WO2024051132A1 - 一种秋水仙碱水凝胶微针及其制备方法 - Google Patents

一种秋水仙碱水凝胶微针及其制备方法 Download PDF

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WO2024051132A1
WO2024051132A1 PCT/CN2023/081347 CN2023081347W WO2024051132A1 WO 2024051132 A1 WO2024051132 A1 WO 2024051132A1 CN 2023081347 W CN2023081347 W CN 2023081347W WO 2024051132 A1 WO2024051132 A1 WO 2024051132A1
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colchicine
hydrogel
microneedles
microneedle
col
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PCT/CN2023/081347
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English (en)
French (fr)
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彭灿
刘欢欢
江素萍
柯寄明
张静
左池靖
方灿灿
桂双英
彭代银
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安徽中医药大学
安徽百草精研医药科技有限公司
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Publication of WO2024051132A1 publication Critical patent/WO2024051132A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
    • A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C35/00—Heating, cooling or curing, e.g. crosslinking or vulcanising; Apparatus therefor
    • B29C35/02—Heating or curing, e.g. crosslinking or vulcanizing during moulding, e.g. in a mould
    • B29C35/08—Heating or curing, e.g. crosslinking or vulcanizing during moulding, e.g. in a mould by wave energy or particle radiation
    • B29C35/0805—Heating or curing, e.g. crosslinking or vulcanizing during moulding, e.g. in a mould by wave energy or particle radiation using electromagnetic radiation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C37/00—Component parts, details, accessories or auxiliary operations, not covered by group B29C33/00 or B29C35/00
    • B29C37/006—Degassing moulding material or draining off gas during moulding
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C41/00—Shaping by coating a mould, core or other substrate, i.e. by depositing material and stripping-off the shaped article; Apparatus therefor
    • B29C41/02—Shaping by coating a mould, core or other substrate, i.e. by depositing material and stripping-off the shaped article; Apparatus therefor for making articles of definite length, i.e. discrete articles
    • B29C41/04—Rotational or centrifugal casting, i.e. coating the inside of a mould by rotating the mould
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
    • A61M2037/0053—Methods for producing microneedles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C35/00—Heating, cooling or curing, e.g. crosslinking or vulcanising; Apparatus therefor
    • B29C35/02—Heating or curing, e.g. crosslinking or vulcanizing during moulding, e.g. in a mould
    • B29C35/08—Heating or curing, e.g. crosslinking or vulcanizing during moulding, e.g. in a mould by wave energy or particle radiation
    • B29C35/0805—Heating or curing, e.g. crosslinking or vulcanizing during moulding, e.g. in a mould by wave energy or particle radiation using electromagnetic radiation
    • B29C2035/0827—Heating or curing, e.g. crosslinking or vulcanizing during moulding, e.g. in a mould by wave energy or particle radiation using electromagnetic radiation using UV radiation

Definitions

  • the invention relates to the field of medical technology, and in particular to a colchicine hydrogel microneedle and a preparation method thereof.
  • Gout is a common inflammatory arthritis in developed and developing countries, caused by the accumulation of sodium urate (MSU) crystals.
  • a form of acute gouty arthritis (AGA) characterized by extreme pain, swelling, fever, and difficulty moving the affected joints, seriously affecting daily life. Therefore, AGA has always been the focus of basic research in the entire medical field.
  • TNF tumor necrosis factor
  • IL-6 interleukin-6
  • IL-1 ⁇ IL-1 ⁇
  • Cell function including non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids and colchicine and other inflammatory factors to reduce the inflammatory response.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • Microneedle is a new painless transdermal drug delivery technology with broad application prospects and high safety. Compared with other MNs, hydrogel microneedles have higher drug loading capacity and adjustable drug release, thereby avoiding toxic reactions caused by burst release of colchicine. Generally speaking, hydrogel microneedles have a certain hardness in a dry state, and after entering the skin, they absorb interstitial fluid and expand into a 3D network, thereby releasing drugs. These factors are often directly related to the polymer cross-linking ratio, but the uneven distribution of cross-linking points in the polymer network makes traditional hydrogel microneedles less brittle, resulting in thicker cuticles such as elbow joints. Difficult to use on skin. The incompatibility between the mechanical properties and swelling properties of hydrogel microneedles makes it difficult to release drugs within a reasonable application time of the microneedles.
  • the primary purpose of the present invention is to provide a method for preparing colchicine hydrogel microneedles.
  • the colchicine hydrogel microneedles prepared by this method have high swelling properties and high Mechanical toughness.
  • a preparation method of colchicine hydrogel microneedles including the following steps:
  • the mass ratio of acrylamide, N, N'-bis (acryloyl) cysteamine and Irgacure 2959 in the transparent gel solution to the transparent gel solution is 10 to 35wt%, respectively. 0.01 ⁇ 0.1wt% and 0.01 ⁇ 0.2wt%.
  • the speed of the low-speed centrifugation is 3000-4000 rpm/min, and the time is 5-20 min.
  • the irradiation conditions under ultraviolet light are 315-400 nm, 100-500 W, and the irradiation time is 10-60 min.
  • the invention also provides a colchicine hydrogel microneedle, which is prepared by the above preparation method.
  • the present invention has the following beneficial effects:
  • the hydrogel microneedles of the present invention are prepared by a highly hydrophilic monomer (acrylamide) and a cross-linking agent (N, N'-bis(acryloyl)cysteamine) with UV-responsive disulfide bonds. , overcoming the shortcomings of uneven cross-linking points of ordinary hydrogel microneedles, the prepared hydrogel microneedles have a complete needle shape, an orderly matrix arrangement, a uniform distribution of network cross-linking points, and excellent mechanical toughness (>10N/ needle) and super swelling capacity (>2500%);
  • the cumulative release rate of the colchicine hydrogel microneedles prepared by the present invention is greater than 80% within 48 hours.
  • the release curve conforms to the first-order equation and is biocompatible. It can effectively treat inflammation of acute gout in rats. , providing new ideas and strategies for the development and treatment of new colchicine therapeutic dosage forms.
  • Figure 1 is a schematic diagram of continuous single factor optimization
  • Figure 2 shows the gel solution formed by different contents of AM (0.5, 1 and 2g), Irgacure 2959 (1, 5 and 10mg) and BACA (1, 2 and 4mg) in the hydrogel microneedle of the present invention in 5mL water. Study of the influence of microneedle rupture force and swelling behavior of hydrogels.
