Epotacaline microneedle and preparation method thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a gel microneedle and a preparation method thereof. The invention provides an ertakaline gel microneedle. The microneedle comprises three structural parts, namely a needle shell, a needle core and a substrate, the needle shell contains a supporting material, a gel material and a medicine; the needle core contains a sustained-release material and a medicine; the base structure is composed of an adhesive material. According to the invention, the microneedle with good biocompatibility, high hardness and high drug loading capacity is obtained by improving the microneedle material and adjusting the proportion of each component. The microneedle material is of a chiral structure, good in biocompatibility, free of toxic reaction and capable of being kept in vivo for a long time. Meanwhile, compared with oral administration or intravenous drip, the microneedle can reduce indexes such as uric acid and creatinine for a long time, the medicine taking burden of a patient is relieved, and the microneedle can be used as an oral alternative medicine for reducing the uric acid.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to an ipakaline gel microneedle and a preparation method thereof. Background Art

[0002] Patients with kidney disease have reduced glomerular filtration rate and reduced uric acid excretion, leading to elevated uric acid. Therefore, uric acid has always been considered to be only a sign of reduced kidney function. Recent studies have shown that elevated uric acid levels promote the occurrence and development of kidney disease. Among untreated gout patients, 25% of patients die of renal failure. In addition, 25% of patients have proteinuria, 50-65% of patients have reduced inulin clearance, 70-80% of patients have reduced renal blood flow, and 95% of patients have histological changes of chronic renal failure. It can be seen that hyperuricemia can cause, aggravate or predict kidney disease; conversely, kidney damage weakens the kidney's ability to excrete uric acid, forming a vicious cycle between the two.

[0003] High uric acid can be deposited in joints and kidneys, irritating blood vessel walls, causing low glucose utilization, leading to arthritis, uremia and other diseases. Urate crystals in patients with hyperuricemia are deposited in the synovium, bursa, cartilage and other tissues of the joints, causing recurrent inflammatory diseases called gout, manifested as tophi and joint deformities. 36% of asymptomatic hyperuricemia patients can be seen with tophi in their joints under B-ultrasound. In addition, people with high uric acid have a significantly higher risk of hypertension, stroke, type 2 diabetes and renal failure than ordinary people.

[0004] Kidney disease further develops into end-stage renal disease, which imposes a heavy economic burden on patients and society. Today, the United States spends more than $10 billion each year to treat 333,000 patients with end-stage renal disease. Existing treatments are far from enough to protect the kidneys: renin-angiotensin system inhibitors cannot inhibit the deterioration of kidney disease in patients with proteinuria; diuretics, such as low-dose aspirin, increase blood uric acid levels; angiotensin II receptor antagonists, such as losartan, can promote uric acid excretion by inhibiting urate transporter-1, and reduce blood uric acid levels in normal people and patients with hypertension. However, different angiotensin II receptor antagonists have different effects on uric acid, and some drugs have no effect on blood uric acid levels. Therefore, it is necessary to find new ways to inhibit the development of kidney disease.

[0005] Western medicines used clinically to lower uric acid include allopurinol, febuxostat, and benzbromarone. However, these drugs, which only work by reducing the mechanism of uric acid synthesis, have limited effects and contraindications. They cannot be used by patients with coronary heart disease and kidney stones. Colchicine is a drug for the treatment of acute hyperuricemia, but it is highly toxic and can only be used in the acute phase. Developing drugs that improve renal function and can lower uric acid is always meaningful work.

[0006] ATP-sensitive potassium channels (K ATP ) is a hetero-octamer composed of an inward rectifier potassium channel subunit (Kir6) and a sulfonylurea receptor subunit (SUR). The Kir6 subunit constitutes the potassium ion permeability pore of the channel, while the SUR subunit has an ATP binding site and regulates the activity of the channel by sensing changes in the intracellular ATP / ADP concentration ratio. ATP It exists in myocardium, pancreatic B cells, pituitary tissue, skeletal muscle, brain tissue, vascular smooth muscle, uterus, ovary and kidney tissue. The auxiliary subunit represented by sulfonylurea receptor (SUR) and the functional subunit represented by inward rectifier potassium channel kir6.x together constitute K ATP Ch. K ATP It is a functional unit composed of SURx and kir6.x in a 1:1 ratio, i.e. (SURx / kir6.x×40). SUR senses changes in intracellular ATP / ADP concentrations. When the intracellular ATP concentration increases, K ATP When ATP concentration decreases, K ATP Open. SUR has SU, ATP, and Mg 2++ , ADP, potassium channel openers and other insulin secretagogues (such as gliadins) binding sites. SUR is divided into two subtypes, SUR1 and SUR2. According to the difference in the last 48 amino acids at the carboxyl end, SUR2 is further divided into SUR2A and SUR2B. ATP There are at least three types: SUR1 / kir6.2, SUR2A / kir6.2 and SUR2B / kir6.2. SUR1 / kir6.2 has a high affinity for SU and is mainly distributed in pancreatic B cells and the brain, and also exists in cardiac muscle and skeletal muscle. SUR2 / kir6.2 has a lower affinity for SU, of which SUR2A / kir6.2 is mainly distributed in cardiac muscle, brain and skeletal muscle, and SUR2B / kir6.2 is mainly distributed in vascular smooth muscle. ATP The physiological effects of diazoxide include regulating the secretion of insulin, the repolarization process of myocardial action potential, maintaining vascular tone, and regulating the release of certain neurotransmitters in the brain. Diazoxide (Dia) is a classic ATP-sensitive potassium channel opener that can non-selectively activate SUR1, SUR2A, and SUR2B. The order of subtype activation is SUR2A / Kir6.2>SUR2B / Kir6.1>SUR1 / Kir6.2, with a stronger activation effect on SUR2A. Diazoxide is suitable for malignant hypertension and hypertensive crisis. However, it selectively targets the SUR2B / kir6.2 subunit. ATPSensitive potassium channel openers are safe, effective, and potential uric acid-lowering drugs that can protect the cardiovascular system and lower blood sugar.

