Introduction

Cancer is a term used for diseases in which abnormal cells divide without control and are able to invade other tissues. According to the World Health Organization, cancer is a leading cause of death and accounted for 7.6 million deaths (around 13% of all deaths) in 2008. This number is expected to rise to 12 million by 20301. The increase in cancer deaths is signaling the pressing need for newer, even more effective diagnostics and therapies. Recently, “theranostics”, which incorporate both therapy and diagnosis, are attracting significant attention2. The integration of diagnostic imaging capability with therapeutic interventions is critical in addressing the challenges of cancer heterogeneity and adaptation. Therefore, theranostic agents have attracted enormous interest in cancer diagnosis and treatment in recent years.

Albeit substantial progress was accomplished, conventional diagnostic and therapeutic agents, such as iodinated contrast agent, radio-labeled biomolecules, or chemotherapy drugs, are often limited by short blood circulation times and nonspecific biodistribution3. The emergence of nanomaterials offers new opportunities for advanced diagnostics and therapeutics. Nanomaterials provide several potential advantages over conventional agents, including extension of circulating half-life, passive accumulation at tumor sites due to the enhanced permeability and retention (EPR) effect, reduced toxicity and integration of multiple diverse functions in a single complex3. Up to now, several nanomaterials have acted as nanotheranostic agents4,5,6,7, such as magnetic nanoparticles8,9,10, carbon nanotubes11,12, gold-based nanostructures13,14,15 and nanoshells-based nanoparticles3,16. Although impressive advances have been made in nanotheranostic agents, the types of nanomaterials with efficient diagnostic imaging and therapeutic capabilities simultaneously are limited. Therefore, there is still a great demand for developing new nanomaterials for the diagnosis and treatment of different cancers.

Topological insulators are a new class of quantum matter for which their bulk phases are ordinary insulators but possess robust, nontrivial and conducting surface or boundary states with nondegenerate spins17. Beyond the inherent importance of exploring complex phases of quantum matter, topological insulators are of great interest for device applications involving quantum computing and photonics. For their special characteristics, topological insulators have become the rising star in physics and provided challenges and opportunities for chemistry and material science18. Bismuth selenide (Bi2Se3) is theoretically predicted and experimentally observed to be a three-dimensional (3D) topological insulator with a single Dirac cone19. Bi2Se3 has attracted great attention in physics and chemistry because of its remarkable thermoelectric20, optical21 and photoelectric properties22. Although, the synthesis and investigation of the physical properties of Bi2Se3 have been undertaken, little has been done to explore Bi2Se3 in biomedical fields. Bi2Se3 is composed of bismuth (Bi) and selenium (Se). Bi is an environmentally friendly element and has a long history of use as a therapeutic agent with low toxicity23. Furthermore, Bi is an attractive element for X-ray contrast agents due to its high atomic number (Z = 83) and well-known biological tolerance24. Recently, some Bi-based nanomaterials have been explored as high contrast, long-circulating, low-toxicity X-ray contrast agents25,26,27. The other component is Se, which is an essential trace element for human. The recommended dietary allowance of Se is 55 μg per day for healthy human adults28. Meanwhile, Se also plays important biological roles through the selenoproteins, which helps protect tissues and membranes from oxidative stress and controls cell redox status29. In addition, accumulating evidence indicates that Se has benefits in reducing the risk of cancer incidence and mortality in many cancer types and specifically in liver, prostate and lung cancers30. Therefore, Bi2Se3 is presumed to be biocompatible and holds great potential in biomedical fields.

Here we investigate the theranostic properties of Bi2Se3 nanoplates in in vivo and in vitro system for the first time (Fig. 1). We show that Bi2Se3 nanoplates can absorb NIR laser light and effectively convert laser energy into heat. The photothermal conversion properties of topological insulators have not been reported so far, although many excellent physical properties of topological insulators have been extensively investigated. Besides, Bi2Se3 nanoplates also exhibit strong X-ray attenuation for in vivo X-ray computed tomography (CT) imaging. Therefore, topological insulator Bi2Se3 holds promise as a novel theranostic platform.

