**1. Introduction**

Local therapy has recently attracted an increasing amount of attention in tumor treatment due to the advantages of solid selectivity, controllability, and minor systemic side effects [1,2]. Local therapy mainly includes local ablation (radiofrequency ablation [3–5], irreversible electroporation [6–8], high-intensity focused ultrasound [9], local laser ablation [10], cryoablation [11–13], and chemical ablation [14,15]), local phototherapy (PTT [16,17] and photodynamic therapy [16,18,19]), local radioisotope therapy [20,21], local radiotherapy [22–24], and local chemotherapy [25]. An increasing number of studies have shown that local therapy can be applied to many clinical situations, such as in the preservation of tissue and function [26], situations without medical indication for surgery [27], the local treatment of metastatic tumors [28,29], and the salvage treatment of recurrent tumors [30]. Thus, local therapy is sometimes essential to improve the quality of life of patients with tumors, as well as survival time. At the same time, some studies have reported the success of the combination of local therapy and other treatment methods, such as immunotherapy [31,32], which further illustrates the broad prospect of local therapy.

**Citation:** Wang, X.; Yang, Z.; Meng, Z.; Sun, S.-K. Transforming Commercial Copper Sulfide into Injectable Hydrogels for Local Photothermal Therapy. *Gels* **2022**, *8*, 319. https://doi.org/10.3390/ gels8050319

Academic Editors: Kiat Hwa Chan, Yang Liu and Bjørn Torger Stokke

Received: 3 February 2022 Accepted: 11 May 2022 Published: 20 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

<sup>+86-22-8333-6093 (</sup>S.-K.S.)

As a kind of burgeoning local therapy, PTT uses photoabsorbers to transform nearinfrared (NIR) light energy to heat energy, which induces tumor cell necrosis [16,33]. NIR-based PTT is mainly conducted in two biological windows: the first NIR window (NIR-I) and the second NIR window (NIR-II). The wavelength range of NIR-I is from 750 nm to 1000 nm, and the range of NIR-II is from 1000 nm to 1350 nm [34]. Compared with the widely studied NIR-I light, NIR-II light has a stronger penetrating ability and a higher thermal safe power because of its low absorption and scattering in tissue, which has a substantial superiority in curing deeper tumor tissues [34–37]. In addition to the flourishing development of PTT in fundamental studies [38,39], it is very encouraging that PTT based on local administration has also successfully entered a clinical trial for the treatment of prostate cancer [1], which demonstrates the great potential of PTT-based local therapy in clinical transformation.

To date, plenty of biomaterials have been developed as photoabsorbers, such as organic dyes [40–42], organic nanoparticles [43–45], noble metal materials [46–48], carbon materials [49,50], black phosphorus [51,52], and metal oxide and sulfides [53,54]. Among them, metal sulfides, such as CuS, Bi2S3, WS2, CoS, NiS, and FeS, have high photothermal conversion efficiency because of the surface plasmon resonance effect [55,56], and they have been widely used in PTT in recent years [57–62]. In particular, CuS, which possesses strong absorption in the NIR-II bio-window, has been extensively used in NIR-II PTT [63,64]. The current photoabsorbers are mainly obtained using either bottom-up methods or topdown methods, such as coupling thermal oxidation etching and liquid exfoliation to form a solvent-dispersible system [65,66]. However, these methods face several common problems, such as complex steps, high time and energy costs, low raw material utilization, and lacking universal strategy [67].

To avoid the use of metal sulfides using complex synthesis methods, commercial metal sulfides are an excellent choice. The advantage of commercial metal sulfides is that they are mature industrial products with reasonable quality control and low cost, but their disadvantages lie in the raw materials having large particles, being insoluble in water, and not being able to be used for biological applications. Recently, our group proposed a smart "turning solid into gel" strategy [68] by dispersing solid materials in alginate–Ca2+ hydrogel (ACH), which can transform solid materials into an injectable hydrogel, making the solid materials bioavailable. Therefore, it is fascinating to develop versatile commercial metal-sulfide-based hydrogels as novel photoabsorbers without complex synthesis.

