Microneedle for repairing hypertrophic scars, preparation method thereof, and medical device

Abstract

A microneedle loaded with at least curcumin, exosomes, and an acellular matrix. Curcumin is incorporated into the exosomes to form functionalized exosomes (Cur@EV), which serve as the primary active ingredient for the repair of hypertrophic scars. Cur@EV has been shown to exhibit a high affinity for hypertrophic scar fibroblasts, while more efficient delivery of curcumin to HSFs has demonstrated enhanced anti-HSF cell proliferation activity and significant anti-fibrosis effects, potentially meeting clinical needs for the repair of hypertrophic scars.

Description

Technical Field

[0001] The present invention relates to a medical device made of biomaterials, and in particular to a microneedle made of a curcumin compound as an active ingredient, supplemented with exosomes and acellular matrix, for scar repair. Background Art

[0002] Hypertrophic scars (HS) are caused by excessive fibrosis and homeostasis during the skin healing process, typically presenting as raised, firm, and pruritic lesions. HS imposes a significant psychological, cosmetic, and economic burden on patients. In clinical practice, a variety of treatments have been used for HS, including surgical excision, corticosteroid injections, radiation therapy, and pressure therapy. However, the efficacy of these existing treatment strategies is suboptimal, primarily due to their inability to effectively adapt to the complex pathological microenvironment of HS. Therefore, there is an urgent need to develop targeted strategies tailored to the pathological characteristics of HS to improve clinical treatment outcomes and reduce disease recurrence.

[0003] Abnormal activation of hypertrophic scar fibroblasts (HSFs), also known as myofibroblasts, and chronic inflammatory responses are considered to be the most critical factors in the development of HS. Recent in vitro studies have shown that the autophagy pathway is associated with abnormal activation of HSFs. In addition, the initial inflammatory response, mainly involving M1 macrophages, is considered another key pathogenic factor in the development of HS. M1 macrophages can induce the transformation of other cells into HSFs and initiate the excessive fibrotic process. However, the underlying pathological changes and biological processes of HS are still not fully understood, which greatly restricts the development of HS treatment strategies.

[0004] Studies have shown that curcumin (Cur), a natural polyphenolic compound, has a promising therapeutic effect on HS. Its mechanism of action is to inhibit the activation of HSFs, however, its biological mechanism of action remains unclear. Furthermore, curcumin's anti-fibrotic effects are significantly limited by its poor water solubility, lack of target specificity, and low bioavailability. This issue highlights the urgency of developing efficient targeted delivery systems. Summary of the Invention

[0005] One object of the present invention is to provide a composition that targets curcumin to scar fibroblasts to facilitate the repair of hypertrophic scars.

[0006] Another object of the present invention is to provide a microneedle that targets curcumin to scar fibroblasts to facilitate the repair of hypertrophic scars.

[0007] Another object of the present invention is to provide an application of curcumin-containing microneedles in the preparation of medical devices for repairing hypertrophic scars.

[0008] Another object of the present invention is to provide a medical device that uses curcumin as an active ingredient and microneedles (arrays or patches) as carriers to repair hypertrophic scars.

[0009] Combined with the applicant's previous research, the findings of elevated autophagy, suppressed apoptosis, and exacerbated inflammation confirm the pathological characteristics of human HS tissue. Therefore, inhibiting autophagy in HSFs, inducing apoptosis, and simultaneously promoting the transformation of macrophages to the M2 phenotype, thereby addressing the two key issues of abnormal HSF activation and persistent inflammation in HS, represents the optimal therapeutic strategy for HS.

[0010] Exosomes (EVs) have become a highly promising platform for the delivery of lipid-soluble drugs due to their lipid membrane structure, low immunogenicity, and high biocompatibility. Therefore, EVs are incorporated into the compositions of the present invention. Notably, similar membrane proteins and lipids give EVs a natural homologous targeting ability, which can enhance their efficiency in cellular uptake. Therefore, HSF-derived EVs are preferably introduced into the compositions of the present invention to significantly enhance the targeted delivery and therapeutic effect of curcumin on HSFs.

[0011] The composition of the present invention comprises curcumin and exosomes. Curcumin is carried in the exosomes to form curcumin-engineered exosomes (Cur@EV), which act on scar fibroblasts (enter the interior of the cells) to facilitate the repair of hypertrophic scars.

[0012] The composition of the present invention has a curcumin / exosome ratio of 1±0.2 by weight.

[0013] The composition of the present invention effectively loads curcumin into EVs through ultrasonic co-incubation technology, with an average particle size of about 200 nm.

[0014] Exemplarily, Cur@EV of the present invention is prepared as follows:

[0015] Dissolve curcumin powder in anhydrous ethanol to obtain a curcumin ethanol solution (concentration, e.g., 1 mg / mL to 5 mg / mL);

[0016] Curcumin ethanol solution, exosome solution (exosome concentration, e.g., 0.5 mg / mL to 0.6 mg / mL), and DMEM medium were mixed evenly (e.g., the volume ratio of curcumin ethanol solution to exosome solution was 1:1±0.5, and the volume ratio of curcumin ethanol solution to DMEM medium was 1:8±4). The mixed solution was sonicated using an ultrasonic device (e.g., 120 W for 15 seconds, repeated three times), followed by an ice bath (e.g., 5 minutes), and repeated three times. The mixed solution was then incubated at 37°C±1°C (e.g., 1 hour) to allow the EV membrane to stabilize. The mixed solution was then centrifuged once (e.g., 10,000 g for 30 minutes) and then again (e.g., 100,000 g for 90 minutes) to isolate Cur@EVs. Finally, Cur@EVs were resuspended in DMEM medium / PBS solution by pipetting.

[0017] Stem cells, especially umbilical cord mesenchymal stem cells (UCMSCs), are widely used in the field of regenerative therapy because of their strong immunomodulatory ability. Decellularized extracellular matrix (dECM) has excellent biocompatibility and bioactivity and is considered to be an ideal material for tissue repair and regeneration. In addition, cell-derived dECM is reported to retain the natural bioactive factors of the parent cells and minimize the risk of pathogen transmission and immunogenic macromolecules. Previous research results have shown that dECM from UCMSCs (UdECM) can regulate M1 macrophages and reduce their profibrotic effects on HS.

[0018] In the combination of the present invention, dECM, especially UdECM, such as Cur@EV / UdECM, is also added to facilitate the mechanism of immune regulation and synergistic inhibition of fibrosis.

[0019] The dense stratum corneum of HS severely hinders the transdermal delivery of drugs, biomacromolecules, and vesicles (such as exosomes). The present invention also uses microneedles (MNs) as delivery vehicles, which has obvious advantages in achieving transdermal delivery of the above components.

