A double-layer gel patch for diabetic wound surface and a preparation method thereof

Abstract

The application discloses a double-layer gel patch for diabetic wound surface and a preparation method thereof. The double-layer gel patch is composed of an upper gel layer and a lower gel layer, and the lower gel layer is attached to the skin surface during use. The upper gel layer is made of methacrylated gelatin, oxidized hyaluronic acid, a photoinitiator and an anti-inflammatory drug, and the lower gel layer is made of 3-carboxyphenylboronic acid grafted gelatin, dihydrocaffeic acid grafted epsilon-polylysine and an antibacterial agent. The double-layer gel patch has high mechanical strength and absorbability, and can realize a sequential treatment strategy of antibacterial treatment first and then anti-inflammatory treatment.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a double-layer gel patch for diabetic wounds and its preparation method. Background Technology

[0002] Diabetic wounds are one of the most common complications of diabetes, characterized by chronic, slow-healing wounds caused by diabetic peripheral neuropathy, vascular disease, and local tissue infection. Extensive sensory nerve damage in the skin reduces or eliminates basic sensation and pain, leading to insensitivity or even unconsciousness of the nervous system to the wound, thus increasing the risk of ulceration and even amputation. The normal wound healing process typically involves four stages: hemostasis, inflammation, proliferation, and remodeling. However, diabetic wounds are hindered from this normal healing process due to the hyperglycemic environment, lipid metabolism disorders, and insulin resistance. Diabetic wounds often exhibit excessive inflammatory responses, prolonging the transition from the inflammatory stage to the proliferative stage, resulting in a chronic inflammatory state, continuous tissue damage, and difficulty in wound healing. Simultaneously, the hyperglycemic environment makes it easier for bacteria to colonize the wound, leading to persistent infection.

[0003] Currently, routine treatments for diabetic wounds include mechanical debridement, topical antibiotics, hyperbaric oxygen therapy, and wound dressings. Mechanical debridement is crucial for wound closure and reducing the probability of amputation; however, it is often incomplete and prone to recurrence, so it is usually combined with drug therapy. Topical antibiotics are generally only suitable for patients with mild symptoms, and hyperbaric oxygen therapy is expensive and has limited clinical research, making it difficult to assess its actual efficacy. Hydrogel dressings, with their unique three-dimensional porous structure, have stronger water absorption properties, maintaining a moist environment for a longer period and providing a suitable environment for cell attachment, survival, and proliferation, making them ideal dressings for diabetic wounds. However, existing gel dressings often suffer from insufficient mechanical strength and are prone to leakage from the wound. Mechanical tearing during dressing changes and backing bandage changes can easily cause secondary damage and infection, significantly reducing patient compliance. Therefore, developing a gel dressing with strong mechanical strength and absorbability to provide long-term coverage and protection for open wounds while avoiding tearing during dressing changes is of significant clinical importance for the treatment of diabetic wounds.

[0004] Meanwhile, given the complexity of the diabetic microenvironment—including high glucose levels, persistent bacterial infection, and lingering inflammation—most current gel treatments target only a single environmental factor in the wound. This singular approach often leads to poor wound healing or a risk of recurrence. Therefore, this invention employs a dual-layer gel multi-stage combined treatment strategy and a phased sequential treatment approach. The lower layer first rapidly releases an antibacterial agent, using photothermal therapy for sterilization, while the upper gel then releases an anti-inflammatory drug. This achieves a treatment effect of first addressing bacteria and then intensifying inflammation, thereby accelerating the healing process of diabetic wounds. Summary of the Invention

[0005] One objective of this invention is to provide a double-layer gel patch, consisting of an upper gel layer and a lower gel layer, wherein the lower gel layer is applied to the skin surface during use;

[0006] The upper gel layer is made of methacrylamide gelatin, oxidized hyaluronic acid, photoinitiator and anti-inflammatory drug. The mass ratio of methacrylamide gelatin to oxidized hyaluronic acid is 20-30:10-20, the mass percentage of photoinitiator is 0.2 wt.%, and the concentration of anti-inflammatory drug is 1 mg / mL.

