A soluble gel microneedle and its preparation method and application

Abstract

The present invention belongs to the technical field of biomedical microneedles, and discloses a soluble gel microneedle and a preparation method and application thereof. The soluble gel microneedle comprises a backing layer and a needle tip, wherein the backing layer is connected to the bottom of the needle tip; the needle tip and the backing layer both comprise sialic acid and gelatin. The soluble gel microneedle provided by the present invention uses sialic acid and gelatin as the main matrix raw materials, and does not require the addition of additional cross-linking agents. The matrix material is simple, the needle-forming property is good, and the microneedle has good mechanical properties and biocompatibility. After being inserted into the skin, the microneedle can be slowly dissolved, and can significantly improve skin photoaging pigmentation. The soluble gel microneedle has a mild preparation process, does not require high temperature, does not use organic solvents, uses only water as a solvent and an adhesive, and does not require the addition of additional cross-linking agents. The preparation process is controllable, and the preparation and use methods are simple, green, safe, and pollution-free.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical microneedles, and in particular relates to a soluble gel microneedle and a preparation method and application thereof. Background Art

[0002] Skin photoaging causes skin aging and degradation, among which collagen loss is an important cause of skin photoaging. Skin wrinkles, enlarged pores, rough skin, etc. are related to changes in protein content and functional defects. Photoaging pigmentation is caused by the human skin being affected by multiple factors such as ultraviolet rays, hormones in the body, and inflammatory reactions, which causes changes in the microenvironment of melanocytes in the local skin, increases the amount of melanin produced, and presents local color darkening on the skin appearance, i.e., spots, scar areas (acne marks, wounds, surgical incisions), chloasma, and age spots. The uneven skin color caused by pigmentation affects the patient's facial beauty, and then affects their work and social life. With the improvement of living standards, anti-pigmentation has received more and more attention from people, and various freckle removal products and treatment technologies have emerged in an endless stream, but they often cost a lot but have little effect. Therefore, it is of great significance to develop products that improve skin pigmentation.

[0003] The skin is composed of the epidermis, dermis and subcutaneous tissue, among which the epidermis is the main barrier to drug penetration. Traditional syringe needles are long and will pierce the epidermis into the dermis containing nerves and blood vessels, causing pain and bleeding. Microneedles are a safe and effective transdermal drug delivery technology that can effectively overcome the barrier effect of the skin's stratum corneum and increase the transdermal speed and absorption of drugs. Soluble microneedles will not produce solid waste when dissolved in the skin, and have better biocompatibility and drug loading than other types of microneedles. They are currently the most advanced type of microneedle patches. However, the current microneedle patches have complex ingredients, and most require the addition of organic solvents during preparation. Residual organic solvents will impose an additional burden on the skin.

[0004] Therefore, there is an urgent need to provide a microneedle with simple ingredients that can effectively improve skin pigmentation. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a soluble gel microneedle and a preparation method and application thereof. The soluble gel microneedle provided by the present invention has a simple matrix material, good needle-forming property, and can effectively improve skin photoaging pigmentation.

[0006] The invention provides a soluble gel microneedle.

[0007] Specifically, a soluble gel microneedle comprises a backing layer and a needle tip, wherein the backing layer is connected to the bottom of the needle tip; the needle tip comprises sialic acid and gelatin.

[0008] In the above-mentioned soluble gel microneedle, the needle tip is conical, such as a cone, the height of the needle tip is 300-800 microns, and the bottom diameter of the needle tip is 150-400 microns.

[0009] In the above soluble gel microneedle, the backing layer is square.

[0010] In the above soluble gel microneedle, the gelatin is animal gelatin. Preferably, the animal gelatin is selected from at least one of fish gelatin, porcine gelatin and bovine gelatin.

[0011] Preferably, the molecular weight of the gelatin is 10-40 kDa.

[0012] In the above soluble gel microneedle, the needle tip is made of sialic acid, gelatin and water as main raw materials.

[0013] Preferably, the main raw materials include 40%-60% gelatin, 10%-20% sialic acid and 20%-40% water by mass; further preferably, the main raw materials include 40%-60% gelatin, 15%-20% sialic acid and 20%-40% water by mass.

