Collagen type 17 dopamine drug-loaded nanoparticle composite microneedle patch modified by sulfur ketal bond and preparation method and application thereof

Abstract

The present application relates to the field of biological materials, in particular to a kind of thioacetal bond modified 17 collagen dopamine drug-loaded nanoparticle composite microneedle patch and its preparation method and application, the microneedle patch is composed of dopamine drug-loaded nanoparticle, thioacetal bond modified modified recombinant 17 collagen and polymer base.Sulfur ketone bond modified 17 collagen and polyvinyl alcohol are formed into hydrogel by physical and chemical double crosslinking, then loaded drug-loaded nanoparticles, and with polymer base to form ROS and pH value microenvironment double-response collagen microneedle patch loaded with bioactive substances.When it is applied to skin, microneedle tip reaches the area where hair follicle stem cells exist in a painless and minimally invasive manner, due to the change of microenvironment molecules in skin tissue, it can promote the rupture of thioacetal bond in collagen hydrogel, thereby removing active oxygen and releasing encapsulated IGF-1 into hair follicle stem cells, to provide a potential treatment strategy for androgenetic alopecia.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomaterials, in particular to a kind of 17 collagen protein dopamine drug-loaded nanoparticle composite microneedle patch modified by thioacetal bond and its preparation method and application. BACKGROUND

[0002] Hydrogel is a three-dimensional network structure gel with hydrophilic, which can swell rapidly in water and keep a large amount of water insoluble, with high flexibility and water absorption. The preparation of hydrogel microneedle mainly focuses on its application as a transdermal drug delivery technology. By using PDMS mold, hydrogel microneedle can be quickly and low-cost manufactured. Microneedle treatment is favored by more and more people due to its small trauma, quick recovery, non-surgical characteristics, compared with drug treatment which may cause allergy or adverse reactions, long-term multiple laser treatment and hair transplantation surgery which has the risk of infection. The therapeutic drugs or nutritional ingredients introduced by microneedle 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-acting 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 hyperthermia at tumor sites. In addition, PTT can positively affect angiogenesis by promoting local blood circulation and improving tissue oxygen supply. In terms of hair follicle growth, local blood circulation can be promoted through photothermal effect, increasing scalp tissue oxygenation, improving cell activity and metabolism, and stimulating hair growth, providing a new idea for hair loss treatment.

[0004] Hair loss is characterized by gradual miniaturization of hair follicles and shortening of the growth phase, resulting in significant reduction of scalp hair. Treatment methods include drugs (such as minoxidil and finasteride, which need to be used for a long time and the effect varies from person to person), laser and hair transplantation surgery (the effect is affected by the skill of the doctor). Choosing the right treatment method in combination with the patient's condition can help improve the problem of hair loss.

[0005] Hair loss affects the physical and mental health of patients, and early diagnosis and treatment are crucial. Microneedle patch treatment methods include minoxidil and hair growth factor patches. Minoxidil patches may cause skin irritation and have no obvious effect on acute hair loss; hair growth factor patches have unstable effects and high storage and use requirements. Other treatment methods such as laser hair growth devices have limited indications and high costs, and PRP treatment has a long cycle and no effect guarantee. SUMMARY

[0006] The purpose of the present application is to provide a ROS and pH dual-responsive collagen microneedle patch loaded with bioactive substances and its preparation method and application, which combines bioactive substances with modified COL17 to develop a degradable COL17 microneedle patch, and can deliver bioactive substances to the hair follicle stem cells at the site of androgenetic alopecia. The microneedle patch prepared by the present application has a remarkable effect on promoting hair regeneration, and has a wide application prospect in the field of hair follicle regeneration induction treatment.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] The microneedle patch provided by the present application is composed of ROS and pH dual-responsive modified COL17, 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 is as follows: PVA is dissolved in deionized water (4g PVA and 20ml deionized water), and a 20% PVA solution is prepared and stored at room temperature for standby.

[0010] The ROS and pH dual-responsive COL17 (sulfone ketone bond modified 17 type collagen COL17-PTK) is prepared by freeze-drying COL17 and 3-amino-4-methoxybenzoic acid (AMB) after reaction by oxidative polymerization, and then dissolved in PBS with TK-NH2, and EDC and NHS are added and stirred to form, and the specific steps are as follows:

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

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

[0013] 12000rpm, 10min to remove F127 template, then collect MPDAs.