[0007] The lower gel layer is made of 3-carboxyphenylboronic acid grafted gelatin, dihydrocaffeic acid grafted ε-polylysine and an antibacterial agent. The mass ratio of 3-carboxyphenylboronic acid grafted gelatin to dihydrocaffeic acid grafted ε-polylysine is 5:1, and the concentration of the antibacterial agent is 100 μg / mL.

[0008] Furthermore, the upper gel layer and the lower gel layer have the same thickness and an area ratio of 5:1.

[0009] Furthermore, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphite, and the anti-inflammatory drug is nimesulide.

[0010] Furthermore, the antibacterial agent is copper sulfide nanoparticles.

[0011] In embodiments of the present invention, the mass ratio of methacrylamide gelatin to oxidized hyaluronic acid is preferably 25-30:10-15, more preferably 25:15.

[0012] A second objective of this invention is to provide a method for preparing the aforementioned double-layer gel patch, comprising the following steps:

[0013] Step 1: Dissolve methacrylamide gelatin and oxidized hyaluronic acid in water, then mix them together. Add a photoinitiator and an anti-inflammatory drug to the mixture to obtain the upper gel precursor solution.

[0014] Step 2: Inject the upper gel precursor solution into mold A and crosslink and cure it under ultraviolet conditions to obtain the upper gel layer;

[0015] Step 3: Dissolve 3-carboxyphenylboronic acid grafted gelatin in water, and then add an antibacterial agent;

[0016] Step 4: Dissolve dihydrocaffeic acid grafted with ε-polylysine in water, adjust the pH of the solution to 9, add it to the mixture in Step 3, and after gelation, inject it into mold B to obtain the lower gel layer.

[0017] Step 5: Combine the upper gel layer with the lower gel layer for self-healing, allow to stand for cross-linking, and obtain the double-layer gel patch.

[0018] Furthermore, the ultraviolet light condition in step 2 is 405 nm.

[0019] Furthermore, the condition for static crosslinking in step 5 is 4°C.

[0020] A third objective of this invention is to provide the application of the above-mentioned double-layer gel patch in the preparation of topical medications for diabetic wounds.

[0021] This invention selects dihydrocaffeic acid containing catechol groups to graft ε-polylysine, and crosslinks it with 3-carboxyphenylboronic acid grafted gelatin through the formation of phenylboronic ester bonds to form a lower gel. The high-sugar microenvironment of diabetic wounds causes the phenylboronic ester bonds to break, and the lower gel responds by liquefying rapidly. The copper sulfide loaded thereon settles on the wound surface, and then photothermal stimulation by 808 nm near-infrared light generates antibacterial activity. Furthermore, the ε-polylysine in the gel can continuously inhibit bacteria on the wound surface. In the upper gel, methacrylamide gelatin crosslinks under 405 nm ultraviolet light to form a gel with high mechanical strength, elasticity, and adhesion. It can cover and adhere to the wound to prevent secondary infection, and avoid mechanical tearing and secondary damage after medication. Simultaneously, it loads anti-inflammatory drugs as a gel reservoir to exert a long-lasting anti-inflammatory effect. The bilayer gel of this invention combines high mechanical strength and absorbability, and enables a sequential treatment strategy of first treating bacteria and then inducing anti-inflammation. Attached Figure Description

[0022] Figure 1 This is a macroscopic morphological diagram of the double-layer gel patch prepared in Example 1.

[0023] Figure 2 The diagram shows the maximum compressive stress of the double-layer gel patches prepared in Examples 1-3.

[0024] Figure 3 The image shows the adhesion strength of the double-layer gel patches prepared in Examples 1-3.

[0025] Figure 4 This is a graph showing the percentage of drug release from the double-layer gel patches prepared in Examples 1-3.

[0026] Figure 5This is a graph showing the glucose-responsive morphological changes of the double-layer gel patch prepared in Example 1.

[0027] Figure 6 The graph shows the temperature change over time of the copper sulfide solution and the lower gel layer under 808 nm near-infrared light.

[0028] Figure 7 This is a graph showing the residual rate during the gel degradation process.

[0029] Figure 8 The images show the inhibitory effect of the gel on Escherichia coli and Staphylococcus aureus, where: A is the antibacterial effect of plate coating, B is the survival rate of Escherichia coli, and C is the survival rate of Staphylococcus aureus.

