A hydrogel microneedle patch for identifying subtypes of bullous skin diseases, a manufacturing method and a method of use

Abstract

The present invention provides a hydrogel microneedle patch for identifying subtypes of bullous dermatoses, a manufacturing method and a use method, and belongs to the technical field of diagnosis and typing of autoimmune bullous dermatoses; the hydrogel microneedle patch can penetrate the stratum corneum of the skin with the help of a microneedle structure integrated in a microneedle matrix layer, and extract the interstitial fluid of the patient's skin lesions through the hydrophilic property of the hydrogel, so that the disease-related autoantibodies present in the interstitial fluid are combined with the specific antigens of the bullous dermatoses carried in the microbubbles of the microneedle body, and further, the types of specific autoantibodies in the interstitial fluid of the patient are determined by fluoresceinization reaction and immunofluorescence color development, thereby completing further classification and identification of the subtypes of bullous dermatoses.

Description

Technical Field

[0001] The present invention belongs to the technical field of diagnosis and typing of autoimmune bullous skin diseases, and relates to a hydrogel microneedle patch for identifying subtypes of bullous skin diseases, a manufacturing method and a use method. Background Art

[0002] Autoimmune bullous skin diseases are a group of blistering diseases driven mainly by autoantibodies, with typical manifestations of erythema, blisters and erosions occurring sequentially in the skin / mucosa. From the perspective of pathogenesis, this type of disease can be divided into pemphigus, pemphigoid, acquired epidermolysis bullosa and dermatitis herpetiformis. Various types of autoimmune bullous skin diseases have certain characteristics in terms of skin lesions and predilection sites. For example, 60% of patients with pemphigus vulgaris present with thin-walled blisters and erosions on the oral mucosa, while the typical skin lesions of bullous pemphigoid are mostly hemispherical thick-walled bullae scattered on the trunk and other parts. However, in clinical work, due to the heterogeneity of individual patients, the skin lesions of each subtype of disease often overlap, which makes it difficult for dermatologists to make an accurate diagnosis directly based on clinical manifestations. Therefore, the diagnosis of this type of disease often requires the help of histopathology and immunopathology examinations. Among them, direct immunofluorescence is still the gold standard for the diagnosis of this type of disease. Direct immunofluorescence technology accurately classifies diseases through different binding patterns of immune complexes in various autoimmune bullous skin diseases. In bullous pemphigoid, autoantibodies bind to bullous pemphigoid antigens BP180 and BP230 in the epidermal basement membrane zone and further activate complement to mediate the disease. Therefore, the linear deposition of C3 and IgG in the basement membrane zone is the specific direct immunofluorescence pattern of the disease. However, direct immunofluorescence technology requires dermatologists to remove the patient's skin lesions and the surrounding 1~2cm local full-thickness skin tissue for inspection. Since the biopsy site often has problems such as slow wound healing, postoperative infection and inevitable postoperative scar formation, this technology also exacerbates the patient's painful experience to a certain extent. As a supplementary examination method of direct immunofluorescence, indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA) do not require skin biopsy, which to a certain extent makes up for the shortcomings of direct immunofluorescence technology. However, the sensitivity and specificity of indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA) are inferior to those of direct immunofluorescence. At the same time, these two types of serological examination methods also have their own shortcomings. For example, indirect immunofluorescence can display disease-specific fluorescence patterns on specific substrates, but it cannot distinguish the target antigen types of autoantibodies. Although ELISA can identify disease target antigens by antigen-capture antibodies, it cannot display the in situ fluorescence deposition pattern of human tissues like direct immunofluorescence to determine the disease typing. Therefore, in order to achieve accurate immunotyping of autoimmune bullous skin diseases and reduce the impact of examination methods on the quality of life of patients, a minimally invasive / non-invasive detection tool that can accurately reflect the immunofluorescence deposition pattern of such diseases is needed.

[0003] A microneedle array is a tiny needle-like structure made by microfabrication technology. It is micrometer-sized and can be used in a variety of scenarios such as drug delivery and sampling analysis in the biomedical field. The microneedle array can effectively penetrate the stratum corneum of the skin. During the application of microneedles in the human body, the penetration layer is shallow and the skin nerve endings are less distributed, so the subjects feel less pain or even no pain. Therefore, the microneedle array is an ideal tool for the extraction and detection of biomarkers in the lesion area of ​​skin diseases. However, there is no record in the prior art of hydrogel microneedles carrying specific antigens for autoimmune bullous skin diseases, or of using free autoantibodies in interstitial fluid or autoantibodies on free cells for immunopathological typing of autoimmune bullous skin diseases such as pemphigus and pemphigoid. Summary of the invention

[0004] The present invention provides a hydrogel microneedle patch for identifying subtypes of bullous skin diseases, comprising a microneedle matrix layer and a microneedle flexible base layer;

[0005] The material of the microneedle matrix layer is set to be one or a combination of polymethyl vinyl ether / maleic anhydride, maleic anhydride, hyaluronic acid, polyvinyl alcohol, polyethylene glycol, carboxymethyl cellulose, polylactic acid or chitosan;

[0006] Alternatively, the material of the microneedle matrix layer is one or a combination of polymethyl vinyl ether / maleic anhydride, maleic anhydride, hyaluronic acid or polyvinyl alcohol;

[0007] The material of the microneedle flexible base layer is set to be one or a combination of hyaluronic acid, methacrylate hyaluronic acid, polyvinyl pyrrolidone, polyvinyl alcohol or polylactic acid.

[0008] Furthermore, the material of the microneedle matrix layer also includes autoimmune bullous skin disease specific antigens, and the autoimmune bullous skin disease specific antigen is set to be any one of Dsg1, Dsg3, BP180, BP230, type VII collagen, and transglutaminase.

