WO2019007380A1 - 一种含有无机盐的分层溶解微针 - Google Patents

一种含有无机盐的分层溶解微针 Download PDF

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WO2019007380A1
WO2019007380A1 PCT/CN2018/094582 CN2018094582W WO2019007380A1 WO 2019007380 A1 WO2019007380 A1 WO 2019007380A1 CN 2018094582 W CN2018094582 W CN 2018094582W WO 2019007380 A1 WO2019007380 A1 WO 2019007380A1
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stabilizer
inorganic salt
needle
molecular weight
microneedle
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PCT/CN2018/094582
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English (en)
French (fr)
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高云华
杨国忠
贺美琳
赵小峪
张锁慧
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中国科学院理化技术研究所
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61K47/02—Inorganic compounds
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • 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
    • 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
    • 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
    • A61K47/38—Cellulose; Derivatives thereof
    • 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
    • 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
    • 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
    • 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 relates to the field of medical technology. More specifically, it relates to a layered dissolving microneedle containing an inorganic salt.
  • inorganic salts should be added to adjust the ionic strength and pH in the microenvironment in which the molecules are located to maintain stability, and also in the production of microneedles. These inorganic salts are encased in the tip of the needle together with the biomolecule. However, inorganic salts are likely to precipitate during the drying of the microneedles and affect the mechanical properties of the microneedles.
  • the needle tips cannot be formed.
  • chondroitin sulfate chondroitin sulfate, dextran or polyvinylpyrrolidone
  • a polymer material having a stable protein molecular structure such as chondroitin sulfate, dextran or polyvinylpyrrolidone to produce a microneedle affects the formability of the microneedle due to the brittleness of the material itself and the presence of an inorganic salt in the solution. .
  • An object of the present invention is to provide a layered-dissolved microneedle containing an inorganic salt which can improve the formability of a microneedle tip containing an inorganic salt and improve the stability of a biologically active ingredient.
  • Another object of the present invention is to provide a method for preparing the above layered dissolving microneedles.
  • the invention provides a layered dissolved microneedle comprising an inorganic salt, comprising a substrate, a needle body on the substrate and a needle tip on the needle body, the needle tip being composed of an inorganic salt solution, a stabilizer, and a film forming water-soluble polymer
  • the material is made by mixing a biologically active ingredient.
  • the length of the needle tip is less than or equal to two-thirds of the total length of the microneedle (the needle tip and the needle body on the substrate).
  • the stabilizer comprises a small molecule active stabilizer, which may be a mixture of one or more of trehalose, mannitol, sucrose, arginine, calcium glucohepate.
  • the stabilizer further comprises a polymer active stabilizer, which may be one or a mixture of dextran, chondroitin sulfate (CS), polyvinylpyrrolidone (PVP). .
  • the dextran has a molecular weight of 10,000 or more
  • the chondroitin sulfate has a molecular weight of 30,000 to 50,000
  • the polyvinylpyrrolidone has a molecular weight of 10,000 to 1,500,000.
  • the mass ratio of the small molecule active stabilizer to the macromolecular active stabilizer is from 10:1 to 1:10.
  • the small molecule active stabilizer is a hydrophilic substance, has good moisturizing property, and can surround the active ingredient under the action of the intermolecular force between the active ingredients, and has a water-blocking effect;
  • the polymer active stabilizer is a hydrophilic material, which can interact with the active ingredient under the action of intermolecular forces (for example, hydrogen bonding) to stabilize the spatial structure of the active ingredient; and the inorganic salt is used to regulate the surrounding of the active ingredient.
  • the ionic strength keeps the charge of the active ingredient stable, so as to stabilize the activity of the active ingredient.
  • the film-forming water-soluble polymer material is a cellulose derivative, a chitosan derivative, a polyvinyl alcohol and a derivative thereof, PVP-VA64, sodium hyaluronate, seaweed a mixture of one or more of water-soluble polymer materials such as sodium.
  • the cellulose derivative includes, but is not limited to, sodium carboxymethylcellulose, hydroxypropyl methylcellulose;
  • the chitosan derivative includes, but is not limited to, carboxymethyl chitosan;
  • the polyvinyl alcohol derivative Including but not limited to polyvinyl alcohol-polyethylene glycol;
  • sodium carboxymethyl cellulose has a molecular weight of 10,000-100,000 and cross-linked CMC
  • hydroxypropyl methylcellulose (HPMC) has a molecular weight of 5,000-100,000
  • carboxymethyl chitosan has a molecular weight of 10,000. -1 million
  • polyvinyl alcohol (PVA) molecular weight is 5000 or more
  • polyvinyl alcohol-polyethylene glycol (PVA-PEG) molecular weight is 5,000-100,000
  • PVP-VA64 molecular weight is 10,000-100,000
  • sodium hyaluronate (HA) has a molecular weight of 1 to 1 million
  • sodium alginate has a molecular weight of 10,000 to 1,000,000.
  • the mass ratio of the film-forming water-soluble polymer material to the stabilizer is less than 10:1, the ratio of the stabilizer is too small to achieve the purpose of stabilizing the activity of the active ingredient for a long time; when the film forming water is dissolved
  • the mass ratio of the polymer material to the stabilizer is greater than 1:10, since the needle tip layer is mainly composed of small molecules after drying, the needle tip cannot be completely demolded because the brittleness itself is not easy to form a needle.
  • the biologically active ingredient comprises, but is not limited to, a polypeptide drug, a protein drug, a nucleic acid drug, a vaccine having a therapeutic or prophylactic health effect.
  • the polypeptide drug may be interferon, insulin, thymoactive pentapeptide, parathyroid hormone, glucagon, growth factor, lumbrokinase, hexapeptide, oxytocin, leuprolide, peroxidation a compound having therapeutic, prophylactic or immunostimulatory effects synthesized or extracted by dismutase, calcitonin, recombinant human brain peptide, etc.
  • the protein drug may be interleukin, erythropoietin, tumor necrosis factor, streptokinase , a fibroblast growth factor, a recombinant human epidermal growth factor, a recombinant human growth hormone, a monoclonal antibody, a genetically engineered
  • the nucleic acid drug may Is synthesized, recombined or extracted by Fomivirsen, ATP, CoA, deoxynucleotide, CTP, UTP, adenosine, polyinosinic acid, adenosine, cytarabine, etc. a nucleic acid having therapeutic, prophylactic and immunostimulating effects;
  • the vaccine may be influenza vaccine, rabies vaccine, hepatitis B vaccine, hepatitis C vaccine, etc. including treatment or defense The proteins, inactivated virus class, class of DNA vaccines. Further, the vaccine may contain an adjuvant.
