*4.6.* •*OH Generating Capacity*

Firstly, we explored the ability of different concentrations of Fe–Zr@PDA to produce •OH. Specifically, a 3,3<sup>0</sup> ,5,50 -Tetramethylbenzidine (TMB) solution with a concentration of 3.2 mM was prepared using ultrapure water and 300 µL was mixed with different concentrations of Fe–Zr@PDA and H2O2. The total volume of the mixed solution was 1.5 mL, with the final concentration of H2O<sup>2</sup> set at 8 mM and the final concentrations of Fe–Zr@PDA set at 0 µg/mL, 20 µg/mL, 40 µg/mL, 60 µg/mL, 80 µg/mL, and 100 µg/mL. The reaction lasted for 15 min at room temperature accompanied by ultra-high-speed centrifugation (15,000 rpm, 10 min). After the reaction, the supernatant was collected and photographed with a digital camera. At the same time, the absorbance of each sample supernatant at the wavelength of 652 nm was measured by an ultraviolet spectrometer.

The •OH formation ability of Fe–Zr@PDA@CMCS hydrogel was also determined by the color development properties of TMB after oxidation. Fe–Zr@PDA@CMCS hydrogel with different concentrations (1, 2, or 4 mg/mL) was mixed with TMB (mM) and H2O<sup>2</sup> (0.3%). The group of TMB and H2O<sup>2</sup> without Fe–Zr@PDA@CMCS hydrogel was set as the control. After being co-cultured at room temperature for 1 h, the supernatant was accumulated and photographed with a digital camera. The absorption value of the supernatant at λ = 652 nm was measured by UV–Vis–NIR spectroscopy (Lambda 25, Perkin Elmer, Waltham, MA, USA).

### *4.7. Degradation Analysis of Fe–Zr@PDA@CMCS Hydrogel*

To explore the in vitro degradation ability, lyophilized Fe–Zr@PDA@CMCS hydrogel (100 mg) was immersed in 5 mL of PBS (pH = 7.4) or CBS (pH = 5.4). Then, the Fe–Zr@PDA@CMCS hydrogel samples were removed at predetermined time points (1, 3, 7, 14, and 28 days), washed with ultrapure water, and finally lyophilized and weighed in a lyophilizer. The degradation percentage of Fe–Zr@PDA@CMCS hydrogel was once calculated by way of the following formula:

$$\text{Weight remaining ratio} \left(\% \right) = \frac{R\_l}{R\_0} \times 100\% \tag{2}$$

where *R<sup>t</sup>* represents the actual gram weight of Fe–Zr@PDA@CMCS hydrogel and *R*<sup>0</sup> represents the original weight of Fe–Zr@PDA@CMCS hydrogel.

### *4.8. Swelling Analysis of Hydrogel*

The swelling rate (SR) and stability of Fe–Zr@PDA@CMCS hydrogel in different solutions (H2O, PBS, and CBS) were determined by the swelling test. In short, 10 mg of Fe–Zr@PDA@CMCS hydrogel was placed into a sealed centrifuge tube (*n* = 3) and added to 20 mL of H2O, PBS, and CBS respectively. The tubes were placed at 37 ◦C. The hydrogel was removed from the solution at predetermined time points (0.5, 1, 2, 12, and 24 h), and the surface water of the hydrogel was absorbed by filter paper and weighed again. The swelling kinetics curve of Fe–Zr@PDA@CMCS hydrogel was drawn, and the SR of the hydrogel reached swelling equilibrium was calculated according to the following formula:

$$\text{Swelling ratio } (\%) = \frac{m\_t - m\_0}{m\_0} \times 100\% \tag{3}$$

where *m<sup>t</sup>* is the mass of hydrogel after swelling at different points in time and *m*<sup>0</sup> represents the initial condition of this hydrogel.

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

The cell safety of Fe–Zr@PDA@CMCS was evaluated using mouse fibroblasts (L929 cells). First, the L929 cells were inoculated on 96-well clear cell culture plates (5000 cells per well) and cultured for 24 h in a constant temperature incubator with 37 ◦C and 5% CO2. At the same time, Fe–Zr@PDA@CMCS hydrogel was positioned in DMEM cell medium (1, 2.5, or 5 mg hydrogel, in 1 mL DMEM medium) and incubated at 37 ◦C in a single day to attain the extract with the attention of 5 mg/mL. On the second day, the old medium was replaced with 100 µL of the leaching solution, and the control group was added with a fresh DMEM medium. The extract concentration in the leaching solution was 1 mg/mL, 2.5 mg/mL, or 5 mg/mL. Finally, the medium was removed at 24 h or 48 h culture, and the cells were washed with PBS twice. Cell viability was determined by the CCK-8 kit. In addition, staining was performed using an AM-PI staining kit for both living and dead cells, and staining images were collected using an inverted phase contrast microscope (Leica DM IL, Weztlar, Germany).

