WO2021196546A1 - 一种用于蛋白质类药物快速透皮递送的微针阵列及其制备方法 - Google Patents

一种用于蛋白质类药物快速透皮递送的微针阵列及其制备方法 Download PDF

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WO2021196546A1
WO2021196546A1 PCT/CN2020/119364 CN2020119364W WO2021196546A1 WO 2021196546 A1 WO2021196546 A1 WO 2021196546A1 CN 2020119364 W CN2020119364 W CN 2020119364W WO 2021196546 A1 WO2021196546 A1 WO 2021196546A1
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microneedle array
drug
layer
microneedle
acid
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PCT/CN2020/119364
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English (en)
French (fr)
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张拥军
王远鹏
关英
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南开大学
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • 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
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • 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
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • 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
    • A61M2037/0046—Solid microneedles
    • 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

Definitions

  • the invention belongs to the technical field of biomedicine, and specifically relates to a microneedle array for rapid transdermal delivery of protein drugs and a preparation method thereof.
  • Microneedles are generally between 200-4000 microns in length and can pierce the stratum corneum of the skin and deliver protein drugs to the more hydrophilic dermal layer, and then enter the circulatory system in the body.
  • the microneedle is small in size, will not cause obvious wounds, will not touch the nerves and cause pain, and its use is also very simple and convenient.
  • microneedles are mainly divided into four categories: solid microneedles, hollow microneedles, soluble (or swellable) microneedles, and coated microneedles.
  • Solid microneedles are inconvenient to use and difficult to control the dosage; hollow microneedles are expensive to prepare and cause obvious pain; soluble (or swellable) microneedles are easy to absorb moisture, their mechanical strength is unstable, and the administration speed is slow. It is inconvenient to use, and the large accumulation of microneedle materials in the skin may cause safety risks.
  • the traditional coated microneedles have low drug loading, complicated preparation process and slow drug delivery speed.
  • the patent adopts the layer-by-layer self-assembly (LBL) technology to load the microneedles with precision and efficiency simply and conveniently.
  • LBL layer-by-layer self-assembly
  • a pH-sensitive release layer single-layer film
  • a pH-stable drug-loading layer layer-by-layer self-assembled film
  • the release layer is rapidly dissociated due to the pH change of the environment, which in turn causes the drug-carrying layer to peel off the microneedle surface as a whole and slowly dissociate in the skin to release the drug.
  • This scheme is suitable for sustained release of drugs and cannot be used for rapid administration. Therefore, it is still a problem to be solved to develop a microneedle drug delivery carrier that simultaneously meets the requirements of simple preparation, rapid onset, and no safety risks.
  • the purpose of the present invention is to provide a microneedle array for rapid transdermal delivery of protein drugs and a preparation method thereof, which can quickly achieve transdermal delivery of protein drugs.
  • the present invention provides a microneedle array for rapid transdermal delivery of protein drugs.
  • the structure of the microneedle array includes a microneedle array substrate and a drug-loaded release layer deposited on the surface of the microneedle.
  • the drug-loaded release layer It is composed of layers of self-assembled membranes formed by the interaction of protein drugs and anionic polyelectrolytes through electrostatic interaction.
  • the isoelectric point of the protein drug is less than 7.
  • the protein drug includes one or more of insulin, interferon alpha-2b, bovine serum albumin, carbonic anhydrase and corticotropin.
  • the anionic polyelectrolyte includes one or more of polyglutamic acid, polyaspartic acid, hyaluronic acid, chondroitin sulfate, heparin, polyacrylic acid, polymethacrylic acid and polystyrene sulfonic acid .
  • the present invention also provides the method for preparing the microneedle array described in the above technical scheme, which includes: alternately immersing the blank microneedle array in a protein drug solution and an anionic polyelectrolyte solution, and drying to obtain the microneedle array.
  • the concentration of the protein drug solution is 1 ⁇ g/ml to 1 g/ml, and the pH value is 2.5 to 4; the concentration of the anionic polyelectrolyte solution is 1 ⁇ g/ml to 1 g/ml, and the pH value is 2.5 to 4.
  • the temperature of the soaking is 1-37°C, and the time of each soaking is 10s-30min.
  • the present invention provides a microneedle array for rapid transdermal delivery of protein drugs.
