FVIII ice microneedle as well as preparation method and application thereof

Abstract

The invention relates to the technical field of drug delivery systems, in particular to an FVIII ice microneedle as well as a preparation method and application thereof. The preparation method comprises the following steps: providing a mold with a microneedle cavity; a medicine solution containing FVIII is injected into the microneedle cavity of the mold; and freezing the mold into which the medicine solution is injected, so that the medicine solution is solidified and formed in the cavity of the micro-needle, and the FVIII ice micro-needle is obtained. The invention systematically solves a series of problems of easy activity loss, low delivery efficiency, inconvenience in use and the like in transdermal delivery of FVIII protein through a core technical concept of low-temperature freezing forming, and provides a novel hemophilia A treatment dosage form which is high in activity retention rate, safe and convenient to use and suitable for large-scale production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug delivery systems, in particular to an FVIII ice microneedle and a preparation method and application thereof. BACKGROUND

[0002] Hemophilia A is a genetic bleeding disorder caused by mutations in the coagulation factor VIII (FVIII) gene. The current standard treatment regimen is regular intravenous infusion of exogenous FVIII preparations. However, this treatment regimen has obvious deficiencies in practical application: the intravenous administration method is highly professional and painful, resulting in poor patient treatment compliance, and cannot achieve patient self-administration, which seriously affects their daily activities and emergency handling capabilities.

[0003] To overcome the deficiencies of intravenous administration, transdermal microneedle technology has attracted widespread attention as a painless and convenient administration method. Microneedles can penetrate the stratum corneum through their micron-scale structure, delivering drugs directly to the dermis, avoiding liver first-pass effect, and potentially enabling patient self-administration. However, when microneedle technology is applied to FVIII delivery, it still faces severe challenges: FVIII, as a complex macromolecular protein, has extremely poor stability in liquid and room temperature environments, and is prone to denaturation, aggregation, or degradation, resulting in rapid loss of its biological activity. The preparation process of traditional microneedles often involves steps such as heating, ultraviolet irradiation, or chemical crosslinking, which can further exacerbate the degradation of FVIII, making it difficult to ensure the activity of the delivered drug. In addition, even if successful, FVIII-loaded microneedles are difficult to maintain their long-term stability during storage, with rapid activity decay. SUMMARY

[0004] The purpose of the present application is to overcome the problems in the prior art and provide an FVIII ice microneedle and a preparation method and application thereof.

[0005] To achieve the above-mentioned purpose, the present application provides a preparation method of an FVIII ice microneedle, comprising the following steps: S1. Providing a mold with microneedle cavities; S2. Injecting a drug solution containing FVIII into the microneedle cavities of the mold; S3. Freezing the mold injected with the drug solution to solidify the drug solution in the microneedle cavities to form an FVIII ice microneedle.

[0006] Preferably, S1 comprises the following steps: (1) Using 3D printing technology to prepare a microneedle positive mold; (2) Hydrophobizing the microneedle positive mold; (3) After mixing the PDMS prepolymer and curing agent, pour the mixture onto the hydrophobic microneedle positive mold. After curing, demold to obtain a mold with microneedle cavity.

[0007] Preferably, in S2, the drug solution further contains hyaluronic acid, and the mass-volume concentration of hyaluronic acid in the drug solution is 0.5-2%.

[0008] Preferably, in S3, the freezing temperature is -70°C to -90°C, and the time is 20-40 minutes.

[0009] Preferably, in step S3, before freezing, a centrifugation step is also included; the centrifugation speed is 3000-4000 rpm and the time is 2-4 min.

[0010] Preferably, in S2, the concentration of FVIII in the drug solution is 1-10 IU / μL.

[0011] The present invention also provides an FVIII ice microneedle, which is prepared according to the preparation method of the FVIII ice microneedle.

[0012] Preferably, the ice microneedles have a microneedle length of 500-1000 μm, a bottom diameter of 300-500 μm, and a tip diameter of 5-20 μm.

[0013] Preferably, after the ice microneedles are stored at -20°C for 14 days, the FVIII activity retention rate is not less than 85%.

[0014] The present invention also provides the application of the aforementioned FVIII ice microneedles in the preparation of drugs for the prevention or treatment of hemophilia A.

