A nano-material microneedle with high-efficiency antibacterial and anti-inflammatory capability and a preparation method thereof

Abstract

This invention relates to the field of bio-antibacterial materials technology, specifically to a nanomaterial microneedle with highly efficient antibacterial and anti-inflammatory capabilities and its preparation method. 5-Aminolevulinic acid is obtained by dehydrochlorination of 5-aminolevulinic acid salt. 5-Aminolevulinic acid, epigallocatechin gallate, and a ferric iron source are mixed and reacted via metal coordination self-assembly, followed by dialysis purification to obtain an aqueous solution of AFeE. The AFeE aqueous solution is mixed with sodium hyaluronate to prepare an AFeE-loaded sodium hyaluronate hydrogel. The hydrogel is filled into a microneedle mold, centrifuged to remove bubbles, and dried to obtain an array of microneedles containing nanomedicine. The nanomaterial microneedles of this invention not only possess good biological activity but also antibacterial properties. AFeE is internalized by sebaceous cells and undergoes intracellular dissociation; the released ALA is metabolized into protoporphyrin IX in the mitochondria, while EGCG has anti-inflammatory and antioxidant effects.

Description

Technical Field

[0001] This invention relates to the fields of photocatalysis, hydrogel microneedles, and antibacterial biomaterials, specifically to a nanomaterial microneedle with highly efficient antibacterial and anti-inflammatory capabilities and its preparation method. Background Technology

[0002] As the largest organ in the human body, the skin needs to perform a series of metabolic activities to protect the body from external pathogens. Acne is a common multifactorial skin disease whose pathogenesis includes abnormal keratinization of the hair follicle duct, excessive proliferation of Propionibacterium acnes, sebaceous gland hyperfunction, and inflammatory response. Traditional treatments include topical antibiotics and retinoids, but due to the skin barrier, drug penetration is poor, efficacy is limited, and long-term use can easily lead to drug resistance.

[0003] For a long time, people have used antibiotics to treat various infections, but the overuse of antibiotics carries significant risks. Abuse of antibiotics can lead to the development of drug-resistant bacteria and even increase the likelihood of superbugs. Therefore, scientists are dedicated to finding a range of new methods to kill bacteria and replace antibiotic treatment. Common methods include using the active ingredients of traditional Chinese medicine or the release of metal ions as alternatives to antibiotics in clinical treatment.

[0004] Aminolevulinic acid photodynamic therapy (ALA-PDT) is a combined drug-device treatment. ALA, the photosensitizing drug used in ALA-PDT, is a natural, hydrophilic small molecule compound and a precursor in the heme synthesis pathway; it is not photosensitizing itself. When a large amount of exogenous ALA is administered, tumor cells or rapidly proliferating cells preferentially and selectively absorb ALA, and through a series of enzymatic reactions, generate a large amount of the photosensitizing substance protoporphyrin IX in the mitochondria. Due to the increased activity of bilirubinogen deaminase in tumor cells or rapidly proliferating cells, protoporphyrin IX production increases, while ferrochelate activity decreases, reducing the conversion of protoporphyrin IX to heme, leading to its accumulation in diseased cells. Under irradiation with a specific wavelength of excitation light, protoporphyrin IX is activated, absorbing light energy and converting it to surrounding oxygen molecules, generating singlet oxygen and other reactive oxygen species (ROS), exerting a photodynamic effect. ROS can eliminate various bacteria and regulate immune and inflammatory responses. However, excessive ROS can lead to inflammatory reactions and sequelae.

[0005] Epigallocatechin gallate (EGCG), a natural plant secondary metabolite, plays an important role in medical applications due to its anti-inflammatory, antitumor, and antibacterial properties. However, as a small organic molecule found in natural traditional Chinese medicine, it suffers from poor solubility and stability, significantly limiting its use as a therapeutic agent in clinical treatment. With the development and application of synthetic methods, EGCG-based nanoparticles have found widespread application in the medical field, particularly in antibacterial applications. As a major polyphenol in tea, the highly reactive phenolic hydroxyl groups readily bind to metal ions, amino acids, and other antibacterial components to exhibit antibacterial activity or serve as drug delivery systems. This property has been used to develop various bio-nanomaterials for biological effect studies, such as anti-biofilm infection. Furthermore, polyphenols have been shown to possess antioxidant capabilities, helping to alleviate tissue damage caused by phototherapy.

