Injectable hydrogel with efficient ROS (reactive oxygen species) removal and H2S release capabilities as well as preparation method and application of injectable hydrogel

Abstract

The invention discloses injectable hydrogel with efficient ROS (reactive oxygen species) removal and H2S release capabilities, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: respectively synthesizing a cerium-based metal organic framework Ce-MOF and a pH response type H2S donor JK-1; blending and dispersing the two substances into a precursor solution containing polylysine and oxidized sodium alginate, and then adding carboxymethyl chitosan for crosslinking, so as to obtain injectable hydrogel Jamp with efficient ROS removal and H2S release capabilities; cEM (at) HG. Ce-MOF in the hydrogel can efficiently remove reactive oxygen species (ROS) and relieve oxidative stress; the JK-1 responds to release H2S in an acid environment of a psoriasis affected part, induces polarization of M2 type macrophages, reduces expression of inflammatory factors, plays anti-inflammatory and immune regulation roles, and effectively relieves the psoriasis through combination of the JK-1 and the M2 type macrophages.

Description

Technical Field

[0001] This invention belongs to the field of injectable hydrogel preparation, specifically relating to an injectable hydrogel with efficient ROS scavenging and H2S release capabilities, its preparation method, and its application. Background Technology

[0002] Psoriasis is an immune-mediated inflammatory skin disease characterized by chronic, relapsing erythematous plaques covered with silvery-white scales. The pathological mechanisms of psoriasis involve an imbalance between oxidation and antioxidation, systemic immune dysregulation, and the excessive release of pro-inflammatory cytokines. Reactive oxygen species (ROS) are oxygen-containing molecules or atoms produced in redox reactions, including free radicals (superoxide anion radicals) and non-free radicals (hydrogen peroxide). In psoriasis patients, the decreased activity of key antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) leads to insufficient ROS scavenging capacity, resulting in an imbalance between oxidation and antioxidation, thus causing excessive accumulation of ROS and exacerbating oxidative damage. Innate and adaptive immune cells play a crucial role in mediating the immune dysregulation in psoriasis, particularly the infiltration of T cells and neutrophils into psoriatic lesions, inducing the release of inflammatory factors, which is a key pathogenic mechanism of psoriasis. Therefore, effectively removing excess ROS from psoriatic lesions and improving immune infiltration are of great significance for the treatment of psoriasis.

[0003] The pathological process of psoriasis is closely related to chronic inflammatory responses. Pro-inflammatory cytokines (such as IL-17, IL-23, and TNF-α) activate abnormal proliferation of keratinocytes, promote angiogenesis, and recruit immune cell infiltration, forming a vicious cycle of "inflammation-oxidative stress." To address this complex pathology, employing multiple treatment modalities may lead to better efficacy and enhance the comprehensiveness and effectiveness of treatment.

[0004] Studies have shown that antioxidant enzymes are underexpressed in psoriasis patients and cannot effectively scavenge reactive oxygen species (ROS). Therefore, targeting the scavenging of reactive oxygen species, i.e., reducing oxidative stress damage by restoring redox balance, is a key aspect of psoriasis treatment. However, the clinical application of natural antioxidant enzymes is still limited by problems such as limited catalytic activity, low tissue permeability, and high preparation costs.

[0005] Hydrogen sulfide (H2S), as an important gaseous mediator, can penetrate cell membranes, promote the transformation of macrophages to the M2 phenotype, reduce the level of pro-inflammatory cytokines, help regulate the inflammatory state, and protect tissues from damage, thereby controlling the inflammatory response. However, the direct use of gaseous H2S has certain limitations, such as the uncontrollability of the gaseous state, difficulty in dosage control, and poor biostability. Therefore, finding safe and effective H2S donors to replace the direct use of H2S is particularly important. Traditional H2S donors, such as sodium hydrosulfide (NaHS) and sodium sulfide (Na2S), not only have short release times but also uneven release rates. In addition, these donors have poor chemical stability and are easily affected by the external environment, limiting their clinical application.

