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• Reactive oxygen species (ROS) play a crucial role in both physiological and pathological processes in humans [1]. Under normal conditions, ROS contribute to intracellular redox regulation and influence cell and tissue functions [2]. However, during pathological events, such as inflammation or acute myocardial infarction (MI), ROS accumulate excessively intracellularly and extracellularly, leading to oxidative stress and disease progression [3–5]. Excessive ROS induce structural and functional alterations in lipids, proteins, and DNAs, ultimately resulting in cell dysfunction and apoptosis. Furthermore, ROS disrupt cell signaling pathways, disturb cell equilibrium, and impair physiological functions [6]. Therefore, establishing effective antioxidant systems to eliminate ROS, reduce oxidative stress, and maintain normal cell function is essential [7].

  Although the body's intrinsic antioxidant system, including enzymes, such as superoxide dismutase and glutathione peroxidase, helps maintain ROS balance, this equilibrium is often disrupted under pathological conditions, leading to ROS accumulation [5]. To address this issue, various antioxidant therapies have been developed, including antioxidant enzymes [8,9], nanoparticles that upregulate endogenous antioxidants [10], and antioxidant hydrogels [11]. Hydrogels have emerged as versatile biomaterials with extensive applications in biomedicine due to their excellent water absorption, biocompatibility, modifiable physicochemical properties, and capability to deliver bioactive molecules [12]. The unique structures and functionalities allow hydrogels to efficiently capture and neutralize ROS, thereby protecting cells from oxidative stress-induced damage [13]. Poly(amino acid)s, which encompass a variety of amino acids, exhibit diverse functions [14–16]. Methionine residues, with their sulfur ether groups prone to ROS oxidation, experience changes in hydrophilicity upon oxidation, affecting their secondary structures and self-assembly behaviors [17–19]. These changes can be advantageous for drug delivery applications.

  In this study, a poly(amino acid) methoxy poly(ethylene glycol)-block-poly(l-methionine) (mPEG-b-PMet, PM) was synthesized via the ring-opening polymerization of l-methionine N-carboxyanhydride (Met NCA), using amino-terminated mPEG (mPEG-NH₂; number-average molecular weight (M_n) = 2000.0 g/mol) as a macroinitiator (Scheme 1) [20]. PM demonstrated thermo-sensitive sol-to-gel transition properties and remarkable ROS scavenging capability. Its cytoprotective efficacy was validated using hydrogen peroxide (H₂O₂)-induced oxidative stress and oxygen-glucose deprivation (OGD) models. Mechanistic investigations using the OGD model revealed its potential for treating ischemic-related disorders.

  Scheme 1

  

  Scheme 1.  Synthesis of mPEG-PMet and its cytoprotective efficacy through anti-oxidation mechanism.

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  First, the successful synthesis of mPEG-NH₂ was confirmed by changes in the carbon-13 nuclear magnetic resonance (¹³C NMR) spectrum, specifically the disappearance of resonance signal at 61.4 ppm (-CH₂CH₂OH) and the appearance of a resonance signal at 40.4 ppm (-CH₂CH₂NH₂) (Fig. S1 in Supporting information). The successful synthesis of Met NCA was verified by proton nuclear magnetic resonance (¹H NMR) spectroscopy, which showed characteristic resonance signals at 7.0 ppm (-CONH-) and 2.2 and 2.1 ppm (-CH₂CH₂SCH₃) (Fig. S2 in Supporting information). The presence of representative peaks of the methionine residue (4.8, 2.6, and 2.1 ppm) confirmed the successful polymerization of Met NCA (Fig. 1A). The degree of polymerization of PMet was determined to be 30 by analyzing the ratio of methine peak at 4.8 ppm (-COCHNH-) to the methylene peak of mPEG at 3.8 ppm (-CH₂CH₂O-). Fourier transform-infrared (FT-IR) spectroscopy further supported the successful PM synthesis, showing absorption peaks at 3295.0 cm^-1 (secondary amine groups), 1657.5 cm^-1 (carbonyl groups), 1306.0 cm^-1 (ether groups), and 1107.0 cm^-1 (thioether groups) (Fig. 1B).

