Renal Clearable Ag Nanodots for in Vivo Computer Tomography

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Renal Clearable Ag Nanodots for in Vivo CT Imaging and Photothermal Therapy Yanyan Cui, Jian Yang, Qunfang Zhou, Ping Liang, Yaling Wang, Xueyun Gao, and Yongtian Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16133 • Publication Date (Web): 23 Jan 2017 Downloaded from http://pubs.acs.org on January 25, 2017

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ACS Applied Materials & Interfaces

Renal Clearable Ag Nanodots for in Vivo CT Imaging and Photothermal Therapy Yanyan Cui,a* Jian Yang,a* Qunfang Zhou,b Ping Liang,b Yaling Wangc, Xueyun Gaoc and Yongtian Wanga* a

Beijing Engineering Research Center of Mixed Reality and Advanced Display, School of

Optics and Electronics, Beijing Institute of Technology, Beijing, China. b

Department of Interventional Ultrasound, Chinese PLA General Hospital, Beijing, China.

c

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of

High Energy Physics, Chinese Academy of Sciences, Beijing, China KEYWORDS silver nanodots, protein, X-ray CT imaging, photothermal therapy, renal clearable

ABSTRACT Albumin-stabilized Ag nanodots (ANDs) are prepared by a one-step biomineralisation method. The highly-crystallized nanodots have ultra-small sizes (approximately 5.8 nm) and robust X-ray attenuation (5.7313 HU per mM Ag). The unlabeled ANDs are directly excreted from the body via the urine after in vivo X-ray computer tomography (CT) imaging application. ANDs could be used as CT imaging agents and effective photothermal therapy agents. The tumor growth inhibition reaches 90.2% after photothermal treatment with ANDs.

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ANDs are promising tools for in vivo CT imaging and clearable near-infrared-triggered theranostic agents.

1. INTRODUCTION Inorganic nanoparticles are used for biomedical applications in terms of imaging and treatment because of the excellent properties of the metal or compound structure.1–3 Theranostic nanoparticles may lead to the simultaneous diagnosis and therapy of one disease at a curable stage. The personalisation of medicine is crucial. Numerous metallic nanoparticles have been developed for in vivo imaging and photothermal therapy, such as bismuth-based nanoparticles (Bi2S3, Bi2Se3),4 CuS,5 MoS2,6 Au or Ag nanoparticles7–9 and some synergistic nanoparticlebased theranostic platforms.10–13 Computer tomography (CT) is a non-invasive imaging modality, which is usually used for identifying and guiding cancer therapy with the help of nanoparticles. Zhang et al.14 developed GO@Ag composite nanoparticles and studied the application of X-ray imaging and chemo-photothermal therapy. Another composite nanoparticle was designed by Wang et al. 15, Au nanocluster assemblies@polyacrylic acid/calcium phosphate nanoparticles, for near-infrared (NIR)/CT dual-mode image-guided cancer chemotherapy. Silver nanoparticles are developed as powerful antimicrobial agents, thereby improving wound healing or therapy.16–18 An agent should have good biocompatibility and can be extracted out of the body in time to satisfy the biomedical application.19 Bovine serum albumin (BSA) is an essential blood protein that contains sufficient amino acid residues, such as sulfhydryl bonds, which have strong coordination with transition metal element. Thus, BSA is always used to improve water solubility and biocompatibility, as well as the colloidal stability of other nanoparticles.20–22 BSA is also used as a natural biotemplate in the bio-mineralisation of various nanoparticles, such as