[0006] Hydrogels are widely used for drug delivery and wound dressings due to their three-dimensional cross-linked network structure, but they still have the following shortcomings: On the one hand, physically cross-linked hydrogels have poor mechanical properties, are easily broken by mechanical disturbances, and lack self-healing ability; on the other hand, traditional chemically cross-linked hydrogels are usually irreversibly cross-linked, making it difficult to form gels in situ in vivo by injection, and there may be biosafety hazards caused by cross-linking agent residues, and the drug sustained-release performance is insufficient. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an injectable hydrogel, J&CEM@HG, with highly efficient ROS scavenging and H2S release capabilities, along with its preparation method and applications. This invention provides an injectable self-healing hydrogel based on dynamic covalent cross-linking of Schiff base bonds. This hydrogel can rapidly gel in situ at the lesion site after injection, forming a stable structure. Its dynamically reversible chemical bonds endow the gel with excellent self-healing ability and shape adaptability, allowing it to automatically repair itself after external mechanical disturbances, avoiding structural breakage or detachment. Simultaneously, the uniform cross-linked structure within the gel enables long-term sustained drug release and precise local treatment. Compared with existing hydrogels, this invention offers advantages such as simple preparation, controllable gelation, good biocompatibility, and long-lasting therapeutic effects.

[0008] This invention first provides a method for preparing an injectable hydrogel with efficient ROS scavenging and H2S release capabilities, which includes the following steps:

[0009] 1) Dissolve terephthalic acid in dimethylformamide, add cerium ammonium nitrate aqueous solution to it, transfer the resulting mixture to a high-pressure reactor lined with polytetrafluoroethylene for reaction, and obtain cerium-based metal-organic framework Ce-MOF after dialysis and freeze-drying.

[0010] 2) Phosthioyl dichloride was dissolved in anhydrous dichloromethane. 3-hydroxypropionitrile and triethylamine were added sequentially under an inert atmosphere to obtain a reaction mixture. A mixed solution of glycine methyl ester, triethylamine, and dichloromethane was added to the reaction mixture and stirred. After the reaction was completed, the product was post-processed and purified to obtain a precursor. The obtained precursor was dissolved in methanol, and lithium hydroxide aqueous solution was added. The mixture was concentrated to dryness, and the resulting solid was resuspended in anhydrous methanol, filtered, concentrated again, and lyophilized to obtain the pH-responsive H2S donor JK-1.

[0011] 3) Prepare a precursor solution containing polylysine and sodium oxidized alginate, add Ce-MOF and JK-1 to the precursor solution, and then add carboxymethyl chitosan for cross-linking to obtain the injectable hydrogel.

[0012] According to a preferred embodiment of the present invention, in step 1), the mass ratio of terephthalic acid to cerium ammonium nitrate is 1:2 to 4; the concentration of terephthalic acid in dimethylformamide solution is 0.05-0.1 g / mL; and the concentration of cerium ammonium nitrate aqueous solution is 0.05-0.1 g / mL.

[0013] According to a preferred embodiment of the present invention, the reaction temperature in step 1) is 80-120°C and the reaction time is 0.5-1.5h.

[0014] According to a preferred embodiment of the present invention, in the reaction mixture of step 2), the volume ratio of thiophosphonodichloride to anhydrous dichloromethane in the reaction mixture is 1:7-8; the molar ratio of thiophosphonodichloride, 3-hydroxypropionitrile, and triethylamine is 1:(0.8-1.2):(0.8-1.2); and the reaction mixture is stirred and mixed at 0°C under an argon atmosphere.

[0015] According to a preferred embodiment of the present invention, in step 2), the volume ratio of phosthioyl dichloride in the reaction mixture to glycine methyl ester in the mixed solution is 1.5 to 2:1; the volume ratio of glycine methyl ester, triethylamine, and dichloromethane in the mixed solution is 1:(4 to 6):(12 to 14).

[0016] According to a preferred embodiment of the present invention, in step 2), the product is post-processed and purified to obtain the precursor, which includes: after the reaction is completed, diluting the reaction system with dichloromethane, then washing the organic phase with sulfuric acid aqueous solution, concentrating under reduced pressure, and purifying the crude product by column chromatography to obtain a yellow oily compound as the precursor.

[0017] According to a preferred embodiment of the present invention, in the precursor solution of step 3), the concentration of polylysine is 5-10 mg / mL, the concentration of sodium alginate is 30-40 mg / mL, the concentration of Ce-MOF in the precursor solution is 0.5-3 mg / mL, the concentration of JK-1 in the precursor solution is 2-5 mg / mL, and the concentration of carboxymethyl chitosan in the precursor solution is 15-25 mg / mL.

