Ferrous chloride tetrahydrate (FeCl₂ 4H₂O), ferric chloride hexahydrate (FeCl₃ 6H₂O), sodium hydroxide (NaOH) and ALG were purchased from Shanghai Sinopharm Chemical Reagent Co., RPMI 1640 cell culture medium, fetal bovine serum (FBS), and penicillin-streptomycin were obtained from Gibco (Grand Island, NY, United States). Cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). Ultrapure water used in the experiments was prepared using a water purification system (PALL Cascada, MI, United States).

The UV-visible spectra of Fe₃O₄ nanoparticles were characterized by Persee spectrophotometer (TU-1810, Beijing, China). The surface morphologies of Fe₃O₄ hydrogels were observed using a scanning electron microscope (SEM, HITACHI, Japan). The hydrodynamic diameters and zeta potentials of Fe₃O₄ nanoparticles were measured using a Zetasizer Nano-series (Nano-ZS90, Malvern, United Kingdom). The Fe concentrations were measured by using an inductively coupled plasma atomic emission spectroscopy (ICP-AES) system (Hudson, NH, United States).

Fe₃O₄ nanoparticles were synthesized according to a previous report (Hu et al., 2015). In brief, 178 mg FeCl₂ 4H₂O and 314 mg FeCl₃ 6H₂O were dissolved in deionized (DI) water. Then 10 mL of NaOH solution (200 mg/mL) was added to the above solution. After mixing well, the solution was placed in a water bath at 80°C for 30 min or 2 h, respectively. The obtained samples were used to evaluate the influence of the reaction times on the properties of the nanoparticles. Subsequently, the mixed solution was placed on a magnetic stirrer to precipitate the synthesized Fe₃O₄ nanoparticles, and the upper liquid was discarded. Then Fe₃O₄ nanoparticles were dispersed in 10 mL water under sonication. The above steps were repeated at least 5 times to purify the Fe₃O₄ nanoparticles.

To prepare Fe₃O₄ hydrogels, Fe₃O₄ nanoparticles were mixed with ALG solution to obtain AF mixture solution and the solution was then injected into Ca²⁺ (1.8 mM) solution. AF solutions at different concentrations of ALG (0.5, 1, 2.5, 5, 10 μg/mL) were slowly injected into the Ca²⁺ solution (1.8 mM). Then photographs were taken at different times after injection of solutions.

To evaluate the photothermal properties of Fe₃O₄ nanoparticles and Fe₃O₄ hydrogels, 200 μL of Fe₃O₄ solution or Fe₃O₄ hydrogels at the Fe concentration of 200 μg/mL were placed in a 96-well plate. Then, 808 nm laser at different power densities (0.5, 1.0, and 1.5 W/cm²) was used to irradiate the solutions for 5 min. Thermal images of solutions were obtained using a thermal infrared camera and the temperatures of the solution were recorded under laser irradiation. To investigate the effect of Fe concentrations on the photothermal properties, Fe₃O₄ nanoparticles or Fe₃O₄ hydrogels at different Fe concentrations (100, 200, 350, 500 μg/mL) were irradiated by 808 nm laser (1 W/cm²) for 5 min. DI water was used as the control group. These solutions were irradiated by a laser at the power density of 1 W/cm². The laser was turned on/off every 5 min for 50 min to evaluate the photothermal stability of Fe₃O₄ nanoparticles and Fe₃O₄ hydrogels.

CT26 cancer cells were cultured in RPMI 1640 cell medium containing penicillin, streptomycin and 10% FBS at 37°C and 5% CO₂. The cells were incubated with Fe₃O₄ nanoparticles at the Fe concentration of 100 μg/mL for different time. The cellular uptake efficacy was evaluated using ICP-AES.

The cytotoxicity of CT26 cancer cells after incubation with Fe₃O₄ nanoparticles or Fe₃O₄ hydrogels was investigated using the CCK-8 assay. CT26 cancer cells were cultured with 100 μL of fresh cell culture medium in 96-well plates (10,000 cells per well) and incubated for 24 h. The cell culture medium was then discarded and Fe₃O₄ solutions or Fe₃O₄ hydrogels (1 μg/mL for ALG) at different Fe concentrations (25, 50, 100, 200 and 400 μg/mL) were added into the cell culture medium. Meanwhile, 1 μL of Ca²⁺ solution (180 mM) was added into the wells containing Fe₃O₄ nanoparticles and ALG to form Fe₃O₄ hydrogels. After the incubation of cells for 24 h, the cell culture medium was discarded and the cells were carefully washed with PBS to remove free Fe₃O₄ nanoparticles. Cell culture medium containing 10% CCK-8 agent was then added into each well. After incubation of the cells for 2 h, the absorbance value of each well at 450 nm was detected using a microplate reader. The cells treated with PBS were used as a control. The ratio of absorbance values was used to calculate cell viability.

