Rapid Degradation and High Renal Clearance of Cu3BiS3 Nanodots

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Rapid Degradation and High Renal Clearance of Cu3BiS3 Nanodots for Efficient Cancer Diagnosis and Photothermal Therapy in vivo Jing Liu, Pengyang Wang, Xiao Zhang, Liming Wang, Dongliang Wang, Zhanjun Gu, Jinglong Tang, Mengyu Guo, Mingjing Cao, Huige Zhou, Ying Liu, and Chunying Chen ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.6b00745 • Publication Date (Web): 25 Mar 2016 Downloaded from http://pubs.acs.org on March 26, 2016

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Rapid Degradation and High Renal Clearance of Cu3BiS3 Nanodots for Efficient Cancer Diagnosis and Photothermal Therapy in vivo Jing Liu†, Pengyang Wang†,‡, Xiao Zhang†, Liming Wang†,*, Dongliang Wang†, Zhanjun Gu†, Jinglong Tang†, Mengyu Guo†, Mingjing Cao†, Huige Zhou†, Ying Liu†,* and Chunying Chen†,* †

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety &

CAS Center for Excellent in Nanoscience, Beijing Key Laboratory of Ambient Particles Health Effects and Prevention Techniques, National Center for Nanoscience and Technology of China and Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China ‡

College of Materials Science and Opto-Electronic Technology, University of

Chinese Academy of Sciences, Beijing, China

*Corresponding Authors： E-mail address: [email protected]. Tel: +86 10 82545560; fax: +86 10 62656765. E-mail addresses: [email protected] or [email protected].


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ABSTRACT: A key challenge for the use of inorganic nanomedicines in clinical applications is their long-term accumulation in internal organs, which raises the common concern on the risk of adverse effects and inflammatory responses. It is thus necessary to rationally design inorganic nanomaterials with proper accumulation and clearance mechanism in vivo. Herein, we prepared ultra-small Cu3BiS3 nanodots (NDs) as a single-phased ternary bimetal sulfide for photothermal cancer therapy guided by multispectral optoacoustic tomography (MSOT) and X-ray computed tomography (CT) due to excellent X-ray attenuation coefficient of Bi element. We then monitored and investigated their absorption, distribution, metabolism and excretion as well. We also used CT imaging to demonstrate that Cu3BiS3 NDs can be quickly removed through renal clearance, which may be related to their small size, rapid chemical transformation and degradation in acidic lysosomal environment as characterized by synchrotron radiation-based X-ray absorption near edge structure (SR-XANES) spectroscopy. These results reveal that Cu3BiS3 NDs act as a simple but powerful “theranostic” nanoplatform for MSOT/CT imaging-guided tumor ablation with excellent metabolism and rapid clearance that will improve safety for clinical applications in the future. KEYWORDS: Cu3BiS3 nanodots, Multispectral optoacoustic tomography, X-ray computed tomography, Photothermal therapy, Clearance, Degradation, Chemical transformation


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Nanotechnology is a rapidly expanding field and many nanoparticles are being tested as candidates for multifunctional, molecular and physically targeted contrast agents for clinical diagnosis and therapy.1-3 Materials at the nanometer scale have very different physical and biochemical properties that make nanomaterials attractive in diagnostics and therapy.4 This approach, referred to as “theranostics”, holds great promise for cancer diagnosis and therapy, and “theranostic” nanomedicine is an important direction in which nanotechnology is progressing at this time.1, 5, 6 Even more attractive is combining different modalities (targeting, imaging, and therapy) in one particle to make multifunctional platforms that can both detect and treat tumors.7, 8

Such complex nanosystems are the basis of intelligent detect-kill platforms.

Recently many such nanoparticles have been applied in active fields of research such as drug delivery,2,

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cancer diagnostics10-12 and therapeutics13-15. However, a most

worrisome problem has been safety. Most nanoparticles can accumulate in the vital organs, leading to acute toxicity, a long-term inflammatory response, or even fibrosis and cancer. Therefore, a general but elusive goal to strive for is to ensure the safety of multifunctional “theranostic” contrast agents. In addition to safety, these agents must be effective. For effective phototherapy, nanoparticle platforms must absorb in an appropriate therapeutic window of laser irradiation. As the tissue is mostly transparent to near-infrared NIR (700 ~ 1400 nm) light, especially the second NIR window (1000 ~ 1400 nm), NIR absorption by the contrast agent results in deeper penetration and eliminates the absorption of laser light by the tissue around the target region. Thereby, the absorption band of the nanomdicine should be best in the NIR region. Therefore, the best position for the absorption band of a nanomedicine is in the NIR region. Up to now, a large number of nanoparticles absorbing in the first biological window (700 ~ 950 nm), including gold nanospheres, nanorods, nanocages and others7, 8, 13, 16, 17 as candidates for diagnosis and therapy by many research groups, including our previous publications have been developed.18-21 However, less attention has been paid to the development of nanoprobes responding to the second near-infrared region (NIR-II). Among these investigations, imaging-guided photothermal therapy (PTT) has drawn considerable


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attention because a specific amount of photoenergy is delivered directly into the tumor mass without causing systemic side effects, and using NIR laser light-generated heat to destroy tumors is highly efficient. Furthermore, imaging techniques not only provide PTT with guidance for integrating diagnosis, monitoring therapeutic response, and treating cancers with higher specificity and sensitivity 22-25, but also as a favorable “weapon” to track nanoparticle metabolism in vivo and in real time. For tracking nanoparticle metabolism, it is especially useful to have a three-dimensional (3D) imaging technology for the whole body, such as X-ray computed tomography. CT as a noninvasive diagnostic imaging technique has been widely used in clinics because of its high resolution and deep penetration of tissues and organs.26-28 Moreover, it can provide 3D structural details of internal tissues. However, each type of imaging mode displays not only distinct advantages but also intrinsic limitations – in the case of CT, poor soft tissue contrast, low throughput capacity, limited accessibility, and exposure to ionizing radiation.28,

