Ambient Aqueous Synthesis of Ultrasmall Ni0.85Se Nanoparticles for

Nov 17, 2017 - Large-size-induced long-term retention in the body has hampered the translational applications of many reported nanomedicines. Herein, ...
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Ambient Aqueous Synthesis of Ultrasmall Ni0.85Se Nanoparticles for Non-Invasive Photoacoustic Imaging and Combined Photothermal-Chemo Therapy of Cancer Xianwen Wang, Fei Li, Xu Yan, Yan Ma, Zhaohua Miao, Liang Dong, Huajian Chen, Yang Lu, and Zhengbao Zha ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15780 • Publication Date (Web): 17 Nov 2017 Downloaded from http://pubs.acs.org on November 19, 2017

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Ambient Aqueous Synthesis of Ultrasmall Ni0.85Se Nanoparticles for Non-Invasive Photoacoustic Imaging and Combined Photothermal-Chemo Therapy of Cancer Xianwen Wang#†, Fei Li#‡, Xu Yan#‡, Yan Ma*†, Zhao-Hua Miao†, Liang Dong‡, Huajian Chen†, Yang Lu*‡ and Zhengbao Zha*† †

School of Biological and Medical Engineering, Hefei University of Technology, Hefei, Anhui

230009, P. R. China. ‡

School of Chemistry and Chemical Engineering, Hefei University of Technology, Hefei, Anhui

230009, P. R. China. Corresponding author: [email protected], [email protected] and [email protected]; #

These authors contributed equally to this work.

KEYWORDS: Ni0.85Se nanoparticles, photoacoustic imaging, photothermal therapy, chemotherapy, cancer

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ABSTRACT Big size induced long-term retention in the body has hampered the translational applications of many reported nanomedicine. Herein, we reported a multifunctional theranostic agent composed of ultrasmall polyacrylic acid functionalized Ni0.85Se nanoparticles (PAANi0.85Se NPs), which were successfully obtained through a facile ambient aqueous precipitation strategy. Without exhibiting any noticeable toxicity, the as-prepared PAA-Ni0.85Se NPs (average diameter of 6.40 ± 1.89 nm) showed a considerable absorption in near-infrared (NIR) region and high photothermal conversion efficiency (PTCE) of 54.06%, which could induce remarkable photoacoustic signals for tumor imaging and heat for localized ablation of cancerous cells upon exposure to NIR light. Notably, the ultrasmall PAA-Ni0.85Se NPs, unlike conventional nanomaterials with larger sizes, showed reasonable body clearance within 8 h after intravenous injection. Furthermore, ascribed to protonation process of amino groups in DOX molecules and carboxyl groups in PAA molecules at acidic microenvironment, the drug-loaded (doxorubicin hydrochloride, DOX·HCl) PAA-Ni0.85Se NPs (PAA-Ni0.85Se-DOX NPs) revealed promoted drug release at acidic pH, which could be useful for acidic tumor microenvironment responsive drug delivery. Evident from the results of cell-killing assay in vitro and tumor treatment study in vivo, PAA-Ni0.85Se-DOX NPs exhibited evident synergistic effects on killing 4T1 breast cancer cells. Thus, this study presents a multifunctional theranostic agent composed of ultrasmall PAANi0.85Se NPs for potential cancer treatment without long-term toxicity concerns.

Introduction Up to now, cancer is still devastating to human body and become an agonizing worldwide health challenge.1-3 Compared to clinically used cancer treatments, photothermal therapy (PTT), which could employ PTT agents to convert light energy into hyperthermia to precisely ‘cook’

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cancer cells with minimal damages to healthy tissues, has attracted considerable interest in noninvasive oncotherapy.4-7 To become a potential promising PTT agent, it should possess several features at the same time, including good biocompatibility, small and uniform diameter, considerable absorption in the tissue-penetrable near-infrared (NIR) region (λ = 650-1000 nm), high photothermal conversion efficiency (PTCE) and stability, etc.8 Although numerous PTT agents have been explored ascribed to the prosperous development of materials science and nanotechnology, they usually suffer from their own drawbacks. For example, despite inorganic PTT agents are usually photostable (except some gold-based PTT agents), large size and nondegradable property in vivo which would bring long-term toxicity concerns hinder their further applications.9 In the contrary, organic dyes (i.e., indocyanine green, ICG) with relatively ultrasmall size would be eliminated from body quickly without showing long-term toxicity, but they are easily to be photo-bleached exposure to continuous laser illumination. Thus, developing ultrasmall PTT agents (< 10 nm) with high PTCE and stability would become a feasible strategy to overcome the abovementioned deficiency of reported PTT agents.10-12 In addition, it is not easy to thoroughly eliminate cancer cells by PTT alone, especially deepseated cells, owing to the unavoidable light scattering and absorption effect in vivo.13 Encouraged by the phenomenon that hyperthermia would promote the cellular uptake of chemotherapeutic drug, the combination of PTT and chemotherapy can leading to a noteworthy enhanced therapeutic outcome.14 Thereafter, plenty of PTT agents, including gold nanostructures,15-17 carbon-based nanomaterials,10, 18-21 palladium nanosheets,22 transition-metal chalcogenides,23-24 and numerous organic polymeric nanoparticles,6, 14, 25-26 have been developed for synergistic PTT-chemo cancer treatment. However, limited nanomaterials have been

