Magnesium-loaded decellularized amnion microneedle and preparation method thereof

Abstract

The invention discloses a magnesium-loaded decellularized amnion microneedle and a preparation method thereof.The magnesium-loaded decellularized amnion microneedle comprises a needle tip part and a needle bottom part, and the needle tip part and the needle bottom part each comprise a photo-crosslinking gelatin matrix; the needle tip part further comprises magnesium ions; the needle bottom part further comprises decellularized amnion particles; the substitution degree of the methacrylated gelatin of the needle tip part is higher than that of the methacrylated gelatin of the needle bottom part. Through the puncture effect of the microneedle, magnesium ions for promoting angiogenesis are accurately delivered to the deep part of a wound surface, and meanwhile, decellularized amnion particles are slowly released by the needle bottom part so as to provide a long-acting regeneration microenvironment, so that functional synergy of vascularization promotion and tissue regeneration is realized. The microneedle can significantly enhance the vascularization level of the wound surface, effectively accelerate the healing process of the chronic refractory wound surface, and improve the repair quality.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomaterials and tissue engineering, and particularly relates to a magnesium-loaded acellular amniotic membrane microneedle and a preparation method thereof. BACKGROUND

[0002] With the aggravation of population aging and the rising incidence of underlying diseases such as diabetes and vascular diseases, trauma and chronic wounds have become common problems affecting people's health. Skin and soft tissue defects caused by accidental injury, surgical incision and chronic diseases often lead to slow tissue healing or even long-term non-healing, forming chronic refractory wounds such as diabetic foot, pressure sores and venous ulcers. These chronic wounds have a huge impact on the quality of life and psychological state of patients, and in severe cases can even lead to infection, amputation, and even threaten life.

[0003] Under the background of multidisciplinary diagnosis and treatment mode, various tissue injuries such as trauma, burns and chronic wounds often need to be treated with basic treatment, surgery and dressings. Dressings play a crucial role in wound repair. Among them, acellular amniotic membrane, as one of the skin biological dressings applied in clinical for many years, retains the natural extracellular matrix by removing the cell components, has excellent biocompatibility, regenerative ability and antibacterial activity, and has been used for the treatment of burn repair and donor site wounds. During the wound repair process, an internal blood vessel network needs to be formed to provide necessary pathways and nutrients for cell migration, proliferation and differentiation, thereby realizing tissue regeneration. Therefore, promoting vascularization is considered to be a key link to achieve wound repair. Magnesium element, as the fourth most abundant cation in the body, exists in large quantities in the body and is involved in many physiological activities. In recent years, a large number of studies have shown that magnesium element has potential utility in promoting angiogenesis. SUMMARY

[0004] The present application aims to at least partially solve one of the technical problems in the related art. To this end, the present application aims to provide a magnesium-loaded acellular amniotic membrane microneedle and a preparation method thereof.

[0005] To achieve the above-mentioned object, according to a first aspect of the present application, the magnesium-loaded acellular amniotic membrane microneedle provided by the present application comprises a needle tip portion and a needle base portion, wherein the needle tip portion and the needle base portion each comprise a photo-crosslinked gelatin matrix.

[0006] The needle tip portion further comprises magnesium ions, and the needle base portion further comprises acellular amniotic membrane particles.

[0007] According to an embodiment of the present application, the photo-crosslinked gelatin is methacrylated gelatin.

[0008] According to an embodiment of the present application, the degree of substitution of the methacrylated gelatin in the needle tip portion is higher than that of the methacrylated gelatin in the needle base portion.

[0009] According to one embodiment of the present application, the degree of substitution of the methacrylated gelatin of the tip portion is 50%-70%; and the degree of substitution of the methacrylated gelatin of the base portion is 20%-40%.

[0010] According to one embodiment of the present application, the amount of the methacrylated gelatin of the tip portion is 350 to 500 microliters, the magnesium ions are provided by a soluble magnesium ion solution, and the amount of the magnesium ion solution of the tip portion is 3 to 5 microliters.

[0011] The amount of the methacrylated gelatin of the base portion is 700 to 900 microliters, and the amount of the decellularized amniotic membrane particles of the base portion is 4 to 6 milligrams.