[0014] In the above soluble gel microneedle, for the convenience of preparation, the needle tip and the backing layer both include sialic acid and gelatin.

[0015] In the above-mentioned soluble gel microneedle, the needle tip and the backing layer are made of sialic acid, gelatin and water as main raw materials. Preferably, by mass fraction, the main raw materials include 40%-60% gelatin, 10%-20% sialic acid and 20%-40% water; further preferably, by mass fraction, the main raw materials include 40%-60% gelatin, 15%-20% sialic acid and 20%-40% water.

[0016] In the above-mentioned soluble gel microneedle, the amino and carboxyl groups of the gelatin interact with the carboxyl and hydroxyl groups of sialic acid through hydrogen bonds. The hardness of the needle tip of the soluble gel microneedle prepared by gelatin and sialic acid is 10N-15N. Studies have shown that a minimum force of 5N is required to penetrate the skin. The microneedle provided by the present invention has sufficient mechanical strength and can withstand a force of more than 10N. The microneedle can slowly dissolve after penetrating the skin, and has good thermal stability and can be stored at room temperature (5-35°C) for a long time.

[0017] The present invention also provides a method for preparing the soluble gel microneedle.

[0018] Specifically, the method for preparing the above-mentioned soluble gel microneedle comprises the following steps:

[0019] Gelatin, sialic acid and water are mixed, heated and dissolved to obtain a matrix solution; the matrix solution is added to a microneedle array mold, centrifuged, dried and peeled off to obtain soluble gel microneedles.

[0020] In the above preparation method, the temperature of the heating and dissolving is 50-60°C.

[0021] In the above-mentioned preparation method, the heating and dissolving process is carried out by homogeneous heating and dissolving with the assistance of ultrasound.

[0022] In the above preparation method, the centrifugal speed is 2500-3500 r / min, and the centrifugal time is 5-30 min; preferably, the centrifugal speed is 2800-3200 r / min, and the centrifugal time is 10-30 min.

[0023] In the above preparation method, the drying process is: drying at 20-30°C for 8-48h.

[0024] The present invention also provides the application of the soluble gel microneedle.

[0025] Specifically, the soluble gel microneedle is used in the preparation of a transdermal drug delivery preparation.

[0026] The transdermal preparation can be used to treat and / or improve skin photoaging pigmentation.

[0027] Animal gelatin is a natural polymer with a wide range of sources and low cost. It has the characteristics of extremely low immunogenicity and low rejection reaction. It has been used in food, pharmaceuticals, medical and cosmetics. It has the characteristics of biodegradability and high solubility, and is easily absorbed and utilized by the human body. In addition, gelatin has the ability to repair and moisturize, which can delay and reduce skin aging and wrinkles. Sialic acid is a derivative of neuraminic acid, an acidic amino sugar containing 9 carbon atoms and a pyranose structure. Sialic acid has multiple physiological functions and plays a very important role in regulating the physiological and biochemical functions of the human body. The active hydroxyl group of sialic acid can provide active hydrogen and superoxide anion free radicals (O 2- ) and hydroxyl radicals (-OH), playing an anti-free radical oxidation role. Sialic acid can effectively inhibit the activity of tyrosine hydroxylase and dopa hydroxylase, preventing the formation of melanin. Transdermal delivery of gelatin and sialic acid through microneedles is easier to be absorbed by the skin, improving the effectiveness and safety of skin photoaging.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] (1) The soluble gel microneedles provided by the present invention use sialic acid and gelatin as the main matrix materials and can be formed without adding additional cross-linking agents. The matrix material is simple, the needle-forming property is good, and it has good mechanical and biocompatibility. After being inserted into the skin, it can slowly dissolve and can significantly improve skin photoaging pigmentation.

[0030] (2) The soluble gel microneedles provided by the present invention have a mild preparation process, do not require high temperature, do not use organic solvents, only use water as a solvent and adhesive, and do not need to add additional cross-linking agents; the preparation process is controllable, the preparation and use methods are simple, and the microneedles are green, safe and pollution-free.