[0020] This invention specifically designs a functionalized microneedle therapy system. Curcumin-engineered exosomes (Cur@EV) inhibit the activation of myofibroblasts (HSFs) and UdECM exerts immune regulation and synergistic inhibition of fibrosis. Finally, Cur@EV / UdECM-functionalized microneedles promote regenerative repair of hypertrophic scars. For example: the needle tips are in an N×M array (N and M are independently selected from integers greater than 10, such as: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30), and the height of each needle tip is greater than 200μm, especially greater than 400μm, such as: 450μm±50μm, 550μm±50μm, 650μm±50μm, 750μm±50μm, 850μm±50μm and 850μm±50μm. The curcumin content in the microneedle patches is greater than 1μg, especially greater than 3μg, such as: 3μg~4μg, and the Cur@EV content is greater than 3μg, especially greater than 6μg, such as: 6μg~8μg.

[0021] The Cur@EV provided by the present invention, as the active ingredient, is carried within the microneedles and promotes regenerative repair of hypertrophic scars. Supplemented with UdECM, it facilitates immune regulation and synergistic inhibition of fibrosis. UdECM is present in amounts exceeding 10 μg, particularly exceeding 30 μg, for example, 30 to 50 μg.

[0022] The present invention forms individual microneedles into an array or an array patch to form a medical device, which acts on the affected area, allowing Cur@EV or Cur@EV / UdECM to act on scar fibroblasts, thereby facilitating the repair of hypertrophic scars.

[0023] The microneedles of the present invention are made of hyaluronic acid (HA), or hydroxypropyl-β-cyclodextrin (HP-β-CD) is added. The mass ratio of hyaluronic acid to hydroxypropyl-β-cyclodextrin is greater than 5, especially greater than 9, such as 9 to 19. HA and HP-β-CD have intermolecular hydrogen bonds to form a complex. For example, HA is added to a 5mM to 10mM HP-β-CD solution to a concentration of 15% w / v, and then Cur@EV (content such as 6 to 8μg) or Cur@EV / UdECM (Cur@EV content such as 6 to 8μg, UdECM content such as 30μg to 50μg) is added. After that, the microneedles are poured into a PDMS microneedle mold and dried in a 37°C oven, and then demolded to obtain a microneedle or microneedle array (patch) carrying the Cur@EV active ingredient.

[0024] It has been verified that the functionalized exosomes loaded with curcumin (Cur@EV) provided by the present invention exhibit high affinity for HSFs, more efficiently deliver curcumin to HSFs, and exhibit anti-HSFS cell proliferation activity and significant anti-fibrosis effect, which can be used as an active ingredient to meet the clinical needs for the repair of hypertrophic scars.

[0025] By adding UdECM, the polarization of macrophages to the M2 phenotype was promoted, and a significant synergistic anti-fibrosis effect was also exhibited, effectively playing an immunomodulatory and synergistic anti-fibrosis role.

[0026] Verified in porcine skin and rabbit ear HS lesion models, the HA / HP-β-CD composite MNs of the present invention meet the mechanical requirements for effective skin penetration, enabling efficient transdermal drug delivery. Cur@EV / UdECM MNs play a positive and significant role in regulating scar status and promoting scar improvement, with a significant reduction in scar thickness and collagen deposition. The arrangement of collagen fibers becomes more orderly, with a morphology and structure more similar to normal skin tissue.

[0027] By simultaneously regulating multiple key biological pathways such as inflammation, autophagy, and apoptosis, the Cur@EV / UdECM MNs provided by the present invention effectively inhibited scar formation and demonstrated excellent therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Figure 3 is a diagram showing the preparation and characterization of curcumin-engineered exosomes (Cur@EV); A is a schematic diagram of the isolation of exosomes and the subsequent curcumin loading process; B is a protein immunoblot result for identifying Alix and TSG101 in exosomes (calnexin was used as a negative control); C is a transmission electron microscopy (TEM) image of exosomes (EV) and curcumin-engineered exosomes (Cur@EV); D is a nanoparticle tracking analysis (NTA) result, showing the particle size distribution of EV and Cur@EV; E is a high-performance liquid chromatography (HPLC) result, confirming the successful loading of curcumin into EV; F is the loading efficiency of Cur@EV at various curcumin concentrations; G is a fluorescence image of cellular uptake in human skin fibroblasts (HSFs) and macrophages (Raw) samples (nuclei stained blue, cytoskeleton stained green, EV and Cur@EV labeled with PKH26 marked red);

[0029] Figure 2The results of the in vitro anti-fibrotic effect of Cur@EV on human skin fibroblasts (HSFs); A is the detection of cell viability of HSFs after exposure to different concentrations of Cur@EV on the 1st, 3rd and 5th days; B is the Ki67 staining image in HSFs after incubation with curcumin (Cur), exosomes (EV) and Cur@EV (cell nuclei are blue, Ki67-positive cells are red); C is the verification image of the migration ability of HSFs evaluated by Transwell experiment after treatment of each group (cells are stained purple); D is the statistical graph of transforming growth factor-β (TGF-β) expression level in each experimental group; E is the statistical graph of α-smooth muscle actin (α-SMA) expression level in each experimental group; F is the statistical graph of type I collagen (Col I) expression level in each experimental group; G is the statistical graph of type III collagen (Col III) Statistical graph of expression levels; H is the staining image of α-SMA in HSFs of each group (nuclei stained blue, cytoskeleton stained green, and α-SMA stained red); I is a schematic diagram of the inhibitory effect of Cur@EV on fibrosis;

[0030] Figure 3 Figure 1 shows the results of the in vitro validation of the anti-inflammatory and anti-fibrosis effects of umbilical cord mesenchymal stem cell-derived decellularized extracellular matrix (UdECM); A is a staining image of type I collagen (COL1A1) in human umbilical cord mesenchymal stem cells (hUCMSCs) and UdECM (nuclei stained blue, COL1A1 stained green); B is a statistical graph of the DNA, glycosaminoglycan (sGAG) and collagen content between the hUCMSCs group and the UdECM group; C is a statistical graph of the macrophages (Raw) after treatment with UdECM solutions at various concentrations. ARG-1 staining in samples; D is a heat map of the expression levels of inflammatory biomarkers in the raw samples after treatment; E is a schematic diagram of the co-culture model of macrophages (raw) and human skin fibroblasts (HSFs); F is a staining image of α-SMA in HSFs after treatment (nuclei stained blue, cytoskeleton stained green, α-SMA stained red); G is a statistical graph of the expression levels of fibrosis, mitochondrial autophagy, and apoptosis-related biomarkers in HSFs of each group; H is a schematic diagram of the immunomodulatory and synergistic anti-fibrotic effects of UdECM;

[0031] Figure 4Preparation and characterization results of Cur@EV / UdECM functionalized microneedle patch; A is a schematic diagram of the microneedle patch preparation process; B is a bright field photograph of the prepared microneedle patch; C is a representative scanning electron microscope (SEM) image of the microneedle array and a single microneedle; D is a Fourier transform infrared spectroscopy (FTIR) analysis confirming the molecular interaction between hyaluronic acid (HA) and hydroxypropyl-β-cyclodextrin (HP-β-CD); E is a mechanical analysis of the prepared microneedle patch; F is a confocal image confirming the uniform distribution of Cur@EV in a single microneedle, with Cur@exosomes stained red; G is a trypan blue staining image of pig skin tissue after microneedle treatment; H is an H&E staining image of rabbit hypertrophic scar (HS) lesions after microneedle patch implantation;