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

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

[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 the hydrogel is placed on the PDMS mold, and the hydrogel is pressed into the needle hole of the mold using a small spoon, the mold is centrifuged at 2200rpm for 15min, then the excess hydrogel is scraped out of the cavity and collected for reuse, then the pressed mold is placed in a vacuum drying box and vacuumed repeatedly three times; secondly, 20% PVA solution is poured onto the mold loaded with the microneedle patch to form a backing, and finally the covered mold is dried in a well-ventilated place for one day, and the MNs are separated from the PDMS micromold. The obtained microneedle patch is stored in a sealed well plate for further study.

[0017] The present application uses TK-NH2 for modification of COL17, adds a thio ketal bond to COL17, and further constructs COL17 modified with both a thio ketal bond and an amide bond through EDC / NHS crosslinking, so that it can respond to changes in ROS and pH in the microenvironment to reversibly break and recombine.

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

[0019] (1) In androgenetic alopecia (AGA), the skin microenvironment changes significantly, with increased ROS and decreased pH value, which makes the microneedle respond to environmental changes and adjust its structure and performance. Reactive oxygen species (ROS) in the skin tissue can promote the cleavage of thio-ketone bonds, thereby triggering changes in the internal structure of the hydrogel, and the decrease in pH value can promote the cleavage of amide bonds. Moreover, with the cleavage of the dynamic cross-linking chemical bonds in the microneedle, the active substances originally wrapped in the microneedle are released, which helps to promote the proliferation and differentiation of hair follicle stem cells, providing a new way for the treatment of alopecia.

[0020] (2) The microneedle material contains IGF-1 and collagen, which stimulates cell proliferation and differentiation, and promotes the activation and growth of hair follicles. COL17 is of great concern due to its significant ability to induce hair follicle regeneration, and TK-NH2 can respond to ROS produced by folliculitis. In drug-loaded nanoparticles, IGF-1 exhibits the ability to induce hair follicle regeneration. These components work together to promote the regeneration and health of hair follicles.

[0021] (3) The microneedle patch of the present application is simple to prepare and low in cost, laying a foundation for large-scale preparation. The microneedle patch made of collagen hydrogel has a certain sustained-release effect and slowly releases in the body for several days after use. BRIEF DESCRIPTION OF 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) FTIR spectra of COL17 and COL17-PTK. 1 HNMR spectra; (e) FTIR spectra of COL17 and COL17-PTK.

[0023] Figure 2 Construction of modified COL17 microneedles. (a) Photographs of COL17-PTK, PVA and COL17 hydrogels; (b) Injection evaluation of hydrogels; (c) Schematic diagram of microneedle making process; (d) Representative SEM micrographs of COL17 MNs and MPDA-COL17 MNs; (e) Representative front and side views of COL17 MNs and MPDA-COL17 MNs; (f) Distribution of drugs at the tip of the needle represented by methylene blue.

[0024] Figure 3Properties of microneedles. (a) Viscosity study of modified COL17 hydrogel; (b) Force-displacement curve of microneedles; (c) Mechanical properties of microneedles; (d) COL17 MNs and MPDA-COL17 MNs penetration of aluminum foil; (e) Penetration of COL17 MNs and MPDA-COL17 MNs into pig skin; (f) DPPH scavenging rate; (g) Temperature rise curve of MPDA-COL17 MNs under different power laser irradiation; (h) Temperature of mouse back after PTT.

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

[0026] Figure 5 In vitro biological activity of MNs. (a) Representative bright field images of cell proliferation at 0, 12, 24, 36 h after treatment with different MNs; (b-d) Cell proliferation at corresponding time points was determined by CCK-8 method; (e) Representative bright field images of cell migration at 0, 12, 24, 36 h after treatment with different MNs; (f-h) Cell migration was evaluated by quantifying the area occupied by the migrated cells at corresponding time points.

[0027] Figure 6 In vivo therapeutic activity of modified COL17 MNs for AGA treatment. (a) Schematic diagram of establishing AGA mouse model by daily topical testosterone solution for 28 consecutive days and treatment strategy of mice in each group. The control group was only treated with testosterone solution in the depilation area, and the rest of the experimental groups were treated with MNs or minoxidil 4 times, with an interval of 3 days each time; (b) Representative photos of hair regrowth in different groups of mice; (c) Regenerated hair; (d) Diameter of regenerated hair; (e) Quantification of regenerated hair density.

