Preparation method of soluble microneedle for delivering LIF (leukemia inhibitory factor)

Abstract

The invention provides a preparation method of a soluble microneedle capable of realizing painless sustained-release delivery of LIF factors. According to the method, the LIF factors are uniformly loaded on the needle tip (the LIF factors are verified to be uniformly distributed through fluorescence labeling) by optimizing the design and process of the microneedle, and the prepared microneedle is complete and smooth in needle shape (observed and confirmed by an optical microscope). An in-vitro penetrating power test (four layers of sealing films and a mouse skin model) proves that the microneedle has enough mechanical strength to penetrate into the skin. The technology solves the problem of pain of a traditional injection mode, obviously improves the compliance of a patient, and is especially suitable for a scene requiring long-term administration; meanwhile, technical reference is provided for microneedle delivery of biomacromolecules such as protein and nucleic acid.

Description

Technical Field

[0001] This invention relates to a method for preparing soluble microneedles for delivering LIF (leukemia inhibitory factor). Background Technology

[0002] Dissolvable microneedles (DMNs), as an emerging drug delivery technology, are based on the principle of combining drugs with a biodegradable polymer matrix material to create tiny needle-like structures. These microneedles typically have a diameter on the order of tens of micrometers, while their length is strictly controlled within the range of hundreds of micrometers. This design aims to ensure that the microneedles can precisely penetrate the stratum corneum of the skin while avoiding damage to deeper skin tissues. The unique biodegradable matrix material gradually dissolves within the skin and is released into specific skin layers. This release mechanism enables continuous and stable drug delivery over a certain period, thereby optimizing the therapeutic effect. Compared to traditional microneedling techniques, dissolvable microneedles can improve drug absorption efficiency and enhance therapeutic effects, while reducing injection discomfort and side effects. Furthermore, this technology has been applied in various fields such as vaccination, topical treatment, and cosmetic skincare, showing broad application prospects.

[0003] Leukemia inhibitory factor (LIF) is a pleiotropic cytokine expressed in various tissue types. It possesses a wide range of biological activities, including inducing leukemia cell differentiation, regulating bone tissue growth and metabolism, maintaining the basic characteristics of embryonic pluripotent stem cells (ES cells), and promoting the differentiation of cholinergic neurons. LIF primarily exerts its biological functions by activating and regulating the JAK / STAT3, AKT, ERK1 / 2, and mTOR signaling pathways, and is eliminated by SOCS and PIAS proteins. The leukemia inhibitory factor receptor (LIFR) is a class I cytokine receptor belonging to the hematopoietic cytokine receptor superfamily. Beyond its classic hematopoietic function, LIF (Liver-Induced Fertilizer) also influences various endocrine tissues and cell types, including the proliferation of primordial germ cells, maintenance of pluripotent embryonic stem cells, endometrial decidualization and blastocyst implantation, activation of the hypothalamus-pituitary-adrenal (HPA) axis and pituitary development, osteoblast and osteoclast function, adipocyte lipid and energy homeostasis, and autocrine / paracrine growth regulation of endocrine-responsive tumors, playing a crucial role under diverse physiological and pathological conditions. Therefore, LIF has significant value in biomedical research and clinical applications. Summary of the Invention

[0004] The present invention aims to explore the preparation of soluble microneedles for delivering LIF factors and provide a novel method for painless, slow and continuous delivery of LIF factors.

[0005] The key technical solutions adopted in this invention are mainly the preparation of soluble microneedles and the delivery of LIF factors.

