A bioactive microneedle patch for repairing radioactive dermatitis and a preparation method and application thereof

Abstract

The present application relates to a kind of biological active microneedle patch for repairing radiation dermatitis and its preparation method and application, belong to biomedical material technical field.The microneedle patch of the present application includes backing layer and needle tip layer, wherein the material of backing layer is methacrylated hyaluronic acid with photo-crosslinking characteristics, the material of needle tip layer is silk fibroin and hyaluronic acid, and loaded with chlorogenic acid and adipose stem cell exosome as core bioactive component.The microneedle patch of the present application has excellent mechanical properties and biological activity, can minimally invasive penetration skin keratin layer barrier, realize the accurate delivery and sustained release of active ingredient to damaged tissue deep layer.Microneedle patch improves the problem of insufficient mechanical strength of natural polymer microneedle through the composite structure of "methacrylated hyaluronic acid backing + silk fibroin needle tip", realizes the synergistic effect of antioxidant, anti-inflammatory and extracellular matrix homeostasis reconstruction through "chlorogenic acid + adipose stem cell exosome", and can be used for repairing radiation dermatitis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to a bioactive microneedle patch for the repair of radiation dermatitis, its preparation method and application. Background Technology

[0002] Radiation dermatitis is an inflammatory injury caused by various types of ionizing radiation (such as X-rays, alpha, beta, and gamma rays) irradiating the skin and mucous membranes. It can be classified into acute and chronic types based on its course. In patients requiring high-dose radiotherapy, such as those with breast cancer or head and neck tumors, the incidence of acute radiation dermatitis is as high as 98%, with grade 3-4 severe injuries accounting for approximately 30%. Acute injuries typically occur within hours to 90 days after radiotherapy, commonly caused by a single or multiple high-dose radiation exposures within a short period, and are the most common type among cancer radiotherapy patients. Its core characteristics are rapid onset, rapid symptom progression, and a high degree of synchronization with the radiotherapy cycle. Clinical manifestations include erythema, dry desquamation, and moist blisters; in severe cases, it can lead to skin necrosis or dermal ulceration. Chronic injuries have a longer latency period, appearing months or even years after radiotherapy. They generally develop from acute radiation dermatitis or are caused by long-term, repeated exposure to low-dose radiation. Clinical manifestations include skin atrophy, fibrosis, telangiectasia, and even an increased risk of skin cancer.

[0003] Currently, there are five main categories of treatments for radiation dermatitis. The first category is traditional treatments, which use physical methods or moisturizing or repairing ointments to relieve symptoms, prevent infection, and promote basic skin repair. Physical methods involve using mild, non-irritating cleansers to clean the affected area, but the disadvantage is that the treatment effect is generally poor and the treatment period is long. In terms of drug treatment, topical corticosteroids (such as mometasone and dexamethasone) reduce the release of inflammatory factors (TNF-α, IL-6) by inhibiting the NF-κB pathway, thereby relieving redness, swelling, and itching. The disadvantage is that long-term use may lead to skin atrophy. The second category is treatment with bioactive ingredients, such as epidermal growth factor (EGF), adipose-derived mesenchymal stem cells (ADSCs), and bone marrow mesenchymal stem cells (BMSCs), which can reduce the degree of inflammation and fibrosis in acute dermatitis. Exosome therapy is also a common approach; engineered exosomes (such as those loaded with curcumin) can deliver active ingredients in a targeted manner. The third category is biomaterials, such as hydrogels, which, due to their good bioactivity and functional plasticity, have become an effective treatment for radiation dermatitis, enabling sustained drug release, wound protection, and active repair. The fourth category is microneedle patches, which include dissolving microneedles, hydrogel microneedles, and nanocomposite microneedles. These patches can painlessly penetrate the skin surface to achieve controlled drug release, adapting to different pathological stages of radiation dermatitis. The fifth category is emerging combination therapy strategies, which enhance efficacy through the synergistic effect of multiple methods.