  • the osmotic pressure of the inorganic salt solution and the liquid phase of the human body are one or more of inorganic salt solutions such as sodium chloride, potassium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, and zinc chloride. mixture.
  • inorganic salt solutions such as sodium chloride, potassium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, and zinc chloride. mixture.
  • salt concentrations (1) 0.01 mM PBS (pH 7.4): 8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 and 0.24 g KH 2 PO 4 are dissolved in 1000 ml of distilled water; (2) 0.01 mM PBS (pH 7.2): 8.5 g NaCl, 2.86 g Na 2 HPO 4 12H 2 O and 0.312 g Na H 2 PO 4 2H 2 O are dissolved in 1000 ml of distilled water; (3) 0.3 mM NaCl Solution: 18g NaCl is dissolved in 1000ml of distilled water; (4) 0.1mM NaCl solution: 6g NaCl is dissolved in 1000ml of distilled water; (5) 0.022mM ZnCl 2 solution: 3g ZnCl 2 is dissolved in 1000ml distilled water Made.
  • the ratio of the biologically active component to the inorganic salt solution is such that an inorganic salt solution containing a biologically active component which is uniform and transparent can be formed; the inorganic salt solution containing the biologically active component is dissolved in a film-forming water.
  • the proportional relationship between the polymer material and the stabilizer may be such that the water-soluble polymer material and the stabilizer satisfying the film forming property can be dissolved into a uniform transparent solution in the inorganic salt solution containing the biologically active component.
  • the needle body and the substrate are made of a film-forming water-soluble polymer material; preferably, the needle body and the substrate are made of a film-forming water-soluble polymer material and a stabilizer; The mass ratio of the film-forming water-soluble polymer material to the stabilizer in the needle body and the substrate is 1:0-5.
  • the film-forming water-soluble polymer material may be high in water solubility such as cellulose derivatives, chitosan derivatives, polyvinyl alcohol and derivatives thereof, PVP-VA64, sodium hyaluronate, sodium alginate, etc.
  • the cellulose derivative includes, but is not limited to, sodium carboxymethylcellulose, hydroxypropyl methylcellulose;
  • the chitosan derivative includes, but is not limited to, carboxymethyl chitosan;
  • the polyvinyl alcohol derivative These include, but are not limited to, polyvinyl alcohol-polyethylene glycol.
  • the sodium carboxymethyl cellulose has a molecular weight of 10,000-100,000 and cross-linked CMC, the molecular weight of hydroxypropyl methylcellulose is 5,000-100,000, and the molecular weight of carboxymethyl chitosan is 10,000-100,000.
  • the molecular weight of the alcohol is 5000 or more
  • the molecular weight of polyvinyl alcohol-polyethylene glycol is 5,000-100,000
  • the molecular weight of PVP-VA64 is 10,000-100,000
  • the molecular weight of sodium hyaluronate is 1-100,000
  • the molecular weight of sodium alginate is 10,000. -1000000.
  • the stabilizer comprises a small molecule active stabilizer, which may be a mixture of one or more of trehalose, mannitol, sucrose, arginine, calcium glucohepate.
  • the stabilizer further comprises a polymer active stabilizer, which may be a mixture of one or more of dextran, chondroitin sulfate, and polyvinylpyrrolidone.
  • the dextran has a molecular weight of 10,000 or more, a chondroitin sulfate having a molecular weight of 30,000 to 50,000, and a polyvinylpyrrolidone having a molecular weight of 10,000 to 1,500,000.
  • the mass ratio of the small molecule active stabilizer to the macromolecular active stabilizer is from 10:1 to 1:10.
  • the layered dissolving microneedles containing the inorganic salt of the present invention further comprise a backing under the substrate.
  • the invention also provides a preparation method of the above layered dissolved microneedle containing an inorganic salt, comprising the following steps:
  • the biological active component is dissolved in the inorganic salt solution to prepare an inorganic salt solution containing the biological active component; and the film-forming water-soluble polymer material and the stabilizer are added to the biological active component.
  • Inorganic salt solution after dissolution, static at 0-4 ° C for 5-60 min, degas;
  • Microneedle molding the tip solution is added dropwise to each microneedle mold unit, the bottom of the mold is vacuumed for 5 minutes, and the air is blown for 10 minutes, and the needle body and the base solution are applied to each microneedle mold unit. After the bottom part is vacuumed for 10 minutes, the excess part is scraped off, and the air is dried for 60 minutes.
  • the preparation of the microneedle tip by the inorganic salt solution is difficult to form and the stability of the polypeptide and the protein active ingredient is problematic.
  • the present invention enhances the stability of the biologically active ingredient by a small molecule active stabilizer such as trehalose, mannitol, sucrose, arginine or glucoheptonate which has a stabilizing effect on the biologically active ingredient.
  • biocompatible water-soluble film-forming polymer materials such as cellulose derivatives, carboxymethyl shell polymerization Sugar, polyvinyl alcohol or polyvinyl alcohol-polyethylene glycol, sodium hyaluronate, etc., further improve the stability of the bioactive component and improve the formability of the microneedle tip containing the inorganic salt.
  • microneedle made of microneedle material has faster intradermal solubility, generally dissolves in 3-10 minutes, can be quickly separated from the microneedle substrate, and reduces the time for the microneedle patch to be applied to the skin, which is more convenient to use. .
  • Figure 1 shows a schematic of a layered dissolved microneedle containing an inorganic salt.
  • Figure 2 shows the morphology of the whole microneedle observed by a stereomicroscope; A: Example 1, B: Comparative Example 3, C: Comparative Example 4.
  • Figure 3 shows the fluorescence distribution of the Example 45 microneedle observed under a fluorescence microscope.
  • Figure 4 shows the skin puncture ability of the microneedle of Example 45.
  • Figure 5 shows the morphological changes of the rats before and after the microneedle action of Example 2; A: before the skin of the rat, B: after 5 minutes of the skin of the rat.
  • Embodiment 1 Preparation method of layered dissolved microneedle containing inorganic salt using film-forming material sodium carboxymethyl cellulose (CMC) as basic skeleton material
  • CMC carboxymethylcellulose sodium, molecular weight 10,000-100,000
  • CS chondroitin sulfate
  • trehalose 0.004g
  • PVA polyvinyl alcohol, molecular weight 10,000-50,000
  • the tip solution was first added to each microneedle mold unit, and the bottom of the PDMS mold was evacuated for 5 minutes, and then opened for 10 minutes.
