Solution for microneedle, microneedle preparation method and microneedle product

Abstract

The invention discloses a solution for a microneedle and a microneedle body and a substrate for preparing a soluble microneedle, the solution for the microneedle comprises a basic solution, a microneedle framework material in the basic solution comprises carboxymethyl chitosan and sodium hyaluronate, the content percentage of the microneedle framework material is 1.8%-3.6%, preferably, the content percentage of the sodium hyaluronate is 1.8%-3.6%, and preferably, the content percentage of the carboxymethyl chitosan and the sodium hyaluronate is 1%-2%. The content percentage of the microneedle framework material is 1.9%-3.4%, and furthermore, the content percentage of the microneedle framework material is 1.96%-3.22%. The invention also provides a corresponding microneedle, a preparation method and a microneedle product. The soluble microneedle is good in biocompatibility, low in toxicity, good in plasticity and low in cost, the preparation method is particularly suitable for adding temperature-sensitive functional components, and inactivation of active protein and active polypeptide drugs cannot be caused by the temperature needed during preparation and filling.

Description

Technical Field

[0001] This invention relates to the field of microneedle technology, and particularly to microneedle solutions, microneedle preparation methods, and microneedle products. Background Technology

[0002] Microneedling technology has developed rapidly in recent years and has attracted widespread attention in the medical field. Microneedles combine the advantages of injectable and transdermal patches, penetrating the stratum corneum of the skin almost painlessly to dissolve and release drugs, effectively delivering multiple medications deep into skin tissues and allowing them to enter the bloodstream for systemic delivery. Furthermore, because microneedles deliver drugs transdermally, the pharmacokinetics of the drug are altered, accelerating absorption into the bloodstream for faster onset of action, easier accumulation at the lesion site, and more stable blood drug concentrations. Microneedles are classified into five main categories based on function: solid microneedles, hollow microneedles, coated microneedles, hydrogel microneedles, and soluble microneedles. Microneedles made with biodegradable or soluble microneedle framework materials are called soluble microneedles. Compared to other microneedles, soluble microneedles have the advantages of using biodegradable materials, high biocompatibility, and high safety; they dissolve in the skin after insertion.

[0003] Soluble microneedles have various matrix materials, and parameters such as biocompatibility, degradability, solubility, and mechanical properties should be considered when selecting a polymer matrix. Chinese patent CN103893018A involves sodium hyaluronate microneedles, but the addition of hyperbranched polysulfoneamine or polyester reduces their mechanical properties and makes them difficult to demold during preparation, resulting in poor microneedle formation. Furthermore, drug release patterns generally depend on many factors, including drug binding affinity, the molecular weight of the polymer material, and the water solubility rate. It has been reported that the preparation of microneedles composed of polylactic acid, polyglycolic acid, maltose, or galactose requires heating steps exceeding 140°C, and some materials lack sufficient mechanical strength.

[0004] In summary, there is currently no soluble microneedle that is biocompatible, has low toxicity, good plasticity, and low cost, and is suitable for various applications such as medical treatment, skin whitening, and anti-aging. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a microneedle solution for preparing soluble microneedles, including the microneedle body and substrate. The microneedle solution comprises a base solution, in which the microneedle framework material includes carboxymethyl deacetylated chitosan and sodium hyaluronate. The content percentage of the microneedle framework material is 1.8% to 3.6%, preferably 1.9% to 3.4%, and more preferably 1.96% to 3.22%.

[0006] Sodium hyaluronate, the sodium salt of hyaluronic acid, is an essential component of human cells. Compared to hyaluronic acid, which is unstable, sensitive to free radicals, and has poor mechanical strength, sodium hyaluronate exhibits enhanced mechanical strength and stability. Sodium hyaluronate also demonstrates good biodegradability and biocompatibility, and does not cause inflammatory reactions when ingested. Carboxymethyl deacetylated chitosan, a chitosan derivative, possesses excellent properties such as biocompatibility, biodegradability, antibacterial properties, non-cytotoxicity, and protein affinity, making it a promising candidate for applications in biomedicine, particularly as a drug carrier.

[0007] In one embodiment, the mass ratio of the carboxymethyl deacetylated chitosan to the sodium hyaluronate is 1:(0.55-3). Preferably, the mass ratio of the carboxymethyl deacetylated chitosan to the sodium hyaluronate is 1:(0.55-2), 1:(0.6-2), 1:(0.667-2), or 1:1.

[0008] In another embodiment, the degree of deacetylation of the carboxymethyl deacetylated chitosan is ≥80%.

[0009] In another embodiment, the sodium hyaluronate has a molecular weight range of 5 to 1500 kDa. Preferably, the sodium hyaluronate is composed of any one or more of the following: high molecular weight (1200 to 1500 kDa), medium molecular weight (200 to 400 kDa), and low molecular weight (5 to 10 kDa). More preferably, when it is composed of multiple components, they are composed in equal amounts.

[0010] In one embodiment, the microneedle solution further includes a functional component, which is added to the base solution to prepare the microneedle body. The functional component is one or more combinations of water-soluble small molecule and macromolecule compounds. The small molecule compound is selected from pigments, markers, disease compounds, diagnostic compounds, or cosmetic compounds. The macromolecule compound is selected from proteins and peptides, polysaccharides, or nucleic acids.

[0011] In one embodiment, the pigment is selected from Sudan II, calcein, sulfonylrhodamine B, 3H-indocyanine dyes, green fluorescent dyes, indocyanine green, or brilliant blue;

[0012] The marker is selected from fluorescein isothiocyanate (FITC) or lipophilic membrane dye (DiI).

[0013] The compounds used for the disease are selected from ibuprofen sodium, meloxicam, sinomenine hydrochloride, capsaicin, tetracycline, metronidazole, lidocaine, sumatriptan succinate, 5-aminolevulinic acid, porphyrin compounds, itraconazole, ferric pyrophosphate, donepezil hydrochloride, alendronate, or caffeine.

[0014] The diagnostic compounds are selected from allergens or cyanobacterium green ICG;

[0015] The cosmetic compounds are selected from vitamin C glycosides, niacinamide, all-trans retinoic acid, vitamin C, 4-butylresorcinol, epidermal growth factor, collagen, adenosine, dipeptide-1, dipeptide-2, dipeptide-4, dipeptide-15, acetyl dipeptide-1 cetyl ester, palmitoyl dipeptide-7, tripeptide-1, tripeptide-1 copper, tripeptide-2, tripeptide-3, tripeptide-10 citrulline, tripeptide-32, acetyl tripeptide-1, palmitoyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl tripeptide-8, poly(tripeptide-6), trifluoroacetyl tripeptide-2, tetrapeptide-1, tetrapeptide-3, tetrapeptide-4, acetyl tetrapeptide-2, acetyl tetrapeptide-3, acetyl tetrapeptide-5, acetyl tetrapeptide-9, acetyl tetrapeptide-11, palmitoyl tetrapeptide-5, palmitoyl tetrapeptide-7, and palmitoyl tetrapeptide-10. SH-Pentapeptide-1, Pentapeptide-3, Palmitoyl Pentapeptide-4, Palmitoyl Pentapeptide-5, Myristoyl Pentapeptide-4, Hexapeptide-1, Hexapeptide-2, Hexapeptide-3, Hexapeptide-5, Hexapeptide-9, Hexapeptide-11, Acetyl Hexapeptide-1, Acetyl Hexapeptide-7, Acetyl Hexapeptide-8, Palmitoyl Hexapeptide-12, Palmitoyl Hexapeptide-14, Palmitoyl Hexapeptide-15, Myristoyl Hexapeptide-5, Heptapeptide-6, Acetyl Heptapeptide-4, Acetyl Octapeptide-3, Nonapeptide-1, Decapeptide-4, Tridecapeptide-1, Glutathione, Oligopeptide-1, Oligopeptide-2, Oligopeptide-3, Oligopeptide-4, Oligopeptide-5, Oligopeptide-6, Oligopeptide-29, Oligopeptide-32, Carnosine, Yeast Polypeptides, Arginine / Lysine Polypeptides, Retinol / Yeast Polypeptides, Ascorbic Acid Polypeptides, Glutathione or Lactobacillus Peptides.

[0016] In one embodiment, the protein and polypeptide are selected from soluble elastin, soluble collagen, fibronectin, silk fibroin, fibroin, whey protein, lactoglobulin, lactoferrin, ovalbumin, bovine serum albumin, soluble type III collagen, soluble type XVII collagen, viral capsid protein, PGF21 protein, nanobody, papain, lactoperoxidase, bromelain, lipase, superoxide dismutase, hesperidinase, amylase, Bacillus subtilis enzyme, protease, and oxidase. Reductase, catalase, alcohol oxidase, glucose oxidase, glucosyl amylase, α-amylase, lysozyme, sutelanase, asparaginase, bovine pancreatic ribonuclease A, horseradish peroxidase, glutamate oxidase, insulin, desmopressin, glucagon, glucagon-like peptide-1 receptor agonist, parathyroid hormone, growth hormone, etanercept, botulinum toxin, calcitonin, exendin-4 (increased insulin analog), leuprolide acetate, erythropoietin, interferon, fibroblast growth factor;

[0017] More preferably, the glucagon-like peptide-1 receptor agonist is selected from exenatide, lixisenatide, lixisenatide, liraglutide, abiglutide, dulaglutide, lixisenatide, loxenatide, smegglutide, beraglutide, or semaglutide.

