MoS2 nanosheets encapsulated in sodium alginate microcapsules as microwave embolization agents for large orthotopic transplantation tumor therapy

Changhui Fu ab, Fan He c, Longfei Tan a, Xiangling Ren a, Wei Zhang c, Tianlong Liu a, Jingzhuo Wang d, Jun Ren a, Xudong Chen *c and Xianwei Meng *a
aLaboratory of Controllable Preparation and Application of Nanomaterials, CAS Key Laboratory of Cryogenics, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China. E-mail: mengxw@mail.ipc.ac.cn
bUniversity of Chinese Academy of Sciences, Beijing 100049, P. R. China
c2nd Clinical Medical college of Jinan University, Shenzhen People's Hospital, 518020, P. R. China. E-mail: chenxudong9999@126.com
dDepartment of Electronic Engineering, Huaihai Institute of Technology, Lianyungang, 222005, P.R. China

Received 15th June 2017 , Accepted 19th July 2017

First published on 7th August 2017


In recent years, it is prevalent to treat various kinds of the tumors through microwave ablation method. However, it is still very difficult to ablate large tumors by the traditional microwave ablation therapy. In this work, an effective microwave embolization agent designed by encapsulating molybdenum sulfide nanosheets in the sodium alginate microcapsules, denoted as MSMCs, was prepared for the effective therapy of large tumor. The toxicity evaluation showed that MSMC had a good biocompatibility in vitro. The in vitro and in vivo experiments demonstrated that the MSMC was an excellent embolic and microwave susceptible agent that could be used for dual-enhanced microwave ablation therapy. As such, the MSMC showed excellent tumor therapeutic effect with 5 times larger ablation zone observed by magnetic resonance (MR) imaging than the microwave alone after 3 days treating. Besides, the tumor is nearly completely ablated and can not be recurrent due to the persistent hyperthermia. Moreover, MSMCs have a good biocompatibility and can be degraded and cleared from the body. It is believed that the MSMC is demonstrated to be a promising multifunctional theranostic agent used for treating the larger tumor via the synergistic therapy of enhanced microwave ablation and transcatheter arterial embolization (TAE).


Introduction

As we know that, liver cancer is usually at the middle and late period when it is diagnosed, which brings serious pain to people all around the world. Furthermore, the therapy of large live cancer is a tremendous challenge in clinic.1,2 The thermal therapy is becoming a prevalent strategy in the treatment of tumors due to its safety, high efficient and minimally invasive.3–8 In particular, microwave ablation therapy attracts much attention due to its highly active heating efficiency. Despite much advance in recent years, the microwave ablation therapy is still not practicable for the treatment of large tumor due to the dissipation and inadequate accumulation of heat around the whole tumor. To this end, it is desired to introduce microwave susceptible agents to improve the microwave-heat converting efficiency for treating large tumor.9 Recently, we developed several microwave susceptible agents that showed high heating effect in the subcutaneous tumor models.9–11 However, it is still difficult to completely ablate the large orthotopic transplantation tumor or tumor cells remained in the periphery. On the one hand, the blood flow from almost entire host organs provides sufficient supply for a orthotopic transplantation tumor and a lot of blood vessels formed in the periphery of a large tumor, thus resulting in the serious heat-sink effect (HSE) caused by the blood flow. The accumulation of heat in the tumor is decreased. On the other hand, the heat of the microwave is reduced as the distance far away from microwave source, resulting in a limited ablation and remaining tumor cells in the periphery.12–16 Transarterial embolization (TAE) and chemoembolization (TACE) are two common methods for treating the patients with unresectable hepatocellular carcinoma (HCC) in clinic with the advantage of blocking the tumor blood vessels. In order to relieve the HSE, the transarterial embolization (TAE) can be used to block the tumor blood vessel with a specific targeted embolization agent delivered arterially.17,18

Owing to the physiological characteristics of the tumor tissue, we designed a micrometer-sized microwave susceptible agent that can be delivered into the proper artery to stop the blood supply of the tumor and improve the microwave susceptibility in the periphery of a large tumor, thus to complete eradicate the large orthotopic transplantation tumor by combing TAE and microwave abalation methods. On one hand, the micrometer-sized microwave susceptible agents can be well distributed in the margin of tumor after the arterial injection because there are abundant blood vessels in the periphery of large tumor.19 This can greatly enlarge the ablation zone due to the greatly improved microwave sensibility in the periphery of the tumor. On the other hand, the embolic effect greatly improves the microwave ablation efficiency by reducing heat loss and cutting off the feeding of nutrition.