[0004] However, existing treatments have significant clinical limitations: traditional ointments have low transdermal efficiency and long-term use can easily lead to skin atrophy; simple bioactive ingredients are easily inactivated and metabolized in vitro and are difficult to directly penetrate the stratum corneum barrier; while traditional natural polymer microneedles can penetrate the skin, they generally face technical bottlenecks such as insufficient mechanical strength, easy breakage during puncture, and difficulty in synergizing the release rates of multiple components. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a bioactive microneedle patch for the repair of radiation dermatitis, its preparation method and application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a bioactive microneedle patch for the repair of radiation dermatitis. The microneedle patch consists of a backing layer and a needle tip layer. The backing layer is made of methacryloyl hyaluronic acid (HAMA), and the needle tip layer is made of silk fibroin (SF) and hyaluronic acid (HA), and is loaded with dual active components: adipose-derived stem cell exosomes (ADSC-Exos) and chlorogenic acid (CGA). The structural formula of the methacryloyl hyaluronic acid is shown below:

[0007] Where n is an integer greater than or equal to 2; The present invention also provides a method for preparing the above-mentioned microneedle patch, which specifically includes the following steps: (1) Extraction of adipose stem cell exosomes (ADSC-Exos); (2) Synthesis of HAMA: Hyaluronic acid (HA) is reacted with methacrylic anhydride, and HAMA powder is obtained by dialysis and freeze-drying, and HAMA solution is prepared; (3) Extraction of silk fibroin: Silkworm cocoons are degummed and neutralized with alkali solution, and then placed in a ternary solvent system for full dissolution, followed by dialysis, centrifugation purification and freeze-drying to obtain silk fibroin powder. (4) S1: Preparation of needle tip layer mixture: Chlorogenic acid and extracted adipose stem cell exosomes (ADSC-Exos) are added to the pre-crosslinked solution of silk fibroin (SF) and hyaluronic acid (HA), and stirred evenly to obtain needle tip layer mixture; S2: Preparation of backing layer solution: After reacting hyaluronic acid (HA) with methacrylic anhydride, the methacrylated hyaluronic acid (HAMA) powder obtained by freeze-drying is dissolved in deionized water and a photoinitiator is added to prepare a 4% concentration HAMA pre-crosslinked solution. S3: Casting and molding of microneedle patch: The pre-crosslinked solution obtained in step S1 is slowly injected into the PDMS microneedle mold, placed in a vacuum drying oven to remove bubbles, and then the backing layer solution obtained in step S2 is injected to remove bubbles. The microneedle patch is obtained after being irradiated under ultraviolet light to undergo a photocrosslinking reaction and after drying and demolding. Preferably, methacrylamide hyaluronic acid (HAMA) is prepared by the following method: HA is dissolved in deionized water to prepare a 1% mass concentration solution, which is placed in an ice-water bath and sodium hydroxide is added to adjust the pH to 8-9. Methacrylic anhydride is slowly added dropwise, and the reaction is carried out overnight at 4°C. The solution is then transferred to a dialysis bag, dialyzed with a dialysis bag with a molecular weight of 20000 Da, and then freeze-dried to obtain methacrylamide hyaluronic acid (HAMA). Preferred method for preparing silk fibroin: Silkworm cocoons are chopped and added to a 0.5% sodium carbonate solution for degumming at 80°C for 30 minutes. This process is repeated once or twice. After washing, the cocoons are added to a ternary solution of calcium chloride / ethanol / water with a molar ratio of 1:2:8 and dissolved at 70°C for 2 hours. The solution is then placed in an 8000 Da dialysis bag and dialyzed for 3 days. The solution is centrifuged at 4000 rpm for 15 minutes. The supernatant is collected and freeze-dried to obtain silk fibroin powder. Preferred method for preparing microneedle tip casting solution: After mixing the prepared hyaluronic acid solution (HA) and silk fibroin (SF) solution at a mass ratio of 1:3, under gentle stirring, the pH value of the mixture is precisely adjusted to 7.2~7.4 using a trace amount of 0.1 M NaOH solution. After the system is uniform and stable, a certain amount of chlorogenic acid and ADSC-Exos are added, and the mixture is gently stirred to obtain the microneedle casting solution. Preferred method for preparing microneedles: The obtained needle tip mixture is slowly injected into a PDMS microneedle mold, placed in a vacuum drying oven for 15 min to degas, then the backing layer solution obtained in step S2 is injected, air bubbles are removed, and a photocrosslinking reaction occurs under ultraviolet light irradiation. The ultraviolet light irradiation parameters are wavelength 365 nm, power 10 mW / cm², and irradiation time 3 min. After crosslinking, the microneedles are dried in a 37°C oven. After drying and demolding, microneedle patches loaded with Exos and chlorogenic acid are obtained. The preferred method for preparing ADSC-Exos is as follows: Adipose-derived stem cells collected from adipose tissue are cultured to the third generation, and then cultured in serum-free medium for another 48 h. The culture supernatant is collected and centrifuged sequentially at 300×g for 10 min, 2000×g for 10 min, 10000×g for 10 min, and 100000×g ultracentrifugation for 70 min. The precipitate is collected and resuspended in PBS to obtain ADSC-Exos. Furthermore, the application of the aforementioned bioactive microneedle patch for repairing radiation dermatitis in the preparation of drugs for radiation dermatitis; The application of the bioactive microneedle patch for radiation dermatitis repair in the preparation of medical materials for radiation dermatitis repair.