[0009] Furthermore, a plurality of microneedles are arranged on the microneedle matrix layer, the height H1 of a single microneedle is set to 100-1200 μm, the base width D1 of a single microneedle is set to 300 μm, and the needle distance X2 between two adjacent microneedles is set to 600 μm;

[0010] The shape of the microneedle flexible base layer is set to any one of a circle, a diamond or a square; and when the microneedle flexible base layer is set to a square, its side length X1 is set to 16 mm corresponding to 225 microneedles, 30 mm corresponding to 900 microneedles, or 60 mm corresponding to 3600 microneedles; or, when the microneedle flexible base layer is set to a square, its length and width are respectively set to 25 mm×15 mm corresponding to 525 microneedles, 50 mm×15 mm corresponding to 1050 microneedles, or 100 mm×15 mm corresponding to 2250 microneedles.

[0011] Furthermore, a single microneedle is provided with microbubbles, and the walls of the microbubbles are bound with autoimmune bullous dermatosis-specific antigens.

[0012] The present invention also provides a method for manufacturing a hydrogel microneedle patch for identifying subtypes of bullous dermatosis, comprising the following steps:

[0013] (A) Preparation of microneedle patch solution;

[0014] Step 1: Prepare materials for preparing a microneedle matrix layer and a microneedle flexible base layer and lithium phenyl-2,4,6-trimethylbenzoylphosphonate; the preparation process of the microneedle patch solution is as follows:

[0015] S1.1, adding polymethyl vinyl ether / maleic anhydride to ice distilled water, stirring thoroughly until completely dissolved, then heating to 95°C-100°C until a clear solution is obtained, and cooling to obtain a polymethyl vinyl ether / maleic anhydride solution, wherein the mass volume concentration of the polymethyl vinyl ether / maleic anhydride solution is 60%-72%;

[0016] S1.2, dissolving hyaluronic acid in distilled water → slowly adding methacrylic acid → adding sodium hydroxide until the pH is 8-10, then reacting at room temperature for 24 hours, and dialyzing in distilled water at 4°C using a dialysis membrane with a molecular cutoff of 12 kDa-14 kDa for 3 days to obtain a MeHA solution, wherein the concentration of the MeHA solution is 30-36%;

[0017] S1.3, add polyvinyl alcohol into distilled water, stir at 90°C until completely dissolved, and obtain 30% -36% PVA solution;

[0018] S1.4. At room temperature, PMVE / MAH solution, MeHA solution and PVA solution were mixed in a ratio of 1:1:1, and 0.1% lithium phenyl-2,4,6-trimethylbenzoylphosphonate was added, and the mixture was stirred thoroughly to form a uniform solution and the bubbles were removed to obtain a microneedle patch solution in which the ratio of PMVE / MA solution, MeHA solution and PVA solution was 2:1:1;

[0019] Step 2: The material of the microneedle flexible base layer is methacrylated hyaluronic acid, and the preparation process of the microneedle flexible base layer solution is as follows:

[0020] Add methacrylated hyaluronic acid to ice distilled water, stir thoroughly until completely dissolved, then heat to 95°C-100°C until a clear solution is obtained, and after cooling, obtain a MeHA solution with a mass volume concentration of 20%-24%, then add 0.1% lithium phenyl-2,4,6-trimethylbenzoylphosphonate, stir thoroughly to remove bubbles, and obtain a microneedle flexible base layer solution;

[0021] (ii) Preparation of antigen solution;

[0022] Dsg1, Dsg3, BP180, BP230, collagen type VII, and transglutaminase antigen stock solutions were prepared into respective dilution solutions, and then 3N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide in morpholineethanesulfonic acid buffer were added as carbodiimide crosslinker solution to ensure that the antigen concentration in the final solution was 0.5 μg / ml-1.5 μg / ml and the carbodiimide crosslinker concentration was 8-12 mg / ml;

[0023] (iii) Preparation of a microneedle matrix layer containing microbubbles and a microneedle flexible base layer;

[0024] The microneedle matrix layer is manufactured by a mold method, and the mold is an integrated design of a negative mold of the microneedle matrix layer and a negative mold of the microneedle flexible base layer;

[0025] The process of preparing microbubbles using the gel cycle freezing method is as follows:

[0026] S3.1. Take the microneedle patch solution obtained in the above step 1, heat it in a microwave at 90°C for 5 minutes, and after confirming that no solute is precipitated, pour the microneedle patch solution into the PDMS microneedle mold and fill the negative mold part of the microneedle body with the microneedle patch solution; freeze the mold containing the microneedle patch solution at -20°C for 10 hours, then take it out and place it at room temperature for 5 hours to thaw;

[0027] S3.2, after repeating step S3.1 twice, drip the microneedle flexible base layer solution into the thawed horizontal mold to fill the microneedle flexible base layer part of the mold; then freeze it at -80°C for 4-6 hours, and transfer it to a room temperature drying oven to dry for 15-24 hours;

[0028] S3.3, take out the liquid-carrying mold, demould, place the obtained integrated microneedle matrix layer and the microneedle flexible base layer under blue light for curing and cross-linking for 10-15 minutes, and continue to transfer to a room temperature drying oven for drying for 10 hours to obtain a microneedle matrix layer containing microbubbles and a microneedle flexible base layer connected thereto;

[0029] (IV) Preparation of microneedle matrix layer containing antigen;

[0030] The microneedle matrix layer containing microbubbles was immersed in Dsg1, Dsg3, BP180, BP230, type VII collagen and glutamyl transferase antigen solutions for 3-5 minutes, then taken out and placed at room temperature for 1-1.5 hours. The binding reaction between the antigen and the microneedle was mediated by the carbodiimide cross-linking principle. The microneedle matrix layer was then washed with deionized water and placed in a room temperature drying oven for 5-10 minutes to obtain microneedle matrix layers containing Dsg1 antigen, Dsg3 antigen, BP180 antigen, BP230 antigen, type VII collagen antigen and glutamyl transferase antigen, respectively.

