Microneedle of medicine with short half-life period as well as preparation method and application of microneedle

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a microneedle of a drug with a short half-life period as well as a preparation method and application of the microneedle. The bipolar controllable release microneedle is provided for drugs with short half-life periods, long-acting percutaneous minimally invasive drug delivery is achieved, the convenience and comfort of a patient in drug use are greatly improved, controllable plasma concentration maintaining time can be achieved by adjusting the proportion of the first-stage release phase and the second-stage release phase, and the personalized analgesic drug delivery requirement is met. Besides, by taking an opioid analgesic administration strategy as an entry point, the precise controllable two-stage release microneedle of the biphasic matrix carried medicine is innovatively provided, a new dosage form is developed for the opioid analgesic medicine, and compared with an existing intravenous injection administration mode, the analgesic time can be prolonged by 24 times, and the analgesic effect of the medicine is greatly improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a microneedle for a short half-life drug and a preparation method and application thereof. Background Art

[0002] Opioids are a class of drugs widely used in the clinical treatment of moderate to severe pain, including morphine, fentanyl, pethidine, etc. These drugs mediate analgesic effects by stimulating opioid receptors in the central nervous system (Li Wei, Li Fang, Huang Yuhong. Application of opioids in the treatment of chronic pain [J]. Chinese Health and Nutrition, 2016, 26(16): 269.), especially for severe pain caused by cancer. At the same time, opioids can effectively inhibit the stress response caused by tracheal intubation and surgical pain stimulation when used for anesthesia, and maintain the stability of blood pressure and heart rate. It can be said that opioids are irreplaceable in anesthesia and pain treatment. At present, opioids are widely used worldwide to treat acute and chronic pain, cancer-related pain and palliative care. Other studies have shown that 5.5% of people often use opioids, and the dosage increases with age, and 87% of people who use this drug report chronic pain.

[0003] The overall trend of the opioid market is still gradually expanding. The main reasons for the analysis are the increase in the number of surgeries and the increase in the prevalence of chronic pain such as cancer pain. With the advancement of medical technology and the improvement of people's health awareness, the number of surgeries continues to increase. The increase in the number of patients with chronic diseases worldwide has led to increasingly prominent pain problems related to chronic diseases, thereby increasing the demand for opioids. However, the use of opioids is accompanied by a series of side effects and risks, such as respiratory depression, constipation, and addiction (Liu Xiaowen, Liu Zhen, Zhao Jing. Research progress on the mechanism of respiratory depression of opioids [J]. Basic Medicine and Clinic, 2017, 37(3): 422-426. Wu Ge, Lin Shenxian, Zhu Qi, et al. Is opioid a dead end? [J]. Chinese Journal of Pain Medicine, 2021, 27(03): 212-215.). In 2021, the number of deaths in the United States due to overdose of opioids exceeded 107,000, nearly 10 times that of 2000. Compared with the classic strong opioids morphine, oxycodone, and methadone, oxeridine provides equally effective analgesia while causing less respiratory depression side effects, making it a more promising preferential opioid receptor agonist (Hill R, Sanchez J, Lemel L, et al. Assessment of the potential of novel and classical opioids to induce respiratory depression in mice. Br J Pharmacol. 2023 Dec; 180(24): 3160-3174.). Ocellidine, as a selective G protein signaling agonist of μ-opioid receptors, can significantly reduce the side effect of respiratory depression while exerting the same analgesic effect (Zhu Changmao, Xie Li, Wu Zifeng, et al. Clinical application progress of the biased μ-opioid receptor agonist occellidine [J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2024, 29(09): 1057-1061.), and is approved by the US Food and Drug Administration (FDA) for the treatment of severe pain in adults and acute pain that requires intravenous opioid analgesics and is insufficient for alternative therapies (Feng Congrui, Li Lixia, Wang Shuping, et al. Application research progress of occellidine [J]. Clinical Rational Drug Use, 2023, 16(9): 179-181.). Ocellidine fumarate injection produced by Enhua Pharmaceuticals in China was also officially launched in 2023. Currently, there is only one intravenous preparation. Although the intravenous preparation takes effect quickly, it has a short duration and relies on medical staff to administer the drug, which is very inconvenient for patients who use it for a long time or at home.

[0004] Opioids for home use are mainly divided into oral and topical types. Oral preparations (taking morphine sulfate sustained-release tablets and OxyContin hydrochloride sustained-release tablets as examples) can last up to 12 hours, and still need to be taken orally twice a day, and are not suitable for patients with oral restrictions. For topical opioids, only the strong opioid fentanyl transdermal patch and the weak opioid buprenorphine patch can maintain efficacy for 3 days and 7 days respectively, but the fentanyl transdermal patch takes 6-12 hours to take effect, and the buprenorphine patch takes effect more slowly, so these two topical patches often need to be used in combination with fast-acting oral opioids to meet clinical needs. The half-life of a drug is the time required for the plasma drug concentration to decrease by 50%. In general, the half-life of a drug can usually be divided into long-acting, medium-acting and short-acting. The half-life of a long-acting drug is generally 24 hours, the half-life of a medium-acting drug is 8 hours, and the half-life of a short-acting drug is 4 hours. Therefore, there is an urgent need in clinical practice for an opioid preparation that has a rapid onset of action, long duration of effect, and few side effects.