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quantum dots23, 24 and novel metal clusters.25–31 The small silver nanoparticles embedded in or surrounded by the protein may be a candidate agent for various bio-applications. To the best of our knowledge, there was no study on the application of small silver nanoparticles alone as a multifunctional agent for both X-ray CT imaging in vivo and tumour growth inhibition. Albumin-based bio-mineralisation exhibits multiple advantages such as a facile and reproducible methodology, biocompatibility and good stability. The abundant active groups in the BSA molecular, such as the imidazolyl groups in histidine residues and thiol groups in cysteine residues, could complex well with Ag ions.32 In this paper, a facile method was designed to developed silver nanoparticles in the existence of BSA in one pot. The resulting silver nanodots (ANDs) have small sizes but showed high stability and photothermal conversion capability. The nanodots can effectively cause cell death when used with the NIR laser in vitro. The ANDs were very dense at approximately 5.7313 HU per mM Ag and can be a candidate as X-ray CT imaging contrast agent. The three dimensional (3D) CT imaging proved that ANDs can be extracted out of the body by renal clearance after tail vein or intra-tumour injection. Furthermore, the tumour growth inhibition was 90.2% after the intra-tumour injection of ANDs, thereby showing good biocompatibility. 2. RESULTS AND DISCUSSION A bio-minerazation method was developed to synthesise Ag nanoparticles with BSA. As briefly shown in scheme 1, the aqueous BSA solution was mixed with AgNO3 with vigorous stirring. The NaOH solution was introduced to regulate the pH value of the solution up to above 12. The mixture was kept in 80 °C water bath for 1 h. Some amino acid residues of BSA, such as the hydroxyl groups (tyrosine) and the carboxyl groups (aspartic acid, glutamic acid), could reduce


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the Ag+ ions and collected the atoms to form nanoparticles,33 which were surrounded by or embedded in the protein.

Scheme 1. Schematic illustration of the synthesis of silver nanodots. 2.1 Effects of ratio on the optical and photothermal properties. The ratio of Ag+ ions to BSA determined the corresponding optical and photothermal properties. Serial ratios of Ag+ ions to BSA were studied to optimise the optical and photothermal properties. UV–vis spectra were used to determine the absorption property of these silver nanoparticles with the same molar concentration of elemental silver. As shown in Figure 1A, there was no obvious absorption peaks (300 nm to 750 nm) when the ratio of Ag+ ions to BSA was below 13.2, suggesting that the products were mainly formed with particles subnanometre-sized (smaller than or around 1nm) when the molar quantity of Ag+ ions was insufficient..32 One peak at 420 nm emerged when the ratio of Ag+ ions to BSA reached 26.4, which agree with the surface plasmon resonance of silver nanoparticles, suggesting that bigger silver nanoparticles were formed.34, 35 The peak intensity slightly increased with the increasing ratio of Ag+ ions to BSA, thereby illustrating that the silver nanoparticle grew larger when the molar quantity of Ag+ ions increased.


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The capability for photothermal conversion of the silver nanoparticles with different ratios was determined by measuring the thermal behaviour under the irradiation of 808 nm NIR laser at 1.0 W/cm2. The temperature of the solution with a low ratio (such as 1.32 or 2.64) barely changed (less than 2 °C) under irradiation, whereas the temperature of the solution with a high ratio (such as 26.4 or 52.8) strikingly increased by 14°C within a short period of time as shown in Figure 1B. Therefore, the silver nanoparticles with a ratio of 52.8 were chosen for further study. The small particles could be called silver nanodots (named as ANDs). While irradiated by 808 nm NIR laser, the temperature evolvement of the ANDs with different concentrations were investigated (Figure 1C), thereby implying the concentration- and time-dependent mode of the thermal behaviour. The temperature elevation of the ANDs solution was also studied for two irradiation cycles (Figure S1). The high photo-thermal efficiency and thermal stability presented ANDs as excellent candidates for photothermal therapy (PTT) applications. 2.2 Stability of the ANDs in different pH. The stability of the silver nanoparticles in different pH is critical to the bioimaging quality. We studied the optical absorption property of ANDs in different phosphate-buffered solutions at different pH. The digital photos and relative absorption spectra are shown in Figure S2A, Figure 1D and Figure S2B (expanding absorption spectra). The AND solution in neutral pH retained its excellent water-solubility. The intensity of absorption peak around 400 nm was slightly blueshifted and weaker when the solution was alkaline. However, a few flocculent precipitates appeared when the ANDs solution pH was in the range of 4–6, which is consistent with the isoelectric point (pI) of the BSA, thereby suggesting that ANDs were embedded into or surrounded by the BSA.


