3D printed microneedle patch as well as preparation method and application thereof

Abstract

The invention relates to a 3D printed microneedle patch and a preparation method and application thereof, the preparation method comprises the following steps: (1) mixing a gelatin solution with methacrylic anhydride, dialyzing, and freeze-drying to obtain methacrylic acid gelatin; and (2) mixing the medicine solution with methacrylic acid gelatin, and carrying out 3D printing forming. A non-invasive and convenient administration mode is provided, the microneedle patch needle tip has high puncture force, meanwhile, the pain feeling during puncture can be effectively reduced, and the accuracy of drug delivery is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to a 3D printed microneedle patch and a preparation method and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is a serious public health problem worldwide, especially in East Asia and Africa, and is one of the leading causes of cancer-related deaths. Malignant ascites is considered a sign of tumor progression and poor prognosis. About 23% of liver cancer patients develop malignant ascites. If not treated in time, the median survival of liver cancer patients with malignant ascites is only 18 months. Therefore, it is crucial to alleviate ascites symptoms and improve patients' quality of life. Since 2015, significant progress has been made in the management of HCC, especially in the development of systemic therapies, resulting in a significant improvement in patients' overall survival and quality of life.

[0003] The production of ascites stems from the imbalance between fluid production and absorption in the peritoneal cavity. In liver cancer-related ascites, the main causes can be summarized into several aspects. First, hypoalbuminemia caused by liver disease is a key factor in the formation of ascites. When liver function is impaired, albumin levels decrease, resulting in a decrease in colloid osmotic pressure, and fluid is more likely to leak from blood vessels into the abdominal cavity. Secondly, the release of inflammatory factors enhances vascular permeability. Liver cancer patients are often accompanied by inflammatory responses, which promote fluid leakage and aggravate the formation of ascites. Finally, tumor cell growth may block lymphatic vessels, hinder the normal return of lymph fluid, cause lymph accumulation, further interfere with fluid circulation, and aggravate ascites. Taken together, these mechanisms interact and jointly promote the occurrence of liver cancer-related ascites.

[0004] The main treatments for ascites include sodium restriction, diuresis, paracentesis, albumin supplementation, transjugular intrahepatic portosystemic shunt, and liver transplantation. However, these methods are not suitable for or have limited effects on some patients with malignant ascites caused by advanced HCC. Therefore, it is particularly important to find new treatment strategies for malignant ascites caused by HCC.

[0005] In summary, providing an effective method for treating malignant ascites has become one of the problems to be solved urgently in this field. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a 3D printed microneedle patch and its preparation method and application, which uses 3D printing technology to accurately control the shape, size and drug loading of the needle, has a strong puncture force, and can effectively reduce the pain during puncture and improve the accuracy of drug delivery.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a 3D printed microneedle patch, the method for preparing the microneedle patch comprising the following steps:

[0009] (1) mixing a gelatin solution with methacrylic anhydride, dialyzing, and freeze-drying to obtain methacrylated gelatin;

[0010] (2) The drug solution is mixed with methacrylated gelatin and 3D printed.

[0011] The present invention is based on natural high molecular polymers, which are modified by methacrylate to form a network. The 3D printed microneedles contain covalent crosslinking and physical effects, so that the microneedles have high mechanical strength and good toughness to facilitate puncturing the epidermis and relatively non-invasively release drugs. The raw materials for preparing the microneedle patch provided have good swelling properties and degradation capabilities, among which the swelling properties are excellent, and the degradation rate is slower than that of ordinary hydrogels. The microneedle not only has high strength, but also has good toughness, and can be used as a carrier to support drugs, enhancing the skin puncture and drug release effects.

[0012] Microneedle patches are mainly used for drug delivery and skin treatment. With their tiny needles, they break through the pain of traditional injections and provide a non-invasive and convenient way of drug delivery. Through 3D printing technology, personalized microneedle structures can be achieved, and the shape, size and drug loading of the needles can be precisely controlled to improve the pertinence and effectiveness of treatment.