[0018] The present invention further provides an injectable hydrogel prepared by the aforementioned method.

[0019] The present invention further provides the application of the aforementioned injectable hydrogel in the preparation of medicaments for treating psoriasis.

[0020] Compared with the prior art, the beneficial effects of the present invention include:

[0021] (1) The injectable hydrogel prepared in this invention, based on the synergistic effect of a pH-responsive hydrogen sulfide donor and nanozymes, uses natural polymer materials as a framework and loads a cerium-based metal-organic framework (Ce-MOF) with multi-enzyme activity and a pH-responsive hydrogen sulfide donor, JK-1. Ce-MOF can efficiently scavenge reactive oxygen species (ROS) and reduce oxidative stress; JK-1 responds by releasing H2S in the acidic environment of psoriasis lesions, inducing M2 macrophage polarization, reducing the expression of inflammatory factors, and exerting anti-inflammatory and immunomodulatory effects. Together, they effectively alleviate psoriasis and have a therapeutic effect on psoriasis.

[0022] (2) The hydrogel obtained by the present invention has injectability and self-healing ability. The hydrogel can be injected into the body to form a stable gel structure in situ, realize the controlled release of H2S and rapid clearance of ROS, and has excellent antioxidant, anti-inflammatory and tissue repair properties.

[0023] (3) The present invention has low requirements for experimental instruments, the method is simple and easy to operate, and the hydrogel obtained has excellent performance in terms of mechanical properties, biocompatibility and psoriasis treatment. Attached Figure Description

[0024] Figure 1 This is a transmission electron microscope image of Ce-MOF, the product obtained in Example 1 of the present invention.

[0025] Figure 2 The image shows the hydrogen NMR spectrum of product JK-1 obtained in Example 1 of this invention.

[0026] Figure 3 This is a scanning electron microscope image of the product J&CEM@HG obtained in Example 2 of the present invention.

[0027] Figure 4 This is a graph showing the adhesion ability of the product J&CEM@HG obtained in Example 2 of the present invention.

[0028] Figure 5 This is a rheological diagram of the product J&CEM@HG obtained in Example 2 of the present invention.

[0029] Figure 6 This is a graph showing the antioxidant capacity of the product J&CEM@HG obtained in Example 2 of the present invention.

[0030] Figure 7 This image shows the therapeutic effect of the product J&CEM@HG obtained in Example 2 of this invention on psoriasis in mice. Detailed Implementation

[0031] The following embodiments provide those skilled in the art with guidance on how to manufacture and evaluate the present invention. These embodiments are merely illustrative of the present disclosure and do not limit the scope of the invention. While every effort has been made to ensure accuracy regarding numerical values ​​(e.g., quantities, temperatures, etc.), some errors and deviations should be considered. Unless otherwise stated, temperatures are expressed in °C or at ambient temperature.

[0032] Example 1

[0033] 0.177 g of terephthalic acid (H₂BDC) was added to 24 mL of dimethylformamide (DMF) solution and stirred magnetically until completely dissolved. Then, 8 mL of an aqueous solution containing 0.558 g of cerium ammonium nitrate [(NH₄)₂Ce(NO₃)₆] was added. After stirring for 10 minutes, the resulting mixture was transferred to a polytetrafluoroethylene-lined autoclave and sealed. Finally, the autoclave was maintained at 100°C for 1 hour. After natural cooling to room temperature, the resulting precipitate was collected and dialyzed against deionized water at room temperature for 2 days using a dialysis membrane. After dialysis, the solution was concentrated for subsequent use. Lyophilization yielded Ce-MOF powder.

