Microneedle for repairing hypertrophic scars, preparation method of microneedle and medical instrument

Abstract

The invention relates to a microneedle which at least carries curcumin, exosome and an acellular matrix, curcumin is carried in the exosome to form a functionalized exosome (Cur-EV), and the Cur-EV is used as a main active component for repairing hypertrophic scars. Experiments prove that Curat EV shows high affinity to hypertrophic scar fibroblasts, curcumin is delivered into HSFs more efficiently, better anti-HSFs cell proliferation activity is shown, the anti-fibrosis effect is remarkable, and the Curat EV can serve as activity to meet the requirement for repairing hypertrophic scars clinically.

Description

Technical Field

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

[0002] Hypertrophic scar (HS) is caused by excessive fibrosis and homeostasis disorders during the healing process of the skin, and usually manifests as raised, hard lesions with itchy symptoms. Approximately 30 million people worldwide develop HS each year, and the cost of treatment is as high as 12 billion US dollars. HS brings heavy psychological, cosmetic and economic burdens to patients. In clinical treatment, a variety of methods have been used to treat HS, including surgical resection, corticosteroid injection, radiation and pressure therapy. However, the efficacy of these existing treatment strategies is not ideal, and the recurrence rate is as high as 70% to 100%. This is mainly because they cannot effectively adapt to the complex pathological microenvironment of HS. Therefore, there is an urgent need to develop targeted strategies for the pathological characteristics of HS to improve clinical treatment effects and reduce the recurrence rate of the disease.

[0003] The abnormal activation of hypertrophic scar fibroblasts (HSFs), also known as myofibroblasts, and chronic inflammatory response are considered to be the most critical factors in the formation of HS. Recent in vitro studies have shown that the autophagy pathway is associated with the abnormal activation of HSFs. In addition, the initial inflammatory response dominated by M1 macrophages is considered to be another key pathogenic factor in the formation of HS. M1 macrophages can induce other cells to transform into HSFs and initiate the process of excessive fibrosis. 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), as a natural polyphenol compound, has a good therapeutic effect on HS. Its mechanism of action is to inhibit the activation of HSFs, but its biological mechanism of action has not yet been clarified. In addition, the anti-fibrotic effect of curcumin is significantly limited by its poor water solubility, lack of targeting specificity and low bioavailability. This problem highlights the urgency of developing an efficient targeted delivery system. 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 a microneedle containing curcumin in the preparation of a medical device for repairing hypertrophic scars.

[0008] Another object of the present invention is to provide a medical device which 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, it was found that there were phenomena such as increased autophagy levels, suppressed apoptosis, and increased inflammation, thus confirming the pathological characteristics of human HS tissue. Therefore, by inhibiting the autophagy process in HSFs, inducing apoptosis, and promoting the transformation of macrophages to the M2 phenotype, the two key problems of abnormal HSF activation and persistent inflammation in HS are solved, which is the best treatment strategy for HS.

[0010] Exosomes (EVs) have become a highly potential platform for the delivery of lipophilic drugs due to their lipid membrane structure, low immunogenicity and high biocompatibility. Therefore, EVs are included in the composition of the present invention. It is worth noting that similar membrane proteins and lipids give EVs natural homologous targeting capabilities, which can enhance their efficiency of cellular uptake. Therefore, it is preferred to introduce HSFs-derived EVs into the composition 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 (e.g., concentration: 1 mg / mL to 5 mg / mL);

[0016] Take curcumin ethanol solution, exosome solution (exosome concentration, e.g., 0.5 mg / mL to 0.6 mg / mL) and DMEM medium and mix them evenly (e.g., the volume ratio of curcumin ethanol solution to exosome solution is 1:1±0.5, and the volume ratio of curcumin ethanol solution to DMEM medium is 1:8±4). Use an ultrasonic machine to treat the mixed solution with ultrasound (e.g., power 120 W, ultrasound time 15 s, repeated 3 times), followed by ice bath (e.g., 5 min), and repeat this 3 times; after completion, incubate the mixed solution at 37℃±1℃ (e.g., 1 h) to restore the stability of the EV membrane; then, centrifuge the mixed solution for the first time (e.g., 10,000 g, 30 min), followed by a second centrifugation (e.g., 100,000 g, 90 min) to separate Cur@EV. Finally, use DMEM medium / PBS solution to resuspend Cur@EV 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 reportedly retains the natural bioactive factors of parental cells and minimizes 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 seriously hinders the transdermal delivery of drugs, biomacromolecules and vesicles (such as exosomes). The present invention also uses microneedles (MNs) as delivery carriers, which has obvious advantages in achieving transdermal delivery of the above components.

