Hydroxytyrosol-loaded soluble microneedle as well as preparation method and application thereof

Abstract

The invention provides a hydroxytyrosol-loaded soluble microneedle as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. The microneedle comprises a substrate layer and a needle body arranged on the surface of the substrate layer, the needle body comprises 0.01%-20% of hydroxytyrosol and 1%-10% of hyaluronic acid, the molecular weight of the hyaluronic acid is 100-1000 kDa, and the% is mass percentage content w / w or mass volume percentage content w / v. The soluble microneedle is mild and controllable in preparation process, does not need high temperature, is simple in preparation and use method, and is green, safe and pollution-free; the matrix material is simple, no organic solvent is used, only water is used as a solvent and an adhesive, and no extra cross-linking agent needs to be added; the hydrogel is good in needle forming property, good in biocompatibility, high in safety, high in mechanical strength, excellent in transdermal drug delivery effect and capable of remarkably improving skin photoaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a soluble microneedle loaded with hydroxytyrosol, and a preparation method and application thereof. Background Art

[0002] Skin photoaging refers to the aging and degradation phenomena that occur in the skin under the influence of various factors such as ultraviolet rays, hormones in the body, and inflammatory reactions. Long-term exposure to ultraviolet rays is the main cause of photoaging.

[0003] Currently available photoaging treatment methods mainly include externally applied skin care products, injection of hyaluronic acid or botulinum toxin, laser and photon technologies, etc. Although these methods can improve the appearance of the skin to a certain extent, such as reducing wrinkles and age spots, they cannot prevent the real photoaging process, and there may also be side effects such as irritation.

[0004] Transdermal drug delivery is a method for treating photoaging, but the absorption rate of topical drugs is low and the effect is not ideal. As a physical penetration enhancement method, microneedles can improve the transdermal absorption efficiency of drugs and are widely used in the treatment of skin diseases, such as acne marks, pigment deposition, wrinkles and aging. The advantages of microneedle treatment include: low risk, easy to operate, non-invasive, no drugs or surgical means, and high patient compliance. It breaks through the skin barrier, penetrates the stratum corneum, avoids contact with epidermal nerves and blood vessels, realizes efficient and rapid drug delivery, and has no gastrointestinal irritation and first-pass liver effect.

[0005] Some microneedles capable of treating photoaging are provided in the prior art. For example, patents CN118717641A and CN11468134A respectively provide a microneedle that can be used to treat photoaging, and use gelatin, sialic acid and modified resveratrol, collagen, etc. to make the needle body. However, due to the limitations of material properties, drug delivery efficiency and single action mechanism, the above microneedles are difficult to meet the multi-dimensional treatment needs of photoaging.

[0006] Therefore, there is an urgent need for a new type of microneedle patch as an efficient, low recurrence rate, high safety, non-invasive and painless photoaging treatment method. Summary of the Invention

[0007] In order to solve the above problems, the present application aims to provide a soluble microneedle loaded with hydroxytyrosol, with hydroxytyrosol at a relatively high concentration as the main active drug, without the need to add a cross-linking agent, the preparation process is mild, green and safe, can effectively improve skin photoaging, and has broad prospects in the field of medical beauty repair applications.

[0008] On the one hand, the present application provides a soluble microneedle, comprising a base layer and a needle body disposed on the surface of the base layer, wherein the needle body contains 0.01% to 99.99% of hydroxytyrosol, and the % is mass percentage w / w or mass-volume percentage w / v.

[0009] In one embodiment, the content of hydroxytyrosol in the needle body is 0.01% to 50%, preferably 0.01% to 30%.

[0010] In one embodiment, the needle body includes 0.01% to 20% of hydroxytyrosol and 1% to 10% of hyaluronic acid, and the molecular weight of the hyaluronic acid is 100 to 1000 kDa.

[0011] In one embodiment, the content of hydroxytyrosol in the needle body is 0.01% to 15%.

[0012] In one embodiment, the content of hyaluronic acid is 3% to 8%, and the molecular weight is 150 to 300 kDa.

[0013] In one embodiment, the solvent of hydroxytyrosol in the needle body is an aqueous solvent, and the aqueous solvent is selected from one or more of water, PBS buffer solution, and PVA aqueous solution.

[0014] Optionally, the water can be ultrapure water, deionized water, or distilled water.

[0015] In a preferred embodiment, the needle body part is made by dissolving hydroxytyrosol and hyaluronic acid in an aqueous solvent.

[0016] It can be understood that the preparation raw materials of the microneedles of the present application, in addition to hydroxytyrosol that mainly plays an active role, also include materials known to be used for preparing microneedles as a matrix. For example, the base layer can be selected from flexible polymer materials as a matrix, so that the microneedle patch can be deformed arbitrarily to better fit the skin; the needle body can use natural polymer materials with good biocompatibility and high safety as a matrix, and is required to have a certain mechanical strength, so that the needle body layer can effectively pierce the skin site to form a drug delivery channel.

