A ceria composite nanenzyme hydrogel as well as a preparation method and application thereof

Abstract

The application discloses a cerium dioxide composite nano-enzyme hydrogel as well as a preparation method and application thereof. The composite nano-enzyme hydrogel is composed of beta-cyclodextrin modified cerium dioxide composite nano-enzyme loaded with myricetin and a hydrogel matrix. According to mass percentage, the beta-cyclodextrin modified cerium dioxide composite nano-enzyme loaded with myricetin is 0.3%, the hydrogel matrix is 11.4%, and the balance is ultrapure water. The preparation method is ingenious, simple in preparation steps, and does not need complex equipment, and is easy to scale production. The application is suitable for the nursing and treatment of diabetic chronic wounds at different parts and different healing stages, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a cerium dioxide composite nanoenzyme hydrogel, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is a widespread chronic metabolic disease and has become a serious global public health problem. Among its many complications, chronic wounds (especially diabetic foot ulcers) are a leading cause of disability, amputation, and even death. Clinical data shows that approximately 25% of diabetic patients have delayed-healing or non-healing skin wounds each year. These wounds are typically characterized by high morbidity, frequent recurrence, and poor treatment outcomes, placing a heavy burden on global healthcare systems. During wound healing, a moderate amount of reactive oxygen species (ROS) helps the body resist external stimuli, but diabetic patients have reduced antioxidant capacity due to high blood sugar, making them prone to excessive accumulation of ROS at the injury site. Excessive ROS can trigger severe oxidative stress, causing lipid membrane damage, protein oxidation, and nucleic acid damage. This process induces apoptosis in key repair cells such as keratinocytes and fibroblasts, inhibits their proliferation and migration, prolongs the inflammatory response, and hinders wound healing. Therefore, effectively regulating ROS levels is crucial for improving wound healing in diabetic patients.

[0003] To address oxidative stress in diabetic wounds, antioxidant-based strategies have been extensively studied. Polyphenols are commonly used antioxidants due to their powerful free radical scavenging capabilities, rapidly neutralizing reactive oxygen species and reducing oxidative damage. However, polyphenolic antioxidants are easily oxidized or degraded in physiological environments, have short effective durations, and repeated bursts of reactive oxygen species rapidly deplete their phenolic hydroxyl groups, resulting in a lack of sustained protection and limiting their effectiveness in chronic wound environments. Cerium oxide nanozymes, due to their reversible Ce... 3+ / Ce 4+ Redox cycles can continuously scavenge reactive oxygen species and their activity is regenerable. However, pure cerium oxide nanozymes are limited by slow electron transfer and insufficient oxygen vacancies, resulting in low catalytic efficiency. They are unable to quickly alleviate severe oxidative stress in the early stages of diabetic wounds and cannot dynamically adapt to the needs of different healing stages.

[0004] Therefore, there is an urgent need for a hydrogel that can both quickly eliminate the early burst of reactive oxygen species in wounds and provide long-term antioxidant protection for the healing of diabetic wounds. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a cerium dioxide composite nanoenzyme hydrogel, its preparation method, and its application.

[0008] To solve the above technical problems, the present invention provides the following technical solution: a cerium dioxide composite nanoenzyme hydrogel, comprising, (i) by mass percentage of the hydrogel raw material, 0.1~0.5% of β-cyclodextrin-modified cerium dioxide composite nanoenzyme loaded with myricetin, 5.52~12.8% of the hydrogel matrix, and the balance being water; (ii) In the β-cyclodextrin-modified cerium dioxide composite nanozyme loaded with myricetin, the mass content of β-cyclodextrin-modified cerium dioxide is 92.23~98.36%, and the mass content of myricetin is 1.64~7.77%; (iii) The hydrogel matrix is ​​composed of polyvinyl alcohol, xanthan gum and borax in a mass ratio of 25~50:2.5~10:1~4.

[0009] As a preferred embodiment of the cerium dioxide composite nanoenzyme hydrogel of the present invention, wherein, based on the mass percentage of the cerium dioxide composite nanoenzyme hydrogel, the β-cyclodextrin-modified cerium dioxide composite nanoenzyme loaded with myricetin accounts for 0.3%, the hydrogel matrix accounts for 11.4%, and the remainder is water.

[0010] As a preferred embodiment of the cerium dioxide composite nanozyme hydrogel described in this invention, the β-cyclodextrin-modified cerium dioxide composite nanozyme loaded with myricetin also possesses the following staged antioxidant properties: (i) Myricetin achieves rapid ROS clearance in the early stages: in the first three cycles, the DPPH clearance rate drops sharply from 81.8% to 64.5%; (ii) Cerium nanozymes ensure continuous catalytic ROS removal in the later stages: the removal rate decreased by only 5% after the last three cycles.

[0011] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing cerium dioxide composite nanoenzyme hydrogels, characterized by comprising: Polyvinyl alcohol is dissolved in ultrapure water to obtain a polyvinyl alcohol solution; A cerium dioxide composite nanozyme modified with xanthan gum and β-cyclodextrin and loaded with myricetin was added to a polyvinyl alcohol solution and stirred to obtain a homogeneous solution, which was then added to a borax solution to obtain the final product.

[0012] As a preferred embodiment of the cerium dioxide composite nanozyme hydrogel of the present invention, the preparation process of the β-cyclodextrin-modified cerium dioxide composite nanozyme loaded with myricetin includes, Cerium nitrate hexahydrate and β-cyclodextrin were dissolved in ultrapure water and stirred at room temperature. Sodium hydroxide was then added to obtain a mixture. The mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and heated, and then cooled to room temperature. The precipitate was collected by centrifugation and washed alternately with sodium chloride solution and ultrapure water to remove residual ions, thus obtaining β-cyclodextrin-modified cerium dioxide nanozyme. Next, myricetin was added to the β-cyclodextrin-modified cerium dioxide nanozyme suspension, stirred, and centrifuged to obtain a β-cyclodextrin-modified cerium dioxide composite nanozyme loaded with myricetin.

