Nanoparticle hydrogel microneedle with microenvironment responsiveness regulation function and preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a nano-particle hydrogel microneedle with a microenvironment responsive regulation function as well as a preparation method and application of the nano-particle hydrogel microneedle. The nanoparticle hydrogel microneedle comprises a needle tip layer and a backing layer, the needle point layer is located on the backing layer, and the needle point layer and the backing layer form an integrated double-layer structure through photo-crosslinking; the needle tip layer is formed by compounding methylacryloylated gelatin and nanoparticles of a Prussian blue nano-enzyme modified by glucose oxidase; and the backing layer is composed of methacrylated gelatin. The nanoparticle hydrogel microneedle provided by the invention can penetrate through a skin barrier and a bacterial biofilm structure during use, delivers functional nanoparticles to deep tissues of a wound surface, and aims at different requirements of an infection stage, an inflammation stage and a repair stage in a healing process of the infectious diabetes wound surface; and ordered regulation and control of microenvironment response and treatment functions of infectious diabetes mellitus wounds in different repair stages are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to nanoparticle hydrogel microneedles with microenvironment responsive regulation function, their preparation methods, and applications. Background Technology

[0002] Skin wounds in chronic diabetes are prone to persistent inflammation, oxidative stress, and bacterial infection under long-term hyperglycemia. The wound microenvironment is complex and dynamically changing, resulting in a long healing period and great difficulty in treatment.

[0003] To address the aforementioned issues, existing research has proposed various treatment strategies, including antibacterial drugs, anti-inflammatory agents, and nanomaterials with enzyme-like catalytic activity. Among these, nanozymes, due to their high structural stability, tunable catalytic performance, and ability to mimic the activities of various natural enzymes, have shown potential in antibacterial therapy, inflammation regulation, and metabolic intervention. However, existing nanozymes and related therapeutic systems still have the following limitations: most nanozyme systems have relatively simple modes of action, making it difficult to achieve therapeutic effects tailored to the different needs of the infection, inflammation, and repair phases of diabetic wound healing based on changes in the microenvironment. Summary of the Invention

[0004] To address the issue that most existing nanozyme systems operate on a relatively singular mechanism, making it difficult to achieve therapeutic effects tailored to the different needs of the infection, inflammation, and repair phases in the healing process of diabetic wounds based on changes in the microenvironment, this invention provides nanoparticle hydrogel microneedles with microenvironment-responsive regulation capabilities, along with their preparation method and applications. The nanoparticle hydrogel microneedles provided by this invention can achieve phased functional regulation based on the microenvironmental characteristics of different healing stages of infectious diabetic wounds, thereby providing an orderly and synergistic treatment approach for the repair of infectious diabetic wounds. To achieve the above objectives, this invention employs the following technical solution.

[0005] This invention provides a nanoparticle hydrogel microneedle with microenvironment-responsive regulation function, the nanoparticle hydrogel microneedle comprising a tip layer and a backing layer. The tip layer is located on top of the backing layer, and the two are photocrosslinked to form an integrated bilayer structure.

[0006] The needle tip layer is composed of methacrylated gelatin and Prussian blue nanoparticles modified with glucose oxidase; wherein the mass ratio of the glucose oxidase-modified Prussian blue nanoparticles to methacrylated gelatin is 3:2000~3000; the glucose oxidase-modified Prussian blue nanoparticles have a core-shell structure, with Prussian blue nanoparticles as the core and ε-polylysine and glucose oxidase sequentially modified on the surface.

[0007] The backing layer is composed of methacrylated gelatin; the methacrylated gelatin is obtained by an amidation reaction of gelatin and methacrylic anhydride.