  • the needle and substrate solutions are then applied to each microneedle mold unit. After vacuuming the bottom of the PDMS mold for 10 minutes, the excess part was scraped off. After drying for 60 minutes, the mold was placed in a 20% humidity drying oven for 6 hours, and the lining was stripped to prepare the microneedle shown in Fig. 1.
  • Microneedle needle shape The whole microneedle morphology was observed by a stereo microscope.
  • Instrument Computer power stroke tester (model: 1220SB), set the upper limit of force to 5N, use the probe (2mm ⁇ 2mm), the number of micro-needle test each time is 16. Fix the microneedle to the stage with the needle tip facing up and set the stage movement speed to 1.1mm/s. After the test was completed, a stereo microscope was used to observe whether or not the needle tip was broken.
  • Example 2 According to the method of Example 1, the microneedles of Comparative Examples 1-5, Examples 2-10 were prepared according to the weight percentages of the materials given in Table 1, and the biomolecule activity, microneedle integrity and mechanical properties were examined ( See Table 1 and Figure 2).
  • Comparative Example 1 is that the needle tip solution uses only the water-soluble polymer material CMC containing a film forming property, and the activity of the microneedle active ingredient produced is only the control group (the same concentration of the active ingredient salt solution). 25%, the activity loss is a lot, but the needle shape is complete after demolding, the single needle is not broken after 0.32N; and the contrast of 2-5 is only the stabilizer (the film formation is poor).
  • the activity of the active ingredient of the needle accounted for 80%-100% compared with the control group, which proved that the stabilizer can keep the active ingredient active, but the needle shape is incomplete after demolding, and the single needle has a broken needle after 0.32N (Fig.
  • Examples 1-10 use both CMC and sodium alginate (content 1%-10% in aqueous solution) and stabilizer (CS, dextran, PVP, trehalose, refined ammonia)
  • CS dextran
  • PVP trehalose
  • CS dextran
  • PVP trehalose
  • refined ammonia One or several of acid, sucrose, and glucoheptonate, the results showed that the activity of the microneedle active ingredient was 75%-95% compared with the control group, and the needle shape was intact after demolding. There was no broken needle after the needle was subjected to a force of 0.32 N (Fig. 2A).
  • Table 1 The film forming material CMC was used as the basic skeleton material, and the specific ratios of Comparative Examples 1-5 and Examples 1-10 were shown.
  • Embodiment 11 Method for preparing layered dissolved microneedle containing inorganic salt using film-forming material hydroxypropylmethylcellulose (HPMC) as basic skeleton material
  • HPMC hydroxypropylmethylcellulose
  • HPG hydroxypropyl methylcellulose, molecular weight 10,000-100,000
  • CS 0.004g trehalose 0.004g
  • 3mg/mL hepatitis B DNA vaccine PBS (0.01M, pH 7.2) salt solution 0.912ml stirred and dissolved, allowed to stand at 2 ° C for 30 min, centrifuged and degassed for use.
  • HPMC molecular weight 10,000-100,000
  • the tip solution was first added to each microneedle mold unit, and the bottom of the PDMS mold was evacuated for 5 minutes, and then opened for 10 minutes.
  • the needle and substrate solutions are then applied to each microneedle mold unit. After the vacuum was applied to the bottom of the PDMS mold for 10 minutes, the excess was scraped off. After drying for 60 minutes, the liner was stripped and released.
  • Example 12-21 the microneedles of Comparative Example 6, Examples 12-21 were prepared according to the weight percentages of the materials given in Table 2, and the activity of the biomolecules and the integrity of the microneedles were examined according to the methods described in the Examples. And mechanical properties (see Table 2).
  • Comparative Example 6 is that the needle tip solution matrix uses only the water-soluble polymer material HPMC containing the film forming property, and the activity of the microneedle active ingredient produced is only the control group (the same concentration of the active ingredient salt solution) 25%, the activity loss is a lot, but the needle shape is complete after demolding, and the single needle is not broken after 0.32N;
  • Examples 11-21 use HPMC and PVP-VA64 (solution solution) with good film formation. Medium content of 1%-10%) and one or more of stabilizers (CS, dextran, PVP, trehalose, arginine, sucrose, calcium glucoheptonate), the results show that the microneedles The activity of the active ingredient was 75%-95% compared with the control group. The needle shape was intact after demolding, and the needle was not broken after the single needle was subjected to 0.32N.
  • Embodiment 22 Preparation method of inorganic salt-containing layered dissolved microneedle using film-forming material sodium hyaluronate (HA) as basic skeleton material
  • HA sodium hyaluronate, molecular weight 40,000-60,000
  • CS 0.004g trehalose 0.004g
  • 0.912ml of 2mg/mL insulin PBS (0.01M, pH 7.2) salt solution stirred and dissolved After that, it was allowed to stand at 4 ° C for 60 min, and then degassed by centrifugation.
  • the tip solution was first added to each microneedle mold unit, and the bottom of the PDMS mold was evacuated for 5 minutes, and then opened for 10 minutes.
  • the needle and substrate solutions are then applied to each microneedle mold unit. After the vacuum was applied to the bottom of the PDMS mold for 10 minutes, the excess was scraped off. After drying for 60 minutes, the liner was stripped and released.
  • microneedles of Comparative Example 7 were prepared according to the method of Example 22 according to the weight percentages of the materials given in Table 3, and the biomolecule activity, microneedle integrity and Mechanical properties (see Table 3).
  • Comparative Example 7 is that the needle tip solution matrix uses only the water-soluble polymer material HA containing a film-forming property, and the activity of the microneedle active ingredient produced is only in the control group (the same concentration of the active ingredient salt solution) 30%, the activity loss is a lot, but the needle shape is complete after demolding, the single needle is not broken after 0.32N; the examples 22-32 use HA or HA with good film formation and CMC, HPMC , a mixture of PVP-VA64 (content of 1%-10% in aqueous solution) and one or more of stabilizers (CS, dextran, PVP, trehalose, arginine, sucrose, calcium glucoheptonate), The results showed that the activity of the microneedle active ingredient was 80%-95% compared with the control group. The needle shape was intact after demolding, and there was no broken needle after the single needle was subjected to 0.32N.