[0008] The beneficial effects of this invention are as follows: This invention innovatively designs a bioactive microneedle patch co-loaded with chlorogenic acid (CGA) and adipose-derived stem cell exosomes (ADSC-Exos). The "backing (HAMA)-needle tip (silk fibroin)" structure of the microneedles effectively solves the problem of insufficient mechanical strength in traditional natural polymer microneedles, achieving both good bioactivity and excellent mechanical properties for microneedle puncture, with transdermal efficiency 6-7 times higher than traditional ointments. In terms of synergistic activity, chlorogenic acid activates the Nrf2 pathway to scavenge ROS and inhibit the release of inflammatory factors (TNF-α, IL-6, etc.); exosomes target and regulate ECM synthesis and degradation, promoting the proliferation and migration of fibroblasts and endothelial cells. The two synergistically intervene in the NF-κB and Nrf2 pathways, rapidly restoring ECM homeostasis. This invention demonstrates safe and efficient radiation dermatitis repair capabilities in both in vivo and in vitro models, showing great promise for clinical application.

[0009] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0010] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 The following diagrams illustrate the preparation process and mechanism of action of the bioactive microneedle patch for the repair of radiation dermatitis according to the present invention. Figure A is a schematic diagram of the preparation of adipose stem cell exosomes (ADSC-Exos), Figure B is a schematic diagram of the microneedle patch preparation process, and Figure C is a schematic diagram of the mechanism of action of the bioactive microneedle patch for the repair of radiation dermatitis.

[0011] Figure 2 A schematic diagram illustrating the preparation process of a bioactive microneedle patch for the repair of radiation dermatitis; Figure 3 The results show the NMR absorption peaks and characteristic proton peaks of the synthesized methacryloyl hyaluronic acid (HAMA) (A) and silk fibroin (SF) (B).

[0012] Figure 4Figure 1 shows the morphology and physicochemical characterization of bioactive microneedle patches for radiation dermatitis repair. Figure A is a SEM image of the bioactive microneedle patch for radiation dermatitis repair prepared in Example 1, with the test screen on a 5-star universal testing machine. Figure B shows the hydrogel. Figure C shows the Young's modulus of hydrogel microneedle patches with different concentrations. Figure D shows the rheological analysis of different hydrogel microneedle patches (G' represents storage modulus, G'' represents loss modulus).

[0013] Figure 5 To demonstrate the bioactivity of the bioactive microneedle patch used for the repair of radiation dermatitis, Figure A shows the live / dead staining results of the composite microneedle patch on L929 cells and HaCaT cells; Figures B and C show the corresponding quantitative fluorescence analysis.

[0014] Figure 6 The antioxidant capacity test results of the bioactive microneedle patch used for radiation dermatitis repair: Figures A and B show the cck-8 activity test results of the bioactive microneedle patch used for radiation dermatitis repair on mouse fibroblasts and human epidermal immortalized cells. Figures C and D show the statistics of DPPH and ABTS free radical scavenging rates, respectively.

[0015] Figure 7 To test the anti-inflammatory effect of bioactive microneedle patches for the repair of radiation dermatitis: Figures A and B show the expression of related factors after macrophage immune activation by ELISA; Figures C, D, E, and F show the detection of RNA expression of related factors after macrophage immune activation.