  • the structure of the microneedle array includes a microneedle array substrate and a drug-loaded release layer deposited on the surface of the microneedle.
  • the drug-loaded release layer It is composed of layers of self-assembled membranes formed by the interaction of protein drugs and anionic polyelectrolytes through electrostatic interaction.
  • the invention makes use of the electrostatic effect between the protein drug and the anionic polyelectrolyte. Under low pH conditions, through simple alternate soaking, the self-assembly of the protein drug and the anionic polyelectrolyte on the surface of the blank microneedle array is carried out to form a pH-sensitive The layer-by-layer self-assembled membrane, that is, the drug-loaded layer.
  • the net charge of the loaded protein drugs is reversed due to changes in the pH of the environment where the drug-loading layer is located, resulting in layer-by-layer self-assembled film Rapidly dissociate and release protein drugs, thereby achieving safe and rapid transdermal delivery of protein drugs.
  • the microneedle array prepared by the invention is simple and convenient to use, has a short application time, only 10s-1min, and can be taken away after application, which is convenient for patients to self-administer.
  • the net charge of the protein drug is electrically reversed, and the anionic polyelectrolyte changes from mutual attraction to mutual repulsion, resulting in instantaneous dissociation of the drug-carrying layer and immediate release of the protein drug .
  • microneedle array prepared by the invention is used for protein drug administration, and the drug is released rapidly.
  • the pharmacokinetics is comparable to ordinary subcutaneous injection.
  • microneedle array prepared by the invention is used to administer protein drugs, and the drugs take effect quickly.
  • the pharmacodynamic curve is comparable to ordinary subcutaneous injection.
  • FIG. 1 is a schematic diagram of the microneedle array prepared in the present invention for transdermal delivery of protein drugs.
  • A is the combination of protein drugs with a positive net charge and a polyelectrolyte with a negative net charge in an acidic environment.
  • the upper layer of the microneedle is self-assembled to form a drug-loading layer (layer-by-layer self-assembly membrane);
  • B in Figure 1 is the microneedle array loaded with the drug-loading layer (layer-by-layer self-assembly membrane) inserted into the skin, protein drugs are clean
  • the electrical charge reverses and repels each other with the polyelectrolyte, causing the drug-loaded release layer to quickly dissociate and release protein drugs into the skin;
  • Figure 2 shows the morphological difference between polymethyl methacrylate microneedles and blank microneedles loaded with drug-loaded release layers ("insulin/poly-L-glutamic acid" layer-by-layer self-assembled film) under scanning electron microscope observation.
  • a in 2 is the surface morphology of the blank polymethyl methacrylate microneedle array after 100 times magnification;
  • Figure 2 B is the surface morphology of the blank polymethyl methacrylate microneedle array after 1000 times magnification;
  • Figure 2 C is the surface morphology of the polymethyl methacrylate microneedle array loaded with "insulin/poly-L-glutamic acid” layers of self-assembled film after 100 times magnification;
  • D in Figure 2 is the surface of the "insulin/poly” -The surface morphology of polymethyl methacrylate microneedles of L-glutamic acid self-assembled film after 1000 times magnification;
  • Figure 3 is a cumulative meter release curve of insulin released in vitro by a microneedle array loaded with a drug-loaded release layer (“insulin/poly-L-glutamic acid” layer-by-layer self-assembled membrane);
  • Figure 4 is a polymethyl methacrylate microneedle array loaded with a drug-loaded release layer ("insulin/poly-L-glutamic acid" layer-by-layer self-assembled film) and subcutaneous injection to give insulin to type I diabetic SD rats After the drug is administered, the comparison chart of the change curve of the blood drug concentration in the rat, and the blank microneedle array is applied to the rat as a control.
  • a drug-loaded release layer ("insulin/poly-L-glutamic acid" layer-by-layer self-assembled film)
  • Figure 5 is a polymethyl methacrylate microneedle array loaded with a drug-loaded release layer ("insulin/poly-L-glutamic acid" layer-by-layer self-assembled film) and subcutaneous injection to give insulin to type I diabetic SD rats After the medicine, the comparison chart of the change curve of the blood glucose level of the rat, and the blank microneedle array was applied to the rat as a control.
  • a drug-loaded release layer ("insulin/poly-L-glutamic acid" layer-by-layer self-assembled film)
  • the present invention provides a microneedle array for rapid transdermal delivery of protein drugs.