[0015] The beneficial effects of this invention are as follows: This invention provides a method for preparing FVIII ice microneedles, comprising the following steps: providing a mold with a microneedle cavity; injecting a drug solution containing FVIII into the microneedle cavity of the mold; and freezing the mold containing the drug solution to solidify the drug solution within the microneedle cavity, thereby obtaining FVIII ice microneedles. This invention, through the core technical concept of "low-temperature cryo-forming," systematically solves a series of problems in transdermal delivery of FVIII protein, such as easy loss of activity, low delivery efficiency, and inconvenience of use, providing a new dosage form for the treatment of hemophilia A with high activity retention, safe and convenient use, and suitable for large-scale production. Specifically: 1. The entire preparation and storage process of this invention is completed in a low-temperature environment, avoiding the heating, ultraviolet irradiation and cross-linking steps that are unavoidable in the traditional microneedle preparation. This provides an activity protection solution for FVIII from production to use throughout the entire cycle. FVIII can still maintain more than 88% of its activity after being stored at -20℃ for 14 days.

[0016] 2. The ice microneedles prepared by this invention have an ice crystal structure formed under specific processes, which endows the microneedle array with excellent overall mechanical properties. This structure ensures that the microneedles have sufficient axial and lateral strength, enabling them to effectively and reliably penetrate the stratum corneum barrier of human skin, laying a physical foundation for the efficient transdermal delivery of subsequent drugs.

[0017] 3. Based on the skin-melting property of the ice matrix, the microneedles provided by this invention do not need to be removed after drug delivery, eliminating the risk of sharp object residue. The low-temperature stimulation caused during the action is brief and localized, allowing the skin barrier function to quickly repair itself. Simultaneously, this drug delivery method significantly reduces the professional skill required for operation and the associated pain, making it possible for patients to safely, painlessly, and autonomously administer drugs.

[0018] 4. By delivering FVIII directly to the rich capillary network in the superficial dermis, this invention is expected to bypass the first-pass effect of the liver and the delay of traditional injection, thereby achieving rapid absorption and distribution of therapeutic drugs in the body, thus meeting the clinical needs for rapid onset of action in acute bleeding or routine preventive treatment of hemophilia.

[0019] 5. The core steps of the preparation method of this invention are clearly defined, mainly involving mold replication, solution filling, and low-temperature freezing, without requiring complex precision equipment or harsh chemical reaction conditions. The entire process is mild and highly controllable, and easy to standardize and scale up, providing favorable conditions for the industrialization and widespread clinical application of the product. Attached Figure Description

[0020] Figure 1 This is a morphological characterization diagram of the microneedle positive mold in Embodiment 1 of the present invention. Figure 1 In the image, 'a' is a top view of the microneedle array obtained under a high-resolution microscope. Figure 1 In the image, b is a frontal view of the microneedle array obtained under a high-resolution microscope. Figure 1 In the image, 'c' represents a top view of a single microneedle via SEM. Figure 1 In the image, d represents a front view of a single microneedle via SEM. Figure 2 This is a schematic diagram of the preparation process of FVIII ice microneedles in Embodiment 1 of the present invention; Figure 3 This is a graph showing the activity change trend of FVIII ice microneedles in Example 1 and Comparative Examples 1-3 of the present invention in an environment of -20℃. Figure 4 This is a graph showing the axial destructive force test of the ice microneedle array (8×8) in Experimental Example 3 of this invention; Figure 5 This is a graph showing the transverse destructive force test of the ice microneedle array (1×8) in Experiment Example 3 of this invention; Figure 6 This is a test diagram of the ice microneedle insertion performance in Experiment Example 3 of this invention;Figure 6 In the image, 'a' represents the first test image. Figure 6 In the image, b represents the second test image. Figure 6 In the image, 'c' represents the third test image. Figure 7 This is a schematic diagram of the Franz diffusion device and a drug release curve from Experimental Example 4 of this invention. Figure 7 In the diagram, 'a' is a schematic diagram of the Franz diffusion device, and 'b' is a drug release curve. Figure 8 This is a schematic diagram of the changes in rat skin temperature in Experiment Example 5 of this invention; Figure 8 In the diagram, 'a' represents the skin temperature at 10 seconds. Figure 8 In the diagram, b represents the skin temperature at 20 seconds. Figure 8 The diagram shows the skin temperature at 30 seconds (c). Figure 8 The diagram shows the skin temperature at 60 seconds (d). Figure 9 This is a schematic diagram of the recovery of pinholes in the rat skin in Experiment Example 5 of this invention; Figure 9 In the diagram, 'a' represents the initial micropore closure state. Figure 9 In the diagram, b represents the micropore closure status over 1 minute. Figure 9 In the diagram, 'c' represents the micropore closure status after 2 minutes. Figure 9 In the diagram, d represents the micropore closure status over 3 minutes. Figure 10 This is a characterization image of a rat skin section from Experimental Example 5 of this invention. Figure 10 Image (i) shows a representation of a rat skin section on a 500-micrometer scale. Figure 10 (ii) is a characterization of a rat skin section on a 250-micrometer scale. Figure 10 (iii) in the figure is a characterization of a rat skin section on a 100-micrometer scale. Figure 11 This is a graph showing the change in FVIII levels in rats during Experiment Example 6 of this invention. Detailed Implementation