[0017] In one embodiment, the base layer of the microneedle is a polymer matrix material, and the polymer matrix material is selected from one or several of hyaluronic acid, chitosan, silk fibroin, or polylactic acid, polyethylene glycol, polycaprolactone, polyurethane, polyglycolic acid, polyhydroxybutyrate, polyvinyl alcohol, polyvinylpyrrolidone, poly(lactic acid-glycolic acid) copolymer, poly(lactic acid-caprolactone) copolymer, or poly(β-hydroxybutyrate-hydroxyvalerate).

[0018] In one embodiment, the polymer matrix material of the base layer is polyvinylpyrrolidone PVP. For example, it can be PVPK12, PVPK15, PVPK17, PVPK25, PVPK30, PVPK45, PVPK60, PVPK80, PVPK90, PVPK120, and preferably PVPK90.

[0019] In one embodiment, the base layer is made by dissolving PVPK90 in an aqueous solvent, where the mass percentage of PVPK90 is 5% - 30%, preferably 15% - 25%.

[0020] In one embodiment, in the microneedle, the thickness of the base layer is 1 - 10 mm.

[0021] In one embodiment, the height of the needle body is 500 - 1000 μm, and the bottom width is 200 - 500 μm.

[0022] In one embodiment, the base layer of the microneedle can be square, with a side length of 10 - 20 mm and a thickness of 1 - 5 mm.

[0023] In one embodiment, the needle body of the microneedle can be a regular square pyramid structure, with a height of 800 μm, a bottom width of 360 μm, and the distance between the tips of adjacent microneedles is 720 μm.

[0024] On the other hand, the present application provides a method for preparing the soluble microneedle, and the method includes:

[0025] Step 1: After dissolving hydroxytyrosol and hyaluronic acid in an aqueous solvent, inject them into a microneedle mold to form the needle body;

[0026] Step 2: After dissolving the polymer matrix material in an aqueous solvent, inject it into the microneedle mold of Step 1 to form the base layer;

[0027] Step 3: Dry the microneedle mold of Step 2, peel off the mold, and obtain the soluble microneedle.

[0028] In one embodiment, the polymer matrix material is polyvinylpyrrolidone.

[0029] In one embodiment, both Step 1 and Step 2 use heating for dissolution, where the heating temperature in Step 1 is 50 - 60 °C, and the heating temperature in Step 2 is 90 - 95 °C.

[0030] In one embodiment, after injecting the solution into the mold, place the mold in a negative pressure environment for defoaming, and the negative pressure condition is to place it at -0.7 MPa for 15 min.

[0031] In one embodiment, in step three, the mold is placed in a constant temperature incubator for drying, and the drying condition is drying at 37 °C for 12 h.

[0032] On the other hand, the present application provides the use of the soluble microneedles or the microneedles prepared by the method in the preparation of transdermal drug delivery preparations for treating skin photo-damage, antioxidant, anti-wrinkle, and anti-aging.

[0033] In one embodiment, the transdermal drug delivery preparation includes a transdermal drug, a skin repair dressing, an implant, a biomaterial, a medical device or a medical aesthetic product.

[0034] The present application has at least the following beneficial effects:

[0035] 1. The microneedles provided by the present application are made of hyaluronic acid, hydroxytyrosol and polyvinylpyrrolidone as raw materials, with simple composition and good biocompatibility. No additional cross-linking agent is required during the preparation process. They are inserted into the skin in the form of a microneedle patch, and can be degraded after playing a therapeutic role at the lesion site, which is green, safe and pollution-free.

[0036] 2. The microneedles provided by the present application introduce hydroxytyrosol into the tip component. As a natural antioxidant, hydroxytyrosol can exert antioxidant capacity, scavenge free radicals, effectively inhibit the generation of reactive oxygen species, protect cells from oxidative damage, and increase the content of hyaluronic acid and collagen, thereby improving skin photoaging symptoms, such as skin relaxation, increased wrinkles, age spots, etc. Moreover, the in vitro and in vivo experimental results show that the hydroxytyrosol soluble microneedles can significantly improve the skin state of photoaged mice, making the skin smooth, elastic, with fewer wrinkles and reduced relaxation. Compared with sunscreen, it can carry out antioxidant and stimulate skin repair while preventing ultraviolet damage, and the effect is more comprehensive.

[0037] 3. The microneedles provided by the present application, after introducing hydroxytyrosol, select hyaluronic acid and polyvinylpyrrolidone with specific content and molecular weight as matrix components, which can better balance the needle-forming property, solubility and mechanical properties, so as to better deliver drugs to the treatment area and promote skin absorption of drugs. At the same time, the skin wound caused after microneedle patch administration can be re-closed within 15 minutes, the skin damage is reversible, the harm is small, and it can recover quickly.

[0038] 4. The microneedles provided in this application, compared with injecting hyaluronic acid, botulinum toxin, etc., have no risks of trauma and infection, are safe, efficient, portable, fast, painless, non-invasive, easy to operate, can greatly reduce the pain brought by treatment, and have a high acceptability among patients; compared with applying medicaments, the microneedle technology can penetrate the stratum corneum of the skin, can deliver the antioxidant hydroxytyrosol to the deep layer of the skin, can be naturally degraded compared with solid microneedles, has a larger drug loading capacity compared with coated microneedles, has a simpler preparation process compared with hollow microneedles, has good needle formation, and has good mechanical properties; the preparation process is controllable, the drug loading capacity is stable, and the process is stable and feasible, with low cost, and is suitable for industrial-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The drawings here are incorporated into the specification and form a part of this specification, showing embodiments in line with the present invention, and are used together with the specification to explain the principles of the present invention.