[0032] Figure 5 Figure 3 is a diagram showing the therapeutic effect of Cur@EV / UdECM functionalized microneedle patch on rabbit hypertrophic scar (HS) model; A is a schematic diagram of the establishment of hypertrophic scar model, microneedle patch intervention and treatment effect evaluation, B is a photograph of hypertrophic scar in each group before and after treatment, C is the H&E staining and Masson staining of hypertrophic scar tissue after the third evaluation; D is a statistical graph of scar severity index (SEI) among all groups (* indicates statistical difference compared with the Normal skin group, # indicates statistical difference compared with the Control group, @ indicates statistical difference compared with the Cur@EV / UdECM MNs group); E is an immunohistochemical staining of type I collagen (Col I) and type III collagen (Col III) in normal ear skin and hypertrophic scar tissue that received or did not receive different treatments; F is a polarizing microscope image of picric acid Sirius red staining in all samples;

[0033] Figure 6 The figures show the verification results of Cur@EV / UdECM functionalized microneedle patch promoting regeneration and repair in rabbit hypertrophic scar model; among them, A is a cytokeratin staining image, highlighting the regeneration of newly formed hair follicles; B is a statistical graph of scar hardness assessment in each experimental group; C is a statistical graph of IL-1β expression level in each experimental group; D is a statistical graph of LGR6 expression level in each experimental group; E is a statistical graph of CD34 expression level in each experimental group; F is a statistical graph of white blood cell (WBC), red blood cell (RBC) aspartate aminotransferase (AST) and creatinine (CREA) after the third evaluation (the yellow area indicates the normal range); G is a schematic diagram of the therapeutic effect of Cur@EV / UdECM microneedle patch on hypertrophic scars (* indicates statistical difference compared with the Nornaml skin group, # indicates statistical difference compared with the Control group, and @ indicates statistical difference compared with the Cur@EV / UdECM MNs group). DETAILED DESCRIPTION

[0034] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are intended only to illustrate the technical solution of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention may be modified or replaced with equivalents without departing from the spirit and scope of the technical solution of the present invention, and all such modifications or equivalents should be included in the scope of the claims of the present invention.

[0035] The sources of the main materials used in the following examples of the present invention are as follows:

[0036] Hyaluronic acid was purchased from Dalian Meilun Biotechnology Co., Ltd. (China).

[0037] Hydroxypropyl-β-cyclodextrin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (China).

[0038] Curcumin (HPLC ≥ 98%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (China).

[0039] The microneedle mold was purchased from Shanghai Qifa Experimental Reagent Co., Ltd. (China), and the needle tip was a 15 × 15 array with 200 μm × 200 μm × 600 μm (length × width × height) MN needle tips.

[0040] DMEM cell culture medium (Dulbecco's Modified Eagle Medium) was purchased from Gibco (UK).

[0041] The various test methods used in the following examples of the present invention are specifically described as follows:

[0042] 1) Extraction of exosomes from human myofibroblasts

[0043] HSF cells were cultured in DMEM complete medium without EV for 2 days. The cell culture supernatant was collected and filtered through a 0.22 μm filter to remove cell debris. The filtered supernatant was then centrifuged at 10,000 g for 30 min in an ultracentrifuge. The supernatant was then transferred to a new ultracentrifuge tube and continued to be centrifuged at 100,000 g for 90 min in an ultracentrifuge. The supernatant was discarded and the EVs were resuspended by pipetting using DMEM medium / PBS solution and stored in aliquots.

[0044] 2) Preparation of curcumin-loaded exosomes

[0045] Curcumin was loaded into the extracted EVs by ultrasonic co-incubation. The specific experimental steps were as follows: first, curcumin powder was dissolved in anhydrous ethanol to prepare curcumin solutions with concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL; the protein concentration of the extracted EVs was determined using a BCA protein quantification kit; 100 μL of curcumin solutions of different concentrations, 100 μL of exosome solution, and 800 μL of DMEM culture medium (i.e., the volume ratio of curcumin ethanol solution, exosome solution and DMEM culture medium is 1:1:8) was mixed evenly; the mixed solution was ultrasonically treated using an ultrasonic machine, with the ultrasonic power set to 120 W and the ultrasonic time set to 15 s, repeated 3 times, followed by an ice bath for 5 minutes, and the above operation cycled 3 times; after the ultrasonic treatment was completed, the mixed solution was incubated at 37°C for 1 hour to restore the stability of the EV membrane; then, the sample was centrifuged at 10,000 g for 30 minutes and 100,000 g for 90 minutes to separate the curcumin-loaded exosomes; finally, the EVs were resuspended by blowing with DMEM culture medium / PBS solution and aliquoted for storage.

[0046] 3) Identification of curcumin-loaded exosomes

[0047] In order to explore the parameters such as the morphology, size and drug loading efficiency of the prepared curcumin-loaded exosomes (Cur@EV), a series of experimental analyses were performed on Cur@EV, including protein immunoblotting (WB), transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and high performance liquid chromatography (HPLC).

[0048] 4) Endocytosis of curcumin-loaded exosomes

[0049] Take 10μL EV and Cur@EV and dissolve them in Diluent C solution. Dissolve 1μL PKH26 dye in an equal volume of Diluent C solution and mix well by pipetting. Add the exosome solution to the dye solution and mix well by pipetting quickly. Incubate the mixed solution at room temperature in the dark for 10 minutes. Add an equal volume of exosome-free serum to terminate the staining reaction. Use an ultracentrifuge to separate the labeled exosomes. Next, HSFs and Raw cells were inoculated in 24-well plates for overnight culture. Fluorescently labeled EV and Cur@EV were added to the well plates and co-cultured with the cells. After culturing for 24 hours, the culture medium was discarded, the cells were washed with PBS solution, and the cells were fixed with 4% PFA solution for 30 minutes. The cell membrane was permeabilized with immunostaining permeabilization solution for 10 minutes. QuickBlock TM The cells were blocked with immunostaining blocking solution for 15 min, diluted phalloidin-AF488 labeling dye was added and incubated in the dark for 30 min, diluted DAPI solution was added for nucleus staining and incubated in the dark for 5 min, and finally observed and photographed under a confocal microscope.

[0050] 5) CCK-8 assay

[0051] HSFs cells were seeded into 96-well plates and cultured overnight. Cur@EV solution at varying concentrations was added to the plates. The culture medium was replaced every two days. After one, three, and five days of culture, the original culture medium was aspirated and 100 μL of freshly prepared CCK-8 working solution was added to each well. The cells were incubated in a 37°C incubator in the dark for one hour. At the indicated time points, the OD value of each well was measured at a wavelength of 450 nm using a microplate reader.