  • Figure 3 shows the appearance characterization of MN of the present invention, (a) photo of microneedles; (b) optical microscopy image of microneedles, 4 ⁇ ; (c) optical microscopy of microneedles (bottom, 4 ⁇ ) and calcein Comparison of fluorescence inverted microscopy of microneedles (top, 5 ⁇ ); (d, e) scanning electron microscopy images of microneedles; (f) SEM image of freeze-dried microneedles showing porous structure;
  • FIG. 4 is an energy spectrum analysis (EDS) image of the microneedle of the present invention.
  • Figure 5 is a microscope picture of the colchicine microneedles of the present invention.
  • Figure 6 shows the in vitro characterization of the microneedles of the present invention, (a) force-displacement curves of microneedles prepared with different cross-linking agents and initiators, (i) prepared using BACA as the cross-linking agent and Irgacure 2959 as the photoinitiator. Microneedles; (ii) Blank-MN prepared by using MBA as cross-linking agent and Irgacure 2959 as photoinitiator; (iii) Blank-MN prepared by using BACA as cross-linking agent and K 2 O 8 S 2 as thermal initiator.
  • Figure 7 is a study on the insertion of microneedles of the present invention, (a) Membrane layer insertion rate; (b) methylene blue staining image of rat skin insertion hole; (c) confocal microscope image of calcein microneedle skin insertion;
  • Figure 8 is a diagram showing the in vitro and in vivo biocompatibility studies of microneedles of the present invention.
  • Figure 9 shows the swelling and inflammatory cytokine levels of rat ankle joints.
  • MSU monosodium urate
  • MSU monosodium urate
  • Rat Changes in foot volume over time after ankle joint injection of monosodium urate (MSU) crystal suspension and normal saline (n 6)
  • MSU monosodium urate
  • d-f Levels of serum inflammatory factors IL-1 ⁇ (d), IL-6(e), and tumor necrosis factor- ⁇ (f) in rats after treatment;
  • the standard mechanical test of MN was performed using the compression mode of TMS-Pilot texture analyzer. Take out a needle from the MN and place it on the stainless steel bottom plate of the texture analyzer with the needle tip facing up. The cylindrical probe with a diameter of 6mm falls at a speed of 30mm/min. When it comes into contact with the needle array, a trigger force of 0.03N is applied and the force is maintained until a displacement of 0.3mm is reached.
  • SPSSAU software https://spssau.com/ was used to normalize the data, eliminate the unit dimension, and calculate the variability, conflict, information content, weight coefficient and comprehensive score between indicators.
  • a preparation method of colchicine hydrogel microneedles including the following steps:
  • step (1) N,N-methylenebisacrylamide (MBA) is used instead of N,N'-bis(acryloyl)cysteamine (BACA) as the cross-linking agent .
  • MCA N,N-methylenebisacrylamide
  • BACA N,N'-bis(acryloyl)cysteamine
  • step (1) 30 mg of potassium persulfate (K 2 O 2 S 2 ) is used instead of Irgacure-2959 as the initiator, and in step (3), heating in an oven for 6 hours is used instead of 20 min of UV irradiation. .
  • Example 1 The microneedles prepared in Example 1 and Comparative Examples 1-2 were subjected to the standard mechanical test of MN using the compression mode of the TMS-Pilot texture analyzer. The results are shown in Figure 6a.
  • the microneedle rupture force of Example 1 is 11.53 N/needle
  • the microneedle rupture force of Comparative Example 1 is 8.11N/needle
  • the microneedle rupture force of Comparative Example 2 is 3.61N/needle.
  • Example 1 requires greater force to achieve the same compression amount, indicating that the mechanical strength of Example 1 is higher than Comparative Examples 1 and 2.
  • Previous studies have determined that each needle requires more than 0.058N to insert the microneedle into the skin.
  • the displacement of the microneedle in Example 1 under the required force is less than 0.1mm, indicating that the mechanical strength of the microneedle prepared by the present invention is large enough. Enough to penetrate skin.
  • the present invention found through research that the contents of acrylamide monomer (AM), cross-linking agent (BACA) and photoinitiator have a relatively large impact on the physical and chemical properties of hydrogel microneedles.
  • AM acrylamide monomer
  • BACA cross-linking agent
  • photoinitiator have a relatively large impact on the physical and chemical properties of hydrogel microneedles.
  • the present invention optimizes the process parameters of microneedles through continuous single-factor experiments, and studies different contents of AM (0.5, 1 and 2g), Irgacure 2959 (1, 5 and 10mg) and BACA (1, 2 and 4 mg) in 5 mL of pure water to form a pre-gel solution on the rupture force of Blank-MN and the swelling of the hydrogel respectively.
  • the factor level table is shown in Table 1 below, and the comparison is carried out according to the order in Figure 1:
  • the three-dimensional structure of the microneedles and the surface morphology of the microneedles after freeze-drying were observed using optical microscopy and scanning electron microscopy (SEM). Use an inverted fluorescence microscope to observe the fluorescence distribution at the tip of the calcein microneedle. Scanning microneedle tips with energy dispersive analyzer (EDS) C, H, O, S elements.
  • EDS energy dispersive analyzer
  • the hydrogel microneedle is the product (Blank-MN) obtained in steps (1-3) of Example 1.
  • the difference between the preparation of freeze-dried microneedles and the steps (1-3) of Example 1 is that freeze-drying is used instead of air-drying in step (3).
  • the difference between the preparation of calcein microneedles and steps (1-4) of Example 1 is that calcein is used instead of colchicine in step (1).
  • the uniformity of electric potential may be the reason for the uniform mechanical properties, or it may be the fundamental reason that determines its macroscopic mechanical behavior.
  • the surface has uniform pores (Figure 3f), and this porous structure is beneficial to the dissolution and diffusion of drugs.
  • Energy spectroscopy (EDS) results showed that the disulfide bonds in the hydrogel microneedles were evenly distributed ( Figure 4).
  • the colchicine hydrogel microneedle is the product (COL-MN) obtained in steps (1-4) of Example 1.
  • the colchicine hydrogel microneedle prepared in Example 1 has a good apparent structure, has a sharp tip, the array is evenly arranged, the tip structure is uniform, and there is no structural damage compared with the hydrogel microneedle.
  • PBS phosphate buffer solution
  • the hydrogel microneedle is the product (Blank-MN) obtained in steps (1-3) of Example 1.
  • the swelling rate of the hydrogel microneedles (Blank-MN) prepared by the present invention reaches equilibrium in 8 hours, and the maximum swelling degree is 2708%.
  • the colchicine hydrogel microneedle is the product (COL-MN) obtained in steps (1-4) of Example 1.
