*4.3. Stability Assessment of CSH*

The homogeneous stability of CSH was evaluated for 2 weeks. Briefly, different concentrations of CSH (15, 30, 60, 90, 120, 240, 480 mg CuS/mL) were placed in vials, respectively. If the stability time was less than 10 min or the sulfide could not be dispersed in ACH, the mixture was regarded as overloaded. The stable concentrations of CSH were monitored for 14 days and photographed at different time points (10 min, 20 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 24 h, and then every day). During the monitoring period, if the hydrogel was found to be layered, it was regarded as precipitation.

### *4.4. Syringeability of CSH*

The syringeability of CSH was evaluated using four sizes of syringe needles (26 G, 0.45 mm; 25 G, 0.5 mm; 23 G, 0.6 mm; 18 G, 1.2 mm). Different concentrations of CSH were extruded through the various sizes of syringe needles. For every size of syringe, the maximum injectable concentration was recorded, and a "TMU" (an abbreviation of "Tianjin Medical University") was written by its maximum injectable concentration. Then, a "TMU" was formed by CSH (20 mg CuS/mL) through a 0.45 mm syringe needle, which was used for in vivo PTT.

### *4.5. Characterization*

Rheology experiments of ACH and CSH (20 mg CuS/mL) were conducted on a DHR-2 rheometer (TA Instruments), with a strain amplitude of 1% and an angular frequency of 10 rad/s for dynamic oscillatory time sweep measurements.

The swelling ratio and degradation behavior of ACH were investigated according to a previous study [70]. To calculate the swelling ratio, lyophilized ACH was weighed (recorded as M0), immersed in PBS (pH = 7.4), and incubated in an incubator shaker at a shaking speed of 100 rpm at 37 ◦C. The swelled ACH was removed, and the surface water was wiped out. Then, the collected ACH was weighed at a specific time interval (recorded as Mt). The swelling ratio (%, *w*/*w*) was calculated using the equation (M<sup>t</sup> − M0)/M<sup>0</sup> × 100. The experiments were carried out in triplicate to obtain an average value. The degradation behavior of ACH was assessed in PBS (pH = 7.4). The lyophilized ACH was weighed (recorded as Mi) and completely immersed in PBS, and then it was degraded in an incubator shaker at 37 ◦C and 100 rpm. After different time intervals (1, 3, or 7 days), ACH was

washed with ultrapure water to remove PBS, freeze-dried, and weighed (recorded as M<sup>f</sup> ). The degradation rate (%, *w*/*w*) was calculated using the equation (M<sup>i</sup> − M<sup>f</sup> )/M<sup>i</sup> × 100.

Field-emission scanning electron microscopy (FE-SEM) images of ACH, CuS powder, and CSH (20 mg CuS/mL) were acquired under a 2 kV accelerating voltage on a Gemini SEM 300 (ZEISS, Germany) microscope.

### *4.6. Photothermal Performance In Vitro*

In order to evaluate the photothermal efficacy of CSH in vitro, PBS or different concentrations of CSH (0, 1, 2.5, and 5 mg CuS/mL) with a volume of 1 cm<sup>3</sup> were placed in cuvettes with a base area of 1 cm<sup>2</sup> . Then, cuvettes were irradiated with a 1064 nm laser (1 W/cm<sup>2</sup> ) for 5 min, and temperature elevations were recorded using an infrared thermal camera. In order to test its photothermal stability, CSH (5 mg CuS/mL, 1 mL) was put into a cuvette and irradiated using NIR-II (1064 nm) laser with a power density of 1 W/cm<sup>2</sup> for 5 min, and then the system was cooled for 10 min to bring the temperature close to room temperature; the process was repeated three times.

### *4.7. Cell Culture and Animals*

The growth and metastasis of 4T1 cells in BALB/c mice are similar to those of human breast cancer, making the cells a relatively classical and widely used cell line to test the therapeutic effects on tumors [71]. Therefore, the 4T1 cell line was used to study CSH in vitro and in vivo. 4T1 cells were cultured in a culture medium with 90% DMEM and 10% FBS. Cells were cultured in a humidified incubator (5% CO<sup>2</sup> and 37 ◦C), and the culture medium was refreshed at 1–2 day intervals. Kunming mice and BALB/c mice were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). All animal experiments were performed according to the protocols established by the Animal Care and Use Committee of Tianjin Medical University, and all experimental operations were approved by the Animal Care and Use Committee.