Figure 1
figure 1

Schematic representation of topological insulator bismuth selenide as a theranostic platform.

Bismuth selenide is able to perform for simultaneous X-ray CT imaging and photothermal therapy.

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Results

Synthesis and characterization of Bi2Se3 nanoplates

Bi2Se3 nanoplates were prepared by a simple synthetic route and coated with polyvinylpyrrolidone (PVP)31. PVP is a biodegradable and water-soluble biocompatible polymer and is frequently employed as binding, dispersing and stabilizing agent in pharmaceutical tablets and injectables32. Moreover, PVP has also been used to stabilize a variety of nanoparticles to expand their biological applicability25,33,34. The synthesized Bi2Se3 nanoplates showed relatively uniform sizes with an average diameter of about 90 nm (Fig. 2a) based on transmission electron microscope (TEM) images. In addition, the as-prepared Bi2Se3 has a layered structure based on AFM analysis. The thickness of the outer layer was about 3.9 nm and inner layer was about 21.5 nm (Supplementary Fig. S1). The phase analysis of the as-prepared product was determined by powder X-ray diffraction (XRD) (Fig. 2b). All peaks in the pattern correspond to the reflections of the rhombohedral phase of Bi2Se3, which match well with the reported value (JCPDS Card No. 33-0214)31. Furthermore, Bi2Se3 nanoplates exhibited good dispersion in a range of physiological solutions including saline, cell medium and serum (Supplementary Fig. S2).

Figure 2
figure 2

Characterization of Bi2Se3 nanoplates.

(a) TEM image and (b) XRD pattern of Bi2Se3.

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Cytotoxicity and cell internalization of Bi2Se3 nanoplates

Prior to using Bi2Se3 nanoplates for in vivo imaging and therapy, we tested their cell cytotoxicity using the methyl thiazolyl tetrazolium (MTT) assay at first. Encouragingly, cell viability was not hindered by Bi2Se3 nanoplates up to a concentration of 200 μg ml−1 (Fig. 3a). The results suggest that Bi2Se3 nanoplates possess low cell cytotoxicity and good biocompatibility. To study the mechanism of the cellular uptake of Bi2Se3 nanoplates, we incubated the H22 cells with Bi2Se3 nanoplates at 4°C and 37°C, respectively. The uptake efficiency of Bi2Se3 nanoplates was investigated using inductively coupled plasma mass spectrometry (ICP-MS). The results shown in Fig. 3b reveal that the cellular uptake of Bi2Se3 nanoplates is significantly decreased at 4°C. This result implies cellular uptake of Bi2Se3 nanoplates is through an energy-dependent endocytosis35.

Figure 3
figure 3

Cell cytotoxicity and cell internalization of nanoplates.

(a) Cell viability of H22 cells after incubation with increased concentration of Bi2Se3 nanoplates. (b) ICP-MS results of H22 cells incubated with Bi2Se3 nanoplates at 37°C and 4°C. Error bars correspond to mean ± standard deviations.

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In vitro and in vivo X-ray computed tomography (CT) imaging

Bi2Se3 nanoplates hold great promise for CT contrast agents because Bi possesses the large X-ray attenuation coefficient (Bi: 5.74, Au: 5.16, Pt: 4.99, Ta: 4.3 and I: 1.94 cm2kg−1 at 100 keV). In this work, the Hounsfield Units (HU) of Bi2Se3 nanoplates was evaluated by a clinical CT. Figure 4a displays the CT images in the range from 0 to 0.0488 mol Bi l−1 of Bi2Se3 nanoplates. It can be observed that CT signal intensity continuously increases with the increasing of Bi2Se3 nanoplates concentrations, resulting in brighter images. To further investigate the CT contrast efficacy, we next compared the X-ray absorption of Bi2Se3 to that of Iopamidol in vitro (Fig. 4a). Iopamidol is one of most widely used CT contrast agents in current clinical applications. The results demonstrate that Bi2Se3 produces higher contrast than Iopamidol. The HU values increase linearly with concentration for both Bi2Se3 and Iopamidol (Fig. 4b). Nevertheless, the HU values of Bi2Se3 are higher than that of Iopamidol at equivalent concentrations due to the fact that the attenuation coefficient of Bi is larger than that of iodine (I). Thus, Bi2Se3 provides an equivalent contrast at a lower dose compared to the clinical iodinate agent. The reduced dose is highly beneficial because it reduces potential side effects and complications in clinical applications. Furthermore, in vitro CT imaging of H22 cells incubated with different concentrations of Bi2Se3 nanoplates was also investigated. Figure 4a (last row) shows the transection CT images of H22 cells with or without incubation of the Bi2Se3 nanoplates. It is clear that with the incubated concentration of Bi2Se3 nanoplates increasing, H22 cells gradually display brighter CT images.