[0021] This invention provides a method for preparing FVIII ice microneedles, comprising the following steps: S1. Provide a mold with a microneedle cavity; S2. Inject the drug solution containing FVIII into the microneedle cavity of the mold; S3. Freeze the mold into which the drug solution is injected to solidify the drug solution within the microneedle cavity, thus obtaining the FVIII ice microneedle.

[0022] In this invention, S1 includes the following steps: (1) Microneedle positive molds were prepared using 3D printing technology; (2) The microneedle positive mold is hydrophobically treated; (3) After mixing the PDMS prepolymer and curing agent, pour the mixture onto the hydrophobic microneedle positive mold. After curing, demold to obtain a mold with microneedle cavity.

[0023] In this invention, in step (1), the microneedle array on the microneedle male mold is an 8×8 array, the microneedle length is 500-1000μm, the bottom diameter is 300-500μm, and the tip diameter is 5-20μm.

[0024] In this invention, in step (2), 1H,1H,2H,2H-perfluorooctyltrichlorosilane (PFTS) is used to hydrophobize the microneedle anode.

[0025] In this invention, in step (3), the volume ratio of PDMS prepolymer to curing agent is 8-12:1.

[0026] In this invention, in step (3), the curing temperature is 60-80℃ and the time is 1-3h.

[0027] In this invention, in S2, the drug solution further contains hyaluronic acid, and the mass-volume concentration of hyaluronic acid in the drug solution is 0.5-2%.

[0028] Mass volume concentration refers to the amount of hyaluronic acid contained in 100 milliliters (mL) of drug solution, ranging from 0.5 grams to 2 grams.

[0029] In this invention, in S3, the freezing temperature is -70°C to -90°C, and the time is 20-40 minutes.

[0030] In this invention, step S3, before freezing, includes a centrifugation process; the centrifugation speed is 3000-4000 rpm and the time is 2-4 min; after centrifugation, excess solution is scraped off the surface of the mold.

[0031] In this invention, in S2, the concentration of FVIII in the drug solution is 1-10 IU / μL.

[0032] The present invention also provides an FVIII ice microneedle, which is prepared according to the preparation method of the FVIII ice microneedle.

[0033] In this invention, the ice microneedles have a microneedle length of 500-1000 μm, a bottom diameter of 300-500 μm, and a tip diameter of 5-20 μm.

[0034] In this invention, after the ice microneedles are stored at -20°C for 14 days, the FVIII activity retention rate is not less than 85%.

[0035] The present invention also provides the application of the aforementioned FVIII ice microneedles in the preparation of drugs for the prevention or treatment of hemophilia A.

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. The features mentioned above or in the specific examples mentioned in this invention can be combined arbitrarily, and these specific embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention.

[0037] In the following embodiments of the present invention, FVIII lyophilized powder was purchased from Shenzhou Cell Engineering Co., Ltd. (Beijing, China); PDMS prepolymer and curing agent (SYLGARD 184) were purchased from Dow Chemical Company (Dow Corning, Michigan).

