A light-controlled single-atom nanozyme hydrogels for glutathione depletion mediated low-dose radiotherapy

Due to the unique ability to mimic natural enzymes, single-atom nanoenzymes (SAE) have garnered significant attention and research in tumor therapy. However, their efficacy often faces challenges in terms of drug delivery methods, and the research regarding their applications in radiotherapy is scarce. Herein, we introduce a light-controlled SAE hydrogel platform (SH) for glutathione-depletion-mediated low-dose radiotherapy. The SH incorporates a Cu single-atom enzyme (CuSA), and upon irradiation with 1064 nm near-infrared light, the CuSA can convert light energy into heat, which in turn degrades the hydrogel, enabling the release of CuSA into tumor cells or tissues. The diffused CuSA not only can facilitate the conversion of H2O2 into hydroxyl radicals (•OH), but also can effectively depletes cellular glutathione. This leads to increased sensitivity of tumor cells to radiotherapy, resulting in enhanced cytotoxicity even at low doses. The animal study results further confirmed the good tumor-killing efficacy of this SH system. To the best of our knowledge, this stands as the pioneering report on leveraging a single-atom enzyme for GSH depletion-mediated low-dose radiotherapy.


Introduction
Radiotherapy, which uses the energy of photon, proton, or carbon ion radiation to destroy malignant cells, remains one of the most frequently utilized methods for treating cancer [1,2].However, the effectiveness of radiotherapy is often limited by the side-effect damage to surrounding healthy tissues, or the radio-resistance of cancer cells that can lead to potential recurrence [3,4].Nanomaterials have emerged as a promising solution to these challenges due to their unique properties and versatility [5].They can enhance the precision and efficacy of radiotherapy by enhancing local dose at cellular level or modulating cellular response to radiation by regulating the micro-environment [6,7].Despite the potential, certain drawbacks still limit the application of nanomaterials in radiotherapy.On the one hand, due to the low drug concentration and limited reaction cross-section, the dose enhancement is actually limited [8,9].On the other hand, Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence.Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.nanomaterials with higher chemical activity, such as oxides and alloy nanomaterials, will be easily dissociated by the cells into ions.These ions will then diffuse into the interstitial fluid, causing great ionic toxicity [10,11].Therefore, the exploration of novel nanomedicines remains an urgent issue to be addressed.
Building on the this, a particular area of interest has been the development of nanoenzymes, especially single atom nanoenzymes (SAE), for tumor therapy.SAE is characterized by the dispersion of individual atoms on a suitable substrate, and mimics the catalytic activities of natural enzymes but with enhanced stability and tunable catalytic properties [12][13][14].They exhibit superior catalytic efficiency and selectivity due to their maximized atom utilization and unique electronic structure [12,15].These properties make them highly effective in catalyzing therapeutic reactions in tumor cells, thereby enhancing the therapeutic outcomes [16][17][18][19].Compared to traditional nanoenzymes, such as Fe 3 O 4 or Horseradish Peroxidase, SAE exhibits higher unit activity due to its dispersed metal structure.Moreover, the chemical stability of SAE ensures that it does not release metal ions within cells or organisms, thereby reducing potential toxicity caused by diffused metal ion.Generally, catalytic reactions for SAE in tumor therapy can encompass a range of processes, including the catalysis of hydroxyl radicals, also known as peroxidase (POD)-like enzyme, as well as glutathione (GSH) depletion and so on [13].
Glutathione (GSH) plays a crucial role in maintaining cellular redox homeostasis and in the detoxification of chemotherapeutic drugs, which often leads to drug resistance in cancer treatment [20].Depleting GSH in cells can therefore enhance the sensitivity of these cells to therapy [21].In the context of radiation therapy, GSH depletion can augment the production of radiation-induced reactive oxygen species (ROS), subsequently leading to enhanced cell damage and death [22,23].However, the application of SAE in this aspect remains unexplored.
Although SAE show promise for biomedical applications, the inherent hydrophobicity limits their in vivo transportation efficacy [24,25].Attempts have been made to modify SAE with biocompatible or targeting coatings such as cellular membranes or polyethylene glycol (PEG), yet these endeavors often face challenges related to low drug loading content or decreased encapsulation efficiency (EE) [26,27].A potential solution is localized intravenous delivery, allowing for extended and controlled drug release without the worry of systemic toxicity [28].In fact, therapeutic regimes for radiosensitization agents have adopted such methods [29].However, the requirement for multiple injections can be problematic due to potential pain and postoperative complications for patients.In light of these challenges, light-activated injectable hydrogels can be an attractive method for consistent, intratumoral delivery of SAE, presenting a patientfriendly approach [30,31].This hydrogel system can steadily release SAE to boost the effectiveness of subsequent radiotherapy.Incorporating this method can significantly improve therapeutic outcomes while minimizing patient discomfort.
In this study, we introduced a light-controlled release SAE hydrogel (SH) platform designed for GSH depletion-mediated low-dose radiotherapy (scheme 1).Initially, a copper-based SAE (CuSA) was synthesized using the adsorption-pyrolysis method.This synthesized SAE functions dually as a photothermal agent and a peroxidase-like enzyme.Upon encapsulating CuSA within the hydrogel, exposure to 1064 nm nearinfrared light causes the hydrogel to undergo hydrolysis due to the heat generated by CuSA.Thereafter, CuSA diffuses into the tumor cells, where it acts as a peroxidase-like enzyme, facilitating the conversion of H 2 O 2 into hydroxyl radicals (•OH).Notably, CuSA also has the ability to deplete intracellular GSH.A decrease in GSH levels enhances radiationinduced oxidative damage to cancer cells, thus overcoming tumor radioresistance.As supported by both in vivo and in vitro experimental results, the SAE effectively induces substantial cancer cell death.To our knowledge, this is the first report of employing an SAE-based stimulating hydrogel system in GSH depletion-mediated low-dose radiotherapy.We believe that our findings can pave the way for future development of SAE-mediated therapeutic strategies.

