*2.3.* •*OH Generating Capacity of Fe–Zr@PDA@CMCS Hydrogel*

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Therapeutic modalities based on reactive oxygen radicals and their derivatives have been demonstrated as emerging therapeutic strategies for tumors [43,51,52]. Among these, Fenton-based and Fenton-like responses are considered to be potential tumor cell therapy modalities and there are now several studies demonstrating the use of the Fenton effect to cause tumor-killing effects [44–47,53]. We first verified the ability of Fe–Zr@PDA to produce •OH; specifically, •OH can rapidly oxidize colorless TMB to a blue–green oxidized TMB compound (oxTMB), and further scanned the absorbance of the reacted solution at 652 nm by UV spectrophotometry. The magnitude of the absorbance gave a side view of the ability of the Fenton reaction to produce •OH. As shown in Figure 3a, the concentration of Fe–Zr@PDA was increased from 0 µg/mL to 100 µg/mL, and the results of the absorbance of the final solution after reaction with TMB were gradually increased, indicating a positive correlation between its ability to produce •OH and the concentration of Fe–Zr@PDA. The reaction of Fe–Zr@PDA with H2O<sup>2</sup> shows that the colorless TMB is oxidized to the blue oxTMB, which further demonstrates the ability of Fe–Zr@PDA to generate •OH (Figure 3a). Further, we tested the •OH generation ability of Fe–Zr@PDA@CMCS hydrogel. The control hydrogel without Fe–Zr@PDA@CMCS had an absorbance value of almost zero at 652 nm and the mixture remained transparent (Figure 3b). In contrast, the absorbance of Fe–Zr@PDA@CMCS hydrogel increased with the concentration of Fe–Zr@PDA and the color of the solution deepened, which further indicated that the catalytic ability of Fe–Zr@PDA was well-preserved after doping in the hydrogel (Figure 3b). *2.3. •OH Generating Capacity of Fe–Zr@PDA@CMCS Hydrogel* Therapeutic modalities based on reactive oxygen radicals and their derivatives have been demonstrated as emerging therapeutic strategies for tumors [43,51,52]. Among these, Fenton-based and Fenton-like responses are considered to be potential tumor cell therapy modalities and there are now several studies demonstrating the use of the Fenton effect to cause tumor-killing effects [44–47,53]. We first verified the ability of Fe–Zr@PDA to produce •OH; specifically, •OH can rapidly oxidize colorless TMB to a blue–green oxidized TMB compound (oxTMB), and further scanned the absorbance of the reacted solution at 652 nm by UV spectrophotometry. The magnitude of the absorbance gave a side view of the ability of the Fenton reaction to produce •OH. As shown in Figure 3a, the concentration of Fe–Zr@PDA was increased from 0 μg/mL to 100 μg/mL, and the results of the absorbance of the final solution after reaction with TMB were gradually increased, indicating a positive correlation between its ability to produce •OH and the concentration of Fe–Zr@PDA. The reaction of Fe–Zr@PDA with H2O<sup>2</sup> shows that the colorless TMB is oxidized to the blue oxTMB, which further demonstrates the ability of Fe–Zr@PDA to generate •OH (Figure 3a). Further, we tested the •OH generation ability of Fe– Zr@PDA@CMCS hydrogel. The control hydrogel without Fe–Zr@PDA@CMCS had an absorbance value of almost zero at 652 nm and the mixture remained transparent (Figure 3b). In contrast, the absorbance of Fe–Zr@PDA@CMCS hydrogel increased with the concentration of Fe–Zr@PDA and the color of the solution deepened, which further indicated that the catalytic ability of Fe–Zr@PDA was well-preserved after doping in the hydrogel (Figure 3b).

**Figure 3.** (**a**) Experimental results and corresponding digital camera photos of the reaction of different concentrations of Fe–Zr@PDA nanoenzymes with TMB and H2O<sup>2</sup> to produce •OH; (**b**) absorbance values of different concentrations of Fe–Zr@PDA@CMCS after co-incubation with TMB and H2O2, and digital photographs of the supernatant photographic images; (**c**) degradation rates of Fe– **Figure 3.** (**a**) Experimental results and corresponding digital camera photos of the reaction of different concentrations of Fe–Zr@PDA nanoenzymes with TMB and H2O<sup>2</sup> to produce •OH; (**b**) absorbance values of different concentrations of Fe–Zr@PDA@CMCS after co-incubation with TMB and H2O<sup>2</sup> , and digital photographs of the supernatant photographic images; (**c**) degradation rates of Fe–Zr@PDA@CMCS hydrogel in CBS and PBS solutions; (**d**) swelling rate of Fe–Zr@PDA@CMCS hydrogel in ultrapure water, CBS solution, and PBS solution.