### *4.8. Cytotoxicity and Cellular Uptake Assay*

To determine the potential cytotoxic effects of CSH, 4T1 cells (1 <sup>×</sup> <sup>10</sup><sup>4</sup> per well) were cultured in 96-well plates with 200 µL of cell culture medium per well for 24 h. Then, after the exchange of the cell medium, PBS or different concentrations of CSH (0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1 mg CuS/mL) were added to the wells. After 24 h incubation, cell viabilities were evaluated via a standard MTT test. After the cells were washed with PBS, new cell medium and MTT (10 µL, 5 mg/mL) were added and incubated with the cells for 4 h; then, the supernatant was discarded, and 120 µL of DMSO per well was added. Finally, the wells' absorptions at 490 nm were measured using a microplate reader.

The cellular uptake mechanism of CSH was also investigated. In brief, 4T1 cells (1 <sup>×</sup> <sup>10</sup><sup>4</sup> per well) were cultured in 96-well plates. After 24 h, PBS or different concentrations of CSH (0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1 mg CuS/mL) were added and co-incubated with the cells for another 24 h. Then, the cells were washed with PBS, and 120 µL of PBS per well was added. Finally, the cells were observed under a microscope.

### *4.9. In Vitro Photothermal Cytotoxicity Study*

The photothermal cell killing ability of CSH under 1064 nm laser irradiation was evaluated using the MTT assay. 4T1 cells (1.4 <sup>×</sup> <sup>10</sup><sup>4</sup> per well) were incubated in a 96 well plate for 24 h. After being washed with PBS, the 4T1 cells were treated with PBS or different concentrations of CSH (0.5 or 0.8 mg CuS/mL) for 1 h, and they were irradiated with varying densities of power of 1064 nm laser (0, 2, or 3 W/cm<sup>2</sup> ) for 5 min. Then, cell viabilities were measured using the MTT assay, and the absorption of each well at 490 nm was recorded using a microplate reader.

### *4.10. Live/Dead Cells Staining Test*

To further investigate the PTT efficacy in vitro, 4T1 cells (1.4 <sup>×</sup> <sup>10</sup><sup>4</sup> per well) were incubated in 96-well plates for 24 h, and CSH (0.5, 0.8 mg CuS/mL) or PBS was added and co-incubated with 4T1 cells for 1 h. Then, 4T1 cells were exposed to NIR-II laser (1064 nm, 0, 2, or 3 W/cm<sup>2</sup> ) for 5 min, thoroughly washed with PBS twice, and stained with calcein AM and PI. Fluorescent images were recorded with an inverted luminescence microscope.

### *4.11. Intratumoral Retention Test of CSH*

In vitro and in vivo CT scans were carried out via a clinical X-ray CT (SOMATOM Force, Siemens healthineers, Erlangen, Germany) under a clinical voltage (120 kV) [72]. CSHs with different concentrations (0, 1, 2.5, 5, 10, 15, 30 mg CuS/mL) were prepared, and then CT images of CSH were collected. For in vivo CT imaging, 50 µL of CSH (20 mg CuS/mL) was intratumorally injected into BALB/c mice (*n* = 3). Then, the mice were scanned pre-injection and after injection at different time points (0 h, 24 h, and 48 h). CT values were measured using Radiant DICOM Viewer software.

### *4.12. Anti-Tumor Assessment In Vivo*

To ensure biosafety, a mild laser power (0.3 W/cm<sup>2</sup> ) and CSH with a concentration of 20 mg CuS/mL were used for in vivo PTT. To verify the in vitro heating effect, CSH (20 mg CuS/mL) or PBS was made into 50 µL droplets, and they were irradiated using a 1064 nm laser (0.3 W/cm<sup>2</sup> ) for 5 min. Thermal images of them were taken, and photothermal heating curves were obtained. Then, to explore the anti-tumor ability of CSH with 1064 nm laser irradiation, tumor-bearing BALB/c mice were divided into 4 groups (*n* = 5) as follows: (1) only PBS, (2) only CSH, (3) PBS + laser, and (4) CSH + laser. Mice in Group 1 were intratumorally injected with PBS (50 µL). Mice in Group 2 were intratumorally injected with CSH (20 mg CuS/mL, 50 µL). Mice in Group 3 were intratumorally injected with PBS and exposed to 1064 nm laser irradiation (0.3 W/cm<sup>2</sup> ) for 10 min. Mice in Group 4 were intratumorally injected with CSH (20 mg CuS/mL, 50 µL) and exposed to 1064 nm laser irradiation (0.3 W/cm<sup>2</sup> ) for 10 min. The hyperthermia effect on tumor site was carefully recorded using an infrared thermal camera. Then, tumor sizes were measured and recorded every 2 days. Tumor volume was calculated using the following formula: V = a × b <sup>2</sup>/2, where a and b mean the longest and shortest diameters, respectively. The relative volume of the tumors was the ratio of the day's volume to the initial volume. Photos of tumors in all groups were taken every 2 days, and the tumors were removed and weighed on day 15 after the treatment.