Herein, we introduced a simple and powerful ACH platform to load commercial CuS as a representative sample for local tumor NIR-II PTT (Figure 1). The ultra-simple synthesis, 100% loading efficiency, good biocompatibility, low cost, outstanding photothermal capacity, and extreme flexibility allow this platform to provide more options for highly efficient PTT. The CuS hydrogel (CSH) can be simply obtained through mixing and stirring steps. In vitro experiments indicated that CSH exhibits good syringeability and intense NIR-II absorption (1064 nm). Then, CSH was employed for in vivo PTT studies. The results confirm that this hydrogel not only performs well in killing tumor cells under mild laser irradiation but that it also shows low toxicity in vitro and in vivo. To the best of our knowledge, this is the first time that commercial CuS was elegantly employed for highly efficient PTT in vivo.

**Figure 1.** Schematic representation of the synthesis of CSH as a PTT agent for local NIR-II PTT in **Figure 1.** Schematic representation of the synthesis of CSH as a PTT agent for local NIR-II PTT in vivo.

### vivo. **2. Results and Discussion**

### **2. Results and Discussion**  *2.1. Synthesis and Characterization of CSH*

*2.1. Synthesis and Characterization of CSH*  Firstly, alginate solution and Ca2+ were mixed to produce ACH within 1 min based on their strong coordination interaction (Figure S1). Then, CSH was obtained by dispersing commercial CuS powder into ACH. To investigate the loading capacity of ACH, increasing concentrations of CSH were employed. The maximum loading capacity was 480 mg CuS/mL (Figure 2A). The long-term stability of CSH was also monitored (Table S1 and Figure S2). All concentrations of CSH were stable for more than 4 days, and concentrations of 90 mg CuS/mL and below were still stable after 14 days. Considering that excellent syringeability is essential to potential biological applications, we investigated the maximum loading capacity of CSH capable of fluently being injected with different diameters of syringe needles. The results showed that the maximum injectable concentrations for 0.45, 0.5, 0.6, and 1.2 mm syringe needles were 120, 240, 480, and 480 mg CuS/mL, respectively, and a "TMU" pattern could be written by a 0.45 mm syringe with 20 mg CuS/mL Firstly, alginate solution and Ca2+ were mixed to produce ACH within 1 min based on their strong coordination interaction (Figure S1). Then, CSH was obtained by dispersing commercial CuS powder into ACH. To investigate the loading capacity of ACH, increasing concentrations of CSH were employed. The maximum loading capacity was 480 mg CuS/mL (Figure 2A). The long-term stability of CSH was also monitored (Table S1 and Figure S2). All concentrations of CSH were stable for more than 4 days, and concentrations of 90 mg CuS/mL and below were still stable after 14 days. Considering that excellent syringeability is essential to potential biological applications, we investigated the maximum loading capacity of CSH capable of fluently being injected with different diameters of syringe needles. The results showed that the maximum injectable concentrations for 0.45, 0.5, 0.6, and 1.2 mm syringe needles were 120, 240, 480, and 480 mg CuS/mL, respectively, and a "TMU" pattern could be written by a 0.45 mm syringe with 20 mg CuS/mL CSH (Figure 2B), which proved its excellent syringeability due to the shear-dependent and reversible gel–sol transition (Figure S3) [69].

CSH (Figure 2B), which proved its excellent syringeability due to the shear-dependent and reversible gel–sol transition (Figure S3) [69]. Rheological experiments showed that the storage moduli (G') of ACH and CSH were higher than their loss moduli (G''), demonstrating that ACH and CSH were in a gel state with a relatively weak mechanical strength and flexible shape, which made them easily injectable (Figure S4). As the essential components of CSH, the swelling ratio and degradation behavior of ACH were further investigated. The swelling test showed that ACH could reach swelling equilibrium in 10 min in PBS (pH = 7.4). The swelling ratio of ACH was as high as 13,342.6% (Figure S5), which suggested that the internal cross-linking Rheological experiments showed that the storage moduli (G') of ACH and CSH were higher than their loss moduli (G"), demonstrating that ACH and CSH were in a gel state with a relatively weak mechanical strength and flexible shape, which made them easily injectable (Figure S4). As the essential components of CSH, the swelling ratio and degradation behavior of ACH were further investigated. The swelling test showed that ACH could reach swelling equilibrium in 10 min in PBS (pH = 7.4). The swelling ratio of ACH was as high as 13,342.6% (Figure S5), which suggested that the internal crosslinking points of ACH were relatively few, the cross-linking density was low, and the water absorption capacity was strong. According to the ACH degradation curve (Figure S6),