[0018] The polysaccharide is selected from heparin, dextran, or hyaluronic acid;

[0019] The nucleic acid is selected from RNA or DNA.

[0020] In one embodiment, the mass ratio of the functional component to the microneedle base solution is 1:(20-1050000), more preferably, the mass ratio of the functional component to the microneedle base solution is 1:(30:60000) or 1:(50-50000), and even more preferably 1:(70-35000) or 1:(75-30000).

[0021] The second aspect of this invention is a method for preparing microneedles, wherein any one of the microneedle solutions in the aforementioned technical solutions is used as a preparation substrate solution A, and functional components are added to the same solution A to prepare the microneedle body solution B.

[0022] In one implementation, the method includes: Step 1) Filling the microneedle body, as follows:

[0023] A layer of solution B is spread at the bottom of the mold. The mold is then placed in a vacuum stirring degassing machine. The vacuum stirring degassing machine is operated in five stages for filling the microneedles: the vacuum level of the first stage is greater than that of the second stage, the vacuum level of the second stage is greater than that of the third stage, the vacuum level of the third stage is less than that of the fourth stage, and the vacuum level of the fourth stage is less than that of the fifth stage. The vacuuming time for each of the above five stages is 30-300 seconds, and the stirring speed is set to 0 r / min. The above five stages of vacuuming are repeated 3 times to complete the filling of the microneedles. After each run, the solution B at the bottom of the mold is scraped off with a scraper and a new layer is spread.

[0024] Furthermore, the vacuum level of the first segment is not greater than that of the fifth segment, and the vacuum level of the second segment is not greater than that of the fourth segment;

[0025] Furthermore, the vacuum degree of the first stage is 30-90 kPa or 30-60 kPa; the vacuum degree of the second stage is 1-30 kPa or 1-20 kPa or 1-10 kPa; the vacuum degree of the third stage is 0.1-5 kPa or 0.1-3 kPa; the vacuum degree of the fourth stage is 1-30 kPa or 3-30 kPa; and the vacuum degree of the fifth stage is 30-90 kPa or 30-60 kPa.

[0026] In one implementation, the vacuuming time for the first stage is 30-120s, the vacuuming time for the second stage is 30-120s, the vacuuming time for the third stage is 30-120s, the vacuuming time for the fourth stage is 30-120s, and the vacuuming time for the fifth stage is 30-120s. Preferably, the vacuuming time for the five stages is the same.

[0027] In one implementation, step 2) substrate filling is included, as follows:

[0028] After filling the microneedle body, scrape off the solution at the bottom of the mold with a scraper, fill the mold groove with solution A, and place the mold in a vacuum stirring degassing machine. The vacuum stirring degassing machine is operated in two stages for substrate filling: the vacuum degree of the first stage is greater than that of the second stage. The time for each of the two stages of vacuuming is 30 to 300 seconds, and the stirring speed is set to 0 r / min. The two stages of vacuuming are repeated twice to complete the substrate filling.

[0029] Furthermore, the vacuum degree of the first stage is 30-90 kPa and the vacuuming time is 60-180 s; the vacuum degree of the second stage is 10-45 kPa and the vacuuming time is 60-180 s.

[0030] In one implementation, step 3) drying is included, as follows:

[0031] Carefully remove the microneedle mold filled with substrate from the vacuum stirring degassing machine and place it in a constant temperature and humidity drying oven for 24–36 hours, with the temperature set at 25°C and the humidity at 50%.

[0032] In one implementation scheme, step 4) the finished product is as follows:

[0033] (1) Carefully remove the dried microneedle sample from the mold and cut it into the appropriate size as needed;

[0034] (2) Attach a backing label to the back of the cut microneedle sample and package it for later use.

[0035] The third aspect of this invention is a microneedle product, prepared using the microneedle solution of the aforementioned technical solution or the preparation method of the aforementioned technical solution.

[0036] In one embodiment, the microneedle body is a single microneedle shape or a microneedle shape consisting of a tip and a base.

[0037] The single microneedle shape includes any of the following: regular triangular pyramid, regular pentagonal pyramid, regular hexagonal pyramid, regular heptagonal pyramid, regular octagonal pyramid, regular octagonal pyramid, regular nonagonal pyramid, regular decimal pyramid, and conical shape;

[0038] The shape of the microneedle combination of the needle tip and the bottom includes any of the following: regular triangular pyramid + regular triangular prism, regular square pyramid + regular square prism, regular pentagonal pyramid + regular pentagonal prism, regular hexagonal pyramid + regular hexagonal prism, regular heptagonal pyramid + regular heptagonal prism, regular octagonal pyramid + regular octagonal prism, regular nonagonal pyramid + regular nonagonal prism, regular decimal pyramid + regular decimal prism, cone + cylinder.

[0039] In another embodiment, the microneedle has a height ranging from 25 to 1500 μm; a base side length of 50 to 500 μm; a tip diameter of 0 to 30 μm; and a microneedle spacing of 50 to 1000 μm.

[0040] In another embodiment, an optional backing for medical adhesive tape is also included.

[0041] The beneficial effects of this invention are as follows:

[0042] 1) The microneedle matrix material includes carboxymethyl deacetylated chitosan and sodium hyaluronate, which have good biocompatibility, low toxicity, are soluble in the skin, have high safety, and are simple in composition and low in dosage.

[0043] 2) The mechanical strength of the microneedle body is qualified, and it can penetrate the skin without deformation;

[0044] 3) The microneedle matrix material is stable and has advantages such as rapid dissolution of microneedles, improved drug release rate, and no impact on the activity of the loaded drug;

[0045] 4) Functional ingredients can be added as needed, and the application range is wide;

[0046] 5) The preparation method is particularly suitable for adding temperature-sensitive functional components. The required temperature during preparation and filling will not cause the active protein or active peptide drug to become inactive.

[0047] 6) The preparation method is simple and low-cost, making it suitable for large-scale production. Attached Figure Description

[0048] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0049] Figure 1 These are an overall drawing and a partial enlarged drawing of a microneedle mold design provided by this invention (Note: all measurements in the drawings are in mm).

[0050] Figure 2 These are overall and enlarged views of the microneedle sample prepared according to formulation 13 in Example 2.

[0051] Figure 3 The image shows the results of an in vitro simulated skin puncture experiment on the microneedle sample prepared by formulation 17 in Example 4. The left side shows the puncture results, and the right side shows the transdermal results.

[0052] Figure 4 This is an animal transdermal experiment diagram of the microneedle sample prepared by formulation 17 in Example 5. The left side shows that needle holes can be observed on the skin of the mouse back, and the right side shows that the microneedle tip of the bright blue-marked microneedle sample was completely dissolved after being torn off the mouse back.

[0053] Figure 5 The image shows the results of an in vitro simulated skin puncture experiment on the microneedle sample containing bovine serum albumin prepared in Example 6. The left side shows the puncture results, and the right side shows the transdermal results.

[0054] Figure 6 This is an image of the soluble type III collagen microneedle sample prepared in Example 8.

[0055] Figure 7 This is an image of the soluble elastin microneedle sample prepared in Example 9.

[0056] Figure 8 This is an image of a soluble type XVII collagen microneedle sample prepared in Example 10.

[0057] Figure 9 This is an image of the superoxide dismutase protein microneedle sample prepared in Example 11.

[0058] Figure 10 This is an image of the recombinant type A botulinum toxin microneedle sample prepared in Example 12.

[0059] Figure 11 This is an image of the VTNF1 antibody protein microneedle sample prepared in Example 13.

[0060] Figure 12 This is an image of the Her2VHH2A2 antibody protein microneedle sample prepared in Example 14.

[0061] Figure 13 This is an image of the Q1 protein microneedle sample prepared in Example 15.

[0062] Figure 14 This is an image of the GM2 protein microneedle sample prepared in Example 16.

[0063] Figure 15 This is an image of the PGF242 protein microneedle sample prepared in Example 17.

[0064] Figure 16 This is a graph showing the results of the first batch of glucose tolerance tests in Example 18.

[0065] Figure 17This is a graph showing the results of the second batch of glucose tolerance tests in Example 18. Detailed Implementation

[0066] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0067] Example 1: Design of Microneedle Mold

[0068] There are currently many methods for preparing soluble microneedles, including micromolding, solvent casting, photolithography, 3D printing, thermoforming, and ultrasonic welding. Among these, micromolding is the most commonly used method. This method mainly consists of three steps: first, a master mold is made from a robust material (such as metal or silicon) and used as the mother mold; then, a female mold (such as polydimethylsiloxane) is made from the mother mold; finally, the final structure of the microneedle is formed within the female mold.