[0031] Furthermore, the mass volume concentrations of the PMVE / MAH solution, the MeHA solution, and the PVA solution in the microneedle patch solution are 20%-24%, 10%-12%, and 10%-12%, respectively;

[0032] Alternatively, the mass volume concentration ratio of the PMVE / MA solution, the MeHA solution, and the PVA solution in the microneedle matrix layer solution is set to 1:1:1;

[0033] Alternatively, the mass volume concentration ratio of the PMVE / MA solution, the MeHA solution, and the PVA solution in the microneedle matrix layer solution is set to 1:2:1.

[0034] Furthermore, the material of the microneedle matrix layer is selected from polymethyl vinyl ether / maleic anhydride, maleic anhydride, hyaluronic acid and polyvinyl alcohol in a ratio of 6:1:2:3;

[0035] Alternatively, the material of the microneedle matrix layer is selected from a mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and polyethylene glycol, wherein the volume concentration ratio between the mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and polyethylene glycol is set to 2:1;

[0036] Alternatively, the material of the microneedle matrix layer is selected from a mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and polylactic acid, wherein the volume concentration ratio between the mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and the polylactic acid is set to 1:1;

[0037] Alternatively, the material of the microneedle matrix layer is selected from a mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and chondroitin sulfate, wherein the volume concentration ratio between the mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and chondroitin sulfate is set to 1:2;

[0038] Alternatively, the material of the microneedle matrix layer is selected as a mixture of any two combinations of PMVE / MA, PVA, CMC, PEG, PLA, and CS, and the corresponding mass volume concentration (w / v) ratio can be 2:1:1, 1:1:1 or 1:1:2 after any two combinations.

[0039] The present invention also provides a method for using the hydrogel microneedle patch for identifying subtypes of bullous dermatosis, comprising the following steps:

[0040] (A) Sampling;

[0041] The microneedle patches carrying different antigens are respectively introduced into the junction of the patient's blisters and normal skin, so that the microneedle matrix layer enters the deep layer of the epidermis and the microneedle flexible base layer is tightly attached to the skin. The microneedle matrix layer absorbs the interstitial fluid in the skin and allows the antigens carried by the microneedles to combine with the autoantibodies.

[0042] (B) Diagnosis;

[0043] The diagnostic process includes incubation of biotinylated detection antibodies, incubation of fluorophores containing streptavidin, and observation with a fluorescence microscope, wherein:

[0044] ①, Incubation of biotinylated detection antibody;

[0045] The microneedle patch after sampling was incubated with 5 ml of BSA solution containing biotinylated detection antibody at 22° C. for 2 h, and then washed with PBS solution; the biotinylated detection antibody was of the same type as the antigen carried by the microneedle patch, and the concentration was set to 2 μg / ml-4 μg / ml;

[0046] ②, Incubation of streptavidin fluorophore;

[0047] The microneedle patch washed with the PBS solution in step ① is placed in 5 ml of a BSA solution containing a streptavidin fluorophore and incubated at 22° C. for 30 min, and then washed with a PBS solution; the concentration of the solution containing the streptavidin fluorophore is set to 3 μg / ml-5 μg / ml;

[0048] ③ Observation by fluorescence microscope;

[0049] The microneedle patch after being washed with the PBS solution in step ② is observed using a fluorescent microscope, and a corresponding diagnosis result can be obtained based on whether fluorescence is observed.

[0050] Furthermore, the method for determining whether fluorescence is observed is as follows:

[0051] Using a microscope, it is observed that the microneedle patch carrying the Dsg1 antigen and / or the Dsg3 antigen emits strong fluorescence, indicating that the patient has autoantibodies against the Dsg1 antigen and / or the Dsg3 antigen in the skin, and it is judged that the patient has pemphigus;

[0052] Using a microscope, it is observed that the microneedle patch carrying BP180 antigen and / or BP230 antigen emits strong fluorescence, indicating that the patient has autoantibodies against BP180 antigen and / or BP230 antigen in the skin, and it is judged that the patient suffers from bullous pemphigoid;

[0053] Using a microscope, it was observed that the microneedle patch carrying type VII collagen emitted strong fluorescence, indicating the presence of type VII collagen autoantibodies in the patient's skin, and it was determined that the patient suffered from acquired epidermolysis bullosa;

[0054] Using a microscope, it was observed that the microneedle patch carrying glutaminase emitted strong fluorescence, indicating the presence of autoantibodies to glutaminase in the patient's skin, and that the patient was diagnosed with dermatitis herpetiformis.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] (1) The present invention provides a hydrogel microneedle patch for identifying subtypes of bullous dermatoses. The hydrogel microneedle patch temporarily and reversibly destroys the stratum corneum of the skin, absorbs interstitial fluid and swells, and binds to free autoantibodies in the interstitial fluid and autoantibodies on free cells through specific antigens of autoimmune bullous dermatoses carried in the microbubbles of the needle body, and further binds to fluorescein-labeled detection antibodies and streptavidin-fluorophores, emitting strong fluorescence visible under a microscope under light excitation.

[0057] (2) The present invention uses microneedles as the test carrier and ELISA technology as the basis, combining the advantages of indirect immunofluorescence and ELISA, and realizes the determination of autoimmune disease target antigens and the imprinting of the in situ fluorescent deposition pattern of the disease under the premise of rapid, minimally invasive and painless examination.