points of ACH were relatively few, the cross-linking density was low, and the water

the degradation rate of ACH in PBS (pH = 7.4) was 51% after 7 days, which showed its excellent degradability. powder were characterized, and they indicated that the CuS particles were dispersed in the ACH with a porous structure (Figure 2C).

absorption capacity was strong. According to the ACH degradation curve (Figure S6), the degradation rate of ACH in PBS (pH = 7.4) was 51% after 7 days, which showed its excel-

The scanning electron microscope (SEM) images of ACH, CSH, and commercial CuS

*Gels* **2022**, *8*, x FOR PEER REVIEW 4 of 16

**Figure 2.** (**A**) Standing and oblique photos of different concentrations of CSH taken immediately after the preparation. (**B**) "TMU" formed by CSH (20 mg CuS/mL) through a 0.45 mm syringe needle. (**C**) SEM images of ACH, CuS particles, and CSH (20 mg CuS/mL). **Figure 2.** (**A**) Standing and oblique photos of different concentrations of CSH taken immediately after the preparation. (**B**) "TMU" formed by CSH (20 mg CuS/mL) through a 0.45 mm syringe needle. (**C**) SEM images of ACH, CuS particles, and CSH (20 mg CuS/mL).

*2.2. Photothermal Performance of CSH In Vitro*  To evaluate the photothermal efficiency of CSH in vitro, different concentrations of CSH were treated with NIR-II laser irradiation (1064 nm, 1 W/cm2) for 5 min, and an in-The scanning electron microscope (SEM) images of ACH, CSH, and commercial CuS powder were characterized, and they indicated that the CuS particles were dispersed in the ACH with a porous structure (Figure 2C).

### frared thermal camera was used to record the temperature elevations. Under NIR-II laser irradiation, CSH showed good photothermal capacity (Figure 3A). The temperature en-*2.2. Photothermal Performance of CSH In Vitro*

lent degradability.

hancement of CSH with different concentrations increased from 17.3 °C to 38.1 °C, while the temperature increase of ACH and PBS was just 6.4 °C. The thermal images also demonstrate the outstanding photothermal ability of CSH (Figure 3B). After undergoing the heating–cooling process three times, the heating capacity of CSH did not significantly change (Figure 3C), which indicates that CSH has good photothermal stability under NIR-II laser irradiation. Therefore, not only can the prepared CSH efficiently transform NIR laser energy to heat energy, but it can also remain stable after repeated laser illumination. To evaluate the photothermal efficiency of CSH in vitro, different concentrations of CSH were treated with NIR-II laser irradiation (1064 nm, 1 W/cm<sup>2</sup> ) for 5 min, and an infrared thermal camera was used to record the temperature elevations. Under NIR-II laser irradiation, CSH showed good photothermal capacity (Figure 3A). The temperature enhancement of CSH with different concentrations increased from 17.3 ◦C to 38.1 ◦C, while the temperature increase of ACH and PBS was just 6.4 ◦C. The thermal images also demonstrate the outstanding photothermal ability of CSH (Figure 3B). After undergoing the heating–cooling process three times, the heating capacity of CSH did not significantly change (Figure 3C), which indicates that CSH has good photothermal stability under NIR-II laser irradiation. Therefore, not only can the prepared CSH efficiently transform NIR laser energy to heat energy, but it can also remain stable after repeated laser illumination.