[0034] 0.45 mL (3 mmol) of phosphonothioic dichloride was dissolved in 3.5 mL of anhydrous dichloromethane (CH₂Cl₂). Under an argon atmosphere at 0 °C, 0.2 mL (3 mmol) of 3-hydroxypropionitrile and 0.45 mL (3 mmol) of triethylamine were added sequentially to this solution. The reaction mixture was stirred at 0 °C for 15 minutes, and then stirred at room temperature for 3 hours. Subsequently, a mixture of glycine methyl ester (0.414 g, 3.3 mmol), triethylamine (1.25 mL, 9 mmol), and 3.5 mL of dichloromethane was added to the system, and the reaction was stirred for another 3 hours. After the reaction was complete, the mixture was diluted with 10 mL of dichloromethane, washed with 10 mL of 2M sulfuric acid aqueous solution, dried (anhydrous MgSO₄), and concentrated under reduced pressure. The crude product was purified by column chromatography (flash chromatography) to obtain a yellow oily compound as a precursor, in a yield of 554 mg (65%). The obtained precursor (150 mg, 0.5 mmol) was dissolved in 2 mL of methanol, and 2 mL of freshly prepared 1 M lithium hydroxide aqueous solution was added. The reaction solution was stirred at room temperature for 12 hours, and then concentrated to dryness. The resulting solid was resuspended in anhydrous methanol, filtered (using a 0.2 μm polytetrafluoroethylene membrane), concentrated again, and lyophilized to obtain JK-1 powder.

[0035] Transmission electron microscopy of the Ce-MOF product obtained in Example 1 is shown below. Figure 1 As shown, the product can be observed to be nanoparticles of approximately 2 nm in size. Figure 2 This is the hydrogen NMR spectrum of JK-1.

[0036] Example 2

[0037] A precursor solution containing polylysine (ε-PL) and oxidized sodium alginate (OSA) was prepared, with polylysine concentration of 8 mg / mL and oxidized sodium alginate concentration of 33 mg / mL. Ce-MOF and JK-1 were added to the precursor solution to make Ce-MOF final concentration of 1 mg / mL and JK-1 final concentration of 3.75 mg / mL. Then, carboxymethyl chitosan was added for cross-linking, with carboxymethyl chitosan final concentration of 20 mg / mL. After mixing, the system was allowed to stand at room temperature for 5 minutes to form a hydrogel simultaneously loaded with JK-1 and Ce-MOF, denoted as J&C@HG.

[0038] The scanning electron microscope image of the product J&CEM@HG obtained in Example 2 is shown below. Figure 3 As shown, the product J&CEM@HG has a relatively uniform pore structure.

[0039] To further verify the effectiveness of the present invention, the following control group was set up:

[0040] 1) HG group, i.e., the gel group without drug loading. Its preparation process is carried out according to Example 2, except that JK-1 solution and Ce-MOF solution are not added.

[0041] 2) JK-1@HG group. Preparation process: The procedure was carried out according to Example 2, except that Ce-MOF solution was not added.

[0042] 3) CeM@HG group. Preparation process: The procedure was carried out according to Example 2, except that JK-1 solution was not added.

[0043] The adhesion ability of the product J&CEM@HG obtained in Example 2 and the control group product is as follows: Figure 4 As shown, J&CEM@HG exhibits good adhesion, achieving a long-lasting drug release effect through prolonged adhesion to the skin surface. The rheological properties of the product J&CEM@HG obtained in Example 2 are as follows: Figure 5 As shown, the critical strain point of J&CEM@HG is approximately 70%, indicating that the material maintains a gel-like structure even when this strain threshold is reached. Under high strain (100%), the storage modulus (G') of the J&CEM@HG hydrogel decreases significantly, indicating a temporary disruption of the internal network. Notably, when the strain recovers to 1%, the G' value rapidly returns to its original level, demonstrating the effective reconstruction of the hydrogel network. This showcases the excellent self-healing ability of J&CEM@HG. Furthermore, G' exceeds G" and exhibits frequency-dependent properties, indicating the formation of an elastic network. In addition, rheological analysis confirms the shear-thinning behavior, suggesting excellent injectability of J&CEM@HG.

[0044] The antioxidant capacity of the products obtained in Example 2 and each control group was determined by... Figure 6 As shown, the product J&CEM@HG obtained in Example 2 exhibits excellent SOD and CAT activities, with a superoxide anion scavenging capacity of approximately 70% and a hydrogen peroxide scavenging capacity of approximately 80%, while also demonstrating time-dependent oxygen generation. Its DPPH radical scavenging capacity is approximately 80%, and its ABTS radical scavenging capacity is approximately 95%, indicating excellent antioxidant capabilities.