[0020] The present invention specifically designs a functionalized microneedle treatment 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 200 μm, especially greater than 400 μm, such as 450 μm ± 50 μm, 550 μm ± 50 μm, 650 μm ± 50 μm, 750 μm ± 50 μm, 850 μm ± 50 μm and 850 μm ± 50 μm. The curcumin content in the microneedle patches is greater than 1 μg, especially greater than 3 μg, such as 3 μg to 4 μg, and the Cur@EV content is greater than 3 μg, especially greater than 6 μg, such as 6 μg to 8 μg.

[0021] The Cur@EV provided by the present invention, as an active ingredient carried in the microneedle, has the effect of promoting the regenerative repair of hypertrophic scars, and is supplemented with UdECM to facilitate immune regulation and synergistic inhibition of fibrosis. UdECM is greater than 10μg, especially greater than 30μg, for example: 30μg to 50μg.

[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 to facilitate the repair of hypertrophic scars.

[0023] The microneedle of the present invention is 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 HP-β-CD solution with a concentration of 5mM to 10mM 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, and then poured into a PDMS microneedle mold, 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 (Cur@EV) loaded with curcumin provided by the present invention exhibited a high affinity for HSFs, delivered curcumin to HSFs more efficiently, and exhibited anti-HSFS cell proliferation activity and significant anti-fibrosis effect, which can be used as an active substance 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] The HA / HP-β-CD composite MNs of the present invention meet the mechanical requirements for effective skin penetration and achieve transdermal drug delivery in a highly efficient manner, as verified by pig skin tissue and rabbit ear HS lesion models. Cur@EV / UdECM MNs play a positive and significant role in regulating scar status and promoting scar improvement. It can be clearly seen that scar thickness is significantly reduced, collagen deposition is also greatly reduced, and the arrangement of collagen fibers becomes more orderly, which is more similar to normal skin tissue in morphology and structure.

[0027] Since multiple key biological pathways such as inflammation, autophagy and apoptosis are regulated simultaneously, the Cur@EV / UdECM MNs provided by the present invention effectively inhibit the formation of scars and show excellent therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Figure 2 is the preparation and characterization diagram of curcumin engineered exosomes (Cur@EV); A is a schematic diagram of the separation 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, which shows the particle size distribution of EV and Cur@EV; E is a high-performance liquid chromatography (HPLC) result, which confirms that curcumin is successfully loaded into EV; F is the loading efficiency of Cur@EV at various concentrations of curcumin, and G is a fluorescent image of cellular uptake in human skin fibroblasts (HSFs) and macrophages (Raw) samples (nuclei are stained blue, cytoskeleton is stained green, and EV and Cur@EV labeled with PKH26 are marked red);

[0029] Figure 2The results of the in vitro anti-fibrotic effect of Cur@EV on human skin fibroblasts (HSFs); A is the 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 and Ki67-positive cells are red); C is the Transwell experiment to evaluate the migration ability of HSFs after treatment in each group (cells are stained purple); D is the statistical graph of transforming growth factor-β (TGF-β) expression levels in each experimental group; E is the statistical graph of α-smooth muscle actin (α-SMA) expression levels in each experimental group; F is the statistical graph of type I collagen (Col I) expression levels 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 (nucleus stained blue, cytoskeleton stained green, α-SMA stained red); I is a schematic diagram of the inhibitory effect of Cur@EV on fibrosis;