[0069] Design of microneedle molds:

[0070] We commissioned Jiangsu Jicui Functional Materials Research Institute Co., Ltd. to manufacture the mold based on the microneedle mold design information we provided: the microneedle is a regular square pyramid shape, the height of the microneedle body is 600μm; the side length of the bottom surface of the pyramid is 300μm×300μm; the microneedle spacing is 600μm; the total side length of one mold is 5cm×5cm.

[0071] Overall and enlarged views of the microneedle mold design are as follows: Figure 1 As shown. The microneedles are in the shape of a regular square pyramid, with a height ranging from 25 to 1500 μm; a base side length of 50 to 500 μm; a tip diameter of 0 to 30 μm; and a spacing of 50 to 1000 μm.

[0072] The designs for other shaped microneedle molds are as follows:

[0073] Other single microneedle shapes include: regular triangular pyramid, regular pentagonal pyramid, regular hexagonal pyramid, regular heptagonal pyramid, regular octagonal pyramid, regular octagonal pyramid, regular nonagonal pyramid, regular decimal pyramid, and cone.

[0074] Other microneedle shapes for needle and base combinations include: regular triangular pyramid + regular triangular prism, regular square pyramid + regular square prism, regular pentagonal pyramid + regular pentagonal prism, regular hexagonal pyramid + regular hexagonal prism, regular heptagonal pyramid + regular heptagonal prism, regular octagonal pyramid + regular octagonal prism, regular nonagonal pyramid + regular nonagonal prism, regular decimal pyramid + regular decimal prism, and cone + cylinder.

[0075] Other microneedle designs have a needle height range of 25–1500 μm; a base side length or diameter range of 50–500 μm for cones or cylinders; a tip diameter range of 0–30 μm; and a microneedle spacing range of 50–1000 μm.

[0076] Example 2: Formulation Screening of Basic Solutions

[0077] 1. Composition of formulas 1-16:

[0078] Formula 1: 74.5g purified water, 0.5g carboxymethyl deacetylated chitosan, 1g sodium hyaluronate, 10g polyvinylpyrrolidone K30, 3g glycerin, 10g PVA 0588, 801g polysorbate.

[0079] Formula 2: 74.5g purified water, 0.5g carboxymethyl deacetylated chitosan, 1g sodium hyaluronate, 5g polyvinylpyrrolidone K30, 3g glycerin, 15g HPMC, 801g polysorbate.

[0080] Formula 3: 75g purified water, 1g hyaluronic acid, 3g glycerin, 0.5885g PVA, 15g HPMC, 801g polysorbate.

[0081] Formula 4: 75g purified water, 25g Pullulan, 5g HPMC, 2g glycerin, 801g polysorbate.

[0082] Formula 5: 80g purified water, 1g hyaluronic acid, 3g glycerin, 0.5885g PVA, 10g HPMC, 801g polysorbate.

[0083] Formula 6: 100g purified water, 1g hyaluronic acid, 25g Pullulan, 5g HPMC, 3g glycerin, 801g polysorbate.

[0084] Formula 7: 100g purified water, 5g carboxymethyl deacetylated chitosan, 1g sodium hyaluronate, 30g polyvinylpyrrolidone K30.

[0085] Formula 8: 100g purified water, 2g sodium hyaluronate, 20g polyvinylpyrrolidone K30, 5g HPMC, 2.5g PVA0588.

[0086] Formula 9: 168g purified water, 2g carboxymethyl deacetylated chitosan, 1g sodium hyaluronate, 15g polyvinylpyrrolidone K30, 2g glycerin, 801g polysorbate.

[0087] Formula 10: 168g purified water, 1.5g carboxymethyl deacetylated chitosan, 1g sodium hyaluronate, 20g polyvinylpyrrolidone K30, 2g glycerin, 801g polysorbate.

[0088] Formula 11: 100g purified water, 1g carboxymethyl deacetylated chitosan, 0.3g sodium hyaluronate, 8g polyvinylpyrrolidone K30, 2g glycerin.

[0089] Formula 12: 100g purified water, 1g carboxymethyl deacetylated chitosan, 0.3g sodium hyaluronate, 12g polyvinylpyrrolidone K30, 2g glycerin.

[0090] Formula 13: 100g purified water, 1g carboxymethyl deacetylated chitosan, 1g sodium hyaluronate.

[0091] Formula 14: 100g purified water, 1g sodium hyaluronate, 1g polyvinylpyrrolidone K30.

[0092] Formula 15: 100g purified water, 1g carboxymethyl deacetylated chitosan, 1g sodium hyaluronate, 0.5g polysorbate-800.

[0093] Formula 16: 100g purified water, 1g carboxymethyl deacetylated chitosan, 1g sodium hyaluronate, 5g HPMC.

[0094] In the above formula: the degree of deacetylation of carboxymethyl deacetylated chitosan is ≥80%, and the molecular weight of sodium hyaluronate is 1200-1500 kDa.

[0095] 2. Preparation method:

[0096] 1) Take 16 250ml beakers as stirring containers, weigh the above reagent components according to formulas 1 to 16 in sequence using an electronic balance, add them to their respective beakers, stir for 30 to 60 minutes to obtain 16 completely dissolved basic solutions.

[0097] 2) Take 16 female molds made of polydimethylsiloxane (PDMS) material provided in Example 1, and lay a layer of 16 different base solutions on the bottom of each mold. Place the molds in a vacuum stirring degassing machine (TMV-700T). For the 16 base solutions, the vacuum stirring degassing machine is operated in five stages: Stage 1: Time 30-300s, Rotation speed 0r / min, Vacuum degree 30-90Kpa; Stage 2: Time 30-300s, Rotation speed 0r / min n: vacuum degree 1~30Kpa; third stage: time 30~300s, rotation speed 0r / min, vacuum degree 0.1~5Kpa; fourth stage: time 30~300s, rotation speed 0r / min, vacuum degree 1~30Kpa; fifth stage: time 30~300s, rotation speed 0r / min, vacuum degree 30~90Kpa. Repeat the above five stages three times to complete the filling of the microneedle body. After each run, scrape off the solution at the bottom of the mold with a scraper and re-lay a layer.

[0098] 3) After filling the microneedles, scrape off the solution at the bottom of the mold with a scraper. Fill the corresponding mold grooves with the 16 base solutions respectively. Place the mold in a vacuum stirring degassing machine (TMV-700T). The vacuum stirring degassing machine is run in two stages: Stage I: time 30-300s, speed 0r / min, vacuum degree 30-90Kpa; Stage II: time 30-300s, speed 0r / min, vacuum degree 10-45Kpa. Repeat the above two stages twice to complete the filling of the substrate.

[0099] 4) Carefully remove the microneedle mold from the substrate of the vacuum stirring degassing machine and place it in a constant temperature and humidity drying oven for 24 to 36 hours. The temperature is set to 25°C and the humidity is set to 50%.

[0100] 5) Carefully remove the dried microneedle sample from the mold and cut it to the appropriate size as needed.

[0101] 6) Attach a backing label to the back of the cut microneedle sample and package it for later use.

[0102] 3. Experimental results of formulas 1-16

[0103] The experimental results for formulas 1-16 are shown in Table 1 below:

[0104] Table 1. Experimental results of formulas 1-16

[0105]

[0106] Remark:

[0107] The key indicators are: needle shape, needle body hardness, and needle body toughness, among which:

[0108] (1) Needle shape: The needle body has no empty needle and the needle shape is clear and without deformation, which means the needle shape is obvious; otherwise, the needle shape is not obvious.

[0109] (2) Needle tip hardness: If the needle tip is easily deformed when the finger is pressed lightly, the hardness is too soft; if the needle tip is not easily deformed, the hardness is qualified.

[0110] (3) Needle toughness: If the needle body is bent by applying external force, and the needle body can recover without fragments after the external force is removed, it is considered tough; otherwise, it is considered brittle and lacks toughness.

[0111] (4) Viscosity of the formulation solution: The viscosity of the solution is moderate if it is within the set range, low if it is below the set range, and high if it is above the set range. Solutions with high viscosity are prone to causing production inconvenience.

[0112] (5) Vacuuming conditions: Bubbles may be generated during the preparation of the base solution for microneedles and during the vacuuming process. However, residual bubbles after vacuuming will affect the performance of the microneedle body and the substrate, such as the needle shape, needle body hardness, and substrate flatness. There are no residual bubbles if there are no bubbles in the microneedle body and the substrate; there are residual bubbles if there are bubbles in the microneedle body and / or a large number of bubbles in the substrate; there are slight residual bubbles if there are no bubbles in the microneedle body but a small number of bubbles in the substrate.