[0008] The nanoparticle hydrogel microneedles provided by this invention can penetrate the skin barrier and bacterial biofilm structure to deliver functional nanoparticles to deep wound tissues. In the microenvironment of infectious diabetic wounds, the nanoparticle hydrogel microneedles can regulate local glucose levels and pH levels, thereby electrostatically adsorbing onto bacterial surfaces and inducing the nanoparticles to exert corresponding POD-like (peroxidase) catalytic functions. This, combined with the photothermal conversion ability of the nanoparticles, produces an inhibitory effect on bacterial growth. When wound infection is controlled and the local microenvironment tends towards neutral conditions, the nanoparticles of this invention can further exert CAT-like (catalase) and SOD-like (superoxide dismutase) activities to scavenge excess reactive oxygen species, alleviate inflammatory responses, and generate oxygen to alleviate hypoxic microenvironments, thus helping to improve the wound microenvironment. Through the above methods, the nanoparticle hydrogel microneedles of this invention can achieve phased functional regulation according to the microenvironmental characteristics of different stages of infectious diabetic wounds, thereby providing an orderly and synergistic treatment approach for the repair of infectious diabetic wounds.

[0009] Preferably, the mass ratio of gelatin to methacrylic anhydride is 1:0.7~0.9.

[0010] Preferably, the gelatin is fish gelatin.

[0011] Preferably, the amidation reaction is carried out at 48°C to 52°C for 3.5 to 4.5 hours.

[0012] The present invention also provides a method for preparing the aforementioned nanoparticle hydrogel microneedles, comprising the following steps: The glucose oxidase-modified Prussian blue nanozyme nanoparticles and methacrylamide gelatin were dispersed in water to obtain mixed solution 1.

[0013] The methacrylated gelatin was dissolved in water to obtain mixed solution 2.

[0014] The photoinitiator is mixed with mixed solution 1 to obtain mixed solution 3, and the photoinitiator is mixed with mixed solution 2 to obtain mixed solution 4; the mass ratio of the photoinitiator to mixed solution 1 and mixed solution 2 is 0.14%~0.16%.

[0015] The mixed solutions 3 and 4 are centrifuged to remove air bubbles, and then injected into the microneedle mold as the tip layer (tip solution) and backing layer (backing solution) in sequence.

[0016] After drying, the nanoparticle hydrogel microneedles are demolded and cured under ultraviolet light to obtain the nanoparticle hydrogel microneedles.

[0017] Preferably, the concentration of the glucose oxidase-modified Prussian blue nanozyme nanoparticles in mixed solution 1 is 295 μg / mL to 305 μg / mL.

[0018] Preferably, the glucose oxidase-modified Prussian blue nanozyme nanoparticles are obtained through the following process.

[0019] Potassium ferricyanide and polyvinylpyrrolidone were added to a hydrochloric acid solution and then heat-treated to obtain Prussian blue nanozyme.

[0020] The Prussian blue nanozyme was dispersed in water and reacted with ε-polylysine by stirring to obtain ε-polylysine-modified Prussian blue nanozyme; wherein the mass ratio of the Prussian blue nanozyme to ε-polylysine was 1:30~40.

[0021] The Prussian blue nanozyme modified with ε-polylysine was dispersed in ultrapure water and reacted with a solution of glucose oxidase in a light-protected environment by stirring to obtain nanoparticles of the glucose oxidase-modified Prussian blue nanozyme; wherein the mass ratio of the Prussian blue nanozyme modified with ε-polylysine to glucose oxidase was 1:1 to 1.5.

[0022] Preferably, the mass fraction of the methacrylamide gelatin in both mixed solution 3 and mixed solution 4 is 18% to 22%.

[0023] The cross-linking reaction was carried out at room temperature for 35 to 45 minutes.

[0024] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid.

[0025] The present invention also provides the application of the aforementioned nanoparticle hydrogel microneedles in the preparation of products for treating infectious diabetic wounds.

[0026] Preferably, the product includes wound dressings.

