Beta-1, 3 / alpha-1, 3-glucan microneedle as well as preparation method and application thereof

Abstract

The invention provides a preparation method of a beta-1, 3 / alpha-1, 3-glucan microneedle, which comprises the following steps: dissolving low-molecular-weight sodium hyaluronate and high-molecular-weight sodium hyaluronate in a weight ratio of 5: 1 in deionized water, and fully stirring to obtain a hyaluronic acid hydrogel solution with the concentration of weight relative to volume of 20%; adding beta-1, 3 / alpha-1, 3-glucan into the obtained hyaluronic acid hydrogel solution, and fully stirring to obtain a uniform solution; carrying out continuous twice vacuum treatment and bubble pre-removal treatment on the prepared uniform solution; adding the uniform solution into a polydimethylsiloxane female die, and carrying out vacuum treatment and bubble removal treatment twice continuously; shoveling off the solution of the substrate of the polydimethylsiloxane female mold, and adding a pure sodium hyaluronate solution; drying treatment is conducted at the drying temperature within the normal body temperature range of the human body, and the microneedles are stripped from the female die after drying is completed; the microneedle obtained through the steps can meet the requirement of serving as a percutaneous drug delivery instrument.

Description

Technical Field

[0001] The present invention relates to the technical field of microneedles, and in particular to a preparation method and application of β-1,3 / α-1,3-glucan microneedles. Background Art

[0002] Human skin is the largest organ, covering 20 square feet of our body surface area, and has important functions. The skin plays a role like a wall for the human body, but a large number of studies have shown that the skin is not completely impermeable. Transdermal drug delivery is a non-invasive drug delivery system used to replace traditional injection methods. It is a new way to deliver drugs on the skin using the osmotic effect. However, due to the existence of the stratum corneum, a skin barrier, most drugs are isolated from the dense epidermis, and the dose that can penetrate into the skin accounts for a very low proportion of the total drug library, sometimes less than 0.1%. Microneedles are favored for their convenience, portability, miniaturization, low user learning cost, high safety, and low damage to the skin. Microneedle technology uses microscopic needles to deliver drugs through the SC layer to the bottom layer with minimal invasiveness. After years of development, microneedles have evolved from instruments that increase the degree of transdermal drug delivery to innovative medical technologies with applications as the core and multiple application scenarios. In the current stage of scientific research, researchers start from the three main application scenarios of drug delivery, fluid extraction and biological monitoring, and design and manufacture microneedle-based devices to solve problems in specific problem scenarios.

[0003] With the aging of the global population and the increase in the number of patients with chronic wounds, wound management has brought a huge economic burden to society, and the situation tends to worsen further. The microscale needle tips of microneedles can pass through the physical barriers of the wound site, such as clots, scars, and exudates, and release drugs sustainably. Ordinary delivery methods use polysaccharides to degrade EPS, but their effectiveness in killing bacteria is limited, and glucose and enzymatic products may promote bacterial growth and cause the risk of reinfection. Microneedles essentially provide mechanical stimulation for wound healing and tissue regeneration. On the one hand, they stimulate and induce collagen deposition and reorganization through tissue penetration; on the other hand, microneedle arrays can support the insertion area and change the local stress environment.

[0004] As a miniaturized mechanical structure that can be used for transdermal drug delivery, vaccination, extraction, monitoring and beauty, microneedles have very high potential in reducing patient pain, improving compliance, overcoming first-pass effect and precise treatment, making them a hot topic in therapeutic technology research in the past two decades.

[0005] The present invention aims to provide a microneedle that can meet the needs of transdermal drug delivery. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a preparation method and application of β-1,3 / α-1,3-glucan microneedles to meet the requirements of transdermal drug delivery devices.

[0007] To achieve the above-mentioned and other related purposes, the present invention provides a method for preparing β-1,3 / α-1,3-glucan microneedles in a first aspect, comprising: S100, dissolving low molecular weight sodium hyaluronate and high molecular weight sodium hyaluronate in a weight ratio of 5:1 in deionized water, and stirring thoroughly to obtain a hyaluronic acid hydrogel solution with a concentration of 20% by weight relative to volume; S200, adding β-1,3 / α-1,3-glucan to the obtained hyaluronic acid hydrogel solution, and stirring thoroughly to obtain a uniform solution; wherein the mass ratio of hyaluronic acid to β-1,3 / α-1,3-glucan is 10:1.5; S300, performing two consecutive vacuum and pre-bubble removal treatments on the uniform solution prepared in step S200; S400, adding the uniform solution treated in step S300 into a polydimethylsiloxane female mold, and performing vacuum and degassing treatments twice in succession; S500, scraping off the solution at the base of the polydimethylsiloxane negative mold, and adding pure sodium hyaluronate solution; S600, drying the female mold processed in step S500 at a drying temperature within the normal body temperature range of the human body, and peeling off the microneedles from the female mold after drying.