[0007] Studies have shown that hyperuricemia can cause renal-cardiovascular damage, leading to renal damage caused by hypertension and endothelial dysfunction. The pathogenesis of hyperuricemia, endothelial dysfunction, hypertension and renal damage is progressive and forms a vicious cycle. We have reason to believe that an antihypertensive drug with endothelial protection may block this vicious cycle. Iptakalim (Ipt) and its derivative natakalim are new structural types of ATP-sensitive potassium channel openers that can improve endothelial dysfunction, prevent hypertension in hyperuricemic rats, and delay the occurrence of endothelial dysfunction and renal damage. (Zhao Ying, Wang Hai. Intervention effect and mechanism of activating SUR2B / Kir6.1 subtype KATP channels on renal cell damage [J]. Chinese Journal of Applied Physiology, 2022, 38(6): 604-616) However, animal experiments have found that the dosage of iptakalim is greater than 3 mg / kg to exert the uric acid-lowering effect, and high doses will cause some systemic secondary side effects. Drugs that lower uric acid and improve renal blood flow are best administered topically, which can reduce systemic secondary effects. For example, overdose of diazoxide carries the risk of hypoglycemia and congestive heart failure. Although selective targeting of the SUR2B / kir6.2 subunit K ATP Sensitive potassium channel openers are more targeted than non-selective ATP-sensitive potassium channel openers, but local administration is still the best approach.

[0008] Microneedles (MNs) refer to micro-scale needles used for transdermal drug delivery, tissue fluid sampling, signal detection, biosensing and other purposes, with the advantages of painlessness, minimal invasion and easy operation. The typical length of MNs is 25-2000 μm, and the tip size is 10-20 μm. The tip is much sharper than the hypodermic needle so that it can easily pierce the stratum corneum and form a micro delivery channel without touching the nerve fibers and blood vessels of the epidermis and skin layer. In 1976, the preparation technology of micro-nanoparticles was first proposed to overcome the limitations of traditional transdermal drug delivery. Since the 1990s, with the development of modern micro-fabrication technology, various nanomaterials have been successfully prepared and have been widely used in the field of biomedicine.

[0009] At present, there are many types of microneedles, including solid microneedles, hollow microneedles, coated microneedles, gel microneedles, dissolving microneedles, etc. Solid microneedles are hard but brittle, and are prone to breakage during transportation and use. Hollow microneedles are miniature hypodermic needles that need to be connected to a device that provides an external driving force on the base to inject liquid or drugs through the internal cavity. Coated microneedles are easy to fall off during the insertion of the skin, and the dissolution of the coating in the skin is difficult to control. Gel microneedles have a high drug loading capacity and a controllable drug release effect to prevent the needle tip from breaking. The disadvantage is that the hardness of the gel microneedle is not high. When the dissolving microneedle is inserted into the skin, the needle tip will be dissolved by the tissue fluid, and the encapsulated drug will be naturally released, thereby achieving the purpose of drug administration. However, this type of microneedle has a low drug load, poor skin penetration, and poor drug activity release effect.

[0010] The ideal microneedle dot matrix transdermal patch has the properties of being biocompatible with skin and proteins, maintaining a continuous diffusion channel, allowing for larger doses of drug administration, and being able to control release as needed, and is easy to prepare. Microneedles used for medical and aesthetic purposes only need to penetrate the epidermis of the skin, but microneedles used as an alternative to injections need to penetrate the dermis in order for the drug to penetrate into the tissue, which requires the microneedles to have both a certain length and a certain hardness. Safe biomaterials are not hard enough, so inorganic materials must be added. Therefore, it is necessary to select safe inorganic materials with good biocompatibility.

[0011] In recent years, silk fibroin has been considered as a suitable biomedical material with good biocompatibility and biodegradability, and is suitable for making biomedical materials. Silk fibroin is water-soluble, and enzymes or drugs can be encapsulated by it under mild conditions. The bioactive components can be kept for a certain period of time in the dry silk fibroin and can be released. However, the hardness of silk fibroin is poor. The current silk fibroin microneedle length is less than 3mm, which can only penetrate the epidermis. The preparation of injection-substitute microneedles needs to penetrate the dermis, and the length should be about 5mm. Therefore, it is necessary to find a formula to increase the hardness of silk fibroin gel. Summary of the invention

[0012] The present applicant has discovered that the use of materials containing chiral structures in the preparation of microneedles can not only improve the hardness of the microneedles, but also does not affect the release of active drugs in the microneedles. Based on this, the present invention has been completed.

[0013] In the first aspect, the present invention provides an ipakaline gel microneedle, wherein the microneedle comprises three structural parts: a needle shell, a needle core and a substrate; the needle shell comprises a supporting material, a gel material and a drug, wherein the mass ratio of the supporting material to the gel material is 1-2:9-8, and the needle shell has a thickness of 10 μm; the needle core comprises a sustained-release material and a drug; the substrate structure is composed of an adhesive material; wherein the drug concentration in the needle shell and the needle core is 0.5-1.0 mg / ml.

[0014] Furthermore, the supporting material is a chiral calcium phosphide material.

[0015] Furthermore, the chiral calcium-phosphorus material is synthesized by reacting a chiral inducer with a calcium-phosphorus inorganic salt, and the chiral inducer is a chiral amino acid.