To evaluate the therapeutic effect of Fe₃O₄ nanoparticles and Fe₃O₄ hydrogels, CT26 cancer cells were seeded in 96-well plates (10,000 cells per well) and incubated at 37°C and 5% CO₂ for 24 h. For Fe₃O₄ nanoparticle treatment group, the cell culture medium was discarded and 10 μL Fe₃O₄ nanoparticles at the Fe concentration of 200 μg/mL was added into each well containing 190 μL cell culture medium. For Fe₃O₄ hydrogel treatment group, the cell culture medium was discarded, and 10 μL solution of Fe₃O₄ nanoparticles (200 μg/mL) and ALG (the concentration of ALG was 1 μg/mL) was added into each well containing 189 μL cell culture medium, and then 1 μL of Ca²⁺ solution (180 mM) was added into the wells to form Fe₃O₄ hydrogels. The formed hydrogels could stick to cells for cell incubation. After incubation of cells for 12 h, the cells were irradiated by 808 nm laser (1 W/cm²) for 5 min. After that, the cells were incubated for another 12 h and the hydrogels were removed, and then cell viability was detected by CCK-8 assay.

Significant difference between the experimental statistics is analyzed by One-way ANOVA and indicated as (*), p < 0.01 by (**) and p < 0.001 by (***).

To investigate the effect of different reaction times on the properties of Fe₃O₄ nanoparticles, the hydrodynamic sizes and surface zeta potentials of the Fe₃O₄ nanoparticles formed after the reaction for 30 min or 2 h were measured. The hydrodynamic diameter of Fe₃O₄ nanoparticles with 30 min of reaction was 61.3 nm, which was much smaller than that of 2 h (1,624 nm) (Figures 1A, B). Meanwhile, the surface zeta potential of Fe₃O₄ nanoparticles formed via a 30 min reaction (−21.4 mV) was lower than that of 2 h (−6.8 mV) (Figure 1C). These results indicated that the Fe₃O₄ nanoparticles obtained by reacting for 30 min had a smaller diameter and a more suitable surface potential. The Fe₃O₄ nanoparticles formed via a 30 min of reaction had a smaller size, and thus they would show a higher stability. Stronger steric stabilization and less electrostatic stabilization may lead to their lower surface potential (Shah et al., 2014). Therefore, the reaction time was set at 30 min in the following study. In addition, the UV-Vis absorption spectra of Fe₃O₄ nanoparticles were evaluated (Figure 1D). The absorbance value at 850 nm increased with increasing Fe concentrations measured using ICP-AES, which could enable their PTT applications (Yang et al., 2017).

To evaluate the photothermal conversion efficacy, the Fe₃O₄ nanoparticle solutions were irradiated by an 808 nm laser. The thermal images were captured and the temperatures of the solutions were recorded. At the same Fe concentration, the temperatures of the solutions gradually increased with increasing laser time, which reached a maximum after 5 min of laser irradiation (Figure 2A). In order to evaluate the relationship between different power densities and the temperature increases, lasers at different power densities (0.5, 1, 1.5 W/cm²) were used. Higher power densities achieved a greater increase in solution temperatures. The solution temperature increased to 35.5, 42.3, and 43.5°C after 5 min of laser irradiation at the power densities of 0.5, 1, and 1.5 W/cm², respectively (Figure 2B). The solutions at different Fe concentrations showed different degrees of temperature increases after irradiation by 808 nm laser (1 W/cm²) for the same time (Figure 2C). The temperature of solutions at Fe concentrations of 100, 200, 350, and 500 μg/mL increased to 37.0, 42.3, 47.3, 55.0°C, respectively (Figure 2D). In contrast, the temperature of PBS solution showed no significant change after laser irradiation. The photothermal stability of Fe₃O₄ nanoparticles was then evaluated. After five cycles of heating and natural cooling, the temperature increases of the Fe₃O₄ nanoparticle solutions did not change significantly, indicating their good photothermal stability.

