A double-layer soluble microneedle patch and a preparation method and application thereof

Abstract

The application discloses a double-layer soluble microneedle patch, which comprises a backing layer and a needle body layer arranged on the backing layer, wherein the needle body layer is a microneedle array composed of a plurality of microneedles; components of the needle body layer comprise rabies human immunoglobulin and a water-soluble polymer material; the water-soluble polymer material is at least one of hyaluronic acid and sodium hyaluronate; components of the backing layer comprise a drug and a carrier material; the carrier material is any one of polyvinylpyrrolidone and methacrylated gelatin; and the drug is any one of tazarotene or proangiogenic peptide. The double-layer soluble microneedle patch has the characteristics of resisting rabies virus and promoting skin wound repair, can effectively prevent rabies and promote wound healing, and provides an effective and patient-friendly alternative new scheme for emergency prevention and treatment of rabies exposure.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a double-layer soluble microneedle patch with anti-rabies virus and skin wound repair promoting function, and a preparation method and application thereof. BACKGROUND

[0002] Rabies is a zoonosis caused by rabies virus infection. The exposure factors mainly cause skin damage, tissue laceration, bleeding and infection to human body by teeth, claws and other of the host animals with rabies, suspected rabies or uncertain rabies, and the virus invades the nervous system of human body to cause the disease. The current clinical post-exposure prophylaxis of rabies mainly includes arm injection of rabies vaccine and injection of rabies human immunoglobulin at the wound. Among them, the injection of rabies vaccine can produce rabies virus neutralizing antibodies through active immunization. However, studies have shown that even if the effective wound flushing and rabies vaccination are performed in time after the exposure of rabies, some people still get infected with rabies. The reason is that at least 7 days (up to 14 days) are needed for the antibodies in the body to reach the protection level (antibody > 0.5 IU / mL) after the patient is inoculated with the first dose of rabies vaccine. Therefore, there is a window period from the inoculation of the vaccine to the antibodies reaching the protection level, and if the virus has invaded the central nervous system at this time, there is still a possibility of disease.

[0003] Rabies human immunoglobulin is a specific high-titer antibody against rabies virus extracted from the plasma of healthy people immunized with rabies vaccine, which can effectively neutralize free viruses and clear viruses through phagocytosis of macrophages or complement lysis, so as to prevent the infection of viruses to host cells. After the exposure of rabies, the local infiltration injection of rabies human immunoglobulin injection around the wound can provide immunoprotection for the high-risk period when the vaccination has not reached the protection level. However, the operation process of the traditional rabies human immunoglobulin administration is complex, which needs multiple infiltration injections at the wound site, has poor timeliness, and may cause secondary trauma to the surrounding tissues, has very high requirements for the professional skills of medical personnel, and the administration mode will cause severe pain to the patients, and the compliance of the patients is low. At present, there is no more moderate and effective disposal scheme for the prevention of rabies exposure. SUMMARY

[0004] In view of this, the purpose of the present application is to provide a double-layer soluble microneedle patch with anti-rabies virus and skin wound repair promoting function.

[0005] The present application is realized by the following technical scheme:

[0006] The double-layer soluble microneedle patch comprises a backing layer and a needle body layer arranged on the backing layer, and the needle body layer is a microneedle array composed of a plurality of microneedles.

[0007] The components of the needle body layer include rabies human immunoglobulin and water-soluble polymer material; the water-soluble polymer material is at least one of hyaluronic acid and sodium hyaluronate;

[0008] The components of the backing layer include a drug and a carrier material; the carrier material is any one of polyvinylpyrrolidone and methacrylated gelatin; the drug is any one of tazarotene or pro-angiogenic peptide.

[0009] Preferably, the water-soluble polymer material in the needle body layer is hyaluronic acid and sodium hyaluronate, and the mass ratio of hyaluronic acid to sodium hyaluronate is 30:(1-10).

[0010] Further preferably, the molecular weight of the hyaluronic acid is less than 5000 Da.

[0011] Preferably, the shape of the needle body of the microneedle is conical or pyramidal. Further preferably, the height of the single needle body of the microneedle is 800-2000 μm, the diameter of the bottom is 400-1000 μm, and the radius of the tip is 10-30 μm.

[0012] As a preferred embodiment, the needle body layer of the double-layer soluble microneedle patch is composed of a 20x20 single needle array.

[0013] Preferably, the thickness of the backing layer is 1000-3000 μm.

[0014] Preferably, the loading amount of rabies human immunoglobulin in the double-layer soluble microneedle patch is 10-30 IU / patch, and the loading amount of tazarotene or pro-angiogenic peptide is 0.01-1 mg / patch.

[0015] Preferably, the K value of the polyvinylpyrrolidone is 80-96, and the grafting rate of the methacrylated gelatin is 60-90%.