### *2.3. Cytotoxicity and Cellular Uptake of CSH*

CSH, which had a great photothermal efficacy under 1064 nm irradiation, super-large loading capacity, and excellent stability, was capable of being used for further studies. To evaluate its cytotoxicity, different concentrations of CSH were added to 4T1 cells in 96-well plates, and the cells were continued to be cultured for 24 h. Then, the cell viabilities were calculated through a standard MTT assay [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT]. The cell viability was as high as 88.8% after incubation with a high concentration of CSH (1 mg CuS/mL), which indicated the low cytotoxicity of CSH (Figure 4A). The cellular uptake experiment proved that CuS particles in CSH could not be uptaken by cells due to their

big size (Figure S7), which illustrates that the mechanism of PTT based on CHS is deduced heat conduction instead of the direct interaction of cell and CSH. *Gels* **2022**, *8*, x FOR PEER REVIEW 5 of 16

**Figure 3.** (**A**) Photothermal heating curves of PBS, ACH, and CSH irradiated by NIR-II laser (1064 nm, 1 W/cm2). (**B**) The thermal images of PBS, ACH, and CSH under NIR-II laser irradiation taken by an infrared thermal camera. (**C**) The photothermal stability of CSH. **Figure 3.** (**A**) Photothermal heating curves of PBS, ACH, and CSH irradiated by NIR-II laser (1064 nm, 1 W/cm<sup>2</sup> ). (**B**) The thermal images of PBS, ACH, and CSH under NIR-II laser irradiation taken by an infrared thermal camera. (**C**) The photothermal stability of CSH. *Gels* **2022**, *8*, x FOR PEER REVIEW 6 of 16

**Figure 4.** (**A**) Cytotoxicity of 4T1 cells incubated with different concentrations of CSH or PBS. (**B**) Viability of 4T1 cells incubated with PBS or CSH (0.5, 0.8 mg CuS/mL) and irradiated by NIR-II laser (1064 nm: 0, 2, or 3 W/cm2) (\* *p* < 0.05, \*\* *p* < 0.01). (**C**) Dead/live cell staining test of 4T1 cells treated with PBS or CSH (0.5, 0.8 mg CuS/mL) and irradiated by NIR-II laser (1064 nm: 0, 2, or 3 W/cm2). *2.4. In Vitro PTT of CSH*  **Figure 4.** (**A**) Cytotoxicity of 4T1 cells incubated with different concentrations of CSH or PBS. (**B**) Viability of 4T1 cells incubated with PBS or CSH (0.5, 0.8 mg CuS/mL) and irradiated by NIR-II laser (1064 nm: 0, 2, or 3 W/cm<sup>2</sup> ) (\* *p* < 0.05, \*\* *p* < 0.01). (**C**) Dead/live cell staining test of 4T1 cells treated with PBS or CSH (0.5, 0.8 mg CuS/mL) and irradiated by NIR-II laser (1064 nm: 0, 2, or 3 W/cm<sup>2</sup> ).

Due to the low cytotoxicity of CSH, PTT of 4T1 cells using the hydrogel was investigated with an MTT assay and live and dead cell staining. As the standard procedure, 4T1

1 h, the cells were irradiated by a 1064 nm laser (0, 2, or 3 W/cm2) for 5 min. The 4T1 cell viabilities showed CSH-concentration- and laser-power density-dependent deceases. After being treated with both 0.8 mg CuS/mL of CSH and 1064 nm laser irradiation (3 W/cm2), 4T1 cell viability dropped to less than 4%. However, the viability of 4T1 cells treated with only CSH or laser irradiation remained approximately 100% (Figure 4B). The fluorescent images of live and dead cells, which were stained by calcein acetoxymethyl ester (calcein AM) and propidium iodide (PI), respectively, also showed that the 4T1 cells were significantly destructed after the combined treatments (Figure 4C). These results prove that CSH has an excellent photothermal effect on tumor cells with 1064 nm laser

In order to assess the intratumoral retention ability of CSH, computer tomography (CT) scans were carried out in vitro and in vivo under a voltage of 120 kV (clinical use). Although CSH was considered to have a weak CT value attenuation (Figure 5A,B), after intratumoral injection, it could still be found at the tumor site with CT scans. During the 2 days of CT monitoring that followed, no significant change was found in the CT value

*2.5. Intratumoral Retention Test of CSH* 

irradiation.