Results and discussion
The production of Cu Single Atom (CuSA) with atomically dispersed copper was achieved through an initial anchoring of copper ions to the carbon sphere forerunners, and followed by a pyrolytic procedure at 600 °C for 4 h under an argon atmosphere.
The spherical shape of the as-prepared CuSA can be seen in transmission electron microscopy (TEM) images (figure 1(B)), same morphology as NC nanosphere without Cu after calcination (figure 1(A)).The energy dispersive spectroscopy mapping demonstrated a consistent distribution of Cu, N, and C elements, and no apparent nanoparticle can be seen in the TEM and STEM images (figure 1(C)).In addition, the AC-HAADF-    1(E)), which can be indexed to the carbon plane of (002) and (100), respectively.Interestingly, no traces of iron species were found in the prepared CuSA, this is in sound agreement with the XRD results.Both the Raman spectra of NC and CuSA exhibited two distinct patterns, characterized by two peaks at approximately 1350 cm −1 for the D band, which is related with the disorder of carbon structure, and 1580 cm −1 for the G band, indicating sp2-hybridized carbon structures of graphitized carbon (figure 1(F)).The Raman spectroscopy results indicated that the introduction of copper single atoms did not significantly alter the framework of the carbon-nitrogen material.In the XPS spectrum results, prominent peaks corresponding to N, C, and Cu are clearly observed (figure 1(G)).Furthermore, through a more detailed analysis of the Cu 2P high-resolution spectrum, the peak positions of Cu suggest Cu species only exists in their oxidation states (figure 1(H)).All the experimental data suggests that the Cu atoms have been successfully integrated into the Nitrogen-Carbon framework and formed Cu-N-C coordination structure (figure 1(I)), which can potentially serve as efficient catalytic sites to transform H 2 O 2 to •OH.
To verify the POD-like enzyme performance of CuSA, an electron paramagnetic resonance (EPR) experiment was conducted.It could be seen that obvious •OH generation in the CuSA and H 2 O 2 group (figure 2(A)).In addition, the POD characteristic was also assessed in different pH conditions by 3, 3″, 5, 5″-Tetramethylbenzidine (TMB) as a chromogenic substrate, the intensity of characteristic peaks (652 nm) in a more acidic condition (figure 2(B)).This is good since the pH of the tumor TME is biased toward weakly acidic.After verifying its robust POD activity, CuSA was encapsulated by 2% agarose hydrogel, forming a SAE-based hydrogel (SH) system.The morphology of this system can subsequently be imaged using SEM (figure 2(C)).Additionally, rheological analyzes of the SH formulation revealed that the storage modulus values of SH decreased with rising temperature, an advantageous for subsequent heat-dependent tumor therapies (figure 2(D)).The temperature profiles of SH infused with varying concentrations of CuSA nanoparticles over time revealed that temperatures could exceed 50 °C within a span of 5 min; this temperature threshold is sufficiently high for effective tumor cell ablation (figure 2(E)).The infrared thermal imaging also indicated a significant increasing in the temperature of SH upon irradiation (figure 1(F)).