[0030] Figure 9 The effect on the healing of infected wounds in diabetic rats after 14 days. Detailed Implementation

[0031] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[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] In this invention, the methacrylamide gelatin, oxidized hyaluronic acid, 3-carboxyphenylboronic acid-grafted gelatin, dihydrocaffeic acid-grafted ε-polylysine, and copper sulfide nanoparticles can be prepared using methods reported in existing literature. In the following examples, they were prepared using the following methods.

[0035] Preparation of methacrylamide gelatin: 10 g of gelatin was dissolved in 200 mL of PBS by stirring at 50 °C. 6 mL of methacrylic anhydride was slowly added dropwise to the gelatin solution, and the mixture was stirred vigorously at 50 °C for 2 hours. 50 mL of PBS was preheated to 50 °C and added to the concentrated solution for dilution, followed by stirring at 50 °C for 10 min. The solution was then dialyzed for 7 days at 40 °C using a 12-14 kDa dialysis bag. After dialysis, 100 mL of preheated ultrapure water was added to the solution for dilution, and the mixture was stirred at 40 °C for 15 min. The solution was then quickly transferred to a tray and lyophilized for 2 days to obtain a white, porous, foamy product.

[0036] Preparation of oxidized hyaluronic acid: Weigh 2 g of hyaluronic acid and dissolve it in 200 mL of deionized water, stirring until completely dissolved. Dissolve 1.072 g of sodium periodate in 10 mL of deionized water, then add it dropwise to the above hyaluronic acid solution, stirring in an ice bath in the dark for 24 h. Then add 1 mL of ethylene glycol to terminate the reaction for 1 h. After the reaction, transfer the reaction solution to an 8k-14kD dialysis bag and dialyze for three days to obtain the final product.

[0037] Preparation of 3-carboxyphenylboronic acid-grafted gelatin: 4 g of gelatin was dissolved in 100 mL of pure water at 40 °C. Simultaneously, 1 g of 3-carboxyphenylboronic acid was dissolved in 20 mL of methanol, and 1 g of EDC and 575 mg of NHS were dissolved in 20 mL of water. The mixture was then activated in an ice bath for 30 min. The activated 3-carboxyphenylboronic acid solution was then added dropwise to the gelatin aqueous solution, and the reaction was carried out in a 37 °C water bath for 48 h. The product was dialyzed against deionized water at pH 7.4 for 3 days using a dialysis bag, and then freeze-dried to obtain a white solid.

[0038] Preparation of ε-polylysine grafted with dihydrocaffeic acid: 1.2 g of ε-polylysine and 167 mg of NHS were dissolved in 40 mL of deionized water. Then, 427 mg of dihydrocaffeic acid was dissolved in 40 mL of acetonitrile. The acetonitrile solution of dihydrocaffeic acid was added to the ε-polylysine aqueous solution and stirred until homogeneous. 500 mg of EDC was weighed and dissolved in 20 mL of deionized water, and added dropwise to the above solution. The reaction was carried out at room temperature in the dark for 24 h. The resulting solution was then dialyzed in a 1 kDa dialysis bag for 3 days and finally lyophilized to obtain the final product.

[0039] Preparation of copper sulfide nanoparticles: 2.4 g PVP-K30 was dissolved in 250 mL of water and stirred until dissolved. 1 mL of 0.5 mol / L copper chloride solution was added dropwise, and the mixture was stirred vigorously at room temperature for 5 min. 250 mL of sodium hydroxide (pH=9) was added, and the mixture was stirred until homogeneous. 80 μL of 80% hydrazine hydrate was added dropwise, and the mixture was stirred vigorously at room temperature for 10 min. Finally, 4 mL of 160 mg / mL sodium sulfide solution was added. The mixture was heated in a 60℃ water bath and stirred vigorously for 2 h. After centrifugation at 10000 rpm for 10 min, the nanoparticles were washed three times with ultrapure water until neutral, and then lyophilized for 2 days. Example 1

[0040] This embodiment describes a method for preparing a double-layer gel patch consisting of a blank upper gel layer and a blank lower gel layer. The specific steps are as follows:

[0041] Step 1: Dissolve 1 g of methacrylamide gelatin (GelMA) in 2 mL of deionized water and heat and stir at 40°C until dissolved; dissolve 600 mg of oxidized hyaluronic acid (OHA) in 2 mL of deionized water and stir until completely dissolved; mix the two solutions thoroughly; add 8 mg of phenyl-2,4,6-trimethylbenzoyl lithium phosphite to the above mixed solution and stir thoroughly to obtain a blank upper gel precursor solution;

[0042] Step 2: Take 1 mL of blank upper hydrogel precursor solution and inject it into a 20 mm * 20 mm * 2 mm square polydimethylsiloxane mold. Cure it by photocrosslinking under 405 nm ultraviolet light for 10 min until a blank upper hydrogel (UPgel) is formed.