[0016] Figure 8 Figure 1 shows the results of treating mice with radiation dermatitis using bioactive microneedle patches for radiation dermatitis repair in Example 1. Figure A is a schematic diagram of the animal experiment process. Figure B is a comparison of the appearance of wound healing in the mouse radiation dermatitis model on days 3, 7, and 14 using microneedle patches. Figure C shows the radiation dermatitis score results for each group of mice. Figure D shows the experimental results of Western blot detection of the Nrf2 oxidative stress pathway in animal protein samples after treatment. Detailed Implementation

[0017] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0018] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0019] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0020] Example 1: A method for preparing a bioactive microneedle patch for the repair of radiation dermatitis 1. The specific method for preparing methacrylamide hyaluronic acid is as follows: Hyaluronic acid (HA) was dissolved in deionized water to prepare a 1% (w / w) HA solution. The HA solution was placed in an ice-water bath, and sodium hydroxide was added to adjust the pH to 8-9. Methacrylic anhydride was slowly added dropwise to achieve a 1:1 molar ratio of HA to methacrylic anhydride, and the reaction was allowed to proceed overnight. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 20,000 Da and dialyzed against deionized water for 3 days, changing the water 3 times daily to remove unreacted reagents. After dialysis, the solution was freeze-dried to obtain HAMA powder. HAMA was characterized by Fourier transform infrared spectroscopy (FT-IR) and ¹H-NMR, observing the characteristic absorption peaks and proton peaks related to the methacryloyl group to confirm successful grafting of the methacryloyl group onto the hyaluronic acid molecular chain. The NMR results are as follows: Figure 3 As shown in Figure A.

[0021] 2. Extraction and characterization of silk fibroin (SF) The silkworm cocoons were cut into small pieces, and a 0.5% sodium carbonate solution was added. The mixture was heated and stirred at 80°C for 30 minutes to degumme the silk fibers. This process was repeated twice to remove the sericin. The degummed silk fibers were then rinsed with deionized water until neutral.

[0022] Washed silk fibroin fibers were added to a ternary solution of calcium chloride / ethanol / water in a molar ratio of 1:2:8, and stirred at 70°C for 2 hours to dissolve. The resulting solution was placed in a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed with deionized water for 3 days, changing the water 3 times a day. The dialyzed solution was centrifuged at 4000 rpm for 15 min to remove insoluble matter, yielding a silk fibroin solution. This solution could be freeze-dried to obtain silk fibroin powder if needed. The secondary structure characteristics of the silk fibroin were analyzed using nuclear magnetic resonance and other methods to verify the successful acquisition of the silk fibroin. The results are as follows: Figure 3 As shown in B.

[0023] 3. Isolation, collection and characterization of ADSC-Exos Mouse inguinal adipose tissue was harvested, rinsed with PBS to remove blood, minced, and then digested with 0.1% collagenase I at 37°C for 1 h with shaking. After digestion, the digestion was terminated with DMEM medium containing 10% fetal bovine serum. The cells were filtered through a 200-mesh sieve and collected by centrifugation at 1000 rpm for 5 min. The resulting cells were seeded in culture flasks and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%-90%, the cells were passaged, and cells from passages 3-5 were used for subsequent exosome collection.

[0024] The third-generation ADSCs cultured to the logarithmic growth phase were replaced with serum-free medium and cultured for another 48 h. The culture supernatant was collected and centrifuged sequentially at 300×g for 10 min to remove cell debris, 2000×g for 10 min to remove dead cells, 10000×g for 30 min to remove large vesicles, and finally ultracentrifuged at 100000×g for 70 min to collect the precipitate. The precipitate was resuspended in PBS to obtain ADSC-Exos.

[0025] 4. Preparation method of microneedle patch The prepared hyaluronic acid solution and silk fibroin solution were mixed at a ratio of 1:3 and stirred until homogeneous. Then, a certain concentration of exosomes and chlorogenic acid were added to prepare the needle tip layer casting solution. This casting solution was slowly injected into a PDMS microneedle mold, ensuring the solution fully filled the needle holes. Vacuum degassing was then performed to remove air bubbles, completing the needle tip layer molding. Subsequently, HAMA solution was added above the mold as a backing layer, covering the bottom of the needle tip and forming an integral support structure. Vacuum degassing was performed again. After UV cross-linking, the mold was placed in an oven to dry until the microneedles were stable and cured. The mold was then carefully demolded to obtain a bioactive microneedle patch for radiation dermatitis repair. The entire process ensured the integrity of the needle tip layer structure, excellent mechanical properties of the microneedles, and that the active ingredients maintained their bioactivity, making it suitable for transdermal drug delivery and tissue repair.

