WO2019076125A1 - 负载治疗性蛋白的纳米粒及其微胶囊 - Google Patents

负载治疗性蛋白的纳米粒及其微胶囊 Download PDF

Info

Publication number
WO2019076125A1
WO2019076125A1 PCT/CN2018/101106 CN2018101106W WO2019076125A1 WO 2019076125 A1 WO2019076125 A1 WO 2019076125A1 CN 2018101106 W CN2018101106 W CN 2018101106W WO 2019076125 A1 WO2019076125 A1 WO 2019076125A1
Authority
WO
WIPO (PCT)
Prior art keywords
nanoparticles
nanoparticle
insulin
cpp
therapeutic protein
Prior art date
Application number
PCT/CN2018/101106
Other languages
English (en)
French (fr)
Inventor
何治宇
陈永明
刘利新
毛海泉
梁锦荣
Original Assignee
中山大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中山大学 filed Critical 中山大学
Publication of WO2019076125A1 publication Critical patent/WO2019076125A1/zh

Links

Images

Classifications

    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • A61K9/5042—Cellulose; Cellulose derivatives, e.g. phthalate or acetate succinate esters of hydroxypropyl methylcellulose
    • A61K9/5047—Cellulose ethers containing no ester groups, e.g. hydroxypropyl methylcellulose
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin

Definitions

  • the invention belongs to the technical field of nanomedicine, and relates to a nanoparticle loaded with therapeutic protein, a microcapsule containing the nano particle, a method for preparing the nano particle or micro capsule, and a pharmaceutical composition containing the nano particle or micro capsule. And the use of the nanoparticles or microcapsules.
  • Insulin is a protein hormone secreted by islet beta cells in the pancreas, involved in regulating sugar metabolism and controlling blood sugar balance. Exogenous insulin is mainly used to treat diabetes. The traditional route of administration of insulin is subcutaneous injection, however, long-term injections reduce patient compliance and produce many side effects.
  • the oral administration route of insulin Compared with subcutaneous injection, the oral administration route of insulin has the advantages of good patient compliance, economy, convenience and safety. After oral administration, insulin can enter the systemic circulation through the portal vein and directly participate in the metabolism of glucose in the liver, which can effectively simulate the secretion mode and physiological function of endogenous insulin.
  • insulin when insulin is administered by the oral route, there are the following problems to be solved: first, insulin is easily degraded in the stomach due to the acidic environment in the stomach; second, insulin may be deactivated by enzyme degradation in the digestive tract; Due to the high molecular weight and low fat solubility of insulin, its permeability to intestinal epithelial cells is low, resulting in low oral bioavailability.
  • nanoparticles loaded with protein drugs such as insulin have been used for oral delivery of these drugs.
  • the use of nanoparticles as a carrier helps to reduce the effects of the acidic environment in the stomach and/or the enzymatic environment on the drug.
  • these nanoparticles often have problems such as wide particle size distribution, uneven surface morphology, low bioavailability, or low efficacy.
  • the surface of the intestinal cells is covered with a mucus layer, and the mucin in the mucus layer can be complexed with the nanoparticles by electrostatic or hydrophobic action to form larger aggregates in situ, and the nanoparticles are anchored.
  • the rapid irregular movement of the nanoparticles in the mucus layer is affected, thereby hindering the penetration efficiency of the nanoparticles in the mucus layer. Therefore, the presence of a mucus layer becomes a bottleneck that limits the efficiency of oral protein drug delivery.
  • the inventors obtained a nanoparticle loaded with a therapeutic protein through intensive research and creative labor.
  • the inventors coated the positively charged drug-loaded nanocore with a negatively charged polyanion to form a nanoparticle having a core-nuclear structure with a negatively charged polyanion on the outer layer and a drug-loaded nanocore inside.
  • the nanoparticles of the present invention can reduce the interaction between themselves and the mucin in the mucus layer by electrostatic repulsion, increase the permeability of the nanoparticles in the mucus layer, and thereby deliver more drugs to the intestinal epithelial cells.
  • the application provides a nanoparticle comprising a core and a polyanion coated on the core; the core comprising a therapeutic protein, further comprising a cell penetrating peptide (CPP).
  • CPP cell penetrating peptide
  • the polyanion is selected from the group consisting of sodium tripolyphosphate, alginic acid, heparin, hyaluronic acid (HA), hyaluronate, chondroitin sulfate, polyacrylic acid polymers, polystyrene sulfonic acid Polymers of the class or any combination thereof.
  • the polyanion is selected from the group consisting of hyaluronic acid, hyaluronate (eg, sodium hyaluronate), or a combination thereof.
  • Hyaluronic acid has the characteristics of wide source, non-toxicity, biodegradability and good biocompatibility, and is suitable for the preparation of drug-loaded nanoparticles.
  • Hyaluronic acid can be converted to hyaluronate under alkaline conditions.
  • the polyanion has a weight average molecular weight of from 4 kDa to 200 kDa (eg, 4 kDa to 10 kDa, 10 kDa to 50 kDa, from 50 kDa to 100 kDa, from 100 kDa to 150 kDa, or from 150 kDa to 200 kDa).
  • the core comprises a therapeutic protein which may be a hormone, a hormone analog, an enzyme, an enzyme inhibitor or an antibody.
  • the therapeutic protein is insulin.
  • the core contains CPP which can function as a carrier.
  • CPP is thought to have the ability to load pharmaceutically active molecules and enhance their transcellular transport.
  • the CPP comprises an arginine residue.
  • the N-terminus of the CPP is an arginine residue.
  • the CPP is a Penetratin.
  • Penetratin is derived from the homeodomain of the Drosophila elegans protein, consisting of 16 amino acid residues, and its amino acid sequence is shown in SEQ ID NO: 1. Penetratin can mediate a variety of hydrophobic macromolecules into the living cytoplasm without destroying the integrity of the cell membrane.
  • the CPP is modified with an alkyl group.
  • alkylated CPP as a carrier makes it easier to form stable, dispersible nanoparticles.
  • the alkylated CPP can be obtained by a process comprising the steps of condensing a carboxyl group of a saturated fatty acid with an amino group (for example, an N-terminal amino group) on the CPP to form an amide bond.
  • the condensation reaction is in a condensing agent (for example, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU)), a solvent (for example, N, N) It is carried out in the presence of dimethylformamide (DMF) and/or an alkaline reagent (for example, N,N-diisopropylethylamine (DIEA)).
  • a condensing agent for example, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU)
  • TBTU O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate
  • N, N tetramethyluronium tetrafluoroborate
  • DMF dimethylformamide
  • DIEA alkaline reagent
  • the alkyl group is C 12 -C 18 alkyl (e.g., C 12 alkyl, C 14 alkyl, C 16 alkyl or C 18 alkyl group). In certain embodiments, the alkyl group is a linear alkyl group.
  • the N-terminus of the CPP is modified with an alkyl group.
  • the CPP is a Penetratin whose N-terminal is modified with n-octadecyl.
  • the nanoparticles of the present invention may be labeled with a fluorescent compound, and thus, in certain embodiments, the nanoparticles further comprise a chromophore of a fluorescent compound or fluorescent compound.
  • the fluorescent compound includes, but is not limited to, fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), 3H-phthalocyanine dye (eg Cy3, Cy5) or rhodamine (eg rhodamine 6G). , Luo Danming 123, Luo Danming B).
  • the fluorescent compound can be labeled on a polyanion, a therapeutic protein, and/or a CPP.
  • the nanoparticles of the invention and the cores comprised thereof can be of any shape.
  • the nanoparticles are spherical.
  • the core is spherical.
  • the nanoparticles of the invention have a narrow particle size and/or a uniform particle size distribution.
  • the nanoparticles have a particle size between 100 nm and 900 nm (eg, 100 nm to 200 nm, 200 to 300 nm, 300 to 400 nm, 400 to 500 nm, 500 to 600 nm, 600 to 700 nm, 700 to 800 nm, or 800-).
  • 900nm the nanoparticle size has a polydispersity index (PDI) of from 0.05 to 0.5 (eg, 0.05-0.1, 0.1-0.2, 0.2-0.3, 0.3-0.4, or 0.4-0.5).
  • PDI polydispersity index
  • the nanoparticles of the present invention comprise a core having a narrow particle size and/or a uniform particle size distribution.
  • the core has a particle size of from 30 nm to 500 nm (eg, 30 nm to 100 nm, 100 to 200 nm, 200 to 300 nm, 300 to 400 nm, or 400 to 500 nm).
  • the core has a particle size polydispersity index (PDI) of from 0.1 to 0.5 (eg, from 0.1 to 0.2, from 0.2 to 0.3, from 0.3 to 0.4, or from 0.4 to 0.5).
  • PDI particle size polydispersity index
  • the surface of the nanoparticles of the invention is electronegative.
  • the nanoparticles have a zeta potential of from -10 mV to -50 mV (eg, -10 mV to -20 mV, -20 mV to -30 mV, -30 mV to -40 mV, or -40 mV to -50 mV).
  • the nanoparticles of the invention have a higher encapsulation efficiency and/or drug loading.
  • the encapsulation efficiency of the nanoparticles is from 90% to 99% (eg, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).
  • the nanoparticles are loaded with a drug loading of 50% to 90% (eg, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%- 75%, 75%-80%, 80%-85% or 85%-90%).
  • the encapsulation efficiency (EE) and drug loading (LC) of the nanoparticles can be measured by a method comprising the steps of: 1) preparing nanoparticles using fluorescently labeled therapeutic proteins, 2) containing the The suspension of nanoparticles is placed in an ultrafiltration tube for centrifugation to obtain a filtrate; 3) the fluorescence intensity of the therapeutic protein in the filtrate is determined, and the concentration of the therapeutic protein in the filtrate is calculated to determine the amount of free therapeutic protein.
  • the encapsulation efficiency (EE) and drug loading (LC) were calculated as follows:
  • the therapeutic protein: polyanion: CPP has a mass ratio of 1:0.5-0.9:0.1-0.5 (eg, 1:0.5:0.1-0.5, 1:0.6:0.1- in the nanoparticles). 0.5, 1:0.7:0.1-0.5, 1:0.8:0.1-0.5 or 1:0.9:0.1-0.5, for example 1:0.5-0.9:0.1, 1:0.5-0.9:0.2, 1:0.5-0.9:0.3 , 1:0.5-0.9:0.4 or 1:0.5-0.9:0.5). Further, in certain embodiments, the sum of the mass of polyanion and CPP: the therapeutic protein has a mass ratio of 1:1. In certain embodiments, the mass ratio of polyanion:CPP:therapeutic protein is 0.9:0.1:1, 0.8:0.2:1, 0.7:0.3:1, 0.6:0.4:1, or 0.5:0.5:1.