[0013] Compared with traditional manufacturing, 3D printing usually uses only the required materials, which helps reduce waste, significantly shortens the product development cycle, and quickly generates usable prototypes. In small-batch production and complex designs, 3D printing is cheaper than traditional manufacturing methods, reduces the need for molds and equipment, and has high production flexibility. 3D printing can produce at the location of demand, reduce transportation costs and time, simplify the supply chain, and has a high degree of automation, which can reduce human intervention and reduce labor costs.

[0014] Preferably, the concentration of the gelatin solution in step (1) is 80-120 mg / mL (for example, it can be 80 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 105 mg / mL, 110 mg / mL or 120 mg / mL, etc.).

[0015] Preferably, the weight average molecular weight of the gelatin in step (1) is 50,000 to 100,000 Da (for example, 50,000 Da, 60,000 Da, 70,000 Da, 80,000 Da, 90,000 Da or 100,000 Da, etc.).

[0016] Preferably, the ratio of gelatin to methacrylic anhydride in the gelatin solution of step (1) is 1 g: (0.5-1) mL.

[0017] The specific point value of the above (0.5-1) can be selected from 0.5, 0.6, 0.7, 0.8, 0.9 or 1, etc.

[0018] Preferably, the mixing of the gelatin solution and methacrylic anhydride in step (1) specifically comprises: adding methacrylic anhydride dropwise to the gelatin solution and stirring for 12 to 18 hours (for example, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours or 18 hours, etc.).

[0019] Preferably, the drug in step (2) includes rhubarb.

[0020] Rhubarb not only has important functions such as purgation, removing stasis and promoting menstruation, and removing dampness and jaundice, but also has multiple pharmacological effects such as purgative, anti-inflammatory and diuretic. Specifically, its diuretic effect can effectively remove the water retained in the lesion site, while its anti-inflammatory effect helps to inhibit the progression of local lesion inflammation, thereby achieving the purpose of eliminating edema.

[0021] Preferably, the method for preparing the drug solution in step (2) comprises: taking 8% to 12% (for example, 8%, 9%, 10%, 11% or 12%, etc.) w / v supernatant of rhubarb decoction, centrifuging at 2000 to 4000 rpm (for example, 2000, 2500, 2800, 3000, 3200, 3500 or 4000, etc.) for 10 to 20 min (for example, 10 min, 12 min, 14 min, 15 min, 16 min, 18 min or 20 min, etc.), and collecting the supernatant.

[0022] Preferably, after the drug solution in step (2) is mixed with methacrylated gelatin, the content of methacrylated gelatin is 15% to 25% (for example, 15%, 17%, 19%, 20%, 21%, 23% or 25%, etc.) w / v.

[0023] Preferably, the 3D printing method in step (2) comprises a projection stereolithography method.

[0024] Preferably, the platform temperature of the 3D printing in step (2) is 35-40°C (for example, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, etc.), the trough temperature is 35-40°C (for example, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, etc.), and the light intensity is 10-20 mW / cm 3 (For example, it can be 10mW / cm 3 , 12mW / cm 3 、14mW / cm 3 、15mW / cm 3 、16mW / cm 3、18mW / cm 3 or 20mW / cm 3 The exposure time of the microneedle array is 10 to 15 s (for example, it can be 10 s, 11 s, 12 s, 13 s, 14 s or 15 s, etc.), and the exposure time of the base layer is 5 to 12 s (for example, it can be 5 s, 7 s, 8 s, 9 s, 10 s or 12 s, etc.).

[0025] The present invention ensures the accuracy and consistency of microneedles by selecting specific 3D printing parameters and methods. This process requires controlling the temperature, rate and material viscosity during the printing process to obtain the ideal finished product quality.

[0026] Preferably, after the 3D printing molding in step (2), the step also includes a step of drying at 35-40°C (for example, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, etc.) for 20-30h (for example, 20h, 22h, 24h, 26h, 28h or 30h, etc.).

[0027] In a second aspect, the present invention provides a 3D printed microneedle patch, which is prepared by the preparation method described in the first aspect.