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required to be used in the description of the embodiments or the prior art. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. In the drawings:

[0041] Figure 1 is a schematic diagram of the preparation process of the microneedles in this application;

[0042] Figure 2 is the template mold (A) diagram used in the preparation of the microneedles in this application and the appearance morphology (B) diagram of the microneedles;

[0043] Figure 3 is the scanning electron microscope (SEM) diagram of the overall array (A, B) and a single microneedle (C) of the microneedles in this application;

[0044] Figure 4 is a schematic diagram of the detection results of the drug content of the microneedles in this application, where Figure A is the high-performance liquid chromatography diagram of the hydroxytyrosol standard product, and Figure B is the high-performance liquid chromatography diagram of the microneedle sample;

[0045] Figure 5 is the pressure-strain curve diagram of the microneedles in this application;

[0046] Figure 6 is a schematic diagram of the drug delivery process of the microneedles in this application acting on the separated back skin of a mouse;

[0047] Figure 7 is a schematic diagram of the result of H&E staining of the skin section after the microneedle patch in this application is inserted;

[0048] Figure 8 is a schematic diagram of the result of trypan blue staining of the skin after the microneedle patch in this application is inserted;

[0049] Figure 9 It is a schematic diagram of the wound healing process within 15 minutes after the microneedles of this application are pressed on the surface of mouse skin;

[0050] Figure 10 It is a graph showing the survival rate of human dermal fibroblasts under the action of different concentrations of hydroxytyrosol in the microneedles of this application;

[0051] Figure 11 It is a graph showing the survival rate of human dermal fibroblasts in different treatment groups;

[0052] Figure 12 It is a graph of the ROS fluorescence results of human dermal fibroblasts in different treatment groups. Among them, Figure A is the Normal group, Figure B is the Model group, Figure C is the HA MNs group, and Figure D is the HT MNs group;

[0053] Figure 13 It is a graph of the ROS content of human dermal fibroblasts in different treatment groups;

[0054] Figure 14 It is a graph of the β-galactosidase staining results of human dermal fibroblasts in different treatment groups. Among them, Figure A is the Normal group, Figure B is the Model group, Figure C is the HA MNs group, and Figure D is the HT MNs group;

[0055] Figure 15 It is a graph of the β-galactosidase activity of human dermal fibroblasts in different treatment groups;

[0056] Figure 16 It is a graph of the treatment effect of different treatment groups on ultraviolet-induced photoaged mice;

[0057] Figure 17 It is a graph of the appearance score of mouse skin after different treatments;

[0058] Figure 18 It is an H&E staining graph of the wound on the back skin of mice after different treatments;

[0059] Figure 19 It is a graph of the epidermal thickness of mouse skin tissue sections after different treatments;

[0060] Figure 20 It is a Masson staining graph of the wound on the back skin of mice after different treatments;

[0061] Figure 21 It is a graph of the collagen fiber content of mouse skin tissue sections after different treatments;

[0062] Figure 22 It is a graph recording the weekly body weight changes of mice after different treatments;

[0063] Figure 23These are the H&E staining diagrams of the major organs of mice after different treatments. Detailed implementation manners

[0064] In order to more clearly illustrate the overall concept of this application, the following will be described in detail by way of examples. In the following description, a large number of specific details are given to provide a more thorough understanding of this application. However, it is obvious to those skilled in the art that this application can be implemented without one or more of these details. In other examples, in order to avoid confusion with this application, some well-known technical features in the art are not described.

[0065] In the embodiments of the present invention, the production processes, experimental methods or detection methods involved, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the art, and are very clear and definite in the relevant application fields. Those skilled in the art can understand the conventional process steps according to the name and apply the corresponding equipment, and implement them under conventional conditions or conditions recommended by the manufacturer.

[0066] There are no special restrictions on the sources of various instruments, equipment, raw materials or reagents used in the embodiments of the present invention. Unless otherwise specified, they can all be obtained from conventional commercial channels or prepared according to the conventional methods well-known to those skilled in the art.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field of the present invention. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0068] The information on the main reagents and instruments involved in the following examples is shown in Table 1:

[0069] Table 1

[0070]

[0071]

[0072] Example 1: Preparation process of hydroxytyrosol microneedles

[0073] This example provides a preparation method for soluble microneedles loaded with hydroxytyrosol, which specifically includes the following steps:

[0074] 1. Preparation of matrix solution: Weigh 10 g of hydroxytyrosol and 5 g of hyaluronic acid (with a molecular weight of 200 kDa), place them in 85 g (i.e., 85 mL) of distilled water, heat to 50 - 60 °C, and stir until completely dissolved to obtain a needle matrix solution with a mass percentage of hydroxytyrosol of 10%.

[0075] 2. Preparation of microneedle mold: Use a PDMS microneedle mold. The needle body of the mold is designed as a regular square pyramid structure, with a microneedle height of 800 μm, a bottom width of 360 μm, a distance between adjacent microneedle tips of 720 μm, an array number of 15×15, and a needle distribution area of 10.8×10.8 mm 2 , and the overall patch size is 14.5×14.5 mm 2 , and the groove depth (i.e., the thickness of the base layer) is 2 mm.

