PH (Potential of Hydrogen) and ROS (Reactive Oxygen Species) dual-response hydrogel based on probiotic microcapsule combined with prebiotics as well as preparation method and application of pH and ROS dual-response hydrogel

Abstract

The invention discloses a pH and ROS double-response hydrogel based on probiotic microcapsules combined with prebiotics and a preparation method and application thereof, and belongs to the field of biomedical materials, the hydrogel is composed of the probiotic microcapsules, ROS response release mangiferin fragments and pH sensitive hydrogel; specifically, amino groups rich in the surface of the probiotic gelatin microcapsule are crosslinked with aldehyde groups contained in oxidized dextran and 4-formylphenylboronic acid to form a Schiff base bond with pH response. Mangiferin is combined with a boric acid group in 4-formylphenylboronic acid through a catechol structure and is crosslinked in the hydrogel to form a boric acid ester bond with ROS (reactive oxygen species) response. According to the hydrogel prepared by the preparation method disclosed by the invention, the probiotic microcapsules and the ROS responsively released prebiotics are combined, so that the synergistic antibacterial and antioxidant effects are achieved, and the effect of treating diabetic wounds is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a pH and ROS dual-responsive hydrogel based on probiotic microcapsules combined with prebiotics, its preparation method, and its application. Background Technology

[0002] Diabetic wounds are chronic ulcerative diseases caused by persistent hyperglycemia and are a common complication of diabetes, with a prevalence of 19%-34% in diabetic patients. The main characteristic of diabetic wounds is long-term chronic low-grade inflammation, often considered the root cause of their non-healing nature. Under normal physiological conditions, wound healing involves four stages: hemostasis, inflammation, cell migration and proliferation, and tissue remodeling. However, in diabetic patients, hyperglycemia causes excessive protein glycation and the excessive accumulation of advanced glycation end products (AGEs), leading to excessive oxidative stress at the wound site, thereby disrupting the normal repair process. Excessive oxidative stress accelerates the invasion of pathogens and the formation of biofilms, causing severe bacterial infection and impaired angiogenesis at the wound site. Currently, there are various clinical treatments for diabetic wounds, ranging from debridement surgery and local oxygen therapy to the use of antibiotics. However, single-modality treatments cannot address the complex microenvironment of diabetic wounds. Therefore, there is an urgent need to develop multifunctional delivery systems that can reduce inflammation and eliminate multiple pathogens.

[0003] Probiotics are beneficial microorganisms, especially lactobacilli, which have multiple functions including inhibiting inflammatory signaling pathways, promoting macrophage polarization, and killing pathogenic bacteria. However, the application of probiotics in the treatment of diabetic wounds is limited by defects such as low survival rate and uncontrolled proliferation. Microencapsulation technology is a technique that encapsulates probiotics using natural or synthetic polymers. Microencapsulation of probiotics can improve bacterial survival rates under harsh wound conditions while preventing leakage and minimizing disruption to the wound microbiome. Mangiferin, as a prebiotic, can be used as a nutrient for probiotics to promote their proliferation. Simultaneously, mangiferin and probiotics can synergistically inhibit the NF-κB signaling pathway to reduce the production of inflammatory factors and alleviate oxidative stress at the wound site.

[0004] Hydrogel dressings are polymeric materials with a three-dimensional network structure formed by physical or chemical cross-linking of high molecular weight polymers. Due to their porous structure, hydrogels can absorb excess exudate from wounds while ensuring breathability, avoiding the drawbacks of traditional wound dressings that only provide physical isolation. Common dynamic chemical bonds in hydrogels include Schiff base bonds (imine bonds), borate ester bonds, and disulfide bonds. These dynamic chemical cross-linking bonds endow hydrogels with excellent self-healing and injectability, which are indispensable for wound repair. Borate ester bonds are dynamic responsive bonds formed by phenylboronic acid and catechol structures. In the presence of reactive oxygen species (ROS), borate ester bonds can break to achieve precise drug release. Therefore, the design and development of probiotic and prebiotic-based hydrogel dressings is of great significance for the clinical treatment of diabetic wounds. Summary of the Invention

[0005] This invention aims to provide a pH and ROS dual-responsive hydrogel based on probiotic microcapsules combined with prebiotics and its preparation method, and also provides the application of this pH and ROS dual-responsive hydrogel in the treatment of diabetic wounds.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: A pH- and ROS-responsive hydrogel delivery system based on probiotic microcapsules combined with prebiotics comprises probiotic microcapsules, a ROS-responsive mangiferin fragment, and a pH-sensitive hydrogel. The probiotic microcapsules are prepared by encapsulating probiotics with amino-containing polymers via phase separation. The ROS-responsive mangiferin fragment is prepared by forming ROS-responsive borate ester bonds between mangiferin and a compound containing borate groups. The pH-sensitive hydrogel is prepared by cross-linking probiotic microcapsules with amino groups on their surface, a polymer compound with aldehyde groups on its surface, and the ROS-responsive mangiferin fragment; the cross-linking network is constructed based on pH-sensitive Schiff base bonds. This hydrogel exerts its antibacterial and antioxidant capabilities through the synergistic effect of the probiotic microcapsules and prebiotics, thereby promoting wound healing in diabetic patients.