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explored as both PTT agents and drug carriers up to now for combined photothermal-chemo cancer therapy. Among various transition-metal chalcogenide nanomaterials, nickel chalcogenides which are non-noble metal based nanoparticles have shown great potentials in various applications, including superconductors, biomedical sensors, catalysis, solar cells, lithium-ion batteries, and so on.27 Due to their interesting magnetic properties, Cai et. al. fabricated a Ni-integrated CuS nanostructure through a chelator-free doping method as a multifunctional contrast agent for photoacoustic (PA)/magnetic resonance (MR) imaging.16 As an important class of nickel chalcogenides, nickel selenides have been applied in many fields recently due to their distinctive electronic configurations and comparatively high catalytic activities, as well as straightforward synthesis.28 However, rare works have been done to explore the application of nickel selenides in biomedicine.

Scheme 1 Schematic illustration of preparing PAA-Ni0.85Se-DOX NPs for combined photothermal-chemo cancer therapy.

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Herein, we report the synthesis of polyacrylic acid functionalized Ni0.85Se nanoparticles (PAANi0.85Se NPs) with an ultrasmall dimension through a facile ambient aqueous precipitation method and then investigate their theranostic properties for cancer treatment (Scheme 1). The asprepared PAA-Ni0.85Se NPs exhibit a considerable NIR absorption as well as high PTCE, which would be well suited for PA imaging and photothermal treatment of cancer cells by exposing to NIR laser (808 nm) irradiation. Moreover, PAA-Ni0.85Se NPs can also load chemotherapeutic drug (Doxorubicin hydrochloride, DOX·HCl) through electrostatic attraction to obtain PAANi0.85Se-DOX NPs, which shows an acidic pH-promoted drug release property. Furthermore, the as-prepared PAA-Ni0.85Se-DOX NPs can realize combined PTT and chemotherapy upon NIR laser irradiation, which shows an enhanced cell-killing effect in both cell culture assay and tumor inhibition experiment after intravenous injection. Thus, PAA-Ni0.85Se NPs reported here has great potential as a promising theranostic nanoagent for cancer treatment.

Materials and Methods Materials. Selenium powder (~100 mesh, ≥99.5%), NiCl2·6H2O (≥99%), sodium borohydride (NaBH4, 99%) were purchased from Aladdin Industrial Corporation. DOX·HCl was obtained from Beijing Huafeng United Technology Co, Ltd. Polyacrylic acid (PAA, Mw = 3000~5000, 99%) was bought from Tianjin Kemiou Chemical Reagent Co., Ltd. Deionized water (DI water) with a resistivity of 18.2 MΩ·cm was used. Ambient Aqueous Synthesis of PAA-Ni0.85Se NPs. In a typical synthesis, selenium precursor solution was firstly prepared by mixing NaBH4 (0.3 mmol) and Se powder (0.1 mmol) with 9.0 mL DI water under inert atmosphere at room temperature. On the other side, 100 µL PAA was dispersed in 90 mL of DI water and added with 1.0 mL of NiCl2·6H2O (0.1 M) solution. After 5 min stirring, the selenium precursor was dropwise added into the above mixture, and then a black