[0058] In addition to the above-described purposes, features and advantages, the present invention has other purposes, features and advantages. The present invention will be further described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0060] Figure 1 Schematic diagram of the overall structure of a hydrogel microneedle patch for identifying subtypes of bullous dermatoses in an embodiment of the present invention;

[0061] Figure 2 yes Figure 1 Schematic diagram of the planar structure of the two microneedles;

[0062] Figure 3 yes Figure 1 Schematic diagram of the size parameters of a single microneedle;

[0063] Figure 4 yes Figure 1 Axial schematic diagram of a single microneedle;

[0064] Figure 5 yes Figure 1 Schematic diagram of a single microneedle from above;

[0065] Figure 6 It is an integrated mold of a microneedle matrix layer and a microneedle flexible base layer for preparing a hydrogel microneedle patch for identifying subtypes of bullous skin diseases in an embodiment of the present invention;

[0066] Figure 7 It is a schematic flow chart of a method for preparing a hydrogel microneedle patch for identifying subtypes of bullous skin diseases in an embodiment of the present invention;

[0067] Figure 8 This is a schematic flow chart of a method for using a hydrogel microneedle patch for identifying subtypes of bullous skin diseases according to an embodiment of the present invention;

[0068] Fig. 9 This is a schematic diagram of the application process of a hydrogel microneedle patch for identifying subtypes of bullous dermatoses in an embodiment of the present invention.

[0069] in:

[0070] 1. microneedle matrix layer, 11. microneedles, 12. microbubbles, 13. autoimmune bullous dermatosis specific antigens, 2. microneedle flexible base layer. DETAILED DESCRIPTION

[0071] In order to make the above-mentioned purposes, features and advantages of the present invention more clear and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings. It should be noted that the drawings of the present invention are all simplified and use non-precise proportions, which are only used to conveniently and clearly assist in explaining the implementation of the present invention; the "number" mentioned in the present invention is not limited to the specific number in the examples in the accompanying drawings; the directions or positional relationships indicated by "front", "middle", "back", "left", "right", "up", "down", "top", "bottom", "middle", etc. mentioned in the present invention are based on the directions or positional relationships shown in the drawings of the present invention, and do not indicate or imply that the devices or components referred to must have a specific direction, nor can they be understood as limitations on the present invention. Embodiment 1:

[0072] See also Figures 1 to 5 As shown, a hydrogel microneedle patch for identifying subtypes of bullous skin diseases provided by the present invention comprises a microneedle matrix layer 1 and a microneedle flexible base layer 2;

[0073] The material of the microneedle matrix layer 1 is not limited to one or a combination of polymethyl vinyl ether / maleic acid (anhydride) (PMVE / MA(H)), methacrylic acid (MA), hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), carboxymethyl cellulose (CMC), polylactic acid (PLA) and chondroitin sulfate (CS); the material of the microneedle preformed base layer is methacrylated hyaluronic acid (MeHA).

[0074] Furthermore, the material of the microneedle matrix layer 1 also includes autoimmune bullous skin disease specific antigens, and the autoimmune bullous skin disease specific antigen 13 is set to be any one of Dsg1, Dsg3, BP180, BP230, type VII collagen, and transglutaminase.

[0075] Further, the microneedle matrix layer 1 is provided with a plurality of microneedles 11, and the number of the microneedles 11 is preferably set to 225 (15*15), 900 (30*30) or 3600 (60*60). Further preferably, the number of the microneedles 11 is preferably set to 225 (15*15). Alternatively, the microneedle matrix layer 1 is provided with a plurality of microneedles 11, and the number of the microneedles 11 is preferably set to 675 (45×15), 1350 (90×15), and 2250 (150×15). Further preferably, the number of the microneedles 11 is preferably set to 675 (45×15).

[0076] Further, the shape of the microneedle 11 is set to be conical, pyramidal, cylindrical, tower-shaped, prism-shaped, truncated cone-shaped, prism-shaped, truncated cone-shaped or hemispherical. Further preferably, considering the stability, penetration and feasibility of preparation of the microneedle 11, the shape of the microneedle 11 is preferably set to be pyramidal or conical. Further preferably, the shape of the microneedle 11 is preferably set to be conical.

[0077] Further, the height H1 of the microneedle 11 is set to 100-1200 μm, the base width D1 of the microneedle 11 is set to 300 μm, and the needle distance X2 between two adjacent microneedles 11 is set to 600 μm. Further preferably, considering the thickening of the local skin of the patient's skin lesions, the height H1 of the microneedle 11 is preferably 400-1200 μm. Further preferably, the height H1 of the microneedle 11 is preferably 600-700 μm.

[0078] Furthermore, the microneedle 11 contains a microbubble 12 , and the wall of the microbubble 12 is bound with an autoimmune bullous dermatosis-specific antigen 13 .

[0079] In addition to the above structure, the material of the microneedle matrix layer 1 is preferably one or a combination of PMVE / MA(H), MA, HA and PVA.

[0080] In addition to the above structure, the microneedle matrix layer 1 is provided with a plurality of microneedles 11, and the number of the microneedles 11 is preferably set to 525 (35×15), 1050 (70×15) or 2250 (150×15). Further preferably, the number of the microneedles 11 is preferably set to 525 (35×15).

[0081] The material of the microneedle flexible base layer 2 is one or more of hyaluronic acid, methacrylic hyaluronic acid, polyvinyl pyrrolidone, polyvinyl alcohol and polylactic acid. Preferably, the material of the microneedle flexible base layer 2 is preferably methacrylic hyaluronic acid.

