Biomaterials 57 (2015) 12e21

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A facile synthesis of versatile Cu2
xS nanoprobe for enhanced MRI and infrared thermal/photoacoustic multimodal imaging Juan Mou a, 1, Chengbo Liu b, 1, Pei Li c, Yu Chen a, Huixiong Xu c, Chenyang Wei a, Liang Song b, *, Jianlin Shi a, Hangrong Chen a, * a State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, PR China b Research Lab for Biomedical Optics and Molecular Imaging, Shenzhen Key Lab for Molecular Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, PR China c Tenth People's Hospital of Tongji University, Shanghai 200072, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 January 2015 Received in revised form 3 April 2015 Accepted 8 April 2015 Available online 22 April 2015

A novel type of intelligent nanoprobe by using single component of Cu2
xS for multimodal imaging has been facilely and rapidly synthesized in scale via thermal decomposition followed by biomimetic phospholipid modification, which endows them with uniform and small nanoparticle size (ca.15 nm), well phosphate buffer saline (PBS) dispersity, high stability, and excellent biocompatibility. The assynthesized Cu2
xS nanoprobes (Cu2
xS NPs) are capable of providing contrast enhancement for T1weighted magnetic resonance imaging (MRI), as demonstrated by the both in vitro and in vivo imaging investigations for the first time. In addition, due to their strong near infrared (NIR) optical absorption, they can also serve as a candidate contrast agent for enhanced infrared thermal/photoacoustic imaging, to meet the shortfalls of MRI. Hence, complementary and potentially more comprehensive information can be acquired for the early detection and accurate diagnosis of cancer. Furthermore, negligible systematic side effects to the blood and tissue were observed in a relatively long period of 3 months. The distinctive multimodal imaging capability with excellent hemo/histocompatibility of the Cu2
xS NPs could open up a new molecular imaging possibility for detecting and diagnosing cancer or other diseases in the future. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Cu2
xS nanoprobe Magnetic resonance imaging Infrared thermal imaging Photoacoustic imaging Contrast agent

1. Introduction Over the past few decades, cancer has become one of the most dominant causes of mortality and morbidity of human beings worldwide [1]. Fortunately, various diagnostic imaging technologies have been emerging to assist cancer management in terms of early detection, therapy guidance, and response monitor [2e4]. However, so far, no single imaging modality is competent enough to provide satisfactorily accurate and comprehensive information under the complex scenarios of cancer. Therefore, multimodal imaging technologies and strategies are attracting much interest as

* Corresponding authors. E-mail addresses: [email protected] (H. Chen). 1 These authors contributed equally.

(L.

http://dx.doi.org/10.1016/j.biomaterials.2015.04.020 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

Song),

[email protected]

they hold great promising potential to offer ultimate the solutions for addressing this challenges [5e10]. Currently, MRI [11e13] has been widely used for cancer diagnosis in clinic, attributing to its high soft tissue contrast. However, MRI has certain limitations, such as relatively slow imaging speed, high cost, and inconvenience for general intra-operative use. Fortunately, many kinds of imaging modalities have been emerging to assist MRI to overcome its shortfalls [14e17]. Recently, infrared thermal imaging [18e20] and the newly developed photoacoustic imaging [21e23] have attracted much interest for their respective imaging advantages. Therefore, the combination of MR/infrared thermal/photoacoustic multimodal imaging may offer new opportunity for cancer detection and diagnosis, by providing potentially more comprehensive and accurate information, attributing to the complementary features of these technologies. Many multimodal imaging contrast agents show great potential applications in medical diagnosis and imaging [24e29]. Nevertheless, these reported multimodal probes are generally