[0043] Step 3: Take 200 mg of 3-carboxyphenylboronic acid grafted gelatin and dissolve it in 3 mL of deionized water by heating and stirring at 37°C.

[0044] Step 4: Dissolve 40 mg of dihydrocaffeic acid-grafted ε-polylysine in 1 mL of deionized water and stir until completely dissolved. Adjust the pH to 9 with sodium hydroxide and add it dropwise to the solution in Step 3 while stirring to obtain the gel precursor solution. A blank lower layer gel (BT gel) is formed within 1 min. Inject the blank lower layer gel into a circular polydimethylsiloxane mold with a diameter of 10 mm and a thickness of 2 mm and continue crosslinking at 4 °C for 10 min.

[0045] Step 5: Combine the blank upper gel and the blank lower gel for self-healing, crosslink at 4°C for 20 min, and demold to obtain the blank bilayer gel (UP / BT gel).

[0046] like Figure 1 As shown, the bilayer gel has a square upper layer and a circular lower layer. The upper layer has a larger area, which is conducive to completely covering the wound, while the lower layer directly contacts the wound to exert the drug effect. Moreover, the cross-linking effect between the two layers is excellent. Example 2

[0047] The difference between this embodiment and Embodiment 1 is that the amount of methacrylamide gelatin and oxidized hyaluronic acid in the upper gel precursor solution is 20% by mass and volume, respectively; the rest is the same as in Embodiment 1. Example 3

[0048] The difference between this embodiment and Embodiment 1 is that the amount of methacrylamide gelatin used in the upper gel precursor solution is 30% by mass and the amount of oxidized hyaluronic acid used is 10% by mass and volume, while the rest is the same as in Embodiment 1. Example 4

[0049] The difference between this embodiment and Embodiment 1 is that the amount of methacrylamide gelatin used in the upper gel precursor solution is 30% by mass and the amount of oxidized hyaluronic acid used is 10% by mass and volume, while the rest is the same as in Embodiment 1. Example 5

[0050] The difference between this embodiment and Embodiment 1 is that the amount of 3-carboxyphenylboronic acid grafted gelatin in the lower gel is 10% by mass and the amount of dihydrocaffeic acid grafted ε-polylysine is 2% by mass and volume. The rest is the same as in Embodiment 1.

[0051] During the preparation process, it was observed that the lower gel formed at this ratio had a longer glucose response time, which was (19.49±0.61) min. Example 6

[0052] The difference between this embodiment and Example 1 is that the gel precursor solution in step 2 contains 1 mg / mL of nimesulide solution, and an upper gel loaded with nimesulide and a blank lower gel (NIM@UP / BT gel) are prepared. The rest is the same as in Example 1. Example 7

[0053] The difference between this embodiment and Example 1 is that the gel precursor solution in step 4 contains 100 μg / mL ultrasonically dispersed copper sulfide (CuS) solution to prepare a blank upper gel and a lower gel loaded with copper sulfide (UP / CuS@BT gel). The rest is the same as in Example 1. Example 8

[0054] The difference between this embodiment and Embodiment 1 is that the gel precursor solution in step 2 contains 1 mg / mL of nimesulide solution, and the gel precursor solution in step 4 contains 100 μg / mL of ultrasonically dispersed copper sulfide (CuS) solution, thus obtaining an upper gel loaded with nimesulide and a lower gel loaded with copper sulfide (NIM@UP / CuS@BT gel). The rest is the same as in Embodiment 1.

[0055] The gels obtained in the above examples were then measured:

[0056] 1. Determination of gel mechanical properties

[0057] The compressive force of the gel patches in Examples 1-3 was tested using a universal testing machine, and the mechanical strength of the prepared gels was analyzed.