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Figure 1. Optical and photothermal properties of the silver nanodots. (A) Absorption spectra and (B) temperature elevation of different ratios of Ag to BSA. (C) Temperature elevation of silver nanodots in different concentration (silver element concentration). (D) Absorption spectra of silver nanodots in different pH buffer solution. 2.3 Size and structure the ANDs. Images from the high-resolution transmission electron microscopy (HRTEM) showed that ANDs had a mono-dispersive nanostructure (Figure 2A). The mean diameter size was 5.80 ± 0.5 nm based on counting approximately 340 particles (Figure 2C). An image of a single AND (Figure 2B) showed a lattice spacing of approximately 2.36 Å, which is close to the distance (2.37 Å) of the [1 1 1] facet of silver in a face-centred cubic. Electron diffraction pattern from the particles also showed that the crystal structure of ANDs was face-centred cubic (Figure S2). To


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further illustrate the phase and the crystallographic structure of the ANDs, X-ray diffraction (XRD) of the sample was processed. The XRD pattern of the ANDs (Figure 2D) showed several broadening peaks positioned at 2θ = 38.17°, 64.64°, 77.44°; these patterns corresponds to the [111], [220] and [311] directions and agrees with the Joint Committee on Powder Diffraction Standards (JCPDS) file (No. 01-1164). The reflection characteristic of the cubic phase further confirmed the results from the HRTEM. ANDs were determined by X-ray photoelectron spectroscopy (XPS; Figures 2E and S4) to reveal the composition of silver element species in the ANDs. Two peaks, 373.98 and 367.94 eV, appeared in the Ag 3d spectrum (Figure 2E), which can be attributed to the Ag 3d3/2 and 3d5/2 of the Ag0 configuration, respectively.36 The peaks indicated the small amount of elemental silver present in the AND oxidation state.


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Figure 2. (A) TEM, (B) HRTEM and (C) statistical graph of the diameter size of ANDs by counting approximately 340 particles. (D) XRD and (E) XPS of silver nanodots. 2.4 Cell cytotoxicity and location of the ANDs. To evaluate the potential biological applications, we firstly studied the cell cytotoxicity of the ANDs. The Human Oral Epithelial Cell Line (KB) cells were chosen as the model cell line. After co-cultured with different doses of ANDs for 24h, the viability of KB cell was determined by a Cell Counting Kit (CCK-8) assay. The ANDs showed little toxicity to the KB cell at the experiment does (Figure 3B); the cell viability was above 85% even with 500 µg/mL concentration. When the ANDs were modified with Fluorescein isothiocyanate (FITC), the onsite AND location could be observed by the inverted fluorescence microscope system. The fluorescence images clearly displayed that FITC-modified ANDs had entered the cytoplasm of KB cells as shown in Figure 3A.


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Figure 3. (A) Fluorescence images of the FITC-modified ANDs and (B) cell cytotoxicity of the ANDs. 2.5 X-ray CT imaging of ANDs in vitro and in vivo. According to the X-ray absorption coefficient rule, to be better absorbing X-rays, the materials should have higher density or high atomic numbers. Few reports have studied the silver nanoparticles for X-ray CT imaging.37 Herein, we evaluated the feasibility of ANDs for CT imaging based on the X-ray absorption characteristics as compared with iopromide (ultravist@370, a commercial CT contrast agent). The X-Ray CT imaging measurements demonstrated that the X-ray CT signal intensity was enhanced with the ANDs concentrations