J. Mou et al. / Biomaterials 57 (2015) 12e21

constructed of multi-functional nano-composites via complicated structure/composition design and time-consuming synthesis, which involves multiple distinct components, such as magnetic, fluorescent and NIR absorption ingredients [26,30e36]. For example, a kind of composite nanoparticle has been constructed for triple-modality MR/photoacoustic/Raman imaging, which was composed of a 60-nm gold core for photoacoustic imaging, following covered with a thin layer of Raman molecular for Raman imaging and Gd3þ ion for MRI [37]. Very recently, multi-functional Fe5C2 nanoparticles were reported for targeted dual-modal MR and photoacoustic imaging-guided photothermal therapy, in which all the functionalities were integrated within one structure or component, reducing the toxicity due to avoiding the overdose of applying multiple composites/moieties [38]. Therefore, multifunctional and multimodal imaging contrast agents constructed by single component are highly preferred owing to the simplification of structure design and material synthesis, which is a new tendency for the development of low-toxic multimodal probes. Copper (II) complexes have been considered to have magnetic character owing to the presence of unpaired electrons [39e42], which can be served as a potential contrast agent for T1-weighted MRI. However, their prospect as MRI contrast agent has not been previously demonstrated and documented neither in vitro nor in vivo. Meanwhile, copper sulfide nanocrystal (Cu2
xS NCs) has been demonstrated to be a potential contrast agent for infrared thermal/photoacoustic imaging [43,44], since it is a kind of p-type semiconductor (copper deficiencies), thus the high concentration of free carriers benefits the localized surface plasmon resonances (LSPRs), arising from excess holes in the valence band [45e47], which enables the Cu2
xS NPs having strong absorption in the near infrared (NIR) region, in particular, around 1000 nm in the second NIR window (l ¼ 1000e1350 nm), and converting the laser energy into hyperthermia efficiently and rapidly. However, previously reported Cu2
xS NCs were often restricted from wide biological applications, primarily due to their large size (e.g., >100 nm) [48e50], hydrophobicity [46,51], and relatively poor biocompatibility [52e54]. As far as we know, their prospect as a contrast agent for simultaneous triple-modality MR/infrared thermal/photoacoustic imaging has not been found. Herein, we report the facile and successful design and synthesis of a novel type of contrast agent based on single component Cu2
xS nanoprobes (Cu2
xS NPs) for enhanced T1-weighted MR/infrared thermal/photoacoustic multimodal imaging. These versatile Cu2
xS NPs can be facilely and rapidly prepared via thermal decomposition followed by biomimetic phospholipid modification, which not only ensures many excellent properties such as good dispersity, high stability in normal physiological environment, prolonged circulation in blood, and effective passive accumulation within tumor through the enhanced penetration and retention (EPR) effect, but also provides a potential for mass production in the future clinic translation. Importantly, the Cu2
xS NPs are found to possess enhanced contrast properties for MRI both in vitro and in vivo for the first time. In addition, these prepared Cu2
xS NPs also are endowed with strong NIR optical absorption, attributing to the LSPRs, which enables them serving as an excellent candidate contrast agent for infrared thermal/photoacoustic imaging. Therefore, in principle, the as-synthesized Cu2
xS NPs could be able to serve as a multimodal contrast agent for simultaneous MR/infrared thermal/photoacoustic imaging, which, for the first time, had been indeed well demonstrated and validated through both in vitro and in vivo imaging experiments in this work. Additionally, in a study across a relatively long period of 3 months, negligible systematic side effects to the blood and tissue were observed, demonstrating high hemo/histocompatibility for potential translation into the clinic. To the best of our knowledge, this is the first report of pure

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single component Cu2
xS NPs for enhanced MR/infrared thermal/ photoacoustic multimodal imaging, which can potentially provide more comprehensive information with complementary features and may open up new possibilities for better detection and diagnosis of cancer in the future medical imaging. 2. Results and discussion 2.1. Preparation and characterization of versatile Cu2
xS NPs The synthesis of pure single component Cu2
xS NPs-based contrast agent for enhanced MR/infrared thermal/photoacoustic multimodal imaging is illustrated in Scheme 1. Uniform hydrophobic oleylamine-capped Cu2
xS NPs were facilely and rapidly fabricated via a modified mass producible thermal decomposition process [46]. These hydrophobic oleylamine-capped Cu2
xS NPs were then transformed into hydrophilic ones through lipid PEGylation by biomimetic phospholipid (DSPE-PEG2000-NH2). Representative transmission electron microscopy (TEM) images reveal that the as-prepared oleylamine-capped Cu2
xS NPs are monodisperse and disk-like, with an average diameter of 15 nm and an average thickness of 3.8 nm, as shown in Fig. 1a and Fig. S1a. The microstructure information of Cu2
xS NPs was further investigated by high-resolution TEM (HRTEM) image and selective area electron diffraction (SAED) pattern. The HRTEM image (Fig. S1c) shows a polycrystal structure with resolved an interplanar d-spacing of 0.190 nm, corresponding to the (1, 1, 0) lattice fringe of a rhombohedral phase of Cu9S5. The SAED (Fig. S1d) of Cu2
xS NPs shows distinctive spots forming complete rings, which is indexed as crystallographic planes of (110). Energy dispersive X-ray (EDX) spectrum (Fig. S2) confirms the existence of all expected elements (Cu, S) in Cu2
xS NPs. The well-defined peaks in the XRD pattern (Fig. S3a, top curve) indicate the formation of pure rhombohedral phase of Cu9S5 with high crystallinity in comparison with the standard Cu9S5 powders on the JPCD card (no. 47e1748). Fig. S3b shows the high-resolution X-ray photoelectron spectra (XPS) of the Cu2
xS NPs with all binding energies being calibrated by referencing the C 1s (284.8 eV). As illustrated in Fig. S3b, two peaks at 933.8 eV and 954.0 eV corresponding to Cu(II) 2p3/2 and Cu(II) 2p1/2 were found, which are essentially identical binding energies for the Cu 2p orbital in accordance with Cu2þ [55]. And the two separate peaks locating at the binding energies of 932.5 eV and 952.4 eV are corresponding to Cu 2p3/2 and Cu 2p1/2, belonging to Cuþ [56]. Hence, the above results prove that the Cu2
xS NPs contain both Cu2þ and Cuþ ions. In addition, the molar ratio of Cu/S is 1.8 obtained by analyzing the integrated areas of Cu and S according to XPS spectra, indicating that the Cu2
xS NPs are the digenite (Cu9S5) phase, which is in good consistent with XRD results. The chloroform containing oleylamine-capped Cu2
xS NPs, which was floating above the PBS solution in a quartz cuvette (the inset in Fig. 1a), shows a transparent green color (in the web version), indicative of well dispersity of oleylamine-capped Cu2
xS NPs in chloroform. In order to transfer hydrophobic oleylamine-capped Cu2
xS NPs into hydrophilic ones for biological application, biomimetic phospholipid (DSPE-PEG2000-NH2) possessing special amphiphilic structure and excellent biocompatible property was used for further surface modification. The representative TEM images of Cu2
xS NPs in Fig. 1b and Fig. S1b show no significant morphology and size change after lipid PEGylation. The as-prepared Cu2
xS NPs can be well dispersed in PBS solution for up to 3 months without obvious aggregations (the inset in Fig. 1b), which was attributed to the successful surface lipid PEGylation for excellent dispersity and high stability. A hydrodynamic diameter of 17 nm (Fig. S4a) and a positive Zeta potential of 12 mV (Fig. S4b) resulted from biomimetic