[0052] 6) Ki-67 immunofluorescence staining

[0053] HSFs cells were seeded in 24-well plates and cultured overnight. Cur@EV, EV, and Cur solutions were added to the plates. After 24 hours of culture, the original culture medium was removed. Immunofluorescence staining was performed using diluted Anti-Ki67 primary antibody solution and corresponding secondary antibody solution. Finally, the cells were observed and photographed under an inverted fluorescence microscope.

[0054] 7) Cell migration ability

[0055] To evaluate the effects of Cur@EV, EV, and Cur treatment on the migration ability of HSFs cells, a cell migration experiment was performed using a Transwell chamber. The specific steps were as follows: HSFs cells were evenly resuspended in Cur@EV, EV, Cur, and serum-free DMEM medium, and the above cell suspensions were evenly inoculated into the upper chamber of the Transwell chamber. 600 μL of DMEM complete medium was added to the lower chamber of the Transwell. After culturing in a 37°C incubator for 24 hours, the cells were fixed with 4% PFA solution for 30 minutes, stained with crystal violet solution for 20 minutes, and then washed with PBS to remove excess dye. A small cotton swab was used to gently wipe one side of the Transwell upper chamber to remove the non-migrated cells on the filter membrane surface. The Transwell chamber was inverted and observed and photographed using a stereo microscope. Finally, the number of migrated cells was quantitatively counted using ImageJ software.

[0056] 8) Cell fibrosis-related gene expression

[0057] HSFs cells were seeded into 6-well plates and cultured overnight. Cur@EV, EV, and Cur solutions were added to the plates, respectively. After 72 hours of culture, 1 mL of TRIzol solution was added to each well to lyse the cells. Subsequent cell total RNA extraction, cDNA reverse transcription, and qRT-PCR experimental steps were performed.

[0058] 9) Cellular α-SMA protein expression

[0059] HSFs cells were seeded in 24-well plates and cultured overnight. Cur@EV, EV, and Cur solutions were added to the plates, respectively. After 72 hours of culture, the cells were fixed with 4% PFA solution for 30 minutes and immunofluorescence staining was performed using diluted Anti-α-SMA primary antibody solution and corresponding secondary antibody solution. Finally, the cells were observed and photographed under an inverted fluorescence microscope.

[0060] 10) Preparation of decellularized extracellular matrix (UdECM) derived from human umbilical cord mesenchymal stem cells (hUCMSCs)

[0061] hUCMSCs were cultured in an incubator at 37°C with 5% CO2. When the cell confluence reached approximately 90% to 100%, 50 μg / mL of ascorbic acid was added to the culture medium to promote the production of extracellular matrix. After 2 weeks of culture, the original culture medium was removed and the cells were washed three times with PBS solution. 1% Triton X-100 solution was added to the culture dish and incubated at room temperature for 30 minutes. The solution was removed and the cells were washed three times with PBS solution. Then, ammonium hydroxide (NH4OH) solution was added and incubated at room temperature for 30 minutes. The culture dish was frozen at -20°C overnight, the ammonium hydroxide solution was removed by aspiration, and the cells were washed with PBS solution. Finally, the samples were freeze-dried in a freeze dryer overnight and stored in a -80°C refrigerator.

[0062] 11) UdECM identification - immunofluorescence staining

[0063] After decellularization, 4% PFA solution was added for 30 min, and the cell membrane was permeabilized with immunostaining permeabilization solution for 10 min. TM Block with immunostaining blocking solution for 15 minutes, add diluted Anti-Collagen I primary antibody solution, incubate at 4°C overnight in the dark, recover the primary antibody solution, wash three times with PBS solution, add the corresponding secondary antibody solution, incubate at 37°C for 1 hour, wash three times with PBS solution, add diluted DAPI solution for cell nucleus staining, incubate in the dark for 5 minutes, and finally observe and photograph under an inverted fluorescence microscope.

[0064] 12) UdECM Identification - DNA Quantification

[0065] Papain was dissolved in the extract to prepare papain extract. 1 mL of the extract was added to 50 mg of decellularized extracellular matrix and 50 mg of undecellularized cell samples, respectively. The samples were incubated in a water bath at 65 °C for 3 h, followed by centrifugation at 10,000 g for 10 min. 10 μL of Qubit TM Standard, 10 μL sample, and 180 μL Qubit TM 1X dsDNA BR working solution, vortex the sample for 3-5 seconds, incubate at room temperature for 2 minutes, and finally TM The DNA content of the samples was measured on a fluorometer.

[0066] 13) UdECM Identification - Collagen Quantification

[0067] Pepsin was dissolved in acetic acid to prepare a pepsin extract. This extract was added to the decellularized extracellular matrix and undecellularized cell samples at a ratio of 1:20 tissue to extract, and digested overnight at 4°C with shaking. After digestion, the samples were centrifuged at 3000g for 10 minutes. 100 μL of the supernatant was aspirated, and 1 mL of Sircol dye reagent was added to the supernatant. After inversion and mixing, the samples were shaken at room temperature for 30 minutes. The samples were then centrifuged at 13000g for 10 minutes, the supernatant was aspirated, and the pellet was washed with 750 μL of acid-salt. The pellet was then centrifuged at 12000g for 10 minutes, the supernatant was aspirated, and 250 μL of Alkali reagent was added to resolubilize the bottom pellet. The samples were shaken at room temperature for 5 minutes. The OD value of each well was measured at a wavelength of 555 nm using a microplate reader, and the collagen content in the samples was calculated based on the standard curve.

[0068] 14) UdECM Identification - Quantification of Sulfated Glycosaminoglycans

[0069] Papain was dissolved in the extraction solution to prepare the papain extract. 1 mL of the extract was added to 50 mg of the decellularized extracellular matrix and the undecellularized cell sample, and the samples were incubated in a water bath at 65°C for 3 h. The samples were then centrifuged at 10,000 g for 10 min, 100 μL of the supernatant was aspirated, 1 mL of Blyscan dye reagent was added to the sample and mixed by inversion. The sample was shaken at room temperature for 30 min, followed by centrifugation at 13,000 g for 10 min, the supernatant was aspirated, 500 μL of dissociation reagent was added to redissolve the bottom precipitate, the sample was shaken at room temperature for 10 min, and then centrifuged at 13,000 g for 5 min. Finally, 200 μL of the supernatant was aspirated, and the OD value of each well was measured at a wavelength of 656 nm using a microplate reader, and the content of sulfated aminoglycans in the sample was calculated based on the standard curve.

[0070] 15) Verification of the anti-inflammatory effect of UdECM in vitro - immunofluorescence staining

[0071] Raw cells were seeded into 24-well plates and cultured overnight. 1 μg / mL LPS solution was added to the culture medium for stimulation for 24 h. Different concentrations of UdECM solution were added to the well plates, and the culture medium was replaced every 2 days. After 72 h of culture, 4% PFA solution was added to fix the cells for 30 min. Immunofluorescence staining was performed using diluted Anti-ARG-1 primary antibody solution and corresponding secondary antibody solution. Diluted DAPI solution was added for cell nucleus staining. The cells were incubated in the dark for 5 min. Finally, the cells were observed and photographed under an inverted fluorescence microscope.