[0012] According to a second aspect of the present application, the present application provides a preparation method of the magnesium-loaded decellularized amniotic membrane microneedle, comprising:

[0013] a) preparing a tip precursor solution comprising a first photo-crosslinked gelatin and magnesium ions;

[0014] b) preparing a base precursor solution comprising a second photo-crosslinked gelatin and decellularized amniotic membrane particles; the degree of substitution of the first photo-crosslinked gelatin is higher than that of the second photo-crosslinked gelatin;

[0015] c) adding the tip precursor solution into a microneedle mold and performing a first concentration treatment;

[0016] d) adding the base precursor solution on top of the tip precursor solution after the first concentration treatment, and performing a second concentration treatment;

[0017] e) performing ultraviolet light irradiation on the mixture after the second concentration treatment to achieve crosslinking;

[0018] f) drying and demolding the crosslinked product, thereby obtaining the magnesium-loaded decellularized amniotic membrane microneedle.

[0019] According to one embodiment of the present application, the first photo-crosslinked gelatin is a methacrylated gelatin with a degree of substitution of 50%-70%, and the second photo-crosslinked gelatin is a methacrylated gelatin with a degree of substitution of 20%-40%.

[0020] According to one embodiment of the present application, the first concentration treatment in step c) comprises drying in a drying oven.

[0021] The second concentration treatment in step d) comprises oven drying at a temperature of 30-40°C; and the base precursor solution is added in batches.

[0022] According to one embodiment of the present application, the photo-crosslinking in step e) is performed when the mixture is in a semi-cured state that is not completely dried and the surface is not tacky.

[0023] According to one embodiment of the present application, the preparation of the needle tip precursor solution comprises mixing a first photo-crosslinking gelatin solution with a volume of 350-500 microliters and a magnesium ion solution with a volume of 3-5 microliters.

[0024] The preparation of the needle base precursor solution comprises adding decellularized amniotic membrane particles with a mass of 4-6 milligrams to a second photo-crosslinking gelatin solution with a volume of 700-900 microliters.

[0025] Through the above technical solutions, the magnesium-loaded decellularized amniotic membrane microneedle and the preparation method thereof, by combining magnesium ions with the function of promoting angiogenesis and decellularized amniotic membrane particles providing a natural regeneration scaffold, achieve functional synergy and jointly promote wound healing. At the same time, the microneedle structure is adopted, and in particular, the magnesium ions are loaded on the needle tip part, which can penetrate the surface layer of the wound, accurately and efficiently deliver the angiogenesis factor to the deep tissue, and act on the area that needs to be vascularized, overcoming the defect of insufficient penetration depth of traditional surface dressings. Therefore, the magnesium-loaded decellularized amniotic membrane microneedle of the present application significantly enhances the vascularization level of the wound, thereby effectively accelerating the healing process of the wound, especially the difficult-to-heal chronic wound, and improving the repair quality.

[0026] Other features and advantages of the present application will be described in detail in the following specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a scanning electron microscope image of the magnesium-loaded decellularized amniotic membrane microneedle array from one perspective;

[0028] Figure 2 is a scanning electron microscope image of the magnesium-loaded decellularized amniotic membrane microneedle array from another perspective;

[0029] Figure 3 is a fluorescence microscope image of the magnesium-loaded decellularized amniotic membrane microneedle. DETAILED DESCRIPTION

[0030] The specific embodiments of the present application are described in detail below. It should be understood that the specific embodiments described herein are only for illustration and explanation of the present application, and are not intended to limit the present application.

[0031] The magnesium-loaded decellularized amniotic membrane microneedle provided by the embodiment of the present application comprises a needle tip part and a needle base part, which form a conical structure as a whole, with a total height of generally 600-800 micrometers and a needle tip bottom length and width of about 300-400 micrometers. The microneedle structure enables it to effectively penetrate the surface layer of the wound tissue and deliver functional components to the deep layer.

[0032] Specifically, the tip portion and the base portion of the microneedle each comprise a photo-crosslinked gelatin matrix. The tip portion further comprises magnesium ions, and the base portion further comprises acellular amniotic membrane particles.

[0033] That is, the tip portion and the base portion of the microneedle each use photo-crosslinked gelatin as the matrix material to provide the necessary strength and biocompatibility. Photo-crosslinked gelatin is a chemically modified gelatin derivative, usually methacrylated gelatin (GelMA), which contains functional groups capable of crosslinking under ultraviolet light. After photo-crosslinking, the gelatin matrix forms a stable three-dimensional network structure, which not only retains the original biological activity of gelatin, but also has enhanced mechanical properties and resistance to enzymatic degradation, and can maintain structural stability for a certain period of time in a physiological environment.