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Thus the

necessary information cannot be obtained from a single modality; it is better to combine two imaging techniques that can take advantage of each other’s best features while avoiding the shortcomings of both. Ultrasound (US) imaging is real time and can likely be well complemented by multispectral optoacoustic tomography (MSOT).12,

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MSOT is a hybrid imaging modality that combines the spectral

selectivity of molecular excitation by laser light with the high resolution of ultrasound detection, thereby overcoming the limitations of pure optical and ultrasound-based methods. In photoacoustic imaging, a short NIR laser pulse incident on the target tissue causes heating and thermal expansion, resulting in generation of acoustic waves that are capable of being detected by an acoustic transducer.31 The use of NIR to create the laser pulse allows for deeper penetration into the tissue. MSOT carries the promise of a real-time tool for in vivo imaging, and can be used to guide effective surgery and remove completely metastasized regions. In conclusion, multimodal imaging is obligatory to diagnostics and therapeutics in cancer treatment for their ability of exhibiting detailed and exact information about the occurrence, development and migration of tumors associated with the microenvironment. 19, 32-34


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A key challenge for the application of inorganic nanomedicines in clinic is their long-term accumulation in internal organs, which introduces the risk of toxicity and inflammatory responses.35 Historically, the US Food and Drug Administration has required that agents injected into the human body be cleared completely in a reasonable period of time. Therefore, it is essential to have a nanoprobe that has both “theranostic” capabilities and optimal in vivo metabolism.36,

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Until now, most

research has agreed that the biodistribution, clearance, and pharmacokinetics of nanoprobes depend mainly on their particle sizes and surfaces.37-41 It has been reported that nanoparticles smaller than 5.5 nm can be rapidly removed by renal clearance, and sizes ranging from 15 to 50 nm will be absorbed by the reticuloendothelial system (RES), while those larger than 50 nm can be partially cleared by liver metabolism.42-45 Of course, smaller particles are more easily and rapidly removed by renal filtration. The actual situation, however, is far more complex than this. The metabolism of nanoparticles not only depend on their sizes and surfaces, but also their morphology and components. For example, carbon materials can be gradually cleared by the kidney and liver when the size was up to 10-30 nm.46 Even so, their toxicity is a significant problem.46-48 Fortunately, ultrasmall and rapidly degradable nanoparticles which can be metabolized to remove them quickly from the body will serve as safe platforms and are likely to be transformative for multimodal imaging and tumor therapy in vivo. In order to achieve all these goals, we here demonstrate the potential use of ultrasmall Cu3BiS3 nanodots (NDs) as a novel, simple but powerful, and most importantly, safe contrast agent, which can be used not only for multimodal imaging with CT/MSOT/IR (infrared thermal) and for selective photothermal therapy of cancer using a near-infrared low-energy laser, but can also be removed from the whole body quickly through renal clearance and degradation in an acidic environment. Bi-based nanoparticles have been proposed as CT contrast agents (Bi2S3 nanodots, Bi2Se3 nanoplates, Bi2S3 nanorods and some ternary semiconductor nanomaterials) due to their high X-ray attenuation coefficient (Z =83, K-edge value=90.5 keV), cost effectiveness, and low toxicity.19, 49-52 Ultrasmall Cu3BiS3 NDs are well suited for


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imaging and treating tumors, by reason of their many copper vacancies, exhibition of NIR absorption as with self-doped copper chalcogenides, and their much stronger absorption coefficient (> 105 cm-1) than other Cu-S based materials like CuInS2 and Cu2ZnSnS4.52, 53 In addition, they transform absorbed energy into efficient PT, and are accompanied by other phenomena such as photoacoustic (PA) waves.52 Other attractive features include photostability, small diameter, good biocompatibility and low toxicity. But their most important characteristic is their rapid degradation and elimination from the body for visceral accumulation (mainly in the liver and spleen) is often a big drawback for inorganic nanoparticles. Finally, ultrasmall Cu3BiS3 NDs were applied as a single-phase bimetal sulphide rather than as nanocomposites as before,54-57 the composites tended to have large scales, complex synthesis processes, and difficulty being metabolized. This single-phase formulation allows combining the capability of multimodal imaging and therapy without any functional components, but with a powerful “theranostic” platform, to provide the potential use of a novel, simple but powerful, and safe agent in the clinic. RESULTS AND DISCUSSION Characterization of Cu3BiS3 NDs. Typical transmission electron microscope (TEM) image of the Cu3BiS3 NDs as synthesized showed a diameter about 10 nm and the hydrodynamic size of the nanodots after tween-functionalization is about 21.95 nm that is larger than the size of NDs in the TEM image. (Figure 1A; Figure S1, supporting information). The structure of the NDs as synthesized was confirmed by X-ray diffraction (XRD) as the result of Cu3BiS3 phase (JCPDS No. 71-2115) (Figure S2B). To improve their biocompatibility and dispersibility in physiological solutions, the synthesized Cu3BiS3 NDs were functionalized with a commercially available surfactant, Tween-20. Tween not only enabled Cu3BiS3 NDs to have good dispersibility, but also protected them from nonspecific protein absorption. To determine the success of OA-Cu3BiS3 functionalized with Tween-20, Fourier transform infrared (FTIR) spectra was conducted to confirm (Figure S2C). As a result, Tween-20-modified Cu3BiS3 showed good dispersibility in various physiological


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liquids (Figure S2A). As shown in Figure 1B, the aqueous solution of Tween-Cu3BiS3 NDs was black, and Cu3BiS3 NDs had a broad absorption in the NIR region since Cu3BiS3 is an indirect band gap semiconductor with a fundamental band gap energy estimated to be about 1.5–1.7 eV, which expands the light absorption to the NIR region.52,