Figure 4
figure 4

Results of X-ray CT imaging in vitro and in vivo.

(a) In vitro CT images of Bi2Se3 nanoplates and Iopamidol with different concentrations. Cell analysis: H22 cells incubated without contrast agent (1), with increased concentration of Bi2Se3 nanoplates (0.02 and 0.05 mol Bi l-1 for 2 and 3, respectively). (b) CT value (HU) of Bi2Se3 nanoplates () or Iopamidol () as function of the concentration. (c) CT coronal views of a mouse following an intratumoral injection of 100 μl of Bi2Se3 nanoplates solution (0.2 mol Bi l−1) (top). The corresponding 3D rendering of in vivo CT images above (bottom). The position of tumor is marked by red circles.

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The excellent in vitro performance of Bi2Se3 nanoplates as a potential CT imaging agent encouraged us to pursue their applicability in CT imaging in vivo. The feasibility of Bi2Se3 nanoplates as CT contrast agents in vivo was demonstrated by intratumoral injection of Bi2Se3 nanoplates. Figure 4c shows the tumor CT images before and after intratumoral injection. The subcutaneous injection site displays a brighter CT signal than other soft tissues, which is attributed to the strong X-ray attenuation induced by Bi. After analysis of the CT values of the tumor area at 30 min and 120 min post-injection, we found that the CT value decreased slightly (627 ± 22.8 versus 545 ± 13.6). It is because with the passage of time, Bi2Se3 nanoplates could diffuse in the tumor. Therefore, the CT values of the tumor area would decrease gradually. However, the CT value of tumor area even at 120 min post-injection was still significantly higher than that of the tumor area before injection (545 ± 13.6 versus 85 ± 5.2). As control, the mice were injected with Iopamidol at the central region of the tumor under similar conditions. The inject region showed a white spot with brightness lower than the case of Bi2Se3 nanoplates (Supplementary Fig. S3). These results provide strong evidence that Bi2Se3 may be a promising contrast agent for CT imaging in future clinical applications.

In vitro and in vivo photothermal therapy

Photothermal therapy (PTT) is a hyperthermia therapeutic approach that employs photoabsorbing agents to burn cancer cells by heat generated from optical energy36. Deep penetration and little nonspecific photothermal heating in the near-infrared (NIR) window are due to the transparency and low absorption of light by tissues in this optical window37. Compared with traditional cancer therapies such as radiotherapy and chemotherapy, PTT cancer treatment is a minimally invasive or noninvasive technique. In recent years, some NIR light absorbing nanomaterials have been developed for PTT. At present, there are four main types of NIR laser-driven photothermal agents have been extensively developed, resulting in great contributions to progress in PTT. The first type is organic compounds38,39,40,41. The second type is carbon-based materials, including carbon nanotubes42,43,44,45 and graphene46,47,48. The third type is metal nanostructures, such as Au nanostructures13,14,15, palladium nanosheets49 and chemically exfoliated MoS250. The last type is copper chalcogenide semiconductors51,52,53,54. As far as we know, the photothermal conversion properties of topological insulators have not been reported so far.

