Multimodal Imaging-Guided Antitumor Photothermal Therapy and

Sep 30, 2016 - Multimodal Imaging-Guided Antitumor Photothermal Therapy and Drug Delivery Using Bismuth Selenide Spherical Sponge ... Citing Articles;...
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Multimodal Imaging-Guided Antitumor Photothermal Therapy and Drug Delivery Using Bismuth Selenide Spherical Sponge Zhenglin Li,†,‡,⊥ Jing Liu,∥ Ying Hu,§ Kenneth A. Howard,⊥ Zhuo Li,† Xuelei Fan,† Manli Chang,¶ Ye Sun,*,‡ Flemming Besenbacher,*,⊥ Chunying Chen,∥ and Miao Yu*,† †

State Key Laboratory of Urban Water Resource and Environment, School of Chemical Engineering and Technology, ‡Condensed Matter Science and Technology Institute, and §School of Life Science and Technology, Harbin Institute of Technology, Harbin 150001, China ⊥ Interdisciplinary Nanoscience Center (iNANO) and Department of Physics and Astronomy, Aarhus University, Aarhus 8000, Denmark ∥ National Center for Nanoscience and Technology, Chinese Academy of Sciences, Beijing 100190, China ¶ Department of Lab Medicine, The Second Affiliated Hospital of Harbin Medical University, Harbin 150001, China S Supporting Information *

ABSTRACT: Elaborately designed biocompatible nanoplatforms simultaneously having diverse therapeutic and imaging functions are highly desired for biomedical applications. Herein, a Bi2Se3 nanoagent with a special morphology as a nanoscale spherical sponge (NSS) has been fabricated and investigated in vitro and in vivo. The highly porous NSS exhibits strong, steady, and broad-band absorbance in the nearinfrared range as well as high efficiency and stability of photothermal conversion, resulting in high antitumor efficacy for photothermal therapy (PTT). Together with a high X-ray attenuation coefficient (218% that of the clinically used iopromide), the NSS shows excellent performance on triple-modal high-contrast imaging, including X-ray-computed tomography, multispectral optoacoustic tomography, and infrared thermal imaging. Furthermore, the high surface area and porous structure impart the NSS a competent drug loading capability as high as 600% of that on Bi2Se3 nanoplates, showing a bimodal pH/photothermal sensitive drug release and pronounced synergetic effects of thermo-chemotherapy with a tumor inhibition ratio even higher than that of PTT alone (∼94.4% vs ∼66.0%). Meanwhile, the NSS is highly biocompatible with rather low in vitro/in vivo toxicity and high stability, at variance with easily oxidized Bi2Se3 nanoagents reported previously. Such biocompatible single-component theranostic nanoagents produced by a facile synthesis and highly integrated multimodal imaging and multiple therapeutic functions may have substantial potentials for clinical antitumor applications. This highly porous nanostructure with a large fraction of void space may allow versatile use of the NSS, for example, in catalysis, gas sensing, and energy storage, in addition to accommodating drugs and other biomolecules. KEYWORDS: bismuth selenide, porous, multimodal imaging, photothermal therapy, drug delivery inherent limitations of each single modality.12,13 Drawbacks of such integration have been revealed, such as inconsistent dose required for imaging and therapy, the uncertain stability and compatibility of the resultant composites, the increased potential toxicity, and the different dissociation rate of each component upon circulation in vivo.14−18 It is, therefore, crucial to achieve multifunctional single nanoagents intrinsically having both imaging and therapeutic capabilities.

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maging-guided photothermal therapy (PTT) that allows simultaneous monitoring and eradication of tumors has become considerably attractive due to the simplicity, efficacy, spatial selectivity, and remote-control properties.1−6 However, a sophisticated, expensive, and challenging synthesis is normally required by integrating PTT agents with additional components, such as magnetic materials (e.g., Fe3O4 and MnSe) for magnetic resonance imaging (MRI), high-Z elements (e.g., gold and rare-earth elements) for X-ray-computed tomography (CT), and fluorescence molecules or upconversion luminescence nanoparticles (NPs) for fluorescence imaging.7−11 In some cases, even multiple components corresponding to different imaging modes are required to compensate the © 2016 American Chemical Society

Received: August 12, 2016 Accepted: September 30, 2016 Published: September 30, 2016 9646