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solution was immediately generated, which indicated the formation of PAA-Ni0.85Se NPs. The black solution was subsequently purified by dialysis for 24 h and stored at 4 oC for further use. Characterization of PAA-Ni0.85Se NPs. The hydrodynamic diameter of PAA-Ni0.85Se NPs were measured by a NanoBrook-90 Plus instrument. A JEM-2100F transmission electron microscope (TEM) and atomic force microscope (AFM, Bruker Dimension Icon.) were used for acquiring the morphology of as-prepared PAA-Ni0.85Se NPs. Powder X-ray diffraction (XRD) patterns (PANalytical B. V.) were recorded to characterize the phase purity and crystallographic structure of PAA-Ni0.85Se NPs. X-ray photoelectron spectroscopy (XPS) of PAA-Ni0.85Se NPs was also characterized by ESCALab 250Xi spectrometer. The absorption spectra of PAANi0.85Se NPs were collected by a U-5100 UV-vis-NIR spectrophotometer. The exact content of nickel and selenium element in nickel selenide NPs were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES, Atomscan Advantage). Temperature Elevation of PAA-Ni0.85Se NPs under NIR Laser Irradiation. 3.0 mL of PAA-Ni0.85Se NPs solution was illuminated for 10 min by a continuous NIR laser (MDL-III-8082, China). The solution temperature was monitored and recorded every 10 s. Moreover, the photostability of PAA-Ni0.85Se NPs under NIR laser irradiation was performed by using LASER ON/OFF cycles. And then, UV-vis-NIR spectra of PAA-Ni0.85Se NPs were performed to investigate the photostability and the PTCE was also calculated according to the reported papers.29-30 PA Imaging. Both in vitro and in vivo PA imaging enhanced ability of PAA-Ni0.85Se NPs were acquired by a commercialized scanner (Nexus 128, USA) with a laser source wavelength of 808 nm. The PA signals of PAA-Ni0.85Se NPs aqueous solutions (0, 0.3125, 0.625, 1.25, 2.5 and 5.0 mM) were recorded in vitro to evaluate the PA imaging enhanced ability. Moreover, Balb/c

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nude mouse (weight ~20 g, n = 5) with 4T1 tumor xenograft was intravenously injected with PAA-Ni0.85Se NPs dispersions (100 µL, 5.0 mM) followed acquiring PA imaging of tumor at predetermined time intervals. The signal intensity of PA imaging was measured by the own software of photoacoustic computed tomography scanner. All the animal handlings were performed ethically and guided by Xiamen University Laboratory Animal Center. In Vitro DOX Loading and Release Profiles. DOX·HCl, as a model chemotherapeutic drug, was used here to incubated with PAA-Ni0.85Se NPs to form PAA-Ni0.85Se-DOX NPs through electrostatic interaction. Briefly, a certain amount of DOX·HCl were incubated with PAANi0.85Se NPs (1.0 mg mL-1, 3.0 mL) overnight under magnetic stirring. PAA-Ni0.85Se-DOX NPs was then collected by three ultrafiltration (9000 rpm, 5 min)/washing cycles. The discarded solutions were kept and used for calculating DOX loading capacity (LC) and loading efficiency (LE). After that, the final product was dispersed in phosphate buffer solution (PBS, pH=7.4). The LC and LE of DOX were calculated according to equations as below: LC = LE =

    –           . ! "      –             

× 100%

(1)

× 100%

(2)

PAA-Ni0.85Se-DOX NPs (1.0 mg mL-1, 1.0 mL) was packaged with a dialysis membrane (MW=3500) and then submerged in 30 mL PBS (pH = 5.0 or 7.4) which was shaken with a constant speed at 25 oC, 37 oC or 50 oC. To monitor the releasing process, the release medium (3.0 mL) was collected at predetermined time interval, and then fresh PBS with same volume was added to maintain the releasing system unchanged. NIR laser irradiation was also carried out to evaluate the influence of light/heat on drug release properties of PAA-Ni0.85Se-DOX NPs. Cell Cytotoxicity Study. The biocompatibility, localized photothermal cell-killing ability and synergistic cell-killing efficiency of as-prepared PAA-Ni0.85Se NPs was evaluated according to

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our previously published work.24 Fluorescence Activated Cell Sorting (FACS) technology was further used to quantitatively investigate the intracellular uptake of DOX.24 Pharmacokinetics and Biodistribution Assay. All animal handlings were approved by Anhui Medical University Animal Use Committee (LLSC20150134). ICR mice were intravenously injected with as-prepared PAA-Ni0.85Se NPs (2.0 mM, 200 µL per mouse), and then the animals were sacrificed at predetermined time points(n = 5 per group). Blood and major organs were collected, weighed and digested with nitric acid. The limpid solution was diluted to 10 mL for measuring the content of nickel by ICP-AES, and distributions of PAA-Ni0.85Se NPs were expressed as percentages of injected dose per gram of tissue (% ID/g). In Vivo Tumor Treatment Study. Female Balb/c mice were subcutaneously injected with murine 4T1 cancer cells (1 × 106 cells per mouse, volume: 100 µL) to their breasts and randomly grouped when tumor grew to ~30 mm3. Each group of mice were treated with 200 µL of PAANi0.85Se NPs (2.0 mM) or PAA-Ni0.85Se-DOX NPs (2.0 mM) through tail vein injection. PBS and equivalent amount of free DOX was used as control. After 4 h injection, tumors were selectively illuminated with/without NIR laser (1.0 W cm-2) for 10 min. An IR thermal camera (Fluke, USA) was used here to record the temperature change of tumor. After various treatments, tumor volume (width2 × length/2) and body weight of mice were measured every other day for two weeks. After two weeks, mice with tumors were sacrificed and vital tissues were collected for analysis.