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(Ag concentrations: from 0 to over 200 mM as dissolved in H2O). The X-ray attenuation (in hounsfield unit (HU)) of each concentration was calculated. As shown in Figures 4A and 4B, monotonic linearity between the HU intensity and the concentration of ANDs was present. The X-ray absorption coefficient was calculated as 5.7313 HU per mM Ag, higher than the commercial CT contrast agent iopromide (4.316 HU per mM I). The results proved that ANDs were a promising candidate contrast agent for 3D X- ray CT imaging. The blood hemolysis of the ANDs was studied to further explore its potential applications in vivo. As shown in Figure S5, no megascopic hemolysis was observed even when the incubation concentration of AND nanoparticles reached 500 µg/mL, thereby indicating the good blood compatibility of ANDs. Further, we studied the performance of ANDs as X-ray CT contrast agents in vivo. The mouse breast cancer cell line (4T1) tumour-bearing mice were anesthetised and intratumourally injected with the ANDs (10 mg/mL, 20 µL). The mice were imaged with an animal micro-CT system. The mean CT value in the tumour region was higher than the control, thereby suggesting that imaging contrast was greatly enhanced by the injected ANDs as shown in Figure 4C. The CT images of the tumour-bearing mice when the ANDs were intravenously injected were taken at varied time points as presented in Figure S6. From the supine position view, the black dash circle marked the position of bladder; ANDs can be observed in the bladder immediately after intratumour administration (Figure 4C, upper row). The signal intensity of bladder decreased with increasing time. Simultaneously, the signal of the tumour site gradually weakened (Figure 4C; lower row). These data directly proved that ANDs can easily be extracted out from the body via urine, thereby decreasing the potential toxicity of ANDs to the body caused by long term organ accumulation. In summary, ANDs can be promising candidate imaging contrast agents for CT imaging in vivo.


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Figure 4. (A) Phantom images of ANDs and the iopromide solution at different element concentrations (Ag concentration: 6.25, 12.5, 25, 50, 100 and 200 mM; I concentration: 1.0, 5.0, 10, 20, 50 and 100 mM). (B) Plot of the CT value with different concentrations of Ag and I. (C) 3D CT images in vivo of mice at different time point after intratumour injection of ANDs. Upper row: supine position; B indicates bladder. Lower row: prone position; T indicates tumour. 2.6. Photothermal therapy applications in vitro and in vivo. Our earlier data suggested that ANDs could enhance the photothermal effect. Furthermore, the feasibility of ANDs as a photothermal agent in vitro was investigated. KB cells were exposed to the NIR laser for 10min after 24 h cocultured with or without ANDs (1.0 mL per well, 50 µg/mL). After that, the cells were stained with special fluorochrome, calcein acetoxymethyl ester (Calcein AM) for viable cells and propidium iodide (PI) for dead, and then visualised under a fluorescence microscope. The fluorescence images clearly indicated that the samples treated with the laser without ANDs or ANDs without the laser did not present green no red fluorescence in


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the whole well as shown in Figures 5A–C. The cells exposed to the NIR laser with ANDs had a high death rate. These results indicated that ANDs can effectively destroy cells when used with the NIR laser. The potential photothermal therapy usage of ANDs in vivo was further studied in addition to the capability of photothermal agent. The mice bearing 4T1 tumour (tumours with the mean diameter of approximately 8−10 mm) were divided into four groups: (1) the control group with PBS (named as M-PBS); (2) the NIR group (named as M-IR); (3) AND group (named as M-AND); and (4) the AND + NIR group (named as M-AND-IR). ANDs were injected into the model mouse for groups M-AND and M-AND-IR. 10 min later, the tumours of the groups M-IR and M-AND-IR were sited under 808 nm laser for 5 min (1.2 W/cm2); meanwhile, the temperature variations of the tumour site were also recorded by an IR thermal camera. As shown in Figures 5D and 5F, the temperature of the tumour surface under the NIR irradiation of group M-AND-IR rapidly increased from approximately 32 °C to 52 °C, which was sufficient to induce hyperthermia and kill tumour cells. By contrast, the temperature of the tumour region for the mice injected with PBS only (group M-IR) increased slightly (approximately 5 °C, Figures 5E and 5F) during the whole irradiation process.