[0006] Meanwhile, due to the skin's barrier function, most drugs cannot be delivered through the skin to achieve therapeutic effects. In recent years, many researchers have investigated the use of microneedles (MNs) for drug delivery. Due to their unique morphology, nanoparticles can easily penetrate the stratum corneum and enter the epidermis. Therefore, the main benefits of using MNs are painless delivery and the delivery of active ingredients to the dermis for treating skin diseases. Summary of the Invention

[0007] This invention addresses the problems of poor drug penetration, easy development of drug resistance, and imprecise effects of photodynamic therapy in existing acne treatments. It provides a nanomaterial microneedle with highly efficient antibacterial and anti-inflammatory capabilities, along with its preparation method. The microneedle utilizes the endogenous synthesis of the photosensitizer PpIX. Under visible light irradiation, PpIX generates reactive oxygen species (mainly singlet oxygen), killing bacteria surrounding the cells and disrupting biofilms. Simultaneously, the released EGCG exerts anti-inflammatory and antioxidant effects, regulating inflammatory factors and reducing tissue damage.

[0008] One of the objectives of this invention is achieved through the following technical solution: a method for preparing nanomaterial microneedles with highly efficient antibacterial and anti-inflammatory capabilities, comprising the following steps: Step 1: Dehydrochlorination of 5-aminolevulinic acid salt to obtain 5-aminolevulinic acid; Step 2: The 5-aminolevulinic acid, epigallocatechin gallate obtained in Step 1 and the ferric iron source are mixed and reacted through metal coordination self-assembly. After dialysis purification, an aqueous solution of the 5-aminolevulinic acid-iron-epigallocatechin gallate nano-coordination complex AFeE is obtained. Step 3: Mix the AFeE aqueous solution obtained in Step 2 with sodium hyaluronate to obtain sodium hyaluronate hydrogel loaded with AFeE. Step four: Fill the microneedle mold with the hydrogel obtained in step three, and after centrifugation to remove bubbles and drying, obtain an array of microneedles containing nanomedicine.

[0009] Preferably, the specific steps of step one are as follows: neutralize and dehydrochlorinate 5-aminolevulinate salt with an alkaline solution to form 5-aminolevulinic acid, wherein the molar ratio of 5-aminolevulinate salt to the alkaline solution is 1:1, and the alkaline solution used is NaOH or ammonia water with a concentration of 0.05-0.1M.

[0010] Preferably, in step one, 5-aminolevulinate salt is neutralized with alkali to a pH of 6.0-8.0.

[0011] Preferably, in step two, 5-aminolevulinic acid, epigallocatechin gallate, and Fe... 3+ The molar ratio is 18-22:3-5:1.

[0012] Preferably, in step two, the concentration of 5-aminolevulinic acid is 1-20 mM, and the concentrations of epigallocatechin gallate and the ferric iron source are each independently 1-20 mM.

[0013] Preferably, in step two, the ferric source includes at least one of ferric chloride, ferric nitrate, ferric sulfate, and ferric citrate.

[0014] Preferably, in step three, the mass-to-volume ratio of sodium hyaluronate is 10%-20%, and the concentration of the diluted AFeE nanomedicine is 20 mM.

[0015] Preferably, in step four, the microneedle mold is a polydimethylsiloxane mold.

[0016] Preferably, in step four, the drying temperature is 30-40 ℃, the drying time is 20-48 h, and the solution evaporation is observed every 12-24 hours during the drying process, and the solution is replenished and air bubbles are removed.

[0017] The second objective of this invention is achieved by the following technical solution: a nanomaterial microneedle with highly efficient antibacterial and anti-inflammatory capabilities, prepared by the aforementioned method.