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Scheme 1. Schematic illustration for the facile synthesis procedure of single component Cu2
xS NPs constructed as an excellent contrast agent for enhanced MR/infrared thermal/ photoacoustic multimodal imaging.

phospholipid capping were found from the Dynamic Light Scattering (DLS) analysis. Fourier transform infrared (FTIR) spectra also prove the success of lipid PEGylation. As shown in Fig. S5, a broad band at 3450 cm
1 corresponding to OeH stretching vibration, the transmission bands at 2924 and 2854 cm
1 assigning to the asymmetric (nas) and symmetric (ns) stretching vibrations of methylene (CH2) in the long alkyl chain, and two bands at 1634 and 1454 cm
1 attributing to C]C bending and CH3 stretching modes, respectively, and 1008 cm
1 indexing to CeN stretching mode, were observed in both samples before and after lipid PEGylation. Comparatively, the characteristic peak at 1739 cm
1 assigning to NeC]O of DSPE-PEG2000-NH2 was only observed after lipid PEGylation [57], indicative of the successful lipid PEGylation as well. Copper ion (II) is regarded to be an alternative candidate contrast agent for MRI as it possesses unpaired electrons and magnetic property, which, however, has not been verified experimentally. Herein, for the first time as far as we know, the promising potential of Cu2
xS NPs as a contrast agent for enhanced T1weighted MRI application was investigated in detail. In order to study the MRI contrast properties of Cu2
xS NPs, proton T1 relaxation measurements as well as phantom images of Cu2
xS NPs at varied Cu concentration were performed on a commercial MRI instrument (3.0 T). The concentration-normalized relaxivity value and linear correlation between MR signal intensity and Cu concentration are calculated to be r1 ¼ 0.26 mM
1 s
1 and R2 ¼ 0.99, respectively (Fig. 1c), indicating an effective longitudinal (r1) relaxation enhancement of water protons. Meanwhile, progressively brighter images of the phantoms containing Cu2
xS NPs can be observed in the inset of Fig. 1c, showing the significant T1weighted signal enhancement effect induced by the Cu2
xS NPs. Note that although the r1 relaxivity value appears to be modest compared to the clinic Gd3þ-based contrast agent, however, compared with prussian blue [58], this kind of Cu2
xS NPs shows great potential of contrast capability for enhanced T1-weighted MRI and opens up a new avenue for the design and exploration of novel MRI contrast agent based on Cu class compounds.