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ACS Nano Bismuth selenide (Bi2Se3) nanomaterials have stimulated considerable interest, mainly due to their intriguing physical properties as typical topological insulators.19−21 Interestingly, Bi2Se3 is also promising for biomedical applications given the high X-ray attenuation coefficient of Bi as well as the important function of Se element on reducing cancer incidence or mortality.22 The high biocompatibility, metabolizability, and photothermal conversion capability of Bi2Se3 in a nanoplate form have been demonstrated.23,24 However, the efficiency and stability of photothermal conversion of Bi2Se3-containing nanoagents as well as the possibility to apply them on multispectral optoacoustic tomography (MSOT) still remain unexplored, although MSOT based on the optoacoustic effect from light absorption and subsequent thermal expansion has quickly emerged as an important imaging technology to provide high spatial resolution of soft tissues25,26 and remedy the inherent limitations of other imaging modes, such as CT. Despite the known advantages of CT (i.e., high resolution and facile three-dimensional visualization on tissues of interest), its drawback of low sensitivity and poor contrast in soft tissues requires a large dose of agents to get adequate imaging.27,28 In fact, even for the PTT applications, the potential of Bi2Se3 nanoagents was severely limited by the poor in vivo stability;9,23 thus, most reported experiments were performed by intratumor (i.t.) injection instead of intravenous (i.v.) injection. Moreover, although the synergistic therapeutic effect combining PTT with chemotherapy is well-recognized,29,30 the difficulty of drug loading upon the reported Bi2Se3 nanostructures largely restricted the possibility for drug delivery, as well. In the recent work of our group, we designed a Bi2Se3 nanoplate into a drug delivery vehicle with high in vivo stability by sequential coating of polydopamine and human serum albumin/ doxorubicin (DOX)31 and realized a pH/thermal-sensitive drug release. However, confined by the limited specific surface area (SSA), the drug loading capability on the nanoplate was not significant. Even an effective thermo-chemotherapy was still achieved; a new morphology of Bi2Se3 nanoagents with a largely enhanced SSA and, hence, loading capability as well as simple fabrication and loading procedures would be apparently preferred for practical applications. With respect to this aspect, highly porous nanoagents may hold notable superiorities. Moreover, as demonstrated from a large variety of lightharvesting metallic and semiconducting materials in the literature,32 a porous structure may largely improve the light absorbance due to the pore-induced multiple scattering and absorption together with increased SSA (relative to the solid NPs in the same size). A strong absorbance in the near-infrared (NIR) range is indispensable for a good PTT nanoagent. In this work, nanoscale spherical sponges (NSS) of Bi2Se3 successfully coordinating diagnostic and therapeutic functions are synthesized via a facile hydrothermal method, using the Bi2O3 NPs as the precursor and template (Scheme 1). The resultant NSS exhibits distinct porous structures with large SSA and plentiful mesopores, as well as high stability, no/low toxicity, strong and steady absorbance in the entire NIR range, high X-ray attenuation ability, and high photothermal conversion efficiency and stability, leading to high performance in therapeutic PTT, drug delivery, and trimodal MSOT/CT/ infrared thermal (IRT) imaging. Compared with the reported Bi2Se-containing antitumor agents, the NSS owns distinct superiorities: (1) the high in vivo stability to allow practical applications by i.v. injection; (2) the competent drug loading capability, 600% higher than that of Bi2Se3 nanoplates, showing

Scheme 1. Illustration of the Synthesis and Multifunction of the Bi2Se3 NSS

a bimodal pH/thermal-sensitive drug release and pronounced synergetic effects of thermo-chemotherapy with a much higher tumor inhibition ratio than that of PTT alone (∼94.4% vs ∼66.0%); (3) the high scalability enabled by the highly porous nanostructure with large fraction of void space allows versatile use in catalysis, gas sensing, and energy storage, in addition to accommodating drugs and other biomolecules for drug delivery; (4) the steady and strong broad-band NIR adsorption may allow PTT with a wavelength >808 nm. Such biocompatible single-component theranostic nanoagents produced by a facile synthesis and integrated multiple highcontrast imaging modals with multiple effective therapeutic functions may have substantial potential for clinical antitumor applications.

RESULTS AND DISCUSSION Synthesis and Characterization. First, the Bi2O3 sample was produced via a one-pot solvothermal method33 using Bi(NO3)3·5H2O, HNO3, NaOH, and polyvinylpyrrolidone (PVP), together with ethylene glycol. As seen in Figure S1, transmission electron microscopy (TEM) images show that the as-grown Bi2O3 NPs were spherical with an average diameter of ∼125 nm. The varied contrast in the high-resolution TEM image of an individual Bi2O3 NP indicates its porous structure (Figure S1b). The Bi2Se3 product was subsequently obtained by a simple hydrothermal process from the Bi2O3 NPs by utilizing the as-grown Bi2O3 NPs as the precursor and template together with D-(+)-glucose as the reducing agent and surface stabilizer. This method can endow the resulting product high water solubility. TEM (Figure 1a) and backscattered electron scanning electron microscopy (SEM) (inset) results reveal its highly porous spherical-sponge-like structure with an average diameter of ∼131 nm. The infrared spectrum (Figure S2) of these NSS shows features corresponding to abundant functional groups, such as hydroxyl and carbonyl groups. Especially, the presence of C−H (∼2900 cm−1) and C−O/C−C bonds (1000−1100 cm−1) indicates the organic stabilizing coating derived from glucose decomposition. This is further supported by the dynamic light scattering (DLS) results (Figure S3), where the Bi2Se3 NSS showed a hydrodynamic diameter slightly larger than that determined by TEM and negatively charged with a zeta-potential (ζ) of approximately −18.80 mV. Energy-dispersive spectroscopy (EDS) analysis indicates the presence of Bi and Se (Figure 1b). All the diffraction peaks of the Bi2Se3 NSS in the X-ray diffraction (XRD) pattern (Figure 1c) can be well indexed to Bi2Se3 (JCPDS 12-732), indicative of its high purity. The porosity was investigated via Brunauer− 9647

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Figure 1. Characterization of the as-grown Bi2Se3 NSS. (a) TEM image (inset: backscattered electron scanning electron microscopy image); (b) EDS analysis; (c) XRD analysis; (d) N2 adsorption−desorption isotherm (inset: corresponding pore size distribution); (e) UV−vis−NIR absorption spectra of Bi2Se3 NSS and Bi2Se3 nanoplate suspensions (100 μg·mL−1); (f) temperature increase at various concentrations; (g) IR thermal images; (h) heating and cooling curves of deionized water and the Bi2Se3 NSS suspension (50 μg·mL−1); (i) plot of cooling time versus negative natural logarithm of the temperature driving force. Time constant for heat transfer of the system is τs = 394.7 s.

pores31 instead of Sref because it has been demonstrated that nanoparticles in a thin flake shape can largely benefit the NIR light absorbance.36 Also considering the smaller size and higher SSA of the nanoplate relative to the Sref, the nanoplate is supposed to have a significantly higher absorbance than the latter. Even so, it was found that the absorbance of the NSS was higher than that of the nanoplate in the >770 nm range. Such improved absorbance may benefit the photothermal effect of the NSS. Moreover, the absorption profile of the NSS was different from that of the nanoplate. The NSS showed a broad, strong, and steady absorption between 400 and 900 nm, whereas the nanoplate presented stronger absorption in the short-wavelength range but with a sharp decline in the NIR range, similar to the MnSe@Bi2Se3 core−shell nanoparticles.9 The NSS may, therefore, be promising for broad-band PTT applications, and the superiority may become more pronounced when the irradiation wavelength is larger (>800 nm).