[0082] Further, considering the polymorphism and extensiveness of the patient's skin lesions, the shape of the microneedle flexible base layer 2 is set to be rectangular, circular or diamond. Further preferably, the shape of the microneedle flexible base layer 2 is set to be rectangular or circular. Further preferably, the shape of the microneedle flexible base layer 2 is set to be square.

[0083] Further, the side length X1 of the microneedle flexible base layer 2 is set to 14mm, 28mm or 56mm to match the number of microneedles 11 of 225 (15*15), 900 (30*30) or 3600 (60*60) respectively. Further preferably, the side length X1 of the microneedle flexible base layer 2 is set to 14mm; the thickness Z1 of the microneedle flexible base layer 2 is set to 200μm. Alternatively, the side length combination of the microneedle flexible base layer 2 can be set to 40mm×15mm, 80mm×15mm, 120mm×15mm to match the number of microneedles 11 of 675 (45×15), 1350 (90×15), and 2250 (150×15) respectively. Further preferably, the length X1 of the long side of the rectangular microneedle flexible base layer 2 is set to 45 mm; and the thickness Z1 of the microneedle flexible base layer 2 is set to 200 μm.

[0084] In addition to the above structure, the side length combination of the microneedle flexible base layer 2 can be set to 25mm×15mm, 50mm×15mm, 100mm×15mm, to match the number of microneedles 11 of 525 (35×15), 1050 (70×15) or 2250 (150×15) respectively. Further preferably, the side length X1 of the microneedle flexible base layer 2 is set to 25mm. Further preferably, the side length X1 of the microneedle flexible base layer 2 is 10mm. Embodiment 2:

[0085] See also Figure 6 As shown, the present invention also provides a method for manufacturing a hydrogel microneedle patch for identifying subtypes of bullous skin diseases, comprising the following steps:

[0086] (A) Preparation of microneedle patch solution;

[0087] Step 1: Prepare materials for preparing the microneedle matrix layer 1 and the microneedle flexible base layer 2 and lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP); specifically, the materials for the microneedle matrix layer 1 are PMVE / MA(H), MA, HA and PVA, and PMVE / MA(H):MA:HA:PVA=6:1:2:3; the preparation process of the microneedle patch solution is as follows:

[0088] S1.1. Add PMVE / MA(H) to ice distilled water, stir thoroughly until completely dissolved, then heat to 95°C-100°C until a clear solution is obtained. After cooling, a PMVE / MA solution is obtained, wherein the mass volume concentration (w / v) of the PMVE / MA solution is 60%-72%.

[0089] S1.2. Dissolve HA in distilled water → slowly add MA → add sodium hydroxide until pH 8-10, then react at room temperature for 24 hours, and dialyze in distilled water at 4°C using a dialysis membrane with a molecular cutoff of 12 kDa-14 kDa for 3 days to obtain a MeHA solution with a concentration of 30-36% (w / v).

[0090] S1.3. Add PVA to distilled water and stir at 90°C until completely dissolved to obtain a 30% -36% (w / v) PVA solution.

[0091] S1.4. At room temperature, PMVE / MAH solution, MeHA solution and PVA solution were mixed in a ratio of 1:1:1, and 0.1% (w / v) LAP was added. The mixture was stirred thoroughly to form a uniform solution with bubbles separated, and a microneedle patch solution with a ratio of PMVE / MA, MeHA and PVA of 2:1:1 was obtained, wherein the mass volume concentrations (w / v) of PMVE / MAH, MeHA and PVA were 20%-24%, 10%-12% and 10%-12%, respectively.

[0092] In a further embodiment, the mass volume concentration (w / v) ratio of PMVE / MA, MeHA, and PVA in the microneedle matrix layer 1 solution can also be set to 1:1:1 or 1:2:1.

[0093] In a further embodiment, the material of the microneedle matrix layer 1 can also be selected from PMVE / MA and any one of CMC, PEG, PLA, and CS, and the corresponding mass volume concentration (w / v) ratio can be set to 2:1, 1:1, or 1:2. Specifically, the material of the microneedle matrix layer is selected from a mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and polyethylene glycol, wherein the volume concentration ratio between the mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and polyethylene glycol is set to 2:1;

[0094] Alternatively, the material of the microneedle matrix layer is selected from a mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and polylactic acid, wherein the volume concentration ratio between the mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and the polylactic acid is set to 1:1;

[0095] Alternatively, the material of the microneedle matrix layer is selected from a mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and chondroitin sulfate, wherein the volume concentration ratio between the mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and chondroitin sulfate is set to 1:2.

[0096] In a further embodiment, the material of the microneedle matrix layer 1 can also be selected as a mixture of any two combinations of PMVE / MA, PVA, CMC, PEG, PLA, and CS, and the corresponding mass volume concentration (w / v) ratio can be 2:1:1, 1:1:1 or 1:1:2 after any two combinations.

[0097] Step 2: The material of the microneedle flexible base layer 2 is MeHA, and the preparation process of the microneedle flexible base layer 2 solution is as follows:

[0098] Add MeHA to ice distilled water, stir thoroughly until completely dissolved, and then heat to 95°C-100°C until a clear solution is obtained. After cooling, a MeHA solution with a mass volume concentration (w / v) of 20%-24% is obtained. Then, 0.1% (w / v) LAP is added, stirred thoroughly and bubbles are removed to obtain a microneedle flexible base layer 2 solution.

[0099] (ii) Preparation of antigen solution;

[0100] The Dsg1, Dsg3, BP180, BP230, type VII collagen, and transglutaminase antigen stock solutions were prepared into respective dilution solutions, and then 3N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) in morpholineethanesulfonic acid (MES) buffer were added as carbodiimide crosslinker solution to ensure that the antigen concentration in the final solution was 0.5μg / ml-1.5μg / ml and the carbodiimide crosslinker concentration was 8-12mg / ml.

