Seeded Growth of Cu2âxSe Nanocrystals and Their Size-Dependent

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Seeded Growth of Cu Se Nanocrystals and Their Size-dependent Phototherapeutic Effect Xianguang Ding, Dongdong Fu, Ye Kuang, Yu Zou, Xiuzhu Yang, Liangzhu Feng, Xia Sun, Haiyan Wu, and Jiang Jiang ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00516 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 26, 2018

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ACS Applied Nano Materials

Seeded Growth of Cu2-xSe Nanocrystals and Their Size-dependent Phototherapeutic Effect Xianguang Ding, †,‡ Dongdong Fu, †,‡,§ Ye Kuang, † Yu Zou, † Xiuzhu Yang, †,§ Liangzhu Feng, ⊥ Xia Sun, † Haiyan Wu, † and Jiang Jiang*† †

i-Lab and Division of Nanobiomedicine, CAS Key Laboratory of Nano-Bio Interface, CAS

Center for Excellence in Nanoscience, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China §

University of Chinese Academy of Sciences, Beijing, China 100049

⊥

Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for

Carbon-Based Functional Materials & Devices, Soochow University, 199 Ren’ai Road, Suzhou 215123, China

Corresponding author: * [email protected]

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Abstract Doped semiconductors supporting localized surface plasmon resonances, such as nonstoichiometric copper chalcogenides Cu2-xSe, have been intensively researched for their potential applications in photoacoustic imaging and photothermal therapy. For these self-doped nanoparticles, their physicochemical attributes such as size and surface chemistry will not only affect their cellular uptake and biodistribution, but also influence their own optical properties. Thus, optimization of the physicochemical properties is crucial for their nano-bio applications. Through a seeded growth approach, aqueous phase synthesis of monodisperse Cu2-xSe with various sizes has been developed for the first time. Taking three different sized Cu2-xSe NPs (35, 65, and 105 nm in diameter) as examples, their size dependent optical cross section, photothermal property, and cellular cytotoxicity have been investigated, and the 65 nm one displays the largest optical cross section at 21.57 L g-1 cm-1 and best photothermal property per unit mass. In addition, Cu2-xSe exhibits a size dependent ROS generation effect that is inversely proportional to their diameters. Finally, on the basis of optical property and cytotoxicity optimizations, the photothermal cancer cell ablation ability and potential use of Cu2-xSe NPs as photoacoustic contrast enhancing agent has been demonstrated in vivo.

KEYWORDS copper chalcogenide nanocrystals, photothermal, photoacoustic, ROS, seeded growth, size effect.


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Introduction A family of self-doped semiconducting nanocrystals, nonstoichiometric copper chalcogenide, has drawn intense research interests since their discovery in the past decade, as they support localized surface plasmon resonance (LSPR),1-3 a property which is traditionally linked to noble metal nanoparticles (NPs). LSPR of nonstoichiometric copper chalcogenides NPs lies in the NIR region, an important optical window for bio-related applications, where light encounters minimal absorption and scattering in tissues.4 The optical cross sections of these nonstoichiometric copper chalcogenides can be close to noble metals, due to their high doping level. Furthermore, the LSPR resonance frequency can be easily modulated over a broad spectral window by adjusting their chemical stoichiometry and redox states.5-6 Due to their unique tunable NIR optical resonances and ability to efficiently convert photon energy to heat, copper chalcogenides and their nanocomposites have been extensively studied in the research for cancer cell ablation,7-14 controlled drug release,10, 15-16 photoacoustic imaging,17-21 photothermal catalysis,22-23 as well as for solar water evaporation.24 In nano-bio applications, physical property of nanomaterials is a critical and yet complex factor affecting nanomaterials-cell interactions as well as nanotoxicity.25-27 For example, nanomaterials size, shape, and surface chemistry can significantly affect their in vivo tumor uptake and biodistribution.28-31 An additional complexity comes into play when using nanomaterials such as nonstoichiometric copper chalcogenides, as their LSPR also depends on the nanocrystal morphology and surface ligands.32-36 Therefore, it is of great importance to investigate the size and surface effect of these self-doped semiconductor NPs on their LSPR and photothermal conversion properties, as well as the optimized physicochemical properties for their nano-bio applications.


