Glucose-Derived Carbonaceous Nanospheres for Photoacoustic

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Glucose-derived Carbonaceous Nanospheres for Photoacoustic Imaging and Photothermal Therapy Zhao-Hua Miao, Hui Wang, Huanjie Yang, Zhenglin Li, Liang Zhen, and Cheng-Yan Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03652 • Publication Date (Web): 09 Jun 2016 Downloaded from http://pubs.acs.org on June 13, 2016

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ACS Applied Materials & Interfaces

Glucose-derived Carbonaceous Nanospheres for Photoacoustic Imaging and Photothermal Therapy Zhao-Hua Miao, †, ‡, #Hui Wang, §, # Huanjie Yang, § Zhenglin Li, ¶ Liang Zhen, †, ‡ and Cheng-Yan Xu*, †, ‡ †

School of Materials Science and Engineering, Harbin Institute of Technology,

Harbin 150001, Peoples’ Republic of China ‡

MOE Key Laboratory of Micro-system and Micro-structures Manufacturing, Harbin

Institute of Technology, Harbin 150080, Peoples’ Republic of China §

School of Life Science and Technology, Harbin Institute of Technology, Harbin

150080, Peoples’ Republic of China ¶

Condensed Matter Science and Technology Institute, School of Science, Harbin

Institute of Technology, Harbin 150000, Peoples’ Republic of China E-mail: [email protected] #

These authors contributed equally to this work.

ABSTRACT Carbon nanomaterials with small size and unique optical properties have attracted intensive interest for their promising biomedical applications. In this work, glucose-derived carbonaceous nanospheres (CNSs) with

high

photothermal

conversion efficiency up to 35.1%, are explored for the first time as a novel carbon-based theranostic agent. Different from most other carbon nanomaterials, the obtained CNSs are highly biocompatible and nontoxic because of intrinsic hydrophilic

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property and the use of glucose as raw materials. Under near- infrared laser irradiation (808 nm, 6 W cm-2) for 10 min, less than 15% of PC-3M-IE8 cells exposed to CNSs aqueous dispersions (0.16 mg/mL) remained alive. After intravenous administration of CNSs aqueous dispersions into nude mice, the photoacoustic intensity of tumor region is about 2.5 times higher than that of pre-injection. These results indicate that CNSs are suitable for simultaneous photoacoustic imaging and photothermal ablation of cancer cells, and can serve as promising biocompatible carbon-based agents for further clinical trials. KEYWORDS: carbonaceous nanospheres; biocompatibility; photoacoustic imaging; photothermal therapy; theranostic agents 1. Introduction Near-infrared (NIR) light absorbing nanomaterials that can effectively convert light energy into thermal energy are gaining popularity for their promising biomedical applications1-2. Owing to the rapid temperature elevation effect upon laser irradiation, nanoscaled NIR absorbers, such as gold nanorods3, copper sulfide (CuS) nanoparticles4, and conjugated polymers5-6, have been employed as therapeutic agents for enhancing the efficacy of photothermal therapy (PTT), a minimally invasive and potentially effective treatment alternative to conventional approaches (e.g., radiotherapy and chemotherapy). In fact, these photothermal agents can also be used as contrast agents for photoacoustic (PA) imaging, an emerging non-invasive modality based on the detection of ultrasonic waves induced by biomolecules’ absorption of photons through the PA effect, providing higher spatial resolution and deeper tissue penetration compared with most optical imaging techniques7-10. A typical example is highly biocompatible polydopamine (PDA) nanoparticle, which was initially reported as a novel photothermal agent for cancer treatment11, and later it was found that PDA


