dodd, N. Bouraoud, A.P. Koretsky, and K.M. Krishnan, Biomaterials 33, 3560(2012). DOI:10.1016/j.biomaterials.2012.01.062
[9]
D. Liu, W. Wu, J. Ling, S. Wen, N. Gu, and X. Zhang, Adv. Funct. Mater. 21, 1498(2011). DOI:10.1002/adfm.v21.8
[10]
B.H. Kim, N. Lee, H. Kim, K. An, Y.I. Park, Y. Choi, K. Shin, Y. Lee, S.G. Kwon, H.B. Na, J.G. Park, T.Y. Ahn, Y.W. Kim, W.K. Moon, S.H. Choi, and T. Hyeon, J. Am. Chem. Soc. 133, 12624(2011). DOI:10.1021/ja203340u
[11]
M.A. Sieber, T. Steger-Hartmann, P. Lengsfeld, and H. Pietsch, J. Magn. Reson. Imaging 30, 1268(2009). DOI:10.1002/jmri.v30:6
[12]
H.B. Na, J.H. Lee, K. An, Y.I. Park, M. Park, I.S. Lee, D.H. Nam, S.T. Kim, S.H. Kim, S.W. Kim, K.H. Lim, K.S. Kim, S.O. Kim, and T. Hyeon, Angew. Chem. 119, 5493(2007). DOI:10.1002/(ISSN)1521-3757
[13]
H. Yang, Y. Zhuang, Y. Sun, A. Dai, X. Shi, D. Wu, F. Li, H. Hu, and S. Yang, Biomaterials 32, 4584(2011). DOI:10.1016/j.biomaterials.2011.03.018
[14]
Z. Zhou, D. Huang, J. Bao, Q. Chen, G. Liu, Z. Chen, X. Chen, and J. Gao, Adv. Mater. 24, 6223(2012). DOI:10.1002/adma.v24.46
[15]
W.S. Seo, J.H. Lee, X. Sun, Y. Suzuki, D. Mann, Z. Liu, M. Terashima, P.C. Yang, M.V. McConnell, D.G. Nishimura, and H. Dai, Nat. Mater. 5, 971(2006). DOI:10.1038/nmat1775
[16]
L.Q. Xiong, Z.G. Chen, M.X. Yu, F.Y. Li, C. Liu, and C.H. Huang, Biomaterials 30, 5592(2009). DOI:10.1016/j.biomaterials.2009.06.015
[17]
M.Z. Koylu, S. Asubay, and A. Yilmaz, Molecules 14, 1537(2009). DOI:10.3390/molecules14041537
[18]
S. Aime, S. Canton, S.G. Crich, and E. Terreno, Magn. Reson. Chem. 40, 41(2002). DOI:10.1002/(ISSN)1097-458X
[19]
G.Z. Zhao, G. Zhen, and Q.W. Chen, Chin. J. Chem. Phys. 31, 717(2018). DOI:10.1063/1674-0068/31/cjcp1805100
[20]
A.C. Silva, J.H. Lee, L. Aoki, and A.R. Koretsky, Nmr. Biomed. 17, 532(2004). DOI:10.1002/(ISSN)1099-1492
[21]
D.P. J. Pan, A.H. Schmieder, S.A. Wickline, and G.M. Lanza, Tetrahedron 67, 8431(2011). DOI:10.1016/j.tet.2011.07.076
[22]
D.A. Cory, D.J. Schwartzentruber, and B.H. Mock, Magn. Reson. Imaging 5, 65(1987). DOI:10.1016/0730-725X(87)90485-1
[23]
L.D. Yu, Y. Chen, M.Y. Wu, X.J. Cai, H.L. Yao, L.L. Zhang, H.R. Chen, and J.L. Shi, J. Am. Chem. Soc. 138, 9881(2016). DOI:10.1021/jacs.6b04299
[24]
Y.Q. Wang, G.Z. Wang, H.Q. Wang, C.H. Liang, W.P. Cai, and L.D. Zhang, Chem-Eur. J. 16, 3497(2010). DOI:10.1002/chem.v16:11
[25]
J. Chen, Z. Guo, H.B. Wang, M. Gong, X.K. Kong, P. Xia, and Q.W. Chen, Biomaterials 34, 571(2013). DOI:10.1016/j.biomaterials.2012.10.002
[26]
J. Chen, W.J. Zhang, M. Zhang, Z. Guo, H.B. Wang, M.N. He, P.P. Xu, J.J. Zhou, Z.B. Liu, and Q.W. Chen, Nanoscale 7, 12542(2015). DOI:10.1039/C5NR02402A
[27]
S. Wang, and T. D. Westmoreland, Inorg. Chem. 48, 719(2008).
[28]
K.X. Yao, and H. C. Zeng, Chem. Mater. 24, 140(2011).