  • the structure of the microneedle array includes a microneedle array substrate and a drug-loaded release layer deposited on the surface of the microneedle.
  • the drug-loaded release layer It is composed of layers of self-assembled membranes formed by the interaction of protein drugs and anionic polyelectrolytes through electrostatic interaction.
  • the isoelectric point of the protein drug is preferably less than 7.
  • the protein drug preferably includes one or more of insulin, interferon alpha-2b, bovine serum albumin, carbonic anhydrase and corticotropin.
  • the source of the above-mentioned drugs is not particularly limited, and conventional commercial products can be used.
  • the anionic polyelectrolyte preferably includes polyglutamic acid, polyaspartic acid, hyaluronic acid, chondroitin sulfate, heparin, polyacrylic acid, polymethacrylic acid, and polystyrene sulfonic acid.
  • the material of the microneedle array substrate preferably includes metal, silicon, silicon dioxide, glass or high molecular polymer.
  • the metal preferably includes stainless steel, copper, copper alloy, aluminum, aluminum alloy, titanium, titanium alloy, nickel or nickel alloy.
  • the high molecular polymer preferably includes polymethyl methacrylate, polystyrene, polylactic acid or polylactic acid-glycolic acid copolymer.
  • the present invention also provides the method for preparing the microneedle array described in the above technical scheme, which includes: alternately immersing the blank microneedle array in a protein drug solution and an anionic polyelectrolyte solution, and drying to obtain the microneedle array.
  • the concentration of the protein drug solution is preferably 1 ⁇ g/ml to 1 g/ml, and the pH value is preferably 2.5 to 4; the concentration of the anionic polyelectrolyte solution is preferably 1 ⁇ g/ml to 1 g/ml, and the pH value is Preferably it is 2.5-4.
  • the isoelectric point of the anionic polyelectrolyte solution is lower than the isoelectric point of the protein drug solution.
  • the temperature of the soaking is preferably 1 to 37°C, more preferably 10 to 25°C, most preferably 15 to 20°C; the time of each soaking is preferably 10s to 30min, more preferably 4 to 20min , Most preferably 5 to 6 minutes.
  • the drying time is preferably 10s to 48h, and the drying temperature is preferably 1 to 37°C.
  • the method of using the microneedle array preferably includes: inserting the microneedle array into a subject.
  • the insertion time is preferably 10 s to 1 min.
  • the net charge of the protein drug is electrically reversed, the layers of self-assembled membranes are rapidly dissociated, and the protein drug is released.
  • a polymethyl methacrylate microneedle array loaded with drug-loading layers (“insulin/poly-L-glutamic acid” layer-by-layer self-assembled film) was prepared.
  • the polymethyl methacrylate microneedle array (the needle is 600 ⁇ m long, 10 ⁇ 10 array) is alternately immersed in the two solutions for 5 minutes each time, and 1 cycle is 1 layer.
  • the temperature of the control system is 25°C, and the cycle is repeated 20 times, and it is obtained after drying.
  • a polylactic acid-glycolic acid copolymer (PLGA) microneedle array loaded with a drug-loaded release layer ("interferon ⁇ -2b/polyaspartic acid" layer-by-layer self-assembled film) was prepared.
  • the PLGA microneedle array (the needle is 700 ⁇ m long, 15 ⁇ 15 array) is alternately immersed in the two solutions for 4 minutes each time, and 1 cycle is 1 layer.
  • the temperature of the control system is 20°C, and the cycle is repeated 15 times, and it is obtained after drying.
  • a stainless steel microneedle array loaded with a drug-loaded release layer (“Bovine Serum Albumin/Hyaluronic Acid” self-assembled film layer by layer) was prepared.
  • the microneedle array material of 304 stainless steel, 800 ⁇ m long needle body, 20 ⁇ 20 array
  • 1 cycle is 1 layer.
  • the temperature of the control system is 30°C, and the cycle is repeated 10 times, and it is obtained after drying.
  • the aluminum microneedle array (needle body 500 ⁇ m long, 8 ⁇ 10 array) is alternately immersed in two solutions for 10 minutes each time, and 1 cycle is 1 layer.
  • the temperature of the control system is 15°C, and the cycle is repeated 30 times, and it is obtained after drying.