[0101] (iii) Preparation of a microneedle matrix layer 1 containing microbubbles and a microneedle flexible base layer 2;

[0102] The microneedle matrix layer 1 is manufactured by a mold method, and the mold is an integrated design of a negative mold of a microneedle matrix layer and a negative mold of a microneedle flexible base layer. Specifically, the mold material is polydimethylsiloxane (PDMS);

[0103] The microneedle matrix layer 1 containing microbubbles 12 is prepared by using a gel cycle freezing method and a mold method, which is specifically as follows:

[0104] Take the microneedle patch solution obtained in the above step 1, heat it in a microwave at 90°C for 5 minutes, and after confirming that no solute is precipitated, pour the solution into the PDMS microneedle mold until it just fills the negative mold part of the microneedle body; place the mold containing the solution at -20°C and freeze it for 10 hours, take it out, and place it at room temperature for 5 hours to thaw it. Repeat the above operation twice to obtain a molded microneedle matrix layer 1 containing microbubbles 12.

[0105] The microneedle flexible base layer 2 integrally formed with the microneedle matrix layer 1 is prepared by a mold method, which is specifically:

[0106] Continue to drip the microneedle flexible base layer solution into the mold containing the formed microneedle matrix layer 1 obtained in the previous step to fill the microneedle flexible base layer part of the mold; place the liquid-carrying mold in a -80°C environment and freeze it for 4-6 hours, and transfer it to a room temperature drying oven for drying for 15-24 hours; take out the liquid-carrying mold, demould it, place the obtained integrated microneedle matrix layer and microneedle flexible base layer under blue light for curing and cross-linking for 10-15 minutes, and continue to transfer it to a room temperature drying oven for drying for 10 hours to obtain a microneedle matrix layer 1 containing microbubbles and a microneedle flexible base layer 2 connected thereto;

[0107] (iv) Preparation of microneedle matrix layer 1 containing antigen;

[0108] The microneedle matrix layer 1 containing microbubbles 12 is immersed in a certain antigen solution for 3-5 minutes, then taken out and placed at room temperature for 1-1.5 hours, and the binding reaction between the antigen and the microneedle 11 is mediated by the carbodiimide cross-linking principle, and then the microneedle matrix layer 1 is washed with deionized water and placed in a room temperature drying oven for 5-10 minutes to obtain the antigen-containing microneedle matrix layer 1;

[0109] A microneedle matrix layer 1 containing Dsg1, Dsg3, BP180, BP230, type VII collagen, and transglutaminase antigen was manufactured in sequence. Embodiment three:

[0110] The present invention also provides a method for using the microneedle patch for diagnosing pemphigus, including a sampling method and subsequent diagnostic steps.

[0111] like Figure 7 As shown, the sampling method of the microneedle patch for diagnosing pemphigus comprises the following process:

[0112] The microneedle patches carrying different antigens are introduced into the junction of the patient's blisters and normal skin or other parts of the skin selected by the physician manually or using a microneedle 11 introduction instrument, so that the microneedle matrix layer 1 enters the deep layer of the epidermis and the microneedle flexible base layer 2 is tightly applied to the skin, which is beneficial for the microneedle matrix layer 1 to absorb the interstitial fluid in the skin and allow the antigens carried by the microneedles 11 to combine with the autoantibodies.

[0113] Specifically, the residence time of the microneedle patch in the skin can be selected within a range of 5-30 minutes according to needs, and is preferably 10 minutes in this embodiment.

[0114] like Figure 7 As shown, the subsequent diagnostic steps of the microneedle patch for diagnosing pemphigus include incubation of biotinylated detection antibodies, incubation of fluorophores containing streptavidin, and observation with a fluorescence microscope.

[0115] ①Incubation of biotinylated detection antibody

[0116] The microneedle patch was incubated with 5 ml of BSA solution containing biotinylated detection antibody at 22°C for 2 h and washed with PBS; the biotinylated detection antibody was of the same type as the antigen carried by the microneedle patch, and the concentration could be 2 μg / ml-4 μg / ml, preferably 3 μg / ml in this embodiment.

[0117] ②Incubation of streptavidin fluorophore

[0118] The microneedle 11 is placed in 5 ml of BSA solution containing streptavidin fluorophore and incubated at 22° C. for 30 min, and then washed with PBS; the concentration of the solution containing streptavidin fluorophore can be 3 μg / ml-5 μg / ml, and preferably 4 μg / ml in this embodiment.

[0119] ③. Observation with fluorescence microscope

[0120] The microneedle patch is observed using a fluorescence microscope, and a corresponding diagnosis result can be obtained based on whether fluorescence is observed.

[0121] In a further embodiment, a microscope was used to observe that the microneedle patch carrying Dsg1 and Dsg3 antigens emitted strong fluorescence, indicating the presence of anti-Dsg1 and Dsg3 autoantibodies in the patient's skin, and the patient may have pemphigus.

[0122] In a further embodiment, it was observed under a microscope that the microneedle patch carrying BP180 and BP230 emitted strong fluorescence, indicating the presence of autoantibodies against BP180 and BP230 in the patient's skin, and that the patient may suffer from bullous pemphigoid.

[0123] In a further embodiment, it was observed under a microscope that the microneedle patch carrying type VII collagen emitted strong fluorescence, indicating the presence of autoantibodies against type VII collagen in the patient's skin and the patient may suffer from acquired epidermolysis bullosa.