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nanoparticle could also serve as an excellent PA contrast agent for disease diagnosis12-13. As a result, NIR light absorbing nanomaterials are highly desirable in order to take full advantages of PA imaging and PTT technologies. Recent studies have shown that carbon nanomaterials with robust π–π conjugated structure could act as excellent NIR light absorbing agents for PA imaging and PTT14. The majority of currently used carbon nanomaterials are carbon nanotubes15, graphene nanosheets16-17 and some amorphous carbon nanoparticles18-19. However, due to the intrinsic hydrophobic property, these carbon nanomaterials have to be treated with concentrated sulphuric acid/nitric acid or modified by hydrophilic molecules for biological applications18-20, raising the concern of additional potential toxicity. In addition, the anisomerous structure of one-dimensional carbon nanotubes and two-dimensional graphene may not be beneficial for the treatment of solid tumors because of the high rotational energy in relative rigid tissue, causing heterogeneous existence in margin21-22. Very recently, carbon dots have been reported as novel activatable theranostic agents for fluorescent and photoacoustic imaging-guided PTT23. But the precursor molecule of such type of carbon dots, namely, polythiophene phenylpropionic acid, was insoluble and required complex synthetic procedures23. Another example is carbon nanoparticles from food-grade honey24. Due to rapid clearance properties and one-pot “green” synthesis method, the obtained carbon nanoparticles can serve as a biocompatible photoacoustic contrast agent for imaging sentinel lymph nodes24. Hence, more attention should be paid to the development of novel carbon-based nanomaterials through straightforward synthetic route but with excellent biocompatibility and significant photothermal effect to overcome aforementioned issues. Hydrothermal treatment of glucose has been widely adopted to synthesize



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colloidal carbon nanospheres (denoted as CNSs) with tunable diameter. Since the use of glucose as carbon source and water as reaction solvent, the obtained CNSs were highly biocompatible, nontoxic and could continuously be removed from tissues over time as previously reported25. For example, glucose-derived carbon shell was recently demonstrated to be a viable alternative to polyethylene glycol (PEG) molecule to reduce the toxicity of gold nanorods26. However, although glucose-derived CNSs have been widely used in many fields, including energy storage and conversion27-28, environment29 and biomedicine25, the trial of CNSs as photoacoustic and photothermal agents has not yet been reported. Herein, we demonstrated, for the first time, that glucose-derived CNSs can be employed as a novel near-infrared light absorbing agent for simultaneous photoacoustic imaging and photothermal ablation of cancer cells. Compared with graphene nanosheets, carbon nanotubes, and some other carbon nanomaterials18-20, glucose-derived CNSs as theranostic agents have several advantages: (1) The obtained CNSs are intrinsically biocompatible due to the hydrophilic surface, and thus no additional treatment or modification is needed. (2) The fabrication procedure is an absolutely “green” method. The colloidal CNSs are obtained in glucose aqueous solution, and no toxic reagents are used. (3) The spheres shape of CNSs may be beneficial for the treatment of solid tumors, causing homogeneous existence in margin. All these features benefit the safety of CNSs for further clinical applications. 2. Experimental section 2.1 Materials Glucose and NH3·H2O (28–30%) were purchased from Sinopharm Chemical Reagent Co.. Dopamine hydrochloride (98%), MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and calcein AM were purchased from Sigma-Aldrich


