A cation-exchange controlled core–shell MnS@Bi2S3 theranostic platform for multimodal imaging guided radiation therapy with hyperthermia boost

Yuhao Li *a, Yun Sun *bcdef, Tianye Cao cdef, Qianqian Su g, Zili Li bcdef, Mingxian Huang a, Ruizhuo Ouyang a, Haizhou Chang a, Shuping Zhang a and Yuqing Miao a
aCollege of Science, University of Shanghai for Science and Technology, Shanghai 200093, China. E-mail: yhli@usst.edu.cn
bDepartment of Nuclear Medicine, Shanghai Proton and Heavy Ion Center, Shanghai 201321, China. E-mail: yun.sun@sphic.org.cn
cDepartment of Nuclear Medicine, Fudan University Shanghai Cancer Center, Shanghai 200032, China
dDepartment of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China
eCenter for Biomedical Imaging, Fudan University, Shanghai 200032, China
fShanghai Engineering Research Center for Molecular Imaging Probes, Shanghai 200032, China
gInstitute of Nanochemistry and Nanobiology, Shanghai University, Shanghai 200444, China

Received 4th April 2017 , Accepted 25th June 2017

First published on 26th June 2017

Overtreatment as a crucial modern medicine issue needs to be urgently addressed. Theranostic agents supply a unique platform and integrate multiple diagnosis and therapies to deal with this issue. In this study, a core–shell MnS@Bi2S3 nanostructure was fabricated via two step reactions for tri-modal imaging guided thermo-radio synergistic therapy. The mass ratio between the core and shell of the constructed MnS@Bi2S3 can be precisely controlled via cation exchange reaction. After surface PEGylation, MnS@Bi2S3-PEG nanoparticles exhibited excellent aqueous medium dispersibility for bioapplications. Based on the r1 and r2 relaxivity obtained from the MnS core and the strong near-infrared absorption and X-ray attenuation abilities of the Bi2S3 shell, the intratumoral injected MnS@Bi2S3-PEG can realize in vivo magnetic resonance, computer tomography, and photoacoustic tumor imaging under a single injection dose. Hyperthermia significantly boosts the efficacy of radiation therapy, showing synergistic tumor treatment efficacy. No obvious toxicity is monitored for the treated mice. Our study not only provides a new way to precisely construct the core–shell nanocomposite, but also presents a unique theranostic platform and unifies the solutions for the challenges related with high injection dose and overtreatment.


Overtreatment such as the repeated injection of an imaging probe and therapeutic drugs has recently attracted significant concern.1–3 Decreasing the injected dose of drugs (or probes) while retaining its effectiveness is beneficial for patients. One of the attractive advantages of nanoparticles is that they are a platform to integrate different functions in a particle without changing individual functions and thus can significantly decrease the dose of materials in the human body and reduce the side effects.4–7 Nanomaterials containing high-Z elements such as bismuth chalcogenides (Bi2M3, M = S, Se, and Te) have proved their effectivities in various applications for X-ray computed tomography (CT) imaging, photoacoustic (PA) imaging, photothermal therapy (PTT), and radiotherapy (RT).8–11 Moreover, the fabricated composite nanostructure not only can introduce different imaging and therapy functions, but also can dramatically decrease the interference between different functions while enhancing its effectiveness.12–16

Despite these exciting results reported in the last few years, different functions of reported multifunctional materials have affected the injection dose according to different imaging models and therapy types.17,18 Thus, the issues of controllable synthesis of nano-reagents, the synergistic theranostic effects, or the short-term and long-term toxicities should be carefully investigated as a whole to realize every function (diagnosis and therapy) and the lowest side effect with single injection.

Among bismuth chalcogenides, bismuth sulfide has been considered as an effective and flexible nanoplatform with minimal toxicity, which can be easily constructed to form composite nanostructures.19–21 For example, doping of Mn element into bismuth sulfide could provide T1 and T2-weighted magnetic resonance (MR) imaging function to improve the soft-tissue imaging resolution, which overcomes the limitations of CT imaging.22–24 CT imaging and RT function are mainly attributed to the strong X-ray attenuation ability of Bi that can reduce the X-ray penetrability to increase the CT imaging signal and concentrate the X-ray energy inside the tumor to enhance the RT curative effect.9,14 PA imaging diagnostic ability and PTT therapeutic functions were achieved due to high absorbance of bismuth sulfide in the near-infrared region (NIR) and high photothermal conversion efficiency.25–27 Via the introduction of PA imaging, the tumor microenvironment could be non-destructively and locally observed in real time while CT and MR imaging confirmed the tumor location. These three imaging methods in one nano-platform are complementary and can completely acquire the details of the tumor. The introduced PTT therapeutic function could supplement RT to enhance the therapeutic effect of the radiation-resistant tumor.13,28

Based on the abovementioned imaging and therapy methods, herein, we report a hydrophilic polyethylene glycol (PEG) surface-modified core–shell MnS@Bi2S3 nano reagent (MnS@Bi2S3-PEG, as shown in Scheme 1) that can serve as an efficient diagnostic and therapeutic combinational reagent. MnS core was first synthesized and utilized as a template. Then, Mn on the surface of the nanoparticles (NPs) could be exchanged with Bi to form a Bi2S3 shell. Under the controllable synthesis process, the thickness of the Bi2S3 shell can be precisely adjusted, and the ratio of Bi and Mn can also be controlled and balanced to realize different functions in one theranostic platform. In MnS@Bi2S3-PEG NPs, the MnS core was in charge of both T1 and T2-weighted MR imaging. Moreover, the Bi2S3 shell qualified intense X-ray absorption ability and broad absorbance in the NIR window, which could be implemented in CT imaging, PA imaging, as well as PTT and enhanced RT therapies functions. We believe that this theranostic platform can supply a nanostructure to combine the useful diagnostic and therapeutic functions together, with a single injection, through precise control of the ratio of different functionalized elements in one theranostic platform.

image file: c7nr02384g-s1.tif
Scheme 1 A schematic of the composite nanostructure MnS@Bi2S3-PEG for multimodal imaging and synergetic photothermia-radiation tumor therapy.