[0058] Experimental results are as follows Figure 2 As shown, the gel prepared in Example 1 has the highest maximum compressive stress, indicating that it has the best mechanical strength and can better meet the design requirements.

[0059] 2. Determination of gel adhesion force

[0060] The adhesion force of the gel patches in Examples 1-3 was determined by shear tensile test, and the adhesion strength was calculated by the formula "σ=F / (Lb)" (where σ represents tensile shear strength, MPa; F represents failure load, N; L represents adhesive surface length, mm; b represents adhesive surface width, mm).

[0061] Experimental results are as follows Figure 3 As shown, the adhesive strength increases with increasing methacrylamide gelatin content, depending on the bioadhesive properties of the methacrylamide gelatin itself. The gel prepared in Example 3 exhibits the strongest adhesive strength, while the gel prepared in Example 1 has moderate adhesive strength.

[0062] 3. Determination of drug release performance of the gel

[0063] Using a membrane-free dissolution model and pH 7.4 PBS solution as the release medium, the in vitro drug release behavior of the gel patches in Examples 1-3 was investigated. Precursor solutions containing 1 mg / mL nimesulide were prepared according to the proportions described in Examples 1-3. The demolded gel was placed in a beaker containing 50 mL of pH 7.4 PBS solution, and the beaker was placed in a 37 ºC constant-temperature shaker and shaken at 100 rpm. At predetermined time points (0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h), 1 mL of release medium was collected, and 1 mL of release medium was added simultaneously. After centrifuging the collected solutions at 10000 rpm for 10 min, the supernatant was used to determine the nimesulide concentration using UV spectrophotometry, and the cumulative release percentage of nimesulide at each time point was calculated.

[0064] Experimental results are as follows Figure 4 As shown, the cumulative release of nimesulide decreased with increasing content of methacrylamide gelatin. This may be because the increased content of methacrylamide gelatin leads to increased gel cross-linking density, resulting in a smaller pore size in the three-dimensional gel network and thus a decrease in the cumulative drug release. The release rate showed an inverse trend to the cumulative release amount. The gel prepared in Example 1 exhibited a sustained-release effect within the first 4 hours, with a moderate release rate. In Example 2, the gel released nimesulide rapidly, and the gel skeleton almost dissolved after release, which was not conducive to achieving sustained drug release. In Example 3, the gel released slowly, but its cumulative release rate was only 47%. Considering mechanical strength, adhesion, and drug release performance, the preferred ratio of methacrylamide gelatin to oxidized hyaluronic acid is 25:15.

[0065] 4. Glucose Response Performance Determination

[0066] The gel prepared in Example 8 was placed on a clean glass slide with the lower gel layer facing upwards. A 9 mg / mL glucose solution was added to the surface of the gel, and the liquefaction time of the lower gel layer was recorded.

[0067] Experimental results are as follows Figure 5 As shown, the lower gel liquefies after 12 minutes under the action of glucose solution, changing from a solid to a liquid state, while the upper gel maintains its shape. This indicates that the lower layer of the double-layer gel patch can quickly respond to the high-sugar environment of the diabetic wound, liquefying to fill the wound shape and releasing copper sulfide nanoparticles, which is beneficial for subsequent infrared photothermal sterilization. The upper layer stably covers the wound to prevent the liquefaction and loss of the lower gel.

[0068] 5. Determination of photothermal properties of the gel

[0069] The lower gel (CuS@BT gel) prepared in Example 8 was placed in a 1.5 mL ep tube. Deionized water (DI water), 100 μg / mL CuS solution, and the blank lower gel (BT gel) prepared in step 4 of Example 1 were set up as control groups. The sample was irradiated with 808 nm near-infrared light for 10 min, and the temperature was recorded every 30 s using an infrared thermal imager to observe the temperature change over time.

[0070] like Figure 6 As shown, CuS@BT gel exhibits good photothermal properties, reaching a temperature of 55℃ after 10 minutes of 808 nm near-infrared light irradiation, which is beneficial for wound antibacterial activity. The CuS solution reaches a temperature of 50.7℃ after 10 minutes, demonstrating excellent photothermal conversion performance. The temperature of BT gel also increases under near-infrared light irradiation compared to the control group, possibly due to the free radical scavenging ability of the catechol structure in dihydrocaffeic acid, which endows the gel with certain photothermal conversion properties.