Molybdenum sulfide nanosheets have attracted a great deal of interests due to their unique physicochemical property and good biocompatibility in the application of nanomedicine.20–25 Alginates are widely used in the pharmaceutical application due to natural, nontoxic and biodegradable properties.26–29 Inspired by these, we develop an unique microwave embolization agent by encapsulating molybdenum sulfide nanosheets in the sodium alginate microcapsule, named as MSMC, for the effective treatment of large tumor by combing TAE and microwave ablation methods. As expected, the resultant MSMC shows excellent microwave susceptive properties in vitro and in vivo. More importantly, the MSMC has a good embolization effect due to its proper size distribution. As such, due to the synergistic effect of microwave ablation and TAE treatments, a much larger ablation zone was found in the liver implanted VX2 tumor in rabbits after an arterial delivery of MSMC under microwave irradiation. Interesting, the presence of molybdenum sulfide nanosheets also enables the MSMC to be suitable for CT imaging due to its high mass density. The MSMC is demonstrated to be a promising multifunctional theranostic agent that can be realized for an effective destruction of large orthotopic transplantation tumor by dual-enhanced microwave ablation therapy.

Results and discussion

Microwave ablation is commonly used in the liver cancer therapy due to its safety, efficacy and minimally invasive treatment. However, it is still relatively difficult for the ablation of large hepatic tumors. As shown in Scheme 1, a combined therapy is first proposed based on the synergistic effect of microwave ablation and TAE therapy using microwave embolization agent to improve the therapeutic efficiency of larger liver tumor. In our strategy, the well-designed microwave embolization agent, MSMC, was injected to the VX2 liver tumor through hepatic artery. Due to the high microwave susceptibility of MSMC, a quick increase of the temperature in the tumor region occurred and resulted in the destruction of the tumor cells under microwave irradiation. Moreover, the ablation area of the tumor will be significantly increased due to the synergistic effect. Thus, this combined therapy based on MSMC can achieve a large ablation zone and show a high efficiency of cancer microwave ablation.
image file: c7nr04274d-s1.tif
Scheme 1 Schematic illustration of the MSMC used for the microwave ablation and the TAC therapy.

Recently, we have demonstrated that the ions confined in the microcapsule (MC) possessed a much higher efficiency of microwave-heat conversion than the dissociative ions due to the lamellar structures. This will results in a much higher elevated temperature of the surrounding solution. Therefore, we designed one kind of microwave embolization agent by encapsulating molybdenum sulfide nanosheets in alginate microcapsule. According to this theory, it is assumed that the molecular collision and friction inside the microcapsule can be fiercer than that of liquid alone under the microwave electric field if the microcapsule was further segmented into smaller spaces by lamellar structures. It will result in a much higher elevated temperature of the surrounding solution. Therefore, we designed one kind of microwave embolization agent by encapsulating molybdenum sulfide nanosheets in alginate microcapsule. Firstly, MoS2 nanosheets dispersed in the sodium alginate were dropped in the atolin to form a W/O emulsion. Afterwards, isopropanol was used to demulsified to obtain the microcapsules. As shown in Fig. 1A and B, an obvious spherical structure of the MSMC with the rough surface can be clearly observed by SEM imaging, agreeing with the morphology observed from optical microscopy in Fig. S1. An analysis of the optical and SEM images showed that the average size of MC and MSMC is about 2.5 ± 1.1 μm and the 5.6 ± 1.8 μm, respectively, suggesting that the MSMC became bigger after adding MoS2 nanosheets. The chemical composition analyzed by EDS spectra indicates that both MC and MSMC mainly contain C, O and Ca elements. However, Mo and S elements were found to be existed in MSMC with the weight ratio of 4.53% and 7.92%, respectively (Fig. S2). The contrastive analysis of elemental mappings of MC and MSMC also confirmed the uniform distribution of Mo and S elements in the MSMC, as shown in Fig. 1F. The presence of MoS2 in the MSMC was further confirmed by TEM observation. As shown in Fig. 1C, there are many dark corrugated nanosheets embedded in the metrix MC. High-resolution TEM (HRTEM) images of the nanosheets clearly reveals the typical lattice spacing of 0.65 nm, corresponding to the interlayer spacing of (002) in MoS2. This is also consistent with previous report.30 We also explored thermogravimetric analysis to determine the exact weight content of MoS2 in the MSMC. The TGA curves shown in Fig. S3 revealed that the content of MoS2 is about 12%. All these results demonstrate the MoS2 nanosheets have been encapsulated in the resultant MSMC.


