Separable frozen microneedle with hierarchical structure as well as preparation method and application of separable frozen microneedle

Abstract

The invention discloses a separable frozen microneedle with a hierarchical structure and a preparation method and application thereof, based on a 3D printing material, a microneedle-shaped male mold main structure with the hierarchical structure is obtained, a polymer template is prepared from the main structure through an impression method, gaps of microneedle holes in the template are filled with a pre-gel solution containing functional cells, and the separable frozen microneedle with the hierarchical structure is obtained. And filling other parts of the template with the non-gel solution. And after solidification and freezing, removing the template to obtain the separable frozen microneedle which is similar to a main structure and has a hierarchical structure. The frozen microneedle has a three-layer structure of the microneedle array, the frostbite-preventing microcolumn array and the back plate, and the frozen microneedle can be smoothly punctured into the skin to realize percutaneous delivery by utilizing the high hardness of the frozen microneedle. In the pricking process, the microcolumn array prevents the frozen back plate from making contact with the skin, and frostbite is effectively prevented. After the tip of the microneedle is inserted, the frozen microcolumn array and the back plate are melted into liquid, so that safe, simple and convenient percutaneous cell delivery is realized.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, specifically relating to a separable cryogenic microneedle with a hierarchical structure and its preparation method, which can be used for transdermal cell delivery. Background Technology

[0002] Cell therapy is a cutting-edge medical strategy that utilizes living cells as therapeutic agents to treat diseases by transplanting, replacing, or repairing damaged cells and tissues. In the field of dermatology, cell therapy has shown enormous application potential, widely used in wound healing, tissue regeneration, scar repair, and the treatment of pigmented diseases. For example, skin-derived cells such as hair follicle stem cells, skin fibroblasts, and keratinocytes, as well as mesenchymal stem cells with multi-lineage differentiation potential, have been shown to effectively promote angiogenesis, regulate the local immune microenvironment, and secrete various growth factors, thereby accelerating skin repair and functional reconstruction. However, the efficient, safe, and in-situ delivery of therapeutic cells to the target skin layer remains a key technological bottleneck in achieving its therapeutic effects. Traditional methods such as injection or surgical implantation often suffer from high invasiveness, poor patient compliance, low cell survival rates, and poor targeting, severely limiting the widespread application and maximization of efficacy of cell therapy in clinical dermatology.

[0003] To overcome the limitations of traditional delivery methods, microneedling technology has emerged as an innovative percutaneous delivery platform. Microneedle arrays can penetrate the outermost layer of the skin, the stratum corneum, in a minimally invasive manner, forming micron-sized channels. This opens up an effective pathway for macromolecules, including cells, and active ingredients to directly enter the deeper layers of the skin. Compared to syringes, microneedle delivery of cells can significantly reduce pain, minimize tissue damage, and improve positioning accuracy. However, conventional cell-loaded soluble or hydrogel microneedles often face problems such as insufficient mechanical strength, difficulty in effectively penetrating the skin, and low cell release efficiency due to uncontrollable dissolution or degradation processes. In recent years, cryo-microneedling technology, which freezes cell-containing solutions in a mold, has provided a new approach to solving these problems. These microneedles, with their ice crystal matrix, possess excellent mechanical properties, ensuring smooth insertion; simultaneously, their biocompatible matrix can gently melt within the skin, achieving targeted and rapid cell release. Nevertheless, existing cryo-microneedles are still relatively simple in structural design, and there is still room for further optimization and improvement in terms of controllability of separation from the base after insertion, protection of surrounding tissues, and maintenance of cell activity during freeze-thaw processes. Summary of the Invention