  Figure 1

  

  Figure 1.  Characterizations of PM. (A) ¹H NMR spectrum of PM. (B) FT-IR spectrum of PM. (C) Phase diagram of sol-to-gel transition of PM. (D) Modulus of PM changes with temperature. (E) CD spectra of PM as a function of temperature. (F) Quantitative analysis of CD spectra.

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  The PM aqueous solution displayed thermo-sensitive behavior, transitioning from sol to gel as temperature increased. This transition was characterized by the critical gelation temperature (CGT), which decreased as PM concentration increased. Specifically, at a 6.0 wt% PM concentration, CGT was 31.0 ℃, whereas, at 9.0 wt%, it decreased to 15.0 ℃ (Fig. 1C). CGTs are below body temperature, ensuring that the PM precursor solution forms a gel smoothly upon injection into the body. Rheological experiments supported these observations, demonstrating a rapid increase in the storage modulus of PM aqueous solution, escalating sharply from 5.3 Pa at 19.9 ℃ to 1020.0 Pa at 36.9 ℃ (Fig. 1D), indicating the transition from a sol to a gel phase. Before reaching CGT, PM aqueous solution exhibited lower storage moduli, facilitating thorough mixing with drugs and highlighting its potential as a drug delivery carrier. By adjusting the polymer's polymerization ratio and concentration, its mechanical properties can be finely tuned to better meet the physiological needs of various affected areas.

  To elucidate the mechanism underlying PM gelation, circular dichroism (CD) spectroscopy was employed to monitor temperature-induced alterations in the secondary structure (Fig. 1E). Initially, at 10.0 ℃, PM exhibited approximately half of its secondary structure in a random coil conformation, with around 10.0% in α-helix and the remainder in β-sheet structure. As the temperature increased from 10.0 ℃ to 60.0 ℃, the proportion of α-helix decreased from 10.0% to 3.0%, while β-sheet content increased from 43.0% to 50.0%. The content of the random coil remained unchanged (Fig. 1F). These shifts in the secondary structure are likely attributed to the dehydration effects of PEG segments as the temperature rises, promoting intermolecular interactions among PM molecules and facilitating micelle formation and aggregation, thereby driving the sol-to-gel phase transition [17]. β-Sheet, common in protein secondary structures, is crucial for regulating the properties of hydrogels [21]. In biomedical applications, adjusting the polymer concentration and β-sheet density allows hydrogels to mimic the physiological characteristics of specific pathological sites better.

  Scanning electron microscopy revealed that PM hydrogel exhibited a well-ordered internal network structure with a pore area percentage of approximately 31.6% (Fig. S3 in Supporting information). This porous network endows PM hydrogel with notable water absorption, air permeability, and stability, highlighting its potential for biomedical applications, particularly in drug delivery systems and tissue engineering.

  H₂O₂ serves as a primary ROS in vivo [22]. This study used H₂O₂ to investigate its interaction with PM. The thioether group in methionine confers PM with the ability to interact with ROS. Upon oxidation, PM hydrogel becomes more hydrophilic, leading to a transition from a gel to a solution state (Fig. 2A). Adding H₂O₂ to PM aqueous solution resulted in multiple resonance signals in the ¹H NMR spectra, spanning 4.4–4.3, 3.0–2.5, and 2.3–1.9 ppm. The intensities of these peaks positively correlated with both oxidation time and H₂O₂ concentration (Fig. 2B and Fig. S4 in Supporting information). Quantitative analysis revealed that with 40.0 mmol/L H₂O₂, complete PM oxidation required approximately five days, whereas 20.0 mmol/L H₂O₂ achieved only 65.3% oxidation over the same period. Lower H₂O₂ concentration reduced the oxidation rate, resulting in 22.7%–23.6% oxidation over five days (Fig. 2C).