image file: c7nr04274d-f1.tif
Fig. 1 Morphology and chemical composition of MC and MSMC: (A) low and (B) high-resolution SEM images of MSMC; (C) low and (D) high-resolution TEM images of MSMC; (E) high-resolution TEM images showing the interlayer spacing of MoS2 nanosheets in the MSMC; (F) EDS elemental mapping of MC and MSMC.

The previous report suggested that combination of computational modeling, in vitro testing, and in vivo validation should be carried out for the rational design of the properties of nanomaterials.31 Based on this theory, the microwave susceptibility of two kinds of MC models designed ideally were simulated by computer and compared in detail. One model was a MC containing three slices of MoS2 nanosheets. This was used for the simulation of MSMC. Another model was a MC with pure liquid. The microwave condition was 500 W for 8 seconds at the frequency of 2450 MHz. As shown in Fig. 2A and B, under microwave irradiation, the temperature of MSMC increased to 360–380 K, which is much higher than that of MC without MoS2. This is also confirmed by the simulated temperature–time curve shown in Fig. 2C. The loading of MoS2 nanosheets in the MC is very helpful for temperature elevation under microwave irradiation. It is indicated that the as-designed MSMC is an efficient microwave susceptible agent.


image file: c7nr04274d-f2.tif
Fig. 2 The microwave susceptibility and ablation of MSMC under microwave irradiation: (A and B) the simulated microwave susceptibility for MC and MSMC under the microwave irradiation with the frequency of 2450 MHz at the outpower of 500 W; (C) the corresponding simulated temperature–time curves of MC and MSMC; (D) the thermal infrared images of the pure saline, the saline containing NaMC, and MSMC after MW irradiation for 5 min; (E) the temperature difference of pure saline, the saline containing MC, and MSMC at the concentration of 10 mg mL−1 after MW irradiation for different time; (F) near-infrared thermography images and (G) the ablation diameter of the gelatin water film with different MW susceptible agents.

Microwave heating experiment of the MSMC was then carried out in vitro. Fig. 2D shows the temperature change reflected by the near-infrared images of the saline solution and the saline solution containing MSMC and MC loading with 3% NaCl (NaMC) under the microwave irradiation of 2 W for 5 min. It can be clearly seen that the temperature of saline solution increased gradually as increasing the microwave irradiation time. With the assistance of NaMC and MSMC, the temperature increased more rapidly. Particularly, the saline solution containing MSMC shows a higher value of 4.5 °C than that of the saline containing NaMC after microwave irradiation for 5 min at 10 mg mL−1, suggesting that MSMC can perform a higher heat conversion efficiency than that of the NaMC. It can be seen from Fig. 2E that the temperature elevation for other groups are only about 18 °C while the solution containing MSMC increased more than 25 °C, agreeing with the above theory simulation results. Meanwhile, the elevated temperature of the saline became much higher as the concentration of MSMC increased (Fig. S4).

Furthermore, the gelatin water film could be used to simulate the conductive and dielectric properties of human muscle.32 Therefore, saline, MoS2 nanosheets, and MSMC were well dispersed in the gelatin solution to evaluate the susceptible effect of MSMC under microwave irradiation at a microwave frequency of 2450 MHz, which is available in clinic. As shown in Fig. 2F, the temperature of the gelatin water film containing MoS2 nanosheets at the concentration of 1.5 mg mL−1 is little higher than that of the control group without adding anything after microwave irradiation for 5 min at 5 W. The temperature of gelatin water film containing MSMC at the concentration of 0.5 and 20 mg mL−1 quickly reached to 40 °C after microwave irradiation for only 1 min, which is corresponding to the concentration of MoS2 of 0.06 and 2.4 mg mL−1. The temperature increased continually to about 44 °C after 5 min. The diameter of the ablation region of the gelatin water film was also measured in detail. Due to the presence of MSMC, the diameter of ablation region of the gelatin water film reached 2.4 cm, which is much larger than those of the control group and the group containing MoS2 nanosheets (Fig. 2G). This suggests that MSMC could potentially be used as effect microwave susceptible agent for treating large tumors due to its large ablation area. These results also confirmed that MSMC can efficiently promote the conversion the microwave energy into thermal energy due to the microwave susceptibility.