[0028] Preferably, the microneedle patch comprises a substrate and a microneedle array, wherein the microneedle array is protrudingly disposed on one surface of the substrate;

[0029] Preferably, the microneedle array is obtained by arranging needles in a rectangular array;

[0030] Preferably, the needle body is a quadrangular pyramid.

[0031] The quadrangular pyramid needle tip provided by the present invention has a strong puncture force, and can effectively reduce the pain during puncture and improve the accuracy of drug delivery.

[0032] Preferably, the row spacing and column spacing of the microneedle array are independently 1.8 to 2.2 mm (for example, 1.8 mm, 1.9 mm, 1.95 mm, 2 mm, 2.05 mm, 2.1 mm or 2.2 mm, etc.),

[0033] Preferably, the needle body is a regular quadrangular pyramid.

[0034] Preferably, the base of the regular quadrangular pyramid is a square with a side length of 0.8 to 1.2 mm (for example, 0.8 mm, 0.9 mm, 0.95 mm, 1 mm, 1.05 mm, 1.1 mm or 1.2 mm, etc.).

[0035] Preferably, the height of the regular tetrahedron is 1 to 2 mm (for example, 1 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm or 2 mm, etc.), and more preferably 1.3 to 1.7 mm (for example, 1.3 mm, 1.35 mm, 1.4 mm, 1.45 mm, 1.5 mm, 1.55 mm, 1.6 mm, 1.65 mm or 1.7 mm, etc.).

[0036] Preferably, the number of the needles is 20 to 100 (for example, 20, 30, 40, 50, 60, 70, 80, 90 or 100, etc.).

[0037] Preferably, the number of rows and columns of the microneedle array are independently 5 to 10 (eg, 5, 6, 7, 8, 9 or 10).

[0038] Preferably, the number of rows and columns of the microneedle array is equal.

[0039] The specific microneedle shape, size and array structure provided by the present invention achieve the best puncture effect and drug release efficiency, and are not easy to fall off after piercing the skin.

[0040] Preferably, the thickness of the substrate is 0.8-1.2 mm (for example, 0.8 mm, 0.9 mm, 0.95 mm, 1 mm, 1.05 mm, 1.1 mm or 1.2 mm, etc.).

[0041] Preferably, the bottom surface of the substrate is rectangular, more preferably square.

[0042] The function of the substrate in the present invention is to load the microneedle array, so the bottom surface area of ​​the substrate needs to be larger than the area of ​​the microneedle array. For the purpose of saving materials, those skilled in the art can adjust the bottom surface area of ​​the substrate to be slightly larger than the area of ​​the microneedle array according to actual conditions, without wasting materials on an operational basis.

[0043] In a third aspect, the present invention provides use of the microneedle patch as described in the second aspect in preparing an apparatus for abdominal transdermal drug delivery.

[0044] The microneedle drug delivery method provided by the present invention has the advantages of high-precision manufacturing, rapid prototyping, personalized design, and Chinese medicine loading and release control. However, the abdominal skin is thicker, and ordinary microneedles are difficult to penetrate the epidermis, which places high mechanical requirements on the microneedles. 3D printing and photocuring treatment can significantly enhance the mechanical properties of the microneedles, and the unique quadrangular pyramid microneedles of the present invention can effectively fix the microneedle patch to prevent it from falling off.

[0045] Preferably, the transdermal drug comprises rhubarb.

[0046] Preferably, the subject of transdermal administration includes mice.

[0047] In a fourth aspect, the present invention provides use of the microneedle patch as described in the second aspect in the preparation of a drug for treating malignant ascites and / or liver cancer.

[0048] The present invention applies rhubarb microneedles to KM mice with liver cancer ascites caused by H22 cells to explore the efficacy of rhubarb microneedles in treating liver cancer ascites. After 3D printing and light curing treatment, the mechanical properties of the microneedles are significantly enhanced. Compared with ordinary microneedles, this unique quadrangular pyramid microneedle is not only easier to penetrate the thicker abdominal epidermis of mice, but also can be effectively fixed to prevent falling off. For patients with advanced liver cancer and malignant ascites, this relatively non-invasive 3D microneedle technology can effectively avoid the adverse complications caused by multiple abdominal punctures, providing a new way to administer rhubarb.