[0013] In a preferred embodiment of the preparation method described in this invention, polyvinyl alcohol is dissolved in ultrapure water at a dissolution temperature of 90-95°C and a stirring time of 1-2 hours.

[0014] As a preferred embodiment of the preparation method described in this invention, the cerium dioxide composite nanozyme modified with xanthan gum and β-cyclodextrin and loaded with myricetin is added to a polyvinyl alcohol solution and stirred at a temperature of 60-70°C for 0.5-1 h.

[0015] As a preferred embodiment of the preparation method described in this invention, cerium nitrate hexahydrate and β-cyclodextrin are dissolved in ultrapure water and stirred at room temperature. Then sodium hydroxide is added to obtain a mixture in which the molar ratio of cerium nitrate hexahydrate to β-cyclodextrin is 1:1 and the mass fraction of sodium hydroxide in the mixture is 3.5%.

[0016] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of cerium dioxide composite nanoenzyme hydrogel in promoting the healing of diabetic wounds.

[0017] Beneficial effects of this invention: (1) The hydrogel wound dressing provided by the present invention has a polyvinyl alcohol / xanthan gum / borax matrix forming a dynamic cross-linked network through borate ester bonds, which gives the hydrogel good flexibility and self-healing ability, can closely fit the wound and adapt to dynamic changes such as skin stretching, reduce dressing displacement, and create a stable and moist microenvironment for wound healing.

[0018] (2) The hydrogel wound dressing provided by the present invention combines the rapid antioxidant function of myricetin with the continuous antioxidant function of cerium oxide nanoenzyme, which solves the problem of short-term effect and single function of single active ingredients, and achieves the therapeutic effect of "rapid removal of reactive oxygen species and long-term continuous antioxidant".

[0019] (3) The hydrogel wound dressing provided by the present invention has catalase-like, superoxide dismutase-like and natural antioxidant activities, which can effectively remove excess reactive oxygen species in the wound microenvironment, effectively reduce oxidative stress and inflammatory response, and create a good microenvironment for wound healing.

[0020] (4) The hydrogel wound dressing provided by the present invention contains polyvinyl alcohol and xanthan gum, which are natural or synthetic polymers with excellent biocompatibility. The amount of borax is controlled within a safe range. The overall system has no obvious cytotoxicity and can promote cell adhesion and proliferation.

[0021] (5) The hydrogel wound dressing provided by the present invention has an ingenious preparation method, simple preparation steps, no need for complex equipment, and is easy to mass-produce. It is suitable for the care and treatment of diabetic chronic wounds in different locations and at different healing stages, and has broad clinical application prospects. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This is a schematic diagram of the synthesis path of CCMNRs in Embodiment 1 of the present invention.

[0023] Figure 2 This is a schematic diagram of the preparation process of PBX@CCM hydrogel in Example 5 of the present invention.

[0024] Figure 3 These are transmission electron microscope images of CCMNRs in Example 1 and CCNRs in Comparative Example 1 of the present invention.

[0025] Figure 4 The images shown are XRD images of the synthesized CCMNRs and CCNRs in Embodiment 1 and Comparative Example 1 of this invention.

[0026] Figure 5 The Fourier transform infrared spectra of β-cyclodextrin, myricetin, and CCMNRs in Example 1 of this invention are shown.

[0027] Figure 6 The images shown are actual photographs of the Ce3d peak spectrum in XPS before and after the addition of hydrogen peroxide to CCMNRs in Example 1 of this invention, as well as the color changes of the CCNRs and CCMNRs dispersion.

[0028] Figure 7 The image shows the XPS Ce3d peak spectrum of CCNRs in Comparative Example 1 of this invention.

[0029] Figure 8 The above figures show the simulated activity statistics of superoxide dismutase (SOD) and catalase (CAT) and the DPPH clearance statistics of CCMNRs in Example 1 and CCNRs in Comparative Example 1 of this invention.

[0030] Figure 9 This is a statistical chart showing the cyclic DPPH clearance rate of CCMNRs in Example 1 and CCNRs in Comparative Example 1 of the present invention.

[0031] Figure 10 P in embodiments 6, 7, and 8 of this invention 10 B 0.4 X1, P5B 0.2 X 0.5 and P 10 B 0.8 Images of the macroscopic morphology and self-healing properties of X2 hydrogel.

[0032] Figure 11 This is a statistical chart showing the cell survival rate of L929 cells after incubation with different concentrations of PBX@CCM hydrogel extract in Examples 5, 6, 9, 10, 11, and 12 of this invention.

[0033] Figure 12 The images are scanning electron microscope images of PBX@CCM in Example 5 of the present invention and PB and PBX in Comparative Examples 2 and 6.

[0034] Figure 13 The Fourier transform infrared spectra of PVA, xanthan gum, borax, and PBX@CCM in Example 5 of this invention are shown.

[0035] Figure 14 The rheological properties of PBX@CCM in Example 5 of this invention are obtained after 5 cycles under alternating low strain (1%) and high strain (600%) conditions.

[0036] Figure 15 The images show the color changes of PBX@CCM in Example 5 and PBX@CC hydrogel in Example 13 before and after the addition of hydrogen peroxide.

[0037] Figure 16 The above are statistical charts showing the simulated activities of superoxide dismutase (SOD) and catalase (CAT) and the ABTS clearance rate of PBX@CCM in Example 5 of the present invention and PB, PBX, PBX@CC, and PBX@MYR in Comparative Examples 2, 6, 13, and 14.