Based on above results, cell experiments were carried out to further confirm that MSMC is an effective susceptible agent under microwave irradiation. Firstly, the cytotoxicity of MSMC was evaluated by the methyl thiazolyltetrazolium (MTT) assay. As shown in Fig. 3, no obvious toxicity was found in the HepG2 cells incubated with MSMC at the concentration ranging from 15 to 500 μg mL−1 for 24 h. It is also found that MSMC caused no obvious toxicity to the normal cells of fibroblast L929 and Raw 264.7 macrophages. The cell viability of these two cells was more than 80% after incubating with MSMC even at the 500 μg mL−1. These results demonstrate that MSMC have a good biocompatibility. Afterwards, the microwave susceptible property of the MSMC in vitro was examined. HepG2 cells were incubated with the MSMC at the concentration of 0.5 mg mL−1 or MoS2 nanosheets at 0.05 mg mL−1 under microwave irradiation at 3 W for 3 min. A control experiment with only microwave irradiation was also conducted. As shown in Fig. 3C, the viability of HepG2 is more than 80% in the control group and MoS2 nanosheet-treated group. However, the group incubated with MSMC exhibits obvious cell inhibition with only 50% of cell viability under microwave irradiation. This is confirmed by the calcein-AM and propidium iodide (PI) staining experiment that can help us to clearly distinguish the live and dead cells induced by microwave irradiation with the assistance of MSMC. As shown in Fig. 3B, most cells showed green fluorescence in the control group and the group treated with MoS2 nanosheets, suggesting that the microwave irradiation have little effect on the cells. In comparison, almost all the cells in the group incubated with MSMC exhibits red fluorescence, indicating that most of the cancer cells were destroyed after the irradiation with microwave in the present of MSMC. The in vitro cell experiments further confirm that MSMC is an effective microwave susceptible agent that results in the death of the HepG2 cells due to the obvious increase of temperature under microwave irradiation.


image file: c7nr04274d-f3.tif
Fig. 3 Biocompatible and microwave susceptible evaluation of MSMC in vitro: (A) effect of MSMC on the viability of HepG2, fibroblast L929 and RAW264.7 macrophage after incubating with MSMC at different concentration for 24 h; (B) cytotoxic evaluation of MSMC under the MW irradiation by staining live and dead cell with calcein-AM and PI. Green fluorescence refers to the live cells and red fluorescence represents the dead cells. The scale bar is 50 μm. (C) Cytotoxic evaluation of HepG2-cells exposed with MSMC after microwave (MW) irradiation at 3 W for 3 min; **p < 0.01.

In vivo microwave therapy was further conducted in mice model subcutaneously bearing H22 tumor injected with MSMC based on the previous reports. H22-bearing mice were divided into four groups: (1) only microwave (MW) irradiation; (2) MC combined with MW irradiation; (3) MSMC plus MW irradiation; (4) control group without MW and any susceptive agent. Since our previous reports have demonstrated that the tumor in the mice showed no obvious destruction after injecting with MC and MoS2 nanosheets only. These groups treated with MC, MoS2 nanosheets and MSMC without MW irradiation were not performed for the animal welfare in our study. During our experiments, after intratumorally injecting the different susceptible agents for 1 h, the mice were exposed with MW irradiation at 2450 Hz at 2 W for 5 min. The temperature at the tumor site was monitored using the IR thermal camera. The temperature around the microwave needle of the mice in the control group only increased to 50 °C (Fig. 4A). The temperature near microwave needle area of the MC and MoS2 nanosheet group exhibited a mild increase to about 53 °C after MW irradiation for 5 min. In contrast, the temperature of the tumor site injected with MSMC rapidly increased to 50 °C after MW irradiation for 1 min and reached to about 60 °C for 5 min. Such a high and persistent hyperthermia can completely kill the cancer cells and avoid the recurrence. This is because the protein denatures quickly at such a temperature above 60 °C. This results in the immediately cytotoxic and coagulative necrosis.33 Although previous reports indicated that hyperthermia temperature range from 41 to 45 °C induced irreversible cell damage, it required a prolonged exposure time ranging from 30 to 60 min. However, the sublethal and reversible damage happened after a short exposure time, resulting in recurrence and metastasis.34 Moreover, the completed tumor necrosis can be only achieved when the temperature at least reached 54 °C for 3 min or 60 °C during the microwave treatment process in clinic. Therefore, the temperature of 60 °C is required for the tumour destruction. However, the application of MW in the tumor treatment is also limited by targeting the tumor. The tumor in the special location, such as near to the bile duct or intestinal tract, the overheating hyperthermia may cause some damage to the important tissues. In such case, the treated temperature should be little lower than 60 °C.35