[0034] The tip portion of the microneedle further comprises magnesium ions (Mg 2+ ) in addition to the photo-crosslinked gelatin matrix. As an important bioactive factor, magnesium ions play an important role in the process of angiogenesis, and can promote the proliferation, migration of endothelial cells and the formation of vascular-like lumens. By loading magnesium ions in the tip portion of the microneedle, they can be quickly released after the microneedle is inserted into the wound, directly acting on the tissues deep in the wound, thereby rapidly promoting the process of angiogenesis. The optimal concentration range of magnesium ions is determined to be 1-3 M / L through experimental screening, within which range the magnesium ions can exert a significant pro-angiogenic effect without causing other side effects to the surrounding cells.

[0035] The base portion of the microneedle is also composed of a photo-crosslinked gelatin matrix, but it is loaded with acellular amniotic membrane particles. Acellular amniotic membrane is an amniotic membrane tissue that has been subjected to decellularization treatment, removing cellular components that can cause immune rejection, while retaining abundant extracellular matrix (ECM) proteins such as collagen, elastin, laminin, and fibronectin. These natural ECM components not only provide a scaffold for cell attachment and migration, but also release various growth factors to promote tissue regeneration. Preferably, the acellular amniotic membrane is subjected to a micro-pulverization treatment to form particles, making it easier to mix with photo-crosslinked gelatin and facilitating the increase of specific surface area to promote the release of active ingredients.

[0036] When the magnesium-loaded acellular amniotic membrane microneedle is applied to a wound, the tip portion can penetrate the surface layer of the wound and deliver magnesium ions directly to the deep tissues, rapidly promoting the process of angiogenesis. At the same time, the base portion adheres to the wound and gradually releases the bioactive components in the acellular amniotic membrane particles, providing a natural scaffold and nutrients for tissue regeneration. This synergistic structure and function supports the repair process of the wound, especially for chronic non-healing wounds with insufficient blood supply, significantly improving the healing speed and quality.

[0037] The magnesium-loaded acellular amniotic membrane microneedle according to the present application realizes functional synergy by combining magnesium ions with the function of promoting angiogenesis and acellular amniotic membrane particles providing a natural regeneration scaffold, and promotes wound healing together. At the same time, the microneedle structure is adopted, and in particular, the magnesium ions are loaded on the needle tip part, which can penetrate the surface layer of the wound, accurately and efficiently deliver the angiogenesis factor to the deep tissue, and act on the area that needs to be vascularized, overcoming the defect of insufficient penetration depth of traditional surface dressings. Therefore, the magnesium-loaded acellular amniotic membrane microneedle of the present application significantly enhances the vascularization level of the wound, thereby effectively accelerating the healing process of the wound, especially the refractory chronic wound, and improving the repair quality.

[0038] In some embodiments of the present application, the degree of substitution of the methacrylated gelatin in the needle tip part is higher than that of the methacrylated gelatin in the needle base part. Preferably, the degree of substitution of the methacrylated gelatin in the needle tip part is 50%-70%; the degree of substitution of the methacrylated gelatin in the needle base part is 20%-40%. More preferably, the degree of substitution of the methacrylated gelatin in the needle tip part is 60%; the degree of substitution of the methacrylated gelatin in the needle base part is 30%.

[0039] The degree of substitution is a percentage of amino groups on the gelatin molecular chain being substituted by methacryl groups, which directly determines the density of the hydrogen gel network, mechanical strength, degradation rate and biocompatibility of the methacrylated gelatin after light crosslinking. The present application realizes the optimization and synergy of the functions of each part of the microneedle by precisely regulating the degrees of substitution of different parts.

[0040] In the present embodiment, the needle tip part uses GelMA with a high degree of substitution, which has more light-sensitive crosslinking sites distributed on the molecular chain. When subjected to ultraviolet light, these sites can form a more dense and solid three-dimensional crosslinking network. The needle tip part after solidification has significantly enhanced mechanical strength, which is sufficient to enable it to effectively penetrate the stratum corneum or the surface tissue of the wound when applied to the wound. This excellent piercing ability ensures that the magnesium ions loaded on the needle tip can be successfully and reliably delivered to the target area in the deep layer of the wound.