[0038] Example 1 This embodiment provides a method for preparing FVIII ice microneedles, including the following steps. A microneedle positive mold was fabricated using 3D printing technology. The microneedle array was 8×8, with a length of 800 μm, a bottom diameter of 400 μm, and a tip diameter of 10 μm. 100 μL of 1H,1H,2H,2H-perfluorooctyltrichlorosilane was added to the microneedle positive mold and subjected to hydrophobication treatment in a 70℃ oven for 6 hours. Subsequently, PDMS prepolymer and curing agent were mixed uniformly at a volume ratio of 10:1, allowed to stand to eliminate air bubbles, and then poured onto the hydrophobicated microneedle positive mold. The mixture was then cured in a 70℃ oven for 2 hours, cooled, and demolded to obtain a mold with microneedle cavities.

[0039] Deionized water was added to the lyophilized FVIII powder and mixed thoroughly to completely reconstitute it, yielding an FVIII solution with a final concentration of 1 IU / μL. Hyaluronic acid was then added to the FVIII solution and stirred until completely dissolved, yielding a drug solution with a hyaluronic acid concentration of 1% (w / v). 100 μL of the drug solution was injected into the microneedle cavity of the mold.

[0040] The mold containing the drug solution was centrifuged at 3500 rpm for 3 minutes to ensure the solution fully filled the microneedle chambers and removed air bubbles. Excess solution was then gently scraped off the surface of the mold. The handheld ice microneedle delivery device was placed on the mold and frozen at -80°C for 20 minutes to allow the drug solution to solidify within the microneedle chambers, resulting in a mold containing an FVIII ice microneedle array. This mold was then stored at -20°C. When needed, the FVIII ice microneedles were removed from the mold and demolded to obtain the final product.

[0041] Example 2 This embodiment provides a method for preparing FVIII ice microneedles, which differs from Example 1 in that the final concentration of the FVIII solution is modified to 5 IU / μL.

[0042] Comparative Example 1 This comparative example provides a method for preparing FVIII ice microneedles, which differs from Example 1 in that hyaluronic acid is replaced with glycerin.

[0043] Comparative Example 2 This comparative example provides a method for preparing FVIII ice microneedles, which differs from Example 1 in that hyaluronic acid is replaced with maltose.

[0044] Comparative Example 3 This comparative example provides a method for preparing FVIII ice microneedles, which differs from Example 1 in that: the addition of hyaluronic acid is omitted, and 100 μL of 1 IU / μL FVIII solution is directly injected into the microneedle cavity of the mold.

[0045] Experimental Example 1 The morphology of the microneedle positive mold prepared in Example 1 was characterized using a high-resolution microscope and a scanning electron microscope, resulting in morphology characterization images of the microneedle positive mold in Example 1, as shown below. Figure 1 As shown; Figure 1 In the image, 'a' is a top view of the microneedle array obtained under a high-resolution microscope. Figure 1 In the image, b is a frontal view of the microneedle array obtained under a high-resolution microscope. Figure 1 In the image, 'c' represents a top view of a single microneedle via SEM. Figure 1 In the diagram, d is a front view of a single microneedle via SEM. Among them, a and b clearly show the overall array arrangement and the three-dimensional outline of the microneedles, while c and d reveal in detail the sharpness of the microneedle tip and the surface morphology of the sidewalls.

[0046] A schematic diagram of the preparation process of FVIII ice microneedles in Example 1 is shown below. Figure 2 As shown.

[0047] Experiment Example 2 The changes in FVIII activity of the FVIII ice microneedles in Examples 1 and Comparative Examples 1-3 during storage at -20℃ were detected using a UV-Vis-NIR spectrophotometer. First, deionized water and FVIII powder were mixed to prepare a 1 IU / μL FVIII solution, which was then serially diluted to obtain working solutions of 0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 IU / μL. Subsequently, using an ELISA kit and following the instructions, the optical density of each working solution at 405 nm was measured, and a standard curve was plotted. Based on this, the ice microneedle samples of Examples 1 and Comparative Examples 1-3 were tested. Samples were taken on days 0, 3, 7, 10, and 14 of storage, and the FVIII activity was calculated using the aforementioned standard curve. The trend graphs of FVIII ice microneedles activity in Examples 1 and Comparative Examples 1-3 at -20℃ were obtained, as shown below. Figure 3 As shown in the figure. Experimental results show that when the ice microneedles of Example 1 were stored at -20°C for 14 days, their FVIII activity could still be maintained at more than 88% of the initial activity, which is better than that of Comparative Examples 1-3. This result confirms that the ice microneedle system prepared in this invention has excellent activity protection ability for FVIII.