[0007] Preferably, the amino-containing polymeric compound is any one of gelatin, chitosan, or carboxymethyl chitosan.

[0008] Preferably, the ROS-responsive mangiferin-releasing fragment is composed of 4-formylphenylboronic acid and mangiferin. In the presence of high levels of ROS in a diabetic wound, the boronic acid ester bond can cleave to release mangiferin. Its structural formula is as follows:

[0009] Preferably, the polymeric compound with an aldehyde group is any one of oxidized dextran, oxidized hyaluronic acid, oxidized konjac glucomannan, and oxidized starch.

[0010] The method for preparing a pH and ROS dual-responsive hydrogel based on probiotic microcapsules and prebiotics, as described in this invention, includes the following steps: Step 1: Preparation of probiotic microcapsules, using the single aggregation method to prepare microcapsules; A polymeric compound containing amino groups (such as gelatin) is dissolved in water by heating to obtain solution a; anhydrous sodium sulfate is added to water and ultrasonically vibrated to obtain a clear and transparent sodium sulfate solution; probiotic culture is added to solution a and mixed evenly to form solution b; the prepared sodium sulfate solution is added to solution b in small, uniform flow rates, and heated and stirred to react and form capsules; finally, a curing agent is added to the formed probiotic microcapsule solution, and the mixture is stirred and cured under ice bath conditions to form stable probiotic microcapsules.

[0011] Step 2: Preparation of pH and ROS dual-responsive hydrogels; The probiotic microcapsules formed in step 1 were washed with PBS solution and centrifuged to obtain microcapsule precipitate; a polymer compound with aldehyde group (such as oxidized dextran), 4-formylphenylboronic acid and mangiferin were mixed to obtain a composite solution, and the pH of the solution was adjusted to alkaline; the composite solution was mixed with the microcapsule precipitate to obtain a hydrogel precursor solution, the pH of the precursor solution was adjusted to 9, and the mixture was uniformly mixed to obtain a hydrogel.

[0012] In the above preparation method, wherein: In step 1, the mass-volume percentage of amino-containing polymers in solution a is 1%–10% (w / v); the mass-volume percentage of sodium sulfate solution is 20%–40% (w / v); and the selected probiotic is any one of Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus bulgaricus, with an addition amount of 1×10⁻⁶. 9 CFU / mL ~9×10 9 CFU / mL; the reaction time after adding sodium sulfate solution is 0.5h to 2h, and the reaction temperature is 30℃ to 40℃; the curing agent used is any one of formaldehyde, glutaraldehyde, glucose and oxidized dextran, with a mass-volume percentage of 1% to 6% (w / v); the reaction time after adding curing agent is 0.5h to 2h, and the reaction temperature is 0℃ to 5℃.

[0013] Preferably, the method for preparing the oxidized dextran is as follows: dextran is dissolved in water, sodium periodate is added, and the mixture is stirred at room temperature in the dark to carry out the reaction; ethylene glycol is added to the reaction solution, and the oxidation reaction is terminated by continuing to stir; the obtained oxidized dextran solution is placed in a dialysis bag and dialyzed in pure water, and finally freeze-dried to obtain a solid, which is stored at -20°C.

[0014] More preferably, in the method for preparing oxidized dextran, the molar ratio of dextran to sodium periodate is 1:(0.5-5); the reaction time of dextran and sodium periodate is 1h-8h; the stirring time after adding ethylene glycol is 10min-30min; the dialysis time of the oxidized dextran solution is 24h-96h; and the molecular weight cutoff of the dialysis bag used is 1000Da-4000Da.

[0015] In step 2, the composite solution contains 5%–15% (w / v) of aldehyde-containing polymers, 0.05%–0.2% (w / v) of 4-formylphenylboronic acid, and 0.1%–0.4% (w / v) of mangiferin. The volume ratio of the composite solution to the microcapsule precipitate is 1:(0.5–5). The pH of the composite solution and the hydrogel precursor solution is adjusted to 9, and the alkaline pH adjuster used is sodium hydroxide, potassium hydroxide, or triethylamine, with a concentration of 1M–4M.

[0016] This invention also provides the application of the pH and ROS dual-responsive hydrogel based on probiotic microcapsules combined with prebiotics as a dressing for diabetic wound treatment. This dressing is a multifunctional wound care material that integrates antibacterial, antioxidant and repair-promoting properties, and can specifically solve the core problems of chronic inflammation, infection and slow healing of diabetic wounds.