### *4.13. Statistics*

The differences between groups were studied using one-way ANOVA, and "*p*" value < 0.05 was considered as statistically significant. All analyses were conducted using GraphPad Prism 8.0.2 software.

### *4.14. In Vivo Biosafety Analysis*

To evaluate the biosafety of CSH in vivo, weight monitoring, blood biochemistry analysis, and H&E staining were conducted on Kunming mice. To monitor the body weight change, CSH (50 µL, 20 mg CuS/mL) or PBS was subcutaneously injected into Kunming mice (*n* = 5, respectively), and their body weights were recorded every two days until the 15th day. For blood biochemistry analysis and H&E staining, Kunming mice were subcutaneously injected with PBS (*n* = 5) or CSH (50 µL, 20 mg CuS/mL) (*n* = 15). Mice in the hydrogel-injected group were dissected on the 1st, 7th, and 15th days (*n* = 5 every time), and mice in the PBS group were dissected on the 15th day. After the mice were dissected, their major organs (i.e., heart, lung, spleen, liver, and kidney) were removed and stained with hematoxylin and eosin, and blood samples were collected. The blood samples were centrifugated at 3000 rpm for 10 min to separate and collect the supernatant serum. Then, the blood biochemistry biomarkers were analyzed, which included albumin (ALB), total

bile acid (TBA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) for liver function assessment, and uric acid (UA), urea nitrogen (BUN), and serum creatinine (Cr) for kidney function evaluation.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/gels8050319/s1, Figure S1: Photos of ACH stirred for different times after mixing alginate solution and Ca2+ solution; Figure S2: Standing and oblique photos of different concentrations of CSH taken on day 7 and day 14; Figure S3: The maximum injectable concentration of CSH through various size of syringes and "TMU" written by them; Figure S4: Rheological properties of ACH and CSH (20 mg CuS/mL); Figure S5: Curve of swelling ratio of ACH in PBS (pH = 7.4); Figure S6: Degradation curve of ACH in PBS (pH = 7.4); Figure S7: Photos of 4T1 cells incubated with different concentrations of CSH or PBS for 24 h; Figure S8: Thermal images and photothermal heating curves of CSH (20 mg CuS/mL) and PBS under 1064 nm laser irradiation in vitro; Table S1: The stability of different concentrations of CSH.

**Author Contributions:** Conceptualization, S.-K.S. and Z.M.; methodology and validation, X.W. and Z.Y.; writing—original draft preparation, X.W. and Z.Y.; writing—review and editing, S.-K.S. and Z.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the following foundations: the National Natural Science Foundation of China (21874101, 21934002, and 82071982 to S.-K.S.; 81571709 and 81971650 to Z.M.); the Natural Science Foundation of Tianjin City (19JCJQJC63700 to S.-K.S.); Young Elite Scientists Sponsorship Program by Tianjin (TJSQNTJ-2018-08 to S.-K.S.); Key Project of Tianjin Science and Technology Committee Foundation grant (16JCZDJC34300 to Z.M.), Tianjin Medical University General Hospital New Century Excellent Talent Program (to Z.M.); Young and Middle-aged Innovative Talent Training Program from the Tianjin Education Committee (to Z.M.); Talent Fostering Program (the 131 Project) from the Tianjin Education Committee; and the Tianjin Human Resources and Social Security Bureau (to Z.M.).

**Institutional Review Board Statement:** The animal study protocol was approved by the Ethics Committee of Tianjin Medical University (SYXK-2019-0004).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