Emmett−Teller analysis of nitrogen adsorption−desorption isotherms (Figure 1d), showing that the Bi2Se3 NSS sample has a surface area of 67.83 m2·g−1 and a total pore volume of 0.478 cm3·g−1, combining both mesopores and macropores. Such a highly porous nanostructure may allow the versatile use of the Bi2Se3 NSS, for example, in catalysis, gas sensing, energy storage, and drug delivery. High optical absorption capability in the NIR range is essential for PTT agents.34,35 Ultraviolet−visible−near-infrared (UV−vis−NIR) absorption spectrum of the Bi2Se3 NSS sample is presented in Figure 1e. Given that Bi2Se3 is a typical lightharvesting material, the highly porous structure of the NSS is, therefore, anticipated to induce an improved absorbance due to the pore-induced multiple scattering and absorption together with increased specific surface area. An ideal reference to explore the influence of the porous structure is to compare the absorbance of the NSS with a solid Bi2Se3 sphere of the same size (Sref). We employed the solid Bi2Se3 nanoplate without 9648

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Figure 2. TEM images of HeLa cells (a) before and (b) after incubation with Bi2Se3 NSS for 6 h. Scale bar is 500 nm. (c) Time-dependent cellular uptake measured by ICP-MS. (d) Cell viability after incubation with the Bi2Se3 NSS at different concentrations for 24 h. (e) Hemolytic percent of red blood cells incubated with the Bi2Se3 NSS at various concentrations for 4 h. (f) Fluorescence images of HeLa cells upon NIR irradiation with or without the Bi2Se3 NSS. Diameter of the laser spot was ∼2.0 mm. Scale bar is 1000 μm. (g) Cell viability after treated by the Bi2Se3 NSS at various concentrations and irradiation times.

comparison, the temperature of the culture medium was barely changed (1.3 °C) upon an identical irradiation. Given that the body temperature is normally 36−37 °C, it is rational that tumors can be easily heated to 43 °C or even over 50 °C at a much lower concentration within 5 min under irradiation. Meanwhile, the high thermal contrast produced by the Bi2Se3 NSS suspensions (Figure 1g) proved the high IR thermal imaging properties, which were further confirmed by the in vitro thermal imaging results on human HeLa cervical cancer cells (Figure S6). In addition, the photothermal conversion efficiency (η) of Bi2Se3 NSS was determined to be ∼31.1% (Figure 1h,i), which is comparable to that of the Bi2Se3 nanoplate (∼34.7%, Figure S7) and much higher than that of widely used Au nanoshells (13%) and Au nanorods (21%).37 Moreover, it was found that the temperature increase (Figure S8) and optical absorption (Figure S9) of the Bi2Se3 NSS remained unchanged after five repeated irradiation cycles,

Likely due to the surface coating, the Bi2Se3 NSS exhibited high stability in water (Figure S4) and various physiological solutions, such as 1640 culture medium, fetal bovine serum (FBS), and phosphate-buffered saline (PBS), without any macroscopic aggregates. The high stability was further supported by the linearly increasing absorbance with increased Bi2Se3 concentrations (Figure S5). To investigate the photothermal performance, the Bi2Se3 NSS suspensions at different concentrations (0, 10, 50, 100, and 200 μg·mL−1 in culture medium) were irradiated with a NIR laser (808 nm, 1.0 W·cm−2) for 5 min, and the temperature was monitored by a digital thermometer. As shown in Figure 1f, the photothermal effect of the Bi2Se3 NSS was pronounced, and the temperature increase followed a concentration- and irradiation duration-dependent manner. For example, at 200 μg·mL−1, the temperature was increased from 21.6 to 68.4 °C after only 5 min irradiation. As a sharp 9649

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Figure 3. (a) In vitro CT images and (b) CT values of the Bi2Se3 NSS aqueous dispersions at the indicated concentrations (in mg·mL−1). (c) In vivo three-dimensional CT images of a mouse before (Pre) and after (Post) i.t. injection of the Bi2Se3 NSS (10.0 mg·mL−1, 200 μL). The CT contrast at the tumor site was largely enhanced after injection. (d) In vitro MSOT images and (e) MSOT intensity of the Bi2Se3 NSS suspension at indicated concentrations (in mg·mL−1). (f) Representative MSOT images of tumors on a mouse before (Pre) and 1 and 6 h after i.v. injection with the Bi2Se3 NSS (2.0 mg·mL−1, 200 μL). The tumor area is marked by the circles. (g) Blood circulation and (h) biodistribution of the Bi2Se3 NSS after i.v. injection, determined by Bi3+ concentrations in tissue lysates.

indicating a high photothermal conversion stability, which is important for the PTT applications. The strong NIR absorbance and the high photothermal conversion efficiency and stability make the Bi2Se3 NSS highly promising as a PTT nanoagent. In Vitro Cytotoxicity and Photothermal Effect. Efficient cellular uptake and internalization by cancer cells are paramount for therapeutic effects. TEM was used to study the intracellular localization and distribution of the Bi2Se3 NSS in cancer cells. As expected, for the control results on HeLa cells without incubation with the NSS, only a typical cellular micromorphology with obvious cellular microstructures was observed (Figure 2a). After incubation with the Bi2Se3 NSS for 6 h, it is revealed that the Bi2Se3 NSS were localized in the vesicles within the cytoplasm (Figure 2b), consistent with the cellular uptake via endocytosis reported previously for other NPs.38,39 Furthermore, quantification of cellular internalization was performed by inductively coupled plasma mass spectrometry (ICP-MS) (Figure 2c), demonstrating that the Bi2Se3 NSS could be effectively internalized by cancer cells, and such uptake was steadily improved with the NSS concentration and incubation duration. It is noted that, at the same concentrations, the cellular uptake by human umbilical vein endothelial cells (HUVEC, a normal cell line) was much less than that by HeLa cells, which is likely attributed to the specific alteration of cancer cells, such as improved cell membrane