[0016] Preferably, the pro-angiogenic peptide is vascular endothelial growth factor mimetic peptide IGKYKLQYLEQWTLK (QK peptide), and the amino acid sequence is Lle-Gly-Lys-Tyr-Lys-Leu-Gln-Tyr-Leu-Glu-Gln-Trp-Thr-Leu-Lys, as shown in SEQ ID NO. 1.

[0017] The application also provides a preparation method of the double-layer soluble microneedle patch, comprising the following steps:

[0018] (1) preparing a needle body solution, injecting the needle body solution into a microneedle mold, removing air bubbles by vacuumizing, making the needle body solution completely fill the needle body cavities of the microneedle mold and removing the excess needle body solution, and standing for solidification and molding to obtain a microneedle mold containing molded microneedle bodies;

[0019] The preparation method of the needle body solution comprises the following steps: adding water-soluble polymer material into the aqueous solution of rabies human immunoglobulin, stirring and dissolving, removing bubbles by ultrasonic, and preparing the needle body solution;

[0020] (2) preparing a backing solution, injecting the backing solution into the microneedle mold containing the shaped microneedle needle body of step (1), making the backing solution completely cover the bottom of all microneedle needle bodies, removing bubbles by vacuum and retaining the thickness of the backing solution to be 2-3 mm, and standing and curing to form, demolding, and preparing a double-layer soluble microneedle patch;

[0021] The preparation method of the backing solution comprises the following steps:

[0022] When polyvinylpyrrolidone is selected as the carrier material of the backing layer, polyvinylpyrrolidone and tazarotene drug are added into anhydrous ethanol, stirred and dissolved, bubbles are removed by ultrasonic, and the backing solution is prepared;

[0023] When methacrylated gelatin is selected as the carrier material of the backing layer, methacrylated gelatin is added into the aqueous solution of pro-angiogenic peptide, stirred and dissolved at 50-80°C, LAP photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate salt) is added, stirred and dissolved, bubbles are removed by ultrasonic, and the backing solution is prepared.

[0024] Preferably, the mass ratio of the water-soluble polymer material and the aqueous solution of rabies human immunoglobulin in the needle body solution is (1-5):10.

[0025] Preferably, the potency of rabies human immunoglobulin in the aqueous solution of rabies human immunoglobulin is 50-300 IU / mL. Further preferably, the aqueous solution of rabies human immunoglobulin is rabies human immunoglobulin injection;

[0026] Preferably, the mass concentration of tazarotene or pro-angiogenic peptide drug in the backing solution is 0.01-1 mg / mL.

[0027] When polyvinylpyrrolidone is selected as the carrier material of the backing layer, the mass ratio of polyvinylpyrrolidone to anhydrous ethanol in the backing solution is (1-5):10.

[0028] When methacrylated gelatin is selected as the carrier material of the backing layer, the mass ratio of methacrylated gelatin to the aqueous solution of pro-angiogenic peptide in the backing solution is (1-5):10; preferably, the mass concentration of LAP photoinitiator in the backing solution is 1-10 mg / mL.

[0029] Preferably, the standing and curing operation is drying and curing at a temperature of 20-30°C for 6h-18h.

[0030] Preferably, the microneedle mold is a PDMS microneedle negative mold.

[0031] The application provides a preparation method of a PDMS microneedle negative mold, which comprises the following steps: mixing polydimethylsiloxane and a curing agent at a mass ratio of 10:1, uniformly stirring, vacuum degassing bubbles, immediately pouring on the surface of a stainless steel microneedle master template for curing and drying, demolding after drying, and obtaining the PDMS microneedle negative mold.

[0032] The application also provides application of the double-layer soluble microneedle patch in preparation of a medicine for preventing rabies virus infection.

[0033] The application has the following beneficial effects:

[0034] The double-layer soluble microneedle patch of the application uses hyaluronic acid and sodium hyaluronate as microneedle body materials for loading rabies human immunoglobulin, and uses polyvinylpyrrolidone or methacrylated gelatin as backing layer materials for loading skin wound repair promoting drugs, so that the rabies human immunoglobulin / skin wound repair promoting drugs are released in a layered and differential manner. When used, the microneedle patch is covered on a wound site, and the needle tip is pressed into the skin, so that the needle tip can be quickly dissolved, the rabies human immunoglobulin can quickly release to neutralize the rabies virus, and the infection risk can be reduced. The backing layer has a slow-release property, avoids the wound hemostasis stage, targets the wound inflammation and proliferation stage, and delivers the skin wound repair promoting drugs, so that the function of promoting skin wound vascularization and accelerating wound healing is realized. Compared with the traditional rabies exposure prevention and treatment mode, the timeliness of drug administration is greatly improved, the rabies infection is effectively prevented, the severe pain caused by the drug administration process is significantly reduced, and the patient compliance is improved. An effective patient-friendly alternative new scheme for using rabies human immunoglobulin to neutralize the virus and wound management is provided.