[0018] The microneedle (MN) patch of the present invention provides a nano-coordination complex (AFeE) that reprograms overactive sebaceous cells into endogenous photosensitizer factories for precise photodynamic immunotherapy. AFeE is generated through a one-pot coordinated process involving 5-aminolevulinic acid (ALA), Fe... 3+Designed for the self-assembly of epigallocatechin gallate (EGCG), AFeE is delivered painlessly intradermally via soluble MN. AFeE is internalized by sebaceous cells and undergoes intracellular dissociation. The released ALA is metabolized in the mitochondria to protoporphyrin IX, while EGCG possesses anti-inflammatory and antioxidant properties. Under 660 nm laser irradiation, these reprogrammed sebaceous cells generate potent reactive oxygen species, achieving ~99.94% acne eradication and strong biofilm disruption in vivo. This cell reprogramming platform demonstrates superior efficacy and disease specificity, establishing a new paradigm for targeted therapy of multifactorial inflammatory skin diseases.

[0019] The present invention has the following beneficial effects: The preparation method of this invention synthesizes an AFeE (an abbreviation for ALA-Fe-EGCG ternary complex, i.e., 5-aminolevulinic acid-iron-epigallocatechin gallate nanocomplex) nanomedicine, which possesses photodynamic antibacterial and antioxidant / anti-inflammatory capabilities. Further, the AFeE nanomedicine is compounded with easily soluble sodium hyaluronate to form a hydrogel, which is then molded using a microneedle mold to obtain an AFeE-loaded nanomedicine array microneedles, i.e., an MN patch. AFeE reaches the site of action through the penetrating power of MN, is internalized by sebaceous cells, and undergoes intracellular dissociation. The released ALA is metabolized into protoporphyrin IX in the mitochondria, while EGCG has anti-inflammatory and antioxidant effects. Under 660 nm laser irradiation, these reprogrammed sebaceous cells generate powerful reactive oxygen species, achieving highly efficient acne eradication and strong biofilm disruption in vivo. The preparation method described in this invention is simple and quick to synthesize AFeE nanoparticles, requires little equipment investment and uses few consumables, and can achieve large-scale production. The use of epigallocatechin gallate, a traditional Chinese medicine, can inhibit bacterial growth to a certain extent and also has good antioxidant effects.

[0020] The nanomaterial microneedles of this invention not only possess excellent bioactivity but also antibacterial properties. After painless intradermal delivery via soluble MN, AFeE is internalized by sebaceous cells and undergoes intracellular dissociation. The released ALA is metabolized into protoporphyrin IX within the mitochondria, while EGCG exhibits anti-inflammatory and antioxidant effects. Attached Figure Description

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments: Figure 1-1 This is a field emission scanning electron microscope image of the nanomedicine synthesized in Example 2; Figure 1-2 This is a field emission scanning electron microscope image of the microneedles synthesized in Example 3; Figure 2 Images showing the dissolution effect of the microneedles synthesized in Example 3; Figure 3-1 This is an electron spin resonance spectroscopy image of the singlet oxygen produced after the material synthesized in Example 2 was internalized by sebaceous gland cells; Figure 3-2 This is a fluorescent image of cellular oxidative stress generated after the material synthesized in Example 2 was internalized by sebaceous gland cells; Figure 4-1 The images show the antibacterial effect and anti-biofilm properties of the MRSA colonies of the material synthesized in Example 2 after irradiation with an exogenous source. Figure 4-2 This is a graph showing the antibacterial efficiency of the MRSA colonies of the material synthesized in Example 2 after irradiation with an exogenous source. Figure 4-3 This is a graph showing the antibacterial efficiency of the microneedles synthesized in Example 3 after exogenous irradiation of MRSA colonies. Figure 4-4 This is a graph showing the antibacterial efficiency of the Propionibacterium acnes colonies synthesized in Example 3 after exogenous irradiation. Figure 4-5 The image shows the fluorescent staining of live and dead Propionibacterium acnes bacteria on the microneedles synthesized in Example 3. Figure 5-1 This is a fluorescence image of cell growth of the microneedles synthesized in Example 3; Figure 5-2 The graph shows the cell viability of the microneedles synthesized in Example 3 after different number of days of culture. Figure 6 The mRNA expression levels of inflammatory factors were detected by real-time quantitative reverse transcription polymerase chain reaction using the microneedles synthesized in Example 3. Detailed Implementation

[0022] To better understand the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the scope of protection of the present invention is not limited to the scope shown in the embodiments.

