Sulfur ketal bond modified 17-type collagen dopamine drug-loaded nanoparticle composite microneedle patch as well as preparation method and application thereof

Abstract

The invention relates to the field of biological materials, in particular to a thioketal bond modified 17-type collagen dopamine drug-loaded nanoparticle composite microneedle patch and a preparation method and application thereof, and the microneedle patch is composed of dopamine drug-loaded nanoparticles, thioketal bond modified recombinant 17-type collagen and a polymer substrate. The thioketal bond modified 17-type collagen and polyvinyl alcohol are subjected to physical and chemical double-crosslinking to form hydrogel, then the hydrogel is loaded with drug-loaded nanoparticles, and the hydrogel and a polymer substrate form the ROS and pH value microenvironment double-response collagen microneedle patch loaded with bioactive substances. When the collagen hydrogel is applied to the skin, the tip of the microneedle reaches the area where the hair follicle stem cells exist in a painless and minimally invasive mode, and due to the change of microenvironment molecules in the skin tissue, the breakage of thioketal bonds in the collagen hydrogel can be promoted, so that active oxygen is removed, and the wrapped IGF-1 is released into the hair follicle stem cells; and a potential treatment strategy is provided for androgenetic alopecia.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and in particular to a thioketal bond-modified type 17 collagen dopamine drug-loaded nanoparticle composite microneedle patch, and a preparation method and application thereof. Background Art

[0002] Hydrogel is a hydrophilic three-dimensional network structure gel that can swell rapidly in water and keep a large amount of water insoluble. It is highly flexible and absorbent. The preparation of hydrogel microneedles mainly focuses on its application as a transdermal drug delivery technology. Hydrogel microneedles can be manufactured quickly and at low cost by using PDMS molds. Microneedle treatment is favored by more and more people because of its characteristics of less trauma, fast recovery, and non-surgical. Compared with drug treatments that may cause allergies or adverse reactions, long-term and multiple laser treatments, and hair transplantation surgery with infection risks, microneedle treatments are favored by more and more people. The therapeutic drugs or nutrients introduced by microneedles can penetrate deeper into the skin, thereby improving skin conditions, such as fading acne marks, reducing wrinkles, and improving skin firmness.

[0003] Low-temperature photothermal therapy (PTT) is a minimally invasive, long-term and safe treatment strategy. PTT can generate mild heat energy through laser irradiation, destroy cancer cells while avoiding damage to healthy tissues, and achieve low-temperature thermal therapy at the tumor site. In addition, PTT can have a positive effect on angiogenesis by promoting local blood circulation and improving tissue oxygen supply. In terms of hair follicle growth, the photothermal effect can promote local blood circulation, increase scalp tissue oxygenation, improve cell activity and metabolism, stimulate hair growth, and provide new ideas for the treatment of hair loss.

[0004] Hair loss is characterized by the gradual miniaturization of hair follicles and a shortened growth period, resulting in a significant reduction in scalp hair. Treatments include medications (such as minoxidil and finasteride, which require long-term use and the effects vary from person to person), lasers, and hair transplant surgery (the effects are affected by the doctor's skills). Choosing the right treatment method, combined with the patient's condition, can help improve the problem of hair loss.

[0005] Hair loss affects the patient's physical and mental health, so early diagnosis and treatment are crucial. Microneedle patch treatments include minoxidil and hair growth factor patches. Minoxidil patches may cause skin irritation and are not effective for acute hair loss; hair growth factor patches are unstable and require high storage and use. Other treatments, such as laser hair growth devices, have limited indications and are expensive, and PRP treatment cycles are long and there is no guarantee of effectiveness. Summary of the invention

[0006] The purpose of the present invention is to provide a dual-responsive collagen microneedle patch of ROS and pH loaded with bioactive substances, and a preparation method and application thereof, and to develop a degradable COL17 microneedle patch by combining the bioactive substances with modified COL17, which can deliver the bioactive substances to the hair follicle stem cells in the androgenic alopecia area. The microneedle patch prepared by the present invention has a significant effect in promoting hair regeneration and has a wide range of application prospects in the treatment field of inducing hair follicle regeneration.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The microneedle patch provided by the present invention is composed of ROS- and pH-responsive modified COL17 encapsulating drug-loaded nanoparticles I@M-COL17 and a polymer matrix.