[0035] The double-layer soluble microneedle patch of the application has high stability, can be stably stored at room temperature by using conventional blister packaging (the titer of the rabies human immunoglobulin still reaches more than 90% of that of fresh rabies human immunoglobulin injection after being stored at room temperature of 25 DEG C for 30 days), has good portability, and can be used as an ideal choice for emergency prevention and treatment of rabies exposure. The application widens the potential application of microneedles in emergency treatment. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 It is a preparation process of the double-layer soluble microneedle patch of Example 1.

[0037] Figure 2 It is a diagram of the double-layer soluble microneedle patch of Example 1.

[0038] Figure 3 The preparation process of the bilayer soluble microneedle patch in Example 2 is as follows;

[0039] Figure 4 This is an image of the bilayer soluble microneedle patch from Example 2;

[0040] Figure 5 SEM images of the needle morphology of the bilayer soluble microneedle patch in Examples 1 and 2;

[0041] Figure 6 This is a fluorescent drug distribution map of the bilayer soluble microneedle patch in Example 1;

[0042] Figure 7 This is a fluorescent drug distribution map of the bilayer soluble microneedle patch in Example 2;

[0043] Figure 8 The force-displacement curve is the mechanical strength test result of Example 6;

[0044] Figure 9 This is a graph showing the transdermal performance test results of Example 7;

[0045] Figure 10 The graph shows the solubility test results for Example 8;

[0046] Figure 11 This is a graph showing the results of the in vitro drug release test in Example 9;

[0047] Figure 12 Body mass index and survival status of mice used in Example 10 to test their resistance to rabies virus infection;

[0048] Figure 13 The images and area statistics of mouse wounds used in the skin wound repair ability test of Example 11 are shown.

[0049] Figure 14 This is an illustration of the blister packaging effect of Example 12;

[0050] Figure 15 The graph shows the stability test results for Example 12;

[0051] Figure 16 The loading of tazarotene on the monolithic microneedle patch of Comparative Example 1 and the single tazarotene patch of the bilayer microneedle patch of Example 1;

[0052] Figure 17 The titers of rabies immunoglobulin from a single piece of the integrated microneedle patch in Example 2 and the double-layer microneedle patch in Example 1 were compared. Detailed Implementation

[0053] To make the technical contents of the present application, the purposes and effects achieved clear, the technical solutions of the present application are described below in detail in connection with the embodiments and the accompanying drawings. However, the described embodiments are only some of the embodiments of the present application, and the implementation and protection of the present application are not limited thereto. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. It should be noted that, if there are processes not specifically described below, they can be implemented or understood by those of ordinary skill in the art with reference to the prior art. If the reagents or instruments used are not specified by the manufacturer, they are considered to be conventional products that can be purchased on the market.

[0054] The reagents used in the examples and comparative examples of the present application are described as follows, but are not limited thereto:

[0055] Rabies human immunoglobulin injection: rabies human immunoglobulin titer is 100 IU / mL, purchased from Hualan Biotechnology Co., Ltd., drug base code: 86903115000425;

[0056] Hyaluronic acid: super-active hyaluronic acid, molecular weight <5000 Da, purchased from Aladdin Reagent (Shanghai) Co., Ltd., product code: U293516;

[0057] Sodium hyaluronate: purchased from Aladdin Reagent (Shanghai) Co., Ltd., product code: E293520;

[0058] Polyvinylpyrrolidone K90: purchased from Aladdin Reagent (Shanghai) Co., Ltd.;

[0059] Tazarotene: purchased from Aladdin Reagent (Shanghai) Co., Ltd.;

[0060] Methacrylate gelatin: grafting rate is 80%, purchased from Aladdin Reagent (Shanghai) Co., Ltd., product code: M398247;

[0061] LAP photoinitiator: purchased from Aladdin Reagent (Shanghai) Co., Ltd., product code: L157759;

[0062] QK peptide: purchased from Suzhou Qiangyao Biotechnology Co., Ltd.; amino acid sequence is Lle-Gly-Lys-Tyr-Lys-Leu-Gln-Tyr-Leu-Glu-Gln-Trp-Thr-Leu-Lys.