image file: c7nr04274d-f4.tif
Fig. 4 (A) Infrared thermal imaging of mice bearing H22 cells after the intratumoral injection of MSMC, MoS2 nanosheets, and saline at 50 mg kg−1 under the MW irradiation for 5 min at 5 W. (B) The changes of tumor volume and (C) body weight of the mice in different groups. (D) Tumor tissues removed from the mice and (E) tumor inhibition rate of different groups at 23 day. **p < 0.01.

The tumor size and body weight were recorded during the whole experiment. It is found that the tumor size in the group with only MW, MC and MoS2 plus microwave irradiation decreased compared with that of the control group. An appreciable inhibitory effect on the tumor growth was observed in the mice irradiated with MW in the presence of MSMC (Fig. 4B). A scab was observed in the tumor region after 1 day post-injection of MSMC with microwave irradiation. However, a tumor recurrence occurred and the tumors grown fast from the 13th day in the groups injected with MC and MoS2 nanosheets. In comparison, the mice subjected with MSMC showed a thorough ablation of the tumor under MW irradiation. The tumor inhibition rate in the group of MW, MC + MW, MoS2 nanosheets + MW, and MSMC + MW group was 59%, 69%, 79% and 100%, respectively, as shown in Fig. 4E. This indicates that MW ablation is an effective method to kill the tumor in clinic owing to its faster heating effect.36,37

Although the traditional MW ablation can only inhibit the growth of tumor, the recurrence rate is also very high.38 One of the main factors is the limited ablation area of the tumor caused by the complex microenvironment. The existence of the MSMC in the tumor induced a quick temperature rising, resulting in a very large ablation area of the tumor and also causing persistent hyperthermia to destructive the tumor thoroughly. No significant change was found in body weight of different groups, indicating MSMC was low toxicity (Fig. 4C).

In order to evaluate the embolic effect of MSMC, the rabbit was used as the animal model according to previous study.39–41 The MSMC was first administrated intra-arterially to the liver of the normal rabbit at the concentration of 10 mg kg−1 by digital subtraction angiography (DSA) that is a common evaluation means for the availability of the embolization. As shown in Fig. 5A and B, we can observe significant differences for the visibility of hepatic vessel before and after the embolization with MSMC. The blood vessels disappeared after embolizing by MSMC, indicating that MSMC can block up the blood vessels of the liver. Furthermore, it is also found that the vessels of the kidney can be effectively occluded by MSMC, suggesting the embolic feasibility of MSMC to other organs (Fig. S5).


image file: c7nr04274d-f5.tif
Fig. 5 Embolization and microwave ablation effect of MSMC on the liver in normal rabbit: (A–B) DSA images of the liver before and after the embolization of MSMC; (C) the maximum diameter of the liver embolized with MSMC following MW irradiation ablation; (D) the anatomy pictures in the group with MSMC plus MW; (E) the CT values and (F) images of MSMC at different concentration (1.25, 3.25, 7.5, and 15 mg mL−1); (G) the CT image of the tumor after the embolization with MSMC. *p < 0.05, **p < 0.01.

