Materials

Calcium chloride (CaCl2) anhydrous was purchased from Macklin (Shanghai, China). Ammonium bicarbonate (NH4HCO3) was obtained from Tianjin Chemical Three Plant (Tianjin, China). Curcumin (Cur) was obtained from Shanghai Yuanye Bio-Technology (Shanghai, China). Indocyanine green (ICG) was purchased from Meryer (Shanghai, China). Methyl thiazolyl tetrazolium (MTT) was purchased from BioFroxx (Guangzhou, China). Sodium alginate (SA), rhodamine B (RhB), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and Fluo-3, AM were purchased from Solarbio Science & Technology (Beijing, China). Calcein-AM/PI kit, ATP detection kit and MMP assay kit with JC-1were purchased from Solarbio Science & Technology (Beijing, China). Coumarin 6 (C6) was obtained from Macklin (Shanghai, China). Lyso-Tracker Red was purchased from Beyotime (Shanghai, China).

Cell lines and animals

4T1 cells were cultured in RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin. The medium was then placed in a humidified incubator with 5% carbon dioxide (CO2) at 37 °C.

Female BALB/c mice came from GemPharmatech (Chengdu, China), and were kept under standard conditions. All animal experiments were approved by the Animal Care and Ethics Committee of Southwest Medical University. In situ mouse breast cancer model was established by injecting 5 × 105 4T1 cells into the right breast pad of BALB/c mice (6 weeks old). When the tumor volume was around 100 mm3, animal studies started. The tumor volume can be calculated using the following formula:

Preparation of Cur@CaCO3-ICG (CCI) and SCCI nanoparticles

CCI and SCCI nanoparticles were synthesized by the one-pot gas diffusion process. In general, three beakers were placed in an airtight container. The first beaker contained 10.0 g of NH4HCO3, which was used to produce large amounts of CO2. 100.0 mL of ethanol dissolved with 50.0 mg CaCl2, 8.0 mg Cur and 5.0 mg ICG was added to another beaker and covered with a membrane with several holes. The CO2 produced by NH4HCO3 continually diffused into ethanol solution containing CaCl2, Cur, and ICG as a source of carbonate ions (CO32−). Dissolved Ca2+ reacted with CO32− to form CaCO3 nanoparticles, enabling encapsulation of the above drugs. The last beaker was filled with 250.0 mL of deionized water to absorb the excess NH3 produced by NH4HCO3. After reaction at room temperature for 12 h, the collected CCI was repeatedly centrifuged at 10,000 rpm and washed twice with ethanol. The prepared nanoparticles were added into SA dissolved deionized water and stirred at room temperature for 2 h. The mass ratio of CCI and SA was 1:10. The collected SCCI nanoparticles were centrifuged twice at 10,000 rpm and washed with deionized water. Finally, the nanoparticles were freeze-dried for further use.

Characterization of nanoparticles

The morphology of CCI and SCCI were characterized by transmission electron microscopy (TEM, JEM 1200EX; JEOL, Japan). In addition, the zeta potential of the preparations was further analyzed using a Malvern Zetasizer (Nano ZS90, Malvern Instruments, UK). Energy dispersive X-ray spectroscopy (EDX, FEI Tecnai G2 F30, USA) and X-ray photoelectron spectroscopy (XPS, Thermo escalab 250XI, America) were used to determine the element composition and visual distribution of SCCI. X-ray diffractometer (XRD, Brucker D8 Advance, Germany) was used to evaluate the crystal properties of the samples. The freeze-dried samples were scanned using a Fourier Transform infrared spectrometer (FT-IR, Nicolet 6700, USA) with a scanning range of 4000–500 cm−1. The optical properties of the nanoparticles were then characterized using an ultraviolet visible (UV–vis) spectrophotometer (Shimadzu UV-3600Plus, Japan), and the ICG loading efficiency (LE) was verified at 780 nm. The loading LE of ICG is calculated as follows:

$$LE\, of\, ICG \left(\%\right)=\frac \times 100\%$$

The loading content (LC) and LE of Cur were determined by HPLC according to the following conditions. The mobile phase was mixed with 4% glacial acetic acid and acetonitrile (55:45, v/v). The flow rate was 1.0 mL/min, the detection wavelength was set to 426 nm, and the column temperature was 30 °C. The LC and LE for Cur are calculated as follows:

$$LC\, of\, Cur \left(\%\right)=\frac \times 100\%$$

$$LE \,of \,Cur \left(\%\right)=\frac \times 100\%$$

Hemolysis assay

Red blood cells (RBC) were obtained from BALB/c mice and diluted with saline to 2% suspension. Cur, ICG, CCI, and SCCI were added to 2% red blood cell suspension to the desired concentrations (5, 25, 50, and 200 μg/mL). It was then incubated at 37 °C for 3 h. An identical red cell suspension incubated with saline and ultrapure water was used as a negative and positive control under the same conditions. All samples were centrifuged at 3,000 rpm for 10 min before accurately absorbing the same volume of the supernatant into a 96-well plate. The hemoglobin release at 540 nm was measured by Varioskan Flash microplate reader.

Acid-responsive release of Cur in the SCCI

The SCCI (2 mg/mL) was dispersed in PBS solution with pH 5.0, 6.5, and 7.4 for 1 h. The sample was centrifuged at 10,000 rpm for 10 min and the color depth and precipitation of the supernatant were observed.

Cumulative drug release from Cur in vitro was measured by dialysis. Briefly, SCCI (containing 1 mg of Cur, 2 mL) was added to dialysis bags with a molecular weight cutoff of 3500 Da. The dialysis bags were immersed in 500 mL PBS solution with 0.5% Tween at pH 5.0, 6.5, and 7.4. After incubation and shaking at 37 °C, aliquots of release medium (500 μL) were collected at 0.5, 1, 2, 4, 6, 8, 10, and 12 h, and equal amounts of fresh release medium were replaced. The concentration of Cur was determined by HPLC.

In vitro photothermal imaging

For the photothermal imaging experiments, PBS, Cur, ICG (10 μg/mL), CCI, and SCCI with the same ICG equivalent concentration were added to 1.5 mL of centrifuge tubes and exposed to laser irradiation (808 nm, 0.75 W/cm2) for 5 min. The temperature variation was recorded by the FLIR C3-X photothermal camera (FLIR Systems, Estonia). Subsequently, the prepared SCCI (5, 10, 25, 50, and 100 μg/mL) solution was added to 1.5 mL of centrifuge tubes and irradiated for 5 min under the same conditions. Furthermore, the photothermal stability of the nanoparticles was verified by repeated "turn-off" laser irradiation and the temperature variation of each sample over time was recorded by the FLIR C3-X photothermal camera.

In vitro cytotoxicity

The MTT assay was carried out to evaluate the cytotoxicity of Cur, ICG, CCI, and SCCI with NIR. Briefly, 4T1 cells were seeded in 96-well plates at a density of 5,000 per well and incubated in 200.0 µL of medium at 37 °C for 24 h. After removal of the medium, 200.0 µL of the medium containing different concentrations of Cur, ICG, CCI, and SCCI were added to the wells. After incubation for 4 h, After incubation for 4 h, the NIR group was irradiated with laser (808 nm, 0.75 W/cm2) in the dark for 3 min, and the cells were further incubated for 20 h. After the medium was removed, 20.0 µL of MTT solution (5 mg/mL) and 180 µL of complete medium were added to the wells and incubated for 4 h at 37 °C. The medium was then replaced with 150.0 µL dimethyl sulfoxide to solubilize the formazan crystals. Absorbance at 490 nm was measured by microplate reader (Thermo Fisher, USA).