[0076] 3. Filling the mold: Drop the prepared needle matrix solution into the PDMS microneedle mold to form a needle body layer, and then place the mold in a negative pressure environment of -0.7 MPa for 15 minutes to eliminate the bubbles in the solution.

[0077] 4. Preparation of the base layer: Weigh 10 g of polyvinylpyrrolidone PVPK90, place it in 90 g (i.e., 90 mL) of distilled water, heat to 90 - 95 °C to dissolve, obtain a base layer solution with a PVP mass percentage of 10%, and then add the base layer solution to the PDMS microneedle mold that has been filled with the needle matrix solution until it is flush with the highest surface of the mold to form the base layer.

[0078] 5. Drying and forming: Place the filled PDMS microneedle mold in an incubator at 37 °C for drying for 12 hours. After drying, peel off the mold to obtain a soluble microneedle array.

[0079] Using the above method, while keeping the mass percentages of hyaluronic acid in the needle body layer and PVP in the base layer unchanged, microneedles with mass percentages of hydroxytyrosol in the needle body layer of 0.1%, 0.5%, 1%, 5%, 10%, and 15% are prepared, and are respectively denoted as microneedles 1#, 2#, 3#, 4#, 5#, and 6#.

[0080] Among them, the schematic diagram of the preparation process of the above microneedles can be referred to Figure 1 . The mold used in step 2 is as shown in Figure A of Figure 2 , and the microneedles obtained after peeling off the mold in step 5 are as shown in Figure B of Figure 2 .

[0081] Example 2: Optimization of the microneedle preparation method

[0082] In this example, the prepared microneedles were comprehensively evaluated with needle formation, mechanical strength, and the amount of bubbles as evaluation indicators. The effects of solvent type, tip material composition, backing material ratio, vacuum time, and drying method on the performance of microneedles were systematically investigated.

[0083] 1. Effect of hydroxytyrosol content on needle formation of microneedles

[0084] In this example, a kind of microneedles was prepared. Its preparation method was roughly the same as that of the 5# microneedles in Example 1, except that the mass percentage of hydroxytyrosol in the needle body was 0%, denoted as D1#; the mass percentage of hydroxytyrosol in the needle body was 20%, denoted as D2#.

[0085] Results: D1# had good needle formation, but its mechanical hardness was lower than that of Example 1; D2# had good needle formation, but due to excessive drug loading, it was prone to breakage and had poor mechanical strength.

[0086] 2. Effect of hyaluronic acid on the solubility of microneedles

[0087] In this example, a kind of microneedles was prepared. Its preparation method was roughly the same as that of the 5# microneedles in Example 1, except that the molecular weight of hyaluronic acid was 1200 kDa and the mass percentage was 5%, denoted as D3#; the molecular weight of hyaluronic acid was 200 kDa and the mass percentage was 10%, denoted as D4#.

[0088] Results: The viscosity of the needle body matrix solution of D3# and D4# was too high to effectively fill the mold, and the needle formation of the microneedles was lower than that of Example 1.

[0089] 3. Effect of microneedle components on the effect of hydroxytyrosol microneedles

[0090] In this example, a kind of microneedles was prepared. Its preparation method was roughly the same as that of the 5# microneedles in Example 1, except that the matrix layer component used sodium carboxymethylcellulose to replace hyaluronic acid, denoted as D5#.

[0091] Results: The compatibility between hydroxytyrosol and sodium carboxymethylcellulose in the matrix solution of D5# was poor, causing the water-soluble sodium carboxymethylcellulose to precipitate and unable to form needles, that is, the needle formation of the microneedles was lower than that of Example 1.

[0092] 4. Effect of polyvinylpyrrolidone content on the performance of hydroxytyrosol microneedles

[0093] In this example, a kind of microneedles was prepared. Its preparation method was roughly the same as that of the 5# microneedles in Example 1, except that the base layer component used hyaluronic acid to replace PVPK90, denoted as D6#; the base layer component used 20% of PVPK90, denoted as D7#.

[0094] Results: The integrity between the basal layer and the needle body layer of D6# is good, but its mechanical hardness is lower than that of Example 1; the integrity between the basal layer and the needle body layer of D7# microneedles is poor, and the needle-forming property is lower than that of Example 1.

[0095] 5. Other influencing factors in the preparation process of microneedles

[0096] In this example, a kind of microneedles was prepared. The preparation method was roughly the same as that of the 5# microneedles in Example 1, except that the vacuum defoaming time was 5 min, denoted as D8#; the drying method was natural air drying, denoted as D9#.

[0097] Results: Serious bubble residues were found in D8# microneedles, and the needle-forming property was lower than that of Example 1; the drying of D9# microneedles was uneven, and the needle-forming property was lower than that of Example 1.