  • the present application also provides a microcapsule comprising a wall layer, and a nanoparticle of the invention embedded in a wall layer, the wall layer comprising or consisting essentially of an enteric material.
  • the nanoparticle loaded with the protein drug is made into an enteric microcapsule, which can improve the stability of the nanoparticle in the gastrointestinal tract, improve the specificity of drug release in the intestinal tract, improve the pharmacokinetic behavior, and thereby improve the drug. Bioavailability in the body, improving pharmacodynamic results in vivo.
  • the enteric material is selected from the group consisting of cellulose and derivatives thereof, such as hypromellose phthalate (HPMCP), cellulose acetate phthalate (CAP), 1, 2,4-benzenetricarboxylic acid cellulose acetate (CAT) and the like.
  • HPMCP hypromellose phthalate
  • CAP cellulose acetate phthalate
  • CAT 1, 2,4-benzenetricarboxylic acid cellulose acetate
  • the enteric material is HPMCP.
  • the microcapsules have a particle size of 1-10 ⁇ m (eg, 1 ⁇ m-2 ⁇ m, 2 ⁇ m-3 ⁇ m, 3 ⁇ m-4 ⁇ m, 4 ⁇ m-5 ⁇ m, 5 ⁇ m-6 ⁇ m, 6 ⁇ m-7 ⁇ m, 7 ⁇ m-8 ⁇ m, 8 ⁇ m- 9 ⁇ m or 9 ⁇ m - 10 ⁇ m).
  • the microcapsules are spherical in shape.
  • the encapsulation efficiency of the microcapsules is from 30% to 95% (eg, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%).
  • the encapsulation efficiency (EE) of the microcapsules can be measured by a method comprising the steps of: 1) preparing nanoparticles using fluorescently labeled therapeutic proteins, 2) forming the nanoparticles into microcapsules, 3
  • the suspension containing the microcapsules is placed in an ultrafiltration tube for centrifugation to obtain a filtrate; 4) the fluorescence intensity of the therapeutic protein in the filtrate is measured, and the concentration of the therapeutic protein in the filtrate is calculated to determine the free treatment.
  • the amount of sex protein Calculate the encapsulation ratio (EE) as follows:
  • the application provides a pharmaceutical composition comprising a nanoparticle or microcapsule of the invention.
  • the pharmaceutical composition is for preventing or treating a disease that is preventable or treatable by a therapeutic protein contained in the nanoparticle or microcapsule.
  • the therapeutic protein is insulin
  • the pharmaceutical composition for preventing or treating hyperglycemia in a subject is insulin
  • the pharmaceutical composition for preventing or treating hyperglycemia comprises stress-induced hyperglycemia, diabetes (including type 1 diabetes and type 2 diabetes), and impaired glucose tolerance.
  • the pharmaceutical composition comprises a prophylactically or therapeutically effective amount of nanoparticles or microcapsules.
  • the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers.
  • Pharmaceutically acceptable carriers for use in the present invention include, but are not limited to, fillers, diluents, binders, wetting agents, disintegrants, lubricants, surfactants, preservatives, colorants, flavoring agents, fragrances, Effervescent agent, emulsifier, flocculant, deflocculant, bacteriostatic agent, solubilizer.
  • the pharmaceutically acceptable carrier is selected from the group consisting of: an ion exchanger, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), glycerin, sorbic acid, potassium sorbate, water, Protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic material, polyethylene glycol, sodium carboxymethyl cellulose , polyacrylate, beeswax, polyethylene-polyoxypropylene block polymer, lanolin, and any combination thereof.
  • the pharmaceutical composition comprises a lyoprotectant.
  • the lyoprotectant is an alcoholic lyoprotectant such as xylitol, mannitol or sorbitol.
  • compositions of the present invention can be formulated into a variety of suitable dosage forms including, but not limited to, oral dosage forms, injectable dosage forms (eg, dosage forms suitable for subcutaneous, intramuscular or intravenous injection), inhaled dosage forms, mucosal administration forms or Topical dosage form.
  • the pharmaceutical composition is formulated into an oral dosage form, such as a tablet, capsule, granule, oral solution, oral suspension, pellet, or microtablet.
  • the application provides the use of a nanoparticle or microcapsule of the invention for the preparation of a pharmaceutical composition for preventing or treating a therapeutic protein contained in the nanoparticle or microcapsule A disease that is prevented or treated.
  • the therapeutic protein is insulin and the disease is hyperglycemia.
  • the hyperglycemia comprises stress-induced hyperglycemia, diabetes (including type 1 diabetes and type 2 diabetes), and impaired glucose tolerance.
  • the application provides a method of preventing or treating a disease comprising administering to a subject in need thereof a nanoparticle, microcapsule or pharmaceutical composition of the invention, the disease being the nanoparticle, micro A disease in which a therapeutic protein contained in a capsule or a pharmaceutical composition is capable of being prevented or treated.
  • the therapeutic protein is insulin and the disease is hyperglycemia.
  • the hyperglycemia comprises stress-induced hyperglycemia, diabetes (including type 1 diabetes and type 2 diabetes), and impaired glucose tolerance.
  • the subject is preferably a mammal, such as a bovine, equine, ovine, porcine, canine, feline, rodent, primate Animal; for example, the subject is a human.
  • the present application provides a method of making the nanoparticles of the present invention, the method comprising the steps of:
  • Step 1 preparing a nanoparticle A, the nanoparticle A being a nanoparticle comprising a therapeutic protein and CPP;
  • Step 2 The nanoparticles A obtained in the step 1 were coated with a polyanion.
  • the step 1 comprises mixing a solution comprising a therapeutic protein with a solution comprising CPP.
  • the step 1 further comprises the steps of:
  • Step 1-1 providing a solution comprising a therapeutic protein and a solution comprising CPP;
  • Step 1-2 providing means comprising a vortex mixing zone and a plurality of passages for fluid flow to the vortex mixing zone;
  • Step 1-3 The solution containing the therapeutic protein and the solution containing CPP are passed through different channels to the vortex mixing zone, and mixed to obtain a suspension containing the nanoparticles A.
  • the CPP-containing solution of step 1-1 has a 0.1-0.5 mg/mL (eg, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, or 0.5 mg/mL) The mass concentration.
  • the solution comprising CPP has a mass concentration ratio of from 0.1 to 0.5:1 (eg, 0.1:1, 0.2:1, 0.3:1, 0.4: 1 or 0.5:1).
  • the solution comprising a therapeutic protein is an aqueous solution.
  • the solution comprising CPP is an aqueous solution.
  • the mass concentration of the solution containing the therapeutic protein refers to the mass concentration of the therapeutic protein in the solution.
  • the mass concentration of the solution containing CPP means the mass concentration of CPP in the solution.
  • the step 1-1 further comprises: adjusting the pH of the therapeutic protein-containing solution to 6.5-7.0 using an acidic solution (eg, hydrochloric acid) or an alkaline solution (eg, sodium hydroxide solution). (eg 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0).
  • an acidic solution eg, hydrochloric acid
  • an alkaline solution eg, sodium hydroxide solution
  • the device in steps 1-2 is a multi-inlet vortex mixer (MIVM).
  • MIVM multi-inlet vortex mixer
  • a solution comprising a therapeutic protein, and a solution comprising CPP flows at a constant rate in the channel at the same flow rate.
  • the flow rate is 1-50 mL/min (eg, 1-15 mL/min, 15-25 mL/min, or 25-50 mL/min).
  • the nanoparticles A prepared in step 1 have a narrow particle size and/or a uniform particle size distribution.
  • the nanoparticles A have a particle size of from 30 nm to 500 nm (eg, 30 nm to 100 nm, 100 to 200 nm, 200 to 300 nm, 300 to 400 nm, or 400 to 500 nm).
  • the particle size of the nanoparticles A has a polydispersity index (PDI) of from 0.1 to 0.5 (e.g., from 0.1 to 0.2, from 0.2 to 0.3, from 0.3 to 0.4, or from 0.4 to 0.5).
  • PDI polydispersity index
  • the nanoparticles A prepared in step 1 are electropositive.
  • the nanoparticle A has a zeta potential of +10 mV to +50 mV (eg, +10 mV to +20 mV, +20 mV to +30 mV, +30 mV to +40 mV, or +40 mV to +50 mV).
  • the nanoparticles A prepared in step 1 have a higher encapsulation efficiency and/or drug loading.
  • the encapsulation efficiency of the nanoparticles A is from 90% to 99% (eg, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%) Or 99%).
  • the nanoparticle A has a drug loading of from 60% to 90% (eg, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%). -85% or 85%-90%).
  • the encapsulation efficiency (EE) and drug loading amount (LC) of the nanoparticles A can be measured by a method comprising the steps of: 1) preparing a nanoparticle A using a fluorescently labeled therapeutic protein; 2) comprising The suspension of the nanoparticles A is placed in an ultrafiltration tube for centrifugation to obtain a filtrate; 3) the fluorescence intensity of the therapeutic protein in the filtrate is measured, and the concentration of the therapeutic protein in the filtrate is calculated to determine the free therapeutic protein. The amount.
  • the encapsulation efficiency (EE) and drug loading (LC) were calculated as follows:
  • the step 2 comprises mixing a solution comprising a polyanion with a suspension comprising nanoparticles A.
  • the step 2 further comprises the steps of:
  • Step 2-1 providing a solution comprising a polyanion and a suspension comprising the nanoparticles A;
  • Step 2-2 providing means comprising a vortex mixing zone and a plurality of passages for fluid flow to the vortex mixing zone;
  • Step 2-3 The solution containing the polyanion and the suspension containing the nanoparticles A are passed through different channels to the vortex mixing zone, and mixed to obtain a suspension comprising the nanoparticles of the present invention.
  • the suspension comprising nanoparticles A in step 2-1 is obtained by a process comprising steps 1-1, 1-2, and steps 1-3.
  • the solution comprising polyanion of step 2-1 has a concentration of 0.5-0.9 mg/mL (eg, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, or 0.9 mg/mL).
  • concentration 0.5-0.9 mg/mL (eg, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, or 0.9 mg/mL).
  • the mass concentration 0.5-0.9 mg/mL (eg, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, or 0.9 mg/mL).
  • the solution comprising the polyanion has a mass concentration ratio of from 0.5 to 0.9:1 (eg, 0.5:1, 0.6:1, 0.7:1) , 0.8:1 or 0.9:1).