[0006] The specific operation of this invention includes the following steps: (1) A PDMS microneedle mold with a needle length of 800μm, a microneedle bottom diameter of 300μm, a needle tip distance of 650μm, a needle tip number of 10×10, a needle distribution area of ​​6.5mm×6.5mm, and an overall patch size of 8.9mm×8.9mm was customized. (2) Preparation of soluble microneedle material: Weigh 102.8 mg of 2-aminopyridine, add 10 ml of N,N-dimethylformamide (DMF) to dissolve it. After it is completely dissolved, add 54 mg of 1-hydroxybenzotriazole (HOBT) and 77 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (HOBT), and incubate at 37°C for 1 h to ensure that the components react fully. (3) Preparation of 3% GelMA solution (containing LIF): Weigh 0.3g GelMA and dissolve it in 10ml ddH2O. Incubate at 40℃ for 25min, then transfer to a 50℃ water bath and keep for 5min to ensure complete dissolution. Filter through a 0.45μm filter membrane for sterilization, add 500μl Leukemia inhibitory factor (LIF) and mix well. Centrifuge at 3000rpm for 2min to remove air bubbles. (4) Transfer the solution prepared in step (2) to the solution in step (3) and react at 37°C overnight; (5) Adjust the pH to 8, add 1g of sodium chloride (NaCl), and perform dialysis using a 3500 dialysis bag; on the first day of dialysis, change the water every 2 hours and add 5g of NaCl to the beaker; on the second and third days, change the water every 2 hours, but do not add NaCl; on the fourth day, perform freeze drying. (6) Preparation of 10% precursor solution: Weigh 0.3g of the above-obtained lyophilized powder and dissolve it in 3ml ddH2O. Incubate at 40℃ for 25min, then transfer to a 50℃ water bath and keep for 5min to ensure complete dissolution. Filter with a 0.45μm filter membrane for sterilization, and centrifuge at 3000rpm for 2min to remove air bubbles. (7) PDMS microneedle membranes are sterilized by UV irradiation. 600 μl of precursor solution is added to the mold, and vacuum is applied to remove bubbles 3 times, 2 min each time. The bubbles are gently scraped off with a pipette tip. The membrane is then placed in a 30-35℃ drying oven and dried for 2-3 h. After heating and concentrating to a viscous state (avoiding over-drying and exposing the mold substrate), 600 μl of 10% precursor solution is added again, and the membrane is dried for 3-4 h (repeated 2-3 times). (8) Then place it at 4℃ for rapid cooling to remove the microneedles; (9) Take pictures of the demolded microneedles and observe and photograph them under a microscope to check whether the microneedle tips are intact; (10) Place four sealing films on a table, insert the microneedle into the sealing film, and observe whether the microneedle can penetrate the sealing film and the changes in the needle tip before and after the microneedle is inserted. (11) Mice were euthanized by cervical dislocation. Hair removal cream was used to remove hair from the back of the mice. Microneedles were inserted into the back of the mice to observe whether the microneedles were inserted into the skin and to stain the needle holes with trypan blue. (12) Detection of LIF factor distribution in microneedles: LIF factor is linked to fluorescent dye CY5. When making microneedles, LIF factor linked to CY5 is added. After the microneedles are made, the distribution of LIF factor in microneedles is observed under a fluorescence microscope.

[0007] The present invention has the following advantages: (1) High safety: The microneedles have a small diameter and moderate length, which only penetrate the stratum corneum and do not damage the deep tissues. The matrix material has good biocompatibility and is biodegradable, avoiding the risks of traditional microneedle tip breakage and drug leakage. (2) Stable drug release: After the microneedles are inserted into the skin, they gradually dissolve, allowing the drug to be slowly released into a specific skin layer, achieving continuous and stable drug delivery, improving drug absorption efficiency and therapeutic effect. (3) High comfort: Continuous and stable delivery reduces discomfort and potential side effects during the injection process, improving the patient's user experience. (4) High efficiency of absorption: Microneedles can penetrate the stratum corneum of the skin and deliver drugs directly to specific layers of the skin (such as the epidermis or dermis), thereby significantly improving the absorption efficiency of drugs. Attached Figure Description

[0008] like Figure 1 Figure A shows a schematic diagram of the microneedles after curing and demolding, illustrating that Xinhecheng's materials can be used to make microneedles; for example... Figure 1 The image shown in Figure B, taken under a microscope, reveals that the microneedle tip is intact and sharp.

