# A core-shell Au@Cu2-xSe heterogeneous steel nanocomposite for photoacoustic and computed tomography dual-imaging-guided photothermal boosted chemodynamic remedy | Journal of Nanobiotechnology

### Supplies

Hydrogen Tetrachloroaurate(III) Trihydrate (HAuCl4·3H2O) and methylene blue (MB) have been obtained from Shanghai Titan Expertise Co., Ltd. Copper(II) sulfate pentahydrate (CuSO4·5H2O), selenium dioxide (SeO2), sodium citrate dihydrate, ascorbic acid, and hydrogen peroxide (H2O2) have been bought from Sinopharm Chemical Reagent Co., Ltd. Polyvinylpyrrolidone (PVP, Mw ~55,000), 2′,7′- dichlorofluorescein diacetate (DCFH-DA), and Calcein AM have been supplied by Sigma Aldrich. Roswell Park Memorial Institute 1640 (RPMI-1640) was supported by Corning Included. Fetal bovine serum (FBS), trypsin, and penicillin-streptomycin resolution have been provided from Gibco Firm. Cell Counting Equipment-8 (CCK-8) resolution was bought from Dojindo Laboratories. Hoechst 33,342, 4′,6-diamidino-2-phenylindole (DAPI), and propidium iodide (PI) have been acquired from Beyotime Biotechnology. Mitochondrial hydroxyl radical detection assay, anti-gamma H2AX (phospho S139) antibody [EP854(2)Y], and goat polyclonal secondary antibody to rabbit IgG – H&L (Alexa Fluor® 647) have been bought from Abcam Firm. Accutase™ Cell Detachment Answer and FITC Annexin V Apoptosis Detection Equipment have been provided from BD Bioscience. All chemical substances and reagents have been used as acquired with out additional purification.

### Synthesis of Au NPs

HAuCl4 aqueous resolution ready by mixing 1.215 mL of 10 mM HAuCl4 resolution and 50 mL of deionized water was heated to slight boiling. A condenser was utilized to forestall the evaporation of the water. Then 50 mL of 10 mg mL−1 sodium citrate resolution was added into the boiling HAuCl4 resolution. After conserving boiling for 15 min below steady stirring, the combination was allowed to chill to room temperature. The resulted Au NPs have been collected by centrifuging the suspension at 15,000 rpm for 15 min.

### Synthesis of Au@Cu2-xSe NPs

5.5 mL of Au NPs redispersed in deionized water was blended with 1.6 mL of 10 mg mL−1 PVP resolution and stored stirring at 30 °C for 1 h, adopted by the addition of 0.1 mL of 0.1 M SeO2 and 0.3 mL of 0.2 M ascorbic acid resolution. After response for 30 min, a blended resolution of 0.1 mL of 0.2 M CuSO4 and 0.4 mL of 0.2 M ascorbic acid was added. After stirring the suspension at 30 °C for an additional 10 h, the Au@Cu2-xSe NPs have been collected by centrifuging the suspension at 15,000 rpm for 15 min and washed 3 times with deionized water.

### Synthesis of Cu2-xSe NPs

1.6 mL of 10 mg mL−1 PVP resolution was added into 5.5 mL of deionized water and stored stirring at 30 °C, adopted by the addition of 0.1 mL of 0.1 M SeO2 and 0.3 mL of 0.2 M ascorbic acid resolution. After response for 30 min, a blended resolution of 0.1 mL of 0.2 M CuSO4 and 0.4 mL of 0.2 M ascorbic acid was added. After stirring the suspension at 30 °C for an additional 10 h, the Cu2-xSe NPs have been collected by centrifuging the suspension at 15,000 rpm for 15 min and washed 3 times with deionized water.