Results and discussion

The core–shell structure of MnS@Bi2S3 has been synthesized via two step reactions that are presented in Fig. 1a. First, the MnS core was synthesized by a solvothermal method.29 Under the condition of 1-octadecene (ODE) as a solvent, manganese oleate reacted with sulfur to form spheroidal MnS NPs. TEM images and corresponding energy dispersive spectrometer (EDS) maps are shown in Fig. 1b and Fig. S1 in the ESI. The MnS NPs with an average diameter of ca. 20 nm were highly dispersed in nonpolar solvents. The EDS data (Fig. S2) and elemental mapping images confirmed that the atomic ratio of Mn and S was close to 1[thin space (1/6-em)]:[thin space (1/6-em)]1. MnS exists in three distinct crystalline forms, α-MnS (cubic), β-MnS (defect spinel), and γ-MnS (layered structure), in which α-MnS is the most stable form.30 The synthesized MnS NPs were characterized by X-ray diffraction (XRD) to monitor their crystalline forms (Fig. S3). Compared to those of the cubic structure of MnS (PDF#65-2919, 2θ = 29.61°, 34.32°, 49.32°, 58.59°, 61.47°, 72.33°, and 82.56°), the peaks of the synthesized MnS were well-matched and could be attributed to the (111), (200), (220), (311), (222), (400), and (420) crystalline phases.29 According to the Bragg equation, crystal lattice spacing (d) could be associated with the data of 2θ. By investigating the high resolution TEM images (HR-TEM), a crystal lattice spacing of ca. 0.18 nm can be found, which is consistent with the corresponding (220) plane of MnS (d = 0.184 nm). All these results verified that the synthesized MnS was a stable cubic α-MnS.
image file: c7nr02384g-f1.tif
Fig. 1 (a) A schematic of the synthesis of MnS@Bi2S3-PEG. TEM images of (b) MnS NPs and (c) MnS@Bi2S3 nanocomposite structure (shell was constructed at 160 °C for 30 min). (d) An HRTEM image of MnS@Bi2S3. (e) Powder XRD pattern of MnS@Bi2S3 and XRD PDF patterns of α-MnS and Bi2S3. The corresponding MnS@Bi2S3 EDS element line (f) and map (g) scanning of Bi (red), Mn (green), and S (yellow). Mn(OA)2 for manganese oleate.

Subsequently, the synthesized MnS NPs were redispersed in ODE. Bismuth neodecanoate dissolved in oleylamine was injected into the reaction system at 160 °C and allowed to react for further 30 min. The reaction mixture turned from yellow-green to dark brown,11 which indicated that Bi2S3 shell was coated on MnS core NPs. As shown in Fig. 1c, the core–shell structure of the obtained MnS@Bi2S3 with the diameter of ca. 20 nm and shell thickness of ca. 2.1 nm was clearly monitored via TEM images. The high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) image shown in Fig. S4 further confirmed the core–shell structure of the synthesized products. By selecting the XRD results and comparing with the PDF data of MnS and Bi2S3 (PDF#43-1471), we clearly found that Bi2S3 diffraction peaks emerged together with the MnS peaks and verified that the NPs contained MnS and Bi2S3, as shown in Fig. 1e. The crystal lattice spacings of ca. 0.18 nm and 0.26 nm for the core and 0.28 nm for the shell were measured via the HR-TEM, as shown in Fig. 1d, which corresponded to the lattice planes of (111) and (200) for MnS and (221) for Bi2S3, respectively. The fast Fourier transform (FFT) image of MnS@Bi2S3 shown in Fig. S5 clearly verified that the obtained interplanar spacing was 0.301 nm, which corresponded to the MnS XRD (111) crystalline phases. Moreover, the obtained X-ray photoelectron spectroscopy (XPS) spectra shown in Fig. S6 characterized the existence of Bi, Mn, and S.31–33 Further, the EDS lines and mapping scan images (Fig. 1f and g) also specifically indicated that Bi2S3 was uniformly coated on MnS to form a core–shell MnS@Bi2S3 nanostructure.

To investigate the formation of the nanocomposite, the synthesis parameters were investigated, and the results are shown in Fig. 2. At the same reaction molar ratio (Mn[thin space (1/6-em)]:[thin space (1/6-em)]Bi = 1[thin space (1/6-em)]:[thin space (1/6-em)]0.5) and reaction time (30 min), reaction temperature was adjusted to 140 °C or 180 °C, the core–shell structure could also be fabricated, and the morphology showed a slight change. At the same molar ratio and identical reaction temperature (140 °C), the reaction time was prolonged from 30 min to 1 h. No obvious morphology change of the composite structure was monitored. However, when the diameters of MnS@Bi2S3 NPs and Bi2S3 shell thickness were monitored and determined, a clear trend was observed. When the reaction temperature was increased from 140 °C to 180 °C, the average diameters of the NPs decreased from ca. 20 nm to 18 nm, and the average shell thickness increased from ca. 1.9 nm to 3.1 nm (Fig. S7). Compared to the average diameters of the MnS NPs, at higher reaction temperatures, the average diameter of the core–shell structure reduced, and the surface of the MnS@Bi2S3 NPs became rough. Furthermore, as shown in Table S1, the EDS elemental analysis verified that the proportion of Bi in the final products became higher with an increase in Bi mass fraction from 27% to 57%. The acquired ICP-OES results, as shown in Table S2, were also in line with the EDS results. This obviously confirmed that the cation exchange reaction occurred between Bi and Mn, which was mainly driven and controlled by thermodynamics. This phenomenon has also been monitored in other sulfides and selenides involving CdS or CdSe NPs with the cation exchange of Pb2+, Ag+, Cu+, Cu2+, or Hg2+ cations.34–37