[0030] Figure 3 The results of the verification of the in vitro inflammation and fibrosis inhibition effects of umbilical cord mesenchymal stem cell-derived decellularized extracellular matrix (UdECM); A is a staining diagram 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 diagram of the DNA, glycosaminoglycan (sGAG) and collagen content between the hUCMSCs group and the UdECM group; C is a statistical diagram of the macrophages (Raw) after treatment with UdECM solutions of various concentrations. ARG-1 staining in samples; D is a heat map of the expression levels of inflammation-related biomarkers in 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 of α-SMA in HSFs after treatment (nuclei are stained blue, cytoskeleton is stained green, and α-SMA is stained red); G is a statistical chart 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-fibrosis effects of UdECM;

[0031] Figure 4The preparation and characterization results of Cur@EV / UdECM functionalized microneedle patch; A is a schematic diagram of the preparation process of microneedle patch; B is a bright field photo 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, which confirms the uniform distribution of Cur@EV in a single microneedle, and Cur@exosomes are 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 The figure shows 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 photo of hypertrophic scar before and after treatment in each group, 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 with Normall skin group, # indicates statistical difference with Control group, @ indicates statistical difference with Cur@EV / UdECM MNs group); E is an immunohistochemical staining of type I collagen (Col I) and type III collagen (ColIII) 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 figure shows the verification results of Cur@EV / UdECM functionalized microneedle patch promoting regeneration and repair of 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 evaluation 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 with Nornaml skin group, # indicates statistical difference with Control group, @ indicates statistical difference with 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 only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is 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 can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0035] The sources of the main materials used in the following embodiments 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, 200 μm × 200 μm × 600 μm (length × width × height) MN needle tip.

[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, and 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 in an ultracentrifuge for 30 min. The supernatant was then transferred to a new ultracentrifuge tube and continued to be centrifuged at 100,000 g in an ultracentrifuge for 90 min. The supernatant was discarded, and finally 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 solutions, 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 with an ultrasonic machine, the ultrasonic power was set to 120 W, the ultrasonic time was 15 s, repeated 3 times, and then ice bathed for 5 min, and the above operation was cycled 3 times; after the ultrasonic treatment, the mixed solution was incubated at 37°C for 1 h to restore the stability of the EV membrane; then, the sample was centrifuged at 10000g for 30 min and 100000g for 90 min to separate the curcumin-loaded exosomes; finally, the EV was resuspended by blowing with DMEM culture medium / PBS solution, and stored in aliquots.

[0046] 3) Identification of curcumin-loaded exosomes

[0047] In order to explore 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 Western Blot (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 by pipetting, add the exosome solution to the dye solution and mix quickly by pipetting, 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, and use an ultracentrifuge to separate the labeled exosomes. Next, HSFs and Raw cells were inoculated in 24-well plates for overnight culture, and fluorescently labeled EVs and Cur@EVs were added to the well plates for co-culture with the cells. After 24 hours of culture, the culture medium was discarded, the cells were washed with PBS solution, 4% PFA solution was added to fix the cells for 30 minutes, the cell membrane was permeabilized with immunostaining permeabilization solution for 10 minutes, and QuickBlock was used. 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 experiment

[0051] HSFs cells were inoculated in a 96-well plate for overnight culture, and different concentrations of Cur@EV solution were added to the plate. The culture medium was replaced every 2 days. After 1, 3, and 5 days of culture, the original culture medium was removed, and 100 μL of freshly prepared CCK-8 working solution was added to each well, and incubated in a 37°C incubator in the dark for 1 hour. After reaching the time point, the OD value of each well was measured at a wavelength of 450 nm using an enzyme reader.