### Characterization

The morphology and construction have been characterised by transmission electron microscopy (TEM, JEOL JEM-2100 F). The crystal construction was decided by X-ray diffraction (XRD, Rigaku D/max2500). The chemical state was analyzed by X-ray photoelectron spectroscopy (XPS, Thermo Escalab 250), and the binding vitality of C 1 s peak at 284.8 eV was taken as an inside customary. Fourier remodel infrared (FTIR) spectrum was recorded on Bruker Tensor-27 spectrometer. Ultraviolet-visible-infrared (UV-vis-NIR) absorption spectrum was scanned on Persee TU-1901 spectrometer. The steel component content material was decided by inductively coupled plasma optical emission spectroscopy (ICP-OES, Varian 710ES). Hydrodynamic dimension and zeta potential have been characterised by a particle dimension zeta potential analyzer (Malvern ZEN3690).

### Photothermal efficiency

An 808 NIR laser was used to estimate the photothermal efficiency of Au@Cu2-xSe NPs. The NIR energy density was maintained at 1.0 W cm−2 in all exams. The amount of Au@Cu2-xSe aqueous dispersions in every take a look at was 1.5 mL. Temperature adjustments have been recorded by a thermal digicam. For concentration-dependent photothermal efficiency, numerous Au@Cu2-xSe aqueous dispersions in a quartz cuvette with concentrations of 0, 25, 50, 100, and 200 µg mL−1 have been uncovered to laser for 10 min. To judge the photothermal stability, Au@Cu2-xSe aqueous dispersion with a focus of fifty µg mL−1 was irradiated by laser for 10 min (laser on) adopted by pure cooling with out irradiation for an additional 10 min (laser off). Such heating/cooling processes have been repeated 6 instances. Heating-cooling curve was obtained by constantly irradiating 50 µg mL−1 dispersion with laser for 15 min, after which turning off the laser to permit the dispersion to naturally cool to room temperature. Following beforehand reported methodology [8, 20, 28], the photothermal conversion effectivity was calculated based on Eqs. 14.

$$eta =frac{{hS({T_{hbox{max} }} – {T_{hbox{max} ,{{textual content{H}}_{textual content{2}}}{textual content{O}}}})}}{{I(1 – {{10}^{ – {A_lambda }}})}}$$

(1)

$$hS=frac{{m{C_{textual content{p}}}}}{{{tau _s}}}$$

(2)

$$t= – {tau _{textual content{s}}}ln theta$$

(3)

$$theta =frac{{T – {T_{{textual content{sur}}}}}}{{{T_{hbox{max} }} – {T_{{textual content{sur}}}}}}$$

(4)

the place η is the photothermal conversion effectivity, h is the heat-transfer coefficient, S is the floor space of container, T is the temperature of Au@Cu2-xSe aqueous dispersion, Tmax is the utmost temperature of Au@Cu2-xSe aqueous dispersion, Tmax,H2O is the utmost temperature of pure water, Tsur is the ambient temperature of environment, I is the laser energy, Aλ is the absorbance of Au@Cu2-xSe aqueous dispersion on the wavelength of 808 nm, m and Cp are the mass (1.5 g) and warmth capability (4.2 J g−1) of solvent (water), t is the cooling time, τs is the time fixed of pattern system, θ is the driving pressure temperature. τs may be obtained by making use of the linear time knowledge from the cooling interval vs. –lnθ (Fig. 2D, E).

### Photothermal enhanced Fenton-like catalytic efficiency

An MB degradation assay was used to watch the ·OH era. First, blended aqueous options of Au@Cu2-xSe and MB remained static state at the hours of darkness for 30 min at 25, 37, and 42 °C, respectively. Second, after including H2O2, the mixtures have been then remained static state at the hours of darkness for an additional 30 min at 25, 37, or 42 °C, respectively. The ultimate concentrations of Au@Cu2-xSe NPs, MB, and H2O2 in mixtures have been set at 100 µg mL−1, 10 µg mL−1, and 10 mM, respectively. Lastly, after eradicating Au@Cu2-xSe NPs through centrifugation at 15,000 rpm for 15 min, the absorbance of every supernatant at 664 nm was measured. In these experiments, the pure MB resolution (10 µg mL−1), and the combination of MB (10 µg mL−1) and H2O2 (10 mM) resolution, have been chosen as management.

