**1. Introduction**

The effective treatment of tumors remains a challenge for the biomedical field today [1]. Traditional clinical treatments include surgery [2], chemotherapy [3], radiotherapy [4,5], immunotherapy [6,7], etc. However, all these methods have certain limitations such as the risk of metastasis or infection due to the surgical trauma [8], the lack of targeting of some chemotherapeutic drugs to the lesion, and the killing of normal tissue cells [9,10]. These shortcomings have limited the prospects for their use in oncology treatment. Therefore, there is a pressing want to develop more effective therapeutic strategies to tackle the difficulties in tumor therapy.

In recent years, studies have proven that various kinds of tumor cells show off multiplied ranges of reactive oxygen species (ROS) and altered redox status due to genetic, metabolic, and microenvironmental alterations [11–14]. Stimulated by high ROS levels, oncogenes can induce the activation of various downstream signaling pathways to adapt

**Citation:** Zheng, X.; Wu, H.; Wang, S.; Zhao, J.; Hu, L. Preparation and Characterization of Biocompatible Iron/Zirconium/Polydopamine /Carboxymethyl Chitosan Hydrogel with Fenton Catalytic Properties and Photothermal Efficacy. *Gels* **2023**, *9*, 452. https://doi.org/10.3390/ gels9060452

Academic Editor: Avinash J. Patil

Received: 6 May 2023 Revised: 25 May 2023 Accepted: 29 May 2023 Published: 31 May 2023

**Copyright:** © 2023 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/).

to the oxidative stress environment [15–17]. This further leads to cell immortalization and contributes to the necessary conditions for the cells to reach a malignant growth state [18,19]. As a result, ROS-based antitumor strategies have gained widespread attention. Chemodynamic therapies (CDT) is a novel strategy for cancer treatment that involves multiple transition metal ions, such as iron (Fe), silver (Ag), molybdenum (Mo), ruthenium (Ru), cerium (Ce), and zinc (Zn) [20,21]. These metal ions can react with the endogenous hydrogen peroxide (H2O2) in the tumor microenvironment (TME) and produce a large number of hydroxyl radicals (•OH) to kill tumor cells [22–25]. Inspired by this promising therapeutic paradigm, iron-based nanoplatforms have been frequently studied for the treatment of tumors. Specifically, due to the anaerobic conditions at the tumor site, anaerobic enzymolysis and proton pumping at the cell membrane result in an acidic environment in the TME, and some TMEs exhibit high H2O<sup>2</sup> expression [26]. CDT has several advantages over other therapies in that it is tumor-targeted, has few side effects on normal tissues, and can selectively kill tumor cells without relying on exogenous stimuli [23].

Photothermal therapy (PTT) has promising applications in the treatment of tumors. PTT refers to the use of photothermal agents with photothermal conversion efficiency and converts the near-infrared laser into heat [27–29]. This warmness can enlarge tumor temperature, cause irreversible injury to tumor cell membranes, and set off protein denaturation, leading to irreversible cell harm and subsequent tumor regression [30,31]. Currently, the main conjugated polymers (CPs) available for tumor PTT include polydopamine (PDA), polypyrrole, and polyaniline (PAI) [32–35]. Among them, PDA is a novel polymer assembled from dopamine monomers through oxidative autopolymerization [36,37]. It has been broadly used in tumor-targeted drug delivery systems due to its advantages such as good biocompatibility, excellent photothermal conversion properties, and multiple drug release response possibilities [36,38,39].