  • the cumulative release rate of colchicine hydrogel microneedles can reach more than 80% within 48 h, and the first-order kinetic equation is used to bridge the release curve, indicating that it is consistent with colchicine hydrogel microneedles ( COL-MN) has a sustained release effect.
  • This method uses calcein instead of colchicine as a simulated drug, and uses the fluorescence distribution of calcein in the skin to characterize the diffusion behavior of the drug.
  • the rest is the same as in Example 1.
  • the isolated rat abdominal skin was taken from the -20°C refrigerator, thawed, equilibrated in physiological saline for 30 minutes, and then cut appropriately according to the diameter of the diffusion pool.
  • the bottom of the plastic wrap is the dermis layer, the top is the epidermis layer, and the skin is covered on the plastic wrap.
  • the epidermis was dried with filter paper. Apply microneedles for 30 seconds.
  • the receptor chamber is filled with fluid until it contacts the skin, and a timer is started.
  • the skin was removed at 2, 6, 12, 24, and 48 hours, washed, and frozen sections were observed under a laser scanning confocal microscope.
  • the drug penetration process includes the penetration process of the drug from the microneedle to the skin and the diffusion process of the drug from the epidermis to the dermis. According to the fluorescence distribution, the former process dominates within 12 hours, the latter process dominates within 12 to 24 hours, and calcein is still continuously released in the skin within 24 to 48 hours.
  • the colchicine hydrogel microneedle is the product (COL-MN) obtained in steps (1-4) of Example 1.
  • the membranes are arranged layer by layer with a depth of approximately 1mm. Place it on a piece of dental wax as a mechanical support and apply "thumb pressure" to each array prototype of Example 1 colchicine hydrogel microneedles. The penetration of the microneedles was assessed by counting the pores produced in each layer using a light microscope. Calculate according to formula (3): the percentage of pores in each layer of membrane.
  • FIG. 7a the penetration results of colchicine hydrogel microneedles (COL-MN) are shown.
  • the insertion rate of colchicine hydrogel microneedles (COL-MN) is close to 100%, indicating that the insertion rate of colchicine hydrogel microneedles (COL-MN) prepared by the present invention is Colchicine hydrogel microneedles (COL-MN) have sufficient ability to penetrate the skin.
  • the colchicine hydrogel microneedle is the product (COL-MN) obtained in steps (1-4) of Example 1.
  • FIG. 7b it shows that the colchicine hydrogel microneedles (COL-MN) prepared by the present invention can penetrate the skin with an efficiency close to 100%.
  • Calcein was used instead of colchicine as the simulated drug.
  • Blank-MN is the product obtained in steps (1-3) of Example 1.
  • COL-MN is the product obtained in steps (1-4) of Example 1.
  • CCK-8 kit was used to detect the cytotoxicity of 5 mg COL, Blank-MN and COL-MN (containing 5 mg COL) on the activity of human keratinocytes (HaCaT).
  • HaCaT human keratinocytes
  • three samples were soaked in 10 mL of DMEM complete medium for 24 hours.
  • HaCaT cells were seeded on a 96-well plate at a density of 1 ⁇ 10 6 cells/well and cultured overnight. Then, the cells were incubated in different sample soaking solutions for 24 h. The treated cells were incubated with 10% CCK-8 at 37°C for 1 h, and the absorbance was measured at 450 nm using a microplate reader. Measure three times. Results are expressed as percentage of cell viability compared with untreated control cells.
  • Blank-MN and COL-MN showed higher cell safety compared with the COL group.
  • Blank-MN is the product obtained in steps (1-3) of Example 1.
  • COL-MN is the product obtained in steps (1-4) of Example 1.
  • IL-1 ⁇ is the first and most important event in gout inflammation.
  • TNF- ⁇ enhances neutrophil activity.
  • the pro-inflammatory cytokine IL-6 is key to initiating the innate immune response.
  • Exposure of synovial fluid to sodium urate crystals results in necrosis, macrophage release, neutrophil death, and inflammation.
  • MSU sodium urate crystals
  • COL tablet solution i.g. COL
  • mice Male SD rats (weight 180g-200g) aged 6-8 weeks were provided by the Animal Experiment Center of Anhui University of Traditional Chinese Medicine. All animal experiments were approved by the Ethics Committee of Anhui University of Traditional Chinese Medicine. Animal ethics number: AHUCM-rats-2019001. Rats were raised under standardized conditions in animal facilities and had no food or water for 12 hours before the experiment.
  • MSU Sodium urate
  • the rats were randomly divided into 4 groups: 1) blank group (Blank), 2) model group (Model), 3) colchicine hydrogel microneedle group (COL-MN), 4) colchicine tablet solution irrigation Gastric group (i.g.COL), 10 animals in each group. Except for the blank group, acute gout models were established in other rats. 2 hours later, COL-MN containing 1 mg of COL was administered to the ankles of the rats in the COL-MN group and fixed with medical tape. Rats in i.g. COL group were given COL tablet aqueous solution (1mg/mL) 0.5mg/kg.
  • the initial volume of rat toes was measured using a toe volume measuring instrument. After 24 hours, the toe volume of rats in each group was measured.
  • the swollen foot volume of rats in the model control group increased by 80%.
  • the relational change in foot swelling volume increased to about 50% and gradually decreased to less than 37% of the thickness of the model group (Fig. 9c).
  • rat abdominal aortic blood let it stand for 2 hours and then centrifuge. Take the upper serum and store it at -80°C.
  • the levels of inflammatory factors such as IL-1 ⁇ , TNF- ⁇ , and IL-6 in the four groups were analyzed and measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. Measure the absorbance value (OD) using a microplate reader.
  • ELISA enzyme-linked immunosorbent assay
  • the synovial cavity of the injected ankle joint was flushed with 30 ⁇ L (10 ⁇ L, 3 times) of PBS to obtain synovial lavage samples.
  • the total protein content was determined using the Bradford (1976) colorimetric method with bovine serum albumin (BSA) as the standard.
  • MPO Myeloperoxidase
  • the MN and COL-MN prepared by the present invention have complete needle shapes, neat matrix arrangement, uniform distribution of network cross-linking points, and excellent mechanical properties and swelling properties.
  • the drug loading capacity of COL-MN can reach an effective therapeutic dose, and transdermal diffusion has a sustained-release effect.
  • COL-MN can exert a good anti-inflammatory effect through transdermal administration and effectively treat acute gout induced by sodium urate crystals in rats.