Furthermore, the optical property of aqueous dispersions of Cu2
xS NPs at varied Cu concentration was examined by using UVvis spectroscopy. Obviously elevated absorption can be observed with the increase of wavelength and Cu concentration (Fig. 1d). Interestingly, all the absorption peaks corresponding to different Cu concentration are located in the second NIR window (l ¼ 1000e1350 nm) around 1160 nm, which is much beneficial for deep tissue imaging [59e62]. Moreover, the absorbance of the aqueous solution containing Cu2
xS NPs at 1160 nm is linearly enhanced with Cu concentration (Fig. 1e). The strong NIR optical absorption of Cu2
xS NPs motivates us to investigate their potential application as a contrast agent for enhanced infrared thermal/ photoacoustic imaging. For photoacoustic imaging, the linearly enhanced photoacoustic signals of Cu2
xS NPs with increased Cu concentration can be observed in Fig. 1f, and the inset shows the corresponding gradually brightened photoacoustic images of agar gel cylinders containing Cu2
xS NPs at different Cu concentration. In addition, for infrared thermal imaging, the temperature increase of water droplet and aqueous droplet containing Cu2
xS NPs (Cu concentration: 100 ppm) at different time intervals after 980 nm laser irradiation with different power densities were recorded by an infrared thermal imaging instrument. It can be seen that the temperature rise of the aqueous droplet containing Cu2
xS NPs can reach up to 20.5 and 21.5  C in 30 s, with a power density of 0.21 W cm
2 and 0.38 W cm
2, respectively, and longer irradiation time (60 s) leads to more significant temperature increase (Fig. 1geh). In contrast, the pure water shows no obvious temperature increase under the same experimental condition. The in vitro results reveal that the as-obtained Cu2
xS NPs could serve as an excellent candidate for enhanced infrared thermal and photoacoustic imaging. 2.2. Intracellular uptake and in vivo biodistribution In order to assess the cellular uptake and intracellular distribution of Cu2
xS NPs at varied time intervals, confocal laser scanning microscopic (CLSM) observation of HeLa cells was performed

J. Mou et al. / Biomaterials 57 (2015) 12e21

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Fig. 1. Representative TEM images of the hydrophobic oleylamine-capped Cu2
xS NPs dispersing in CHCl3 (a) and hydrophilic lipid PEGylated Cu2
xS NPs in PBS (b), the insets show the corresponding photographs, respectively. (c) Plots of the 1/T1 value vs Cu concentration of aqueous solution containing Cu2
xS NPs. (d) UV-vis absorbance spectra of aqueous solutions containing Cu2
xS NPs at varied Cu concentration and (e) the corresponding linear plot of absorbance vs Cu concentration at 1160 nm. (f) Plots of the photoacoustic amplitude vs Cu concentration of aqueous solution containing Cu2
xS NPs under 1064 nm laser irradiation. Infrared thermal images of water droplet and aqueous droplet containing Cu2
xS NPs (Cu concentration: 100 ppm) under 980 nm laser irradiation for 60 s at varied power densities of 0.21 (g) and 0.38 (h) W cm
2, respectively.

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after 1 h and 4 h incubation with fluorescein isothiocyanate (FITC)labeled Cu2
xS NPs, by examining the green fluorescence originating from FITC groups. To verify the intracellular localization of FITC-labeled Cu2
xS NPs, cell nuclei were stained in blue with 40 , 6diamidino-2-phenylindole (DAPI). As shown in Fig. S6aeb, bright green signals were observed around the perinuclear regions both in 1 h and 4 h incubation, indicative of the presence of high accumulation of Cu2
xS NPs. In addition, brighter green fluorescence was observed in 4 h incubation, which shows the enhanced accumulation of the FITC-labeled Cu2
xS NPs within the cytoplasm with prolonged incubation time. Furthermore, blood circulation time and accumulation efficiency of nanoparticles within tumor are of great importance as to evaluate the diagnostic or therapeutic performance of nanomaterials. The circulation of Cu2
xS NPs in blood was investigated and calculated to be as long as 2.35 h (Fig. S7a), exhibiting great advantages for prolonged imaging and enhanced accumulation within tumor through EPR effect. The biodistribution of Cu2
xS NPs in main organs and tumors was investigated after 24 h intravenous injection of the Cu2
xS NPs into nude mice bearing HeLa tumor, by analyzing Cu content with inductively coupled plasma atomic emission spectroscopy (ICP-AES) measurement. As shown in Fig. S7b, approximate 2% of Cu2
xS NPs were passively accumulated within the tumor through enhanced EPR effect attributing to the prolonged circulation in blood, which could be further increased via targeting modification. 2.3. In vivo MR/Infrared thermal/photoacoustic imaging In vivo MRI experiments were performed to further assess the contrast capability of Cu2
xS NPs for MR imaging. As shown in Fig. 2a, T1-weighted MR images of tumor site were acquired sequentially on mice treated with PBS, intravenous and intratumoral injection of Cu2
xS NPs, respectively, in 30 min. Compared with the mouse treated with PBS as a control, moderately brightened T1-weighted MR image (Fig. 2a2) was observed in the region of interest (ROI) within tumor in 30 min after intravenous injection of Cu2
xS NPs, and much more significant signal enhancement