  Figure 2

  

  Figure 2.  Oxidation process of PM. (A) Oxidation of PM hydrogel with H₂O₂. (B) ¹H NMR spectra of PM oxidized by H₂O₂ in D₂O. (C) Quantitative analysis of ¹H NMR spectra of PM oxidized by varying concentrations of H₂O₂ for different durations. (D) CD spectra of PM incubation with H₂O₂ for various time intervals. (E) Quantitative analysis of CD spectra.

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  Changes in the secondary structure of PM during oxidation were examined using CD spectroscopy. As oxidation time increased, α-helix content significantly increased. In contrast, β-sheet content decreased markedly (Fig. 2D). After 72 h of oxidation with 0.5 mmol/L H₂O₂, α-helix content increased from 5.0% to 34.0%, and β-sheet content dropped from 48.0% to 17.0%. The random coil content remained nearly unchanged (Fig. 2E). After oxidation, the hydrophilicity of PM side chains increased, resulting in a shift in secondary structure contrary to the gelation process, indicating a gel-to-sol transition.

  The in vitro degradation behavior of PM hydrogel was examined under varying concentrations of H₂O₂ and elastase. Exposure to 500.0 µmol/L H₂O₂ for 18 days resulted in a 93.8% degradation of the hydrogel. The degradation rate decreased progressively with lower H₂O₂ concentrations. In PBS without H₂O₂, only 35.1% of the hydrogel mass was lost. Meanwhile, 0.2 mg/mL of elastase led to nearly complete degradation of the hydrogel over 17 days (Fig. S5 in Supporting information). These results demonstrate that the degradation rate of the hydrogel is directly correlated with the concentration of H₂O₂, and elastase also accelerates hydrogel degradation.

  These findings indicate that the PM hydrogel underwent controlled degradation in ROS and elastase environments, facilitating on-demand drug release and confirming its potential for biomedical applications. The ability of PM to interact with ROS prompts further investigation into its protective effects against oxidative stress and OGD conditions.

  In vitro assays with L929 cells and H9c2 cells cultured in media containing varying concentrations of PM demonstrated that, after 24, 48, and 72 h of co-incubation, PM initially promoted cell proliferation. Although this effect diminished with prolonged culture time, PM still exhibited favorable cytocompatibility after 72 h (Fig. 3A and Figs. S6 and S7 in Supporting information). Live cell staining confirmed these observations (Fig. S8 in Supporting information). The hemolysis assay showed that at a PM concentration of 20.0 mg/mL, the hemolysis rate was 0.8%, and it continued to decrease with further reductions in PM concentration, indicating excellent hemocompatibility (Fig. S9 in Supporting information).

  Figure 3

  

  Figure 3.  Cytoprotective effect of PM against H₂O₂. (A) Cell viability of L929 cells cultured with various concentrations of PM for 72 h. (B) Cell viability of L929 cells under standard and H₂O₂ conditions and protective effect of PM against oxidation. (C) Relative LDH release in cell culture medium after oxidation. (D) Live cell staining of L929 cells under standard and H₂O₂ conditions and protective effect of PM against oxidation. (E) Quantitative analysis results of D. The statistical data are represented as mean ± standard deviation (SD; n = 8 for A, n = 6 for B, n = 3 for C and E). NS: no significance. P < 0.05, ***P < 0.001.

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  Subsequent experiments induced oxidative stress in L929 cells using H₂O₂ to assess the protective effect of PM. After 12 h of co-culture with H₂O₂, cell viability decreased by 42.5% when exposed to 100.0 µmol/L H₂O₂, further decreasing as H₂O₂ concentration increased. The addition of PM significantly improved cell viability, reaching 91.5% in cells treated with 100.0 µmol/L H₂O₂ (Fig. 3B). Measurement of lactate dehydrogenase (LDH) level in the culture medium to assess cell damage showed that treatment with 100.0 µmol/L H₂O₂ increased LDH content by 45.3%. In contrast, PM reduced LDH level by 11.6% (Fig. 3C). Live cell staining corroborated these findings, showing that PM effectively mitigated H₂O₂-induced cell damage, notably increasing viable cell count (Fig. 3D). Specifically, in the 100.0 µmol/L H₂O₂ treatment group, viable cell count increased by 1.7 times, and in the 200.0 µmol/L H₂O₂ treatment group, it increased by 2.6 times (Fig. 3E).