### *2.4. Evaluation of Hydrogel Degradation and Swelling Properties*

The ability to swell and degrade swelling properties are important physical parameters of hydrogels, which affect the rate of release of loaded compounds and the absorption of tissue exudate, among other things [54–56]. In this section, we first investigated the degradation properties of Fe–Zr@PDA@CMCS hydrogel under different pH conditions. As shown in Figure 3c, Fe–Zr @PDA@CMCS hydrogel showed a fast weight loss in both PBS and CBS at the beginning and then degraded by more than 45% in the first three days. The degradation rate increased gradually with time, but the degradation rate was slower in the CBS than in the PBS. It is worth mentioning that the TME is acidic due to the altered metabolism of the tumor cells; therefore, the Fe–Zr@PDA@CMCS hydrogel can be retained in the acidic TME for a longer period to achieve prolonged drug release and tumor therapy.

Then, we evaluated the swelling properties of Fe–Zr@PDA@CMCS hydrogel by immersing them in ultrapure water, CBS, or PBS for 24 h. As shown in Figure 3d, the freeze-dried hydrogel immersed in PBS buffer and ultrapure water swelled hastily inside a brief length when they come upon the liquid. However, the swelling state of the hydrogel immersed in the PBS buffer solution also increased slowly with time. At the same time, the swelling of the hydrogel in pure water remained almost constant, and in the final experimental phase, the freeze-dried hydrogel reached swelling equilibrium. Interestingly, compared with the hydrogel soaked in PBS and pure water, Fe–Zr@PDA@CMCS hydrogel soaked in CBS showed little change in the swelling degrees. This will facilitate the presence of Fe–Zr@PDA@CMCS hydrogel in a slightly acidic environment.

### *2.5. In Vitro Cytocompatibility Results of Fe–Zr@PDA@CMCS Hydrogel*

Biocompatibility refers to the ability of a material to elicit appropriate host and material responses in a given application environment [57,58]. The ideal composite hydrogel requires good biocompatibility both in vitro and in vivo [59,60]. We evaluated the cell safety of Fe–Zr@PDA@CMCS hydrogel through a series of in vitro cell experiments using the mouse fibroepithelial L929 cell line as a model. Adherently grown L929 cells were cocultured with 1 mg/mL, 2.5 mg/mL, and 5 mg/mL of hydrogel extracts for 24 h and 48 h, and the results are shown in Figure 4a. Even at concentrations up to 5 mg/mL, the viability of L929 cells at 24 h and 48 h was higher than 90%, indicating that Fe–Zr@PDA@CMCS hydrogel has good cytocompatibility (Figure 4a). Following the CCK-8 cell viability assay, we further stained cells in each experimental and control group using AM-PI stain and observed the distribution of live and dead cells using inverted phase contrast fluorescence microscopy. As can be seen in Figure 4b, in the 24 h and 48 h images, both in the control and experimental groups, the l929 cells, which represent viable cells, show green fluorescence, while the red fluorescence, which represents dead cells, is negligible in the images. It is worth noting that there is no significant difference in the observation of cell morphology between the experimental group and the control group. The above staining results were consistent with the results of CCK-8 experiments, which further demonstrated that Fe–Zr@PDA@CMCS hydrogel has no significant effect on the cell morphology of L929 cells and has good cell safety.

### *2.6. In Vitro Hemocompatibility Results of Fe–Zr@PDA@CMCS Hydrogel*

The validation of in vitro biocompatibility experiments is crucial to ensure the biomedical applications of the Fe–Zr@PDA@CMCS hydrogel. The hemocompatibility test was used to assess whether Fe–Zr@PDA@CMCS hydrogel would cause hemolysis. Briefly, Fe–Zr@PDA@CMCS hydrogel was co-cultured with rat red blood cells to investigate their hemocompatibility. As shown in Figure 4c, the hemolysis rate of erythrocytes was 2.56 ± 3.31%, 3.35 ± 0.41%, 3.98 ± 0.43%, and 4.61 ± 0.18% after being cultured with Fe–Zr@PDA@CMCS hydrogel with concentrations of 10, 20, 50, and 100 mg/mL respectively, which were all less than 5%. This result indicated that Fe–Zr@PDA@CMCS hydrogel has good hemocompatibility. In addition, we recorded digital photographs of the erythrocyte solution after co-culture and, in agreement with the previous results, the positive