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Interestingly, our Cu3BiS3 NDs as synthesized had a broad absorption from 800 nm to 1300 nm with a peak at 1100 nm (Figure 1B). This absorption range fits well with the most common lasers used to investigate photothermal effects, with wavelengths of 808 nm, 915 nm, and 980 nm. Light that excites the peak absorbance at 1100 nm would penetrate tissues more deeply while generating lower temperature elevation from absorption by water acting as an NIR-II photothermal agent.58-60 Thus, an 1100 nm laser would provide potential photothermal therapy with Cu3BiS3 NDs. The high NIR absorption of Cu3BiS3 NDs motivated us to explore their NIR photothermal properties. In our experiment, we used an 880 nm laser to provide strong evidence for their potential use as an NIR-II “theranostic” agent in the clinic. To examine the photothermal properties of Cu3BiS3 NDs, we measured temperature trends in aqueous dispersions of different concentrations of Cu3BiS3 NDs (0.025, 0.05, 0.1, 0.25, 0.5 and 1 mg mL-1) under the irradiation of an 880 nm NIR laser (0.75 W cm-2); pure water was used as the control (Figure 1C). The temperature of Cu3BiS3 ND aqueous dispersions increased strongly with the increase of concentration and irradiation time, while the temperature of DI water exhibited little change. Thus Cu3BiS3 NDs have a good ability to convert NIR energy into thermal energy efficiently. For instance, the temperature increased by about 40 ℃ within 8 min with a concentration of 1 mg mL-1 (Figure S2D). In addition, it is nice to see that Cu3BiS3 NDs have good photostability, which is critical for their future application (Figure S3): their absorption had no obvious decrease after continuous irradiation for 30 min. Thus Cu3BiS3 NDs exhibit a strong absorbance in the NIR region, especially in the NIR-II range, possess high photothermal conversion efficiency, and have the potential for applications in PTT and MSOT. In vitro Biological Characterization. Next, with biomedical applications in mind, we


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assessed the cytotoxicity of Cu3BiS3 NDs. Because cells must be able to internalize these NDs for effective cytotoxicity, we used dark-field scattering microscopy to detect their uptake. Of the two different concentrations (50 and 100 µg mL-1) of Cu3BiS3 NDs incubated for the same time (24 h), the higher one exhibited more internalization (insert one shows the control), indicating the efficient uptake of Cu3BiS3 NDs by cells (Figure 1E and F). This also indicated that Cu3BiS3 NDs may be a good dark-field scattering imaging probe due to its high spatial resolution, good photostability, and none-blink.61,

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Next, the cell viability was assessed with a

standard cell counting kit-8 (CCK-8) assay. Cu3BiS3 NDs without irradiation exhibited little toxicity in human breast cancer Michigan Cancer Foundation -7 (MCF-7) cells over a wide range of concentrations (Figure 1D). This is a crucial factor in determining their suitability for applications in experimental small animals and ultimately in the clinic. In vitro Photothermal Therapy. To evaluate the photothermal ability of Cu3BiS3 NDs after an NIR irradiation in vitro, an Annexin V-FITC/PI kit was used together with flow cytometry. Figure 2A shows the flow cytometry graphs of cells incubated with 20 and 50 µg mL-1 Cu3BiS3 NDs for 24 h, with and without different power densities of laser irradiation. The Annexin V-FITC emission signal was plotted on the x-axis, while the PI emission signal was plotted on the y-axis. The quantities of living cells, early apoptosis cells, and late apoptosis/necrosis cells were determined by the percentages of Annexin V-/PI-, Annexin V+/PI-, Annexin V+/PI+ and Annexin V-/PI+. Few apoptotic or necrotic cells were observed in the NIR (1.5 W cm-2) only or Cu3BiS3 NDs (50 µg mL-1) only groups. In contrast, when cells were treated with both Cu3BiS3 NDs and NIR irradiation, the cell death rate increased rapidly with increased concentration and power density. According to the statistics (Figure 2B), when cells were treated with 50 µg mL-1 Cu3BiS3 NDs and NIR irradiation (1.5 W cm-2), the late apoptotic and necrotic cell count was about 84.2%, while the early apoptotic cells were about 11%. These results revealed that PTT treatment induced significant MCF-7 cell death. X-ray Computed Tomography for Angiogenesis. Since angiogenesis can serve as a


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marker for cancer recognition, one method of curing solid tumors is to attack the vessels supplying them rather than the cancer cells themselves.63 Therefore, we next employed the Cu3BiS3 NDs as prepared as an efficient CT contrast agent for angiogenesis imaging. These NDs make a good contrast agent because their Bi atoms have a strong X-ray attenuation capability and their HU values are much higher than the clinical iodine-based CT contrast agent (iopromide), which was shown in our previous work.19 For this purpose, Cu3BiS3 NDs dispersed in physiological saline were intravenously injected into 4T1 tumor-bearing mice (200 µL, 10 mg mL-1). We first conducted CT imaging at 5 min post injection. Interestingly, the vasculature was clearly observed when it filled with Cu3BiS3 NDs, including: 1, pulmonary vein plexus; 2, vena cava; 3, nephric vein plexus; 4, vena epigastrica; 5, tumor surface vascularity; and 6, tumor microvessel (TMV) (Figure 3, Video S1 in the Supporting Information). The tumor vessels possessing irregular and superficial spatial distribution spring from the branches of the abdominal veins. It is well-known that tumor vessels grow rapidly, resulting in the tortuous, leaky, and irregular architecture of tumor angiogenesis. This defective vasculature induces the enhanced permeability and retention (EPR) effect, which allows the NDs to accumulate in the tumor site preferentially.64-66 In addition, the incidence of vessel-associated disease such as embolism is increasing in the population. Obtaining such a 3D image of tumor-associated vasculature will also be useful for designing an effective precision nanomedicine for vascular disease diagnosis. Multispectral Photoacoustic Tomography in vivo. Multispectral optoacoustic tomography (MSOT) is a hybrid imaging modality that detects absorbed photons ultrasonically through the photoacoustic effect with high sensitivity and specificity. It overcomes the depth- and time-spatial resolution problem in optical imaging and the lack of sensitivity and poor soft tissue contrast in CT imaging; moreover, MSOT provides real-time monitoring.30,

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Herein, we used Cu3BiS3 NDs as an MSOT

contrast agent to monitor tumors in mice because of the NDs’ high NIR absorption and efficient NIR photothermal conversion abilities. In order to trace Cu3BiS3 NDs in the tumor region, 200 µL of 2 mg mL-1 Cu3BiS3 NDs was i.v. injected into the tail