[0063] Example 1

[0064] (1) hyaluronic acid, sodium hyaluronate, rabies human immunoglobulin injection were added into a container according to a mass ratio of 30:3:100, and magnetic stirring was performed for 4 h. Air bubbles were removed by ultrasonic treatment to prepare a needle body solution. The needle body solution was injected into a PDMS microneedle negative mold, and vacuum was applied to remove air bubbles. The needle body solution was allowed to completely fill the needle body cavities of the microneedle mold and the excess needle body solution was removed. Drying was performed at a temperature of 25 °C for 8 h to solidify and form the microneedle mold containing the formed microneedle needle bodies;

[0065] (2) Polyvinylpyrrolidone, tazarotene, anhydrous ethanol were added into a container according to a mass ratio of 30:0.5:100, and magnetic stirring was performed for 4 h. Air bubbles were removed by ultrasonic treatment to prepare a backing solution (the mass concentration of tazarotene in the backing solution was 5 mg / mL). The backing solution was injected into the microneedle mold containing the formed microneedle needle bodies of step (1) to completely cover the bottoms of all the microneedle needle bodies. Vacuum was applied to remove air bubbles and the thickness of the backing solution was maintained at 2-3 mm. Drying was performed at a temperature of 25 °C for 8 h to solidify and form the microneedle mold, and the mold was demolded to prepare a double-layer dissolvable microneedle patch (MN-H-T).

[0066] Example 2

[0067] (1) hyaluronic acid, sodium hyaluronate, rabies human immunoglobulin injection were added into a container according to a mass ratio of 30:3:100, and magnetic stirring was performed for 4 h. Air bubbles were removed by ultrasonic treatment to prepare a needle body solution. The needle body solution was injected into a PDMS microneedle negative mold, and vacuum was applied to remove air bubbles. The needle body solution was allowed to completely fill the needle body cavities of the microneedle mold and the excess needle body solution was removed. Drying was performed at a temperature of 25 °C for 8 h to solidify and form the microneedle mold containing the formed microneedle needle bodies;

[0068] (2) Methacrylated gelatin and QK peptide aqueous solution were added into a container according to a mass ratio of 10:100, and magnetic stirring was performed at 60 °C to dissolve. LAP photoinitiator was added, and the mass ratio of LAP photoinitiator to methacrylated gelatin was 0.25:10. Magnetic stirring was performed to dissolve, air bubbles were removed by ultrasonic treatment, and a backing solution (the mass concentration of QK peptide in the backing solution was 10 mg / mL) was prepared. The backing solution was injected into the microneedle mold containing the formed microneedle needle bodies of step (1) to completely cover the bottoms of all the microneedle needle bodies. Vacuum was applied to remove air bubbles and the thickness of the backing solution was maintained at 2-3 mm. Blue light of 405 nm was used for irradiation to allow the methacrylated gelatin to undergo crosslinking reaction. Drying was performed at a temperature of 25 °C for 8 h to solidify and form the microneedle mold, and the mold was demolded to prepare a double-layer dissolvable microneedle patch (MN-H-Q).

[0069] Example 3

[0070] The present example is a scanning electron microscope experiment of the double-layer soluble microneedle patch of Example 1 and Example 2, and the experimental method is as follows: the prepared microneedle patch is fixed on a 45° slope sample table with conductive glue, after gold spraying, an electron scanning microscope (SEM) is used to set the working voltage to 5kV, the morphology of the microneedle patch is observed, and the image is collected.

[0071] As shown in Figure 5 , the microneedle patch base is flat, the microneedle body is complete and conical, the single needle body is 1200μm high, the bottom diameter is 600μm, and the tip radius is 20μm, and the arrangement is neat.

[0072] Example 4

[0073] The present example is a drug loading experiment of the double-layer soluble microneedle patch of Example 1, and the difference between Example 4 and Example 1 is only that in step (1), fluorescein isothiocyanate (FITC) is used as a substitute for rabies human immunoglobulin; in step (2), rhodamine B is used as a substitute for tazarotene; and the rest of the preparation steps are the same.

[0074] Figure 6 The present example is the morphology and schematic diagram of the microneedle patch of Example 4 under confocal laser scanning microscope (CLSM), in order to observe the spatial distribution of the two drugs in the microneedle patch, the picture shows that the microneedle tip uniformly loads green fluorescence representing rabies human immunoglobulin, and red fluorescence instead of tazarotene is mainly concentrated in the base of the microneedle.

[0075] Example 5

[0076] The present example is a drug loading experiment of the double-layer soluble microneedle patch of Example 2, and the difference between Example 5 and Example 2 is only that in step (1), rhodamine B is used as a substitute for rabies human immunoglobulin; in step (2), fluorescein isothiocyanate modified QK peptide (QK-FITC) is used as a substitute for QK peptide; and the rest of the preparation steps are the same.

[0077] Figure 7 The present example is the morphology and schematic diagram of the microneedle patch of Example 5 under confocal laser scanning microscope (CLSM), in order to observe the spatial distribution of the two drugs in the microneedle patch, the picture shows that the microneedle tip uniformly loads green fluorescence representing rabies human immunoglobulin, and red fluorescence instead of QK peptide is distributed throughout the microneedle.