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Abstract

一种秋水仙碱水凝胶微针的制备方法，包括步骤：将丙烯酰胺、N，N'-双(丙烯酰基)半胱胺和Irgacure 2959溶于超纯水中，得到透明凝胶溶液；将得到的透明凝胶溶液倒入PDMS模具中，低速离心，然后超声，消除气泡；将PDMS模具置于紫外光下照射，然后在烘箱中风干，得到水凝胶微针；在水凝胶微针中加入秋水仙碱溶液，溶胀后风干，脱模，即得。克服了普通水凝胶微针交联点不均匀的缺点，制备得到的水凝胶微针的针型完整，矩阵排列整齐，网络交联点分布均匀，具有优异的机械韧性和超强溶胀能力。

Description

一种秋水仙碱水凝胶微针及其制备方法 技术领域
本发明涉及医药技术领域，具体涉及一种秋水仙碱水凝胶微针及其制备方法。
背景技术
痛风是发达国家和发展中国家常见的炎症性关节炎，由尿酸钠(MSU)晶体积累引发。一种急性痛风关节炎(AGA)，表现为极度疼痛、肿胀、发烧和受影响关节活动困难，严重影响日常生活。因此，AGA始终是整个医学领域的基础研究焦点。目前，有几种有效的治疗方法可以缓解AGA的疼痛和炎症反应，即通过抑制肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6、IL-1β等多种炎症因子，抑制炎症细胞功能，包括非甾体抗炎药(NSAID)、皮质激素和秋水仙碱等多种炎症因子来降低炎症反应。然而，经常口服COL可导致不良反应，如胃肠道副反应。2007年秋水仙碱贴剂被注册为药品，这可能为透皮给药领域开辟新的机遇。虽然透皮方法可以绕过上述限制，但是秋水仙碱的高水溶性和较差的皮肤渗透性使透皮给药具有挑战性。
微针(MN)是一种具有广阔应用前景的新型无痛透皮给药技术，具有较高的安全性。与其他MN相比，水凝胶微针具有更高的载药能力和可调的药物释放，从而避免了秋水仙碱爆发释放引起的毒性反应。一般来说，水凝胶微针在干燥状态下具有一定的硬度，并在进入皮肤后吸收间质液体膨胀成3D网络，从而释放药物。这些因素往往与聚合物交联比直接相关，但交联点在聚合物网络中的不均匀分布，使得传统的水凝胶微针具有较弱的脆性，导致在肘关节等较厚的角质层皮肤中使用困难。水凝胶微针的力学性能和膨胀性能的不兼容，使得药物难以在微针合理应用时间内释放。
因此，开发一种具有高溶胀性和高机械韧性的秋水仙碱水凝胶微针具有重要的前景。
发明内容
为了克服现有技术的缺点与不足，本发明的首要目的在于提供一种秋水仙碱水凝胶微针的制备方法，该方法制备得到的秋水仙碱水凝胶微针具有高溶胀性和高机械韧性。
本发明是通过以下技术方案实现的：
一种秋水仙碱水凝胶微针的制备方法，包括如下步骤：
(1)将丙烯酰胺、N，N’-双(丙烯酰基)半胱胺和Irgacure 2959溶于超纯水中，得 到透明凝胶溶液；
(2)将得到的透明凝胶溶液倒入PDMS模具中，低速离心，然后超声，消除气泡；
(3)将盛有透明凝胶溶液的PDMS模具置于紫外光下照射，然后在烘箱中风干，得到水凝胶微针；
(4)在水凝胶微针中加入秋水仙碱溶液，溶胀后风干，脱模，得到秋水仙碱水凝胶微针。
优选地，步骤(1)中，所述透明凝胶溶液中丙烯酰胺、N，N’-双(丙烯酰基)半胱胺和Irgacure 2959与透明凝胶溶液的质量比分别为10～35wt％、0.01～0.1wt％和0.01～0.2wt％。
优选地，步骤(2)中，所述低速离心的速度为3000～4000rpm/min，时间为5～20min。
优选地，步骤(3)中，所述紫外光下照射的条件为315～400nm，100～500W，照射时间为10～60min。
本发明还提供一种秋水仙碱水凝胶微针，由上述制备方法制备得到。
与现有技术相比，本发明具有如下有益效果：
(1)本发明水凝胶微针通过高度亲水的单体(丙烯酰胺)与具有紫外光响应性二硫键的交联剂(N，N’-双(丙烯酰基)半胱胺)制备，克服了普通水凝胶微针交联点不均匀的缺点，制备得到的水凝胶微针的针型完整，矩阵排列整齐，网络交联点分布均匀，具有优异的机械韧性(＞10N/针)和超强溶胀能力(＞2500％)；
(2)本发明制备得到的秋水仙碱水凝胶微针在48h内的累积释放率大于80％，释放曲线符合一级方程，且具有生物相容性，可以有效治疗大鼠急性痛风的炎症，为新型秋水仙碱治疗剂型的开发和治疗提供新的思路和策略。
附图说明
图1为连续单因素优化示意图；
图2为本发明水凝胶微针中不同含量的AM(0.5、1和2g)、Irgacure 2959(1、5和10mg)和BACA(1、2和4mg)在5mL水形成的凝胶溶液对微针的破裂力和水凝胶的溶胀行为的影响的研究。其中，(a,b)为单体丙烯酰胺(AM)对力-位移曲线、溶胀比(n＝3)的影响；(c,d)为光引发剂(Irgacure 2959)对MN的力-位移曲线、溶胀比的影响(n＝3)；(e,f)为交联剂(BACA)对MN的力-位移曲线、溶胀比的影响(n＝3)；
图3为本发明MN的表观表征，(a)微针的照片；(b)微针的光学显微图像，4×；(c)微针的光学显微镜(下，4×)和钙黄绿素微针的荧光倒置显微镜(上，5×)的比较；(d，e)微针的扫描电镜图像；(f)显示多孔结构的冻干微针的SEM图像；
图4为本发明微针的能谱分析(EDS)图像；
图5为本发明秋水仙碱微针的显微镜图片；
图6为本发明的微针的体外表征，(a)不同交联剂和引发剂制备的微针的力-位移曲线，(i)以BACA为交联剂，以Irgacure 2959为光引发剂制备的微针；(ii)以MBA为交联剂，Irgacure 2959为光引发剂制备的Blank-MN；(iii)以BACA为交联剂，K2O8S2为热引发剂制备的Blank-MN；(b)本发明微针的溶胀率-时间曲线(n＝3)；(c)秋水仙碱微针的药物累积透皮释放与一级动力学拟合曲线(n＝6)；(d)钙黄绿素微针体外皮肤释放的皮肤渗透行为在共聚焦显微镜下成像；
图7为为本发明微针的插入研究，(a)膜层插入率；(b)大鼠皮肤插入孔的亚甲蓝染色图；(c)钙黄绿素微针皮肤插入物的共聚焦显微镜图像；