Results and discussion Characterization of PAA-Ni0.85Se NPs Ultrasmall PAA-Ni0.85Se NPs was developed by using a facile one-pot aqueous precipitation method and functionalized with biocompatible PAA molecules. After removing the superfluous

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PAA molecules and unreacted ions by dialysis method, well-dispersed PAA-Ni0.85Se NPs was finally obtained. As shown in Figure 1a, the as-prepared PAA-Ni0.85Se NPs displayed a monodisperse, dot-like morphology with an average diameter of 6.40 ± 1.89 nm (Figure 1b). AFM images revealed that most PAA-Ni0.85Se NPs had a height of about 6.5 nm, the slight bigger diameter determined from AFM images could be ascribed to the PAA surface layer which was hardly seen in the TEM images (Figure S1). Compared with Ni0.85Se precipitate without stabilizer or post-modified with PAA molecules, the PAA-Ni0.85Se NPs showed better stability and smaller size which are important for biomedical applications (Figure S2). XRD analysis was carried out to characterize the component and crystal structure of obtained nickel selenide NPs (Figure 1c), and characteristic diffraction peaks at 33.1o, 44.9o, 50.4o, 60.2o, 61.8o, and 71.4o that can clearly confirm the composition of hexagonal phase Ni0.85Se crystal (JCPDS No.18-0888). Moreover, the well-defined crystalline lattice with a 0.181 nm spacing in the typical high resolution TEM image (Figure 1d) matches well with (110) plane of Ni0.85Se, which can be further confirmed by the fast Fourier transform (FFT) pattern. Energy dispersive X-ray (EDX) elemental mapping images of the PAA-Ni0.85Se NPs have been shown in Figure 1e, further illustrating the component containing Ni and Se elements. ICP-AES was utilized to determine the exact element content in nickel selenide NPs. As 2.0 mM (500 µg mL-1) PAANi0.85Se NPs, the concentration of nickel and selenium element were determined to be 119.1 and 148.4 µg mL-1, respectively, indicating a Ni/Se stoichiometry of 0.80:1 which confirmed the formation of quasi-stoichiometric Ni0.85Se NPs. Therefore, all of the above results suggested that the PAA-Ni0.85Se NPs had been prepared successfully through this facile ambient aqueous solution approach.

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Figure 1. Characterization of as-prepared PAA-Ni0.85Se NPs. TEM image a) and the diameter distribution b) of PAA-Ni0.85Se NPs; c) XRD pattern collected on PAA-Ni0.85Se NPs, which exhibits a good match with hexagonal phase Ni0.85Se (JCPDS No.18-0888); d) High resolution TEM image, inset: FFT pattern of PAA-Ni0.85Se NPs; e) EDX elemental mapping images of the PAA-Ni0.85Se NPs. We further used the XPS characterization to confirm the composition of as-prepared NPs. The XPS characterization yields a Ni/Se stoichiometry of 0.81:1, which indicates the formation of quasi-stoichiometric Ni0.85Se. As shown in Figure 2a, the Ni 2p spectrum could be best fitted with two spin-orbit doublets, characteristic of Ni2+ and Ni3+, as well as two shake-up satellites

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(Sat.).20 The binding energy of ca. 55.0 eV was attributed to Se 3d (Figure 2b). In Se 3d region one small peak probably arise due to oxidation of Se2- on the surface of PAA-Ni0.85Se NPs. Meanwhile, PAA molecules which guaranteed the stability Ni0.85Se NPs would generated O 1s and C 1s peaks during XPS analysis (Figure 2c and 2d). Thus, based on the results of XPS characterization, the as-prepared NPs are consisted of Ni2+, Ni3+, and Se2-, further supporting the purified Ni0.85Se phase.

Figure 2. XPS characterization of PAA-Ni0.85Se NPs. Core level spectrum of a) Ni 2p; b) Se 3d and c) C 1s; d) the survey spectrum. Maintaining the hydrodynamic diameter of NPs below 500 nm is crucial for applying NPs in biomedical field.31 As seen from Figure 3a and 3b, PAA-Ni0.85Se NPs could be dispersed well in both DI water and DMEM medium without aggregates, as well as linearly growing absorbance with incremental NPs dose (Figure S3a and S3b). Owing to the hydrophilic PAA surface layer to weaken adsorption of proteins, no distinct change in size distribution was found for PAA-

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Ni0.85Se NPs dispersed in various medium (Figure S4). In addition, the long-term stability of PAA-Ni0.85Se NPs was further evaluated by incubating NPs in DI water, PBS and DMEM medium for more than one week. As seen from Figure S5, no observable difference was observed in hydrodynamic diameter of PAA-Ni0.85Se NPs, demonstrating the excellent stability of as-prepared PAA-Ni0.85Se NPs.