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Among nonstoichiometric copper chalcogenides family, copper selenide (Cu2-xSe) has less structural variations compared to copper sulfides, which exist in a wide range of stable and metastable compositions,37-38 making it a simpler model for systematic investigation of size and surface effect on photothermal properties. Despite Cu2-xSe nanocrystals have shown good potential as photothermal and photoacoustic imaging agents due to their intrinsic LSPR in the second near-infrared (NIR-II) region,39-43 there has been no report on their aqueous phase size controlled synthesis to the best of our knowledge. It is thus critical to develop an effective synthetic protocol for obtaining monodisperse Cu2-xSe NPs of various sizes and surfaces, which will then enable research on correlating the photothermal properties with their physicochemical attributes. To address this problem, a facile method of synthesizing various sized Cu2-xSe NPs of narrow dispersions has been developed by seeded growth of elemental Se NP, followed by Cu+ catalyzed disproportion reaction.44-46 Using spherical Cu2-xSe NPs of three different diameters (~35, 65, and 105 nm) as examples, their size dependent optical cross section, photothermal property, and cellular cytotoxicity have been investigated. 65 nm Cu2-xSe NPs are found to be better photothermal agents per unit mass compared with the other two NPs, which also show good biocompatibility versus their smaller counterparts. In vivo photoacoustic imaging and photothermal therapy (PTT) treatment of tumors are then carried out using intravenously injected Cu2-xSe NPs, and a potent therapeutic effect in suppressing tumor growth has been achieved without any signs of nanotoxicity for over 2 weeks, demonstrating their potential application in imaging guided phototherapy. Experimental procedures Materials


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Selenium dioxide (SeO2, 99.9%), L(+)-ascorbic acid (AA, 99.7%), copper sulfate pentahydrate (CuSO4•5H2O, 99%), and poly(styrene sulfonic acid) sodium salt (PSSNa) were purchased from Alfa Aesar Chemicals Co. Ltd (Shanghai, China). Polyvinylpyrrolidone (PVP) was purchased from Sinopharm Chemical Reagent Co. Ltd. Poly(allylamine hydrochloride) (PAH) was purchased from J&K Scientific. 2’7’-dichlorodihydrof-Luorescein (DCFH-DA) was purchased from Sigma-Aldrich. Calcein AM and propidium iodide (PI) were purchased from Thermo Fisher Scientific. All chemicals were used as received without further purifications. Deionized (DI) water (Millipore, Milli-Q grade) was used in all experiments. Cells were obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology. Female nude mice used in the experiment were obtained from Shanghai Sushang Bio-tech Company Ltd. Synthesis of PSS capped Cu2-xSe NPs A seed solution was first made by dissolving PSSNa (0.08 g) in DI water (8 mL) at room temperature, followed by adding SeO2 (0.5 mL, 0.1 M) and ascorbic acid (1.5 mL, 0.2 M), and the solution quickly turned to orange red in color. The mixture was used for subsequent Cu2-xSe NPs growth after reacting for 10 min. For the synthesis of ~35 nm diameter Cu2-xSe NPs, a mixed solution of CuSO4•5H2O (0.5 mL, 0.2 M) and ascorbic acid (2 mL, 0.2 M) was added to the seed solution. After left reacting at room temperature over night, final products were collected by centrifuging and washing with DI water 2-3 times at 8000 rpm for 10 min, and then redispersed in 2 mL water for further usage. To obtain ~65 nm diameter Cu2-xSe NPs, PSSNa (0.064 g) was first dissolved in DI water (2 mL). Then the seed solution (2 mL) was added in, followed by 3 mL each of 0.012 M SeO2 and 0.1 M ascorbic acid, injected by syringes at a rate of 90 µL min-1. After several hours, a mixture solution of CuSO4•5H2O (0.5 mL, 0.184 M) and ascorbic acid (2 mL, 0.2 M) was added in. After