  In pathological conditions, such as ischemia-reperfusion, the H₂O₂ concentration at the lesion site may reach 100.0 µmol/L [23]. PM hydrogel has shown effectiveness in reducing oxidative stress-induced cell death under conditions similar to pathological H₂O₂ concentrations, confirming its potential therapeutic applications in conditions associated with ROS-mediated injury.

  Further studies employed OGD to simulate the ischemic pathological process in vivo, aiming to investigate the protective effects of PM against OGD-induced damage in H9c2 cells and the underlying mechanisms. 10 h of OGD resulted in a substantial 52.4% decrease in cell survival rate, significantly mitigated by PM, increasing cell survival to 81.2% (Fig. S10 in Supporting information). Live cell staining further validated these findings, showing that OGD reduced live cells to 43.6%, whereas PM increased cell survival to 63.8% (Fig. 4A and Fig. S11 in Supporting information). Moreover, OGD markedly increased LDH release by 82.3%, which PM reduced by 5.1% (Fig. 4B), indicating PM reduced OGD-induced cell damage.

  Figure 4

  

  Figure 4.  Cytoprotective effect and mechanism of PM. (A) Live cell staining of H9c2 cells under standard and OGD conditions and protective effect of PM against OGD. (B) Relative LDH release in cell culture medium after OGD. (C) Anti-apoptotic effect of PM on H9c2 cells after OGD. (D) Quantitative analysis results of C. (E, F) ROS scavenging ability of PM upon H9c2 cells under OGD condition. (G) Representative confocal images of JC-1 staining in H9c2 cells after OGD. (H) Quantitative analysis results of G. The statistical data are represented as mean ± SD (n = 3). P < 0.05, **P < 0.01, ***P < 0.001.

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  Under various pathological conditions, including cancer and ischemia, apoptosis regulation is frequently disrupted [24,25]. Following MI, the overexpression of apoptosis-related proteins is commonly observed in the ischemic myocardial tissue. Therapeutic strategies aimed at reducing apoptosis effectively alleviate tissue damage. Apoptosis analysis revealed that 10 h of OGD induced a 99.6% increase in apoptotic cells, reduced by 29.4% with PM intervention, highlighting its anti-apoptotic effect (Figs. 4C and D) and offering a unique mechanism for treating ischemic diseases.

  Intracellular ROS levels were assessed during OGD to evaluate the ROS scavenging ability of PM. Fluorescence staining revealed that the cells cultured under standard conditions produced minimal ROS, while OGD significantly elevated ROS levels. PM effectively scavenged ROS, reducing their levels by 35.8% (Fig. 4E and Fig. S12 in Supporting information). Flow cytometry corroborated these results (Fig. 4F and Fig. S13 in Supporting information), confirming the ROS clearance capability of PM hydrogel.

  Mitochondrial damage is a critical consequence of oxidative stress, leading to reduced mitochondrial membrane potential (MMP), opening of the mitochondrial permeability transition pore, and release of cytochrome c, ultimately resulting in cell apoptosis. MMP changes were monitored using JC-1 staining. Under normal conditions, JC-1 emits red fluorescence due to its aggregated form. Decreased MMP causes JC-1 to exist more in its monomeric form, emitting green fluorescence (Fig. 4G). OGD significantly reduced the red/green mean fluorescence intensity (MFI) ratio by 97.3%, indicative of MMP reduction, while PM treatment increased the ratio by 5.5-fold in the OGD group (Fig. 4H). ROS induce oxidative damage to mitochondria, which, in turn, produce more ROS, creating a vicious cycle [26]. PM effectively scavenged ROS, reduced mitochondrial damage, and broke this cycle, significantly enhancing cell protection.