[0005] Microneedles are a new transdermal drug delivery method that can overcome the problems of oral administration and direct access to the blood. The microneedle system was first introduced in 1976 and successfully manufactured and applied in 1998 (S. Henry, D. McAllister, et al. "Microfabricated microneedles: a novel approach to transdermal drug delivery.." Journal of pharmaceutical sciences (1998).). Five types have been developed according to their functions: solid, coated, hollow, dissolved and hydrogel forms (Ping Yang, Wang Yan, Wang Lihong, et al. Modern research progress on transdermal drug-loaded microneedles [J]. Chinese Journal of New Drugs, 2023.). An important advantage of microneedle technology is its ability to reduce pain and tissue damage during drug delivery. Because the size of microneedles is very small, they can avoid touching the pain nerves under the skin, thereby reducing pain (Zhao Xiao, Li Xinfang, Zhang Peng, et al. Research on polymer microneedle-mediated transdermal drug delivery [J]. Progress in Chemistry, 2017.). In addition, microneedles can also be designed to achieve sustained release of drugs, which is an important means of administering drugs with short half-lives. Compared with intravenous administration, microneedles are simpler, non-invasive and convenient. It can also minimize needlestick injuries, avoid risks such as needle phobia, drug waste and blood-borne pathogens. These advantages allow microneedle preparations encapsulated with drugs to be used by patients themselves, avoiding reliance on medical staff for drug administration and saving public medical resources.

[0006] For example, although oxeridine, a drug with a short half-life, is the newest and safest strong opioid, its clinical use is limited due to its low oral bioavailability (5.77%). A better drug delivery system needs to be established. Summary of the invention

[0007] In view of the limitations of the use of drugs with short half-lives, the present invention proposes a dual-phase matrix to carry active drugs with short half-lives, and uses two carrying methods to realize precise and controllable dual-stage release microneedles, thereby achieving rapid and sustained release of short half-life drugs and extending the blood drug cycle of the drugs, thereby completing the present invention.

[0008] In the first aspect, the present invention provides a drug microneedle, wherein the drug is a short half-life drug, and the microneedle comprises two parts: a microneedle body and a substrate; the microneedle body can be a mixed structure comprising a primary release phase and a secondary release phase, or a separated structure comprising a microneedle body I and a microneedle body II; the microneedle body I comprises a primary release phase, and the microneedle body II comprises a secondary release phase; the primary release phase comprises a primary release matrix and the drug, and the secondary release phase comprises a secondary release matrix and the drug.

[0009] Further, the primary release matrix is ​​selected from one or more of uncrosslinked or weakly crosslinked hyaluronic acid, uncrosslinked or weakly crosslinked chitosan, HAMA (Hyaluronic Acid Methacrylate), GelMA (Gelatin Methacryloyl Gelatin Methacrylamide) and / or F127 (polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer).

[0010] Furthermore, the concentration of the primary release matrix is ​​5%-20%.

[0011] Furthermore, when the microneedle is a mixed structure, the secondary release matrix is ​​selected from one or more of bovine serum albumin (BSA) nanoparticles, liposome nanoparticles and / or nanofibers.

[0012] Furthermore, the diameter of the BSA nanoparticles is 50-300 nm.

[0013] Furthermore, the liposome nanoparticles are prepared from one or more selected from 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine sodium salt (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and / or cholesterol (CHOL).

[0014] Furthermore, the main material of the nanofiber is a biodegradable material, and the biodegradable material is selected from one or more of polylactic acid (PLA), polyketone ester, polylactic acid-polycaprolactone and / or polyhydroxyalkanoate.

[0015] Furthermore, the mass ratio of the drugs in the primary release phase and the secondary release phase of the hybrid structure microneedle is 1-2:3-5.

[0016] Preferably, the mass ratio of the drugs in the primary release phase and the secondary release phase of the hybrid structure microneedle is 1:3.

[0017] Furthermore, when the microneedles are separated structures, the secondary release matrix is ​​selected from one or more of medium-strength to strong-crosslinked gelatin and / or hydrogel microspheres.

[0018] Furthermore, the concentration of the moderately to strongly cross-linked gelatin or hydrogel microspheres is 0.05-0.5 g / ml.

[0019] Furthermore, the hydrogel is prepared by adding a photoinitiator and mineral oil to liposome nanoparticles and gelatin methacrylamide (GelMA) solution.

[0020] Furthermore, the photoinitiator is phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP for short).

[0021] Furthermore, the photoinitiator is added in an amount of 1%.

[0022] Furthermore, the GelMA and mineral oil are mixed by microfluidics at a volume ratio of 1:1, and the liposome nanoparticles are added at a volume ratio of 1:10-100.

[0023] Furthermore, the short half-life drug is a drug with a half-life of less than 4 hours, and the drug is selected from one or more of oxeridine, oxycodone, dezocine, fentanyl, morphine, remifentanil, hydromorphone and / or tramadol.

[0024] Preferably, the drug is oxalidine.

[0025] Furthermore, the substrate is an organic material, selected from one or more monomers or cross-linked bodies of hyaluronic acid, gelatin, chitosan, polyethylene glycol, polyvinyl alcohol and / or polyvinyl pyrrolidone (PVP).