  • the solution comprising a polyanion is an aqueous solution.
  • the dispersion medium is water.
  • the mass concentration of the solution containing the polyanion means the mass concentration of the polyanion in the solution.
  • the mass concentration of the suspension containing the nanoparticles A refers to the mass concentration of the therapeutic protein contained in the nanoparticles A in the suspension.
  • the device in step 2-2 is a multi-inlet vortex mixer.
  • the solution comprising the polyanion, and the suspension comprising the nanoparticles A flow at a constant flow rate at the same flow rate in the channel,
  • the flow rate is 1-50 mL/min (eg, 1-15 mL/min, 15-25 mL/min, or 25-50 mL/min).
  • the present application also provides a method of preparing the microcapsules of the present invention, the method comprising the steps of:
  • Step 1' preparing the nanoparticles of the present invention
  • Step 2' The nanoparticles obtained in the step 1' are coated with an enteric material.
  • step 1&apos comprises: preparing the nanoparticles of the invention in a method of preparation as described above.
  • the step 2' further comprises the steps of:
  • Step 2'-1 providing a suspension comprising the nanoparticles of the invention and a solution comprising an enteric material;
  • Step 2'-2 providing means comprising a vortex mixing zone and a plurality of channels for fluid flow to the vortex mixing zone;
  • Step 2'-3 mixing a suspension comprising the nanoparticles of the invention, a solution comprising an enteric material, and optionally an acidic solution (such as hydrochloric acid) through different channels into the vortex mixing zone, mixing, to obtain the invention comprising A solution of microcapsules.
  • an acidic solution such as hydrochloric acid
  • the solution comprising enteric material of step 2'-1 has from 0.25-1 mg.mL -1 (eg, 0.25 mg/mL, 0.3 mg/mL, 0.35 mg/mL, 0.4 mg/mL, 0.45) Mg/mL, 0.5 mg/mL, 0.55 mg/mL, 0.6 mg/mL, 0.65 mg/mL, 0.7 mg/mL, 0.75 mg/mL, 0.8 mg/mL, 0.85 mg/mL, 0.9 mg/mL, 0.95 Mass concentration of mg/mL or 1 mg/mL).
  • 0.25-1 mg.mL -1 eg, 0.25 mg/mL, 0.3 mg/mL, 0.35 mg/mL, 0.4 mg/mL, 0.45
  • Mg/mL 0.5 mg/mL, 0.55 mg/mL, 0.6 mg/mL, 0.65 mg/mL, 0.7 mg/mL, 0.75 mg/mL, 0.8 mg/mL, 0.85 mg/mL, 0.9
  • a solution comprising an enteric material a suspension comprising the nanoparticles of the invention has a mass concentration ratio of 0.25-1:1 (eg, 0.25:1, 0.3:1) 0.35:1, 0.4:1, 0.45:1, 0.5:1, 0.55:1, 0.6:1, 0.65:1, 0.7:1, 0.75:1, 0.8:1, 0.85:1, 0.9:1, 0.95: 1 or 1:1).
  • the solution comprising enteric material is an aqueous solution.
  • the dispersion medium is water.
  • the mass concentration of the solution containing the enteric material means the mass concentration of the enteric material in the solution.
  • the mass concentration of the suspension comprising the nanoparticles of the present invention refers to the mass concentration of the therapeutic protein contained in the nanoparticles of the present invention in the suspension.
  • the device in step 2'-2 is a multi-entry vortex mixer.
  • a solution comprising an enteric material, and a suspension comprising the nanoparticles of the invention are flowed at a uniform rate in the channel at the same flow rate.
  • the flow rate is 1-50 mL/min (eg, 1-15 mL/min, 15-25 mL/min, or 25-50 mL/min).
  • Figure 1 exemplarily shows a process for preparing the nanoparticles and microcapsules of the present invention, wherein the therapeutic protein for preparing the nanoparticles is insulin, and the polyanion is hyaluronic acid (HA) for preparing enteric of microcapsules.
  • the material is HPMCP.
  • the preparation process is as follows: In the first step, the CPP solution and the insulin solution are mixed in a multi-inlet vortex mixer to form a surface positively charged nanoparticle A (NP-A); the second step is to mix the NP-A.
  • NP-A surface positively charged nanoparticle A
  • the suspension is mixed with a solution containing HA to form a nanoparticle (NP-B) having NP-A as a core and surface-coated with HA; and a third step, a suspension containing NP-B, a solution containing HPMCP, and The diluted hydrochloric acid having a pH of 2.5 was mixed, and HPMCP was coated on the nanoparticles to obtain enteric microcapsules.
  • the liquid is rapidly mixed using a multi-inlet vortex mixer.
  • therapeutic protein refers to a protein that can be used to prevent or treat a disease, including but not limited to hormones, hormone analogs, enzymes, enzyme inhibitors, and antibodies.
  • cell penetrating peptide refers to a short peptide constructed from generally no more than 30 amino acids that can enter the cell through the cell membrane and can be used to exogenous molecules. Carry into the cell.
  • the cell penetrating peptide can be a naturally occurring peptide or a synthetic peptide.
  • Common cell penetrating peptides include: cationic CPP, for example: TAT (48-60), Penetratin, polyarginine, Oct4, WT1-pTj, DPV3; amphiphilic CPP, for example: Transportan, MAP, VP22, Pep1 KW; hydrophobic CPP, for example: KFGF, FGF12, Integrin ⁇ 3Peptide, C105Y, TP2.
  • Sources and sequences of common cell penetrating peptides can be found, for example, in Joshua D. Ramsey, Nicholas H. Flynn. Cell-penetrating peptides transport therapeutics into cells, Pharmacology & Therapeutics 154 (2015) 78-86.
  • the cell penetrating peptide can be modified, for example, at the C-terminus or N-terminus of the CPP (e.g., alkylation modification).
  • saturated fatty acid refers to a saturated hydrocarbon chain having a carboxyl group at least at one end, wherein the saturated hydrocarbon chain is mostly linear, and the number of carbon atoms may be less than 6, 6-12 or More than 12 (for example, 12-18).
  • saturated fatty acids include, but are not limited to, caproic acid, caprylic acid, capric acid, lauric acid (dodecanoic acid), myristic acid (myristic acid), palmitic acid (hexadecanoic acid), stearic acid (ten Octaic acid), arachidic acid (eicosanic acid).
  • nanoparticle refers to a particle of a size (ie, the diameter in the longest dimension of the particle) at the nanometer scale, such as a size of 1-100 nm, 100-500 nm, 500-1000 nm, or 1000-2000 nm. Particles.
  • particle size or “equivalent particle size” means that when a physical property or physical behavior of a particle to be measured is closest to a homogenous sphere (or combination) of a certain diameter, The diameter (or combination) of the sphere is taken as the equivalent particle size (or particle size distribution) of the particles to be tested.
  • the term "average particle size" means, for an actual population of particles consisting of particles of different sizes and shapes, compared to a hypothetical population of particles consisting of uniform spherical particles, if both When the total length of the particle diameter is the same, the diameter of the spherical particles is referred to as the average particle diameter of the actual particle group.
  • Methods of measuring the average particle size are known to those skilled in the art, such as light scattering; and measuring instruments of average particle size include, but are not limited to, Malvern particle size meters.
  • microcapsule refers to a solid particle having a wall layer and a content embedded in the wall layer.
  • the substance constituting the wall layer is usually a polymer.
  • the microcapsules can be of various shapes, such as spheres, typically having a diameter on the order of microns or millimeters.
  • enteric microcapsule refers to a microcapsule made of an enteric material as the main material of the wall layer, which is resistant to gastric acid and capable of disintegrating and releasing the contents upon entering the intestinal tract. .
  • enteric material refers to a material that is insoluble or nearly insoluble in gastric fluid and that disintegrates or dissolves in intestinal fluid.
  • enteric materials varies with pH.
  • Enteric materials useful in the present invention include, but are not limited to, cellulose and derivatives thereof, such as hypromellose phthalate (HPMCP), cellulose acetate phthalate (CAP), 1, 2, 4-Benzene tricarboxylate acetate (CAT) and the like.
  • the term "suspension” refers to a liquid dispersion formed by the dispersion of solid particulates in a liquid dispersion medium, including but not limited to water.
  • prevention refers to preventing or delaying the onset of a disease.
  • treating refers to curing or at least partially arresting the progression of a disease, or alleviating the symptoms of a disease.
  • the present invention provides a nanoparticle loaded with a therapeutic protein having a core-shell structure in which the core comprises a CPP loaded with a therapeutic protein and the shell layer comprises a polyanion.
  • the nanoparticles of the invention achieve higher mucus permeability and intestinal epithelial cell transport efficiency, and ultimately improve the oral bioavailability and efficacy of the therapeutic protein.
  • the microcapsules comprising the nanoparticles of the present invention can protect the nanoparticles in gastric acid and rapidly release the drug in the intestinal tract, and the drug effect continues to be stable.
  • Figure 1 exemplarily shows a process for preparing the nanoparticles and microcapsules of the present invention, wherein the therapeutic protein for preparing the nanoparticles is insulin, and the polyanion is hyaluronic acid (HA) for preparing enteric of microcapsules.
  • the material is HPMCP.
  • the preparation process is as follows: In the first step, the CPP solution and the insulin solution are mixed in a multi-inlet vortex mixer to form a surface positively charged nanoparticle A (NP-A); the second step is to mix the NP-A.
  • NP-A surface positively charged nanoparticle A
  • the suspension is mixed with a solution containing HA to form a nanoparticle (NP-B) having NP-A as a core and surface-coated with HA; and a third step, a suspension containing NP-B, a solution containing HPMCP, and The diluted hydrochloric acid having a pH of 2.5 was mixed, and HPMCP was coated on the nanoparticles to obtain enteric microcapsules.
  • the liquid is rapidly mixed using a multi-inlet vortex mixer.
  • Figure 2B shows the particle size ( ⁇ ) and dispersion ( ⁇ ) of NP-A prepared at different flow rates of 1 under the initial pH of the CPP solution of 8.0.
  • the particle size of NP-A in the range of flow rate 1 of 1 to 50 mL ⁇ min -1 , by adjusting the flow rate, the particle size of NP-A can be controlled within the range of about 75 nm to about 480 nm, and the dispersion degree is controlled within the range of 0.12 to 0.45.
  • the particle size and dispersion of NP-A decrease sharply and reach a plateau after the flow rate increases to 30 mL ⁇ min -1 .