[0027] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a nanoparticle hydrogel microneedle with microenvironment-responsive regulation function. The nanoparticle hydrogel microneedle provided by this invention comprises a tip layer and a backing layer; the tip layer is located above the backing layer, and the two are photocrosslinked to form an integrated bilayer structure; the tip layer is composed of methacrylated gelatin and Prussian blue nanoparticles modified with glucose oxidase; wherein the mass ratio of the glucose oxidase-modified Prussian blue nanoparticles to the methacrylated gelatin is 3:2000~3000; the glucose oxidase-modified Prussian blue nanoparticles have a core-shell structure, with Prussian blue nanoparticles as the core, and the surface sequentially modified with ε-polylysine and glucose oxidase; the backing layer is composed of methacrylated gelatin. The nanoparticle hydrogel microneedles provided by this invention have antibacterial, anti-inflammatory, and wound microenvironment-improving effects. By constructing a three-in-one intelligent regulation mechanism of "multi-enzyme cascade-charge reversal-microenvironment response", it solves the problem that most nanoenzyme systems in the prior art have relatively simple modes of action and are difficult to achieve therapeutic effects according to the different needs of the infection, inflammation and repair phases in the healing process of diabetic wounds based on changes in the microenvironment. Specifically, glucose oxidase (GOx) and Prussian blue nanozyme (PBzyme) are linked by ε-polylysine electrostatic bridging to form composite nanoparticles. In the hyperglycemic microenvironment of diabetic wounds, GOx catalyzes the oxidation of glucose to produce gluconic acid and H2O2, thus lowering the local pH. Prussian blue nanozyme then undergoes a pH-dependent enzyme activity conversion—exhibiting peroxidase activity under acidic conditions during the infection phase, catalyzing the generation of ·OH from H2O2 for efficient sterilization. Simultaneously, the dissociation of GOx on the nanoparticle surface exposes the ε-polylysine-modified Prussian blue nanozyme. Due to the positive charge on the ε-polylysine surface, the nanoparticles can bind to bacterial surfaces, synergistically enhancing photothermal conversion capabilities and thus strengthening the bactericidal effect. During the inflammatory and repair phases, as the pH rises, it exhibits catalase and superoxide dismutase activity, scavenging ROS, alleviating oxidative stress, and simultaneously generating oxygen to improve the hypoxic microenvironment. Combined with the deep delivery function of methacrylated gelatin hydrogel microneedles, nanoparticles can be delivered to the deep part of the wound through the skin barrier and bacterial biofilm to exert their effects. This allows the system to intelligently sense changes in the wound microenvironment and dynamically switch between antibacterial, anti-inflammatory, and repair-promoting functions, achieving precise sequential treatment for the entire healing process of infectious diabetic wounds.

[0028] 2. In preparing nanoparticle hydrogel microneedles with microenvironment-responsive regulation, this invention uses methacrylated gelatin (GelMA) as the main material and loads glucose oxidase-modified Prussian blue nanozyme nanoparticles (PB@EPL@GOx) at the needle tip. Methacrylated gelatin is cross-linked into a hydrogel through free radical polymerization, thus loading the nanoparticles into the hydrogel network.

[0029] This invention combines functional nanoparticles with a hydrogel microneedle structure, enabling the nanoparticles to effectively penetrate the skin barrier and bacterial biofilms, achieving delivery to deep tissues of the wound. By integrating multiple functions into a single nanoparticle hydrogel microneedle system, and through the regulation of the microenvironment in infectious diabetic wounds, the nanoparticle hydrogel microneedles can achieve phased functional regulation according to the microenvironmental needs at different stages of infectious diabetic wounds. During the active infection stage, the system can regulate the microenvironment, generating reactive oxygen species (ROS) and specifically binding with bacteria to provide a synergistic photothermal effect, thus offering a beneficial antibacterial effect. During the inflammatory stage, the system can scavenge ROS, alleviate inflammation, and generate oxygen to relieve wound hypoxia, thereby further improving the microenvironment and promoting wound healing. Through phased functional regulation, the coordination and treatment efficiency of each stage of wound healing can be improved, which is beneficial for accelerating tissue repair and restoring microenvironmental homeostasis, thus providing a comprehensive and synergistic treatment plan for infectious diabetic wounds.