[0017] (1) Inhibition of bacterial infection in diabetic wounds: It has a significant inhibitory effect on common clinical pathogens (Gram-positive bacteria: Staphylococcus aureus; Gram-negative bacteria: Escherichia coli), with inhibition rates of 98.23% (Staphylococcus aureus) and 92.27% (Escherichia coli), respectively. It can effectively reduce bacterial colonization and biofilm formation at the wound site, and reduce the risk of delayed healing of diabetic wounds due to infection.

[0018] (2) Clears excess ROS from wounds and alleviates chronic inflammation: It has a high antioxidant capacity and can specifically clear excess •OH, H2O2 and O2• from the microenvironment of diabetic wounds. - The ROS clearance rates were 49.55%, 81.36%, and 75%, respectively. This can reduce wound inflammatory infiltration, regulate the inflammatory response process, and break the vicious cycle of chronic low-grade inflammation in diabetic wounds.

[0019] (3) Constructing a suitable microenvironment for wound healing: The porous mesh structure can absorb excess exudate from the wound while ensuring oxygen flow, avoiding the problems of moisture and hypoxia caused by the physical isolation of traditional dressings. Excellent mechanical properties, self-healing and injectability, adaptable to changes in wound shape, and can continuously maintain wound stability, providing good conditions for cell migration and proliferation.

[0020] (4) Promotes rapid healing of diabetic wounds: It can induce macrophages to polarize to the M2 type, accelerating the transition of wound inflammation to the proliferative and tissue remodeling phases. Preclinical experiments have shown that it can reduce wound inflammation infiltration within 3 days after use and achieve complete healing of 8mm diameter diabetic wounds within 14 days, which is significantly better than ordinary dressings.

[0021] The pH and ROS dual-responsive hydrogel delivery system based on probiotic microcapsules and prebiotics provided by this invention has the following advantages compared with the prior art: (1) The hydrogel prepared in this invention is composed of pH-responsive Schiff base bonds formed between the amino groups on the surface of gelatin microcapsules and the aldehyde groups contained in oxidized dextran and 4-formylphenylboronic acid, and ROS-responsive borate ester bonds formed by the boric acid groups and catechol groups in 4-formylphenylboronic acid, thus endowing the hydrogel with the ability to respond to both pH and ROS.

[0022] (2) The present invention is based on a probiotic microcapsule combined with a prebiotic hydrogel delivery system. The gelatin microcapsule not only ensures the activity of the probiotics, but also prevents the risk of leakage. At the same time, in the diabetic wound with high reactive oxygen species, the borate ester bond will break rapidly to release mangiferin, which can not only exert its antibacterial and antioxidant advantages, but also provide nutrition to promote the proliferation of probiotics and produce a synergistic effect.

[0023] (3) The hydrogel prepared by the present invention has excellent mechanical properties, self-healing properties and injectability. Its porous network structure can absorb excess exudate at the wound site while ensuring oxygen content, providing a good repair environment for the healing of diabetic wounds.

[0024] (4) The hydrogel prepared by the present invention has excellent antibacterial properties and has a significant inhibitory effect on both Gram-positive and Gram-negative bacteria. At the same time, the hydrogel shows that its antibacterial effect on Gram-positive bacteria is stronger than that on Gram-negative bacteria.

[0025] (5) The hydrogel prepared by the present invention has the function of removing excess ROS and reducing wound inflammation infiltration.

[0026] (6) The hydrogel prepared by the present invention has excellent therapeutic effect in the diabetic wound model. After 3 days of treatment, the wound inflammation infiltration decreased, after 7 days of treatment, the number of macrophages polarized to M2 type increased, and after 14 days of treatment, the wound was completely healed, thus expanding the application of probiotic hydrogel in diabetic wounds. Attached Figure Description

[0027] Figure 1 This is a flowchart illustrating the preparation process of the pH and ROS dual-responsive hydrogel based on probiotic microcapsules and prebiotics according to the present invention.

[0028] Figure 2 It is the oxidized dextran (OD) in Example 1 of this invention. 1 H NMR spectrum.

[0029] Figure 3 This is the Fourier transform infrared spectrum of oxidized dextran, gelatin and hydrogel (OPM@GLB) in Example 1 of the present invention.

[0030] Figure 4 This is a scanning electron microscope image of the hydrogel (OPM@GLB) in Example 1 of the present invention.

[0031] Figure 5 This is a graph showing the release of mangiferin from ROS in response to the hydrogel in Example 1 of this invention.

[0032] Figure 6 This is an in vitro antibacterial diagram of the hydrogel in this invention; wherein, (Ⅰ) is the hydrogel group of Example 1; (Ⅱ) is the hydrogel group of Example 2; and (Ⅲ) is the hydrogel group of Example 3.