  • Figure 3 shows the particle size ( ⁇ ) and dispersion ( ⁇ ) of NP-B3 prepared at different flow rates of 2, using NP-A at an initial pH of 8.0 in CPP solution and a flow rate of 1 at 50 mL ⁇ min - Made under the conditions of 1 .
  • the particle size of NP-B decreases from about 219 nm to about 105 nm, and the dispersion decreases from 0.46 to 0.067.
  • Figure 4 shows the morphology of NP-A, NP-B1, NP-B2, and NP-B3 in Table 1.
  • all the nanoparticles are approximately spherical, uniform in particle size and uniform in dispersion. Due to the use of phosphotungstic acid staining, the dark portion should be the enriched region of the positively charged cell-penetrating peptide.
  • Figures A1-A3 are images of NP-A with scales of 1 ⁇ m, 200 nm, and 100 nm, respectively. It can be clearly seen that the vast majority of cell-penetrating peptides are located on the surface of NP-A, forming a fluffy thin layer.
  • Figures A4-A6 are images of NP-B1, NP-B2, and NP-B3, respectively, with scales of 200 nm.
  • a visible light gray layer appeared on the surface of the nano core, indicating that the nano core surface was successfully coated with HA, forming a core-shell structure.
  • the inner nano-core structure becomes denser, indicating that the surface of the nano-core is coated, and the internal structure can be compacted to make the structure of the whole nano-particle more stable.
  • FIG. 5 shows the test results of the FRET experiment.
  • Single fluorescently labeled nanoparticles (Rho123-HA/(CPP/Insulin) and HA/(CPP/RITC-Insulin)) were observed at 450 nm excitation light, and respective emission peaks were observed at 530 nm and 580 nm, respectively.
  • the emission peak (530 nm) of Rho123-HA was significantly reduced after excitation with 450 nm excitation, while the emission of RITC-Insulin was observed.
  • a significant increase in peak (580 nm) means that energy is transferred from the donor to the acceptor, indicating that HA was successfully coated on the nanocore carrying insulin.
  • Figure 6 shows the particle size and particle size distribution of NP-A and NP-B3 in Table 1 at low temperature (4 ° C) or normal temperature (25 ° C).
  • NP-A was stable for 9 h at 25 °C, and the particle size and dispersion increased significantly after 9 h.
  • the stability of the nanoparticles was significantly improved, and the particle size and dispersion were almost unchanged within 48 hours.
  • the stability of nanoparticles of NP-A and NP-B3 was higher, and the particle size and PDI value did not change significantly.
  • the above results show that the stability of the nano core after hydrophilic HA coating is significantly improved.
  • Figure 7 shows the percentage of insulin loss in NP-A, NP-B1, NP-B2, NP-B3 in simulated gastric fluid (pH 2.5) and simulated intestinal fluid (pH 7.0).
  • NP-A in the simulated gastric fluid (1h) and simulated intestinal fluid (2h) insulin loss was as high as 24% and 21%, respectively.
  • the insulin loss can be significantly reduced.
  • the above results indicate that the coating of HA can improve the stability of the nanoparticles and reduce the damage of the gastrointestinal digestive juice to the nanoparticles, and the higher the molecular weight of HA, the better the protective effect.
  • Figure 8 shows insulin release profiles of NP-A and NP-B3 in PBS containing hyaluronidase (0.01 mg/mL) at pH 7.4, and NP-A and NP-B3 at pH 7.4. Insulin release profile in PBS without hyaluronidase. As shown in the figure, NP-A released insulin at the fastest rate with or without hyaluronidase, and there was a significant burst in the first 2 hours (accumulated release rate was 45%). Upon coating with the HA surface, the rate of insulin release is slowed and the rate of insulin release decreases as the molecular weight of the surface HA increases.
  • Figure 9 shows the insulin content in the aggregate precipitate formed after the nanoparticles were incubated with different concentrations of mucin solution for 1 h at 37 ° C in Example 9.
  • the fluorescence intensity of the precipitate obtained by NP-A and 0.5% mucin was used as a control and normalized; # indicates p ⁇ 0.01 compared with the other 0.5% mucin group; * indicates the same with other 1.0% mucin group In comparison, p ⁇ 0.001.
  • Figure 10 shows the toxicity of NP-A, NP-B1, NP-B2, and NP-B3 on HT29-MTX cells. As shown in the figure, the nanoparticles have no obvious cytotoxicity to HT29-MTX and have good safety.
  • Figure 11 is a photograph of a confocal microscope in Example 11, showing free insulin (Figure 11A), NP-A (Figure 11B), NP-B1 (Figure 11C), NP-B2 ( Figure 11D), NP-B3 ( Figure 11E) The penetration in the mucus layer and the integrity of the core structure during penetration through the mucus layer.
  • the scale in the figure is 10 ⁇ m, green is FITC-CPP, red is Cy-5-insulin, and blue is DIPA-stained nuclei.
  • the nanoparticles were sorted according to the order of low to high intake: NP-A ⁇ NP-B1 ⁇ NP-B2 ⁇ NP-B3.
  • NP-B3 had the highest cellular uptake, 11 times and 1.9 times that of free insulin and NP-A, respectively.
  • Figure 13 shows the apparent permeation constant (Papp) of the free insulin group and nanoparticles in the Caco-2/mucin model in Example 13.
  • ⁇ represents p ⁇ 0.01; * represents p ⁇ 0.001.
  • the Papp value of each nanoparticle group was significantly higher, the Papp value of NP-B was increased compared with NP-A, and with the increase of HA molecular weight, Papp increased, indicating that the molecular weight of HA increased with High, the higher the transfer rate.
  • Figure 14 is a fluorescence confocal micrograph of the cross section of jejunum tissue of each group of mice in Example 14, in which free insulin and each nanoparticle are red (fluorescent color of Cy-5), and the mucus layer in the tissue is Green (fluorescent color of WGA-647), the nucleus is blue (DAPI color).
  • the scale in the figure is 50 ⁇ m.
  • NP-B1 has the strongest signal in the small intestine villi, and the fluorescence is mostly located in the presence of villus capillaries, indicating that NP-B3 can be effectively absorbed by epithelial cells and transported to the blood circulation.
  • the above results demonstrate that the ability of the anti-mucous layer of the nanoparticles coated with HA to retain and rapidly osmotic transport is significantly enhanced relative to the positively charged and unmodified nanocores on the surface, thereby increasing the intestinal absorption efficiency of insulin in vivo. improve.
  • Figure 15 is a graph showing changes in blood glucose levels over time in each group of rats in Example 15 - Test 1.
  • # represents relative to the NP-B1 group, p ⁇ 0.05; * represents relative to the NP-B2 group, p ⁇ 0.05.
  • the blood glucose level of the rats orally administered with deionized water was slightly lowered within eight hours after administration.
  • the blood glucose level of the subcutaneous group dropped sharply to 25% of the initial level within 1 h, and the blood glucose remained at a low level for the next 4 h.
  • the blood glucose levels of the rats given the nanoparticles were significantly but slowly decreased within 8 h after administration.
  • NP-A After oral administration of NP-A, NP-B1 and NP-B2 nanoparticles, there was no significant difference in the hypoglycemic effect at the initial stage of administration (0-5 h). During 5 to 8 hours, the hypoglycemic effect of NP-B2 was more pronounced than that of NP-A and NP-B1, and the blood glucose level was reduced to 45% of the initial value. The NP-B3 group had the most significant and long-lasting hypoglycemic effect, and the blood glucose level was slowly and steadily reduced to 40% of the initial value within 8 hours.
  • Figure 16 shows the changes in blood glucose levels over time in each group of rats in Example 15 - Test 2.
  • * represents p ⁇ 0.05 relative to the NP-B1 microcapsule group; # represents relative to the NP-B2 microcapsule group, p ⁇ 0.05.
  • the hypoglycemic effect of the NP-B1 and NP-B2 microcapsule groups was significant.
  • the blood glucose levels of these two groups of rats continued to decrease steadily.
  • the blood glucose levels of the rats decreased to 50% and 40% of the initial levels after 8 hours of administration.
  • the anti-hyperglycemic effect of the NP-B3 microcapsule group was extremely significant, which could reduce the blood glucose level of rats to 20% of the initial value within 8 hours.
  • the blood glucose level of diabetic rats reached the normal range (3.5-5 mM). .
  • Figure 17 is a graph showing the relationship between serum insulin concentration and time in each group of rats in Example 16.
  • the insulin concentration in the rat serum increased sharply and peaked at 1 h ( ⁇ 120 mIU ⁇ L -1 ).
  • serum insulin levels in rats increased slowly, reaching a peak time of 4 h.
  • the relative bioavailability of NP-B1 microcapsules, NP-B2 microcapsules and NP-B3 microcapsules was calculated to be 5.3%, 7.4% and 11%, respectively.
  • Figure 18 shows the contents of ALP, AST, ALT, ⁇ -GT in the serum of each group of rats in Example 17.
  • the negative control group normal rats with oral PBS
  • the positive control group model rats with oral PBS
  • the activity of (ALP, AST, ALT, ⁇ -GT) was not significantly elevated, indicating that oral delivery of the nanoparticles of the present invention and their enteric microcapsules were not significantly toxic in animals.
  • Figure 19 is a photomicrograph showing HE staining sections of liver tissues of each group in Example 17, A1: normal rats orally administered with PBS; A2: model rats orally administered with PBS; A3: large model of oral NP-B3 Rat; A4: Oral HPMCP-coated model rats of NP-B3. Similar to the situation of the control group rats, the liver structure of the rats in the experimental group was intact, and the liver cells were arranged neatly, indicating that the nanoparticles and their enteric microcapsules of the present invention did not cause obvious liver after repeated oral administration of multiple doses. damage.
  • Sequence 1 (SEQ ID NO: 1): 16aa
  • Fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), rhodamine 123 (Rho123) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Cy5 was purchased from Wuhan Little-PA Sciences Co., Ltd.; -Dimethylaminopropyl)carbodiimide hydrochloride (EDC ⁇ HCl), N-hydroxysuccinimide (NHS) was purchased from Shanghai Aladdin Reagent Co., Ltd.; CPP (Ste-RQIKIWFQNRRMKWKK, N-terminal modification was ten Octadecene Penetratin was purchased from Nanjing Peptide Biotechnology Co., Ltd., item number: NJP12879; mucin, hyaluronidase (HAase) was purchased from Shanghai Protoplast Bio Co., Ltd.; sodium hyaluronate (Mw was 4.7KDa, respectively).