### *4.10. In Vitro Blood Compatibility of Fe–Zr@PDA@CMCS Hydrogel*

The feeding and testing of animals were conducted at Changhai Hospital of Naval Medical University in strict accordance with the program and policies of the Ministry of Health. To test whether Fe–Zr@PDA@CMCS can cause rupture of red blood cells and hemolysis, the blood compatibility of these cells was evaluated by using rat red blood cells. Firstly, blood was collected from the anesthetized heart of SD rats into anticoagulant collection vessels and centrifuged (4000 rpm, 5 min) to collect the red blood cells. Finally, the purified rat red blood cells were diluted to 2% with PBS and stored in the refrigerator at 4 ◦C for subsequent use. For the hemolysis experiment, we first mixed previously stored diluted erythrocyte suspensions with PBS buffer containing 20, 40, 100, and 200 mg of Fe–Zr@PDA@CMCS hydrogel, respectively. In addition, a negative control group (specifically, 1 mL of diluted PBS buffer mixed with 1 mL blood cells) and a positive control group (that is, 1 mL of diluted red cell suspension mixed with 1 mL ultrapure water) were set up, respectively. Finally, the above-mixed solutions were incubated in a water bath at 37 ◦C for 2 h and then centrifuged immediately to obtain the supernatant (3000 rpm, 5 min). The absorbance at 541 nm was measured with the UV–visible–near-infrared spectrometer (Lambda 25, Perkin Elmer, Waltham, MA, USA) of all the supernatants collected. Then, the hemolysis percentage (HP) was calculated according to the following formula, and the supernatant was photographed with a camera.

$$\text{Hemployment ratio}(\%) = \frac{B\_{sample} - B\_{negative}}{B\_{positive} - B\_{negative}} \times 100\% \tag{4}$$

where *Bsample* is the absorbance at 541 nm of red blood cell suspension after treatment with hydrogel extract, *Bnegative* is the absorbance of erythrocyte suspension dealt with PBS buffer, and *Bpositive* is the absorbance of blood cell suspension after treatment with ultrapure water.

### *4.11. In Vivo Animal Tissue Safety of Fe–Zr@PDA@CMCS Hydrogel*

To explore the safety of Fe–Zr@PDA@CMCS hydrogel in animals, KM mice were used as the animal model. Fe–Zr@PDA@CMCS hydrogel was embedded under the skin of mice to observe whether it would cause damage to mice. Specifically, KM mice (from the Laboratory in Shanghai Changhai Hospital, China) were randomly divided into control and experimental groups with three animals in each group (*n* = 3). Mice anesthetized with pentobarbital sodium were subcutaneously implanted with 200 mg of Fe–Zr@PDA@CMCS hydrogel in the experimental group. Normal healthful mice were used in the control group. The mice were weighed every 2 days after embedding to investigate the adjustments in body weight. The mice were sacrificed on the 7th, 14th, and 28th days, respectively. We collected blood by removing the eyes of mice, and major organs such as lungs, spleen, liver, heart, and kidney were collected and fixed in 4% paraformaldehyde for HE staining. Meanwhile, the collected blood was used to measure the values of the blood routine indexes of the two groups by the blood routine analyzer. An ELISA kit was used to detect the related indexes of kidney function and liver function in the serum. HE-stained sections of major organs were used to observe whether there were lesions such as inflammation and necrosis. All animal experimental operations were carried out in strict accordance with the protocols authorized by the hospital's Comprehensive Laboratory Animal Centre.

### *4.12. In Vitro Antitumor Effects of Fe–Zr@PDA@CMCS Hydrogels*

We selected the human pancreatic cancer cell line SW1990 as a model to assess the in vitro therapeutic effect of Fe–Zr@PDA@CMCS. SW1990 cells were firstly inoculated into the 96-well culture plate at a density of 9000 cells/well. After 24 h of incubation, the original medium was replaced with 100 µL of fresh DMEM solution containing different substances and subjected to various treatments. The treatments were as follows: (a) control group (fresh DMEM), (b) Fe–Zr@PDA group (100 µg/mL Fe–Zr@PDA), (c) Fe–Zr@PDA@CMCS group (100 µg/mL Fe–Zr@PDA hydrogel extract), (d) laser group (808 nm NIR, 1 W/cm<sup>2</sup> , 5 min), (e) Fe–Zr@PDA@CMCS + laser group (100 µg/mL Fe–Zr@PDA hydrogel extract, 808 nm NIR, 1 W/cm<sup>2</sup> , 5 min). The groups were treated as described above and incubation was continued for 12 h, after which the survival of SW1990 cells was assessed by the CCK-8 method and the live–dead cell assay.

### *4.13. Statistical Analysis*

All results are expressed as mean ± standard deviation and one-way ANOVA statistical analysis was used to assess the significance of the experimental data. The value of 0.05 was used as the significance level data, with probabilities less than 0.05 (*p* < 0.05), 0.01 (*p* < 0.01), and 0.001 (*p* < 0.01) indicated by (\*), (\*\*), and (\*\*\*), respectively. The sample size was 3 unless stated (*n* = 3).

**Author Contributions:** Conceptualization, L.H., J.Z. and S.W.; methodology, X.Z. and H.W.; software, J.Z.; validation, X.Z., H.W. and L.H.; formal analysis, X.Z., J.Z., S.W. and L.H.; investigation, S.W.; resources, L.H. and J.Z.; data curation, H.W. and X.Z.; writing—original draft preparation, X.Z. and H.W.; visualization, J.Z. and S.W.; writing—review and editing, supervision, project administration, and funding acquisition, X.Z., H.W., S.W., J.Z. and L.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** National Natural Science Foundation of China (82103696).

**Institutional Review Board Statement:** The animal study protocol was approved by the Institutional Review Board of The First Affiliated Hospital of Naval Medical University of the People's Liberation Army (SYXK (Shanghai) 2020-0033).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that support the findings of this study are available from the corresponding author upon reasonable request.

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