Rosa roxburghii tratt exosome microneedle patch for promoting skin wound healing and preparation method
By preparing microneedle patches containing prickly pear exosomes, and utilizing the microneedle array to penetrate the skin barrier and uniformly release the prickly pear exosomes, the problem of insufficient application of prickly pear exosomes was solved, promoting rapid healing of skin wounds.
Patent Information
- Application Number
- CN202610206648.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-12
- Publication Date
- 2026-03-24
AI Technical Summary
Existing technologies lack research on the efficacy of prickly pear exosomes, making it difficult to effectively apply them and failing to fully utilize their potential to promote skin wound healing.
A biodegradable microneedle patch was prepared by mixing prickly pear exosomes with silk fibroin and polyvinyl alcohol. The microneedle array was distributed on the surface of the patch substrate. The microneedles penetrated the skin barrier to construct a prickly pear exosome delivery channel and uniformly released the prickly pear exosomes to the skin wound site after the microneedles degraded.
It accelerated the migration of immortalized human keratinocytes, fibroblasts, and human umbilical vein endothelial cells, promoted the repair of epidermis, dermis, and subcutaneous tissues, and expanded the application scope of prickly pear exosomes in skin wound healing.
Smart Images
Figure CN121714508A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological materials, and particularly relates to a Rosa roxburghii exosome microneedle patch for promoting skin wound healing and a preparation method. BACKGROUND
[0002] The microneedle patch is a transdermal drug delivery device, and the shape of the base body is usually a circular or square patch. The microneedle array is distributed on the surface of the patch base body, and is used to penetrate the skin barrier, reach the epidermis, and build a delivery channel for drugs and active factors. Compared with the traditional injection or external preparation, the microneedle patch has the advantages of minimally invasive, painless, efficient delivery of drugs and active factors, and gradually becomes one of the research hotspots in the field of drug delivery and tissue repair.
[0003] According to different characteristics of the microneedles, the microneedle patch can be further divided into hollow microneedle patches, degradable microneedle patches and responsive microneedle patches. The degradable microneedle patch is mainly made of biodegradable polymer materials. After penetrating the skin, the active ingredients loaded will be gradually released with the degradation or swelling of the microneedle patch, and the corresponding therapeutic effect will be achieved. Silk fibroin (SF) is a natural high molecular protein derived from the silkworm, which has good biocompatibility, biodegradability and mechanical properties, and is widely used in tissue engineering and regenerative medicine. At the same time, by compounding with other functional materials such as polyvinyl alcohol (PVA), the formability and mechanical properties of silk fibroin can be further optimized. Its good film-forming property and structural adjustability make it one of the ideal materials for preparing microneedle patches.
[0004] The therapeutic effect of the degradable microneedle patch is closely related to the active ingredients loaded. Natural active ingredients refer to active ingredients extracted and separated from natural sources such as plants, animals, microorganisms and minerals, which have the advantages of mild therapeutic effect, good biocompatibility and low risk of side effects. Plant exosomes are a kind of membrane vesicles with a diameter of about 30-150 nm, which are secreted by cells and can be used as natural active ingredients. Rosa roxburghii is a high-nutrition fruit with medicinal and edible properties, which is rich in active substances such as vitamin C, polyphenols, flavonoids and triterpenoids, and has the effects of antioxidant, anti-inflammatory and immune regulation. After oral administration, it can reduce inflammation in the oral cavity and gastrointestinal tract. At present, Rosa roxburghii has been widely used, such as direct consumption, juice or preserved fruit, etc. However, the Rosa roxburghii exosomes extracted from Rosa roxburghii are still in the early stage of research and industrialization, and the efficacy lacks research, making it difficult to fully utilize the Rosa roxburghii exosomes. SUMMARY
[0005] Therefore, the application provides a Rosa roxburghii exosome microneedle patch for promoting skin wound healing and a preparation method, which solves the technical problem that the efficacy of the Rosa roxburghii exosomes lacks research and it is difficult to effectively use the Rosa roxburghii exosomes in the prior art.
[0006] The first aspect of this application provides a prickly pear exosome microneedle patch for promoting skin wound healing, comprising: an integrally formed patch substrate and a microneedle array, wherein the microneedle array is distributed on the surface of the patch substrate;
[0007] The patch substrate and the microneedle array are made of a biodegradable material doped with prickly pear exosomes.
[0008] Preferably, the microneedles in the microneedle array are arranged in one of the following array structures: square array, rectangular array, or circular array.
[0009] Preferably, in the square array structure arrangement, the number of microneedles arranged horizontally is 10 to 20, the number of microneedles arranged vertically is 10 to 20, and the number of microneedles arranged horizontally and vertically is the same.