[0009] The polymer substrate is composed of PVA with good biocompatibility, and the preparation method thereof is as follows: PVA is dissolved in deionized water (4 g PVA is added to 20 ml deionized water) to prepare a 20% PVA solution, which is stored at room temperature for later use.

[0010] ROS and pH dual-responsive COL17 (thioketal-modified type 17 collagen COL17-PTK) is prepared by reacting COL17 with 3-amino-4-methoxybenzoic acid (AMB) by oxidative polymerization under APS catalysis, followed by freeze-drying, and then dissolving it in PBS with TK-NH2, adding EDC and NHS and stirring. The specific steps are as follows:

[0011] First, 1 g of COL17 and 100 mg of 3-amino-4-methoxybenzoic acid (AMB) were dissolved in 20 ml of deionized water, and 136.5 mg of ammonium persulfate (APS) was added as an oxidative polymerization catalyst. After 24 h, the reaction solution was dialyzed in deionized water for at least 3 days (Mw = 3500) and then lyophilized to obtain COL17-PAMB. Then 300 mg of COL17-PAMB and 100 mg of propane-2, 2-diylbis(sulfonamide)diethylamine (TK-NH2) were dissolved in PBS. Next, 0.25 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.09 g of N-hydroxysuccinimide (NHS) were added and the mixture was stirred at 37 ° C for 24 h to obtain COL17-PTK.

[0012] Secondly, synthesis of MPDAs: Poloxamer (F127) (0.36 g) and 1,3,5-trimethylbenzene (TMB) (417 μL) were dissolved in 64 mL of deionized water, and then 60 mL of ethanol was added to the solution. After 30 min, 60 mg of dopamine hydrochloride and 90 mg of Tris were added to the mixed solution. After stirring for 24 h, the crude product was collected.

[0013] The F127 template was removed at 12000 rpm for 10 min, and then the MPDAs were collected.

[0014] Preparation of MPDAs@IGF-1 microspheres: MPDAs (100 mg) and IGF-1 (65 μg) were suspended in deionized water. After 24 h, IGF-1-loaded MPDAs (M@I) were collected.

[0015] Finally, M@I was first dissolved in deionized water, M@I (200 μg / mL) was directly dissolved in COL17-PTK (30 wt%) solution (volume ratio 1:1) at 30 °C, and then dissolved in 800 μL PBS to obtain a clear solution A, and 15 mg PVA was dissolved in 200 μL PBS to obtain a clear solution B. Then, solution B was added to solution A, and the resulting mixed solution was kept at 37 °C for a short period of time to form a ROS-responsive hydrogel (I@M-COL17) with thiol bonds.

[0016] The preparation method of the microneedle patch is as follows: after eliminating the trapped air in the hydrogel by centrifugation (4000rpm, 10min), 50μL of hydrogel is placed on the PDMS mold, and the hydrogel is pressed into the pinhole of the mold using a small medicine spoon. After the mold is centrifuged at 2200rpm for 15min, the excess hydrogel is scraped out of the cavity and collected for reuse, and then the pressed mold is placed in a vacuum drying oven and repeatedly vacuumed three times; secondly, 20% PVA solution is poured onto the mold carrying the microneedle patch to form a backing, and finally the covered mold is placed in a ventilated place to dry for one day, and MNs are separated from the PDMS micromold. The resulting microneedle patch is stored in a sealed well plate for further study.

[0017] The present invention uses TK-NH2 to modify COL17, adds a thioketal bond to COL17, and further constructs COL17 modified with both a thioketal bond and an amide bond through EDC / NHS cross-linking, so that it can respond to ROS and pH changes in the microenvironment to undergo reversible breakage and recombination.

[0018] The present invention has the following beneficial effects:

[0019] (1) In androgenic alopecia (AGA), the skin microenvironment changes significantly, with an increase in ROS and an inflammatory decrease in pH. This property enables the microneedles to respond to environmental changes and adjust their structure and performance. Reactive oxygen species (ROS) in skin tissue can promote the breakage of thioketal bonds, thereby triggering changes in the internal structure of the hydrogel, and the decrease in pH can promote the breakage of amide bonds. In addition, as the dynamic cross-linking chemical bonds in the microneedles break, the active substances originally encapsulated in the microneedles are released, which helps promote the proliferation and differentiation of hair follicle stem cells and provides a new way to treat hair loss.