[0078] Example 6

[0079] The present example is a mechanical strength test experiment of the double-layer soluble microneedle patch of Example 1 and Example 2, and the test method is as follows:

[0080] The breaking force was accurately measured by a universal material testing machine. The microneedle patch sample was fixed on the hard stainless steel flat plate base of the testing machine, and the upper sensor was moved downward at a speed of 1 mm / min. Once the needle tip of the microneedle patch contacted the upper sensor platform, the sensor continuously recorded the force and displacement, and a force-displacement curve was drawn. The results are shown in Figure 8 The force-displacement curve generally reflects the sudden drop in force when the microneedle breaks, and the force at the moment of sudden drop can be considered as the breaking force of the microneedle. By analyzing the curve, the breaking force of the microneedle can be obtained.

[0081] According to Figure 8 It can be seen that the breaking force of each needle in Example 1 is 0.57N, and the breaking force of each needle in Example 2 is 0.77N, both of which are higher than the force required for the microneedle to pierce the skin (0.2N), indicating that the needle body layer of the double-layer dissolvable microneedle patch prepared in Example 1 and Example 2 is sufficient to pierce the skin.

[0082] Example 7

[0083] This example is a transdermal performance test experiment of the double-layer dissolvable microneedle patch of Example 1, and the test method is as follows: the microneedle patch is pressed on the surface of the isolated pig skin for 2 minutes, then the microneedle patch is removed, the cross section of the pig skin is sliced and stained to evaluate the transdermal performance of the microneedle. The results are shown in Figure 9 The left graph is the situation after the microneedle patch is pressed on the surface of the pig skin for 2 minutes and then removed, and the skin surface forms an ordered 20x20 micropore structure, indicating that the microneedle has sufficient strength to pierce the skin; the right graph is a hematoxylin-eosin (H&E) staining image of the cross-sectional morphology of the skin after the microneedle patch is removed, further confirming the micropores formed due to the rupture of the skin after using the microneedle patch, indicating that the double-layer dissolvable microneedle patch of the application has good ability to pierce the skin.

[0084] Example 8

[0085] This example is a dissolution performance test experiment of the double-layer dissolvable microneedle patch of Example 1, and the measurement method is as follows:

[0086] The microneedle patch is manually inserted into the skin, and after pressing for 30s, the insertion state is maintained, and the dissolution status of the microneedle is observed at 30s, 60s and 120s, respectively. As shown in Figure 10 The microneedle has completely dissolved at 120s, and has good solubility.

[0087] Example 9

[0088] The in vitro drug release curve of the double-layer dissolvable microneedle patch of Example 1 and Example 2' was determined. The double-layer dissolvable microneedle patch of Example 2' is different from Example 2 only in that the QK peptide modified with fluorescein isothiocyanate (QK-FITC) is used instead of the QK peptide in step (2), and the rest of the steps are the same, to prepare a double-layer dissolvable microneedle patch (MN-H-Q). The measurement steps are as follows:

[0089] First, the Kunming white mouse was shaved and the subcutaneous fat layer and connective tissue were carefully peeled off. After washing with normal saline, the skin was placed in normal saline and stored in a low-temperature refrigerator for later use. Before the experiment, the skin was naturally thawed and soaked in 0.9% normal saline for 30 minutes. The skin surface was then dried with filter paper and prepared for use.

[0090] Second, the transdermal diffusion device was a modified Franz diffusion device, and the exposed skin area in the diffusion cell was 1.77 cm 2 . The receiving chamber had a volume of 8 mL. The microneedle patch prepared in Example 1 and Example 2' was pressed vertically into the skin surface, and 8.5 mL of receiving liquid was added to the receiving cell (absorption liquid control, absorption liquid level, accurate height above the in vitro skin surface by about 1 mm, to ensure stable contact between the liquid surface and the skin during sampling). The receiving cell was placed in a constant-temperature water bath at 32±0.5°C, and the receiving liquid was continuously stirred with a magnetic stirrer to allow the drug to be released naturally. The stirring speed was 300 rpm / min.

[0091] Third, sampling was performed at 0 min, 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h after the microneedle was inserted into the in vitro skin, and 0.2 mL of receiving liquid was taken at each time point. Each time point was repeated six times. After sampling, 0.2 mL of receiving liquid was added to the receiving chamber, filtered through a 0.22 μm microporous filter, and stored in the freezer for testing. After the transdermal absorption experiment was completed, the skin was dried with filter paper, the microneedle was removed, and the skin was cut into small pieces. 2 mL of pH 7.4 phosphate buffer was added to the grinding tank, and the uniform grinders were thoroughly ground. After centrifugation, 0.5 mL of supernatant was taken and stored in the freezer for testing.

[0092] Fourth, the double-layer microneedle patch of Example 1 and Example 2' was completely dissolved in 10 mL of deionized water, filtered through a 0.22 μm microporous filter, and the content of rabies human immunoglobulin was measured using a rabies human immunoglobulin enzyme-linked immunosorbent assay kit. The content of tazarotene and QK-FITC was determined using a microplate reader at a detection wavelength of 351 nm for tazarotene and 494 nm for QK-FITC. The total amount of drugs in the double-layer microneedle patch of Example 1 and Example 2 was calculated.