Figure 3. Characterization of PAA-Ni0.85Se NPs: UV-vis-NIR spectra in a) DI water and b) DMEM medium (insets: photographs of NPs dispersions with gradient concentrations); Temperature changes of PAA-Ni0.85Se NPs solution upon NIR laser irradiation with c) gradient solution concentrations; and d) different power density of laser; e) LASER ON/OFF cycles; f) photothermal profile of PAA-Ni0.85Se NPs aqueous solution (inset: τs =711.4 s).

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Photothermal and Photoacoustic Imaging Effect of PAA-Ni0.85Se NPs Considerable NIR absorption and high PTCE are two basal preconditions for NPs used for PTT. Fortunately, PAA-Ni0.85Se NPs possess considerable absorption in the NIR region both in DI water and DMEM medium (Figure 3a and 3b, the strong absorption around 560 nm in Figure 3b was ascribed to the absorption of DMEM medium), suggesting the potential of PAA-Ni0.85Se NPs as a PTT agent. Thus, exposing PAA-Ni0.85Se NPs aqueous solution (400 µM, 3.0 mL) to a NIR laser irradiation (2.0 W, 10 min) would increase the solution temperature from 23.0 oC to 51.2 oC, while only 2.0 oC for DI water (Figure 3c). The elevated magnitude of solution temperature depended on not only the NPs concentration (Figure S6a), but also the power density of NIR laser (Figure 3d and S6b). Due to the importance of photostability for PA imaging and PTT, five NIR LASER ON/OFF irradiation cycles were further used. No significant decrease in either the elevated magnitude of solution temperature (Figure 3e) or the spectra of PAA-Ni0.85Se NPs before and after NIR laser irradiation cycles (Figure S7), implying the excellent photothermal stability of as-prepared PAA-Ni0.85Se NPs. Moreover, based on reported method,32 the PTCE of PAA-Ni0.85Se NPs was calculated to be 54.06%. These data suggested that PAA-Ni0.85Se NPs could be utilized as a promising PTT agent for cancer treatment. To monitor the in vivo behaviour of PAA-Ni0.85Se NPs, we studied the pharmacokinetics of PAA-Ni0.85Se NPs in female ICR mice and the blood activity-time profile was presented in Figure 4a. After single intravenous injection, the blood activity-time course curve for PAANi0.85Se NPs showed a bi-exponential disposition. Thus, the pharmacokinetic parameters for each mouse were determined with a two-compartment model, and the mean and standard deviations of pharmacokinetic parameters are summarized in Table S1. Due to the good hydrophilic property of PAA surface layer and similar small diameters, the mean systemic

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clearance (0.52 mL h-1) and mean volume of distribution at steady state (Vd) of PAA-Ni0.85Se NPs (7.87 mL) were comparable to that of reported ultrasmall PEG-[64Cu]CuS NPs (0.48 mL h-1 and 3.79 mL, respectively).2 As shown in Table S1, the mean blood distribution half-life, tα1/2, and blood terminal elimination half-life, tβ1/2, was 0.17 h and 10.77 h, respectively, confirming the in vivo quick elimination ability of these ultrasmall PAA-Ni0.85Se NPs which behaved like small-molecular-weight compounds that can be cleared through the renal-urinary system.5 Furthermore, the biodistribution of PAA-Ni0.85Se NPs was also investigated in ICR mice (Figure 4b). After immediate intravenous injection, the liver, kidney and spleen had the dominant PAANi0.85Se NPs content and then significantly decreased during the following 6~8 h, indicating that the majority of PAA-Ni0.85Se NPs might be efficiently eliminated through the reticuloendothelial systems and renal-urinary system after 6 h intravenous injection.5 Among various medical imaging modalities, PA imaging is an emerging cancer imaging technology with several merits, including good spatial resolution, reasonable penetration depth, and so on.33 To demonstrate the capacity of PAA-Ni0.85Se NPs as a novel PA imaging contrast agent, both in vitro and in vivo photoacoustic signals of PAA-Ni0.85Se NPs were collected. As shown in Figure 4c, PAA-Ni0.85Se NPs could generate strong photoacoustic signal upon exposure to pulsed NIR laser, exhibiting a concentration-dependent manner. Moreover, the contrast of tumor region was gradually enhanced in the first 4 h after intravenous injection of PAA-Ni0.85Se NPs, indicating a continuous accumulation of PAA-Ni0.85Se NPs (Figure 4d). Moreover, the maximum PA imaging signal enhancement of the tumor site by PAA-Ni0.85Se NPs was achieved at 4 h post injection, attributed to their ultrasmall size and leaky vasculature of tumor site. At the final time point (8 h after intravenous injection), a negligible signal enhancement in the tumor was observed, indicating the successful excretion of most PAA-

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Ni0.85Se NPs from the tumor region (Figure 4d and 4e) which was consistent with the biodistribution results. These results demonstrate that PAA-Ni0.85Se NPs showing impressive improvement on PA imaging, which could illuminate the tumor region easily.