[0030] The present invention relates to nanoparticle hydrogel microneedles with microenvironment responsive regulation function, which are prepared from methacrylated gelatin (GelMA) and Prussian blue nanozyme nanoparticles modified with glucose oxidase (PB@EPL@GOx). The resulting hydrogel microneedles have good biocompatibility, mechanical properties, hypoglycemic ability, antibacterial, anti-inflammatory and wound healing promotion ability, and have a significant effect on the treatment or improvement of infectious diabetic wounds. Attached Figure Description

[0031] Figure 1 This is the NMR spectrum of the methacrylated gelatin used in this invention.

[0032] Figure 2 This is a potential diagram of the Prussian blue nanoparticles modified with glucose oxidase in this invention.

[0033] Figure 3 This is an electron microscope image of the nanoparticle hydrogel microneedles with microenvironment responsive regulation function in this invention.

[0034] Figure 4 This is a diagram showing the mechanical properties of the nanoparticle hydrogel microneedles with microenvironment responsive regulation function in this invention.

[0035] Figure 5 This is a diagram showing the hypoglycemic performance of the nanoparticle hydrogel microneedles with microenvironment responsive regulation function in this invention.

[0036] Figure 6 This is a diagram illustrating the antibacterial properties of the nanoparticle hydrogel microneedles with microenvironment-responsive regulation function in this invention.

[0037] Figure 7To verify the wound healing of rats in infectious diabetic rats, the present invention utilizes nanoparticle hydrogel microneedles with microenvironment-responsive regulation function.

[0038] Figure 8 This is a schematic diagram illustrating the material composition and function of the nanoparticle hydrogel microneedles with microenvironment responsive regulation function in this invention. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0040] Example 1 The present invention provides a nanoparticle hydrogel microneedle with microenvironment responsive regulation function, comprising a tip layer and a backing layer; the tip layer is composed of nanoparticles of methacrylated gelatin and Prussian blue nanozyme modified with glucose oxidase; the backing layer is composed of methacrylated gelatin.

[0041] The nanoparticle hydrogel microneedles of this invention with microenvironment-responsive regulation function are prepared using the following methods: The raw materials (methacrylated gelatin (GelMA) and glucose oxidase-modified Prussian blue nanozyme nanoparticles (PB@EPL@GOx)) are prepared as follows: Step 1, Preparation of Methacrylamide Gelatin (GelMA): At 50°C, 10 g of fish gelatin was added to 100 mL of PBS solution and stirred continuously in a 50°C water bath until completely dissolved. Then, 8 mL of methacrylic anhydride was gradually added to the solution, and the mixture was stirred vigorously at 50°C for 4 hours. After the reaction was complete, PBS solution was added to increase the system volume to five times its original volume to terminate the reaction. The resulting liquid was transferred to a dialysis bag with a molecular weight cutoff of 8000 Da for dialysis to remove unreacted impurities and byproducts. Finally, the dialyzed liquid was lyophilized to obtain methacrylamide gelatin (GelMA).

[0042] Fish gelatin (CAS: 9000-70-8) was purchased from Aladdin Reagent (Shanghai) Co., Ltd.

[0043] Methacrylic anhydride (CAS: 760-93-0) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0044] The PBS solution has a pH of 7.4 and a concentration of 10 mM.

[0045] The obtained product, methacrylated gelatin, was analyzed using nuclear magnetic resonance (NMR). The results of the ¹H NMR spectrum (¹H NMR) of the obtained product (methacrylated gelatin) are as follows: Figure 1 As shown, 1 In the H NMR, a clear double peak was observed at 5.3 ppm and 5.6 ppm, which is attributed to the olefinic signal of the -CH2=C(CH3)- structure in the methacryloyl group, indicating that the methacryloylated gelatin (GelMA) was successfully prepared.