[0052] 6) Ki-67 immunofluorescence staining

[0053] HSFs cells were inoculated in a 24-well plate for overnight culture. Cur@EV, EV and Cur solution were added to the plate. 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 the Transwell chamber. The specific steps are as follows: HSFs cells were evenly resuspended in Cur@EV, EV, Cur and serum-free DMEM culture medium, and the above cell suspensions were evenly inoculated in the upper chamber of the Transwell chamber. 600 μL of DMEM complete culture 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 stain for 20 minutes, and then the excess stain was washed with PBS. 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) Expression of cell fibrosis-related genes

[0057] HSFs cells were inoculated in a 6-well plate for overnight culture. Cur@EV, EV and Cur solutions were added to the wells respectively. After 72 h 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 inoculated in a 24-well plate for overnight culture. Cur@EV, EV and Cur solutions were added to the wells respectively. After 72 h of culture, the cells were fixed with 4% PFA solution for 30 min. 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 cells were cultured in an incubator at 37°C and 5% CO2. When the cell confluence reached about 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 3 times with PBS solution. 1% Triton X-100 solution was added to the culture dish, incubated at room temperature for 30 minutes, the solution was removed, and then washed 3 times with PBS solution. Then, ammonium hydroxide (NH4OH) solution was added and incubated at room temperature for 30 minutes. The culture dish was frozen overnight at -20°C, the ammonium hydroxide solution was removed, and washed with PBS solution. Finally, the sample was freeze-dried overnight in a freeze dryer and stored in a -80°C refrigerator.

[0062] 11) UdECM identification - immunofluorescence staining

[0063] After decellularization, 4% PFA solution was added for fixation for 30 min, and the cell membrane was permeabilized with immunostaining permeabilization solution for 10 min. TM The cells were blocked with immunostaining blocking solution for 15 min, and diluted Anti-Collagen I primary antibody solution was added. The cells were incubated at 4°C overnight in the dark. The primary antibody solution was recovered, and the cells were washed three times with PBS solution. The corresponding secondary antibody solution was added and the cells were incubated at 37°C for 1 h. After washing three times with PBS solution, diluted DAPI solution was added for nucleus staining. The cells were incubated in the dark for 5 min. Finally, the cells were observed and photographed 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, and then centrifuged at 10000 g for 10 min. 10 μL of Qubit TM Standards, 10 μL samples, 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 solution to prepare pepsin extract. The pepsin extract was added to the decellularized extracellular matrix and non-decellularized cell samples at a ratio of 1:20 between tissue volume and extract, and digested overnight at 4°C on a shaker. After digestion, the sample was centrifuged at 3000g for 10 minutes, 100μL of supernatant was aspirated, 1mL of Sircol dye reagent was added to the supernatant, inverted and mixed, and shaken at room temperature for 30 minutes, then centrifuged at 13000g for 10 minutes, the supernatant was aspirated, 750μL of Acid-salt was added to wash the precipitate, and then centrifuged at 12000g for 10 minutes, the supernatant was aspirated, 250μL of Alkali reagent was added to re-dissolve the bottom precipitate, and shaken at room temperature for 5 minutes. Finally, the OD value of each well was measured at a wavelength of 555nm using an ELISA reader, and the collagen content in the sample was calculated according to the standard curve.

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

[0069] Papain was dissolved in the extract to prepare papain extract, 1 mL of the extract was added to 50 mg of the decellularized extracellular matrix and the cell sample without decellularization, and the sample was incubated in a water bath at 65°C for 3 h. Then the sample was centrifuged at 10000 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, and the sample was shaken at room temperature for 30 min, followed by centrifugation at 13000 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 13000 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 an enzyme reader, and the content of the sulfated aminoglycan in the sample was calculated according to the standard curve.

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

[0071] Raw cells were seeded in 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 plate. 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 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) Verify the anti-inflammatory effect of UdECM in vitro - expression of inflammation-related genes

[0073] Raw cells were inoculated in 6-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 plate, and the culture medium was replaced every 2 days. After 72 h of culture, 1 mL of TRIzol solution was added to each well to lyse the cells, and total cell RNA was extracted, cDNA reverse transcribed and qRT-PCR experiments were performed subsequently.

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

[0075] A co-culture model of Raw and HSFs cells was constructed using Transwell chambers. 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 performed as follows.