[0038] Figure 17The results of this invention, using an inverted fluorescence microscope, verify the antioxidant stress capacity of RAW 264.7 macrophages induced by lipopolysaccharide (LPS) in PBX@CCM in Example 5, PBX in Example 6, and PBX@MYR in Example 14.

[0039] Figure 18 The results of this invention, using an inverted fluorescence microscope, verify the anti-inflammatory ability of PBX@CCM in Example 5, PBX in Example 6, and PBX@MYR in Example 14 on RAW 264.7 macrophages induced by lipopolysaccharide (LPS).

[0040] Figure 19 This is a schematic diagram illustrating the effects of PBX@CCM and PBX hydrogel in promoting wound healing in diabetic rats in Examples 5 and 6 of the present invention. Detailed Implementation

[0041] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0042] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0043] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0044] Unless otherwise specified, all raw materials used in the embodiments of this invention are commercially available. See Table 1 for details.

[0045] Table 1

[0046] The cerium dioxide composite nanoenzyme hydrogel of this invention was characterized and its performance was determined according to the following methods: 1. TEM: TEM was used to observe the surface morphology of the nanozyme sample. 10 μL of the uniformly dispersed nanozyme dispersion, which had been ultrasonically treated, was slowly added dropwise to the surface of a copper mesh. After drying naturally at room temperature for 12 h, the sample-carrying copper mesh was placed into the sample holder of the TEM, the sample was injected, and representative images of the nanozyme from different fields of view were captured.

[0047] 2. SEM: SEM is used to observe the surface and cross-sectional morphology of hydrogel samples. When observing the cross-sectional morphology, the sample is freeze-dried and then fractured in liquid nitrogen. The sample is then placed on the conductive adhesive of the sample stage with the surface to be observed facing upwards, and platinum is sprayed on it before observation.

[0048] 3. XRD: The lyophilized nanozyme powder was placed in the test stage groove of the X-ray diffractometer, ensuring that the sample was aligned with the groove plane. The scanning parameters were set as follows: accelerating voltage 40kV, current 40mA, scanning range 5°-80°, and scanning speed 8° / min, to analyze the crystal structure of the nanozyme.

[0049] 4. FT-IR: At room temperature, Fourier transform infrared spectroscopy was used to record the hydrogel and its main components at 4000-500 cm⁻¹. -1 Infrared spectrum within the range.

[0050] 5. Antioxidant performance testing 1) The sustained antioxidant properties of nanozymes and hydrogel samples were analyzed using XPS and dispersion color change analysis.

[0051] i) The XPS spectrum of the Ce3d peak of CCMNRs was directly determined by XPS. Another 10 mg of CCMNRs was treated with 0.15 mM H2O2, and the XPS spectra of the Ce3d peak were recorded on day 0 and day 6 for comparison. The Ce3d peak in the CCMNRs was analyzed. 3+ With Ce 4+ Its self-regenerating redox capability.

[0052] ii) 1 mM H2O2 was added to CCNRs, CCMNRs dispersions (5 mM), and PBX@CC and PBX@CCM hydrogels, and photographs were taken to record the color changes. Six days later, 1 mM H2O2 was added again, and images were obtained to record the changes after the second reaction. The sustained antioxidant properties of CCNRs, CCMNRs, PBX@CC, and PBX@CCM were visually observed through the color changes before and after the reaction.

[0053] 2) The in vitro antioxidant properties of nanozymes and hydrogel samples were analyzed by testing DPPH, ABTS free radical scavenging efficiency, SOD and CAT enzyme simulated activities.

[0054] i) Place 100 mL of 5 mM CCNRs and CCMNRs dispersion in 2 mL of 0.1 mM 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) solution and react at room temperature for 30 min under light-protected conditions. Measure the absorbance of the solution at 517 nm using a UV spectrophotometer and calculate the DPPH free radical scavenging rate: DPPH scavenging rate = (Ablank - Asample) / Ablank × 100%, where Ablank is the absorbance of the DPPH solution before reacting with the hydrogel, and Asample is the absorbance of the DPPH solution after reacting with the hydrogel for 30 min.

[0055] ii) Prepare an ABTS working solution with an absorbance of 0.70±0.02. Then, place 100 mg of PB, PBX, PBX@CC, and PBX@CCM into 2 mL of the ABTS working solution and react at room temperature for 30 min under light-protected conditions. Measure the absorbance of the solution at 734 nm using a UV spectrophotometer and calculate the ABTS free radical scavenging rate: ABTS scavenging rate = (Ablank - Asample) / Ablank × 100%, where Ablank is the absorbance of the ABTS working solution before reacting with the hydrogel, and Asample is the absorbance of the ABTS working solution after reacting with the hydrogel for 30 min.

[0056] iii) The SOD-like activity of 1.0 mM CCNRs, CCMNRs and 100 mg PB, PBX, PBX@CC, and PBX@CCM hydrogels was evaluated by recording absorbance at 450 nm using the WST-1 assay kit.

[0057] iv) Assess CAT-like activity by monitoring hydrogen peroxide consumption. Mix 1 mL of 5 mM CCNRs, CCMNRs, and 1 g of PB, PBX, PBX@CC, and PBX@CCM with 10 mL of 10 mM hydrogen peroxide and incubate for 30 min. Then, take 1 mL of the mixture and add it to 2 mL of titanium sulfate solution and mix for 30 min. Measure the absorbance of the solution at 415 nm using a UV spectrophotometer. Further confirm the consumption of hydrogen peroxide by nanozymes and hydrogel samples by using a standard curve for quantitative hydrogen peroxide analysis using titanium sulfate.

