Nanozyme hydrogel for enhanced alkyl radical generation and potent antitumor therapy

Alkyl radicals (R˙), which do not rely on oxygen generation for causing cellular stress, have been applied in tumor treatment, but a large amount of glutathione (GSH) in the tumor cells reacts with alkyl radicals, thereby reducing their antitumor effect. In this study, an enhanced alkyl radical generation system responsive to near-infrared light was designed. The alkyl radical trigger 2,2′-azobis[2-(2-imidazolin-2-yl)propane]-dihydrochloride (AIPH) and nanozyme pyrite (FeS2) were encapsulated in agarose hydrogel to prepare the AIPH–FeS2–hydrogel (AFH) system. FeS2 can be used as a photothermal agent to convert near-infrared light energy into heat energy, leading to the dissolution of the hydrogel. AIPH is simultaneously induced to produce alkyl radicals. FeS2 can also be used as an oxidative stress amplifier to reduce intracellular GSH content, thereby boosting the therapeutic effect of alkyl radicals. Eventually, the oxygen-independent free radicals generated by the AFH system under near-infrared laser irradiation and photothermal treatment can kill cancer cells through the synergistic oxidation/photothermal effect. The AFH system developed herein provides new insights into enhancing the therapeutic effect of alkyl radicals.


Introduction
In recent years, free radical-based treatments such as photodynamic therapy, sonodynamic therapy, and electrodynamic therapy have become popular choices for the treatment of breast cancer. [1][2][3] External stimuli (laser, ultrasound, electric elds) promote the production of reactive oxygen species (ROS) that induce cell death by causing severe oxidative damage or cell metabolism dysfunction. [4][5][6] Unfortunately, the hypoxic tumor environment and rapid tumor cell proliferation reduce the therapeutic outcomes of such treatments that rely on ROS production especially in the treatment of solid tumors. 7-10 Alkyl radicals are a new type of free radicals that do not depend on oxygen generation to kill cells and have been shown to be effective in the treatment of tumors in normoxic and hypoxic environments. 11 The released alkyl radicals can induce apoptosis of cancer cells by increasing oxidative stress, leading to cellular lipid and DNA damage. As a typical free radical initiator, 2,2 0 -azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (AIPH) can generate alkyl radicals at high temperatures. 12 It does so even in the absence of oxygen. The generated free radicals are toxic to cells and will immediately oxidize cell elements or interact with oxygen to produce secondary toxic substances. 13 Even in a hypoxic tumor microenvironment, AIPH can produce alkyl radicals to increase intracellular lipid hydroperoxide and further trigger tumor cell apoptosis. 14 All these indicate that Rc is a promising cancer treatment drug.
Light or heat is used as an external stimulus in photothermal therapy. [15][16][17] Photothermal therapy (PTT) based on near-infrared (NIR) laser irradiation has been used to prevent tumor cell aggregation; it relies on local thermal effects to remove the tumors. [18][19][20] Several photothermal agents (PTAs) such as gold nanocrystals and molybdenum disulde are used in combination with AIPH to release alkyl radicals in response to infrared laser irradiation. 19,[21][22][23] However, the tumor microenvironment (TME) usually has a high level of glutathione (GSH) expression in solid tumors because GSH plays an important role in resisting radiotherapy through a spontaneous reaction or a GSH Stransferase catalyzed reaction with xenogenics. 24,25 In addition, as a reducing agent, GSH can directly remove alkyl radicals. 21,26 This reduces the effectiveness of alkyl radical-based therapies.
Pyrite (FeS 2 ) nano-enzyme, as a novel photothermal nanomaterial, not only has a good photothermal effect under NIR light but also has a nano-enzyme activity. 27 The FeS 2 nanase has demonstrated similar activity to glutathione oxidase (GSH-OXD), oxidizing GSH to oxidized glutathione (GSSG). 28 In the TME, GSH depletion was found to disrupt the REDOX balance of cells and cause oxidative stress. 29 Therefore, FeS 2 is expected to achieve a good synergistic effect with AIPH.
Minimally invasive photoresponsive hydrogels have recently become popular as a controlled drug release platform. [30][31][32] Hydrogels coagulate as they are injected into tumor tissue and serve as a long-term depot. 33 This topical form of administration can be repeated aer a single injection. In addition, parameters such as laser power and irradiation time can be modied to change the drug release rate, thus extending the applicability of this treatment method. 34,35 Given these advantages, it was hypothesized that the use of hydrogels to deliver FeS 2 and AIPH to the TME would improve the efficacy of free radical-based therapies.
In this study, a method using intratumoral delivery of an injectable hydrogel containing FeS 2 nanozyme and the free radical initiator AIPH was designed. First, a hybrid system of AIPH-FeS 2 -hydrogel (AFH) was prepared by loading FeS 2 nanoparticles and AIPH into agarose hydrogel. The hydrogel is solid at room temperature, and FeS 2 and AIPH are encapsulated within its matrix. Once irradiated by near-infrared (NIR) light, FeS 2 converts NIR light energy into heat energy, which causes the AFH system to heat up, and then the hydrogel melts and releases FeS 2 and AIPH. At high temperatures, AIPH decomposes to produce alkyl radicals. Subsequently, FeS 2 nanozyme, which mimics the activity of GSH-OXD, reduces the intracellular GSH content. The destruction of GSH will promote the killing effect of alkyl radicals. Because AFH can reside at the tumor site for a long time, the AFH system can allow precise control over the release of alkyl radicals by changing the laser intensity and radiation time. Finally, the oxygen-independent free radicals generated by the AFH system under NIR irradiation and photothermal treatment can synergistically kill cancer cells through the synergistic oxidation/photothermal effect, and tumor growth during the treatment cycle is well inhibited. In short, the AFH system designed herein further amplies the therapeutic effect of AIPH by changing the redox homeostasis (Scheme 1).