[0093] The fifth step involves determining the levels of rabies immunoglobulin, tazarotene, and QK-FITC in the receiving fluid and supernatant obtained at different time points in the third step, and plotting the in vitro release curve of the microneedles. Figure 11 As shown, the different spatial distributions of rabies immunoglobulin (PIG) ​​and tazarotene or QK peptide within the microneedle patch lead to different release kinetics. PIG located at the needle tip exhibits a rapid release curve, releasing over 50% within 1 hour. This rapid release is attributed to the rapid dissolution of hyaluronic acid in the tip region, which quickly neutralizes the virus at the wound site upon application. In contrast, tazarotene or QK peptide, mainly concentrated in the microneedle base and backing layer, exhibits a significantly slower release rate, releasing less than 15% in the initial 1 hour and requiring 12 hours to reach 50% release. This slow-release curve facilitates the gradual administration of tazarotene or QK peptide after the rapid action of PIG, thereby promoting the action of more wound-healing drugs during the inflammatory phase of wound repair.

[0094] Example 10

[0095] This embodiment describes the determination of the anti-rabies virus infection ability of the double-layer microneedle patches of Examples 1 and 2 in a mouse skin trauma rabies exposure model. The determination method is as follows:

[0096] Mice were weighed before the experiment. After hair removal, two circular wounds with a diameter of 0.8 cm were made on the back of each mouse, and rabies virus strain CVS-11 (5 × 10⁻⁶) were injected into the wounds. 5 FFU / mL, 100μL). Mice were placed individually in cages and divided into 5 groups (n=10 per group), including 3 control groups: (1) uninfected back wound group (rabies virus challenge negative, CVS-), (2) untreated post-infection group (rabies virus challenge positive, CVS+), and (3) post-infection conventional rabies patient immunoglobulin infiltration treatment group (200IU / mL, 100μL). The other two groups were treated with MN-HT double-layer microneedle patch (MN-HT) of Example 1 and MN-HQ double-layer microneedle patch (MN-HQ) of Example 2, respectively. Treatment began 4 hours after wound establishment and infection to simulate the typical delay in receiving medical care after rabies exposure. The patches were fixed with medical tape and removed after 2 days. The weight and survival status of the mice were recorded daily.

[0097] like Figure 12 As shown, there were significant differences in survival rates among the groups after 14 days. In the CVS+ group, 90% of the mice experienced weight loss and rabies virus infection, with a mortality rate of 80%, thus validating the effectiveness of the infection model. In contrast, the mortality rate in the conventional rabies patient immunoglobulin infiltration treatment group after infection was only 10%, with two mice experiencing weight loss and dying from infection.

[0098] The mice treated with microneedle patches showed better survival results compared to the control groups. In the MN-H-T group, all 10 mice maintained their body weight and survived, and in the MN-H-Q group, the survival rate was 90%, which was comparable to the traditional infiltration treatment group. These results highlight the good efficacy of microneedle patches in delivering rabies human immunoglobulin to neutralize the rabies virus.

[0099] Example 11

[0100] This example is a determination of the skin wound repair ability of the double-layer microneedle patches of Example 1 and Example 2 in a mouse skin wound rabies exposure model, and the determination method is as follows:

[0101] The mice were weighed before the experiment, and after being shaved, two circular wounds with a diameter of 0.8 cm were created on the back of each mouse, and the CVS-11 strain of the rabies virus (5 x 10 5 FFU / mL, 100 μL) was injected into the wounds. The mice were individually placed in cages and divided into 5 groups (10 mice per group), including 3 control groups: (1) non-infected back wound group (rabies virus challenge negative, CVS-), (2) infected but untreated infected group (rabies virus challenge positive, CVS+), (3) traditional rabies human immunoglobulin infiltration treatment group after infection (200 IU / mL, 100 μL), and the remaining two groups were treated with the double-layer microneedle patch of Example 1 (MN-H-T) and the double-layer microneedle patch of Example 2 (MN-H-Q), respectively. On days 3, 7, and 14, the wounds were photographed and their areas were measured, and a wound area statistical graph was drawn.

[0102] As shown in Figure 13 the comparison of the CVS- and CVS+ two untreated groups, it was found that the wound healing rate after rabies virus infection was significantly slowed down. By day 14, the wound size of the 3 surviving mice in the CVS+ group was 2.69 times that of the 10 surviving mice in the CVS- group, indicating that RABV infection impairs wound repair.

[0103] The wound repair rate of the infiltration treatment group was significantly faster than that of the untreated CVS group, which was similar to that of the CVS- group, indicating that viral inhibition significantly improved the wound repair rate. However, compared with the MN patch groups (MN-H-T and MN-H-Q), the wound repair rate of the infiltration group was still relatively slow, with wound sizes of 7.04 and 5.3 times that of the CVS- group, respectively. This delay was due to the secondary damage to the surrounding tissue caused by repeated injections of immunoglobulin during the infiltration treatment.