image file: c7nr02384g-f2.tif
Fig. 2 (a) Schematic and TEM images of the MnS@Bi2S3 nanostructures prepared at different reaction temperatures and time. (b) Particle size statistical figures of the MnS core and MnS@Bi2S3 composites obtained at diverse reaction temperatures (reaction time = 30 min).

On further investigating the atomic number change of dissimilar elements, it was observed that Mn exchanged three cations with two cations of Bi, and S anion retained the same atomic ratio. The core–shell formula ratio between MnS and Bi2S3 changed from ca. 11[thin space (1/6-em)]:[thin space (1/6-em)]1 to 5[thin space (1/6-em)]:[thin space (1/6-em)]2 on increasing the reaction temperature. These results were consistent with the phenomena of increased Bi2S3 shell thickness at higher reaction temperatures. All the abovementioned findings clearly guided us to conclude that the molar or the mass ratio of core and shell could be precisely controlled through the thermodynamic process-driven cation exchange reaction for adjusting the ratio of separate functional substances in one single platform. For further study, the core–shell MnS@Bi2S3 NPs were fabricated through the cation exchange reaction at 160 °C in 30 min

Then, the reported amphipathic PMHC18-mPEG2000 (Fig. S8) was decorated on the surface of MnS@Bi2S3 to form hydrophilic MnS@Bi2S3-PEG NPs.38 The structure of the synthesized PMHC18-mPEG2000 has been verified via1H NMR characterization (Fig. S9) as the surface ligand of the synthesized MnS@Bi2S3 NPs can be either the hydrophobic ODE or OAm. Based on the hydrophobic–hydrophobic interaction, the amphipathic PMHC18-mPEG2000 could be easily modified on the surface of NPs, and the hydrophilic PEG2000 part of PMHC18-mPEG2000 made NPs well dispersed in the aqueous system. After PEGylation, the obtained MnS@Bi2S3-PEG NPs could pass through the 0.2 μm millipore filter and well disperse in the aqueous medium such as saline, phosphatic buffer solution (PBS), fetal bovine serum (FBS) solution, etc. By checking its dynamic light scattering (DLS) consequence, the mean hydrated diameters of MnS@Bi2S3-PEG NPs were found to be ca. 136 nm, 131 nm, 140 nm, and 131 nm, and the corresponding zeta potentials were ca. 0 mV, −1.16 mV, −1.02 mV, and 0 mV in H2O, saline, PBS, and FBS solution, respectively (Fig. S10). No aggregation was observed in these simulated physiological solutions. The amphipathic PMHC18-mPEG2000 is a electric neutral polymer. Thus, the measured zeta potential of MnS@Bi2S3-PEG NPs in these solutions exhibited electric neutrality. The admirable water dispersibility and the stability in different simulated physiological solutions made MnS@Bi2S3-PEG a promising candidate for the following biotechnological applications.

The synthesized MnS@Bi2S3-PEG NPs contained the MnS core. Mn2+ with five unpaired 3d electrons has been taken into account to be a T1,T2-weighted MR imaging contrast agent. The corresponding MR images and the longitudinal relaxivity (r1) and transverse relaxivity (r2) of MnS@Bi2S3-PEG are shown in Fig. 3a–d. On increasing the concentration of MnS@Bi2S3-PEG, the T1-weighted images became brighter, whereas the T2-weighted images became more darker. In the Mn concentration range from 0 to 1.2 mM, the linear relationship between 1/T1 or 1/T2 and Mn concentration was fitted, and the corresponding r1 = 5.33 mM−1 s−1 or r2 = 24.08 mM−1 s−1 were determined, which were comparable to those of manganese oxide and manganese selenide NPs.13,23,39,40 Even MnS was coated with Bi2S3 to form a core–shell structure. Inner MnS still qualified T1 and T2-weighted MR imaging ability, which was similar to other magnetic NPs coated with Au, ZnS, and better than the Gd-contained NPs (Magnevist, 4.25 mM−1 s−1).41–44

image file: c7nr02384g-f3.tif
Fig. 3 (a) T1-Weighted MR images and (b) relaxation rates; (c) T2-weighted MR images and (d) relaxation rates. (e) CT images and (f) HU values. (g) PA images and (h) PA intensity of MnS@Bi2S3-PEG solutions with different concentrations. (i) UV-vis absorption spectra of MnS@Bi2S3-PEG (inset is the image of MnS@Bi2S3-PEG in H2O, 0.2 mg mL−1). (j) Photothermal images and (k) the temperature change curves of MnS@Bi2S3-PEG solutions with different concentrations after 808 nm NIR laser irradiation (power density = 800 mW cm−2).