Hydrogels are three-dimensional porous structures produced from hydrophilic polymers by physical or chemical cross-linking methods [40]. Hydrogels can effectively encapsulate molecules and control their concentration at the lesion site [41]. In addition, hydrogels can respond to physical, biological, chemical, or external stimuli [42,43]. In this study, Fe ions are used as the classical Fenton agents [44,45], which can react with H2O<sup>2</sup> at the tumor site to produce highly reactive •OH for tumor cells killing. Moreover, Zr ions that can synergize with Fe ions were introduced [46]. As a ligand for metal ions, PDA was employed to form noncovalent bonds with the nanoparticle surfaces and modulate the photothermal properties [47]. Then, the EDC/NHS activated carboxymethyl chitosan (CMCS) is used to combine with Fe–Zr@PDA nanoparticles to form a drug reservoir. Together with the CMCS hydrogel drug reservoir, this nanoplatform can be locally implanted to the target site to react with the H2O2, transform the laser to heat, and release the drug. Specifically, we introduced PDA with Fe–zirconium (Zr) to impart unique photothermal properties to the nanomedicine. In brief, Fe–Zr@PDA nanoparticles were mixed with carboxymethyl chitosan (CMCS) activated by 1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide (EDC)/N-hydroxysuccinimide (NHS) to form a hydrogel.

In this work, an Fe-ions-mediated Fenton reaction was combined with the synergistic effect of Zr ions and the photothermal effect of PDA. Together with the CMCS hydrogel drug reservoir, this nanoplatform can be locally implanted to the target site to react with the H2O2, transform the laser to heat, and release the drug. Therefore, the composite hydrogel is expected to improve the efficacy of oncology treatments via preventing the postoperative tumor recurrence and increase patient compliance in future potential clinical applications. In summary, our aim is to combine the classical Fenton effect of Fe-based nanomaterials, the synergistic effect of Zr, the photothermal properties of PDA, and the bicompatible advantages of hydrogels to form a composite hydrogel. The composite system provides a new idea in broadening the application potential of hydrogels. While combining the Fenton effect and photothermal efficacy, the functionalized hydrogel can not only function as a tumor suppressor, but also plays an important role in preventing the recurrence of residual tumor tissue after local surgical excision (Scheme 1). The novelty of our work

lies in the use of the classical Fenton reaction, combined with metal ion properties, to complement the hydrogel to form a composite multifunctional hydrogel, circumventing the disadvantages of applying nanoparticles alone and amplifying their advantages to provide a novel strategy in the field of tumor killing and prevention of local tumor recurrence. venting the disadvantages of applying nanoparticles alone and amplifying their advantages to provide a novel strategy in the field of tumor killing and prevention of local tumor recurrence.

bicompatible advantages of hydrogels to form a composite hydrogel. The composite system provides a new idea in broadening the application potential of hydrogels. While combining the Fenton effect and photothermal efficacy, the functionalized hydrogel can not only function as a tumor suppressor, but also plays an important role in preventing the recurrence of residual tumor tissue after local surgical excision (Scheme 1). The novelty of our work lies in the use of the classical Fenton reaction, combined with metal ion properties, to complement the hydrogel to form a composite multifunctional hydrogel, circum-

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**Scheme 1.** A practical and working schematic of a multifunctional hydrogel that can be injected for future use in killing tumors and inhibiting their postoperative recurrence (CDT: chemodynamic therapies; PTT: photothermal therapy). **Scheme 1.** A practical and working schematic of a multifunctional hydrogel that can be injected for future use in killing tumors and inhibiting their postoperative recurrence (CDT: chemodynamic therapies; PTT: photothermal therapy).