[0058] 3) The phased antioxidant mechanism was directly verified by testing the cyclic DPPH scavenging of CCNRs and CCMNRs.

[0059] In the cyclic DPPH scavenging experiment, 2 mL of 0.1 mM DPPH solution was mixed with 100 μL of nanozyme dispersion, incubated in the dark for 30 min, and then centrifuged to obtain the supernatant. The absorbance at 517 nm was measured. After each cycle, the supernatant was discarded, and 2 mL of fresh DPPH solution was added. The above incubation and absorbance measurement were repeated for six consecutive cycles, and the DPPH radical scavenging efficiency for each cycle was calculated.

[0060] 6. Biocompatibility testing Mouse L929 fibroblasts were divided into 1×10⁻⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells per well in 96-well plates and cultured at 37°C (in a humid environment containing 5% carbon dioxide) for 24 h. The culture medium in the 96-well plates was replaced with equal volumes of CCMNRs extract at concentrations of 1, 2, 3, 4, and 5 mg / mL, and cultured for another 24 h. Finally, the liquid in the 96-well plates was removed, and 100 μL of 10% (V / V) CCK-8 solution was added and incubated for 2 h. The absorbance at 450 nm was measured using a microplate reader. The group containing only culture medium was used as a negative control, and the group containing 0.01 g / mL zinc diethyldithiocarbamate was used as a positive control. The survival rate of L929 fibroblasts was calculated using the following formula (1): Cell viability (%) = (A t -A0) / (A c -A0)×100% formula (1); Among them, A t Indicates the ultraviolet absorption of the experimental group; A c A represents the UV absorption of the negative control group; A0 represents the UV absorption of the positive control group.

[0061] 7. Intracellular reactive oxygen species scavenging capacity of PBX@CCM hydrogel RAW264.7 cells were spaced at 5 × 10⁶ cells per dish. 5 Cells were seeded at a density of [number] cells per confocal culture dish and cultured at 37°C (in a humidified environment containing 5% carbon dioxide) for 24 h. Subsequently, cells were co-treated with lipopolysaccharide (5 μg / mL) and either PBX hydrogel extract or PBX@CCM hydrogel extract for 24 h. After treatment, cells were incubated with a DCFH-DA probe (10 mM) for 30 min. Intracellular reactive oxygen species levels were assessed by recording fluorescence intensity using confocal laser scanning microscopy. 8. Intracellular anti-inflammatory capacity of PBX@CCM hydrogel RAW264.7 cells were cultured and stimulated with LPS (5 mg / mL) under PBX or PBX@CCM hydrogel incubation (as described above). After treatment, cells were fixed with 4% paraformaldehyde for 30 minutes, washed three times with PBS, and blocked with 5% goat serum for 1 hour at room temperature. Primary antibodies against CD86, CD163, and CD206 were diluted in blocking buffer according to the manufacturer's instructions and incubated with cells overnight at 4°C in the dark. Cells were then washed three times with PBS and incubated with the corresponding secondary antibodies for 2 hours at room temperature in the dark. Cell nuclei were counterstained with DAPI for 20 minutes. After washing three times with PBS, fluorescence images were acquired using a confocal laser scanning microscope.

[0062] 9. Observation on the effect of promoting the healing of diabetic wounds Male Sprague-Dawley rats weighing 220-250 grams were selected. After a one-week acclimatization period, a diabetic model was induced by intraperitoneal injection of STZ (40 mg / kg) for three consecutive days. Successful induction was confirmed by a non-fasting blood glucose level (16.7 mM and a stable hyperglycemic state for one week). The experimental animals were randomly divided into three groups (n=8 per group): a control group (500 mL saline), a PBX group, and a PBX@CCM group. All procedures were performed under isoflurane anesthesia. After diagnosis of diabetes, the hair on the back was shaved and the skin was cleaned and disinfected. An 8 mm diameter full-thickness excision wound was created on the back. To reduce wound contraction, a 12 mm inner diameter silicone ring was used to fix the area around the wound. Subsequently, 500 mL of saline (control group), PBX hydrogel, or PBX@CCM hydrogel were applied to the wound surface. The wound was covered with a 3 MTegaderm film to prevent scratching or biting until the end of the experiment. The hydrogel was replaced on days 3, 7, and 10. Digital photos were taken on days 0, 3, 7, 10, and 14.

[0063] Example 1 (1) Dissolve 3 mmol of cerium nitrate hexahydrate and 3 mmol of β-cyclodextrin in 45 mL of ultrapure water and stir at room temperature.

[0064] (2) Then, 1.8 g of sodium hydroxide was slowly added while stirring continuously. After 10 minutes, the mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and heated at 120°C for 6 hours, and then cooled to room temperature.

[0065] (3) The precipitate was collected by centrifugation and washed alternately with 2% sodium chloride solution and ultrapure water to remove residual ions, yielding β-cyclodextrin-modified cerium dioxide nanozymes (CCNRs). Then, 25 mg of myricetin was added to a dispersion containing 300 mg CCNRs, and the mixture was stirred for 6 hours. Finally, β-cyclodextrin-modified cerium dioxide composite nanozymes loaded with myricetin (CCMNRs) were collected by centrifugation and dispersed in water for subsequent use.

[0066] Example 2 The difference between this embodiment and embodiment 1 is that step 3) is adjusted so that 5 mg of myricetin is added to the suspension containing 300 mg CCNRs, wherein the mass fraction of myricetin is 1.64%. The remaining steps are the same as in embodiment 1.

