Figure 1A shows a transmission electron microscope image of FeGA. The results show better dispersibility and smaller size of FeGA, with an average of 2.36 ± 1.2 nm. The nanoparticles of smaller than 10 nm in size are easily cleared by the kidney, which limits its therapeutic efficacy. Therefore, the applicability of FeGA can be greatly improved through the hydrogel delivery system. The particle size of FeGA was measured for three consecutive days ( Figure 1C ), and the size fluctuation range was found small, reflecting the good stability of FeGA. Thus, in turn makes it a good system in clinical application prospects. Even though many materials have a beneficial biological impact, their high instability restricts their future value (36). The agarose hydrogel has been authorized by the FDA as a safe material that does not cause any toxicity in the body and is metabolized by the body’s natural processes following dissolution. The scanning electron microscope (SEM) image of the agarose hydrogel is shown in Figure 1B . The pore size of the hydrogel is larger and the AES system was then developed by encapsulating FeGA and α-CHCA in a hydrogel. Figure 1D demonstrates the good photothermal heating potential of AES while the developed hydrogel remains solid. The hydrogel gradually dissolves after 10 minutes of 808 nm laser irradiation. The drug and nanoparticles in the gel are almost totally dissolved and are released. Infrared thermal imaging further indicated that following irradiation, the temperature of AES increased dramatically. Figure 1E shows the ultraviolet-visible absorption of FeGA. FeGA has a quite high absorption bandwidth in the 600-800 nm range, with a distinctive peak near 600 nm. X-ray photoelectron spectroscopy (XPS) was used to measure the Fe 2p spectrum in FeGA ( Figure 1F ). Zeta potential of FeGA was detected to be -18.1 ± 6.57mV. The rheological value of AES was evaluated at various temperatures ( Figure 1G ), and the results revealed that as the temperature increases, AES rapidly dissolves, accompanied by a gradual decrease in storage modulus. This is in line with the hydrogel’s rheological properties. We then put AES to the test to see if it could regulate the release of materials and the results are described in Figure 1H . Laser irradiation can partially disintegrate AES and liberate the α-CHCA within it. The hydrogel becomes cool and hardens after the laser irradiation is stopped, and the drug will remain protected. The drug is usually released completely after four laser switching cycles. This also demonstrates that our AES system has a strong ability to control drug release, which inhibits lactic acid efflux, strengthens the tumor’s acidic environment, and keeps the cells in an acidic environment, which is likely to promote FeGA-mediated CDT.

One of the most essential factors for evaluating PTA is photothermal stability. A powerful photothermal treatment can be assisted by a good photothermal agent. To test the photothermal performance of FeGA nanoparticles, FeGA solutions were prepared with different concentrations (0, 25, 50, 100, 200 μg/mL). Figure 2A shows that assuming all other parameters remain constant, the heating impact of the solution increases as the FeGA concentration rises. The temperature of 100 μg/mL FeGa increased by roughly 16.6°C after 5 min of laser irradiation. For 5 minutes, the 200 μg/mL FeGA solution was repeatedly heated with the 808 nm NIR laser ( Figures 2B, D, E ), then the solution was allowed to cool to ambient temperature. The heating curves of each cycle were identical, and the variations in peak temperature changes were minor, demonstrating that the FeGA nanoparticles’ photothermal conversion capability was found to be stable and reproducible over repeated 4 heating and cooling cycles. These findings suggest that the FeGA nanoparticles have good photothermal stability. Furthermore, the photo-thermal conversion efficiency (η) of the FeGA was calculated from the data of Figures 2C, F , and was found as 42.6%, which was higher than various materials such as Au nanorods (21%) and Ti₃C₂ nanosheets (30.6%) (37, 38). Above all, FeGA nanoparticles are an excellent PTA for photothermal conversion to anti-tumor.