[0010] Preferably, the microneedles in the microneedle array are evenly distributed with a spacing of 0.5~1.5mm.
[0011] Preferably, the microneedles in the microneedle array are in the shape of a regular square pyramid, with a base side length of 0.15~0.75mm, a height of 0.5~3mm, and a tip radius of 20~40μm.
[0012] Preferably, the microneedles in the microneedle array are conical in shape, with a base radius of 0.15~0.75mm, a height of 0.5~3mm, and a tip radius of 20~40μm.
[0013] Preferably, the shape of the patch substrate is one of a square, a rectangle, or a circle; wherein the side length of the square patch substrate is 30mm to 100mm, the length of the rectangular patch substrate is 30mm to 100mm, the width is 20mm to 80mm, and the width is less than the length, and the radius of the circular patch substrate is 15mm to 50mm.
[0014] Preferably, the biodegradable material doped with prickly pear exosomes comprises, by mass, silk fibroin, polyvinyl alcohol and prickly pear exosomes in a mass ratio of 80~90:10~20:0.2~2.
[0015] Preferably, the biodegradable material doped with prickly pear exosomes comprises, by mass, silk fibroin, polyvinyl alcohol and prickly pear exosomes in a mass ratio of 80~90:10~20:0.5~2.
[0016] Preferably, the biodegradable material doped with prickly pear exosomes comprises, by mass, silk fibroin, polyvinyl alcohol and prickly pear exosomes in a mass ratio of 80~90:10~20:0.8~1.2.
[0017] The second aspect of this application provides a method for preparing a prickly pear exosome microneedle patch that promotes skin wound healing, which can prepare the prickly pear exosome microneedle patch for promoting skin wound healing as described in the first aspect. The preparation method includes the following steps:
[0018] The steps for preparing prickly pear exosomes are as follows: prickly pear juice is subjected to differential ultracentrifugation combined with sucrose density gradient centrifugation to obtain prickly pear exosomes;
[0019] The steps for preparing silk fibroin are as follows: the silkworm cocoons are boiled to degummify, the degummed product is dissolved and purified to obtain a silk fibroin solution;
[0020] The steps for preparing microneedle patches are as follows: A microneedle patch precursor solution containing 80-90 parts by weight of silk fibroin, 10-20 parts by weight of polyvinyl alcohol, and 0.2-2 parts by weight of prickly pear exosomes is mixed evenly and then added into a microneedle patch mold and solidified to obtain a prickly pear exosome microneedle patch that promotes skin wound healing.
[0021] Preferably, in the step of preparing prickly pear exosomes, the differential ultracentrifugation combined with sucrose density gradient centrifugation method,
[0022] The differential ultracentrifugation process includes: sequentially centrifuging at 400-600g for 5-15 minutes at 4℃, centrifuging at 800-1200g for 5-15 minutes, centrifuging at 2500-3500g for 20-40 minutes, centrifuging at 5000-15000g for 40-80 minutes, and centrifuging at 100000-200000g for 80-100 minutes to obtain a mixture of prickly pear exosome precipitates;
[0023] The process of sucrose density gradient centrifugation includes: adding the PBS resuspension solution of the prickly pear exosome precipitate mixture to a centrifuge tube pre-filled with sucrose solutions of varying concentrations, centrifuging at 100,000-200,000 g for 80-120 minutes at 4°C, taking the solution between the 30 wt% sucrose solution layer and the 45 wt% sucrose solution layer in the sucrose concentration gradient solution to obtain the prickly pear exosome extract, and centrifuging to obtain the prickly pear exosome precipitate.
[0024] Preferably, the pre-filling process of the centrifuge tube containing the concentration gradient sucrose solution includes: adding sucrose solutions with mass concentrations of 60wt%, 45wt%, 30wt%, and 15wt% sequentially into the centrifuge tube in a volume ratio of 1:1:1:1 to obtain centrifuge tubes pre-filled with the concentration gradient sucrose solution.
[0025] Preferably, in the step of preparing silk fibroin, the boiling degumming process includes: adding silkworm cocoons to a sodium carbonate solution with a concentration of 0.01~0.05M and boiling degumming for 15~60 minutes to obtain the boiled degummed product, silk fibroin fiber bundles.
[0026] The dissolution process includes: adding the boiled degummed silk fibroin fiber bundles to a lithium bromide solution with a concentration of 5-15M, and dissolving them at 50-70℃ for 2-6 hours to obtain a crude silk fibroin extract solution.
[0027] The purification process includes: dialyzing the crude silk fibroin extract solution with ultrapure water using a dialysis bag with a molecular weight cutoff of 3000~4000 Da, and centrifuging to obtain the silk fibroin solution.