[0076] Immunofluorescence staining: After 72 h of culture, the 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 cell 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 5mM and 10mM 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 (5mM) and HA / HP-β-CD (10mM) solutions, respectively. Cur@EV and UdECM were added to the above composite solution, 150μL of the mixed solution was drawn and added to the PDMS microneedle mold, the microneedle mold was placed in a vacuum drying oven for vacuuming to remove bubbles in the solution, and the mold was placed in a 37°C oven for drying for 2h. Subsequently, 150μL of the same solution was added to the mold, and the mold was continued to be 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 observed and characterized by SEM. The microneedles were placed under a stereo microscope to observe their overall morphology and microneedle array. The microneedles were gold-sprayed using a coating instrument, and the microneedles were attached to the sample stage with conductive gel for SEM scanning to characterize the structure and integrity of the microneedles.

[0082] 20) Characterization of microneedle mechanical properties

[0083] The microneedle sample was fixed in the center of the mechanical testing table, 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] First, fluorescently labeled Cur@EVs were prepared, and then added to the microneedles during preparation. After the microneedles were dried and de-molded, the distribution of Cur@EVs in the microneedles was observed under a confocal microscope.

[0086] 22) Fourier 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 spectrum 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 the composite microneedle and remove the membrane. The ex vivo pig skin was fixed on the laboratory table and the prepared composite microneedle was vertically pressed into the pig skin sample. After 30 minutes, the microneedle base was removed and the pig skin sample was observed and photographed under a stereo microscope.

[0090] 24) Microneedle transdermal puncture experiment on hypertrophic scars in rabbit ears

[0091] The rabbit ear scar sample was fixed on the laboratory table, and the prepared composite microneedle was 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. Then, the fixed sample was dehydrated, paraffin-embedded, sliced, and H&E stained. Finally, the sample was sealed with neutral gum and observed under a microscope.

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

[0093] One month after the rabbit ear hypertrophic scar model was established, microneedle intervention treatment was performed on it. The experimental group was divided into 6 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), and normal rabbit ear skin was used as a blank group (Normal skin). The frequency of microneedle treatment was once a week for 3 consecutive weeks. The scar area of ​​the rabbit ear was cleaned with alcohol cotton balls, and the macroscopic morphology of the scar was recorded with a camera. The prepared composite microneedle was vertically pressed into the scar tissue, and the microneedle base was removed after 30 minutes. The above operation was repeated one week later, and a total of three treatments were performed. After the treatment, the local scar tissue was excised, one part was stored in 4% PFA solution, and the other part was quickly frozen in liquid nitrogen, followed by 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 analyses, and 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-test was used for both groups. Statistical analysis and graphics were drawn using GraphPad Prism 8.0. Image J software (NIH, Bethesda, MD, USA) was used for image analysis and statistics. *, #, and @ all 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), effectively loading curcumin into EVs and identifying them. First, surface specific proteins of EVs and curcumin-loaded EVs (Cur@EVs) were identified by immunoblotting (WB). Figure 1As shown in B, the presence of EV-specific proteins Alix and TSG101 were detected in both EV and Cur@EV groups. At the same time, Calnexin was used as a negative control. Calnexin is not a specific surface protein of EV and is not detected under normal circumstances. The experimental results were as expected. No Calnexin bands were detected in the EV and Cur@EV groups, proving the successful extraction and preparation of EV and Cur@EV.

[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 from the TEM image of C that after loading with curcumin, the EV nanovesicle structure was well preserved, showing a typical round or oval outline with clear boundaries. At the same time, the average particle size of Cur@EV was 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 are distributed in a relatively concentrated 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 ability of each component in a complex mixture. First, pure curcumin samples were subjected to HPLC analysis, and their characteristics such as 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 times 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 intuitively shows that the loading efficiency of Cur@EV for curcumin changes with the change of the initial concentration of curcumin. The experimental results show that the loading efficiency is the highest when the curcumin is 400μg. Therefore, Cur@EV with a loading efficiency of about 9% was selected for subsequent experiments.