  • Malvern laser particle size analyzer Malvern laser particle size analyzer (Malvern Zetasizer Nano ZS, Malvern, UK); freeze dryer (Alpha 1-2LD Plus, Christ, Germany); pH meter (Seven Compact pH Meter, Mettler, Switzerland); automatic double pure water distillation Device American Millipore Company); High Speed Centrifuge (5810R, Eppendorf, Germany); UV-Vis Spectrophotometer (Evo Lution, Thermo, USA); Fluorescence Spectrometer (FS-980, Edinburgh, UK); Laser Confocal Microscopy (SP8, German Leica company); TEM (JEOL-1400, Japan JEOL company); multi-function microplate reader (Synergy 2, Biotek, USA); cell culture incubator (Forma TM Steri-Cycle TM , Thermo Company, USA); blood glucose meter ( UltraVue, Johnson, USA).
  • Malvern laser particle size analyzer Malvern Zetasizer Nano ZS, Malvern, UK
  • freeze dryer
  • Preparation solution Dissolve the cell penetrating peptide powder in double distilled water to prepare a solution with a concentration of 0.3 mg ⁇ mL -1 , and adjust the desired pH value with a 3 M sodium hydroxide solution (5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0).
  • the insulin powder was dissolved in a 0.01 M dilute hydrochloric acid solution to prepare a solution having a concentration of 1 mg ⁇ mL -1 , and the pH was adjusted to its pI with a 3 M sodium hydroxide solution, and slowly adjusted to 6.8 to form a transparent clarification again. Uniform solution.
  • CPP-insulin nano core (nanoparticle A): the cell penetrating peptide solution was injected from the inlets 2 and 3 of the multi-inlet vortex mixer, and the insulin solution was injected from the inlets 1, 4 to keep the flow rates of the solutions uniform (flow rate) 1) A suspension containing nanoparticles A (NP-A) was obtained.
  • HA-coated CPP-insulin nanoparticles The obtained NP-A-containing suspension was injected from the inlets 1, 2, and 3, and the HA solution was injected from the inlet 4 to keep the flow rates of the solutions uniform (flow rate 2).
  • a suspension containing CPP-insulin nanoparticles (NP-B) coated with HA A suspension containing CPP-insulin nanoparticles (NP-B) coated with HA.
  • Mw 4.7 KDa, 35 KDa, 190 KDa
  • the prepared nanoparticles were named NP-B1, NP-B2, and NP-B3, respectively.
  • enteric microcapsules a suspension containing NP-B was injected from the inlet 1 of the multi-inlet vortex mixer, and an ethanol solution (concentration of 0.75 mg ⁇ mL -1 ) of the HPMCP was injected into the inlet 2, and the inlets 3 and 4 were injected. Dilute hydrochloric acid (pH 2.5) gave a suspension containing enteric microcapsules.
  • Fig. 1 exemplarily shows the above preparation process.
  • Rhodamine 123-labeled hyaluronic acid (Rho123-HA)
  • the nanoparticles were prepared using fluorescently labeled starting materials.
  • the preparation conditions were as follows: the initial pH of the cell penetrating peptide solution was 8.0, and the flow rate 1 and the flow rate 2 were both 50 mL ⁇ min -1 .
  • the particle size, dispersion and surface potential of the nanoparticles were determined using a Malvern laser particle size analyzer.
  • the measurement method is as follows: 1) directly measuring the prepared suspension containing the nanoparticles; 2) ultrafiltration of the suspension with an ultrafiltration tube having a molecular weight cut off of 100 kDa at a speed of 300 ⁇ g, for the concentrate 0.01 M phosphate buffer (PBS, pH 6.6) was resuspended and subjected to subsequent assays. The sample was placed in a sample cell and equilibrated at 25 ° C for 120 s. Each sample was measured in parallel three times.
  • PBS phosphate buffer
  • Figure 2B shows the particle size ( ⁇ ) and dispersion ( ⁇ ) of NP-A prepared at different flow rates of 1 under the initial pH of the CPP solution of 8.0.
  • the particle size of NP-A in the range of flow rate 1 of 1 to 50 mL ⁇ min -1 , by adjusting the flow rate, the particle size of NP-A can be controlled within the range of about 75 nm to about 480 nm, and the dispersion degree is controlled within the range of 0.12 to 0.45.
  • the particle size and dispersion of NP-A decrease sharply and reach a plateau after the flow rate increases to 30 mL ⁇ min -1 .
  • the above results indicate that the method of the present invention can obtain NP-A having different particle diameters and dispersions by adjusting the flow rate.
  • Figure 3 shows the particle size ( ⁇ ) and dispersion ( ⁇ ) of NP-B3 prepared at different flow rates of 2, using NP-A at an initial pH of 8.0 in CPP solution and a flow rate of 1 at 50 mL ⁇ min - Made under the conditions of 1 .
  • the particle size of NP-B decreases from about 219 nm to about 105 nm, and the dispersion decreases from 0.46 to 0.067.
  • the above results indicate that the method of the present invention can obtain NP-B having different particle diameters and dispersions by adjusting the flow rate.
  • NP-A and NP-B were prepared under the conditions that the initial pH of the CPP solution was 8.0 and the flow rates 1 and 2 were both 50 mL ⁇ min -1 , and the particle size, particle dispersibility and surface potential were as shown in Table 1.
  • the obtained NP-A had a small particle size ( ⁇ 75 nm) and a narrow distribution (PDI: ⁇ 0.12), the surface is positively charged ( ⁇ +22mV).
  • the particle size increases ( ⁇ 100 nm) and the surface exhibits a negative charge ( ⁇ -20 mV).
  • the reversal of charge indicates that HA was successfully coated on the NP-A surface.
  • the particle size distribution of the nanoparticles is still relatively uniform (PDI: ⁇ 0.1).
  • NP-A was prepared according to the method of Example 2 using RITC-insulin, and a suspension containing NP-B was further obtained.
  • the suspension containing NP-B was placed in an ultrafiltration tube (molecular weight cutoff of 100 KDa) and centrifuged at 300 x g for 20 min at 4 °C.
  • the fluorescence intensity of insulin in the filtrate was measured by a multi-function microplate reader, and the insulin concentration in the filtrate was calculated to determine the amount of free insulin.
  • EE encapsulation efficiency
  • LC loading content
  • the encapsulation efficiency of each nanoparticle is above 90%, and the drug loading is more than 55%.
  • the total yield of NP-B3 nanoparticles can reach 6.6 g ⁇ h -1 , which is equivalent to an insulin yield of about 4.5 g ⁇ h -1 (calculated by the total yield of nanoparticles multiplied by the drug loading), indicating the present invention.
  • the method can be applied to the continuous and large-scale production of insulin nanoparticles.
  • the morphology of the nanoparticles was observed by transmission electron microscopy. Take a suspension containing nanoparticles, drop on a 200 mesh copper mesh covered with a carbon support film, stay for 20 min, use a filter paper to remove excess liquid, add 2% phosphotungstic acid solution for 2 min, and then use a filter paper to remove The excess phosphotungstic acid solution was naturally dried and observed and photographed by transmission electron microscopy.
  • Figure 4 shows the morphology of NP-A, NP-B1, NP-B2, and NP-B3 in Table 1.
  • all the nanoparticles are approximately spherical, uniform in particle size and uniform in dispersion. Due to the use of phosphotungstic acid staining, the dark portion should be the enriched region of the positively charged cell-penetrating peptide.
  • Figures A1-A3 are images of NP-A with scales of 1 ⁇ m, 200 nm, and 100 nm, respectively. It can be clearly seen that the vast majority of cell-penetrating peptides are located on the surface of NP-A, forming a fluffy thin layer.
  • Figures A4-A6 are images of NP-B1, NP-B2, and NP-B3, respectively, with scales of 200 nm.
  • a visible light gray layer appeared on the surface of the nano core, indicating that the nano core surface was successfully coated with HA, forming a core-shell structure.
  • the inner nano-core structure becomes denser, indicating that the surface of the nano-core is coated, and the internal structure can be compacted to make the structure of the whole nano-particle more stable.
  • the fluorescence resonance energy transfer (FRET) technique is used to confirm the interaction between hyaluronic acid and insulin.
  • Example 2 using Rho 123 (ex: 490 nm, em: 530 nm) labeled HA, and RITC (ex: 540 nm, em: 580 nm) labeled insulin, double fluorescently labeled nanoparticles NP-B3 were prepared and formed. Fluorescence resonance energy pair. Nanoparticles prepared by separately labeling Rho123 hyaluronic acid or insulin labeled with RITC alone as a control were used to measure the change in fluorescence spectrum of each sample at an excitation wavelength of 450 nm. The test results are shown in Figure 5.
  • Example 7 Storage stability of nanoparticles and stability in simulated gastrointestinal fluids
  • the NP-A and NP-B3 in Table 1 were placed at low temperature (4 ° C) or normal temperature (25 ° C), and the nanoparticles were monitored at specific time points (1, 3, 6, 9, 12, 18, 24, 48 h). The particle size and particle size distribution of the particles were used to initially evaluate the stability of the nanoparticles during short-term storage.
  • NP-A was stable for 9 h at 25 °C, and the particle size and dispersion increased significantly after 9 h.
  • the stability of the nanoparticles was significantly improved, and the particle size and dispersion were almost unchanged within 48 hours.
  • the stability of nanoparticles of NP-A and NP-B3 was higher, and the particle size and PDI value did not change significantly.
  • the above results show that the stability of the nano core after hydrophilic HA coating is significantly improved.
  • NP-A, NP-B1, NP-B2 and NP-B3 were prepared according to the method of Example 2 using RITC-insulin, dispersed in simulated gastric juice (diluted hydrochloric acid solution, pH 2.5) and simulated intestinal fluid (0.01 M PBS, pH 7.0).
  • Figure 7 shows the percentage of insulin loss in NP-A, NP-B1, NP-B2, NP-B3 in simulated gastric fluid (pH 2.5) and simulated intestinal fluid (pH 7.0).
  • NP-A in the simulated gastric fluid (1h) and simulated intestinal fluid (2h) insulin loss was as high as 24% and 21%, respectively.
  • the insulin loss can be significantly reduced.
  • the above results indicate that the coating of HA can improve the stability of the nanoparticles and reduce the damage of the gastrointestinal digestive juice to the nanoparticles, and the higher the molecular weight of HA, the better the protective effect.
  • NP-A and NP-B3 were prepared according to the method of Example 2 using RITC-insulin.