图8为本发明微针的体内外生物相容性研究图，(a)空白微针、秋水仙碱微针和秋水仙碱溶液对人类永生化表皮细胞(HaCaT)存活率的影响；(b)各组微针应用于大鼠腹部皮肤后的HE染色切片图混合TUNEL染色切片图；
图9为大鼠踝关节肿胀及炎性细胞因子水平，(a)左踝关节注射尿酸单钠(MSU)晶体混悬液与右踝关节注射生理盐水后的水肿图像比较；(b)大鼠踝关节注射尿酸单钠(MSU)晶体混悬液和生理盐水后足部体积随时间的变化(n＝6)；(c)COL-MN治疗后急性痛风大鼠的足趾肿胀度；(d-f)治疗后大鼠血清炎性因子IL-1β(d)、IL-6(e)、肿瘤坏死因子-α(f)水平；
图10为经COL-MN治疗后血浆外渗(a)和髓过氧化物酶活性(b)的反应(n＝6)。
具体实施方式
下面通过具体实施方式来进一步说明本发明，以下实施例为本发明较佳的实施方式，但本发明的实施方式并不受下述实施例的限制。
性能测试方法：
1.1、质构仪表征
采用TMS-Pilot质构仪的压缩模式进行MN的标准力学测试。从MN取出一根针，放置在质构仪的不锈钢底板上，针尖朝上。直径为6mm的圆柱形探头以30mm/min的速度落下，与针阵接触时，施加0.03N的触发力，并持续保存力，直到达到0.3mm的位移。
1.2、溶胀度表征
根据Mt-M0/M0方程计算微针的溶胀能力，其中M0和Mt代表各配方水凝胶在磷酸盐缓冲 液(PBS 7.4)中浸泡0-24h前后的质量。
1.3、CRITIC权重分析
利用SPSSAU软件(https://spssau.com/)对数据进行归一化处理，剔除单位维，计算指标间的变异性、冲突性、信息量、权重系数和综合得分。
实施例1：
一种秋水仙碱水凝胶微针的制备方法，包括如下步骤：
(1)将1g丙烯酰胺(AM)、2mg N，N’-双(丙烯酰基)半胱胺(BACA)和5mg Irgacure 2959溶于5mL超纯水中，得到透明凝胶溶液；
(2)将得到的透明凝胶溶液倒入PDMS模具中，低速离心(3500rpm，5min)，然后超声，消除气泡；
(3)将盛有透明凝胶溶液的PDMS模具置于紫外光(365nm，300w)下照射20min，然后在烘箱中风干，得到水凝胶微针(Blank-MN)；
(4)在水凝胶微针中加入秋水仙碱溶液，溶胀12h后风干，脱模，得到秋水仙碱水凝胶微针(COL-MN)。
对比例1：
本对比例与实施例1的区别为步骤(1)中用N，N-亚甲基双丙烯酰胺(MBA)代替N，N’-双(丙烯酰基)半胱胺(BACA)作为交联剂。
对比例2：
本对比例与实施例1的区别为步骤(1)中用30mg过硫酸钾(K2O2S2)代替Irgacure-2959作为引发剂，步骤(3)用在烘箱中加热6h代替紫外光照20min。
将实施例1和对比例1-2制备得到的微针，采用TMS-Pilot质构仪的压缩模式进行MN的标准力学测试，结果如图6a所示，实施例1的微针破裂力为11.53N/needle，对比例1的微针破裂力为8.11N/needle，对比例2的微针破裂力为3.61N/needle。相比于对比例1和2，实施例1需要更大的力，才能达到相同的压缩量，说明实施例1的机械强度高于对比例1和2。以前的研究已经确定每根针需要大于0.058N才能使微针插入皮肤，实施例1的微针在所需的力下的位移小于0.1mm，表明本发明制备得到的微针机械强度足够大，足以穿透皮肤。
另外，本发明通过研究发现，丙烯酰胺单体(AM)、交联剂(BACA)及光引发剂的含量对水凝胶微针的理化性能有比较大的影响。为此，本发明通过连续单因素实验优化微针的工艺参数，研究了不同含量AM(0.5、1和2g)、Irgacure 2959(1、5和10mg)和BACA(1、 2和4mg)在5mL纯水中形成的预凝胶溶液分别对Blank-MN的破裂力和水凝胶的溶胀的影响。因素水平表见下表1，根据图1中顺序实施对比：
表1
结合图2与表2-3，当微针溶液中AM的含量为0.5g时，由于溶胀超过了固有弹性强度，造成了力学断裂，水凝胶网的结构完整性丧失(图2b)。然后，除该条件外，通过标准间相关(critical)加权法对破裂力和溶胀度这两个指标的权重系数进行加权，得到综合以上两个性质的综合得分。破裂力的变异性、冲突、信息含量和重量系数与溶胀度的几乎相等(表2)，说明两项指标的重复性低，冲突性高。
各组指标及综合得分见表3。综合分数的结果显示“AM1mg”处方的微针与“AM2mg”处方的微针同等重要。因此选择了溶胀度较高的微针“AM 1mg”用于后续筛选。其次，Irgacure 2959浓度对凝胶溶胀度的影响不显著(图2d)，而破裂力的权重较高(表2)。根据综合评分，筛选出破裂力更高的“Irgacure-2959 5mg”微针(表3)。同理，最终以综合评分较高的“BACA 2mg”微针为最优配方(表3)。
表2
表3
实施例2表观表征
2.1、水凝胶微针的表观表征。
用光学显微和扫描电子显微镜(SEM)观察了微针的三维结构和冻干后微针的表面形貌。用倒置荧光显微镜观察钙黄绿素微针尖端的荧光分布。用能谱分析仪(EDS)扫描微针针尖 的C、H、O、S元素。
水凝胶微针为实施例1步骤(1～3)得到的产品(Blank-MN)。冻干后微针的制备与实施例1步骤(1～3)中不同的是步骤(3)中用冷冻干燥代替风干。钙黄绿素微针的制备与实施例1步骤(1～4)中不同的是步骤(1)中用钙黄绿素代替秋水仙碱。
结合图3-4，制备了均匀排列的400个MN的阵列(图3a)。光学显微镜显示了高约500μm、底宽约300μm的样品的尺寸(图3b)。钙黄绿素在微针针尖中分布均匀，表明亲水性小分子化合物可以通过溶胀和消溶胀的方法均匀分布在微针中(图3c)。扫描电子显微镜显示MN呈规则的圆锥形，尖端锋利，表面具有类似于人皮肤的多层结构(图3e，d)。这可能是因为均聚丙烯酰胺很难质子化，不会形成局部势能。电势的均匀可能是力学性能均匀的原因，也可能是决定其宏观力学行为的根本原因。表面具有均匀的孔隙(图3f)，这种多孔结构有利于药物的溶解和扩散。能谱(EDS)结果表明，水凝胶微针中二硫键分布均匀(图4)。
2.2、秋水仙碱水凝胶微针的表观表征
用光学显微镜观察秋水仙碱微针的表观形态。