[0104] The wound healing rates of the two microneedle patch treatment groups were the fastest among all groups. By day 14, the wounds entered the late healing stage, with healing rates of 97.87% and 97.17%, respectively. This indicates that the wounds of both MN patch treatment groups were significantly healed.

[0105] Example 12

[0106] This example provides a blister packaging storage method for the double-layer microneedle patch of Example 1 and stability determination of the storage method. The blister packaging includes a polyethylene tray with a size corresponding to the microneedle patch and an aluminum plastic film covering the tray, as shown in Figure 14 The microneedle patch can be stored in the blister packaging.

[0107] The double-layer microneedle patch was stored in the blister packaging at 25°C for 30 days, the packaging was opened and the double-layer microneedle patch was reconstituted in 10 mL of deionized water, and the relative potency of the rabies human immunoglobulin was determined, as shown in Figure 15 The drug stability test of the microneedle patch in the packaging showed that the HRIG in MN-H-T remained stable at room temperature for at least 30 days and did not require cold chain storage,

[0108] Comparative Example 1

[0109] This comparative example provides a comparison between a one-piece microneedle patch loaded with rabies human immunoglobulin and tazarotene and the double-layer microneedle patch of Example 1 of the present application. The preparation method of the one-piece microneedle patch of Comparative Example 1 is as follows:

[0110] Hyaluronic acid, sodium hyaluronate, rabies human immunoglobulin injection, and tazarotene were added to a container in a mass ratio of 30:3:100:0.5, magnetically stirred for 4 h, and ultrasonically degassed to prepare a needle body solution. The needle body solution was injected into a PDMS microneedle negative mold, vacuum degassed to completely fill the needle body cavity of the microneedle mold with the needle body solution and leave a backing layer with a thickness of 2-3 mm, dried at a temperature of 25°C for 8 h to solidify and form, demolded, and a one-piece microneedle patch was prepared.

[0111] The content of tazarotene in the microneedle patch was determined, and the results are shown in Figure 16 It was determined that the content of tazarotene in a single one-piece microneedle was 50.93 μg, and the content of tazarotene in a single double-layer microneedle patch was 413.76 μg. It can be seen that when the carrier materials of the needle body layer and the backing layer are both hyaluronic acid and sodium hyaluronate, tazarotene cannot be effectively loaded, and the drug loading is low.

[0112] Comparative Example 2

[0113] This comparative example provides a comparison between a one-piece microneedle patch loaded with rabies human immunoglobulin and tazarotene and the double-layer microneedle patch of Example 1 of the present application. The preparation method of the one-piece microneedle patch of Comparative Example 2 is as follows:

[0114] Polyvinylpyrrolidone, tazarotene, anhydrous ethanol, rabies human immunoglobulin injection were added into a container according to a mass ratio of 30:0.5:100:100, and magnetic stirring was carried out for 4h, and ultrasonic was used to remove air bubbles, so that a needle body solution was prepared; the needle body solution was injected into a PDMS microneedle negative mold, vacuum was applied to remove air bubbles, the needle body solution was completely filled into the needle body cavity of the microneedle mold and a backing layer with a thickness of 2-3mm was reserved, drying was carried out at a temperature of 25 DEG C for 8h, solidification was formed, demolding was carried out, and an integrated microneedle patch was prepared.

[0115] The titer of rabies human immunoglobulin in the microneedle patch was determined, and the results are shown in Table 1. Figure 17 It can be seen that when the carrier materials of the needle body layer and the backing layer are both hyaluronic acid and sodium hyaluronate, tazarotene cannot be effectively loaded, and the drug loading amount is low.

[0116] Since the rabies human immunoglobulin is inactivated by precipitation in ethanol, the rabies human immunoglobulin in the integrated microneedle patch loses the ability to neutralize the rabies virus, as shown in Table 1. Figure 17 It can be seen that when the carrier materials of the needle body layer and the backing layer are both polyvinylpyrrolidone, the rabies human immunoglobulin in the prepared microneedle patch is inactivated, and the microneedle patch cannot realize the anti-rabies virus characteristics.

[0117] The above results show that the double-layer soluble microneedle patch of the application has good biocompatibility and the ability to promote cell migration in vitro, and has the characteristics of anti-rabies virus and promoting skin wound repair, and can effectively prevent rabies and promote wound healing. The portability, stability and effectiveness of the patch make it an ideal choice for emergency situations. Compared with the traditional rabies exposure prevention and treatment method, the patch greatly reduces the severe pain caused by the administration process and improves patient compliance. This also broadens the potential application of microneedles in emergency.

[0118] The above only describes the embodiments of the application, and does not limit the patent scope of the application, and any equivalent transformation or direct or indirect application in related technical fields based on the content of the specification and drawings of the application is also included in the patent protection scope of the application.