[0008] To implement the above technical solution, a solution formed by mixing two sodium hyaluronates with different molecular weights is used as a base material, so that while the viscosity of the solution is reduced, the hardness of the microneedle body obtained is greatly increased due to the presence of β-1,3 / α-1,3-glucan, thereby improving the structural stability of the microneedle body; the needle base of the microneedle is formed by using a sodium hyaluronate solution without β-1,3 / α-1,3-glucan, which makes the needle base quite flexible and facilitates skin adhesion.

[0009] Furthermore, step S400 also includes: S401, using 3D printing technology to make a microneedle positive mold, and making a polydimethylsiloxane negative mold based on the microneedle positive mold; S402, adding the uniform solution treated in step S300 into the polydimethylsiloxane female mold, and performing vacuum and degassing treatments twice in succession.

[0010] Furthermore, the printing material of the microneedle positive mold is a thermoplastic photosensitive resin.

[0011] To implement the above technical solution, the entire structure of the microneedle positive mold is formed by using 3D layer-by-layer printing technology, which can accurately shape the needle tip of the microneedle.

[0012] Furthermore, the tip size of the microneedle positive mold is 15-20 μm.

[0013] By implementing the above technical solution, the needle tip size meets the requirement of piercing the skin.

[0014] Furthermore, the microneedle positive mold includes a needle base with a double-layer substrate design and a needle body formed on the top of the needle base.

[0015] To implement the above technical solution, the needle base of the microneedle positive mold adopts a double-layer substrate design, which can leave space on the bottom substrate when making the polydimethylsiloxane negative mold, so that the liquid is restricted when making the microneedle.

[0016] Furthermore, the needle body includes a cylindrical lower portion formed at the top of the needle base and a conical upper portion formed at the top of the cylindrical lower portion.

[0017] Furthermore, the height of the lower part of the cylinder is 250 μm, and the height of the upper part of the cone is 200 μm.

[0018] Furthermore, the needle bodies are formed on the needle base in a 15 by 15 square array.

[0019] The above technical solution is implemented to ensure a certain depth-to-width ratio while taking into account the rigidity of the entire needle, so that the needle body of the microneedle can be easily inserted into the skin and the needle body will not bend due to being too thin and long.

[0020] In a second aspect, the present application provides a β-1,3 / α-1,3-glucan microneedle prepared according to any of the above preparation methods.

[0021] By implementing the above technical solution, the drug will not be denatured due to high temperature.

[0022] In a third aspect, the present application provides the use of hyaluronic acid / β-1,3 / α-1,3-glucan microneedles prepared according to any of the above preparation methods as a transdermal drug delivery device.

[0023] By implementing the above technical solution, no organic reagents are used in the preparation process of the transdermal drug delivery device, which can fully meet the biosafety requirements; and the device has sufficient strength and hardness to be inserted into the skin.

[0024] As described above, the preparation method and application of β-1,3 / α-1,3-glucan microneedles provided by the present invention have the following beneficial effects: The present invention provides a method for preparing β-1,3 / α-1,3-glucan microneedles, comprising S100, dissolving low molecular weight sodium hyaluronate and high molecular weight sodium hyaluronate in a weight ratio of 5:1 in deionized water, and fully stirring to obtain a hyaluronic acid hydrogel solution with a concentration of 20% by weight relative to volume; S200, adding β-1,3 / α-1,3-glucan to the obtained hyaluronic acid hydrogel solution, and fully stirring to obtain a uniform solution; wherein the mass ratio of hyaluronic acid to β-1,3 / α-1,3-glucan is in the range of 10:1 to 10:2; S300 , subjecting the homogeneous solution prepared in step S200 to two consecutive vacuum and pre-debubbling treatments; S400, adding the homogeneous solution treated in step S300 to the polydimethylsiloxane negative mold, and subjecting it to two consecutive vacuum and debubbling treatments; S500, scraping off the solution at the base of the polydimethylsiloxane negative mold, and adding pure sodium hyaluronate solution; S600, drying the negative mold treated in step S500 at a drying temperature within the normal body temperature range of the human body, and peeling off the microneedles from the negative mold after drying; the microneedles obtained through the above steps can meet the requirements as a transdermal drug delivery device. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shown is a schematic diagram of the steps of the preparation method of an embodiment of the present invention; Figure 2 Shown is a schematic structural diagram of a microneedle positive mold according to an embodiment of the present invention; Figure 3 It is a schematic diagram showing the structure of the needle body of the microneedle positive mold according to an embodiment of the present invention; Figure 4 Shown is a scanning electron microscope image of a microneedle according to an embodiment of the present invention; Figure 5 Shown is a stress-strain diagram of mechanical testing of an embodiment of the present invention.