秋水仙碱水凝胶微针为实施例1步骤(1～4)得到的产品(COL-MN)。
如图5，实施例1制得的秋水仙碱水凝胶微针表观结构良好，具有尖锐的针尖，阵列排列均匀，针尖结构均一，与水凝胶微针相比没有结构损坏。
实施例3水凝胶微针的体外表征
3.1、微针的溶胀度表征
用天平称量实施例1称量微针的初始重量m0，随后将其浸入50mL pH 7.4的磷酸盐缓冲液(PBS)中室温溶胀24h。定时取出，用滤纸除去多余的表面水，并称重，记为mt，使用Mt-M0/M0方程计算。由于PBS非常类似皮肤间质液，所以选择其作为溶胀介质，并在其他类似的研究中用于模拟皮肤间质液。
水凝胶微针为实施例1步骤(1～3)得到的产品(Blank-MN)。
结合图6b，本发明制备得到的水凝胶微针(Blank-MN)的溶胀率在8h达到平衡，最高溶胀度为2708％。
3.2、体外透皮扩散实验
从-20℃冰箱取离体大鼠腹部皮肤，解冻，在生理盐水中平衡30min，然后根据扩散池直径进行适当裁剪。保鲜膜底部为真皮层，顶部为表皮层，皮肤覆盖在保鲜膜上。表皮用滤纸 干燥。应用微针30s。将搅拌器加入到接收单元中。转速为600rpm，温度为(32±0.2℃)。扩散池中加入搅拌器和10mL PBS(pH 7.4)，恒温32℃。磁性搅拌器以600转/分的速度连续搅拌。感受器腔内充满液体，直到接触到皮肤，然后启动计时器。
分别在1、2、4、6、8、10、12、24、36、48h自动采样1mL，同时自动填充1mL液体。采用HPLC(Waters Corporation,USA)，采用Unitaryl C18(4.6mm×250mm,5μm)色谱柱，定量分析样品的COL浓度。流动相为甲醇-水(60:40)。柱温30℃，检测波长254nm，进样量10μL，流速1.0ml/min。计算秋水仙碱的浓度。
秋水仙碱水凝胶微针为实施例1步骤(1～4)得到的产品(COL-MN)。
如图6c，秋水仙碱水凝胶微针(COL-MN)在48h内累积释放率可达80％以上，用一级动力学方程弥合释放曲线，表明与秋水仙碱水凝胶微针(COL-MN)具有缓释作用。
3.3、载药微针的体外透皮扩散行为研究
本方法用钙黄绿素代替秋水仙碱作为模拟药物，借钙黄绿素在皮肤中的荧光分布来表征药物的扩散行为，其余同实施例1。
采用钙黄绿素微针、巴马微型猪皮和自动透皮扩散仪，从-20℃冰箱取离体大鼠腹部皮肤，解冻，在生理盐水中平衡30min，然后根据扩散池直径进行适当裁剪。保鲜膜底部为真皮层，顶部为表皮层，皮肤覆盖在保鲜膜上。表皮用滤纸干燥。应用微针30s。将搅拌器加入到接收单元中。转速为600rpm，温度为(32±0.2℃)。扩散池中加入搅拌器和10mL PBS(pH 7.4)，恒温32℃。磁性搅拌器以600rpm的速度连续搅拌。感受器腔内充满液体，直到接触到皮肤，然后启动计时器。在2、6、12、24、48h分别取皮，清洗、冷冻切片，在激光扫描共聚焦显微镜下观察。
如图6d，药物渗透的过程包括药物从微针到皮肤的渗透过程和药物从表皮向真皮扩散的过程。根据荧光分布，前一过程在12h内占主导地位，后一过程在12～24h内占主导地位，24～48h内钙黄绿素仍在皮肤内持续释放。
3.4、膜插入研究
秋水仙碱水凝胶微针为实施例1步骤(1～4)得到的产品(COL-MN)。
八层膜一层一层排列，深度约为1mm。将其放置在一片牙蜡上作为机械支撑，使用“拇指压力”应用于实施例1秋水仙碱水凝胶微针每个阵列原型。通过使用光学显微镜对每层上产生的孔进行计数，评估微针的穿透力。根据公式(3)计算：每层膜的孔数百分比。
如图7a，展示了秋水仙碱水凝胶微针(COL-MN)的穿透结果，秋水仙碱水凝胶微针(COL-MN)的插入率接近100％，表明本发明制备得到的秋水仙碱水凝胶微针(COL-MN)有足够的能力穿透皮肤。
3.5、离体大鼠腹部皮肤插入研究
秋水仙碱水凝胶微针为实施例1步骤(1～4)得到的产品(COL-MN)。
取大鼠腹部皮肤，去除皮下粘连，用生理盐水清洗，滤纸干燥，用保鲜膜包裹，20℃冰箱保存。手动将微针插入大鼠皮肤10min后取出。然后，用1％亚甲蓝溶液(100μL)均匀分布于孔部10min。用滤纸吸收多余溶液，用生理盐水浸泡洗净皮肤，用滤纸干燥表面，成像。
如图7b，表明本发明制备得到的秋水仙碱水凝胶微针(COL-MN)能够以接近100％的效率穿透皮肤。
3.6、猪皮插入研究
采用钙黄绿素代替秋水仙碱作为模拟药物。
将钙黄绿素微针应用于猪皮上48h后，立即用低温超薄切片机将猪皮切成5μm厚的薄片，在激光共聚焦扫描显微镜下观察。如图7c，表明微针有效穿透猪皮并形成微通道。
实施例4微针的体内外生物相容性研究
4.1、细胞毒性研究
Blank-MN为实施例1步骤(1～3)得到的产品。COL-MN为实施例1步骤(1～4)得到的产品。
用CCK-8试剂盒检测5mgCOL、Blank-MN和COL-MN(含5mg COL)对人角质形成细胞(HaCaT)活性的细胞毒性。研究前，3个样品在10mL DMEM完全培养基中浸泡24h，将HaCaT细胞按1×106个/孔的密度接种于96孔板上，培养过夜。然后，细胞在不同样品浸泡液中孵育24h。处理后的细胞在37℃下与10％CCK-8孵育1h，使用酶标仪在450nm处测量吸光度。测量三次。结果以细胞活力百分比表示，与未处理的对照细胞比较。
如图8a，与COL组相比，Blank-MN和COL-MN显示出更高的细胞安全性。
4.2、组织病理学实验和皮肤组织细胞凋亡实验
Blank-MN为实施例1步骤(1～3)得到的产品。COL-MN为实施例1步骤(1～4)得到的产品。
大鼠腹部皮肤剃毛、脱毛，应用Blank-MN 24或48h，COL-MN 24或48h，用医用胶带 固定。分别于24、48h取腹部皮肤组织，10％中性福尔马林固定18h，石蜡包埋。制作4μm厚切片，分别用苏木精-伊红(H&E)和原位末端标记法(TUNEL)染色观察组织病理学改变和细胞凋亡情况。显微镜下可见炎性细胞，激光扫描共聚焦显微镜下可见凋亡细胞。