[0048] Experimental Example 3 To evaluate whether the mechanical properties of the prepared ice microneedles meet the requirements for transdermal drug delivery, a systematic mechanical test was conducted on the ice microneedle array.

[0049] First, hyaluronic acid and deionized water were mixed to obtain a hyaluronic acid solution with a mass-volume concentration of 1%. Taking the mold with microneedle cavities prepared in Example 1, 100 μL of the above solution was injected into the microneedle cavities of the mold. The mold was centrifuged at 3500 rpm for 3 minutes to ensure the solution fully filled the microneedle cavities and removed air bubbles. Then, excess solution was gently scraped off the surface of the mold with a scraper. The mold was then frozen at -80°C for 20 minutes to allow the solution to solidify within the microneedle cavities, resulting in a mold containing an ice microneedle array.

[0050] (1) Axial compressive strength test After obtaining the mold containing the ice microneedle array, the microneedle array was immediately demolded and tested. The ice microneedles were fixed parallel to the sensor's direction of motion, and an axial compressive force was applied to a polymethyl methacrylate (PMMA) plate at a constant speed of 0.6 mm / s. The computer recorded the force value every 0.005 seconds. The axial destructive force test curve of the ice microneedle array (8×8) in Experiment Example 3 was obtained, as shown below. Figure 4 As shown in the figure. Test results indicate that the total axial destructive force of this 8×8 microneedle array is 23.8 N. Based on this, the average axial compressive force of a single ice microneedle is calculated to be approximately 0.372 N.

[0051] (2) Lateral compressive strength test A 1×8 ice microneedle array was fixed perpendicular to the sensor's direction of motion, and a lateral compressive force was applied to the PMMA plate at a speed of 0.6 mm / s. The lateral destructive force test curve of the 1×8 ice microneedle array in Experiment Example 3 was obtained, as shown in the figure. Figure 5 As shown. Figure 5 The display shows that the total lateral compressive force of the array is 5.08N, and the average lateral compressive force of a single needle is approximately 0.635N.

[0052] (3) Skin penetration performance test To verify its actual penetration ability, an ex vivo skin insertion experiment was conducted. SD rat skin was laid flat on a 37°C constant-temperature metal block, and an array of ice-microneedles was inserted into the skin, followed by staining with methylene blue solution. After 5 minutes, excess dye was washed off, and the blue spots formed on the skin due to the microneedle channels were observed. Each microneedle patch contained 64 needles, and the insertion percentage was calculated using the formula (number of blue channels / 64) × 100%. After three parallel experiments, the ice-microneedle insertion performance test diagram in Experiment 3 is shown below. Figure 6 As shown; Figure 6 In the image, 'a' represents the first test image. Figure 6 In the image, b represents the second test image. Figure 6 Figure 'c' in the image represents the third test. Statistics show that the average penetration rate of the ice-micro needle on detached skin is as high as 98%.

[0053] The above three test results show that the ice microneedle has excellent mechanical properties: its single needle axial and transverse load-bearing capacity is 0.372N and 0.635N, respectively, and it can efficiently penetrate the stratum corneum of the skin (average penetration rate of 98%), fully meeting the mechanical requirements of percutaneous drug delivery devices.

[0054] Experiment Example 4 To evaluate the transdermal drug delivery capability of ice microneedles, nicotinamide was used as a model drug, and its in vitro release behavior was tested using a Franz diffusion cell device.

[0055] First, a mold with microneedle cavities was prepared according to Example 1. 0.01 g of hyaluronic acid was dissolved in 1 mL of deionized water, and 0.45 g of nicotinamide was added and mixed thoroughly to obtain a solution. 100 μL of this solution was injected into the microneedle cavity of the mold. The mold containing the drug solution was centrifuged at 3500 rpm for 3 min to ensure the solution fully filled the microneedle cavity and removed air bubbles. Excess solution was then gently scraped off the surface of the mold with a scraper. The ice-microneedle handheld delivery device was placed on the mold and frozen at -80°C for 20 min to allow the drug solution to solidify within the microneedle cavity. Finally, the nicotinamide ice microneedles were demolded.

