Composite nanohydrogel and application thereof in repair of diabetic wound surface

Abstract

The application belongs to the technical field of biomedical materials and regenerative medicine, and provides a composite nano hydrogel and application thereof in repair of diabetic wound surface. The three-dimensional porous nano hydrogel framework formed by composite crosslinking of HA-3APBA, PVA and lithium clay nanosheet is used as a carrier, antioxidant small molecules NAC and VC are double-loaded, and inactivated umbilical cord serum is dispersed and compounded. In view of the pathological characteristics of high oxidative stress and sustained inflammation of the diabetic wound surface, the application removes active oxygen through the antioxidant small molecules, remodels the wound microenvironment, protects and cooperates with the umbilical cord serum to play the roles of promoting angiogenesis, collagen deposition and re-epithelialization, and solves the problems of easy inactivation of growth factors, prolonged inflammation and healing stagnation of the diabetic wound surface. Animal experiments show that the composite nano hydrogel significantly improves the healing speed and quality of the diabetic wound surface, and can be used for preparing drugs for treating diabetic foot ulcers, diabetic chronic ulcers and high oxidative stress difficult-to-heal wound surfaces.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and regenerative medicine technology, specifically relating to a composite nanohydrogel and its application in the repair of diabetic wounds. Background Technology

[0002] Diabetic foot ulcers, as one of the most serious chronic complications of diabetes, are characterized by high disability and mortality rates, seriously affecting patients' quality of life and placing a heavy burden on the clinical medical system.

[0003] The core reason for the difficulty in self-healing and stagnation of diabetic foot wounds lies in the imbalance of the local microenvironment at the wound site. Prolonged hyperglycemia induces the production of large amounts of reactive oxygen species (ROS) in the wound tissue, leading to persistent oxidative stress damage and mediating macrophage polarization towards the M1 pro-inflammatory phenotype, thus triggering the continuous activation of the inflammatory cascade. This abnormal pathological microenvironment not only causes degradation and functional impairment of the extracellular matrix but also leads to the loss of endogenous growth factor activity and significant inhibition of angiogenesis, ultimately resulting in refractory ulcers.

[0004] Current clinical and existing technologies for repairing diabetic foot ulcers have significant technical shortcomings: First, while the use of growth factor-based biological repair agents such as umbilical cord serum alone possesses the biological potential to promote wound tissue regeneration and repair, the high oxidative stress environment of diabetic wounds rapidly destroys the molecular structure of growth factors, leading to their inactivation. Furthermore, these agents lack physical support and barrier protection, making them prone to loss and depletion within the wound, significantly reducing their actual repair effect. Second, conventional hydrogel dressings only provide the basic conditions for moist healing, offering only basic functions of physical moisturization and isolation from external stimuli. They lack the ability to actively remove excess reactive oxygen species from the wound and regulate the wound's immune and inflammatory microenvironment, failing to address the root cause of impaired ulcer healing.

[0005] Given the limitations of existing treatments, such as limited single-component repair efficacy, inability to synergistically improve oxidative stress and the inflammatory microenvironment, and the tendency for bioactive agents to be inactivated and lost, there is an urgent clinical need to develop a synergistic treatment strategy that combines antioxidant intervention and biological tissue repair. Developing a composite repair system that integrates antioxidant function and the sustained-release properties of bioactive factors, by targeting and eliminating excess ROS in the wound and inhibiting excessive inflammatory responses, and creating a suitable microenvironment with low oxidative stress for bioactive components such as umbilical cord serum, thus ensuring the stable performance of their biological repair functions, is a key research direction for solving the problem of difficult-to-heal diabetic foot ulcers. Summary of the Invention

[0006] The purpose of this invention is to provide a composite nano-hydrogel and its application in the repair of diabetic wounds. By introducing specific antioxidant small molecules and umbilical cord serum into the nano-hydrogel framework, a composite repair system with dual effects of "antioxidant-biological repair" is constructed. This system targets and removes excess reactive oxygen species (ROS) from diabetic wounds, reduces persistent inflammatory responses, and regulates the wound's immune microenvironment. At the same time, it provides a physical barrier and protection for umbilical cord serum, reducing its loss and preventing its oxidative inactivation. This addresses the problems in existing technologies, such as the easy inactivation and loss of simple growth factors, the lack of active regulation of the wound microenvironment in ordinary hydrogel dressings, and the stagnation of healing in diabetic wounds, thereby achieving efficient repair of refractory diabetic wounds.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: a composite nanohydrogel, comprising a nanohydrogel framework, antioxidant small molecules loaded in the nanohydrogel framework, and umbilical cord serum dispersed in the nanohydrogel framework; the antioxidant small molecules are NAC and VC; the nanohydrogel framework is a three-dimensional porous network structure formed by the composite cross-linking of HA-3APBA, PVA and lithium diatomite nanosheets.