[0026] Example 2 Evaluation of microneedle morphology, mechanical properties, degradation and release performance The morphology of the composite hydrogel microneedle patch was observed using scanning electron microscopy (SEM), including needle tip shape, array integrity, needle tip breakage, and microneedle surface structure. The results are as follows: Figure 4 As shown, the prepared microneedle array is regularly arranged and uniformly distributed, with intact needles and no obvious collapse, breakage, or defects (Figure A, leftmost). High-magnification observation reveals that the microneedles have a regular quadrangular pyramidal structure with sharp tips and good geometric morphology (Figure A, leftmost second and third), indicating that the PDMS mold replication process can effectively ensure the complete formation of the microneedle structure. Further observation of the internal structure of the microneedles (Figure A, rightmost) shows that a continuous and uniform layered porous network structure is formed inside the material, indicating that a stable composite system is formed between SF and HA. This structure is beneficial for drug loading, tissue fluid absorption, and continuous release of active ingredients.

[0027] Compression tests were conducted on the microneedles using a universal testing machine to evaluate their mechanical properties. The results showed that the overall structure of the microneedles remained intact under external force, with no obvious breakage or damage (Figure B). The microneedles were able to withstand a large load during compression, indicating that the constructed SF / HA composite system possesses good mechanical strength and structural stability, meeting the mechanical requirements for microneedles to penetrate the stratum corneum and ensuring subsequent transdermal delivery. Furthermore, as shown in Figure C, compression performance tests were conducted on composite microneedles with different HA:SF mass ratios of 1:1, 1:2, and 1:3. The results showed that the mechanical properties of the material significantly improved with increasing SF content. The HA:SF=1:1 group had the lowest compressive stress, while the HA:SF=1:2 group showed a significant increase; when HA:SF=1:3, the compressive stress reached its highest value, approaching 150 MPa at 80% compressive strain, significantly higher than the other two groups. The results show that silk fibroin, as the main reinforcing phase, can effectively improve the load-bearing capacity and deformation resistance of the composite system. Among them, HA:SF=1:3 has the best mechanical properties and can provide more reliable mechanical support for microneedle puncture.

[0028] Frequency sweep tests were performed on the SF / HAMA composite system using a rotational rheometer. Figure 4 As shown in Figure D, the storage modulus (G′) consistently exceeded the loss modulus (G″) across the entire testing frequency range, indicating that the system exhibits predominantly elastic behavior and typical hydrogel characteristics. The storage modulus of the SF / HAMA composite system remained between approximately 1400 and 1600 kPa, while the loss modulus was approximately 300 to 600 kPa, with minimal fluctuations with frequency, indicating the formation of a stable three-dimensional cross-linked network structure. The high G′ value indicates that the material possesses good structural stability and resistance to deformation, maintaining the integrity of the microneedles during puncture and providing a stable carrier environment for sustained drug release.

[0029] Example 3: Evaluation of the biosafety of microneedles HaCaT cells and L929 cells were selected as in vitro evaluation cells. Cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator until they reached the logarithmic growth phase, at which point they were used for experiments.

[0030] Cell compatibility of the materials was evaluated using Calcein-AM / PI live / dead staining. After incubation with a mixture of Calcein-AM and PI according to the kit instructions for 30 min, the cells were washed with PBS and observed under a laser confocal microscope. Quantitative fluorescence analysis was performed (results are shown in the figure). Figure 5 ).like Figure 5 As shown in Figure A, all groups of cells exhibited abundant and uniformly distributed green fluorescence, with only a very small amount of red fluorescence signal observed, indicating that the cells maintained a high survival rate. Compared with the blank control group, no significant cell death was observed in the blank microneedle group, the chlorogenic acid-loaded microneedle group, the exosome-loaded microneedle group, and the microneedle group co-loaded with chlorogenic acid and exosomes. The cells had normal morphology and spread well, indicating that the constructed microneedle material has good biological activity and will not produce significant toxic effects on cells.

[0031] Further quantitative analysis of live cell fluorescence intensity ( Figure 5 B and Figure 5 (C) The results showed that the relative fluorescence intensity of L929 cells and HaCaT cells in each experimental group remained at around 100%, with no statistically significant difference compared with the blank control group (ns, P>0.05). Among them, the cell viability of the group co-loaded with chlorogenic acid and exosome microneedles was basically the same as that of the control group, indicating that the introduction of chlorogenic acid and ADSC-Exos does not reduce the cell compatibility of the material.