  • Embodiment 40 Preparation method of layered dissolved microneedle containing inorganic salt using film-forming material carboxymethyl chitosan as basic skeleton material
  • the tip solution was first added to each microneedle mold unit, and the bottom of the PDMS mold was evacuated for 5 minutes, and then opened for 10 minutes.
  • the needle and substrate solutions are then applied to each microneedle mold unit. After the vacuum was applied to the bottom of the PDMS mold for 10 minutes, the excess was scraped off. After drying for 60 minutes, the liner was stripped and released.
  • Example 40 the microneedles of Comparative Example 8, Examples 33-39, 41-43 were prepared according to the weight percentages of the materials given in Table 4, and the biomolecule activity, microneedle integrity and mechanics were examined. Performance (see Table 4).
  • Comparative Example 8 is that the needle tip solution matrix uses only the water-soluble polymer material carboxymethyl chitosan containing a film forming property, and the activity of the microneedle active ingredient produced is only in the control group (the same concentration). 40% of the active ingredient salt solution), the activity loss is a lot, but the needle shape is intact after demolding, and the single needle is not broken after 0.32 N; the examples 33-39 and 41-43 are used together.
  • Example 44 Preparation method of layered dissolved microneedle containing inorganic salt
  • CS chondroitin sulfate, molecular weight 30,000-50,000
  • CMC cross-linked CMC
  • trehalose 0.1g add 3mg/mL of hepatitis E vaccine (cracked vaccine containing aluminum hydroxide) Solution: 0.835 ml of NaCl (0.3 mM) salt solution, stirred and dissolved, allowed to stand at 4 ° C for 10 min, centrifuged and degassed for use.
  • the tip solution was first added to each microneedle mold unit, and the bottom of the PDMS mold was evacuated for 5 minutes, and then opened for 10 minutes.
  • the needle and substrate solutions are then applied to each microneedle mold unit. After the vacuum was applied to the bottom of the PDMS mold for 10 minutes, the excess was scraped off. After drying for 60 minutes, the liner was stripped and released.
  • Example 45 Microneedle puncture detection of a layered dissolved microneedle containing an inorganic salt
  • the tip solution was first added to each microneedle mold unit, and the bottom of the PDMS mold was evacuated for 5 minutes, and then opened for 10 minutes.
  • the needle and substrate solutions are then applied to each microneedle mold unit. After the vacuum was applied to the bottom of the PDMS mold for 10 minutes, the excess was scraped off. After drying for 60 minutes, the liner was stripped and released.
  • the prepared microneedle was observed under a fluorescence microscope to observe the fluorescence distribution. As shown in Fig. 3, it was obvious that the drug was concentrated on the tip portion. It was applied to the surface of the pig skin using a force of 40 N, and the skin puncture property was observed by trypan blue staining. As a result, as shown in Fig. 4, it can be clearly seen from the figure that the microneedle acts on the skin and the blue-stained array pinhole.