[0041] The needle base part uses GelMA with a relatively low degree of substitution. The use of GelMA with a low degree of substitution forms a relatively loose hydrogen gel network after crosslinking, which has a softer texture. The lower crosslinking density makes the needle base matrix have better biocompatibility and hydrophilicity, which can create a microenvironment closer to the natural extracellular matrix, which is conducive to the adhesion, infiltration and growth of surrounding tissue cells; secondly, the relatively loose network structure makes its degradation rate in the body relatively fast, which helps the acellular amniotic membrane particles to be released in a more gradual and sustained manner, and stably play their role of providing biological scaffolds and nutritional factors in wound repair.

[0042] By the above-mentioned differential design of the substitution degree of the needle tip and the needle bottom part, a micro-needle structure of "hard on the top and soft on the bottom" is constructed. The high hardness of the needle tip ensures the success of delivery, while the softness of the needle bottom ensures the biocompatibility of tissue integration. Thus, the two core functions of the micro-needle, i.e. the rapid delivery of magnesium ions and the sustained release and support of the acellular amniotic membrane particles, can be optimally matched, so as to better exert the synergistic therapeutic effect of each component.

[0043] In some embodiments of the present application, the amount of methacrylated gelatin in the needle tip part is 350 to 500 microliters, and the magnesium ions are provided by a soluble magnesium ion solution, and the amount of magnesium ion solution in the needle tip part is 3 to 5 microliters. The amount of methacrylated gelatin in the needle bottom part is 700 to 900 microliters, and the amount of acellular amniotic membrane particles in the needle bottom part is 4 to 6 milligrams.

[0044] By the amount ratio of methacrylated gelatin in the needle tip part and the magnesium ion solution, the mechanical strength and magnesium ion release characteristics of the micro-needle are ensured; by the amount of methacrylated gelatin and the content of acellular amniotic membrane particles in the needle bottom part, good tissue fitting and sustained support are achieved.

[0045] The present application also provides a magnesium-loaded acellular amniotic membrane micro-needle preparation method, comprising:

[0046] a) preparing a needle tip precursor solution comprising a first photo-crosslinked gelatin and magnesium ions. Preferably, the first photo-crosslinked gelatin is methacrylated gelatin with a substitution degree of 50%-70%.

[0047] In this step, the preparation of the needle tip precursor solution can specifically include: mixing a first photo-crosslinked gelatin solution with a volume of 350-500 microliters with a magnesium ion solution with a volume of 3-5 microliters.

[0048] That is, this step is to prepare the needle tip precursor solution. The needle tip precursor solution is the basis for forming the puncture part of the micro-needle. Its preparation process includes: dissolving the first photo-crosslinked gelatin, i.e. methacrylated gelatin (GelMA), as the matrix material in a suitable solvent to form a solution, and then adding magnesium ions as the pro-angiogenic functional component to the solution. In this step, the above-mentioned solvent can be prepared by diluting 0.25% photo-initiator LAP by 100 times.

[0049] In the present embodiment, the first photo-crosslinked gelatin preferably uses GelMA with a higher degree of substitution, which ranges from 50% to 70%, and more preferably, GelMA with a degree of substitution of 60%. A higher degree of substitution means that its molecular chain contains more methacryloyl functional groups, which provides a material basis for the subsequent formation of a high-density cross-linked network and thus a needle tip with high mechanical strength. Magnesium ions are usually added in the form of a soluble magnesium salt solution, such as magnesium chloride or magnesium sulfate solution. For example, 2g of magnesium chloride is weighed and dissolved in 4.9261ml of deionized water to make a 2M / L magnesium chloride solution. In a preferred example, 350 to 500 microliters (e.g., 450 microliters) of the first photo-crosslinked gelatin solution and 3 to 5 microliters (e.g., 3.85 microliters) of the magnesium ion solution are uniformly mixed to form a needle tip precursor solution.

[0050] b) preparing a needle base precursor solution comprising a second photo-crosslinked gelatin and acellular amniotic membrane particles; the degree of substitution of the first photo-crosslinked gelatin is higher than that of the second photo-crosslinked gelatin. Preferably, the second photo-crosslinked gelatin is a methacrylated gelatin with a degree of substitution of 20% to 40%.