[0004] Purpose of the invention: In order to solve the problems of poor mechanical properties, complex composition, and unsatisfactory delivery and therapeutic effects of traditional microneedles used for transdermal cell delivery, this invention provides a separable cryo-microneedle with a hierarchical structure and its preparation method.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing separable cryoneedles with a hierarchical structure, comprising a microneedle array, a frostbite-resistant micropillar array, and a backplate, is obtained through the following steps: (1) A microneedle-shaped male mold main structure with a hierarchical structure was obtained by 3D printing; (2) A polymer template was made by means of a microneedle-shaped positive mold main structure by means of PDMS template replication method, and then repeatedly washed with anhydrous ethanol and ultrapure water; (3) Fill the micropores in the polymer template prepared in step (2) with the pregel solution, and then fill the other parts of the template with the nongel solution; after photocuring and gradient freezing, remove the template to obtain the final product.

[0006] Specifically, in step (1), the microneedle-shaped male mold main structure has a microneedle array number of 3×3~10×10, a microneedle height of 500~1000 µm, a bottom radius of 300~700 µm, and a needle tip angle of 30~40°.

[0007] Preferably, the microneedle array consists of a cylindrical micropillar array connected to the microneedle array and a cuboid array centrally arranged within the cylindrical micropillar array; wherein, the bottom radius of the cylindrical array is the same as the bottom radius of the microneedle, and the height is 50-100% of the bottom radius; the side length of the cuboid array is 200-300 μm, and the height is the same as that of the cylindrical array.

[0008] Specifically, in step (3), the pregel solution is a mixture of methacrylate gelatin, cell cryopreservation solution and photoinitiator, and contains cells.

[0009] Preferably, in the pregelation solution, the concentration of methacrylate gelatin is 10-15% v / v, the methacrylate gelatin is mixed with the cell cryopreservation solution at a volume ratio of 1:1, and the photoinitiator accounts for 1-2% of the volume of the pregelation solution.

[0010] Preferably, the cells are selected from at least one of hair follicle stem cells, skin fibroblasts, keratinocytes, and mesenchymal stem cells; the cell addition density in the pregel solution is 1~4×10⁻⁶. 5 per mL.

[0011] Specifically, in step (3), the non-gel solution is selected from any one of deionized water, cell cryopreservation solution, or DMSO aqueous solution (5%-10% v / v).

[0012] Preferably, in step (3), the irradiation time of the photocuring is 30-90s; the gradient freezing method is to first store at 4℃ for 2 hours, then store at -20℃ for 2 hours, then freeze at -80℃ for 2 hours, and finally place in liquid nitrogen for 1 hour.

[0013] Furthermore, the separable cryogenic microneedles with hierarchical structures prepared by the above preparation method are also within the scope of protection of this invention.

[0014] Furthermore, the present invention also claims the use of the above-described separable cryo-microneedles with a hierarchical structure in the preparation of transdermal drug delivery.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention integrates a microneedle array, a frostbite-resistant micropillar array, and a backplate into a single unit through a sophisticated structural design. This allows the microneedles to reliably detach and remain within the skin after completing their cell delivery mission, while the base portion can be completely removed, thereby reducing unnecessary stimulation of the epidermis and the potential risk of frostbite. In terms of materials, we preferentially use biocompatible methacrylate gelatin and cell cryopreservation solution to construct the cell-loading matrix. Through an optimized gradient freezing process, we maximize the cell activity during preparation and storage, achieving effective cell therapy.

[0016] (2) The present invention utilizes 3D printing to fabricate the main structure of the microneedle positive mold. The method is simple, easy to operate, and the morphology of the microneedle array can be easily controlled.

[0017] (3) The separable cryo-microneedle with a hierarchical structure designed in this invention can be easily inserted into the skin to achieve percutaneous delivery by utilizing the high hardness of the cryo-microneedle. During the insertion process, the microcolumn array prevents the frozen backplate from contacting the skin, effectively preventing frostbite.

[0018] (4) After the microneedles prepared by the present invention are inserted into the needle tip, the frozen microcolumn array and backplate melt into liquid, thereby realizing safe and convenient percutaneous cell delivery and is highly practical. Attached Figure Description

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0020] Figure 1 This is a schematic diagram of the preparation process of the separable cryogenic microneedles with a hierarchical structure according to the present invention.