[0031] (3) The soluble gel microneedles provided by the present invention have good thermal stability and can be stored at room temperature for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a morphological image of the soluble gel microneedle provided in Example 1;

[0033] Figure 2 is a diagram of the wound healing process formed after the soluble gel microneedle provided in Example 2 was pressed on the surface of mouse skin for 3 minutes;

[0034] Figure 3 is a graph showing the height and dissolution trend of the soluble gel microneedles provided in Example 3 after the microneedles were pressed into the skin of an isolated rat;

[0035] Figure 4 This is a diagram showing the skin color change after the soluble gel microneedles provided in Example 4 were manually inserted into the photoaged pigmented skin of mice. DETAILED DESCRIPTION

[0036] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0037] Unless otherwise specified, the raw materials, reagents or devices used in the following examples or experiments can be obtained from conventional commercial sources or by existing known methods.

[0038] Experiment 1

[0039] The effect of reagent ratio on the formation of gel microneedles was studied. The composition, concentration, and needle formation results of each group of reagents are shown in Table 1.

[0040] Table 1 Composition and concentration of different reagents, state of solution

[0041] Reagent Set Gelatin / sialic acid mass ratio Water content (%) Is it a gel microneedle? Microneedle hardness (N) Ⅰ 55％ / 35％ 10％ no / Ⅱ 60％ / 20％ 20％ yes 15.89 Ⅲ 50％ / 30％ 20％ no / Ⅳ 55％ / 25％ 20％ no / Ⅴ 50％ / 20％ 30％ yes 12.26 Ⅵ 55％ / 15％ 30％ yes 14.38 Ⅶ 40％ / 20％ 40％ yes 11.68 Ⅷ 30％ / 20％ 50％ no /

[0042] According to the data in Table 1, in terms of mass percentage, 40%-60% gelatin, 15%-20% sialic acid and 20%-40% water, the soluble gel microneedles formed in this range have good mechanical properties and needle-forming characteristics, and the hardness of the microneedle tip is greater than 11N. The combination of gelatin and sialic acid, the amino and carboxyl groups in gelatin interact with the carboxyl and hydroxyl groups in sialic acid through hydrogen bonds, and further the water molecules interact with their hydrophilic groups, and can form hydrogel microneedles without adding additional cross-linking agents. Studies have shown that the higher the water content, the lower the viscosity of the reagent, the looser the gel formed, which is not conducive to microneedle formation; the higher the gelatin content, the higher the viscosity of the reagent, the denser the gel formed, and the higher the hardness of the microneedle. However, when the gelatin content is too high (greater than 60%), the microneedles formed are brittle and easy to break, which is not conducive to insertion into the skin and affects use.

[0043] The following is further described by examples.

[0044] Example 1

[0045] A soluble gel microneedle comprises a backing layer and a needle tip, wherein the backing layer is connected to the bottom of the needle tip; both the needle tip and the backing layer comprise sialic acid and gelatin.

[0046] The preparation method of the soluble gel microneedle comprises the following steps:

[0047] (1) 42% gelatin, 18% sialic acid and 40% water were mixed uniformly in proportion by mass, and heated and dissolved at 50° C. with the aid of ultrasound until the mixture was completely dissolved to obtain a matrix solution;

[0048] (2) Add the matrix solution to the microneedle array mold and then centrifuge at 3000 r / min for 10 min;

[0049] (3) The microneedle array mold after centrifugation is then dried at 25° C. for 24 h, and finally the microneedles are peeled off from the mold to obtain soluble gel microneedles.

[0050] After testing, the hardness of the obtained soluble gel microneedle is 13.56N, and it can be stored at room temperature (5-35°C) for 10-12 months.

[0051] The appearance of the soluble gel microneedles was observed, and the experimental results are as follows: Figure 1 As shown, the appearance of the microneedles is uniform. Figure 1 A is a plan view of the soluble microneedle prepared in this example; Figure 1 Middle B is a stereoscopic image of the soluble microneedle; Figure 1 Middle C is a partial image of the back of the microneedle under an optical microscope; Figure 1 D in the middle is a partial picture of the microneedle. Figure 1It can be seen that the microneedle is conical, with a height of 600μm, a bottom diameter of 250μm, and a uniform morphology, which provides a guarantee for the microneedle to penetrate the skin.