[0026] In a second aspect, the present invention provides a method for preparing a microneedle of a short half-life drug, the method comprising the following steps:

[0027] 1) Preparation of hybrid structure microneedles

[0028] S1. Preparing a negative microneedle mold;

[0029] S2. Preparation of primary release phase: dissolving the primary release matrix and the drug in a solvent to form a primary release phase;

[0030] S3. Preparation of secondary release phase: mixing one or more of the secondary release matrix bovine serum albumin (BSA) nanoparticles, liposome nanoparticles and / or nanofibers with the drug to form a secondary release phase;

[0031] S4. Mixing the primary release phase prepared in step S2 and the secondary release phase prepared in step S3 to obtain a microneedle tip blend, applying the microneedle tip blend on a microneedle negative mold, injecting a substrate material, and obtaining a hybrid structure microneedle;

[0032] 2) Preparation of separation structure microneedles

[0033] N1. Prepare a negative microneedle mold;

[0034] N2. Preparation of primary release phase: dissolving the primary release matrix and the drug in a solvent to form a primary release phase;

[0035] N3. Preparation of secondary release phase: Mixing the secondary release matrix of moderately to strongly cross-linked gelatin and / or hydrogel microspheres with the drug to form the secondary release phase;

[0036] N4. Apply the primary release phase prepared in step N2 and the secondary release phase prepared in step N3 to the microneedle negative mold respectively, inject the substrate material, and form separated microneedles.

[0037] Further, in steps S2 and N2, the primary release matrix is ​​selected from one or more of uncrosslinked or weakly crosslinked hyaluronic acid, uncrosslinked or weakly crosslinked chitosan, HAMA (Hyaluronic Acid Methacrylate), GelMA (Gelatin Methacryloyl Gelatin Methacrylamide) and / or F127 (polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer).

[0038] Furthermore, in steps S2 and N2, the solvent is a buffer such as water, physiological saline or glucose aqueous solution.

[0039] Furthermore, in steps S2 and N2, the concentration of the primary release matrix is ​​5%-20%.

[0040] Furthermore, the drug is a drug with a half-life of less than 4 hours, and the drug is selected from one or more of oxeridine, oxycodone, dezocine, fentanyl, morphine, remifentanil, hydromorphone and / or tramadol.

[0041] Furthermore, in step S3, the particle size of the BSA nanoparticles in the secondary release phase is 50-300 nm.

[0042] Further, in step S3, the liposome nanoparticles are prepared from one or more selected from 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine sodium salt (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and / or cholesterol (CHOL).

[0043] Furthermore, in step S3, the main material of the nanofiber is a biodegradable material selected from one or more of polylactic acid (PLA), polyketone ester, polylactic acid-polycaprolactone and / or polyhydroxyalkanoate.

[0044] Furthermore, in steps S4 and N4, the substrate material is selected from one or more monomers or cross-linked bodies of hyaluronic acid, gelatin, chitosan, polyethylene glycol, polyvinyl alcohol and / or polyvinyl pyrrolidone (PVP).

[0045] Furthermore, in steps S4 and N4, the mass ratio of the drugs in the primary release phase and the secondary release phase is 1-2:3-5.

[0046] Preferably, in steps S4 and N4, the mass ratio of the drug in the primary release phase and the secondary release phase is 1:3.

[0047] Further, in step N3, the concentration of the medium-strength cross-linked gelatin and hydrogel microspheres is 0.05-0.5 g / ml.

[0048] Furthermore, in step N3, the hydrogel is prepared by adding a photoinitiator and mineral oil to a solution of liposome nanoparticles and gelatin methacrylamide (GelMA).

[0049] Furthermore, the photoinitiator is phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP for short).

[0050] Furthermore, the photoinitiator is added in an amount of 1%.

[0051] Furthermore, the GelMA and mineral oil are mixed by microfluidics at a volume ratio of 1:1, and the liposome nanoparticles are added at a volume ratio of 1:10-100.

[0052] In a third aspect, the present invention provides an application of a drug microneedle in the preparation of a drug for treating a disease; the drug is a drug with a short half-life, and the microneedle comprises a microneedle body and a substrate; the microneedle body can be a mixed structure comprising a primary release phase and a secondary release phase, or a separated structure comprising a microneedle body I and a microneedle body II; the microneedle body I comprises a primary release phase, and the microneedle body II comprises a secondary release phase; the primary release phase comprises a primary release matrix and a drug, and the secondary release phase comprises a secondary release matrix and a drug.

[0053] Furthermore, the drug is a drug with a half-life of less than 4 hours, selected from one or more of oxeridine, oxycodone, fentanyl, morphine, remifentanil, hydromorphone, tramadol and / or dezocine.

[0054] Preferably, the drug with a short half-life is oxeridine.

[0055] Furthermore, the disease is selected from one or more of painful diseases such as neuropathic pain, joint pain, traumatic pain and / or cancer pain.

[0056] Beneficial Effects

[0057] 1. The present invention provides bipolar controlled-release microneedles for drugs with short half-lives, which have two forms: a mixed structure and a separated structure, to achieve long-acting transdermal minimally invasive drug delivery, which not only greatly improves the convenience and comfort of patients using drugs, but also can achieve controllable blood drug concentration maintenance time by adjusting the ratio of the primary release phase and the secondary release phase to meet the needs of personalized analgesic drug delivery.

[0058] 2. The present invention optimizes the opioid analgesic drug delivery strategy for acute and chronic pain management as the entry point, and innovatively proposes a biphasic matrix-loaded microneedle with precise and controllable two-stage release of oxycellidine, and develops a microneedle dosage form for oxycellidine, a new and safe opioid analgesic drug. Compared with the existing intravenous injection method, it can increase the analgesic time by up to 24 times, greatly improving the analgesic effect of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Schematic diagram of the structure and effect of hybrid microneedles. Note: A. Hybrid microneedle structure; B. Schematic diagram of the effect of hybrid microneedles.