[0028] Preferably, in the step of preparing the microneedle patch, the curing process includes: adding the microneedle patch precursor solution into the microneedle patch mold, evacuating the vacuum, and then placing it in a ventilated drying oven at 25~37°C to evaporate moisture and cure the patch.
[0029] Compared with existing technologies, the prickly pear exosome microneedle patch for promoting skin wound healing provided in this application has at least the following beneficial effects:
[0030] This application provides a prickly pear exosome microneedle patch that promotes skin wound healing. This patch can penetrate the skin barrier and construct prickly pear exosome delivery channels. These channels are evenly distributed, allowing the prickly pear exosomes to be uniformly released to the skin wound site after the microneedle patch degrades. This accelerates the migration of cells such as immortalized human keratinocytes, fibroblasts, and human umbilical vein endothelium, thereby speeding up the repair and healing of skin tissues such as the epidermis, dermis, and subcutaneous tissue. This application demonstrates the efficacy of prickly pear exosomes in promoting the migration of cells such as immortalized human keratinocytes, fibroblasts, and human umbilical vein endothelium. Furthermore, this efficacy has been creatively applied to microneedle patches, resulting in a prickly pear exosome microneedle patch that promotes skin wound healing. This expands the application scope of prickly pear exosomes and fully utilizes their benefits. Attached Figure Description
[0031] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0032] Figure 1 This is a schematic diagram showing the process of preparing a prickly pear exosome microneedle patch for promoting skin wound healing, as provided in Example 1 of this application, after centrifugation in a sucrose solution with a concentration gradient of 15wt% to 60wt%.
[0033] Figure 2The particle size distribution diagram of the prickly pear exosomes prepared in the preparation method of the prickly pear exosome microneedle patch for promoting skin wound healing provided in Embodiment 1 of this application;
[0034] Figure 3 The Zeta potential distribution diagram of the prickly pear exosomes prepared in the method for preparing a skin wound healing microneedle patch for promoting skin wound healing provided in Embodiment 1 of this application;
[0035] Figure 4 The prickly pear exosome protein electrophoresis image is shown in the preparation method of the prickly pear exosome microneedle patch for promoting skin wound healing provided in Embodiment 1 of this application.
[0036] Figure 5 The prickly pear exosome lipid thin-layer chromatogram is shown in the preparation method of the prickly pear exosome microneedle patch for promoting skin wound healing provided in Example 1 of this application.
[0037] Figure 6 A schematic diagram of the first polydimethylsiloxane microneedle patch mold in the preparation method of a prickly pear exosome microneedle patch for promoting skin wound healing provided in Embodiment 1 of this application;
[0038] Figure 7 The image shows the microneedle patch-rhodamine under a fluorescence microscope, prepared in the method for preparing a prickly pear exosome microneedle patch to promote skin wound healing provided in Embodiment 1 of this application.
[0039] Figure 8 In the preparation method of the prickly pear exosome microneedle patch for promoting skin wound healing provided in Embodiment 1 of this application, the pressure-displacement curve of the prepared microneedle patch at -0.2% is shown.
[0040] Figure 9 In the preparation method of the prickly pear exosome microneedle patch for promoting skin wound healing provided in Example 1 of this application, the biodegradability test results of the prepared microneedle patch-0.2% are shown in the figure. (a) is a microscope image of the microneedle patch-0.2% when it is first inserted into the pig skin, and (b) is a microscope image of the microneedle patch-0.2% after pressing for 2 minutes.
[0041] Figure 10 The results of the test on the release uniformity of the prepared microneedle patch-trypan blue in the method for preparing a skin wound healing prickly pear exosome microneedle patch provided in Embodiment 1 of this application.
[0042] Figure 11 The image shows the experimental results of the prickly pear exosome microneedle patch for promoting skin wound healing provided in Example 1 of this application, which shows the preparation method of the prickly pear exosome for promoting the migration of human immortalized keratinocytes.
[0043] Figure 12 The image shows the experimental results of the prickly pear exosomes promoting fibroblast migration in the preparation method of the prickly pear exosome microneedle patch for promoting skin wound healing provided in Embodiment 1 of this application.
[0044] Figure 13 The figure shows the experimental results of the prickly pear exosomes promoting the migration of human umbilical vein endothelial cells in the preparation method of the prickly pear exosome microneedle patch for promoting skin wound healing provided in Embodiment 1 of this application. Detailed Implementation
[0045] This application provides a prickly pear exosome microneedle patch for promoting skin wound healing and its preparation method, which addresses the technical problem in the prior art of lacking research on the efficacy of prickly pear exosomes and making it difficult to effectively apply prickly pear exosomes.