[0072] 16) Verification of UdECM's anti-inflammatory effect in vitro - expression of inflammation-related genes

[0073] Raw cells were seeded into 6-well plates and cultured overnight. 1 μg / mL LPS solution was added to the culture medium for stimulation for 24 hours. Different concentrations of UdECM solution were added to the well plates, and the culture medium was replaced every 2 days. After 72 hours of culture, 1 mL of TRIzol solution was added to each well to lyse the cells. Total cell RNA was then extracted, cDNA was reverse transcribed, and qRT-PCR experiments were performed.

[0074] 17) Verify the anti-fibrotic effect of UdECM in vitro

[0075] A co-culture model of Raw and HSFs cells was established using a Transwell chamber. Raw cells were seeded in the upper chamber of the Transwell chamber and UdECM solution was added. HSFs cells were evenly seeded in the lower chamber of the Transwell chamber and fluorescence color and gene expression were analyzed as follows.

[0076] Immunofluorescence staining: After 72 h of culture, HSFs cells in the lower chamber were fixed with 4% PFA solution for 30 min, and immunofluorescence staining was performed using diluted Anti-α-SMA primary antibody solution and corresponding secondary antibody solution. Finally, the cells were observed and photographed under an inverted fluorescence microscope.

[0077] Expression of cell fibrosis and autophagy-related genes: After 72 h of culture, 1 mL of TRIzol solution was added to the lower chamber of the Transwell to lyse the cells, followed by total RNA extraction, cDNA reverse transcription, and qRT-PCR experiments.

[0078] 18) Preparation of microneedles

[0079] HP-β-CD was dissolved in PBS solution to prepare 5 mM and 10 mM HP-β-CD solutions. Hyaluronic acid was added to PBS and HP-β-CD solutions of different concentrations at a ratio of 15% w / v to prepare HA, HA / HP-β-CD (5 mM) and HA / HP-β-CD (10 mM) solutions, respectively. Cur@EV and UdECM were added to the above composite solution. 150 μL of the mixed solution was drawn into the PDMS microneedle mold. The microneedle mold was placed in a vacuum drying oven for evacuation to remove bubbles in the solution. The mold was placed in a 37°C oven to dry for 2 h. Subsequently, 150 μL of the same solution was added to the mold, and the mold was dried in a 37°C oven overnight. Finally, the microneedle mold was demolded and temporarily stored at 4°C.

[0080] 19) Characterization of microneedle surface morphology

[0081] After the composite soluble microneedles were prepared, their surface morphology and microneedle structure were grossly observed and characterized using SEM. The microneedles were placed under a stereomicroscope to observe their overall morphology and microneedle array. The microneedles were then gold-sprayed using a coating apparatus and affixed to a sample stage with conductive gel for SEM scanning to characterize their structure and integrity.

[0082] 20) Characterization of microneedle mechanical properties

[0083] The microneedle sample was fixed in the center of the mechanical testing platform, and the upper pressure plate was adjusted to make it evenly contact with the microneedle test surface. The mechanical testing parameters were set, the displacement speed of the upper pressure plate was 3 mm / min, and the data sampling frequency was 10 times per second. The mechanical-displacement curve was drawn according to the test data to evaluate the mechanical properties of the composite microneedle.

[0084] 21) Characterization of Cur@EV loading in microneedles

[0085] Fluorescently labeled Cur@EVs were first prepared and then added to the microneedles during fabrication. After drying and debonding, the distribution of Cur@EVs within the microneedles was observed under a confocal microscope.

[0086] 22) Fourier transform infrared

[0087] The UdECM sample was placed on an attenuated total reflectance (ATR) test bench, and the spectrometer was started to collect spectra. The chemical composition of the sample was analyzed based on the spectra to identify specific chemical bonds and functional groups.

[0088] 23) Microneedle transdermal puncture experiment on pig skin

[0089] An appropriate amount of methylene blue was added to the microneedle solution to prepare composite microneedles and remove the membrane. The ex vivo pig skin was fixed on the laboratory table and the prepared composite microneedles were vertically pressed into the pig skin sample. After 30 minutes, the microneedle base was removed. Finally, the pig skin sample was observed under a stereo microscope and photographed.

[0090] 24) Microneedle transdermal puncture experiment on rabbit ear hypertrophic scar

[0091] A rabbit ear scar sample was fixed on a laboratory bench. The prepared composite microneedles were vertically pressed into the scar sample. After 30 minutes, the microneedle base was removed and the sample was fixed in 4% PFA solution for 48 hours. The fixed sample was then dehydrated, paraffin-embedded, sectioned, and stained with H&E. Finally, the sample was mounted with neutral gum and observed under a microscope.

[0092] 25) In vivo experiments on microneedle treatment of scars

[0093] One month after establishing a rabbit ear hypertrophic scar model, microneedle treatment was performed. The experimental group was divided into six groups: control group (Control), medical anti-scar hydrogel group (Scar gel), blank microneedle group (Free MNs), Cur@EV microneedle group (Cur@EV MNs), UdECM microneedle group (UdECM MNs), and Cur@EV / UdECM microneedle group (Cur@EV / UdECM MNs). Normal rabbit ear skin was also used as a blank control (Normal skin). Microneedle treatment was performed once a week for three consecutive weeks. The scar area was cleaned with an alcohol cotton ball, and the macroscopic morphology of the scar was recorded with a camera. The prepared composite microneedles were vertically pressed into the scar tissue. After 30 minutes, the microneedle base was removed. This procedure was repeated one week later, for a total of three treatments. After treatment, the scar tissue was excised, one portion stored in 4% PFA solution, and the other portion was quickly frozen in liquid nitrogen and subsequently subjected to histological staining.

[0094] 26) In vivo biosafety assessment

[0095] After completing the microneedle treatment, blood was drawn from the experimental rabbits for routine blood tests and biochemical analysis. At the same time, samples of the animals' major organs were collected for H&E staining to comprehensively evaluate the in vivo biosafety of the prepared microneedles.

[0096] 27) Statistical analysis

[0097] All quantitative data are expressed as mean ± standard deviation. Unpaired t-tests were used for both groups. Statistical analysis and graphics were performed using GraphPad Prism 8.0. Image J software (NIH, Bethesda, MD, USA) was used for image analysis and statistics. *, #, and @ indicate p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0098] Example 1 Preparation and Characterization of Homologous Targeted Curcumin Engineered Exosomes (Cur@EV)

[0099] Given that EVs secreted by HSFs have homologous targeting properties, they are preferred as curcumin targeted delivery carriers for loading curcumin, a natural compound with anti-fibrotic properties. Figure 1 A) Curcumin was effectively loaded into EVs and identified. First, surface-specific proteins of EVs and curcumin-loaded EVs (Cur@EVs) were identified by immunoblotting (WB). Figure 1As shown in Figure B, the EV-specific proteins Alix and TSG101 were detected in both the EV and Cur@EV groups. Calnexin was used as a negative control. Calnexin is not a specific EV surface protein and is normally undetectable. As expected, no calnexin bands were detected in either the EV or Cur@EV groups, demonstrating the successful extraction and preparation of EVs and Cur@EVs.