[0016] Furthermore, the chiral inducing agent has a concentration of 1 mol / L, and a volume ratio of the chiral inducing agent to the calcium phosphate inorganic salt solution is 1:1.

[0017] Furthermore, the chiral amino acid is selected from one or more of arginine, phenylalanine, leucine, tryptophan, methionine, proline, cysteine, glutamine, tyrosine, aspartic acid, glutamic acid and / or threonine.

[0018] Furthermore, the calcium-phosphorus inorganic salt is selected from one or more of a calcium-phosphorus mixture, β-tricalcium phosphate, calcium silicate, calcium borate, calcium carbonate, calcium sulfate, diammonium phosphate and / or calcium chloride.

[0019] Furthermore, the molar ratio of calcium to phosphorus in the calcium-phosphorus material is 1:1.67.

[0020] Furthermore, the gel material includes one or more of silk fibroin, collagen, gelatin, hyaluronic acid, chondroitin sulfate and / or sodium alginate.

[0021] Furthermore, the drugs in the needle shell and the needle core are iptakalim and its derivative natakalim.

[0022] Furthermore, the sustained-release material includes a gel material and a photogel cross-linked polymer material.

[0023] Furthermore, the mass ratio of the gel material and the photogel cross-linked polymer material is adjustable. The higher the ratio of the photogel cross-linked polymer material, the longer the drug release time.

[0024] Furthermore, when the photogel cross-linked polymer material is 100%, the drug release time is 4 weeks.

[0025] Furthermore, the photo-crosslinked polymer material includes one or more of methacryloyl gelatin, methacryloyl hyaluronic acid, methacryloyl sodium alginate, methacryloyl silk fibroin and / or methacryloyl chondroitin sulfate.

[0026] Furthermore, the photo-crosslinked polymer material is cured by a photoinitiator.

[0027] Furthermore, the photoinitiator is selected from one or more of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), disodium tetrabromofluorescein (DY) and / or ruthenium-based photoinitiators.

[0028] Furthermore, the volume ratio of the photoinitiator to the photocrosslinking polymer material is 1:50.

[0029] Furthermore, the substrate adhesive material is polyvinyl alcohol.

[0030] Furthermore, the substrate adhesive material is a mixed solution of polyvinyl alcohol K30 (PVPK30) and polyvinyl alcohol 1788 (PVA1788).

[0031] In a second aspect, the present invention provides a method for preparing the ipakalim microneedle as described in the first aspect, the method comprising the following steps:

[0032] S01. Microneedle Shell Preparation

[0033] S011, dissolving the gel material in the ternary solution to form a mixed co-solution;

[0034] S012, adding calcium-phosphorus material and chiral inducer to the mixed co-solvent formed in S011 at a Ca / P molar ratio of 1.67, and drying to obtain a chiral gel material calcium-phosphorus mixed powder;

[0035] S013, dissolving the chiral gel material calcium-phosphorus mixed powder obtained in S012 again, adding drug powder, and obtaining an ipakalin rapid release phase;

[0036] S014, pouring the ipakalim rapid release phase obtained in S013 into the microneedle female mold, inserting the microneedle male film, drying, and pulling out the microneedle male film to obtain a microneedle shell;

[0037] S02. Microneedle Body Preparation

[0038] S021, mixing the photo-crosslinked polymer material powder and the gel material, dissolving the mixture in a ternary solution, and adding ipakalin to obtain an ipakalin slow-release phase;

[0039] S022, after adding a photoinitiator to the ipakalim slow-release phase obtained in S021, pouring it into a microneedle mold containing a microneedle shell, removing bubbles, ultraviolet irradiation, and drying, to obtain a microneedle body;

[0040] S03, Iptakalim microneedle preparation

[0041] S031, pour the mixed base material into the mold where the microneedle body is cast, and obtain the ipakalim microneedle after degassing, drying and demolding.

[0042] Further, in step S011, the gel material includes one or more of silk fibroin, collagen, gelatin, hyaluronic acid, chondroitin sulfate and / or sodium alginate.

[0043] Further, in step S011, the ternary solution is a 40% calcium chloride / ethanol / water ternary solution, and the ratio of the ethanol to the pure water is 3:7.

[0044] Further, in step S012, the calcium-phosphorus material includes one or more of a calcium-phosphorus mixture, β-tricalcium phosphate, calcium silicate, calcium borate, calcium carbonate, calcium sulfate, diammonium phosphate and / or calcium chloride.

[0045] Furthermore, in step S012, the chiral inducing agent is a chiral amino acid.

[0046] Furthermore, the chiral amino acid is selected from one or more of arginine, phenylalanine, leucine, tryptophan, methionine, proline, cysteine, glutamine, tyrosine, aspartic acid, glutamic acid and / or threonine.

[0047] Furthermore, the concentration of the chiral inducer is 1 mol / L, and the volume ratio of the chiral inducer to the calcium phosphate material solution is 1:1.

[0048] Further, in step S013, the drug is iptakalim and its derivative natakalim.

[0049] Further, in step S021, the photo-crosslinked polymer material includes one or more of methacryloyl gelatin, methacryloyl hyaluronic acid, methacryloyl sodium alginate, methacryloyl silk fibroin and / or methacryloyl chondroitin sulfate.

[0050] Further, in step S021, the gel material includes one or more of silk fibroin, collagen, gelatin, hyaluronic acid, chondroitin sulfate and / or sodium alginate.

[0051] Further, in step S022, the photoinitiator is selected from one or more of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), disodium tetrabromofluorescein (TEFY) and / or ruthenium-based photoinitiators.