[0020] (2) The microneedle material contains IGF-1 and collagen, which stimulate cell proliferation and differentiation, and promote the activation and growth of hair follicles. COL17 has attracted much attention for its significant activity in inducing hair follicle regeneration. At the same time, TK-NH2 can respond to ROS produced by folliculitis. In the drug-loaded nanoparticles, IGF-1 exhibits the ability to induce hair follicle regeneration. These components work synergistically to promote the regeneration and health of hair follicles.

[0021] (3) The microneedle patch of the present invention is simple to prepare and has low cost, which lays a foundation for large-scale preparation. The microneedle patch made of collagen hydrogel has a certain sustained release effect and is slowly released in the body for several days after use. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Characteristics of MPDAs and COL17-PTK. (a) Representative SEM images of MPDAs; (b) Representative size distribution of MPDAs and IGF-1@MPDAs; (c) Zeta potential of MPDAs and IGF-1@MPDAs; (d) 1 HNMR spectrum; (e) FTIR spectra of COL17 and COL17-PTK.

[0023] Figure 2 Construction of modified COL17 microneedles. (a) Photos of COL17-PTK, PVA, and COL17 hydrogels; (b) Evaluation of the injectability of hydrogels; (c) Schematic diagram of the microneedle fabrication process; (d) Representative SEM micrographs of COL17 MNs and MPDA-COL17 MNs; (e) Representative front and side views of COL17 MNs and MPDA-COL17MNs; (f) The distribution of drugs at the needle tip is indicated by methylene blue.

[0024] Figure 3Characteristics of microneedles. (a) Viscosity study of modified COL17 hydrogel; (b) Force-displacement curve of microneedles; (c) Mechanical properties of microneedles; (d) Penetration of aluminum foil by COL17 MNs and MPDA-COL17 MNs; (e) Penetration of pig skin by COL17 MNs and MPDA-COL17MNs; (f) DPPH clearance; (g) Temperature increase curve of MPDA-COL17 MNs under different laser powers; (h) Back temperature of mice after PTT.

[0025] Figure 4 Degradation and small molecule release from MNs. (a) In vitro degradation of COL17 MNs; (b) Drug diffusion of COL17 MNs through agarose; (c) In vivo degradation of COL17 MNs; (d) Rhodamine release from COL17 MNs in vivo; (e) Relative fluorescence intensity of rhodamine release over time after administration of COL17 MNs; (f) Rhodamine release from COL17 MNs in vitro; (g) Statistical analysis of rhodamine release in vitro.

[0026] Figure 5 In vitro biological activity of MNs. (a) Representative bright field images of cell proliferation at 0, 12, 24, and 36 h after treatment with different MNs; (bd) CCK-8 assay to measure cell proliferation at the corresponding time points; (e) Representative bright field images of cell migration at 0, 12, 24, and 36 h after treatment with different MNs; (fh) Cell migration was assessed by quantifying the area occupied by migrated cells at the corresponding time points.

[0027] Figure 6 In vivo therapeutic activity of modified COL17 MNs for AGA treatment. (a) Schematic diagram of the establishment of the AGA mouse model by applying testosterone solution topically every day for 28 consecutive days and the treatment strategy of each group of mice in the established model. The control group was treated with testosterone solution only on the hair removal area, while the other experimental groups were treated with MNs or minoxidil 4 times, with an interval of 3 days each time; (b) Representative photos of hair regeneration in mice in different groups; (c) Regenerated hair; (d) Regenerated hair diameter; (e) Quantification of regenerated hair density.

[0028] Figure 7 Histological evaluation of the therapeutic effect of AGA. (a) H&E staining of skin after treatment in different groups; (b) Representative fluorescence staining of CD31 in skin after hair removal in different groups; (c) Representative fluorescence staining of VEGF in hair removal skin in different groups; (d) Representative fluorescence staining of Ki67 in hair removal skin in different groups; (e) Representative fluorescence staining of β-catenin in hair removal skin in different groups; (f) Representative fluorescence staining of DHE in hair removal skin in different groups; (gl) Quantitative analysis of fluorescence images on day 25 in each group (n=6).