[0098] The scoring of needle-forming property, mechanical strength and bubble amount of the above examples is shown in Table 2:

[0099] Table 2

[0100] Example Needling property (0 - 5 points) Mechanical strength (0 - 5 points) Amount of bubbles (0 - 5 points) Example 1 (1# - 6#) 5 5 5 D1# (without hydroxytyrosol) 5 4 5 D2# (high - concentration hydroxytyrosol) 5 1 5 D3# (high - molecular - weight hyaluronic acid) 0 0 0 D4# (high - concentration hyaluronic acid) 0 0 0 D5# (sodium carboxymethyl cellulose) 0 0 0 D6# (without PVP) 5 3 5 D7# (high - concentration PVP) 5 2 3 D8# (short vacuum - pumping time) 2 5 0 D9# (natural air - drying) 3 3 2

[0101] Example 3: Performance detection of hydroxytyrosol microneedles

[0102] In this example, the performance of the hydroxytyrosol microneedles prepared in Example 1 was detected, including the following specific indicators and detection methods:

[0103] 1. Morphological characterization of microneedles

[0104] SEM was used to analyze the morphology of the microneedle patch to determine the shape and size of the needles. Taking the 5# microneedles in Example 1 as an example. The microscopic morphologies of the whole and single needle bodies of the prepared microneedles under the scanning electron microscope are as Figure 3 shown.

[0105] It can be Figure 3 seen that the preparation method of Example 1 successfully prepared microneedles with the same shape and size as the mold, and had good needle-forming property and formability.

[0106] 2. Determination of drug loading of microneedles

[0107] HPLC was used to analyze the content of hydroxytyrosol in the microneedles. The specific parameters are as follows:

[0108] Chromatographic conditions: Chromatographic column: AQ-C18 (4.6x150 mm, 5μm); Detector wavelength: 276 nm; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Column temperature: 35 °C; Running time: 20 min; Running mode: Isocratic elution.

[0109] Reagents and Solutions: Glacial acetic acid: Analytical reagent; Methanol: Chromatographic reagent; Mobile phase: Mobile phase A: Mobile phase B = 80:20; Mobile phase A: Pipette 1.0 mL of acetic acid into 1000 mL of water to prepare 0.1% acetic acid in water, filter through a 0.22 μm filter membrane for later use; Mobile phase B: Methanol; Diluent: Mobile phase.

[0110] Solution Preparation: Standard solution: Weigh 0.1 g of HT and place it in a 100 mL volumetric flask. Add mobile phase to make up to 100 mL, sonicate to dissolve, cool to room temperature, and prepare a test solution containing 1 mg / mL of HT. Filter through a 0.22 μm organic filter membrane, and take the subsequent filtrate and place it in an injection vial. Test solution: Take one 5# microneedle (drug loading 10%) from Example 1 and place it in a 50 mL beaker. Add 10 mL of diluent solution, sonicate to dissolve the needle tip, remove the base, cool to room temperature, and prepare a test solution. Filter through a 0.22 μm organic filter membrane, and take the subsequent filtrate and place it in an injection vial.

[0111] Injection Procedure: Pipette 10 μL each of the standard solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram for 20 min.

[0112] Expression of Analysis Results: Based on the peak time and peak area in the HPLC chromatogram as the judgment basis, compare the analysis results with the standard product chromatogram. The obtained results are as Figure 4 shown. From Figure 4 the results, it can be seen that the microneedle body contains hydroxytyrosol, and the drug loading of the microneedle is 6.215 mg, which is close to the theoretical design value.

[0113] 3. Pressure Deformation Test of Microneedles

[0114] Use a universal tensile testing machine to measure the mechanical strength of the microneedles. The specific operation is as follows:

[0115] Place the microneedle horizontally on a stainless - steel plane with the needle tip perpendicular upward. Use a pressure sensor to move vertically downward at a constant speed (1 mm / s), apply an axial force orthogonal to the array axis to the microneedle and gradually increase the force. Stop applying pressure after reaching the preset height, and continuously record the compression displacement and the change in the pressure borne by the microneedle during the compression process. The obtained results are as Figure 5 shown.

[0116] From Figure 5From the results in , it can be seen that the data recording of the universal testing machine shows that when the compression displacement is 0.6 mm, the high-concentration hydroxytyrosol (100 mg / mL, that is, the needle body part is 10% hydroxytyrosol + 5% hyaluronic acid (200 kDa)) microneedles can withstand an axial force of 12 N, and the low-concentration hydroxytyrosol microneedles (50 mg / mL, that is, the needle body part is 5% hydroxytyrosol + 5% hyaluronic acid (200 kDa)) can withstand an axial force of 10 N without breaking. When the displacement of the hyaluronic acid microneedles (that is, the needle body part is 5% hyaluronic acid (200 kDa)) is 2 mm, they can only withstand an axial force of 4 N and break, proving that the mechanical strength of the hydroxytyrosol microneedles is higher than that of the hyaluronic acid microneedles. The addition of hydroxytyrosol can significantly increase the hardness of the microneedles and reduce the deformation degree of the microneedles caused by external forces.