control group of erythrocytes co-cultured with ultrapure water appeared bright red. This demonstrated that the erythrocytes have all ruptured, whereas the supernatant of the experimental group was clearer and more transparent, indicating that most of the erythrocyte structure was still intact (Figure 4d). *Gels* **2023**, *9*, x FOR PEER REVIEW 8 of 18

**Figure 4.** (**a**) Cell viability of Fe–Zr@PDA@CMCS hydrogel extracts at 1 mg/mL, 2.5 mg/mL, and 5 mg/mL co-cultured with mouse fibroblasts (L929 cell line) for 24 h and 48 h; (**b**) AM-PI stained images of L929 cell lines co-cultured with Fe–Zr@PDA@CMCS hydrogel extracts at 1 mg/mL, 2.5 mg/mL, and 5 mg/mL for 24 h and 48 h; (**c**) hemolysis ratio of rat erythrocytes after co-culture with different concentrations of Fe–Zr@PDA@CMCS hydrogel; (**d**) photos of rat erythrocytes after centrifugation. **Figure 4.** (**a**) Cell viability of Fe–Zr@PDA@CMCS hydrogel extracts at 1 mg/mL, 2.5 mg/mL, and 5 mg/mL co-cultured with mouse fibroblasts (L929 cell line) for 24 h and 48 h; (**b**) AM-PI stained images of L929 cell lines co-cultured with Fe–Zr@PDA@CMCS hydrogel extracts at 1 mg/mL, 2.5 mg/mL, and 5 mg/mL for 24 h and 48 h; (**c**) hemolysis ratio of rat erythrocytes after coculture with different concentrations of Fe–Zr@PDA@CMCS hydrogel; (**d**) photos of rat erythrocytes after centrifugation.

### *2.6. In Vitro Hemocompatibility Results of Fe–Zr@PDA@CMCS Hydrogel 2.7. In Vitro Biocompatibility Assessment Results of Fe–Zr@PDA@CMCS Hydrogel*

The validation of in vitro biocompatibility experiments is crucial to ensure the biomedical applications of the Fe–Zr@PDA@CMCS hydrogel. The hemocompatibility test was used to assess whether Fe–Zr@PDA@CMCS hydrogel would cause hemolysis. Briefly, Fe–Zr@PDA@CMCS hydrogel was co-cultured with rat red blood cells to investigate their hemocompatibility. As shown in Figure 4c, the hemolysis rate of erythrocytes was 2.56 ± 3.31%, 3.35 ± 0.41%, 3.98 ± 0.43%, and 4.61 ± 0.18% after being cultured with Fe– Zr@PDA@CMCS hydrogel with concentrations of 10, 20, 50, and 100 mg/mL respectively, which were all less than 5%. This result indicated that Fe–Zr@PDA@CMCS hydrogel has good hemocompatibility. In addition, we recorded digital photographs of the erythrocyte solution after co-culture and, in agreement with the previous results, the positive control group of erythrocytes co-cultured with ultrapure water appeared bright red. This demon-After establishing the excellent in vitro cytocompatibility of Fe–Zr@PDA@CMCS hydrogel, the safety of the hydrogel was further assessed in vivo by subcutaneously embedding Fe–Zr@PDA@CMCS hydrogel in KM mice and recording the changes in body weight of the mice. The body weight of mice in both the experimental and control groups increased normally throughout the feeding cycle, and the body weight of mice in the experimental group fluctuated slightly for 21 days after encapsulation in the hydrogel; however, there was no statistical difference compared to the control group (*p* > 0.05) (Figure 5a). Further, we measured the blood biochemical parameters of the experimental and control mice on day 7 and 14 of the hydrogel implantation to assess whether the liver and kidney functions were affected. The serum biochemical parameters measured included TB (total bilirubin), ALT (alanine aminotransferase), AST (aspartate aminotransferase), UREA (urea), and CREA (creatinine). As shown in Figure 5b, the experimental and control groups of

strated that the erythrocytes have all ruptured, whereas the supernatant of the experimental group was clearer and more transparent, indicating that most of the erythrocyte