[0045] BALB / c mice aged 6-8 weeks were randomly divided into six groups, with six mice in each group (n=6). Back hair was removed with depilatory cream. Except for the Blank group, each mouse in the other five groups received 62.5 mg of 5% imiquimod (IMQ) cream (MedShine, China) on its back for seven consecutive days. Six hours after each IMQ application, mice underwent HG, JK-1@HG, CeM@HG, and J&CEM@HG application tests, i.e., 0.5 ml of each group's gel was applied to the lesion site on each mouse. For the PBS group, 0.5 ml of PBS was applied to the lesion site six hours after each IMQ application. The therapeutic effects of each group on psoriasis in mice were determined by... Figure 7 As shown in the figure, based on the experimental grouping and PASI scoring results, different gels exhibited significant differences in their effects on promoting wound healing. J&CEM@HG demonstrated the best therapeutic effect, and the figure shows that J&CEM@HG can effectively treat psoriasis.

[0046] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing an injectable hydrogel with efficient ROS scavenging and H2S release capabilities, characterized in that, Includes the following steps: 1) Dissolve terephthalic acid in dimethylformamide, add cerium ammonium nitrate aqueous solution to it, transfer the resulting mixture to a high-pressure reactor lined with polytetrafluoroethylene for reaction, and obtain cerium-based metal-organic framework Ce-MOF after dialysis and freeze-drying. 2) Phosthioyl dichloride was dissolved in anhydrous dichloromethane. 3-hydroxypropionitrile and triethylamine were added sequentially under an inert atmosphere to obtain a reaction mixture. A mixed solution of glycine methyl ester, triethylamine, and dichloromethane was added to the reaction mixture and stirred. After the reaction was completed, the product was post-processed and purified to obtain a precursor. The obtained precursor was dissolved in methanol, and lithium hydroxide aqueous solution was added. The mixture was concentrated to dryness, and the resulting solid was resuspended in anhydrous methanol, filtered, concentrated again, and lyophilized to obtain the pH-responsive H2S donor JK-1. 3) Prepare a precursor solution containing polylysine and sodium oxidized alginate, add Ce-MOF and JK-1 to the precursor solution, and then add carboxymethyl chitosan for cross-linking to obtain the injectable hydrogel.

2. The preparation method according to claim 1, characterized in that, In step 1), the mass ratio of terephthalic acid to cerium ammonium nitrate is 1:2 to 4; the concentration of terephthalic acid in dimethylformamide solution is 0.05-0.1 g / mL; and the concentration of cerium ammonium nitrate aqueous solution is 0.05-0.1 g / mL.

3. The preparation method according to claim 1, characterized in that, The reaction temperature in step 1) is 80-120℃, and the reaction time is 0.5-1.5h.

4. The preparation method according to claim 1, characterized in that, In the reaction mixture of step 2), the volume ratio of thiophosphonodichloro to anhydrous dichloromethane in the reaction mixture is 1:7-8; the molar ratio of thiophosphonodichloro, 3-hydroxypropionitrile, and triethylamine is 1:(0.8-1.2):(0.8-1.2); the reaction mixture is stirred and mixed at 0°C under an argon atmosphere.

5. The preparation method according to claim 1, characterized in that, In step 2), the volume ratio of phosthioyl dichloride in the reaction mixture to glycine methyl ester in the mixed solution is 1.5 to 2:1; the volume ratio of glycine methyl ester, triethylamine, and dichloromethane in the mixed solution is 1:(4 to 6):(12 to 14).

6. The preparation method according to claim 1, characterized in that, In step 2), the product is post-processed and purified to obtain the precursor, including: after the reaction is completed, dichloromethane is added to the reaction system for dilution, then the organic phase is washed with sulfuric acid aqueous solution and concentrated under reduced pressure. The crude product is purified by column chromatography to obtain a yellow oily compound as the precursor.

7. The preparation method according to claim 1, characterized in that, In the precursor solution of step 3), the concentration of polylysine is 5-10 mg / mL, the concentration of sodium alginate is 30-40 mg / mL, the concentration of Ce-MOF in the precursor solution is 0.5-3 mg / mL, the concentration of JK-1 in the precursor solution is 2-5 mg / mL, and the concentration of carboxymethyl chitosan in the precursor solution is 15-25 mg / mL.

8. An injectable hydrogel prepared by the method according to any one of claims 1-7.

9. The use of the injectable hydrogel of claim 8 in the preparation of a medicament for treating psoriasis.

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