[0113] As shown in Table 1 above, formulations 1, 2, 5, 7, 8, 11, 12, and 14 all have key indicators that fail to meet standards and are therefore not qualified formulations. Formulations 3, 4, 9, 10, 15, and 16 all exhibit residual air bubbles after vacuuming. Formulation 6 has a high viscosity solution that can negatively impact production. Overall, formulation 13 is the best. The overall image and magnified views of the microneedle sample prepared with formulation 13 are shown below. Figure 2 As shown.

[0114] Example 3: Process Optimization of Microneedle Filling Molding

[0115] Microneedle filling molding uses a negative pressure filling method, which involves pouring the matrix into the mold and then immediately applying a vacuum to allow the matrix solution to exchange positions with the air in the needle cavity, thus completely filling the needle cavity in the mold.

[0116] 1. The optimal formulation 13 selected from Table 1 in Example 2 was adopted.

[0117] 2. Process optimization methods:

[0118] 1) Take a 1000ml beaker as a stirring container, use an electronic balance to weigh the reagent according to formula 13 and add it to the beaker to prepare 500ml of solution. Stir for 30-60 minutes to obtain a completely dissolved solution.

[0119] 2) Take seven female molds made of polydimethylsiloxane (PDMS) material provided in Example 1, and lay a layer of the solution from step 1) on the bottom of each mold. Place the molds in a vacuum stirring degassing machine (TMV-700T). The following vacuum filling process is designed for the microneedle body, as detailed in Table 2, with a rotation speed of 0 r / min:

[0120] Table 2. Optimization of microneedle filling process

[0121]

[0122]

[0123] The above processes 1 to 7 are repeated 3 times. The above five stages of vacuuming are used to complete the filling of the microneedle body. After each run, the solution at the bottom of the mold is scraped off with a scraper and a new layer is laid.

[0124] 3) After filling the microneedle body, scrape off the solution at the bottom of the mold with a scraper, and fill the corresponding groove of the mold with the solution of Formula 17. Place the mold in a vacuum stirring degassing machine (TMV-700T). The vacuum stirring degassing machine is run in two stages for substrate filling: Stage I: time 120s, speed 0r / min, vacuum degree 60Kpa; Stage II: time 120s, speed 0r / min, vacuum degree 30Kpa. Repeat the above two stages twice to complete the substrate filling.

[0125] 4) Carefully remove the microneedle mold after substrate filling in the vacuum stirring degassing machine and place it in a constant temperature and humidity drying oven for 24 to 36 hours. The temperature is set to 25°C and the humidity is set to 50%.

[0126] 5) Carefully remove the dried microneedle sample from the mold and cut it to the appropriate size as needed.

[0127] 6) Attach a backing label to the back of the cut microneedle sample and package it for later use.

[0128] 3. Experimental results of process optimization

[0129] The experimental results of process optimization are shown in Table 3 below:

[0130] Table 3. Experimental results of process optimization

[0131] Microneedle needle filling Result description Process 1 Air bubbles were generated during the process, the filling was incomplete, and air bubbles remained after extraction. Process 2 Air bubbles were generated during the process, the filling was incomplete, and air bubbles remained after extraction. Process 3 Air bubbles are generated during the process, and some air bubbles remain after extraction. Process 4 Air bubbles are generated during the process, and a few air bubbles remain after extraction. Process 5 Bubbles are generated during the process, and a bunch of dense bubbles remain after extraction. Process 6 Air bubbles are generated during the process, and a few air bubbles remain after extraction. Process 7 No air bubbles were generated during the process, and no air bubbles remained after extraction.

[0132] As shown in Table 3 above, the filling process of microneedles needs to be optimized, and different processes will have different effects:

[0133] Processes 1 through 3 are all substandard processes, each consisting of only three stages. In processes 1 and 2, the vacuum level decreases and then increases, with the only difference being the vacuuming time. However, due to the small decrease and increase, a large number of air bubbles generated during vacuuming remain in the mold, resulting in incomplete filling. Appropriately extending the vacuuming time, as in process 2, does not solve the problem. In process 3, the vacuum level decreases sequentially without increasing. Although the decrease is larger, causing some air bubbles to grow larger under pressure changes and eventually burst, some air bubbles still remain.

[0134] Processes 4 through 7 each have five stages. Theoretically, stages one through three involve a gradual decrease in vacuum, during which some bubbles grow larger under pressure changes and eventually burst. Stages three through five involve a gradual increase in vacuum, during which the remaining bubbles shrink further until they disappear. In practice, the vacuum level in the third stage of process 5 was set too high, resulting in smaller changes in vacuum level from stage one to stage three and from stage three to stage five. Consequently, many bubbles remained unbroken, making process 5 a substandard process. Processes 4, 6, and 7 are all satisfactory processes, with process 7 being the optimal process.

[0135] Example 4: In vitro simulated skin puncture experiment and optimization of microneedle solution formulation

[0136] 1. Based on the optimal formula 13 selected in Example 2, formulas 17 to 29 were designed as shown in Tables 4 to 7 below.

[0137] Table 4. Effect of Moisture Content

[0138]

[0139] Table 5. Influence of the proportion of microneedle matrix material

[0140]

[0141] Table 6. Effects of Single Components

[0142] Carboxymethyl deacetylated chitosan Sodium hyaluronate Purified water Microneedle matrix material ratio Formula 24 / 2.66g 100g 2.59％ Formula 25 2.66g / 100g 2.59％

[0143] In Tables 4-6: the degree of deacetylation of carboxymethyl deacetylated chitosan is ≥80%, and the molecular weight of sodium hyaluronate is 1200-1500 kDa.

[0144] Table 7. Effects of sodium hyaluronate with different molecular weights

[0145]

[0146] In Table 7: the degree of deacetylation of carboxymethyl deacetylated chitosan is ≥80%, the high molecular weight of sodium hyaluronate is 1200-1500 kDa, the medium molecular weight of sodium hyaluronate is 200-400 kDa, and the low molecular weight of sodium hyaluronate is 5-10 kDa.

[0147] 2. Microneedle sample preparation method:

[0148] 1) Take 14 250ml beakers as stirring containers, weigh the above reagent components according to formulas 13 and 17-29 in sequence using an electronic balance, add them to their respective beakers in sequence, stir for 30-60 minutes, and obtain 14 completely dissolved microneedle solutions A in sequence.

[0149] 2) Take 14 250ml beakers as stirring containers. Use an electronic balance to weigh the above reagent components according to formulas 13 and 17-29. Add 1% by volume of brilliant blue to each beaker and stir for 30-60 minutes to obtain 14 completely dissolved microneedle solutions B.

[0150] 3) Take 14 negative molds made of polydimethylsiloxane (PDMS) material provided in Example 1, and sequentially lay a layer of 14 microneedle solutions B on the bottom of the corresponding negative molds. Place the molds in a vacuum stirring degassing machine (TMV-700T). For the above 14 microneedle solutions B, the vacuum stirring degassing machine is set according to process 7 in Example 3 and run in five stages: Stage 1: Time 60s, Rotation speed 0r / min, Vacuum degree 30Kpa; Stage 2: Time 60s, Rotation speed 0r / min, Vacuum degree 3Kpa; Stage 3: Time 60s, Rotation speed 0r / min, Vacuum degree 0.3Kpa; Stage 4: Time 60s, Rotation speed 0r / min, Vacuum degree 30Kpa; Stage 5: Time 60s, Rotation speed 0r / min, Vacuum degree 60Kpa. Repeat the above five stages three times to complete the filling of the microneedles. After each run, scrape off the solution at the bottom of the mold with a scraper and lay a new layer.

[0151] 4) After filling the microneedle body, scrape off the solution B at the bottom of the mold with a scraper, and sequentially fill the corresponding 14 microneedle solutions A in step 1) in the mold groove. Place the mold in a vacuum stirring degassing machine (TMV-700T). The vacuum stirring degassing machine is set according to Example 3 and runs in two stages: Stage I: time 120s, speed 0r / min, vacuum degree 60Kpa; Stage II: time 120s, speed 0r / min, vacuum degree 30Kpa. Repeat the above two stages twice to complete the filling of the substrate.

[0152] 5) Carefully remove the microneedle mold after substrate filling in the vacuum stirring degassing machine and place it in a constant temperature and humidity drying oven for 24 to 36 hours. The temperature is set to 25℃ and the humidity is set to 50%.

[0153] 6) Carefully remove the dried microneedle sample from the mold and cut it to the appropriate size as needed.

[0154] 7) Attach a backing label to the back of the cut microneedle sample and package it for later use.

[0155] 3. In vitro simulated skin puncture and transdermal experimental methods:

[0156] Prepare 10% by weight of pigskin gelatin blocks (gelatin strength ≥250g Bloom), and conduct puncture and transdermal experiments on the microneedles prepared according to formulas 13 and 17-29.

[0157] For the procedures of puncture and transdermal experiments, please refer to the notes in Table 8 below.