[0023] Example 1

[0024] Step 1: Dehydrochlorination treatment of 5-aminolevulinate ALA·HCl (5-aminolevulinate) was neutralized with NaOH to pH = 7.4 and then dehydrochlorinated to ALA. Step 2: Metal coordination self-assembly for the preparation of nanomedicines The 5-aminolevulinic acid obtained in step one, epigallocatechin gallate, and ferric chloride were mixed in a molar ratio of 20:4:1 and stirred for 0.5 h via metal self-assembly. After dialysis for three days, AFeE was obtained. o C is stored in the dark; Step 3, preparing sodium hyaluronate hydrogel loaded with AFeE, includes the following steps: Sodium hyaluronate (15%) was added to the AFeE aqueous solution obtained in step two to prepare an AFeE-loaded sodium hyaluronate hydrogel. The specific steps are as follows: (1) Dilute the AFeE solution to the required concentration (10 mM), weigh out 15% sodium hyaluronate by mass and add it to the solution and mix well; (2) Place the mixed solution on a magnetic stirrer and stir at room temperature to obtain a sodium hyaluronate hydrogel solution containing nanomedicine; Step four involves using nanomaterial microneedles with highly effective antibacterial and anti-inflammatory properties. The specific steps are as follows: The hydrogel solution obtained in step 3 was filled into the microneedle mold. It was centrifuged at 4000 rpm for 6 min at room temperature to remove the generated air bubbles. The above operation was repeated until the solution completely filled the mold. The mold was dried in an oven at 37℃ for 24-48 h. The evaporation of the solution was observed during the process, and the solution was replenished and air bubbles were removed in time. After drying and shaping, an array of microneedles containing nanomedicine was obtained.

[0025] Example 2

[0026] Step 1: Dehydrochlorination treatment of 5-aminolevulinate ALA·HCl (5-aminolevulinate) was neutralized with NaOH to pH = 7.4 and then dehydrochlorinated to ALA. Step 2: Metal coordination self-assembly for the preparation of nanomedicines The 5-aminolevulinic acid obtained in step one, epigallocatechin gallate, and ferric chloride were mixed in a molar ratio of 20:4:1 and stirred for 0.5 h via metal self-assembly. After dialysis for three days, AFeE was obtained. o C is stored in the dark; Step 3, preparing sodium hyaluronate hydrogel loaded with AFeE, includes the following steps: Sodium hyaluronate (15%) was added to the AFeE aqueous solution obtained in step two to prepare an AFeE-loaded sodium hyaluronate hydrogel. The specific steps are as follows: (1) Dilute the AFeE solution to the required concentration (20 mM), weigh out 15% sodium hyaluronate by mass and add it to the solution and mix well; (2) Place the mixed solution on a magnetic stirrer and stir at room temperature to obtain a sodium hyaluronate hydrogel solution containing nanomedicine; Step four involves using nanomaterial microneedles with highly effective antibacterial and anti-inflammatory properties. The specific steps are as follows: The hydrogel solution obtained in step 3 was filled into the microneedle mold and centrifuged at 4000 rpm for 6 min at room temperature to remove the generated air bubbles. The above operation was repeated until the solution completely filled the mold. The mold was then dried in an oven at 37℃ for 24-48 h. The evaporation of the solution was observed during the drying process, and the solution was replenished and air bubbles were removed in a timely manner. After drying and shaping, an array of microneedles containing nanomedicine was obtained.

[0027] Example 3

[0028] Step 1: Dehydrochlorination treatment of 5-aminolevulinate ALA·HCl (5-aminolevulinate) was neutralized with NaOH to pH = 7.4 and then dehydrochlorinated to ALA. Step 2: Metal coordination self-assembly for the preparation of nanomedicines The 5-aminolevulinic acid obtained in step one, epigallocatechin gallate, and ferric chloride were mixed in a molar ratio of 20:4:1 and stirred for 0.5 h via metal self-assembly. After dialysis for three days, AFeE was obtained. o C is stored in the dark; Step 3, preparing sodium hyaluronate hydrogel loaded with AFeE, includes the following steps: Sodium hyaluronate (15%) was added to the AFeE aqueous solution obtained in step two to prepare an AFeE-loaded sodium hyaluronate hydrogel. The specific steps are as follows: (1) Dilute the AFeE solution to the required concentration (20 mM), weigh out 15% sodium hyaluronate by mass and add it to the solution and mix well; (2) Place the mixed solution on a magnetic stirrer and stir at room temperature to obtain a sodium hyaluronate hydrogel solution containing nanomedicine; Step four involves using nanomaterial microneedles with highly effective antibacterial and anti-inflammatory properties. The specific steps are as follows: The hydrogel solution obtained in step three was filled into the microneedle mold, and centrifuged at 3000 rpm for 8 min at room temperature to remove air bubbles. The above operation was repeated until the solution completely filled the mold. o Dry in an oven at C for 24-48 hours, observing the evaporation of the solution during the drying process, replenishing the solution and removing air bubbles as needed. After drying and shaping, an array of microneedles containing nanomedicines is obtained.