[0071] 6. Determination of gel degradation performance

[0072] The in vitro degradation performance of the upper hydrogel from step 2 and the lower hydrogel from step 4 in Example 1 was investigated using a gravimetric method. The prepared gel was weighed and recorded as m1, then immersed in PBS solution and placed in a 37°C incubator. On days 1, 2, 4, 6, 8, 10, 16, and 20, the hydrogel was removed from the PBS solution and weighed, recorded as m2. The percentage of residual hydrogel degradation was calculated using the formula: Residual percentage = m2 / m1 × 100%.

[0073] Residual rate results as follows Figure 7As shown, the upper gel swells in the initial stage and degrades slowly, reaching complete degradation after 20 days. The lower gel, however, degrades more rapidly, almost completely degrading by day 10. This is presumably because the lower gel has weaker mechanical strength than the upper gel, making it more easily degraded. The experimental results indicate that both the upper and lower layers of the gel possess good biodegradability and can be degraded and absorbed by the wound, avoiding damage during dressing changes.

[0074] 7. In vitro antibacterial properties of the gel

[0075] Six experimental groups were set up: PBS group, 3-carboxylated phenylboronic acid grafted group (PBA), blank lower gel obtained in step 4 of Example 1 (BT gel), infrared-illuminated CuS solution group (CuS / NIR+), non-infrared-illuminated CuS solution group (CuS / NIR-), and lower gel obtained in Example 8 (CuS@BT gel).

[0076] Escherichia coli or Staphylococcus aureus were incubated overnight in a shaker at 220 rpm and 37°C. Bacteria in the logarithmic growth phase were then collected and the bacterial suspension was diluted to 10⁻¹⁰ with LB medium. 6 CFU / mL; 5% PBA solution, 100 μg / mL CuS solution, BT gel, and CuS@BT gel were prepared in advance. 500 μL of the prepared sample was added to a 1.5 mL ep tube, followed by 500 μL of diluted bacterial solution. After irradiation with 808 nm near-infrared light for 10 min, 100 μL of supernatant bacterial solution was taken from each group, diluted 100 times, and then spread on agar plates. The spread plates were incubated overnight at 37℃, and the colony count was observed.

[0077] Experimental results are as follows Figure 8 As shown in Figure A, the growth of *Escherichia coli* and *Staphylococcus aureus* on agar plates is as follows: Compared with the control group PBS, both the CuS / NIR+ group and the BT gel group showed good antibacterial effects. This is attributed to the photothermal effect of CuS and the cationic antibacterial effect of ε-polylysine in BT gel. Figures B and C show the survival rates of *Escherichia coli* and *Staphylococcus aureus*, respectively. It can be seen that CuS@BT gel has the strongest antibacterial effect, indicating that the antibacterial effect of the gel itself obtained in Example 1, combined with the photothermal properties imparted by CuS, works synergistically to inhibit pathogens in wounds, achieving a synergistic antibacterial effect.

[0078] 8. Internal wound healing effect

[0079] Establishment of a type 2 diabetes and infected wound model in SD rats: Thirty SD rats were acclimatized to a normal diet for one week, followed by a high-sugar, high-fat diet for four weeks. SD rats were intraperitoneally injected with 1% streptozotocin at a dose of 35 mg / kg, while continuing the high-sugar, high-fat diet. Successful modeling of diabetes was defined as a fasting blood glucose level of 13.5 mmol / L seven days after injection, accompanied by diabetic symptoms such as polydipsia, polyphagia, and loose, yellowing fur. Rats successfully modeled according to the above criteria were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution. After anesthesia, the rats were fixed to the operating table, all hair on their backs was removed, and a 10 mm diameter wound was created. Subsequently, 50 μL of Staphylococcus aureus suspension (10 μL / kg) was dripped into the wound. 8 (CFU / mL) to establish a wound infection model in diabetic rats.