[0032] Cytotoxicity was detected using the CCK-8 assay. HaCaT cells and L929 cells were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at different densities in 96-well plates and cultured for 24 h. Afterward, microneedle extracts of different treatments were added, resulting in a blank control group, a blank microneedle group, a chlorogenic acid group, an ADSC-Exos group, and a chlorogenic acid + ADSC-Exos composite microneedle group. After further culture for 24 h, 48 h, and 72 h, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 2 h. The OD value was measured at 450 nm using a microplate reader, and cell viability was calculated (results are shown in the figure). Figure 5 The experimental results also showed that after homogenization, the relative cell viability of L929 cells and HaCaT cells in each experimental group was maintained at around 100%, and there was no statistically significant difference compared with the blank control group.

[0033] Example 4: Microneedle DPPH and ABTS Free Radical Antioxidant Capacity Test Antioxidant capacity of ABTS free radicals: First, prepare an ABTS stock solution: Mix 7 mM ABTS solution with 2.45 mM potassium persulfate (K2S2O8) solution at a volume ratio of 1:1, and let it react at room temperature for 12–16 h under light-protected conditions to form stable ABTS. +- Free radical working solution. Then, ABTS was treated with anhydrous ethanol or PBS. +- The working solution was diluted to adjust its absorbance at 734 nm to 0.70 ± 0.02. Different sample groups (including blank, CGA, Exos, and MN@CGA+Exo groups) were added to 96-well plates, with a certain volume of ABTS added to each well. + • The working solution was incubated at room temperature in the dark for 10 min. After the reaction, the absorbance of each group was measured at a wavelength of 734 nm using an ELISA reader.

[0034] Antioxidant capacity of DPPH free radicals: DPPH powder was dissolved in anhydrous ethanol to prepare a 0.1 mM DPPH solution, which was then stored in the dark for later use. Subsequently, different groups of sample solutions (including blank group, CGA group, Exos group, and MN@CGA+Exo group) were taken and mixed with DPPH working solution at a certain volume ratio, and incubated at room temperature in the dark for 30 min to allow the antioxidant components in the samples to fully react with DPPH free radicals.

[0035] After the reaction was completed, the absorbance of each group was measured at a wavelength of 517 nm using an ELISA reader.

[0036] The free radical scavenging rate is calculated using the following formula:

[0037] Where A0 represents the absorbance of the control group, A1 represents the absorbance of the experimental group, and vitamin C is used as a positive control. Results are as follows: Figure 6 As shown in the figure, the blank microneedle group (without active substances) exhibited a relatively low free radical scavenging rate, indicating that the SF / HA composite microneedles themselves possess certain background antioxidant activity. The free radical scavenging rate of the chlorogenic acid-loaded microneedle group was significantly increased, approaching the level of vitamin C, indicating that chlorogenic acid can effectively maintain its antioxidant activity within the microneedles. The scavenging rate of the exosome-loaded microneedle group was slightly higher than that of the blank microneedle group, but lower than that of the chlorogenic acid-loaded group, suggesting that exosomes contribute only a limited amount to the antioxidant effect. The scavenging rate of the microneedles co-loaded with chlorogenic acid and exosomes was close to that of the chlorogenic acid-loaded microneedle group, indicating that the synergistic loading of the two active substances does not weaken the antioxidant effect of chlorogenic acid.

[0038] Example 5: Microneedle Inflammatory Factor Detection Changes in inflammatory factors were detected using ELISA and qPCR. Macrophages were irradiated with X-rays to induce an inflammatory response, then treated with microneedle extract. Cell culture supernatants were collected to detect the levels of inflammatory factors such as TNF-α, IL-1β, and IL-6. Simultaneously, total RNA was extracted from the cells, and the expression of inflammation-related genes was detected using real-time quantitative PCR (results are shown in the figure). Figure 7 (As shown in Figures A and B). ELISA results (Figures A and B) showed that the secretion levels of IL-6 and TNF-α in the blank microneedle group were slightly lower than those in the radiation damage group, but the difference was not significant. The chlorogenic acid-loaded microneedles and exosome-loaded microneedles had certain inhibitory effects on inflammatory factors. The microneedles co-loaded with chlorogenic acid and exosomes significantly reduced the levels of IL-6 and TNF-α, showing the strongest inhibitory effect compared with the radiation damage group.

[0039] qPCR analysis (Figures C–F) further showed that co-loaded microneedles significantly inhibited radiation-induced IL-1β, IL-6, and TNF-α mRNA expression while increasing TGF-β mRNA expression, suggesting that co-loaded microneedles promote tissue repair while alleviating inflammatory responses. Compared with the blank control, the radiation-induced inflammatory factor expression was significantly upregulated in all groups; chlorogenic acid-loaded or exosome-loaded microneedle groups inhibited inflammation to some extent; the co-loaded microneedle group showed the closest inflammatory factor expression to the blank control, and the expression of the repair factor TGF-β was significantly increased.