The outer shell Bi2S3 was distinct from the inner MnS core, which contained high-Z Bi element. Compared to the commercialized iodine containing CT agent, the reported Bi qualified large X-ray attenuation coefficient, even larger than that of the Au element (Bi: 5.74 and Au: 5.16, I: 1.94 cm−2 kg−1 at 100 keV).45 Thus, MnS@Bi2S3-PEG could be used as a CT contrast imaging agent. As shown in Fig. 3e and f, the significant contrast enhancement in CT imaging was monitored with the increase in the Bi concentration. Hounsfield unit (HU) value also linearly increased, and the HU value was larger than 1000 HU when the Bi concentration was higher than 9 mg mL−1 at 70 keV energy. These observations clearly revealed that MnS@Bi2S3-PEG could be applied as a CT contrast imaging agent.

Based on the photoacoustic effect and photothermal conversion effect of the reported Bi2S3 NPs, we speculated that MnS@Bi2S3-PEG could qualify both functions. By investigating its ultraviolet-visible (UV-vis) absorption spectra (Fig. 3i), it was observed that MnS@Bi2S3-PEG NPs in H2O exhibited broad absorption ranging from UV-vis to NIR region and showed transparent chestnut brown color. Compared with the equivalent mass of MnS, the light absorption ability increased by the formation of the core–shell MnS@Bi2S3 structure (Fig. S11), and the increased linear absorption was monitored by the increased concentration of the MnS@Bi2S3-PEG NPs. PA signal was then acquired under different wavelengths of the laser light source ranging from 680 nm to 970 nm in nanoseconds, as shown in Fig. S12. With the increasing excitation wavelength, the average PA signal gradually decreased, and the trend was consistent with the light absorption ability. PA imaging was then acquired by exciting the light source in the NIR region (892 nm) to decrease the PA signal interference of hemoglobin, as shown in Fig. 3g and h. With an increasing concentration of the MnS@Bi2S3-PEG NPs from 0 to 1.8 mg mL−1, increased PA signal was observed with a direct ratio relationship, and PA imaging was also observed with an enhanced intensity. The increased PA signal could be easily distinguish from the background for further bioimaging application. Next, as shown in Fig. 3j and k, under the irradiation of 808 nm NIR laser (800 mW cm−2), the solution temperature of the MnS@Bi2S3-PEG solutions with different concentrations (0, 0.2, 0.4, 0.6, and 0.8 mg mL−1) rapidly increased, accompanied by the increase of irradiation time in first several minutes, and then tended to decrease. After irradiating for 10 min, the temperature increased between 12 and 34 °C for the concentration between 0.2 and 0.8 mg mL−1 (Fig. S13). In comparison, the temperature of pure water only increased by 3.2 °C. The study of photostability was also conducted. After 5 times of laser on–off cycles irradiation lasting for 50 min, no obvious photothermal convention decrease was monitored for the MnS@Bi2S3-PEG solution (Fig. S14), which presented good photostability. Therefore, MnS@Bi2S3-PEG NPs have remarkable potential to be used as a PA imaging and PTT therapeutic agent.

Furthermore, taking all the abovementioned physical properties into consideration, MnS@Bi2S3-PEG NPs could be used as a tri-modal imaging contrast agent. BALB/c mice bearing 4T1 tumor on the back-leg served as the in vivo model. For MR imaging, the superior spatial resolution of excellent soft-tissue morphological details could be monitored. Before and after the intratumoral injection of MnS@Bi2S3-PEG at the dose of 10 mg kg−1, the T1 and T2-weighted MR signals were obtained using a 7T small animal MR scanner. In Fig. 4a and b, T1 and T2-weighted MR images are shown in the coronal plane and transverse plane. After the intratumoral injection, a remarkable signal enhancement was observed in the tumor region using the T1-weighted model, whereas a dark signal in the tumor region was found using the T2-weighted model. The signal in the tumor region was improved in contrast, which could be distinguished. The signal comparison is shown in Fig. 4e. The signal in the T1-weighted MR images is more distinct than the signal in the T2-weighted MR images.

image file: c7nr02384g-f4.tif
Fig. 4 In vivo (a and b) MR images, (c) CT images, and (d) PA images and ultrasound B-mode of the 4T1 tumor mouse intratumorally injected with MnS@Bi2S3-PEG solutions. The corresponding comparison of the (e) MR signal, (f) HU values, and (g) PA signal intensity of the mouse 4T1 tumor region with or without intratumoral injection of the MnS@Bi2S3-PEG solutions.

For CT imaging, the imaging quality of bone is much better than that of the soft-tissues and viscera. However, 3D reconstruction capability of CT imaging could provide a guidance for clinical surgery. Via comparing the signals obtained before and after the intratumoral injection of MnS@Bi2S3-PEG (10 mg kg−1), an in vivo CT signal was gathered and reconstructed, as shown in Fig. 4c. The obvious signal enhancement was found in the tumor region, which could be distinguish from other soft-tissues. In Fig. 4f, no obvious change in the HU value was found in the bone, muscle, and viscera regions after the intratumoral injection of MnS@Bi2S3-PEG, whereas the HU value enhancement of ca. 6 times was monitored in the tumor region. The obvious change in the signal intensity in the CT imaging and the enhanced HU values in the tumor region clearly proved that they originated from the injected MnS@Bi2S3-PEG.

PA imaging was also applied for the imaging of the 4T1 tumor tissue, which not only provided a non-invasive detection method, but also supplied a high spatial resolution of regional tissue than the optical imaging method. Based on the strong NIR absorbance after the intratumoral injection of MnS@Bi2S3-PEG (10 mg kg−1), a strong PA signal in the tumor group was monitored, whereas the control group was injected with saline and showed a weak PA signal (Fig. 4d). The acquired PA imaging was colocalized by the B-mode ultrasound image, which verified the PA signal increase in the tumor region. In Fig. 4g, the comparison of PA signals directly revealed that the monitored PA signal was almost twice that of the control group.