[0052] Furthermore, the volume ratio of the photoinitiator to the photocrosslinking polymer material is 1:50.

[0053] Further, in step S031, the base material is polyvinyl alcohol.

[0054] Furthermore, the base material is a mixed solution of polyvinyl alcohol K30 (PVP K30) and polyvinyl alcohol 1788 (PVA1788).

[0055] Beneficial Effects

[0056] 1. The microneedle prepared in the present application has the advantages of good biocompatibility, high hardness and high drug loading. The present invention adds a chiral support material to the gel material, and the chiral support material has porosity, which can enhance the hardness of the gel material while not affecting the dissolution and release of the gel material.

[0057] 2. The chiral support material selected in this application has good biocompatibility, strong plasticity, uniform particles and dense texture.

[0058] 3. After adding the gel material and the photo-crosslinked polymer material mixture to the microneedle core of the present application, the drug release rate can be controlled, and the longest release time is 4 weeks.

[0059] 4. The ipakalim gel microneedle prepared in the present application can be locally administered to the kidneys to reduce systemic secondary drug reactions. The administration concentration is higher than that of oral and infusion dosage forms, and can permanently reduce blood uric acid and blood creatinine, making it more suitable for preventing renal failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The schematic diagram of the microneedle structure and preparation and the example of the chiral calcium-phosphorus mixture-silk fibroin-iptakalim gel microneedle. 1. Polydimethylsiloxane membrane (PDMS) microneedle negative mold; 2. Adding chiral calcium-phosphorus mixture-silk fibroin-iptakalim solution to prepare the needle shell; 3. Adding the microneedle positive mold; 4. Adding methacrylylated silk fibroin-iptakalim solution to prepare the needle core.

[0061] Figure 2 This is a fluorescence microscope image of FITC fluorescent microneedles.

[0062] Figure 3 This is the hardness test curve of the chiral silk fibroin calcium-phosphorus mixture-iptakaline-methacryloyl silk fibroin gel microneedle.

[0063] Figure 4 Schematic diagram of the in vitro skin microneedle release efficiency detection device. 1. The upper cover glass of the in vitro simulated skin device; 2. The upper chamber; 3. The microneedle; 4. Fresh pig skin; 5. The lower chamber; 6. The sampler.

[0064] Figure 5 This is the blood concentration curve of ipakalim microneedle.

[0065] Figure 6 This is the plasma uric acid curve of the iptakalim microneedle, oral, and intravenous formulation treatment groups.

[0066] Figure 7 This is the plasma creatinine curve of the iptakalim microneedle, oral, and intravenous formulation treatment groups.

[0067] Figure 8 This is the blood glucose curve of the groups treated with ipakalim microneedle, oral, and intravenous preparations.

[0068] Fig. 9 This is the total cholesterol curve of the groups treated with iptakalim microneedle, oral, and intravenous formulations.

[0069] Fig.10 This is the urine protein curve of the iptakalim microneedle, oral, and intravenous preparation treatment groups. DETAILED DESCRIPTION

[0070] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0071] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0072] Examples 1 to 9 Preparation of Iptakalim Rapid Release Phase

[0073] Prepare a ternary solution. Add 40 g of calcium chloride to 30 ml of ethanol and 70 ml of pure water to obtain a 40% calcium chloride ethanol-water ternary solution, filter the solution, and store it.

[0074] 20g, 40g, and 80g of silk fibroin were dissolved in 100ml of calcium chloride / ethanol / water ternary solution at 95°C and cracked for 30min; stirred to obtain mixed co-solutions of silk fibroin with mass concentrations (g / ml) of 20%, 40%, and 80%, respectively. In the ternary solution, the binding of silk fibroin with water gradually increased, the silk fibroin fibers swelled, and then gradually broke into lamellar shapes, the crystalline structure was destroyed, and the regenerated silk fibroin changed from β-folding to irregular curling, resulting in improved solubility. 240ml of 1mol / L diammonium hydrogen phosphate solution was added dropwise to the mixed co-solution at a Ca / P molar ratio of 1.67, the Ph value of the reaction solution was adjusted to 10 with ammonia water, and in an alkaline environment, 400ml of 1mol / L chiral inducers L-glutamic acid, D-glutamic acid, and racemic glutamic acid were added according to Table 1, wherein L-glutamic acid, D-glutamic acid, and racemic glutamic acid were mixed with diammonium hydrogen phosphate and Ca 2+The molar ratio is 1:1:1.67. Stir the reaction for 4 hours, age it statically for 24 hours, centrifuge and dry it to obtain nine kinds of chiral silk fibroin calcium-phosphorus composite powders, namely, left-handed silk fibroin calcium-phosphorus mixed powder, right-handed silk fibroin calcium-phosphorus mixed powder and racemic silk fibroin calcium-phosphorus mixed powder. Add 1g of each of the above chiral silk fibroin calcium-phosphorus composite powders to 90ml of distilled water, then add 10ml of acetic acid, stir at 4°C to dissolve the powder, then add ipakalin powder to make the final concentration of ipakalin 0.5μg / μl, and ultrasonically disperse for 20min to obtain the ipakalin rapid release phase.

[0075] Table 1 Ratio of components, chiral structure and drug content of Examples 1 to 9

[0076]

[0077] Examples 10-20 Preparation of Iptakalim Slow Release Phase

[0078] 1) Preparation of methacryloyl silk protein

[0079] The free amino groups of the side chains of silk fibroin are reacted with methacrylic anhydride to introduce carbon-carbon double bonds into the side chains to form a network structure, and then a free radical polymerization reaction is initiated by ultraviolet radiation to prepare methacryloyl cross-linked silk fibroin, which has good mechanical properties. Specifically, 2g of silk fibroin is fully dissolved in 40ml of hexafluoroisopropanol, dissolved, the pH value is adjusted to 7.0, the solution is filtered to remove impurities and microorganisms, 6ml of methacrylic anhydride is added under ice-water bath conditions, stirred, and reacted for 8h to covalently fix methacrylic anhydride and silk fibroin. After the reaction, it is dialyzed in deionized water in an ice-water bath for 7 days. The product is freeze-dried to obtain a highly modified methacryloyl silk fibroin.