[0033] Figure 7 This is a graph showing the in vitro DPPH clearance of hydrogels at different concentrations in Example 1 of this invention.

[0034] Figure 8 These are images of the dorsal wounds of mice on days 0, 3, 7, and 14 after treatment of diabetic wounds in this invention. (I) is the hydrogel group of Example 1; (II) is the hydrogel group of Example 2; and (III) is the hydrogel group of Example 3. Detailed Implementation

[0035] The present invention will be further described below through specific embodiments, but the present invention is not limited thereto.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0037] Example 1

[0038] A pH- and ROS-responsive hydrogel delivery system based on probiotic microcapsules combined with prebiotics comprises probiotic gelatin microcapsules, a ROS-responsive mangiferin-releasing fragment, and a pH-sensitive hydrogel. The probiotic gelatin microcapsules (i.e., probiotic-loaded gelatin microcapsules) are prepared by encapsulating *Lactobacillus bulgaricus* in amino-containing gelatin using a phase separation method. The ROS-responsive mangiferin-releasing fragment is prepared by forming ROS-responsive borate ester bonds between mangiferin and 4-formylphenylboronic acid, enabling responsive release of mangiferin. The pH-sensitive hydrogel is prepared by cross-linking probiotic gelatin microcapsules, oxidized dextran, and the ROS-responsive mangiferin-releasing fragment; its cross-linking network depends on pH-sensitive Schiff base bonds. The dynamic Schiff base bonds and borate ester bonds endow the hydrogel with self-healing and injectability capabilities, while simultaneously enabling responsive release of mangiferin at the wound site. The synergistic effect of the probiotic microcapsules and prebiotics enhances the hydrogel's antibacterial and antioxidant capabilities, thereby promoting wound healing in diabetic patients.

[0039] The above-mentioned method for preparing pH and ROS dual-responsive hydrogels based on probiotic microcapsules and prebiotics, such as... Figure 1 As shown, it includes the following steps: Step 1: Preparation of oxidized dextran; 5 g of dextran (Dextran, Dex) (unit glucose molecular weight) was dissolved in 50 mL of water and stirred evenly at room temperature to obtain a dextran solution. 8.36 g of sodium periodate was added to the dextran solution, and the mixture was stirred at room temperature in the dark for 4 hours to obtain an oxidized dextran solution. 5 mL of ethylene glycol was added to the oxidized dextran solution, and the oxidation reaction was terminated by stirring for 15 minutes. The prepared oxidized dextran solution was placed in a dialysis bag (3500 Da) and dialyzed with pure water at room temperature for 72 hours. Finally, it was freeze-dried to obtain a white solid and stored at -20°C. Figure 2 As shown, oxidized dextran (OD) 1 The H NMR spectrum shows weak resonances at 9.18 ppm and 8.38 ppm, which are characteristic proton signal peaks of aldehyde groups. These peaks are absent in the dextran spectrum, confirming the oxidation of dextran. Furthermore, the significant decrease in the hydroxyl signal (3.51 ppm–3.84 ppm) in oxidized dextran also indicates that the hydroxyl group was successfully oxidized to an aldehyde group. Figure 3 In the Fourier transform infrared spectrum, oxidized dextran appears at 1732 cm⁻¹. -1 There is a characteristic peak at this point, which is the (-C=O) stretching vibration peak of the aldehyde group, proving the successful synthesis of the aldehyde group.

[0040] Step 2: Preparation of probiotic microcapsules (preparation of microcapsules by single-cell aggregation method); 0.428 g of gelatin was dissolved in 20 mL of water under a 40°C water bath to obtain a gelatin solution; 32.13 g of anhydrous sodium sulfate was added to 100 mL of water and sonicated to obtain a clear and transparent sodium sulfate solution; 1 mL of a 5×10⁻⁶ solution was added to the solution. 9 CFU / mL of Lactobacillus bulgaricus bacterial culture was added to the gelatin solution and mixed thoroughly. Then, the prepared sodium sulfate solution was added to the reaction system in small, uniform flow rates, and the mixture was heated and stirred at 37°C for 1 hour. Finally, 4.21% (w / v) oxidized dextran was added as a curing agent to the formed microcapsule solution, and the mixture was stirred and cured for another 2 hours under an ice-water bath at 4°C to form probiotic gelatin microcapsules.