[0056] Ex vivo SD rat skin was used as a permeability barrier in the experiment. Before the experiment, subcutaneous tissue was carefully removed using ophthalmic scissors, and the skin was then washed with phosphate-buffered saline (PBS, pH 7.4), with residual water absorbed using filter paper. The skin was then laid flat on aluminum foil, and nicotinamide microneedles were inserted. The skin was then fixed to the opening of the receiving cell of a Franz diffusion cell, with the epidermal side exposed and the dermal side in contact with the receiving solution (PBS, pH 7.4). The device was tilted to remove air bubbles and ensure full contact between the skin and the receiving solution. The diffusion cell was placed in a 37°C water bath of a TP-6 ​​intelligent transdermal analyzer, and the experiment was conducted with stirring at 350 rpm.

[0057] At 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours and 24 hours after drug administration, 0.8 mL of sample solution was taken from the receiving pool for testing, and isothermal and equal volume of PBS was immediately added to maintain a constant receiving liquid volume.

[0058] Finally, the schematic diagram of the Franz diffusion device and the drug release curve for Experiment 4 were obtained, as follows: Figure 7 As shown; Figure 7 In the diagram, 'a' is a schematic diagram of the Franz diffusion device, and 'b' is a drug release curve. From... Figure 7 It can be seen that the cumulative penetration rate of nicotinamide can reach about 50% within 24 hours, which indicates that the ice microneedles prepared by the present invention can effectively disrupt the skin barrier structure and have the ability to efficiently deliver drug molecules in vitro.

[0059] Experimental Example 5 To evaluate the in vivo safety of the FVIII ice microneedles prepared in Example 2, the following animal experiment was conducted: Healthy rats were selected, anesthetized by intraperitoneal injection, and their back hair was shaved and cleaned to expose the skin. The FVIII ice microneedles were applied to the hair removal area, and uniform pressure was applied until the ice microneedles were completely melted. The handheld device was then carefully removed.

[0060] The changes in skin temperature in rats in Experiment 5 were obtained by dynamically monitoring skin temperature using an infrared thermal imager, as shown in the diagram. Figure 8 As shown; Figure 8 In the diagram, 'a' represents the skin temperature at 10 seconds. Figure 8 In the diagram, b represents the skin temperature at 20 seconds. Figure 8 The diagram shows the skin temperature at 30 seconds (c). Figure 8 The diagram shows that d represents the skin temperature at 60 seconds. The results show that 10 seconds after the application of ice microneedling, the local skin temperature dropped to 11.9℃, and then recovered to about 20℃ within 60 seconds, indicating that the low-temperature stimulation is short-lived and reversible.

[0061] The closure of micropores on the skin surface was observed under an optical microscope, resulting in a schematic diagram of the recovery of pinholes in the rat skin in Experiment 5. Figure 9 As shown; Figure 9 In the diagram, 'a' represents the initial micropore closure state. Figure 9 In the diagram, b represents the micropore closure status over 1 minute. Figure 9 In the diagram, 'c' represents the micropore closure status after 2 minutes. Figure 9 In the diagram, d represents the micropore closure status over 3 minutes. Figure 9 The scale is 4mm. It can be seen that the micropores left by the microneedling largely close within 3 minutes, indicating that the skin barrier function can quickly repair itself.

[0062] To further analyze the safety at the tissue level, full-thickness skin samples were taken from the backs of rats after the intervention. These samples were fixed in 4% paraformaldehyde solution (containing 4 grams of paraformaldehyde per 100 ml of solution), embedded in paraffin, and then vertical sections were prepared and stained with hematoxylin and eosin (H&E). The resulting skin section characterization images from Experiment 5 are shown below. Figure 10 As shown, Figure 10 Image (i) shows a representation of a rat skin section on a 500-micrometer scale. Figure 10 (ii) is a characterization of a rat skin section on a 250-micrometer scale. Figure 10 Image (iii) shows a rat skin section visualized on a 100-micrometer scale. At different magnifications, the epidermis and dermis remained intact, with no signs of frostbite or damage such as separation of the basal layer from the dermis, epidermal edema, necrosis, blistering, or connective tissue fibrosis. These results indicate that the FVIII ice microneedles have good safety in in vivo applications, with a short-lived hypothermic effect, rapid micropore closure, and no damage to skin tissue structure.