[0046] The technical solutions of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0047] Example 1
[0048] This embodiment provides a method for preparing a prickly pear exosome microneedle patch that promotes skin wound healing, including the steps of preparing prickly pear exosomes, preparing silk fibroin, and preparing the microneedle patch.
[0049] The steps for preparing prickly pear exosomes include:
[0050] Take an appropriate amount of fresh prickly pear, remove the core, and juice it for 2 minutes (3 times) using a juicer. Let it stand at 4℃ for 15 minutes, and then filter it through 80~120 mesh gauze to obtain prickly pear juice.
[0051] The prickly pear juice was centrifuged at 500g for 10 minutes at 4°C to obtain supernatant A; supernatant A was centrifuged at 1000g for 10 minutes at 4°C to obtain supernatant B; supernatant B was centrifuged at 3000g for 30 minutes at 4°C to obtain supernatant C; supernatant C was centrifuged at 10000g for 60 minutes at 4°C to obtain supernatant D; supernatant D was centrifuged at 150000g for 90 minutes at 4°C to obtain a prickly pear exosome precipitate mixture; the prickly pear exosome precipitate mixture and PBS buffer were mixed and resuspended at a mass ratio of 1:75 to obtain a PBS resuspension solution of the prickly pear exosome precipitate mixture.
[0052] First, prepare sucrose-PBS solutions with mass fractions of 60%, 45%, 30%, and 15% by mass, and add them sequentially to centrifuge tubes at a volume ratio of 1:1:1:1 to obtain centrifuge tubes pre-filled with sucrose solutions of varying concentrations. Next, add the PBS resuspension of the prickly pear exosome precipitate mixture to the centrifuge tubes pre-filled with sucrose solutions of varying concentrations, with a volume ratio of 4:1 between the total volume of the sucrose solutions of varying concentrations and the volume of the added PBS resuspension of the prickly pear exosome precipitate mixture. Then, slowly place the centrifuge tubes into an ultracentrifuge and centrifuge at 150,000g for 90 minutes at 4°C. A schematic diagram of the centrifuged product is shown below. Figure 1 As shown,
[0053] Take the solution between the 30wt% sucrose solution layer and the 45wt% sucrose solution layer in the centrifuge tube, that is, between the second to third layer from the bottom. Figure 1 The solution in the square box was used to obtain a refined extract of prickly pear exosomes; then, it was centrifuged at 150,000g for 120 minutes at 4°C to obtain the prickly pear exosome precipitate.
[0054] The steps for preparing silk fibroin include:
[0055] Take an appropriate amount of silkworm cocoons, cut them into small pieces, rinse them with water, and let them air dry at room temperature to prepare as silkworm cocoon samples.
[0056] The silkworm cocoon sample was boiled in a 0.02M sodium carbonate solution (purchased from Sigma Aldrich) for 45 minutes to efficiently hydrolyze and remove sericin, resulting in a silk fibroin fiber bundle. The bundle was then thoroughly rinsed with ultrapure water to remove residual alkali and gum impurities, and dried overnight at room temperature for later use.
[0057] The dried silk fibroin fiber bundles were added to a 9.3M lithium bromide (LiBr) solution and dissolved in a 60°C water bath for 4 hours until completely dissolved to form a homogeneous crude silk fibroin extract solution.
[0058] The crude silk fibroin extract was transferred into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in 1 L of ultrapure water. The water was changed sequentially at 1 hour, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, and 40 hours after the start of dialysis to remove small molecule impurities such as lithium bromide. After 40 hours of dialysis, the silk fibroin solution was centrifuged to remove insoluble impurities, and a purified silk fibroin solution was obtained. The concentrated silk fibroin solution was controlled to have a mass fraction of 10% wt and stored at 4°C for later use.
[0059] The steps for preparing microneedle patches include:
[0060] Weigh the silk fibroin solution, polyvinyl alcohol, and prickly pear exosomes at a mass ratio of 850:15:0.2 and mix them evenly to obtain a microneedle patch precursor solution with a prickly pear exosome doping amount of 0.2%; at the same time, weigh the silk fibroin solution, polyvinyl alcohol, and prickly pear exosomes at a mass ratio of 850:15:1 and mix them evenly to obtain a microneedle patch precursor solution with a prickly pear exosome doping amount of 1%.
[0061] Prepare the first polydimethylsiloxane microneedle patch mold, such as Figure 6 As shown, the needle cavities in the mold are distributed in a square array; there are 15 needle cavities arranged horizontally and 15 needle cavities arranged vertically. The shape of the needle cavities is a regular square pyramid with a base side length of 0.5 mm, a height of 1 mm, a tip radius of 36 μm, and a spacing of 0.72 mm between the needle cavities.