To evaluate the microwave susceptibility of the MSMC after the embolization, the rabbit injected with 10 mg MSMC through hepatic artery was irradiated with microwave in the mode of continuous impulse at 15 W for 2 min. As shown in Fig. 5C, the ablation zone of the liver in the group embolized with MSMC is much bigger than that in group administrated with MC after microwave irradiation. The maximal diameter of the ablation in the liver is about 10.3 mm after a treatment by microwave alone. After the liver was embolized with MC and MSMC, the diameter of the ablation zone increased from 12.3 mm to 16.8 mm (Fig. 5C). It is known that the liver is an organ with a large amount of large vessels that will result in an obvious heat-sink effect.42 Awad et al. showed that the susceptibility of microwave ablation can be decreased by vascular cooling effect and thus the use of occlusion could create an increased size of microwave ablation for the liver.43 This is consistent with our observation. The ablation zone in the group treated by the combined therapy of TAE and microwave irradiation is larger than that of the group with microwave irradiation alone. It can be seen that the MSMC injected arterially blocked the arteries and was in favor of heat deposition and thus enlarge the ablation area. More importantly, the susceptibility of MSMC to microwave irradiation results in more heat energy to create a larger ablation zone than the blank MC. In addition, an ablation zone for the kidney of the rabbits was observed. The diameter of 16.5 mm was observed in the group injected with MSMC after a combined treatment with microwave and TAE while near 12 mm of the ablation zone was found in the group with only microwave irradiation and the group irradiated with MC after TAE (10 W, 2 min) (Fig. S6), indicating the excellent microwave susceptibility of the MSMC. It can be seen that the as-obtained MSMC showed synergy effects of embolization and microwave susceptibility to other organs.

It has been reported that MoS2 is a promising CT contrast agent owing to the outstanding X-ray attenuation ability caused by its high mass density. Fig. 5E shows the CT images and the Hounsfield unit values of the as-prepared MSMC. It can be seen that the enhancement of CT contrast increased as the concentration of MSMC increased in vitro. Furthermore, we evaluated the feasibility of MSMC used as contrast agent for CT imaging of tumor in vivo. In our experiment, MSMC was in vivo injected to the liver tumor of VX2 in rabbits via transcatheter arterial route. As shown in Fig. 5G, some blurry signal was observed in the tumor site of the liver before treatment due to the low-density (in Fig. S6), while the hyperintense signal of the tumor was visualized after 3 days post-injection with MSMC, suggesting that MSMC can be also used as promising contrast agent for CT imaging of tumors due to the presence of MoS2.

As a proof of concept, we further used the MSMC to treat the VX2 liver tumor in vivo by combining microwave ablation therapy and TAE owing to its good performance in normal liver of the rabbit and in rodent models. The embolic effect of MSMC on the blood vessels in the liver tumor was first studied by digital subtraction angiography (DSA) after injecting arterially. As shown in Fig. 6A and B, most tumor-feeding arteries were effectively blocked after the release of MSMC to the hepatic vessels compared with the vessels before embolization.


image file: c7nr04274d-f6.tif
Fig. 6 The therapy effect of MSMC for the rabbit hepatic VX2 tumor after treating with transcatheter arterial delivery under microwave irradiation: (A and B) DSA images of the liver before and after embolization of MSMC; (C and D) the near-infrared imaging of tumor treated without and with MSMC under microwave irradiation; (E and F) the pictures obtained by DWI of the liver in the groups of MW and MSMC plus MW; (G) the ablation area of the tumor obtained from by DWI in different groups after treatment for 3 days; **p < 0.01.

After an arterial injection with MSMC, abdominal surgery was performed to expose the VX2 tumor in the liver. Then the microwave needle were inserted into the center of the tumor and irradiated with a continuous microwave at the outpower of 15 W for 2 min. The temperature change is observed directly through near-infrared imaging technology. As shown in Fig. 6C, in the control group, the temperature in the center of the microwave is higher than 100 °C while that of the region away from the central area become lower in the microwave irradiation alone. As a result, the tumor showed a limited ablation zone due to the slow heat dispersal. While the increased temperature in the tumor region containing MSMC is much higher than that of the control group under the microwave irradiation alone (6D).

After 3 days of post-injection of MSMC, the ablation area was clearly observed by diffusion weighted imaging (DWI) (Fig. 6E and F). Owing to the synergistic effect of the embolization with MSMC under microwave irradiation, the shape of ablation zone of the tumor is irregular and necrosis occurred in the whole tumor. In comparison, a regular rotund necrosis was found around the needle without MSMC, indicating that MSMC can greatly improve the thermal sensitivity of the tumor under microwave treatment. The ablation area in the group of MSMC + MW is nearly 5 times bigger than that of MW alone analyzed by DWI. The larger ablation area demonstrated that the MSMC exhibits tremendous advantages as microwave embolic agent for the cancer treatment and shows promising application in clinic.