[0029] Figure 8 Gene expression analysis of treated skin. (a) Venn diagram of differentially expressed genes between groups; (b) Heat map of differentially expressed genes between groups (red: high expression, blue: low expression); (c) Radar chart showing the relationship between different treated skins; (d) GO level term classification of differentially expressed genes; (e) KEGG pathway analysis of gene enrichment; (f) Reactome enrichment analysis of gene enrichment; (g) Wiki pathway analysis of gene enrichment.

[0030] Fig. 9 The potential mechanism of hair regeneration by the COL17-based MNs system. (a) Immunoblotting to detect the expression of hair regeneration-related proteins in dorsal skin tissue; (bg) Immunoblotting protein quantification of dorsal skin tissue. DETAILED DESCRIPTION

[0031] The present invention is described in detail below with reference to the embodiments. However, these embodiments are only for illustration and should not be construed as limiting the implementation of the present invention.

[0032] Example 1 Preparation of COL17-PTK

[0033] 1 g COL17 and 100 mg 3-amino-4-methoxybenzoic acid (AMB) were dissolved in 20 ml deionized water, and 136.5 mg ammonium persulfate (APS) was added as an oxidative polymerization catalyst. After 24 h, the reaction solution was dialyzed in deionized water for 3 days (Mw=3500) and then freeze-dried to obtain COL17-PAMB.

[0034] 300 mg COL17-PAMB and 100 mg TK-NH2 were dissolved in 5 ml PBS, 0.25 g EDC, 0.09 g NHS were added and the mixture was stirred at 37°C for 24 h to obtain COL17-PTK.

[0035] Example 2 Encapsulation of IGF-1@MPDAs

[0036] Synthesis of MPDAs: F127 (0.36 g) and TMB (417 μL) were dissolved in 64 mL of deionized water, and then 60 mL of ethanol was added to the solution. After 30 min, 60 mg of dopamine hydrochloride and 90 mg of Tris were added to the mixed solution. The crude product was collected after stirring for 24 h. The F127 template was removed at 12000 rpm for 10 min, and then the MPDAs were collected.

[0037] Preparation of MPDAs@IGF-1 microspheres: MPDAs (100 mg) and IGF-1 (65 μg) were suspended in 20 mL of deionized water. After 24 h, IGF-1-loaded MPDAs (M@I) were collected.

[0038] Example 3 Preparation of ROS and pH dual-responsive collagen microneedle patch

[0039] M@I was first dissolved in deionized water, and then a solution containing 20 μg of M@I (200 μg / mL) was directly dissolved in a solution containing 30 μg of COL17-PTK (30 wt%) at 30°C (the volume ratio of the two was 1:1, i.e., 100 μL:100 μL). It was then dissolved in 800 μL of PBS to obtain a clear solution A, and 15 mg of PVA was dissolved in 200 μL of PBS to obtain a clear solution B. Then, solution B was added to solution A (the volume ratio of solution A to solution B was 10:1), and the resulting mixed solution was kept at 37°C for 1 h to form a ROS-responsive hydrogel with thioketone bonds (I@M-COL17, i.e., modified collagen hydrogel).

[0040] I@M-COL17 was then placed on the microneedle PDMS mold, and the gel was pressed into the pinholes of the mold using a small medicine spoon. The mold was centrifuged at 2200 rpm for 15 min, and the excess hydrogel was scraped out of the cavity and collected for reuse. The pressed mold was then placed in a vacuum drying oven and vacuumed three times repeatedly, and 20% PVA solution was poured onto the back of the mold carrying the microneedle patch to prepare the microneedle patch.

[0041] Example 4 Determination of Antioxidant Capacity of Modified Type XVII Collagen Microneedles

[0042] Modified collagen hydrogel (I@M-COL17 MNs) with a Wt% of 30 was prepared, 3 50 mg hydrogels were weighed and placed in a 5 ml centrifuge tube, and then the antioxidant capacity of the hydrogel was tested using the DPPH kit of Shanghai Yuanye Biotechnology Co., Ltd.