[0021] Figure 2 This is a design drawing of the main structure of a microneedle male mold designed in an example.

[0022] Figure 3 This is a morphology characterization diagram of separable cryogenic microneedles with hierarchical structure obtained in an example.

[0023] Figure 4 This is a schematic diagram of the frostbite prevention mechanism of separable cryo-microneedles with a hierarchical structure obtained in an example.

[0024] Figure 5 This is a schematic diagram illustrating the application of separable cryo-microneedles with a hierarchical structure in an example.

[0025] Figure 6 This is an example of the mechanical stress that a single separable cryogenic microneedle with a hierarchical structure can withstand. Detailed Implementation

[0026] The present invention can be better understood from the following embodiments.

[0027] Example 1: Preparation of separable cryogenic microneedles with hierarchical structure like Figure 1 As shown, the preparation process of the separable cryogenic microneedles with hierarchical structure of the present invention is as follows: (1) A microneedle-shaped male mold main structure with a hierarchical structure was obtained by 3D printing; (2) A polymer template was made by means of a microneedle-shaped positive mold main structure by means of PDMS template replication method, and then repeatedly washed with anhydrous ethanol and ultrapure water; (3) Fill the micropores in the polymer template prepared in step (2) with the pregel solution, and then fill the other parts of the template with the nongel solution; after photocuring and gradient freezing, remove the template to obtain the final product.

[0028] Figure 1 In Figure F, A represents the main structure of the 3D-printed positive mold, B represents the positive mold replicated using PDMS, C represents the polymer template obtained through template replication, D represents the addition of a cell-containing pre-gel solution to the microneedle pores of the template, E represents the addition of a non-gel solution to the remaining portion of the template, and F represents the separable cryogenic microneedles with a hierarchical structure obtained after UV curing and gradient freezing. Specifically, 1 represents the main structure of the 3D-printed positive mold, 2 represents the PDMS polymer, 3 represents the polymer template replicated using the template, 4 represents the pre-gel solution, 5 represents the cells, and 6 represents the non-gel solution. In the cryogenic microneedles shown in Figure F, 7 represents the array of cell-containing microneedle tips, 8 represents the array of cylindrical micropillars connected to the microneedle array, 9 represents the array of cuboids centrally arranged within the cylindrical micropillar array, and 10 represents the backplate.

[0029] Example 2: Separable cryoneedles with a hierarchical structure (1) Design the main structure of the male mold, with a microneedle array of 5×5, a microneedle height of 1200μm, a bottom radius of 800μm, a cylindrical array height of 1000μm, and a cuboid array height of 1000μm with a side length of 300μm. The design drawing is as follows. Figure 2 As shown, the microneedle array consists of a cylindrical micropillar array connected to the microneedle array and a cuboid array centrally arranged within the cylindrical micropillar array.

[0030] A hierarchical microneedle-shaped male mold master structure was obtained by 3D printing. A polymer template was then fabricated from the master structure using a PDMS template replication method, and repeatedly washed with anhydrous ethanol and ultrapure water.

[0031] (2) Prepare a 10% (v / v) GelMA solution, mix it 1:1 with the cell cryopreservation solution, add 1% (v / v) photoinitiator, mix the hair follicle stem cells with this pre-gel solution, add it to the microneedle holes in the template prepared in step 1, let it stand for 10 minutes, and fill the rest of the template with pure cell cryopreservation solution. Cure by UV irradiation for 30 seconds. The microneedles were subjected to a gradient freezing process: 4°C for 2 hours, -20°C for 2 hours, -80°C for 2 hours, and then placed in liquid nitrogen for 1 hour. The solidified cryogenic microneedles were carefully peeled from the template under low-temperature conditions and stored in liquid nitrogen. Their morphology is characterized as follows: Figure 3 As shown in the figure, the cryo-microneedle consists of a cell-containing microneedle tip array, a cylindrical micropillar array connected to the microneedle array, and a cuboid array centrally arranged within the cylindrical micropillar array. It has an ideal hierarchical and porous structure, which is beneficial for achieving frostbite-resistant cell transplantation.