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

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

[0030] Figure 9 Potential mechanism of COL17-based MNs system for hair regeneration. (a) Immunoblotting detection of hair regeneration-related protein expression in dorsal skin tissue; (b-g) Immunoblotting protein quantification of dorsal skin tissue. DETAILED DESCRIPTION

[0031] The present application is described in detail below with reference to Examples, which are intended to be illustrative only and should not be construed as limiting the application.

[0032] Example 1 Preparation of COL17-PTK

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

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

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

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

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

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

[0039] First, M@I was dissolved in deionized water, and then the solution containing 20 μg M@I (200 μg / mL) was directly dissolved in the solution containing 30 μg COL17-PTK (30 wt%) at 30°C (volume ratio of 1:1, i.e. 100 μL:100 μL). Then it was dissolved in 800 μL PBS to obtain a clear A solution, and 15 mg PVA was dissolved in 200 μL PBS to obtain a clear B solution. Then, solution B was added to solution A (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 thioether bonds (I@M-COL17, i.e. modified collagen hydrogel).

[0040] Then I@M-COL17 was placed on the microneedle PDMS mold, and the gel was pressed into the needle holes of the mold using a small 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. Then the pressed mold was placed in a vacuum drying oven and repeatedly vacuumed three times. A 20% PVA solution was poured onto the back of the mold loaded with the microneedle patch to prepare the microneedle patch.

[0041] Example 4 Determination of the antioxidant capacity of modified collagen XVII microneedles

[0042] Prepare modified collagen hydrogel (I@M-COL17 MNs) with a Wt% of 30. Weigh 3 50 mg hydrogels into 5 ml centrifuge tubes, then use the DPPH kit from Shanghai Yuan Ye Biological Technology Co., Ltd. to test the antioxidant capacity of the hydrogel.

[0043] Example 5 In vitro and in vivo degradation capacity of microneedle patch

[0044] Weigh 3 g of agarose into a 50 ml beaker and add 50 ml of deionized water to make a 6% agarose solution. Then heat the agarose solution to boiling in a microwave oven, pour it into a cell culture dish three times, and then freeze it in a 4°C refrigerator for 1 h. Use an inverted fluorescence camera to take a picture of the microneedle patch (Example 3) at 0 min in bright field to observe the shape of the needle tip. Place the microneedle on the 6% agarose and press it with a 1 kg weight for 1 min. Then take a picture of the microneedle degradation at 1 min, and finally take a picture of the microneedle patch completely degraded at 4 min.

[0045] Example 6 Microneedles for promoting cell proliferation and migration experiments

[0046] Cell plating: 12h, 24h, 36h, the next morning to add micro-needle extraction solution, culture 12h, 24h, 36h after inverted microscope and add MTT, 4h after adding 150μDMSO, shaking bed shaking 5min, using microplate reader to measure OD value.

[0047] Label 6-hole plate: draw parallel lines with a spacing of about 0.5cm on the outer bottom surface to ensure consistent photographing position. Preparation of cell suspension: trypsinize cells to prepare suspension. Plating: plate after cell counting, control full monolayer the next day. Scoring: use scoring gun head to score cells. Washing: aspirate culture medium, wash with PBS for 3 times. Change liquid and take pictures: change serum-free medium and samples (micro-needle extraction solution), select time 0h, 8h, 24h, 36h to take pictures.

[0048] Example 7 Micro-needle in vitro release experiment

[0049] The micro-needles loaded with rhodamine B dyed bovine serum albumin (BSA) were placed in 15mL centrifuge tubes and divided into four groups: the first group was added with 10mL of 0.2mM H2O2+ pH 7.4 PBS solution, the second group was added with equal amount of 0.2mM H2O2+ pH 5 PBS solution, the third group was added with equal amount of pH 7.4 PBS solution, and the fourth group was added with equal amount of pH 5 PBS solution. Shake in a 37℃ shaking bed, sample every other time and replenish. After 24h, transfer the samples to a 96-hole plate and take pictures using a live imaging instrument.

[0050] Example 8 Micro-needle in vivo release experiment

[0051] After pressing the rhodamine B dyed micro-needles on the back of mice for 30min, take pictures of the release in a live imaging instrument every day.