[0043] Example 5 Degradability of microneedle patch in vivo and in vitro

[0044] Weigh 3g of agarose in a 50ml beaker and add 50ml of deionized water to make a 6% agarose solution, then heat the agarose solution in a microwave oven until boiling, pour it into a cell culture dish three times, and then put it in a 4°C refrigerator for 1h to freeze and solidify. Use inverted fluorescence to shoot the needle tip morphology of the microneedle patch (Example 3) at 0min in the bright field, place the microneedle on 6% agarose, press with a 1kg weight and time for 1min, then shoot the degradation of the microneedle for 1min, and finally shoot that the microneedle patch is completely degraded at the 4th minute.

[0045] Example 6 Microneedles used to promote cell proliferation and migration experiments

[0046] Cell plating: 12h, 24h, 36h, add microneedle extraction solution on the next morning, culture for 12h, 24h, 36h, take pictures under an inverted microscope and add MTT, add 150μDMSO after 4h, shake on a shaker for 5min, and measure OD value with an enzyme marker.

[0047] Mark the 6-well plate: Draw parallel lines with a spacing of about 0.5 cm on the outer bottom surface to ensure that the photo positions are consistent. Prepare cell suspension: Digest the cells with trypsin and prepare the suspension. Plate: Plate the cells after counting and control the growth of a single layer on the second day. Scratch: Use a scratch gun tip to scratch the cells. Wash: Aspirate the culture medium and wash 3 times with PBS. Change the medium and take pictures: Change the serum-free culture medium and sample (microneedle extraction solution), and select the time of 0h, 8h, 24h, and 36h to take pictures.

[0048] Example 7 Microneedle in vitro release experiment

[0049] Microneedles loaded with bovine serum albumin (BSA) stained with rhodamine B were placed in 15 mL centrifuge tubes and divided into four groups: the first group was added with 10 mL of 0.2 mM H2O2 + pH 7.4 PBS solution, the second group was added with an equal amount of 0.2 mM H2O2 + pH 5 PBS solution, the third group was added with an equal amount of pH 7.4 PBS solution, and the fourth group was added with an equal amount of pH 5 PBS solution. Shake in a 37 ° C shaker, take samples and replenish the solution at regular intervals. After 24 hours, transfer the samples to a 96-well plate and use a live imaging device to capture images.

[0050] Example 8 Microneedle in vivo release experiment

[0051] After the rhodamine B-stained microneedles were pressed on the back of mice for 30 minutes, the release was imaged in an in vivo imager every day.

[0052] Example 9 Hair follicle regeneration experiment

[0053] The specific experimental steps are:

[0054] (1) Mouse model of androgenic alopecia

[0055] Twenty-four male 6-week-old C57BL / 6 mice were injected subcutaneously with testosterone solution (0.2%, w / v, 0.1, mL / cm 2) for 35 consecutive days to induce the AGA model; during the entire experimental period (35 days), each animal was injected with 0.2 mL of 0.2% testosterone solution every day. On the 14th day after injection, the hair on the back skin was removed by shaving an area of ​​2×3 cm, and the mice were randomly divided into 5 groups: ① PBS group ② Minoxidil group ③ COL17 MNs group ④ I@M-COL17 MNs group ⑤ NIR+I@M-COL17 MNs group. After modeling, the skin of the mice turned pink and thickened, indicating that the modeling was successful.

[0056] (2) Animal grouping

[0057] On day 14 after injection, the hair on the dorsal skin was removed by shaving a 2 × 3 cm area and the mice were randomly divided into 4 groups:

[0058] Group ①, 200 μl PBS, applied on the back

[0059] ②Minoxidil group, 200 μl minoxidil, applied on the back

[0060] ③COL17 MNs group, back pressing for 15 minutes

[0061] ④I@M-COL17 MNs group, back pressing for 15 minutes.

[0062] ⑤NIR+I@M-COL17 MNs group. (808nm, 1.5W, 10min)

[0063] (3) On the 14th day after androgen modeling, shave a 2×3 cm area to remove the hair on the back skin, and start drug administration on the 14th day, and give the drug every two days. Take photos to record the hair growth on the back of the mice starting on the 15th day, and take digital photos of the skin and hair at 0, 5, 10, 15, 20, and 25 every five days.

[0064] After observation, as time goes by after drug administration, it can be seen from the figure that the hair growth in the NIR+I@M-COL17MNs group is obvious. On the 20th day, the skin of ③④ shows slight signs of blackening. On the 25th day, hair follicles grow out of the skin of ②③④. The results are shown in the attached figure, which shows that the speed of promoting hair follicle regeneration by using the drug-loaded microneedle patch of the present invention is significantly greater than that of the control group.