Live/Dead cytotoxicity kit was used to further visualize the viability and survival rates of 4T1 cells. Briefly, 4T1 cells were seeded into 12-well plates at a density of 5 × 104 per well and incubated overnight. After discarding the medium, the culture-medium containing Cur, CCI and SCCI were added to the plates and incubated for 6 h. After 4 h of co-incubation, cells in the NIR group were irradiated with laser (808 nm, 0.75 W/cm2) for 3 min and cultured for another 2 h. After removal of the culture medium, the cells were washed three times with PBS. 5 µL Calcein-AM solution (2 mM) and 15 µL propyl iodide solution (1.5 mM) was added to 5 mL 1 × Assay Buffer to obtain the staining working solution. Each well was added with 200 µL staining working solution and incubated at 37 °C for 15 min. The stained cells were observed by fluorescence microscopy.

Cellular uptake of SCCI

To assess the uptake of nanoparticles in 4T1 cells, we replaced ICG with RhB. 4T1 cells were seeded into confocal dishes at a density of 4 × 104 and cultured for 24 h. Cells were treated with free Cur, free RhB, Cur@CaCO3-RhB, and SA/Cur@CaCO3-RhB for 4 h. Subsequently, we washed the cells with PBS for 3 times, fixed the cells with 4% paraformaldehyde for 10 min, and then stained the nuclei with DAPI. Cellular uptake ability was assessed by confocal laser-scanning microscopy (CLSM, Leica Microsystems, Wetzlar, Germany).

Furthermore, to further quantify cellular uptake by flow cytometry, we replaced Cur with coumarin 6 (C6). 4T1 cells were seeded into 12-well plates (5 × 104 cells/well) and cultured for 12 h. We treated the cells with free C6, C6@CaCO3-ICG, and SA/C6@CaCO3-ICG for 4 h. Cells were subsequently washed twice with PBS and collected. Cellular uptake of nanoparticles was quantified by flow cytometry.

Biodistribution of SCCI in lysosomes

To detect the biodistribution of nanoparticles in lysosomes, 4T1 cells were seeded in 12-well plates at a density of 5 × 104 per well and cultured in medium for 24 h. The cells were treated with medium containing SCCI for 1, 2, and 4 h. After washing the cells with PBS for 3 times, the lysosomes were stained with Lyso-Tracker Red probe. Lysosomes and Cur were visually characterized by fluorescence microscopy.

In vitro ROS detection

DCFH-DA serves as a sensor for intracellular ROS detection. Briefly, 4T1 cells were seeded into 6-well plates at a density of 1 × 105 per well and cultured for 24 h. After removing the medium, cells were then incubated with medium containing ICG, CCI, and SCCI for 4 h. Then, the NIR group was irradiated with a laser (808 nm, 0.75 W/cm2) for 3 min. The cells were incubated with DCFH-DA (10 μM) for 20 min. Finally, the ROS production was observed by fluorescence microscope.

Furthermore, 4T1 cells were seeded at a density of 5 × 103 per well in 96-well plates and incubated in 200.0 µL of medium for 24 h. Cells were then incubated with medium containing ICG, CCI and SCCI for 4 h. After the incubation, the NIR group was irradiated with NIR laser (808 nm, 0.75 W/cm2) in the dark for 3 min. Cells were washed three times with PBS and then incubated with DCFH-DA (10 μM) for 20 min. Fluorescence intensity at 525 nm was detected with a microplate reader at 488 nm excitation wavelength.

Finally, 4T1 cells were seeded at a density of 5 × 104 per well in 24-well plates and incubated in 500.0 µL of medium for 24 h. The cells were treated with the same method as described above and incubated with DCFH-DA (10 μM) for 20 min. The fluorescence intensity of each group was determined by flow cytometry.