[0080] 2) A mixed solution of silk fibroin and methacryloyl silk fibroin containing etalin is prepared.

[0081] Prepare mixed solutions of silk fibroin and methacryloyl silk fibroin with different mass fractions. Prepare 200 ml of 11 mixed solutions of silk fibroin and methacryloyl silk fibroin with different mass percentages, respectively, according to the mass percentages of silk fibroin and methacryloyl silk fibroin of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%. Then add ipakalin powder respectively to make the final concentration of ipakalin 0.5 μg / μl, and ultrasonically disperse for 20 minutes to obtain ipakalin slow release phase solution.

[0082] 3) Preparation of photoinitiator LAP standard solution.

[0083] 250 mg of LAP, a water-soluble photoinitiator, was placed in 100 ml of PBS, stirred and dissolved to prepare a 0.25% (w / v) LAP standard solution, and the solution was filtered and stored.

[0084] 4) Preparation of ipakalim slow-release gel.

[0085] 2 ml of the filtered LAP standard solution was added to each 100 ml portion of the above-mentioned mixed solution of silk fibroin and methacryloyl silk fibroin with different mass fractions of iptakalin, heated to dissolve, and cross-linked and cured for 10 min under ultraviolet (405 nm) irradiation to form iptakalin-silk fibroin and methacryloyl silk fibroin gel.

[0086] Example 21 Slow release phase release fluorescent dye isothiocyanate (FITC) test

[0087] In order to visually observe the drug release effect of mixed gels of silk fibroin and methacryloyl silk fibroin with different mass fractions, green fluorescein (FITC) was used instead of ipakalim, and the fluorescence intensity of microneedles prepared only with the slow release phase was observed using a fluorescence microscope. The specific steps are as follows:

[0088] 1) Preparation of silk fibroin and methacryloyl silk fibroin gels containing different mass percentages of FITC. Silk fibroin and methacryloyl silk fibroin mixtures containing 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% silk fibroin were added to a 40% calcium chloride ethanol-water ternary solution, and FITC powder was added to a final concentration of 1 ug / ml, and stirred at 50°C for 2 hours to dissolve. The mixed solution was poured into the microneedle mold, and the mold was placed in a vacuum dryer, and vacuumed for 30 minutes to remove the bubbles remaining in the mixed solution in the mold and allow the solution to reach the bottom of the mold needle tip. The mold was cross-linked and cured for 10 minutes under ultraviolet (405nm) irradiation, and then placed in a constant temperature drying oven for drying, then demoulding, peeling off from the mold, and then placed in a dryer for storage, to prepare silk fibroin and methacryloyl silk fibroin gel microneedles containing FITC with a mass percentage of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, which were used to detect the diffusion effect of fluorescent dyes mixed in different proportions of two silk fibroin. After adding FITC fluorescent dye, the whole process was kept away from light.

[0089] 2) Preparation of 5% methylcellulose semisolid gel. Used to observe the mass percentage of FITC is 0%, 10%,

[0090] Release of FITC in 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% silk fibroin and methacryloyl silk fibroin gel microneedles. Weigh 5 g of methylcellulose, add 50 ml of ultrapure water, mix well and sterilize by high pressure. After sterilization, wait until the temperature drops below 37°C, add 50 ml of IMDM cell culture medium, shake, place at 4°C for 15 minutes, shake again, repeat several times until there is no clot, place at -20°C overnight, shake again the next day, and store at -20°C.

[0091] 3) The mass percentage is 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,

[0092] 90%, 100% FITC-containing silk fibroin and methacrylylated silk fibroin gel microneedles were placed in a 48-well cell culture dish containing 0.5 ml of 5% methylcellulose semi-solid culture medium in each well, and placed at room temperature away from light. At different time points, the cell culture dish containing fluorescent gel microneedles was placed under a fluorescence microscope for observation. The results are as follows: Figure 1 The expansion degree and time of FITC were observed and recorded, and the results are shown in Table 2.

[0093] The results show that with the increase of methacryloyl silk fibroin content, the duration of fluorescence intensity of silk fibroin and methacryloyl silk fibroin gel microneedles gradually increases. Among them, FITC in 100% methacryloyl silk fibroin gel microneedles can be maintained for 48 hours. Because FITC itself also has a quenching time, the fluorescence disappearance time observed in this example cannot represent the actual release time of the drug in the microneedle. According to the fluorescence duration, in the following examples, Example 20 containing 100% methacryloyl silk fibroin gel is selected as the sustained-release phase.

[0094] The present embodiment is used to judge the drug release trend of silk fibroin and methacryloyl silk fibroin gel microneedles of different mass percentages. After freeze drying, the size of the holes of the silk fibroin solution of the same concentration will decrease with the increase of temperature, but the shape will not change. The hole size will decrease with the increase of the concentration of the silk fibroin solution at the same temperature, and the shape will also change. Therefore, the gel hole size can be adjusted by adjusting the ratio of methacryloyl silk fibroin and silk fibroin, and the release time of the drug can be controlled to prepare gel microneedles with different release times.