[0046] Step 2: Preparation of glucose oxidase-modified Prussian blue nanozyme nanoparticles (PB@EPL@GOx): 0.396 g of potassium ferricyanide (K3[Fe(CN)6]) and 8.5 g of polyvinylpyrrolidone K30 (PVP) were added to 500 mL of 1 mol / L hydrochloric acid and completely dissolved under magnetic stirring. The dissolved liquid was then placed in a vacuum drying oven at 80 °C for 18 h. After the reaction, the product was washed successively with ultrapure water and ethanol, and then freeze-dried to obtain Prussian blue nanozyme (PB). The Prussian blue nanozyme was dispersed in ultrapure water and stirred with 30 mg / mL ε-polylysine (EPL) for 6 h. After washing with ultrapure water and freeze-drying, ε-polylysine-modified Prussian blue nanozyme (PB@EPL) was obtained. Subsequently, PB@EPL was dispersed in ultrapure water and mixed with 1 mg / mL glucose oxidase (GOx) solution and placed in a light-protected environment at 4 °C overnight with stirring. After washing, the product was freeze-dried to obtain glucose oxidase-modified Prussian blue nanozyme nanoparticles (PB@EPL@GOx).

[0047] The preparation method for the glucose oxidase (GOx) solution is as follows: Dissolve glucose oxidase in water at a concentration of 1 mg / mL and stir for 1 hour to ensure uniform dispersion.

[0048] Overnight stays refer to stays of 12 hours or more.

[0049] The Zeta potentials of the products at each stage were measured. The components were assembled sequentially, and the results are as follows: Figure 2 As shown, the surface charge of the nanoparticles exhibits a regular "negative → positive → negative" reversal: PB is negatively charged, becomes positively charged after modification with positively charged ε-polylysine, and finally becomes negatively charged after adsorbing negatively charged GOx. This indicates that the nanoparticles of Prussian blue nanozyme modified with glucose oxidase (PB@EPL@GOx) were successfully prepared.

[0050] Step 3, the preparation method of nanoparticle hydrogel microneedles with microenvironment responsive regulation function includes the following steps: Step 3.1: Disperse glucose oxidase-modified Prussian blue nanozyme nanoparticles (PB@EPL@GOx) at a concentration of 300 µg / mL and methacrylamide gelatin (GelMA) at a concentration of 20 wt% in deionized water to prepare mixed solution 1.

[0051] Step 3.2: Disperse methacrylamide gelatin (GelMA) at a concentration of 20 wt% in deionized water to prepare mixed solution 2.

[0052] Step 3.3: Add the photoinitiator to the mixed solution 1 obtained in Step 3.1 to obtain mixed solution 3 containing the initiator (0.15 wt%). Add the photoinitiator to the mixed solution 2 obtained in Step 3.2 to obtain mixed solution 4 containing the initiator (0.15 wt%). The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphine (CAS: 85073-19-4), purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0053] Step 3.4: Centrifuge the mixed solutions 3 and 4 obtained in step 3.3 at 5000 rpm for 5 min in a centrifuge to remove air bubbles and obtain the hydrogel microneedle solution.

[0054] Step 3.5: Inject the hydrogel microneedle solution obtained in step 3.4 into the mold respectively, wherein mixed solution 3 is the needle tip layer (needle tip solution) and mixed solution 4 is the backing layer (backing solution).

[0055] Step 3.6: Place the product obtained in step 3.5 into an oven at 37°C and dry for 9 hours.

[0056] Step 3.7: After completely demolding the product obtained in Step 3.6, place the dried microneedles in a UV curing chamber for 40 minutes to obtain nanoparticle hydrogel microneedles (PBEG@GelMA MN) with microenvironment responsive regulation function. Nanoparticle hydrogel microneedles with microenvironment responsive regulation function are simply referred to as nanoparticle hydrogel microneedles or hydrogels.