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keeping the solution reacting at room temperature over night, the final products were collected in a similar way as the 35 nm NPs. Cu2-xSe NPs of diameter of ~105 nm were prepared under the following conditions: PSSNa (0.063 g) was first dissolved in DI water (1.5 mL). Then the seed solution (2 mL) was added in, followed by 3.5 mL each of 0.014 M SeO2 and 0.114 M ascorbic acid, injected at a rate of 60 µL min-1 by syringes, forming the second seed solution. Then, another growth solution was made by dissolving PSS (0.04 g) in DI water (1 mL). 4 mL of the second seed solution was added in, followed by 3.5 mL each of 0.014 M SeO2 and 0.114 M ascorbic acid, injected by syringes at a rate of 35 µL min-1. A mixture solution of CuSO4•5H2O (0.5 mL, 0.296 M) and ascorbic acid (2 mL, 0.3 M) was added last. The solution was allowed to react at room temperature over night. Final products were obtained in the same way as described above. Photothermal property characterization To evaluate the photothermal performance of different sized Cu2-xSe NPs, a cuvette containing 1.5 mL Cu2-xSe NPs aqueous dispersion of various concentrations were exposed to a laser (1064 nm) at varied power densities for 10 min. The solution temperature was measured and recorded every 20 s by a thermocouple, while a thermal imaging camera was used to capture thermal images. In vitro cytotoxicity CT26 (murine colon cancer cells) and HUVEC (human umbilical vein endothelial cells) were chosen as cancerous and normal cell examples for the evaluation of Cu2-xSe NPs cytotoxicity. Cells in logarithmic phase were seeded at a density of 7 × 103 cells per well in 96-well plates, and incubated in DMEM culture medium at 37 °C for 24 h for cell attachment. Through a series dilution, Cu2-xSe NPs in DMEM (100 µL) were added to the cells. After incubating with various


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sized Cu2-xSe NPs (~35, 65, and 105 nm in diameter) at concentrations of 200, 100, 50, 25, 12.5, 6.25, and 3.125 ppm (Cu) for 24-72 h, the NP containing media were changed to fresh DMEM to remove unbound NPs, and methyl thiazolyl tetrazolium (MTT) assays were then used to evaluate cell viability. ROS detection ROS level was measured by using a fluorescence dye indicator 2’7’-dichlorodihydrof-Luorescein (DCFH-DA). After CT26 or HUVEC cells were seeded the same way as described above, they were incubated with Cu2-xSe NPs (100 µg mL-1 Cu) for 24 h, and then washed 2-3 times by phosphate-buffered saline (PBS). Final analysis was conducted by culturing the cells with DCFH-DA (10 µM, 50 µL) at 37 °C for 30 min followed by washing. In vitro photothermal cell ablation CT26 cells were cultured at a density of 4 × 104 cells per well in 24-well plates for 24 h. After washing 2-3 times using PBS, the cells were cultured with fresh media (without NPs) and different sized Cu2-xSe NPs (100 µg mL-1 Cu) respectively, followed by laser irradiation (1064 nm, 1 W cm-2) for 10 min. After cells were cultured for additional 4 h, cell viability was evaluated by MTT assays as well as live/dead cell staining using Calcein AM and propidium iodide (PI). Photoacoustic (PA) imaging Cu2-xSe NPs (200 µL, 2 mg mL-1) was intravenously (i.v.) administered to CT26 tumor bearing mice through tail vein injection. PA images (excitation wavelength 950 nm) were then captured at different time points post i.v. injection of the Cu2-xSe NPs. In vivo photothermal therapy


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Female nude mice bearing CT26 tumors reaching a volume of ~100 mm3 were selected for in vivo experimental study. For photothermal therapy, 65 nm Cu2-xSe NPs were administered either intratumorally (100 µL, 100 µg mL-1) or intravenously (200 µL, 2 mg mL-1) to the mice. After 4 h, mice tumor area was exposed to a laser (1064, 1.0 W cm-2) for 10 min, during which the temperature was monitored using an IR thermal imaging camera. The weight of mice and the tumor sizes were measured every other day for a period of two weeks. Characterizations FEI Technai G2 S-Twin was used to take transmission electron microscopy (TEM) images. UVvis-NIR spectra were taken on a PerkinElmer Lambda 750 spectrophotometer. Inductively Coupled Plasma Optical Emission Spectrometer (PerkinElmer ICP-OES 2100 DV) was used to quantify Cu concentrations. Bruker D8 Advance X-ray diffractometer (Cu Ka, 0.15418 nm) was used to conduct powder X-ray diffraction (XRD) measurements. MTT assay was performed using a PerkinElmer Victor X4 Reader. Nikon Laser scanning microscope (Eclipse Ti, Japan) was used to capture fluorescence images. PA images were captured using a preclinical photoacoustic computerized tomography scanner (inVision 256, iThera Medical). Infrared thermal imaging was taken using an IR thermal camera (Fluke, USA). Results and discussion Seeded growth of Cu2-xSe NPs Previously, aqueous phase synthesis of Cu2-xSe NPs has been conducted by first forming Se NPs, followed by Cu+ catalyzed disproportion reaction of elemental Se.44 In this way, Cu2-xSe NPs of fixed sizes were obtained depending on the surfactant molecules used,45 which could be tuned to a limited extent by varying the surfactant molecule concentrations.43 Seeded growth strategy can separate nucleation and growth stage temporally, which is a very powerful method to control NP


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size and shape.47 By adopting a seeded growth approach, we have achieved controlled synthesis of monodisperse Cu2-xSe nanoparticles with different sizes and surfactant molecules for the first time by changing the molar ratio of growth solution to seeds, under suitable experimental conditions (Figure 1).

