Repsox-loaded microneedle as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a Repsox-loaded microneedle as well as a preparation method and application thereof. The method specifically comprises the following steps: (1) mixing water with polyvinyl alcohol with low polymerization degree and low alcoholysis degree, then sequentially adding Repsox and hyaluronic acid, and mixing to obtain a needle tip solution; (2) mixing water with high-polymerization-degree and high-alcoholysis-degree polyvinyl alcohol, then adding a bacteriostatic agent, and mixing to obtain a substrate solution; and (3) adding the needle tip solution into a mold, carrying out primary centrifugation and curing, then adding the substrate solution, and carrying out secondary centrifugation, drying and demolding. The microneedle prepared by the invention has the technical effect of improving the thickness of the aged skin, so that a specific action way of the microneedle in a skin regeneration process is disclosed, and a more convenient method is provided for improving the thickness of the skin.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a Repsox-loaded microneedle and a preparation method and application thereof. Background Art

[0002] With the improvement of medical level, the average life expectancy of human beings is increasing year by year, the problem of population aging is aggravated, and the research of anti-aging beauty technology has become a hot field in the international medical and beauty circles. The most intuitive manifestation of aging is skin aging, which is the result of the combined action of various factors. It is mainly divided into two types: one is endogenous skin aging, also known as natural aging, which is mainly affected by genetic factors and is a natural and gradual aging process; the other is exogenous skin aging caused by external environmental factors such as ultraviolet radiation, gravity, smoking, chemicals, etc., also known as photoaging. More than 80% of facial skin aging is caused by ultraviolet radiation. The characteristics of this type of skin aging are rough skin surface, wrinkles, pigmentation, dark skin color, decreased skin elasticity, and reduced facial subcutaneous fat. It is also a characteristic of skin aging. Whether it is endogenous or exogenous aging, the loss of facial skin fat tissue, thinning of the skin and increased wrinkles need to be solved urgently.

[0003] To solve this series of problems, the commonly used methods are surgical treatment and drug treatment. Surgical treatment mainly includes autologous fat transplantation, which can avoid repeated and continuous drug administration and has immediate effects. However, the preoperative preparation and postoperative care steps are complicated and may cause wound infection, allergic reaction, microcysts, foreign body reaction, chronic inflammation, fibrosis and other problems. Drug treatment can be divided into oral administration, injection, dressing and smear administration, and microneedle administration. The analysis is as follows:

[0004] a. Oral administration: The advantages are avoidance of pain, easy swallowing, various dosage forms, autonomous administration, and high patient compliance; the disadvantages are slow and irregular absorption, first-pass effect, and the efficacy is easily affected by gastrointestinal function and gastrointestinal contents. Some drugs can also cause adverse irritation to the gastrointestinal tract.

[0005] b. Injection: The advantages are that it can quickly reach blood drug concentration, high drug utilization, and can accurately control the dosage; the disadvantages are that it requires the help of professional medical institutions or professionals, is invasive and painful, has many contraindications, and is prone to redness, swelling, pain, and allergic reactions.

[0006] c. Dressing and smear administration: The advantages are convenient self-administration and local painless administration; the disadvantage is that it is difficult to penetrate the epidermis, especially the stratum corneum, which seriously hinders the transdermal absorption of drugs. Even with the action of various penetration enhancers, only some small molecule drugs can pass through the epidermis to reach effective drug concentrations. For most drugs, especially protein macromolecule drugs, the transdermal penetration rate is difficult to meet the needs of treatment.

[0007] d. Microneedle drug delivery: The advantages are that it can avoid the first-pass effect of the liver and gastrointestinal inactivation of drugs, improve the therapeutic effect, can deliver drugs through the skin epidermis, is not prone to redness, swelling and infection, is convenient for self-administration and adjustment of the delivery area, and has no postoperative inflammation, infection and allergic reactions. The disadvantages are low drug loading, relatively expensive cost and high technical requirements.

[0008] As a new treatment technology, microneedle drug delivery has not been widely studied and applied in the field of skin beauty and medicine. Therefore, how to provide a microneedle preparation that can be used in skin beauty and provide a new treatment strategy for the treatment of aging skin is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0009] The purpose of the present invention is to provide a Repsox-loaded microneedle and a preparation method and application thereof.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides a method for preparing a Repsox-loaded microneedle, comprising the following steps:

[0012] (1) After mixing water and polyvinyl alcohol with low polymerization degree and low alcoholysis degree, Repsox and hyaluronic acid are added in sequence and mixed to obtain a needle tip solution;

[0013] (2) mixing water with polyvinyl alcohol with a high degree of polymerization and a high degree of alcoholysis, and then adding an antibacterial agent to obtain a substrate solution;

[0014] (3) Add the needle tip solution into the mold, centrifuge once, solidify, then add the substrate solution, centrifuge twice, dry, and demold.