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vein and the photoacoustic (PA) signals at the tumor sites were recorded as images at different time intervals. As shown in Figure 4, before injection, a weak signal was observed in the tumor region. At 2 h post injection, signals in the tumors were remarkably enhanced, indicating the gradual homing of Cu3BiS3 NDs to the tumor region; moreover, signals lasted for up to 24 h in tumors, although weaker than those at 2 h. All the above clearly demonstrate that not only can the Cu3BiS3 NDs effectively target the tumor site, but they also undergo a metabolism process in the tumor region. Photothermal Therapy in vivo. Once we had demonstrated that Cu3BiS3 NDs are excellent diagnostic agents, the nice PTT effect in vitro motivated us to investigate their photothermal ablation of cancer cells in vivo. When the tumors reached approximately 100 mm3, the 4T1 tumor-bearing mice were randomly divided into four groups: (a) PBS, (b) Laser only, (c) Cu3BiS3 NDs only and (d) Cu3BiS3 NDs (i.v. injection) + laser (n=6). Tumor-bearing mice of groups (c) and (d) were i.v. injected with 20 mg kg-1 Cu3BiS3 NDs, which is much lower than the reference dosage of 75 mg kg-1,52 while groups (b) and (d) were exposed to an 880 nm laser (1 W cm-2) for 10 min. The temperature change of the tumor region was recorded by an IR thermal camera at specific time intervals (Figure 5A). As expected, the temperature change of the tumor surface under NIR irradiation of group (d) rapidly increased from 34 ℃ to 55 ℃, which was capable of inducing sufficient hyperthermia to kill tumor cells. For the mice injected with PBS only, the surface temperature of the tumor site had a small change of about 6 ℃ during the whole irradiation process (Figure 5B). The tumor volume of each mouse was measured every other day after the treatments described above. Figure 5D shows the tumor volume change of each group as a function of time. It was found that group (d) showed efficient tumor inhibition and complete ablation without recurrence. In contrast, tumors of groups (a), (b) and (c) showed indistinguishable growth rates, suggesting that laser irradiation or Cu3BiS3 ND injection alone did not affect tumor development. As a result, the surviving proportion of groups (a), (b) and (c) decreased rapidly in the range from days 36 to 49 post-injection because of the huge tumors and metastasis. In striking contrast, mice of


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group (d) had only one mouse die, at day 45 (Figure 5C). Coincidentally, each group had a mouse die on the 45th day post-injection, which enabled us to directly compare the state of their major tissues at this time. As shown in Figure 5E, the liver and lungs (white arrows show metastasis sites) were stained with Bouin’s liquid and photographed. Pulmonary and hepatic metastasis sites can be seen distinctly in the three control groups, compared with none in group (d), this was also demonstrated by Hematoxylin and Eosin (H&E) staining. Moreover, the spleen photographs showed obvious enlargement in groups (a), (b) and (c). These results reveal that Cu3BiS3 NDs could be an excellent PTT agent with complete tumor destruction and inhibition of metastasis. We also investigated toxicity in mice not injected with tumor cells. In a long-term (45 day) investigation of toxicity, the liver, lung and spleen, and other major organs including the heart and kidney were stained with H&E. As shown by the histological examination (Figure 5E and S4), there was no tissue damage or adverse effect associated with the administration of the Cu3BiS3 NDs. Moreover, no obvious signs of abnormal mouse behavior or weight loss were noticed (Figure S5). In addition, we performed the blood biochemistry analysis with the mice treated with the Cu3BiS3 NDs (20 mg kg-1) at the different time points of 10 min, 2 h, 6 h, and 24 h (Figure S6). The biochemical parameters including amino transferase (ALT), amino transferase (AST), total bilirubin (TBIL), blood urea nitrogen (BUN), uric acid (UA), creatinine (CREA), creatine kinase (CK). We emphasize ALT, AST, and BUN because they are closely related to the functions of the liver and kidney of mice. Meaningful changes were not observed in all parameters after 10 min and 2 h. The NDs presented appreciable liver accumulation after 2 h, but did not cause acute injury at this time point. After 6 h treatment, ALT and BUN significantly increased in the NDs-treated mice. After 24 h, ALT and BUN has changed back to a normal level indicating that the Cu3BiS3 NDs caused slight and transient damage for liver and kidney after 6 h, which can be recovered after 24 h. This is because of the rapid degradation and high renal clearance of Cu3BiS3 NDs, which is consistent with biodistribution and clearance patterns of the NDs (Figure 6B and C). These preliminary results prove that Cu3BiS3


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NDs at the dose given are not noticeably toxic in vivo. Metabolism and Degradation. For a potential medical application, it is also critical to investigate the clearance of Cu3BiS3 NDs. If nanomaterials are not metabolized or cleared quickly through the liver and kidneys, they will be cleared only by slow first-pass extraction from the reticuloendothelial system (RES) clearance processes, resulting in potential health hazards due to accumulation and thus limiting their clinical application.36 For example, like other inorganic nanomaterials, Bi2S3 nanorods in our previous research accumulated in the liver and spleen.19 The advantage of Cu3BiS3 NDs is that they not only have a relatively short time scale for renal clearance, but they are also degraded and metabolized out of the whole body more thoroughly, thus reducing the toxicity. As shown in Figure 6A (Video S2), we observed their clearance by CT with 3D imaging of the whole body. Ten min after they were intravenously injected (100 µL, 10 mg mL-1), Cu3BiS3 NDs were observed remarkably clearly in the liver and bladder, suggesting that they are quickly cleared, just like small gold nanoparticles, through renal filtration and urinary excretion. 38 As time went on, the signal from the liver became stronger, and the Cu3BiS3 NDs began homing in on the tumor site. At 6 h post-injection, the liver attained its strongest signal and we began to see the spleen as well. At the same time, the bladder was filled with Cu3BiS3 NDs. By 24 h after the injection, there was almost no signal in the liver and little in the spleen, thus little residual in the whole body. To further verify our CT imaging results, we then studied the in vivo behaviors of Cu3BiS3 NDs by measuring Bi element levels in various organs, urinary and feces using inductively coupled plasma mass spectrometry (ICP-MS), which is consistent with the CT imaging results (Figure 6B and C). Following this, the excellent clearance ability of Cu3BiS3 NDs motivated us to further investigate the process of their metabolism, to see whether there was another mechanism besides renal clearance. Since we noticed that the color of Cu 3BiS3 NDs in DI water changed over the course of 2 months (shown in Figure S7), we guessed that they might been oxidized. It is well known that Cu+ is not a stable form in biological systems: It is easily oxidized and reacts with ligands like Cl -, S2-, thiols,