[0049] Preferably, the malignant ascites includes liver cancer ascites.

[0050] Preferably, the microneedle patch contains rhubarb.

[0051] Preferably, the administration method of the drug includes abdominal transdermal administration.

[0052] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] (1) The microneedle patch provided by the present invention is prepared by a 3D printing method, which can accurately control the shape, size and drug loading of the needle. The quadrangular pyramid needle tip has a strong puncture force and can effectively reduce the pain during puncture. By further optimizing the raw materials, size and array structure of the microneedles, the best puncture effect and drug release efficiency are obtained, and it is not easy to fall off after piercing the skin.

[0055] (2) The microneedle patch provided by the present invention can be used for abdominal transdermal drug delivery and can penetrate the thick abdominal epidermis to effectively fix the microneedle patch and prevent it from falling off.

[0056] (3) The microneedle patch provided by the present invention can be loaded with the Chinese medicine rhubarb and administered transdermally in the abdomen to treat liver cancer and malignant ascites caused by liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a physical picture of the 3D printed microneedle patch of the present invention.

[0058] Figure 2 This is the Giemsa staining result of the ascites smear in Test Example 5.

[0059] Figure 3This is the H&E staining result of liver tissue in Test Example 5.

[0060] Figure 4 This is the result of Ki67 tissue immunofluorescence staining of liver tissue in Test Example 5.

[0061] Figure 5 This is the result of Ki67 tissue immunofluorescence staining of ascites smear in Test Example 5. DETAILED DESCRIPTION

[0062] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0063] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0064] The instruments and reagents used in the following examples are:

[0065] 3D printer: projection-type multi-material light-curing biological 3D printer (Suzhou Yongqinquan Intelligent Equipment Co., Ltd.).

[0066] Gelatin: Macklin, biotech grade, Cas number 9000-70-8.

[0067] Methacrylic anhydride: Macklin, 94%, stabilized with 0.2% topanol, Cas No. 760-93-0.

[0068] Rhubarb: Authentic medicinal rhubarb from Chengkou County, Chongqing.

[0069] Preparation Example 1

[0070] This preparation example provides a method for preparing a 3D printed microneedle patch, comprising the following steps:

[0071] (1) 10% w / v rhubarb water decoction was prepared using authentic medicinal rhubarb from Chengkou County, Chongqing. The supernatant was transferred to a new centrifuge tube and centrifuged at 3000 rpm for 15 min. The supernatant was collected to obtain a rhubarb water extract.

[0072] (2) Gelatin was dissolved in deionized water until fully dissolved to prepare a solution with a concentration of 100 mg / mL, and methacrylic anhydride (MA) was then added dropwise to the solution and stirred for 14 h. The amount of MA added per gram of gelatin was 0.8 mL. The solution after the reaction was then dialyzed, and the liquid in the dialysis bag was freeze-dried to obtain methacrylated gelatin (GelMA).

[0073] (3) The rhubarb water extract obtained in step (1) and the GelMA obtained in step (2) are mixed in a beaker and stirred until fully mixed. The content of GelMA is 20% w / v, and rhubarb bio-ink is obtained.

[0074] (4) Create a three-dimensional model, use projection stereolithography technology to perform layer-by-layer molding, and dry at 37°C for 24 hours to obtain a 3D printed microneedle patch. The process parameters of 3D printing are as follows: a 37°C platform and a 37°C material tank are used for projection stereolithography printing, and the light intensity is 15mW / cm 3 The exposure time of the microneedle array is 10-15s, the exposure time of the base layer is 5-12s, the slice layer height is 50μm, the number of base layers is 20, the peeling distance is 4mm, the peeling speed is 25mm / min, the peeling return speed is 180mm / min, the lifting height is 0mm, and the lifting speed is 100mm / min.

[0075] Comparative Preparation Example 1

[0076] This preparation example provides a method for preparing a microneedle patch, comprising the following steps:

[0077] (1) Preparation of microneedle body: 5% gelatin + 0.5% hyaluronic acid are mixed to form a microneedle base material mixed solution, and then the mixed solution is evenly filled into the microneedle mold and the bubbles are removed by vacuum treatment; then it is solidified at an appropriate temperature and the residual moisture is removed by vacuum drying or freeze drying.