Figure 4. Pharmacokinetic profiles (a) and (b) biodistribution of PAA-Ni0.85Se NPs (n = 5) after intravenous injection in ICR mice. In vitro c) and in vivo d) photoacoustic imaging ability of PAA-Ni0.85Se NPs; e) quantitative photoacoustic signal intensity of tumor region before and after PAA-Ni0.85Se NPs intravenous injection. Investigation of Drug Loading and Release Behaviour in vitro

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Forced by the fact that deep-seated tumor cells could not be erased completely by PTT alone due to the shortage of heat, DOX·HCl was used here to test the possibility of PAA-Ni0.85Se NPs as stimuli-responsive drug carriers to realize synergistic effect of combined PTT-chemo cancer treatment. Upon mixing DOX and PAA-Ni0.85Se NPs overnight and ultrafiltration process, PAANi0.85Se-DOX NPs was successfully obtained. Notably, PAA-Ni0.85Se-DOX NPs occupied characteristic absorption of both PAA-Ni0.85Se NPs and free DOX (Figure 5a), indicating successful DOX loading. Not surprisingly, the LC and LE of DOX onto PAA-Ni0.85Se NPs depended on the concentration of DOX molecules (Table S2) through the electrostatic interactions between DOX and the PAA-Ni0.85Se NPs.33

Figure 5. a) UV-vis-NIR spectra (inset: photographs of solutions); in vitro DOX release from PAA-Ni0.85Se-DOX NPs in PBS buffer with different pH under NIR laser irradiation b) and under different temperatures c); d) mechanism for acidic pH-promoted drug release property of PAA-Ni0.85Se-DOX NPs.

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A typical dialysis method was utilized to figure out the in vitro DOX release profile. Briefly, PAA-Ni0.85Se-DOX NPs (with weight ratio of 1:10 for DOX to PAA-Ni0.85Se NPs) were dispersed in PBS (pH = 7.4 or 5.0). The release amount of DOX at predetermined time intervals was calculated from fluorescent peak (580 nm) intensity of DOX in released medium. As shown in Figure 5b, only 19.4% of DOX was released from PAA-Ni0.85Se-DOX NPs at pH 7.4 over 10 h, while 55.7% of DOX was released at pH 5.0. The acidic pH-promoted drug release phenomenon may be caused by the protonation of amino group in DOX molecules and carboxylate radical in PAA molecules at pH 5.0, which weakens the electrostatic attraction interactions between PAA-Ni0.85Se NPs and DOX molecules while the repulsive force is increased (Figure 5d). The acidic pH-promoted drug release profile of PAA-Ni0.85Se-DOX NPs was similar to other previously reported PAA-functionalized drug carriers.33 In contrast, the release velocity and amount of DOX are slightly changed at both pH 7.4 and 5.0 in spite of NIR laser irradiation, which may be attributed to no thermo-/light-sensitive groups existed in PAANi0.85Se-DOX NPs. We further investigated the influence of temperature on drug release profiles by incubating the system in the heating water bath with setting temperature of 25 oC and 50 oC. As seen from Figure 5c, there was no significant difference in release drug amount when the system temperature at either 25 oC or 50 oC, indicating negligible effect of temperature on drug release profiles of PAA-Ni0.85Se-DOX NPs. Therefore, any potential synergistic effect of combined PTT-chemo treatment delivered by PAA-Ni0.85Se-DOX NPs is maybe resulted from heat-promoted cellular uptake of NPs/drug rather than enhanced drug release. Synergistic PTT-Chemo Treatment of Cancer Cells An ideal PTT agent for biomedical applications should be nontoxic or low-toxic. Therefore, we used a standard MTT assay to evaluate the cytotoxicity of PAA-Ni0.85Se NPs on HUVECs

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which is a commonly used normal cell line (Figure 6b). The percentage of viable HUVECs was still 84.1 ± 5.3% after incubating with PAA-Ni0.85Se NPs (3.0 mM) for 24 h, indicating the good biocompatibility of PAA-Ni0.85Se NPs.