[0015] Preferably, in step (1), the volume percentage concentration of the low-polymerization degree and low-alcoholysis degree polyvinyl alcohol in the needle tip solution is 2.5-4.0%; the volume percentage concentration of the hyaluronic acid in the needle tip solution is 7-13%; and the final concentration of Repsox in the needle tip solution is 400-600 μM.

[0016] Preferably, in step (2), the volume percentage concentration of the high polymerization degree and high alcoholysis degree polyvinyl alcohol in the substrate solution is 14-20%; the mass percentage concentration of the antibacterial agent in the substrate solution is 0.05-0.15%; and the antibacterial agent is doxycycline hydrochloride.

[0017] Preferably, the molecular weight of the hyaluronic acid is ≥ 2 million.

[0018] Preferably, the speed of the first centrifugation in step (3) is 4000-4400 rpm, and the time is 20-26 min; the speed of the second centrifugation is 3500-4000 rpm, and the time is 15-20 min; the model of the low-polymerization-degree and low-alcoholysis-degree polyvinyl alcohol is 0588; the model of the high-polymerization-degree and high-alcoholysis-degree polyvinyl alcohol is 1797.

[0019] Preferably, the curing time in step (3) is 40 to 50 seconds; the drying time is 60 to 80 hours, and the relative humidity is 4 to 6%; and the ambient temperature needs to be maintained at 20 to 24° C. during the preparation of the Repsox-loaded microneedles.

[0020] The present invention also provides the Repsox-loaded microneedles prepared by the preparation method of the Repsox-loaded microneedles.

[0021] The present invention also provides the use of the Repsox-loaded microneedles in preparing a drug for improving aged skin.

[0022] The present invention also provides the use of Repsox in preparing a drug for improving aged skin.

[0023] The invention also provides the use of Repsox in preparing cosmetics for improving skin aging.

[0024] Preferably, the improvement of skin aging is to increase skin thickness; the improvement of skin aging is to increase skin thickness and restore skin elasticity; the increase in skin thickness is to increase the proportion of white fat cells; the restoration of skin elasticity is to change elastic fibers from being coarse and few to being thin and many, and the ratio of type III to type I collagen is increased.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] Repsox used in the present invention has the technical effect of promoting adipogenesis, and the specific mechanism is as follows: Figure 1 As shown:

[0027] In the absence of Repsox, TGF-β binds to TGF-β receptor II to activate the TGF-β pathway, and TGF-β dimers bind to the extracellular domains of two type II receptor kinases (TGF-βRII), which then recruit two type I receptor kinases (TGF-βRI). At this time, one dimeric TGF-β molecule binds to two TGF-βRI and two TGF-βRII molecules to form a symmetrical 2:2:2 complex. Both receptors include an extracellular domain, a transmembrane region, and an intracellular domain that is mainly occupied by the kinase domain. Biochemical and single-molecule imaging studies have shown that both TGF-βRII and TGF-βRI can remain monomers or dimers in the resting state, and they form tetramers together when bound to TGF-β dimers. In the receptor complex, TGF-βRII trans-phosphorylates serine and threonine residues in the membrane-proximal GS domain of TGF-βRI, leading to the activation of T TGF-βRI kinase. Activated TGF-βRI phosphorylates smad2 / 3 under the action of ATP. Phosphorylated smad2 / 3 forms a complex with smad4 and enters the cell nucleus. Under the joint action of general transcription factors (GTFs) auxiliary factors, it promotes the expression of fibrosis genes and inhibits the expression of adipogenic genes.

[0028] After adding Repsox, Repsox acts as a selective inhibitor of TGF-β type I receptor kinase (ALK5 / TGF-βRI), inhibiting ATP binding to ALK5 and inhibiting ALK5 autophosphorylation, thereby preventing the activation of the TGF-β signaling pathway, and ultimately inhibiting the expression of fibrosis genes, thereby promoting the expression of adipogenic genes.