Claims

1. A dual-layer dissolvable microneedle patch, characterized by, The double-layer soluble microneedle patch comprises a backing layer and a needle body layer arranged on the backing layer, wherein the needle body layer is a microneedle array composed of a plurality of microneedles. The components of the needle body layer comprise rabies human immunoglobulin and water-soluble polymer material; the water-soluble polymer material is at least one of hyaluronic acid and sodium hyaluronate. The components of the backing layer comprise a drug and a carrier material; the carrier material is any one of polyvinylpyrrolidone and methacrylated gelatin; the drug is any one of tazarotene or pro-angiogenic peptide.

2. The dual-layer dissolvable microneedle patch of claim 1, wherein, The water-soluble polymer material in the needle body layer is hyaluronic acid and sodium hyaluronate, and the mass ratio of hyaluronic acid to sodium hyaluronate is 30:(1-10).

3. The dual-layer dissolvable microneedle patch of claim 1, wherein, The molecular weight of the hyaluronic acid is less than 5000 Da.

4. The dual-layer dissolvable microneedle patch of claim 1, wherein, The shape of the microneedle body is conical or pyramidal; the height of a single needle body of the microneedle is 800-2000 μm, the diameter of the bottom is 400-1000 μm, and the radius of the tip is 10-30 μm; the thickness of the backing layer is 1000-3000 μm.

5. The dual-layer dissolvable microneedle patch of claim 1, wherein, The loading amount of rabies human immunoglobulin in the double-layer soluble microneedle patch is 10-30 IU / patch, and the loading amount of tazarotene or pro-angiogenic peptide is 0.01-1 mg / patch.

6. The dual-layer dissolvable microneedle patch of claim 1, wherein, The K value of the polyvinylpyrrolidone is 80-96; the grafting rate of the methacrylated gelatin is 60-90%; and the pro-angiogenic peptide is a vascular endothelial growth factor mimetic peptide.

7. The method of making a dual-layer dissolvable microneedle patch according to any one of claims 1-6, wherein, The method comprises the following steps: (1) preparing a needle body solution, injecting the needle body solution into a microneedle mold, removing air bubbles by vacuumizing, making the needle body solution completely fill the needle body cavities of the microneedle mold and removing excess needle body solution, and standing for solidification molding to obtain a microneedle mold containing molded microneedle bodies; The preparation method of the needle body solution comprises the following steps: adding water-soluble polymer material into rabies human immunoglobulin aqueous solution, stirring and dissolving, and removing air bubbles by ultrasonic to prepare the needle body solution; (2) preparing a backing solution, injecting the backing solution into the microneedle mold containing molded microneedle bodies in step (1) to make the backing solution completely cover the bottoms of all microneedle bodies, removing air bubbles by vacuumizing and keeping the thickness of the backing solution at 2-3 mm, standing for solidification molding, and demolding to prepare a double-layer soluble microneedle patch; The preparation method of the backing solution comprises the following steps: When polyvinylpyrrolidone is selected as the carrier material of the backing layer, polyvinylpyrrolidone and tazarotene are added into anhydrous ethanol, stirred and dissolved, and air bubbles are removed by ultrasonic to prepare the backing solution; When methacrylated gelatin is selected as the carrier material of the backing layer, methacrylated gelatin is added into a pro-angiogenic peptide aqueous solution, stirred and dissolved at 50-80℃, LAP photoinitiator is added, stirred and dissolved, and air bubbles are removed by ultrasonic to prepare the backing solution.

8. The method for preparing the double-layer soluble microneedle patch according to claim 7, wherein The mass ratio of the water-soluble polymer material in the needle body solution and the rabies human immunoglobulin aqueous solution is (1-5):10; the potency of the rabies human immunoglobulin in the rabies human immunoglobulin aqueous solution is 50-300 IU / mL; and the rabies human immunoglobulin aqueous solution is a rabies human immunoglobulin injection; The mass concentration of tazarotene or pro-angiogenic peptide in the backing solution is 0.01-1 mg / mL; When polyvinylpyrrolidone is selected as the carrier material of the backing layer, the mass ratio of polyvinylpyrrolidone to anhydrous ethanol in the backing solution is (1-5):10; When methacrylated gelatin is selected as the carrier material of the backing layer, the mass ratio of methacrylated gelatin to the pro-angiogenic peptide aqueous solution in the backing solution is (1-5):

10.

9. The method of making a dual-layer dissolvable microneedle patch according to claim 7, wherein, The microneedle mold is a PDMS microneedle negative mold; and the standing and curing forming operation is drying and curing forming at a temperature of 20-30℃ for 6-18 hours.

10. Use of the double-layer soluble microneedle patch according to any one of claims 1-6 in the preparation of a drug for preventing rabies virus infection.

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