  • Example 46 Morphological changes of rat abdominal skin before and after microneedle action
  • the microneedles prepared in Examples 1-45 were demolded and observed under a light microscope to observe the morphology of the microneedles, and then the microneedles were attached to the abdominal skin of the rat which had been depilated 24 hours in advance, and pressed with a finger for 5 minutes. The remainder of the microneedles were removed and observed under an optical microscope.
  • the results showed that the layered microneedles prepared in Example 2 were taken as an example (Fig. 5). After the microneedle was released from the mold, the needle shape was intact (Fig. 5A), and after about 5 minutes on the rat skin, the bottom of the needle tip was about three-thirds. The second has been dissolved (Fig. 5B) and it can be considered that most of the active drug has been delivered to the skin.

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Abstract

一种含有无机盐的分层溶解微针及其制备方法，所述含有无机盐的分层溶解微针包括基底、基底上的针体和针体上的针尖；所述针尖由无机盐溶液、稳定剂、成膜性的水溶性高分子材料和生物活性成分混合制成；所述针体和基底由成膜性的水溶性高分子材料或成膜性的水溶性高分子材料与稳定剂制成，所述含有无机盐的分层溶解微针改善了含无机盐的微针针尖的成形性，提高了生物活性成分的稳定性。

Description

一种含有无机盐的分层溶解微针 技术领域
本发明涉及医药技术领域。更具体地，涉及一种含有无机盐的分层溶解微针。
背景技术
随着溶解微针技术的发展，将具有生物活性的多肽、蛋白等大分子包载到微针中，通过微针将其输送到皮内以达到治疗效果逐渐成为人们研究的热点。在疫苗等蛋白类药物的制作过程中，为了维持生物大分子的活性，要加入无机盐以调整分子所处的微环境中的离子强度和酸碱度使其保持稳定性，也不得不在微针的制作过程中将这些无机盐同生物分子一起包载到针尖中。但无机盐会在微针干燥的过程中容易析出而影响微针的机械性能，严重地，则使针尖无法成型。
使用硫酸软骨素、葡聚糖、聚乙烯吡咯烷酮等具有稳定蛋白分子结构的高分子材料制作微针，由于其本身材料的脆性，再加上溶液中的无机盐的存在，影响微针的成形性。
因此，需要提供一种有效解决含无机盐溶液制作微针针尖不易成型以及多肽和蛋白类活性成分的稳定性问题的微针。
发明内容
本发明的一个目的在于提供一种含有无机盐的分层溶解微针，该微针能改善含无机盐的微针针尖的成形性和提高生物活性成分的稳定性。
本发明的另一个目的在于提供上述分层溶解微针的制备方法。
为达到上述目的，本发明采用下述技术方案：
本发明提供了一种含有无机盐的分层溶解微针，包括基底、基底上的针体和针体上的针尖，所述针尖由无机盐溶液、稳定剂、成膜性的水溶性高分子材料和生物活性成分混合制成。其中，所述针尖的长度小于等于微针总长(针尖和基底上的针体)的三分之二。
在本发明优选的实施方式中，所述稳定剂包含小分子活性稳定剂，可以为海藻糖、甘露醇、蔗糖、精氨酸、葡庚糖酸钙中一种或几种的混合物。
在本发明更优选的实施方式中，所述稳定剂还包含高分子活性稳定剂， 可以为葡聚糖、硫酸软骨素(CS)、聚乙烯基吡咯烷酮(PVP)中一种或几种的混合物。其中，所述葡聚糖分子量为1万以上，硫酸软骨素分子量为3万-5万，聚乙烯吡咯烷酮分子量为1万-150万。
在本发明优选的实施方式中，所述小分子活性稳定剂与大分子活性稳定剂的质量比为10:1-1:10。
在本发明中，小分子活性稳定剂为亲水性物质，具有良好的保湿性，能与活性成分之间在分子间作用力的作用下，环绕在活性成分周围，具有锁水保湿的作用；而高分子活性稳定剂为亲水性材料，能在分子间作用力(例如氢键)的作用下与活性成分之间相互作用，稳定活性成分的空间结构；而无机盐用于调节活性成分周围的离子强度，使活性成分的电荷保持稳定，从而达到稳定活性成分活性的目的。
在本发明优选的实施方式中，所述成膜性的水溶性高分子材料为纤维素衍生物、壳聚糖衍生物、聚乙烯醇及其衍生物、PVP-VA64、透明质酸钠、海藻酸钠等水溶性高分子材料中的一种或几种的混合物。所述纤维素衍生物包括但不限于羧甲基纤维素钠、羟丙基甲基纤维素；所述壳聚糖衍生物包括但不限于羧甲基壳聚糖；所述聚乙烯醇衍生物包括但不限于聚乙烯醇-聚乙二醇；
其中，羧甲基纤维素钠(CMC)分子量为1万-100万以及交联CMC，羟丙基甲基纤维素(HPMC)分子量为5000-100万，羧甲基壳聚糖分子量为1万-100万，聚乙烯醇(PVA)分子量为5000以上，聚乙烯醇-聚乙二醇(PVA-PEG)分子量为5000-10万，PVP-VA64分子量为1万-100万，透明质酸钠(HA)分子量为1-100万，海藻酸钠分子量为1万-100万。
在本发明优选的实施方式中，所述成膜性的水溶性高分子材料与稳定剂的质量比与二者的分子量相关，更优选的，所述成膜性的水溶性高分子材料与稳定剂的质量比为10:1-1:10。当所述成膜性的水溶性高分子材料与稳定剂的质量比小于10:1时，稳定剂的比例过小，无法达到长时间稳定活性成分活性的目的；当所述成膜性的水溶性高分子材料与稳定剂的质量比大于1:10时，由于干燥后针尖层主要是由小分子构成，由于其本身的脆性不易成针，针尖无法完整脱模。