Results and discussion
A low-melting-point agarose hydrogel was used to prepare a composite hydrogel containing FeS 2 and AIPH. FeS 2 was mixed with an aqueous agarose solution at 60 C and then loaded with the alkyl radical trigger AIPH, followed by quickly cooling to room temperature. Thus, the AFH hydrogel matrix was formed. Transmission electron microscopy was used to characterize the morphology of FeS 2 (Fig. 1A). The FeS 2 nanozyme showed sphere-like morphology with an average particle size of about 148 nm (Fig. S1 †). The UV-vis absorption spectra of FeS 2 (Fig. 1B) showed that FeS 2 strongly absorbed in the NIR range at 808 nm. This property makes FeS 2 a good PTA. The photothermal performance of FeS 2 nanozyme at different concentrations (0, 25, 50, and 100 mg mL À1 ) was studied by irradiating the solution with a 0.5 W cm À2 808 nm laser system (Fig. 1C). The heating effect of the solution was directly proportional to the FeS 2 concentration. The temperature of a 100 mg mL À1 FeS 2 solution increased by about 17.5 C in just 5 min of laser irradiation, demonstrating the good photothermal performance of FeS 2 . Scanning electron microscopy also showed that the AFH hydrogel has a complex network of pore structure and pore size distribution (Fig. 1D), which makes the hydrogel suitable for drug loading for delivery. Photothermal stability is also an important criterion for evaluating the quality of a PTA. 36 A 200 mg mL À1 FeS 2 solution was heated with an 808 nm NIR laser for 5 min, then the laser was turned off, and the FeS 2 solution was allowed to cool naturally to room temperature. This process was repeated several times to evaluate the photothermal stability of FeS 2 (Fig. 1E). There was no obvious change in the heating curve of each photothermal cycle, and only a slight difference was observed in the peak temperature achieved aer 5 min of irradiation, verifying the photothermal stability of FeS 2 . Rheological analysis also revealed a high storage modulus of the prepared hydrogel in the solid state at room temperature (Fig. 1F). As the temperature increased, the hydrogel gradually soened and dissolved, and the storage modulus gradually decreased. Next, a photothermal dissolution experiment was conducted on the FeS 2 hydrogel. The solidied AFH (FeS 2 contained in the hydrogel) was placed in a glass dish containing deionized water. At room temperature, the prepared AFH hydrogel maintained its solidied shape, but aer 10 min of irradiation with an 808 nm laser, the hydrogel almost completely dissolved, and the FeS 2 nanomaterial was released and dissolved into the water in the glass dish (Fig. 1G). A thermal infrared imager also conrmed the signicant increase in temperature of AFH during the irradiation (Fig. 1H). X-ray photoelectron spectroscopy (XPS) spectrum of FeS 2 aer reaction with hydrogel showed that FeS 2 contained Fe and S elements (Fig. S2 †). As shown in Fig. S3, † GSH content was signicantly decreased aer the co-incubation of FeS 2 and GSH, it has a positive correlation curve with time and concentration. Furthermore, the POD-like activity of the FeS 2 was sizedependent. Nanozymes with a size of 150 nm showed higher activity than those with sizes of 280 and 687 nm (Fig. S4 †).
Based on the high-performance characteristics of AFH, its antitumor effects were evaluated. Initially, the uorescein diacetate/propidium iodide live/dead cell staining kit was used to explore the killing effect of the prepared AFH hydrogel combined with NIR irradiation. The PBS + NIR group and the AFH only group showed strong green uorescence, whereas AIPH hardly showed any killing effect ( Fig. 2A). We prepared hydrogel only containing FeS 2 (FH) to verify relative experiments results. The prepared hydrogel containing only FeS 2 (FH) combined with NIR had a moderate killing effect. Notably, AFH + NIR treatment had the best cytotoxic effect.
The dichlorouorescein diacetate probe was used to detect the ability of AIPH to produce ROS. AIPH showed a weaker ability to induce ROS in the absence of laser irradiation, while FH loaded with FeS 2 showed a moderate degree of ROS production aer NIR irradiation (Fig. 2B). The bright green uorescence observed in the AFH + NIR treatment group could be attributed to the release of FeS 2 and AIPH aer laser irradiation (Fig. 2C). Furthermore, the groups of AIPH and AIPH + NIR showed very little green uorescence (Fig. S5 †). The CCK-8 assay also showed the same results. The cell viability of the AFH + NIR group was about 8.5%, which was signicantly different Scheme 1 Nanozyme hydrogel for enhanced alkyl radical generation and potent antitumor therapy. from that of the other groups (Fig. 2D). High temperature will promote the decomposition of AIPH to produce alkyl radicals. At the same time, FeS 2 can exert GSH-OXD activity to reduce intracellular GSH. GSH, as a ubiquitous thiol-containing tripeptide, is synthesized from its constituent amino acids (glutamic acid, cysteine, and glycine) in many cells. GSH is produced abundantly in various types of cancer cells. GSH in cells generally exists in the reduced form, which can react with oxidizing substances such as alkyl groups while getting oxidized to its oxidized form glutathione disulde (GSSG), thereby reducing the antitumor effect based on free radicals. 