[0067] Example 3 The difference between this embodiment and embodiment 1 is that step 3) is adjusted so that 15 mg of myricetin is added to the suspension containing 300 mg CCNRs, wherein the mass fraction of myricetin is 4.76%. The remaining steps are the same as in embodiment 1.

[0068] Example 4 The difference between this embodiment and embodiment 1 is that step 3) is adjusted so that 35 mg of myricetin is added to the suspension containing 300 mg CCNRs, wherein the mass fraction of myricetin is 10.45%. The remaining steps are the same as in embodiment 1.

[0069] Comparative Example 1 The difference between this comparative example and Example 1 is that myricetin was not added after obtaining CCNRs in step 3), while the remaining steps were the same as in Example 1.

[0070] The DPPH radical scavenging rate and loading efficiency of the above embodiments and comparative examples were tested. Three parallel experiments were set up for each sample. The results are shown in Table 2. Table 2

[0071] The results showed that the introduction of MYR could significantly enhance the antioxidant activity of nanozymes. When the amount of MYR added was 25 mg, the antioxidant activity and loading efficiency of the composite nanozymes were close to saturation. Further increasing the amount of MYR could not achieve a significant improvement in antioxidant performance, and the loading efficiency also decreased.

[0072] Figure 1 This is a schematic diagram of the synthesis path of CCMNRs in Embodiment 1 of the present invention. Figure 3The images show TEM images of CCMNRs in Example 1 and CCNRs in Comparative Example 1. It can be seen that both CCNRs and CCMNRs exhibit uniformly dispersed short rod-shaped structures with a length of approximately 50-70 nm.

[0073] Figure 4 The XRD images of the CCMNRs and CCNRs synthesized in Example 1 and Comparative Example 1 are shown. The results show that CCNRs and CCMNRs have diffraction peaks at 2θ values ​​of 28.5, 33.1, 47.5, 56.3, 59.1, 69.4, 76.7 and 79.1, which correspond to the (111), (200), (220), (311), (222), (400), (331) and (420) crystal planes of the cerium nanoenzyme cubic fluorite structure, confirming that both CCNRs and CCMNRs have the typical cubic fluorite structure of cerium nanooxide.

[0074] Figure 5 The Fourier transform infrared (FT-IR) spectra of β-cyclodextrin, myricetin, and CCMNRs in Example 1 are shown. It can be seen in the FT-IR spectra of the CCMNRs that at 556 cm⁻¹... -1 Characteristic Ce-O vibrational peaks of nano-cerium dioxide can be observed at 1022 cm⁻¹. -1 A β-CD-related peak (COC) appears at 1655 cm⁻¹. Additionally, a peak at 1655 cm⁻¹ is observed. -1 (C=O) and 1594cm -1 A MYR-related peak appears at (C=C), and at 3391 cm⁻¹ -1 The broad OH stretching vibration band at the location was clearly identified by comparison with the spectra of β-CD and MYR, confirming their presence in CCMNRs and demonstrating the successful synthesis of CCMNRs.

[0075] Figure 6 The images show the Ce3d peak spectrum in XPS and actual photos of the dispersion of CCMNRs before and after the addition of H2O2 in Example 1. Figure 7 The XPS Ce3d peak spectrum of CCNRs in Comparative Example 1 is shown. The XPS results show ( Figure 6 a) Ce in CCMNRs 3+ The component accounts for 48.48% of the total cerium. After adding H2O2, Ce... 3+ The proportion dropped to 34.18% (a decrease of approximately 14.3%), indicating that Ce... 3+ Oxidized to Ce 4+ After standing for 6 days, Ce 3+ The content recovered to 45.81%, indicating that CCMNRs can partially recover their reduced state over time and possess self-regenerating redox capabilities. The color change results of the nanozyme dispersion are consistent with the spectral results. Figure 6(b) After treatment with H2O2, the color of the CCNRs dispersion changed from milky white to yellow, and the CCMNRs dispersion changed from light brown to brownish-yellow. Both gradually returned to their initial colors after standing, visually reflecting the color change of Ce. 3+ and Ce 4+ Reversible redox transition. XPS comparison of CCNRs and CCMNRs shows that MYR, with its strong reducing ability, can further reduce Ce when loaded onto CCMNRs. 4+ Restored to Ce 3+ This leads to Ce on the surface of CCMNRs nanozymes 3+ / Ce 4+ The valence ratios significantly increased from 40.92% / 50.08% to 48.48% / 51.52%. This indicates that CCMNRs can maintain active site regeneration even under repeated oxidation challenges, and MYR loading can promote the regeneration of their active sites, demonstrating excellent sustained antioxidant performance.

[0076] Figure 8 The charts show the simulated activities of superoxide dismutase (SOD) and catalase (CAT) in CCMNRs of Example 1 and the DPPH scavenging rate in CCNRs of Comparative Example 1. It is evident that the MYR-loaded CCMNRs exhibit excellent antioxidant capacity. Both the DPPH free radical scavenging rate and the activities of CAT and SOD enzymes are significantly superior to those of CCNRs, indicating that MYR loading can further enhance the antioxidant performance of CCNRs, highlighting their rapid antioxidant capacity.

[0077] Table 3. DPPH clearance rates of CCNRs and CCMNRs after 6 cycles.