To prepare Fe₃O₄ hydrogels, Fe₃O₄ nanoparticles were added to ALG solutions at different concentrations (0.5, 1, 2.5, 5, 10 mg/mL) and the solutions were slowly injected into 10 mL Ca²⁺ solution (1.8 mM). The hydrogels could be formed via cross-linking of ALG by Ca²⁺. The rate of hydrogel formation increased with the increasing of ALG concentrations (Figure 3A). When the solution with a ALG concentration of 0.5, 1 or 2.5 mg/mL was injected into the Ca²⁺ solution, hydrogels could be formed. However, the formed hydrogels disintegrated rapidly in solution due to the low cross-linkage of the formed hydrogels. When the concentration of ALG was 5 or 10 mg/mL, the formed hydrogels were able to maintain stability state for a long time without significant morphological changes due to the high cross-linking degree. Therefore, the concentration of ALG was set at 5 mg/mL in the following experiments. The SEM images showed that nanoparticles were attached to the surface of the hydrogels, which proved that Fe₃O₄ nanoparticles could be effectively encapsulated into the hydrogels (Figures 3B, C).

The Fe₃O₄ hydrogels were irradiated using an 808 nm laser to study their photothermal conversion properties. The thermal images were captured and temperatures of the hydrogels were recorded. The temperatures of Fe₃O₄ hydrogel solutions gradually increased with the increasing of laser irradiation time, which reached the maximum after laser irradiation for 5 min (Figure 4A). Meanwhile, the temperatures of the hydrogel solutions irradiated by 808 nm laser at different power densities (0.5, 1, 1.5 W/cm²) for 5 min were different. The solution temperature increased to 35.1, 41.3, and 43.9°C after 5 min of laser irradiation at power densities of 0.5, 1, and 1.5 W/cm², respectively (Figure 4B), indicating that higher power densities could achieve better photothermal effects. The temperature increases of the Fe₃O₄ hydrogel solutions were not significantly different for power density of 1 and 1.5 W/cm², so the power density used in the subsequent experiments was set at 1 W/cm². The photothermal performance of Fe₃O₄ hydrogels at different Fe concentrations was also evaluated. After 5 min of 808 nm (1 W/cm²) laser irradiation, the temperature of the hydrogel solutions at Fe concentrations of 100, 200, 350, and 500 μg/mL increased to 34.6, 41.3, 46.4, and 55.2°C, respectively (Figures 4C, D). These concentrations were used for photothermal effect evaluation because the Fe₃O₄ nanoparticles at these concentration ranges could obviously increase temperatures under laser irradiation (Chen et al., 2023). Meanwhile, the photothermal stability of the Fe₃O₄ hydrogels was evaluated (Figure 4E). The temperature increase did not change significantly after five cycles of heating/cooling, indicating that the Fe₃O₄ hydrogels had good photothermal stability. There was no significant difference between the photothermal performance of Fe₃O₄ hydrogels and Fe₃O₄ nanoparticles, indicating that the loading of Fe₃O₄ nanoparticles into hydrogels did not affect their photothermal performance.

The in vitro cellular uptake of Fe₃O₄ nanoparticles by cancer cells was evaluated using ICP-AES. The cellular uptake of Fe₃O₄ nanoparticles was pivotal to induce therapeutic effect for Fe₃O₄ nanoparticle-treated cells. After incubation the cells with Fe₃O₄ nanoparticles, the Fe uptake in cancer cells gradually increased in a time dependent manner (Figure 5A). After 24 h, the cellular Fe level increased by 6.6-fold. These results suggested the effective cellular uptake of Fe₃O₄ nanoparticles by cancer cells. To evaluate the cytotoxicity, CT26 cancer cells were co-incubated with Fe₃O₄ nanoparticles or Fe₃O₄ hydrogels for 24 h. The cell viability of CT26 cells was higher than 92.5% after incubation with Fe₃O₄ nanoparticles or Fe₃O₄ hydrogels even when the Fe concentration was as high as 400 μg/mL (Figure 5B), indicating that both Fe₃O₄ nanoparticles and Fe₃O₄ hydrogels had good biosafety and cytocompatibility. The in vitro therapeutic effects of Fe₃O₄ nanoparticles and Fe₃O₄ hydrogels were then evaluated using CCK-8 assay. CT26 cancer cells were irradiated by 808 nm laser (1 W/cm²) for 5 min, and the cell viability was not significantly reduced compared to that in the control group, indicating that cancer cells were not killed by laser irradiation alone (Figure 5C). When CT26 cells were treated with Fe₃O₄ nanoparticles or Fe₃O₄ hydrogels plus laser irradiation, the cell activity of cells decreased to 16.1% and 15.4%, respectively. The cell vitality of cells in Fe₃O₄ nanoparticles + laser and Fe₃O₄ hydrogels + laser was similar. These results verified the therapeutic effect of Fe₃O₄ hydrogels. Although the therapeutic efficacy of Fe₃O₄ hydrogels was similar to that of Fe₃O₄ nanoparticles, the Fe₃O₄ hydrogels could maintain a high concentration at injected sites and obviously reduce systemic toxicity in the form of intravenous administration, which would contribute to their future in vivo studies.