  • the nanoparticles were dispersed in PBS containing pH 7.4 containing hyaluronidase (0.01 mg ⁇ mL -1 ), and shaken at 37 ° C, 100 rpm. Timed sampling, ultrafiltration (molecular weight cut-off is 100KDa), the fluorescence intensity of the filtrate (ex: 540nm, em: 580nm) was measured by a multi-function microplate reader, and the insulin content was calculated to obtain the cumulative drug release percentage.
  • the nanoparticles were dispersed in a dispersion medium containing no hyaluronidase for in vitro release studies and used as a control experiment.
  • Figure 8 shows insulin release profiles of NP-A and NP-B3 in PBS containing hyaluronidase (0.01 mg/mL) at pH 7.4, and NP-A and NP-B3 at pH 7.4. Insulin release profile in PBS without hyaluronidase. As shown in the figure, NP-A released insulin at the fastest rate with or without hyaluronidase, and there was a significant burst in the first 2 hours (accumulated release rate was 45%). Upon coating with the HA surface, the rate of insulin release is slowed and the rate of insulin release decreases as the amount of surface HA molecules increases.
  • Example 9 In vitro mimic mucin-nanoparticle adsorption
  • This example examines the interaction of nanoparticles with mucin in vitro. Fluorescently labeled nanoparticles were prepared using RITC-insulin according to the method of Example 2. Different concentrations of mucin solution and nanoparticles were incubated for a certain period of time, and the interaction between the nanoparticles and mucin was investigated by monitoring the insulin content in the final formed mucin-nanoparticle aggregate precipitate.
  • HT29-MTX cells in logarithmic growth phase were seeded at a density of 1.0 ⁇ 10 4 cells/well in 96-well plates, and cultured in 200 ⁇ L medium for 24 hours, then the culture solution was aspirated, and 200 ⁇ L of free insulin, NP-A, and NP were added.
  • the medium of -B1, NP-B2 or NP-B3 was incubated at 37 ° C for 24 h. Subsequently, 20 ⁇ L of MTT solution (5 mg ⁇ mL -1 , pH 7.4 PBS) was added to continue incubation for 4 h.
  • the OD sample is the absorbance of the experimental group
  • the OD control is the absorbance of the blank culture medium containing the cells
  • the OD 0 is the absorbance of the background.
  • each of the nanoparticles showed no significant toxicity to the growth of HT29-MTX cells, and the safety was good.
  • This example investigates the ability of nanoparticles to penetrate in the mucus layer and the integrity of the nanocore structure during penetration through the mucus layer.
  • Double fluorescently labeled nanoparticles were prepared using FITC-CPP and Cy5-insulin according to the method of Example 2, and co-incubated with mucus-secreting HT29-MTX cells and observed by confocal microscopy.
  • HT29-MTX cells were seeded at a density of 2 ⁇ 10 4 /well in a dedicated confocal dish (1 ⁇ 1 cm) and cultured for 2 to 3 days. Double fluorescently labeled nanoparticles were added. After incubation for 3 h, the medium was removed and gently washed twice with PBS to remove the sample adsorbed on the surface of the mucus layer, and the cells were fixed with 4% paraformaldehyde for 10 min and washed twice with PBS. Subsequent scanning was performed layer by layer under a confocal microscope. Cy-5-labeled free insulin was used as a control group.
  • Figure 11 shows the penetration of free insulin (Figure 11A), NP-A (Figure 11B), NP-B1 (Figure 11C), NP-B2 ( Figure 11D), NP-B3 ( Figure 11E) in the mucus layer.
  • the scale in the figure is 10 ⁇ m, green is FITC-CPP, red is Cy-5-insulin, and blue is DIPA-stained nuclei.
  • the overall fluorescence intensity of insulin was significantly smaller than that of each nanoparticle group, and most of them stayed in the mucus layer (see photo at 15 ⁇ m), and finally passed through the mucus layer.
  • the amount reaching the cell surface is small. The reason may be that the insulin molecules are directly exposed to the mucus environment and are easily destroyed by enzymatic degradation in the mucus, thus exhibiting a lower overall fluorescence intensity.
  • insulin is a hydrophilic macromolecule that is difficult to penetrate through the mucus layer and is taken up by intestinal epithelial cells.
  • NP-A For the NP-A group, a large number of mucin-nanoparticle aggregates were formed at the initial stage of penetration of the nanoparticles in the mucus layer, which was difficult to pass down and stay in place (see photo at 0 ⁇ m).
  • the possible reason is NP-
  • the surface of A has a positive charge and is susceptible to electrostatic interaction with various mucins in the mucus layer.
  • the fluorescence intensity at the cell level is increased. The reason may be that NP-A has a certain protective effect on the insulin inside, and it is easy to enter the cell with the negatively charged cell membrane.
  • NP-B For NP-B, as the molecular weight of HA increases, the retention of nanoparticles in the mucus layer gradually decreases, and the signal reaching the cell surface gradually increases, indicating that the hydrophilic, negatively charged HA coating does increase the nanometer.
  • the stability of the granules reduces the affinity for mucin in the mucus layer, and the protective effect is related to the molecular weight of HA.
  • HT29-MTX cells were seeded at a density of 5 x 10 4 cells/well in 24-well plates, and fluorescently labeled nanoparticles prepared using RITC-insulin were added. After 3 h of incubation, the medium was removed and gently washed 3 times with PBS to remove residual mucus and samples adsorbed on the cell surface. Cell number was determined using Scepter TM 2.0 with an automatic cell counter. After the cells were fully lysed and disrupted with the cell lysate, the fluorescence intensity of the cell lysate was measured using a multi-function microplate reader. The relative quantification of cell uptake was unified to the fluorescence intensity value corresponding to 5.0 x 10 3 cells. RITC-labeled free insulin was used as a control group.
  • Figure 12 shows the relative uptake of free insulin and each group of nanoparticles by HT29-MTX cells.
  • the intake of each nanoparticle group was significantly higher than that of the free insulin group.
  • the nanoparticles were sorted according to the order of low to high intake: NP-A ⁇ NP-B1 ⁇ NP-B2 ⁇ NP-B3.
  • NP-B3 had the highest cellular uptake, 11 times and 1.9 times that of free insulin and NP-A, respectively.
  • the uptake of nanocore NP-A is lower, probably because the positively charged nanocore NP-A is easily aggregated with negatively charged mucin into larger particles.
  • the permeation in the mucus layer is hindered, the amount reaching the cell surface is significantly reduced, and the cell uptake efficiency is lowered.
  • the HA-coated nanoparticles can efficiently permeate through the mucus layer and are easily taken up by the cells.
  • Fluorescently labeled nanoparticles were prepared using RITC-insulin according to the method of Example 2.
  • the Caco-2 cell model with artificial mucin added on the surface was used to investigate the trans-cell transport ability of the nanoparticles and the effect of the mucus layer on the nanoparticle transport efficiency.
  • Cell culture was placed in a 37 ° C, 5% CO 2 incubator with 10% fetal bovine serum, 1% L-glutamine, 1% cyan-streptomycin (100 IU ⁇ mL - 1 ), 1% non-essential amino acid, 10 mg ⁇ mL -1 mucin in DMEM high glucose medium.
  • Caco-2 cells were seeded on a polyester membrane of a Costar Transwell 12-well plate (polyester membrane pore size 0.4 ⁇ m, diameter 12 mm, cell growth region 1.1 cm 2 ), every 2 days
  • the Trans-epithellal Electric Resistance (TEER) value was monitored using a Millicell ERS-2 type resistance meter; and the culture medium was changed every two days. After 2 to 3 weeks of culture, when the TEER value is higher than 700 ⁇ cm 2 , it can be used in subsequent experiments.
  • the culture medium of the upper and lower chambers of Transwell was replaced with Hanks balanced salt solution (HBSS) and equilibrated at 37 ° C for 30 min.
  • the upper chamber was changed to 200 ⁇ L of HBSS containing fluorescently labeled nanoparticles (wherein insulin was labeled at a concentration of 0.1 mg ⁇ mL -1 ).
  • an appropriate amount of the lower chamber solution was taken, and the fluorescence intensity in the solution was measured by a multi-function microplate reader to determine the insulin concentration, and the apparent permeation constant (Papp) was calculated.
  • Fluorescently labeled free insulin was used as a control group.
  • Q is the total amount of drug permeated (ng); c is the initial concentration of the upper chamber drug (ng ⁇ cm -3 ); A is the membrane area (cm 2 ); t is the test time (s).
  • Figure 13 shows the apparent permeation constant (Papp) of the free insulin group and nanoparticles in the Caco-2/mucin model.
  • ⁇ represents p ⁇ 0.01; * represents p ⁇ 0.001.
  • the Papp value of each nanoparticle group was significantly higher, the Papp value of NP-B was increased compared with NP-A, and with the increase of HA molecular weight, Papp increased, indicating that the molecular weight of HA increased with High, the higher the transfer rate.
  • Fluorescently labeled nanoparticles were prepared in accordance with the method of Example 2 using Cy5-insulin to trace the absorption behavior of insulin in rat intestinal villi.
  • Several normal male SD rats (230-250g) were divided into 5 groups: insulin solution group, NP-A group, NP-B1 group, NP-B2 group, NP-B3 group (insulin dose: 1.5 mg ⁇ kg) -1 ).
  • insulin solution group a free insulin solution was administered; for each nanoparticle group, the suspension containing the nanoparticles prepared in Example 1 was lyophilized by centrifugation (4000 rpm), and the obtained solid was resuspended in double distilled water. The resulting suspension was administered to rats. Animals were fasted overnight before administration and were given free access to water.
  • the rats were anesthetized with sodium pentobarbital (0.04 mg ⁇ kg -1 ), the rat abdominal cavity was opened along the midline of the abdomen, the intestinal tissue was gently removed, and the jejunum segment near the duodenum was taken. ( ⁇ 10cm), cut longitudinally along the lumen of the intestine, gently rinse the surface-adhered nanoparticles and visible impurities with PBS, then gently crimp it and quickly place it in liquid nitrogen-cooled OCT (optimum cutting temperature) ) fixed in the compound.
  • sodium pentobarbital 0.4% mg ⁇ kg-1
  • the rat abdominal cavity was opened along the midline of the abdomen, the intestinal tissue was gently removed, and the jejunum segment near the duodenum was taken. ( ⁇ 10cm), cut longitudinally along the lumen of the intestine, gently rinse the surface-adhered nanoparticles and visible impurities with PBS, then gently crimp it and quickly place it in liquid nitrogen-cooled OCT (optimum cutting temperature) ) fixed in the compound
  • Tissue sections (8 ⁇ m) were sectioned using a cryostat, and mucin and nuclei in the mucus layer of the cross section of the tissue were stained with Alexa Fluor 647-labeled wheat germ agglutinin (WGA) and DAPI dye, respectively, and observed by confocal microscopy.