[0057] The surface of the prepared hydrogel was observed using scanning electron microscopy (SEM), and the results are as follows: Figure 3 As shown, the microneedle array is well-organized and uniform in size. The needle tips are typically pyramidal in shape, with a height of 750µm, a base width of about 300µm, and a spacing of 300µm between needles. This indicates that the nanoparticle hydrogel microneedles with microenvironment responsive regulation function have been successfully prepared.

[0058] The nanoparticle hydrogel microneedles with microenvironment responsive regulation function prepared in Example 1 all have good biocompatibility, mechanical properties, hypoglycemic ability, antibacterial, anti-inflammatory and wound healing promotion ability. The following description uses Example 1 as an example.

[0059] 1) The mechanical properties of nanoparticle hydrogel microneedles with microenvironment-responsive regulation were evaluated using skin from SD rats. After pressing rhodamine B-loaded microneedles into the dorsal skin of SD rats for 1 minute, the needle tips successfully penetrated the stratum corneum, forming clear needle-shaped imprints. Hematoxylin-eosin (H&E) staining was performed on the skin in the needle-penetration area, and the results are as follows: Figure 4 As shown, the microneedles were inserted into the skin of SD rats to a depth of approximately 500 μm, verifying that the nanoparticle hydrogel microneedles with microenvironment-responsive regulation function have certain mechanical properties and can achieve efficient transdermal drug delivery.

[0060] The SD rats were purchased from the Air Force Medical University.

[0061] 2) By co-culturing with L929 cells, we evaluated the ability of nanoparticle hydrogel microneedles with microenvironment-responsive regulation to regulate glucose levels in the cellular environment.

[0062] L929 cells were cultured in medium for 24 h, then transferred to medium containing 5 mg / mL glucose and cultured for another 24 h. In 6-well plates, medium extracts containing GelMA hydrogel microneedles (GelMA MN) and PBEG@GelMA hydrogel microneedles (PBEG@GelMAMN) were added respectively, and cultured for 24 h. Glucose consumption of L929 cells was then measured using a glucose analysis kit. Cells in medium extracts containing PBEG@GelMA hydrogel microneedles were designated as the group with microenvironment-responsive regulation (PBEG@GelMA MN), abbreviated as PBEG@GelMA MN group; cells in medium extracts containing GelMA hydrogel microneedles were designated as the group without microenvironment-responsive regulation (GelMA MN), abbreviated as GelMA MN group.

[0063] The L929 cells were purchased from Shanghai Fuheng Biotechnology Co., Ltd.

[0064] The results are as follows Figure 5 As shown, the nanoparticle hydrogel microneedles (PBEG@GelMAMN) with microenvironment-responsive regulation function can significantly increase the glucose consumption rate in the culture system. These microneedles can effectively reduce the high glucose level in the cell microenvironment, thereby improving the pathological microenvironment of diabetic wounds and promoting subsequent tissue repair and regeneration.

[0065] 3) The antibacterial effect of nanoparticle hydrogel microneedles with microenvironment-responsive regulation function was evaluated by crystal violet staining experiment.

[0066] 5×10 8 CFU / mL bacterial suspensions (Staphylococcus aureus and Escherichia coli, respectively) were inoculated into 48-well plates and cultured for 3 days. After incubation, the plates were air-dried for 20 minutes to dehydrate the biofilms. 500 μL of 0.1 wt% crystal violet staining solution was added to each well, and the plates were stained at room temperature for 15 minutes. After removing the staining solution, the plates were gently washed three times with physiological saline to remove unbound dye. Then, 1 mL of 33% glacial acetic acid solution was added, and the plates were incubated at 37°C with shaking for 30 minutes to ensure complete dissolution of the crystal violet adhering to the cell surface. An untreated bacterial suspension served as a control group.