[0041] Step 3: Preparation of pH and ROS dual-responsive hydrogel (OPM@GLB); The probiotic gelatin microcapsules formed in step 2 were washed with PBS solution and centrifuged to obtain microcapsule precipitate. 5g of oxidized dextran (OD), 0.05g of 4-formylphenylboronic acid (4-FPBA), and 0.1g of mangiferin (MG) were dissolved in 50mL of water to obtain a composite solution (OD, 4-FPBA, and MG mass / volume fractions were 10% (w / v), 0.1% (w / v), and 0.2% (w / v), respectively). 1M sodium hydroxide solution was added to adjust the pH of the solution to 9. The composite solution and the microcapsule precipitate were mixed at a volume ratio of 1:5 to obtain a hydrogel precursor solution. 1M sodium hydroxide solution was added to adjust the pH of the hydrogel precursor solution to 9, and the mixture was thoroughly mixed to obtain a hydrogel. Figure 3 As shown, oxidized dextran at 1732 cm⁻¹ -1 The aldehyde peak at 1684 cm⁻¹ disappeared significantly, while the hydrogel (OPM@GLB) showed a peak at 1684 cm⁻¹. -1 The characteristic absorption peak representing -C=N- at the position confirms the successful synthesis of the Schiff base bond.

[0042] The morphology and mechanical properties of the hydrogel prepared in Example 1 were tested, as follows: (1) Morphological characterization of hydrogel: The prepared hydrogel was freeze-dried and then quickly immersed in liquid nitrogen to break it and expose the cross-section of the hydrogel. Subsequently, its surface and cross-section were sprayed with gold, and its microstructure was characterized by scanning electron microscopy.

[0043] Scanning electron microscopy (SEM) results of the hydrogel showed ( Figure 4 The hydrogel sample exhibits an irregular porous network structure, giving the hydrogel sufficient air permeability and the ability to absorb exudate.

[0044] (2) Rheological testing of the hydrogel: The rheological properties of the hydrogel were evaluated using a TA rheometer (DHR-2). The hydrogel was placed between parallel plates with a diameter of 40 mm and a gap of 1 mm to measure the changes in storage modulus (G') and loss modulus (G''). Time-frequency and oscillation frequency scans of the hydrogel were performed at a stress value of 1% and a frequency of 1 Hz. Strain scans of the hydrogel were performed to measure the critical strain value between stress values ​​of 0.1% and 1000%. To test the self-healing properties of the hydrogel, 1% and 1000% were set as low and high stresses, respectively, and the changes in G' and G'' were measured three times. The viscosity of the hydrogel was measured at shear rates from 0.1 / s to 100 / s to evaluate the shear thinning properties of the hydrogel.

[0045] Experimental results show that rheological studies can evaluate the self-healing behavior of hydrogels. Time-scan and frequency-scan observations of the hydrogel revealed that its elastic modulus (G') remained higher than the storage modulus (G'') and maintained a relatively stable value over a time range of 5 min and a frequency range of 0.1 rad / s to 100 rad / s, indicating structural stability. Strain-scan results showed that when the stress value was below the critical strain value, its elastic modulus (G') remained higher than the storage modulus (G''), indicating a gel state. When the stress value exceeded the critical strain value, its elastic modulus (G') rapidly decreased until it fell below the storage modulus (G''), indicating that the gel network was disrupted and the hydrogel was in a sol state. Alternating strain scans showed that the elastic modulus (G') recovered to a state greater than the storage modulus (G'') after three cycles, indicating good healing properties. The viscosity of the hydrogel decreased rapidly with increasing shear rate, demonstrating typical shear-thinning properties.

[0046] (3) ROS-responsive release of mangiferin: The ROS-responsive release of the hydrogel was tested by detecting the release of mangiferin from the hydrogel in PBS and PBS solution containing H2O2 (50 mmol / L). First, 1 mL of hydrogel was placed in 30 mL of PBS containing H2O2 and incubated at 100 rpm and 37 °C. After 1, 2, 3, 4, 8, 12, 18, 24, 36, 48 and 60 h, 5 mL of the solution was taken out and detected at 368 nm using a UV spectrophotometer. At the same time, 5 mL of release medium under the same conditions was added, and the cumulative release amount was calculated.

[0047] Experimental results show that: Figure 5As shown, after 12 hours in PBS solution containing H2O2, the release rate of mangiferin from the hydrogel reached 43.3%, and in PBS solution alone, the release rate reached 7.6%. After 60 hours in PBS solution containing H2O2, the release rate of mangiferin reached 85.41%, with a release amount of 23.43% in PBS solution. This may be because the neutral environment of PBS disrupted a small portion of the borate ester bonds, leading to the release of mangiferin. These results indicate that the hydrogel can release mangiferin in response to ROS, thereby achieving the goal of precise drug release.

[0048] (4) In vitro antibacterial activity of the hydrogel: To study the antibacterial properties of the hydrogel, we used the plating method to test Escherichia coli and Staphylococcus aureus. Escherichia coli and Staphylococcus aureus were cultured in LB medium at 37℃ and 100 rpm for 12 h. The sterilized hydrogel was added to a 24-well plate, and 1 mL of 10... 4 A bacterial suspension of CFU / mL was incubated at 37°C for 24 h. After 24 h, 100 μL of the solution was taken from each well of a 24-well plate and spread onto LB agar. After another 24 h of incubation, colony-forming units (CFU) were counted and photographed. The inhibition rate was calculated using the following formula:

[0049] Among them, CFU control The bacterial colony count (CFU) is the control group. hydrogel This represents the number of bacterial colonies in the hydrogel group.