[0117] 4. Detection of the puncture ability of microneedles

[0118] The specific operation is as follows: Wipe the moisture on the surface of the mouse skin with filter paper, and then make the stratum corneum of the skin face upward. After inserting the No. 5 microneedles (drug loading 10%) in Example 1, immediately fix the punctured skin with 4% paraformaldehyde. Remove the microneedles, section the skin and stain it with hematoxylin-eosin (H&E). After wiping off the excess dye, the appearance of the needle hole array on the skin can be used to determine the puncture performance of the microneedles. Then, study the penetration depth of the microneedles in the skin through tissue sections. Observe the skin structure and penetration depth using a microscope.

[0119] Among them, the puncture process is as Figure 6 shown, and the staining result is as Figure 7 shown.

[0120] From Figure 7 the histological changes of the skin observed microscopically in , it can be seen that after inserting the No. 5 microneedles (drug loading 10%) of Example 1 into the skin, the microneedles have an appropriate penetration depth into the skin and form micropores that can supply drugs.

[0121] 5. Transdermal performance test of microneedles

[0122] The specific operation is as follows: Take a female ICR mouse. After the mouse is anesthetized, most of the back hair of the mouse is removed with hair removal cream. After sacrificing the mouse, remove the back skin, strip the adipose tissue and fascia, wash it repeatedly with normal saline, dry it with filter paper, wrap it with tin foil paper, and then put it into a -80°C refrigerator for later use. Take out the skin wrapped with tin foil paper from the -80°C refrigerator, soak it in normal saline for 30 min and then use it. After drying the mouse skin with filter paper, place it on a clean tabletop, vertically press the No. 5 hydroxytyrosol microneedles (drug loading 10%) prepared in Example 1 on the mouse skin, remove the microneedles after 10 min, stain them with trypan blue for 5 min, and wash the excess solution with PBS buffer. Observe and take pictures of the number of holes with a camera, and calculate the penetration efficiency through the formula "penetration efficiency = number of stained holes / number of needle holes".

[0123] The obtained results are as Figure 8 shown. From the results in Figure 8 , it can be calculated that its penetration efficiency is 90 ± 5%, indicating that after the microneedle patch of Example 1 is inserted into the skin, the staining rate of the skin pinholes is high, and the microneedles have good puncture ability.

[0124] 6. Microneedle wound healing experiment

[0125] The specific operation is as follows: To evaluate skin recovery and irritation, the wound healing of microneedles loaded with hydroxytyrosol (No. 5 microneedles prepared in Example 1) puncturing the skin of mice was recorded by continuous video, and a screenshot was taken every 60 seconds. The results are as Figure 9 shown.

[0126] From the results in Figure 9 , it can be seen that the small pores on the skin gradually recover over time after the action of the microneedles, and no irritation reactions such as erythema and swelling occur during the observation period. The micropores generated by the insertion of the microneedles close again after 15 minutes, proving that the damage caused by the microneedles to the skin is reversible and the recovery is good.

[0127] 7. Cytotoxicity evaluation of microneedles

[0128] The No. 5 microneedles prepared in Example 1 were immersed in serum-free medium to prepare extraction media with different concentrations (0 - 9 mg / mL). The extraction media were co-incubated with HSF (human skin fibroblasts) cells, and the cell viability was measured using the cck-8 method. The obtained results are as Figure 10 shown.

[0129] From the results in Figure 10 , it can be seen that at concentrations of 0 - 9 mg / mL, the cell survival rate increased significantly, indicating that the hydroxytyrosol microneedles prepared in Example 1 did not show toxicity to skin cells.

[0130] In summary, the soluble microneedle patch has sufficient mechanical strength to pierce the skin and achieve the retention of the needle tip in the skin to release the loaded hydroxytyrosol.

[0131] Example 3: In vitro application study of hydroxytyrosol microneedles in the treatment of photoaging

[0132] In this example, by constructing a UVA-induced HSF cell photoaging model and combining cell viability detection, ROS quantitative analysis, and SA-β-Gal staining, the regulatory effects of hydroxytyrosol microneedles on cell proliferation, oxidative stress, and the aging process were explored.

[0133] HSF cells in the logarithmic growth phase were digested with trypsin to prepare a concentration of 1×10 5A single-cell suspension of cells / mL. The 5# microneedles (drug loading 10%) and D1# microneedles (i.e., the needle body part is only 5% hyaluronic acid (200 kDa)) in Example 1 were respectively dissolved in the culture medium to prepare hydroxytyrosol and hyaluronic acid solutions with a concentration of 9 mg / mL. Four groups were set up: blank control group (Normal), UVA irradiation group (Model), hydroxytyrosol microneedle group (HT MNs, 9 mg / mL), and hyaluronic acid microneedle group (HA MNs, 9 mg / mL). Add 1 mL of culture medium to each well of a 6-well plate and culture for 24 h.

[0134] 1. Effect of microneedles on cell viability

[0135] Aspirate the culture medium, add 100 μL of culture medium to all wells, add 10 μL of cck-8 solution, and incubate in the incubator for 1 h. Measure the absorbance value at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The culture medium without cells was used as the blank group. Calculate the cell survival rate according to the following formula to determine the optimal dose of UVA irradiation. Cell survival rate (%) = (control group - experimental group) / (control group - blank group) × 100%. The results are as Figure 11 shown.