[0065] Figure 1a SEM image of MPDAs; b particle size results of MPDAs and M@I; c potential results of MPDAs and M@I; d H NMR spectra of COL17 and COL17-TK; e FTIR spectra of COL17 and COL17-TK. This proves that MPDAs, M@I and COL17-TK were successfully synthesized.

[0066] Figure 2 In Figure 2a, the EDC / NHS-mediated reaction successfully produced a dual-responsive hydrogel that was sensitive to both pH and ROS. COL17-PTK was modified with TK-NH2 to enhance its responsiveness to the microenvironment. Figure 2 As shown in b, the injectable properties of the hydrogel were studied. Figure 2 c shows a schematic diagram of the microneedle fabrication process. Figure 2 d shows the scanning electron microscopy (SEM) images of the prepared COL17 nanoparticles and MPDA-COL17 nanoparticles. Figure 2 e Confocal fluorescence microscopy images showed that MPDAs were evenly distributed on MNs.

[0067] Figure 3 When the MN is grasped with forceps, Figure 3 As shown in (c), MN bends without cracking, indicating its strong mechanical properties and ductility, and good damage resistance. Figure 3 d, the generated transparent pores indicate that the MN tips have high density and strong penetration ability. Figure 3 e shows that after MN was inserted into pig skin for 10 minutes, obvious pinholes appeared on the skin surface, indicating successful puncture with a puncture efficiency of over 90%. COL17 MNs showed time-dependency in scavenging DPPH freedom, demonstrating its superior antioxidant capacity. By implanting MNs into the back skin of rats and irradiating them with laser, which reached 40°C after 5 minutes, angiogenesis can be promoted.

[0068] Figure 4 In Figure 4 As shown in a, agar was initially used to simulate in vivo degradation, and the microneedle tip gradually degraded over time, and was almost completely degraded after 120 s. Figure 4 As shown in Figure c, MNs were completely degraded about 20 minutes after application to pig skin. The results further showed that MNs have good degradability. Figure 4 d and 4e indicate that COL17 MNs have strong drug loading and delivery capabilities. Figure 4 f and 4g show that MNs have dual response behaviors to ROS and pH.

[0069] Figure 5In order to further confirm the biocompatibility of MNs, bright field microscopy and CCK-8 assay were used to measure the cell viability of different MNs extracts and cells at different co-culture times. Figure 5 As shown in a, compared with the PBS group (as a control), each experimental group showed good biocompatibility after 12, 24, and 36 hours of co-culture with cells. Representative cell migration images are shown in Figure 5 The results shown in e show that MNs in different groups have good activity in promoting cell migration. The cell rate of the IGF-1@MPDA-COL17MNs group reached 96.20% after 36 hours, which was the most significant result compared with other groups.

[0070] Figure 6 To evaluate the potential of COL17 MNs to promote hair regeneration, we established a testosterone-induced AGA mouse model and subjected it to various treatments ( Figure 6 a). At day 25, NIR+I@M-COL17 MNs showed better hair quality compared with minoxidil, COL17 MNs and I@M-COL17 MNs, which could be attributed to the enhanced regenerated hair coverage ( Figure 6 c) Increase hair diameter ( Figure 6 d) and greater hair density ( Figure 6 e) to prove.

[0071] Figure 7 In the H&E staining histological analysis, the NIR+I@M-COL17 MNs group showed significantly more anagen follicles than the other groups, indicating that MNs can activate telogen follicles in the hair loss area. The CD31 expression in the I@M-COL17MNs group and the NIR+I@M-COL17MNs group was enhanced, indicating increased vascularity. The Ki67 expression in the specific treatment group was increased, indicating enhanced cell proliferation. DHE staining ( Figure 7 f) Evaluation of oxidative stress The treatment groups containing I@M-COL17 MNs and NIR+I@M-COL17 MNs may have a protective effect against oxidative stress.

[0072] Figure 8 middle, Figure 8 The Venn diagram in a shows the differential expression of genes in the two groups, indicating different transcriptional profiles. Figure 8 The heatmap in b further visualizes these differences. Figure 8 The radar plot in c shows the relationship between different treatment groups. Figure 8 The classification of differentially expressed genes in d helps to understand the affected biological processes. Figure 8 KEGG pathway analysis in e, Figure 8 Reactome enrichment analysis in f and Figure 8Wiki pathway analysis in g further elucidates the molecular pathways involved.