[0050] Experimental results show that: Figure 6 Compared with the control group, the bacterial colonies in the hydrogel group of Example 1 were significantly reduced. The inhibition rates of the hydrogel against Escherichia coli and Staphylococcus aureus were 92.27% and 98.23%, respectively. The hydrogel showed a higher inhibition rate against Staphylococcus aureus, which may be due to the strong acid resistance and adaptability of Escherichia coli. In contrast, although the hydrogel groups of Examples 2 and 3 showed some antibacterial ability, their antibacterial ability was significantly lower than that of the hydrogel group of Example 1. The results indicate that the hydrogel prepared by the method of the present invention has a significant antibacterial effect.

[0051] (5) In vitro antioxidant capacity assessment of hydrogels: The antioxidant capacity of hydrogels was assessed using the DPPH scavenging method. First, 2 mL of hydrogel was added to DPPH solution and incubated in the dark for 30 min. Then, 200 μL of the solution was added to a 96-well plate, and the absorbance at 517 nm was measured using a microplate reader. Additionally, the antioxidant capacity of the hydrogels was assessed using hydroxyl (•OH), H₂O₂, and O₂• - The assay kit was used to determine the ability of the hydrogel to scavenge ROS.

[0052] Experimental results show that: Figure 7 As shown, the DPPH scavenging rate increased from 44.32% to 83.16% with increasing hydrogel concentration. To further investigate the antioxidant capacity of the hydrogel, the scavenging of •OH, H₂O₂, and O₂• by the hydrogel was studied. - The study investigated the effects of hydrogels on reactive oxygen species. Compared to the control group, the hydrogels showed a significantly improved scavenging rate for •OH, H2O2, and O2•. - The clearance rates were 49.55%, 81.36%, and 75%, respectively.

[0053] (6) Therapeutic effect of hydrogel on diabetic wounds: ICR mice were selected to establish a diabetic wound model and the wound healing ability of hydrogel was evaluated. First, ICR mice were injected with streptozotocin (STZ) (50 mg / kg) daily for 5 consecutive days to induce type 1 diabetes until blood glucose exceeded 16.7 mmol / L. Under anesthesia, a circular wound with a diameter of 8 mm was made on the back of each mouse. Then, the mice were divided into three groups (n=6): Example 1 hydrogel group, Example 2 hydrogel group and Example 3 hydrogel group, and the groups were changed every two days. Photographs of the wound on the back of each mouse were taken on days 0, 3, 7 and 14.

[0054] Experimental results show that: Figure 8 As shown, the hydrogel group exhibited better wound healing on days 3 and 7. After 14 days of treatment, the wound treated with the hydrogel group in Example 1 had completely healed. Wounds treated with the hydrogel groups in Examples 2 and 3 still showed partial unhealed areas. In conclusion, the hydrogel containing probiotic microcapsules and prebiotics demonstrates superior ability to treat diabetic wounds.

[0055] Example 2

[0056] A pH- and ROS-responsive hydrogel delivery system based on probiotics (directly cross-linked in a hydrogel) combined with prebiotics comprises gelatin microcapsules, a ROS-responsive mangiferin fragment, and a pH-sensitive hydrogel. The gelatin microcapsules are prepared from gelatin via phase separation. The ROS-responsive mangiferin fragment is prepared by forming a ROS-responsive borate ester bond between mangiferin and 4-formylphenylboronic acid. The pH-sensitive hydrogel is prepared by cross-linking gelatin microcapsules, oxidized dextran, and the ROS-responsive mangiferin fragment; its cross-linking network is constructed based on pH-sensitive Schiff base bonds, and it is simultaneously loaded with probiotics (Lactobacillus bulgaricus).

[0057] The preparation method of the above-mentioned pH and ROS dual-responsive hydrogel based on probiotics and prebiotics includes the following steps: Step 1: Preparation of oxidized dextran; Dissolve 5g of dextran in 50mL of water and stir until homogeneous at room temperature to obtain a dextran solution. Add 8.36g of sodium periodate to the dextran solution and stir for 4h at room temperature in the dark to obtain an oxidized dextran solution. Add 5mL of ethylene glycol to the oxidized dextran solution and continue stirring for 15min to terminate the oxidation reaction. Place the prepared oxidized dextran solution in a dialysis bag (3500Da) and dialyze it in pure water at room temperature for 72h. Finally, freeze-dry to obtain a white solid and store at -20℃.