Subsequent, ESR was used to substantiate the era of ·OH in Fenton-like response. DMPO was used as a spin entice agent for ·OH. After conserving the blended aqueous options of Au@Cu2-xSe, H2O2, and DMPO for five min at 25, 37, and 42 °C, the mixtures have been used to report ESR spectra by a spectrometer (Bruker ELEXSYS E500, CW X-band), respectively. The ultimate concentrations of Au@Cu2-xSe, H2O2, and DMPO have been set at 50 µg mL−1, 100 µM, and 50 mM, respectively. The combination of Au@Cu2-xSe (50 µg mL−1) and DMPO (50 mM), and the combination of H2O2 (100 µM) and DMPO (50 mM), have been chosen as management.

To show the photothermal accelerated ·OH era in Fenton-like response, 1.5 mL of blended aqueous resolution of Au@Cu2-xSe NPs, MB, and H2O2 was irradiated with or with out 808 nm NIR laser at 1.0 W cm−2 for 10 min. The ultimate concentrations of Au@Cu2-xSe NPs, MB, and H2O2 in combination have been set at 100 µg mL−1, 10 µg mL−1, and 10 mM, respectively. The Temperature rise was recorded by thermal digicam. Lastly, after eradicating Au@Cu2-xSe NPs through centrifugation at 15,000 rpm for 15 min, the absorbance of supernatant at 664 nm was measured.

### Detection of intracellular ROS

CLSM (Zesis LSM800) was adopted to detect the intracellular ROS by utilizing DCFH-DA (Sigma-Aldrich) as ROS probe. Cells have been divided into six teams, together with (1) management, (2) H2O2, (3) Au@Cu2-xSe, (4) Au@Cu2-xSe + H2O2, (5) Au@Cu2-xSe + NIR, and (6) Au@Cu2-xSe + H2O2 + NIR. First, 2 × 105 4T1 tumor cells have been seeded into the CLSM-specific tradition disk after which cultured at 37 °C below 5% CO2 for twenty-four h. Second, after changing the tradition medium with 1 mL of FBS-free RPMI-1640 medium containing Au@Cu2-xSe (50 µg mL−1) or H2O2 (100 µM), the cells in group (5) and (6) have been irradiated with NIR laser (1.0 W cm−2) for five min, respectively. After irradiation, the cells continued to incubate for 30 min. Third, after washing the cells 3 times with PBS, the cells have been incubated with 1 mL of FBS-free RPMI-1640 medium containing DCFH-DA (10 µM) and Hoechst 33,342 (5 µg mL−1) for 20 min. Fourth, the cells have been washed 3 times with PBS adopted by the addition of 1 mL of FBS-free RPMI-1640 medium. Then the fluorescence photos of cells have been collected by CLSM.

### Detection of intracellular ·OH radicals

Mitochondrial hydroxyl radical detection assay package (Abcam) was used to detect the intracellular ·OH. First, 2 × 105 4T1 tumor cells have been seeded into the CLSM-specific tradition disk after which cultured for twenty-four h. Second, after changing the tradition medium with 200 µL of the ready OH580 stain working resolution, the cells continued to incubate for 1 h with out mild. Third, after washing the cells one time with PBS, the cells have been incubated with 1 mL of RPMI-1640 full medium containing Au@Cu2-xSe (50 µg mL−1) or H2O2 (100 µM), after which irradiated with or with out NIR laser (1.0 W cm−2) for five min. After irradiation, the cells continued to incubate for 30 min. Fourth, the cells have been incubated with 1 mL of PBS containing Hoechst 33,342 (5 µg mL−1) for 10 min. Fifth, the cells have been washed 3 times with PBS adopted by the addition of 1 mL assay buffer. Then the fluorescence photos of cells have been collected by CLSM.