[0009] like Figure 2 Figures A and B show the front and back sides of the sealing film after the soluble microneedles have been inserted; as shown... Figure 2 Figures C and D show the changes in the microneedle tip before and after the soluble microneedles are inserted into the sealing film, illustrating that the microneedles have a certain degree of hardness.

[0010] like Figure 3 Figure A shows a microneedle inserted into the skin on the back of a mouse; as shown in Figure A. Figure 3 As shown in Figure B, trypan blue staining of the microneedle puncture sites indicates that the microneedles can penetrate mouse skin and that the microneedles are sufficiently rigid.

[0011] like Figure 4The distribution of LIF factors in microneedles containing LIF factors connected to CY5, as shown in Figures A and B, is observed under a fluorescence microscope, indicating that LIF is uniformly distributed in the microneedles. Detailed Implementation

[0012] Example (1) Microneedle mold preparation a. A custom PDMS microneedle mold with a needle length of 800μm, a microneedle bottom diameter of 300μm, a needle tip distance of 650μm, 10×10 needle tips, a needle distribution area of ​​6.5mm×6.5mm, and an overall patch size of 8.9mm×8.9mm; b. Preparation of soluble microneedle materials: Weigh 102.8 mg of 2-aminopyridine and dissolve it in 10 ml of N,N-dimethylformamide (DMF). After complete dissolution, add 54 mg of 1-hydroxybenzotriazole (HOBT) and 77 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (HOBT). Incubate at 37°C for 1 h to ensure the material is ready. c. Preparation of 3% GelMA solution (containing LIF): Weigh 0.3g GelMA and dissolve it in 10ml ddH2O. Incubate at 40℃ for 25min, then transfer to a 50℃ water bath and keep for 5min to ensure complete dissolution. Filter through a 0.45μm filter membrane for sterilization, add 500μl of Leukemia inhibitory factor (LIF) and mix well. Centrifuge at 3000rpm for 2min to remove air bubbles. d. Transfer the solution prepared in step (2) to the solution prepared in step (3) and react at 37°C overnight; e. Adjust the pH to 8, add 1g of sodium chloride (NaCl), and perform dialysis using a 3500 dialysis bag; on the first day of dialysis, change the water every 2 hours and add 5g of NaCl to the beaker; on the second and third days, change the water every 2 hours, but do not add NaCl; on the fourth day, perform freeze-drying. f. Preparation of 10% precursor solution: Weigh 0.3g of the above-obtained lyophilized powder and dissolve it in 3ml of ddH2O. Incubate at 40℃ for 25min, then transfer to a 50℃ water bath and keep for 5min to ensure complete dissolution. Filter through a 0.45μm filter membrane for sterilization, and centrifuge at 3000rpm for 2min to remove air bubbles. g. After sterilizing the PDMS microneedle membrane with ultraviolet light, add 600 μl of precursor solution to the mold, vacuum and defoam three times for 2 minutes each time, gently scrape off the air bubbles with a pipette tip, and dry in a 30-35℃ drying oven for 2-3 hours. After heating and concentrating to a viscous state (avoid over-drying and exposing the mold substrate), add 600 μl of 10% precursor solution again and dry for 3-4 hours (repeat 2-3 times). h. Then, it is placed at 4℃ for rapid cooling to remove the microneedles.

[0013] Microneedle performance testing: (1) Morphological observation: The soluble microneedles prepared above were observed using a Leica DMIL inverted microscope. The results showed that the microneedles were in the shape of a rectangular patch, with conical microneedles of 800 μm height and sharp tips evenly distributed on the surface. (2) Hardness testing of soluble microneedle tips, the specific method is as follows: a. Insert the microneedle into the sealing film and observe whether the microneedle can penetrate the sealing film. Figure 2 A, Figure 2 B), and record the changes at the needle tip ( Figure 2 C Figure 2 D); b. The mice were euthanized by cervical dislocation, and the hair on the back of the mice was removed using hair removal cream. c. Insert the microneedles into the mouse skin and observe whether the microneedles penetrate the skin. Figure 3 A), and stain the pinholes with trypan blue solution. Figure 3 B).