In this work, we demonstrate the Bi2Se3 nanoplates display high optical absorption in the NIR range (Supplementary Fig. S4). And they can effectively convert NIR laser radiation into heat. We examined the temperature increase of Bi2Se3 nanoplates aqueous solution with various concentrations by the 808 nm laser (Fig. 5a). The temperature of the Bi2Se3 nanoplates solution increased rapidly, while pure water showed a negligible change. The temperature of the solution containing 50 μg ml−1 Bi2Se3 nanoplates rose from 24.4°C to 50.2°C after 5 min irradiation. We presume that this highly efficient photothermal conversion property may be due to the unique physical properties of topological insulators.

Figure 5
figure 5

Photothermal therapy in in vitro and in vivo system.

(a) Photothermal effect of pure water and Bi2Se3 nanoplates with different concentrations (a–e: 0, 5, 10, 20, 50 μg ml−1) upon the irradiation of 1 W cm−2 808 nm laser. (b) H22 cell viabilities cultured with Bi2Se3 nanoplates at different concentrations with or without laser irradiation. (c) Growth of H22 tumors in different groups of mice after treatment. The relative tumor volumes were normalized to their initial sizes. (d) Representative photos of a Bi2Se3 nanoplates-injected mouse at day 0 before PTT treatment and at day 20 after treatment. Error bars correspond to mean ± standard deviations.

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Next, we evaluated in vitro photothermal ablation capacity of Bi2Se3 nanoplates with H22 cells. The MTT results showed that H22 cells treated with Bi2Se3 nanoplates without laser irradiation remain more than 90% viable at Bi2Se3 nanoplates concentrations up to 50 μg ml−1. The cell viability significantly decreased when they were simultaneously treated with Bi2Se3 nanoplates and laser irradiation. And only about 25% of H22 cells remained viable at a Bi2Se3 nanoplates concentration of 50 μg ml−1 (Fig. 5b).

After confirming the photothermal effect of Bi2Se3 nanoplates, we attempted in vivo therapeutic examinations against Balb/c mice bearing H22 cancer tumors. After the tumor sizes became approximately 100 mm3, the mice were divided into four groups (n = 7): (a) sterile water; (b) sterile water + laser; (c) Bi2Se3 nanoplates; (d) Bi2Se3 nanoplates + laser. For group (d), each mouse was treated with an intratumoral injection of Bi2Se3 nanoplates followed by NIR irradiation. One noticeable observation is that there were small black round marks on the skin after treatment. This black mark is a skin burn scar which is a direct evidence of the generation of excessive local heating from Bi2Se3 nanoplates by NIR. Under the same irradiation conditions, in contrast, there was no noticeable sign of temperature increase in the mice treated with sterile water followed by NIR irradiation (group b). Another two control groups (a and c) of mice with sterile water or Bi2Se3 nanoplates injection were not irradiated.

Tumor sizes were measured every 2 days after the above treatments. Figure 5c shows tumor volume changes of representative mice treated in different groups as a function of time. Tumors that received only Bi2Se3 nanoplates injection (group c) or laser irradiation (group b) showed growth rates indistinguishable from control tumors (group a). This result demonstrated that only NIR laser irradiation or Bi2Se3 nanoplates injection did not affect the tumor development. In marked contrast, tumors of group (d) were completely eliminated one day after treatment, leaving the original tumor site with black scars which fell off about 1 week later (Fig. 5d). In addition, NIR at power density of 2 W/cm2 alone did not cause damage to cancer tissues (Supplementary Fig. S5). No tumor regrowth was noted in this treated group over a course of 45 days, after which the study was ended. In addition, the toxicity of the as-prepared Bi2Se3 nanoplates was examined in vivo. We did not find any obvious sign of toxic side effects for Bi2Se3 nanoplates injected mice within 45 days. Neither death nor significant body weight drop was observed in all experimental groups (Supplementary Fig. S6). Furthermore, major organs of Bi2Se3 nanoparticles + laser treated mice (group d) whose tumors were eliminated by the photothermal therapy were collected 45 days after the treatment for histology analysis. No evident tissue damage from the group (d) compared to the control group (a) (Supplementary Fig. S7). Our results suggest that Bi2Se3 nanoplates may be a powerful agent for in vivo photothermal cancer therapy.