37 The depletion of GSH will disrupt the redox balance of cells, cause oxidative stress, and ultimately lead to cell apoptosis. 38 Ellman's reagent was used to test the ability of each group to deplete GSH. AFH combined NIR group showed the best GSH-depleting ability (Fig. 2E).
In view of its good in vitro performance as PTA and GSH-OXD-mimicking enzyme, the effect of AFH on the NIR light-toheat conversion was studied in vivo. BALB/c mice were injected subcutaneously with 4T1 cells to form tumors. Fig. 3A shows the temperature change curve of the PBS group and the AFH group aer 808 nm NIR laser 0.5 W cm À2 irradiation for 10 min. The temperature of the AFH group increased by about 17.6 C aer the irradiation, while the temperature of the PBS group hardly increased. High temperature can change the uidity of the tumor cell membrane, thereby increasing the permeability of the cell membrane, which in turn causes thermal damage to proteins. 39,40 Eventually, the cancer cells lose the ability to proliferate. Next, AFH-mediated antitumor activity was evaluated in 4T1 tumor-bearing mice. Tumor-bearing mice were divided randomly into ve groups. The tumor volume of mice in the PBS + NIR group and AIPH group increased rapidly during the two-week treatment period, and the AFH group showed a slight tumor suppressor effect (Fig. 3C). This was because agarose was slowly metabolized, and some drugs were slowly released. The AFH + NIR group had the strongest effect on tumor growth. During the treatment, the tumor volume of the mice was signicantly suppressed. Aer the treatment period, the mice were sacriced, and the tumors were isolated and weighed. Results for tumor weight were consistent with results for tumor volume (Fig. 3D).
Importantly, during the entire study, no changes in body weight were observed in the treatment group, which indicates that the treatment did not cause any signicant systemic toxicity to the mice (Fig. S6 †). This result is very encouraging because although many materials have shown good experimental results, they also caused serious side effects, which seriously hinder their clinical prospects. 41,42 2 0 ,7 0 -Dichlorouorescin diacetate was used to measure the intratumoral ROS generation in the treated mice. The staining was signicantly enhanced in the tumors subjected to the combination therapy of AFH + NIR (Fig. 3E). The enhanced free radical production led to an enhanced therapeutic effect in the animal model. TUNEL and Ki-67 staining analyses were used to verify cell apoptosis and proliferation (Fig. 3E). Tumor tissues in the AFH + NIR group were massively necrotic without signicant proliferation.
Aer treatment, the vital organs (heart, liver, spleen, lung, and kidney) did not have any inammation or damage. The liver and kidney indexes were normal (Fig. 4A-D and S7 †). Comprehensive in vivo experimental data show that synergistic AFH and PTT therapy has good therapeutic effects and biocompatibility. It has good prospects for clinical medical applications in the future.

Conclusion
We designed an injectable photo-responsive hydrogel that can simultaneously achieve free radical therapy and PTT by encapsulating the nanozyme FeS 2 and the alkyl radical source AIPH into agarose hydrogel. Under 808 nm laser irradiation, the PTA FeS 2 promotes the heating of the AFH system and leads to the release of AIPH and its decomposition to produce alkyl radicals. At the same time, FeS 2 can reduce the intracellular GSH content, thereby further destroying the redox homeostasis. The dissolution rate of the hydrogel can be changed by parameters such as laser power density and spot size. Both in vivo and in vitro studies have shown that the combination of AFH with NIR irradiation can achieve powerful tumor-killing effects with insignicant adverse effects. The hydrogel system designed in this study provides a strategy to enhance therapeutic systems based on alkyl radicals.

Conflicts of interest
The authors declare that there are no conicts of interest related to this article.