[0032] This separable cryo-microneedle successfully transplanted hair follicle stem cells subcutaneously into nude mice, achieving in vivo hair regeneration. Simultaneously, it prevents the cryo-backplate from contacting the skin, thus preventing frostbite. A schematic diagram illustrating its frostbite prevention effect is shown below. Figure 4 As shown in the figure, during the process of microneedling into the skin, once the microneedle tip enters the skin, the presence of the cuboid array prevents the cylindrical array from penetrating further, thus preventing contact between the skin and the backplate.

[0033] Example 3 Separable cryoneedles with hierarchical structure (1) The main structure of the male mold was designed with a microneedle array of 5×5, a microneedle height of 1000μm, a bottom radius of 700μm, a cylindrical array height of 500μm, and a cuboid array height of 500μm with a side length of 200μm. The hierarchical microneedle-shaped male mold main structure was obtained by 3D printing. A polymer template was made from the main structure using the PDMS template replication method and repeatedly washed with anhydrous ethanol and ultrapure water.

[0034] (2) Prepare a 15% (v / v) GelMA solution, mix it 1:1 with the cell cryopreservation solution, add 1% (v / v) photoinitiator, mix the hair follicle stem cells with this pre-gel solution, add it to the microneedle holes in the template prepared in step 1, let it stand for 10 minutes, and fill the rest of the template with pure cell cryopreservation solution. Cure by UV irradiation for 45 seconds. The microneedles were subjected to a gradient freezing process: 4°C for 2 hours, -20°C for 2 hours, -80°C for 2 hours, and then placed in liquid nitrogen for 1 hour. The solidified cryogenic microneedles were carefully peeled off from the template under low-temperature conditions and stored in liquid nitrogen.

[0035] This separable cryo-microneedle successfully transplanted hair follicle stem cells subcutaneously into nude mice, achieving in vivo hair regeneration. Specifically, the frozen microneedles were inserted into the skin of the nude mice. Once the microneedle tip entered the skin, the presence of a cuboid array prevented further penetration by the cylindrical array, thus preventing contact between the skin and the dorsal plate. Subsequently, within the frozen cylindrical microneedle array, the cuboid array and the dorsal plate thawed, leaving only the gel-crosslinked needle tip remaining within the skin. The successfully delivered hair follicle stem cells differentiated and matured within the nude mouse skin, achieving hair regeneration. Its cell delivery efficiency and final in vivo hair regrowth effect are as follows: Figure 5 As shown in the figures, Figure A shows the skin puncture effect of the microneedles; Figure B shows the transdermal cell delivery achieved using the cryo-microneedles; and Figure C shows the final hair growth effect. It can be seen that the microneedles have good mechanical properties and can be successfully inserted into the skin of nude mice. After insertion, fluorescently labeled hair follicle stem cells can be found in the skin. In summary, the graded cryo-microneedles prepared in this invention can achieve safe and convenient percutaneous cell delivery and are highly practical.

[0036] Example 4 Separable cryoneedles with hierarchical structure (1) The main structure of the male mold was designed with a microneedle array of 10×10, a microneedle height of 700μm, a bottom radius of 400μm, a cylindrical array height of 500μm, and a cuboid array height of 500μm with a side length of 200μm. The hierarchical microneedle-shaped male mold main structure was obtained by 3D printing. A polymer template was made from the main structure using the PDMS template replication method and repeatedly washed with anhydrous ethanol and ultrapure water.