[0060] Figure 2 Schematic diagram of the structure and effect of separated structure microneedle. Note: A. Separated structure microneedle structure, B. Schematic diagram of the effect of separated structure microneedle; 1. Rapid release needle body, 2. Slow release needle body, 3. Microneedle substrate.

[0061] Figure 3 This is the preparation process of oxycellidine@BSA-NP.

[0062] Figure 4 The preparation process of hybrid structure microneedles.

[0063] Figure 5 The process of preparing microneedles for separation structure.

[0064] Figure 6 Transmission electron microscopy (TEM) detection of nanoparticles / nanofibers / hydrogel microspheres. Note: A. TEM photo of nanoparticles; B. Particle size analysis of nanoparticles, 15% of the nanoparticles are 100 nm in size; C. FITR results of nanoparticles.

[0065] Figure 7 This is the microneedle displacement-force curve.

[0066] Figure 8 To simulate the principle of skin diffusion experiment.

[0067] Fig. 9 Schematic diagram of the Franz diffusion cell structure.

[0068] Fig.10 is the cumulative release rate of BSA+HA.

[0069] Fig.11 Comparison of in vitro release efficiency.

[0070] Fig.12 Mouse behavioral experiments. DETAILED DESCRIPTION

[0071] 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.

[0072] 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.

[0073] Explanation of terms

[0074] "Cross-linking" refers to the reaction of linear or branched polymers bonding to form a three-dimensional network or a relatively stable molecule structure, thereby obtaining higher physical and mechanical properties. Cross-linking includes physical cross-linking and chemical cross-linking. Physical cross-linking is formed by the interaction of physical forces such as hydrogen bonds and ions, while chemical cross-linking is formed by connecting two or more molecules together through covalent bonds.

[0075] "Uncross-linked" means that the polymer chains are only connected by physical entanglement and mutual attraction to form a linear structure, without forming a three-dimensional network or body structure, and the physical and mechanical properties are relatively weak.

[0076] "Medium to strong crosslinking" refers to a high crosslinking density of polymer materials, with more crosslinks per unit volume. The crosslinking density refers to the fraction of crosslinked structural units to the total structural units of the polymer. The crosslinking density of medium to strong crosslinking is 20-60%, and the three-dimensional structure formed has high strength and high elasticity.

[0077] "Weak cross-linking" means that the cross-linking density of the polymer material is low, and the three-dimensional structure formed is prone to bond breakage when subjected to stress impact, and has poor mechanical properties. The cross-linking density of weak cross-linking is 0-20%.

[0078] Oxelidine@BSA-NP: BSA nanoparticles containing oxelidine

[0079] Sustained-release needle body: Made of biopolymer materials with slow dissolution rate and high drug-loading capacity, the microneedle slowly releases the drug after piercing the skin, completing the second stage of sustained release;

[0080] Microneedle body: It can pierce the keratin of human skin and open up a drug delivery channel. At the same time, the needle body material is composed of fast-dissolving biomaterials carrying drugs. After piercing the skin, the needle body will dissolve quickly, releasing the drugs carried therein and completing the primary drug release.

[0081] Microneedle substrate: It is composed of soluble organic matter. While fixing the microneedles, it has a certain viscosity, allowing the microneedle patch to adhere to the skin. At the same time, after contacting the skin for a period of time, it will automatically dissolve, and the user only needs to rinse it.

[0082] As used herein, the term "fine electrospinning" and the term "nanofiber" are interchangeable.

[0083] As used herein, the term "matrix" and the term "support" are interchangeable.

[0084] As used herein, the term "primary" and the terms "immediate release" and "rapid" are interchangeable.

[0085] As used herein, the term "secondary" and the terms "sustained release" and "slow" are used interchangeably.

[0086] The terms "microneedles loaded with oxytocin for precise controlled release" and "microneedles with mixed structure" and "microneedles with separated structure" described herein are interchangeable.

[0087] Example 1 Design of mixed structure microneedle and separated structure microneedle products

[0088] 1. Hybrid structure microneedle, the microneedle patch is composed of several single microneedle bodies arranged in an array, each microneedle monomer is composed of a needle body and a substrate, each microneedle tip is set back to the substrate, and the microneedle tip has a certain hardness and sharpness. The needle body is a mixture of a fast-release primary release phase and a slow-release secondary release phase. The structure and effect diagram of the hybrid structure microneedle are shown in the figure. Figure 1 shown.

[0089] 2. Separate structure microneedles. This microneedle patch is composed of several single microneedle bodies arranged in an array. The tip of any microneedle is set back to the substrate. The tip of the microneedle has a certain hardness and sharpness. The microneedles are divided into two groups: Group A is a drug-loaded microneedle made of a fast-dissolving polymer material, and Group B is a drug-loaded microneedle made of a slow-dissolving polymer material. The two groups of microneedles are integrated on the same microneedle patch. The schematic diagram of the structure and effect of the separated structure microneedle is shown in the figure. Figure 2 shown.