[0062] A precursor solution of prickly pear exosomes with a doping concentration of 0.2% was added to the first polydimethylsiloxane microneedle patch mold. The mold was placed in a vacuum environment and vacuumed for 10 minutes (5 times) to ensure that the precursor solution fully entered the needle cavity. After vacuuming, the mold was placed in a ventilated drying oven and dried at room temperature (25-37°C) for 12-24 hours until the microneedle patch was completely cured and formed, avoiding damage to the microneedle patch structure caused by high-temperature drying. After drying, the cured microneedle patch was carefully removed from the mold to obtain a prickly pear exosome microneedle patch with a prickly pear exosome doping concentration of 0.2%, named microneedle patch-0.2%, and used as a sample for later use.
[0063] A precursor solution of prickly pear exosomes with a doping concentration of 0.2% and an appropriate amount of rhodamine dye were mixed and added to a first polydimethylsiloxane microneedle patch mold. The mold was placed in a vacuum environment and vacuumed for 10 minutes (5 times) to ensure that the precursor solution fully entered the needle cavity. After vacuuming, the mold was placed in a ventilated drying oven and dried at room temperature (25-37°C) for 12-24 hours until the microneedle patch was completely cured, avoiding damage to the microneedle patch structure caused by high-temperature drying. After drying, the cured microneedle patch was carefully removed from the mold to obtain a prickly pear exosome microneedle patch loaded with rhodamine dye, with a prickly pear exosome doping concentration of 0.2%, named Microneedle Patch-Rhodamine, and used as a sample for later use.
[0064] A precursor solution of prickly pear exosomes with a doping concentration of 0.2% and an appropriate amount of trypan blue dye were mixed and added to a first polydimethylsiloxane microneedle patch mold. The mold was placed in a vacuum environment and vacuumed for 10 minutes (5 times) to ensure that the precursor solution fully entered the needle cavity. After vacuuming, the mold was placed in a ventilated drying oven and dried at room temperature (25-37°C) for 12-24 hours until the microneedle patch was completely cured, avoiding damage to the microneedle patch structure caused by high-temperature drying. After drying, the cured microneedle patch was carefully removed from the mold to obtain a prickly pear exosome microneedle patch loaded with trypan blue dye, with a prickly pear exosome doping concentration of 0.2%, named microneedle patch-trypan blue, and used as a sample for later use.
[0065] A 1% prickly pear exosome doping precursor solution was added to a polydimethylsiloxane (PDMS) microneedle patch mold. The mold was placed in a vacuum environment and vacuumed for 10 minutes (5 times) to ensure that the precursor solution fully entered the microneedle cavity. After vacuuming, the mold was placed in a ventilated drying oven and dried at room temperature (25-37°C) for 12-24 hours until the microneedle patch was completely cured, avoiding damage to the microneedle patch structure caused by high-temperature drying. After drying, the cured microneedle patch was carefully removed from the microneedle patch mold to obtain a prickly pear exosome microneedle patch with a prickly pear exosome doping concentration of 1%, named Microneedle Patch-1%, and used as a sample for later use.
[0066] Prepare a second polydimethylsiloxane microneedle patch mold. The needle cavities in the mold are arranged in a square array. There are 15 needle cavities arranged horizontally and 15 needle cavities arranged vertically. The shape of the needle cavity is conical, with a base radius of 0.5 mm, a height of 0.8 mm, a tip radius of 36 μm, and a spacing of 0.72 mm between the needle cavities.
[0067] A 1% prickly pear exosome-doped microneedle patch precursor solution was added to a second polydimethylsiloxane microneedle patch mold. The mold was placed in a vacuum environment and vacuumed for 10 minutes (5 times) to ensure that the precursor solution fully entered the needle cavity. After vacuuming, the mold was placed in a ventilated drying oven and dried at room temperature (25-37°C) for 12-24 hours until the microneedle patch was completely cured, avoiding damage to the microneedle patch structure caused by high-temperature drying. After drying, the cured microneedle patch was carefully removed from the mold to obtain a prickly pear exosome microneedle patch with conical microneedles.
[0068] Experimental Example 1
[0069] This experimental example describes the preparation method of the prickly pear exosome microneedle patch for promoting skin wound healing provided in Example 1. The prickly pear exosomes prepared were analyzed for particle size, zeta potential, protein and lipid composition. The microneedle patch-rhodamine sample of the prepared prickly pear exosome microneedle patch sample was also tested.