[0029] Figure 1-1 The image shows the FE-SEM image of the nanomedicine synthesized in Example 2. As can be seen from the image, AFeE has an irregular spherical outline with small particle size and clear boundaries between particles.

[0030] Figure 1-2As shown in Example 3, the hydrogel microneedles prepared in this invention contain nanomedicines and are completely filled. Combined with the synthesis process described, this method is simple to prepare and highly feasible.

[0031] Take the sodium hyaluronate hydrogel microneedle array loaded with AFeE prepared in Example 3, fix the microneedle patch in a small dish, and add simulated tissue fluid (PBS buffer, pH = 7.4) at time points of 0 s, 10 s, 20 s and 30 s at room temperature. At the same time, observe the dissolution of the microneedle tips with an optical microscope and take pictures to record. Figure 2 For the dissolution test of the hydrogel microneedles in Example 3, the results showed that the sodium hyaluronate hydrogel microneedles had good solubility. The needle tip almost completely dissolved within 30 seconds after contacting the simulated tissue fluid, indicating that the microneedles can rapidly release the loaded AFeE nanomedicine in the interstitial fluid of skin tissue.

[0032] The AFeE nanomedicine prepared in Example 2 was added to the culture medium of sebaceous gland cells (such as SZ95 cells) and incubated for 4 hours to allow for full cell internalization. The cell suspension was collected, and the cells in the light-treated group were irradiated with visible light (wavelength 660 nm, 258 mW, irradiation time 2 minutes), while the control group was compared with ultrapure water. Singlet oxygen was detected using electron spin resonance (ESR) spectroscopy: the treated cells were collected, the singlet oxygen scavenger TEMPO was added, and the mixture was transferred to a quartz capillary. ESR spectra were recorded at room temperature, and the intensity of the TEMPO characteristic signal characterized the level of singlet oxygen production. Figure 3-1 This is an ESR detection image of singlet oxygen produced after the material synthesized in Example 2 is internalized by sebaceous gland cells.

[0033] Intracellular reactive oxygen species were detected using the DCFH-DA fluorescent probe: AFeE-treated cells were treated with DCFH-DA (10 μM) and then... o Incubate at C in the dark for 30 minutes, wash with PBS, and observe and photograph using a fluorescence microscope (excitation wavelength 488 nm, emission wavelength 525 nm). The intensity of green fluorescence reflects the level of reactive oxygen species in the cells. Figure 3-2 This is a fluorescent image of cellular oxidative stress generated after the material synthesized in Example 2 was endocytosed by sebaceous gland cells.

[0034] Depend on Figure 3-1 , Figure 3-2 It can be seen that after AFeE is internalized by sebaceous gland cells, it produces singlet oxygen, which produces more obvious and stronger ROS after irradiation with exogenous sources.

[0035] Figure 4-1Images show the antibacterial effect and antibiofilm formation of MRSA colonies after irradiation with exogenous material, as shown in Example 2. In vitro antibacterial activity assay: The antibacterial effect of different samples was evaluated using methicillin-resistant Staphylococcus aureus (MRSA). SZ95 cells (sebaceous gland cells) were first cultured until adherent growth, then co-cultured with AFeE prepared in Example 2 for 4 h. Cells and cell culture medium were then collected for subsequent antibacterial applications. A 660 nm laser was used with parameters set to 0.8 A for 20 min. The bacterial suspension was diluted to 10... 5 At the desired concentration, take 800 μL of the sample and add 200 μL of diluent, then mix thoroughly in a 2 mL centrifuge tube. This mixture should be irradiated under 660 nm excitation light for 20 min. Afterward, use 20 μL of the treated bacterial suspension to perform a plate spread method, evenly distributing it on the surface of an LB agar plate. Incubate at 37°C. o The culture was carried out in a constant temperature incubator at C for 24 hours, and then the number of colonies on each plate was counted to determine whether there was an antibacterial effect.