[0090] Example 2 Preparation of microneedle negative mold

[0091] Polydimethylsiloxane (PDMS) was used to make the concave mold. It is a hydrophobic material with stable chemical properties, low adhesion and good transcription ability, which is conducive to material demolding. The microneedle array can be dried or polymerized in the PDMS mold with a more complex structure constructed by the subsequent addition of functional solutions. The parametrically designed microneedle positive mold (15×15 square pyramid array, height 700μm, bottom side length 300μm) was used for PDMS mold casting to prepare the microneedle negative mold. The negative mold is a parting mold with a concave cavity in the mold, and the positive mold is convex.

[0092] Example 3 Preparation of primary release phase

[0093] The w / v uncrosslinked or weakly crosslinked hyaluronic acid (HA) powder was dissolved in an oxycellidine solution with a concentration of 1 mg / ml to obtain a final concentration of 10% w / v uncrosslinked or weakly crosslinked hyaluronic acid to prepare a primary release needle tip material.

[0094] Example 4 Preparation of BSA Nanoparticles Containing Oxelidine (Oxelidine@BSA-NP)

[0095] like Figure 3 As shown, a certain amount of sodium hydroxide (NaOH) powder is added to a 1mg / ml oliceridine solution to make the sodium hydroxide concentration 0.1M, and stirred to dissolve to obtain solution A. BSA is added to 0.6% acetic acid (CH3COOH) to make the BSA concentration 1.575mg / mL to obtain an equal volume of solution B. Stir solution A, add solution B, and after both are dissolved, add DDH2O. Mix the solutions and centrifuge. Discard the supernatant, keep the granules, wash with DDH2O, and obtain oliceridine@BSA-NP.

[0096] Example 5 Preparation of cross-linked gelatin containing oxycellidine

[0097] 1. Prepare cross-linked gelatin. Add 4 g of fish glue into 8 ml of deionized water to obtain a gelatin solution. Then, dissolve 20 mg of genipin in 1 ml of ultrapure water containing 10% alcohol. Add 0.2 ml of the genipin solution into 5 g of the prepared gelatin solution to obtain a 0.24% t mixed solution. Stir the mixed solution, cross-link it, dialyze it in deionized water, and freeze-dry the dialyzed solution to obtain a cross-linked gelatin freeze-dried powder.

[0098] 2. Prepare cross-linked gelatin containing oxytocin by dissolving 0.5 g of cross-linked gelatin lyophilized powder in 5 ml of oxytocin solution (1 mg / ml).

[0099] Example 6 Preparation of Oxelidine@BSA-NP Hybrid Structure Microneedles

[0100] Preparation process Figure 4 shown.

[0101] 1. According to the mass ratio of oxycellidine of 1:3, the primary release phase prepared in Example 3 and the BSA nanoparticles containing oxycellidine prepared in Example 4 were mixed to obtain a microneedle tip blend.

[0102] 2. The microneedle tip blend is evenly applied to the microneedle female mold, and the material is filled by differential centrifugation (4000 rpm, 3 mins) / vacuum degassing. After filling, the excess material is scraped off and dried.

[0103] 3. After drying, PVP is injected, vacuum degassing (-0.8 bar, 30 minutes), drying and forming are performed, and finally demolding is performed to obtain a mixed structure microneedle.

[0104] Example 7 Preparation of Separation Structure Microneedles

[0105] Preparation process Figure 5 shown.

[0106] The primary release phase prepared in Example 3 and the cross-linked gelatin containing oxycellidine prepared in Example 5 were respectively applied to the microneedle negative mold, and the materials were filled by differential centrifugation (4000 rpm, 3 mins) / vacuum degassing (-0.8 bar, 30 mins). After filling, the excess material was scraped off and dried; after drying, PVP was injected, and vacuum degassing (-0.8 bar, 30 mins) and drying were performed (37°C, 12h), and finally demolding was performed to obtain a separated structure microneedle.

[0107] Example 8 Detection of Nanoparticles by Transmission Electron Microscopy

[0108] Through transmission electron microscopy, the microscopic morphology of nanoparticles can be seen ( Figure 6 a), and the particle size analysis of the nanoparticles was performed ( Figure 6 b), the results show that the nanostructure is uniform.

[0109] Further testing of the displacement of the mixed solution of oceridine@BSA-NP hybrid microneedle (HA&BSA-NP@Oliceridine), HA&BSA-NP, and HA, the results are shown in Figure 6 c. First, the curve of HA was analyzed: the peak at about 3385cm-1 is the hydroxyl absorption peak, indicating that the intramolecular hydroxyl of HA is related to the intramolecular or intermolecular hydrogen bonds. The peaks at 1615 and 1407cm-1 are the relative and symmetrical stretching vibration peaks of the carboxyl group. The absorption peak is about 1047cm-1, which is the characteristic absorption peak of sugar. Secondly, the FTIR of the precursor was detected with BSA-NP or unmixed HA, and it was found that the peak of HA&Oliceridine@BSA-NP at 700cm-1 is the bending vibration peak of the CS bond, and the peak at 2926cm-1 is the stretching vibration peak of the CN bond, which corresponds to certain structures of the organic alkane molecule. That is, after drug loading, the displacement of HA&BSA-NP@Oliceridine is the smallest, indicating that oceridine is encapsulated in BSA-NP and added to the precursor, proving that the sample does contain the component oceridine.

[0110] Example 9 Mechanical Strength Test of Oxelidine-Carrying Microneedles with Precise Release Control

[0111] Mechanical strength is the key mechanical indicator of whether the microneedle can pierce the skin to release the drug. The microneedle is placed horizontally on a stainless steel platform with the needle tip pointing vertically upward. A mechanical sensor that moves vertically downward at a constant speed (10μm / s) is used to apply force to the microneedle. The changes in displacement and force are continuously recorded until the maximum preset load reaches 10N, 20N, 30N and 80N. Finally, a scanning electron microscope is used to observe the final deformation of the microneedle under different pressures.