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Co.. Hemoglobin from Swine was obtained from J&K Scientific Ltd.. Deionized water was supplied by a Milli-Q water system. 2.2 Synthesis of CNSs CNSs with diameters ranging from 30 to 100 nm were prepared by a modified method according to the previous literature30. In a typical synthesis, 1.8 g of glucose was added into 35 mL deionized water, and stirred for 20 min. Then the obtained solution was transferred into 50 mL Teflon-sealed autoclave and maintained at 180 ºC for 4 h. The puce products were isolated by centrifugation at 10, 500 rpm for 15 min and washed with ethanol and deionized water for 4 times. The finally obtained CNSs were redispersed in 20 mL deionized water for further use. 2.3 Synthesis of PDA nanoparticles PDA nanoparticles with an average diameter of 160 nm were prepared according to the previous literature11. Typically, 40 mL ethanol and 90 mL deionized water were premixed together for 10 min, followed by the addition of 2 mL NH3.H20 solution (28%). The mixed solution was further stirred for 20 min at 30 ºC. Then, 10 mL dopamine aqueous solution (50 mg/mL) was added. After continuous stirring (about 500 rpm) for 24 h at 30 ºC, PDA nanoparticles were isolated by centrifugation at 10, 500 rpm for 15 min and washed with water for 3 times. 2.4 Characterization of CNSs Scanning electron microscope (FEI Quanta 200F) and transmission electron microscope (JEOL JEM-2100) were used to observe the morphology and size distribution of CNSs. A PALS/90Plus instrument (Brookhaven USA) was used to determine the Zeta potentials of CNSs aqueous dispersions. The vis-NIR spectra of CNSs were obtained by a Shimadzu UV-2550 spectrophotometer, the Raman spectrum of CNSs was acquired by a LabRAM XploRA laser Raman spectroscopy,



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and the FTIR spectrum of CNSs was acquired by a Varian 3000 FTIR spectrophotometer. The water contact angle of CNSs was measured by a contact angle meter VAF-30. Thermogravimetric analysis was performed using a Netzsch TG 209F3 apparatus at a ramping rate of 10 K min-1 under N2. 2.5 Measurement of photothermal performance of CNSs To evaluate the photothermal performance, 3 mL deionized water and CNSs aqueous dispersions with different concentrations were irradiated for 10 min by an 808 nm NIR laser (2 W). A thermocouple probe was inserted into the aqueous dispersions and the temperature evolution of the aqueous dispersions under irradiation was recorded by a digital thermometer every 10 s. The corresponding infrared thermal images were also monitored by an infrared thermal imager (Ti25, Fluke, USA). To calculate the photothermal conversion efficiency of CNSs, 3 mL of CNSs aqueous dispersions (0.2 mg/mL) was continuously irradiated under the same condition until reaching a steady-state temperature. The laser was then shut off and the temperature decrease process was also recorded. the photothermal conversion efficiency (η) was calculated by equation (1) as previously described31: η=

୦ௌሺ்೘ೌೣ ି்ೞೠೝೝ ሻିொೞ ூሺଵିଵ଴షಲఴబఴ ሻ

(1)

where h and S is the heat transfer coefficient and the surface area of the container, respectively. Tmax and Tsurr represent the maximum system temperature and the ambient surrounding temperature, Qs is the heat associated with the light absorbance of the solvent, I is the laser power (2 w) and A808 is the absorbance of CNSs at 808 nm. The value of hS is derived according to equation (2): τs =

௠ವ ஼ವ ௛ௌ

(2)

where τs is the sample system time constant, mD and CD are the mass (3 g) and heat capacity (4.2 J/ (g. ºC)) of water, respectively. Qs is measured independently to be


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12.6 mW using pure water without CNSs. In order to get the value of hS, we further introduce θ, which is defined as follows: θ=்

்ି்ೞೠೝೝ

೘ೌೣ ି்ೞೠೝೝ

(3)

where T is the solution temperature. Thus, hS can be determined by applying the linear time data from the cooling period vs –lnθ. 2.6 Cytotoxicity assay A standard MTT assay was applied to evaluate the cytotoxicity of CNSs using human prostate cancer PC-3M-IE8 cell line and mouse breast cancer 4T1 cell line as models. The cells were seeded into 96-well plates. When the cell density was about 1×104 cells per well, triplicate wells were treated with the indicated concentrations of CNSs (0, 0.01, 0.02, 0.04, 0.08, 0.16, 0.32 mg/mL) for 24 h or 48 h. Subsequently, MTT with a final concentration of 1mg/mL was added into the medium, and incubated for 4 h at 37 ºC to allow the formation of formazan. Dimethyl sulfoxide was added to each well to dissolve formazan. The absorbance was measured by microplate reader (Infinite 200 NanoQuant, TECAN) at 570 nm to determine the relative cell viability. 2.7 In vitro photothermal ablation of PC-3M-IE8 cells PC-3M-IE8 cells were selected for the photothermal ablation experiment. The cells were seeded into 96-well plates for 24 h before the experiments. For qualitative analysis, the cells were incubated in medium containing different concentrations of CNSs (0.05, 0.10 and 0.20 mg/mL) for 4 h, followed by an 808 nm NIR laser (6 W cm-2) irradiation for 10 min. Then the cells were stained with calcein acetoxymethyl ester (calcein AM), and visualized under fluorescence microscope (Olympus, Japan). To quantitatively evaluate the photothermal cytotoxicity of CNSs on PC-3M-IE8 cells, the cells were incubated with fresh medium containing different concentrations of