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

### **2. Results and Discussion** *2.1. Preparation and Characterization of Fe–Zr@PDA and Fe–Zr@PDA@CMCS Hydrogel*

*2.1. Preparation and Characterization of Fe–Zr@PDA and Fe–Zr@PDA@CMCS Hydrogel* To examine the microstructural morphology, the nanoparticle was investigated with a scanning electron microscope (SEM, Zeiss Sigma 300, Oberkochen, Germany). Fe– Zr@PDA was synthesized by a hydrothermal one-pot method. The Fe–Zr@PDA NPs exhibited a homogeneous spherical morphology with a particle size of approximately 42.4 ± 9.2 nm (Figure 1a). The hydrodynamic diameters of NPs dispersed in different media were measured using DLS. As shown in Figure 1b, the mean hydrodynamic diameters of Fe– Zr@PDA in water was 164 nm. The results of DLS also revealed that Fe–Zr@PDA tended to undergo certain aggregation. More importantly, the hydrodynamic diameters of Fe– Zr@PDA hardly changed within 24 h. To verify the existence of iron, Fe–Zr@PDA was mixed with the o-phenanthroline solution at room temperature. It was observed that the supernatant changed swiftly from colorless to orange–red and showed the characteristic absorption at 510 nm. However, there was no detectable color change when cultured without Fe–Zr@PDA (Figure 1c). These results suggest the presence of Fe in the composite. Fe– Zr@PDA@CMCS hydrogel was prepared by mixing the Fe–Zr@PDA with the EDC/NHSactivated CMCS. SEM analysis showed that the CMCS hydrogel showed a smooth surface (Figure 1d). Compared to the unactivated CMCS [48], a new peak at 1650 cm−1 assigned to the -C=N was observed in the FT-IR spectrum of CMCS hydrogel (Figure 1e). These results suggest that CMCS forms amide bonds through the activation of EDC/NHS, which facilitated the formation of hydrogels. A digital camera was used to record the changes of the To examine the microstructural morphology, the nanoparticle was investigated with a scanning electron microscope (SEM, Zeiss Sigma 300, Oberkochen, Germany). Fe–Zr@PDA was synthesized by a hydrothermal one-pot method. The Fe–Zr@PDA NPs exhibited a homogeneous spherical morphology with a particle size of approximately 42.4 ± 9.2 nm (Figure 1a). The hydrodynamic diameters of NPs dispersed in different media were measured using DLS. As shown in Figure 1b, the mean hydrodynamic diameters of Fe–Zr@PDA in water was 164 nm. The results of DLS also revealed that Fe–Zr@PDA tended to undergo certain aggregation. More importantly, the hydrodynamic diameters of Fe–Zr@PDA hardly changed within 24 h. To verify the existence of iron, Fe–Zr@PDA was mixed with the o-phenanthroline solution at room temperature. It was observed that the supernatant changed swiftly from colorless to orange–red and showed the characteristic absorption at 510 nm. However, there was no detectable color change when cultured without Fe–Zr@PDA (Figure 1c). These results suggest the presence of Fe in the composite. Fe–Zr@PDA@CMCS hydrogel was prepared by mixing the Fe–Zr@PDA with the EDC/NHS-activated CMCS. SEM analysis showed that the CMCS hydrogel showed a smooth surface (Figure 1d). Compared to the unactivated CMCS [48], a new peak at 1650 cm−<sup>1</sup> assigned to the -C=N was observed in the FT-IR spectrum of CMCS hydrogel (Figure 1e). These results suggest that CMCS forms amide bonds through the activation of EDC/NHS, which facilitated the formation of hydrogels. A digital camera was used to record the changes of the black solution (Figure 1f, g), indicating the solution state and solid state of the hydrogel. The macroscopic and microscopic appearance of hydrogel was then recorded, which uncovered that the hydrogel is large and in macro scale. PDA is capable of generating thin film coatings on the surface of multiple materials. As shown in Figure 1d, h, CMCS hydrogel showed a smooth surface, while the Fe–Zr@PDA@CMCS hydrogel showed some loose porous pores.

black solution (Figure 1f, g), indicating the solution state and solid state of the hydrogel.

This may be attributed to the formation of hydrogen bonds between PDA and CMCS. The element distribution diagram clearly showed that Zr were evenly distributed in the hydrogel, and there were no excess impurity elements in the hydrogel (Figure 1i). PDA and CMCS. The element distribution diagram clearly showed that Zr were evenly distributed in the hydrogel, and there were no excess impurity elements in the hydrogel (Figure 1i).