Figure 1. Schematic illustration of seeded growth of various sized Cu2-xSe NPs. Taking PSS capped NPs as an example, Se nanospheres of diameters 31.6 ± 2.3 nm were obtained in the first step (Figure S1a, Supporting Information), which were used as seeds for the subsequent growth processes. By carefully choosing the growth solution to seeds molar ratios, larger diameter Se NPs (65.3 ± 5.5 and 96.4 ± 5.3 nm) could then be obtained (Figure S1b-c). Good monodispersity was achieved via sequential seeded growth steps. The conversion of different sized Se to Cu2Se was then conducted by adding Cu+ generated in situ through reduction of CuSO4 by ascorbic acid, whereby the solution color changed quickly into dark brown from orange red (Figure S1d), with NP size only increased slightly from the staring Se nanospheres. After slow oxidation under ambient conditions for 10 h, solution color turned into


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dark green, in which monodisperse Cu2-xSe NPs of different sizes were obtained (Figure 2a-c). They will be referred to as 35, 65, and 105 nm NPs in the following text for simplicity.

Figure 2. (a-c) TEM images of various sized Cu2-xSe NPs with size distribution histogram in the insets, and all the scale bars represent 200 nm; (d) optical absorbance (normalized to Cu mass) and solution visual appearance of different sized NP dispersions. TEM images show that these Cu2-xSe NPs were spherical in shape with narrow size distributions. By counting over 1000 Cu2-xSe NPs for many samples from TEM images, the size distribution of the Cu2-xSe NPs was obtained and shown in the insets of Figure 2a-c. The mean hydrodynamic diameters of these NPs were determined to be 45.7, 71.8, and 109.1 nm by dynamic light scattering measurements (Figure S2), which were slightly larger than the values obtained by counting TEM images, due to surface hydration layers. Their measured surface charges were all around -25 mV (Figure S3) due to the surface PSS molecules. XRD patterns


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identified that the initial Se seeds were amorphous (Figure S4), while the as-prepared Cu2-xSe NPs were in cubic berzelianite phase with well-defined crystalline form (JCPDS 71-0044, Figure S5). High resolution TEM and corresponding electron diffractions from single Cu2-xSe NP have also been performed (Figure S6), and the observed lattice spacing matches well with berzelianite structure. These obtained Cu2-xSe NPs form stable dispersions in water, and their UV-vis-NIR absorption spectra display well defined absorption in NIR-II (Figure 2d). LSPR of copper chalcogenides nanocrystals depends critically on their stoichiometry.3 With increasing NP dimensions from 35 to 65 and 105 nm, the LSPR peaks display a continuous redshift from 1003 to 1017 and 1090 nm, with the Cu/Se ratio found to be 1.22, 1.38 and 1.61, respectively. The lower Cu/Se ratio indicates smaller NPs have higher copper deficiency, resulting in the observed LSPR blue shift. The absorbance of different sized Cu2-xSe NPs at 1064 nm is linear to the measured Cu2+ concentrations (Figure S7a-d), which was quantitatively determined by ICP measurements. According to Beer-Lambert law, the extinction coefficients at 1064 nm for the three different sized Cu2-xSe NPs (x ~ 0.78, 0.62, and 0.39) are then calculated to be 14.86, 21.57, and 17.50 L g-1 cm-1 for NPs of increasing sizes, respectively (see Supporting Information for calculation details). When the concentration is given in terms of the NPs themselves (using sizes estimated from TEM images), the calculated molar extinction coefficients are 1.40×109, 1.36×1010, 4.24×1010 M-1 cm-1, respectively. The larger optical cross section per unit weight of 65 nm sized NPs is thus a balanced effect of NP cross section and number of NPs in the solution at the same mass concentration. The measured optical cross sections of these Cu2-xSe NPs are comparable to the reported values of noble metal nanocrystals, such as gold nanorods (1.9×109 at 650 nm)48 and gold nanoshells (2 × 1011 at 800 nm),49 demonstrating their strong interaction with NIR light.