The ability of SH to deplete GSH can be verified by measuring intracellular GSH levels.Upon 1064 nm laser exposure, SH rapidly induced hyperthermia, leading to immediate and reversible hydrogel hydrolysis.Subsequently, CuSA gradually diffused into the tumor cells, exerting its cellular effects.As depicted in figure 3(A), GSH levels in the cells of the NIR+SH group showed a notable decrease.In contrast, the other three groups (PBS, SH, NIR) displayed no significant changes in their GSH levels.These findings not only suggest that SH can effectively release its CuSA content after NIR irradiation, but also indicate that CuSA can proficiently diminish intracellular GSH contents, which lays a solid foundation for next exploration of this controllable drugrelease system in radiation therapy.
The experimental evaluation of cell survival under radiotherapy conditions serves as a critical method to guarantee the efficacy of the treatment modalities, we thus treated tumor cells across five distinct groups: (1) PBS, (2) radiotherapy (RT), (3) SH, (4) SH + NIR and (5) SH+NIR+RT.Both the cell surviving experiment and monoclonal assay demonstrated that the group treated with SH+NIR+RT had the highest cytotoxic effect against cancer cells, while SH alone did not exhibit significant cytotoxicity (figures 3(B), (C)).Tumor cells contain a strongly reducing environment compared to normal cells due to intracellular GSH being overproduced which would subsequently consume ROS.In our design, the CuSA depletes the glutathione (GSH) content within the cancer cells and simultaneously enhances the ROS content.Through this reciprocal interaction, the combined effect significantly amplifies the intracellular ROS concentration within the cancer cells.This is aligning with the observation where cell GSH content and ROS expression the five different groups, as depicted in figures 3(A) and (D).Subsequently, the γ-H 2 AX assay findings substantiate that an increase in ROS concentrations results in amplified DNA damage.In the wake of x-ray irradiation, 4T1 cells subjected to SH+NIR+RT treatment exhibit the most pronounced γ-H 2 AX expression, as demonstrated in figure 3(D).
In light of their remarkable in vitro antitumor efficacy and other advantageous properties, our subsequent objective was to evaluate the in vivo tumor ablation potential of SH.Consequently, we administered various treatments to tumorafflicted BALB/c mice, which included: Subsequently, to assess the extent of tumor apoptosis and proliferation, tumor tissues were extracted and subjected to staining using Ki-67, TUNEL, ROS and HE staining.Results from the TUNEL and ROS assay highlighted extensive necrosis and ROS production within tumor cells in the SH+NIR+RT group (figure 4(E)).The GSH results of tumor tissue showed that after the SH+NIR+RT group, the GSH content was the lowest (figure S1).This is the main reason for the increase in ROS generation.Similarly, Ki-67 staining revealed a marked reduction in fluorescence intensity within the SH+NIR+RT group.Furthermore, the most significant loss of tumor tissue can be observed in SH+NIR+RT group, as confirmed by H&E staining tumor sections.These findings underscored that the SH+NIR+RT group experienced the highest degree of tumor apoptosis when compared to the other treatment groups, The histological examination from major organs, including the heart, lung, liver, kidneys, and spleen, were also conducted to assess potential systemic toxicity induced by the proposed treatment approach.The results revealed no significant abnormalities in any of the treatment groups throughout the duration of the mice treatment, which indicated the absence of systemic toxicity (figure 5).