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CNSs for 4 h, then irradiated by using a NIR laser (808 nm, 6 W cm-2) for 10 min. The cell viabilities were determined by MTT assay as described above. Similar with the process, the infrared thermal imaging in vitro was also conducted for cell culture medium containing CNSs with a concentration of 0.16 mg/mL. 2.8 In vitro and in vivo photoacoustic imaging Photoacoustic imaging in vitro and in vivo were both performed under Nexus 128 photoacoustic computed tomography scanner (Ann Arbor, MI, USA) using 808 nm as the working laser wavelength. For in vitro imaging, the photoacoustic signals (averaged value within the whole pseudocolor region) of different concentrations of CNSs aqueous dispersions (0.1, 0.2, 0.4, 0.6 and 0.8 mg/mL) were recorded. Besides, deionized water and PDA nanoparticles aqueous dispersions at a concentration of 0.2 mg/mL were used as control samples. For in vivo photoacoustic imaging of tumor, nude mouse bearing 4T1 tumor was used as a model. CNSs aqueous dispersion (0.1 mL,5 mg/mL) was injected into nude mouse through intravenous tail injection. Then photoacoustic images of tumor section were acquired by the photoacoustic scanner at different time periods (0, 2, 6 and 12 h). During the experiment, the body temperature of the mice was maintained by using a water heating system at 37.5 °C. 3. Results and discussion 3.1 Synthesis and characterization of CNSs Fig. 1a shows the typical scanning electron microscope (SEM) image of CNSs which are obtained by hydrothermal treatment of 1.8 g glucose in 35 mL water at 180 ºC for 4 h. The insert is detailed size distribution through the statistics of diameters of two hundred CNSs from several SEM images, indicating the obtained CNSs have an average diameter of 60 nm (ranging from 30 to 100 nm). Transmission electron microscope (TEM) image (Fig. 1b) further depicts that the carbonaceous nanospheres


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consist of dark core and light-coloured shell, indicating the formation of carbonized core and hydrophilic surface due to the full or part dehydration of oligosaccharides during hydrothermal process30. The as-prepared CNSs can be easily dispersible in aqueous environment, and the Zeta potential of CNSs dispersed in deionized water is as high as -28.1 ± 4.37 mV due to the existence of rich functional groups. Fourier transform infrared (FT-IR) spectroscopy was used to identity the group types. The characteristic FT-IR bands of CNSs at 1703, 1612 and 3334 cm-1 are assigned to C=O and C=C groups, respectively(Fig. S1)30. Particularly, the bands in the range of 1000-1300 cm-1 for C-OH stretching and OH bending vibrations imply the existence

Fig. 1 (a and b) SEM and TEM image of CNSs with diameters ranging from 30 to 100 nm. The insert is the size distribution of CNSs based on SEM images. (c) The vis-NIR absorption spectra of CNSs, the path length of the cuvette is 1 cm. (d) The fitting curve of the absorbance of CNSs aqueous dispersions at 808 nm as a function of CNSs concentrations and the correlation coefficient (R2) is 0.9996. The insert is the digital photographs of the corresponding aqueous dispersions, showing their good dispersibility.

of numerous residual hydroxyl groups. These functional groups are covalently bonded to the carbonized core and can improve the hydrophilicity and stability of CNSs.