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The synthetic protocol developed here is not restricted to PSS capped NPs, as similar seeded growth processes have also been conducted using PVP and PAH as surfactant molecules (Figure S8a-b). Surface potentials of the resulting NPs changed from -25 to -12 and +23 mV when PSS was substituted by PVP and PAH (Figure S8c). Cu2-xSe NPs of the same size but with different capping molecules show that PSS capped NPs display highest absorbance per unit Cu mass (Figure S8d), which we speculate is due to differences in the ability of surface capping molecules to donate or withdraw electrons.33,

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For the following study, we will focus only on PSS

capped Cu2-xSe NPs. Unless otherwise specified, all Cu2-xSe NPs refer to PSS capped ones in the texts to follow. Photothermal property of Cu2-xSe NPs Owing to their strong absorption in the NIR-II window originating from copper vacancies, Cu2-xSe NPs are of great interests for their potential usage in photothermal tumor ablation applications. Thus, the photothermal characteristics of the as-prepared Cu2-xSe NPs in various sizes were firstly evaluated using their aqueous dispersions. When being illuminated by a laser (1064 nm, 0.75 W cm-2), the solution temperature elevations of different sized Cu2-xSe NPs at various concentration (from 6.25 to 100 ppm of Cu) were measured and recorded (Figure 3a-c). In general, the temperature elevation profiles flattens out at high nanomaterials concentrations for all NP sizes, due to logarithmic dependence of absorption on the incident laser intensity (vide infra), which has been observed in other nanomaterials systems as well. At the end of 10 min light exposure, a steady state with ~ 12 °C above room temperature was achieved, while under the same experimental conditions, temperature of pure water (without Cu2-xSe NPs) only increased by less than 3.0 °C. For clearer comparison, the final steady state temperature elevation is plotted versus NPs concentration in Figure 3d. The 100 ppm 65 nm Cu2-xSe NPs displayed


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largest temperature rise of 12.6 °C, compared to 11.5 and 11.8 °C for 35 and 105 nm Cu2-xSe NPs after irradiation by 1064 nm laser under the same conditions. Moreover, infrared thermographs were collected for three NPs at 50 ppm concentration after light irradiation of different power densities of 0 (no light), 0.5, 1.0, and 1.5 W cm-2 for 10 min (Figure 3e). Again, 65 nm Cu2-xSe NPs were demonstrated to be much more efficient nanoheaters than their 35 and 105 nm counterparts, in line with their measured extinction coefficients per unit mass.

Figure 3. (a-c) Photothermal heating curves of 35, 65, and 105 nm Cu2-xSe NPs solution at various concentrations when being exposure to light irradiation (1064 nm, 0.75 W cm-2, 10 min); (d) plots of steady state temperature changes versus Cu2-xSe NPs concentration under light irradiation, * means P < 0.05; (e) IR thermal images of three different sized Cu2-xSe NPs (50 ppm Cu) under laser irradiation at various power densities for 10 min, with the scale bars represent 2 mm. Following a modified method reported by Roper et al.,50 the photothermal transduction efficiency of Cu2-xSe NPs is calculated based on their heating and cooling curve. Under laser irradiation, the solution energy balance is given as


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∑m C i

i

dT = Qin − Qout dt

p ,i

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(1)

where the mass (m) and heat capacity (Cp) terms on left side of the equation are generally approximated to be dominated by the solvent water. The system heat dissipation term Qout is linear with system temperature as

Qout = hS (T − Tsurr )

(2)

in which h denotes the heat transfer coefficient, S represents the exposed surface area of the cuvette. At equilibrium, solution temperature reaches a plateau at Tmax, when the heat input and output are balanced as

Qin = Qout = hS (Tmax − Tsurr )

(3)

With incident laser power I in mW, η as the photothermal transduction efficiency, and A1064 is the NP solution absorption at 1064 nm, the heat input due to NP absorption can be expressed as

Qin , NP = I (1 − 10 − A1064 )η = hS (Tmax − Tmax,H 2O )

(4)

By defining a thermal time constant τs and a dimensionless term θ

τs = θ=

∑mC i

i

p ,i

hS T − Tsurr Tmax − Tsurr

(5)

(6)

the heat dissipation term hS can be obtained by a linear fitting of time t versus –ln(θ) during the system cooling stage when the light source is turned off (Figure S9)

t = −τ s ln(θ )

(7)