(Fig. 2a3) was observed in the mouse treated with intratumoral administration of Cu2
xS NPs. In detail, the signal intensities within ROI could be quantitatively characterized with 15.5% and 51.4% enhancement after intravenous and intratumoral injection of Cu2
xS NPs, respectively. To our best knowledge, this is the first report that Cu2
xS NPs are capable of acting as a new type of T1weighted MRI contrast agent for in vivo tumor imaging. The potential of Cu2
xS NPs acting as a multimodal contrast agent for infrared thermal and photoacoustic imaging in vivo was further investigated. Mice bearing HeLa tumor were intravenously injected with Cu2
xS NPs (10 mg kg
1) and then exposed to NIR laser. Infrared thermal images and photoacoustic images were acquired at different time intervals, respectively. For infrared thermal imaging, the center temperatures of tumor with and without Cu2
xS NPs injection increased by 12.9  C and 3.9  C, respectively, within 30 s of laser irradiation (Fig. 2b2ec2). Moreover, the temperature of the mice group treated with Cu2
xS NPs shows a faster temperature rise rate than that of control. Meanwhile, gradually brightened infrared thermal images were obtained along with laser irradiation time (Fig. 2bec). These results confirm that the Cu2
xS NPs are capable of serving as a sensitive infrared thermal imaging contrast agent for in vivo tumor imaging application. To further evaluate the contrast enhancement capability of Cu2
xS NPs for in vivo photoacoustic imaging, three-dimensional photoacoustic images along with ultrasound images of a mouse tumor were acquired at different intervals before and after intravenous injection of Cu2
xS NPs on a home-developed photoacoustic imaging system. As the Cu2
xS NPs' peak optical absorption is at 1160 nm with a relatively broad absorption spectrum, which is located in the second NIR window, a 1064 nm laser was applied in this experiment. The using of easily accessible 1064 nm ns-pulsed Nd: YAG lasers not only reduce the cost of photoacoustic imaging, but also significantly reduce optical scattering for a possibly extended optical/photoacoustic imaging depth. As shown in Fig. 3, the ultrasound images are utilized to visualize the tumor anatomy (e.g., skin and inclusion edges), while the photoacoustic images can reveal the accumulation of Cu2
xS NPs

Fig. 2. In vivo T1-weighted MR images of tumor sites in 30 min after treated with PBS injection (a1), intravenous (a2) and intratumoral (a3) injection of Cu2
xS NPs. The values in yellow show the T1-weighted MR imaging intensity of the region of interest (ROI) marked with red circles. Infrared thermal images of mice groups treated with (b) and without (c) Cu2
xS NPs at different time intervals (i.e., 0, 30, 60, and 180 s) under laser irradiation.

J. Mou et al. / Biomaterials 57 (2015) 12e21

within the tumor. It can be seen that before the injection of Cu2
xS NPs, the photoacoustic signals remain at a relatively low level, presumably due to the background signal originating from the blood absorption under NIR laser irradiation (Fig. 3a). Upon injection (5 s), the photoacoustic signals in the tumor site even deep to 1.5 cm are instantaneously and significantly enhanced with the quantified photoacoustic signal being 170 times stronger than that of the pre-injection (Fig. 3b, and Fig. S8). This enhancement can be mainly attributed to the strong NIR absorption of Cu2
xS NPs and prolonged circulation time in the blood vessels. The photoacoustic signal intensity in the tumor region shows gradual decrease at 1 h, 4 h and 24 h post-injection (Fig. 3cee, Fig. S8) compared with that at 5 s, mainly attributing to the clearance of Cu2
xS NPs from blood. However, the photoacoustic signal intensity still maintains to be higher, approximately 80, 72, and 21 times stronger than that of pre-injection, respectively. Obviously, the results further confirm that the Cu2
xS NPs are capable of enhancing the contrast of photoacoustic imaging both in vitro and in vivo. 2.4. Toxicity investigation of Cu2
xS NPs both in vitro and in vivo The in vitro cytotoxicity of Cu2
xS NPs at varied Cu concentration on HeLa cells was evaluated via a standard 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT) cell viability assay. It can be seen that the viabilities of HeLa cells treated with Cu2
xS NPs are all higher than 85% (Fig. S9) even at the Cu concentration up to 100 mg mL
1, demonstrating the Cu2
xS NPs are hardly cytotoxic, which was further proved by the unchanged cell morphology (Fig. S10) in 24 h incubation with and without Cu2
xS NPs. In addition, for in vivo application, it is essential to investigate the hemo/histocompatibility of the nanoparticles before they can be considered for intravenous administration. Hemolysis and coagulation assays of human red blood cells (RBCs) were performed to assess the impact of Cu2
xS NPs on blood in vitro. Hemolytic behavior of the Cu2
xS NPs was investigated at varied Cu concentration up to 800 mg mL
1, by comparing with positive and negative control groups using deionized water and PBS, respectively. Negligible hemolysis of the RBCs was detected with the hemolysis percentage of 2.6%, as shown in Fig. S11a, which was much lower than the threshold value of 5%. The inset of Fig. S11a shows the direct observation of hemolysis, with the far right and left plastic tube containing deionized water and PBS, respectively. As for evaluating the coagulation behavior of Cu2
xS NPs, major factors of