  • the copper microneedle array (needle body 900 ⁇ m long, 10 ⁇ 15 array) was alternately immersed in the two solutions for 20 minutes each time, and 1 cycle was 1 layer.
  • the temperature of the control system is 25°C, and the cycle is repeated 50 times, and it is obtained after drying.
  • a silica microneedle array loaded with a drug-loaded release layer (“interferon ⁇ 2b/chondroitin sulfate" self-assembled film layer by layer) was prepared.
  • the silicon dioxide microneedle array 400 ⁇ m long needle body, 10 ⁇ 10 array was alternately immersed in the two solutions for 6 min each time, and 1 layer was formed in 1 cycle.
  • the temperature of the control system is 10°C, and the cycle is repeated 25 times, and it is obtained after drying.
  • a polymethyl methacrylate microneedle array loaded with a drug-loaded release layer ("insulin/poly-L-glutamic acid" layer-by-layer self-assembled film).
  • Example 2 Using the same method as in Example 1, a polymethyl methacrylate microneedle array loaded with a drug-loaded release layer ("insulin/poly-L-glutamic acid" layer-by-layer self-assembled film) was prepared. The surface of the microneedle array was dried and sprayed with gold for scanning electron microscopic observation. The surface morphology of the microneedle array as shown in Figure 2 was seen. The cracked deposit layer indicated that the polymethylmethacrylate microneedle array had been successfully loaded with drug release. Layers, namely "insulin/poly-L-glutamic acid" layer by layer self-assembled membrane.
  • the microneedle array (prepared by the same method as in Example 1) loaded with a drug-loaded release layer ("insulin/poly-L-glutamic acid" layer-by-layer self-assembled film) is immersed in phosphate buffer (pH 7.4) . Detect the content of insulin in the soaking liquid by high performance liquid chromatography. The cumulative release of insulin is shown in Figure 3. It can be seen that the drug-loaded release layer on the microneedle array ("insulin/poly-L-glutamic acid" layer-by-layer self-assembled membrane) dissociates rapidly under a pH environment of 7.4, 30 All insulin is released within seconds.