Combining Eq. 1-7, the photothermal transduction efficiency of the 65 nm PSS capped Cu2-xSe NPs is determined to be 23.0%. This value is slightly lower than Cu2-xS,8 but it is similar to the reported value of Cu2-xSe in smaller dimensions.39 In addition, Au nanorods absorbing in the


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same optical window have been synthesized, and their photothermal conversion efficiency was determined to be 25.0% (Figure S10). For nanomaterials to be used as phototherapeutic agents, photothermal stability is of great importance. To evaluate stability of these Cu2-xSe NPs under light irradiation, their solution was repeatedly exposed to laser light at 0.75 W cm-2 for five times with the solution temperatures recorded accordingly. No significant variations of the heating-cooling curves were observed, suggesting these Cu2-xSe NPs are photo stable (Figure S11). TEM and DLS characterizations of Cu2-xSe NPs (Figure S12) have also confirmed that there were no morphology changes after laser irradiation, demonstrating their potential for photothermal imaging and therapy applications Cellular toxicity, ROS Level and in vitro photothermal cell ablation Before these Cu2-xSe NPs can be used for photothermal therapy, their biocompatibility has to be investigated. For this purpose, CT26 and HUVEC cells were selected as model system for cancerous and normal cells, and the cytotoxicity of different sized Cu2-xSe NPs was characterized by standard MTT assays (Figure 4a and Figure S13a). After 24 h of incubation, significant absorbance increases in the MTT assays were observed for larger sized NPs, indicating sizedependent cytotoxicity where large Cu2-xSe NPs are more biocompatible in general. When looking at the measured size dependence on cytotoxicity, it shows that CT26 cells viability remained over 80% for 65 and 105 nm Cu2-xSe NPs at a concentration of 100 ppm, while cell viability dropped to near 50% for 35 nm NPs at the same dosage. It is worth noting that the size dependence of cytotoxicity on normal HUVEC cells is less striking. At 100 ppm level, the difference in cell viability between small and large NPs was much smaller, and ~70% cells were still viable after incubating with 35 nm Cu2-xSe NPs (Figure S13a). Upon increasing incubation


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time, cytotoxicity of the smaller Cu2-xSe NPs on CT26 cells was even more significant than their larger counterparts (Figure S14).

Figure 4. (a) MTT viability assays of CT26 cells after being treated with different concentrations of Cu2-xSe NPs for 24 h, statistical significance was obtained by performing t-tests, as * means P < 0.05 and ** means P < 0.01; (b) normalized levels of ROS in CT26 cells after being treated with various sized Cu2-xSe NPs at a fixed Cu concentration of 100 ppm; (c) fluorescence images of calcein AM and PI co-stained CT26 cells treated with different sized Cu2-xSe NPs without and with laser irradiation (1 W cm-2, 10 min), the scale bars represent 100 µm. Copper is a redox active transition metal, and it accelerates ROS generation in biological system through Fenton and Haber-Weiss reactions.51-52 The generated ROS will then cause damage to protein, lipid, and DNA inside the cells and induce cell apoptosis. To investigate whether the cellular cytotoxicity is linked to ROS levels, a fluorescence dye indicator DCFH-DA was applied for ROS detection, whose fluorescence will be turned on when being oxidized by free radicals. As indicated in Figure 4b, at an incubation concentration of 100 ppm with CT26


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cells, 35 nm Cu2-xSe NPs generated ROS that was twice as high, while 65 nm NPs was only 25% higher and 105 nm NPs fell to the same level as control sample. ROS generation when incubated with HUVEC cells also showed similar trend (Figure S13b). Furthermore, the generation of ROS was also dose-dependent. When the cells were incubated with 50 ppm 35 nm Cu2-xSe NPs, the ROS level was about 1.5 times as that of the control, which increased further when 100 ppm NPs were used (Figure S15). Thus, the dependence of cell viability on NP dosage and size can be correlated to the amount of generated ROS by Cu2-xSe NPs. In general, optimal NP size for cellular uptake is ~ 50-60 nm.31 If cytotoxicity originates solely from the amount of NPs uptake, then 65 nm Cu2-xSe NPs would be expected to be more toxic to cells, which differs from the experimental observations. Thus, we tentatively ascribe the observed size dependent cytotoxicity to a surface effect, as the surface copper atoms per unit weight NPs will be inversely proportional to the particle diameter. It also explains the difference on cell cytotoxicity between normal and cancerous cells, as cancer cell environment is H2O2 rich, which generally serves as the precursor for ROS generation. The observed cell specific toxicity could be useful in designing NPs with ability to kill cancer cells while being benign to normal cells in future studies. Although larger 105 nm NPs were not as stable in PBS buffer or DMEM medium as the smaller ones (Figure S16) due to limited electrostatic repulsion provided by surface PSS molecules, this does not affect the conclusion drawn on the relative size effect on their cytotoxicity. In vitro photothermal cell ablation was then evaluated for different sized Cu2-xSe NPs. To