[0095] Table 2 FITC release time in silk fibroin and methacrylylated silk fibroin gel microneedles of Examples 10 to 20

[0096]

[0097] Examples 22-30 Preparation of Iptakalim Gel Microneedles

[0098] 1) Preparation of microneedle shells. The quick-release phases prepared in Examples 1 to 9 were poured into the microneedle negative grooves, pressed into the needle body positive mold, and the silk fibroin calcium phosphate mixture / ipakalim composite solution was extruded to make the solution in the mold a thin film with a thickness of 10 μm. The solution was placed at 4° C. for 2 to 8 hours to gel, precooled, freeze-dried, and the upper needle body positive mold was pulled out.

[0099] 2) Preparation of microneedle core. The slow-release phase prepared in Example 20 was poured into a mold containing a needle shell, and the poured PDMS mold was placed in a vacuum drying oven with a vacuum degree of more than 0.09 MPa for 20 to 30 minutes to remove the bubbles in the solution and the micropores of the mold and allow the solution to enter the micropores of the mold. Cross-linking and curing were performed under ultraviolet (405 nm) irradiation for 10 minutes.

[0100] 3) Preparation of microneedle basement membrane. A basement mixed solution was prepared at a ratio of 5% polyvinyl alcohol K30 and 10% polyvinyl alcohol 1788, and the basement mixed solution was poured into the cast microneedle mold. After degassing, the prepared microneedle PDMS system was placed in a constant temperature and humidity environment (humidity of 65%, pressure of 278 kPa) for drying and equilibrium for more than 36 hours, and then demolded to obtain ipakalim microneedles with a shell-core structure. A total of 9 types of microneedles were obtained, and the specific types are shown in Table 3. Figure 2 This is the preparation process and microneedle example of chiral silk fibroin calcium phosphate mixture-iptakaline-methacryloyl silk fibroin gel microneedle.

[0101] Example 31 Determination of drug content in microneedle body and substrate

[0102] Cut the prepared microneedle sample needle body and collect it in a centrifuge tube, and put the corresponding substrate into another centrifuge tube and mark it. Add an appropriate amount of PBS solution to the centrifuge tube, shake for 1 hour, filter, use high-performance liquid chromatography to analyze the corresponding drug content of the needle body and substrate, and calculate the drug content of the needle body. The calculation formula is: drug content of the needle body = drug content in the needle body / (drug content in the needle body + drug content in the substrate) × 100%

[0103] The results are shown in Table 3. The results show that the needle body drug of the gel microneedle with a chiral structure fast release phase is 96%, 98% and 99%, and as the proportion of silk protein increases, the proportion of needle body drug increases. The fast release phase microneedle containing 80% silk protein by mass has a needle body drug proportion of 99%. The needle body drug proportion of the fast release phase gel microneedle containing a racemic structure is 80%, 82%, and 85%.

[0104] Example 32 Microneedle Mechanical Properties Test

[0105] Use a computer force-stroke tester (model: 1220SB) to detect the yield stress of the microneedle, set the upper limit of the force to 5N, and use a probe (2mm×2mm). Fix the microneedle to the stage with the needle tip facing up, and set the stage movement speed to 1.1mm / s. Use pressure-sensitive adhesive to fix the microneedle patch on the test platform below the force-stroke tester, with the needle tip facing up and vertically aligned with the mechanical sensor probe above. When the test starts, the sensor probe moves vertically downward at a longitudinal speed of 1.1mm / s to apply a longitudinal force to the microneedle array. The upper limit of the force is 10N, and the computer synchronously records the curve of the longitudinal yield stress of the microneedle and the distance the probe moves. Place the tested patch under a microscope, record the number of microneedles under force, and plot the obtained data into a mechanical property curve. After the test is completed, use a microscope to observe whether the needle tip is broken.

[0106] As shown in Table 3, the microneedles with a chiral structure in the rapid release phase have a complete needle shape after demolding, and a single needle does not break after a force of 0.5N. This mechanical index enables it to effectively pierce the skin. The yield stress of the microneedles with a racemic structure in the rapid release phase is only 0.1N and 0.3N. In Examples 22 to 27, as the proportion of silk fibroin increases, the yield stress decreases, but it is all above 0.5N, and the changes in the yield stress and the moving distance are linearly distributed. Figure 3 Shown is a graph of yield stress and travel distance for Example 27.

[0107] Table 3

[0108]

[0109] Example 33 In vitro drug release assay of iptakalim gel microneedle

[0110] The in vitro skin microneedle release efficiency detection device detects the drug release efficiency within 24 hours of Examples 22 to 30. The schematic diagram of the in vitro skin microneedle release efficiency detection device is as follows Figure 4 As shown. The drug loading of each microneedle shell was 10 μg. After 16 right-handed calcium-phosphorus mixture-silk fibroin-iptakalim gel microneedles were inserted into fresh pig skin, the release efficiency within 24 hours was tested. The active ingredient iptakalim was released into the lower collection tank through the pig skin tissue fluid. The lower exudate was collected regularly, and the amount of iptakalim was detected by liquid-mass spectrometry high-performance chromatography. The results are shown in Table 4.

[0111] Table 4 Iptakalim release efficiency in 24h release test in vitro in Examples 22-30

[0112]

[0113]

[0114] As can be seen from the above table, Examples 22 to 27 contain microneedles with a chiral structure and a rapid release phase, which can well control the release rate of ipakalin, and the release efficiency is above 95%. Moreover, as the content of silk fibroin increases, the release efficiency increases. The release efficiency of the microneedles with a rapid release phase of 80% silk fibroin in Example 27 is as high as 99%. However, Examples 28 to 30 have a racemic structure and a rapid release phase. Not only is the release rate of ipakalin not stable within 24 hours, but after concentrated release in the first 6 hours, no drug release is detected in 12 hours, 18 hours, and 24 hours. Moreover, the release efficiency is low. The larger the amount of drug released in Examples 29 and 30 before 6 hours, the lower the release efficiency. The ipakalin in Examples 28 to 30 is not fully released into the tissue.