[0078] Figure 9The table above shows the cyclic DPPH scavenging efficiency of CCMNRs in Example 1 and CCNRs in Comparative Example 1. As shown in the table, the DPPH scavenging efficiency of CCNRs gradually decreased with increasing cycle number, from 62.2% in the first cycle to 49.4% in the sixth cycle, reflecting the sustained catalytic antioxidant properties of cerium nanozymes. In contrast, CCMNRs exhibited a biphasic decay pattern: in the first three cycles, the scavenging rate dropped sharply from 81.8% to 64.5%, due to the gradual consumption of MYR phenolic hydroxyl groups caused by irreversible free radical scavenging; from the fourth to the sixth cycle, the rate of decline slowed significantly (only a 5% decrease), and its decay trend was similar to that of CCNRs, indicating that the contribution of MYR had been largely exhausted, and the catalytic activity of cerium nanozymes became the main source of antioxidant activity. This unique biphasic characteristic provides direct experimental evidence for the staged antioxidant design of CCMNRs: MYR achieves rapid ROS scavenging in the early stage, while cerium nanozymes ensure sustained catalytic ROS scavenging in the later stage.

[0079] Example 5 2g of polyvinyl alcohol (PVA) was added to 15mL of ultrapure water and stirred at 90°C for 1 hour until completely dissolved. Then, after the solution temperature dropped to 55°C, 0.2g of xanthan gum (XG) and 60mg of CCMNRs were added, and the mixture was heated and stirred for 1 hour until a homogeneous solution was obtained. 80mg of borax was completely dissolved in 5mL of ultrapure water and added to the above prepolymer solution. The mixture was gently stirred to obtain the cerium dioxide composite nanoenzyme hydrogel of this embodiment, denoted as PBX@CCM. The mass concentrations of PVA, XG, CCMNRs, and Borax were 10wt.%, 1wt.%, 0.3wt.%, and 0.4wt.%, respectively.

[0080] Figure 2 This is a schematic diagram of the preparation process of PBX@CCM hydrogel in Example 5 of the present invention.

[0081] Example 6 The difference between this embodiment and Example 5 is that CCMNRs were not added, the concentration of the remaining components remained the same as in Example 5, and the steps and processes were all the same as in Example 5. The resulting polyvinyl alcohol-xanthan gum-borax hydrogel was denoted as P. 10 B 0.4 X1.

[0082] Example 7 The difference between this embodiment and Example 6 is that the concentration of PVA in the prepolymer solution is adjusted to 5 wt.%, the concentration of xanthan gum to 0.5 wt.%, and the concentration of 5 mL of borax to 0.8 wt.%, while the remaining steps and processes are the same as in Example 6. This yields the polyvinyl alcohol-xanthan gum-borax hydrogel of this embodiment, denoted as P5B. 0.2 X0.5 .

[0083] Example 8 The difference between this embodiment and Example 6 is that the concentration of xanthan gum in the prepolymer solution is adjusted to 2 wt.%, and the concentration of borax in 5 mL is adjusted to 3.2 wt.%. All other steps and processes are the same as in Example 6, resulting in the polyvinyl alcohol-xanthan gum-borax hydrogel of this embodiment, denoted as P. 10 B 0.8 X2.

[0084] Figure 10 P in embodiments 6, 7, and 8 of this invention 10 B 0.4 X1, P5B 0.2 X 0.5 and P 10 B 0.8 Images of the macroscopic morphology and self-healing properties of the X2 hydrogel. P5B 0.2 X 0.5 The hydrogel did not form a stable three-dimensional cross-linked network structure, and was in a fluid state with high fluidity, making it unable to self-support and mold. 10 B 0.4 X1 hydrogel possesses a certain degree of plasticity and excellent self-healing properties. 10 B 0.8 The cross-linking density and network rigidity of the X2 hydrogel are significantly enhanced. The hydrogel changes from a viscoelastic material with dynamic response to a brittle material, and it cannot achieve effective bonding and self-repair of the damaged interface.

[0085] Example 9 The difference between this embodiment and Example 5 is that 20 mg of CCMNRs is added to make the concentration of CCMNRs in the prepolymer solution 0.1 wt.%, while the concentrations of other components remain unchanged. The remaining steps and processes are the same as in Example 5, resulting in the cerium dioxide composite nanoenzyme hydrogel of this embodiment, denoted as PBX@CCM-1.

[0086] Example 10 The difference between this embodiment and Example 5 is that 40 mg of CCMNRs was added to make the concentration of CCMNRs in the prepolymer solution 0.2 wt.%, while the concentrations of other components remained unchanged. The remaining steps and processes were the same as in Example 5, resulting in the cerium dioxide composite nanoenzyme hydrogel of this embodiment, denoted as PBX@CCM-2.

[0087] Example 11 The difference between this embodiment and Example 5 is that 80 mg of CCMNRs was added to make the concentration of CCMNRs in the prepolymer solution 0.4 wt.%, while the concentrations of other components remained unchanged. The remaining steps and processes were the same as in Example 5, resulting in the cerium dioxide composite nanoenzyme hydrogel of this embodiment, denoted as PBX@CCM-4.

[0088] Example 12 The difference between this embodiment and Example 5 is that 100 mg of CCMNRs is added to make the concentration of CCMNRs in the prepolymer solution 0.5 wt.%, while the concentrations of other components remain unchanged. The remaining steps and processes are the same as in Example 5, resulting in the cerium dioxide composite nanoenzyme hydrogel of this embodiment, denoted as PBX@CCM-5.

[0089] Figure 11 The cell viability of L929 cells after incubation with different concentrations of PBX@CCM hydrogel extract was shown. Both the PBX hydrogel and the low-concentration drug-loaded groups (PBX@CCM-1~3) exhibited good cell compatibility (viability ≥80%). With increasing nanozyme concentration, the cell viability of PBX@CCM-4 and PBX@CCM-5 gradually decreased, with PBX@CCM-5 showing a significantly lower viability than 80%, indicating a marked cell inhibitory effect.

[0090] Example 13 The difference between this embodiment and Example 5 is that CCMNRs are replaced with CCNRs, the concentration of the remaining components remains the same as in Example 5, and the remaining steps and processes are all the same as in Example 5, so that the cerium dioxide nanoenzyme hydrogel of this comparative example is obtained, denoted as PBX@CC.