Figure 6 a) Characterization of localized photothermal cell-killing effect of PAA-Ni0.85Se NPs upon NIR laser irradiation; b) cell viability of HUVECs; cell viability of 4T1 cells treated with laser irradiation for c) 0 min; d) 3 min and e) 5 min. Encouraged by its good biocompatibility, the localized photohyperthermic effect of PAANi0.85Se NPs was evaluated by killing 4T1 breast cancer cells under NIR laser irradiation, following a typical Live/Dead cell staining process. Similar to the negative control group

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(without any treatment), free DOX plus NIR laser irradiation (2.0 W, 5 min) would not induce acute cell death due to deficient DOX uptake and heat. Also, either PAA-Ni0.85Se NPs or laser irradiation alone showed no influence on cell viability (Figure 6a), indicating the safety of PAANi0.85Se NPs and laser power density used here. In the contrary, a combined treatment of NIR laser irradiation (2.0 W, 5 min) and PAA-Ni0.85Se NPs (200 µM, 400 µL) resulted in obvious cell death of 4T1 cells, suggesting the good localized photothermal cell-killing effect. However, no observable cell death was found with short-time NIR laser irradiation (3 min) due to insufficient generated-heat. Moreover, the zone of cell death would expand beyond the region of NIR laser spot as higher concentration of PAA-Ni0.85Se NPs (400 µM, 400 µL) was used, even under 3 min NIR laser irradiation. Thus, the localized photothermal cell-killing effect of PAA-Ni0.85Se NPs could be adjusted by tuning either the dose of NPs or intensity of NIR laser irradiation to realize desired cancer therapy. The synergistic PTT-chemo treatment resulted from PAA-Ni0.85Se-DOX NPs with NIR laser irradiation was further quantitatively investigated by using a typical MTT assay. The PAANi0.85Se NPs showed little toxicity to 4T1 cells in dark (Figure 6c), while the cell survival decreased as the dose of NPs/laser irradiation increased when cells were treated with PAANi0.85Se-DOX NPs (Figure 6d and 6e). Without NIR laser irradiation, although about only 55.7% of DOX would release from PAA-Ni0.85Se-DOX NPs under acidic condition (acidic lysosome), the enhanced cellular uptake of PAA-Ni0.85Se-DOX NPs which was facilitated by endocytotic transport would supply comparable intracellular DOX in comparison to free DOX group which are passively diffused into 4T1 cells through the cell membrane, leading to a similar cell-killing efficiency (Fig. 6c). Upon 5 min of NIR laser illumination (2.0 W), the cell viability was 75.6% and 64.5% when 4T1 cells were treated with PAA-Ni0.85Se NPs (49.1 µM) and free DOX (1.0 µg

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mL-1), respectively. Surprisingly, a much lower cell viability (25.7%) was reached when PAANi0.85Se-DOX NPs was used. Namely, the cell-killing efficiency of PAA-Ni0.85Se-DOX NPs under 5 min laser irradiation (74.3%) is evidently higher than the overlay of single PTT by PAANi0.85Se NPs (24.4%) and chemotherapy by free DOX (35.5%), indicating an obvious synergistic cell-killing effect. Thus, this experimental finding demonstrated that PAA-Ni0.85Se-DOX NPs were promising for combined PTT-chemo cancer treatment with impressive synergistic effect.

Figure 7. FACS analysis of DOX uptake by 4T1 cells treated with a) free DOX, b) 0.4 mM and c) 0.8 mM PAA-Ni0.85Se-DOX NPs under 808 nm laser irradiation (1.0 W cm-2); d) quantitative assessment of intracellular DOX. FACS technology was further used to quantitatively investigate the heat-promoted DOX cellular uptake process which were induced by PAA-Ni0.85Se-DOX NPs under NIR laser illumination (Figure 7a, 7b, and 7c). In comparison to free DOX, no significant enhanced DOX

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accumulation in 4T1 cells was found when cells were incubated with PAA-Ni0.85Se-DOX NPs (either 0.4 mM or 0.8 mM) shortly (Figure 7d), while both NPs and laser irradiation dose dependent manners were observed for DOX cellular uptake in 4T1 cells. Specifically, there is a greatly enhanced intracellular DOX when cells treated with 0.8 mM PAA-Ni0.85Se-DOX NPs plus 5 min NIR laser illumination, indicating a heat-promoted DOX cellular uptake process occurred. Therefore, these data demonstrated that enhanced DOX cellular uptake would be resulted from incubation with PAA-Ni0.85Se-DOX NPs plus NIR laser irradiation which may be the reason for synergistic effect of combined photothermal chemotherapy of cancer cells. Motivated from the synergistic cell-killing effect of in vitro combined photothermal-chemo therapy, in vivo tumor inhibition experiments were further carried out in 4T1 tumor bearing mice with tail vein injection of PAA-Ni0.85Se-DOX NPs and NIR laser irradiation. While the volume of tumor reached ~30 mm3, randomly grouped mice were injected with 200 µL of PBS, free DOX (50 µg mL-1, 200 µL), PAA-Ni0.85Se NPs (2.0 mM, 200 µL) or PAA-Ni0.85Se-DOX NPs (2.0 mM, 200 µL), as well as no treatment for control group. After 4 h of materials injection (according to the results of in vivo tumor PA imaging), tumors were locally irradiated with 808 nm laser (1.0 W cm-2) for 10 min and their temperatures were simultaneously monitored (Figure 8a). Upon 10 min of NIR laser irradiation, the temperature of tumor injected with PAA-Ni0.85Se NPs or PAA-Ni0.85Se-DOX NPs was locally reached to about 56.2 oC or 54.8 oC, respectively (Figure 8b). On the contrary, the tumor temperature in PBS injection group was only slightly increased even with the same NIR laser irradiation for 10 min, implying the safety of NIR laser intensity used here.