[0115] Example 34 Detection of blood drug concentration of iptakalim gel microneedle in vivo

[0116] According to the hardness of the ipakalim gel microneedle, the needle body drug ratio, and the 24h in vitro drug release effect in the above embodiments, the microneedles with chiral structure rapid release phase in Examples 22 to 27 have good hardness, needle body drug ratio and 24h in vitro release effect, so this embodiment uses Examples 22 to 27 to detect the blood drug concentration in vivo. Specifically, 18 SD male rats, 3 weeks old, were randomly divided into 6 groups, namely Example 22, Example 23, Example 24, Example 25, Example 26, and Example 27 groups, 3 rats in each group, and were marked and caged. One day before the experiment, all the hair on the back of the rats was removed. After the rats were anesthetized, a patch (100 pieces) of microneedles was pressed on the skin of the kidney area on the back of the rats, and the drug was continuously administered for 35 days. The blood of the rats at different time points was collected, and the serum was separated and stored at -80°C for blood drug concentration determination. During the experiment, food and water were freely available, the temperature of the breeding environment was 22 to 24°C, and the humidity of the breeding environment was 40 to 70%. After 35 days, the ipakalim gel microneedles were pulled out from the back skin of the rats and collected in a centrifuge tube. The residual drugs in the microneedles were dissolved with 40% ethanol solution, and the drug content was analyzed by liquid-mass spectrometry and high performance chromatography.

[0117] Blood was collected before microneedle application as a negative control. After microneedle application, 20 μl of blood was collected from the tail vein or orbital cavity at 1h, 6h, 12h, 24h (1d), 3d, 7d, 14d, 28d, and 35d. The rats were anesthetized each time. The whole blood was collected in an EDTAK2 centrifuge tube, shaken, and centrifuged at 1600xg for 10min. The supernatant was taken and the content of iptakalim in the blood sample was analyzed by liquid chromatography-mass spectrometry, and the blood drug concentration curve was drawn. The results are shown in Figure 5As shown in the figure, the blood drug concentration of rats in Examples 22 to 27 can be seen in both the rapid release phase and the sustained release phase, and can reach the peak of the rapid release phase in 6 hours and the peak of the sustained release phase in 14 days, indicating that Examples 22 to 27 have good rapid release, sustained release and controlled release effects, and the blood drug concentration can still reach 100 μg / ml concentration in 28 days, and after 28 days, the blood drug concentration decreases rapidly as the diffusion of ipakalin in the microneedles is completed. In practical applications, the proportion of silk fibroin can be increased to adjust the release time of the sustained release phase.

[0118] Each microneedle contains 10 μg of ipakalin in the needle shell and 100 μg of ipakalin in the needle core. The residual ipakalin in the ipakalin microneedle after 35 days is shown in Table 5. The results show that the microneedle dosage form can release all the drugs into the body, and the residual ipakalin in the needle body is at the nanogram level. Among them, Example 27 has the best drug release effect. As the silk protein content in the rapid release phase continues to increase, the time to reach the peak of the rapid release phase is faster, and the residual amount of microneedle drug is also lower. In practical applications, by adjusting the dosage of ipakalin in the microneedle, the ideal blood drug concentration can be achieved.

[0119] Table 5 Iptakalim Residue in Iptakalim Microneedles

[0120]

[0121] Example 35 Pharmacodynamics experiment of ipakalim gel microneedle

[0122] 1) Establishment of acute hyperuricemia rat model. Clean-grade SD male rats, 5 weeks old and weighing 150±50g, were selected. SD rats are commonly used experimental animals in medical and biological research, with a high degree of standardization, convenient for drug administration and easy sampling. The temperature of the breeding environment is 24°C, and the humidity of the breeding environment is 40%. The experimental rats are free to eat and drink water. Uric acid was given at 3 mg / kg / d, gavage for 3 days, once a day, for 5 consecutive weeks, and blood uric acid levels were tested regularly. When the blood uric acid level was higher than 7mmol / L, serum BUN (blood urea nitrogen), creatinine content and proteinuria were measured at the same time to evaluate renal function. Model rats with serum BUN and creatinine more than 2 times higher than normal values ​​were selected for subsequent ipakalim gel microneedle pharmacodynamics tests.

[0123] 2) Pharmacodynamics detection of ipakalim gel microneedle. The acute hyperuricemia rat model was divided into a non-administered group, a microneedle group, an oral group, and an intravenous injection group, with 6 rats in each group. Example 27 was used to perform Example 35. The back hair of the rats in the microneedle group was shaved, and the ipakalim gel microneedles were pressed into the skin in the kidney area of ​​the rats to allow the microneedles to enter the muscle tissue. The dose was 100 ipakalim gel microneedles per rat, and the microneedles were fixed. The oral group was administered by gavage at a dose of 1000ug / kg / day; the intravenous administration was by tail vein injection at a dose of 1000ug / kg / day, and the administration was continued for 7 days.

[0124] Orbital venous blood was drawn on the 1st, 2nd, 3rd, 4th, 5th, 6th and 7th day after administration to test indicators such as blood uric acid, blood creatinine, blood sugar, total cholesterol and proteinuria.