[0070] The analysis results of particle size and zeta potential distribution of the prepared prickly pear exosomes are as follows: Figure 2 and Figure 3 As shown; from Figure 2 and Figure 3 The analysis results of the particle size and zeta potential distribution of the prickly pear exosomes show that the average particle size of the prickly pear exosomes is about 222.4 nm, the polydispersity index is 0.167, and the zeta potential is -6.03 mV. This indicates that the prepared prickly pear exosomes have small and uniform particle size and good dispersibility.
[0071] The results of protein and lipid component analysis are as follows: Figure 4 and Figure 5 As shown; from Figure 4 and Figure 5 The protein electrophoresis and lipid thin-layer chromatography images show that the prickly pear exosomes contain proteins of various molecular weights, mainly concentrated between 15-25 kDa and 35-45 kDa. The results of parallel samples S1 and S2 are similar, indicating the reliability of the protein electrophoresis analysis. Meanwhile, the lipid thin-layer chromatography shows multiple separation spots, indicating the presence of various lipid components. Proteins and lipids are the main components of skin. Furthermore, some proteins, such as superoxide dismutase-like enzymes, can reduce the expression of pro-inflammatory factors in macrophages and promote fibroblast proliferation and endothelial cell angiogenesis. Therefore, prickly pear exosomes containing various protein and lipid components have the potential for anti-inflammatory effects and promoting skin wound healing.
[0072] Images of rhodamine under a fluorescence microscope in the prickly pear exosome microneedle patch sample, showing the rhodamine content of the microneedle patch. Figure 7 As shown; from Figure 7 It can be seen that the red fluorescence brightness and signal intensity of the prickly pear exosome microneedle patch are relatively consistent, with no local differences of excessive or weak fluorescence. The use of rhodamine as the tracer component indicates that the prickly pear exosome microneedle patch prepared in this application can effectively disperse the loaded components into the prickly pear exosome microneedle patch.
[0073] Experiment Example 2
[0074] This experimental example tests the performance of the prickly pear exosome microneedle patch samples prepared in Example 1 (microneedle patch-0.2%) and microneedle patch-trypan blue to promote skin wound healing.
[0075] The test of the prickly pear exosome delivery capability of the sample microneedle patch included:
[0076] The ability to deliver prickly pear exosomes requires the microneedle patch to have certain mechanical properties to penetrate the skin barrier and construct a prickly pear exosome delivery channel. It also needs to be biodegradable to release the loaded prickly pear exosomes into the delivery channel. In addition, the release of prickly pear exosomes needs to be as uniform as possible to fully exert the activity of prickly pear exosomes. Therefore, this experimental example conducted mechanical property tests, biodegradability tests, and release uniformity tests.
[0077] Mechanical performance testing employed a texture analyzer for compression testing. The sample microneedle patch -0.2% was cut into circles approximately 0.5 cm in diameter for testing. During the test, a flat stainless steel probe (P / 2) was used, with a compression speed set to 0.5 mm / s, a trigger force of 0.05 N, and a maximum compression distance of 800 μm. The sample microneedle patch -0.2% was placed on the test platform with the needle tip facing upwards. After ensuring the patch's stability, the test began, and the pressure-displacement curve during compression was recorded. The fracture strength and structural integrity of the microneedles were analyzed based on the curves. A single needle withstanding a force of not less than 0.05 N was used as the acceptable standard for penetrating the stratum corneum. The results are as follows: Figure 8 As shown; from Figure 8 It can be seen that the microneedle patch provided in this application has good mechanical properties and meets the requirements for penetrating the skin barrier and constructing a prickly pear exosome delivery channel.
[0078] Degradability was assessed using ex vivo pig skin as a skin model. A 0.2% sample microneedle patch was gently pressed onto the pig skin surface with pressure to ensure complete microneedle insertion. After 2 minutes, the morphological changes of the microneedles were observed under a microscope, and the dissolution process was recorded. The results are as follows: Figure 9 As shown; from Figure 9 It can be seen that the microneedle patch provided in this application has good biodegradability, which helps to smoothly release the loaded prickly pear exosomes into the prickly pear exosome delivery channel.
[0079] Release uniformity testing was conducted using ex vivo porcine skin as a skin model. The porcine skin was cleaned, moistened with sterile PBS buffer, and laid flat on a support platform with the dermis facing upwards, ensuring a smooth, bubble-free surface. A microneedle patch-trypan blue was applied to the designated area of the porcine skin and pressed for 2 minutes. After pressing, the microneedle patch-trypan blue was carefully removed, and the formation of regular micropores on the porcine skin surface was immediately observed. The results are as follows: Figure 10 As shown; from Figure 10It can be seen that during the pressing process, the microneedle array of the microneedle patch-trypan blue degrades, causing the loaded trypan blue to be released and forming regular square microneedle array blue micropores on the pigskin surface. This indicates that the microneedle patch provided in this application can uniformly release the loaded prickly pear exosomes into the skin after piercing the skin barrier and undergoing degradation, so as to avoid the situation where the local concentration is too low and affects the efficacy, and to give full play to the activity of prickly pear exosomes.