[0052] Example 2

[0053] A soluble gel microneedle comprises a backing layer and a needle tip, wherein the backing layer is connected to the bottom of the needle tip; both the needle tip and the backing layer comprise sialic acid and gelatin.

[0054] The preparation method of the soluble gel microneedle comprises the following steps:

[0055] (1) 44% gelatin, 20% sialic acid and 36% water were mixed uniformly in proportion by mass, and heated and dissolved at 55° C. with the aid of ultrasound until the mixture was completely dissolved to obtain a matrix solution;

[0056] (2) Add the matrix solution to the microneedle array mold and then centrifuge at 3000 r / min for 15 min;

[0057] (3) The microneedle array mold after centrifugation is then dried at 25° C. for 36 h, and finally the microneedles are peeled off from the mold to obtain soluble gel microneedles.

[0058] After testing, the hardness of the obtained soluble gel microneedle is 14.28N, and it can be stored at room temperature (5-35°C) for 10-12 months.

[0059] Figure 2 This is a diagram of the wound healing process formed after the soluble gel microneedle prepared in this embodiment was pressed on the mouse skin surface for 3 minutes. After the mouse was euthanized, a square skin tissue was cut from the back, the skin was attached to a clean filter paper, and fixed overnight (20 hours, <24 hours) with at least 20 times the volume of 4% paraformaldehyde. Note: When attaching the skin to the filter paper, use tweezers to gently unfold the skin, neither leaving wrinkles nor pulling too hard to deform the tissue structure. In the experiment, the ex vivo skin of mice with and without microneedle puncture at different times was removed, and then immersed in a tissue fixative for 24 hours to prepare skin pathology sections, and then a microscope was used to observe the skin of mice with and without microneedle puncture at different times. Figure 2 A in the middle shows the appearance of the skin surface after the microneedles are inserted into the skin for different times. Figure 2 B shows the results of skin pathology sections after the microneedles were inserted into the skin for different times. Figure 2 It can be observed that after the microneedles were pulled out of the skin, clear micro-wounds in the shape of the microneedle array were left behind, showing that the stratum corneum of the mouse skin was obviously broken after the microneedles were inserted, which further proved that the microneedles had sufficient mechanical strength to penetrate the skin. After 30 minutes, the skin surface was completely restored and the micro-wounds in the shape of the microneedle array disappeared, meeting the requirements of microneedle treatment.

[0060] The rats were anesthetized and skin was taken out, and then microneedles were inserted into the skin to study the dissolution ability of the thermoreversible supramolecular gel microneedles in the skin. The microneedles prepared in this example were pressed into the skin of the rats for a series of time periods (0-60 min), and then the height and dissolution trend of the microneedles were observed under a microscope. Figure 3 A in the middle shows the dissolution shape of the microneedle before and after microneedle insertion for 0-60 minutes. Figure 3 Figure B shows the height change of the microneedle from 0 to 60 minutes after the microneedle was pressed into the skin. Figure 3 It can be observed that as time goes by, the microneedles gradually dissolve, the needles become rounded, and the height of the microneedles gradually decreases.

[0061] Example 3

[0062] A soluble gel microneedle comprises a backing layer and a needle tip, wherein the backing layer is connected to the bottom of the needle tip; both the needle tip and the backing layer comprise sialic acid and gelatin.

[0063] The preparation method of the soluble gel microneedle comprises the following steps:

[0064] (1) 60% gelatin, 20% sialic acid and 20% water were mixed uniformly in proportion by mass, and heated and dissolved at 55° C. with the aid of ultrasound until the mixture was completely dissolved to obtain a matrix solution;

[0065] (2) Add the matrix solution to the microneedle array mold and then centrifuge at 3000 r / min for 15 min;

[0066] (3) The microneedle array mold after centrifugation is then dried at 25° C. for 24 h, and finally the microneedles are peeled off from the mold to obtain soluble gel microneedles.

