Ce6 was purchased from America J&K Scientific Ltd. (United States). Bovine serum albumin (BSA), N-(3-dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride crystalline (EDC), N-hydroxysuccin-imide (NHS), singlet oxygen sensor green (SOSG), 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) and calcein-AM/propidium iodide (PI) double staining kit were purchased from Sigma Aldrich (United States). FeCl₃.6H₂O and FeCl₂.4H₂O were obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). CCK-8 was purchased from Dojindo Laboratories (Japan). All the other chemicals were purchased from National Pharmaceutical Corporation (Shanghai, China).

Transmission electron microscopy (TEM) images were obtained using Tecnai G2 20 TWIN TEM (FEI, United States). Hydrodynamic sizes and zeta potential values were measured using a Zetasizer (Nano S90, UK). UV-vis absorptions were measured using persee UV-vis spectrophotometer (TU-1810, China). Fluorescence spectra were recorded using fluorescence spectrophotometer (Shimadzu RF-6000, Japan).

FeCl₂.4H₂O (89.0 mg) and FeCl₃.6H₂O (157.0 mg) were dissolved in 8.0 mL water, and then 5 mL aqueous solution containing NaOH (1.0 g) and BSA (20.0 mg) was dropped into above solution. The resulted solution was stirred at 80°C for 30 min and black products were formed. Then the solution was cooled to room temperature and the formed products were precipitated by using magnetic beads. After purification through water washing, BSA-coated Fe₃O₄ nanoparticles were obtained.

Ce6 (12.0 mg), EDC (24.6 mg) and NHS (23.0 mg) were dissolved in 5 mL dimethyl sulfoxide and the solution were stirred at room temperature for 3 h. Then above solution was dropped into 5 mL solution of BSA-coated Fe₃O₄ nanoparticles, and the reaction was contained at room temperature for 24 h. The products were collected using magnetic beads and then further washed with water. After that, Fe₃O₄-Ce6 nanoparticles were obtained.

The solution of Fe₃O₄-Ce6 nanoparticles were mixed with SOSG, and the resulted solutions were irradiated by 660 nm laser (0.3 W/cm²) for different times. The fluorescence spectra of solutions without or with laser irradiation were recorded. The fluorescence intensities of solutions at 525 nm were used to evaluate the ¹O₂ generation by calculating the fluorescence enhancement (F/F₀).

The solutions of Fe₃O₄-Ce6 nanoparticles at different concentrations were irradiated by 808 nm laser (1.0 W/cm²), and the temperatures of solutions under laser irradiation were measured. To evaluate the photothermal stability, the nanoparticle solutions were irradiated by 808 nm laser (1.0 W/cm²) for five times and the temperatures of solutions were measured.

The cell lines (brain endothelial bEnd.3 cells and rat C6 glioma cells) presents in this study were obtained from American Type Culture Collection (ATCC, United States). The bEnd.3 and C6 cancer cells were incubated with Fe₃O₄-Ce6 nanoparticles at different concentrations for 24 h, and then the cells were washed with PBS. The cells were then incubated in cell culture medium containing CCK-8 agent for 2 h. The supernatant of treated cells was collected to measure the absorbance at 450 nm using a Thermo Scientific Multiskan MK3 ELISA reader (Thermo scientific, United States), and then the cell viabilities were calculated.

C6 cancer cells were incubated with Fe₃O₄-Ce6 nanoparticles for 24 h and then the cells were irradiated by 808 nm laser (1.0 W/cm²) for 5 min and 660 nm laser (0.3 W/cm²) for 5 min. The cells were further incubated for 6 h and then the cell viabilities of cells were measured using CCK-8 analysis.

C6 cancer cells were incubated with Fe₃O₄-Ce6 nanoparticles for 24 h and then the cells were cultured in cell culture medium containing calcein-AM/PI double staining agent. The 808 nm laser (1.0 W/cm², 5 min) and 660 nm laser (0.3 W/cm², 5 min) was used to treat the cells. The fluorescence images of cells in various treatment groups were captured using a fluorescence microscope.

C6 cancer cells were incubated with Fe₃O₄-Ce6 nanoparticles for 24 h and then the cells were further cultured in cell culture medium containing H₂DCFDA for 30 min. The cells were then irradiated by 660 nm laser (1.0 W/cm²) for 5 min. Fluorescence images of cells in various treatment groups were captured using a fluorescence microscope.

C6 cancer cells were incubated with Fe₃O₄-Ce6 nanoparticles at different concentration for 12 h, and then the cells were washed with PBS to remove free nanoparticles. The contents of nanoparticles inside cells were evaluated by measuring intracellular Fe concentration using inductively coupled plasma optical emission spectroscopy (ICP-OES).

The data were provided as mean ± standard deviation (SD). Statistical analysis was carried out using one-way ANOVA statistical analysis. Statistical significance was indicated as (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001.

TEM image showed that the formed Fe₃O₄-Ce6 nanoparticles had a spherical morphology and their size distribution was homogeneous (Figure 1A). The hydrodynamic size and zeta potential of Fe₃O₄-Ce6 nanoparticles was measured to be 80.0 nm and −15.4 mV, respectively (Figure 1B). As shown in UV-vis spectra, the characteristic peaks of Ce6 at 400 nm and 641 nm could be detected in the absorbance spectrum of Fe₃O₄-Ce6 nanoparticles (Figure 1C), which however could not be detected in absorbance spectrum of Fe₃O₄ nanoparticles, confirming the conjugation of Ce6 to Fe₃O₄ nanoparticles. In addition, Fe₃O₄-Ce6 nanoparticles showed a fluorescence emission at around 670 nm (Figure 1D), and the fluorescence signal was also observed for Ce6. However, Fe₃O₄ nanoparticles did not have fluorescence property. These results suggested that Fe₃O₄-Ce6 nanoparticles showed the optical properties of Ce6.