[0073] Fig. 9 The proteins of the microneedle administration site (mouse back) of each group were extracted, and the expression levels of CD68, P-P38, VEGF and P-AKT were detected by Western blotting. Compared with the control group, the protein expression of the Minoxidil group and COL17MNs group was slightly upregulated, while the expression of the I@M-COL17 MNs group and NIR+I@M-COL17 MNs group was significantly upregulated, indicating that I@M-COL17 MN has an important role in promoting angiogenesis and cell proliferation.

[0074] Based on the above ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above description. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A thioketal-modified type 17 collagen dopamine-loaded nanoparticle composite microneedle patch, characterized in that: The microneedle patch is composed of dopamine-loaded nanoparticles M@I, modified recombinant type 17 collagen COL17-PTK modified with thioketal bonds, and a polymer substrate.

2. The composite microneedle patch according to claim 1, characterized in that: The preparation method of COL17-PTK is as follows: firstly dissolving COL17 and 3-amino-4-methoxybenzoic acid in deionized water, adding ammonium persulfate, dialyzing the reaction solution in deionized water after 24 hours, and then freeze-drying to obtain COL17-PAMB; COL17-PAMB and TK-NH2 were then dissolved in PBS, EDC and NHS were added, and the mixture was stirred at 37°C for 24 h to obtain a COL17-PTK solution.

3. The composite microneedle patch according to claim 2, characterized in that: The mass ratio of COL17 to 3-amino-4-methoxybenzoic acid is 10:1, and the amount of ammonium persulfate is 13.65% of the mass of COL17; The mass ratio of COL17-PAMB and TK-NH2 is 3:

1.

4. The composite microneedle patch according to claim 1, characterized in that: The preparation method of the dopamine drug-loaded nanoparticles M@I is as follows: F127 and TMB are dissolved in deionized water, and then ethanol is added. After 30 minutes, dopamine hydrochloride and Tris are added to the mixed solution, and the crude product is collected after stirring for 24 hours, F127 is removed, and then mesoporous polydopamine nanoparticles MPDAs are collected; MPDAs and IGF-1 are suspended in deionized water, and after 24 hours, IGF-1-loaded MPDAs are collected.

5. The composite microneedle patch according to claim 4, characterized in that: The mass ratio of the MPDAs to IGF-1 is 100 mg:65 μg.

6. A method for preparing the composite microneedle patch according to claim 1, characterized in that: The preparation method of the composite microneedle patch is: firstly preparing a ROS-responsive hydrogel with thioketal bonds; and then preparing the composite microneedle patch.

7. The method for preparing the composite microneedle patch according to claim 6, characterized in that: The preparation method of the ROS-responsive hydrogel with thioketal bonds is as follows: M@I is first dissolved in deionized water, and then the M@I solution is dissolved in COL17-PTK solution at 30°C, and then dissolved in PBS to obtain a clear A solution, and then PVA is dissolved in PBS to obtain a clear B solution; then, solution B is added to solution A, and the resulting mixed solution is maintained at 37°C to form a ROS-responsive hydrogel I@M-COL17 with a thioketal bond.

8. The method for preparing the composite microneedle patch according to claim 7, characterized in that: The volume ratio of M@I solution to COL17-PTK solution was 1:1, and the volume ratio of solution A to solution B was 10:

1.

9. The method for preparing the composite microneedle patch according to claim 6, characterized in that: The preparation method of the composite microneedle patch is as follows: after eliminating the trapped air in the hydrogel by centrifugation, the hydrogel is placed on the microneedle PDMS mold, and the hydrogel is pressed into the pinhole of the mold using a small medicine spoon. After the mold is centrifuged at 2200rpm for 15 minutes, the excess hydrogel is scraped out of the cavity and collected for reuse, and then the pressed microneedle mold is placed in a vacuum drying oven and repeatedly evacuated three times; then 20% PVA solution is poured onto the mold carrying the microneedle patch to form a backing, and finally the covered mold is placed in a ventilated place to dry for one day, and MNs are separated from the PDMS micromold to obtain the microneedle patch.

Images