Based on the abovementioned imaging results, the synthesized MnS@Bi2S3-PEG could be applied as tri-modal imaging contrast agent in T1, T2-weighted MR imaging, CT imaging, and PA imaging. These imaging methods supplied different valuable information including anatomic information, spatial information, and detailed and localized information. Therefore, the designed MnS@Bi2S3-PEG could be used for the precise diagnosis of multi-modal tumors.

To further investigate the bioapplication of MnS@Bi2S3-PEG, 4T1 cells were incubated with MnS@Bi2S3-PEG at the concentrations ranging from 20 to 200 μg mL−1 for 24 h. No observable adverse effects were monitored on the viabilities of the 4T1 cells, which were evaluated by the cell counting kit-8 (CCK-8) assay (Fig. 5a). Then, 4T1 cells were incubated with MnS@Bi2S3-PEG at the same concentration and irradiated with 808 nm laser at the power density of 800 mW cm−2 for 5 min. In Fig. 5a, a positive cancer cell killing effect was monitored. With the increase of MnS@Bi2S3-PEG NPs concentration, the cell viability considerably decreased. Fluorescence imaging of the cells supplied a visualized imaging to investigate the PTT effect in vitro. Hoechst 33342 (weak and blue color) and propidium iodide (PI, red color) have been used to co-stain cells for the distinction of live cells, apoptotic cells, and dead cells, as shown in Fig. 5b and c. Under the same irradiation conditions, the number of dead cells (red) at higher NP concentrations was much higher, and the amount of apoptotic cells also increased (strong blue color). The cells incubated with only PBS group (control), cells incubated with only MnS@Bi2S3-PEG group (MnS@Bi2S3-PEG), and cells incubated with only irradiated laser group (laser) were also studied to prevent the interference. Clear cell staining was monitored only in the treatment group, indicating that the PTT effect could directly induce the cell death and apoptosis.

image file: c7nr02384g-f5.tif
Fig. 5 (a) Relative viabilities of 4T1 cells after incubation with different concentrations of MnS@Bi2S3-PEG solutions without or with being exposed to the 808 nm laser. Fluorescent images of the Hoechst (weak or strong blue) and PI (red) co-stained cells for (b) incubation with different concentrations of MnS@Bi2S3-PEG solutions + laser, and (c) PBS control, MnS@Bi2S3-PEG solutions (0.1 mg mL−1), laser, and combined MnS@Bi2S3-PEG solutions (0.1 mg mL−1) + laser groups. (d) Fluorescence images of γ-H2AX and DAPI co-stained 4T1 cells treated with the PBS control, MnS@Bi2S3-PEG (0.1 mg mL−1), X-ray alone, MnS@Bi2S3-PEG (0.1 mg mL−1) + X-ray (4 Gy). (e) Clonogenic survival assay of 4T1 cells treated with or without MnS@Bi2S3-PEG (0.1 mg mL−1) under a series of X-ray radiation doses at 0, 2, 4, and 6 Gy (808 nm laser at the power density of 800 mW cm−2 and irradiation to 4T1 cells for 5 min).

Previous studies revealed that Bi element qualified the intense X-ray attenuation ability.8,9 We speculated that whether MnS@Bi2S3-PEG could be utilized to enhance the radiotherapy effect. Amongst the distinctive markers of deoxyribonucleic acid (DNA) double strand break (DSB), one of the most well characterized method is the γ-phosphorylation of the histone H2AX (γ-H2AX). It is widely accepted that a γ-H2AX focus indicates the presence of DNA DSB. Therefore, 4T1 cells were treated with PBS (control group), only with MnS@Bi2S3-PEG (MnS@Bi2S3-PEG group), only with X-ray (4 Gy, X-ray group), and with MnS@Bi2S3-PEG + X-ray (4 Gy, MnS@Bi2S3-PEG + X-ray group). After different treatments, immunofluorescent labeled γ-H2AX method was utilized to investigate the DSBs of DNA in the cell nucleus. As shown in Fig. 5d, obvious DSB was observed after 4 Gy X-ray irradiation, which was compared with the cells treated with only NPs and only X-ray irradiated groups. Then, as shown in Fig. 5e, the clonogenic survival assay was also explored. The results proved that the reduced viable cell colonies occurred with the increased X-ray irradiation dose. The relative biological effectiveness (RBE) value could be used for investigating the RT enhancement effect. Depending on the formula of RBE10 (RBE10 = dose at 0.1 survival fraction of X-ray control group cells/dose at 0.1 survival fraction of Mn@Bi2S3-PEG + X-ray group cells), the calculated RBE10 value is 1.30. It has been suggested that Bi-contained MnS@Bi2S3-PEG exhibited robust RT enhancement effect.