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Besides, the Raman spectrum of CNSs has two broad bands at 1369 and 1592 cm-1, which are assigned to the in-plane vibration of disordered amorphous and crystalline graphite carbon, respectively (Fig. S2)32-33. The energy dispersive spectrometer (EDS) spectrum of CNSs (Fig.S3) indicates that the obtained nanospheres have relative high oxygen content (about 21.4%). The TGA result in Fig. S4 shows huge weight loss (about 48%) during thermal decomposition of CNSs samples under nitrogen. Due to the existence of functional groups on the surface, the water contact angle of CNSs is 19.6° (Fig. S5), indicating the excellent hydrophilicity. Similar with other carbon nanomaterials, the colloidal CNSs aqueous dispersions exhibit broad absorption in the vis-NIR region (Fig. 1c and Fig. S6). As shown in Fig. S7, compared with hemoglobin molecules (the most dominant endogenous absorber in tissues), CNSs show much stronger absorbance in vis-NIR region. Fig. 1d displays the absorption at 808 nm linearly increased with the increase of CNSs concentrations. The insert is the digital photographs of the corresponding aqueous dispersions, demonstrating the good dispersibility of CNSs with concentration up to 0.4 mg/mL. The dispersion prosperity of CNSs in different solution is also studied. As shown in Fig.S8, CNSs disperse well in deionized water, PBS solution and cell culture medium, and no precipitation or aggregation is found. Furthermore, according to Lambert–Beer law (A/L = αC, where α is the mass extinction coefficient), the mass extinction coefficient of CNSs is estimated to be 4.38 Lg-1cm-1, which is comparable to that of graphene oxide (GO) nanosheets (3.6 L g-1 cm-1)34, but lower than that of polydopamine (PDA) nanoparticles (7.96 L g-1 cm-1) determined via the same method (Fig. S11). Of note, PDA is typically used as a control for further comparison because that PDA has been recently reported as good photothermal and photoacoustic agents by our and other groups11-12, 35, as well as similar optical absorbance spectrum with


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CNSs (Figs. S9 and S10). 3.2 Measurement of photothermal performance of CNSs The strong NIR absorption motivates us to evaluate the photothermal effect of CNSs. The photothermal temperature elevation of CNSs aqueous dispersions was first measured with deionized water as a control. As shown in Fig. 2a, under continuous laser irradiation (808 nm, 2 W) for 10 min, CNSs aqueous dispersions (3 mL) present dramatically temperature elevation while no significant temperature change is observed for deionized water. The corresponding infrared thermal images in Fig. 2b also show that the temperature increases with the increase of CNSs concentration and irradiation time. The photothermal conversion efficiency of CNSs is measured according to a previously reported method31. Based on the obtained data in Fig. 3a and 3b, the photothermal conversion efficiency of CNSs is calculated to be 35.1%, which is comparable to that of PDA nanoparticles (40%)11, but much higher than Cu9S5 nanoparticles (25.7%)36 and gold nanorods (21%)37. Particularly, a direct comparison of the photothermal performance of CNSs and PDA nanoparticles is shown in Fig. S12. The value of temperature change of 0.2 mg/mL of CNSs aqueous

Fig. 2 (a) Temperature elevation of water and CNSs aqueous dispersions with different concentrations as a function of irradiation time upon exposure to 808 nm laser. (b) The corresponding infrared thermal mages of water and CNSs aqueous dispersions with different concentrations at different time points.



(b)3000 Time (s)

30

o

∆T ( C)

(a) 40

20

2500

τs = 831.5 s

2000

t = -τs Ln (θ)

1500 1000

10

500 0

0 0

1000

2000

3000

0.0

4000

0.5

1.0

2.0

2.5

3.0

3.5

(d) 1.5 Absorbance (a.u.)