  These comprehensive findings demonstrate that PM protects against cell apoptosis by clearing ROS and mitigating mitochondrial oxidative damage, suggesting its potential application for cell protection in ischemic conditions.

  In conclusion, a thermo- and ROS-responsive PM hydrogel was prepared, capable of transitioning from sol to gel in response to temperature and reverting from gel to solution in the presence of ROS. The hydrogel exhibited excellent biocompatibility and effectively shielded cells against oxidative stress induced by H₂O₂ and damaged caused by OGD. Its protective mechanism involved scavenging ROS, mitigating mitochondrial oxidative stress, and inhibiting cell apoptosis. Overall, the hydrogel holded potential for treating diseases associated with excessive ROS production, such as neurodegenerative diseases, MI, and cancer. In addition, this dual responsiveness enabled the hydrogel to adapt its release profile according to environmental cues, establishing a theoretical basis for its application as a drug delivery system.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Qiang Luo: Writing – original draft, Validation, Investigation, Conceptualization. Jinfeng Sun: Writing – review & editing. Zhibo Li: Investigation, Funding acquisition, Conceptualization. Bin Liu: Writing – review & editing, Supervision, Conceptualization. Jianxun Ding: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

  Acknowledgments

  This work was financially supported by the National Key R&D Program of China (No. 2022YFB3808000), the National Natural Science Foundation of China (No. U21A2099), the Science and Technology Development Program of Jilin Province (No. 20240101002JJ), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (No. Y2023066), and the Plan for Enhancing Health Science and Technology Capacity in Jilin Province (No. 2020J041).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110433.

  
  
• 1. [1]

     H. Wang, N. Wang, D. Xu, et al., Autophagy 16 (2020) 1683–1696. doi: 10.1080/15548627.2019.1704104

  2. [2]

     G.S. Shadel, T.L. Horvath, Cell 163 (2015) 560–569.

  3. [3]

     D.M. Yellon, D.J. Hausenloy, N. Engl. J. Med. 357 (2007) 1121–1135.

  4. [4]

     J. Liu, X. Han, T. Zhang, et al., J. Hematol. Oncol. 16 (2023) 116.

  5. [5]

     H.J. Forman, H. Zhang, Nat. Rev. Drug Discov. 20 (2021) 689–709. doi: 10.1038/s41573-021-00233-1

  6. [6]

     S. Kishi, H. Nagasu, K. Kidokoro, et al., Nat. Rev. Nephrol. 20 (2024) 101–119. doi: 10.1038/s41581-023-00775-0

  7. [7]

     S. Wang, Y. Yao, L. Song, et al., Biomaterials 307 (2024) 122534.

  8. [8]

     Q. Wang, C. Cheng, S. Zhao, et al., Angew. Chem. Int. Ed. 61 (2022) 202201101.

  9. [9]

     W. Le, Z. Sun, T. Li, et al., Adv. Funct. Mater. 34 (2024) 2314328.

  10. [10]

      G.-B. Im, Y.G. Kim, T.Y. Yoo, et al., Adv. Mater. 35 (2023) 2208989.

  11. [11]

      X. Liu, Y. Sun, J. Wang, et al., Bioact. Mater. 34 (2024) 269–281.

  12. [12]

      Q. Wang, J. Luan, Z. Zhao, et al., Chin. Chem. Lett. 34 (2023) 108060.

  13. [13]

      W. Yang, W. Zhong, S. Yan, et al., Adv. Mater. 36 (2024) 2312740.

  14. [14]

      H. Hu, Z. Zhang, Y. Fang, et al., Chin. Chem. Lett. 34 (2023) 107953.

  15. [15]

      Y. Zhang, Z. Zou, S. Liu, et al., Asian J. Pharm. Sci. 19 (2024) 100886.

  16. [16]