### *2.4. In Vitro PTT of CSH*

Due to the low cytotoxicity of CSH, PTT of 4T1 cells using the hydrogel was investigated with an MTT assay and live and dead cell staining. As the standard procedure, 4T1 cells were cultured in a 96-well plate at 37 ◦C for 24 h, and different concentrations of CSH (0.5 and 0.8 mg CuS/mL) or PBS were added and incubated with the cells at 37 ◦C. After 1 h, the cells were irradiated by a 1064 nm laser (0, 2, or 3 W/cm<sup>2</sup> ) for 5 min. The 4T1 cell viabilities showed CSH-concentration- and laser-power density-dependent deceases. After being treated with both 0.8 mg CuS/mL of CSH and 1064 nm laser irradiation (3 W/cm<sup>2</sup> ), 4T1 cell viability dropped to less than 4%. However, the viability of 4T1 cells treated with only CSH or laser irradiation remained approximately 100% (Figure 4B). The fluorescent images of live and dead cells, which were stained by calcein acetoxymethyl ester (calcein AM) and propidium iodide (PI), respectively, also showed that the 4T1 cells were significantly destructed after the combined treatments (Figure 4C). These results prove that CSH has an excellent photothermal effect on tumor cells with 1064 nm laser irradiation.

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

In order to assess the intratumoral retention ability of CSH, computer tomography (CT) scans were carried out in vitro and in vivo under a voltage of 120 kV (clinical use). Although CSH was considered to have a weak CT value attenuation (Figure 5A,B), after intratumoral injection, it could still be found at the tumor site with CT scans. During the 2 days of CT monitoring that followed, no significant change was found in the CT value or in the morphology of CSH (Figure 5C,D), proving its good retention ability at tumor sites. *Gels* **2022**, *8*, x FOR PEER REVIEW 7 of 16 or in the morphology of CSH (Figure 5C,D), proving its good retention ability at tumor sites.

**Figure 5.** (**A**) CT images of CSH at different concentrations. (**B**) CT value curve of CSH. (**C**) CT scan of BALB/c mice before and 0, 24, and 48 h after being intratumorally injected with 20 mg CuS/mL of CSH (*n* = 3). (**D**) CT value (Hounsfield, HU) changing curves on the tumor site of BALB/c mice. **Figure 5.** (**A**) CT images of CSH at different concentrations. (**B**) CT value curve of CSH. (**C**) CT scan of BALB/c mice before and 0, 24, and 48 h after being intratumorally injected with 20 mg CuS/mL of CSH (*n* = 3). (**D**) CT value (Hounsfield, HU) changing curves on the tumor site of BALB/c mice.

### *2.6. In Vivo PTT of CSH 2.6. In Vivo PTT of CSH*

To minimize the damage to surrounding tissues, a mild laser power (0.3 W/cm2) was employed for in vivo PTT. CSH with a concentration of 20 mg CuS/mL, which can cause a significant temperature rise in vitro, was used to guarantee effective tumor ablation (Figure S8). To evaluate the in vivo photothermal tumor therapy efficacy of CSH in the NIR-II bio-window (1064 nm), BALB/c mice were grouped according to different treatments (*n*  = 5) as follows: (1) only PBS; (2) only CSH; (3) PBS + laser; and (4) CSH + laser. In comparison with the control, there was a noticeable temperature increase at the tumor site after being injected with CSH and irradiated with a 1064 nm laser (Figure 6A,B). Tumor sizes were measured every 2 days to evaluate the anti-tumor capacity. The results showed that To minimize the damage to surrounding tissues, a mild laser power (0.3 W/cm<sup>2</sup> ) was employed for in vivo PTT. CSH with a concentration of 20 mg CuS/mL, which can cause a significant temperature rise in vitro, was used to guarantee effective tumor ablation (Figure S8). To evaluate the in vivo photothermal tumor therapy efficacy of CSH in the NIR-II bio-window (1064 nm), BALB/c mice were grouped according to different treatments (*n* = 5) as follows: (1) only PBS; (2) only CSH; (3) PBS + laser; and (4) CSH + laser. In comparison with the control, there was a noticeable temperature increase at the tumor site after being injected with CSH and irradiated with a 1064 nm laser (Figure 6A,B).