Discussion

The development of new nanomaterials for simultaneous cancer diagnosis and treatment has been receiving great attention in recent years and has now become an important field in medical research. Bi2Se3, as a three-dimensional topological insulator, causes wide attention recently. It has been proved that Bi2Se3 has many excellent physical properties, such as thermoelectricity and photoelectricity. In present work, we investigate the theranostic properties of Bi2Se3 nanoplates in in vivo and in vitro system. We prove that Bi2Se3 nanoplates have the X-ray CT imaging and photothermal therapeutic capabilities simultaneously. On one hand, as an X-ray contrast agent, Bi2Se3 nanoplates provide higher contrast efficacy than a clinical iodinate agent. The xenograft tumor model could be imaged using CT after intratumoral administration of Bi2Se3 nanoplates. On the other hand, as a photothermal therapeutic agent, Bi2Se3 nanoplates can absorb NIR laser and effectively transfer laser energy into heat, then successfully induce complete eradications of solid malignant tumors in mice by hyperthermia. More importantly, the photothermally treated mice were all healthy without toxic effects, abnormal behavior, or recurrence of tumors over a long period of time. Our results clearly indicate that Bi2Se3 nanoplates may be a promising platform for simultaneous diagnosis and therapy and bring novel opportunities in theranostics.

Furthermore, bismuth complexes and selenium complexes have a long history of use as therapeutic agents with low toxicity. In addition, Se has benefits in reducing the risk of cancer incidence and mortality in many cancer types. Therefore, Bi2Se3 is presumed to be biocompatible. In our preliminary experiments, the results also prove that Bi2Se3 nanoplates have a low toxicity to cells and no observable toxicity in mice.

Although potential applications of Bi2Se3 in biomedical fields are shown in this paper, a number of issues demand clarification and further researches. Potential toxicity, the long-term biodistribution and quantitative pharmacokinetics in vivo are extremely important and need to be addressed. On the other hand, the incorporation of tumor targeting ligands with Bi2Se3 to achieve active targeting is another interesting issue. These works are underway now in our laboratory and will be communicated in due course.

Methods

Synthetic procedure and characterization of Bi2Se3 nanoplates

The Bi2Se3 nanoplates were synthesized according to the method published previously31. In brief, A reaction batch (100 ml two-neck round-bottom flask) contained ethylene glycol (40 ml), Na2SeO3 (99%, Aldrich, 0.06 g), Bi(NO3)3·5H2O (99.99%, Aldrich, 0.1125 g) and polyvinylpyrrolidone (PVP) (Mw = 55, 000, Aldrich, 0.25 g) as surfactant. The reaction was triggered by rapid injection of 0.6 ml of hydroxylamine solution (NH2OH, 50% in H2O, Aldrich) at 140°C. Then, the reaction mixture turned brown red immediately, which indicated the formation of Bi2Se3 nanoplates. The reaction was allowed to process for 10 min for a complete reaction and cooled down to room temperature. The size and morphology of the Bi2Se3 nanoplates were characterized using transmission electron microscopy (JEM 1010, JEOL, Japan), at an accelerating voltage of 100 kV. X-ray diffraction (XRD) patterns were obtained by using an X-ray power diffraction meter (D/MAX-3C, Rigaku Co., Japan). UV-vis absorption spectra were measured using a TU-1950 UV-Vis spectrophotometer (Beijing Perkinje General Instrument Co., Ltd., Beijing, China) at room temperature.

Mice and cell lines

Female Balb/c mice (weight about 20 g) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China). Animal experiments and animal care were carried out according to protocols approved by the institutional committee for animal care and also in accordance with the policy of the National Ministry of Health. Murine hepatocarcinoma cell line H22 was purchased from China Centre for Type Culture Collection (CCTCC, Wuhan, China) and cultured according to the guidelines given.