[0112] A universal tensile and compression tester was used to test the mechanical properties of the microneedle patch. The test results were displacement-force curves, with a final displacement of 550 μm, measuring the maximum stress of all microneedle tips at 550 μm, and then dividing it by the number of needle tip arrays to obtain the average stress of a single microneedle tip.

[0113] After testing, the yield stress of each microneedle of the hybrid microneedle of Example 6 is about 0.5N, and this mechanical index enables it to effectively pierce the skin. Figure 7 Shown is the displacement-force curve of the microneedle prepared in Example 6.

[0114] Example 10 Detection of in vitro drug release distribution of oxalidin-containing microneedles with precise controlled release

[0115] The microneedles of Example 6 were prepared by adding fluorescent dyes at the same time, and the green dye FITC was encapsulated in the primary release phase, and the red dye Rho.B was encapsulated in the secondary release phase. The simulated skin was prepared by GelMA (gelatin methacrylamide) + HAMA (hyaluronic acid methacrylate) blended hydrogel, and the microneedle diffusion experiment was carried out using microneedles prepared with fluorescent materials. After the microneedles were inserted into the hydrogel, the simulated skin was confocally scanned at different depths at different time points to qualitatively analyze the dissolution and diffusion of the needle body material of the microneedle. The principle of the simulated skin diffusion experiment is as follows: Figure 8 shown.

[0116] Based on the diffusion of microneedles obtained by confocal microscopy, the average fluorescence intensity of the needle material in the GelMA+HAMA simulated skin was calculated over time. The results are shown in Table 1.

[0117] Table 1 Fluorescence intensity values ​​at different times for simulated skin diffusion

[0118]

[0119]

[0120] Example 11 Testing of drug sustained release performance of oxalidin-loaded microneedles in vitro

[0121] 11.1 Microneedle Preparation

[0122] The F127+HA system microneedles and GelMA system microneedles were prepared by referring to the BSA+HA system microneedle preparation method. The only difference is the preparation of the secondary release phase, and the rest of the steps are the same. The details are as follows:

[0123] Preparation of the secondary release phase of F127+HA system microneedles: Weigh an appropriate amount of F127 powder, add it to the oxycellidine injection solution, and mix it on a shaker at 4°C to obtain a F127 hydrogel with a final concentration of 30%, and maintain the concentration of oxycellidine at 1 mg / ml;

[0124] Preparation of the secondary release phase of GelMA system microneedle: Weigh an appropriate amount of GelMA and add it to the oxeridine injection solution, mix it in a 37°C incubator shaker to obtain a final concentration of 8% GelMA hydrogel, and maintain the concentration of oxeridine at 1 mg / ml.

[0125] 11.2 Drug Release Testing

[0126] A Franz diffusion cell is used for precise in vitro detection of drug release. The schematic diagram of the Franz diffusion cell is shown in the figure. Fig. 9 As shown, the following tests were performed using the microneedles prepared in Example 6:

[0127] 1) Using suckling pig skin as a carrier, fixing it between the supply chamber and the receiving chamber of a Franz diffusion cell, and fixing the diffusion cell in a transdermal absorption diffusion instrument;

[0128] 2) With the magnetic stirrer and constant temperature water bath turned on, insert the microneedle sample into the skin surface in the chamber;

[0129] 3) At 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 24h, 48h, 72h, and 96h after insertion, 5ml of solution sample was taken from the diffusion cell, and the oxycellidine concentration in the solution samples at different times was measured by high performance liquid chromatography.

[0130] like Fig.10 As shown, in the in vitro release experiment of the microneedles of Example 6, the BSA+HA system can stably release for 72-96 hours.

[0131] like Fig.11 As shown, compared with F127 (polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer) + HA system microneedles and GelMA system microneedles, in the in vitro release test, the sustained release time of BSA + HA system microneedles was longer and smoother.

[0132] Example 12 Biocompatibility Verification of Oxelidine-Carrying Microneedles with Precise Controlled Release

[0133] The following tests were performed using the microneedles prepared in Example 6:

[0134] 1.1 Intradermal irritation test

[0135] 1) HA was prepared into a sample solution with a concentration of 0.01 g / ml using physiological saline, and another physiological saline solution was taken as a negative control. The injection instrument was sterilized and set aside for use;

[0136] 2) Before the experiment, the mice were depilated on their backs and divided into two groups, one as a control group and the other as a HA group;

[0137] 3) Take 5 points on the back of each mouse, take the sample solution (or control solution) and inject 0.1 ml of the sample solution (or control solution) subcutaneously at each point for testing;

[0138] 4) Observe the skin reaction on the back of the mice 24, 48, and 72 hours after injection and score according to the skin irritation reaction scoring criteria (as shown in Table 1);

[0139] 5) Calculate the average score of each sample per day (Formula 1),

[0140]

[0141] Where: D s-nd is the average score of a sample on day n; D niis the average score of the i-th point of a sample on the nth day; D cj is the average score of the control group at the jth point on the nth day.

[0142] The average score of the three days (D s-nd ) is taken as the average, which is the final score of the sample stimulation response (D s ). Refer to the relevant national standards and determine the irritation level of each sample according to Table 3.