(c) 45 o

1.5

-Ln (θ)

Time (s)

Temperature ( C)

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40

35

30

Before irradiation After irradiation 1.0

0.5

25

0.0 0

1000

2000

3000

4000

5000

Time (s)

6000

650

700

750

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850

Wavelength (nm)

Fig. 3 (a) The photothermal response of CNSs aqueous dispersion (3 mL, 0.2 mg/mL) under laser irradiation (2 W, 808 nm) and then the laser was shut off. (b) Linear time data versus –lnθ obtained from the cooling period in Fig. 2a, and the time constant (τs) for heat transfer from the CNSs system is determined to be 831.5 s. (c) Temperature changes of CNSs aqueous dispersion with a concentration of 0.10 mg/mL after repeatedly turning on or off the laser for 3 times, (d) Vis-NIR absorption of CNSs aqueous dispersion before and after irradiation.

dispersion under laser irradiation for 10 min is 28.9 ºC (from 25.2 to 54.1 ºC), which is higher than that of 0.1 mg/mL of PDA aqueous dispersion (26.2 ºC), but lower than that of 0.2 mg/mL of PDA aqueous dispersion (41.1 ºC). This result is in accordance with the difference of their extinction coefficients and photothermal conversion efficiencies. Similar with some previous reports38-40, the π-plasmon of graphic carbon with large π-conjugated aromatic structure in the carbonized core should be responsible for the effective light-to-heat conversion in our CNSs system. In addition, the photothermal stability of CNSs is also evaluated. As shown in Fig. 3c and 3d, the temperature change of CNSs has no obvious reduction after repeatedly turning on or off the laser for 3 times, and the absorbance of CNSs aqueous dispersion before irradiation and after irradiation were almost the same, implying that the colloidal


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CNSs had good photothermal stability. The excellent photothermal effect and good photothermal stability indicate the great potential of CNSs for photothermal ablation and photoacoustic imaging. 3.3 Assessment of cytotoxicity of CNSs The excellent biocompatibility of nanomaterials is a critical factor for their further clinical application. Despite CNSs were demonstrated to be highly biocompatible and nontoxic in vitro and in vivo by previous reports25-26, a standard MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay is conducted here to further confirm the biocompatibility of carbonaceous nanospheres

(c)

(d)

100

100

Cell Viabllity (%)

Cell Viabllity (%)

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80 60 40 20

80 60 40 20

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0

0.04

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0.16

Concentration (mg/mL)

0.32

0.02

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0.16

Concentration (mg/mL)

Fig. 4 (a) Fluorescence microscopy images of PC-3M-IE8 cells treated with different concentrations of CNSs (0, 0.05, 0.10 and 0.2 mg/mL) under laser irradiation (808 nm, 2 W). Scale bar is 1 mm. (b) General digital images and infrared thermal images of a 96-well cell-culture plate containing PC-3M-IE8 cells and CNSs (well nos.1, 2, 5, and 6) or PC-3M-IE8 cells only (nos. 3 and 4) at different irradiation time points (0, 5 and 10 min), the irradiated region is marked by the circle. (c) Cell viability of PC-3M-IE8 cells exposed to CNSs aqueous dispersions with different concentrations for 24 h. (d) Cell viability of PC-3M-IE8 cells exposed to CNSs aqueous dispersions with different concentrations under laser irradiation (808 nm, 6 W cm-2) for 10 min.