The macroscopic and microscopic appearance of hydrogel was then recorded, which uncovered that the hydrogel is large and in macro scale. PDA is capable of generating thin film coatings on the surface of multiple materials. As shown in Figure 1d, h, CMCS hydrogel showed a smooth surface, while the Fe–Zr@PDA@CMCS hydrogel showed some loose porous pores. This may be attributed to the formation of hydrogen bonds between

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**Figure 1.** (**a**) SEM image of Fe–Zr@PDA nanoparticle; (**b**) the hydration kinetic diameter of Fe– Zr@PDA in H2O; (**c**) the light absorption of phenanthroline solution with and without co-incubation with Fe–Zr@PDA; (**d**) SEM image of CMCS hydrogel; (**e**) FTIR spectra of CMCS hydrogel; (**f**) flowing solution state of Fe–Zr@PDA@CMCS hydrogel; (**g**) solidified state of Fe–Zr@PDA@CMCS hydrogel; (**h**) SEM image of Fe–Zr@PDA@CMCS hydrogel; (**i**) elemental distribution of Zr in Fe– Zr@PDA@CMCS hydrogel. **Figure 1.** (**a**) SEM image of Fe–Zr@PDA nanoparticle; (**b**) the hydration kinetic diameter of Fe–Zr@PDA in H2O; (**c**) the light absorption of phenanthroline solution with and without coincubation with Fe–Zr@PDA; (**d**) SEM image of CMCS hydrogel; (**e**) FTIR spectra of CMCS hydrogel; (**f**) flowing solution state of Fe–Zr@PDA@CMCS hydrogel; (**g**) solidified state of Fe–Zr@PDA@CMCS hydrogel; (**h**) SEM image of Fe–Zr@PDA@CMCS hydrogel; (**i**) elemental distribution of Zr in Fe–Zr@PDA@CMCS hydrogel.

### *2.2. Photothermal Conversion Evaluation of Fe–Zr@PDA@CMCS Hydrogel 2.2. Photothermal Conversion Evaluation of Fe–Zr@PDA@CMCS Hydrogel*

In recent years, great efforts have been made in developing photothermal agents for the therapy of various types of tumors [28]. PDA has good photothermal properties, can enhance the biocompatibility of nanomaterials, improve hydrophilicity, reduce cytotoxicity, and its many other advantages are widely used in the field of nanomaterials research [49,50]. The doping of PDA gives Fe–Zr@PDA@CMCS hydrogel the photothermal conversion properties to convert the absorbed NIR laser mild into heat for action. In this study, the temperatures of the hydrogel were recorded for the different groups of treatments to determine the overall photothermal performance of the hydrogels (Figure 2a–d). As shown in Figure 2a, as the concentration of Fe–Zr@PDA in the hydrogel increased (from 2 mg/mL to 8 mg/mL), and the ΔT gradually increased, with ΔT of 24.5 °C, 30 °C, and 42.1 °C, respectively, while the temperature of the control group only increased by 4.2 °C. The corresponding thermographs, likewise, illustrated that the concentration of In recent years, great efforts have been made in developing photothermal agents for the therapy of various types of tumors [28]. PDA has good photothermal properties, can enhance the biocompatibility of nanomaterials, improve hydrophilicity, reduce cytotoxicity, and its many other advantages are widely used in the field of nanomaterials research [49,50]. The doping of PDA gives Fe–Zr@PDA@CMCS hydrogel the photothermal conversion properties to convert the absorbed NIR laser mild into heat for action. In this study, the temperatures of the hydrogel were recorded for the different groups of treatments to determine the overall photothermal performance of the hydrogels (Figure 2a–d). As shown in Figure 2a, as the concentration of Fe–Zr@PDA in the hydrogel increased (from 2 mg/mL to 8 mg/mL), and the ∆T gradually increased, with ∆T of 24.5 ◦C, 30 ◦C, and 42.1 ◦C, respectively, while the temperature of the control group only increased by 4.2 ◦C. The corresponding thermographs, likewise, illustrated that the concentration of PDA is closely related to the photothermal effect (Figure 2b). Further, the hydrogel was irradiated with an 808 nm NIR laser of exceptional energy densities (the specific power densities of the