[0063] Experimental Example 6 To evaluate the therapeutic effect of the FVIII ice microneedles prepared in Example 2, the following in vivo pharmacodynamic experiments were conducted: Four hemophilia A FVIII gene knockout rats (FVIII-KO) were randomly divided into a control group and a treatment group. All rats were anesthetized via intraperitoneal injection, and their back hair was shaved and cleaned to expose the skin. In the treatment group, FVIII ice microneedles were applied to the shaved area, with uniform pressure applied until the microneedles completely melted before removal. The control group received no treatment. Blood samples (400 μL each time) were collected from rats before administration and at 1, 4, 12, and 24 hours after administration. The samples were immediately injected into anticoagulant tubes containing 3.2% sodium citrate solution (containing 3.2 g of sodium citrate per 100 mL of solution) and gently mixed. Subsequently, the plasma FVIII activity level was measured using an FVIII activity assay kit. The in vivo FVIII level change curve of rats in Example 6 was obtained, as shown in the figure below. Figure 11As shown, the FVIII activity in the treatment group rats increased rapidly within 4 hours after administration, and then gradually decreased; while the FVIII activity in the control group remained at baseline throughout the observation period. These results demonstrate that the FVIII ice microneedles of this invention can rapidly increase FVIII activity in hemophilia model animals and maintain this effect for a certain period of time, thus verifying its feasibility and reliability for the treatment of hemophilia A.

[0064] Therefore, this invention adopts the above-mentioned FVIII ice microneedles and their preparation method and application. Through the core technical concept of "low-temperature cryo-forming", it systematically solves a series of problems such as easy loss of activity, low delivery efficiency and inconvenience in transdermal delivery of FVIII protein, and provides a new dosage form for the treatment of hemophilia A with high activity retention rate, safe and convenient use and suitable for large-scale production.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing FVIII ice microneedles, characterized by, The method comprises the following steps: S1. providing a mold with micro-needle cavities; S2. injecting a drug solution containing FVIII into the micro-needle cavities of the mold; S3. freezing the mold injected with the drug solution to solidify the drug solution in the micro-needle cavities to obtain FVIII ice micro-needles.

2. The process for the preparation of FVIII ice microneedles as claimed in claim 1 wherein, S1. comprises the following steps: (1) preparing a micro-needle male mold by using 3D printing technology; (2) hydrophobizing the micro-needle male mold; (3) mixing PDMS pre-polymer and curing agent, pouring them on the hydrophobized micro-needle male mold, and demolding after curing to obtain a mold with micro-needle cavities.

3. The process for the preparation of FVIII ice microneedles as claimed in claim 1 wherein, In S2, the drug solution further comprises hyaluronic acid, and the mass-volume concentration of hyaluronic acid in the drug solution is 0.5-2%.

4. The process for the preparation of FVIII ice microneedles as claimed in claim 1 wherein, In S3, the freezing temperature is-70℃ to-90℃, and the freezing time is 20-40min.

5. The process for the preparation of FVIII ice microneedles as claimed in claim 1 wherein, In S3, before freezing, a centrifugal treatment step is further included; the centrifugal treatment speed is 3000-4000rpm, and the centrifugal treatment time is 2-4min.

6. The process for the preparation of FVIII ice microneedles as claimed in claim 1 wherein, In S2, the concentration of FVIII in the drug solution is 1-10IU / μL.

7. An FVIII ice microneedle, characterized in that, The FVIII ice micro-needles are prepared by the preparation method according to any one of claims 1-6.

8. The FVIII ice microneedle according to claim 7, characterized in that, The length of the micro-needles of the ice micro-needles is 500-1000μm, the bottom diameter is 300-500μm, and the tip diameter is 5-20μm.

9. The FVIII ice microneedle according to claim 7, characterized in that, After the ice micro-needles are stored at-20℃ for 14 days, the FVIII activity retention rate is not less than 85%.

10. Use of the FVIII ice micro-needles according to any one of claims 7-9 in the preparation of a drug for preventing or treating hemophilia A.

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