[0026] Description of technical feature numbers in the accompanying drawings: 1. Needle base; 2. Needle body; 21. Lower part of cylinder; 22. Upper part of cone. DETAILED DESCRIPTION

[0027] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0028] See also Figure 1-5. It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0029] This application provides a β-1,3 / α-1,3-glucan microneedle, please refer to Figure 1 The present invention provides a method for preparing β-1,3 / α-1,3-glucan microneedles, comprising: S100, dissolving low molecular weight sodium hyaluronate and high molecular weight sodium hyaluronate in a weight ratio of 5:1 in deionized water, and stirring thoroughly to obtain a hyaluronic acid hydrogel solution with a concentration of 20% by weight relative to volume; Specifically, in this embodiment, 1.0 g of low molecular weight sodium hyaluronate (molecular weight MV≈3000) and 0.2 g of high molecular weight sodium hyaluronate (molecular weight MV≈100000) were dissolved in 6 ml of deionized water and stirred for 1 hour to obtain a hyaluronic acid hydrogel solution with a concentration of 20% by weight relative to volume.

[0030] S200, adding β-1,3 / α-1,3-glucan to the obtained hyaluronic acid hydrogel solution, and stirring thoroughly to obtain a uniform solution; wherein the mass ratio of hyaluronic acid to β-1,3 / α-1,3-glucan is 10:1.5; Among them, β-1,3 / α-1,3-glucan is a polysaccharide made from sucrose through fermentation with Rhizobiumpusense, alcohol precipitation, filtration, separation, drying, and crushing. It is composed of glucose molecules alternately connected by β-1,3 and α-1,3 glycosidic bonds, with a clear structure and molecular formula: (C6H10O5)n, n≥615, purity ≥90. This structure gives it unique biological activity and physical properties. The source of β-1,3 / α-1,3-glucan used in the present invention: Sichuan Hetai Xinguang Biotechnology Co., Ltd. uses patented strains of marine microorganisms and sucrose as the main raw material to produce β-1,3 / α-1,3-β-1,3 / α-1,3-glucan with a brand-new structure through biological transformation. It is currently a global first, with an overall level reaching the internationally advanced and domestically leading level. This product is an original achievement achieved with the support of the National Natural Science Foundation key project, and solves the problems of water insolubility, low purity and low activity of traditional β-1,3 / α-1,3-glucan.

[0031] S300, performing two consecutive vacuum and pre-bubble removal treatments on the uniform solution prepared in step S200; Specifically, the prepared uniform solution is placed in a vacuum drying oven, and the bubbles are sucked and pre-removed twice in succession.

[0032] S400, adding the uniform solution treated in step S300 into a polydimethylsiloxane female mold, and performing vacuum and degassing treatments twice in succession; Wherein, step S400 further includes: S401, using 3D printing technology to make a microneedle positive mold, and making a polydimethylsiloxane negative mold based on the microneedle positive mold; S402, the uniform solution after the treatment in step S300 is added to the polydimethylsiloxane female mold, and the vacuum and degassing treatment is performed twice in succession; See also Figure 2-3 Specifically, the positive mold of the microneedle is made of thermoplastic photosensitive resin using 3D printing technology. The printer resolution in this embodiment is 10 μm. The entire structure is formed by printing layer by layer. After the base is formed, the photosensitive resin can be accurately formed at the tip of the microneedle. The size of the needle tip is about 15-20 μm, which can fully meet the requirements of piercing the skin.

[0033] Specifically, the positive mold of the microneedle includes a needle base 1 with a double-layer substrate design and a needle body 2 formed on the top of the needle base 1. The needle body 2 includes a cylindrical lower part 21 formed on the top of the needle base 1 and a conical upper part 22 formed on the top of the cylindrical lower part 21. Specifically, in this embodiment, the height of the cylindrical lower part 21 is 250 μm, the height of the conical upper part 22 is 200 μm, and the needle body 2 is formed on the needle base 1 in a 15 by 15 square array, wherein the width of the substrate connected to the cylindrical lower part 21 in the needle base 1 is 300 μm, so that the microneedle can ensure a certain depth-to-width ratio while taking into account the rigidity of the entire needle. The needle body 2 of the microneedle can be easily inserted into the skin, and the needle body 2 will not be bent due to being too slender. The needle base 1 of the positive mold of the microneedle adopts a double-layer substrate design. When making a polydimethylsiloxane negative mold, space for the substrate located at the bottom layer can be left, so that the liquid is restricted when making the microneedle.

[0034] Specifically, in this embodiment, 0.5 ml of the uniform solution treated in step S300 was added to the polydimethylsiloxane female mold, which was placed in a vacuum drying oven, and vacuumed and air bubbles were removed twice in succession, each time for 5 minutes.