Furthermore, we assessed the ROS content of several formulations. There is no green fluorescence in the control group, the NIR group, or the AES group when treated alone. The AES + NIR group had high fluorescence, whereas the FeGA + NIR group had much lower fluorescence. This could be because α-CHCA alters the tumor acid microenvironment, and FeGA reacts with enough H₂O₂ to produce increased ROS. ROS can cause cellular protein and DNA damage, resulting in the death of tumor cells (39). In several experimental groups, we continued to evaluate the lactic acid concentration of the TME. The results revealed that, despite the presence of α-CHCA in the AES group, α-CHCA was unable to affect the cells due to the hydrogel’s encapsulation, but the AES + NIR group had increased lactic acid concentration. α-CHCA can be released after laser irradiation, changing the lactic acid ecological microenvironment of tumor cells. Hence, the AES + NIR group was able to produce good lactate accumulating effect. The acidic environment is conducive to the subsequent FeGA-mediated Fenton reaction. FeGA was incubated with 4T1 cells at various concentrations (0, 10, 20, 30, 40 μg/mL) for 24 hours. Even at high concentrations, cell viability did not decrease significantly. The findings suggested that FeGA NPs are highly biocompatible. The cell viability of the control group was largely unaffected by the MTT experiment, whereas the NIR with FeGA group demonstrated a little inhibition in tumor growth ( Figure 2G ). AES + NIR system exhibited the best tumor growth inhibition rate (about 88.5%), with significant differences compared to other experiment groups indicating that AES mediated controlled release of α-CHCA can effectively increase the acidity of TME, thereby enhancing the CDT effect and inhibiting tumor growth. These findings motivate our ongoing research to proceed with the developed formulation for anti-tumor efficacy in vivo.

As stated earlier, the good performance of AES in vitro as a PTA and acid boosting system has prompted us to investigate the photothermal conversion effect of FeGA in vivo. BALB/c mice were used to establish 4T1 subcutaneous tumor models. After invading the tumor tissue, we measured the temperature rise of AES under laser irradiation. The PBS group rarely heated up after laser irradiation, as seen in Figures 3A, B , whereas the temperature of the tumor in the AES group increased dramatically, showing that AES has strong photothermal performance. This finding is similar to the in vitro photothermal findings, demonstrating that AES can provide in vivo photothermal therapy and drug release. Tumor tissues’ heat resistance was lowered when compared to normal cells, resulting in tumor cell-selective apoptosis at high temperatures (42 – 47°C) (17). Following that, we measured the amount of lactic acid in the tumor after various treatments. The lactic acid level of the tumor increased considerably after 4 hours of AES plus NIR treatment, as seen in Figure 3C . However, no obvious signals of increase were observed in the other groups, demonstrating that the released -CHCA can effectively prevent tumor cell lactic acid efflux and intracellular accumulation, leading to tumor acidosis ( Figure 3C ). The anti-tumor efficacy of AES-mediated anti-tumor therapy was next evaluated in mice with 4T1 tumors. BALB/c mice were injected subcutaneously with 1 × 10⁶ 4T1 cells in the right flank to assess the main effect of AES. The mice were treated after reaching the primary tumor volume to 200 mm³. Tumor-bearing mice were randomly divided into 4 groups (5 mice per group): (1) PBS; (2) NIR; (3) AES and (4) AES + NIR. The equivalent FeGA dose was 5 mg/kg in groups 3 and 4. For 16 days, treatment was given every 4 days. The body mass of treated and control mice remained normal after treatment, showing the safety of our technique ( Figure 3F ). This is quite interesting because many treatments are associated with severe systemic toxicity, which is detrimental to the material’s future clinical application (40). The tumor volumes of the PBS and NIR treated groups increased substantially during the 2 weeks of treatment, as illustrated in Figure 3D . In addition, the AES group had essentially no tumor-suppressive effect. The hydrogel entrapped FeGA and CHCA are unable to kill tumors. The AES + NIR system, which included both FeGA, had the most potent therapeutic response as the tumor volume growth curves being nearly reduced during therapy ( Figures 3D, E ). When the AES hydrogel is exposed to laser radiation after intratumoral injection, it dissolves and releases FeGA and α-CHCA in FH. α-CHCA would raise the tumor’s lactic acid concentration. FeGA then combines with intratumoral H₂O₂ to produce •OH in situ, which kills the tumor. We tested the fluorescence intensity of ROS in different groups in vivo, and the results also showed that AES + NIR group showed the strongest green fluorescence, and produced a large amount of ROS to promote tumor apoptosis. The tumor mass of mice was also in agreement with the volume curve ( Figure 3E ). We obtained slices of tumor tissue for staining. TUNEL and H&E staining ( Figures 3G, H ) also confirmed the large amount of cell necrosis in the AES combined NIR treatment group.

Furthermore, FeGA activation did not cause any loss to systems, as shown in Figure 4 . After the treatment of mice’s vital organs (heart, liver, spleen, lungs, and kidney), there was no inflammation, and damage in the body, and the liver, and kidney indexes were also normal. As many nanomaterials possess great therapeutic efficacy, they are also associated with systemic toxicity, which limits their future clinical applications (41). In vivo data show that our novel combination therapy not only achieves a high level of biological safety but also increases tumor •OH content, thus enhancing the effect of FH-enhanced therapy.