  • a microneedle array loaded with a drug-loaded release layer (“insulin/poly-L-glutamic acid” layer-by-layer self-assembled film) delivers insulin to rats through the skin.
  • Eighteen type I diabetic SD rats (body weight around 300g) shaved their back hair and fasted for 8 hours, and were randomly divided into three groups: 1) Using a drug-loaded release layer ("Insulin/Poly-L-glutamate" layer) Layer self-assembled membrane) microneedle array (prepared by the same method as in Example 1) was applied to the skin for 1 min; 2) by subcutaneous injection of human recombinant insulin (0.25 IU/each); 3) using blank polymethylmethacrylate The ester microneedle array was applied to the skin for 1 min.

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Abstract

本发明提供了一种用于蛋白质类药物快速透皮递送的微针阵列及其制备方法，属于生物医药技术领域，所述微针阵列的结构包括微针阵列基底与沉积在微针表面的载药释放层，所述载药释放层由蛋白质药物与阴离子聚电解质通过静电相互作用形成的层层自组装膜组成。本发明制备的微针阵列使用简单方便，施用时间短，仅为10s～1min，施用后即取走，方便患者自我给药，具有微创、无痛的特点，可提高病人依顺性。药物释放迅速，可获得与普通皮下注射相当的药代动力学和药效学。

Description

一种用于蛋白质类药物快速透皮递送的微针阵列及其制备方法
本申请要求于2020年04月03日提交中国专利局、申请号为202010259644.8、发明名称为“一种用于蛋白质类药物快速透皮递送的微针阵列及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物医药技术领域，具体涉及一种用于蛋白质类药物快速透皮递送的微针阵列及其制备方法。
背景技术
很多疾病的患者需要频繁注射或输注蛋白质类药物以治疗或缓解症状。传统的皮下注射给药方式会引起疼痛，因而降低患者依从性，并且容易引发局部不良反应；同时蛋白质类药物在消化系统内易降解、难吸收，并不适于口服给药；而且由于蛋白质类药物较高的分子量与较强的亲水性，传统的透皮、透粘膜的给药效率极低。近二十年来，微针透皮传输药物的研究取得了较大进展，有望取代传统的注射给药方式。微针一般长度在200-4000微米之间，可以刺穿皮肤角质层，将蛋白质类药物传送至较亲水的真皮层，进而进入体内循环系统。微针尺寸较小，不会造成明显创口，也不会触及神经造成疼痛，其使用也非常简单方便。
目前微针主要分为四类：固体微针、空心微针、可溶解(或可溶胀)微针以及涂层微针。固体微针使用不便，且难以控制给药量；空心微针制备成本高，且会引发明显疼痛感；可溶解(或可溶胀)微针易吸潮，机械强度不稳定，给药速度缓慢导致使用不方便，而且微针材料在皮内大量积累可能引发安全风险。传统的涂层微针药物负载量低，制备工艺复杂，给药速度缓慢。专利(申请号US：201715437927：A)通过层层自组装(LBL)技术，简单方便的在微针上精准而高效地负载药物。该专利在微针表面先后沉积了pH敏感的释放层(单层膜)与pH稳定的载药层(层层自组装膜)。在微针施用时由于所处环境pH变化导致释放层迅速解离，进而导致载药层从微针表面整体剥离并在皮肤内缓慢解离释放药物。该方案适用 于药物缓释而无法用于快速给药。因此，开发出一种同时满足制备简单、起效迅速、无安全风险等要求的微针给药载体依然是一个需要解决的难题。