  • WGA Alexa Fluor 647-labeled wheat germ agglutinin
  • DAPI DAPI dye
  • Tissue sections were stained with nuclear DAPI staining solution for 5 min in the dark, and the cells were washed twice.
  • Figure 14 is a fluorescence confocal micrograph of the cross section of jejunum tissue of each group of mice.
  • free insulin and each nanoparticle are red (fluorescent color of Cy-5), and the mucus layer in the tissue is green (WGA-647). Fluorescent color), the nucleus is blue (DAPI color).
  • the scale in the figure is 50 ⁇ m.
  • NP-B3 has the strongest signal in the small intestine villi, and the fluorescence is mostly located in the presence of villus capillaries, indicating that NP-B3 can be effectively absorbed by epithelial cells and transported to the blood circulation.
  • the above results demonstrate that the ability of the anti-mucous layer of the nanoparticles coated with HA to retain and rapidly osmotic transport is significantly enhanced relative to the positively charged and unmodified nanocores on the surface, thereby increasing the intestinal absorption efficiency of insulin in vivo. improve.
  • Example 15 Establishment of a rat model of type I diabetes and hypoglycemic test in vivo
  • Fifty-six type I diabetic rats were divided into 7 groups: negative control group (oral deionized water), insulin aqueous solution (insulin concentration 1 mg ⁇ mL -1 ) subcutaneous (sc) injection group (5 IU ⁇ kg -1 ), And an aqueous solution of insulin, NP-A, NP-B1, NP-B2, NP-B3 (insulin concentration 1 mg ⁇ mL -1 ) orally administered group (80 IU ⁇ kg -1 ).
  • Each of the nanoparticle suspensions was obtained by the following method: The suspension containing the nanoparticles prepared in Example 1 was lyophilized by centrifugation (4000 rpm), and the obtained solid was resuspended in double distilled water. Animals were fasted overnight before administration and were given free access to water. Blood samples were collected from the tail veins before administration and at 1, 2, 3, 4, 5, 6, 7, and 8 hours after administration, and blood glucose levels were measured by a blood glucose meter.
  • Figure 15 shows changes in blood glucose levels over time after administration of rats in each group.
  • # represents relative to the NP-B1 group, p ⁇ 0.05; * represents relative to the NP-B2 group, p ⁇ 0.05.
  • the blood glucose level of the rats orally deionized water was slightly lowered, possibly because the diabetic rats were in a fasting state during administration, and there was a basic decrease.
  • the blood glucose level of rats with oral insulin solution was not significantly different from that of the negative control group.
  • the blood glucose level of the subcutaneous group dropped sharply to 25% of the initial level within 1 h, and the blood glucose remained at a low level for the next 4 h.
  • blood glucose levels reach a lower level of dip in a relatively short period of time, which is highly risky for diabetics, severely causing hypoglycemia, coma, shock, and even death.
  • the blood glucose levels of the rats given the nanoparticles were significantly but slowly decreased within 8 h after administration.
  • NP-A, NP-B1 and NP-B2 nanoparticles there was no significant difference in the hypoglycemic effect at the initial stage of administration (0-5 h).
  • the hypoglycemic effect of NP-B2 was more pronounced than that of NP-A and NP-B1, and the blood glucose level was reduced to 45% of the initial value.
  • the NP-B3 group had the most significant and long-lasting hypoglycemic effect, and the blood glucose level was slowly and steadily reduced to 40% of the initial value within 8 hours.
  • NP-B1 microcapsule group Eighteen type I diabetic rats were divided into three groups: HPMCP-coated NP-B1 microcapsule group, NP-B2 microcapsule group and NP-B3 microcapsule group (insulin concentration 1 mg ⁇ mL -1 ). The capsules were resuspended in double distilled water and administered orally (80 IU ⁇ kg -1 ).
  • Figure 16 shows the changes in blood glucose levels over time after oral administration of microcapsules in each group of rats.
  • * represents p ⁇ 0.05 relative to the NP-B1 microcapsule group; # represents relative to the NP-B2 microcapsule group, p ⁇ 0.05.
  • the hypoglycemic effect of the NP-B1 and NP-B2 microcapsule groups was significant.
  • the blood glucose levels of these two groups of rats continued to decrease steadily.
  • the blood glucose levels of the rats decreased to 50% and 40% of the initial levels after 8 hours of administration.
  • the anti-hyperglycemic effect of the NP-B3 microcapsule group was extremely significant, which could reduce the blood glucose level of rats to 20% of the initial value within 8 hours.
  • the blood glucose level of diabetic rats reached the normal range (3.5-5 mM). .
  • the microcapsule of the invention can be used for oral administration, can slow down, stably and persistently lower blood sugar, has high safety, avoids the risk of sudden drop of blood sugar, and is more acceptable to the majority of diabetic patients.
  • Twenty-four type I diabetic rats were divided into 4 groups: insulin injection group (sc5IU ⁇ kg -1 ), NP-B1 microcapsule group, NP-B2 microcapsule group and NP-B3 microcapsule group (insulin concentration 1 mg). ⁇ mL -1 ). The microcapsules were resuspended in double distilled water and administered orally (80 IU ⁇ kg -1 ).
  • Blood samples were collected from the tail veins at 0.3, 0.6, 1, 2, 3, 4, 5, 6, 7 and 8 h after administration, placed in a 1.5 mL collection tube, centrifuged at 3000 rpm for 15 min after coagulation, and the upper serum was carefully collected. Store at -80 °C for backup to avoid repeated freezing and thawing.
  • the insulin concentration in the serum was determined using a porcine insulin ELISA kit using a dual solid phase enzyme immunoassay. The specific operations are as follows:
  • washing the plate Turn the enzyme labeling plate to discard the reaction solution, add 350 ⁇ L of the cleaning solution to each well, shake for a few seconds, discard the cleaning solution, and pat the peeling cleaning solution vigorously on the adsorption paper, and repeat the washing plate 5 times;
  • BA relative bioavailability
  • AUC oral gavage and AUC subcutaneous injection represent the total area under the serum insulin concentration versus time curve after oral gavage and subcutaneous injection , respectively.
  • Figure 17 shows the changes in serum insulin concentration over time in each group of rats.
  • the insulin concentration in the rat serum increased sharply and peaked at 1 h ( ⁇ 120 mIU ⁇ L -1 ), which was compared with that in Example 15, in the diabetic rat model.
  • serum insulin levels in rats increased slowly, reaching a peak time of 4 h.
  • the relative bioavailability of NP-B1 microcapsules, NP-B2 microcapsules and NP-B3 microcapsules was calculated to be 5.3%, 7.4% and 11%, respectively.
  • mice Six normal rats were used as a negative control group, and 18 type I diabetic rats were divided into three groups: oral PBS group, oral NP-B3 group, and oral HPMCP-coated NP-B3 group (insulin). The concentration of 1 mg ⁇ mL -1 ), both the nanoparticles and the microcapsules were resuspended in double distilled water (80 IU ⁇ kg -1 ).
  • blood samples were collected from the tail vein, placed in a 1.5 mL collection tube, and centrifuged at 3000 rpm for 15 min after coagulation. The upper serum was carefully collected and stored at -80 °C to avoid repeated freezing and thawing.
  • alkaline phosphatase ALP
  • aspartate aminotransferase AST
  • alanine aminotransferase ALT
  • ⁇ -glutamyltranspeptidase ⁇ - in serum
  • the animals were anesthetized with sodium pentobarbital (0.04 mg ⁇ kg -1 ), the abdominal cavity was opened along the midline of the abdomen, and the residual blood was washed with PBS for cardiac perfusion.
  • the liver tissue was gently removed and fixed with 4% paraformaldehyde.
  • the cells were embedded in paraffin, sectioned with a cryostat (8 ⁇ m), and subjected to HE staining.
  • Figure 18 shows the contents of ALP, AST, ALT, ⁇ -GT in the serum of each group of rats.
  • the negative control group normal rats with oral PBS
  • the positive control group model rats with oral PBS
  • the activity of (ALP, AST, ALT, ⁇ -GT) was not significantly elevated, indicating that oral delivery of the nanoparticles of the present invention and their enteric microcapsules were not significantly toxic in animals.