[0143] Table 3. Skin reaction grading standards corresponding to sample scores

[0144] Reaction classification Primary irritation index P Non-irritating 0.0-0.4 Slight irritation 0.5-1.9 Moderate irritation 2.0-4.9 Severe irritation 5.0-8.0

[0145] 1.2 Acute toxicity test

[0146] 1) Randomly group healthy mice and record their initial body weight;

[0147] 2) HA solution and saline were injected into the tail vein of mice, with an injection volume of 1 mL per mouse;

[0148] 3) Weigh the rats 24, 48 and 72 hours after injection, observe the reactions and record the results.

[0149] If there is no significant change in the weight of the mice, no death or poisoning reaction, and no difference from the negative control, it indicates that the HA solution has a negative reaction in the acute systemic toxicity test.

[0150] 1.3 Cytotoxicity test (MTT method)

[0151] 1) The 4th generation well-grown human fibroblast (208F) and macrophage (Raw246.7) cell lines were inoculated into well plates, and each well plate was randomly divided into a control group (200 μL of 10% fetal bovine serum DMEM culture medium was added to each well) and an experimental group (200 μL of HA extract was added to each well);

[0152] 2) Perform cell culture. Add 20 μL of MTT solution to each well at 1, 3, 5, and 7 days after induction culture, with 8 wells at each time point;

[0153] 3) After incubation at 37°C for 4 h, add DMSO (150 μL per well) and rotate at 600 r / min -1 After shaking for 10 min, the absorbance was measured at 490 nm using an enzyme-labeled instrument, and the average was calculated and the results were recorded to obtain the cell growth curve of each group;

[0154] 4) Calculate the relative growth rate (RGR) of cells according to the following formula:

[0155] RGR = A value of experimental group / A value of blank control group × 100%.

[0156] According to the cytotoxicity grading standard (RGR ≥ 100%, toxicity grade is O; when RGR is 75%-99%, 50%-74%, 25%-49%, 1%-24%, and 0%, the toxicity grades are 1, 2, 3, 4, and 5, respectively), the RGR values ​​of each group were converted into material toxicity ratings of 0 to 5.

[0157] 1.4 Cell apoptosis detection

[0158] 1) Take the 4th generation well-grown human fibroblasts and macrophages and place them in a culture dish, add DMEM containing 10% fetal bovine serum, and culture them;

[0159] 2) When the cells grew to 50%, the experimental group was replaced with HA as the culture medium, and the control group was replaced with DMEM culture medium containing 10% fetal bovine serum;

[0160] 3) Continue culturing, and when the cells are 80% confluent, use flow cytometry to detect cell survival rate;

[0161] 4) If the survival rates are all within the normal range, it indicates that the HA extract has no significant effect on the growth of human fibroblasts.

[0162] 1.5 Test results

[0163] The results of various biocompatibility tests showed that the microneedles of Example 6 did not cause skin irritation, mouse toxicity, cytotoxicity, or cell apoptosis, indicating that the microneedles were non-toxic.

[0164] Example 14 Detection of sustained-release performance of oxeridine by microneedles in vivo (animal behavior test)

[0165] A. Experimental Animals

[0166] C57 / BL6 male mice

[0167] B. Selective sciatic nerve branch injury (SNI) model

[0168] Anesthetize mice and sterilize them;

[0169] Make precise incisions at designated locations on the left lower limb to fully expose the sciatic nerve trunk and its three important branches - the tibial nerve, the common peroneal nerve, and the sural nerve;

[0170] Using a micro glass needle, the tibial and common peroneal nerves are gently lifted and carefully ligated with sutures;

[0171] Then, the ligated nerve is cut at its distal end and the stump is shortened by 1-2 mm to ensure that the severed nerve cannot heal, while taking care to protect the sural nerve;

[0172] The wound was carefully sutured and the mouse was returned to the home cage to be allowed to recover on its own;

[0173] C. Selective sciatic nerve injury (SNI) grouping

[0174] Twenty-four male C57 / BL6 mice were randomly divided into four groups: SNI (n=6), SNI+BSA-Oliceridine.ad (n=6), SNI+Oliceridine.H (n=6) and Sham (n=6).

[0175] The sham group (Sham) received the same superficial skin incision as the surgical group (SNI), but no nerve injury was performed. In the oliquidine microneedle treatment group (SNI+BSA-oliquidine.ad), microneedles loaded with oliquidine were applied to the shaved area on the back of SNI mice. At the same time, the oliquidine injection subcutaneous injection group (SNI+oliquidine.H) was injected subcutaneously with an equal dose of oliquidine injection in the same skin area of ​​SNI mice. Subsequently, the changes in mechanical pain threshold of each group were measured at different time points after treatment.

[0176] D. Determination of Model Success

[0177] Seven days after modeling, the mechanical pain threshold of the neuralgia model mice was tested by the up-down method. The significant decrease in the pain threshold on the lateral side of the left plantar indicated that the modeling was successful.

[0178] E. Microneedle Use

[0179] After the model was successfully established, the mice in the (SNI+BSA-Oliceridine.ad) group were given the microneedles of Example 6. The microneedles were evenly pressed on the target skin to ensure that all the needle tips of the microneedle array penetrated the stratum corneum of the mouse skin.