[0029] The microneedles prepared by the present invention utilize the principle mechanism of Repsox. The substrate is prepared using insoluble polyvinyl alcohol (PVA) with high alcoholysis degree and high molecular weight, and the needle tip is prepared using hyaluronic acid and a small amount of easily soluble PVA with low alcoholysis degree. The substrate and the needle tip are respectively loaded with the antibacterial drug doxycycline hydrochloride and the specific inhibitor of transforming growth factor-β (TGF-β) receptor I, Repsox. The prepared microneedles have the effect of improving the thickness of aged skin, which helps to reveal its specific action pathway in the process of skin regeneration and provide a more convenient method for improving skin thickness.

[0030] The present invention loads Repsox into hyaluronic acid microneedles for the first time, and adjusts the physicochemical properties of the microneedles, explores its application effect in the field of improving aging skin, and studies its effect on the transdifferentiation of skin fibroblasts into adipocytes after being applied to the skin. The hyaluronic acid microneedles provided by the present invention, as an innovative technology for improving aging skin, have the functions of increasing the ratio of type III collagen to type I collagen, restoring skin elasticity, and reducing skin wrinkles. Repsox (2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]-1,5-naphthyridine) used in the present invention is a specific inhibitor of TGF-β receptors, which has biological functions such as promoting stem cell proliferation, regulating cell cycle / inhibiting cell apoptosis, and is expected to become a new strategy for treating aging skin.

[0031] The present invention develops a multifunctional drug-loaded hyaluronic acid microneedle. The microneedle is prepared by a PDMS (polydimethylsiloxane) mold method. Compared with a vacuum method and a photoetching method, the microneedle prepared by the mold method has a more uniform morphology and a more complete needle tip. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0033] Figure 1 Diagram of the mechanism by which Repsox promotes adipogenesis.

[0034] Figure 2 Schematic diagram of the preparation process of Repsox-loaded microneedles.

[0035] Figure 3 The appearance image of the hyaluronic acid microneedle of Experimental Example 3; the upper image is a digital camera image, and the lower image is a scanning electron microscope image. Scale bar: 500μm; one microneedle sheet contains 100 small microneedles.

[0036] Figure 4 The mechanical properties of the microneedle in Experimental Example 3.

[0037] Figure 5 This is the cell activity of experimental example 3MNs@Repsox / DH microneedles co-cultured with cells for 1 day or 3 days, n = 6. NS indicates p ≥ 0.05, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0038] Figure 6 This is the cytotoxicity test of Experimental Example 3 (scale bar is 200 μm).

[0039] Figure 7 This is a diagram of the antibacterial effect of Experimental Example 3 (scale bar is 1 cm).

[0040] Figure 8 This is a diagram showing the effect of improving skin thickness in Experimental Example 3 (scale bar is 500 μm). DETAILED DESCRIPTION

[0041] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] Example 1

[0043] A method for preparing Repsox-loaded microneedles, comprising the following steps:

[0044] (1) Preparation of needle tip solution

[0045] During the preparation process, the ambient temperature was always maintained at 20°C; deionized water was prepared, polyvinyl alcohol 0588 was added while stirring, and the mixture was heated in a water bath at 88°C until completely dissolved, and the mixture was cooled to room temperature and Repsox solution was added and mixed evenly, followed by addition of hyaluronic acid powder (molecular weight ≥ 2 million), and continuous stirring was performed to allow the mixture to fully dissolve.

[0046] The volume percentage concentration of the polyvinyl alcohol 0588 in the needle tip solution is 2.5%; the volume percentage concentration of the hyaluronic acid in the needle tip solution is 7%; and the final concentration of the Repsox in the needle tip solution is 400 μM.

[0047] (2) Preparation of substrate solution

[0048] Prepare deionized water, add polyvinyl alcohol 1797 while stirring, and heat in a water bath at 88°C until completely dissolved. Cool to room temperature, add doxycycline hydrochloride powder, and stir to fully dissolve.

[0049] The volume percentage concentration of the high-polymerization-degree and high-alcoholysis-degree polyvinyl alcohol in the substrate solution is 14%; the mass percentage concentration of the antibacterial agent in the substrate solution is 0.05%;

[0050] (3) Add the needle tip solution to the PDMS mold, centrifuge at 4000 rpm for 20 min, scrape off the excess needle tip solution after centrifugation, recycle it, solidify it for 40 s, then add the substrate solution, centrifuge at 3500 rpm for 15 min, take out and scrape off the excess solution.