permeability due to less glycoproteins and higher metabolic rate.40−43 Biocompatibility is a prerequisite for biomedical applications of nanoagents. Cell counting kit-8 (CCK-8) assay was employed to evaluate the potential cytotoxicity of the Bi2Se3 NSS to HUVEC and HeLa cells, respectively. As shown in Figure 2d, after incubation with the Bi2Se3 NSS for 24 h, no significant cytotoxicity (cell viability is above 90%) was observed on either cells at a NSS concentration as high as 400 μg·mL−1. Moreover, to further examine the biocompatibility of the Bi2Se3 NSS, we also investigated their influence on hemolysis of red blood cells, using deionized (DI) water (positive) and PBS (negative) as the control groups. As demonstrated in Figure 2e, negligible hemolysis was observed at all indicated concentrations of the Bi2Se3 NSS, indicating excellent blood compatibility. All these results suggest the good biocompatibility of the Bi2Se3 NSS. To evaluate the photothermal effect in vitro, HeLa cells were incubated with the Bi2Se3 NSS suspensions (40.0 μg·mL−1, 1.0 mL per well) and then exposed to an 808 nm laser (1.0 W· cm−2, diameter of the laser spot: ∼2.0 mm) for 0, 3, 5, and 10 min. Fluorescence staining using propidium iodide (PI) and calcein acetoxymethyl ester was carried out for visualization of the live cells (vivid green) and dead cells (red). Compared with the negative control group (no laser and no Bi2Se3 NSS), HeLa cells treated with either the NIR laser or the Bi2Se3 NSS alone 9650

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Figure 4. (a) In vivo IR thermal images of mice i.v. injected with PBS or the Bi2Se3 NSS upon laser irradiation. (b) Tumor temperature variation for different groups as the function of laser irradiation time. (c) Tumor growth curves for different treatments. (d) Average tumor weights collected from the mice 12 days after treatments. Error bars correspond to mean ± standard deviations. (e) Representative photos of tumors 12 days after treatments. (f) Histology analysis of the mice major organs 12 days after various treatments.

plus irradiation. Especially, at the concentration as low as 20 μg· mL−1, the cell viability upon 0, 5, and 10 min irradiation was 106.6 ± 10.4, 65.6 ± 7.9, and 14.7 ± 3.6%, respectively. These experimental data indicate that the Bi2Se3 NSS has an efficient PTT effect on cancer cells. In Vitro and In Vivo CT and MSOT. Multimodal imaging, especially the combination of CT and MSOT, could provide improved feasibility, precision, and efficacy for diagnosis and, thus, has stimulated considerable interest in the biomedical field toward imaging with higher sensitivity and resolution.27 We first investigated the potential of the Bi2Se3 NSS for CT imaging. The CT contrast of the Bi2Se3 NSS was evaluated by acquiring the phantom images of the NSS aqueous solutions at different concentrations in vitro (Figure 3a). It was found that the CT images of the Bi2Se3 NSS dispersions became progressively brighter along with the increasing concentrations, indicating the incremental CT signal intensity at increased NSS concentrations. The calculated CT value (Figure 3b) reveals a linear increase with the NSS concentration, showing a X-ray absorption coefficient of 35.7 HU·mL·mg−1, largely higher than

exhibited vivid green, and no red fluorescence of PI was observed (Figure 2f), indicating that such treatments did not compromise cell viability. Interestingly, HeLa cells treated with the Bi2Se3 NSS and NIR laser irradiation can induce cell death dependent on the irradiation time. While only a small amount of HeLa cells were killed upon 3 min irradiation, the cells within the irradiation zone were effectively destroyed upon 5 min irradiation. When the irradiation was extended to 10 min, obvious cell destruction was further expanded beyond the irradiation zone due to heat transfer. PTT efficacy of the Bi2Se3 NSS was further evaluated on the HeLa cells incubated with the Bi2Se3 NSS suspensions at different concentrations and irradiated for 0, 5, and 10 min using a CCK-8 assay. As seen from Figure 2g, the therapeutic effect on HeLa cells was dependent on the dose and the irradiation time. The cell viability was barely decreased after exposure to the Bi2Se3 NSS suspensions without laser irradiation, confirming the low toxicity of the nanoagents again. In contrast, the cell viability decreased rapidly with increased concentrations for the treatment of the Bi2Se3 NSS 9651