Based on the PTT and RT therapy effect in vitro, we wanted to use MnS@Bi2S3-PEG for PTT and RT treatment in vivo. After anesthetizing the mouse bearing 4T1 tumor, MnS@Bi2S3-PEG was intratumorally injected. Mouse was then irradiated by 808 nm laser at the power density of 800 mW cm−2. During the irradiation process, an infrared imaging device was used to obtain the thermal images. As shown in Fig. 6a and b, the temperature in the tumor region quickly increased from ca. 27 °C to 40 °C within 2 min. The increased temperature further reached 48 °C within 5 min that was maintained for 5 min. In the control group, mouse was injected with PBS and then irradiated under the same condition; no significant photothermal effect was monitored.

image file: c7nr02384g-f6.tif
Fig. 6 (a) IR thermal images of 4T1 tumor-bearing mice without or with intratumoral injection of MnS@Bi2S3-PEG under the 808 nm laser irradiation taken at different time intervals. (b) Temperature changes of the tumor regions were monitored by the IR thermal camera during laser irradiation. The mice tumor volume growth curves (c) and the mice relative survival rate (d) for various treatment groups (five mice for each group). The dose of MnS@Bi2S3-PEG was 10 mg kg−1, PTT was conducted by the 808 nm laser at 800 mW cm−2 for 10 min, and the irradiation dose of RT was 4 Gy. (e) H&E and TUNEL staining of the tumor slices after 2 d various treatment (scale bar = 200 μm).

Then, the efficacy of the combined PTT and RT therapy was evaluated. Mice bearing 4T1 tumors were randomly separated into seven groups including the control group (1) without any treatment, only MnS@Bi2S3-PEG group (2), only laser group (3), only X-ray group (4), MnS@Bi2S3-PEG + laser group (5), MnS@Bi2S3-PEG + X-ray group (6), and MnS@Bi2S3-PEG + laser + X-ray group (7). Each group included 5 mice, and MnS@Bi2S3-PEG was intratumorally injected. The 808 nm laser at the power density of 800 mW cm−2 for 10 min was used for the PTT therapy. The X-ray dose was 4 Gy for the RT treatment. The therapy was replicated every two days. The relative tumor volume and relative survival rate reflected the synergistic therapy effect, which is shown in Fig. 6c and d. No significant therapeutic effect was observed in group 1 to 4. After injecting MnS@Bi2S3-PEG, the efficacy of PTT and RT in group 5 and 6 partly enhanced. As expected, the synergistic PTT and RT treatment under the injection of MnS@Bi2S3-PEG in group 7 presented remarkable inhibition of tumor growth and prolonged life-span for more than 36 days. The result is greater than that of the other control groups.

To further confirm the anti-tumor effects of different treatments, hematoxylin and eosin (H&E) staining and terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining were used to check the tumors (Fig. 6e). Herein, two days after different treatments, H&E stained tumor slices revealed that the tumor cells were significantly damaged in group 7 (MnS@Bi2S3-PEG + laser + X-ray), whereas the tumor tissues in other groups were undamaged or only partially destroyed. TUNEL staining images of tumor slices also showed the most significant damage in group 7, and the partial ruin in group 4–6, which clearly stated that the DNA had broken in the separate treatment process and inhibited or eliminated the enlarged tumors. All these results illustrated that the combined PTT and RT treatment qualified for the synergistic therapeutic effects.

Finally, it was important to investigate the toxicity of the new nanomaterial for possible bioapplication. To further estimate the biosafety of MnS@Bi2S3-PEG, we carried out the H&E staining study of diverse organs including heart, liver, spleen, lung, and kidney. Different organs of healthy mice were acquired after intravenous (i.v.) injection of MnS@Bi2S3-PEG (20 mg kg−1) at 24 h or 21 d. Note that the mouse injected with PBS (200 μL) was used as a control. Based on the obtained H&E staining of different organs at 24 h or 21 d, as shown in Fig. 7 and S15, compared to the H&E sections of the control organs, no obvious difference and damage was observed in the 100× and 400× magnified images. Serum biochemistry assays and complete blood panel tests were also carried on MnS@Bi2S3-PEG NPs (20 mg kg−1) i.v.-injected mice at 24 h and 21 d (Fig. S16). Herein, four function indeces about alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) were measured to be normal, which indicated that no detectable dysfunction of liver or kidney was induced by the MnS@Bi2S3-PEG NPs. Other various vital hematology marker indeces were then measured that fell well within the normal ranges for the MnS@Bi2S3-PEG-injected mice.46 These results collectively evidenced that MnS@Bi2S3-PEG qualified for lower toxicity, and no detectable side effects for mice were observed.

image file: c7nr02384g-f7.tif
Fig. 7 The H&E stained slices of different organs for a control group, and intravenously (i.v.) injected MnS@Bi2S3-PEG (20 mg kg−1) after 24 h or 21 d including 400× magnifications.



All chemicals were obtained from commercial sources: manganese chloride (MnCl2·4H2O, 99%, J&K), sodium oleate (98%, J&K), 1-octodecene (ODE, 90%, Acros), sulfur (99.99%, Aladdin), oleylamine (OAm, 70%, Aldrich), and bismuth neodecanoate (Aldrich). All chemicals were used as received.

Material characterization

TEM and EDS data were obtained using an FEI Tecnai F20 S-Twin. XRD was conducted via a Bruker D8 advance X-ray diffractometer using the Kα line of a Cu source. XRD patterns were compared with powder diffraction files (PDF's) from the ICDD database. XPS data of samples were acquired under Ar and investigated using a Thermo Scientific K-Alpha system (Al Kα, = 1486.6 eV). ICP-OES data was obtained using the ThermoFisher iCAP 7200 ICP-OES. UV-vis spectra were obtained using a Hitachi U-2910. Photothermal images were acquired using the FLIR E60.

Synthesis of manganese dioleate [Mn(OA)2]

Mn(OA)2 was synthesized according to a method reported in literature with some modification.47,48 In a typical synthesis of Mn(OA)2, 40 mmol MnCl2 and 120 mmol sodium oleate were dissolved in the mixed solvent composed of 80 mL ethanol, 60 mL H2O, and 140 mL cyclohexane. The resulting solution was heated to 70 °C and kept at that temperature for 4 h. When the reaction was complete, the upper organic layer containing the Mn(OA)2 was washed three times with 30 mL H2O. Then, the residue was removed from the solvent to afford a waxy solid.