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peptides, and proteins. In order to detect the chemical species in the metabolic process, we employed SR-XANES, a powerful technique to characterize elemental speciation in complicated samples. We tested two environments: Cu3BiS3 NDs in DI water solution, exposed to air for two months to detect its oxidation level, and an acidic environment (pH = 4.5) to simulate lysosomes. On the basis of copper K-edge XANES, nine reference samples for copper were used to fit the XANES results of the test samples: Cu foil (elemental Cu), Cu2S, CuS, CuCl, CuCl2, Cu(CH3COO-)2, Cu2O, CuO and Cu citrate (for the Cu+ and Cu2+ forms). We observed that the shapes of the XANES spectra were quite different for various copper reference samples. For example, the peak shapes for Cu2S (a flat peak) and Cu(CH3COO-)2 (a sharp peak) were quite distinct. The shape of the XANES peak for Cu3BiS3 NDs was similar to Cu2S, as proved by the least-squares fitting results (the original Cu3BiS3 NDs had a composition similar to Cu2S), as shown in Figure 6D. As time went on, XANES results and the data fitting clearly showed changes in the copper chemical forms (Figure 6E, Table 1), indicating that Cu3BiS3 NDs were gradually transformed into Cu-COO- species in the artificial lysosomal fluid (ALF). As shown in Table 1, there was 40% Cu2S form in the sample at 30 min, and none after 24 h, indicating a rapid degradation of Cu3BiS3 NDs in ALF. Moreover, the CuCl ratio fluctuated, suggesting that CuCl existed as an intermediate product between Cu2S and Cu-COO- forms. Consequently, there was little CuS (2.5%) or CuCl2 (6.7%) form and almost of them existed in Cu-COO- (90.8%) species in the 24 h ALF solution. Moreover, after kept in DI water for two months, the sample consisted of various forms of 46.7% Cu-COO-, 12.6% CuS, 20.6% Cu2S and, not surprisingly, 20% CuO from being oxidized in a non-acidic environment. All these results indicate a possibility for degradation and metabolism of Cu3BiS3 NDs in an acidic environment, suggesting a potential application of Cu3BiS3 NDs as a purgeable nanomedicine in multimodal imaging-guided PTT. Conclusion In summary, compared with reported “theranostic” nanocomposites, which require


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complicated synthesis processes, we have successfully prepared a single-phased ternary bimetal sulphide nanomaterial, Cu3BiS3 NDs, that serves as a powerful, degradable and safe nanomedicine for MSOT/CT/IR imaging-guided photothermal therapy. The characteristics of Cu3BiS3 NDs as synthesized include good dispersibility, biocompatibility, and photostability, the large X-ray attenuation coefficient of bismuth, and strong NIR-II absorbance, which can not only be the basis of MSOT/CT/IR imaging, but also provide a potential application for NIR-II therapy in the future. As we expected, ultra-small Cu3BiS3 NDs are capable of being metabolized from the whole body by renal filtration and rapid degradation in acidic environments such as the lysosomes, as identified with 3D CT imaging and SR-XANES. Consequently, Cu3BiS3 NDs exhibit an excellent response to the challenge of the optimal in vivo metabolism problem, with low toxicity. Overall, although more details are waited to be investigated, Cu3BiS3 NDs are a simple but powerful and degradable “theranostic” agent, with the potential to be used for cancer diagnosis and precise ablation of primary tumors while inhibiting metastasis. This may provide some insight into a new way to exploit inorganic nanomedicine. MATERIALS AND METHODS Materials. Calcein-AM (CA), Annexin V-FITC, and propidium iodide (PI) were supplied by Dojindo Laboratories in Japan. Deionized (DI) water was obtained by an 18-MΩ (SHRO-plus DI) system. All the chemicals, unless specified otherwise, were acquired from Sigma-Aldrich and used without further purification. Characterization. 0.5 mmol of bismuth chloride and 1.5 mmol of copper acetylacetonate were dispersed in 4 mL of oleic acid (OA) and 10 mL of 1-octadecene (ODE) in a 50 mL two-necked flask equipped with a condenser. The solution was stirred and heated to 220 °C and then kept at that temperature for 30 min. The entire procedure was carried out under a continuous flow of N2. Next a solution of 3 mmol of sulfur in 5 mL of oleylamine (OM) was swiftly injected into the flask. The solution turned black immediately after injection, and the nanocrystals were allowed to grow for 10 min at this temperature. The reaction was then quenched with 20 mL of cold