[0136] From Figure 11 the results, it can be seen that the cell survival rate of the model group decreased significantly. After treatment with hydroxytyrosol microneedles, the cell survival rate recovered significantly, which was better than that of the hyaluronic acid microneedle group. It shows that hydroxytyrosol microneedles have a promoting effect on the proliferation of photoaged cells.

[0137] 2. Effect of microneedles on the content of ROS

[0138] Dilute DCFH-DA with PBS at a ratio of 1:1000 to make the final concentration 10 μM. Remove the cell culture medium and add 1 mL of DCFH-DA. Incubate in a 37 °C cell culture incubator for 20 min. Wash the cells three times with PBS to fully remove the DCFH-DA that has not entered the cells. Observe the cells with a fluorescence microscope and capture fluorescence images. The results are as Figure 12 and Figure 13 shown.

[0139] From Figure 12 and Figure 13 the results, it can be seen that UVA irradiation treatment significantly enhanced the green fluorescence in the cells and greatly increased the generation of ROS; compared with the model group, the green fluorescence in the hydroxytyrosol microneedle group was significantly weakened and the ROS content was significantly reduced, which was better than that of the hyaluronic acid microneedle group. It shows that hydroxytyrosol microneedles have an antioxidant effect on photoaged cells.

[0140] 3. Effect of microneedles on the activity of β-galactosidase

[0141] Pour out the culture medium, wash with PBS, add 1 mL of β-galactosidase staining fixative, and fix at room temperature for 15 min. Wash with PBS 3 times, 3 min each time. Add an appropriate amount to each well, and prepare the working solution according to the ratio (A solution 1:B solution 1:C solution 93:X-Gal solution 5), and incubate overnight in a 37 °C water bath. Wash with PBS and observe under the microscope. The results obtained are as Figure 14 and Figure 15 shown.

[0142] From Figure 14 and Figure 15 The results show that the cells in the blank control group showed a small amount of blue staining, the cells in the model group showed obvious blue staining, the blue staining in the hyaluronic acid microneedle group decreased, and the hydroxytyrosol microneedle group did not show blue staining. It indicates that hydroxytyrosol microneedles have an anti-aging effect on photoaged cells.

[0143] Experimental Example 5: In Vivo Application Study of Hydroxytyrosol Microneedles in the Treatment of Photoaging

[0144] In this example, a photoaging model of ICR mice induced by UVA / UVB was constructed to systematically evaluate the in vivo efficacy of HT MNs. The changes in wrinkle depth, epidermal thickness and collagen content were analyzed quantitatively by skin appearance scoring and histological staining; the biosafety of HT MNs was verified by pathological analysis of major organs and monitoring of physiological indexes.

[0145] Experimental protocol: 30 mice were fed at normal temperature (23±2 °C), humidity (55%±10%) and light (12 hours of light / 12 hours of darkness, no ultraviolet irradiation). After feeding for one week, when they adapted to the environment, the mice were anesthetized with an anesthetic, then the hair on the back skin of the mice was removed with a hair clipper, and then the depilatory cream was evenly applied to the back of the mice. After 5-8 min, the depilatory cream was removed and the back of the mice was wiped clean. Except for 5 control groups (Normal) that were not treated, the other 25 were randomly divided into the following 5 groups: model group (Model), hyaluronic acid microneedle group (HA, containing 5% HA), low-concentration hydroxytyrosol microneedle group (HT-L, containing 5% HT), high-concentration hydroxytyrosol microneedle group (HT-H, containing 10% HT), high-concentration hydroxytyrosol topical application group (TM, containing 10% HT). At a distance of 30 cm from the mouse cage, the power was 40 W / m 2, irradiated with UVA and UVB lamps with wavelengths of 365 nm and 308 nm respectively. Irradiate once a day. In the first week, irradiate at a dose of 1 MED, and the irradiation dose increases by 1 MED per week compared to the previous week for 4 consecutive weeks. Obvious thickening, deep and thick wrinkles, and relaxation and other photoaging skin manifestations appeared on the back skin of the model group, indicating successful establishment of the skin photoaging model. Four weeks after ultraviolet modeling, each treatment group was administered drugs for 4 weeks. Each time, the microneedles or the applied drugs were applied to the same part of the back skin of each mouse. On the 28th day of treatment, gross observation was performed on the back skin, and the skin surface texture was graded and recorded. A skin tester was used to evaluate the moisture and elasticity of the mouse skin. After cleaning and disinfecting the test probe, gently place it on the depilated skin surface of the mouse, start the instrument, measure the skin moisture and elasticity according to the set program, and record the measurement data. The mice were sacrificed, and the back skin tissues were fixed with tissue fixative for H&E section staining and Masson staining to compare the muscle fiber morphology and quantity. The obtained results are as Figures 16 - 23 shown.

[0146] As can be seen from Figure 16 and Figure 17 the results in: The skin of the control group mice was smooth, firm, elastic, without wrinkles and without relaxation. After UVA radiation, the skin of the model group mice became dry and rough, with wrinkles and obvious relaxation, indicating successful establishment of the photoaging mouse model. The skin condition of the blank microneedle group mice was not significantly improved compared with the model group, the skin was dry and rough, and there was relaxation and sagging. The skin condition of the low-dose hydroxytyrosol microneedle group mice was improved to a certain extent, the skin wrinkles were reduced compared with the model group, and the relaxation was alleviated. The skin condition of the high-dose hydroxytyrosol microneedle group mice was significantly improved, the skin became smooth and elastic, the wrinkles were significantly reduced compared with the model group, and the relaxation was greatly alleviated.