[0102] The cellular uptake in HSFs and RAW cells was characterized to evaluate whether the prepared Cur@EV had the homologous targeting ability to HSFs. Figure 1As shown in G, the nuclei and cytoskeleton of the two cells were stained blue and green, respectively, and Cur@EV and EV were marked in red, making it possible to clearly and intuitively track the internalization of vesicles in the cells. Cur@EV showed a high affinity for HSFs, and a large number of Cur@EVs were observed to successfully enter the cells. However, no obvious Cur@EV was observed in RAW cells. These results indicate that Cur@EV can accurately recognize and bind to HSFs as an efficient targeted delivery platform with its unique properties, 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 biological effect of Cur@EV was evaluated and the experimental concentration was optimized by detecting the viability and proliferation of HSFs cells under different Cur@EV concentration gradients. Figure 2 As shown in A, when Cur@EV is in the dilution concentration range of 1 / 4 to 1 / 16, it can significantly inhibit the proliferation of HSFs. As the dilution concentration decreases, the inhibitory effect weakens to a certain extent. At the same time, when the Cur@EV concentration is 1 / 16, HSFs cells still have good cell viability in the early stage, so this dilution concentration is selected as the optimal concentration for subsequent experiments. It not only ensures that the proliferation of HSFs has sufficient inhibitory effect, but also takes into account its feasibility and safety in experimental operations.

[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 B, it can be clearly observed that the number of Ki67-positive cells (dyed red) in the Cur@EV group was significantly reduced compared with other groups. This result is consistent with the conclusions obtained from the cell proliferation experiment, further confirming that Cur@EV has satisfactory anti-HSFS cell proliferation activity. At the same time, neither the pure EV nor the curcumin group showed obvious anti-proliferation 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 group alone showed a certain degree of inhibitory effect on migration. At the same time, the migration ability of HSFs treated with Cur@EV was significantly inhibited, and the number of cell migration was significantly reduced. This shows that Cur@EV effectively delivers curcumin to the interior of cells and exerts a good anti-fibrosis effect.

[0107] The expression of fibrotic 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, in the Cur@EV treatment group, the expression levels of fibrosis markers such as TGF-β, α-SMA, Col I and Col III were significantly reduced. In contrast, in the Cur group and EV group alone, the expression of the above fibrosis markers did not decrease. These results indicate that EV and drug treatment alone cannot effectively inhibit the fibrosis level of HSFs. At the same time, Cur@EV may also reduce the degree of 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, in cells treated with Cur@EV, the fluorescence density of α-SMA 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, the in vitro results showed that Cur@EV exerted anti-fibrotic effects by inhibiting HSFs cell proliferation and migration and reducing the expression of key fibrotic 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 example, a biomaterial with immunomodulatory properties, UdECM, was used to achieve this goal. It is crucial to evaluate the presence of cell residues in UdECM, because these residues may trigger cytotoxic reactions, immune rejection, and directly affect the final repair effect. Figure 3 As shown in A, the successful removal of the nuclear components was confirmed by DAPI staining, 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 retention of major active ingredients 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) The immunomodulatory ability of UdECM was further verified. Under the action of UdECM, the expression of anti-inflammatory factors (such as IL-4 and TGF-β) increased, while the expression of pro-inflammatory factors (IL-1 and iNOS) decreased accordingly. At the same time, based on the above experimental results, the 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 immunoregulatory 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 immunomodulatory role and promotes the polarization of macrophages to the 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 treatment, this example prepared a hyaluronic acid (HA)-based functionalized microneedle (MNs) patch ( 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) has a complete structure and a square shape, where 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 tetrahedral 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 evenly 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) is 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, when HA and HP-β-CD formed a complex, obvious spectral shifts appeared at 1556cm-1, 1611cm-1 and 1641cm-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 E, the introduction of HP-β-CD has a significant effect on the stress tolerance of MNs and significantly improves 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 microneedle successfully penetrated the stratum corneum of HS and could 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 B shows the appearance photos of HS before and after various treatments. After comparative analysis, it can be found that the treatment effects of the Scargel group and the Free MNs group were not significant. The boundaries of the rabbit ear HS after treatment in these two groups were blurred, and the improvement of the overall appearance and characteristics was not significant. However, the treatment effect of the Cur@EV / UdECM MNs group was completely different. The surface of the scar after treatment was relatively flat and the boundaries became very blurred. This series of intuitive changes fully demonstrated that Cur@EV / UdECMMNs played a positive and significant role in regulating the state of scars 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, which can intuitively reflect the 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, especially the Cur@EV / UdECM MNs group, showed a significant decrease in SEI compared with the control group. Previous studies have shown that multi-drug combination therapy can exert a synergistic effect, thereby improving the therapeutic effect. In this experiment, the Cur@EV / UdECM microneedle patch was based on this multi-drug synergistic effect, and by simultaneously regulating multiple key biological pathways such as inflammation, autophagy and apoptosis, it effectively inhibited the formation of scars and showed excellent therapeutic effects.