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Figure 5. Fluorescence images of KB cells after various treatments. (A) PBS, with NIR light exposure; (B) ANDs without NIR light exposure; (C) ANDs with NIR light exposure (scale bar: 100 µm). Infrared thermal images of 4T1 tumour-bearing mice at different time point after intravenously injected with (D) ANDs and (E) PBS. (F) Temperature variation of tumours at at different time point of the two groups under laser irradiation. Following the treatments, the tumour volumes were recorded every 2–3 days for 22 days. Figure 6C shows Tumour growth curves for the groups. The control group with (group M-IR) and without (group M-PBS) irradiation showed indistinguishable increasing rates of tumour volumes. In these groups, the tumours of groups M-IR and M-PBS were all grewing rapidly during the experiment time, At 21 days after treatment, the relative tumour volume variations (V/V0) was 10.06 ± 0.96 and 9.28 ± 0.53, respectively, thereby indicating that irradiation had minimal influence on tumour development. The tumour growth rates of ANDs without irradiation (group M-AND, with relative tumour volume variations (V/V0) of 9.14 ± 1.29) are similar to those of the control group, thereby suggesting that ANDs without irradiation slightly


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affect the tumour growth. By contrast, data from ANDs with irradiation (group M-AND-IR) showed conspicuous tumour inhibition; the tumour volumes slightly increased at 21 days after treatment (V/V0 of 1.86 ± 0.44). The body weights of the mice in all groups are presented in Figures 6B and few obvious signs of weight loss or abnormal mouse behaviour were observed. When the experiment ended, all 4T1 tumour-bearing mice were killed. Necropsy was performed on mice from the treatment and control groups. We collected and weighed the tumour tissues. Tumour growth inhibition (TGI) data was obtained from the average weight of the tumours from different groups. Results showed that the TGI was 90.2% for group M-AND-IR, thereby indicating the efficient sensitisation effect of ANDs on PTT in vivo.

Figure 6. (A) Photographs of the tumor tissue. (B) Corresponding average body weight variation curves for the four groups of 4T1 tumour-bearing mice. (C) Tumour growth curves for the PBS, 808 nm laser, ANDs and ANDs+808 nm laser treatment groups of mice.


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During necropsy, the tumour, as well as all major organs including the liver, lung, spleen, heart and kidney, were collected and stained with H&E. Figure S7 shows no apparent tumour necrosis in the ANDs group without irradiation and the PBS group with or without irradiation. By contrast, ANDs with irradiation caused severe cell necrosis of the tumour issue. Furthermore, as the groups treated with ANDs (with or without irradiation), there was no significant organ damage or inflammation in all the normal tissues, such as heart, liver, spleen, lung and kidney, thereby confirming the very low toxicity of ANDs in vivo at the tested dosage. 3. CONCLUSIONS We successfully developed a facile BSA-mediated method to synthesise silver nanodots. The molar ratio of BSA and elemental silver could affect the optical and photothermal properties of the resultant products. The optimised silver products were small with high stability and photothermal conversion capability. When modified with FITC, ANDs in the cytoplasm of KB cells showed low cytotoxicity to the KB cell. ANDs effectively caused cell death only with 808 nm laser irradiation in vitro. The good X-ray attenuation of ANDs enabled us to find the urine extraction route of ANDs after intratumour administration. Furthermore, ANDs showed efficient sensitisation effects of PTT with low toxicity in vivo, thereby suggesting that ANDs are promising agents for both in vivo CT imaging and photothermal therapy. 4. EXPERIMENTAL SECTION 4.1 Materials. Silver nitrate (AgNO3, ≥99.8%, Aladdin), sodium hydroxide (NaOH, ≥97.0%, Aladdin), Bovine Serum Albumin (BSA, molecular biology grade, Aladdin), Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology), calcein acetoxymethyl ester (Calcein AM, Dojindo