[0158] 4. Experimental results of formulas 13 and 17–29

[0159] The experimental results for formulas 13 and 17–29 are shown in Table 8 below:

[0160] Table 8. Experimental results of formulas 13 and 17–29

[0161] Experimental results Needle success rate Needle hardness Needle toughness Puncture rate Transdermal rate Viscosity of the formulation solution Formula 13 qualified qualified have qualified qualified Moderate Formula 17 qualified qualified have qualified qualified Moderate Formula 18 qualified qualified have qualified qualified Moderate Formula 19 qualified qualified none qualified qualified High Formula 20 qualified Soft have qualified qualified Low Formula 21 qualified qualified have qualified qualified Moderate Formula 22 qualified qualified have qualified qualified Moderate Formula 23 qualified qualified have qualified qualified High Formula 24 qualified qualified none qualified qualified High Formula 25 qualified Soft have qualified qualified Low Formula 26 qualified qualified have qualified qualified Moderate Formula 27 qualified qualified have qualified qualified Moderate Formula 28 qualified qualified have qualified qualified Moderate Formula 29 qualified qualified have qualified qualified Moderate

[0162] Remark:

[0163] Key indicators include: needle success rate, needle tip hardness, and needle body toughness, among which:

[0164] (1) Needle success rate: The proportion of qualified microneedles to the total number of microneedles. Qualified microneedles are defined as microneedles without empty needles, obvious breaks, clear needle tip shape, and no bending or deformation of the needle body. If the needle success rate is lower than the set value of 95%, the needle success rate is considered unqualified.

[0165] (2) Needle tip hardness: If the needle tip bends and deforms when pressed lightly with a finger, the hardness is too soft; if the needle tip does not easily deform, the hardness is qualified.

[0166] Note: The microneedles selected in this round of screening are softer than the microneedles in Example 1.

[0167] (3) Needle toughness: If the needle body is bent by applying external force, and the needle body can recover without fragments after the external force is removed, it is considered tough; otherwise, it is considered brittle and lacks toughness.

[0168] (4) Puncture rate: After pressing the microneedles vertically onto a 10% by weight block of pigskin gelatin (gelatin strength ≥250g Bloom) for 2 seconds with a certain force, observe under a microscope the proportion of microneedles that form holes in the pigskin gelatin block after puncture to the total number of microneedles. A puncture rate lower than 95% is considered unqualified.

[0169] (5) Transdermal penetration rate: After pressing the microneedles vertically onto a 10% (by weight) block of pigskin gelatin (gelatin strength ≥ 250g Bloom) for 10 seconds with a certain force, observe under a microscope the proportion of microneedles with color diffusion in the holes formed on the pigskin gelatin block after puncture. A transdermal penetration rate of less than 95% is considered unqualified.

[0170] (6) Viscosity of the formulation solution: The viscosity of the solution is moderate if it is within the set range, low if it is below the set range, and high if it is above the set range. Solutions with high viscosity are prone to causing production inconvenience.

[0171] As shown in Table 8 above, the microneedle framework material, with a degree of deacetylation of carboxymethyl deacetylated chitosan ≥80% and a molecular weight of sodium hyaluronate of 1200–1500 kDa, demonstrates the following:

[0172] 1) Except for formula 19, all formulas in Table 4 are suitable. That is, when the mass ratio of carboxymethyl deacetylated chitosan to sodium hyaluronate is 1:1, the microneedles of formula 19 are brittle and lack toughness. The water content of each formula needs to be greater than 96.57% to avoid brittleness and achieve toughness. Among them, the best formula is formula 17.

[0173] 2) Except for formula 20, all other formulas in Table 5 are suitable, that is, the mass ratio of carboxymethyl deacetylated chitosan to sodium hyaluronate is suitable in the range of 1:(0.667~2). Formula 20 indicates that the needle hardness is too soft when the mass ratio of carboxymethyl deacetylated chitosan to sodium hyaluronate is 1:0.5.

[0174] 3) In Table 6, formulations 24 and 25 are not suitable formulations. Formulation 24, which uses sodium hyaluronate alone, makes the needle body brittle and lacks toughness. Formulation 25, which uses carboxymethyl deacetylated chitosan alone, makes the needle tip too soft. Although the puncture rate and transdermal rate are qualified, they are not suitable for human puncture.

[0175] As shown in Table 8 above, the microneedle framework material, with a deacetylation degree of carboxymethyl deacetylated chitosan ≥80%, a high molecular weight of sodium hyaluronate of 1200–1500 kDa, a medium molecular weight of sodium hyaluronate of 200–400 kDa, and a low molecular weight of sodium hyaluronate of 5–10 kDa, indicates that:

[0176] 4) Formulas 26 to 29 in Table 7 are all suitable formulas, that is, sodium hyaluronate can meet the requirements when composed of any two or three equal amounts of high molecular weight 1200-1500 kDa, medium molecular weight 200-400 kDa and low molecular weight 5-10 kDa.

[0177] The puncture rate and transdermal penetration rate of formulas 13 and 17-29 in the in vitro simulated skin puncture experiment were both qualified. The experimental results are not listed here; only the result of formula 17 is shown. Figure 3 As shown, the left side shows the puncture results, and the right side shows the transdermal results. Both the puncture rate and the transdermal rate reached 100%.

[0178] Example 5: Microneedle Animal Transdermal Test

[0179] 1. Pre-experimental animal hair removal treatment:

[0180] 1) The day before the formal experiment, the mice (weighing 25 grams) need to be shaved. First, the C57 mice are anesthetized with tribromoethanol (220 mg / kg).

[0181] 2) After anesthetizing the mice, use a pet razor to shave the back area (2cm×2cm) of the fully anesthetized mice to remove most of the hair;

[0182] 3) After shaving, apply hair removal cream to the shaved area with a cotton swab, spread it evenly, massage gently for a few seconds, and wait for 1 minute;

[0183] 4) Wet a cotton ball with warm water and apply the hair removal cream in the direction of hair growth to completely remove the fine hairs. If a small amount of hair remains, apply a small amount of hair removal cream to the hair, wait 30 seconds, and then rinse off the cream.

[0184] 5) After completely washing off the hair removal cream, dry the mouse thoroughly and place it in the cage to wait for it to wake up.

[0185] 2. Animal transdermal experiments were conducted using microneedles containing 1% by volume brilliant blue prepared according to formulation 17 in Example 4:

[0186] 1) Anesthetize pre-haired C57 mice with tribromoethanol (220 mg / kg);

[0187] 2) Use surgical scissors to cut the microneedles into 1cm x 1cm pieces for later use;

[0188] 3) Place the cut microneedles on the tape with the microneedle tips facing outwards (towards the mouse skin). Then attach the microneedles to the hair removal area of ​​the C57 mouse, secure them with the tape, and press the microneedles for 15 seconds to ensure they adhere completely to the mouse skin. After the microneedles adhere, wrap an elastic bandage around the area where the microneedles adhere to prevent the mouse from biting the microneedles after waking up.

[0189] 4) Place the mouse in the cage and wait for it to wake up;

[0190] 5) The microneedles were removed the next day to observe how well they were absorbed by the mice.

[0191] 3. Experimental Results:

[0192] Experimental results are as follows Figure 4 As shown, when the microneedles containing 1% by volume of the bright blue marker prepared by Formula 17 were peeled off, it could be observed that the needle tips of the microneedles had dissolved, and needle-shaped hole traces could be observed on the skin of the mouse's back.

[0193] Example 6: Microneedles containing bovine serum albumin were prepared using an optimized process.

[0194] 1. Microneedle formulation containing bovine serum albumin:

[0195] Table 9. Microneedle formulations containing bovine serum albumin

[0196]

[0197] The functional ingredients are 0.35g of bovine serum albumin and 1g of brilliant blue.

[0198] 2. Preparation method:

[0199] 1) Take a 250ml beaker 1 as a stirring container. Use an electronic balance to weigh the reagents according to the drug-loaded microneedle formula to prepare solution A, add it to the beaker, and stir for 30-60 minutes to obtain a completely dissolved solution A.

[0200] 2) Take another 250ml beaker 2 as a stirring container. Use an electronic balance to weigh the reagents according to the drug-loaded microneedle formula to prepare solution B, add it to the beaker, and stir for 30-60 minutes to obtain a completely dissolved solution B.

[0201] 3) Take a negative mold made of polydimethylsiloxane (PDMS) material provided in Example 1, spread a layer of solution B at the bottom of the mold, and place the mold in a vacuum stirring degassing machine (TMV-700T). For filling the microneedles, the vacuum stirring degassing machine is set according to process seven in Example 3, and run in five stages: Stage 1: Time 60s, Rotation speed 0r / min, Vacuum degree 30.0Kpa; Stage 2: Time 60s, Rotation speed 0r / min, Vacuum degree 3.0Kpa; Stage 3: Time 60s, Rotation speed 0r / min, Vacuum degree 0.3Kpa; Stage 4: Time 60s, Rotation speed 0r / min, Vacuum degree 30.0Kpa; Stage 5: Time 60s, Rotation speed 0r / min, Vacuum degree 60.0Kpa. Repeat the above five stages of vacuuming 3 times to complete the filling of the microneedles. After each run, scrape off the solution B at the bottom of the mold with a scraper and spread a new layer.