### In vitro cytotoxicity assay

The cytotoxicity of Au@Cu2-xSe towards HEK293 regular cells and 4T1 tumor cells have been examined through a CCK-8 assay. HEK293 regular cells or 4T1 tumor cells have been seeded into 96-well plate at 1 × 104 cells/effectively and incubated for twenty-four h. Then the cells have been incubated in 100 µL of the entire medium containing Au@Cu2-xSe NPs at numerous concentrations of 0, 12.5, 25, 50, 100, and 200 µg mL−1. After 24 h, the tradition medium was eliminated and the cells have been washed 3 times with PBS. Then the cells have been incubated in 100 µL of full medium containing 10 µL CCK-8. After one other 1–2 h, the optical densities of every effectively have been recorded at 450 nm on a microplate reader (BioTek Cytation 3).

### In vitro mobile uptake

First, 4T1 tumor cells have been seeded into 6-well plate at 1.5 × 105 cells/effectively after which cultured at 37 °C below 5% CO2 for twenty-four h. Second, after changing the tradition medium with 1 mL of the entire medium containing Au@Cu2-xSe NPs (50 µg mL−1), the cells have been then incubated for 0.5, 1, 2, 4, and eight h, respectively (three wells at every time level). Third, the tradition medium was eliminated and the cells have been washed 3 times with PBS, digested with 0.25% trypsin (Gibco), and centrifuged at 1500 rpm for 3 min. When the collected cells have been redispersed in 1 mL PBS, the cells have been counted. Lastly, the content material of Au in PBS was analyzed by ICP-MS.

### In vitro PTT and CDT

4T1 tumor cells have been divided into eight teams, together with (1) management, (2) H2O2, (3) NIR, (4) H2O2 + NIR, (5) Au@Cu2-xSe, (6) Au@Cu2-xSe + H2O2, (7) Au@Cu2-xSe + NIR, and (8) Au@Cu2-xSe + H2O2 + NIR. First, 4T1 tumor cells have been seeded into 96-well plate at 1 × 104 cells/effectively and incubated for twenty-four h. Second, after changing the tradition medium with 100 µL of the entire medium containing Au@Cu2-xSe (50 µg mL−1) or H2O2 (100 µM), the cells have been irradiated with or with out NIR laser (1.0 W cm−2) for five min. After irradiation, the cells continued to incubate for twenty-four h. Third, the tradition medium was eliminated and the cells have been washed 3 times with PBS. Then the cells have been incubated in 100 µL of full medium containing 10 µL CCK-8 for an additional 1 h. Lastly, the optical densities of every effectively have been recorded at 450 nm on a microplate reader.

CLSM was then employed to watch the cell dying induced by hyperthermia and ·OH radicals. First, 2 × 105 4T1 tumor cells have been seeded into the CLSM-specific tradition disk after which cultured at 37 °C below 5% CO2 for twenty-four h. Second, after changing the tradition medium with 1 mL of the entire medium containing Au@Cu2-xSe (50 µg mL−1) or H2O2 (100 µM), the cells have been irradiated with or with out NIR laser (1.0 W cm−2) for five min. After irradiation, the cells continued to incubate for 4 h. Third, the tradition medium was changed by 1 mL of PBS containing Calcein AM (2 µM) and PI (4 µM) and co-incubated with the cells for an additional 20 min. Lastly, the fluorescence photos of cells have been collected instantly by CLSM.

### In vitro apoptosis and necrosis evaluation by move cytometry

First, 4T1 tumor cells have been seeded into 12-well plate at 7 × 104 cells/effectively after which cultured at 37 °C below 5% CO2 for twenty-four h. Second, after changing the tradition medium with 1 mL of the entire medium containing Au@Cu2-xSe (50 µg mL−1) or H2O2 (100 µM), the cells have been irradiated with or with out NIR laser (1.0 W cm−2) for five min. After irradiation, the cells continued to incubate for twenty-four h. Third, the cells have been digested with Accutase™ Cell Detachment Answer (BD Biosciences) and re-suspended in PBS. Then, the cells have been stained based on the FITC Annexin V Apoptosis Detection Equipment (BD Biosciences) and quantified by move cytometry (Beckman Cytoflex).