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ACS Nano that of iopromide (16.4 HU·mL·mg−1), a widely used clinical CT contrast agent.27 The high performance of the Bi2Se3 NSS can be attribuated to the higher X-ray attenuation coefficient of Bi [Bi (5.74) > Au (5.16) > Pt (4.99) > Ta (4.3) > I (1.94 cm2· kg−1) at 100 keV).23 To investigate the performance on in vivo CT imaging, tumor-bearing Balb/c mice were intratumorally injected with the Bi2Se3 NSS (10.0 mg·mL−1, 200 μL), anesthetized, and then imaged using a small animal X-ray CT imaging system 20 min after the injection. Compared with the images before injection, where there was no tumor signal, a strong tumor contrast was observed after injection (Figure 3c), confirming the excellent performance of the Bi2Se3 NSS for CT imaging. We also investigated the capacity of the Bi2Se3 NSS as a MSOT contrast agent. As shown by the in vitro results in Figure 3d,e, the Bi2Se3 NSS can induce intense optoacoustic signals, strictly following a concentration-dependent manner. For in vivo MSOT imaging, the mice were i.v. injected with the Bi2Se3 NSS (2.0 mg·mL−1, 200 μL) and imaged by a MSOT system at different time intervals. No obvious signal in the tumor region was seen before or 1 h after the injection, while high MSOT contrast of the tumor was observed 6 h after the injection (Figure 3f), suggesting the promising potential of the NSS on real-time MSOT imaging for tumor diagnosis. Also, the timedependent MSOT contrast indicates the gradual and efficient passively targeted accumulation of the NSS at the tumor site after systemic administration, making it possible to precisely track the agent’s accumulation and evaluate the therapeutic efficacy. The passive tumor site accumulation of the Bi2Se3 NSS after systemic i.v. injection can be explained by the enhanced permeability and retention effect of tumors.44 To examine the blood circulation and biodistribution of the Bi2Se3 NSS, blood samples and major organs were collected after i.v. injection to tumor-bearing mice and then solubilized by HNO3/H2O2 solution (HNO3/H2O2 = 2:1) for ICP-MS measurement of Bi element at selected time points. As shown in Figure 3g, the Bi2Se3 NSS showed a relatively long in vivo blood circulation, showing a high Bi3+ level of ∼5.33% ID/g retained in blood even 24 h after the injection. Moreover, consistent with the MSOT results, a high level of Bi element was detected in the tumors (Figure 3h) and organs of the mononuclear phagocyte system, such as liver and spleen. The tumor uptake reached as high as ∼9.66% ID/g 12 h post-injection. Such prolonged circulation half-life and preferential tumor accumulation are favorable for in vivo applications. In Vivo Photothermal Therapy. Encouraged by the good photothermal property, strong in vitro PTT efficacy, and efficient passively targeted accumulation of the Bi2Se3 NSS to the tumor, we then performed the in vivo PTT experiment. Four groups of tumor-bearing mice were used (three in each group), including (1) PBS only (control group), (2) PBS + laser, (3) Bi2Se3 only, and (4) Bi2Se3 + laser. NIR laser irradiation (10 min, 1.0 W·cm−2) was carried out after i.v. injection with PBS or Bi2Se3 NSS (5.0 mg·mL−1, 80 μL) for 12 h. During irradiation, an IR thermal camera was used to monitor the temperature variation of tumors at selected time points (Figure 4a). Consistent with the in vitro results (Figures 1g and S6), it was revealed that high-contrast real-time infrared thermal imaging can be realized by the Bi2Se3 NSS in vivo and can provide feedback on tumor temperature to guide the PTT process. After 10 min irradiation, there was only a slight tumor temperature increase (∼6.2 °C) for the PBS + laser group. In

contrast, the tumor temperature of Bi2Se3 + laser group rapidly increased. Within 10 min laser irradiation, the tumor temperature reached a plateau of ∼62.3 °C (Figure 4b), which is sufficient to induce hyperthermia and eradicate the tumor immediately. The large temperature increase not only confirms the powerful PTT effect of the Bi2Se3 NSS in vivo but also demonstrates their efficient passive accumulation to tumor sites after i.v. injection. Tumors of the Bi2Se3 + laser group were absent after the treatment, with only black scars observed at the initial tumor sites, whereas tumors after the other treatments kept the natural growth trend. The tumor size was measured every 2 days, and the tumor volume was plotted as the function of time (Figure 4c and Figure 5). It is revealed that tumors in mice injected

Figure 5. Representative mice photos before treatment and 8 days after different treatments.

with the Bi2Se3 NSS were efficiently inhibited after the irradiation, but the tumors treated by either the Bi2Se3 NSS without irradiation or 10 min irradiation alone grew quickly within 12 days, indicating that neither the Bi2Se3 NSS by itself nor laser irradiation at this power density had noticeable antitumor function. Figure 4d,e shows the mean tumor weight and the tumor photograph of each group. Among all the groups, the lowest mean tumor weight was found with the Bi2Se3 NSS + laser group. These results demonstrate the strong PTT therapeutic effect of the Bi2Se3 NSS for in vivo antitumor treatments. Since the potential toxicity of the nanoagents in vivo is of importance for practical applications, the behaviors of Balb/c nude mice after the Bi2Se3 NSS i.v. injection were carefully monitored. No observable sign of toxic effects, including drinking, eating, grooming, exploratory behavior, activity, urination, and neurological status, took place within the entire experimental period. The body weights of these mice also showed no noticeable difference upon various treatments (Figure S10). Moreover, 12 days after the treatments, major organs (i.e., heart, liver, spleen, lung and kidney) were stained with hematoxylin and eosin (H&E). Neither organ damage nor inflammatory lesion in the major organs was observed (Figure 4f), further confirming no/very low in vivo toxicity at least at the tested dose of the NSS. Although further studies on the long-term potential toxicity to animals are needed, these in vivo results demonstrate the safety of the Bi2Se3 NSS for medical application. Drug Loading and Release. Given the intrinsic highly porous nanostructure and large fraction of void space, it is 9652

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Figure 6. (a) UV−vis−NIR absorption spectra of the Bi2Se3, free DOX, and the Bi2Se3 + DOX samples. (b) Fourier transform infrared spectra of the Bi2Se3, the Bi2Se3 + DOX, free DOX, and gelatin. (c) DOX release from the Bi2Se3 + DOX triggered by NIR irradiation at various pH and selected time points. (d) Fluorescence images of HeLa cells treated with the Bi2Se3 NSS and the Bi2Se3 + DOX for various durations. DOX is in red, and cell nuclei are in blue. Scale bar is 100 μm.