[29]
Z.B. Lei, J.T. Zhang, and X.S. Zhao, J. Mater. Chem. 22, 153(2012). DOI:10.1039/C1JM13872C
[30]
A.P. Grosvenor, E.M. Bellhouse, A. Korinek, M. Bugnet, and J.R. Mc Dermid, Appl. Surf. Sci. 379, 242(2016). DOI:10.1016/j.apsusc.2016.03.235
[31]
D. Brousse, L. Bélanger, and J.W. Long, Electrochem. Soc. Interf. 17, 49(2008).
[32]
B.W. Zhang, H.W. Ni, R.S. Chen, W.T. Zhan, C. Zhang, R. Lei, and Y.X. Zha, Appl. Surf. Sci. 351. , 1161(2015).
[33]
J.X. Feng, H. Xu, Y.T. Dong, S.H. Ye, Y.X. Tong, and G.R. Li, Angew. Chem. Int. Edit. 55, 3694(2016). DOI:10.1002/anie.201511447
[34]
Y. Jiang, Z.J. Jiang, L.F. Yang, S. Cheng, and M.L. Liu, J. Mater. Chem. A 3, 11847(2015). DOI:10.1039/C5TA01848J
[35]
H. Tissot, L.F. Li, S. Shaikhutdinov, and H.J. Freund, Phys. Chem. Chem. Phys. 18, 25027(2016). DOI:10.1039/C6CP03460H
[36]
S.D. Conner, and S. L. Schmid, Nature 422, 37(2003). DOI:10.1038/nature01451
[37]
Y.Y. Yuan, D. Ding, K. Li, J. Liu, and B. Liu, Small 10, 1967(2014). DOI:10.1002/smll.201302765
[38]
J.H. Na, S. Lee, H. Koo, H. Han, K.E. Lee, S.J. Han, S.H. Choi, H. Kim, S. Lee, I.C. Kwon, K. Choi, and K. Kim, Nanoscale 8, 9736(2016). DOI:10.1039/C5NR06673E
[39]
M. Longmire, P.L. Choyke, and H. Kobayashi, Nanomedicine-UK 3, 703(2008). DOI:10.2217/17435889.3.5.703
[40]
B.C. Duan, D.D. Wang, H.H. Wu, P.P. Xu, P. Jiang, G.L. Xia, Z.B. Liu, H.B. Wang, Z. Guo, and Q.W. Chen, ACS Biomater. Sci. Eng. 4, 3047(2018). DOI:10.1021/acsbiomaterials.8b00287
具有介孔结构的MnSiO3@Fe3O4@C纳米粒子的制备以及作为pH响应的T1-T2双模MRI造影剂的研究应用
段北晨a , 徐鹏平a , 郭振b , 陈乾旺a     
a. 中国科学技术大学材料科学与工程系,中国科学技术大学功能纳米实验室,合肥 230026;
b. 中国科学技术大学生命科学学院,安徽省细胞动力学和化学生物学重点实验室,合肥 230027
摘要: 本文通过一个简单的、温和的方案制备了平均尺寸为120 nm,介孔结构的纳米粒子MnSiO3@Fe3O4@C.粒子的细胞毒性微小,可以用作T1-T2双模MRI造影剂.酸性条件下MnSiO3@Fe3O4@C释放出大量的Mn2+缩短T1弛豫时间,提高成像分辨率.超顺磁性的Fe3O4可以增强T2对比成像,检测病变组织.类似于肿瘤微环境/细胞器的酸性PBS(pH=5.0)中Mn2+的释放率达到31.66%,约为中性条件(pH=7.4)下的7倍.释放的Mn2+通过内吞作用被细胞摄取,经肾脏排出,细胞毒性实验表明,MnSiO3@Fe3O4@C具有低的细胞毒性,即使高浓度的200 ppm MnSiO3@Fe3O4@C对HeLa细胞的毒性也相对较小.对荷瘤小鼠静脉注射定量MnSiO3@Fe3O4@C后,可以观察到一个快速增强的对比成像,给药24 h后,T1MRI信号显著增强,达到132%,而T2信号则明显降低至53.8%,活体MR成像证明了MnSiO3@Fe3O4@C可以同时作为阳性和阴性造影剂.此外,得益于介孔MnSiO3优秀的酸敏感性,MnSiO3@Fe3O4@C可以作为一种潜在的药物载体,实现肿瘤的诊疗一体化.
关键词: 造影剂    双模    MR成像    MnSiO3@Fe3O4@C