circumvent the interference coming from NP intrinsic cytotoxicity, the experiments were conducted by exposing CT26 cells incubated with 50 ppm of NPs to laser (1064 nm, 1 W cm-2, 10 min). Fluorescent live/dead cell co-staining with calcein AM/PI were applied to visually examine the photothermal ablation effect, where green/red fluorescence indicates live/dead cells.


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The lack of obvious red fluorescent cells in control groups (laser only and NPs only treatment, Figure 4c) suggests almost no cytotoxicity under these circumstances. In contrast, considerable dead cells were detected for the NPs plus laser group. More specifically, nearly all cells were ablated and displayed red fluorescence when being incubated with 65 nm NPs, while there were still a small percentage of cells that remained alive when using 35 nm and 105 nm NPs. Quantitative MTT assays (Figure S17) were also performed to corroborate the above fluorescence live/dead staining results. Under NIR laser irradiation, about 90% of CT26 cells were killed with 65 nm NPs, while cell viability is higher when treated with 35 nm and 105 nm NPs. The trend that Cu2-xSe NPs of 65 nm in diameter are more effective PTT agents for cancer cell ablation than their 35 and 105 nm counterparts is in good agreement with the measured solution photothermal properties. Photoacoustic imaging The feasibility of Cu2-xSe NPs for in vitro photothermal ablation of cancer cells suggests that these NPs could be also used for photoacoustic (PA) imaging, as they both utilize the heat generated from the interaction of NIR light source with photothermal nanoagents. Therefore, in vivo PA imaging performance of Cu2-xSe NPs was evaluated for their application in imaging

guided phototherapy. For this purpose, the best light absorbing 65 nm Cu2-xSe NPs (200 µL, 2 mg mL-1) were applied to CT26 tumor bearing nude mice through intravenous injection, and then a series of tumor region PA images were captured before and after i.v. injection. Figure 5a-b shows the PA images and integrated image intensities at tumor site measured at various time points. The data clearly demonstrated that PA image contrast at tumor region increased gradually post injection of Cu2-xSe NPs, due to NP passive accumulation at tumor region through the enhanced permeability and retention (EPR) effect. The integrated tumor site PA signal reached


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its maximum at 4 h post injection and decreased afterwards, with PA image contrast returned to near baseline level 24 h post injection. Independently, the biodistribution of the Cu2-xSe NPs in tumor bearing mice was also determined quantitatively by ICP at 24 h post injection (Figure S18). The biodegradation of Cu2-xSe NPs was further evaluated by monitoring their accumulated amount in live as a function of time (Figure S19), and ICP analysis indicated that Cu2-xSe NPs were degraded and excreted over the course of several days. In vivo photothermal therapy in the NIR-II window As 65 nm Cu2-xSe NPs display good biocompatibility, the best photothermal property and passive accumulation in tumor, they were further examined for in vivo photothermal therapy. Cu2-xSe NPs dispersion were administered to nude mice bearing CT26 tumor model either intratumorally (100 µL, 100 ppm) or intravenously (200 µL, 2 mg mL-1), while the same amount of PBS was injected in control groups, after which the tumor sites were put under laser irradiation (1064 nm, 1 W cm-2, 10 min), with real-time tumor surface temperature monitored by a thermal imaging camera. Experimental results (Figure 5c-d) show that the temperature in tumors with intravenously administered Cu2-xSe NPs experience a rapid increase from 33 °C to 49 °C in 10 min (47 °C for intratumoral injection, as shown in Figure S20), which was high enough for tumor cells ablation. On the other hand, tumor injected with PBS only showed less than 5 °C temperature rise under the same laser exposure conditions. The therapeutic time window was set at 4 h post i.v. injection, when the nanomaterials accumulation at tumor site reached maximum as suggested by PA imaging results (Figure 5b).