Synthesis of MnS NPs

MnS nanoparticles were synthesized according to a method reported in literature with some modification.30 Typically, 2.5 mmol Mn(OA)2 and 5 mmol S were dissolved in 50 mL 1-octodecene. The resulting solution was subjected to vacuum–argon cycles and further degassed for 15 min at 120 °C. Then, the mixture was heated to 310 °C under argon protection and reacted for 45 min. After being cooled down to room temperature (r.t.), the MnS nanoparticles were precipitated by the addition of 50 mL ethanol and obtained by centrifugation. The obtained MnS NPs were re-dispersed in cyclohexane for the next step.

Synthesis of MnS@Bi2S3 NPs

In general, 0.5 mmol MnS NPs dispersed in cyclohexane were added to 9 mL 1-octodecene. The mixture was heated to 120 °C to remove cyclohexane and further degassed for 30 min at 120 °C under vacuum. Then, the argon-protected mixture was heated to the desired temperature (140 °C, 160 °C, or 180 °C). A solution containing 0.25 mmol bismuth neodecanoate dissolved in 1 mL OAm was injected into the abovementioned mixture and then allowed to react for 30 min. The mixture turned from dark green to dark brown. After the reaction completed, the MnS@Bi2S3 NPs were obtained by centrifugation and then washed with ethanol. The obtained MnS@Bi2S3 NPs were re-dispersed in cyclohexane for the next step.

Fabrication of the PMHC18-mPEG2000 coated MnS@Bi2S3 NPs (MnS@Bi2S3-PEG)

PMHC18-mPEG2000 was synthesized according to the literature reported method and the core–shell MnS@Bi2S3 NPs coated with ODE or OAm ligands were modified through the PMHC18-mPEG2000 polymer coating method.38 Generally, 5 mg MnS@Bi2S3 NPs dispersed in cyclohexane were added to 5 mL chloroform solution containing 5 mg PMHC18-mPEG2000. The resulting mixture was stirred for 30 min. Then, the solvents were removed under vacuum. The residue was re-dispersed in H2O to form the PMHC18-mPEG2000-coated MnS@Bi2S3 NP solution. The water-dispersed MnS@Bi2S3-PEG solution was passed through the 0.2 μm microfiltration membrane before use.

Cellular experiments

Murine breast cancer 4T1 cells were obtained from Shanghai Institutes for Biological Sciences, CAS (China). Herein, 4T1 cells were grown in RPMI-1640 with 10% fetal bovine serum and cultured at 37 °C under 5% CO2.

For in vitro cytotoxicity test

Herein, 4T1 cells were seeded into 96-well cell culture plates and then incubated with different concentrations of MnS@Bi2S3-PEG RPMI-1640 solutions for 24 h. A standard Cell Counting Kit-8 (CCK-8, Sigma) assay was carried out to determine the cell viabilities relative to the control untreated cells.

For in vitro photothermal therapy

In this study, 4T1 cells were seeded into 12-well cell culture and then incubated without or with MnS@Bi2S3-PEG RPMI-1640 solutions (0.1 mg mL−1). After 12 h incubation, the cells were irradiated by 808 nm laser (power density = 800 mW cm−2) for 5 min. Then, the cells were stained with Hoechst 33342 (Beyotime) and propidium iodide (Beyotime) and imaged by a fluorescence microscope to check the live, apoptosis, or dead cells.

For γ-H2AX immunofluorescence analysis

4T1 cells seeded in 12-well plates were incubated without or with MnS@Bi2S3-PEG (0.1 mg mL−1) for 12 h and then irradiated without or with X-ray at the dose of 4 Gy. Then, the cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilizated with 0.1% Triton X-100 for 30 min, and preincubated with PBS containing 1% BSA for 1 h. Then, the cells were incubated with anti-histone γ-H2AX mouse monoclonal antibody (dilution 1[thin space (1/6-em)]:[thin space (1/6-em)]500) at 4 °C for 12 h. After this, the cells were further incubated with sheep anti-mouse secondary antibody (dilution 1[thin space (1/6-em)]:[thin space (1/6-em)]500) for 2 h at r.t. Then, the cells were stained by 4′-6-diamidino-2′-phenylindole (DAPI) for 5 min at r.t. Finally, the cells were imaged by a fluorescence microscope.

For clonogenic assay

4T1 cells were seeded in 60 mm dishes and incubated for 12 h, and MnS@Bi2S3-PEG solution was then added to the dishes and it resulted in MnS@Bi2S3-PEG with the concentration of 0.1 mg mL−1. Cells were incubated for another 24 h before irradiation with a dose of 2, 4, and 6 Gy X-ray. The colony formation was analyzed after 10-d incubation by counting the cells stained with crystal violet.

Tumor model

BALB/c female mice were obtained from Shanghai Lingchang Biotechnology Co., Ltd. All the following in vivo mice experiment protocols were approved by Fudan University Laboratory Animal Center. Herein, 4T1 cells (2 × 106) suspended in 100 μL PBS were grafted into the leg of each BALB/c mouse to develop the tumor model.