Cellular uptake efficiency and cytotoxicity assessment

In a typical experiment, H22 cells (1 × 104 cells per well) were seeded in 96-well plates and incubated overnight at 37°C in a humidified 5% CO2 atmosphere. The cells were incubated with 100 μl of varying concentration of Bi2Se3 nanoplates prepared above for 24 h at 37°C in the dark under the same conditions. Cell viability was determined by the standard 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) (Aldrich) assay. In some cases, cells were collected and digested in HNO3 (65%) for 24 h and then diluted in Milli-Q water for ICP-MS (7500CE, Agilent Technologies, USA) analysis.

CT imaging in vitro

The solution of Bi2Se3 nanoplates with different concentrations in the range from 0 to 0.0488 mol Bi l−1 was directly detected using a 320-detector row scanner (Aquilion ONE; Toshiba Medical Systems, Japan) with the following operating parameters: thickness, 0.5 mm; pitch, 0.5 mm; tube voltage 120 KV; tube current, 50 mA; field of view, 350 mm. CT values were acquired on the same workstation using the software supplied by the manufacturer. Each experiment was carried out in triplicate.

0, 0.02 and 0.05 mol Bi l−1 were incubated with H22 cells (1 × 106 cells per well) for 24 h at 37°C under 5% CO2. After washing with PBS for three times, H22 cells containing Bi2Se3 nanoplates in PBS were precipitated at the bottom of the tube for CT imaging.

CT imaging in vivo

100 μl of 1 × 106 H22 cells (Balb/c background) in PBS was injected into left axilla region of female Balb/c mice. Mice were anesthetized during the CT imaging with 100 μl 20% urethane. CT scanning was performed both before and after intratumoral injection of Bi2Se3 nanoplates (100 μl, 0.2 mol Bi l−1). The mice were scanned by CT after 30 min and 120 min post-injection. As control, CT scanning was performed both before and after intratumoral injection of Iopamidol (100 μl, 0.2 mol I l−1). The mice were scanned by CT after 3 min and 60 min post-injection. All CT scans were performed using the above CT system.

In vitro photothermal therapy effects

An optical fiber coupled 808 nm high power diode-laser (Hi-Tech Optoelectronics Co., Ltd. Beijing, China) was used in the experiments. To study the photothermal effect induced by the NIR absorption, 1 ml aqueous solutions containing different concentration (0, 5, 10, 20 and 50 μg ml−1) of Bi2Se3 nanoplates were irradiated by a NIR laser (808 nm, 1 W) for 5 min. The temperatures of the solutions were monitored by a thermocouple microprobe (ϕ = 0.5 mm) submerged in the solution in a 1-cm square cuvette. The probe was placed at such a position that the direct irradiation of the laser on the probe was avoided.

For the cell toxicity assay, H22 cells were precultured in 96-well cell culture plates (1 × 104 cells per well) for 24 h and then added with Bi2Se3 nanoplates at a series of concentrations. After incubation for 24 h, cells were rinsed with PBS and then were irradiated by the 808 nm laser at a power density of 1 W cm−2 for 5 min. After 24 h incubation, the standard MTT (Sigma) assay was carried out to determine the cell viabilities relative to the control untreated cells (incubated with the same volume of PBS).

In vivo photothermal therapy effects

The H22 tumor models were generated by subcutaneous injection of 1 × 106 H22 cells (Balb/c background) in PBS on the right rear flank of each mouse. The Bi2Se3 nanoplates were injected to the mice when the tumor volumes approached about 100 mm3. For photothermal treatment, the mice of each group were intratumorally injected with 100 μl of each solution (1 mg ml−1 Bi2Se3 nanoplates solution or sterile water) and immediately irradiated with continuous NIR laser to tumor region for 5 min at the power density of 2 Wcm−2. The tumor sizes were measured by a caliper every other day and were calculated using the equation (1): volume = ab2/2, where a is the maximum diameter of tumor and b is the minimum diameter of tumor. Relative tumor volumes were calculated as V/V0 (V0 is the tumor volume when the treatment was initiated).