[0078] (2) Preparation of microneedle base: Weigh 6 g of PVA into a beaker, add 100 mL of ddH2O, turn on the stirrer, and stir for 15-30 minutes without heating. Slowly increase the temperature to 85°C (partial alcoholysis of PVA) or 90-95°C (complete alcoholysis of PVA), and continue stirring until the PVA is completely dissolved to obtain a 6% PVA solution. The microneedle body is joined to the base to form a microneedle patch.

[0079] Example 1

[0080] This embodiment provides a 3D printed microneedle patch, which is prepared by the method provided in Preparation Example 1. The microneedle patch includes a substrate and a microneedle array. The microneedle array is protruding on one side of the substrate. The microneedle array is obtained by arranging needles in a rectangular array, consisting of 49 needles, with 7 needles in each row and column. The row spacing and column spacing of the microneedle array are both 2mm; the needle body is a regular tetrahedron, the side length of the bottom square is 1mm, and the height is 1.5mm; the bottom surface of the substrate is a square with a side length of 15mm and a thickness of 1mm. The actual picture of the 3D printed microneedle patch is as follows: Figure 1 shown.

[0081] Example 2

[0082] This embodiment provides a 3D printed microneedle patch, which is prepared by the method provided in Preparation Example 1. The microneedle patch includes a substrate and a microneedle array, the microneedle array is protrudingly arranged on one side of the substrate, and the microneedle array is obtained by arranging needle bodies in a rectangular array, consisting of 25 needle bodies, 5 needles in each row and column, and the row spacing and column spacing of the microneedle array are both 2.2mm; the needle body is a regular tetrahedron, the side length of the bottom square is 0.8mm, and the height is 1.7mm; the bottom surface of the substrate is a square with a side length of 11mm and a thickness of 0.8mm.

[0083] Example 3

[0084] This embodiment provides a 3D printed microneedle patch, which is prepared by the method provided in Preparation Example 1. The microneedle patch includes a substrate and a microneedle array, the microneedle array is protruding on one side of the substrate, the microneedle array is obtained by arranging needles in a rectangular array, and is composed of 100 needles, with 10 needles in each row and column, and the row spacing and column spacing of the microneedle array are both 1.8mm; the needle body is a regular tetrahedron, the side length of the bottom square is 1.2mm, and the height is 1.3mm; the bottom surface of the substrate is a square with a side length of 19mm and a thickness of 1.2mm.

[0085] Example 4

[0086] This embodiment provides a 3D printed microneedle patch, which differs from Embodiment 1 only in that the row spacing and column spacing of the microneedle array are both 1.5 mm.

[0087] Example 5

[0088] This embodiment provides a 3D printed microneedle patch, which differs from Embodiment 1 only in that the row spacing and column spacing of the microneedle array are both 2.5 mm.

[0089] Example 6

[0090] This embodiment provides a 3D printed microneedle patch, which differs from Embodiment 1 only in that the length of the needle body is 0.5 mm.

[0091] Example 7

[0092] This embodiment provides a 3D printed microneedle patch, which differs from Embodiment 1 only in that the length of the needle body is 2.5 mm.

[0093] Example 8

[0094] This embodiment provides a 3D printed microneedle patch, which is different from Embodiment 1 only in that the bottom surface of the substrate is circular with a diameter of 20 mm.

[0095] Example 9

[0096] This embodiment provides a 3D printed microneedle patch, which is different from Embodiment 1 only in that the needle body is conical, the bottom surface is a circle with a diameter of 1 mm and a height of 1.5 mm.

[0097] Comparative Example 1

[0098] This comparative example provides a microneedle patch, which is different from Example 1 only in that it is prepared by the method provided in Comparative Preparation Example 1.