[0040] Example 6: Treatment and Safety Evaluation of a Microneedle In Vivo Radiation Dermatitis Model Balb / c mice were selected, and after anesthesia, the hair on their right hind limbs was shaved to expose the skin. The right hind limbs of the mice were irradiated with X-rays, with a single irradiation of 35 Gy being preferred, to establish an acute radiation dermatitis model. Skin changes were observed after irradiation, and symptoms such as erythema, edema, and ulceration appeared on day 12, indicating successful model establishment.

[0041] Mice that successfully modeled the virus were randomly divided into several groups, which may include a blank control group, a clinical routine drug group, a blank microneedle group, a chlorogenic acid microneedle group, an ADSC-Exos microneedle group, and a chlorogenic acid + ADSC-Exos combined microneedle group. The specific number of groups and the number of animals in each group will be determined by ethical approval and experimental design. Each group will contain 5 to 6 animals.

[0042] like Figure 8 As shown, microneedle patches were applied to the damaged area of ​​the right hind limb of mice according to the preset treatment time and frequency. Skin appearance changes were recorded periodically by taking photos, and erythema, desquamation, edema, ulceration, and healing were quantitatively assessed according to the radiation dermatitis scoring criteria. Figure 8 As shown in Figure A, the experimental procedure clearly defines the timing points for radiotherapy and microneedle drug delivery. Figure 8B shows the changes in the gross appearance of the right hind limb of mice in each group over time. The results showed that the limbs of the radiotherapy-damaged group were significantly red and swollen, with slow healing and a tendency to worsen; the medical cream and blank microneedle groups alleviated the redness and swelling to some extent, but the improvement was limited; the redness and swelling were significantly reduced in the chlorogenic acid-loaded or exosome-loaded microneedle groups; however, the co-loaded microneedle group recovered the fastest, with the redness and swelling almost completely eliminated by day 14, and the wound closed well.

[0043] Figure C further quantifies the changes in radiation dermatitis scores over time. The radiation injury group had the highest score, indicating the most severe inflammation; both the chlorogenic acid-loaded and exosome-loaded microneedle groups reduced dermatitis scores, while the co-loaded microneedle group had the lowest score, showing a significant anti-inflammatory effect.

[0044] Figure D shows the results of Western blot analysis, evaluating the regulation of antioxidant pathways by microneedles. The results indicated that the co-loaded microneedle group showed significantly increased Nrf2 protein expression and decreased Keap1 expression, while the expression of the downstream antioxidant protein HO-1 was significantly enhanced. This suggests that microneedles can activate the Nrf2 / Keap1 / HO-1 signaling pathway, enhancing local antioxidant defense capabilities and thus reducing radiation-induced skin damage. Other drug-loaded groups also showed some activation effects, but the effects were not as significant as those of the co-loaded microneedle group.

[0045] In summary, this invention provides a bioactive microneedle patch for the repair of radiation dermatitis, its preparation method, and its application. This microneedle patch utilizes a composite hydrogel system constructed from methacrylamide hyaluronic acid (HAMA) and silk fibroin (SF), exhibiting excellent bioactivity, mechanical properties, and biodegradability, enabling stable drug loading and local delivery. Chlorogenic acid rapidly eliminates excess reactive oxygen species (ROS) after radiation injury, alleviating acute inflammatory responses; while adipose-derived stem cell exosomes continuously regulate the immune microenvironment, promote angiogenesis, and tissue repair. The synergistic effect of these two components effectively promotes the transformation of macrophages from pro-inflammatory M1 to reparative M2 types, activates the Nrf2 / HO-1 antioxidant pathway, improves local microcirculation, maintains extracellular matrix (ECM) homeostasis, and reduces post-radiation fibrosis, thereby significantly promoting high-quality repair of radiation dermatitis wounds.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A bioactive microneedle patch for the repair of radiation dermatitis, characterized in that: The microneedle patch includes a backing layer and a needle tip layer; the material of the backing layer is methacryloyl hyaluronic acid. The structural formula of the methacrylated hyaluronic acid is shown below: Where n is an integer greater than or equal to 2; The material of the needle tip layer is silk fibroin, and the needle tip layer is loaded with bioactive components, namely adipose stem cell exosomes and chlorogenic acid.