> the tumor growth of mice treated with both CSH and laser irradiation was effectively inhibited, and the tumors were eliminated after PTT (Figure 6C). There was tumor recur-

> groups multiplied, and the final tumor volumes after 15 days of growth were about 12.9, 14.1, and 12.5 times larger than the initial tumor volume in groups 1, 2, and 3, respectively (Figure 6D). The tumors were dissected and photographed on the 15th day (Figure 6E). The dissected tumors were weighed, and the ratio of tumor weight to mouse body weight in each group was calculated (Figure 6F), which further revealed that the tumors were obviously suppressed by CSH-based PTT. These results illustrate that CSH can wreck tu-

mors entirely due to its high thermal efficiency in vivo.

Tumor sizes were measured every 2 days to evaluate the anti-tumor capacity. The results showed that the tumor growth of mice treated with both CSH and laser irradiation was effectively inhibited, and the tumors were eliminated after PTT (Figure 6C). There was tumor recurrence in only one mouse in the combined treatment group, and the recurred tumor was significantly smaller than that in the other groups. In contrast, the tumors in the other groups multiplied, and the final tumor volumes after 15 days of growth were about 12.9, 14.1, and 12.5 times larger than the initial tumor volume in groups 1, 2, and 3, respectively (Figure 6D). The tumors were dissected and photographed on the 15th day (Figure 6E). The dissected tumors were weighed, and the ratio of tumor weight to mouse body weight in each group was calculated (Figure 6F), which further revealed that the tumors were obviously suppressed by CSH-based PTT. These results illustrate that CSH can wreck tumors entirely due to its high thermal efficiency in vivo. *Gels* **2022**, *8*, x FOR PEER REVIEW 8 of 16

**Figure 6.** (**A**) Thermal images of tumor-bearing mice treated with only PBS or both CSH and NIR-II laser irradiation. (**B**) Photothermal heating curves of tumor sites taken with an infrared thermal camera. (**C**) Tumor-monitoring photography of mice in various groups. (**D**) Relative tumor volume curves of mice in different groups (*n* = 5 in each group, \*\* *p* < 0.01). (**E**) Excised tumors from the mice on the 15th day of the observation period. (**F**) Ratio of final tumor weight to final body weight of mice in different groups (\*\* *p* < 0.01). *2.7. In Vivo Toxicity of CSH*  **Figure 6.** (**A**) Thermal images of tumor-bearing mice treated with only PBS or both CSH and NIR-II laser irradiation. (**B**) Photothermal heating curves of tumor sites taken with an infrared thermal camera. (**C**) Tumor-monitoring photography of mice in various groups. (**D**) Relative tumor volume curves of mice in different groups (*n* = 5 in each group, \*\* *p* < 0.01). (**E**) Excised tumors from the mice on the 15th day of the observation period. (**F**) Ratio of final tumor weight to final body weight of mice in different groups (\*\* *p* < 0.01).

To assess the systemic toxicity of CSH in vivo, the weight monitoring, blood biochemistry analysis, and H&E staining of major organs of the mice were accomplished. The weight monitoring results displayed no noticeable difference in body weight among the mice with various treatments (Figure 7A). The blood biochemistry analysis indicated that

(Figure 7B), and no evident inflammatory lesion or organ damage was found in all major organs of the mice (Figure 7C). All of the above results confirm that CSH has good biocompatibility, which makes it a promising PTT agent with good biosafety and photother-

mal efficacy in vivo.

### *2.7. In Vivo Toxicity of CSH*

To assess the systemic toxicity of CSH in vivo, the weight monitoring, blood biochemistry analysis, and H&E staining of major organs of the mice were accomplished. The weight monitoring results displayed no noticeable difference in body weight among the mice with various treatments (Figure 7A). The blood biochemistry analysis indicated that the liver and kidney function indexes of the mice were entirely within the normal range (Figure 7B), and no evident inflammatory lesion or organ damage was found in all major organs of the mice (Figure 7C). All of the above results confirm that CSH has good biocompatibility, which makes it a promising PTT agent with good biosafety and photothermal efficacy in vivo. *Gels* **2022**, *8*, x FOR PEER REVIEW 9 of 16