[0014] LIF factor in microneedle distribution detection (1) The distribution of LIF factor in microneedles was observed under a fluorescence microscope. The specific method is as follows: a. The LIF factor is linked to the fluorescent dye CY5 and added to the GelMA solution during microneedle fabrication. Microneedles are fabricated according to the steps described above. b. Observe the distribution of LIF factor in the prepared microneedles under a fluorescence microscope. Figure 4 (A and B).

Claims

1. A method for preparing soluble microneedles for delivering LIF (Leukemia inhibitory factor), characterized in that... LIF is slowly and continuously released through soluble microneedles, thus achieving minimally invasive and painless continuous delivery of LIF.

2. The method according to claim 1, characterized in that... The process of creating soluble microneedles to encapsulate LIF for slow and continuous release is as follows; (1) A PDMS microneedle mold with a needle length of 800μm, a microneedle bottom diameter of 300μm, a needle tip distance of 650μm, a needle tip number of 10×10, a needle distribution area of ​​6.5mm×6.5mm, and an overall patch size of 8.9mm×8.9mm was customized. (2) Preparation of soluble microneedle material: Weigh 102.8 mg of 2-pyridine (2-Aminopyridine), add 10 ml of N,N-dimethylformamide (DMF) to dissolve it. After it is completely dissolved, add 54 mg of 1-hydroxybenzotriazole (HOBT) and 77 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (HOBT), and incubate at 37°C for 1 h to ensure that the components react fully. (3) Preparation of 3% GelMA solution (containing LIF): Weigh 0.3g GelMA and dissolve it in 10ml ddH2O. Incubate at 40℃ for 25min, then transfer to a 50℃ water bath and keep for 5min to ensure complete dissolution. Filter with a 0.45μm filter membrane for sterilization, add 500μl LIF factor and mix well. Centrifuge at 3000rpm for 2min to remove air bubbles. Transfer the solution prepared in step (2) to the solution in step (3) and react at 37℃ overnight. (4) Adjust the pH to 8, add 1g of sodium chloride (NaCl), and perform dialysis using a 3500 dialysis bag; perform dialysis for two days, and freeze-dry on the third day; (5) Preparation of 10% precursor solution: Weigh 0.3g of the above-obtained lyophilized powder and dissolve it in 3ml of ddH2O. Incubate at 40℃ for 25min, then transfer to a 50℃ water bath and keep for 5min to ensure complete dissolution. Filter sterilize with a 0.45μm filter membrane and centrifuge at 3000rpm for 2min to remove air bubbles. Sterilize the PDMS microneedle membrane with UV light. Add 600μl of precursor solution to the mold, vacuum and degas 3 times for 2min each time, gently scrape off the air bubbles with a pipette tip, and dry in a 30-35℃ drying oven for 2-3h. After heating and concentrating to a viscous state (avoid over-drying and exposing the mold base), add 600μl of 10% precursor solution again and dry for 3-4h (repeat 2-3 times). (6) Then place it at 4℃ for rapid cooling to remove the microneedles; (7) Test the integrity, mechanical properties and encapsulation ability of the microneedles.

3. The method according to claim 2, characterized in that... Soluble microneedles for delivering LIF were fabricated using newly synthesized materials. The integrity of the needle tip was observed using an optical microscope. The mechanical properties of the microneedles were tested using a sealing film and mouse ex vivo skin. The distribution of LIF factors in the microneedles was observed using a fluorescence microscope, demonstrating that the newly fabricated microneedles can deliver LIF factors.

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