[0051] (4) Place the mold (with solution loaded) after centrifugation in a drying cabinet without wind for 60 hours with relative humidity set to 4%. After drying, use tweezers to gently pick out the microneedle. If the substrate is smooth and free of bubbles and the needle tip is intact and uniform in shape, the drying and demolding is successful.

[0052] Example 2

[0053] A method for preparing Repsox-loaded microneedles, comprising the following steps:

[0054] (1) Preparation of needle tip solution

[0055] During the preparation process, the ambient temperature was always maintained at 24°C; deionized water was prepared, polyvinyl alcohol 0588 was added while stirring, and the mixture was heated in a water bath at 95°C until completely dissolved, and the mixture was cooled to room temperature and Repsox solution was added, mixed evenly, and then hyaluronic acid powder (molecular weight ≥ 2 million) was added, and stirring was continued to allow the mixture to fully dissolve.

[0056] The volume percentage concentration of the polyvinyl alcohol 0588 in the needle tip solution is 4.0%; the volume percentage concentration of the hyaluronic acid in the needle tip solution is 13%; and the final concentration of the Repsox in the needle tip solution is 600 μM.

[0057] (2) Preparation of substrate solution

[0058] Prepare deionized water, add polyvinyl alcohol 1797 while stirring, keep heating in a 95°C water bath until completely dissolved, cool to room temperature, add doxycycline hydrochloride powder, stir to fully dissolve.

[0059] The volume percentage concentration of the high-polymerization-degree and high-alcoholysis-degree polyvinyl alcohol in the substrate solution is 20%; the mass percentage concentration of the antibacterial agent in the substrate solution is 0.15%;

[0060] (3) Add the needle tip solution to the PDMS mold, centrifuge at 4400 rpm for 26 min, scrape off the excess needle tip solution after centrifugation, recycle it, solidify it for 50 s, then add the substrate solution, centrifuge at 4000 rpm for 20 min, take out and scrape off the excess solution.

[0061] (4) Place the mold (with solution loaded) after centrifugation in a drying cabinet without wind for 80 hours with relative humidity set to 6%. After drying, use tweezers to gently pick out the microneedle. If the substrate is smooth and free of bubbles and the needle tip is intact and uniform in shape, the drying and demolding is successful.

[0062] Example 3

[0063] A method for preparing Repsox-loaded microneedles, comprising the following steps:

[0064] (1) Preparation of needle tip solution

[0065] During the preparation process, the ambient temperature was always maintained at 22°C; deionized water was prepared, polyvinyl alcohol 0588 was added while stirring, and the mixture was heated in a water bath at 92°C until completely dissolved, and the mixture was cooled to room temperature and Repsox solution was added and mixed evenly, followed by addition of hyaluronic acid powder (molecular weight ≥ 2 million), and continuous stirring was performed to allow the mixture to fully dissolve.

[0066] The volume percentage concentration of the polyvinyl alcohol 0588 in the needle tip solution is 3%; the volume percentage concentration of the hyaluronic acid in the needle tip solution is 10%; and the final concentration of the Repsox in the needle tip solution is 500 μM.

[0067] (2) Preparation of substrate solution

[0068] Prepare deionized water, add polyvinyl alcohol 1797 while stirring, and heat in a water bath at 93°C until completely dissolved. Cool to room temperature, add doxycycline hydrochloride powder, and stir to fully dissolve.

[0069] The volume percentage concentration of the high-polymerization-degree and high-alcoholysis-degree polyvinyl alcohol in the substrate solution is 16%; the mass percentage concentration of the antibacterial agent in the substrate solution is 0.1%;

[0070] (3) Add the needle tip solution to the PDMS mold, centrifuge at 4200 rpm for 23 min, scrape off the excess needle tip solution after centrifugation, recycle it, solidify it for 45 s, then add the substrate solution, centrifuge at 3800 rpm for 18 min, take out and scrape off the excess solution.

[0071] (4) Place the mold (with solution loaded) after centrifugation in a drying cabinet without wind for 72 hours with relative humidity set to 5%. After drying, use tweezers to gently pick out the microneedle. If the substrate is smooth and free of bubbles and the needle tip is intact and uniform in shape, the drying and demolding is successful.