MR imaging

MR imaging was conducted using a 7-T small animal MR scanner (Bruker Biospin Corporation, Billerica, MA, USA). The parameters for T1 and T2 measurements of MnS@Bi2S3-PEG in vitro were adjusted as follows: TE = 6 ms and TR = 633 ms for T1-weighted imaging and TE = 29 ms and TR = 2500 ms for T2-weighted imaging. The r1 and r2 relaxation rates were obtained from the slope of the linear fitting of 1/T1 and 1/T2 against the different Mn concentration from 0 to 1.2 mM. For in vivo mice MR imaging, MnS@Bi2S3-PEG solution (10 mg kg−1, 50 μL) was intratumorally injected into 4T1 tumor-bearing mice, and then scanned under the same T1 and T2 parameter settings in vitro.

CT imaging

In vitro and in vivo CT imaging were conducted using a Siemens Inveon PET/CT imaging system with 70 keV tube voltage. To evaluate the in vitro CT imaging, different concentrations of MnS@Bi2S3-PEG solutions containing Bi concentration from 0 to 12 mg mL−1 was carried out. For in vivo CT imaging, MnS@Bi2S3-PEG solution (10 mg kg−1, 50 μL) were intratumorally injected into 4T1 tumor-bearing mice and then scanned under the same in vitro scanning parameters.

PA imaging

In vitro and in vivo PA imaging were acquired using the high resolution pre-clinical photoacoustic imaging system (Vevo LAZR, FujiFilm VisualSonics Inc., USA). A series of MnS@Bi2S3-PEG solutions with the concentrations of 0–1.8 mg mL−1 was used to carry out in vitro tests. To evaluate the in vivo imaging performance, MnS@Bi2S3-PEG solution (10 mg kg−1, 50 μL) were intratumorally injected into 4T1 tumor-bearing mice and then scanned under ultrasound imaging B mode and photoacoustic imaging mode using the 892 nm laser as the excitation photosource.

In vivo PTT-RT synergistic therapy

A 808 nm laser (power density = 800 mW cm−2, irradiation time = 10 min) was used for PTT, and 4 Gy X-rays were used for RT. MnS@Bi2S3-PEG solutions (10 mg kg−1, 50 μL) were intratumorally injected into 4T1 tumor-bearing mice. When the volume of 4T1 tumor on BALB/c mouse reached ca. 125 mm3, the mice were separated into seven groups (each group contained 5 mice, n = 5): (1) control (mice without any treatment), (2) MnS@Bi2S3-PEG group (mice only intratumorally injected with MnS@Bi2S3-PEG solution), (3) laser group (mice were only irradiated with laser), (4) X-ray group (mice were only irradiated with 4 Gy X-ray), (5) MnS@Bi2S3-PEG + PTT group (mice were intratumorally injected with the MnS@Bi2S3-PEG solution, and then irradiated with laser), (6) MnS@Bi2S3-PEG + X-ray group (mice were intratumoral injected with the MnS@Bi2S3-PEG solution, and then irradiated with 4 Gy X-ray), and (7) MnS@Bi2S3-PEG + laser + X-ray group (mice were intratumorally injected with the MnS@Bi2S3-PEG solution and then irradiated with 808 nm laser and 4 Gy X-ray). Tumors were treated every 2 days, and the tumor lengths and widths were monitored every 2 days for 16 days. The relative tumor volume was calculated by the formula (width2 × length)/2. The relative survival rate was calculated after the whole treatment (36 days).

Histology measurement and blood analysis

Healthy BALB/c mice without and with i.v. injection of MnS@Bi2S3-PEG solution at a dose of 20 mg kg−1 were sacrificed at 24 h and 21 d. The major organs (lung, liver, heart, spleen, and kidney) were harvested from mice in each group, then dipped into formalin, embedded in paraffin, stained with H&E, and examined under a digital microscope. The blood samples were also obtained for serum biochemistry assay and complete blood panel analysis. On the other hand, tumors acquired from the control and all treatment groups were obtained 2 d post treatment for H&E and TUNEL staining and observed by a digital microscope.

Statistical analysis

All data has been statistically analyzed by a Student's t-test. P < 0.05 was considered as statistically significant. All the presented data has been expressed as mean ± standard deviation.


In summary, we successfully fabricated core–shell structured MnS@Bi2S3 NPs. The mass ratio of Mn and Bi can be precisely controlled by adjusting the reaction temperature via a cation exchange reaction process. After PEGylation, the formed hydrophilic MnS@Bi2S3-PEG NPs can be easily dispersed in the aqueous medium. The MnS@Bi2S3-PEG NPs showed a broad absorbance in the NIR region and contained the functional elements Mn and Bi that made these NPs qualify the r1 and r2 relaxivity, the high NIR optical absorption, and the strong X-ray attenuation ability. All these properties illustrated that MnS@Bi2S3-PEG injected into the tumor model mice could be utilized in a diagnostic contrast agent for MR, CT, and PA tri-modal tumor imaging. The photothermal conversion-induced PTT combined with RT also realized the synergetic enhancement of in vivo tumor therapy efficacy. The toxicity, serum biochemistry assays, and complete blood panel tests proved the minimal toxic side effect of MnS@Bi2S3-PEG NPs for in vivo bioapplications. Therefore, we designed a core–shell nanocomposite structure that supplied a feasible general way to precisely adjust the ratio of various functional parts in one single platform, which combined the diagnosis and therapy functions together and enhanced the treatment efficacy through the single injection dose to reduce overtreatment.


This work was financially supported by the National Natural Science Foundation of China (21501121, 21501029), China Postdoctoral Science Foundation (2015M581635, 2016T90376), and Natural Science Foundation of Shanghai (15ZR1438600).

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Electronic supplementary information (ESI) available: Characterization data and in vivo treatment data of MnS@Bi2S3-PEG. See DOI: 10.1039/c7nr02384g

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