[0202] 4) After filling the mold with microneedles, scrape off the solution at the bottom using a scraper. Fill the mold groove with solution A from step 2). Place the mold in a vacuum stirring degassing machine (TMV-700T). For substrate filling, the vacuum stirring degassing machine is set as in Example 3, running in two stages: Stage I: time 120s, rotation speed 0r / min, vacuum degree 60.0Kpa; Stage II: time 120s, rotation speed 0r / min, vacuum degree 30.0Kpa. Repeat the above two stages of vacuuming twice to complete the substrate filling.

[0203] 5) Carefully remove the microneedle mold after substrate filling in the vacuum stirring degassing machine and place it in a constant temperature and humidity drying oven for 24 to 36 hours. The temperature is set to 25℃ and the humidity is set to 50%.

[0204] 6) Carefully remove the dried microneedle sample from the mold and cut it into 1cm×1cm sizes as needed.

[0205] 7) Attach a backing label to the back of the cut microneedle sample and package it for later use.

[0206] 3. In vitro simulated skin puncture test method:

[0207] Prepare 10% by weight of pigskin gelatin blocks (gelatin strength ≥250g Bloom), and perform puncture and transdermal experiments on the microneedles containing bovine serum albumin.

[0208] 4. Experimental Results:

[0209] Experimental results are as follows Figure 5 As shown, Figure 5 The left side shows the in vitro simulated skin puncture results of the microneedle sample containing bovine serum albumin prepared in this embodiment, and the right side shows the in vitro simulated skin transdermal results of the microneedle sample containing bovine serum albumin prepared in this embodiment. Both the puncture rate and the transdermal rate reached 100%.

[0210] Example 7: Preparation of drug-loaded microneedles using an optimized process

[0211] 1. Drug-loaded microneedle formulation:

[0212] Table 10. Drug-loaded microneedle formulation

[0213]

[0214] 2. Preparation method:

[0215] 1) Take a 250ml beaker 1 as a stirring container. Use an electronic balance to weigh the reagents according to the drug-loaded microneedle formula to prepare solution A, add it to the beaker, and stir for 30-60 minutes to obtain a completely dissolved solution A.

[0216] 2) Take another 250ml beaker 2 as a stirring container. Use an electronic balance to weigh the reagents according to the drug-loaded microneedle formula to prepare solution B, add it to the beaker, and stir for 30-60 minutes to obtain a completely dissolved solution B.

[0217] 3) Take a negative mold made of polydimethylsiloxane (PDMS) material provided in Example 1, spread a layer of solution B at the bottom of the mold, and place the mold in a vacuum stirring degassing machine (TMV-700T). For filling the microneedles, the vacuum stirring degassing machine is set according to process seven in Example 3, and run in five stages: Stage 1: Time 60s, Rotation speed 0r / min, Vacuum degree 30.0Kpa; Stage 2: Time 60s, Rotation speed 0r / min, Vacuum degree 3.0Kpa; Stage 3: Time 60s, Rotation speed 0r / min, Vacuum degree 0.3Kpa; Stage 4: Time 60s, Rotation speed 0r / min, Vacuum degree 30.0Kpa; Stage 5: Time 60s, Rotation speed 0r / min, Vacuum degree 60.0Kpa. Repeat the above five stages of vacuuming 3 times to complete the filling of the microneedles. After each run, scrape off the solution B at the bottom of the mold with a scraper and spread a new layer.

[0218] 4) After filling the mold with microneedles, scrape off the solution at the bottom using a scraper. Fill the mold groove with solution A from step 2). Place the mold in a vacuum stirring degassing machine (TMV-700T). For substrate filling, the vacuum stirring degassing machine is set as in Example 3, running in two stages: Stage I: time 120s, rotation speed 0r / min, vacuum degree 60.0Kpa; Stage II: time 120s, rotation speed 0r / min, vacuum degree 30.0Kpa. Repeat the above two stages of vacuuming twice to complete the substrate filling.

[0219] 5) Carefully remove the microneedle mold after substrate filling in the vacuum stirring degassing machine and place it in a constant temperature and humidity drying oven for 24 to 36 hours. The temperature is set to 25℃ and the humidity is set to 50%.

[0220] 6) Carefully remove the dried microneedle sample from the mold and cut it into 1cm×1cm sizes as needed.

[0221] 7) Attach a backing label to the back of the cut microneedle sample and package it for later use.

[0222] Example 8: Microneedles containing soluble type III collagen were prepared using an optimized process.

[0223] 1. Microneedle formulation:

[0224] Table 11. Microneedle formulations containing soluble type III collagen

[0225]

[0226]

[0227] The functional ingredient, soluble type III collagen, is derived from a soluble fragment with functional structural domains extracted from human tissue, with a total length of 394 amino acids.

[0228] 2. Preparation method: The preparation method is the same as in Example 7, and a series of microneedles containing different concentrations of soluble type III collagen were prepared.

[0229] Figure 6 The microneedle sample containing 2g of soluble type III collagen was shown.

[0230] Example 9: Microneedles containing soluble elastin prepared using an optimized process.

[0231] 1. Microneedle formulation:

[0232] Table 12. Microneedle formulations containing soluble elastin

[0233]

[0234] The functional component, soluble elastin, is derived from a soluble fragment consisting of three connected segments with functional domains, taken from human sources. It has a total length of 118 amino acids.

[0235] 2. Preparation method: The preparation method is the same as in Example 7, and a series of microneedles containing different concentrations of soluble elastin are prepared.

[0236] Figure 7 The sample containing 4.8g of soluble elastin microneedles was shown.

[0237] Example 10: Microneedles containing soluble type XVII collagen were prepared using an optimized process.

[0238] 1. Microneedle formulation:

[0239] Table 13. Microneedle formulations containing soluble type XVII collagen

[0240]

[0241] The functional ingredient, soluble type XVII collagen, is derived from a soluble fragment with functional domains extracted from human sources, with a total length of 144 amino acids.

[0242] 2. Preparation method: The preparation method is the same as in Example 7, and a series of microneedles containing different concentrations of soluble type XVII collagen are prepared.

[0243] Figure 8 The microneedle sample containing 3.2g of soluble type XVII collagen was shown.

[0244] Example 11: Microneedles containing superoxide dismutase protein prepared using an optimized process.

[0245] 1. Microneedle formulation:

[0246] Table 14. Microneedle formulations containing superoxide dismutase protein

[0247]

[0248] Superoxide dismutase is derived from human sources.

[0249] 2. Preparation method: The preparation method is the same as in Example 7, and a series of microneedles containing different concentrations of superoxide dismutase (SOD) were prepared.

[0250] Figure 9 The sample containing 4.4g of superoxide dismutase protein microneedles was shown.

[0251] Example 12: Microneedles containing recombinant botulinum toxin type A were prepared using an optimized process.

[0252] 1. Microneedle formulation:

[0253] Table 15. Microneedle formulations containing recombinant botulinum toxin type A

[0254]

[0255] 2. Preparation method: The preparation method is the same as in Example 7, and a series of microneedles containing different concentrations of recombinant botulinum toxin A are prepared.

[0256] Figure 10 The microneedle sample containing 0.228g of recombinant botulinum toxin type A was shown.

[0257] Example 13: Microneedles containing VTNF1 antibody protein prepared using an optimized process.

[0258] 1. Microneedle formulation:

[0259] Table 16. Microneedle formulations containing VTNF1 antibody protein

[0260]

[0261]

[0262] The functional component, VTNF1 antibody protein, is derived from a shark single-domain antibody.

[0263] 2. Preparation method: The preparation method is the same as in Example 7, and a series of microneedles containing different concentrations of VTNF1 antibody protein were prepared.

[0264] Figure 11 The microneedle sample containing 0.318g of VTNF1 antibody protein was shown.

[0265] Example 14: Microneedles containing Her2VHH2A2 antibody protein were prepared using an optimized process.

[0266] 1. Microneedle formulation:

[0267] Table 17. Microneedle formulations containing Her2VHH2A2 antibody protein

[0268]

[0269] The functional component is derived from a camel antibody targeting Her2 (heavy chain variable region VHH).

[0270] 2. Preparation method: The preparation method is the same as in Example 7, and a series of microneedles containing different concentrations of Her2VHH2A2 antibody protein were prepared.

[0271] Figure 12 The microneedle sample containing 0.066g Her2VHH2A2 antibody protein was shown.

[0272] Example 15: Microneedles containing Q1 protein prepared using an optimized process.

[0273] 1. Microneedle formulation:

[0274] Table 18. Microneedle formulations containing Q1 protein

[0275]

[0276] The functional component Q1 protein is derived from the viral outer shell protein.

[0277] 2. Preparation method: The preparation method is the same as in Example 7, and a series of microneedles containing different concentrations of Q1 protein were prepared.

[0278] Figure 13 The microneedle sample containing 0.24g of Q1 protein was shown.