[0052] Example 9 Hair follicle regeneration experiment

[0053] The specific experimental steps are as follows:

[0054] (1) Androgenic alopecia model of mice

[0055] Twenty-four 6-week-old male C57BL / 6 mice were subcutaneously injected with testosterone solution (0.2%, w / v, 0.1, mL / cm 2), for 35 consecutive days, to induce the AGA model; during the entire experiment (35 days), each animal was injected with 0.2 mL of a 0.2% testosterone solution each day. On day 14 after injection, the mice were randomly divided into 5 groups by shaving a 2x3 cm area to remove the hair on the back skin: ① PBS group ② Minoxidil group ③ COL17 MNs group ④ I@M-COL17 MNs group ⑤ NIR+I@M-COL17 MNs group. The mouse skin became powdery and the skin became thick after modeling, which proved that the modeling was successful.

[0056] (2) Animal grouping

[0057] On day 14 after injection, the mice were randomly divided into 4 groups by shaving a 2x3 cm area to remove the hair on the back skin:

[0058] ① Group, the administration dose was 200 μl PBS, and the back was smeared

[0059] ② Minoxidil group, the administration dose was 200 μl minoxidil, and the back was smeared

[0060] ③ COL17 MNs group, the back was pressed for 15 min

[0061] ④ I@M-COL17 MNs group, the back was pressed for 15 min.

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

[0063] (3) On day 14 after androgen modeling, a 2x3 cm area was shaved to remove the hair on the back skin, and administration was started on day 14, with administration every two days. Photographs were taken to record the hair growth on the back of the mice starting on day 15, with photographs taken every five days on day 0, 5, 10, 15, 20, 25 to take digital photos of the skin and hair.

[0064] After observation, it can be seen from the figure that the hair growth of the NIR+I@M-COL17 MNs group was obvious after administration treatment, and as time went on. By day 20, the skin of ③ and ④ had slight signs of darkening. By day 25, the skin of ②, ③ and ④ had hair follicles growing out. The results are shown in the attached figure, and it can be seen that the speed of hair follicle regeneration using the drug-loaded microneedle patch of the application is significantly greater than that of the control group.

[0065] Figure 1a SEM images of MPDAs; b Particle size results of MPDAs and M@Is; c Zeta potential results of MPDAs and M@Is; d NMR hydrogen spectrum results of COL17 and COL17-TK; e FTIR spectrum of COL17 and COL17-TK. It is proved that MPDAs are successfully synthesized; M@Is are successfully synthesized; COL17-TK is successfully synthesized.

[0066] Figure 2 a, 2a shows that the EDC / NHS-mediated reaction successfully produces a dual-responsive hydrogel, which is sensitive to both pH and ROS. COL17-PTK is modified with TK-NH2 to enhance its responsiveness to the microenvironment. Figure 2 b shows the injectable properties of the hydrogel. Figure 2 c gives a schematic diagram of the MN manufacturing process. Figure 2 d shows scanning electron microscope (SEM) images of the prepared COL17 nanoparticles and MPDA-COL17 nanoparticles Figure 2 e confocal fluorescence microscope images show that MPDAs are uniformly distributed on MNs.

[0067] Figure 3 When the MNs are clamped with tweezers, as shown in Figure 3 c, the MNs bend without breaking, indicating their strong mechanical properties and ductility, and good resistance to damage. As shown in Figure 3 d, the transparent holes produced indicate that the MN tips have high density and strong penetration ability. Figure 3 e shows that after the MNs are inserted into pig skin for 10 min, obvious pinholes appear on the skin surface, indicating successful penetration, with a penetration efficiency of over 90%. COL17 MNs exhibit time-dependent performance in clearing DPPH free radicals, demonstrating their superior antioxidant capacity. By implanting MNs into the skin on the back of rats and irradiating with a laser, a temperature of 40°C is reached after 5 minutes, which can promote angiogenesis.

[0068] Figure 4 As shown in Figure 4 a, agarose was initially used to simulate in vivo degradation, and over time, the MN tips gradually degraded, almost completely degrading after 120 s. As shown in Figure 4 c, the MNs completely degraded after being applied to pig skin for about 20 minutes. The results further indicate that the MNs have good degradability. Figure 4 d and 4e show that COL17 MNs have strong drug loading and delivery capacity. Figure 4 f and 4g show that the MNs have dual-responsive behavior to ROS and pH.

[0069] Figure 5In order to further confirm the biocompatibility of MNs, the cell viability of different MNs extracts and different co-culture time cells were measured by bright field microscope and CCK-8 method. As shown in Figure 5 a, each experimental group showed good biocompatibility after co-cultured with cells for 12, 24, 36 h compared with PBS group (as control). Representative cell migration images are shown in Figure 5 e, the results showed that different groups of MNs all had good activity in promoting cell migration. The cell rate of IGF-1@MPDA-COL17 MNs group reached 96.20% after 36 h, which was the most significant compared with other groups.