如图8b，在显微镜下观察空白微针和秋水仙碱微针处理24h和48h大鼠腹部皮肤HE切片的病理变化，并与空白对照组(未处理的皮肤)比较，研究微针使用的安全性。与空白对照组的皮肤相比，Blank-MN处理的皮肤切片显示几乎相似的细胞完整性。COL-MN处理的皮肤切片无炎症，但由于秋水仙碱与微管的结合作用，在一定程度上抑制了细胞增殖，对皮肤的刺激较小。同时各组TUNEL染色证实微针在载药前后均未引起细胞凋亡。
实施例5药效试验
以往的研究表明，IL-1β的产生和释放是痛风炎症的第一个和最重要的事件。TNF-α可增强中性粒细胞的活性。促炎细胞因子IL-6是启动先天免疫反应的关键。滑液接触尿酸钠晶体导致细胞坏死、巨噬细胞释放、中性粒细胞死亡和炎症。我们对尿酸钠晶体(MSU)诱导的各组大鼠足踝肿胀皮肤施用实施例1的COL-MN或给其灌胃COL片溶液(i.g.COL)，以足肿胀体积、炎症因子水平、血浆外渗、MPO活性等炎症反应评价治疗效果，COL-MN直接下调巨噬细胞ROS的产生，抑制中性粒细胞趋化，从而抑制晶状体诱导的炎症反应。
5.1、动物
6-8周龄雄性SD大鼠(体重180g-200g)由安徽中医药大学动物实验中心提供。所有动物实验均经安徽中医药大学伦理委员会批准。动物伦理号：AHUCM-rats-2019001。大鼠在动物设施标准化条件下饲养，在实验前12h内，禁食不禁水。
5.2、尿酸钠晶体诱导大鼠急性痛风模型的建立
尿酸钠(MSU)晶体悬浮于无菌生理盐水中。手术前使用70％的酒精对脚踝部位进行消毒。将大鼠分为生理盐水组和MSU组，每组6只。注射MSU前在踝关节上方5mm处用不可磨灭的标记物画一条水平线，以统一足趾体积的测量标准。然后，将0.2mL MSU悬液(25mg/mL)通过21号针头插入大鼠胫骨前肌肌腱内侧，针头尖斜置至45°处注入大鼠踝关节。对照组注射0.2mL生理盐水。然后，用足趾体积测量仪测量大鼠足趾体积。
结合图9(a，b)，通过在踝关节腔内注射尿酸钠(MSU)诱导急性痛风性关节炎的速率模型，评估了秋水仙碱微针体内的药效学，以观察其抗炎作用，并与生理盐水比较。24h后，与生理盐水处理大鼠相比，MSU处理大鼠踝关节明显肿胀，爬行受限(图9a)。MSU组大鼠 踝关节出现广泛肿胀，在注射MSU后3h出现肿胀，24h肿胀达到高峰，直至注射MSU后96h(图9b)。
5.3秋水仙碱微针对大鼠急性痛风进展的抑制作用
采用SPSS(26版)软件对数据进行分析。数据以“平均值±标准差”表示。两组之间的数值变量采用无配对t检验。采用单因素方差分析(ANOVA)结合Bonferroni事后验进行多重比较。*P<0.05、**P<0.01、***P<0.001为差异具有统计学意义。
5.3.1、大鼠的分组、造模与给药
将大鼠随机分为4组：1)空白组(Blank)，2)模型组(Model)，3)秋水仙碱水凝胶微针组(COL-MN)，4)秋水仙碱片溶液灌胃组(i.g.COL)，每组10只。除空白组外，其余大鼠均建立急性痛风模型。2h后，COL-MN组大鼠足踝处灌胃含1mg COL的COL-MN，并用医用胶布固定。i.g.COL组大鼠给予COL片水溶液(1mg/mL)0.5mg/kg。
5.3.2、足体积测量
用足趾体积测量仪测量大鼠足趾初始体积。24h后，测量各组大鼠足趾体积。
治疗结束时，模型对照组大鼠足肿胀体积增加80％。相比之下，经COL-MN或i.g.COL处理后，足部肿胀体积的关系变化增加至50％左右，逐渐减少至模型组厚度的37％以下(图9c)。
5.3.3、IL-1β、TNF-α和IL-6等炎症因子水平测定
取大鼠腹主动脉血，静置2h后离心，取上层血清，保存于-80℃。根据制造商的说明书，使用来自酶联免疫吸附试验(ELISA)试剂盒分析和测定四组的IL-1β、TNF-α和IL-6等炎症因子水平。用酶标仪测量吸光度值(OD)。
与模型组比较，COL-MN和i.g.COL处理后大鼠血清中IL-1β、IL-6和TNF-α炎症因子水平降低(图9d～f)。
5.3.4、大鼠踝关节血浆外渗检测
注射的踝关节滑膜腔用30μL(10μL,3次)PBS冲洗，获得滑膜灌洗样品。以牛血清白蛋白(BSA)为标准，采用Bradford(1976)比色法测定总蛋白含量。
MSU注射后24h，炎症引起血浆外渗，可被COL-MN和i.g.COL在一定程度上抑制(图10a)。
5.3.5、大鼠踝关节中性粒细胞检测
通过测量MPO活性评价滑膜灌洗液中中性粒细胞的存在。将踝关节称重，剪碎，匀浆。采用髓过氧化物酶(MPO)测定试剂盒检测各组MPO活性。
MSU注射后24h，踝关节处产生中性粒细胞，可被COL-MN和i.g.COL在一定程度上抑制(图10b)。
综上，本发明制备的MN和COL-MN的针型完整，矩阵排列整齐，网络交联点分布均匀，具有优异的机械性能和溶胀性能。COL-MN的载药量可以达到有效治疗剂量，透皮扩散具有缓释作用。COL-MN可以通过透皮给药进而发挥良好的抗炎作用，有效治疗尿酸钠晶体诱导的大鼠的急性痛风。为新型秋水仙碱治疗剂型的开发和治疗提供新的思路和策略。

Claims (5)

  1. 一种秋水仙碱水凝胶微针的制备方法，其特征在于，包括如下步骤：
    (1)将丙烯酰胺、N，N’-双(丙烯酰基)半胱胺和Irgacure 2959溶于超纯水中，得到凝胶溶液；
    (2)将得到的凝胶溶液倒入PDMS模具中，低速离心，然后超声，消除气泡；
    (3)将盛有凝胶溶液的PDMS模具置于紫外光下照射，然后在烘箱中风干，得到水凝胶微针；
    (4)在水凝胶微针中加入秋水仙碱溶液，溶胀后风干，脱模，得到秋水仙碱水凝胶微针。
  2. 根据权利要求1所述的秋水仙碱水凝胶微针的制备方法，其特征在于，步骤(1)中，所述凝胶溶液中丙烯酰胺、N，N’-双(丙烯酰基)半胱胺和Irgacure 2959与凝胶溶液的质量比分别为10～35wt％、0.01～1wt％和0.01～0.2wt％。
  3. 根据权利要求1所述的秋水仙碱水凝胶微针的制备方法，其特征在于，步骤(2)中，所述低速离心的速度为3000～4000rpm/min，时间为5～20min。
  4. 根据权利要求1所述的秋水仙碱水凝胶微针的制备方法，其特征在于，步骤(3)中，所述紫外光下照射的条件为315～400nm，100～500W，照射时间为10～60min。