[0125] Using rat biochemical instruments, blood uric acid, creatinine, blood sugar, total cholesterol, and urine protein were tested. Figures 6 to 10 As can be seen from the figure, the uric acid, creatinine, total cholesterol and urine protein in the microneedle group continued to decline with the extension of the number of days of administration. At 7 days, blood uric acid, creatinine, total cholesterol and urine protein were close to normal values, and blood sugar and total cholesterol levels did not decrease significantly; the uric acid, creatinine, blood sugar, total cholesterol and urine protein in the oral and intravenous groups did not decrease as much as the microneedle group, and were all higher than normal values ​​at 7 days; the uric acid, creatinine, blood sugar, total cholesterol and urine protein in the non-administration group maintained high values ​​and increased slightly. Therefore, iptakalim gel microneedles can effectively and continuously reduce uric acid, creatinine, proteinuria and other indicators.

[0126] The above data show that by controlling the drug loading of microneedles, the mass fraction of sustained-release silk protein and methacryloyl silk protein or increasing the number of microneedles used, it is possible to provide patients with a constant therapeutic dose of blood drug concentration, avoiding the impact of traditional injection administration due to excessively high blood drug concentration and achieving good controlled release.

[0127] In summary, the microneedles prepared by the present invention have strong plasticity, high hardness, and high drug content in the needle body. After piercing the skin, they can be preserved in the skin for a long time. The microneedles form drug release channels, and the drugs can be released efficiently, lastingly, and stably.

[0128] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. An ipakalim gel microneedle, the microneedle comprising a needle shell, a needle core and a substrate; the needle shell comprises a support material, a gel material and a drug, wherein the mass ratio of the support material to the gel material is 1-2:9-8, and the needle shell thickness is 10 μm; the needle core comprises a sustained-release material and a drug; the substrate structure is composed of an adhesive material; wherein, The drug concentration in the needle shell and needle core is 0.5-1.0 mg / ml.

2. The gel microneedle as claimed in claim 1, wherein the supporting material is a chiral calcium-phosphorus material, wherein the chiral calcium-phosphorus material is synthesized by reacting a chiral inducer with a calcium-phosphorus inorganic salt, and wherein the chiral inducer is a chiral amino acid.

3. The gel microneedle according to any one of claims 1 or 2, wherein the concentration of the chiral inducer is 1 mol / L, and the volume ratio of the chiral inducer to the calcium phosphate inorganic salt solution is 1:

1.

4. The gel microneedle according to any one of claims 1 to 3, wherein the gel material comprises one or more of silk fibroin, collagen, gelatin, hyaluronic acid, chondroitin sulfate and / or sodium alginate.

5. The gel microneedle according to any one of claims 1 to 4, wherein the drug in the needle shell and the needle core is iptakalim and its derivative natakalim.

6. The gel microneedle according to any one of claims 1 to 5, wherein the sustained-release material comprises a gel material and a photogel cross-linked polymer material, the mass ratio of the gel material and the photogel cross-linked polymer material is adjustable, and the higher the ratio of the photogel cross-linked polymer material, the longer the drug release time. 7 . The gel microneedle according to claim 1 , wherein the substrate adhesion material is polyvinyl alcohol.

8. A method for preparing the ipakalim microneedle according to claim 1, comprising the following steps: S01. Microneedle Shell Preparation S011, dissolving the gel material in the ternary solution to form a mixed co-solution; S012, adding calcium-phosphorus material and chiral inducer to the mixed co-solvent formed in S011 at a Ca / P molar ratio of 1.67, and drying to obtain a chiral gel material calcium-phosphorus mixed powder; S013, dissolving the chiral gel material calcium-phosphorus mixed powder obtained in S012 again, adding drug powder, and obtaining an ipakalin rapid release phase; S014, pouring the ipakalim rapid release phase obtained in S013 into the microneedle female mold, inserting the microneedle male film, drying, and pulling out the microneedle male film to obtain a microneedle shell; S02. Microneedle Body Preparation S021, mixing the photo-crosslinked polymer material powder and the gel material, dissolving the mixture in a ternary solution, and adding ipakalin to obtain an ipakalin slow-release phase; S022, after adding a photoinitiator to the ipakalim slow-release phase obtained in S021, pouring it into a microneedle mold containing a microneedle shell, removing bubbles, ultraviolet irradiation, and drying, to obtain a microneedle body; S03, Iptakalim microneedle preparation S031, pour the mixed base material into the mold where the microneedle body is cast, and obtain the ipakalim microneedle after degassing, drying and demolding.

9. The preparation method according to claim 8, wherein in step S011, the gel material comprises one or more of silk fibroin, collagen, gelatin, hyaluronic acid, chondroitin sulfate and / or sodium alginate, the ternary solution is a 40% calcium chloride / ethanol / water ternary solution, and the ratio of ethanol to purified water is 3:7; In step S012, the calcium-phosphorus material includes one or more of a calcium-phosphorus mixture, β-tricalcium phosphate, calcium silicate, calcium borate, calcium carbonate, calcium sulfate, diammonium phosphate and / or calcium chloride, the chiral inducer is a chiral amino acid, the concentration of the chiral inducer is 1 mol / L, and the volume ratio of the chiral inducer to the calcium-phosphorus material solution is 1:1; In step S013, the drug is iptakalim and its derivative natakalim; In step S021, the photo-crosslinked polymer material includes one or more of methacryloyl gelatin, methacryloyl hyaluronic acid, methacryloyl sodium alginate, methacryloyl silk fibroin and / or methacryloyl chondroitin sulfate, and the gel material includes one or more of silk fibroin, collagen, gelatin, hyaluronic acid, chondroitin sulfate and / or sodium alginate; In step S022, the photoinitiator is selected from one or more of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), disodium tetrabromofluorescein (DY) and / or a ruthenium-based photoinitiator; In step S031, the base material is polyvinyl alcohol.

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