      L. Zhang, L. Lei, Z. Zhao, et al., Chin. Chem. Lett. 36 (2025) 110316.

  17. [17]

      Q. Xu, C. He, K. Ren, et al., Adv. Healthc. Mater. 5 (2016) 1979–1990. doi: 10.1002/adhm.201600292

  18. [18]

      T. Zhang, J. Yao, J. Tian, et al., Chin. Chem. Lett. 31 (2020) 1129–1132.

  19. [19]

      J. Liu, X. You, L. Wang, et al., Small 20 (2024) 2401438.

  20. [20]

      Y. Liu, D. Li, J. Ding, et al., Chin. Chem. Lett. 31 (2020) 3001–3014.

  21. [21]

      D.E. Clarke, E.T. Pashuck, S. Bertazzo, et al., J. Am. Chem. Soc. 139 (2017) 7250–7255. doi: 10.1021/jacs.7b00528

  22. [22]

      Q. Luo, W. Sun, Z. Li, et al., Biomaterials 303 (2023) 122368.

  23. [23]

      J.R. Burgoyne, S. Oka, N. Ale-Agha, et al., Antioxid. Redox Signal. 18 (2013) 1042–1052. doi: 10.1089/ars.2012.4817

  24. [24]

      B.A. Carneiro, W.S. El-Deiry, Nat. Rev. Clin. Oncol. 17 (2020) 395–417. doi: 10.1038/s41571-020-0341-y

  25. [25]

      C. Piamsiri, K. Jinawong, C. Maneechote, et al., Eur. Heart J. 44 (2023) 2.

  26. [26]

      D.B. Zorov, M. Juhaszova, S.J. Sollott, Physiol. Rev. 94 (2014) 909–950. doi: 10.1152/physrev.00026.2013

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  Scheme 1  Synthesis of mPEG-PMet and its cytoprotective efficacy through anti-oxidation mechanism.

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  Figure 1  Characterizations of PM. (A) ¹H NMR spectrum of PM. (B) FT-IR spectrum of PM. (C) Phase diagram of sol-to-gel transition of PM. (D) Modulus of PM changes with temperature. (E) CD spectra of PM as a function of temperature. (F) Quantitative analysis of CD spectra.

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  Figure 2  Oxidation process of PM. (A) Oxidation of PM hydrogel with H₂O₂. (B) ¹H NMR spectra of PM oxidized by H₂O₂ in D₂O. (C) Quantitative analysis of ¹H NMR spectra of PM oxidized by varying concentrations of H₂O₂ for different durations. (D) CD spectra of PM incubation with H₂O₂ for various time intervals. (E) Quantitative analysis of CD spectra.

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  Figure 3  Cytoprotective effect of PM against H₂O₂. (A) Cell viability of L929 cells cultured with various concentrations of PM for 72 h. (B) Cell viability of L929 cells under standard and H₂O₂ conditions and protective effect of PM against oxidation. (C) Relative LDH release in cell culture medium after oxidation. (D) Live cell staining of L929 cells under standard and H₂O₂ conditions and protective effect of PM against oxidation. (E) Quantitative analysis results of D. The statistical data are represented as mean ± standard deviation (SD; n = 8 for A, n = 6 for B, n = 3 for C and E). NS: no significance. P < 0.05, ***P < 0.001.

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  Figure 4  Cytoprotective effect and mechanism of PM. (A) Live cell staining of H9c2 cells under standard and OGD conditions and protective effect of PM against OGD. (B) Relative LDH release in cell culture medium after OGD. (C) Anti-apoptotic effect of PM on H9c2 cells after OGD. (D) Quantitative analysis results of C. (E, F) ROS scavenging ability of PM upon H9c2 cells under OGD condition. (G) Representative confocal images of JC-1 staining in H9c2 cells after OGD. (H) Quantitative analysis results of G. The statistical data are represented as mean ± SD (n = 3). P < 0.05, **P < 0.01, ***P < 0.001.

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