[0279] Example 16: Microneedles containing GM2 protein prepared using an optimized process

[0280] 1. Microneedle formulation:

[0281] Table 19. Microneedle formulations containing GM2 protein

[0282]

[0283] The functional component GM2 protein is derived from the viral outer shell protein.

[0284] 2. Preparation method: The preparation method is the same as in Example 7, and a series of microneedles containing different concentrations of GM2 protein were prepared.

[0285] Figure 14 The microneedle sample containing 0.251g of GM2 protein was shown.

[0286] Example 17: Microneedles containing PGF242 protein prepared using an optimized process

[0287] 1. Microneedle formulation:

[0288] Table 20. Microneedle formulations containing PGF242 protein

[0289]

[0290] The functional protein PGF242 is derived from human FGF21.

[0291] 2. Preparation method: The preparation method is the same as in Example 7.

[0292] Figure 15 The microneedle sample containing 0.00012g of PGF242 protein was shown.

[0293] The protein-soluble microneedle samples prepared in Examples 8-17 above are as follows: Figure 6-15 As shown, the tips of the microneedles in each sample are complete and without defects, indicating that the optimized preparation process and formula can successfully produce microneedles, and various functional ingredients can be added as needed.

[0294] Example 18: Animal Experiments on Sugar Tolerance

[0295] 1. Preparation of injection solutions and microneedles for glucose tolerance animal experiments:

[0296] 1) Preparation of semaglutide injection: 1 mg of powdered semaglutide was diluted with PBS buffer to a stock solution of 0.1 mg / mL, aliquoted, and stored at -80°C. Before the experiment, the stock solution was further diluted with PBS buffer (to a final concentration of 30.85 μg / mL). The injection dose was 30 nmol / kg (for a 25g mouse, the injection volume was 100 μL, and the total injection volume of semaglutide was 3.09 μg).

[0297] 2) Preparation of microneedles containing semaglutide: Microneedle samples of 1cm × 1cm size containing an equal amount of semaglutide were prepared using the method of process 7 in Example 3.

[0298] A 1cm x 1cm microneedle has 20 microneedles on one side (theoretically 17, but the number increases to 20 after drying due to the shrinkage in size), for a total of 20 x 20 = 400 microneedles. The body of one microneedle is a regular square pyramid with a volume V = 1 / 3Sh = 1 / 3 x 300μm x 300μm x 600μm = 0.018mm. 3=0.018μl. The total volume of the microneedles on a 1cm×1cm microneedle = 0.018×400 = 7.2μl. The amount of semaglutide added in 100ml of microneedle solution is Xg, then Xg / 100ml = 3.09μg / 7.2μl, X = 0.043g.

[0299] The specific formula is shown in Table 21 below:

[0300] Table 21. Microneedle formulations containing semaglutide

[0301]

[0302] 2. Preparations before the experiment:

[0303] Two batches of mice were prepared: 3 microneedle mice (mice with microneedles containing semaglutide applied) per batch, 3 injection mice (mice injected with the same dose of semaglutide as the microneedles) and 3 control mice per batch.

[0304] The first batch of mice were shaved and fitted with microneedles containing semaglutide (see Example 5 for details) or injected with semaglutide (subcutaneous injection at the back and neck) at 4 PM the day before the experiment (D-1). The mice were then placed in clean cages and fasted for 18 hours until 10 AM on the day of the experiment (D0). During the fasting period, the mice were allowed to drink water normally.

[0305] Two days before the experiment (day D-2), at 4 PM, the mice were shaved and fitted with microneedles containing semaglutide (see Example 5 for details) or injected with semaglutide (subcutaneous injection at the back and neck). After 24 hours of normal feeding, the mice were transferred to clean cages and fasted for 18 hours until 10 AM on the day of the experiment (day D0). During the fasting period, the mice were provided with normal access to water.

[0306] 3. Glucose tolerance test

[0307] 1) Fasting basal blood glucose measurement: One hour before the end of fasting for each batch of experiments, fasting basal blood glucose was measured: The mice were removed from their cages and gently placed on a cage rack. Approximately 1 mm of the tail was cut off from the end of the tail with scissors. The tail was gently squeezed along the tail vein to concentrate the blood into a single drop. The first drop of blood was discarded, and the second drop was used. Fasting blood glucose was measured using a blood glucose meter and blood glucose test strips, and this value was recorded as the blood glucose level at -60 minutes.

[0308] 2) Intraperitoneal injection of glucose: After stabilizing the mice briefly, prepare for intraperitoneal injection of glucose. The glucose concentration for the glucose tolerance test is 2 g / kg. We used pre-prepared glucose injection solution (Heilongjiang Qitai Animal Health Products Co., Ltd.), or a 100 mg / ml glucose solution can be prepared using injection-grade physiological saline. C57 mice weigh 25 grams, so the intraperitoneal injection volume for each mouse is 0.5 ml.

[0309] Gently pick up the mouse and inject it with 0.5 ml of glucose solution using a 1 mL syringe according to standard intraperitoneal injection procedure. Immediately after the injection, measure the mouse's blood glucose level and record it as the blood glucose level at 0 min.

[0310] For the microneedle group, glucose was injected after the microneedles were removed.

[0311] 3) Blood glucose sampling at different time points: Blood glucose values ​​of each mouse were sampled and measured at 15 min, 30 min, 60 min, 90 min and 120 min after injection of glucose solution, following the procedure in step 1).

[0312] 4) After the experiment, feed each cage of mice.

[0313] 4. Results of animal experiments on glucose tolerance

[0314] The blood glucose levels collected and measured at each time point are detailed in Table 22 below. The blood glucose level changes for the first and second batches are shown in the graphs below. Figure 16 , Figure 17 .

[0315] Table 22. Results of animal experiments on glucose tolerance

[0316]

[0317] like Figure 16 As shown, in the first batch of glucose tolerance experiments, the blood glucose lowering effect of the injection group was better than that of the microneedle group at 15 minutes. At 30 minutes, the blood glucose lowering effect of the injection group was basically the same as that of the microneedle group. At 60 minutes, the blood glucose lowering rate of the microneedle group was better than that of the injection group, indicating that the microneedle group can continuously release drugs to achieve the blood glucose lowering effect.

[0318] like Figure 17As shown, in the second batch of glucose tolerance experiments, at 15 minutes, the hypoglycemic effect of the microneedle group was basically the same as that of the injection group and slightly better than that of the injection group. At 30 minutes, 60 minutes, 90 minutes and 120 minutes, the hypoglycemic effect of the microneedle group was better than that of the injection group. This indicates that after a longer period of time in mice (24 hours longer than the first batch), the hypoglycemic effect of the microneedle group was better than that of the injection group. This shows that the microneedle group can maintain the sustained release of the drug, which is conducive to maintaining the blood drug concentration in blood glucose and thus exerting the hypoglycemic effect.

[0319] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A solution for microneedles, characterized in that: A solution is used to prepare the microneedle body and substrate of soluble microneedles. The microneedle solution includes a base solution, in which the microneedle framework material includes carboxymethyl deacetylated chitosan and sodium hyaluronate. The content percentage of the microneedle framework material is 1.8% to 3.6%, preferably 1.9% to 3.4%, and more preferably 1.96% to 3.22%.

2. The solution for microneedles according to claim 1, characterized in that: The mass ratio of the carboxymethyl deacetylated chitosan to the sodium hyaluronate is 1:(0.55-3). Preferably, the mass ratio of the carboxymethyl deacetylated chitosan to the sodium hyaluronate is 1:(0.55-2), 1:(0.6-2), 1:(0.667-2), or 1:

1.

3. The solution for microneedles according to claim 1, characterized in that: The degree of deacetylation of the carboxymethyl deacetylated chitosan is ≥80%.

4. The solution for microneedles according to claim 1, characterized in that: The molecular weight range of the sodium hyaluronate is 5 to 1500 kDa. Preferably, the sodium hyaluronate is composed of any one or more of the following: high molecular weight (1200 to 1500 kDa), medium molecular weight (200 to 400 kDa), and low molecular weight (5 to 10 kDa). More preferably, when it is composed of multiple components, they are composed in equal amounts.

5. The solution for microneedles according to claim 1, characterized in that: The microneedle solution also includes functional components, which are added to the base solution to prepare the microneedle body. The functional components are one or more combinations of water-soluble small molecules and macromolecules. The small molecule compounds are selected from pigments, markers, disease-specific compounds, diagnostic compounds, or cosmetic compounds; the macromolecule compounds are selected from proteins and peptides, polysaccharides, or nucleic acids.