The photothermal property of Fe₃O₄-Ce6 nanoparticles under 808 nm laser irradiation was evaluated. Under laser irradiation, the temperature of solutions containing Fe₃O₄-Ce6 nanoparticles gradually increased, which reached around 58.8°C after 6 min of laser irradiation (Figure 2A). This result verified the good photothermal property of Fe₃O₄-Ce6 nanoparticles. The temperature increase for Fe₃O₄-Ce6 nanoparticles was found to be concentration-dependent, as higher concentration led to a higher temperature (Figure 2B). At the concentration of 500 μg/mL, the temperature increased to 58.8°C after 6 min of laser irradiation. In addition, the temperature increase did not have obvious changes after 5 cycles of laser on and laser off (Figure 2C). These results verified the good photothermal stability of Fe₃O₄-Ce6 nanoparticles. The good photothermal effect and good photothermal stability were similarly observed for Fe₃O₄ nanoparticles as reported in a previous study (Chen et al., 2023).

The photodynamic property of Fe₃O₄-Ce6 nanoparticles was evaluated by measuring the generation of ¹O₂ under 660 nm laser irradiation using SOSG as the ¹O₂ probe. The fluorescence intensity of SOSG for solutions containing Fe₃O₄-Ce6 nanoparticles gradually increased under 660 nm laser irradiation (Figure 3A). This should be because the generated ¹O₂ turned on the fluorescence signals of SOSG. The fluorescence intensity of SOSG for solutions containing Fe₃O₄-Ce6 nanoparticles increased by 1.2-, 1.4-, 1.6-, 1.7-, 1.9-, 2.1-, 2.2-, 2.3-, 2.5-, and 2.6-fold after 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 min of laser irradiation (Figure 3B). These results confirmed the generation of ¹O₂ via photodynamic effect for Fe₃O₄-Ce6 nanoparticles under 660 nm laser irradiation. The ¹O₂ generating efficacy of Fe₃O₄-Ce6 nanoparticles was higher than that of protoporphyrin IX (PpIX)-modified Fe₃O₄ nanoparticles (Ding et al., 2022).

To evaluate the cytotoxicity of nanoparticles to normal cells, bEnd.3 cells were incubated with these nanoparticles. After 24 h of incubation, the cell viability did not have obvious decline (Figure 4A). C6 cancer cells were incubated with Fe₃O₄-Ce6 nanoparticles at different concentrations for 24 h, and the CCK-8 analysis showed that the cell viability of these treated cells was still higher than 85.0% (Figure 4B), which suggested the low cytotoxicity for Fe₃O₄-Ce6 nanoparticles. To evaluate the in vitro therapeutic efficacy, C6 cells were incubated with Fe₃O₄-Ce6 nanoparticles and then treated by 808 and 660 nm laser. The cell viability for PBS + laser and Fe₃O₄-Ce6 nanoparticle-treated groups was similar to that in PBS control group (Figure 4C). These results suggested that laser irradiation and sole Fe₃O₄-Ce6 nanoparticle treatment did not have obvious therapeutic effect. In contrast, the cell viability of C6 cells in Fe₃O₄-Ce6 + laser group was only 19.6%, which suggested the good cell killing effect for Fe₃O₄-Ce6 nanoparticles plus laser irradiation via the combinational effect of PTT and PDT. The therapeutic efficacy of Fe₃O₄-Ce6 nanoparticles via PTT-combined PDT was higher than that of Fe₃O₄ nanoparticles via a sole PTT effect (Chen et al., 2023).

Dead/living staining was then used to evaluate the therapeutic efficacy. As shown in the fluorescence images, only green fluorescence signals (living cells) were observed for cells in PBS + laser and Fe₃O₄-Ce6 nanoparticle-treated groups, which was similar to those in PBS control group (Figure 5A). In contrast, both green and red fluorescence signals could be detected for cells in Fe₃O₄-Ce6 + laser group. The red fluorescence signals in this group indicated the death of cancer cells after treatment. Quantitative analysis showed that the percentage of dead cells and living cells in Fe₃O₄-Ce6 + laser group was 83.6% and 16.4%, respectively (Figure 5B). The percentages of living cells in PBS, PBS + laser and Fe₃O₄-Ce6 nanoparticle-treated groups were around 99.0%. These results further confirmed the good therapeutic efficacy for Fe₃O₄-Ce6 nanoparticles plus laser irradiation.

To confirm the photodynamic effect, the generation of ROS inside cells after treatments was evaluated using H₂DCFHDA as the ROS probe. Obvious green fluorescence signals could be detected in Fe₃O₄-Ce6 + laser group (Figure 6A), which verified the generation of ROS in this group. However, nearly no green fluorescence signals were observed in PBS and PBS + laser group. The very weak green fluorescence signal in Fe₃O₄-Ce6 nanoparticle-treated group may be due to the generation of a little ROS via Fenton reaction. The fluorescence intensity of green signals in Fe₃O₄-Ce6 + laser group was at least 82.0-fold higher than that in the other groups (Figure 6B). These results confirmed the generation of ROS inside cancer cells via photodynamic effect after Fe₃O₄-Ce6 nanoparticle treatment plus 660 nm laser irradiation.

The cellular uptake of Fe₃O₄-Ce6 nanoparticles by C6 cancer cells were investigated. The results showed that the intracellular Fe levels in the treated cells gradually increased in a concentration depend manner (Figure 7). A higher concentration of nanoparticles led to a higher intracellular Fe level. These results confirmed the effective cellular uptake of Fe₃O₄-Ce6 nanoparticles by C6 cancer cells.