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Figure 5. (a) Photoacoustic images and (b) integrated intensities at tumor site before and after i.v. administration of Cu2-xSe NPs, with *** stands for P < 0.001; (c) thermal graphs of CT26bearing mice under 1064 nm laser irradiation (1 W cm-2, 10 min) 4 h post i.v. injection of either PBS (control) or 65 nm Cu2-xSe NPs; (d) recorded temperature rising profiles at mice tumor region during 10 min laser exposure period; (e) hematoxylin and eosin staining of tumor tissue sections from mice one day after treatment, with scale bars represent 100 µm; (f) growth curves of tumor (n = 5) under various treatments; (g) photographs of tumors collected from differently treated mice after 2 weeks. The photothermal ablation effect on the tumor cells was examined by hematoxylin and eosin (H&E) staining, where the histopathology changes of tumor tissues one day after photothermal treatment was taken. Distinct necrosis was observed in the group treated with combined application of laser irradiation and Cu2-xSe NPs, where increased vacuoles, condensed nuclei and changing cell shapes were observed (Figure 5e). In addition, the tumor volumes after different


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treatments were continuously monitored (Figure 5f). Tumor growth has been largely inhibited with Cu2-xSe photothermal treatment, while the control groups showed exponential growth in tumor volumes. Two weeks after treatment, the tumors in different groups were harvested and photographed (Figure 5g), and visual inspection as well as size measurements all showed that tumors treated by Cu2-xSe NPs with 1064 nm laser irradiation became much smaller compared to the control groups. Finally, to assess the in vivo NPs toxicity, behaviors of mice were carefully supervised. No appreciable abnormalities in the animal body weight were observed after photothermal treatment (Figure S21). Moreover, complete necropsies on day 14 after treatment were conducted on humanely euthanized mice of both control and treatment groups, and major organs and tissues of the sacrificed mice were sliced and H&E stained (Figure S22). Only normal tissue structures were observed from the histology analysis, without noticeable organ damage or inflammatory lesions for both PBS and NPs treated groups. These preliminary investigations have verified that Cu2-xSe NPs are rather biocompatible in small animal models. On the basis of developed synthetic method and current experimental findings, further systematic investigation on the nanoparticle size and surface effect on their tumor accumulation and tissue penetration are needed. Our preliminary results have shown that nanoparticle size has little influence in the amount of their tumor accumulations, as there is no statistically significant difference in maximum NP accumulation amount between different sized NPs (Figure S23). Interestingly, the relative accumulation kinetics is size dependent, which is likely related to their blood circulation and tumor clearance behaviors. Moreover, the combined influence of NP ROS generation and photothermal ablation on tumor inhibition, and their long term biodegradation


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pathways are worth exploring further to exploit their full potential in imaging guided phototherapeutic applications. Conclusions A facile aqueous phase synthesis of size tunable Cu2-xSe NPs by seeded growth method has been developed. Both size and surfactant molecules are found to be important factors influencing their NIR surface plasmon resonances, with 65 nm Cu2-xSe NPs capped by PSS possess the largest absorption cross section per unit weight at 21.57 L g-1 cm-1 among the experimental samples, demonstrating their strong interaction with light. Consequently, these 65 nm Cu2-xSe NPs display the best photothermal effect in raising local temperature and killing cancer cells. Taking advantage of their large optical cross section and photothermal conversion ability, in vivo photothermal therapy on mice is carried out successfully within the optimal temporal window guided by photoacoustic imaging, and the tumor growth has been inhibited without introducing significant toxicity over 2 weeks. This study paves the way for future investigations on nanomaterials physicochemical properties on their phototherapeutic effect, which requires balanced consideration on their optical properties as well as on the nano-bio interactions.

ASSOCIATED CONTENT Supporting Information. Extinction coefficient calculation, TEM images and XRD of Se NPs, DLS, XRD, zeta-potential, and high resolution TEM with single NP electron diffraction on Cu2xSe

NPs, photothermal conversion efficiency and stability, cell and dose dependent ROS

generation, tumor accumulation kinetics, biodistribution and degradation of Cu2-xSe NPs, photothermal therapy with intratumorally applied NPs, mice body weight monitoring, and H&E stained slices of major organs after treatment.


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AUTHOR INFORMATION Corresponding Author *[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources This work was funded by National Natural Science Foundation of China (No. 21473243). ACKNOWLEDGMENT This work was funded by National Natural Science Foundation of China (No. 21473243) and supported by Collaborative Innovation Center of Suzhou Nano Science and Technology. REFERENCES (1)

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