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of present work. As expected, no cytotoxicity of CNSs is observed after PC-3M-IE8 cells exposed to CNSs for 24 h (Fig. 4c). Even at a high concentration up to 0.32 mg/mL, the cell viability is still higher than 98%. To further illustrate this, the cell viability of 4T1 cells is also evaluated by a standard MTT assay. As shown in Fig. S13, the cell viability of 4T1 cells after incubation with nanoparticles for 24 h is higher than 96% at the same condition, and this result is similar to the results of PC-3M-IE8 cells. In addition, the 4T1 cell viability for 48 h shows a little decrease, but is still as high as 91.8% after incubation with 0.32 mg/mL of CNSs. Therefore, we believe that CNSs with excellent biocompatibility are suitable for biomedical applications. 3.4 Assessment of photothermal ablation of PC-3M-IE8 cells To visualize the localized photothermal killing of cancer cells, PC-3M-IE8 cells were incubated with CNSs for 4h, followed by the irradiation of a NIR laser (808 nm, 6 W cm-2) for 10 min, and then calcein AM was used to stain PC-3M-IE8 cells for visualization of living cells. As shown in Fig. 4a, the illumination region became dark in the presence of both NIR laser and CNSs, indicating severe cell death caused by the photothermal effect of CNSs mediated by NIR laser. In comparison, vivid green colour is observed in the entire well when cells are treated with only laser irradiation, suggesting that the exposure of cells to laser irradiation alone is safe. In addition, the area of dark region with lower concentrations (0.05 and 0.1 mg/mL) of CNSs is much smaller than that with higher concentration (0.2 mg/mL). This is because that more light energy is changed into heat energy at higher concentration. We further quantitatively evaluated the photothermal cytotoxicity of CNSs on PC-3M-IE8 cells under laser irradiation using a standard MTT assay. The results reveal a significantly negative correlation between cell viability of PC-3M-IE8 cells and the concentration of CNSs in cell culture medium (Fig. 4d). In particular, less than 15% of PC-3M-IE8


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cells exposed to CNSs aqueous dispersion with a concentration of 0.16 mg/mL remain alive under NIR laser irradiation. Of note, the current power density of 6 W cm-2 is suitable for just evaluating the effect of photothermal ablation of cancer cells in vitro because no cell death happened under such laser irradiation alone. However, the power density must be reduced for the concern of safety for further potential clinical application. The infrared thermal imaging in vitro was also conducted under similar condition. As shown in Fig. 4b, PC-3M-IE8 cells are incubated with pure culture medium (well nos. 3 and 4) or CNSs nanoparticles (well nos.1, 2, 5, and 6), and well nos. 3, 4, 5 and 6 are irradiated by an 808 nm laser. As expected, bright thermal mages only appear in the wells (nos. 5 and 6) containing nanoparticles under irradiation, and the thermal images become brighter over time. These results demonstrate that CNSs hold great promise as an efficient photothermal agent for cancer treatment. 3.5 Assessment of photoacoustic imaging in vitro and in vivo Photoacoustic imaging has shown tremendous potential applications in preclinical studies. A scheme to illustrate the process of imaging based on CNSs is displayed in Fig. 6c. CNSs were first intravenously injected into the mouse and then accumulated in tumour tissue through enhanced permeability and retention (EPR) effect. Upon NIR laser irradiation, CNSs can effectively convert light energy into thermal energy, which causes a rapid thermoelastic expansion of tissue. And the rapid thermoelastic expansion further results in the generation of ultrasound wave that can be detected by an ultrasound transducer. To evaluate the photoacoustic properties of CNSs, the photoacoustic amplitudes of CNSs aqueous dispersions with concentrations from 0.1 to 0.8 mg/mL were first determined with water and PDA nanoparticles aqueous dispersion (0.2 mg/mL) as controls. As illustrated in Fig. 5a, CNSs samples generate strong photoacoustic signals upon the irradiation of 808 nm laser pulses. The



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Fig. 5 PA properties of CNSs in vitro. (a) In vitro PA images of water, PDA nanoparticles aqueous dispersion and CNSs aqueous dispersions with different concentrations from 0.1 to 0.8 mg/mL. (b) The corresponding photoacoustic signal intensity of CNSs aqueous dispersions as a function of CNSs concentrations.