Since the location of the embolic agent is important for the susceptible response to the microwave irradiation, we further performed the histological staining and inductively coupled plasma mass spectrometry (ICP-MS) test to study the distribution of MSMC in the tumor. As shown in Fig. 7A and Fig. S7, black dots with a high contrast were found in the tumor regions with the average size of 6.5 μm, analyzed by the Image J software, indicating that MSMC is mainly distributed in the targeted tumor tissue via a catheter infusion in the rabbit model. ICP-MS was further used for the quantificative analysis of MSMC in the tumor. Fig. 7B shows that the amount of Mo element increased in turn from the center of tumor to the normal liver near to the tumor, suggesting that some MSMCs are entrapped in the center of tumor and more MSMC accumulated surrounding the tumor region.


image file: c7nr04274d-f7.tif
Fig. 7 The biodistribution of MSMC in vivo after the combined therapy of TAE and MW thermal treatment: (A) the pathological section of tumor treated with MSMC plus MW; (B) Mo content in the region of the tumor and near to the liver. **p < 0.01.

This agrees with previous result that oil droplets less than 20 μm, especially the diameter between 3–10 μm, were inclined to be trapped in tumor nodules since oil droplets can enter the tumor through the endovascular surface of the portal branches.44 Therefore, MSMC with a diameter about 4–8 μm can enter and accumulate the region of the tumor through the endothelium of vessels, and a small part of the MSMC that have a diameter larger than 20 μm aggregated and occluded microvessels. The different biodistribution of the content of Mo between the tumor and the liver surrounding the tumor is beneficial for the application of MSMC for microwave ablation. Because the characteristic of the microwave irradiation is that the highest temperature is at the center of the thermal ablation region, and gradually decreases far from the center. Therefore, the margin temperature of the tumor is urgent needed to be increased. In our study, the concentration of MSMC is higher at the margin region of the tumor, which can make for the temperature increased in the margin region of the tumor with the help of MSMC with good microwave susceptibility to create a large ablation area. Besides, the obvious decreases in blood flow induced by the embolization of MSMC greatly improved the heating deposition in the tumor and inhibit microscopic intrahepatic metastases that could occur for the microwave ablation therapy alone.36,45,46 Therefore, the MSMC with good microwave susceptibility achieves high therapy efficiency for the treatment of large tumor by combining enhanced microwave ablation therapy and TAE. The distribution of MSMC in other tissues was also detected after an injection after 10 days. The amount of Mo in the liver, spleen, lung, and kidney has been reduced to the normal range, indicating that the MSMC can be degraded and cleared from the body (Fig. S8). It is believed that the biocompatible MSMC is demonstrated to be a promising multifunctional theranostic agent used for treating the larger tumor via the synergistic therapy of microwave ablation and transcatheter arterial embolization.

Conclusions

In summary, a biocompatible microwave susceptible agent designed by encapsulating molybdenum sulfide nanosheets in the sodium alginate microcapsule, MSMC, was developed for effectively treating large tumor by combining microwave ablation therapy and transcatheter arterial embolization. The resultant MSMC exhibited outstanding microwave susceptible properties as well as sensitive contrast effects for CT imaging in vitro and in vivo. By using the rabbit liver VX2 tumor as model, MSMC was demonstrated to be embolic for artery vessels feeding the tumors. The embolic MSMCs were well distributed around the rim and inner regions of the tumors. This is very helpful for improving the therapeutic effect of microwave ablation. Owing to the dual-functions of the microwave susceptibility and the embolic effect, MSMC can produce a larger ablation area and thus achieve significant inhibition of the tumor under microwave irradiation. In addition, the toxicity evaluation showed that the MSMC have a good biocompatibility. These results demonstrate that the MSMC is one promising multifunctional theranostic agent that is practicable for efficient treatment of large tumor via the synergistic therapy of enhanced microwave ablation and TAE.

Acknowledgements

The authors acknowledge funding support from National Natural Science Foundation of China (no. 31400854, 61671435, 81630053, and 81671845), Beijing Natural Science Foundation (no. 4161003), and the Strategic Priority Research Program of the Chinese Academy of Sciences (no. XDA09030301) and CAS-DOE program.

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Footnotes

Electronic supplementary information (ESI) available: Experimental data. See DOI: 10.1039/c7nr04274d
These authors contributed equally to this work.

This journal is © The Royal Society of Chemistry 2017