[0100] After the successful preparation of Cur@EV, TEM and nanoparticle tracking analysis (NTA) were performed to evaluate its morphological characteristics and diameter distribution. Figure 1 It can be clearly seen in the TEM image of C that after loading with curcumin, the EV nanovesicle structure is well preserved, showing a typical round or oval outline with clear boundaries. At the same time, the average particle size of Cur@EV is about 200nm, which is similar to the size range of EV. Subsequently, EV and Cur@EV were analyzed using NTA. Figure 1 The size distribution shown in D shows that both EV and Cur@EV exhibit a relatively concentrated distribution state. Among them, the particle size distribution of Cur@EV is slightly larger than that of EV.

[0101] Cur@EV was tested by high performance liquid chromatography (HPLC) to evaluate whether curcumin was successfully encapsulated in EV and to quantify its encapsulation efficiency. HPLC can accurately identify and measure the content of target substances due to its high-resolution separation capability of each component in a complex mixture. First, pure curcumin samples were subjected to HPLC analysis, and their peak time and peak shape under specific chromatographic conditions were recorded. Subsequently, the Cur@EV samples were analyzed under the same conditions. Figure 1 It can be clearly seen in the chromatogram of E that the peak time of pure curcumin and Cur@EV are consistent, confirming that curcumin has been successfully encapsulated in EV. Subsequently, the loading efficiency of Cur@EV under different curcumin concentrations was further explored, and the relevant results are shown in Figure 1 F. This figure visually demonstrates how the loading efficiency of Cur@EVs for curcumin changes with the initial curcumin concentration. Experimental results indicate that the highest loading efficiency is achieved when the curcumin concentration is 400 μg. Therefore, Cur@EVs with a loading efficiency of approximately 9% were selected for subsequent experiments.

[0102] The cellular uptake in HSFs and RAW cells was characterized to evaluate whether the prepared Cur@EV had the ability to homologously target HSFs. Figure 1As shown in Figure G, the nuclei and cytoskeleton of the two cells were stained blue and green, respectively, while Cur@EV and EV were labeled red, allowing for clear and intuitive tracking of vesicle internalization within the cell. Cur@EV showed a high affinity for HSFs, and a large number of Cur@EVs were observed to successfully enter the cell interior. However, no obvious Cur@EVs were observed in RAW cells. These results indicate that Cur@EV can, with its unique properties, serve as an efficient targeted delivery platform to accurately recognize and bind to HSFs, thereby achieving more efficient delivery of curcumin to HSFs.

[0103] Example 2 Cur@EV exhibits significant anti-fibrotic effect on HSFs cells

[0104] First, the viability and proliferation of HSFs cells under different Cur@EV concentration gradients were detected to evaluate the biological effects of Cur@EV and optimize the experimental concentration. Figure 2 As shown in Figure A, Cur@EV significantly inhibited HSF proliferation at dilutions ranging from 1 / 4 to 1 / 16. The inhibitory effect weakened somewhat with decreasing dilution. Furthermore, at a Cur@EV concentration of 1 / 16, HSF cells maintained good cell viability in the early stages, and this dilution was selected as the optimal concentration for subsequent experiments. This ensured sufficient inhibitory effect on HSF proliferation while also balancing experimental feasibility and safety.

[0105] This example conducted a Ki67 staining experiment to further verify the anti-proliferative effect of Cur@EV. Ki67 is an important biomarker for cell proliferation, and its expression level can directly reflect the proliferation activity of cells. Figure 2 In the experimental results of Figure B, it can be clearly observed that the number of Ki67-positive cells (stained red) in the Cur@EV group was significantly reduced compared to the other groups. This result is consistent with the conclusions of the cell proliferation experiment, further confirming that Cur@EV has satisfactory anti-proliferative activity against HSFS cells. Meanwhile, neither the EV alone nor the curcumin group showed significant anti-proliferative activity.

[0106] Subsequently, the effect of Cur@EV on the migration ability of HSFs was evaluated by Transwell assay. Figure 2 As shown in Figure C, compared with the control group, the Cur@EV-treated group exhibited a moderate degree of migration inhibition. Furthermore, the migration ability of HSFs treated with Cur@EV was significantly inhibited, and the number of migrating cells was significantly reduced. This indicates that Cur@EV effectively delivers curcumin into the cells, exerting a strong anti-fibrotic effect.

[0107] The expression of fibrosis markers in HSFs was evaluated by qRT-PCR and immunofluorescence staining. Figure 2 D. Figure 2 E. Figure 2 F and Figure 2 As shown in Figure G, the expression levels of fibrosis markers such as TGF-β, α-SMA, Col I, and Col III were significantly reduced in the Cur@EV-treated group. In contrast, the expression of these fibrosis markers did not decrease in the Cur and EV groups alone. These results suggest that EV and drug treatment alone cannot effectively inhibit fibrosis in HSFs. Furthermore, Cur@EV may also alleviate fibrosis by regulating these key molecules involved in the fibrosis process.

[0108] In addition, the expression of α-SMA was detected by immunofluorescence staining. As a marker protein of HS, the change of its expression level is crucial for evaluating the occurrence and development of HS. Figure 2 As shown in H, the fluorescence density of α-SMA in cells treated with Cur@EV was significantly weakened, and + The number of cells was significantly reduced. The results showed that Cur@EV can exert its anti-fibrotic effect through the expression of key fibrosis markers.

[0109] Therefore, in vitro studies have shown that Cur@EV exerts anti-fibrotic effects by inhibiting HSFs cell proliferation and migration and reducing the expression of key fibrosis markers ( Figure 2 I).

[0110] Example 3 Umbilical cord mesenchymal stem cell-derived decellularized extracellular matrix (UdECM) exerts immunomodulatory and synergistic anti-fibrotic effects

[0111] In the early stages of HS development, controlling the inflammatory response in local tissues and inducing macrophage polarization to M2 is considered an effective treatment strategy. In this embodiment, a biomaterial with immunomodulatory properties, UdECM, was used to achieve this goal. It is crucial to evaluate the presence of cell residues in UdECM, as these residues may trigger cytotoxic reactions, immune rejection, and directly affect the final repair effect. Figure 3 As shown in A, DAPI staining confirmed the successful removal of nuclear components, while the collagen components (stained green) were well preserved. The DNA, sGAG, and collagen contents between the hUCMSCs group and the UdECM group were then compared and analyzed. Figure 3 The results in B further confirmed the effective removal of DNA components and the retention of the main active components in UdECM.

[0112] First, the ability of UdECM to regulate macrophage phenotypic transformation was evaluated in vitro. ARG-1 is an important biomarker of M2 macrophages, and its expression can directly reflect the phenotypic transformation of macrophages. Figure 3 C shows that after LPS treatment, the expression of ARG-1 in Raw cells was inhibited; however, after UdECM treatment, the expression of ARG-1 increased in a dose-dependent manner. In addition, the results of qRT-PCR ( Figure 3 D) Further validation of UdECM's immunomodulatory capacity. UdECM increased the expression of anti-inflammatory factors (such as IL-4 and TGF-β) while decreasing the expression of pro-inflammatory factors (IL-1 and iNOS). Based on these experimental results, a concentration of 0.15 mg / mL was determined to be the optimal concentration for subsequent experiments.