[0080] Rats that successfully developed the model were divided into 5 groups: PBS group, blank bilayer gel group (UP / BT gel), blank upper gel and copper sulfide-loaded lower gel group (UP / CuS@BT gel), nimesulide-loaded upper gel and blank lower gel group (NIM@UP / BT gel), and the gel prepared in Example 8 (NIM@UP / CuS@BT gel). Images of the wounds in diabetic rats were collected on days 0, 3, 7, and 14 after drug administration.

[0081] Wound healing effect Figure 9 As shown, compared with the control group, all gels effectively promoted wound healing. The NIM@UP / CuS@BT gel group, loaded with nimesulide and copper sulfide, exhibited the best wound healing effect, with a noticeable difference in healing performance observed as early as day 7. Comparing the UP / BT gel group and the UP / CuS@BT gel group, the UP / CuS@BT gel group showed better healing, indicating that the antibacterial effect of copper sulfide promotes wound healing. Comparing the UP / CuS@BT gel group and the NIM@UP / CuS@BT gel group, the NIM@UP / CuS@BT gel group showed a faster healing rate, indicating that the anti-inflammatory effect of nimesulide effectively promotes wound healing. These results suggest that the double-layer gel patch of this invention has potential application value in the treatment of diabetic wounds.

Claims

1. A dual layer gel patch, characterized in that, The double-layer gel patch is composed of an upper gel layer and a lower gel layer, and the lower gel layer is attached to the skin surface when used; the thickness of the upper gel layer and the lower gel layer is the same, and the area ratio is 5:1; The upper gel layer is made of methacrylated gelatin, oxidized hyaluronic acid, a photoinitiator and an anti-inflammatory drug, the mass ratio of methacrylated gelatin and oxidized hyaluronic acid is 20-30:10-20, the mass percentage of the photoinitiator is 0.2wt.%, and the concentration of the anti-inflammatory drug is 1mg / mL; the photoinitiator is phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and the anti-inflammatory drug is nimesulide; The lower gel layer is made of 3-carboxyphenylboronic acid grafted gelatin, dihydrocaffeic acid grafted ε-polylysine and an antibacterial agent, the mass ratio of 3-carboxyphenylboronic acid grafted gelatin and dihydrocaffeic acid grafted ε-polylysine is 5:1, and the concentration of the antibacterial agent is 100μg / mL; the antibacterial agent is copper sulfide nanoparticles; The preparation method of the double-layer gel patch comprises the following steps: Step 1, respectively dissolve methacrylated gelatin and oxidized hyaluronic acid in water, then mix them, add a photoinitiator and an anti-inflammatory drug to the mixed solution to obtain an upper gel precursor solution; Step 2, inject the upper gel precursor solution into mold A, crosslink and solidify under ultraviolet conditions to obtain an upper gel layer; Step 3, dissolve 3-carboxyphenylboronic acid grafted gelatin in water, then add an antibacterial agent; Step 4, dissolve dihydrocaffeic acid grafted ε-polylysine in water, adjust the pH of the solution to 9, and add it to the mixed solution of step 3, then inject the gelled solution into mold B to obtain a lower gel layer; Step 5, combine the last gel layer with the lower gel layer and self-heal, then stand still to crosslink, to obtain the double-layer gel patch.

2. The method of making a double-layered gel patch of claim 1, characterized in that, comprises the following steps: Step 1, respectively dissolve methacrylated gelatin and oxidized hyaluronic acid in water, then mix them, add a photoinitiator and an anti-inflammatory drug to the mixed solution to obtain an upper gel precursor solution; Step 2, inject the upper gel precursor solution into mold A, crosslink and solidify under ultraviolet conditions to obtain an upper gel layer; Step 3, dissolve 3-carboxyphenylboronic acid grafted gelatin in water, then add an antibacterial agent; Step 4, dissolve dihydrocaffeic acid grafted ε-polylysine in water, adjust the pH of the solution to 9, and add it to the mixed solution of step 3, then inject the gelled solution into mold B to obtain a lower gel layer; Step 5, combine the last gel layer with the lower gel layer and self-heal, then stand still to crosslink, to obtain the double-layer gel patch.

3. The production method according to claim 2, characterized by, The ultraviolet conditions in step 2 are 405nm.

4. The preparation method according to claim 2, characterized in that, The crosslinking conditions in step 5 are 4℃.

5. The double-layer gel patch of claim 1 in the preparation of an external drug for diabetic wound surface.

Images