[0147] As can be seen from Figures 18 - 21 the results in: Compared with the control group, after UVA radiation, the epidermis of the model group and blank microneedle group mice was irregularly thickened, the content of dermal fibers was significantly reduced, a large number of collagen fibers were broken, fragmented and arranged in a disorderly and uneven manner, and some glandular structures were damaged, and obvious skin lesions occurred. The degree of irregular thickening of the epidermis in the low-dose hydroxytyrosol microneedle group was reduced, there was infiltration of inflammatory cells in the connective tissue, and the arrangement of collagen fibers was slightly improved. Compared with other groups, the skin lesions of the high-dose hydroxytyrosol microneedle group mice were significantly improved, the structures of each layer of the skin were clear, the connection between the epidermis and the dermis was tight and the boundary was distinct, the thickening of the epidermis was inhibited, the number of dermal collagen fibers increased significantly, and they were arranged orderly, and the subcutaneous hair follicles and glandular structures were normal. The experiment shows that the soluble microneedle patch described in the present invention has obvious curative effect on the healing of skin photoaging.

[0148] As can be seen from Figure 22 and Figure 23It can be seen that the diet and metabolism of the mice were not adversely affected, and the HT MNs had no adverse effects on the tissue morphology of the key immune organs of the mice, namely the heart, liver, spleen, lungs, and kidneys, and there was no obvious inflammatory stimulation.

[0149] As can be seen from the above embodiments, the hydroxytyrosol microneedles provided by this patent optimize the composite matrix material and intervene in the disease process through multiple pathways, significantly improving the mechanical properties, transdermal efficiency, and comprehensive curative effect, filling the gap in the existing technology, effectively treating photoaging, improving the skin texture, making the skin texture delicate, reducing wrinkles, increasing the skin gloss, promoting the synthesis of skin collagen, enhancing skin elasticity, reducing skin inflammation, and reducing skin damage caused by ultraviolet rays, providing a safe and effective treatment method for photoaged skin.

[0150] The above are only the embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A soluble microneedle, comprising a base layer and a needle body disposed on the surface of the base layer, characterized in that: The needle body comprises 0.01% to 20% of hydroxytyrosol and 1% to 10% of hyaluronic acid, the molecular weight of the hyaluronic acid is 100 to 1000 kDa, and the % is the mass percentage content w / w or the mass volume percentage content w / v.

2. The soluble microneedle according to claim 1, characterized in that: The content of hydroxytyrosol in the needle body is 0.1% to 15%; and / or the content of hyaluronic acid in the needle body is 3% to 8%, and the molecular weight is 150 to 300 kDa.

3. The soluble microneedle according to claim 1, characterized in that: The solvent of hydroxytyrosol in the needle body is an aqueous solvent, and the aqueous solvent is selected from one or more of water, PBS buffer solution, and PVA aqueous solution.

4. The soluble microneedle according to claim 1, characterized in that: The base layer of the microneedle is a polymer matrix material, and the polymer matrix material is selected from one or more of polylactic acid, polyethylene glycol, polycaprolactone, polyurethane, polyglycolic acid, polyhydroxybutyrate, polyvinyl alcohol, polyvinyl pyrrolidone, polylactic acid-glycolic acid copolymer, polylactic acid-polycaprolactone copolymer or poly β-hydroxybutyric acid-hydroxyvaleric acid; preferably, it is polyvinyl pyrrolidone.

5. The soluble microneedle according to claim 4, characterized in that: The base layer contains 15% to 25% of polyvinyl pyrrolidone.

6. The soluble microneedle according to claim 1, characterized in that: In the microneedle, the thickness of the base layer is 1 to 10 mm; and / or the height of the needle body is 500 to 1000 μm, and the bottom width is 200 to 500 μm.

7. The method for preparing the soluble microneedle according to any one of claims 1 to 6, characterized in that: The method comprises: Step 1: dissolving hydroxytyrosol and hyaluronic acid in an aqueous solvent and injecting the dissolved hydroxytyrosol and hyaluronic acid into a microneedle mold to form a needle body; Step 2: dissolving the polymer matrix material with an aqueous solvent and injecting it into the microneedle mold of step 1 to form a base layer; Step 3: Dry the microneedle mold of step 2, peel off the mold, and obtain the soluble microneedle.

8. The method according to claim 7, characterized in that The microneedle mold needs to be subjected to negative pressure defoaming treatment after the solution is injected, and the condition is to be placed at -0.7 MPa for 10 to 30 minutes.

9. Use of the soluble microneedle according to any one of claims 1 to 6, or the microneedle prepared by the method according to claim 7 or 8, in the preparation of a transdermal drug delivery preparation for treating skin photodamage, anti-oxidation, wrinkle removal, and anti-aging.

10. The use according to claim 9, characterized in that: The transdermal drug delivery preparations include transdermal drugs, skin repair dressings, implants, biomaterials, medical devices or medical beauty products.

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