[0058] Step 2: Preparation of gelatin microcapsules (preparation of microcapsules via single coagulation method); 0.428 g of gelatin was dissolved in 20 mL of water by heating at 40 °C to obtain a gelatin solution; 32.13 g of anhydrous sodium sulfate was added to 100 mL of water and sonicated to obtain a clear and transparent sodium sulfate solution; the prepared sodium sulfate solution was added to the gelatin solution in small, uniform flow rates, and the mixture was heated and stirred at 37 °C for 1 h; then 4.21% (w / v) oxidized dextran was added as a curing agent to the formed microcapsule solution, and the mixture was stirred and reacted for another 2 h under an ice-water bath at 4 °C to form stable gelatin microcapsules.

[0059] Step 3: Preparation of pH and ROS dual-responsive hydrogels; The gelatin microcapsules formed in step 2 were washed with PBS solution and centrifuged to obtain microcapsule precipitate; 5g of oxidized dextran (OD), 0.05g of 4-formylphenylboronic acid (4-FPBA) and 0.1g of mangiferin (MG) were dissolved in 50mL of water and stirred to obtain a composite solution. 1M sodium hydroxide solution was added to adjust the pH of the solution to 9; the composite solution and the microcapsule precipitate were mixed at a volume ratio of 1:5 to obtain a hydrogel precursor solution; 1mL of a 5×10⁻⁶ solution was added to the hydrogel. 9 Add CFU / mL of probiotic precipitate to the hydrogel precursor solution and mix thoroughly; add 1M sodium hydroxide solution to adjust the pH of the hydrogel precursor solution to 9, and mix thoroughly to obtain the hydrogel.

[0060] Example 3

[0061] A pH- and ROS-responsive hydrogel delivery system comprises gelatin microcapsules, a ROS-responsive mangiferin-releasing fragment, and a pH-sensitive hydrogel. The gelatin microcapsules are prepared from gelatin via phase separation. The ROS-responsive mangiferin-releasing fragment is prepared by forming ROS-responsive borate ester bonds between mangiferin and 4-formylphenylboronic acid. The pH-sensitive hydrogel is prepared by a cross-linking reaction of gelatin microcapsules, oxidized dextran, and the ROS-responsive mangiferin-releasing fragment; its cross-linking network is constructed based on pH-sensitive Schiff base bonds.

[0062] Step 1: Preparation of oxidized dextran; Dissolve 5g of dextran in 50mL of water and stir until homogeneous at room temperature to obtain a dextran solution. Add 8.36g of sodium periodate to the dextran solution and stir for 4h at room temperature in the dark to obtain an oxidized dextran solution. Add 5mL of ethylene glycol to the oxidized dextran solution and continue stirring for 15min to terminate the oxidation reaction. Place the prepared oxidized dextran solution in a dialysis bag (3500Da) and dialyze it in pure water at room temperature for 72h. Finally, freeze-dry to obtain a white solid and store at -20℃.

[0063] Step 2: Preparation of gelatin microcapsules; 0.428 g of gelatin was dissolved in 20 mL of water by heating at 40 °C to obtain a gelatin solution; 32.13 g of anhydrous sodium sulfate was added to 100 mL of water and sonicated to obtain a clear and transparent sodium sulfate solution; the prepared sodium sulfate solution was added to the gelatin solution in small, uniform flow rates, and the mixture was heated and stirred at 37 °C for 1 h; then 4.21% (w / v) oxidized dextran was added as a curing agent to the formed microcapsule solution, and the mixture was stirred and reacted for another 2 h under an ice-water bath at 4 °C to form stable gelatin microcapsules.

[0064] Step 3: Preparation of pH and ROS dual-responsive hydrogels; The gelatin microcapsules formed in step 2 were washed with PBS solution and centrifuged to obtain microcapsule precipitate. 5g of oxidized dextran (OD), 0.05g of 4-formylphenylboronic acid (4-FPBA) and 0.1g of mangiferin (MG) were dissolved in 50mL of water to obtain a composite solution. 1M sodium hydroxide solution was added to adjust the pH of the solution to 9. The composite solution and the microcapsule precipitate were mixed at a volume ratio of 1:5 to obtain a hydrogel precursor solution. 1M sodium hydroxide solution was added to adjust the pH of the hydrogel precursor solution to 9, and the mixture was homogeneously mixed to obtain a hydrogel.