[0036] Crystal violet staining experiment: Take 1×10 8 A bacterial suspension with a concentration of CFU / mL was prepared by adding 400 μL of the suspension to each well of a 48-well plate and incubating at 37°C. o Incubate the bacteria overnight (48 h) in a bacterial incubator until a thin film forms at the bottom. Remove the wells and process the materials from the exogenous treatment group accordingly. Aspirate the bacterial culture from the edge of the wells, wash 1-2 times with PBS buffer, then add 400 μL of 10% methanol solution and fix for 15 min. Allow to air dry, then stain with crystal violet for 15 min. Remove the crystal violet stain and wash 2-3 times with PBS buffer until no floating color remains. Air dry, photograph, then decolorize with 33% glacial acetic acid at 37°C. o The dye was dissolved by incubating in a C incubator for 15 min, and the OD was measured at 595 nm using an ELISA reader.

[0037] Figure 4-2 This is a graph showing the antibacterial efficiency of the MRSA colonies of the material synthesized in Example 2 after irradiation with an exogenous source. The number of colonies on the plate was calculated, and the calculation was repeated multiple times to analyze the error.

[0038] Figure 4-3This is a graph showing the antibacterial efficiency of the microneedles synthesized in Example 3 after exogenous irradiation of MRSA colonies. MN antibacterial procedure: SZ95 cells were first allowed to adhere and grow, then co-cultured with AFeE MN for 4 h. Afterwards, the cells and cell culture medium were removed for antibacterial experiments. The bacterial suspension was diluted 200-fold, and 800 μL of the sample was added to 200 μL of the diluted bacterial suspension in a 2 mL centrifuge tube. The mixture was then irradiated with 660 nm excitation light for 20 min (660 nm, 0.8 A). Subsequently, 20 μL of the treated bacterial suspension was evenly spread onto an agar plate using the plate spreading method and placed at 37°C. o In an environment of C, incubate for 24 hours, and finally check the number of colonies on the plate and calculate the inhibition rate.

[0039] Figure 4-4 This is a graph showing the antibacterial efficiency of the *Propionibacterium acnes* colonies in the microneedles synthesized in Example 3 after exogenous irradiation. (Used...) P. acnes To assess the antibacterial effect of the samples, SZ95 cells were first allowed to adhere and grow, then co-cultured with AFeE MN for 4 h. Afterward, the cells and cell culture medium were collected for antibacterial experiments. The *Propionibacterium acnes* suspension was diluted 200-fold. 800 μL of the sample was then mixed with 200 μL of the diluted bacterial suspension in a 2 mL centrifuge tube. The mixture was then irradiated with 660 nm excitation light for 20 min (660 nm, 0.8 A). Subsequently, 20 μL of the treated bacterial suspension was evenly spread onto a CDC anaerobic blood agar plate using the plate spreading method and incubated at 37°C. o In a vacuum environment of C, culture for 3 days, and finally check the number of colonies on the plate and calculate the inhibition rate.

[0040] Figure 4-5 The image shows the fluorescent staining of live and dead Propionibacterium acnes bacteria using the microneedles synthesized in Example 3. Bacterial live / dead staining: Live bacteria staining or dead bacteria staining was performed using a live bacteria staining kit. After antibacterial treatment, 200 μL of exogenously irradiated bacterial suspension was taken, and dye was added at a ratio of 1:100. The solution was then placed at 4°C. o After 15 minutes in a refrigerator at C, the bacterial cultures from each group were added to a 96-well plate. Once dried, the staining was observed using an inverted fluorescence microscope.