[0072] Experimental Example 1

[0073] Single factor experiment

[0074] Based on the preparation method of Example 3, the effects of different concentrations of polyvinyl alcohol (LPVA) and hyaluronic acid (HA) on the quality of microneedles during the preparation of the needle tip solution were investigated. The dissolution of HA after adding it in step (1), the addition of the needle tip solution into the PDMS mold in steps (3) and (4), the removal of bubbles after centrifugation, and the morphology of the microneedles after drying and demolding were observed and recorded. The results are shown in Table 1.

[0075] Table 1 LPVA+HA concentration screening results

[0076]

[0077]

[0078] As shown in Table 1, different concentrations of polyvinyl alcohol and hyaluronic acid have a significant effect on the quality of microneedles. If the concentration ratio of polyvinyl alcohol to hyaluronic acid is not set properly, it will also affect the quality of the base and needle tip.

[0079] Experimental Example 2

[0080] Single factor experiment

[0081] Based on the preparation method of Example 3, the effect of different drying parameters on the quality of microneedles during the drying process of step (4) was investigated. One group was placed in a constant temperature windless drying oven at 20°C, 25°C, 30°C, 35°C, and 37°C for drying, and the other group was placed in a constant temperature wind drying oven at 20°C, 25°C, 30°C, 35°C, and 37°C for drying. The drying conditions were observed and recorded, and the quality of the microneedles was tested. The results are shown in Table 2.

[0082] in:

[0083] Grade A: The microneedle quality needle tip is complete, the base is flat, sharp, and has clear edges and corners;

[0084] Grade B: The quality of the microneedle refers to the base having slight cracks, the needle tip is intact, sharp, and has clear edges and corners;

[0085] Grade C: The quality of microneedles refers to severe wrinkling of the base, partial bending of the needle tip, unclear edges and corners, and partial breakage.

[0086] Table 2 Drying conditions screening results

[0087]

[0088] It can be seen from Table 2 that only by selecting appropriate drying parameter settings can good quality microneedles be obtained. Moreover, under the same temperature conditions, windless drying has a better effect than wind drying.

[0089] Experimental Example 3

[0090] The microneedles obtained by the method of Example 3 were tested for appearance, mechanical properties, toxicity and antibacterial properties. The results are as follows:

[0091] 1. Preparation of microneedle morphology

[0092] The hyaluronic acid microneedles prepared using the mold have beautiful morphology. Each microneedle is a square sheet with a smooth surface and a complete and uniform needle tip. The needle tip is translucent, while the substrate is a uniform light yellow color due to the addition of dissolved yellow DH powder (doxycycline hydrochloride). Figure 3: Appearance morphology of hyaluronic acid microneedles; the upper image is a digital camera image, and the lower image is a scanning electron microscope image. Scale bar: 500 μm.

[0093] 2. Mechanical properties of microneedles

[0094] A texture analyzer was used to test the mechanical strength of the entire Repsox-loaded HA microneedles.

[0095] The results are as follows Figure 4 As shown in the figure, the experimental results show that the stress (fracture force) corresponding to the yield point of the microneedle patch is greater than 22 N. According to the experimental measurements and theoretical modeling results of other researchers, the microneedle insertion force increases as a linear function of the cross-sectional area of ​​the needle tip. When the needle tip radius is 30 to 80 μm, the measured insertion force is 0.1 to 3 N (10 to 300 g).

[0096] The radius of the microneedle tip prepared by the present invention is 15 μm. According to the functional relationship, it is inferred that the required insertion force must be less than 0.1 N. The ratio of the breaking force to the insertion force is the safety margin. It is believed that when the ratio is greater than 1, the needle tip can be inserted into the skin without breaking. When the safety margin exceeds 5, it is considered that the needle tip will not break at all when inserted into the skin. The breaking force of a single needle of the present invention is much greater than the insertion force, so the mechanical strength of our microneedle can easily achieve transdermal drug delivery. The mechanical properties of the prepared microneedle are good.

[0097] 3. Microneedle cell viability detection experiment

[0098] Mouse fibroblasts (L929) were cultured in a constant temperature incubator containing 5% CO2 and 37°C until the culture dish was fully grown. The cells were then inoculated in a 96-well plate, with 3000 / 2500 / 2000 cells placed in each well. After 12 hours of incubation, 5×5 microneedle square patches were added for co-culture. Among them, complete culture medium was used as the control group, and only CCK-8 was added as the blank group (Control).