laser are 0.5 W/cm<sup>2</sup> , 0.8 W/cm<sup>2</sup> , and 1.0 W/cm<sup>2</sup> ) for 5 min and the ∆T of the hydrogel was measured. The results revealed that the higher the power density of the NIR laser was, the higher the ∆T was (Figure 2c). This suggests that the power density of the NIR laser is another factor affecting its photothermal performance. The thermographic images provided further evidence of the power-density-related photothermal performance (Figure 2d). In addition, Fe–Zr@PDA@CMCS hydrogel has good photothermal stability. After being irradiated by the laser for up to 120 min (six NIR irradiated cycles), the hydrogel showed a stable temperature rise trend, and the maximum ∆T was still greater than 50 ◦C (Figure 2e). Based on linear regression analysis, we calculated that the τs of Fe–Zr@PDA@CMCS hydrogel was 209.22 s (Figure 2f), and the η was 35.68% (Figure 2g), which was higher than the Au@Bi<sup>2</sup> Se<sup>3</sup> core–shell nanoparticle [42]. hydrogel was measured. The results revealed that the higher the power density of the NIR laser was, the higher the ΔT was (Figure 2c). This suggests that the power density of the NIR laser is another factor affecting its photothermal performance. The thermographic images provided further evidence of the power-density-related photothermal performance (Figure 2d). In addition, Fe–Zr@PDA@CMCS hydrogel has good photothermal stability. After being irradiated by the laser for up to 120 min (six NIR irradiated cycles), the hydrogel showed a stable temperature rise trend, and the maximum ΔT was still greater than 50 °C (Figure 2e). Based on linear regression analysis, we calculated that the τs of Fe– Zr@PDA@CMCS hydrogel was 209.22 s (Figure 2f), and the η was 35.68% (Figure 2g), which was higher than the Au@Bi<sup>2</sup> Se<sup>3</sup> core–shell nanoparticle [42].

PDA is closely related to the photothermal effect (Figure 2b). Further, the hydrogel was irradiated with an 808 nm NIR laser of exceptional energy densities (the specific power

, and 1.0 W/cm<sup>2</sup>

) for 5 min and the ΔT of the

, 0.8 W/cm<sup>2</sup>

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densities of the laser are 0.5 W/cm<sup>2</sup>

**Figure 2.** (**a**) Temperature variation of Fe–Zr@PDA@CMCS hydrogel with different Fe–Zr@PDA doping levels under 808 nm laser irradiation; (**b**) infrared thermographs correspond to (**a**), colors indicate different temperatures; (**c**) the temperature profiles of Fe–Zr@PDA@CMCS hydrogel irradiated with NIR laser with different power densities of 0.5, 0.8, and 1.0 W/cm<sup>2</sup> ; (**d**) infrared thermographs correspond to (**c**), colors indicate different temperatures; (**e**) results from photothermal cycling tests results of Fe–Zr@PDA@CMCS hydrogel after 8 irradiation and cooling cycles; (**f**) time constant curves for Fe–Zr@PDA@CMCS hydrogel (808 nm, 1.0 W/cm<sup>2</sup> ); (**g**) in vitro η values for Fe– Zr@PDA@CMCS hydrogel. **Figure 2.** (**a**) Temperature variation of Fe–Zr@PDA@CMCS hydrogel with different Fe–Zr@PDA doping levels under 808 nm laser irradiation; (**b**) infrared thermographs correspond to (**a**), colors indicate different temperatures; (**c**) the temperature profiles of Fe–Zr@PDA@CMCS hydrogel irradiated with NIR laser with different power densities of 0.5, 0.8, and 1.0 W/cm<sup>2</sup> ; (**d**) infrared thermographs correspond to (**c**), colors indicate different temperatures; (**e**) results from photothermal cycling tests results of Fe–Zr@PDA@CMCS hydrogel after 8 irradiation and cooling cycles; (**f**) time constant curves for Fe–Zr@PDA@CMCS hydrogel (808 nm, 1.0 W/cm<sup>2</sup> ); (**g**) in vitro η values for Fe–Zr@PDA@CMCS hydrogel.