[0035] S500, scraping off the solution at the base of the polydimethylsiloxane negative mold, and adding pure sodium hyaluronate solution; Specifically, the solution at the base of the needle base 1 of the polydimethylsiloxane negative mold is scraped off, and a pure sodium hyaluronate solution without β-1,3 / α-1,3-glucan is added. The needle base 1 of the microneedle is formed using a sodium hyaluronate solution without β-1,3 / α-1,3-glucan, so that the needle base 1 has considerable flexibility and is conducive to skin adhesion.

[0036] S600, drying the female mold after the treatment in step S500 at a drying temperature within the normal body temperature range of the human body, and peeling the microneedles from the female mold after the drying is completed. Specifically, in this embodiment, the female mold is placed in a drying oven at a drying temperature of 37° C. for 8 hours, and then the prepared microneedles after drying are peeled from the female mold, and the peeled microneedles are placed in a dryer for subsequent use.

[0037] Please see attached Figure 4 That is a β-1,3 / α-1,3-glucan microneedle provided in this application.

[0038] The β-1,3 / α-1,3-glucan microneedle prepared by the above preparation method can be used as a transdermal drug delivery device. In the preparation method, a solution formed by mixing two sodium hyaluronates with different molecular weights is used as a base material, so that the solution reduces the viscosity of the solution and also greatly increases the hardness of the microneedle body 2 of the obtained microneedle due to the inclusion of β-1,3 / α-1,3-glucan, thereby improving the structural stability of the microneedle body 2 and having sufficient strength and hardness for insertion into the skin (such as the attached Figure 5 No organic reagents were used in the preparation process, which can fully meet the biosafety requirements.

[0039] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for preparing β-1,3 / α-1,3-glucan microneedles, characterized in that: include: S100, dissolving low molecular weight sodium hyaluronate and high molecular weight sodium hyaluronate in a weight ratio of 5:1 in deionized water, and stirring thoroughly to obtain a hyaluronic acid hydrogel solution with a concentration of 20% by weight relative to volume; S200, adding β-1,3 / α-1,3-glucan to the obtained hyaluronic acid hydrogel solution, and stirring thoroughly to obtain a uniform solution; wherein the mass ratio of hyaluronic acid to β-1,3 / α-1,3-glucan is 10:1.5; S300, performing two consecutive vacuum and pre-bubble removal treatments on the uniform solution prepared in step S200; S400, adding the uniform solution treated in step S300 into a polydimethylsiloxane female mold, and performing vacuum and degassing treatments twice in succession; S500, scraping off the solution at the base of the polydimethylsiloxane negative mold, and adding pure sodium hyaluronate solution; S600, drying the female mold processed in step S500 at a drying temperature within the normal body temperature range of the human body, and peeling off the microneedles from the female mold after drying.

2. The method for preparing β-1,3 / α-1,3-glucan microneedles according to claim 1, characterized in that: Step S400 also includes: S401, using 3D printing technology to make a microneedle positive mold, and making a polydimethylsiloxane negative mold based on the microneedle positive mold; S402, adding the uniform solution treated in step S300 into the polydimethylsiloxane female mold, and performing vacuum and degassing treatments twice in succession.

3. The method for preparing β-1,3 / α-1,3-glucan microneedles according to claim 2, characterized in that: The printing material of the microneedle positive mold is a thermoplastic photosensitive resin.

4. The method for preparing β-1,3 / α-1,3-glucan microneedles according to claim 3, characterized in that: The needle tip size of the microneedle positive mold is 15-20 μm.

5. The method for preparing β-1,3 / α-1,3-glucan microneedles according to claim 2, characterized in that: The microneedle positive mold comprises a needle base designed with a double-layer substrate and a needle body formed on the top of the needle base.

6. The method for preparing β-1,3 / α-1,3-glucan microneedles according to claim 5, characterized in that: The needle body comprises a cylindrical lower portion formed on the top of the needle base and a conical upper portion formed on the top of the cylindrical lower portion.

7. The method for preparing β-1,3 / α-1,3-glucan microneedles according to claim 6, characterized in that: The height of the lower part of the cylinder is 250 μm, and the height of the upper part of the cone is 200 μm.

8. The method for preparing β-1,3 / α-1,3-glucan microneedles according to claim 5, characterized in that: The needle bodies are formed on the needle base in a 15 by 15 square array.

9. A β-1,3 / α-1,3-glucan microneedle, characterized in that: Prepared according to any one of the preparation methods of claims 1-8.

10. Use of the β-1,3 / α-1,3-glucan microneedle prepared according to any one of the preparation methods of claims 1 to 8 as a transdermal drug delivery device.

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