After establishing the excellent in vitro cytocompatibility of Fe–Zr@PDA@CMCS hydrogel, the safety of the hydrogel was further assessed in vivo by subcutaneously embedding Fe–Zr@PDA@CMCS hydrogel in KM mice and recording the changes in body weight

*2.7. In Vitro Biocompatibility Assessment Results of Fe–Zr@PDA@CMCS Hydrogel*

the hydrogel did not show a statistically significant difference in liver and kidney function (*p* > 0.05), which could further confirm the safety of the hydrogel. Next, we evaluated H&E staining sections of the major organs after 7 and 14 days of feeding, which showed no significant cellular hypertrophy, atrophy, or necrosis in any of the organ tissue sections. These results further demonstrated that the encapsulated Fe–Zr@PDA@CMCS hydrogel had no significant side effects on the normal physiological function of the major organs and had a good in vivo safety profile (Figure 5c). Finally, we examined the routine blood parameters of each group of mice, and the results are shown in Figure 6a–i. There was no statistical difference (*p* > 0.05) between the experimental and control groups of mice at 7 and 14 days. groups of the hydrogel did not show a statistically significant difference in liver and kidney function (*p* > 0.05), which could further confirm the safety of the hydrogel. Next, we evaluated H&E staining sections of the major organs after 7 and 14 days of feeding, which showed no significant cellular hypertrophy, atrophy, or necrosis in any of the organ tissue sections. These results further demonstrated that the encapsulated Fe–Zr@PDA@CMCS hydrogel had no significant side effects on the normal physiological function of the major organs and had a good in vivo safety profile (Figure 5c). Finally, we examined the routine blood parameters of each group of mice, and the results are shown in Figure 6a–i. There was no statistical difference (*p* > 0.05) between the experimental and control groups of mice at 7 and 14 days.

of the mice. The body weight of mice in both the experimental and control groups increased normally throughout the feeding cycle, and the body weight of mice in the experimental group fluctuated slightly for 21 days after encapsulation in the hydrogel; however, there was no statistical difference compared to the control group (*p* > 0.05) (Figure 5a). Further, we measured the blood biochemical parameters of the experimental and control mice on day 7 and 14 of the hydrogel implantation to assess whether the liver and kidney functions were affected. The serum biochemical parameters measured included TB (total bilirubin), ALT (alanine aminotransferase), AST (aspartate aminotransferase), UREA (urea), and CREA (creatinine). As shown in Figure 5b, the experimental and control

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**Figure 5.** (**a**) Changes in body weight of Fe–Zr@PDA@CMCS hydrogel treated mice and normal mice (control) at day 28; (**b**) results of serum biochemical parameters of Fe–Zr@PDA@CMCS hydrogel treated mice and normal mice (control); (**c**) H&E staining of the main organs of the Fe– Zr@PDA@CMCS hydrogel treated mice and normal mice (control). Bar = 200 μm. **Figure 5.** (**a**) Changes in body weight of Fe–Zr@PDA@CMCS hydrogel treated mice and normal mice (control) at day 28; (**b**) results of serum biochemical parameters of Fe–Zr@PDA@CMCS hydrogel treated mice and normal mice (control); (**c**) H&E staining of the main organs of the Fe–Zr@PDA@CMCS hydrogel treated mice and normal mice (control). Bar = 200 µm.

**Figure 6.** Routine blood test data of Fe–Zr@PDA@CMCS hydrogel-treated mice and normal mice (control): (**a**) white blood cell (WBC), (**b**) red blood cell (RBC), (**c**) hemoglobin (HB), (**d**) red blood cell-specific volume (HCT), (e) mean corpuscular volume (MCV), (**f**) mean corpuscular hemoglobin (MCH), (**g**) mean corpuscular hemoglobin concentration (MCHC), (**h**) red cell volume distribution width (RDW), and (**i**) PLATELET (PLT). **Figure 6.** Routine blood test data of Fe–Zr@PDA@CMCS hydrogel-treated mice and normal mice (control): (**a**) white blood cell (WBC), (**b**) red blood cell (RBC), (**c**) hemoglobin (HB), (**d**) red blood cell-specific volume (HCT), (**e**) mean corpuscular volume (MCV), (**f**) mean corpuscular hemoglobin (MCH), (**g**) mean corpuscular hemoglobin concentration (MCHC), (**h**) red cell volume distribution width (RDW), and (**i**) PLATELET (PLT).