Claims

1. A pH and ROS dual-responsive hydrogel based on probiotic microcapsule combined with prebiotics, characterized in that, The pH and ROS dual-responsive hydrogel based on probiotic microcapsules combined with prebiotics is composed of probiotic microcapsules, ROS-responsive released mangiferin fragments and pH-sensitive hydrogel; the probiotic microcapsules are prepared by wrapping probiotics with high molecular compounds having amino groups through phase separation method; the ROS-responsive released mangiferin fragments are prepared by forming ROS-responsive borate ester bonds between mangiferin and compounds containing boronic acid groups; the pH-sensitive hydrogel is prepared by cross-linking reaction of probiotic microcapsules having amino groups on the surface, high molecular compounds having aldehyde groups on the surface and ROS-responsive released mangiferin fragments, and the cross-linking network of the hydrogel is constructed depending on pH-sensitive Schiff base bonds; the hydrogel has antibacterial and antioxidant abilities through the synergistic effect of probiotic microcapsules combined with prebiotics.

2. The probiotic microcapsule conjugated prebiotic based pH and ROS dual responsive hydrogel according to claim 1, characterized in that, The high molecular compound having amino groups is any one of gelatin, chitosan and carboxymethyl chitosan.

3. The probiotic microcapsule conjugated prebiotic based pH and ROS dual responsive hydrogel according to claim 1, characterized in that, The ROS-responsive released mangiferin fragments have the following structural formula: 。 4. The probiotic microcapsule conjugated prebiotic based pH and ROS dual responsive hydrogel according to claim 1, characterized in that, The high molecular compound having aldehyde groups is any one of oxidized dextran, oxidized hyaluronic acid, oxidized konjac glucomannan and oxidized starch.

5. A method for preparing the probiotic microcapsule-based pH and ROS dual-responsive hydrogel with prebiotics according to any one of claims 1-4, characterized in that, The method comprises the following steps: Step 1: preparation of probiotic microcapsules; dissolve the high molecular compound having amino groups in water by heating to obtain solution a; add anhydrous sodium sulfate to water to prepare a sodium sulfate solution; add probiotic bacterial liquid to solution a and mix uniformly to form solution b; add the sodium sulfate solution to solution b, heat and stir to form capsules; finally, add a solidifying agent to the formed probiotic microcapsule solution, stir and solidify under ice bath conditions to form stable probiotic microcapsules; Step 2: preparation of hydrogel; centrifuge the probiotic microcapsules washed with PBS solution to obtain microcapsule precipitate; mix the high molecular compound having aldehyde groups, 4-formylphenylboronic acid and mangiferin to obtain a composite solution, and adjust the pH of the solution to be alkaline; mix the composite solution with the microcapsule precipitate to obtain a hydrogel precursor solution, adjust the pH of the precursor solution to 9, and obtain the hydrogel.

6. The method of claim 5, wherein the pH and ROS dual-responsive hydrogel based on probiotic microcapsule conjugated prebiotics is prepared by, The mass / volume percentage of the high molecular compound with amino group in solution a is 1%-10% (w / v); the mass / volume percentage of the sodium sulfate solution is 20%-40% (w / v); the selected probiotic is any one of Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus bulgaricus, and the addition amount is 1×10 9 CFU / mL-9×10 9 CFU / mL; the reaction time after adding the sodium sulfate solution is 0.5h-2h, and the reaction temperature is 30°C-40°C; the curing agent used is any one of formaldehyde, glutaraldehyde, glucose and oxidized dextran, and the mass / volume percentage is 1%-6% (w / v); the reaction time after adding the curing agent is 0.5h-2h, and the reaction temperature is 0°C-5°C.

7. The method of claim 6, wherein the pH and ROS dual-responsive hydrogel based on probiotic microcapsule conjugated prebiotics is prepared by, The preparation method of the oxidized dextran is as follows: dissolve dextran in water, add sodium periodate, stir at room temperature in the dark to react, add ethylene glycol to the reaction solution, continue to stir to terminate the oxidation reaction, place the prepared oxidized dextran solution in a dialysis bag, dialyze in pure water, and finally freeze-dry to obtain a solid, which is stored at -20℃.

8. The method of claim 7, wherein the pH and ROS dual-responsive hydrogel based on probiotic microcapsule conjugated prebiotics is prepared by, The molar ratio of dextran to sodium periodate is 1:(0.5-5); the reaction time of dextran and sodium periodate is 1-8 h.

9. The method of claim 5, wherein the pH and ROS dual-responsive hydrogel based on probiotic microcapsule conjugated prebiotics is prepared by, In step 2, the mass / volume percentage of the high molecular compound having aldehyde groups in the composite solution is 5%-15% (w / v), the mass / volume percentage of 4-formylphenylboronic acid is 0.05%-0.2% (w / v), and the mass / volume percentage of mangiferin is 0.1%-0.4% (w / v); the volume ratio of the composite solution to the microcapsule precipitate is 1:(0.5-5); and the pH of the composite solution and the hydrogel precursor solution is adjusted to 9.

10. Use of the pH and ROS dual-responsive hydrogel based on probiotic microcapsules combined with prebiotics as a wound treatment dressing for diabetes according to any one of claims 1-4.

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