  5. 一种秋水仙碱水凝胶微针，由权利要求1-4任一项所述的制备方法制备得到。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100280457A1 (en) * 2007-05-15 2010-11-04 Hisamitsu Pharmaceutical Co., Inc. Method Of Coating Microneedle
US20170119707A1 (en) * 2014-07-18 2017-05-04 Gensco Laboratories, Llc Homeopathic topical gel for transdermal delivery of colchicine formulations and method of use
CN113133991A (zh) * 2020-01-19 2021-07-20 南京大学 秋水仙碱可溶性微针贴片及其制备方法
CN113603826A (zh) * 2021-06-30 2021-11-05 浙江大学 一种丙烯酰基甘氨酰胺-苯硼酸基糖敏微针的制备方法
CN114432275A (zh) * 2022-01-20 2022-05-06 南京医科大学第二附属医院 一种高黏附镇痛水凝胶微针贴片的制备方法及微针贴片
CN115337530A (zh) * 2022-09-05 2022-11-15 安徽中医药大学 一种秋水仙碱水凝胶微针及其制备方法

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090182306A1 (en) * 2006-07-21 2009-07-16 Georgia Tech Research Corporation Microneedle Devices and Methods of Drug Delivery or Fluid Withdrawal
WO2011071287A2 (ko) * 2009-12-07 2011-06-16 에스케이케미칼 주식회사 활성 성분의 흡수 속도가 개선된 마이크로니들
CN103301092B (zh) * 2012-03-06 2014-12-03 中国科学院理化技术研究所 聚合物微针阵列芯片及其制备方法和应用
CN111763333A (zh) * 2019-04-02 2020-10-13 安徽中医药大学 一种自修复型高强度水凝胶的制备方法
CN112402359A (zh) * 2020-11-04 2021-02-26 深圳前海鹰岗生物科技有限公司 一种抑制细胞炎症因子治疗痛风急性发作的聚合物微针及制备方法
CN112842333A (zh) * 2020-12-31 2021-05-28 华中科技大学 一种可视化葡萄糖浓度检测微针贴片、制备方法及应用
CN113563608A (zh) * 2021-07-28 2021-10-29 沈阳药科大学 一种基于组胺衍生物的纳米水凝胶及其制备和应用方法
CN114099936A (zh) * 2021-10-31 2022-03-01 江苏熙美生物科技有限公司 一种长效美容填充和缓释给药用可溶微针及其制备方法
CN114149601B (zh) * 2021-11-19 2024-07-16 深圳大学 一种双网络微针凝胶及其制备方法
CN114773624B (zh) * 2022-03-21 2024-08-02 安徽中医药大学 一种大分子自组装和金属配位作用协同构筑的水凝胶
CN114767618B (zh) * 2022-05-17 2023-02-24 南京鼓楼医院 一种具有结构色的反蛋白石结构微针阵列及其制备方法和应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100280457A1 (en) * 2007-05-15 2010-11-04 Hisamitsu Pharmaceutical Co., Inc. Method Of Coating Microneedle
US20170119707A1 (en) * 2014-07-18 2017-05-04 Gensco Laboratories, Llc Homeopathic topical gel for transdermal delivery of colchicine formulations and method of use
CN113133991A (zh) * 2020-01-19 2021-07-20 南京大学 秋水仙碱可溶性微针贴片及其制备方法
CN113603826A (zh) * 2021-06-30 2021-11-05 浙江大学 一种丙烯酰基甘氨酰胺-苯硼酸基糖敏微针的制备方法
CN114432275A (zh) * 2022-01-20 2022-05-06 南京医科大学第二附属医院 一种高黏附镇痛水凝胶微针贴片的制备方法及微针贴片
CN115337530A (zh) * 2022-09-05 2022-11-15 安徽中医药大学 一种秋水仙碱水凝胶微针及其制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG, JINGWEN ET AL.: "Preparation and Characterization of New Polyacrylamide Hydrogels with Ultrahigh Mechanical Strength and Super Elastic", SYNTHETIC TECHNOLOGY AND APPLICATION, vol. 34, no. 4, 31 December 2019 (2019-12-31), pages 13 - 18, XP055967974, ISSN: 1006-334X *

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