after passive accumulation of the drug-loaded NSS in acidic tumor microenvironment and then be taken by cancer cells into endosomes and lysosomes (pH ∼5−6), thus minimizing the unwanted risk to normal cells during the circulation in vivo after i.v. injection. We then investigated the cellular uptake and internalization of the Bi2Se3 + DOX for the intracellular delivery of DOX. HeLa cells were incubated with the Bi2Se3 and Bi2Se3 + DOX dispersions for 1 and 6 h, respectively. The aggregated dot-like red DOX fluorescence signals in HeLa cells treated with the Bi2Se3 + DOX clearly indicated the effective internalization of the Bi2Se3 + DOX (Figure 6d). Moreover, the cellular uptake was time-dependent, which was much more significant when incubation was extended from 1 to 6 h. These results indicate that the Bi2Se3 NSS may act as an effective drug carrier for thermo-chemotherapy. We further investigated the antitumor effect of the combined PTT and chemotherapy (thermo-chemotherapy) of the Bi2Se3 + DOX in vitro. As shown in Figure S13, upon 10 min NIR irradiation, higher antitumor efficacy was revealed from the thermo-chemotherapy of the Bi2Se3 + DOX at all the tested concentrations compared to that from PTT (the Bi2Se3 NSS) or chemotherapy (free DOX) alone. For instance, when treated with the Bi2Se3 + DOX (30 μg·mL−1) plus 10 min irradiation, the killing efficacy was as high as ∼86.3%. In comparison, the killing efficacy for the Bi2Se3 NSS and free DOX at the same concentration was only ∼49.6 and ∼60.9%, respectively. The largely improved therapeutic effect by thermo-chemotherapy can be attributed to the increased cellular uptake and drug release caused by the local hyperthermia. At low concentrations, the heat induced by the irradiation may not be

important to examine the loading capability of the Bi2Se3 NSS as a drug delivery vehicle. As a proof-of-concept model, DOX, a widely used chemotherapeutic drug, was loaded into the porous Bi2Se3 NSS by using the nanoprecipitation method, followed by gelation coating. The color of the Bi2Se3 NSS was changed from pure black to purple black after DOX loading. As shown in Figure 6a, free DOX showed a characteristic peak at 480 nm, which shifted to 520 nm for the Bi2Se3 + DOX, mainly due to the transformation of DOX from hydrophilicity to hydrophobicity and the strong interaction between the −NH2 group of DOX and the Bi2Se3 NSS. The loading of DOX was further confirmed by Fourier transform infrared spectra (Figure 6b). In addition, a remarkable quenching of the DOX fluorescence (∼79%) was induced in the Bi2Se3 + DOX compared with free DOX (Figure S11). The optimal DOX loading content of the Bi2Se3 + DOX was calculated to be ∼19.1%, which is as high as 6-fold that on Bi2Se3 nanoplates.31 The drug release from the Bi2Se3 + DOX was first investigated in PBS solutions at pH 7.4, 5.5, and 4.5 upon a 5 min NIR irradiation. As shown in Figure 6c and Figure S12, DOX could be efficiently released from the Bi2Se3 + DOX. The rapid increase of local temperature generated from the Bi2Se3 + DOX may increase thermal vibration to weaken the interactions between DOX and the Bi2Se3 NSS, hence accelerating the DOX dissociation. Importantly, such DOX release can be maintained over multiple irradiation cycles and is closely dependent on the pH value. Unlike the limited release in neutral environment (pH 7.4), remarkable release burst occurred under acidic pH values (5.5 and 4.5). Such properties may benefit drug release at the tumor sites, as the Bi2Se3 + DOX can release more drug under targeted NIR irradiation 9653

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Figure 7. Comparative investigation of in vivo combined therapy. (a) In vivo IR thermal images of tumor-bearing mice i.v. injected by PBS, free DOX, Bi2Se3 NSS, or Bi2Se3 + DOX upon NIR laser radiation. (b) Temperature variation of tumors for different groups as the function of irradiation time. (c) Tumor growth curves in different groups. Error bars correspond to standard error of mean values. (d) Average weights and (e) representative tumor photos collected from the mice in different groups. Error bars correspond to mean ± standard deviations. (f) Body weight of mice of different groups.

sufficient to eradicate HeLa cells, but it can enhance membrane permeability and cellular metabolism and hence benefit drug uptake and trigger more drug release. Therefore, the incomplete killing of cancer cells by PTT alone could be nicely solved by the combined therapy in this way.45 In Vivo Combined Therapy. We also performed in vivo experiments to demonstrate the effect of combined PTT and chemotherapy using the Bi2Se3 + DOX. HeLa tumor-bearing mice were randomly divided into five groups (n = 3): (1) untreated group as the control; (2) PBS/laser group; (3) free DOX/laser group; (4) Bi2Se3/laser group; (5) Bi2Se3 + DOX/ laser group. After i.v. injection with 100 μL of PBS, free DOX, the Bi2Se3 NSS, or the Bi2Se3 + DOX (2.0 mg·mL−1, the Bi2Se3 + DOX containing the same equivalent DOX concentration as the group of free DOX), the tumors were irradiated by the 808 nm laser (1.0 W·cm−2, 20 min) and monitored by the IR thermal camera. The results in Figure 7a once again revealed that high-contrast real-time infrared thermal imaging can be realized by the Bi2Se3 NSS and the Bi2Se3 + DOX in vivo. For the groups treated with the Bi2Se3 or the Bi2Se3 + DOX, the tumor site became brighter upon extended irradiation, demonstrating the efficient passive accumulation of the Bi2Se3

or the Bi2Se3 + DOX at the tumor sites. After 20 min irradiation, there was only slight tumor temperature increase for the PBS- or free-DOX-treated group (ΔTPBS ∼ 6.0 °C, ΔTfree DOX ∼ 5.3 °C). In contrast, for the Bi2Se3 or the Bi2Se3 + DOX group, the temperature rapidly increased to 48−49 °C upon identical irradiation, which is sufficient to induce hyperthermia as well as to trigger drug release. After the treatments, the size of the tumors was measured every 2 days and the variation of tumor volume with time was plotted. The tumors of the control, PBS, or free DOX group grew quickly over the following days (Figures 7c and S14). This indicates that treatment with free DOX at such a low dose barely has antitumor function. For the Bi2Se3/laser group, the tumors were noticeably inhibited in the first few days but grew uncontrollably afterward. Compared with the control, the tumor inhibition ratio of the PTT group was ∼66.0%. Remarkably, the tumor growth of the group treated by the Bi2Se3 + DOX upon laser irradiation was effectively inhibited, showing an inhibition ratio as high as ∼94.4% in the entire experimental period. Figure 7d,e shows the mean tumor weights and the tumor photographs of each group. Among all the groups, the mean tumor weight in the group treated by the 9654