在本发明优选的实施方式中，所述生物活性成分包含但不限于具有治疗或修复保健作用的多肽类药物、蛋白类药物、核酸类药物、疫苗。其中，所述多肽类药物可以是干扰素、胰岛素、胸腺活性五肽、去甲状旁腺素、胰高血糖素、生长因子、蚓激酶、六胜肽、催产素、亮丙瑞林、过氧化物歧化酶、 降钙素、重组人脑利肽等合成或者提取的具有治疗、预防和增强免疫作用的多肽；所述蛋白类药物可以是白细胞介素、红细胞生成素、肿瘤坏死因子、链激酶、成纤维细胞生长因子、重组人表皮生长因子、重组人生长激素、单克隆抗体、基因工程乙肝疫苗等合成、重组或者提取的具有治疗、预防和增强免疫作用的蛋白；所述核酸类药物可以是福米韦生(Fomivirsen)、ATP、辅酶A、脱氧核苷酸、CTP、UTP、叠氮腺苷、聚肌胞苷酸、阿糖腺苷、阿糖胞苷等合成、重组或者提取的具有治疗、预防和增强免疫作用的核酸；所述疫苗可以是流感疫苗、狂犬疫苗、乙肝疫苗、丙肝疫苗等包括具有治疗或防御作用的蛋白类、灭活病毒类、DNA类的疫苗。进一步，所述疫苗中可以含有佐剂。
优选地，所述无机盐溶液的渗透压与人体体液相等，为氯化钠、氯化钾、磷酸二氢钠、磷酸氢二钠、氯化锌等无机盐溶液中的一种或几种的混合物。包含但不限于以下几种盐浓度：(1)0.01mM PBS(pH 7.4)：8g NaCl、0.2g KCl、1.44g Na 2HPO 4和0.24g KH 2PO 4溶于1000ml蒸馏水中配制而成；(2)0.01mM PBS(pH 7.2)：8.5g NaCl、2.86g Na 2HPO 412H 2O和0.312g Na H 2PO 42H 2O溶于1000ml蒸馏水中配制而成；(3)0.3mM NaCl溶液：18g NaCl溶于1000ml蒸馏水中配制而成；(4)0.1mM NaCl溶液：6g NaCl溶于1000ml蒸馏水中配制而成；(5)0.022mM ZnCl 2溶液：3g ZnCl 2溶于1000ml蒸馏水中配制而成。
在本发明中，所述生物活性成分与无机盐溶液间的比例关系只要满足能生成均一透明的含有生物活性成分的无机盐溶液即可；含有生物活性成分的无机盐溶液与成膜性的水溶性高分子材料以及稳定剂间的比例关系，只要满足成膜性的水溶性高分子材料以及稳定剂能够在含有生物活性成分的无机盐溶液中溶解成均一透明的溶液即可。
进一步，所述针体和基底采用成膜性的水溶性高分子材料制作而成；优选地，所述针体和基底采用成膜性的水溶性高分子材料与稳定剂制作而成；其中，针体和基底中所述成膜性的水溶性高分子材料与稳定剂的质量比为1:0-5。
其中，所述成膜性的水溶性高分子材料可以是纤维素衍生物、壳聚糖衍生物、聚乙烯醇及其衍生物、PVP-VA64、透明质酸钠、海藻酸钠等水溶性高分子材料中的一种或几种的混合物，以保持微针贴片基底的可弯曲性。所述纤维素衍生物包括但不限于羧甲基纤维素钠、羟丙基甲基纤维素；所述壳聚糖衍生物包括但不限于羧甲基壳聚糖；所述聚乙烯醇衍生物包括但不限于聚乙烯醇-聚乙二醇。所述羧甲基纤维素钠分子量为1万-100万以及交联 CMC，羟丙基甲基纤维素分子量为5000-100万，羧甲基壳聚糖分子量为1万-100万，聚乙烯醇分子量为5000以上，聚乙烯醇-聚乙二醇分子量为5000-10万，PVP-VA64分子量为1万-100万，透明质酸钠分子量为1-100万，海藻酸钠分子量为1万-100万。
在本发明优选的实施方式中，所述稳定剂包含小分子活性稳定剂，可以为海藻糖、甘露醇、蔗糖、精氨酸、葡庚糖酸钙中一种或几种的混合物。
在本发明更优选的实施方式中，所述稳定剂还包含高分子活性稳定剂，可以为葡聚糖、硫酸软骨素、聚乙烯基吡咯烷酮中一种或几种的混合物。所述葡聚糖分子量为1万以上，硫酸软骨素分子量为3万-5万，聚乙烯吡咯烷酮分子量为1万-150万。
在本发明优选的实施方式中，所述小分子活性稳定剂与大分子活性稳定剂的质量比为10:1-1:10。
本发明所述含有无机盐的分层溶解微针还包括基底下的背衬。
本发明还提供了上述含有无机盐的分层溶解微针的制备方法，包括以下步骤：
(1)针尖溶液的制备：将生物活性成分溶于无机盐溶液中，制备得到含有生物活性成分的无机盐溶液；再加入成膜性的水溶性高分子材料、稳定剂到含有生物活性成分的无机盐溶液，溶解后，0-4℃静止5-60min，除气；
(2)针体和基底溶液的制备：成膜性的水溶性高分子材料与水混合，加入或不加入稳定剂，搅拌，溶解后除气；
(3)微针成型：将针尖溶液滴加到每个微针模具单元上，模具底部抽真空5min后，开风吹10min，将针体和基底溶液涂抹到每个微针模具单元上，模具底部抽真空10min后将多余部分刮走，开风干燥60min后，即可。
本发明的有益效果如下：
现有技术中，含无机盐溶液制作微针针尖不易成型以及多肽和蛋白类活性成分的稳定性存在问题。本发明通过对生物活性成分有稳定作用的小分子活性稳定剂如海藻糖、甘露醇、蔗糖、精氨酸、葡庚糖酸钙等，提高生物活性成分的稳定性。通过添加大分子活性稳定剂如硫酸软骨素，葡聚糖、聚乙烯基吡咯烷酮为主材料，通过均匀混合生物相容性的水溶性成膜高分子材料如纤维素衍生物、羧甲基壳聚糖，聚乙烯醇或聚乙烯醇-聚乙二醇、透明质酸钠等，进一步提高了生物活性成分的稳定性和改善含无机盐的微针针尖的成形性。此外，该技术方案相比单纯以一种或多种成膜材料如纤维素衍生物、 羧甲基壳聚糖，聚乙烯醇或聚乙烯醇-聚乙二醇、蚕丝蛋白、透明质酸钠等为微针材料制作的微针，具有更快的皮内溶解性，一般3-10分钟可以溶解，能够快速与微针基底分离，减少微针贴片贴付皮肤上的时间，使用更方便。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1示出含有无机盐的分层溶解微针的示意图。
图2示出体式显微镜观察整片微针形态；A：实施例1，B：对比例3，C：对比例4。
图3示出实施例45微针在荧光显微镜下观察其荧光分布情况。
图4示出实施例45微针的皮肤穿刺能力。
图5示出实施例2微针作用大鼠前后的形态变化；A：扎大鼠皮肤前，B：扎大鼠皮肤5min后。
具体实施方式
为了更清楚地说明本发明，下面结合优选实施例和附图对本发明做进一步的说明。附图中相似的部件以相同的附图标记进行表示。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。
实施例1 一种以成膜材料羧甲基纤维素钠(CMC)作为基本骨架材料的含有无机盐的分层溶解微针的制备方法
1、针尖溶液配制：
称取CMC(羧甲基纤维素钠，分子量1万-10万)0.08g，硫酸软骨素(CS，分子量3万-5万)0.004g，海藻糖0.004g，加入含有1mg/mL的流感疫苗PBS(0.01M,pH 7.2)盐溶液0.912ml，搅拌溶解后，0℃静置5min，离心除气备用。
2、针体和基底溶液：
称取PVA(聚乙烯醇，分子量1万-5万)3g，加入超纯水7mL，在80℃条件下水浴磁搅拌，待其完全溶解后除气，并降到室温备用。
3、微针成型：
先将针尖溶液滴加到每个微针模具单元上，PDMS模具底部抽真空5min后，开风吹10min后。再将针体和基底溶液涂抹到每个微针模具单元上。 PDMS模具底部抽真空10min后将多余部分刮走，开风干燥60min后，将模具放置在20％湿度干燥箱中6h，贴被衬脱模制备出入图1所示的微针。