[0099] Subsequently, CCK-8 reagent was added to the well plate on days 1, 2, and 3, respectively, and incubated for 1 hour. The absorbance (OD value) of the solution was measured.

[0100] The cell viability was calculated according to the following formula: cell viability × (%) - [OD (experimental) - OD (blank)] / [OD (control) - OD (blank)] × 100.

[0101] Results: According to the "Biological Evaluation of Medical Devices" (GB16886.5-2017), the cell activity of all groups in microneedle co-culture was greater than 80%, indicating that our hyaluronic acid microneedles had no inhibitory effect on cell activity. Figure 5As recorded in the table, the cell viability of cells co-cultured with BMNs, MNs@DH, MNs@Repsox and MNs@Repsox / DH microneedles at different concentrations for 1 or 3 days, n = 6. NS indicates p ≥ 0.05, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0102] 4. Cytotoxicity experiment of Repsox-loaded hyaluronic acid microneedles

[0103] Test cell proliferation and apoptosis using a cell live-death staining kit. L929 cells were seeded in 24-well plates (5 × 10 4 cells). One group was added with Repsox (dissolved in DMSO) and DMSO (same volume as Repsox), one group was incubated with microneedle patch extract (100 μL) for 24 hours, and one group was incubated with a 5×5 square patch of microneedles for 24 hours. Then, the cells were stained with live and dead cell staining solution (Calcein-AM / PI), incubated in the dark for 30 minutes, and then observed and photographed under a fluorescence microscope.

[0104] Then, the cells were stained with a live-death staining kit after 24 hours of co-culture to evaluate the cytotoxicity of the hyaluronic acid microneedles. From the fluorescence images, it can be seen that the microneedles did not show any toxicity in the co-culture experiment with cells, which meets the requirements of the "Biological Evaluation of Medical Devices" (GBT16886.5-2017) and can be used as transdermal drug delivery medical devices. Figure 6 Cytotoxicity assay (wherein, the Contr group was untreated, the BMNs group was treated with blank microneedles (no Repsox and doxycycline hydrochloride were added), the MNs@repsox group was treated with microneedles with drug-loaded needle tips and no drug-loaded substrate (no doxycycline hydrochloride was added), and the MNs@repsox / DH group was treated with microneedles with drug-loaded substrates and needle tips, all of which were prepared based on the method of Example 3, the same below).

[0105] 5. Antibacterial properties of microneedles

[0106] The antibacterial properties of the microneedles were tested by bacterial plate counting and observation. Staphylococcus aureus (S. aureus) representing Gram-positive bacteria and Escherichia coli (E. coli) representing Gram-negative bacteria were co-cultured with the microneedles for 12 hours, then coated on solid culture medium, placed in a 37°C constant temperature incubator for 12 hours, and then taken out and photographed. The specific method is:

[0107] (1) Purification and activation of bacteria

[0108] We evaluated the antibacterial properties of Repsox-loaded hyaluronic acid microneedles using Staphylococcus aureus (S. aureus) representing Gram-positive bacteria and Escherichia coli (E. coli) representing Gram-negative bacteria. Before purification, we used the plate streak method to inoculate the bacterial solution, placed it in a constant temperature incubator for 20 hours, and picked a single colony to inoculate it in the liquid culture medium. The liquid culture medium inoculated with a single colony was then placed on a constant temperature shaker at 37°C and 100rpm for activation. After 10 hours of activation, highly active purified bacteria were obtained. A small amount of bacterial solution was taken to measure the absorbance, the concentration of the bacterial solution was calculated, and diluted to 1×10 6 CFU is reserved.

[0109] (2) Counting and observation of coated plates

[0110] In a biosafety cabinet, blank hyaluronic acid microneedles (BMNs), microneedles with DH on the substrate (MNS@DH), and microneedles with Repsox on the tip and DH on the substrate (MNs@Repsox / DH) after UV sterilization were immersed in 900 μL of 24-well plates. The blank group only had 900 μL of liquid culture medium, and three parallel groups were added. Then 100 μL of 1×10 6 CFU and mix well, incubate at constant temperature for 12 hours, take out and measure the absorbance, then dilute 100 times, spread on solid culture medium, put it in a 37℃ constant temperature incubator and invert for 12 hours, take out, take pictures and record observations.