[0051] In this step, the preparation of the needle base precursor solution can include adding 4 to 6 milligrams of acellular amniotic membrane particles to a second photo-crosslinked gelatin solution with a volume of 700 to 900 microliters.

[0052] That is, this step is to prepare a needle base precursor solution. The needle base precursor solution comprises a second photo-crosslinked gelatin that constitutes the base part of the microneedle and acellular amniotic membrane particles as a component of the tissue regeneration promoting function. Unlike the needle tip part, the second photo-crosslinked gelatin used here is GelMA with a lower degree of substitution, which ranges from 20% to 40%, and preferably 30%. A lower degree of substitution results in a relatively loose gel network after cross-linking, which is softer in texture and is beneficial to improve biocompatibility and achieve slow release of the contents. Acellular amniotic membrane particles are obtained by freeze-drying and micronizing the naturally occurring amniotic membrane after decellularization. In a specific example, 4 to 6 milligrams (e.g., 5 milligrams) of acellular amniotic membrane particles are dispersed in a second photo-crosslinked gelatin solution with a volume of 700 to 900 microliters (e.g., 800 microliters), and mixed thoroughly to form a needle base precursor solution.

[0053] c) adding the needle tip precursor solution to the microneedle mold and performing a first step of concentration treatment.

[0054] In this step, the first step of concentration treatment can include drying in an oven.

[0055] Specifically, the prepared microneedle tip precursor solution is added into the microwells of the microneedle mold. Subsequently, the microneedle tip precursor solution in the mold is subjected to a first concentration treatment. The concentration treatment is preferably performed by drying in an oven to form a preliminarily solidified microneedle tip portion with a certain morphological stability.

[0056] d) The prepared microneedle base precursor solution is added on top of the microneedle tip precursor solution after the first concentration treatment, and subjected to a second concentration treatment.

[0057] In this step, the second concentration treatment includes oven drying at a temperature of 30-40°C; and the microneedle base precursor solution is added in batches.

[0058] Specifically, after the first concentration treatment of the microneedle tip portion, the prepared microneedle base precursor solution is added into the mold to cover the preliminarily formed microneedle tip portion. In order to ensure uniform distribution of the decellularized amniotic membrane particles in the microneedle base matrix and avoid uneven distribution due to gravity settling, the microneedle base precursor solution is preferably added in batches, i.e., added dropwise to the mold in multiple quantities, for example, in two batches. After the loading is completed, the two-layer mixture in the mold is subjected to a second concentration treatment. The step is preferably performed by drying in a constant-temperature oven at 30-40°C to further evaporate the solvent by gentle heating, so that the precursors of the entire microneedle structure reach a suitable solidification state.

[0059] e) The mixture after the second concentration treatment is subjected to ultraviolet light irradiation to achieve crosslinking.

[0060] The light irradiation crosslinking in this step is performed when the mixture is in a semi-solidified state that is not completely dried and the surface is not sticky.

[0061] That is, the light crosslinking step is performed when the mixture reaches a semi-solidified state that is not completely dried but the surface is not sticky after the second concentration treatment. The crosslinking is selected to be performed at this time because in this state, the GelMA molecular chain still has a certain activity, which is conducive to efficient crosslinking reaction under ultraviolet light irradiation to form a complete and strong three-dimensional network structure; at the same time, the semi-solidified state also ensures that the structure and morphology of the microneedle do not collapse during the crosslinking process. By using an ultraviolet light source to irradiate the mold, active free radicals are generated by the photoinitiator (which has been previously added to the GelMA solution), which in turn triggers the polymerization reaction of the methacryloyl group on the GelMA molecular chain, thereby solidifying the entire microneedle structure.

[0062] f) The crosslinked product is dried and demolded to obtain the magnesium-loaded decellularized amniotic membrane microneedle.

[0063] That is, the product after sufficient crosslinking is subjected to a final drying treatment to remove residual moisture and enhance its storage stability. After complete drying, the shaped magnesium-loaded acellular amniotic membrane microneedle array is demolded from the mold. Thus, the magnesium-loaded acellular amniotic membrane microneedle product with complete structure and functional partition is successfully prepared.