[0041] Depend on Figure 4-1 , Figure 4-2 , Figure 4-3 , Figure 4-4 , Figure 4-5It can be seen that both AFeE nanomedicine and AFeE-loaded hydrogel microneedles exhibit significant antibacterial activity against MRSA and Propionibacterium acnes under visible light irradiation. The antibacterial efficiency is light-dependent and can effectively remove biofilms. Live and dead bacteria staining further confirmed that the microneedle treatment group was dominated by dead bacteria (red fluorescence), indicating that AFeE endows the hydrogel microneedles with a controllable photodynamic antibacterial mode, and that the AFeE-loaded hydrogel microneedles have good antibacterial effects after co-culturing with cells.

[0042] Figure 5-1 This is a fluorescence image of cell growth of the microneedles synthesized in Example 3. Cell fluorescence procedure: 200 μL of NIH-3T3 was added at 10... 5 Cells were seeded at a density of [insert density here] in 96-well plates. After cell adhesion overnight, AFeE MN medium was added, with pure medium serving as a blank control. Cells were labeled with FITC and DAPI. After drying, fluorescence images of the cells were captured using an inverted fluorescence microscope.

[0043] Figure 5-2 The image shows the cell viability of the microneedles synthesized in Example 3 after different number of days of culture. Cytotoxicity assay: The MTT assay was used to detect the cytotoxicity of the samples against mouse fibroblasts (NIH-3T3). 200 μL of NIH-3T3 cell suspension was injected into 96-well plates at a cell density of 1 × 10⁻⁶ cells / well. 5 Cells were cultured at a density of 100 cells / mL, with 5 replicates per group. After 24 hours of cell attachment, AFeE MN was dissolved in 4 mL of culture medium, with pure culture medium serving as a blank control. Long-term toxicity experiments were conducted using low-density seeding with medium changes every 48 h. A blank control from the same batch was also included to eliminate interference from contact inhibition, natural cell death, and metabolite accumulation on cell viability assays. On days 1, 3, and 5 of co-culture, 150 μL of 0.5 mg / mL MTT solution (dissolved in PBS buffer) was added and the cells were incubated at 37°C. o Incubate the cells in a constant temperature incubator for 4 hours. After 4 hours, remove the MTT solution and then add 200 μL of DMSO solvent. Shake the solution on a shaker for 15 min to ensure thorough mixing and the formation of a purple-red precipitate. Finally, aspirate 100 μL of the supernatant and place it in a multi-purpose microplate. Measure the absorbance (OD) at 490 nm using a spectrophotometer and calculate cell viability.

[0044] Depend on Figure 5-1 , Figure 5-2It can be seen that, compared with the pure sodium hyaluronate microneedle group, the microneedle group loaded with AFeE had a moderate number of fibroblasts and better cell distribution. The cell activity map showed that the microneedles loaded with the nano-drug AFeE improved the biocompatibility of pure sodium hyaluronate microneedles to a certain extent. At the same time, AFeE MN has a certain promoting effect on cell growth, indicating that AFeE microneedles have good biocompatibility.

[0045] Figure 6 Reverse transcription and quantitative PCR were performed on the microneedles synthesized in Example 3. RT-qPCR: Quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression levels of key cytokines involved in inflammation and immune responses. The AFeE-loaded sodium hyaluronate hydrogel microneedles (AFeE MN) prepared in Example 4 were used for in vitro anti-inflammatory activity evaluation. No additional materials were added as the model group, and pure sodium hyaluronate microneedles (Blank MN) served as the control group. Macrophages (RAW 264.7 cells) were introduced at an appropriate density (approximately 1 × 10⁻⁶ cells). 5 Cells / well were seeded in 6-well plates and incubated at 37°C. o C. Incubate overnight in a 5% CO2 incubator. All groups were treated with lipopolysaccharide (LPS, final concentration 1 μg / mL) to induce a cell inflammation model and cultured for another 12 hours. Then, AFeE MN and Blank MN were co-cultured with LPS-induced inflammatory cells for 24 hours. Total RNA was first obtained from RAW cells using TRIzol reagent, followed by cDNA synthesis using a reverse transcription kit. Amplification was then performed using the StepOnePlus real-time quantitative PCR system with SYBR Green Master Mix, and β-actin was used as an endogenous reference gene for normalization. Interleukin-10 (IL-10) was calculated using the 2-ΔΔCt method. IL-10 ), interleukin-6 ( IL-6 ), interleukin-1β ( IL-1β ) and tumor necrosis factor-α ( TNF-α The relative mRNA expression levels of each group were determined, and three or more replicate experiments and independent experiments were conducted for each group.