[0124] In a further embodiment, a microscope was used to observe that the microneedle patch carrying transglutaminase emitted strong fluorescence, indicating the presence of anti-transglutaminase autoantibodies in the patient's skin and the possibility of dermatitis herpetiformis.

[0125] In addition to the above description, the subsequent diagnostic steps of the microneedle patch for diagnosing pemphigus can also be set to include: incubation of biotinylated detection antibodies, incubation of horseradish peroxidase containing streptavidin, and observation with the naked eye / colorimeter.

[0126] ④. Incubation of biotinylated detection antibody

[0127] The microneedle patch is incubated with 5 ml of BSA solution containing biotinylated detection antibody at 22°C for 2 h, and washed with PBS to remove unbound biotinylated detection antibody; the biotinylated detection antibody is of the same type as the antigen carried by the microneedle patch, and the concentration can be 2 μg / ml-4 μg / ml, preferably 3 μg / ml in this embodiment.

[0128] ⑤. Incubation of horseradish peroxidase containing streptavidin

[0129] The microneedle patch was placed in 5 ml of BSA solution containing streptavidin and horseradish peroxidase at 22° C. for 30 min, and then washed with PBS; the concentration of the horseradish peroxidase containing streptavidin can be 3 μg / ml-5 μg / ml, and preferably 4 μg / ml in this embodiment.

[0130] ⑥. Observation with naked eyes / colorimeter

[0131] After the microneedle patch is incubated with the substrate solution (a mixture of hydrogen peroxide and tetramethylbenzidine) for 20 minutes, sulfuric acid is used to terminate the reaction, and the microneedle patch is taken out of the substrate solution. The corresponding diagnosis result can be obtained by observing the solution for coloration with the naked eye and observing the absorbance with a colorimeter.

[0132] In a further embodiment, the solution soaked with the microneedle patch carrying the Dsg1 and Dsg3 antigens was observed to be colored with the naked eye and the absorbance observed with a colorimeter increased, indicating the presence of autoantibodies against Dsg1 and Dsg3 in the patient's skin and the patient may suffer from pemphigus.

[0133] In a further embodiment, the solution soaked with the microneedle patch carrying BP180 and BP230 antigens was observed to be colored with the naked eye and the absorbance observed with a colorimeter increased, indicating the presence of autoantibodies against BP180 and BP230 in the patient's skin and the possibility of bullous pemphigoid.

[0134] In a further embodiment, the solution soaked with the microneedle patch carrying type VII collagen was observed to develop color with the naked eye and the absorbance observed with a colorimeter increased, indicating the presence of autoantibodies against type VII collagen in the patient's skin and the possibility of acquired epidermolysis bullosa.

[0135] In a further embodiment, the solution in which the microneedle patch carrying transglutaminase is immersed is observed to develop color with the naked eye and the absorbance observed with a colorimeter increases, indicating the presence of autoantibodies against transglutaminase in the patient's skin and the possibility of dermatitis herpetiformis.

[0136] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for manufacturing a hydrogel microneedle patch for identifying subtypes of bullous skin diseases, characterized in that: The hydrogel microneedle patch comprises a microneedle matrix layer and a microneedle flexible base layer; The material of the microneedle matrix layer is set to be one or a combination of polymethyl vinyl ether / maleic anhydride, maleic anhydride, hyaluronic acid, polyvinyl alcohol, polyethylene glycol, carboxymethyl cellulose, polylactic acid and chitosan; The material of the microneedle flexible base layer is set to be one or more of hyaluronic acid, methacrylic hyaluronic acid, polyvinyl pyrrolidone, polyvinyl alcohol and polylactic acid; The material of the microneedle matrix layer also includes autoimmune bullous skin disease specific antigens, and the autoimmune bullous skin disease specific antigen is set to any one of Dsg1, Dsg3, BP180, BP230, type VII collagen, and transglutaminase; The microneedle matrix layer is provided with a plurality of microneedles, each of the microneedles is further provided with a microbubble, and the microbubble wall is combined with an autoimmune bullous skin disease specific antigen; The method for manufacturing the hydrogel microneedle patch includes preparing a microneedle patch solution, and the specific steps for preparing the microneedle patch solution are as follows: Step 1: Prepare materials for preparing a microneedle matrix layer and a microneedle flexible base layer and lithium phenyl-2,4,6-trimethylbenzoylphosphonate; the preparation process of the microneedle patch solution is as follows: S1.1, adding polymethyl vinyl ether / maleic anhydride to ice distilled water, stirring thoroughly until completely dissolved, then heating to 95°C-100°C until a clear solution is obtained, and cooling to obtain a polymethyl vinyl ether / maleic anhydride solution, wherein the mass volume concentration of the polymethyl vinyl ether / maleic anhydride solution is 60%-72%; S1.2, dissolving hyaluronic acid in distilled water → slowly adding methacrylic acid → adding sodium hydroxide until pH 8-10, then reacting at room temperature for 24 hours, and dialyzing in distilled water at 4°C using a dialysis membrane with a molecular cutoff of 12 kDa-14 kDa for 3 days to obtain a MeHA solution, wherein the concentration of the MeHA solution is 30-36%; S1.3, add polyvinyl alcohol into distilled water, stir at 90°C until completely dissolved, and obtain 30% -36% PVA solution; S1.