[0067] After testing, the hardness of the obtained soluble gel microneedle is 15.36N, and it can be stored at room temperature (5-35°C) for 10-12 months.

[0068] The soluble hydrogel microneedles provided in this embodiment were applied to the photoaging pigmentation site of mice (mouse back skin) to study the therapeutic effect of the soluble hydrogel microneedles on photoaging skin pigmentation. The specific experiment is as follows: the mice were anesthetized with an anesthetic the day before modeling, and then the hair on the mouse back skin was removed with a shaver, and then the depilatory cream was evenly applied to the mouse back. After 5-8 minutes, the depilatory cream was removed and the mouse back was wiped clean. A mouse skin photoaging pigmentation model was established. When the model was used, it was covered with a blackout curtain. The UVB (310nm) lamp was irradiated at a distance of 15 cm from the guinea pig hair removal site, once a day, for 5 minutes, for two weeks. If the skin is severely damaged, the modeling is stopped. Figure 4As shown in A, before modeling, the guinea pig skin was relatively smooth; after modeling, obvious skin photoaging pigmentation appeared, and a large amount of melanin was produced, proving that the photoaging model was successfully established. 20 mice were divided into a normal group, a model group, a gelatin group, a sialic acid group, and a microneedle group, with 4 mice in each group. The model group and the drug administration group were modeled according to the above method. The normal group was not treated. 5 hours after modeling, the drug administration group was administered at the modeling skin site, and then the soluble gel microneedle was manually inserted into the mouse skin, and then fixed with surgical tape to prevent the drug from struggling and falling off, and to ensure that the microneedle was fully dissolved and removed. Two tablets were administered each time, once every other day, and the administration was continued for 14 days. The gelatin group and the sialic acid group prepared the corresponding solution according to the concentration of gelatin and sialic acid in the microneedle, and evenly applied it to the skin photoaging pigmentation site, once every other day, and the administration was continued for 14 days. Finally, the color changes of the back skin of the mice were observed and recorded with a digital microscope.

[0069] The experimental results are as follows Figure 4 As shown, compared with the model group ( Figure 4 Compared with the gelatin group ( Figure 4 C) and sialic acid group ( Figure 4 Middle D) The skin color of mice recovered slightly, while the soluble gel microneedle group ( Figure 4 Middle E) The color of the mouse skin has been significantly restored and is closer to the original color ( Figure 4 Therefore, the in vivo experimental results show that the soluble gel microneedles provided by the embodiments of the present invention can effectively promote the treatment effect of photoaging skin pigmentation.

Claims

1. A soluble gel microneedle, characterized in that: It comprises a backing layer and a needle tip, wherein the backing layer is connected to the bottom of the needle tip; The needle tip is made of sialic acid, gelatin and water; the gelatin is animal gelatin; Calculated by mass, the raw materials include 40%-60% gelatin, 10%-20% sialic acid and 20%-40% water.

2. The soluble gel microneedle according to claim 1, characterized in that: The needle tip is conical, the height of the needle tip is 300-800 microns, and the bottom diameter of the needle tip is 150-400 microns.

3. The soluble gel microneedle according to claim 1 or 2, characterized in that: The hardness of the needle tip is 10N-15N.

4. The method for preparing the soluble gel microneedle according to any one of claims 1 to 3, characterized in that: The following steps are involved: The matrix solution is prepared by mixing gelatin, sialic acid and water and heating to dissolve the mixture; The matrix solution is added to the microneedle array mold, centrifuged, dried, and peeled off to obtain soluble gel microneedles.

5. The preparation method according to claim 4, characterized in that: The temperature of the heating and dissolving is 50-60°C; the speed of the centrifugation is 2500-3500r / min, and the time of the centrifugation is 5-30min; the drying process is: drying at 20-30°C for 8-48h.

6. Use of the soluble gel microneedle according to any one of claims 1 to 3 in the preparation of a transdermal drug delivery preparation for treating skin photoaging pigmentation.

7. Use of the soluble gel microneedle according to any one of claims 1 to 3 in the preparation of a transdermal drug delivery preparation for improving skin photoaging pigmentation.

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