[0099] Test Example 1

[0100] This test example carried out biocompatibility and safety tests on the microneedle patches provided in the embodiments and comparative examples. In order to evaluate the in vitro toxicity of the rhubarb microneedles, H22 cells and HepG2 cells were inoculated in 48-well plates. After the cells adhered to the wall, they were divided into a control group, a blank microneedle group and a rhubarb microneedle group, and cultured for 24 hours. Cell counting kit-8 (CCK-8, Biyuntian, Shanghai, China) was used to analyze cell activity, and the LIVE / DEAD cell activity / death detection kit (Biyuntian, Shanghai, China) was used to observe cell activity and morphological changes. The test results show that the tested microneedle patch is non-toxic and harmless, and will not cause side effects such as immune rejection of the body.

[0101] Test Example 2

[0102] This test example explores whether the microneedle patch provided in the embodiment and the comparative example can penetrate the abdominal skin, whether it is easy to fall off after penetration, and the pain after penetration. The steps are as follows: Kunming mice are used as experimental subjects. After anesthesia with 1% sodium pentobarbital injected intraperitoneally, the mouse abdomen is depilated with a depilatory cream, the mouse abdominal skin is cleaned and disinfected, and the microneedle is carefully attached vertically to the skin and appropriate pressure is applied to make it completely penetrate. Observe whether there is bleeding or damage at the administration site, lift the mouse vertically to observe the fixation of the microneedle in the abdomen, and regularly monitor the activity and reaction of the mouse to evaluate the pain of microneedle penetration (because there is currently no instrument that directly responds to the pain of mice, the pain reaction can only be observed through the mouse activity and the mouse reaction during microneedle penetration).

[0103] The test results show that Example 4 has a strong piercing pain due to the small row and column spacing; Example 6 has a short needle body, and the microneedle cannot pierce the thicker abdominal skin; Example 7 has a long needle body, resulting in a strong piercing pain; Example 8 has a circular bottom surface, while the microneedle array is rectangular as a whole, and the area on the substrate that is not covered with the needle body accounts for a large proportion, resulting in the microneedle patch not being able to fit well with the skin and being easy to fall off; Example 9 has a conical needle body, and the fixation effect is poor after piercing the skin, and it is easy to fall off; The preparation method of the microneedle patch of Comparative Example 1 is to solidify and mold in a mold, and the mechanical properties of the prepared product are poor, and the microneedle cannot pierce the thicker abdominal skin. The microneedle patches provided in the comparative examples of the above embodiments are no longer tested for subsequent treatment effects.

[0104] Test Example 3

[0105] In this test example, the better 3D printed microneedle patch screened in Test Example 2 was used to treat mouse ascites. 8-week-old KM mice were selected for animal experiments. The experiment was conducted in accordance with the protocol approved by the Medical Ethics Committee of Chongqing Medical University (ACUC-CQMU-2024-0607) and the guidelines of the Chinese Laboratory Animal Management Regulations. The mice were kept at a temperature of 24°C, a relative humidity of 65±15%, a light / dark cycle of 12h, and fed a standard diet and tap water.

[0106] The formation of malignant ascites is closely related to the proliferation ability of tumor cells, the tumor microenvironment, and the systemic inflammatory response. As a liver cancer cell line, the development of H22 cells may directly affect liver function, leading to a decrease in albumin synthesis and changes in liver hemodynamics, thereby inducing ascites. At the same time, the rapid proliferation of H22 cells may oppress and invade surrounding tissues and organs. In addition, H22 cells may also secrete a variety of tumor-promoting factors, further promoting the formation of ascites. Therefore, this test case established a malignant ascites model in tumor-bearing mice by injecting H22 liver cancer cells into the peritoneal cavity of healthy mice.

[0107] Mouse H22 hepatoma cell line was purchased from the National Biomedical Experimental Cell Resource Bank and subcultured until the cells reached the logarithmic growth phase. The cells were resuspended in physiological saline at a concentration of 1×10 6 Thirty-six male KM mice were randomly divided into six groups, each with six mice, namely the experimental group, the model group and the control group. The mice in the experimental group and the model group were injected with 0.2 mL of H22 liver cancer cell suspension (5 × 10 cells / mL per mouse) into the peritoneal cavity. 5 The mice in the experimental group received topical microneedle patch treatment, the mice in the model group received topical saline, and the mice in the control group did not receive any treatment. From the 1st day to the 20th day after tumor inoculation, the abdominal circumference and body weight of the mice were measured regularly. On the 7th day, the abdomen of the mice showed obvious bulges. The drug was started on the 7th day. The changes in the abdominal circumference and body weight of the mice are shown in Table 1.