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Figure 8. PAA-Ni0.85Se-DOX NPs for in vivo combined PTT-chemo tumor treatment. a) IR images and b) tumor temperature changes of 4T1 tumor-bearing mice; growth curves of c) tumor volume and d) body weight of mice after various treatments; e) representive photographs of mice before treatment and 14 days after various treatments as indicated. After various treatments, the therapeutic efficiency was evaluated by measuring the tumor volume with a digital calliper every other day (Figure 8c and 8e). It was found that the tumors treated with free DOX and PBS plus NIR irradiation grew quickly within two weeks, similarly to control group, indicating that either free DOX at this low dose or slightly elevated temperature

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was not effective enough in inhibiting the tumor growth. For the mice injected with PAANi0.85Se NPs and exposed to the NIR laser illumination, localized hyperthermia resulted in notably tumor inhibition in the first four days. However, due to insufficient heat generated in the deep-seated tumor cells, tumors in this group could not be erased completely and became recurrence at later time points. Notably, significant tumor inhibition and no recurrence were realized in mice treated with PAA-Ni0.85Se-DOX NPs and NIR laser exposure, indicating a good synergistic cell-killing effect from combined photothermal chemotherapy which was also observed in cell study (Figure 8c and 8e).33-34 As body weight loss could indicate the toxicity induced by cancer treatment, the body weights of mice were monitored and no significant difference was observed after various different treatments for two weeks (Figure 8d), indicating no unacceptable toxicity. Moreover, compared to age-matched healthy mice, no observable organ damage and inflammation were found in mice treated with PAA-Ni0.85Se-DOX NPs and NIR laser irradiation (Figure 9), indicating that the PAA-Ni0.85Se-DOX NPs based in vivo combined photothermal-chemo therapy induced no significant side effects.

Figure 9. Micrographs of H&E stained organ slices from PAA-Ni0.85Se-DOX NPs plus NIR laser synergistically treated group at day 14 post treatment and healthy control group. Examined organs included heart, liver, spleen, lung and kidney. No observable organ damage was observed for the treatment group.

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Conclusions In summary, ultrasmall PAA-Ni0.85Se NPs were successfully developed through an ambient aqueous solution approach to realize photoacoustic imaging and combined photothermal-chemo treatment of cancer cells both in vitro and in vivo. The as-prepared PAA-Ni0.85Se NPs showed not only promising absorption in the NIR region, but also high photothermal conversion efficiency as high as 54.06%, indicating the great potential of PAA-Ni0.85Se NPs to act as a new type of agent for tumor PA imaging and photothermal ablation. After adsorbing DOX onto the surface of NPs through electrostatic interactions, the formed PAA-Ni0.85Se-DOX NPs showed an acidic pH promoted drug release property and synergistic cell-killing effect to cancer cells with minimal side effects. Therefore, our results highlighted the potential of PAA-Ni0.85Se NPs as a theranostic agent for tumor PA imaging and efficient elimination.

ASSOCIATED CONTENT Supporting Information Available Calculation of photothermal conversion efficiency, AFM image, stability characterization, the linear increased absorbance of PAA-Ni0.85Se NPs as incremental concentration, hydrodynamic size, photothermal elevation ability and stability characterization, pharmacokinetic parameters and summary of DOX loading capacity and efficiency. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected] and [email protected] Notes

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The authors declare no competing financial interest.

Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 81501590; No. 31500808; No. 21501039; No. 51572067), the Anhui Provincial Natural Science Foundation (No. 1608085MH188).

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(34) Liu, T.; Wang, C.; Gu, X.; Gong, H.; Cheng, L.; Shi, X. Z.; Feng, L. Z.; Sun, B. Q.; Liu, Z., Drug Delivery with PEGylated MoS2 Nano-Sheets for Combined Photothermal and Chemotherapy of Cancer. Adv. Mater. 2014, 26, 3433-3440.

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Scheme 1 207x151mm (300 x 300 DPI)

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