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Figure 8. (a) H&E images of major organs collected from different mice groups. (b) Serum biochemistry analysis of mice after various treatments. The results show mean and standard deviation of liver function markers (alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT)), albumin (ALB), total bilirubin (TBIL), renal function markers (creatinine (CREA), uric acid (UA), and blood urea nitrogen (BUN)), creatine kinase (CK), and total protein (TP).

combined therapy was the lightest. These in vivo results directly demonstrate the considerably higher inhibition efficacy and synergistic therapeutic effect of thermo-chemotherapy compared to that with either PTT or chemotherapy alone. In Vivo Toxicity. We then carefully investigated the potential toxicity of the thermo-chemotherapy in vivo after i.v. injection with the Bi2Se3 + DOX, such as monitoring the behaviors and measuring the body weights of Balb/c nude mice. No obvious abnormal signs in drinking, eating, grooming, exploratory behavior, activity, urination, or neurological status took place during the experimental period. Moreover, no apparent variation of the mice body weight was observed for all treatments (Figure 7f), indicating low/no systemic toxicity of the combined therapy in vivo. In addition, mice were sacrificed for necropsy after the treatments. Major organs were collected for histology analysis. As shown in Figure 8a, no evident damage or inflammatory lesion can be found in all of the organs, further confirming no noticeable toxicity in vivo at least at our tested dose. Finally, we used serum biochemistry assay to further assess the potential toxicity in vivo. Three groups of mice, including (1) untreated healthy mice, tumor-bearing mice treated with (2) PTT alone or (3) thermo-chemotherapy, were anesthetized with isoflurane and then sacrificed for blood collection. As

shown in Figure 8b, all measured parameters fell within normal ranges. As mentioned above, the biodistribution results (Figure 3h) indicated that the NSS accumulated considerably into liver and spleen. The values of liver function markers (AST, ALP, ALT) of the group treated with the Bi2Se3 NSS or the Bi2Se3 + DOX, however, were similar to those of the healthy mice, indicating no disturbance of the treatments on the liver function. Moreover, renal function markers (UA, CREA, BUN) and other measured biochemical parameters (ALB, TBIL, CK, TP) of the treated group also fitted in the normal variation ranges, further confirming no noticeable renal dysfunction or other side effects caused by the thermo-chemotherapy of the Bi2Se3 + DOX. Our in vivo results indicate that the Bi2Se3 NSS can act as a competent drug carrier for efficient thermochemotherapy with no detectable toxicity at our tested dose, showing great potential for biomedical applications.

CONCLUSIONS We have designed and synthesized multifunctional Bi2Se3 nanoagents with spherical sponge morphology. The strong NIR absorbance, high photothermal conversion efficiency, and conversion stability together with high X-ray attenuation ability give the NSS powerful capabilities for highly effective therapeutic PTT, high drug loading capability, as well as 9655

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Finally, the obtained product (the Bi2Se3 + DOX) was centrifuged at 4 °C and washed with DI water. For the DOX release experiments, Bi2Se3 + DOX (500 μg) was dispersed in PBS (2.0 mL) at different pH (7.4, 5.5, 4.5) and then stirred in the dark at 37 °C. At predetermined time intervals, the solutions were centrifuged at 4 °C (12 300 rpm, 5 min), and the supernatants were replaced with 2.0 mL of fresh PBS at the same pH. For the photothermally triggered DOX release, the Bi2Se3 + DOX dispersions were irradiated for 5 min at the selected time intervals. After centrifugation, the supernatants were analyzed to determine the released DOX. In Vitro Photothermal Ablation. For qualitative study, HeLa cells (2.5 × 105 cells) were incubated with the Bi2Se3 NSS dispersion (40.0 μg·mL−1 in fresh RPMI-1640 medium, 1.0 mL per well) for 12 h. Then the cells were exposed to an 808 nm laser (1.0 W·cm−2, diameter of the laser spot ∼ 2.0 mm) for 0, 3, 5, and 10 min. To study the photothermal cytotoxicity, HeLa cells (1 × 104 cells) were incubated with the Bi2Se3 NSS dispersions (0, 2, 5, 10, 20, 40, 60 μg·mL−1 in culture medium) for 12 h and then irradiated (1.0 W·cm−2) for 0, 5, and 10 min. The cell viability was analyzed using the CCK-8 assay with triplicate measurements. CT Imaging. The Bi2Se3 NSS dispersions (in PBS) with different concentrations (0, 0.22, 0.88, 3.5, 14.0, 56.0 mg·mL−1) were prepared in Eppendorf tubes (1.5 mL) and swirled for 2 min before imaging. In vitro experiments were carried out by small mice X-ray CT (80 mA, 100 kV, slice thickness of 0.625 mm). For in vivo CT, tumor-bearing nude mice were intratumorally injected with the Bi2Se3 NSS (10 mg· mL−1, 200 μL) prior to imaging. Twenty minutes later, the mice were anesthetized and imaged (270 μA, 80 kV, slice thickness of 154 μm). CT images before and after injection of Bi2Se3 NSS were analyzed using amira 4.1.2. MSOT Imaging. For in vitro measurement, the Bi2Se3 NSS dispersions at different concentrations (0.02, 0.04, 0.08, 0.12, 0.16, 0.24 mg·mL−1 in PBS) were loaded into agar gel cylinders with a diameter of about 1.0 cm. The in vitro MSOT was carried out on the InVision 128 MSOT system (iThera Medical, Germany). After the reconstruction of the MSOT images, MSOT signals were calculated by averaging over the region of interest for each sample. For in vivo MSOT, the Bi2Se3 NSS suspension (2.0 mg·mL−1 in PBS, 200 μL) was intravenously injected into the tumor-bearing Balb/c mice and analyzed using the MSOT InVision 128 system. A water heating system was used to maintain the body temperature of the mice to 37.5 °C. Evaluation of PTT and Thermo-chemotherapy in Vivo. When the tumors grew to 8−10 mm in diameter, the mice were intravenously injected with PBS (80 μL) or the Bi2Se3 NSS (5.0 mg·mL−1) for the PTT experiments and with PBS, free DOX, Bi2Se3 NSS, or the Bi2Se3 + DOX (2.0 mg·mL−1, 100 μL) for the thermochemotherapy experiments. After 12 h, the mice were anesthetized and the tumors were irradiated with/without the 808 nm laser (10 min for PTT, and 20 min for the combined experiments). During the period of treatment, the tumor temperatures were monitored by an IR thermal imager (Ti25, Fluke, USA).