Detection of the MMP

4T1 cells were seeded in 6-well plates at a density of 1 × 105 per well and cultured for 24 h in 2.0 mL of medium. They were then incubated with medium containing Cur, CCI and SCCI for 6 h. For the NIR group, the cells were irradiated with laser (808 nm, 0.75 W/cm2) for 3 min after co-incubation for 4 h and continued to culture for 2 h. Cells were then stained with JC-1 (5 μg/mL) for 30 min and washed 3 times with PBS. Finally, the variation of the MMP was observed by fluorescence microscopy.

Ca2+ production by SCCI

First, 4T1 cells were seeded into 6-well plates at a density of 1 × 105 per well and cultured for 24 h, then the previous medium was replaced with medium containing CCI and SCCI. For the NIR group, the cells were irradiated with NIR laser (808 nm, 0.75 W/cm2) for 3 min after incubation for 4 h, and continued incubation for 2 h. The cells were washed with PBS for three times and stained with Fluo-3 AM Ca2+ fluorescent probe. Finally, visualization of Ca2+ was characterized by fluorescence microscopy. Furthermore, the fluorescence intensity after staining was analyzed by flow cytometry. Briefly, 4T1 cells were seeded into a 12-well plate (5 × 104 cells/well) and cultured for 12 h. The remaining steps were the same as those described above. Cells were collected after staining with Fluo-3 AM Ca2+ fluorescence probe and the intensity of fluorescence signal was analyzed by flow cytometry to determine the intracellular Ca2+ content.

Detection of intracellular ATP content

To observe the variation of ATP levels in 4T1 cells, 4T1 cells were seeded at a density of 2 × 105 per well in 6-well plates containing 2.0 mL of medium and cultured for 24 h. After discarding the medium, the medium containing Cur, Cur@CaCO3, CCI and SCCI was added into the wells and incubated for 6 h. After washing the cells with PBS for 3 times, the cells were treated with ATP extract solution and centrifuged at 10,000 g for 10 min. The supernatant was obtained to determine the content of ATP in cells according to the instructions of the ATP detection kit.

In vivo imaging and biodistribution assays

Two groups of tumor-bearing mice were studied for in vivo imaging and biological distribution. First, free ICG and SCCI were injected into the tail vein. The biodistribution of nanoparticles in tumor-bearing mice was then recorded at 1, 2, 4, 8, and 12 h after drug administration using a small animal imaging system. The mice were subsequently sacrificed and in vitro images of major organs (heart, liver, spleen, lung, kidney) and tumors were obtained.

In vivo photothermal imaging

Tumor-bearing mice were administered with saline, CCI and SCCI (2.3 mg/kg of ICG equivalent, 100 μL) through tail vein injection. After 8 h of injection, the tumor site was irradiated with laser (808 nm, 0.75 W/cm2) for 5 min, and temperature variation was recorded with a photothermal camera.

In vivo antitumor efficiency

When the primary tumor volume reached about 100 mm3, the mice were divided into 6 groups (n = 5): (1) saline, (2) NIR, (3) Cur, (4) CCI, (5) SCCI, and (6) SCCI + L (10 mg/kg of Cur equivalent, 100 μL). At 8 h after administration, NIR and SCCI + L groups were irradiated with NIR laser (808 nm, 0.75 W/cm2) for 5 min. The tumor size was measured by digital caliper every 2 days for 2 weeks.

After the experiment, the mice were euthanized. The tumor was resected and fixed with 4% paraformaldehyde for histological analysis, including hematoxylin–eosin (H&E) staining, TdT-mediated dUTP nick-end labeling (TUNEL) and Ki67 staining. Major organs (liver and kidney) of mice were removed and fixed with 4% paraformaldehyde for H&E staining.

Finally, sera from each group of mice were collected and the hepatotoxicity of SCCI was assessed by measuring the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

Statistical analysis

All data were shown as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, USA). Statistical analysis was conducted with Student’s t-test, one-way analysis of variance and Dunnett’s multiple-comparison test. A P-value < 0.05 was considered to be statistically significant differences.