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Fibrinogen (FIB), prothrombin time (PT) and activated partial thromboplastin time (APTT) were tested to assess the abnormality of coagulation factor I, extrinsic and intrinsic coagulation pathways, respectively (Fig. S11bed). No significant differences over Cu concentration between control and treatment groups were detected, revealing negligible effect of Cu2
xS NPs on blood coagulation. These results demonstrate that the Cu2
xS NPs possess excellent blood compatibility for further biomedical application, presumably attributing to biomimetic phospholipid coating. Furthermore, the in vivo blood biochemistry and hematology investigation along with the Hematoxylin and Eosin (H&E) stain of main tissues were evaluated 3, 30 and 90 days post-injection of Cu2
xS NPs. The key biochemistry parameters and various vital hematology markers show no hepatic dysfunction, renal toxicity and systematical hematological are induced by the Cu2
xS NPs administration within 3 months' investigation (Fig. 4aeo). In addition, the H&E stain results reveal that the main organs of groups treated with Cu2
xS NPs retain similar morphology with that of the control group during 3 months' evaluation, indicative of negligible side effects to the biological tissue (Fig. 4p). All these results demonstrate that the Cu2
xS NPs have excellent hemo/histocompatibility both in vitro and in vivo for potential clinic translation. 3. Conclusion In summary, a novel type of multifunctional contrast agent based on single component Cu2
xS NPs has been rationally constructed for enhanced MR/infrared thermal/photoacoustic multimodal imaging, which is highly desired to cover the shortages of single imaging modality in medical imaging for cancer diagnosis and treatment. Attributing to the intrinsic moderate magnetic property and strong NIR optical absorption, together with uniform and small nanoparticle size, well phosphate buffer saline (PBS) dispersity, and excellent biocompatibility, the Cu2
xS NPs are capable of providing significant contrast enhancement for simultaneous MR/infrared thermal/photoacoustic multimodal imaging both in vitro and in vivo. Moreover, the multifunctional single component Cu2
xS NPs show great advantages, such as less toxicity, compared with those reported nanocomposites with complicated structure/composition, which often bring high toxicity resulting from the overdose for obtaining high effectiveness. Therefore, as we believe, with further optimization (e.g., integration and

Fig. 3. Ultrasound (US) and photoacoustic (PA) images along with their overlay images of the tumor site acquired before and after intravenous injection of Cu2
xS NPs at different time intervals (i.e., pre, 5 s, 1 h, 4 h, and 24 h).

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functionalization with therapeutic moieties), these Cu2
xS NPs will have a broad range of applications in accurate tumor diagnosis and/ or noninvasive cancer treatment (e.g., photothermal therapy). 4. Experimental section 4.1. Chemicals and reagents All reagents were used without further purification. Copper (II) chloride (CuCl2, 99.999%), potassium (I) ethylxanthate (C3H5OS2K, 96%) and oleylamine (C content: 18e19%) were purchased from SigmaeAldrich. Chloroform, ethanol and acetone were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1, 2distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000-NH2) was purchased from Avanti Polar Lipids, Inc.