[0113] Subsequently, a co-culture model of Raw and HSFs was established using Transwell chambers to explore the interaction between UdECM-treated macrophages and HSFs. Figure 3 The ELISA results of E showed that after treatment with UdECM, the level of inflammatory factors in the cell supernatant was significantly reduced, further confirming the immune regulatory ability of UdECM.

[0114] Studies have shown that in physiological processes, the increase in the number of M2 macrophages can effectively reduce inflammatory responses and regulate the development of HS. Figure 3 As shown in the α-SMA immunofluorescence staining image F, the expression of α-SMA in HSFs co-cultured with RAW cells treated with UdECM was significantly reduced, indicating that the fibrotic activity of the cells was significantly inhibited. At the same time, the changes in the expression levels of fibrosis-related biomarkers further confirmed this conclusion. Figure 3 As can be seen in G, the expression levels of TGF-β, α-SMA and Col III were significantly downregulated.

[0115] In the in vitro study of Cur@EV on HSFs, it was found that Cur@EV exerted a biological mechanism of promoting apoptosis, downregulating autophagy and restoring MMP-1 / TIMP1 homeostasis. The same phenomenon was also shown in the RAW / HSFs co-culture model. Figure 3 G It can be found that the autophagy process and the expression of anti-apoptotic genes in HSFs were inhibited; at the same time, the activity of MMP-1 was also increased.

[0116] The above results indicate that UdECM not only plays an effective role in immune regulation and promotes the polarization of macrophages to M2 phenotype, but also exhibits a significant synergistic anti-fibrotic effect ( Figure 3H). In addition, the importance of the "autophagy-apoptosis" pathway in regulating HSFs cells was also confirmed.

[0117] Example 4 Preparation and Characterization of Cur@EV / UdECM Functionalized Microneedle Patch

[0118] In order to further study the in vivo therapeutic effect of Cur@EV / UdECM on HS, a functionalized microneedle (MNs) patch based on hyaluronic acid (HA) was prepared in this example. Figure 4 A). HA was chosen as the substrate due to its good biocompatibility, biodegradability and inherent anti-inflammatory properties. Figure 4 As shown in Figure B, the entire MNs array (15×15) is complete in structure and has a square shape. Each microneedle is 600 μm high, the microneedle base is 200 μm wide, and the adjacent spacing is 500 μm. The MNs were then scanned by SEM to observe the microstructure of the microneedles. Figure 4 As shown in Figure C, each microneedle not only maintains its own integrity but also presents a regular quadrangular pyramid shape. Cur@EV was labeled with fluorescent dye and incorporated into MNs, and the distribution of Cur@EV in MNs was observed under a confocal microscope. Figure 4 As shown in Figure F, Cur@EV is uniformly dispersed in the microneedle structure, indicating that Cur@EV is well integrated in the entire needle matrix, which is of great significance for the subsequent effective release and therapeutic effect.

[0119] Studies have shown that the minimum average force required to effectively pierce the skin is 0.058N. The density and hardness of the HS tissue structure are both increased compared to normal skin. Therefore, it is necessary to improve the mechanical strength of existing MNs, especially the puncture ability, in order to better deliver drugs to the treatment area. Therefore, based on previous studies, hydroxypropyl-β-cyclodextrin (HP-β-CD) was added as a reinforcing agent in this embodiment to improve the mechanical properties of the HA microneedle patch. First, the intermolecular interaction between HA and HP-β-CD was determined by FTIR analysis. Figure 4 As shown in Figure D, after HA and HP-β-CD formed a complex, obvious spectral shifts appeared at 1556 cm-1, 1611 cm-1, and 1641 cm-1, indicating that intermolecular hydrogen bonds were successfully formed between HA and HP-β-CD.

[0120] Subsequently, the mechanical properties of MNs reinforced with HP-β-CD were evaluated. Figure 4As shown in Figure E, the introduction of HP-β-CD has a significant effect on the stress tolerance of MNs, significantly improving their mechanical strength. At the same time, after adding Cur@EV and / UdECM active ingredients, HA / HP-β-CD composite MNs still maintain good mechanical strength. The results show that the MNs prepared in this example can withstand a force of up to 0.1N (single microneedle), far exceeding the mechanical requirements required to effectively pierce the skin. In addition, the mechanical-displacement curve shows that when the displacement exceeds 300μm, the microneedle still does not break and maintains its intact structure.

[0121] Its in vitro penetration ability was further evaluated using porcine skin tissue and rabbit ear HS lesions. Figure 4 As shown in G, the MNs array successfully penetrated the skin, indicating that the MNs achieved efficient transdermal drug delivery. Histological staining and evaluation were performed after MNs puncture on the rabbit ear HS lesions. The results are shown in Figure 4 As shown in Figure 3, a crack with a depth of about 300 μm can be observed, indicating that the prepared microneedles successfully penetrated the stratum corneum of HS and can effectively deliver therapeutic drugs.

[0122] Example 5 Cur@EV / UdECM functionalized microneedle patch has therapeutic effect on hypertrophic scars

[0123] The establishment of hypertrophic scar model, microneedle patch intervention and treatment effect evaluation process are as follows: Figure 5 As shown in A.

[0124] Figure 5 Figure B shows photos of the appearance of HS before and after various treatments. Comparative analysis revealed that the Scargel and Free MNs groups exhibited no significant therapeutic effects. The boundaries of the HS in the rabbit ears after treatment in these two groups were blurred, and the overall appearance and features did not improve significantly. However, the therapeutic effect of the Cur@EV / UdECM MNs group was completely different. The scar surface after treatment was relatively smooth, and the boundaries became very blurred. This series of intuitive changes fully demonstrates that Cur@EV / UdECMMNs plays a positive and significant role in regulating scar status and promoting scar improvement.

[0125] Subsequently, the treated HS tissues were histologically stained and evaluated. Figure 5As shown in C, the control group showed obvious HS pathological characteristics, and the scar thickness increased significantly. At the same time, it can be observed through Masson staining that the collagen fibers were arranged in a rough and disordered state. The two groups treated with Scar gel and Free MNs showed a slight decrease in tissue thickness, but the overall improvement effect was not obvious. In addition, the Cur@EV / UdECM MNs group showed the most ideal therapeutic effect. In the staining results of this group, it can be clearly seen that not only the scar thickness was significantly reduced, but also the collagen deposition phenomenon was greatly reduced. More importantly, the arrangement of collagen fibers became more orderly, and the morphology and structure were more similar to normal skin tissue. This series of changes fully demonstrates that Cur@EV / UdECM MNs has played a significant role in the repair and improvement of HS tissue.