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hexane. The precipitates were removed after centrifugation, and then ethanol was added dropwise until the supernatant became turbid. The mixture was centrifuged again, the supernatant discarded, and the precipitated nanocrystals redispersed in hexane. In order to transform the hydrophobic Cu3BiS3 NDs to hydrophilic ones, the synthesized nanocrystals were modified with Tween-20. In this procedure, 100 mL of Tween-20 was added to a 50 mL flask which contained 20 mg of Cu3BiS3 NDs and 10 mL of hexane. After 1 h ultrasonic treatment at room temperature, 20 mL of deionized water was added and the dispersion was kept in a water bath at 70 oC until the solution became clear. The Tween-20-modified Cu3BiS3 NDs were obtained by centrifugation. Cell Viability Assay. Michigan Cancer Foundation – 7 (MCF-7) cells were maintained in complete RPMI 1640 medium (WISENT Inc.), with 10% (v/v) fetal bovine serum (FBS), at 37 °C, 5% CO2 and 10% humidity. Cell viability was determined with a Cell Counting Kit-8. Firstly, 3000 cells per well were plated into 96-well microplates (Costar, Corning, NY). After incubating (37 °C, 5% CO2 and 10% humidity) for 24 h, the culture medium was removed and replaced with the complete medium with a series of concentrations of Cu3BiS3 NDs (0, 10, 20, 50, 80, 100 and 150 μg mL-1) for 24 h (n = 5). Meanwhile, wells unexposed to samples were regarded as controls. Finally, a mixture of the tetrazolium reagent (from the Cell Counting Kit-8) and the complete medium (1:10) was added into every well of the 96-well microplates (150 μl per well). Then cell viability was calculated at 450 nm (reference wavelength 650 nm) by an Infinite M200 microplate reader (Tecan, Durham, USA). Dark-field Imaging for Cellular Uptake. MCF-7 cells were attached to glass-bottom dishes for 24 h. The culture medium was removed and replaced with new medium containing the final concentrations of 50 and 100 μg mL-1 Cu3BiS3 NDs and co-incubated for 24 h. Afterwards, cells were washed with PBS three times and then fixed with formalin for 15 min. The light scattering images were obtained using a microscope (Nikon Eclipse Ti-S, Nikon Instruments Inc., USA) with a high numerical aperture dark field condenser.


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Photothermal Therapy Evaluation in vitro. In order to detect the photothermal therapy effect of the Cu3BiS3 NDs, MCF-7 cells were seeded into 6-well plates for 24 h with a density of 5 × 104. Meanwhile, they were divided into four groups (control, laser only, Cu3BiS3 NDs only, and Cu3BiS3 NDs + laser (n=3)). When cells grew to 60% confluence, the four groups were treated with the above conditions. The laser only group was irradiated with a power density of 1.5 W cm-2 for 8 min. The Cu3BiS3 NDs only group was incubated with a final concentration of 50 μg mL-1 for 24 h. The Cu3BiS3 NDs + laser group was treated with Cu3BiS3 NDs (20 and 50 μg mL-1) for 24 h, after this, irradiated for 8 min with a power density of 0.75 and 1.5 W cm-2, respectively. Cells without any treatment were regarded as controls. Finally, all the cells were collected, washed three times with PBS, dyed with an Annexin V-FITC/PI kit, and then detected by flow cytometry to make the mode of cell death clear. Animals. Female BALB/c nude mice and their food were purchased from Beijing Vitalriver Experimental Animal Technology Co. Ltd., housed under the standard conditions at a 20 ± 2 oC room temperature and 60 ± 10% relative humidity with a 12 h light/dark cycle. Deionized (DI) water was obtained by an 18-MΩ (SHRO-plus DI) system. Animals were acclimated to this environment for 5 days before treatment. All procedures used were compliant with the local animal ethics committee. CT Imaging in vivo. A 100 µL mixture of 1 × 107 murine 4T1 cells and growth factor (1:1) was inoculated in the right hind leg of each mouse. When tumor vessels were visible, CT imaging was performed on a small mouse X-ray CT (Gamma Medica-Ideas). We regarded the pre-injection mice as controls. After we finished scanning the controls, a 200 µL Cu3BiS3 ND (10 mg mL-1) suspension was i.v. injected into the tail vein of the tumor-bearing BALB/c nude mice. After 5 min, the mice were imaged with a whole body 360o scan. Meanwhile, another mouse was i.v. injected with 100 µL 10 mg mL-1 Cu3BiS3 NDs, and after control scanning was finished, images of the whole body were taken at different time points (10 min, 2 h, 6 h and 24 h). Imaging parameters were as follows: field of view: 78.92 mm × 78.92 mm and 84.56 mm × 84.56 mm; slice thickness: 154 μm and 165 μm; effective pixel size: 50 μm; tube voltage: 80 KV and 60 KV; tube current 270 μA and 250μA. The


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reconstruction was done by the Filtered Back Projection (FBP) method. The reconstruction kernel used a Feldkamp cone beam correction and SheppLogan filter. The CT images were analyzed using amira 4.1.2. MSOT Imaging in vivo. A 100 µL mixture of 1 × 107 murine 4T1 cells and growth factor (1:1) was inoculated in the backside of each mouse. About seven days later, the tumor volume was about 200 mm3 for MSOT imaging. Then a 200 µL Cu3BiS3 ND (2 mg mL-1) suspension was intravenously injected into the tail vein of each tumor-bearing BALB/c nude mouse. After this, the mouse was scanned from 680 nm to 900 nm at different time intervals (10 min, 2 h and 24 h) with the MSOT (MSOT inVision 128, iThera medical, Germany) to collect signals. The main experimental parameters were 10 wavelengths for each slice, with the region of interest (ROI) being 25 mm. We regarded the photoacoustic signals before injection as the controls. Photothermal Therapy Evaluation in vivo. 4T1 tumor-bearing mice were prepared by injecting a 100 µL mixture of 1 × 107 murine 4T1 cells into the right hind leg of each BALB/c female nude mouse. About seven days later, when the tumor volume reached about 100 mm3, the mice were divided into the same four groups as for the cell experiment above (PBS (PBS i.v. injection), laser only, Cu3BiS3 NDs only and Cu3BiS3 NDs (i.v. injection) + laser (n=6)). The 4T1 tumor-bearing mice were i.v. injected with Cu3BiS3 NDs (20 mg kg-1) and irradiated with an 880 nm NIR laser for 10 min. The change of temperature in the tumor region was recorded by an NIR camera. Tumor growth and body weight were measured in the following days. The tumor sizes were measured by a caliper and calculated as follows: V = ab2/2 where V (mm3) is the volume of the tumor, and a (mm) and b (mm) are the tumor length and tumor width, respectively. At 45 days post-injection, every group had a mouse die, and their tumors and major tissues were dissected. The liver and lungs were stained with Bouin’s liquid and other organs were photographed. Blood Biochemistry and Pathology. The analysis of blood biochemical examination was carried out using blood collection by removal eyeball in the different treat mice above. 1 mL of blood was collected from mice, after 3 h standing in room temperature,