发明内容
有鉴于此，本发明的目的在于提供一种用于蛋白质类药物快速透皮递送的微针阵列及其制备方法，能够快速实现蛋白质药物的透皮递送。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了一种用于蛋白质类药物快速透皮递送的微针阵列，所述微针阵列的结构包括微针阵列基底与沉积在微针表面的载药释放层，所述载药释放层由蛋白质药物与阴离子聚电解质通过静电相互作用形成的层层自组装膜组成。
优选的，所述蛋白质药物的等电点小于7。
优选的，所述蛋白质药物包括胰岛素、干扰素α-2b、牛血清白蛋白、碳酸酐酶和促肾上腺皮质激素中的一种或几种。
优选的，所述阴离子聚电解质包括聚谷氨酸、聚天冬氨酸、透明质酸、硫酸软骨素、肝素、聚丙烯酸、聚甲基丙烯酸和聚苯乙烯磺酸中的一种或几种。
本发明还提供了上述技术方案所述的微针阵列的制备方法，包括：将空白微针阵列交替浸泡在蛋白质药物溶液和阴离子聚电解质溶液中，干燥，得到微针阵列。
优选的，所述蛋白质药物溶液的浓度为1μg/ml～1g/ml，pH值为2.5～4；所述阴离子聚电解质溶液的浓度为1μg/ml～1g/ml，pH值为2.5～4。
优选的，所述浸泡的温度为1～37℃，每次浸泡的时间为10s～30min。
本发明提供了一种用于蛋白质类药物快速透皮递送的微针阵列，所述微针阵列的结构包括微针阵列基底与沉积在微针表面的载药释放层，所述载药释放层由蛋白质药物与阴离子聚电解质通过静电相互作用形成的层层自组装膜组成。
本发明利用了蛋白质药物与阴离子聚电解质之间的静电作用，在低pH条件下通过简单的交替浸泡，在空白微针阵列表面进行蛋白质药物与阴离子聚电解质的层层自组装，形成具有pH敏感性的层层自组装膜，即 载药层。本发明所公开的快速透皮递送蛋白质类药物的微针阵列插入皮肤后，由于载药层所处环境pH的变化，所负载的蛋白质药物净电荷电性发生反转，导致层层自组装膜迅速解离并释放出蛋白质药物，从而实现蛋白质类药物安全、快速的透皮递送。
本发明具有如下的有益效果：
本发明制备的微针阵列使用简单方便，施用时间短，仅为10s～1min，施用后即取走，方便患者自我给药。
具有微创、无痛的特点，可提高病人依顺性。
本发明制备的微针阵列刺入皮肤后，由于蛋白质类药物的净电荷发生电性反转，与阴离子聚电解质由相互吸引转为相互排斥，导致载药层瞬时解离，蛋白药物立即得以释放。
利用本发明制备的微针阵列进行蛋白药物给药，药物释放迅速。药代动力学与普通皮下注射相当。
利用本发明制备的微针阵列进行蛋白药物给药，药物起效迅速。药效学曲线与普通皮下注射相当。
附图说明
图1为本发明制备的微针阵列透皮递送蛋白质类药物的示意图，图1中的A是在酸性环境下，净电荷为正的蛋白质类药物与净电荷为负的聚电解质通过静电作用结合，在微针上层层自组装形成载药层(层层自组装膜)；图1中的B是负载了载药层(层层自组装膜)的微针阵列插入皮肤后，蛋白质类药物净电荷发生电性反转，与聚电解质相互排斥，导致载药释放层迅速解离并将蛋白质类药物释放到皮肤内；
图2为负载了载药释放层(“胰岛素/聚-L-谷氨酸”层层自组装膜)的聚甲基丙烯酸甲酯微针与空白微针在扫描电镜观察下的形态差异，图2中的A是空白的聚甲基丙烯酸甲酯微针阵列100倍放大后的表面形态；图2中的B是空白的聚甲基丙烯酸甲酯微针1000倍放大后的表面形态；图2中的C是负载“胰岛素/聚-L-谷氨酸”层层自组装膜的聚甲基丙烯酸甲酯微针阵列100倍放大后的表面形态；图2中的D是负载“胰岛素/聚-L-谷氨酸”层层自组装膜的聚甲基丙烯酸甲酯微针1000倍放大后的表面形态；
图3为负载了载药释放层(“胰岛素/聚-L-谷氨酸”层层自组装膜)的微针阵列体外释放胰岛素的累积计释放曲线；
图4为负载了载药释放层(“胰岛素/聚-L-谷氨酸”层层自组装膜)的聚甲基丙烯酸甲酯微针阵列与皮下注射对I型糖尿病SD大鼠进行胰岛素给药后，大鼠血药浓度的变化曲线对比图，以空白微针阵列施加大鼠作为对照。
图5为负载了载药释放层(“胰岛素/聚-L-谷氨酸”层层自组装膜)的聚甲基丙烯酸甲酯微针阵列与皮下注射对I型糖尿病SD大鼠进行胰岛素给药后，大鼠血糖水平的变化曲线对比图，以空白微针阵列施加大鼠作为对照。