[0126] This example also calculated the scar elevation index (SEI) for all experimental groups to further quantify and verify the treatment effect of each experimental group. SEI is a key indicator for evaluating the effect of scar treatment. It can intuitively reflect the degree of protrusion of the scar relative to the adjacent normal skin. Figure 5 As can be seen from the results of D, the SEI of the Scar gel and Free MNs groups was slightly reduced, further confirming that these two treatment methods were not significantly effective in reducing scars.

[0127] In contrast, the functionalized microneedle patch treatment group, particularly the Cur@EV / UdECM MNs group, showed a significant decrease in SEI compared to the control group. Previous studies have shown that multi-drug combination therapies can exert synergistic effects, thereby enhancing therapeutic efficacy. In this experiment, the Cur@EV / UdECM microneedle patch, leveraging this multi-drug synergy, effectively inhibited scar formation by simultaneously regulating multiple key biological pathways, including inflammation, autophagy, and apoptosis, demonstrating remarkable therapeutic efficacy.

[0128] The abnormal activation of HSFs and the interaction with the persistent inflammatory response together form the pathological microenvironment of HS, which ultimately leads to excessive deposition of ECM and scar thickening. Among the components of ECM, various types of collagen occupy an important position, among which type I and type III collagen are the main ones. Therefore, promoting the degradation of collagens such as type I and type III has become a key indicator for measuring the effectiveness of therapeutic interventions for HS. The immunohistochemical staining results of type I and type III collagen are shown in Figure 2. Figure 5As shown in Figure E, compared to the control group, collagen deposition in HS tissues of all treated groups decreased. This reduction in collagen deposition was particularly pronounced in the Cur@EV / UdECM MNs group. This result intuitively demonstrates that Cur@EV / UdECM MNs exhibit a significant therapeutic effect in inhibiting fibrosis and reducing excessive collagen deposition.

[0129] In addition, the study also found that in HS tissue, there is an imbalance in the ratio of type I and type III collagen. Specifically, there is too much type I collagen and insufficient type III collagen. This imbalance causes scar tissue to become more rigid and less flexible. Therefore, regulating the balance between type I and type III collagen has become an important strategy for treating HS. To this end, Sirius red staining was performed to evaluate the changes in the ratio of type I to type III collagen (Col I / III) after therapeutic intervention. Figure 5 As can be seen in Figure F, after treatment, the collagen fiber arrangement within the scar tissue changed. The previously disordered collagen fiber arrangement improved, with a significantly increased degree of ordered arrangement and a gradual normalization of the Col I / III ratio. Among all treatment groups, the Cur@EV / UdECM MNs group demonstrated the most significant effect, confirming its significant efficacy in treating HS.

[0130] Example 6 Cur@EV / UdECM functionalized microneedle patch promotes regenerative repair of hypertrophic scars

[0131] In the treatment of HS, promoting the regeneration of hair follicles is crucial for restoring the natural function and beauty of the skin. In this experiment, by cytokeratin staining, the appearance of new hair follicles was clearly observed in the samples of the Cur@EV / UdECM MNs group ( Figure 6 A). This phenomenon indicates that the functionalized microneedle patch prepared in this example plays a positive role in promoting the regenerative repair of HS.

[0132] Previous studies have shown that in the pathological state of HS, excessive inflammatory response and high-stress tissue microenvironment are not conducive to hair follicle regeneration. Based on this, we evaluated the hardness and inflammation level of different groups and normal skin tissue. The results showed that untreated HS tissue showed the highest scar hardness value ( Figure 6 B), and IL-1β levels were also high ( Figure 6 C). However, when treated with Cur@EV / UdECM MNs, both tissue stiffness and inflammation levels were significantly reduced.

[0133] Verification found that Cur@EV / UdECM MNs treatment can also significantly increase the expression levels of LGR6 and CD34. LGR6 and CD34 are key markers of hair follicle bulge stem cells. They are important in the identification, characterization and maintenance of stem cell characteristics of hair follicle stem cells. The increase in their expression means that Cur@EV / UdECM MNs treatment creates a good stem cell microenvironment for hair follicle regeneration ( Figure 6 D and Figure 6 E). These results reveal the potential mechanism by which Cur@EV / UdECM MNs promote hair follicle regeneration, providing a new theoretical basis and research direction for the treatment of HS.

[0134] After all treatments were completed, in order to evaluate whether the microneedles prepared in this example would induce immune rejection and cause adverse damage to important organs of the body, routine blood tests and staining and analysis of important organ sections were performed on all experimental animals. Figure 6 As shown in Figure 5, there were no significant deviations in the blood routine and routine blood and serum biochemical indices related to liver and kidney function, and all indices fluctuated within the normal range.

[0135] Application of the Cur@EV / UdECM microneedle patch in a rabbit HS model produced remarkable results, not only reducing scarring but also promoting hair follicle regeneration. This two-pronged strategy addresses both the fibrosis and immune disorders underlying HS.

[0136] Therefore, the present invention validates a synergistic treatment strategy for hypertrophic scars, regulating the "autophagy-apoptosis-inflammation" axis, and provides an innovative approach for the clinical management of HS ( Figure 6 G).

Claims

1. A composition for use in preparing a medical device for repairing hypertrophic scars, the composition comprising curcumin, a decellularized extracellular matrix, and exosomes, wherein the curcumin is contained within the exosomes, and the decellularized extracellular matrix facilitates immune regulation and synergistic inhibition of fibrosis. The medical device is a microneedle or a microneedle array patch; The decellularized extracellular matrix is ​​the decellularized extracellular matrix of umbilical cord mesenchymal stem cells.

2. The use according to claim 1, characterized in that The method for preparing the composition comprises: Curcumin ethanol solution, exosome solution and DMEM culture medium were mixed evenly, and the mixed solution was sonicated using an ultrasonic machine, followed by an ice bath, and this process was repeated three times. After completion, the mixed solution was incubated to allow the EV membrane to recover stability. Subsequently, the mixed solution was centrifuged for the first time and then for the second time to separate Cur@EVs.

3. A microneedle, characterized in that The invention comprises curcumin, exosomes and acellular extracellular matrix. The curcumin is contained in the exosomes to form engineered exosomes Cur@EV, and the acellular extracellular matrix is ​​the acellular extracellular matrix of umbilical cord mesenchymal stem cells.

4. The microneedle according to claim 3, characterized in that The microneedles are mainly made of hyaluronic acid and hydroxypropyl-β-cyclodextrin, and hyaluronic acid and hydroxypropyl-β-cyclodextrin have intermolecular hydrogen bonds.

5. The microneedle according to claim 4, characterized in that The mass ratio of the hyaluronic acid to the hydroxypropyl-β-cyclodextrin is greater than 5.

6. The microneedle according to claim 5, characterized in that The invention has needle tips in an N×M array, each needle tip is greater than 200 μm in height, the curcumin content is greater than 1 μg, the Cur@EV content is greater than 3 μg, and N and M are independently selected from integers greater than 10.

7. The microneedle according to claim 6, characterized in that The content of the decellularized extracellular matrix is ​​greater than 10 μg.

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