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separated by centrifugation into cellular and plasma fractions. Major organs were harvested, fixed in 10 % neutral buffered formalin, processed routinely into paraffin, stained with H&E and pathology were examined by a microscope. ICP-MS for Bi Element Quantification. To study biodistribution of Cu3BiS3 NDs in balb/c mice, 200 µL of Cu3BiS3 NDs (2 mg mL-1) suspension were i.v. injected form the tail vein. At 2h, 6 h and 24 h post administration, mice were sacrificed, organs dissected and weighed. Tissue samples from above were stored at -20 oC before analysis. For ICP-MS experiments, the above each sample was added 5 mL of HNO3, transferred to flasks and seal for pre-digestion overnight, next day, 3 mL of 30 % H2O2 was added to each flask. The flasks were placed onto a hot plate and maintained at 150 oC for 3 h until digestion was complete, and cooled to room temperature. The solution in each flask was diluted to 3 g with 2% HNO3. A series of Bi standard solutions (0, 0.5, 1, 5, 10, 50, and 100 ppb) were prepared with the above solution. Both standard and test solutions were measured by inductively coupled plasma mass spectrometry (ICP-MS, Thermal Elemental X7, Thermal Fisher Scientific Inc, USA). Characterization of Copper Chemical Forms. Cu-based compounds including Cu citrate, Cu foil, Cu2O, CuO, Cu2S, CuS, and Cu(CH3COO-)2 were used as references. Before XANES measurement, all the reference samples were dried and pressed into a uniform pellet adhering to a piece of 3M tape. XANES spectra of Cu K-edge were mainly recorded on an SSRF beamline BL-14W1 in China. The transmission XANES mode was used to measure XANES spectra for the references. Fluorescence XANES mode equipped with a 32-elemental solid detector was used to collect the data for the liquid samples including Cu3BiS3 NDs, Cu3BiS3 NDs in air for two months, and the products of Cu3BiS3 NDs after mixing with ALF. All the liquids were injected into a transparent container to a thickness of 2 mm, which was covered by Mylar film. Data treatment and analysis were based on our previous publications.67, 68 In brief, XANES data were normalized in order to facilitate comparison of spectra from the varied samples, modes, or facilities. The preprocessed data were then analyzed with least-squares fitting (LSF) to calculate the ratio of copper species by using IFEFFIT Athena software (CARS, the Consortium for Advanced Radiation Sources at


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University of Chicago). Conflict of Interest: The authors declare no competing financial interests. Supporting Information Available: Additional figures and movies as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Additional figures and movies, including figures of characterization (TEM, SEM, DLS, XRD and FT-IR), stability (UV-vis absorption spectra before and after laser irradiation), degradation (photographs of different time) and safety evaluation (changes of H&E and body weight) of Cu3BiS3 NDs, and CT information movies. Acknowledgements. This work was financially supported by the Ministry of Science and Technology of China (National Basic Research Program 2012CB934000, Major Equipment Program 2011YQ030134, and International Science & Technology Cooperation Program 2013DFG32340 and 2014DFG52500), the National Science Foundation of China (21320102003, 21403043) and the National Science Fund for Distinguished Young Scholars (11425520). We appreciated the kind help and beam time from SSRF beamline BL-14W1 of China

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Holthoff, K.; Witte, O. W. Intrinsic Optical Signals in Rat Neocortical Slices Measured with Near-Infrared Dark-Field Microscopy Reveal Changes in Extracellular Space. J. Neurosci. 1996, 16, 2740-2749.

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Hill, D. J.; Pinion, C. W.; Christesen, J. D.; Cahoon, J. F. Waveguide Scattering Microscopy for Dark-Field Imaging and Spectroscopy of Photonic Nanostructures. Acs Photonics 2014, 1, 725-731.

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Sreejith, S.; Huong, T.T.M.; Borah, P.; Zhao, Y. Organic-inorganic Nanohybrids for Fluorescence, Photoacoustic and Raman Bioimaging. Sci Bull, 2015, 60, 665-678.

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Barreto, J. A.; O'Malley, W.; Kubeil, M.; Graham, B.; Stephan, H.; Spiccia, L. Nanomaterials: Applications in Cancer Imaging and Therapy. Adv. Mater. 2011, 23, H18-H40.

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Liang, R.; Wei, M.; Evans, D. G.; Duan, X. Inorganic Nanomaterials for Bioimaging, Targeted Drug Delivery and Therapeutics. Chem. Commun. 2014, 50, 14071-14081.

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Kievit, F. M.; Zhang, M. Cancer Nanotheranostics: Improving Imaging and Therapy by Targeted Delivery Across Biological Barriers. Adv. Mater. 2011, 23, H217-H247.

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Wang, L.; Li, J.; Pan, J.; Jiang, X.; Ji, Y.; Li, Y.; Qu, Y.; Zhao, Y.; Wu, X.; Chen, C. Revealing the Binding Structure of the Protein Corona on Gold Nanorods Using Synchrotron Radiation-Based Techniques: Understanding the Reduced Damage in Cell Membranes. J. Am. Chem. Soc. 2013, 135, 17359-17368.

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ACS Nano

C

0.5

Absorbance

B

o

0.4

0.3

1 0.5 0.25 0.1 0.05 0.025 H 2O

70

Temperature ( C)

A

60 50 40 30

100 nm

0.2

700

800

900

1000

1100

1200

1300

0

100

200

300

400

500

600

700 (S)

Wavelenghth (nm)

D

E

100

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 32

50 µg/ml

F

100 µg/ml

80 60 40

Control

20

50 µm

0 ctr

10

20

50

80

100

150 (g/ml)

Figure 1. Characterization, cell viability and cellular uptake of Cu3BiS3 NDs. (A) TEM image of as-prepared Cu3BiS3 NDs. (B) NIR absorbance spectra of Cu3BiS3 NDs. Inset: photo of as-prepared Cu3BiS3 NDs solution（5 mg mL-1）in deionized water. (C) The photothermal profiles of Cu3BiS3 NDs solution in deionized water with different concentrations (mg mL-1) under irradiation from an 880 nm laser with a power density of 0.75 W cm-2. (D) Cell viability was detected using a CCK-8 kit. MCF-7 cells were incubated with various concentrations of Cu3BiS3 NDs for 24 h. (E and F) Cellular uptake of Cu3BiS3 NDs determined by dark-field optical microscopy with different concentrations of Cu3BiS3 NDs for 24 h, with the control shown in the inset.