具体实施方式
本发明提供了一种用于蛋白质类药物快速透皮递送的微针阵列，所述微针阵列的结构包括微针阵列基底与沉积在微针表面的载药释放层，所述载药释放层由蛋白质药物与阴离子聚电解质通过静电相互作用形成的层层自组装膜组成。
在本发明中，所述蛋白质药物的等电点优选小于7。在本发明中，所述蛋白质药物优选包括胰岛素、干扰素α-2b、牛血清白蛋白、碳酸酐酶和促肾上腺皮质激素中的一种或几种。本发明对上述药物的来源没有特殊限定，采用常规市售产品即可。
在本发明中，所述阴离子聚电解质优选包括聚谷氨酸、聚天冬氨酸、透明质酸、硫酸软骨素、肝素、聚丙烯酸、聚甲基丙烯酸和聚苯乙烯磺酸。
在本发明中，所述微针阵列基底的材质优选包括金属、硅、二氧化硅、玻璃或高分子聚合物。在本发明中，所述金属优选包括不锈钢、铜、铜合金、铝、铝合金、钛、钛合金、镍或镍合金。在本发明中，所述高分子聚合物优选包括聚甲基丙烯酸甲酯、聚苯乙烯、聚乳酸或聚乳酸-羟基乙酸共聚物。
本发明还提供了上述技术方案所述的微针阵列的制备方法，包括：将空白微针阵列交替浸泡在蛋白质药物溶液和阴离子聚电解质溶液中，干燥，得到微针阵列。
在本发明中，所述蛋白质药物溶液的浓度优选为1μg/ml～1g/ml，pH值优选为2.5～4；所述阴离子聚电解质溶液的浓度优选为1μg/ml～1g/ml，pH值优选为2.5～4。在本发明中，所述阴离子聚电解质溶液的等电点低于蛋白质药物溶液的等电点。
在本发明中，所述浸泡的温度优选为1～37℃，更优选为10～25℃，最优选为15～20℃；每次浸泡的时间优选为10s～30min，更优选为4～20min，最优选为5～6min。在本发明中，所述干燥的时间按优选为10s～48h，所述干燥的温度优选为1～37℃。
在本发明中，所述微针阵列的使用方法优选包括：将所述微针阵列插入受试者。在本发明中，所述插入的时间优选为10s～1min。在本发明中，所述微针阵列插入受试者后，蛋白质药物的净电荷发生电性反转，层层自组装膜迅速解离，蛋白质药物得以释放。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
制备负载了载药层(“胰岛素/聚-L-谷氨酸”层层自组装膜)的聚甲基丙烯酸甲酯微针阵列。
具体制备步骤为：将胰岛素与聚-L-谷氨酸分别溶于甘氨酸-盐酸缓冲液(pH＝3.5)，浓度均为1mg/mL。将聚甲基丙烯酸甲酯微针阵列(针体600μm长，10×10排列)在两种溶液中交替浸泡，每次5min，1个循环为1层。控制系统温度为25℃，重复循环20次，干燥即得。
实施例2
制备负载了载药释放层(“干扰素α-2b/聚天冬氨酸”层层自组装膜)的聚乳酸-羟基乙酸共聚物(PLGA)微针阵列。
具体制备步骤为：将干扰素α-2b与聚天冬氨酸分别溶于氯化钾-盐酸缓冲液(pH＝3.5)，浓度均为0.5mg/mL。将PLGA微针阵列(针体700μm长，15×15排列)在两种溶液中交替浸泡，每次4min，1个循环为1层。控制系统温度为20℃，重复循环15次，干燥即得。
实施例3
制备负载了载药释放层(“牛血清白蛋白/透明质酸”层层自组装膜) 的不锈钢微针阵列。
具体制备步骤为：将牛血清白蛋白与透明质酸分别溶于磷酸氢二钠/柠檬酸缓冲液(pH＝3.5)，浓度均为2mg/mL。将微针阵列(304不锈钢材质，针体800μm长，20×20排列)在两种溶液中交替浸泡，每次6min，1个循环为1层。控制系统温度为30℃，重复循环10次，干燥即得。
实施例4
制备负载了载药释放层(“碳酸酐酶/肝素”层层自组装膜)的铝微针阵列。
具体制备步骤为：将碳酸酐酶与肝素分别溶于邻苯二甲酸-盐酸缓冲液(pH＝3.0)，浓度均为5mg/mL。将铝微针阵列(针体500μm长，8×10排列)在两种溶液中交替浸泡，每次10min，1个循环为1层。控制系统温度为15℃，重复循环30次，干燥即得。
实施例5
制备负载了载药释放层(“促肾上腺皮质激素/聚丙烯酸”层层自组装膜)的铜微针阵列。
具体制备步骤为：将促肾上腺皮质激素与聚丙烯酸分别溶于乙酸-乙酸钠缓冲液(pH＝4.0)，浓度均为8mg/mL。将铜微针阵列(针体900μm长，10×15排列)在两种溶液中交替浸泡，每次20min，1个循环为1层。控制系统温度为25℃，重复循环50次，干燥即得。
实施例6
制备负载了载药释放层(“干扰素α2b/硫酸软骨素”层层自组装膜)的二氧化硅微针阵列。