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Laboratories), PI (Dojindo Laboratories), Fluorescein 5(6)-isothiocyanate (FITC, ≥95.0%, Aladdin), Cell culture mediums were supported by Hyclone. 4.2 Physical and chemical properties determining instruments. UV–vis spectrophotometer (UV5800, Shimadzu). X-ray photoelectron spectrometer (ESCALAB MK II, using Mg as the exciting source). Transmission Electron Microscopy (TEM) (H-800, Hitachi, accelerating voltage of 200 KV). X-ray diffractometer (XD-3, PERSEE, with 2θ ranging from 10° to 90°). 4.3 Synthesis of silver nanoparticles. In a typical experiment, 250 mg BSA dissolved in 5.0 mL of pure water, and introduced into a 20 mL vial, then 1.0 mL aqueous AgNO3 solution (5, 10, 20, 50, 100, or 200 mM) was slowly added under vigorous stirring. Next, 1.0 mL aqueous NaOH solution (1.0 M) was used to adjust the final pH of the solution to above 12. And the mixture was then was sealed in dark and 80℃ water bath for 1 hours with durative stirring. To further purify the samples, the products were 24 hours. 4.4 Cell culture, cytotoxicity and staining. This part of experiment was proceeded similar to our previous work.39 To identify the location of the ANDs in the cells, we developed the FITC modified ANDs (FITC-ANDs) as follows. 1.0 mL aqueous FITC (1 mg/mL) solution slowly added into 10mL ANDs solution (10mg/mL) under vigorous stirring. After sustained stirring in the dark for 12h, the produce was purified by dialyzing for 24 h, and the FITC-ANDs was obtained. The KB cells that pre-cultured for 24h in a glass bottom culture dish (Mat Tek) were co-cultured with culture medium solution containing FITC-ANDs (10 ug/mL) for 6h. After washed twice with PBS and fixed with paraformaldehyde (Sigma) in mini-Q solution at 4 °C for 30 min, the cells were imaged using an inverted fluorescence microscope system (NIKON TE2000).


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4.5 In vitro photothermal Studies. The capability for photothermal conversion of the silver nanoparticles with different ratios (the ratios of silver to BSA from 1.32 to 52.8) was determined by measuring the thermal behaviour under the irradiation of 808 nm NIR laser (LWIRL808-4W-F, Beijing Laserwave Optoelectronics Technology Co. Ltd.) at 1.0 W/cm2. All the samples were at 5 mM. To study the localized photothermal effect in vitro, KB cells were firstly co-cultured with ANDs at 50 µg/mL for 4 h. After washed with PBS for three times, the cells were irradiated by 808 nm laser (1.0 W/cm2) for 10 min. Following that, cell culture media was added, and the cells were continuing cultured in the incubator for 24 h. Then the cells were all collected by centrifugation, and stained by Calcein AM and PI. The viable or dead cells with green or red fluorescence were visualized using an inverted fluorescence microscope (NIKON TE2000). 4.6 X-ray CT contrast agent in vitro. While iohexol 300 (Iopromide 300 mg I/mL, GE Healthcare) solutions was used as control agent, ANDs with different concentrations were prepared with deionized water and placed into 100 µL tubes. All the sample tubes of ANDs and Iopromide were site into two rows on a scanning holder, and scanned by a Micro-CT imaging system (eXplore Locus, GE). 4.7 Hemolysis Experiment. Firstly, 1 mL of human fresh blood was collected, and Red blood cells (RBCs) were separated by centrifuging at 10,000 rpm for 5 min. Then the RBCs were washed with PBS buffer till no colour could be observed in the supernatant. After the RBCs were suspended in 5 mL PBS, 0.50 mL suspension was mixed with 0.50 mL solution of different concentrations of ANDs at 10, 20, 40, 100, 200, 400 and 1000 µg/mL in PBS buffer, leading the final concentrations of 5, 10, 20, 50, 100, 200 and 500 µg/mL of ANDs, respectively. Meanwhile,