[0080] Experimental Example 3
[0081] This experimental example tests the skin wound healing performance of the prickly pear exosome microneedle patch samples prepared in the preparation method of the skin wound healing method provided in Example 1, specifically the microneedle patch-0.2% and microneedle patch-1%.
[0082] The skin, from the outside in, comprises the epidermis, dermis, and subcutaneous tissue. When the epidermis is damaged, keratinocytes at the wound edge need to migrate to the defect area to initiate the coverage and reconstruction of the epidermal layer. Accelerated migration of human immortalized keratinocytes is a core indicator of the initiation and advancement of epidermal repair. Fibroblasts dominate dermal cells, and their migration to the wound is a key step in dermal repair. Fibroblasts that migrate to the damaged area proliferate and secrete a large amount of extracellular matrix, forming granulation tissue to fill the dermal defect. In addition, human umbilical vein endothelial cells are vascular endothelial cells, and their migration is a core link in angiogenesis. Accelerated migration of human umbilical vein endothelial cells means accelerated angiogenesis, providing oxygen and nutrients to epidermal and dermal repair cells such as keratinocytes and fibroblasts, thus accelerating skin repair. Therefore, this application conducted migration experiments on human immortalized keratinocytes, fibroblasts, and human umbilical vein endothelial cells.
[0083] The process of the human immortalized keratinocyte migration experiment includes:
[0084] Human immortalized keratinocytes were seeded in 6-well plates. Once the cell confluence reached over 90%, a straight line was vertically drawn in the center of the cell monolayer using a sterile 200 μL pipette tip to create a "scratch." The cells were then gently washed twice with PBS to remove detached cells. The cells were divided into a blank control group, experimental group 1, and experimental group 2. The blank control group was cultured in DMEM medium. Experimental group 1 was cultured in DMEM medium mixed with 0.2 wt% *Rhizopus pilosa* exosomes, and experimental group 2 was cultured in DMEM medium mixed with 1 wt% *Rhizopus pilosa* exosomes. Images of the scratched area were captured using an inverted microscope at 0 and 24 hours to assess the effect of *Rhizopus pilosa* exosomes on the migration of human immortalized keratinocytes. The results are as follows: Figure 11 As shown; from Figure 11It can be seen that, compared with the blank control group, the addition of 0.2wt% or 1wt% of prickly pear exosomes to experimental groups 1 and 2 accelerated the migration speed of human immortalized keratinocytes. The addition of 1wt% prickly pear exosomes to experimental group 2 significantly accelerated the migration speed of human immortalized keratinocytes, so 1wt% was selected for subsequent experiments.
[0085] The process of the fibroblast migration experiment includes:
[0086] Fibroblasts were seeded in 6-well plates. Once the cell confluence reached over 90%, a straight line was vertically drawn in the center of the cell monolayer using a sterile 200 μL pipette tip to create a "scratch mark." The cells were then gently washed twice with PBS to remove detached cells. The cells were divided into a blank control group and experimental group 1. The blank control group was cultured in DMEM medium, while experimental group 1 was cultured in DMEM medium mixed with 1 wt% prickly pear exosomes. Images of the scratch mark area were taken using an inverted microscope at 0 and 24 hours to assess the effect of prickly pear exosomes on fibroblast migration. The results are as follows: Figure 12 As shown; from Figure 12 It can be seen that, compared with the blank control group, the addition of 1 wt% prickly pear exosomes to experimental group 1 accelerated the migration of fibroblasts.
[0087] The process of the human umbilical vein endothelial cell migration experiment includes:
[0088] Human umbilical vein endothelial cells (HUVECs) were seeded into 6-well plates. Once the cell confluence reached over 90%, a straight line was vertically drawn in the center of the cell monolayer using a sterile 200 μL pipette tip to create a "scratch." The cells were then gently washed twice with PBS to remove detached cells. The cells were divided into a blank control group and experimental group 1. The blank control group was cultured in DMEM medium, while experimental group 1 was cultured in DMEM medium mixed with 1 wt% *Rhizopus pilosa* exosomes. Images of the scratched area were taken using an inverted microscope at 0 and 24 hours to assess the effect of *Rhizopus pilosa* exosomes on the migration of HUVECs. The results are shown below. Figure 13 As shown; from Figure 13 It can be seen that, compared with the blank control group, the addition of 1 wt% prickly pear exosomes to experimental group 1 accelerated the migration of human umbilical vein endothelial cells.