[0008] Preferably, the antioxidant small molecules NAC and VC are dissolved and dispersed in the pore water of the nano-hydrogel framework, and the umbilical cord serum is physically embedded in the pores of the nano-hydrogel framework and adsorbed on the surface of the lithium diatomite nanosheets.

[0009] Preferably, the HA-3APBA is a copolymer of HA and 3-APBA obtained by stepwise coupling reaction under EDC and NHS activation.

[0010] The present invention further provides a method for preparing the above-mentioned composite nanohydrogel.

[0011] Preferably, the steps include:

[0012] S1. Preparation of HA-3APBA dry powder: Dissolve HA in water, add EDC, NHS and 3-APBA in sequence to carry out stepwise coupling reaction, and obtain HA-3APBA dry powder by dialysis and freeze drying.

[0013] S2. Preparation of antioxidant hydrogel prepolymer: Using PVA aqueous solution as solvent, add NAC, VC and lithium diatomaceous earth, and stir to obtain antioxidant hydrogel prepolymer;

[0014] S3. Preparation of inactivated umbilical cord serum: Collect umbilical cord blood, centrifuge to obtain serum, and obtain inactivated umbilical cord serum by inactivation and filtration;

[0015] S4. Composite loading: At 4 ℃, inactivated umbilical cord serum is added to the antioxidant hydrogel prepolymer solution and mixed well;

[0016] S5. Gel formation: Add HA-3APBA solution to the mixture obtained in S4, mix evenly and allow it to gel naturally to obtain a composite nano-hydrogel.

[0017] Preferably, in step S1, the pH of the stepwise coupling reaction system is controlled at 4.0-7.0, and the reaction time is 4 h; the dialysis uses an 8-14 kDa dialysis bag, and the dialysis time is 72 h.

[0018] Preferably, in step S2, the concentration of the PVA aqueous solution is 30 mg / mL and the volume is 1 mL, the volume of the NAC is 2.5 μL, the volume of the VC is 1.25 μL, and the concentration of the lithium diatomite is 5 mg / mL and the volume is 0.5 mL.

[0019] Preferably, in step S3, the inactivation conditions are 56 °C for 30 min.

[0020] Preferably, in step S4, the volume of the inactivated umbilical cord serum added is 50 μL.

[0021] Preferably, in step S5, the concentration of the HA-3APBA solution is 30 mg / mL and the volume is 1 mL.

[0022] The present invention further provides applications of the above-mentioned composite nanohydrogel.

[0023] Preferably, the composite nanohydrogel is used to prepare a drug for treating diabetic foot, diabetic chronic ulcers, and wounds that are difficult to heal due to high oxidative stress, wherein the effective component of the drug is the composite nanohydrogel.

[0024] The beneficial effects of this invention are:

[0025] 1. Microenvironment remodeling (antioxidant / anti-inflammatory): Two small antioxidant molecules in the nano-hydrogel framework can rapidly clear ROS from the wound, protect cells from oxidative damage, and induce macrophages to polarize to the M2 type (repair type), significantly reducing the levels of inflammatory factors (TNF-α, IL-6).

[0026] 2. Activity protection and synergistic effect: The antioxidant environment effectively protects the biological activity of the loaded umbilical cord serum growth factors, enabling them to more efficiently promote fibroblast proliferation and angiogenesis during subsequent release.

[0027] 3. Comprehensive repair: Experiments have shown that the experimental group of this invention is significantly better than the control group with only biological activity and only antioxidant function in terms of healing speed, granulation tissue thickness, collagen deposition density and vascularization degree (CD31 expression). Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the composite hydrogel in this invention;

[0029] Figure 2 These are the results of the wound healing process in each group of mice in this invention;

[0030] Figure 3 These are the ROS detection results for each group in this invention;

[0031] Figure 4 These are images showing the HE staining results of various groups of skin in this invention. Detailed Implementation

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0035] Example 1: Preparation of Composite Nanohydrogels

[0036] S1. Preparation of HA-3APBA dry powder: Sodium hyaluronate (HA) was dissolved in ultrapure water and stirred for 1 hour to obtain a transparent matrix solution. Simultaneously, aqueous solutions of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), and a water / ethanol mixture of 3-aminophenylboronic acid (3-APBA) were prepared for later use. Then, a stepwise coupling reaction was initiated. Under light-protected stirring, EDC was first added dropwise to the HA solution while maintaining the pH at 4-5 to activate the carboxyl groups. Next, NHS was added dropwise, and the pH was adjusted to 6-6.2 to form a stable ester. Finally, 3-APBA solution was slowly added dropwise while maintaining the pH at 6-6.2 (if hydrogen bonding causes viscosity, dilute NaOH can be added to break the hydrogen bonds). After the addition was complete, the pH was maintained below 7.0, and the reaction continued for 4 hours. After the reaction, the solution was purified and dried at 8-14 °C. Dialyze the product in a kDa dialysis bag for 72 hours (replacing the ultrapure water every 12 hours) to remove unreacted impurities, then dispense it into EP tubes and freeze-dry for 4-5 days to obtain HA-3-APBA dry powder.