[0046] Depend on Figure 6 It can be seen that, compared with the LPS model group, after AFeE MN intervention, pro-inflammatory cytokines... IL-6 The expression was strongly suppressed. TNF-α and IL-1β The expression level of [a substance] also decreased in the AFeE MN treatment group. IL-10 The expression of [acid] continued to be upregulated, demonstrating a dual regulatory characteristic of both inhibiting pro-inflammatory and enhancing anti-inflammatory effects. This indicates that the hydrogel microneedles loaded with AFeE have good anti-inflammatory and antioxidant effects.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing nanomaterial microneedles with highly efficient antibacterial and anti-inflammatory capabilities, characterized in that, Includes the following steps: Step 1: Dehydrochlorination of 5-aminolevulinic acid salt to obtain 5-aminolevulinic acid; Step 2: The 5-aminolevulinic acid, epigallocatechin gallate obtained in Step 1 and the ferric iron source are mixed and reacted through metal coordination self-assembly. After dialysis purification, an aqueous solution of the 5-aminolevulinic acid-iron-epigallocatechin gallate nano-coordination complex AFeE is obtained. Step 3: Mix the AFeE aqueous solution obtained in Step 2 with sodium hyaluronate to obtain sodium hyaluronate hydrogel loaded with AFeE. Step four: Fill the microneedle mold with the hydrogel obtained in step three, and after centrifugation to remove bubbles and drying, obtain an array of microneedles containing nanomedicine.

2. The method for preparing nanomaterial microneedles with highly efficient antibacterial and anti-inflammatory capabilities according to claim 1, characterized in that, The specific steps of step one are as follows: neutralize and dehydrochlorinate 5-aminolevulinate salt with an alkaline solution to form 5-aminolevulinic acid. The molar ratio of 5-aminolevulinate salt to the alkali is 1:

1. The alkaline solution used is NaOH or ammonia water with a concentration of 0.05-0.1M.

3. The method for preparing nanomaterial microneedles with highly efficient antibacterial and anti-inflammatory capabilities according to claim 2, characterized in that, In step one, 5-aminolevulinate salt is neutralized with alkali to a pH of 6.0-8.

0.

4. The method for preparing nanomaterial microneedles with highly efficient antibacterial and anti-inflammatory capabilities according to claim 1, characterized in that, In step two, 5-aminolevulinic acid, epigallocatechin gallate, and Fe... 3+ The molar ratio is 18-22:3-5:

1.

5. The method for preparing nanomaterial microneedles with highly efficient antibacterial and anti-inflammatory capabilities according to claim 1, characterized in that, In step two, the concentration of 5-aminolevulinic acid is 1-20 mM, and the concentrations of epigallocatechin gallate and the ferric iron source are each 1-20 mM independently.

6. The method for preparing nanomaterial microneedles with highly efficient antibacterial and anti-inflammatory capabilities according to claim 1, characterized in that, In step two, the ferric source includes at least one of ferric chloride, ferric nitrate, ferric sulfate, and ferric citrate.

7. The method for preparing nanomaterial microneedles with highly efficient antibacterial and anti-inflammatory capabilities according to claim 1, characterized in that, In step three, the mass-volume ratio of sodium hyaluronate is 10%-20%, and the concentration of the diluted AFeE nanomedicine is 20mM.

8. The method for preparing nanomaterial microneedles with highly efficient antibacterial and anti-inflammatory capabilities according to claim 1, characterized in that, In step four, the microneedle mold is a polydimethylsiloxane mold.

9. The method for preparing nanomaterial microneedles with highly efficient antibacterial and anti-inflammatory capabilities according to claim 1, characterized in that, In step four, the drying temperature is 30-40 ℃ and the drying time is 20-48 h. During the drying process, the evaporation of the solution is observed every 12-24 hours, and the solution is replenished and air bubbles are removed.

10. A nanomaterial microneedle with highly efficient antibacterial and anti-inflammatory capabilities, characterized in that, It is prepared by any one of the preparation methods according to claims 1-9.

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