[0089] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.
Claims
1. A prickly pear exosome microneedle patch for promoting skin wound healing, characterized in that, include: The patch substrate and microneedle array are integrally formed, and the microneedle array is distributed on the surface of the patch substrate; The patch substrate and the microneedle array are made of a biodegradable material doped with prickly pear exosomes.
2. The prickly pear exosome microneedle patch for promoting skin wound healing according to claim 1, characterized in that, The microneedles in the microneedle array are arranged in a square array structure, a rectangular array structure, or a circular array structure.
3. The prickly pear exosome microneedle patch for promoting skin wound healing according to claim 2, characterized in that, In the square array structure arrangement, the number of microneedles arranged horizontally is 10 to 20, and the number of microneedles arranged vertically is 10 to 20, with the number of microneedles arranged horizontally and vertically being the same.
4. The prickly pear exosome microneedle patch for promoting skin wound healing according to claim 1, characterized in that, The microneedles in the microneedle array are in the shape of a regular square pyramid, with a base side length of 0.15~0.75mm, a height of 0.5~3mm, and a tip radius of 20~40μm.
5. The prickly pear exosome microneedle patch for promoting skin wound healing according to claim 1, characterized in that, The microneedles in the microneedle array are conical in shape, with a base radius of 0.15~0.75mm, a height of 0.5~3mm, and a tip radius of 20~40μm.
6. The prickly pear exosome microneedle patch for promoting skin wound healing according to claim 1, characterized in that, The biodegradable material doped with prickly pear exosomes comprises, by mass ratio, silk fibroin, polyvinyl alcohol and prickly pear exosomes in the following proportions: 80-90:10-20:0.2-2.
7. A method for preparing a prickly pear exosome microneedle patch that promotes skin wound healing, characterized in that, A prickly pear exosome microneedle patch for promoting skin wound healing, as described in any one of claims 1-6, can be prepared by the following steps: Prickly pear exosomes were obtained by differential ultracentrifugation combined with sucrose density gradient centrifugation of prickly pear juice. Silkworm cocoons were boiled to degummify them, and the degummed products were dissolved and purified to obtain a silk fibroin solution. A microneedle patch precursor solution containing 80-90 parts by weight of silk fibroin, 10-20 parts by weight of polyvinyl alcohol, and 0.2-2 parts by weight of prickly pear exosomes is mixed evenly and then added to a microneedle patch mold and solidified to obtain a prickly pear exosome microneedle patch that promotes skin wound healing.
8. The method for preparing a prickly pear exosome microneedle patch for promoting skin wound healing according to claim 7, characterized in that, In the differential ultracentrifugation combined with sucrose density gradient centrifugation method The differential ultracentrifugation process includes: sequentially centrifuging at 400-600g for 5-15 minutes at 4℃, centrifuging at 800-1200g for 5-15 minutes, centrifuging at 2500-3500g for 20-40 minutes, centrifuging at 5000-15000g for 40-80 minutes, and centrifuging at 100000-200000g for 80-100 minutes to obtain a mixture of prickly pear exosome precipitates; The process of sucrose density gradient centrifugation includes: adding the PBS resuspension solution of the prickly pear exosome precipitate mixture to a centrifuge tube pre-filled with sucrose solutions of varying concentrations, centrifuging at 100,000-200,000 g for 80-120 minutes at 4°C, taking the solution between the 30 wt% sucrose solution layer and the 45 wt% sucrose solution layer in the sucrose concentration gradient solution to obtain the prickly pear exosome extract, and centrifuging to obtain the prickly pear exosome precipitate.
9. The method for preparing a prickly pear exosome microneedle patch for promoting skin wound healing according to claim 7, characterized in that, The boiling degumming process includes: adding silkworm cocoons to a sodium carbonate solution with a concentration of 0.01~0.05M and boiling degumming for 15~60 minutes to obtain the boiled degummed product, silk fibroin fiber bundles. The dissolution process includes: adding the boiled degummed silk fibroin fiber bundles to a lithium bromide solution with a concentration of 5-15M, and dissolving them at 50-70℃ for 2-6 hours to obtain a crude silk fibroin extract solution. The purification process includes: dialyzing the crude silk fibroin extract solution with ultrapure water using a dialysis bag with a molecular weight cutoff of 3000~4000 Da, and centrifuging to obtain the silk fibroin solution.
10. The method for preparing a prickly pear exosome microneedle patch for promoting skin wound healing according to claim 7, characterized in that, The curing process includes: adding the microneedle patch precursor solution into the microneedle patch mold, vacuuming it, and then placing it in a ventilated drying oven at 25~37℃ to evaporate moisture and cure the patch.