Fig. 6 PA properties of CNSs in vivo. (a) In vivo PA images (2cm×2cm) of tumor region （marked by a black dotted circle）in nude mice before and after intravenous administration of CNSs

aqueous dispersion (0.1 mL, 5 mg/mL) at different time points. The 4T1 tumor was on the upper part of hind leg in mice and the diameter of tumor was about 8.5 mm. (b) The corresponding intensity of photoacoustic signal from tumor at different time points. (c) A scheme to illustrate the process of


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photoacoustic imaging.

photoacoustic intensity (averaged value within the whole pseudocolor region) increases linearly with the increase of CNSs concentrations (Fig. 5b), and the amplitude of generated photoacoustic signal of 0.2 mg/mL of CNSs aqueous dispersion is about 8.5 times higher than that of deionized water. Interestingly, despite PDA nanoparticles have better photothermal performance than CNSs, the photoacoustic signal intensity of CNSs aqueous dispersion is higher than that of PDA nanoparticles aqueous dispersion with the same concentration. The possible reasons for such strong photoacoustic detection signals of CNSs include high heat conductance, high heat capacity and low acoustic absorptivity in contrast to PDA nanoparticles41-43. To further demonstrate the capability of CNSs for photoacoustic imaging in vivo, CNSs aqueous dispersion (0.1 mL, 5 mg/mL) was intravenously injected into the tail vein of 4T1 tumor-bearing nude mice, and then the cross-sectional PA signal of tumor region was collected at different time points upon laser illumination. As shown in Fig. 6a and 6b, a weak photoacoustic signal is observed in the tumor region before injection due to the relatively low intrinsic absorption of hemoglobin molecules inside the blood vessels (Fig. S7). After 6 h post-injection, a detectible enhancement of PA signal is observed, and the enhancement effect is more obvious after 12 h post-injection. Quantitative analysis in Fig. 6b further indicates that the photoacoustic intensity at 12 h after injection is about 2.5 times higher than that of pre-injection. To be addressed, the relative high photoacoustic signal around tumor tissue is attributed to superficial vessels binding with tumor. The obvious enhancement effect should be attributed to the enhanced accumulation and retention of CNSs in the tumor region, making long-term photoacoustic monitoring in vivo possible. These results



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demonstrate that colloidal CNSs could also be used as a promising contrast agent to enhance photoacoustic imaging. 4. Conclusions In conclusion, we demonstrate, for the first time, that glucose-derived CNSs can be employed as a novel light absorbing theranostic agent for cancer diagnosis and treatment. With a high photothermal conversion efficiency up to 35.1%, the as-obtained CNSs can not only have good capability of photothermal destruction of cancer cells, but also exhibit excellent photoacoustic signal enhancement for both in vitro and in vivo photoacoustic imaging. Most importantly, due to intrinsic hydrophilic property and “green” synthesis method, glucose-derived CNSs are highly biocompatible and nontoxic, ensuring their superiority over other carbon-based theranostic agents. Although great efforts are needed to study the biological process, such as photothermal performance in vivo, pharmacokinetics behaviour, long-term toxicity and so on, CNSs have already shown great potential as a novel NIR light absorbing agent for further clinical trials.

ASSOCIATED CONTENT Supporting Information Available EDS, FTIR and Raman spectra of CNSs, TGA curve, contact angle of CNSs, TEM image and Vis-NIR absorption spectra of PDA nanoparticles, the comparison of absorbance of CNSs and hemoglobin from swine, CNSs dispersions in deionized water, PBS solution and cell culture medium, the comparison of temperature elevation of water, CNSs and PDA nanoparticles and additional cell viability of 4T1 cells exposed to CNSs aqueous dispersions for 24 h and 48 h. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION


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Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. Acknowledgements This work was supported by the Fundamental Research Funds for the Central Universities (Grant No. HIT.BRETIII.201203). The authors thank Dr Zhide Guo at the Center for Molecular Imaging and Translational Medicine in Xiamen University for PA imaging in vitro and in vivo. References 1.

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2016, 10(1), 822–831.

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Glucose-Derived Carbonaceous Nanospheres for Photoacoustic

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