[0111] The results are as follows Figure 7 As shown, the plate of the blank microneedle group in the Control group was full of E. coli and S. aureus colonies, while the plate of the drug-loaded microneedle group was as smooth as a mirror, indicating that the drug-loaded microneedle group had a good antibacterial effect (BMNS is a blank microneedle group, and MNS@repsox / DH is a group where both the needle tip and substrate are loaded with drugs).

[0112] Experimental Example 4

[0113] The study on improving skin thickness was conducted using 40-week-old SD rats.

[0114] Divided into Control (control group, blank microneedle treatment) and MNs (experimental group; using the product obtained in Example 3);

[0115] The drugs were administered under anesthesia with a respiratory anesthesia machine for five days, once a day, with one microneedle inserted into the skin at a depth of 600-700μm. The needles were retained for 10 minutes. The animals were killed by dislocation of the neck on the sixth day. The skin tissue at the drug administration site was removed and placed in an embedding box to ensure its elasticity as much as possible. The embedding box containing the skin tissue was immersed in 4% paraformaldehyde and fixed for more than 24 hours. The fixed tissue was dehydrated, embedded, sectioned and stained with hematoxylin-eosin (HE). The hematoxylin stain is alkaline, which mainly stains the chromatin in the cell nucleus and the nucleic acid in the cytoplasm purple-blue; eosin is an acidic dye, which mainly stains the components in the cytoplasm and extracellular matrix red. The HE staining results were imaged using a panoramic scanning microscope. Figure 8 The results showed that the skin thickness and dermal fat tissue of the group using microneedles increased significantly, indicating that the microneedles prepared by the present invention have the technical effect of increasing skin thickness.

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing Repsox-loaded microneedles, characterized in that: The steps include: (1) After mixing water and polyvinyl alcohol with low polymerization degree and low alcoholysis degree, Repsox and hyaluronic acid are added in sequence and mixed to obtain a needle tip solution; (2) mixing water with polyvinyl alcohol with a high degree of polymerization and a high degree of alcoholysis, and then adding an antibacterial agent to obtain a substrate solution; (3) Add the needle tip solution into the mold, centrifuge once, solidify, then add the substrate solution, centrifuge twice, dry, and demold.

2. The method for preparing Repsox-loaded microneedles according to claim 1, characterized in that: In step (1), the volume percentage concentration of the low-polymerization degree and low-alcoholysis degree polyvinyl alcohol in the needle tip solution is 2.5-4.0%; the volume percentage concentration of the hyaluronic acid in the needle tip solution is 7-13%; and the final concentration of Repsox in the needle tip solution is 400-600 μM.

3. The method for preparing Repsox-loaded microneedles according to claim 1, characterized in that: In step (2), the volume percentage concentration of the high-polymerization-degree and high-alcoholysis-degree polyvinyl alcohol in the substrate solution is 14-20%; the mass percentage concentration of the antibacterial agent in the substrate solution is 0.05-0.15%; and the antibacterial agent is doxycycline hydrochloride.

4. The method for preparing Repsox-loaded microneedles according to claim 1, characterized in that: The speed of the first centrifugation in step (3) is 4000-4400 rpm, and the time is 20-26 min; the speed of the second centrifugation is 3500-4000 rpm, and the time is 15-20 min; the molecular weight of the hyaluronic acid is ≥2 million; the model of the low-polymerization-degree and low-alcoholysis-degree polyvinyl alcohol is 0588; the model of the high-polymerization-degree and high-alcoholysis-degree polyvinyl alcohol is 1797.

5. The method for preparing Repsox-loaded microneedles according to claim 1, characterized in that: The curing time in step (3) is 40 to 50 seconds; the drying time is 60 to 80 hours, and the relative humidity is 4 to 6%; and the ambient temperature needs to be maintained at 20 to 24° C. during the preparation of the Repsox-loaded microneedles.

6. The Repsox-loaded microneedles prepared by the method for preparing Repsox-loaded microneedles according to any one of claims 1 to 5.

7. Use of the Repsox-loaded microneedles according to claim 6 in preparing a drug for improving aged skin.

8. Application of Repsox in the preparation of drugs for improving aging skin.

9. Application of Repsox in the preparation of cosmetics for improving skin aging.

10. The use according to any one of claims 7 to 9, characterized in that: The improvement of skin aging is to increase skin thickness and restore skin elasticity; the increase in skin thickness is to increase the proportion of white fat cells; the restoration of skin elasticity is to change elastic fibers from being coarse and few to being thin and many, and the ratio of type III to type I collagen is increased.

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