4、检测微针的各参数
1)检测微针中疫苗活性：将成型脱模后的一片微针溶于相应无机盐溶液后，取200ul加入到96孔板中使用Elisa试剂盒检测活性，并与相同浓度的控制组进行比对。
2)微针针形：通过体式显微镜观察整片微针形态。
3)微针力学性能测试：
采用仪器：电脑力量行程试验机(model：1220SB)，设定力量上限为5N，使用探头为(2mm×2mm)，每次测试微针数目为16根。将微针固定到载物台上，针尖朝上，设定载物台移动速度为1.1mm/s。测试完成后，使用体式显微镜观察是否有针尖断裂的情况。
对比例1-5，实施例2-10
按照实施例1的方法，按照表1中给出的物质重量百分比，制备对比例1-5，实施例2-10的微针并考察其生物分子的活性、微针的完整性和力学性能(见表1和图2)。
从表1中可以看出，对比例1是针尖溶液只使用含有成膜性的水溶性高分子材料CMC，其制作出的微针活性成分的活性只有控制组(相同浓度的活性成分盐溶液)的25％，活性损失很多，但其脱模后针形完整，单根针受力0.32N后无断针；而对比例2-5其针尖组成只有稳定剂(成膜性差)制作出的微针活性成分的活性与控制组相比占80％-100％，证明稳定剂能够使活性成分保持活性，但其脱模后针形不完整，单根针受力0.32N后有断针(图2B、2C)；实施例1-10同时使用含有成膜性好的CMC和海藻酸钠(水溶液中含量1％-10％)以及稳定剂(CS、葡聚糖、PVP、海藻糖、精氨酸、蔗糖、葡庚糖酸钙)的一种或几种，结果表明其制作出的微针活性成分的活性与控制组相比占75％-95％，脱模后针形完整，单根针受力0.32N后也无断针(图2A)。
表1 以成膜材料CMC作为基本骨架材料，对比例1-5、实施例1-10具体配比表
Figure PCTCN2018094582-appb-000001
Figure PCTCN2018094582-appb-000002
Figure PCTCN2018094582-appb-000003
实施例11 一种以成膜材料羟丙基甲基纤维素(HPMC)作为基本骨架材料的含有无机盐的分层溶解微针的制备方法
1、针尖溶液配制：
称取HPMC(羟丙基甲基纤维素，分子量1万-10万)0.08g，CS 0.004g，海藻糖0.004g，加入含有3mg/mL的乙肝DNA疫苗PBS(0.01M,pH 7.2)盐溶液0.912ml，搅拌溶解后，2℃静置30min，离心除气备用。
2、针体和基底溶液：
称取HPMC(分子量1万-10万)3.5g，加入超纯水7.5mL，在80℃条件下水浴磁搅拌，待其完全溶解后除气，并降到室温备用。
3、微针成型：
先将针尖溶液滴加到每个微针模具单元上，PDMS模具底部抽真空5min后，开风吹10min后。再将针体和基底溶液涂抹到每个微针模具单元上。PDMS模具底部抽真空10min后将多余部分刮走，开风干燥60min后，贴被衬脱模。
对比例6，实施例12-21
按照实施例11的方法，按照表2中给出的物质重量百分比，制备对比例6、实施例12-21的微针并根据实施例所述方法考察其生物分子的活性、微针的完整性和力学性能(见表2)。
从表2中可以看出，对比例6是针尖溶液基质只使用含有成膜性的水溶性高分子材料HPMC，其制作出的微针活性成分的活性只有控制组(相同浓度的活性成分盐溶液)的25％，活性损失很多，但其脱模后针形完整，单根针受力0.32N后无断针；实施例11-21同时使用含有成膜性好的HPMC和PVP-VA64(水溶液中含量1％-10％)以及稳定剂(CS、葡聚糖、PVP、海藻糖、精氨酸、蔗糖、葡庚糖酸钙)的一种或几种，结果表明其制作出的微针活性成分的活性与控制组相比占75％-95％，脱模后针形完整，单根针受力0.32N后也无断针。
表2 以成膜材料HPMC作为基本骨架材料，对比例6、实施例11-21具体配比表
Figure PCTCN2018094582-appb-000004
Figure PCTCN2018094582-appb-000005
实施例22一种以成膜材料透明质酸钠(HA)作为基本骨架材料的含有无机盐的分层溶解微针的制备方法
1、针尖溶液配制：
称取HA(透明质酸钠，分子量4万-6万)0.08g，CS 0.004g，海藻糖0.004g，加入含有2mg/mL的胰岛素PBS(0.01M,pH 7.2)盐溶液0.912ml，搅拌溶解后，4℃静置60min，离心除气备用。
2、针体和基底溶液：
称取CMC(分子量1万-10万)1g，PVA-PEG(分子量5000-1万)2g加 入超纯水7mL，在80℃条件下水浴磁搅拌，待其完全溶解后除气，并降到室温备用。
3、微针成型：
先将针尖溶液滴加到每个微针模具单元上，PDMS模具底部抽真空5min后，开风吹10min后。再将针体和基底溶液涂抹到每个微针模具单元上。PDMS模具底部抽真空10min后将多余部分刮走，开风干燥60min后，贴被衬脱模。
对比例7，实施例23-32,
按照实施例22的方法，按照表3中给出的物质重量百分比，制备对比例7、实施例23-32的微针并按照实施例1方法考察其生物分子的活性、微针的完整性和力学性能(见表3)。
从表3中可以看出，对比例7是针尖溶液基质只使用含有成膜性的水溶性高分子材料HA，其制作出的微针活性成分的活性只有控制组(相同浓度的活性成分盐溶液)的30％，活性损失很多，但其脱模后针形完整，单根针受力0.32N后无断针；实施例22-32同时使用含有成膜性好的HA或者HA与CMC、HPMC、PVP-VA64的混合(水溶液中含量1％-10％)以及稳定剂(CS、葡聚糖、PVP、海藻糖、精氨酸、蔗糖、葡庚糖酸钙)的一种或几种，结果表明其制作出的微针活性成分的活性与控制组相比占80％-95％，脱模后针形完整，单根针受力0.32N后也无断针。
表3 以成膜材料HA作为基本骨架材料，对比例7、实施例22-32具体配比表
Figure PCTCN2018094582-appb-000006
Figure PCTCN2018094582-appb-000007
实施例40 一种以成膜材料羧甲基壳聚糖作为基本骨架材料的含有无机盐的分层溶解微针的制备方法
1、针尖溶液配制：
称取羧甲基壳聚糖(分子量2万-25万)0.02g，CMC(分子量1万-10万)0.02g，PVP-VA64 0.01g，CS 0.02g，海藻糖0.005g，蔗糖0.005g，精氨酸0.005g，葡庚糖酸钙0.005g加入含有2mg/mL的福米韦生NaCl(0.1mM)盐溶液0.91ml，搅拌溶解后，4℃静置10min，离心除气备用。
2、针体和基底溶液：
称取PVA(分子量10万)3g，CS0.2g，葡聚糖0.2g(分子量10万以上)，加入超纯水6.1mL，在80℃条件下水浴磁搅拌，待其完全溶解后除气，并降到室温备用。
3、微针成型：
先将针尖溶液滴加到每个微针模具单元上，PDMS模具底部抽真空5min后，开风吹10min后。再将针体和基底溶液涂抹到每个微针模具单元上。PDMS模具底部抽真空10min后将多余部分刮走，开风干燥60min后，贴被衬脱模。