6. The solution for microneedles according to claim 5, characterized in that: in, The pigments are selected from Sudan Red II, Calcein, Rhodamine B sulfonyl, 3H-indocyanine dyes, green fluorescent dyes, indocyanine green, or brilliant blue. The marker is selected from fluorescein isothiocyanate (FITC) or lipophilic membrane dye (DiI). The compounds used for the disease are selected from ibuprofen sodium, meloxicam, sinomenine hydrochloride, capsaicin, tetracycline, metronidazole, lidocaine, sumatriptan succinate, 5-aminolevulinic acid, porphyrin compounds, itraconazole, ferric pyrophosphate, donepezil hydrochloride, alendronate, or caffeine. The diagnostic compounds are selected from allergens or cyanogreen; The cosmetic compounds are selected from vitamin C glycosides, niacinamide, all-trans retinoic acid, vitamin C, 4-butylresorcinol, epidermal growth factor, collagen, adenosine, dipeptide-1, dipeptide-2, dipeptide-4, dipeptide-15, acetyl dipeptide-1 cetyl ester, palmitoyl dipeptide-7, tripeptide-1, tripeptide-1 copper, tripeptide-2, tripeptide-3, tripeptide-10 citrulline, tripeptide-32, acetyl tripeptide-1, palmitoyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl tripeptide-8, poly(tripeptide-6), trifluoroacetyl tripeptide-2, tetrapeptide-1, tetrapeptide-3, tetrapeptide-4, acetyl tetrapeptide-2, acetyl tetrapeptide-3, acetyl tetrapeptide-5, acetyl tetrapeptide-9, acetyl tetrapeptide-11, palmitoyl tetrapeptide-5, palmitoyl tetrapeptide-7, and palmitoyl tetrapeptide-10. SH-Pentapeptide-1, Pentapeptide-3, Palmitoyl Pentapeptide-4, Palmitoyl Pentapeptide-5, Myristoyl Pentapeptide-4, Hexapeptide-1, Hexapeptide-2, Hexapeptide-3, Hexapeptide-5, Hexapeptide-9, Hexapeptide-11, Acetyl Hexapeptide-1, Acetyl Hexapeptide-7, Acetyl Hexapeptide-8, Palmitoyl Hexapeptide-12, Palmitoyl Hexapeptide-14, Palmitoyl Hexapeptide-15, Myristoyl Hexapeptide-5, Heptapeptide-6, Acetyl Heptapeptide-4, Acetyl Octapeptide-3, Nonapeptide-1, Decapeptide-4, Tridecapeptide-1, Glutathione, Oligopeptide-1, Oligopeptide-2, Oligopeptide-3, Oligopeptide-4, Oligopeptide-5, Oligopeptide-6, Oligopeptide-29, Oligopeptide-32, Carnosine, Yeast Polypeptides, Arginine / Lysine Polypeptides, Retinol / Yeast Polypeptides, Ascorbic Acid Polypeptides, Glutathione or Lactobacillus Peptides.

7. The solution for microneedles according to claim 5, characterized in that: in, The proteins and polypeptides mentioned are selected from soluble elastin, soluble collagen, fibronectin, silk fibroin, fibroin, whey protein, lactoglobulin, lactoferrin, ovalbumin, bovine serum albumin, soluble type III collagen, soluble type XVII collagen, viral capsid protein, FGF21 protein, nanobody, papain, lactoperoxidase, bromelain, lipase, superoxide dismutase, hesperidinase, amylase, Bacillus subtilis enzyme, protease, oxidoreductase, and other enzymes. Catalase, alcohol oxidase, glucose oxidase, glucose amylase, α-amylase, lysozyme, sutelanase, asparaginase, bovine pancreatic ribonuclease A, horseradish peroxidase, glutamate oxidase, insulin, desmopressin, glucagon, glucagon-like peptide-1 receptor agonist, parathyroid hormone, growth hormone, etanercept, botulinum toxin, calcitonin, exendin-4 (increased insulin analog), leuprolide acetate, erythropoietin, interferon, fibroblast growth factor; More preferably, the glucagon-like peptide-1 receptor agonist is selected from exenatide, lixisenatide, lixisenatide, liraglutide, abiglutide, dulaglutide, lixisenatide, loxenatide, smegglutide, beraglutide, or semaglutide. The polysaccharide is selected from heparin, dextran, or hyaluronic acid; The nucleic acid is selected from RNA or DNA.

8. The solution for microneedles according to claim 5, characterized in that: The mass ratio of the functional component to the base solution is 1:(20-1050000), more preferably, the mass ratio of the functional component to the base solution is 1:(30:60000) or 1:(50-50000), and even more preferably 1:(70-35000) or 1:(75-30000).

9. A method for preparing microneedles, characterized in that: Solution A is prepared using any one of the microneedle solutions of claims 1 to 8 as the preparation base, and solution B is prepared by adding functional components to the same solution A.

10. The microneedle preparation method according to claim 9, characterized in that: include: Step 1) Filling the microneedle body, as detailed below: A layer of solution B is spread at the bottom of the mold. The mold is then placed in a vacuum stirring degassing machine. The vacuum stirring degassing machine is operated in five stages for filling the microneedles: the vacuum level of the first stage is greater than that of the second stage, the vacuum level of the second stage is greater than that of the third stage, the vacuum level of the third stage is less than that of the fourth stage, and the vacuum level of the fourth stage is less than that of the fifth stage. The vacuuming time for each of the above five stages is 30-300 seconds, and the stirring speed is set to 0 r / min. The above five stages of vacuuming are repeated 3 times to complete the filling of the microneedles. After each run, the solution B at the bottom of the mold is scraped off with a scraper and a new layer is spread. Furthermore, the vacuum level of the first segment is not greater than that of the fifth segment, and the vacuum level of the second segment is not greater than that of the fourth segment; Furthermore, the vacuum degree of the first stage is 30-90 kPa or 30-60 kPa; the vacuum degree of the second stage is 1-30 kPa or 1-20 kPa or 1-10 kPa; the vacuum degree of the third stage is 0.1-5 kPa or 0.1-3 kPa; the vacuum degree of the fourth stage is 1-30 kPa or 3-30 kPa; and the vacuum degree of the fifth stage is 30-90 kPa or 30-60 kPa.

11. The microneedle preparation method according to claim 10, characterized in that: The vacuuming time for the first stage is 30-120s, the vacuuming time for the second stage is 30-120s, the vacuuming time for the third stage is 30-120s, the vacuuming time for the fourth stage is 30-120s, and the vacuuming time for the fifth stage is 30-120s. Preferably, the vacuuming time for the five stages is the same.

12. The microneedle preparation method according to claim 10 or 11, characterized in that: include: Step 2) Base filling, as detailed below: After filling the microneedle body, scrape off the solution at the bottom of the mold with a scraper, fill the mold groove with solution A, and place the mold in a vacuum stirring degassing machine. The vacuum stirring degassing machine is operated in two stages for substrate filling: the vacuum degree of the first stage is greater than that of the second stage. The time for each of the two stages of vacuuming is 30 to 300 seconds, and the stirring speed is set to 0 r / min. The two stages of vacuuming are repeated twice to complete the substrate filling. Furthermore, the vacuum degree of the first stage is 30-90 kPa and the vacuuming time is 60-180 s; the vacuum degree of the second stage is 10-45 kPa and the vacuuming time is 60-180 s.

13. The microneedle preparation method according to claim 10, characterized in that: include: Step 3) Drying, as follows: Carefully remove the microneedle mold filled with substrate from the vacuum stirring degassing machine and place it in a constant temperature and humidity drying oven for 24–36 hours, with the temperature set at 25°C and the humidity at 50%.

14. The microneedle preparation method according to claim 10, characterized in that: include: Step 4) Finished product, as follows: (1) Carefully remove the dried microneedle sample from the mold and cut it into the appropriate size as needed; (2) Attach a backing label to the back of the cut microneedle sample and package it for later use.

15. A microneedle product, characterized in that: The microneedle solution is prepared using any one of claims 1 to 8, or the preparation method is prepared using any one of claims 9 to 14.

16. The microneedle product according to claim 15, characterized in that: The microneedle body is a single microneedle shape or a microneedle shape combining a needle tip and a base, wherein... The single microneedle shape includes any of the following: regular triangular pyramid, regular pentagonal pyramid, regular hexagonal pyramid, regular heptagonal pyramid, regular octagonal pyramid, regular octagonal pyramid, regular nonagonal pyramid, regular decimal pyramid, and conical shape; The shape of the microneedle combination of the needle tip and the bottom includes any of the following: regular triangular pyramid + regular triangular prism, regular square pyramid + regular square prism, regular pentagonal pyramid + regular pentagonal prism, regular hexagonal pyramid + regular hexagonal prism, regular heptagonal pyramid + regular heptagonal prism, regular octagonal pyramid + regular octagonal prism, regular nonagonal pyramid + regular nonagonal prism, regular decimal pyramid + regular decimal prism, cone + cylinder.

17. The microneedle product according to claim 15 or 16, characterized in that: The microneedle has a height range of 25–1500 μm; a base side length of 50–500 μm; a tip diameter of 0–30 μm; and a microneedle spacing of 50–1000 μm.

18. The microneedle product according to any one of claims 15 to 17, characterized in that: It also includes the backing of optional medical tape.

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