### In vitro DNA harm evaluation

First, 2 × 105 4T1 tumor cells have been seeded into the CLSM-specific tradition disk after which cultured for twenty-four h. Second, after changing the tradition medium with 1 mL of RPMI-1640 full medium containing Au@Cu2-xSe (50 µg mL−1) or H2O2 (100 µM), the cells have been irradiated with or with out NIR laser (1.0 W cm−2) for five min. After irradiation, the cells continued to incubate for six h. Third, these cells have been mounted with 4% paraformaldehyde for 10 min, washed a number of instances with PBS, permeabilized with immunostaining permeabilization buffer with triton X-100 (Beyotime Biotechnology) for 10 min, and blocked with QuickBlock blocking buffer (Beyotime Biotechnology) for 10 min at room temperature. Forth, after washing 3 times with PBS, the cells have been stained with anti-gamma H2AX (phospho S139) antibody [EP854(2)Y] (Abcam, dilution 1:400) at 4 °C in a single day. Fifth, after washing 3 times with PBS, the cells have been incubated with the goat polyclonal secondary antibody to rabbit IgG-H&L (Alexa Fluor® 647) (Abcam, dilution 1:400) for 1 h. Sixth, after washing 3 times with PBS, the cells have been stained with DAPI (Beyotime Biotechnology) for five min. Lastly, the cells have been washed 3 times with PBS adopted by the addition of 1 mL PBS. Then the fluorescence photos of cells have been collected by CLSM.

### In vitro hemolysis assay

1 mL of contemporary blood was taken from BALB/c mouse and saved in a tube containing EDTA. 2 mL of saline was added and blended. Crimson blood cells (RBC) have been collected by centrifuging the blood at 5000 rpm for 3 min. Then the RBC was washed 3 times with saline till the supernatant grew to become colorless. After discarding the supernatant, 100 µL of RBC was taken and redispersed into 5 mL saline to organize 2% quantity fraction RBC suspension. Then a sequence of mixtures have been ready by mixing 500 µL RBC suspension and 500 µL saline containing Au@Cu2-xSe with totally different concentrations. The ultimate concentrations of Au@Cu2-xSe within the mixtures have been set at 12.5, 25, 50, 100, and 200 µg mL−1, respectively. The combination of 500 µL RBC suspension and 500 µL saline, and the combination of 500 µL RBC suspension and 500 µL pure water, have been used as detrimental and optimistic controls, respectively. After remaining steady at room temperature for five h, these mixtures have been centrifuged at 15,000 rpm for 15 min. Lastly, the absorbance (A) of supernatant in numerous mixtures was recorded at 570 nm on microplate reader for calculating the hemolysis charge of RBC. Hemolysis charge was calculated based on Eq. 5.

$${textual content{Hemolysis charge}}=frac{{{{textual content{A}}_{{textual content{pattern}}}} – {{textual content{A}}_{{textual content{saline}}}}}}{{{{textual content{A}}_{{{textual content{H}}_{textual content{2}}}{textual content{O}}}}{textual content{-}}{{textual content{A}}_{{textual content{saline}}}}}} instances 100%$$

(5)

### In vivo toxicity

After 200 µL of saline (management group) or 200 µL of Au@Cu2-xSe suspended in saline (2 mg mL−1) have been injected into mice through intravenous injection (n = 3, per group), mice have been sacrificed at 14 d to gather blood for hematological analyses and tissues (together with coronary heart, liver, spleen, lung, and kidney) for H&E stain. In these 14 days, weights of mice have been measured each 2 days.

### Tumor mannequin

100 µL PBS containing 4T1.2 cells (2 × 106 cells mL−1) was subcutaneously injected beside the foreleg of a BALB/c feminine mouse (6-weeks outdated). When tumor grows to ~80 cm3, the tumor-bearing mouse was utilized in subsequent in vivo experiments.