[0180] F. Test Results

[0181] like Fig.12 As shown in the figure, in the mouse neuropathic pain model (SNI) group, the mechanical pain threshold was significantly decreased compared with the sham operation control group, indicating that the model was successfully established. After oxycellidine microneedle treatment, the mechanical pain threshold increased significantly within 1 hour and lasted for nearly 96 hours, showing a good and stable analgesic effect. After subcutaneous injection of oxycellidine, the mechanical pain threshold also increased significantly within 1 hour, but it dropped again to the SNI baseline level after 4 hours, indicating that the analgesic effect disappeared.

Claims

1. A drug microneedle, wherein the drug is a short half-life drug, and the microneedle comprises a microneedle body and a substrate; the microneedle body can be a mixed structure comprising a primary release phase and a secondary release phase, or a separated structure comprising a microneedle body I and a microneedle body II; the microneedle body I comprises a primary release phase, and the microneedle body II comprises a secondary release phase; the primary release phase comprises a primary release matrix and a drug, and the secondary release phase comprises a secondary release matrix and a drug.

2. The drug microneedle according to claim 1, wherein the primary release matrix is ​​selected from one or more of uncrosslinked or weakly crosslinked hyaluronic acid, uncrosslinked or weakly crosslinked chitosan, HAMA (Hyaluronic Acid Methacrylate Hyaluronic Acid Methacrylate), GelMA (Gelatin Methacryloyl Gelatin Methacrylamide) and / or F127 (polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer); the concentration of the primary release matrix is ​​5%-20%.

3. The drug microneedle according to any one of claims 1 or 2, wherein when the microneedle is a mixed structure, the secondary release matrix is ​​selected from one or more of bovine serum albumin (BSA) nanoparticles, liposome nanoparticles and / or nanofibers; when the microneedle is a separated structure, the secondary release matrix is ​​selected from one or more of medium-strength to strong cross-linked gelatin and / or hydrogel microspheres.

4. The drug microneedle according to any one of claims 1 to 3, wherein the short half-life drug is a drug with a half-life of less than 4 hours, and the drug is selected from one or more of oxeridine, oxycodone, dezocine, fentanyl, morphine, remifentanil, hydromorphone and / or tramadol.

5. The drug microneedle according to any one of claims 1 to 4, wherein the substrate is an organic material selected from one or more monomers or cross-linked products of hyaluronic acid, gelatin, chitosan, polyethylene glycol, polyvinyl alcohol and / or polyvinyl pyrrolidone (PVP).

6. A method for preparing a microneedle for a short half-life drug, the method comprising the following steps: 1) Preparation of hybrid structure microneedles S1. Preparing a negative microneedle mold; S2. Preparation of primary release phase: dissolving the primary release matrix and the drug in a solvent to form a primary release phase; S3. Preparation of secondary release phase: mixing one or more of the secondary release matrix bovine serum albumin (BSA) nanoparticles, liposome nanoparticles and / or nanofibers with the drug to form a secondary release phase; S4. Mixing the primary release phase prepared in step S2 and the secondary release phase prepared in step S3 to obtain a microneedle tip blend, applying the microneedle tip blend on a microneedle negative mold, injecting a substrate material, and obtaining a hybrid structure microneedle; 2) Preparation of separation structure microneedles N1. Prepare a negative microneedle mold; N2. Preparation of primary release phase: dissolving the primary release matrix and the drug in a solvent to form a primary release phase; N3. Preparation of secondary release phase: Mixing the secondary release matrix of moderately to strongly cross-linked gelatin and / or hydrogel microspheres with the drug to form the secondary release phase; N4. Apply the primary release phase prepared in step N2 and the secondary release phase prepared in step N3 to the microneedle negative mold respectively, inject the substrate material, and form separated microneedles.

7. The preparation method as claimed in claim 6, wherein the primary release matrix is ​​selected from one or more of uncrosslinked or weakly crosslinked hyaluronic acid, uncrosslinked or weakly crosslinked chitosan, HAMA (Hyaluronic Acid Methacrylate Hyaluronic Acid Methacrylate), GelMA (Gelatin Methacryloyl Gelatin Methacrylamide) and / or F127 (polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer); the drug is a drug with a half-life of less than 4 hours, and the drug is selected from one or more of oxycodone, oxycodone, dezocine, fentanyl, morphine, remifentanil, hydromorphone and / or tramadol; the substrate material is selected from one or more of monomers or crosslinkers of hyaluronic acid, gelatin, chitosan, polyethylene glycol, polyvinyl alcohol and / or polyvinyl pyrrolidone (PVP).

8. The preparation method according to any one of claims 6 or 7, wherein in steps S2 and N2, the concentration of the primary release matrix is ​​5%-20%; In steps S4 and N4, the mass ratio of the drug in the primary release phase and the secondary release phase is 1-2:3-5; In step N3, the concentration of the medium-strength cross-linked gelatin and hydrogel microspheres is 0.05-0.5 g / ml.

9. An application of a drug microneedle in the preparation of a drug for treating a disease; the drug is a drug with a short half-life, and the microneedle comprises a microneedle body and a substrate; the microneedle body can be a mixed structure comprising a primary release phase and a secondary release phase, or a separated structure comprising a microneedle body I and a microneedle body II; the microneedle body I comprises a primary release phase, and the microneedle body II comprises a secondary release phase; the primary release phase comprises a primary release matrix and a drug, and the secondary release phase comprises a secondary release matrix and a drug.

10. The use according to claim 9, wherein the disease is selected from one or more of painful diseases such as neuropathic pain, joint pain, traumatic pain and / or cancer pain.

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