**Figure 7.** (**A**) Weight-changing curves of mice after being subcutaneously injected with PBS and 20 mg CuS/mL of CSH (*n* = 5 in each group). (**B**) Blood biochemical indexes of mice measured after being treated with PBS for 15 days and CSH (20 mg CuS/mL) for 1, 7, and 15 days (*n* = 5 in each group). (**C**) H&E-stained images of major organs of Kunming mice acquired after being treated with PBS for 15 days and CSH (20 mg CuS/mL) for 1, 7, and 15 days. **Figure 7.** (**A**) Weight-changing curves of mice after being subcutaneously injected with PBS and 20 mg CuS/mL of CSH (*n* = 5 in each group). (**B**) Blood biochemical indexes of mice measured after being treated with PBS for 15 days and CSH (20 mg CuS/mL) for 1, 7, and 15 days (*n* = 5 in each group). (**C**) H&E-stained images of major organs of Kunming mice acquired after being treated with PBS for 15 days and CSH (20 mg CuS/mL) for 1, 7, and 15 days.

### **3. Conclusions 3. Conclusions**

In conclusion, according to the "turning solid into gel" strategy, a robust metal sulfide hydrogel system was established to load commercial metal sulfide powders for highefficiency tumor PTT. As a representative metal sulfide, commercial CuS powder was studied in depth. The obtained CSH was verified to have good stability, favorable syringeability, potent photothermal efficacy, and excellent retention capability at the injection site. Due to the deeper tissue penetration of NIR-II light, further studies were investigated using 1064 nm laser irradiation. The follow-up experimentations in vitro and in vivo showed the CSH to have negligible toxicity and a high photothermal killing effect on tumor cells under the irradiation of the 1064 nm laser. Therefore, as a new method of pho-In conclusion, according to the "turning solid into gel" strategy, a robust metal sulfide hydrogel system was established to load commercial metal sulfide powders for highefficiency tumor PTT. As a representative metal sulfide, commercial CuS powder was studied in depth. The obtained CSH was verified to have good stability, favorable syringeability, potent photothermal efficacy, and excellent retention capability at the injection site. Due to the deeper tissue penetration of NIR-II light, further studies were investigated using 1064 nm laser irradiation. The follow-up experimentations in vitro and in vivo showed the CSH to have negligible toxicity and a high photothermal killing effect on tumor cells under the irradiation of the 1064 nm laser. Therefore, as a new method of photothermal agent

> tothermal agent preparation, transforming commercial sulfides into injectable hydrogels can help to save costs, improve accuracy, and raise efficiency without worrying about

> CaCl2 and sodium alginate (200 ± 20 mPa s) were obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). CuS was purchased from Sigma-Aldrich trade Co., Ltd. (Shanghai, China). Fetal Bovine Serum (FBS) was provided by Lanzhou Minhai Bio-Engineering Co., Ltd. Dulbecco's Modified Eagle Medium (DMEM) was obtained

**4. Materials and Methods** 

*4.1. Materials* 

preparation, transforming commercial sulfides into injectable hydrogels can help to save costs, improve accuracy, and raise efficiency without worrying about toxicity, all of which give it great hope for clinical transformation.

### **4. Materials and Methods**

### *4.1. Materials*

CaCl<sup>2</sup> and sodium alginate (200 ± 20 mPa s) were obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). CuS was purchased from Sigma-Aldrich trade Co., Ltd. (Shanghai, China). Fetal Bovine Serum (FBS) was provided by Lanzhou Minhai Bio-Engineering Co., Ltd. Dulbecco's Modified Eagle Medium (DMEM) was obtained from ThermoFisher Instrument Co., Ltd. (Suzhou, China). MTT was bought from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Calcein AM and PI were provided by Dojindo Chemical L.L.C. (Shanghai, China). DMSO was purchased from Concord Technology Co., Ltd. (Tianjin, China). Ultrapure water was bought from Wahaha Group Co., Ltd. (Hangzhou, China).

### *4.2. Synthesis of ACH and CSH*

Typically, 0.5 mL of sodium alginate (10 mg/mL) and 0.05 mL of CaCl<sup>2</sup> (10 mg/mL) were mixed with 0.45 mL H2O to prepare ACH. Then, commercial CuS powder was added to ACH, and the system was stirred for 15 min to obtain CSH.