Page 27 of 32

A

50 µg ml-1

PI

Control

1.5 W cm-2

20 µg ml-1 + 0.75 W cm-2 50 µg ml-1 + 0.75 W cm-2

50 µg ml-1 + 1.5 W cm-2

Annexin V 8 6 4 2 0

early apoptotic cell

late apoptotic and necrotic cell 80 60 40 20 0

Percentage of cells (%)

10


B

100 12


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

100

dead cell

80 60 40 20 0

1 -2 -2 1 -2 -2 -2 -2 -2 -2 -2 -2 -2 -2 ol ol -1 ol ntr g ml cm cm cm cm ontr g ml cm cm cm cm ontr g ml cm cm cm cm o C 0  5 W 5 W 5 W .5 W C 0  5 W 5 W 5 W .5 W C 0  5 W 5 W 5 W .5 W 2 1. 2 1. 2 1. 0.7 0.7-1 + 1 .7 0.7-1 + 1 .7 0.7-1 + 1 -1 + 0 -1 + -1 + -1 + -1 + 0 -1 + l l l l l l l l m m m g m m m g m ml gm  g 0  g 50   g 0  g 50   g 0  g 50  0 0 0 5 2 2 5 2 5

Figure 2. Cell death type of MCF-7 cells after photothermal therapy by flow cytometry. (A) MCF-7 cells were seeded into 6-well plates for 24 h with a density of 5 × 104. When cells grew to 60% confluence, they were treated with different ND concentrations and different power densities of an 880 nm laser. Finally, MCF-7 cells were collected and examined to determine the percentages of early apoptosis and late apoptosis/necrosis cells. (B) Statistical data of percentage of early apoptosis, late apoptosis/necrosis and dead cells under different treatments.


ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

B

A

Page 28 of 32

C

T

6

D 1

2

4

3

5 Figure 3. Angiography by X-ray CT imaging in 4T1 tumor-bearing mice in vivo. (A) CT images of mice before injection. (B and C) CT images of mice 5 min post i.v. injection with 200 µL (10 mg mL-1) Cu3BiS3 NDs (T: tumor). (D) Detailed CT images of the mice 5 min post i.v. injection to highlight the vascular structures: 1, pulmonary vein plexus; 2, vena cava; 3, nephric vein plexus; 4, vena epigastrica; 5, tumor surface vasculaity (from the abdominal venous branch); 6. tumor microvessel (TMV).


Page 29 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Pre

xy

yz xy

10 min

y z high

y

y

x

x

z

z

3 mm

xz xy

2h

3 mm

xz yz xy

y

24 h

yz

y x

x

z

xz

z

3 mm

xz

3 mm

low

Figure 4. 3D optoacoustic imaging in 4T1 tumor-bearing mice in vivo. Optoacoustic images of tumor before and after i.v. injection with 200 µL Cu3BiS3 NDs (2 mg mL-1) at different time points (10 min, 2 h and 24 h).


ACS Nano

A

B 0.5 min

1 min

5 min

8 min

PBS Cu3BiS3

E

PBS Cu3BiS3

55

10 min

Temperature (C)

0 min

Normal control

PBS

Laser only

Cu3BiS3

50

Liver

45

40

35 0

D

100

3

Tumor volume (mm )

C

Propotion surviving (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 32

80 60 40

PBS Laser Cu3BiS3 Cu3BiS3 + laser

20 0

2

4

6

8

10

Laser irradiation Time (min) 2000 1800 1600 1400 1200

Lung

PBS Laser only Cu3BiS3 Cu3BiS3 + laser

1000 800 600 400

Spleen

200 0

0

10

20

30

40

50

60

16

Time (days)

20

24

28

32

Days post injection

Figure 5. Obvious inhibition of tumor growth and metastasis through photothermal therapy in vivo. (A, B) IR thermal images of 4T1 tumor-bearing mice with i.v. injection of Cu3BiS3 NDs (20 mg kg-1) under the 880 nm laser irradiation taken at different time intervals. The laser power density was 1 W cm-2. (C) The surviving proportion of four different groups after various treatments. (D) The tumor growth curves of the four different treatment groups within 32 days post implantation. (E) Photographs and H&E staining of livers, lungs and spleens in the different groups (arrows indicate metastasis).


Cu3BiS3 + laser

Page 31 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

A

Pre

10 min

2h

6h

B

C

D

E

24 h

Figure 6. Metabolism and clearance of Cu3BiS3 NDs. (A) CT images of the whole body before and after i.v. injection with Cu3BiS3 NDs (100 µL, 10 mg mL-1) at different time points of 10 min, 2 h, 6 h and 24 h (blank circles indicate bladder). (B and C) Time-dependent biodistribution and excretion profiles of Cu3BiS3 NDs in balb/c mice. (D) Cu K-edge XANES spectra of reference samples. (E) Cu K-edge XANES spectra of test samples and fitting results.


ACS Nano

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TABLE 1. Time-Dependent Changes in Copper Species in ALF and DI Water Based on Copper K-Edge XANES chemical forms and the ratio (%) Cu2S

CuS

CuCl2

CuCl

Cu-(RCOO-)

CuO

Cu foil

Cu3BiS3 NDs

100

0

0

0

0

0

0

ALF 30 min

40

0

0

22.5

37.5

0

0

ALF 12 h

37.4

0

0

17.9

44.7

0

0

ALF 24 h

0

2.5

6.7

0

90.8

0

0

DI water 2 months

20.6

12.6

0

0

46.7

20

0


Rapid Degradation and High Renal Clearance of Cu3BiS3 Nanodots

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