对比例8，实施例33-39,41-43
按照实施例40的方法，按照表4中给出的物质重量百分比，制备对比例8、实施例33-39、41-43的微针并考察其生物分子的活性、微针的完整性和力学性能(见表4)。
从表4中可以看出，对比例8是针尖溶液基质只使用含有成膜性的水溶性高分子材料羧甲基壳聚糖，其制作出的微针活性成分的活性只有控制组(相同浓度的活性成分盐溶液)的40％，活性损失很多，但其脱模后针形完整，单根针受力0.32N后无断针；实施例33-39、41-43同时使用含有成膜性好的羧甲基壳聚糖或者羧甲基壳聚糖与CMC、PVP-VA64的混合(水溶液中含量0.5％-5％)以及稳定剂(CS、葡聚糖、PVP、海藻糖、精氨酸、蔗糖、葡庚糖酸钙)的一种或几种，结果表明其制作出的微针活性成分的活性与控制组相比占80％-95％，脱模后针形完整，单根针受力0.32N后也无断针。
表4 以成膜材料羧甲基壳聚糖作为基本骨架材料，对比例8、实施例33-43具体配比表
Figure PCTCN2018094582-appb-000008
Figure PCTCN2018094582-appb-000009
实施例44 一种含有无机盐的分层溶解微针的制备方法
1、针尖溶液配制：
称取CS(硫酸软骨素，分子量3万-5万)0.1g，CMC(交联CMC)0.05g，海藻糖0.1g，加入含有3mg/mL的戊肝疫苗(裂解疫苗，含有氢氧化铝佐剂)NaCl(0.3mM)盐溶液0.835ml，搅拌溶解后，4℃静置10min，离心除气备用。
2、针体和基底溶液：
称取羧甲基壳聚糖(平均分子量2万左右)3g，加入超纯水7mL，搅拌均匀待其完全溶解后离心除气，备用。
3、微针成型：
先将针尖溶液滴加到每个微针模具单元上，PDMS模具底部抽真空5min后，开风吹10min后。再将针体和基底溶液涂抹到每个微针模具单元上。PDMS模具底部抽真空10min后将多余部分刮走，开风干燥60min后，贴被衬脱模。
实施例45 一种含有无机盐的分层溶解微针的微针穿刺性检测
1、针尖溶液配制：
称取PVP(分子量1万-2万)0.05g，HPMC(分子量5000-1万)0.15g，海藻糖0.05g，加入含有1mg/mL的罗丹明NaCl(0.3mM)盐溶液0.75ml，搅拌溶解后，4℃静置10min，离心除气备用。
2、针体和基底溶液：
称取CMC(分子量10万-100万)2g，PVA-PEG 1g加入超纯水7mL，搅拌均匀待其完全溶解后减压抽真空除气，备用。
3、微针成型：
先将针尖溶液滴加到每个微针模具单元上，PDMS模具底部抽真空5min 后，开风吹10min后。再将针体和基底溶液涂抹到每个微针模具单元上。PDMS模具底部抽真空10min后将多余部分刮走，开风干燥60min后，贴被衬脱模。
4、微针穿刺性：
将制作成的微针拿到荧光显微镜下观察其荧光分布情况，如图3所示，可以明显的看到药物集中在针尖部分。使用40N的力将其作用于猪皮表面，并使用台盼蓝染色观察其皮肤穿刺性，结果如图4，从图中可以明显的看到微针作用皮肤后台盼蓝染色的阵列针孔。
实施例46 微针作用大鼠腹部皮肤前后的形态变化
将实施例1-45制作的微针脱模后拿到光学显微镜下观察微针形态，然后将此微针贴附到已提前24小时脱毛后的大鼠腹部皮肤上，使用手指按压5min，将微针剩余部分揭下并在光学显微镜下观察。结果表明，以实施例2制作的分层微针为例(图5)，微针脱模后，针形完整(图5A)，在大鼠皮肤上作用5min后，针尖底部的大约三分之二已溶解(图5B)，可以认为大部分活性药物均已输送到皮肤内。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动，这里无法对所有的实施方式予以穷举，凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。

Claims (10)

  1. 一种含有无机盐的分层溶解微针，包括基底、基底上的针体和针体上的针尖，其特征在于：所述针尖由无机盐溶液、稳定剂、成膜性的水溶性高分子材料和生物活性成分混合制成。
  2. 根据权利要求1所述的分层溶解微针，其特征在于：所述稳定剂包含小分子活性稳定剂，所述小分子活性稳定剂为海藻糖、甘露醇、蔗糖、精氨酸、葡庚糖酸钙中一种或几种的混合物。
  3. 根据权利要求2所述的分层溶解微针，其特征在于：所述稳定剂还包含大分子活性稳定剂，所述大分子活性稳定剂为葡聚糖、硫酸软骨素、聚乙烯基吡咯烷酮中一种或几种的混合物；所述葡聚糖分子量为1万以上，硫酸软骨素分子量为3万-5万，聚乙烯吡咯烷酮分子量为1万-150万。
  4. 根据权利要求3所述的分层溶解微针，其特征在于：所述小分子活性稳定剂与大分子活性稳定剂的质量比为10:1-1:10。
  5. 根据权利要求1所述的分层溶解微针，其特征在于：所述成膜性的水溶性高分子材料为羧甲基纤维素钠、羟丙基甲基纤维素、羧甲基壳聚糖、聚乙烯醇、聚乙烯醇-聚乙二醇、PVP-VA64、透明质酸钠、海藻酸钠中的一种或几种的混合物；
    其中，所述羧甲基纤维素钠分子量为1万-100万以及交联CMC，羟丙基甲基纤维素分子量为5000-100万，羧甲基壳聚糖分子量为1万-100万，聚乙烯醇分子量为5000以上，聚乙烯醇-聚乙二醇分子量为5000-10万，PVP-VA64分子量为1万-100万，透明质酸钠分子量为1-100万，海藻酸钠分子量为1万-100万。
  6. 根据权利要求1所述的分层溶解微针，其特征在于：所述无机盐溶液的渗透压与人体体液渗透压相等，所述无机盐溶液为氯化钠、氯化钾、磷酸二氢钠、磷酸氢二钠、氯化锌溶液中的一种或几种的混合物。
  7. 根据权利要求1所述的分层溶解微针，其特征在于：所述成膜性的水溶性高分子材料与稳定剂的质量比为10:1-1:10。
  8. 根据权利要求1所述的分层溶解微针，其特征在于：所述生物活性成分为多肽类药物、蛋白类药物、核酸类药物、疫苗。
  9. 根据权利要求1所述的分层溶解微针，其特征在于：所述针体和基底由成膜性的水溶性高分子材料或成膜性的水溶性高分子材料与稳定剂制成；所述成膜性的水溶性高分子材料与稳定剂的质量比为1:0-5。
  10. 一种制备权利要求1-9任一所述的分层溶解微针的方法，包括以下步骤：
    (1)针尖溶液的制备：将生物活性成分溶于无机盐溶液中，制备得到含有生物活性成分的无机盐溶液；再加入成膜性的水溶性高分子材料、稳定剂到含有生物活性成分的无机盐溶液，溶解后，0-4℃静止5-60min，除气；
    (2)针体和基底溶液的制备：成膜性的水溶性高分子材料与水混合，加入或不加入稳定剂，搅拌，溶解后除气；
    (3)微针成型：将针尖溶液滴加到每个微针模具单元上，模具底部抽真空5min后，开风吹10min，将针体和基底溶液涂抹到每个微针模具单元上，模具底部抽真空10min后将多余部分刮走，开风干燥60min后，即可。
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