具体制备步骤为：将干扰素α2b与硫酸软骨素分别溶于柠檬酸-盐酸缓冲液(pH＝2.5)，浓度均为0.1mg/mL。将二氧化硅微针阵列(针体400μm长，10×10排列)在两种溶液中交替浸泡，每次6min，1个循环为1层。控制系统温度为10℃，重复循环25次，干燥即得。
实施例7
负载了载药释放层(“胰岛素/聚-L-谷氨酸”层层自组装膜)的聚甲基丙烯酸甲酯微针阵列。
采用实施例1相同的方法，制备负载了载药释放层(“胰岛素/聚-L- 谷氨酸”层层自组装膜)的聚甲基丙烯酸甲酯微针阵列。将微针阵列表面干燥后喷金进行扫描电镜观察，可见如图2所示的微针阵列表面形态，裂开的沉积层说明聚甲基丙烯酸甲酯微针阵列上已经成功负载了载药释放层，即“胰岛素/聚-L-谷氨酸”层层自组装膜。
实施例8
负载了载药释放层的微针阵列的体外释放动力学
将负载了载药释放层(“胰岛素/聚-L-谷氨酸”层层自组装膜)的微针阵列(采用实施例1相同的方法制备)浸泡在磷酸盐缓冲液(pH 7.4)中。用高效液相色谱检测浸泡液中胰岛素的含量。胰岛素的累积释放量如图3所示，可见微针阵列上的载药释放层(“胰岛素/聚-L-谷氨酸”层层自组装膜)在pH 7.4的环境下迅速解离，30秒内即释放出全部的胰岛素。
类似地测定实施例2-7制备的载药释放层在pH 7.4磷酸盐缓冲液中的释放动力学。结果负载的蛋白质均在30秒完全释放。
实施例9
负载了载药释放层(“胰岛素/聚-L-谷氨酸”层层自组装膜)的微针阵列对大鼠透皮递送胰岛素。
在由链脲佐菌素(STZ)诱导的I型糖尿病SD大鼠模型中评估负载了载药释放层(“胰岛素/聚-L-谷氨酸”层层自组装膜)的微针阵列的透皮给药效果。18只I型糖尿病SD大鼠(体重300g左右)剃掉背部毛发并禁食8h，随机分为三组：1)使用负载了载药释放层(“胰岛素/聚-L-谷氨酸”层层自组装膜)的微针阵列(采用实施例1相同的方法制备)施用于皮肤1min；2)通过皮下注射人重组胰岛素(0.25IU/每只)；3)使用空白的聚甲基丙烯酸甲酯微针阵列施用于皮肤1min。给药前1min与给药后0.5h、1h、2h、3h、4h、5h、6h，每只大鼠每次各采眼眶静脉血0.1mL，全程禁食并监测大鼠血糖水平。血样品3000rpm离心10min，取上层血清样品，以酶联免疫吸附法检测血清中胰岛素浓度。给药后大鼠血清胰岛素浓度变化如图4所示，可见负载了载药释放层(“胰岛素/聚-L-谷氨酸”层层自组装膜)的微针阵列给药后，大鼠体内胰岛素浓度变化曲线与皮下注射胰岛素的血药浓度曲线基本重合。而给药后大鼠血糖水平变化如图5所示，负载了载药释放层(“胰岛素/聚-L-谷氨酸”层层自组装膜)的微针阵列给药 与皮下注射胰岛素后的血糖水平变化曲线也高度重合。结果显示负载了载药释放层(“胰岛素/聚-L-谷氨酸”层层自组装膜)的微针阵列给药是皮下注射胰岛素的良好替代。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (8)

  1. 一种用于蛋白质类药物快速透皮递送的微针阵列，所述微针阵列的结构包括微针阵列基底与沉积在微针表面的载药释放层，其特征在于，所述载药释放层由蛋白质药物与阴离子聚电解质通过静电相互作用形成的层层自组装膜组成。
  2. 根据权利要求1所述的微针阵列，其特征在于，所述蛋白质药物的等电点小于7。
  3. 根据权利要求1或2所述的微针阵列，其特征在于，所述蛋白质药物包括胰岛素、干扰素α-2b、牛血清白蛋白、碳酸酐酶和促肾上腺皮质激素中的一种或几种。
  4. 根据权利要求1所述的微针阵列，其特征在于，所述阴离子聚电解质包括聚谷氨酸、聚天冬氨酸、透明质酸、硫酸软骨素、肝素、聚丙烯酸、聚甲基丙烯酸和聚苯乙烯磺酸中的一种或几种。
  5. 根据权利要求1所述的微针阵列，其特征在于，所述微针阵列基底的材质包括金属、硅、二氧化硅、玻璃和高分子聚合物中的一种或几种。
  6. 权利要求1～5任一项所述的微针阵列的制备方法，其特征在于，包括：将空白微针阵列交替浸泡在蛋白质药物溶液和阴离子聚电解质溶液中，干燥，得到微针阵列。
  7. 根据权利要求6所述的制备方法，其特征在于，所述蛋白质药物溶液的浓度为1μg/ml～1g/ml，pH值为2.5～4；所述阴离子聚电解质溶液的浓度为1μg/ml～1g/ml，pH值为2.5～4。
  8. 根据权利要求6所述的制备方法，其特征在于，所述浸泡的温度为1～37℃，每次浸泡的时间为10s～30min。
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