[0108] Table 1

[0109]

[0110] Comparing Example 1 with the model group, it can be seen that the 3D printed microneedle patch provided by the present invention can reduce the abdominal circumference and body weight of mice and effectively treat ascites of liver cancer in mice. Comparing Example 1 with Example 5, it can be seen that due to the large spacing between the rows and columns of the microneedle patch in Example 5 and the low drug concentration, the treatment effect is poor.

[0111] Test Example 4

[0112] In this test example, the mice in Test Example 3 were killed on the 21st day, and the ascites volume of the mice, the activity of the alanine aminotransferase (ALT) enzyme and the aspartate aminotransferase (AST) enzyme, which are indicators of liver function in the mice serum, were measured. The results are shown in Table 2.

[0113] Table 2

[0114]

[0115]

[0116] Normal mice usually have less ascites, usually no more than 1 mL, and the cells in their ascites are mainly composed of red blood cells, white blood cells, macrophages and lymphocytes. In malignant ascites, there are often a large number of malignant cells, increased white blood cells and increased protein content. Comparing the experimental group with the model group, it can be seen that the ascites of mice treated with the rhubarb microneedle patch was significantly reduced, and the ALT enzyme activity and AST enzyme activity were lower, and the degree of liver damage was lower.

[0117] Test Example 5

[0118] This test example Example 1: After the ascites smears extracted from the mice in the model group and the control group were placed on glass slides, they were fixed with 4% paraformaldehyde at 4°C for 24 hours and stained with Giemsa (Serbicebio, Wuhan, China). Figure 2 As shown, the smear of the ascites of the model group showed a large number of malignant cell infiltrations, while the number of malignant tumor cells in Example 1 was significantly reduced.

[0119] The liver tissues of mice in Example 1, the model group, and the control group were fixed in 10% buffered formalin and embedded in paraffin. Paraffin sections (4 μm thick) were stained with hematoxylin and eosin (H&E) (H&E, Serbicebio, Wuhan, China) and examined under an optical microscope to determine the cancer cell infiltration of the liver tissue. The results of H&E staining are shown in Figure 2. Figure 3As shown, the diffuse necrosis area of ​​the tumor in Example 1 is smaller, while the necrosis area of ​​the tumor in the model group is larger.

[0120] Liver sections were dewaxed using xylene and graded concentrations of ethanol in deionized water, and ascites smears were directly subjected to antigen retrieval. Sections were incubated in 10 mM citrate buffer (pH 6.0) at 90 °C for 20 min for antigen retrieval. Sections were then incubated with anti-Ki67 (1:200, Proteintech, Wuhan, China) antibodies overnight at 4 °C. Slides were then washed with PBS and incubated with FITC-labeled goat anti-rabbit fluorescent secondary antibodies for 1 h at room temperature (1:200, A0562, Beyotime, Shanghai, China). Slides were washed with PBS, and DAPI (2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride) (C1002, Beyotime, Shanghai, China) was added at a working concentration of 0.5-10 μg / mL, and images were observed using a laser confocal microscope (Olympus, Tokyo, Japan). The results of Ki67 tissue immunofluorescence staining of liver sections and ascites smears are shown in Figure 4 and Figure 5 As shown, Figure 4 Mouse liver tissue slices. Figure 5 This is a smear of mouse ascites. Figure 4 The results showed that the proportion of Ki67-positive cells in the liver tumor tissue of mice in the microneedle intervention group was lower than that in the tumor infiltration area of ​​the model group, indicating that rhubarb extract inhibited the proliferation of tumor cells (H22). Figure 5 The results also showed that there were basically no ascites cells in the control group, and more H22 cells in the model group. The total number of ascites cells and Ki67 expression were significantly reduced after microneedle intervention. In summary, it was confirmed that rhubarb microneedles have anti-tumor activity and reduce ascites caused by malignant tumors.