2. The method for preparing a bioactive microneedle patch for repairing radiation dermatitis as described in claim 1, characterized in that, Includes the following steps: S1: Preparation of microneedle tip casting solution: Hyaluronic acid solution and silk fibroin solution are mixed in proportion. Under gentle stirring, the pH value of the mixture is precisely adjusted to 7.2~7.4 using a trace amount of 0.1M NaOH solution. After the system is uniform and stable, chlorogenic acid and adipose stem cell exosomes are added and gently stirred to obtain microneedle tip casting solution. S2: Preparation of backing layer solution: After reacting hyaluronic acid with methacrylic anhydride, the resulting methacrylated hyaluronic acid powder was freeze-dried, dissolved in deionized water, and a photoinitiator was added to prepare a 4% concentration methacrylated hyaluronic acid pre-crosslinking solution. S3: Preparation of microneedle patch: The microneedle tip casting liquid obtained in step S1 is slowly injected into the PDMS microneedle mold, placed in a vacuum drying oven for degassing for 15 minutes, and then the backing layer solution obtained in step S2 is injected to remove air bubbles. The patch is then irradiated under ultraviolet light to undergo a photocrosslinking reaction. After drying and demolding, the bioactive microneedle patch for the repair of radiation dermatitis is obtained.

3. The preparation method according to claim 2, characterized in that, In step S1, the preparation method of the adipose stem cell exosomes is as follows: Adipose tissue is taken, washed with PBS, minced, and digested with 0.1% collagenase I at 37°C with shaking for 1 hour. Digestion is terminated with DMEM medium containing 10% fetal bovine serum. Cells are collected by filtration and centrifugation and cultured at 37°C and 5% CO2. When the cell confluence reaches 80%-90%, the cells are passaged, and cells from passages 3-5 are used for exosome collection. The third-generation adipose-derived stem cells in the logarithmic growth phase were replaced with serum-free culture medium and cultured for another 48 hours. The culture supernatant was collected and centrifuged sequentially at 300×g for 10 min, 2000×g for 10 min, 10000×g for 10 min, and 100000×g ultracentrifugation for 70 min. The precipitate was collected and resuspended in PBS to obtain the adipose-derived stem cell exosomes.

4. The preparation method according to claim 2, characterized in that, In step S1, the silk fibroin is prepared as follows: After the silkworm cocoons were cut into pieces, they were added to a 0.5% sodium carbonate solution and degummed at 80°C for 30 minutes. This process was repeated once or twice. After washing, the cocoons were added to a calcium chloride / ethanol / water ternary solution and dissolved at 70°C for 2 hours. The solution was then placed in a dialysis bag and dialyzed for 3 days. The cocoons were centrifuged at 4000 rpm for 15 minutes. The supernatant was collected and freeze-dried to obtain the silk fibroin. The molar ratio of the calcium chloride / ethanol / water ternary solution is 1:2:8, and the molecular weight cutoff of the dialysis bag is 8000 Da.

5. The preparation method according to claim 2, characterized in that, In step S1, the mass ratio of the silk fibroin solution to hyaluronic acid is 3:

1.

6. The preparation method according to claim 2, characterized in that: In step S2, the preparation method of the methacrylamide hyaluronic acid is as follows: HA is dissolved in deionized water to prepare a 1% mass concentration solution, placed in an ice-water bath, sodium hydroxide is added to adjust the pH to 8-9, methacrylic anhydride is slowly added dropwise, the reaction is carried out overnight at 4°C, and then transferred to a dialysis bag, dialyzed with water, and then freeze-dried to obtain methacrylamide hyaluronic acid. The molecular weight cutoff of the dialysis bag is 20,000 Da.

7. The preparation method according to claim 2, characterized in that, In step S3, the ultraviolet light irradiation parameters are a wavelength of 365 nm and a power of 10 mW / cm². 2 Irradiation time: 3 minutes; drying temperature in oven: 37℃.

8. A bioactive microneedle patch for the repair of radiation dermatitis prepared by the preparation method according to any one of claims 2-7.

9. The application of the bioactive microneedle patch for repairing radiation dermatitis according to claim 8 in the preparation of a drug for radiation dermatitis.

10. The application of the bioactive microneedle patch for radiation dermatitis repair according to claim 8 in the preparation of medical materials for radiation dermatitis repair.

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