[0037] S2. Preparation of antioxidant hydrogel prepolymer: Select biocompatible polymer materials (HA, lithium diatomite, PVA), add 2.5 μL of two antioxidant small molecules NAC (MCE: HY-B0215) and 1.25 μL of VC (MCE: HY-B0166) to 1 mL of 30 mg / mL PVA, and then add 0.5 mL of 5 mg / mL lithium diatomite and stir evenly to form a hydrogel prepolymer.

[0038] S3. Preparation of inactivated umbilical cord serum: Prepare inactivated umbilical cord serum according to standard procedures. Inactivation conditions are as follows: Collect umbilical cord blood using a gel-promoting blood collection tube, and let it stand at 4 degrees Celsius for four hours until clear stratification occurs; centrifuge at 3000×g for 15 min, and collect the upper serum layer after centrifugation; place the collected serum at 56 ℃ for 30 min for inactivation, and filter the inactivated serum through a 0.22 μm filter sieve.

[0039] S4. Composite loading: Under low temperature (4 °C) conditions, slowly add 50 μL of umbilical cord serum to the antioxidant hydrogel prepolymer solution and mix gently.

[0040] S5. Gel formation: Add 1 mL of 30 mg / mL HA-3APBA to the solution prepared in step 3, mix well, and wait for natural gel formation.

[0041] This invention prepares a "smart" antioxidant bioactive composite hydrogel (HA-3APBA / PVA / lithium diatomite composite gel) cross-linked with a dynamic covalent bond and a physical network of nano-clay. In terms of microstructure, HA-3APBA and PVA are interlocked by dynamic covalent bonds of phenylboronic acid esters, forming a stable three-dimensional porous network (gel framework) together with lithium diatomite nanosheets uniformly dispersed in the gaps and acting as a scaffold through electrostatic interactions. The small-molecule antioxidants NAC and VC are uniformly dissolved in the pore water of the network as free molecules, facilitating rapid diffusion and release. Some of the active protein components inactivated by umbilical cord serum are physically embedded in the network pores, while others are tightly adsorbed onto the surface of the lithium diatomite nanosheets due to electrostatic effects. This unique microstructure protects protein activity while enabling the slow release of biological factors. Figure 1 ).

[0042] Example 2: Verification of wound repair efficacy in diabetic mice

[0043] 1. Modeling: Six-week-old male C57BL / 6 mice were used to induce hyperglycemia (≥16.7 mmol / L) by intraperitoneal injection of streptozotocin (STZ), and a full-thickness skin defect with a diameter of 6 mm was prepared on the back.

[0044] 2. Group intervention and drug administration:

[0045] Group A (young normal mice): normal mice + PBS infusion.

[0046] Group B (control group of diabetic mice): diabetic mice + PBS infusion.

[0047] Group C (serum group): Diabetic mice were infused with a mixture of umbilical cord serum and blood.

[0048] Group D (novel hydrogel group): diabetic mice + blank hydrogel containing dual antioxidant small molecules applied.

[0049] Group E (composite umbilical cord serum nanohydrogel group): diabetic mice + application of the full-component formulation of this invention.

[0050] 3. Detection indicators and results:

[0051] ROS level detection: DHE probe staining was used. The results showed that the ROS fluorescence intensity of the wounds in groups D and E was significantly lower than that in groups B and C, which proved the effectiveness of the antioxidant small molecules.

[0052] HE staining: First, skin from the back of a mouse with a wound is excised, fixed with 4% polyformaldehyde, dehydrated with gradient ethanol, cleared with xylene, and embedded in paraffin. The sections are then cut into 4-5 μm thin slices and baked at 60 ℃ for 1-2 hours to prevent detachment. The slices are then dewaxed and hydrated in xylene and gradient ethanol solutions, followed by immersion in hematoxylin staining for 3-5 minutes. After brief differentiation with 1% hydrochloric acid alcohol and ammonia re-greening, the cell nuclei appear deep blue. Eosin staining is then applied for 30 seconds to 2 minutes, resulting in pink cytoplasm and dermal collagen fibers. Finally, the sections are dehydrated with gradient ethanol, cleared with xylene, and mounted with neutral resin. The blue cell nuclei and pink tissue background can then be clearly observed under a microscope to assess the degree of epidermal re-epithelialization and the repair status of the dermal structure.