Conclusions
In conclusion, we have developed a light-controlled SAE hydrogel system (SH) to facilitate GSH-depletion-mediated low-dose radiotherapy.Upon near-infrared (NIR) light irradiation, the SH undergoes hydrolysis, subsequently releasing the encapsulated CuSA into tumor cells.This liberated CuSA not only augments the ROS within the cells but also significantly diminishes the intracellular GSH levels, markedly amplifying tumor cell apoptosis.To the best of our knowledge, this study offers the inaugural documentation of employing a single-atom based stimuli-responsive hydrogels for GSH-depletion-mediated radiotherapy.We are optimistic that our findings will lay the groundwork for pioneering single-atom based therapeutic methodologies in the future.

Scheme 1 .
Scheme 1. Schematic of copper single atom nanozyme for glutathione depleted enhanced radiotherapy.

Figure 1 .
Figure 1.TEM images of (A) carbon precursor sphere and (B) CuSA nanoparticle.(C) Elemental mapping images of C, N, Cu for CuSA nanoparticle.(D) Atomic-resolution aberration-corrected (AC) HAADF-STEM image of CuSA, showing the atomically dispersed single Cu atom.(E) XRD pattern of the calcined nitrogen-doped carbon (NC) and CuSA nanoparticle.(F) Raman spectra of NC and CuSA nanoparticle.(G) X-ray photoelectron spectroscopy (XPS) and (H) Cu 2p XPS spectra of CuSA nanoparticle.(I) The schematic model for the single-atom structure of CuSA.

Figure 2 .
Figure 2. (A) ESR analysis of •OH production of CuSA with H 2 O 2 .(B) TMB assay for measuring the oxidase-like activity of the CuSA at different pH.The measured absorbance was 652 nm.(C) Representative SEM images cu single atom hydrogen (SH).(D) Temperature dependent rheological analyzes of the SH.(E) Associated temperature increases in SH contained CuSA with different concentrations.(F) The infrared thermal images of the prepared SH following 0.5 W cm −2 1064 nm laser irradiation for 5 min.

Figure 4 .
Figure 4. (A) Changes in the body weight of the mice were recorded every other day with the indicated treatments (n = 5, mean ± SD). (B) Evolution in tumor volume in mice after the indicated treatments (n = 5, mean ± SD). (C) Changes of the tumor weight during therapy (n = 5, mean ± SD). (D) Survival rates of mice during the treatment.(E) ROS, TUNEL, Ki-67 and Hematoxylin and Eosin (H&E) staining of tumor sections from the tumor-bearing mice (scale bars: 40 μm).Student 's t-test ( * P < 0.05, ** P < 0.01, *** P < 0.005).The NIR irradiation was conducted by a beam of 1064 nm and 0.5 W cm −2 .The irradiation dose was 4 Gy.

( 1 )
PBS; (2) RT; (3) SH; (4) SH+NIR; and (5) SH+NIR+RT.Over a two-week treatment duration, the body weight of both the treated and control mice remained consistent, thereby confirming the safety of this formulation (figure 4(A)).Rapid tumor growth was observed in the PBS-treated groups (figure 4(B)), with no discernable reduction in tumor volume even in the RT group.In stark contrast, the SH+NIR+RT group exhibited a substantial decrease in both tumor volume and weight, with tumor growth almost entirely inhibited during the treatment (figures 4(B), (C)).SH+NIR+RT group mice have the longest survival time (figure 4(D)).

Figure 5 .
Figure 5. H&E-stained images of the major organs (heart, lung, liver, kidneys, and spleen) from mice subjected to different treatments.These images were obtained after treated for 16 days (scale bars: 40 μm).