[0091] Example 14 The difference between this embodiment and Example 5 is that CCMNRs are replaced with MYR, the concentration of the remaining components remains the same as in Example 5, and the remaining steps and processes are all the same as in Example 5, so that the cerium dioxide nanoenzyme hydrogel of this comparative example is obtained, denoted as PBX@CC.

[0092] Comparative Example 2 The difference between this comparative example and Example 5 is that XG and CCMNRs were not added, the concentration of the remaining components remained the same as in Example 5, and the remaining steps and processes were all the same as in Example 5. The resulting polyvinyl alcohol-borax hydrogel was denoted as PB.

[0093] Figure 12The images show SEM images of PBX@CCM in Example 5 and PB and PBX in Comparative Examples 2 and 6. It can be seen that PB has a porous structure with a pore size of approximately 10-20 μm and relatively thick pore walls. After the addition of XG, the pore walls of PBX become thinner and interconnected, forming a cross-linked porous network. The addition of CCMNRs to PBX@CCM did not significantly change the pore size or overall network structure of PBX.

[0094] Figure 13 Fourier transform infrared spectra of PVA, xanthan gum, borax, and PBX@CCM in Example 5, at 3316 cm⁻¹. -1 The broad peak observed at 1420 cm⁻¹ is attributed to the OH stretching vibration, reflecting the hydrogen bonding between PVA and XG. Additionally, at 1420 cm⁻¹... -1 and 1334cm -1 The two characteristic peaks appearing at the specified position correspond to the asymmetric stretching vibrations of the borate ester bonds formed between borax, PVA, and XG. These results indicate that the construction of the PBX@CCM network is achieved through the combined action of hydrogen bonds and borate ester bonds.

[0095] Figure 14 The rheological properties of PBX@CCM in Example 5 under alternating low strain (1%) and high strain (600%) conditions for 5 cycles are shown. It can be seen that when PBX@CCM is subjected to large strain, the internal network will temporarily break down, but once the strain is removed, the hydrogel will quickly recover its original mechanical properties, indicating that PBX@CCM has excellent self-healing ability, which makes it significantly advantageous to adapt to wound movement and repeated mechanical disturbances in vivo.

[0096] Figure 15 The images show the color changes of PBX@CCM in Example 5 and PBX@CC hydrogels in Example 13 after the addition of H2O2. After H2O2 treatment, due to Ce... 3+ Oxidized to Ce 4+ The colors of PBX@CC and PBX@CCM hydrogels changed from nearly white and dark brown to yellow and brownish-yellow, respectively, and gradually returned to their initial colors after standing for 1 day, indicating that Ce 3+ Spontaneous regeneration occurred. Upon re-exposure to H2O2, the color changed again, confirming that the CCMNRs loaded in the hydrogel still retained Ce. 3+ / Ce 4+ It has a redox cycle and a continuous ability to scavenge reactive oxygen species.

[0097] Figure 16The figures show simulated activity statistics of superoxide dismutase (SOD) and catalase (CAT) and ABTS scavenging rate of PBX@CCM in Example 5 and PB, PBX, PBX@CC, and PBX@MYR in Comparative Examples 2, 6, 13, and 14. It can be seen that the ABTS free radical scavenging rate, SOD, and CAT enzyme activities of PBX@CCM hydrogel reached the highest levels in all groups, exhibiting the best comprehensive antioxidant performance. MYR mainly relies on rapid and direct free radical scavenging, while CCNRs exhibit the core advantage of long-lasting antioxidant properties through multi-enzyme catalysis. The combination of the two components in PBX@CCM achieves complementary antioxidant performance.

[0098] Figure 17 To verify the antioxidant stress capacity of RAW 264.7 macrophages induced by lipopolysaccharide (LPS) in Examples 5, 6, and 14 using inverted fluorescence microscopy, the results of tests using PBX@CCM, PBX, and PBX@MYR were investigated. Significant intracellular fluorescence signals were observed in the LPS-stimulated group (positive control), indicating a significant increase in intracellular ROS levels. Compared to the LPS group, the PBX-treated group showed no significant difference, indicating that PBX alone failed to effectively reduce ROS levels. In contrast, the fluorescence signal in the PBX@MYR-treated group was significantly weakened, while the ROS level in the PBX@CCM-treated group decreased to a level comparable to the negative control group, indicating that the PBX@CCM group exhibited the strongest intracellular ROS scavenging effect among all treatment groups.

[0099] Figure 18 To verify the anti-inflammatory effects of PBX@CCM, PBX, and PBX@MYR on RAW 264.7 macrophages induced by lipopolysaccharide (LPS) using inverted fluorescence microscopy, the results showed that LPS stimulation significantly upregulated the expression of CD86, a marker of M1 macrophages, while inhibiting the expression of CD163 and CD206, markers of M2 macrophages. The PBX group showed no significant effect on macrophage polarization. The PBX@MYR group partially inhibited CD86 expression while upregulating the expression of CD163 and CD206. The PBX@CCM group significantly reduced CD86 fluorescence intensity and greatly increased the expression levels of CD163 and CD206, exhibiting the strongest M2 polarization induction ability. This indicates that PBX@CCM has the most significant anti-inflammatory and immunomodulatory effects.

[0100] Figure 19This diagram illustrates the effects of PBX@CCM and PBX hydrogel from Examples 5 and 6 on promoting wound healing in diabetic rats. It shows that during the 14-day observation period, the PBX group only showed limited improvement compared to the control group, indicating that the basic hydrogel matrix primarily functions through moisturizing rather than active biological regulation. In contrast, the wound closure rate in the PBX@CCM treatment group was significantly faster than both the control and PBX hydrogel groups, exhibiting significant wound area reduction and accelerated new tissue formation. These results demonstrate that PBX@CCM can significantly accelerate wound healing in diabetic rats, showcasing its potential application value as a chronic wound dressing.