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0.50 mL RBC suspension mixed with 0.50 mL PBS or 0.50 mL distilled water was used as negative and positive controls, respectively. These samples should be gently mixed and kept at room temperature. After 4 h, these samples were centrifuged, 10,000 rpm for 5 min, and the supernatant were collected. Using multiplate reader (Biotek Synergy H1, USA), the absorbance of the hemoglobin in the supernatant was measured at 540 nm. The percentage of hemolysis was calculated as follows: hemolysis % = (sample absorbance − negative control)/(positive control − negative control) ×100% 4.8 X-ray CT imaging in vivo. For the in vivo CT imaging, the mice bearing 4T1 tumors was anesthetized and injected through the tail vein of 200 µL ANDs solution containing 1.0 mg ANDs. Then, the CT imaging was performed at pre-, 10min, 30min, 2, 4 and 24 h post injection using Triumph X-SPECT/X-O CT software, reconstructed and analysed by Amira 4.1.2. The experimental condition was set same as the aforementioned in vitro sample imaging. For the mice of intratumorally injection group, 20 µL ANDs (10 mg/mL) was injected into the tumor issue of the 4T1 tumors bearing mice, then the mice was imaged with an animal micro-CT system. The experimental condition was set same as the former mentioned. 4.9 ANDs -enhanced photothermal therapy (PTT) in vivo. To develop the tumor model, 1.0 × 106 4T1 cells were suspended in 100 µL cell culture mediums (containing fetal calf serum), and then injected into the back of mice subcutaneously. When the mean diameter of tumor reached approximately 8−10 mm, the mice were divided into four groups, based on different treatment: (a) PBS injection; (b) ANDs injection only; (c) IR light only; and (d) ANDs + IR light. For group b, c and d, 20 µL ANDs (10 mg/mL) or phosphate buffer saline was injected into each


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of the 4T1 tumor-bearing mice intratumorally. After 0.5 h, the mice in groups c and d were anesthetized with isoflurane and the tumor area was treated with 808 nm NIR laser (1.0 W/cm2) for 5 min to receive photothermal therapy. During the period of treatment, an IR thermal imager (Ti25, Fluke, USA) was used to record the tumor surface temperatures. After that, every two or three days, the mice weights and tumor size were recorded using an analytical balance and a vernier caliper, respectively. The tumor volume could be obtained as following equation: V = ab2 /2 V (mm3) is the tumor volume, and a (mm) and b (mm) are the tumor length and width, respectively. Relative tumor growth ratio (G) was calculated as follows: G (%) = V/V0 ×100% V and V0 are the tumor volume on the day tumor measured and the day 0, respectively. The tumor growth inhibition (TGI) ratio was calculated as follows: TGI = (1- W/W0) ×100% W and W0 are the average tumor weight of the experiment group (with ANDs and laser) and the control group (with PBS), respectively, the weight of tumor more accurate than volume. 4.10 Histology Analysis in Vivo. When the experiment ended, all the 4T1 tumor-bearing mice were killed and necropsy was performed on the mice from the treatment group and control group. Major organs (include liver, spleen, kidney, heart, and lung) were placed in 4% paraformaldehyde solution. 72h later, these organs were fixed into paraffin, and then cryosectioned into 4µm slices. After stained with hematoxylin and eosin (H&E), these slides


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were further examined using fluorescence microscope with a digital camera for tissue lesion analysis.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available [The temperature elevation, photos and expanding absorption spectra in aqueous with different pH, electron diffraction pattern, X-ray photoelectron spectroscopy, blood hemolysis of the ANDs, and the CT imaging at varied time points and images of the H&E stained major organs of the mice]. See the ACS Publications website at DOI:10.1021/acsami.XXXXXXX.

AUTHOR INFORMATION Corresponding Author * Email: [email protected]. * Email: [email protected]. * Email: [email protected]. Notes The authors declare no competing financial interest.. ACKNOWLEDGMENT This work was supported by the National Basic Research Program of China (2013CB328806), the Key Projects in the National Science & Technology Pillar Program (2013BAI01B01), the


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Renal Clearable Ag Nanodots for in Vivo Computer Tomography

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