[0128] The abnormal activation of HSFs interacts with the persistent inflammatory response, forming the pathological microenvironment of HS, and ultimately leading to excessive ECM deposition 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 with the control group, the collagen deposition in the HS tissues of all treated groups decreased. In the Cur@EV / UdECM MNs group, this phenomenon of reduced collagen deposition was particularly prominent. This result intuitively shows that Cur@EV / UdECM MNs has a significant therapeutic effect in inhibiting the process of fibrosis and reducing excessive collagen deposition.

[0129] In addition, the study also found that there is an imbalance in the ratio of type I and type III collagen in HS tissue. 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 the treatment of 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 It can be observed in F that after treatment, the arrangement of collagen fibers in scar tissue has changed. The originally disordered arrangement of collagen fibers has improved, the degree of orderly arrangement has increased significantly, and the ratio of Col I / III has gradually returned to normal. Among all treatment groups, the Cur@EV / UdECM MNs group showed 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 of vital importance to restore 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 the regeneration of hair follicles. Based on this, we evaluated the hardness and inflammation level of different groups and normal skin tissues. The results showed that untreated HS tissues showed the highest scar hardness value ( Figure 6 B), and IL-1β levels were also high ( Figure 6 C). However, when Cur@EV / UdECM MNs were treated, 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, which are of great significance in the identification, identification 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). The above results reveal the potential mechanism of Cur@EV / UdECM MNs in promoting 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 important organ section staining and analysis were performed on all experimental animals. Figure 6 As shown in Figure F, there were no significant deviations in routine blood tests 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 the regeneration of hair follicles. This two-pronged strategy addresses both the fibrosis and immune disorders of HS.

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

Claims

1. Use of a composition in preparing a medical device for repairing hypertrophic scars, the composition comprising curcumin and exosomes, wherein the curcumin is contained in the exosomes.

2. The use according to claim 1, characterized in that The composition also includes a decellularized extracellular matrix.

3. The use according to claim 2, characterized in that The decellularized extracellular matrix is ​​the decellularized extracellular matrix of umbilical cord mesenchymal stem cells.

4. The use according to claim 1, characterized in that The method for preparing the composition comprises: Take the curcumin ethanol solution, exosome solution and DMEM culture medium and mix them evenly. Use an ultrasonic machine to treat the mixed solution, and then put it in an ice bath. Repeat this process three times. After completion, incubate the mixed solution to allow the EV membrane to recover stability. Subsequently, the mixed solution is centrifuged for the first time and then centrifuged for the second time to separate Cur@EV.

5. The use according to claim 1, characterized in that The medical device is a microneedle or a microneedle array patch.

6. A microneedle, characterized in that It includes curcumin, exosomes and decellularized extracellular matrix, wherein the curcumin is contained in the exosomes to form engineered exosomes Cur@EV.

7. The microneedle according to claim 6, characterized in that The microneedle is mainly made of hyaluronic acid and hydroxypropyl-β-cyclodextrin, and hyaluronic acid and hydroxypropyl-β-cyclodextrin have intermolecular hydrogen bonds.

8. The microneedle according to claim 7, characterized in that The mass ratio of the hyaluronic acid to the hydroxypropyl-β-cyclodextrin is greater than 5.

9. The microneedle according to claim 6, characterized in that It 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.

10. The microneedle according to claim 9, characterized in that The content of the decellularized extracellular matrix is ​​greater than 10 μg.

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