### *2.8. In Vitro Anticancer Potential of Fe–Zr@PDA@CMCS Hydrogel 2.8. In Vitro Anticancer Potential of Fe–Zr@PDA@CMCS Hydrogel*

To investigate their potential effectiveness in antitumor therapy, we selected SW1990 cells for in vitro evaluation of tumor therapy. The CCK-8 results (Figure 7a) showed that there was a decrease in the survival rate of SW1990 cells in the Fe–Zr@PDA nanoparticles group, proving that Fe–Zr@PDA caused some damage to the normal growth of cancer cells. The Fe–Zr@PDA@CMCS hydrogel extract group also showed a decrease in cell survival rate, suggesting that CMCS-doped functionalized hydrogels also have some tumorcell-killing effect. In contrast, the survival rate of SW1990 cells receiving photothermal treatment alone was significantly lower, with almost half of the cells being inactive. Most importantly, the Fe–Zr@PDA@CMCS hydrogel extract combined with photothermal treatment showed an even more pronounced reduction in cell survival, indicating that the synergistic Fe–Zr@PDA@CMCS hydrogel and photothermal treatment can kill even more tumor cells. Afterwards, we further validated the in vitro anticancer effects by live–dead cell staining. The red fluorescence of Fe–Zr@PDA nanoparticles, Fe–Zr@PDA@CMCS hydrogel extracts treated group and photothermal treatment group all showed a significant increase compared to the control group (Figure 7b–e). Notably, there was a significant increase in red fluorescence and a significant decrease in green fluorescence after combining Fe–Zr@PDA@CMCS hydrogel with photothermal treatment, indicating that most of the SW1990 tumor cells were killed (Figure 7f). In conclusion, the combination of multifunctional hydrogel and photothermal therapy can effectively kill tumor cells, which is also expected to further enable tumor treatment at the in vivo level again. To investigate their potential effectiveness in antitumor therapy, we selected SW1990 cells for in vitro evaluation of tumor therapy. The CCK-8 results (Figure 7a) showed that there was a decrease in the survival rate of SW1990 cells in the Fe–Zr@PDA nanoparticles group, proving that Fe–Zr@PDA caused some damage to the normal growth of cancer cells. The Fe–Zr@PDA@CMCS hydrogel extract group also showed a decrease in cell survival rate, suggesting that CMCS-doped functionalized hydrogels also have some tumor-cell-killing effect. In contrast, the survival rate of SW1990 cells receiving photothermal treatment alone was significantly lower, with almost half of the cells being inactive. Most importantly, the Fe–Zr@PDA@CMCS hydrogel extract combined with photothermal treatment showed an even more pronounced reduction in cell survival, indicating that the synergistic Fe–Zr@PDA@CMCS hydrogel and photothermal treatment can kill even more tumor cells. Afterwards, we further validated the in vitro anticancer effects by live–dead cell staining. The red fluorescence of Fe–Zr@PDA nanoparticles, Fe–Zr@PDA@CMCS hydrogel extracts treated group and photothermal treatment group all showed a significant increase compared to the control group (Figure 7b–e). Notably, there was a significant increase in red fluorescence and a significant decrease in green fluorescence after combining Fe–Zr@PDA@CMCS hydrogel with photothermal treatment, indicating that most of the SW1990 tumor cells were killed (Figure 7f). In conclusion, the combination of multifunctional hydrogel and photothermal therapy can effectively kill tumor cells, which is also expected to further enable tumor treatment at the in vivo level again.