[0121] In summary, the 3D printed microneedle patch provided by the present invention can accurately control the shape, size and drug loading of the needle. The quadrangular pyramid needle tip has a strong puncture force and can effectively reduce the pain during puncture. It can be loaded with the traditional Chinese medicine rhubarb and administered transdermally in the abdomen to treat liver cancer and malignant ascites of liver cancer caused by liver cancer.

[0122] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing a 3D printed microneedle patch, characterized in that: The preparation method comprises the following steps: (1) mixing a gelatin solution with methacrylic anhydride, dialyzing, and freeze-drying to obtain methacrylated gelatin; (2) The drug solution is mixed with methacrylated gelatin and 3D printed.

2. The preparation method according to claim 1, characterized in that: The concentration of the gelatin solution in step (1) is 80-120 mg / mL; Preferably, the weight average molecular weight of the gelatin in step (1) is 50,000 to 100,000 Da; Preferably, the ratio of gelatin to methacrylic anhydride in the gelatin solution of step (1) is 1 g: (0.5-1) mL; Preferably, the step (1) of mixing the gelatin solution with methacrylic anhydride specifically comprises: adding methacrylic anhydride dropwise into the gelatin solution and stirring for 12 to 18 hours.

3. The preparation method according to claim 1 or 2, characterized in that: The medicine in step (2) includes rhubarb; Preferably, the preparation method of the drug solution in step (2) comprises: taking the supernatant of 8% to 12% w / v rhubarb decoction, centrifuging at 2000 to 4000 rpm for 10 to 20 min, and collecting the supernatant; Preferably, after the drug solution in step (2) is mixed with methacrylated gelatin, the content of methacrylated gelatin is 15% to 25% w / v.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The 3D printing method in step (2) includes a projection stereolithography method; Preferably, in step (2), the platform temperature of the 3D printing is 35-40°C, the material tank temperature is 35-40°C, and the light intensity is 10-20 mW / cm 3 , the exposure time of the microneedle array is 10 to 15 s, and the exposure time of the base layer is 5 to 12 s; Preferably, after the 3D printing molding in step (2), the step also includes a drying step at 35 to 40° C. for 20 to 30 hours.

5. A 3D printed microneedle patch, characterized in that: The microneedle patch is prepared by the preparation method according to any one of claims 1 to 4.

6. The microneedle patch according to claim 5, characterized in that: The microneedle patch comprises a substrate and a microneedle array, wherein the microneedle array is protrudingly arranged on one surface of the substrate; Preferably, the microneedle array is obtained by arranging needles in a rectangular array; Preferably, the needle body is a quadrangular pyramid; Preferably, the row spacing and column spacing of the microneedle array are independently 1.8 to 2.2 mm.

7. The microneedle patch according to claim 5 or 6, characterized in that: The needle body is a regular quadrangular pyramid; Preferably, the base of the regular quadrangular pyramid is a square with a side length of 0.8 to 1.2 mm; Preferably, the height of the regular quadrangular pyramid is 1 to 2 mm, more preferably 1.3 to 1.7 mm; Preferably, the number of the needles is 20 to 100; Preferably, the number of rows and columns of the microneedle array are independently 5 to 10; Preferably, the number of rows and columns of the microneedle array is equal; Preferably, the thickness of the substrate is 0.8 to 1.2 mm; Preferably, the bottom surface of the substrate is rectangular, more preferably square.

8. Use of the microneedle patch according to any one of claims 5 to 7 in the preparation of an abdominal transdermal drug delivery device.

9. The use according to claim 8, characterized in that: The transdermal drug includes rhubarb; Preferably, the subject of transdermal administration includes mice.

10. Use of the microneedle patch according to any one of claims 5 to 7 in the preparation of a drug for treating malignant ascites and / or liver cancer; Preferably, the malignant ascites includes liver cancer ascites; Preferably, the microneedle patch contains rhubarb; Preferably, the administration method of the drug includes abdominal transdermal administration.

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