4. At room temperature, PMVE / MAH solution, MeHA solution and PVA solution were mixed in a ratio of 1:1:1, and 0.1% lithium phenyl-2,4,6-trimethylbenzoylphosphonate was added, and the mixture was stirred thoroughly to form a uniform solution and the bubbles were removed to obtain a microneedle patch solution in which the ratio of PMVE / MA solution, MeHA solution and PVA solution was 2:1:1; Step 2: The material of the microneedle flexible base layer is methacrylated hyaluronic acid, and the preparation process of the microneedle flexible base layer solution is as follows: Methacrylated hyaluronic acid was added to ice distilled water, stirred thoroughly until completely dissolved, and then heated to 95°C-100°C until a clear solution was obtained. After cooling, a MeHA solution was obtained with a mass volume concentration of 20%-24%. 0.1% lithium phenyl-2,4,6-trimethylbenzoylphosphonate was added, stirred thoroughly to remove bubbles, and a microneedle flexible base layer solution was obtained.

2. The manufacturing method according to claim 1, characterized in that: The following steps are also included: Step 3: Preparation of antigen solution; Dsg1, Dsg3, BP180, BP230, collagen type VII, and transglutaminase antigen stock solutions were prepared into respective dilution solutions, and then 3N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide in morpholineethanesulfonic acid buffer were added as carbodiimide crosslinker solution to ensure that the antigen concentration in the final solution was 0.5 μg / ml-1.5 μg / ml and the carbodiimide crosslinker concentration was 8-12 mg / ml; Step 4: preparing a microneedle matrix layer containing microbubbles and a microneedle flexible base layer; The microneedle matrix layer is manufactured by a mold method, and the mold is an integrated design of a negative mold of the microneedle matrix layer and a negative mold of the microneedle flexible base layer; The process of preparing microbubbles using the gel cycle freezing method is as follows: S3.

1. Take the microneedle patch solution obtained in the above step 1, heat it in a microwave at 90°C for 5 minutes, and after confirming that no solute is precipitated, pour the microneedle patch solution into the PDMS microneedle mold and fill the negative mold part of the microneedle body with the microneedle patch solution; freeze the mold containing the microneedle patch solution at -20°C for 10 hours, then take it out and place it at room temperature for 5 hours to thaw; S3.2, after repeating step S3.1 twice, drip the microneedle flexible base layer solution into the thawed horizontal mold to fill the microneedle flexible base layer part of the mold; then freeze it at -80°C for 4-6 hours, and transfer it to a room temperature drying oven to dry for 15-24 hours; S3.3, take out the liquid-carrying mold, demould, place the obtained integrated microneedle matrix layer and the microneedle flexible base layer under blue light for curing and cross-linking for 10-15 minutes, and continue to transfer to a room temperature drying oven for drying for 10 hours to obtain a microneedle matrix layer containing microbubbles and a microneedle flexible base layer connected thereto; Step 5: Preparation of a microneedle matrix layer containing antigens; The microneedle matrix layer containing microbubbles was immersed in Dsg1, Dsg3, BP180, BP230, type VII collagen and glutamyl transferase antigen solutions for 3-5 minutes, then taken out and placed at room temperature for 1-1.5 hours. The binding reaction between the antigen and the microneedle was mediated by the carbodiimide cross-linking principle. The microneedle matrix layer was then washed with deionized water and placed in a room temperature drying oven for 5-10 minutes to obtain microneedle matrix layers containing Dsg1 antigen, Dsg3 antigen, BP180 antigen, BP230 antigen, type VII collagen antigen and glutamyl transferase antigen, respectively.

3. The manufacturing method according to claim 2, characterized in that: The mass volume concentrations of PMVE / MAH solution, MeHA solution and PVA solution in the microneedle patch solution are 20%-24%, 10%-12% and 10%-12% respectively; Alternatively, the mass volume concentration ratio of the PMVE / MA solution, the MeHA solution, and the PVA solution in the microneedle matrix layer solution is set to 1:1:1; Alternatively, the mass volume concentration ratio of the PMVE / MA solution, the MeHA solution, and the PVA solution in the microneedle matrix layer solution is set to 1:2:

1.

4. The manufacturing method according to claim 3, characterized in that: The material of the microneedle matrix layer is selected from polymethyl vinyl ether / maleic anhydride, maleic anhydride, hyaluronic acid and polyvinyl alcohol in a volume concentration ratio of 6:1:2:3; Alternatively, the material of the microneedle matrix layer is selected from a mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and polyethylene glycol, wherein the volume concentration ratio between the mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and polyethylene glycol is set to 2:1; Alternatively, the material of the microneedle matrix layer is selected from a mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and polylactic acid, wherein the volume concentration ratio between the mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and the polylactic acid is set to 1:1; Alternatively, the material of the microneedle matrix layer is selected from a mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and chondroitin sulfate, wherein the volume concentration ratio between the mixture of polymethyl vinyl ether / maleic acid (anhydride) and carboxymethyl cellulose and chondroitin sulfate is set to 1:2; Alternatively, the material of the microneedle matrix layer is selected as a mixture of any two combinations of PMVE / MA, PVA, CMC, PEG, PLA, and CS, and the corresponding mass volume concentration (w / v) ratio can be 2:1:1, 1:1:1 or 1:1:2 after any two combinations.

5. The manufacturing method according to claim 4, characterized in that: The height H1 of a single microneedle is set to 100-1200 μm, the base width D1 of a single microneedle is set to 300 μm, and the needle distance X2 between two adjacent microneedles is set to 600 μm; The shape of the microneedle flexible base layer is set to any one of a circle, a diamond or a square; and when the microneedle flexible base layer is set to a square, its side length X1 is set to 16 mm corresponding to 225 microneedles, 30 mm corresponding to 900 microneedles, or 60 mm corresponding to 3600 microneedles; or, when the microneedle flexible base layer is set to a square, its length and width are respectively set to 25 mm×15 mm corresponding to 525 microneedles, 50 mm×15 mm corresponding to 1050 microneedles, or 100 mm×15 mm corresponding to 2250 microneedles.

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