**Figure 7.** (**a**) CCK-8 results of cells after different treatments; (**b**–**f**) results of live and dead cell staining of SW1990 cells, morphological staining images of cells corresponding to CCK-8 results (**b**): L929 cells co-incubated with fresh medium; (**c**): L929 cells co-incubated with 100 μg/mL Fe–Zr@PDA nanoparticles; (**d**): L929 cells co-incubated with 100 μg/mL Fe–Zr@PDA@CMCS hydrogel extract; (**e**): L929 cells irradiated by 808 nm NIR, 1 W/cm<sup>2</sup> , 5 min pure photothermal irradiation; (**f**): fluorescent staining images of L929 cells after treatment with 100 μg/ mL Fe–Zr@PDA@CMCS hydrogel extract and 808 nm NIR, 1 W/cm<sup>2</sup> , by 5 min of light. Bar = 100 μm, \*\*\*\* *p* < 0.0001. **Figure 7.** (**a**) CCK-8 results of cells after different treatments; (**b**–**f**) results of live and dead cell staining of SW1990 cells, morphological staining images of cells corresponding to CCK-8 results (**b**): L929 cells co-incubated with fresh medium; (**c**): L929 cells co-incubated with 100 µg/mL Fe–Zr@PDA nanoparticles; (**d**): L929 cells co-incubated with 100 µg/mL Fe–Zr@PDA@CMCS hydrogel extract; (**e**): L929 cells irradiated by 808 nm NIR, 1 W/cm<sup>2</sup> , 5 min pure photothermal irradiation; (**f**): fluorescent staining images of L929 cells after treatment with 100 µg/ mL Fe–Zr@PDA@CMCS hydrogel extract and 808 nm NIR, 1 W/cm<sup>2</sup> , by 5 min of light. Bar = 100 µm, \*\*\*\* *p* < 0.0001.

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

In summary, we successfully designed and prepared multifunctional Fe– Zr@PDA@CMCS hydrogel with the Fenton effect and photothermal conversion properties using Fe, Zr, PDA, and CMCS. Fe–Zr@PDA@CMCS hydrogel combined the advantages of hydrogel and nanoparticles. The photothermal conversion, degradation, and swelling capabilities of the hydrogel and Fenton's catalytic ability under different conditions were investigated. The results showed that the composite hydrogel retains its photothermal and Fentonian catalytic properties while protecting Fe–Zr@PDA nanoparticles from degradation. In addition, the good biocompatibility of the hydrogel was demonstrated at the cellular and animal levels. Further results demonstrated good therapeutic effects at the cellular level, and will be validated at the animal level in the future. Unfortunately, there are limitations to our study and we need further validation at the animal level and clinical In summary, we successfully designed and prepared multifunctional Fe–Zr@PDA@CMCS hydrogel with the Fenton effect and photothermal conversion properties using Fe, Zr, PDA, and CMCS. Fe–Zr@PDA@CMCS hydrogel combined the advantages of hydrogel and nanoparticles. The photothermal conversion, degradation, and swelling capabilities of the hydrogel and Fenton's catalytic ability under different conditions were investigated. The results showed that the composite hydrogel retains its photothermal and Fentonian catalytic properties while protecting Fe–Zr@PDA nanoparticles from degradation. In addition, the good biocompatibility of the hydrogel was demonstrated at the cellular and animal levels. Further results demonstrated good therapeutic effects at the cellular level, and will be validated at the animal level in the future. Unfortunately, there are limitations to our study and we need further

validation at the animal level and clinical level. In summary, multifunctional composite hydrogels can be used as carriers of drugs for multimodal tumor therapy and tissue regeneration for biomedical applications. Based on the good degradability and therapeutic effects of functionalized hydrogels, multifunctional composite hydrogels could provide novel ideas and show great promise for future clinical oncology treatments. The study is also expected to provide a reference for future clinical studies of novel hydrogels against tumor recurrence.

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

### *4.1. Materials*

All chemicals were used without further purification. Zirconium tetrachloride (ZrCl4), ferric chloride hexahydrate (FeCl3-6H2O), CMCS, PDA, ethylene glycol (EG), EDC, and NHS were purchased from Shanghai Aladdin Reagent Co., Ltd. (Shanghai, China). Mouse fibroblast cells (L929) and human pancreatic cancer cell lines (SW1990) were bought from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Dulbecco's Modified Eagle Medium (DMEM), citrate buffer solution (CBS), and phosphate buffer saline (PBS) were procured from Gibco Co., Ltd. (Shanghai, China). The Cell counting kit-8 (CCK-8) was purchased from Vazyme Biotechnology Co., Ltd. (Nanjing, China). Calcein-AM/PI Live/Dead kit was purchased from Solarbio Technology (Beijing, China) Co., Ltd. Kunming (KM) mice (female, 4–6 weeks, 20–25 g) and Sprague-Dawley (SD) rats were ordered from Shanghai Slac Laboratory Animal Center (Shanghai, China). All experimental mice were fed strictly following the rules and regulations of the Ministry of Health of the People's Republic of China.