[0070] Figure 6 In order to evaluate the potential of COL17 MNs to promote hair regeneration, we established a testosterone-induced AGA mouse model and performed various treatments Figure 6 a). On day 25, NIR+I@M-COL17 MNs showed better hair quality compared with minoxidil, COL17 MNs and I@M-COL17 MNs, which could be evidenced by enhanced regenerative hair coverage Figure 6 c), increased hair diameter Figure 6 d) and greater hair density Figure 6 e).

[0071] Figure 7 In the H&E-stained histology section, the NIR+I@M-COL17 MNs group showed significantly more anagen hair follicles than other groups, which indicated that MNs could activate telogen hair follicles in the alopecia area. The I@M-COL17 MNs group and NIR+I@M-COL17 MNs group showed enhanced CD31 expression, indicating increased vascularity. The specific treatment group showed increased Ki67 expression, indicating enhanced cell proliferation. DHE staining Figure 7 f) assessed oxidative stress and the treatment groups containing I@M-COL17 MNs and NIR+I@M-COL17 MNs might have protective effects against oxidative stress.

[0072] Figure 8 In the Venn diagram in Figure 8 a, the differential expression of two groups of genes indicated different transcriptional profiles. Figure 8 The heatmap in Figure 8 c shows the relationship between different treatment groups. Figure 8 The classification of differential genes in Figure 8 e helps to understand the affected biological processes. Figure 8 f, Reactome enrichment analysis in Figure 8 f, Reactome enrichment analysis inWiki pathway analysis in g further elucidated the molecular pathways involved.

[0073] Figure 9 In the middle, the protein of each group of microneedle administration site (mouse back) was 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 Minoxidil group and COL17 MNs group was slightly up-regulated, while the expression of I@M-COL17 MNs group and NIR+I@M-COL17 MNs group was significantly up-regulated, indicating that I@M-COL17 MNs had important promoting effect on angiogenesis and cell proliferation.

[0074] With the above ideal embodiments according to the present application as the inspiration, through the above description, relevant staff can make various changes and modifications without deviating from the technical idea of the present application. The technical scope of the present application is not limited to the content in the specification, and the technical scope must be determined according to the scope of 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; The preparation method of COL17-PTK is as follows: COL17 and 3-amino-4-methoxybenzoic acid are first dissolved in deionized water, ammonium persulfate is added, and after 24 hours, the reaction solution is dialyzed in deionized water and then lyophilized to obtain COL17-PAMB; Then, COL17-PAMB and TK-NH2 were dissolved in PBS, EDC and NHS were added, and the mixture was stirred at 37 °C for 24 h to obtain COL17-PTK solution; The preparation method of the dopamine-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. After stirring for 24 hours, the crude product is collected, the F127 is removed, and then the mesoporous polydopamine nanoparticles MPDAs are collected; MPDAs and IGF-1 are suspended in deionized water. After 24 hours, the IGF-1-loaded MPDAs are collected.

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

1.

3. The composite microneedle patch according to claim 1, wherein The mass ratio of the MPDAs to IGF-1 is 100 mg:65 μg.

4. A method for preparing the composite microneedle patch according to claim 1, characterized in that: The preparation method of the composite microneedle patch comprises the following steps: firstly preparing a ROS-responsive hydrogel having a thioketal bond; and then preparing the composite microneedle patch.

5. The method for preparing the composite microneedle patch according to claim 4, wherein: The preparation method of ROS-responsive hydrogel with thioketal bond 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 solution A, and then PVA is dissolved in PBS to obtain a clear solution B; then, solution B is added to solution A, and the resulting mixed solution is maintained at 37°C to form ROS-responsive hydrogel I@M-COL17 with thioketal bond.

6. The method for preparing the composite microneedle patch according to claim 5, 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.

7. The method for preparing the composite microneedle patch according to claim 5, wherein: 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 2200 rpm for 15 minutes, the excess hydrogel is scraped out of the cavity and collected for reuse. The pressed microneedle mold is then placed in a vacuum drying oven and vacuumed repeatedly three times; 20% PVA solution is then poured onto the mold carrying the microneedle patch to form a backing. Finally, the covered mold is placed in a ventilated place to dry for one day, and then the MNs are separated from the PDMS micromold to obtain the microneedle patch.

Images