[0064] Through the above technical solution, the magnesium-loaded acellular amniotic membrane microneedle and its preparation method can prepare the magnesium-loaded acellular amniotic membrane microneedle. The magnesium-loaded acellular amniotic membrane microneedle realizes functional synergy by combining magnesium ions with the function of promoting angiogenesis and acellular amniotic membrane particles providing a natural regeneration scaffold, and promotes wound healing together. At the same time, the microneedle structure is adopted, especially the magnesium ions are loaded on the needle tip part, which can penetrate the surface layer of the wound, accurately and efficiently deliver the angiogenesis factor to the deep tissue, and act on the area that needs to be vascularized, overcoming the defect of insufficient penetration depth of traditional surface dressings. Therefore, the magnesium-loaded acellular amniotic membrane microneedle of the present application significantly enhances the vascularization level of the wound, thereby effectively accelerating the healing process of the wound, especially the difficult-to-heal chronic wound, and improving the repair quality.

[0065] The above describes the preferred embodiments of the present application, but the present application is not limited to the specific details in the above embodiments. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0066] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present application will not further describe various possible combinations.

[0067] Furthermore, various different embodiments of the present application can also be combined in any manner, as long as they do not deviate from the idea of the present application, and they should also be considered as disclosed by the present application.

Claims

1. A magnesium-loaded acellular amniotic membrane microneedle, characterized by, The needle tip portion and the needle bottom portion each comprise a photo-crosslinked gelatin matrix; wherein the needle tip portion further comprises magnesium ions, and the needle bottom portion further comprises acellular amniotic membrane particles.

2. The Mg-loaded acellular amniotic membrane microneedle according to claim 1, wherein, The photo-crosslinked gelatin is methacrylated gelatin.

3. The Mg-AMMs according to claim 2, wherein, The degree of substitution of the methacrylated gelatin in the needle tip portion is higher than that in the needle bottom portion.

4. The Mg-AMMs according to claim 1, wherein, The degree of substitution of the methacrylated gelatin in the needle tip portion is 50%-70%, and the degree of substitution of the methacrylated gelatin in the needle bottom portion is 20%-40%.

5. The Mg-AMMs according to claim 1, wherein, The amount of the methacrylated gelatin in the needle tip portion is 350-500 microliters, and the magnesium ions are provided by a soluble magnesium ion solution, and the amount of the magnesium ion solution in the needle tip portion is 3-5 microliters. The amount of the methacrylated gelatin in the needle bottom portion is 700-900 microliters, and the amount of the acellular amniotic membrane particles in the needle bottom portion is 4-6 milligrams.

6. A method for preparing a magnesium-loaded acellular amniotic membrane microneedle, characterized by, The method comprises: a) preparing a needle tip precursor solution comprising a first photo-crosslinked gelatin and magnesium ions; b) preparing a needle bottom precursor solution comprising a second photo-crosslinked gelatin and acellular amniotic membrane particles; the degree of substitution of the first photo-crosslinked gelatin is higher than that of the second photo-crosslinked gelatin; c) adding the needle tip precursor solution into a microneedle mold and performing a first concentration treatment; d) adding the needle bottom precursor solution on top of the needle tip precursor solution after the first concentration treatment and performing a second concentration treatment; e) performing UV irradiation on the mixture after the second concentration treatment to achieve crosslinking; f) drying and demolding the crosslinked product to obtain the magnesium-loaded acellular amniotic membrane microneedle.

7. The preparation method according to claim 6, characterized in that, The first photo-crosslinked gelatin is methacrylated gelatin with a degree of substitution of 50%-70%, and the second photo-crosslinked gelatin is methacrylated gelatin with a degree of substitution of 20%-40%.

8. The preparation method according to claim 6, characterized in that, The first concentration treatment in step c) comprises drying in an oven; The second concentration treatment in step d) comprises oven drying at a temperature of 30-40°C; and the needle bottom precursor solution is added in batches.

9. The preparation method according to claim 6, characterized in that, The irradiation crosslinking in step e) is performed when the mixture is in a semi-solid state and the surface is not sticky.

10. The method of claim 6, wherein, The preparation of the needle tip precursor solution comprises mixing a first photo-crosslinked gelatin solution with a volume of 350-500 microliters with a magnesium ion solution with a volume of 3-5 microliters; The preparation of the needle bottom precursor solution comprises adding acellular amniotic membrane particles with a mass of 4-6 milligrams to a second photo-crosslinked gelatin solution with a volume of 700-900 microliters.

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