[0037] (2) Prepare a 10% (v / v) GelMA solution, mix it 1:1 with cell cryopreservation solution, add 1% (v / v) photoinitiator, mix the mesenchymal stem cells with this pre-gel solution, add it to the microneedle pores in the template prepared in step 1, let it stand for 10 minutes, and fill the rest of the template with pure cell cryopreservation solution. Cure by UV irradiation for 30 seconds, followed by gradient freezing at 4℃ for 2 hours, -20℃ for 2 hours, -80℃ for 2 hours, and then placing it in liquid nitrogen for 1 hour. Carefully peel the solidified frozen microneedles from the template under low temperature conditions and store them in liquid nitrogen.

[0038] This separable cryo-microneedle can successfully transplant mesenchymal stem cells into damaged skin sites, and the microneedle's mechanical properties are as follows: Figure 6 As shown, the therapeutic effects of stem cells are used to promote wound healing.

[0039] This invention provides a hierarchical separable cryogenic microneedle, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing separable cryogenic microneedles with a hierarchical structure, characterized in that, Composed of a three-layer structure consisting of a microneedle array, a frostbite-resistant micropillar array, and a backplate, it was prepared through the following steps: (1) A microneedle-shaped male mold main structure with a hierarchical structure was obtained by 3D printing; (2) A polymer template was made by means of microneedle-shaped positive mold main structure by PDMS template replication method, and then repeatedly washed with anhydrous ethanol and ultrapure water; (3) Fill the micropores in the polymer template prepared in step (2) with the pregel solution, and then fill the other parts of the template with the nongel solution; The product is obtained by photocuring, gradient freezing, and then removing the template.

2. The method for preparing separable cryogenic microneedles with a hierarchical structure according to claim 1, characterized in that, In step (1), the microneedle-shaped male mold main structure has a microneedle array number of 3×3~10×10, a microneedle height of 500~1000 µm, a bottom radius of 300~700 µm, and a needle tip angle of 30~40°.

3. The method for preparing separable cryogenic microneedles with a hierarchical structure according to claim 2, characterized in that, The microneedle array consists of a cylindrical micropillar array connected to the microneedle array and a cuboid array centrally arranged within the cylindrical micropillar array; wherein, the bottom radius of the cylindrical array is the same as the bottom radius of the microneedle, and the height is 50-100% of the bottom radius; the cuboid array has a side length of 200-300um, and the height is the same as the cylindrical array.

4. The method for preparing separable cryogenic microneedles with a hierarchical structure according to claim 1, characterized in that, In step (3), the pregel solution is a mixture of methacrylate gelatin, cell cryopreservation solution and photoinitiator, and contains cells.

5. The method for preparing separable cryogenic microneedles with a hierarchical structure according to claim 4, characterized in that, In the pregel solution, the concentration of methacrylate gelatin is 10-15% v / v, the methacrylate gelatin is mixed with the cell cryopreservation solution at a volume ratio of 1:1, and the photoinitiator accounts for 1-2% of the volume of the pregel solution.

6. The method for preparing separable cryogenic microneedles with a hierarchical structure according to claim 4, characterized in that, The cells are selected from at least one of hair follicle stem cells, skin fibroblasts, keratinocytes, and mesenchymal stem cells; the cell addition density in the pregel solution is 1~4×10⁻⁶. 5 per mL.

7. The method for preparing separable cryogenic microneedles with a hierarchical structure according to claim 1, characterized in that, In step (3), the non-gel solution is selected from any one of deionized water, cell cryopreservation solution, and DMSO aqueous solution.

8. The method for preparing separable cryogenic microneedles with a hierarchical structure according to claim 1, characterized in that, In step (3), the irradiation time for photocuring is 30-90s; the gradient freezing method is to first store at 4℃ for 2 hours, then store at -20℃ for 2 hours, then freeze at -80℃ for 2 hours, and finally place in liquid nitrogen for 1 hour.

9. Separable cryogenic microneedles with hierarchical structure prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the separable cryo-microneedles with a hierarchical structure as described in claim 9 in the preparation of transdermal drug delivery.

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