### In vitro and in vivo PA imaging

PA imaging was carried out on an ultrasound-photoacoustic dual-mode imaging system (VEVO LAZR-X, Fujifilm VisualSonics). The excitation wavelength for PA imaging was 808 nm. For in vitro PA imaging, numerous Au@Cu2-xSe aqueous dispersions with concentrations of 0, 25, 50, 100, and 200 µg mL−1 have been injected into polyethylene tubes for PA testing, respectively. For in vivo PA imaging, 200 µL saline containing Au@Cu2-xSe NPs (2 mg mL−1) have been intravenously injected into tumor-bearing mice (n = 3). Then the ultrasound and photoacoustic indicators at tumor web site have been recorded at 1, 2, 4, 6, and eight h post-injection, respectively. The indicators earlier than injection have been used as management.

### In vitro and in vivo CT imaging

CT imaging was collected from the Siemens Inveon PET/CT imaging system. For in vitro CT imaging, numerous Au@Cu2-xSe aqueous dispersions with concentrations of 0, 0.625, 1.25, 2.5, and 5 mg mL−1 have been positioned in small tubes for CT imaging. In vivo CT imaging was performed with tumor-bearing mice (n = 3, per group) after intravenous or intratumoral injection of Au@Cu2-xSe NPs. For intravenous injection, the CT photos have been collected at 1, 2, 4, 6, and eight h post-injection of 200 µL saline containing Au@Cu2-xSe NPs (2 mg mL−1). The CT photos earlier than injection have been used as management. For intratumoral injection, the CT photos have been collected after injection of 150 µL saline containing Au@Cu2-xSe NPs with 0, 6, and eight mg mL−1, respectively.

### In vivo PTT and CDT

Tumor-bearing mice have been divided randomly into 5 teams (n = 5, per group): (1) saline; (2) saline + NIR (1.5 W cm−2); (3) Au@Cu2-xSe; (4) Au@Cu2-xSe + NIR (1.0 W cm−2); (5) Au@Cu2-xSe + NIR (1.5 W cm−2). Mice in group (1) and (2) have been intravenously injected with 200 µL pure saline, whereas mice in group (3), (4), and (5) have been intravenously injected with 200 µL saline containing Au@Cu2-xSe NPs (2 mg mL−1). At 4 h post-injection, tumors in group (2) and (5) have been irradiated with 808 nm NIR laser for five min at 1.5 W cm−2, whereas tumors in group (4) have been irradiated with 808 nm NIR laser for five min at 1.0 W cm−2. The temperature adjustments at tumor websites have been recorded with a thermal digicam. The size and width of tumors and weight of mice have been measured each 2 days. The tumor quantity (V) was calculated based on equation V = (width2 × size)/2. The relative tumor quantity (V/V0) was obtained by normalizing the tumor quantity to the preliminary tumor quantity (V0). Moreover, the tumors at 20 h post-treatment have been collected for H&E and TUNEL analyses. For in vivo ROS detection in tumor, 200 µL saline containing DCFH-DA (100 µM) was intratumorally injected into the tumor-bearing mice at 3.5 h post-injection of saline or Au@Cu2-xSe NPs. 30 min later, NIR irradiation was administrated. Then, tumors in every group have been collected for fluorescence evaluation.

Intratumoral injection of Au@Cu2-xSe NPs was additionally carried out to judge synergistic remedy of PTT + CDT. Tumor-bearing mice have been divided randomly into 4 teams (n = 5, per group): (1) saline; (2) saline + NIR; (3) Au@Cu2-xSe; (4) Au@Cu2-xSe + NIR. After intratumoral injection of 100 µL pure saline or 100 µL saline containing Au@Cu2-xSe NPs (2 mg mL−1), tumors in group (2) and (4) have been then irradiated with 808 nm NIR laser for five min at 0.5 W cm−2. Tumors at 20 h post-treatment have been collected for H&E and TUNEL analyses.

### Statistical evaluation

Bars show imply ± s.d., and statistical evaluation was carried out utilizing Pupil’s t-test and the P values have been supplied (***P < 0.001; **P < 0.01; *P < 0.05).