[0053] The results show:

[0054] Macroscopic wound healing assessment: The diabetic control group showed the slowest healing with significant scab formation. The use of novel hydrogels or umbilical cord serum alone had limited effect on promoting wound contraction. In contrast, the composite umbilical cord serum nanohydrogel group exhibited the fastest wound closure, with the final appearance closely resembling the physiological state of the young, healthy group, confirming its ability to significantly accelerate wound healing in diabetic patients. Figure 2 ).

[0055] ROS level detection: The diabetic control group showed typical localized high ROS accumulation (fluorescence peak shift to the right). The clearance effect of hydrogel or serum alone was not significant. However, the peak position of the composite umbilical cord serum nanohydrogel group shifted significantly to the left, and the overall distribution approached the baseline level of the young normal group, demonstrating that this composite preparation can effectively clear local ROS and improve the high oxidative stress microenvironment of the wound. Figure 3 ).

[0056] Healing rate and histology: On day 14, group E had the highest wound healing rate (>95%), and HE staining showed intact epithelial structure. Figure 4 ).

[0057] In summary, the composite umbilical cord serum nanohydrogel can significantly accelerate the healing of diabetic chronic wounds and the complete regeneration of epithelial tissue by effectively clearing local ROS and improving the microenvironment under high oxidative stress.

[0058] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A composite nanohydrogel, characterized in that, It includes a nano-hydrogel framework, antioxidant small molecules loaded in the nano-hydrogel framework, and umbilical cord serum dispersed in the nano-hydrogel framework; the antioxidant small molecules are NAC and VC; the nano-hydrogel framework is a three-dimensional porous network structure formed by the composite cross-linking of HA-3APBA, PVA and lithium diatomite nanosheets.

2. The composite nanohydrogel of claim 1, wherein, The antioxidant small molecules NAC and VC are dissolved and dispersed in the pore water of the nano-hydrogel framework, and the umbilical cord serum is physically embedded in the pores of the nano-hydrogel framework and adsorbed on the surface of the lithium diatomite nanosheets.

3. The composite nanohydrogel of claim 1, wherein, The HA-3APBA is a copolymer obtained by stepwise coupling reaction of HA and 3-APBA under EDC and NHS activation.

4. The method of claim 1-3, wherein the composite nanohydrogel is prepared by, Includes the following steps: S1. Preparation of HA-3APBA dry powder: Dissolve HA in water, add EDC, NHS and 3-APBA in sequence to carry out stepwise coupling reaction, and obtain HA-3APBA dry powder by dialysis and freeze drying. S2. Preparation of antioxidant hydrogel prepolymer: Using PVA aqueous solution as solvent, add NAC, VC and lithium diatomaceous earth, and stir to obtain antioxidant hydrogel prepolymer; S3. Preparation of inactivated umbilical cord serum: Collect umbilical cord blood, centrifuge to obtain serum, and obtain inactivated umbilical cord serum by inactivation and filtration; S4. Composite loading: At 4 ℃, inactivated umbilical cord serum is added to the antioxidant hydrogel prepolymer solution and mixed well; S5. Gel formation: Add HA-3APBA solution to the mixture obtained in S4, mix evenly and allow it to gel naturally to obtain a composite nano-hydrogel.

5. The method of claim 4, wherein, In step S1, the pH of the stepwise coupling reaction system is controlled at 4.0-7.0, and the reaction time is 4 h; the dialysis uses an 8-14 kDa dialysis bag, and the dialysis time is 72 h.

6. The method of claim 4, wherein, In step S2, the concentration of the PVA aqueous solution is 30 mg / mL and the volume is 1 mL, the volume of the NAC is 2.5 μL, the volume of the VC is 1.25 μL, and the concentration of the lithium diatomite is 5 mg / mL and the volume is 0.5 mL.

7. The method of claim 4, wherein, In step S3, the inactivation conditions are 56 °C for 30 min.

8. The method of claim 4, wherein, In step S4, the volume of the inactivated umbilical cord serum added is 50 μL.

9. The method of claim 4, wherein, In step S5, the concentration of the HA-3APBA solution is 30 mg / mL and the volume is 1 mL.

10. The use of the composite nanohydrogel according to claim 1, characterized in that, The composite nanohydrogel is used to prepare a drug for treating diabetic foot, diabetic chronic ulcers, and wounds that are difficult to heal due to high oxidative stress. The active ingredient of the drug is the composite nanohydrogel.

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