[0101] This invention proposes a phased treatment strategy of early rapid antioxidant activity and long-term sustained antioxidant activity. Using β-cyclodextrin as a carrier, myricetin and cerium oxide nanozymes are combined to form a stable composite nanozyme system (myricetin exerts a short-term potent antioxidant effect, while cerium oxide nanozymes maintain a long-term antioxidant effect). Cyclic DPPH scavenging results of CCNRs and CCMNRs show that the scavenging rate of CCMNRs exhibits a biphasic decay characteristic: in the first three cycles, the scavenging rate decreases sharply due to the gradual consumption of MYR phenolic hydroxyl groups caused by irreversible free radical scavenging; in the last three cycles, the rate of decrease slows significantly, and its decay trend is similar to that of CCNRs, indicating that the contribution of MYR has been largely exhausted, and the sustained catalytic activity of cerium nanozymes becomes the main source of antioxidant activity. This unique biphasic characteristic provides direct experimental evidence for the phased antioxidant design of CCMNRs: MYR achieves rapid ROS scavenging in the early stage, while cerium nanozymes ensure sustained catalytic ROS scavenging in the later stage. By integrating it into a wound-adaptive dynamic cross-linked network to construct a composite nanoenzyme hydrogel, it still maintains rapid and continuous antioxidant capabilities, enabling staged antioxidant treatment of diabetic wounds. This specifically addresses the shortcomings of existing technologies in diabetic wounds, which cannot simultaneously provide rapid short-term relief and long-term continuous protection.

[0102] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A cerium dioxide composite nanoenzyme hydrogel, characterized in that: (i) The hydrogel raw material comprises, by mass percentage, 0.1-0.5% of β-cyclodextrin-modified cerium dioxide composite nanozyme loaded with myricetin, 5.52-12.8% of hydrogel matrix, and the balance being water; (ii) In the β-cyclodextrin-modified cerium dioxide composite nanozyme loaded with myricetin, the mass content of β-cyclodextrin-modified cerium dioxide is 92.23~98.36%, and the mass content of myricetin is 1.64~7.77%; (iii) The hydrogel matrix is ​​composed of polyvinyl alcohol, xanthan gum and borax in a mass ratio of 25~50:2.5~10:1~4.

2. The cerium dioxide composite nanoenzyme hydrogel as described in claim 1, characterized in that: Based on the mass percentage of the cerium dioxide composite nanozyme hydrogel, the β-cyclodextrin-modified cerium dioxide composite nanozyme loaded with myricetin accounts for 0.3%, the hydrogel matrix accounts for 11.4%, and the remainder is water.

3. The cerium dioxide composite nanoenzyme hydrogel as described in claim 1, characterized in that: β-cyclodextrin-modified cerium dioxide composite nanozymes loaded with myricetin also possess the following staged antioxidant properties: (i) Myricetin achieves rapid ROS clearance in the early stages: in the first three cycles, the DPPH clearance rate drops sharply from 81.8% to 64.5%; (ii) Cerium nanozymes ensure continuous catalytic ROS removal in the later stages: the removal rate decreased by only 5% after the last three cycles.

4. The method for preparing cerium dioxide composite nanoenzyme hydrogel according to any one of claims 1 to 3, characterized in that: include, Polyvinyl alcohol is dissolved in ultrapure water to obtain a polyvinyl alcohol solution; A cerium dioxide composite nanozyme modified with xanthan gum and β-cyclodextrin and loaded with myricetin was added to a polyvinyl alcohol solution and stirred to obtain a homogeneous solution, which was then added to a borax solution to obtain the final product.

5. The preparation method according to claim 4, characterized in that: The preparation process of the β-cyclodextrin-modified cerium dioxide composite nanozyme loaded with myricetin includes the following steps: Cerium nitrate hexahydrate and β-cyclodextrin were dissolved in ultrapure water and stirred at room temperature. Sodium hydroxide was then added to obtain a mixture. The mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and heated, and then cooled to room temperature. The precipitate was collected by centrifugation and washed alternately with sodium chloride solution and ultrapure water to remove residual ions, thus obtaining β-cyclodextrin-modified cerium dioxide nanozyme. Next, myricetin was added to the β-cyclodextrin-modified cerium dioxide nanozyme suspension, stirred, and centrifuged to obtain a β-cyclodextrin-modified cerium dioxide composite nanozyme loaded with myricetin.

6. The preparation method according to claim 4, characterized in that: Polyvinyl alcohol is dissolved in ultrapure water to obtain a polyvinyl alcohol solution. The dissolution temperature is 90~95℃, and the stirring time is 1~2h.

7. The preparation method according to claim 4, characterized in that: The xanthan gum and β-cyclodextrin-modified cerium dioxide composite nanozyme loaded with myricetin was added to a polyvinyl alcohol solution and stirred at 60-70℃ for 0.5-1h.

8. The preparation method according to claim 5, characterized in that: Cerium nitrate hexahydrate and β-cyclodextrin were dissolved in ultrapure water and stirred at room temperature. Then sodium hydroxide was added to obtain a mixture in which the molar ratio of cerium nitrate hexahydrate to β-cyclodextrin was 1:1 and the mass fraction of sodium hydroxide in the mixture was 3.5%.

9. The application of the cerium dioxide composite nanoenzyme hydrogel as described in any one of claims 1 to 3 in promoting the healing of diabetic wounds.

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