[0067] Among them, the bacterial solution of Staphylococcus aureus is Staphylococcus aureus ( S.aureus (ATCC25923) was obtained through liquid culture. The bacterial culture of *E. coli* was obtained from *E. coli* (ATCC25923). E. coli (ATCC25922) was obtained by liquid culture. Staphylococcus aureus (ATCC25922) S.aureus ) and Escherichia coli ( E. coli All of them were purchased from Shanghai Sangon Biotech Co., Ltd.

[0068] The results are as follows Figure 6 As shown, compared to the control group, Staphylococcus aureus (Staphylococcus aureus) S.aureus ) and Escherichia coli ( E. coli After being treated with nanoparticle hydrogel microneedles with microenvironment-responsive regulation function and irradiated with 808nm laser in a glucose environment, the biofilm was almost completely removed, which indicates that nanoparticle hydrogel microneedles with microenvironment-responsive regulation function have good antibacterial properties.

[0069] 4) A diabetic rat model was induced by intraperitoneal injection of 1% (wt%) streptozotocin (STZ) solution. An 8mm diameter full-thickness skin defect was created on the rat's back using a ring-blade technique. Subsequently, 50μL of Staphylococcus aureus (STZ) was instilled into the wound. S. aureus Suspension (concentration 1×10) 9 To establish an infected diabetic wound model (CFU / mL), five treatment groups were set up to evaluate the therapeutic effect of hydrogel microneedles: control group, commercial dressing (HeraDerm) group, GelMA MN group, PBEG@GelMA MN group, and PBEG@GelMA MN+NIR group. Details are as follows:

[0070] A diabetic wound infection model was established using the above method. Forty-eight hours after infection, the model rats were treated in different groups (control group, commercial dressing (HeraDerm) group, GelMA MN group, PBEG@GelMA MN group, and PBEG@GelMA MN+NIR group). The wound healing status of the rats was photographed and recorded at the time of infection model establishment (day 2) and after the start of treatment (days 0, 3, 6, 9, and 12).

[0071] The control group consisted of rats that did not receive any treatment.

[0072] The commercial dressing (HeraDerm) group consisted of rats treated with a commercial hydrogel. The commercial hydrogel was purchased from Amway Biomedical Co., Ltd.

[0073] The GelMA MN group consisted of rats treated with hydrogel microneedles without nanoparticles (GelMA MN, hydrogel microneedles without nanoparticles having microenvironment-responsive regulatory functions). GelMA MN was obtained from the Prussian blue nanozyme nanoparticles modified with glucose oxidase in step 3.1 above.

[0074] The PBEG@GelMA MN group consisted of rats treated with nanoparticle hydrogel microneedles (PBEG@GelMA MN) prepared in Example 1.

[0075] The PBEG@GelMA MN+NIR group refers to the application of 808 nm laser irradiation to the nanoparticle hydrogel microneedles (PBEG@GelMA MN) prepared in Example 1.

[0076] The SD rats were purchased from the Air Force Medical University.

[0077] Experimental results are as follows Figure 7 As shown, the results indicated that the wounds of rats in the control group and the commercial dressing (HeraDerm) group healed relatively slowly. The wounds of rats in the PBEG@GelMA MN+NIR group healed significantly faster than those in other groups, and the wounds were almost closed by day 12 with no obvious scars.

[0078] In summary, the nanoparticle hydrogel microneedles (PBEG@GelMAMN) of this invention with microenvironment responsive regulation function have good biocompatibility, mechanical properties, hypoglycemic ability, antibacterial, anti-inflammatory and wound healing ability, and have a significant effect on the treatment or improvement of infectious diabetic wounds.

[0079] The nanoparticle hydrogel microneedles provided by this invention ( Figure 8This invention can penetrate the skin barrier and bacterial biofilm, delivering functional nanoparticles to deep wound tissues. In the microenvironment of infectious diabetic wounds, the microneedles can regulate local glucose levels and pH levels, electrostatically adsorb onto bacterial surfaces, and induce nanoparticles to exert POD-like catalytic activity, achieving highly efficient antibacterial effects through photothermal conversion. When infection is controlled and the wound microenvironment tends to be neutral, the nanoparticles further exert CAT-like and SOD-like enzyme activities, clearing excess reactive oxygen species, alleviating inflammatory responses, and generating oxygen to alleviate hypoxia, thereby improving the wound microenvironment. Thus, this invention achieves orderly regulation of the microenvironment response and function at different stages of repair in infectious diabetic wounds, providing a synergistic and efficient treatment strategy.