4.2. Synthesis of oleylamine-capped Cu2
xS NPs The hydrophobic oleylamine-capped Cu2
xS NPs with strong NIR absorption and magnetic property were prepared via thermal decomposition according to the literature with slight modification. Firstly, copper ethylxanthate (Cu(ex)2) precursor was obtained according to the following procedure. Copper (II) chloride (CuCl2, 10 mmol) and potassium (I) ethylxanthate (C3H5OS2K, 20 mmol) were dissolved in distilled water (40 mL), respectively. To the aqueous solution of CuCl2 was then added the aqueous solution of C3H5OS2K, yellow precipitates formed immediately. Then, the yellow precipitates were centrifuged and washed several times with distilled water. Finally, the product was dried in a freezing dryer for later use. For the synthesis of oleylamine-capped Cu2
xS NPs, oleylamine (8 mL) was poured into a three-neck flask and heated to 150  C to

Fig. 4. The variations of blood index (aeo) and representative H&E stained images (p) of major organs including liver, spleen, and kidney collected from the control untreated mice and mice treated with Cu2
xS NPs at different time intervals (i.e., 0, 3, 30, 90 days post-injection). All the scale bars in (p) are 100 mm.

J. Mou et al. / Biomaterials 57 (2015) 12e21

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Fig. 4. (continued).

degas for 15 min in an argon atmosphere. Then the temperature was heated to 240  C and kept for 15 min. After that, to the hot oleylamine solution, the precursor mixture containing Cu(ex)2 (0.2 mmol) and oleylaimine (4 mL) was injected quickly and kept for 30 s. Subsequently, the reaction mixture was poured into acetone (20 mL) quickly to cool down and quench the reaction. Finally, the resulting oleylamine-capped Cu2
xS NPs were collected and washed three times with ethanol. The product was then dispersed in chloroform (40 mL) for further surface modification.

aqueous solution of lipid PEGylated Cu2
xS NPs were obtained without large aggregates and then kept at 4  C for later use. 4.4. Cell culture Human cervix adenocarcinoma (HeLa) cells were cultured at 37  C under 5% CO2 in standard cell media with Dulbecco's Modified Eagle's Medium (DMEM, high glucose, GIBCO, Invitrogen) and 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin. 4.5. Cytotoxicity assay

4.3. Lipid PEGylation To obtain hydrophilic Cu2
xS NPs with good biocompatibility for biological application, biomimetic phospholipid (DSPE-PEG2000NH2) was used to transfer these hydrophobic Cu2
xS NPs into hydrophilic ones. Typically, to chloroform solution containing oleylamine-capped Cu2
xS NPs (5 mL, 0.05 mM) was then added DSPE-PEG2000-NH2 and sonicated for 5 min to be dissolved. A rotary evaporator was applied to the mixture for evaporating the solvent under vacuum. Subsequently, lipidic film was acquired and physiological saline solution (5 mL) was added followed by 5 min’ sonication. Finally, after filtering through a 0.22 mm membrane,

In vitro cytotoxicity of Cu2
xS NPs was determined by the standard methyl thiazolyl tetrazolium (MTT) viability assay on HeLa cells. Cells were seeded into a 96-well cell culture plate at a density of 3  104 cells/well in standard cell media at 37  C and 5% CO2 for 24 h to allow cells to attach. The culture medium was discarded and cells were incubated with complete medium containing Cu2
xS NPs at varied Cu concentration (i.e. 100, 50, 25, 12.5, 6.25 mg mL
1) at 37  C in the presence of 5% CO2. After incubation for another 24 h, the culture medium was replaced by MTT solution (0.1 mL, 0.5 mg mL
1) and then was incubated for further 4 h. Finally, DMSO (100 mL) was added in the plates and an enzyme-linked