high-contrast CT, MSOT, and IR thermal imaging. Meanwhile, the product shows high biocompatibility and low/no toxicity in vitro/in vivo. Such a single-component theranostic nanoagent coordinated multiple imaging and therapeutic functions may have substantial potential for clinical antitumor applications.

MATERIALS AND METHODS Materials. Polyvinylpyrrolidone (PVP, Mw ≈ 10 000), bismuth nitrate pentahydrate (Bi(NO3)3·5H2O, ≥99.99+%), sodium hydroxide (NaOH, ≥97.0%), ethylene glycol (EG, >99%), D-(+)-glucose (C6H12O6), and doxorubicin hydrochloride (DOX-HCl, 98%) were purchased from Aladdin. Sodium selenite (Na2SeO3, ≥97.0%) was obtained from Shenyang Huadong reagent. Gelatin (gel strength ∼300 g Bloom) and glutaraldehyde solution (50% in H2O) were purchased from Sigma-Aldrich. Calcein acetoxymethyl ester, 4′,6-diamidino-2phenylindole, and propidium iodide were provided by Dojindo Laboratories. Unless otherwise stated, all reagents were of analytical grade and employed without further purification. Synthesis of the Bi2O3 Nanoparticles. The Bi2O3 NPs were fabricated according to the literature33 with small modifications. In a typical experimental procedure, Bi(NO3)3·5H2O (0.364 g) was completely dissolved in HNO3 solution (1 mol·L−1, 10 mL) and mixed with NaOH (0.108 g), PVP (1.2 g), and EG (50 mL). The mixture was transferred into a stainless steel autoclave, heated to 150 °C, and maintained for 3 h and then naturally cooled to room temperature. Finally, the obtained milk-white product was precipitated by centrifugation and washed several times with DI water. Synthesis of the Bi2Se3 Spherical Sponge. The Bi2O3 NPs were utilized as the bismuth precursor and template via the hydrothermal process method. In brief, Na2SeO3 (0.2 g) and D-(+)-glucose (0.614 g) were dissolved in DI water (30 mL), and then the Bi2O3 NPs dispersion (10 mL, DI water) was added. The mixed solution was transferred into an autoclave and hydrothermally treated at 150 °C for 12 h. The obtained product was collected by centrifugation and thoroughly washed several times with DI water. The precipitate was subsequently resuspended with sonication and subsequently dialyzed in a Float-A-Lyzer G2 dialysis tube (approximate molecular weight cutoff 8000−10 000 Da, Spectrum Laboratories Inc.) against DI water to remove excess reagents and small molecular byproduct. Finally, the Bi2Se3 NSS was further washed with absolute ethyl alcohol twice through centrifugation, followed by vacuum drying in an oven at 50 °C for 12 h. Measurement of Photothermal Performance. The Bi2Se3 NSS dispersion at various concentrations (0−200 μg·mL−1, 1.0 mL) was irradiated by the 808 nm laser (1.0 W·cm−2, 5 min). The solution temperature was measured every 1 s by a thermocouple microprobe (Q50.5 mm). To deduce the photothermal conversion efficiency (η), the Bi2Se3 NSS suspension (40 μg·mL−1, 1.0 mL) was irradiated with the 808 nm laser (1.0 W·cm−2) until the solution temperature was steady. The laser was turned off, and the system temperature was cooled to the ambient temperature. The solution temperature was measured every 20 s. To investigate the photothermal conversion stability, the Bi2Se3 NSS dispersion (0.2 mg·mL−1, 1.0 mL) was irradiated for 5 min, and then the laser was switched off and the dispersion allowed to naturally cool. DOX Loading and Release Experiments. Loading of DOX into the Bi2Se3 NSS was accomplished by using the nanoprecipitation method, followed by forming a temperature-induced gelation-capping layer to prevent the release of loaded drug molecules at neutral pH.46 In a typical experiment, the Bi2Se3 NSS (8.0 mg) was dissolved in PBS (35 mL) and mixed with DOX-HCl (2.0 mg·mL−1, 5.0 mL) and NaOH solution (1 mol·L−1, 75 μL). The reaction mixture was then stirred in the dark for 24 h. After the addition of gelatin (200 mg), the obtained solution was allowed to react at 50 °C for 6 h. Afterward, the reaction solution was quickly poured into DI water (4 °C, 100 mL). Precipitates were then centrifuged and washed with DI water. After being redispersed into PBS (pH 7.4, 40 mL), glutaraldehyde solution (wt % = 1%, 2.0 mL) was applied at 4 °C to cross-link the gelatin.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b05427. Additional information as noted in the text (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest. 9656

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