[0080] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0081] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A nanoparticle hydrogel microneedle with microenvironment-responsive regulation function, characterized in that, It includes a needle tip layer and a backing layer; the needle tip layer is located on top of the backing layer, and the two are photocrosslinked to form an integrated double-layer structure; The needle tip layer is composed of methacrylated gelatin and Prussian blue nanoparticles modified with glucose oxidase; wherein the mass ratio of the glucose oxidase-modified Prussian blue nanoparticles to methacrylated gelatin is 3:2000~3000; the glucose oxidase-modified Prussian blue nanoparticles have a core-shell structure, with Prussian blue nanoparticles as the core and ε-polylysine and glucose oxidase sequentially modified on the surface. The backing layer is composed of methacrylated gelatin; the methacrylated gelatin is obtained by an amidation reaction of gelatin and methacrylic anhydride.

2. The nanoparticle hydrogel microneedles according to claim 1, characterized in that, The mass ratio of gelatin to methacrylic anhydride is 1:0.7~0.

9.

3. The nanoparticle hydrogel microneedles according to claim 2, characterized in that, The gelatin mentioned is fish gelatin.

4. The nanoparticle hydrogel microneedles according to claim 1, characterized in that, The amidation reaction is carried out at 48℃~52℃ for 3.5h~4.5h.

5. The method for preparing nanoparticle hydrogel microneedles according to any one of claims 1 to 4, characterized in that, Includes the following steps: The glucose oxidase-modified Prussian blue nanozyme nanoparticles and methacrylated gelatin were dispersed in water to obtain mixed solution 1. The methacrylated gelatin was dissolved in water to obtain mixed solution 2; The photoinitiator is mixed with mixed solution 1 to obtain mixed solution 3, and the photoinitiator is mixed with mixed solution 2 to obtain mixed solution 4; the mass ratio of the photoinitiator to mixed solution 1 and mixed solution 2 is 0.14%~0.16%; The mixed solutions 3 and 4 were centrifuged to remove air bubbles, and then injected sequentially into the microneedle mold. After drying, the nanoparticle hydrogel microneedles are demolded and cured under ultraviolet light to obtain the nanoparticle hydrogel microneedles.

6. The preparation method according to claim 5, characterized in that, The concentration of the glucose oxidase-modified Prussian blue nanozyme nanoparticles in mixed solution 1 is 295 μg / mL to 305 μg / mL.

7. The preparation method according to claim 6, characterized in that, The glucose oxidase-modified Prussian blue nanozyme nanoparticles were obtained through the following process: Potassium ferricyanide and polyvinylpyrrolidone were added to a hydrochloric acid solution and then heat-treated to obtain Prussian blue nanozyme. The Prussian blue nanozyme was dispersed in water and reacted with ε-polylysine by stirring to obtain ε-polylysine-modified Prussian blue nanozyme; wherein the mass ratio of the Prussian blue nanozyme to ε-polylysine was 1:30~40. The Prussian blue nanozyme modified with ε-polylysine was dispersed in water and reacted with a solution of glucose oxidase in a light-protected environment by stirring to obtain nanoparticles of the glucose oxidase-modified Prussian blue nanozyme; wherein the mass ratio of the Prussian blue nanozyme modified with ε-polylysine to glucose oxidase was 1:1 to 1.

5.

8. The preparation method according to claim 5, characterized in that, The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

9. The use of the nanoparticle hydrogel microneedles of claim 1 in the preparation of products for treating infectious diabetic wounds.

10. The application according to claim 9, wherein the product comprises a wound dressing.

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