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immunosorbent assay reader (BioTeck instrument, USA.) was used to measure the optical absorption of formazan at 490 nm. Each treatment group was done in three replicates. 4.6. Assessment of cell uptake in vitro by CLSM The cells were seeded in confocal laser scanning microscope (CLSM) culture dishes and incubated in standard cell media at 37  C in the presence of 5% CO2 for 12 h to allow cells to attach. Upon reaching 70%e80% confluence, FITC-labeled Cu2
xS NPs (100 mL) were added and then incubated for another 1 h and 4 h, respectively. Subsequently, the nuclei were stained in blue with 40 -6diamindino-2-phenylindole (DAPI, lex ¼ 358 nm) for cellular tracking. Finally, the cells were washed three times with PBS before CLSM examination. The green fluorescence originating from FITC and blue fluorescence originating from DAPI were observed under 488 nm light excitation. 4.7. In intro and in vivo magnetic resonance imaging A 3.0 T clinical MRI instrument (GE Signa HDx3.0 T) was applied to evaluate the MR imaging performance both in vitro and in vivo. Aqueous solutions of Cu2
xS NPs at varied Cu concentration were placed in a series of 1.5 mL tubes for T1-weighted MR imaging. For in vivo T1-weighted MR imaging, the mice were first anaesthetized by trichloroacetaldehyde hydrate (10%) at a dosage of 40 mg kg
1 body weight and maintained at normal body temperature. Subsequently, the phosphate-buffered saline (PBS) solution containing Cu2
xS NPs (Cu concentration, 100 ppm) were intravenously and intratumoraly injected into the mice, respectively. Transversal cross-sectional scan images were taken 30 min post-injection for MRI analysis, respectively. Signal intensities in defined regions of interest (ROI) within tumor sites were measured to demonstrate the contrast capability. 4.8. In vitro and in vivo infrared thermal imaging In order to evaluate the contrast capability of Cu2
xS NPs for infrared thermal imaging, water droplet and aqueous droplet containing Cu2
xS NPs were placed on the same plate and a digital infrared thermal image instrument (FLIRA325 sc, USA) was applied to record the temperature evolution and infrared thermal images under a 980 nm laser irradiation. For in vivo study, prior to infrared thermal imaging in vivo, the HeLa tumor-bearing mouse was first anaesthetized and maintained at normal body temperature. The Cu2
xS NPs in PBS solution (Cu concentration: 100 ppm) were then intravenously injected into the mouse. The spatial temperature distributions of the tumor were recorded under a 980 nm laser irradiation for 3 min. 4.9. In vitro and in vivo photoacoustic imaging The home-developed PA/US dual modality imaging system was used to obtain all the photoacoustic data in this paper, which consisted of a pulsed OPO laser (Vibrant 355 II HE, Opotek, Carlsbad, USA) that operated at 1064 nm for photoacoustic excitation, a focused ultrasound transducer (V315-SU, Olympus IMS, Waltham, USA; central frequency: 10 MHz; fractional bandwidth: 6 MHz; N.A.: 0.4) for both ultrasonic firing and photoacoustic detection, and a precision motorized 3D scanning stage (PSA2000-11, Zolix, Beijing, China) to scan the imaging head across the x-y plane for 3D imaging. Note that, as the depth resolution comes from the time of arrival of the received acoustic signals, the 3D stage's z axis is used for positioning the imaging head only. During experiments, the OPO laser works at a repetition rate of 10 Hz and a pulse width of

5 ns. Following the triggering of each laser pulse, the photoacoustic and ultrasound signals were sequently acquired, and then amplified 39 dB and digitized with the DAQ in a personal computer. The photoacoustic segment was compensated for laser intensity variation first, and then Hilbert transformed for envelope detection. The ultrasound segment was processed for envelope detection only. In the end, photoacoustic and ultrasound images were displayed separately or co-registered using Amira software. To investigate the photoacoustic signals generated by Cu2
xS NPs, agar gel cylinders with a diameter of 8 mm containing Cu2
xS NPs at varied Cu concentration were fabricated. Photoacoustic signals of these cylinders were obtained and their intensities were calibrated and quantitatively analyzed based on the reconstructed images. For in vivo photoacoustic imaging, nude mouse (~20 g) bearing HeLa tumor was set on a homemade mount and kept anaesthetized by isoflurane delivered through a nose-cone (2% isoflurane in 100% oxygen, gas flow rate: 2 L min
1) during the scanning. The precision 3D translational stage with a step size of 100 mm was used to move the entire photoacoustic imaging probe for raster scanning in the x-y plane. Photoacoustic images were acquired before and after intravenous injection of Cu2
xS NPs (injection dose: 10 mg Cu kg
1) at different time intervals (5 s, 1 h, 4 h, and 24 h), with a laser energy density of 2 mJ cm
2 (at a wavelength of 1064 nm), well below the 40 mJ cm
2. 4.10. Animal experiments All the nude mice and Kunming mice were purchased and raised at the Laboratory Animal Centre, Shanghai Tenth People's Hospital of Tongji University, China. Cells suspended in PBS were subcutaneously injected into the right hind leg region of the mice to establish HeLa tumor model. The physiological saline solutions containing Cu2
xS NPs were intravenously or intratumoraly injected in nude mice (10 mg Cu kg
1) for MR/infrared thermal/photoacoustic imaging and tissue section studies, while Kunming mice for blood biochemistry/hematology evaluation. All animal experiments were performed in compliance with protocols approved by the Laboratory Animal Centre of Tongji University and carried out ethically and humanely. Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Acknowledgment This work was supported by the National Basic Research Program of China (973 Program, Grant No. 2011CB707905), China National Funds for Distinguished Young Scientists (51225202), National Natural Science Foundation of China (Grant No. 51132009 and 81371570), Shanghai Excellent Academic Leaders Program (Grant No. 14XD1403800). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.04.020. References [1] Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics. CA Cancer J Clin 2014;2014(64):9e29. [2] Brody H. Medical imaging. Nature 2013;502:S81.

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