  • Figure 19 shows photomicrographs of HE stained sections of liver tissue of each group of rats, A1: normal rats with oral PBS; A2: model rats with oral PBS; A3: model rats with oral NP-B3; A4: oral HPMCP coated NP-B3 model rats. Similar to the situation of the control group rats, the liver structure of the rats in the experimental group was intact, and the liver cells were arranged neatly, indicating that the nanoparticles and their enteric microcapsules of the present invention did not cause obvious liver after repeated oral administration of multiple doses. damage.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Nanotechnology (AREA)
  • Optics & Photonics (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Diabetes (AREA)
  • Endocrinology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

一种负载治疗性蛋白的纳米粒，其包含核心和包被在核心上的聚阴离子；所述核心包含治疗性蛋白，还包含细胞穿透肽(CPP)。还包括含有所述纳米粒的微胶囊，制备所述纳米粒或微胶囊的方法，含有所述纳米粒或微胶囊的药物组合物，以及所述纳米粒或微胶囊的用途。所述药物组合物用于预防或治疗所述纳米粒或微胶囊中包含的治疗性蛋白所能够预防或治疗的疾病。优选所述CPP为Penetratin，优选治疗性蛋白为胰岛素，所述聚阴离子为透明质酸，所述疾病为高血糖症。

Description

负载治疗性蛋白的纳米粒及其微胶囊 技术领域
本发明属于纳米医药技术领域，涉及一种负载治疗性蛋白的纳米粒，含有所述纳米粒的微胶囊，制备所述纳米粒或微胶囊的方法，含有所述纳米粒或微胶囊的药物组合物，以及所述纳米粒或微胶囊的用途。
背景技术
胰岛素是一种蛋白质激素，由胰脏内的胰岛β细胞分泌，参与调节糖代谢，控制血糖平衡。外源性胰岛素主要用来治疗糖尿病。胰岛素的传统给药途径是皮下注射，然而，长期注射会使患者的依从性降低，并产生诸多副作用。
相比皮下注射，胰岛素的口服给药途径具有患者依从性好、经济、方便、安全等优点。胰岛素经口服后，可经肝门静脉进入体循环，直接参与肝脏对葡萄糖的代谢，能够有效地模拟内源性胰岛素的分泌模式和生理作用。但是，胰岛素通过口服途径给药时有以下亟待解决的问题：首先，由于胃中的酸性环境，胰岛素在胃中很容易被降解；第二，胰岛素在消化道内可能被酶降解失活；最后，由于胰岛素的高分子量和低脂溶性，其在肠粘膜上皮细胞的渗透性低，造成口服生物利用度较低。
现有技术中，已经出现了负载胰岛素等蛋白类药物的纳米粒，用于这些药物的口服递送。将纳米粒作为载体，有助于减少胃中的酸性环境和/或酶环境对药物的影响。但是，这些纳米粒往往存在粒径分布宽、表面形态不均匀、生物利用度不高或药效不高等问题。特别地，胃肠道系统中，在肠细胞表面覆盖有黏液层，黏液层中的粘蛋白可通过静电或疏水作用与纳米粒络合，在原位形成较大的聚集体，纳米粒锚定在黏液层中，影响了纳米粒在黏液层中的快速无规则运动，进而阻碍纳米粒在黏液层中的渗透效率。因此，黏液层的存在成为限制口服蛋白类药物递送效率的瓶颈问题。
发明内容
本发明人通过深入的研究和创造性的劳动，得到了一种负载治疗性蛋白的纳米粒。本发明人利用带负电的聚阴离子对正电的载药纳米核心进行包被，形成外层为带负电的聚阴离子、内部为载药纳米核心的、具有壳核结构的纳米粒。本发明的纳米粒可以通过静电排斥作用，减少其自身与黏液层中粘蛋白的相互作用，增加纳米粒在黏液层中的渗 透效率，从而将更多的药物递送至肠上皮细胞。
因此，在一个方面，本申请提供了一种纳米粒，其包含核心和包被在核心上的聚阴离子；所述核心包含治疗性蛋白，还包含细胞穿透肽(CPP)。
在某些实施方案中，所述聚阴离子选自三聚磷酸钠、海藻酸、肝素、透明质酸(HA)、透明质酸盐、硫酸软骨素、聚丙烯酸类聚合物、聚苯乙烯磺酸类聚合物或其任意组合。
在某些实施方案中，所述聚阴离子选自透明质酸、透明质酸盐(例如透明质酸钠)或其组合。透明质酸具有来源广泛、无毒、生物可降解、生物相容性好等特点，适于载药纳米粒的制备。透明质酸在碱性条件下可转化为透明质酸盐。
在某些实施方案中，所述聚阴离子的重均分子量为4kDa-200kDa(例如4kDa-10kDa、10kDa-50kDa、50kDa-100kDa、100kDa-150kDa或150kDa-200kDa)。
本发明的纳米粒中，所述核心包含的治疗性蛋白可以是激素、激素类似物、酶、酶抑制剂或抗体。在某些实施方案中，所述治疗性蛋白为胰岛素。
本发明的纳米粒中，所述核心包含的CPP可以起到载体的作用。CPP被认为具有负载药物活性分子并增强其跨细胞转运的能力。在某些实施方案中，所述CPP包含精氨酸残基。在某些实施方案中，所述CPP的N端为精氨酸残基。在某些实施方案中，所述CPP为Penetratin。Penetratin来源于果蝇的触角足蛋白的同源异形域，由16个氨基酸残基组成，其氨基酸序列如SEQ ID NO：1所示。Penetratin可以介导多种疏水大分子进入活体细胞质内而不破坏细胞膜的完整性。
在某些实施方案中，所述CPP被烷基修饰。使用烷基化的CPP作为载体，更容易形成稳定、分散性好的纳米粒。可以通过包含以下步骤的方法获得烷基化的CPP：使饱和脂肪酸的羧基与CPP上的氨基(例如，N端的氨基)发生缩合反应，形成酰胺键。优选地，所述缩合反应在缩合剂(例如，O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸(TBTU))、溶剂(例如，N,N-二甲基甲酰胺(DMF))和/或碱性试剂(例如，N,N-二异丙基乙胺(DIEA))存在的条件下进行。
在某些实施方案中，所述烷基是C 12-C 18烷基(例如C 12烷基、C 14烷基、C 16烷基或C 18烷基)。在某些实施方案中，所述烷基是直链烷基。
在某些实施方案中，所述CPP的N端被烷基修饰。
在某些实施方案中，所述CPP为N端被正十八烷基修饰的Penetratin。
本发明的纳米粒可以被荧光化合物所标记，因此，在某些实施方案中，所述纳米粒还包含荧光化合物或荧光化合物的生色团。所述荧光化合物包括但不限于异硫氰酸 荧光素(FITC)、罗丹明异硫氰酸酯(RITC)、3H-吲哚菁类染料(例如Cy3、Cy5)或罗丹明(例如罗丹明6G、罗丹明123、罗丹明B)。所述荧光化合物可以标记在聚阴离子、治疗性蛋白和/或CPP上。
本发明的纳米粒及其包含的核心可以是任何形状。在某些实施方案中，所述纳米粒为球形。在某些实施方案中，所述核心为球形。
在某些实施方案中，本发明的纳米粒具有窄的粒径和/或均一的粒径分布。在某些实施方案中，所述纳米粒的粒径为100nm-900nm(例如100nm-200nm、200-300nm、300-400nm、400-500nm、500-600nm、600-700nm、700-800nm或800-900nm)。在某些实施方案中，所述纳米粒粒径的多分散指数(PDI)为0.05-0.5(例如0.05-0.1、0.1-0.2、0.2-0.3、0.3-0.4或0.4-0.5)。
在某些实施方案中，本发明的纳米粒所包含的核心具有窄的粒径和/或均一的粒径分布。在某些实施方案中，所述核心的粒径为30nm-500nm(例如30nm-100nm、100-200nm、200-300nm、300-400nm或400-500nm)。在某些实施方案中，所述核心的粒径的多分散指数(PDI)为0.1-0.5(例如0.1-0.2、0.2-0.3、0.3-0.4或0.4-0.5)。
在某些实施方案中，本发明的纳米粒表面呈电负性。在某些实施方案中，所述纳米粒的Zeta电位为-10mV至-50mV(例如-10mV至-20mV、-20mV至-30mV、-30mV至-40mV或-40mV至-50mV)。
在某些实施方案中，本发明的纳米粒具有较高的包封率和/或载药量。在某些实施方案中，所述纳米粒的包封率为90％-99％(例如90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)。在某些实施方案中，所述纳米粒的载药量为50％-90％(例如50％-55％、55％-60％、60％-65％、65％-70％、70％-75％、75％-80％、80％-85％或85％-90％)。
本发明中，纳米粒的包封率(EE)和载药量(LC)可以通过包含以下步骤的方法测得：1)使用荧光标记的治疗性蛋白制得纳米粒，2)将包含所述纳米粒的混悬液置于超滤管中进行离心，得到滤液；3)测定滤液中治疗性蛋白的荧光强度，进而计算滤液中的治疗性蛋白的浓度，以确定游离治疗性蛋白的量。按下式计算包封率(EE)和载药量(LC)：
Figure PCTCN2018101106-appb-000001
Figure PCTCN2018101106-appb-000002
在某些实施方案中，所述纳米粒中，治疗性蛋白：聚阴离子：CPP的质量比为1：0.5-0.9：0.1-0.5(例如1：0.5：0.1-0.5、1：0.6：0.1-0.5、1：0.7：0.1-0.5、1：0.8：0.1-0.5或1：0.9：0.1-0.5，例如1：0.5-0.9：0.1、1：0.5-0.9：0.2、1：0.5-0.9：0.3、1：0.5-0.9：0.4或1：0.5-0.9：0.5)。进一步地，在某些实施方案中，聚阴离子与CPP的质量之和：治疗性蛋白的质量比为1:1。在某些实施方案中，聚阴离子：CPP：治疗性蛋白的质量比为0.9：0.1：1、0.8：0.2：1、0.7：0.3：1、0.6：0.4：1或0.5：0.5：1。
本申请还提供了一种微胶囊，所述微胶囊包含壁层，以及包埋在壁层中的本发明的纳米粒，所述壁层包含肠溶材料或主要由肠溶材料组成。本发明中，将负载蛋白类药物的纳米粒制成肠溶微胶囊，可提高纳米粒在胃肠道中的稳定性、提高肠道部位药物释放的特异性、改善药动学行为，从而提高药物在体内的生物利用度、改善体内药效学结果。
在某些实施方案中，所述肠溶材料选自纤维素及其衍生物，例如羟丙甲纤维素邻苯二甲酸酯(HPMCP)、邻苯二甲酸乙酸纤维素(CAP)、1,2,4-苯三甲酸乙酸纤维素(CAT)等。在某些实施方案中，所述肠溶材料为HPMCP。
在某些实施方案中，所述微胶囊的粒径为1-10μm(例如1μm-2μm、2μm-3μm、3μm-4μm、4μm-5μm、5μm-6μm、6μm-7μm、7μm-8μm、8μm-9μm或9μm-10μm)。
在某些实施方案中，所述微胶囊的形状为球形。
在某些实施方案中，所述微胶囊的包封率为30％-95％(例如30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％或95％)。
本发明中，微胶囊的包封率(EE)可以通过包含以下步骤的方法测得：1)使用荧光标记的治疗性蛋白制得纳米粒，2)将所述纳米粒制成微胶囊，3)将包含所述微胶囊的混悬液置于超滤管中进行离心，得到滤液；4)测定滤液中治疗性蛋白的荧光强度，进而计算滤液中的治疗性蛋白的浓度，以确定游离治疗性蛋白的量。按下式计算包封率(EE)：
在一个方面，本申请提供了一种药物组合物，其包含本发明的纳米粒或微胶囊。
在某些实施方案中，所述药物组合物用于预防或治疗所述纳米粒或微胶囊中包含的治疗性蛋白所能够预防或治疗的疾病。在某些实施方案中，所述治疗性蛋白为胰岛素，所述药物组合物用于预防或治疗受试者中的高血糖症。在某些实施方案中，所述 高血糖症包括应激诱发高血糖症、糖尿病(包括1型糖尿病和2型糖尿病)和葡萄糖耐量受损。
在某些实施方案中，所述药物组合物包含预防或治疗有效剂量的纳米粒或微胶囊。在某些实施方案中，所述药物组合物包含一种或多种药用载体。可用于本发明的药用载体包括但不限于填充剂、稀释剂、粘合剂、润湿剂、崩解剂、润滑剂、表面活性剂、防腐剂、着色剂、矫味剂、芳香剂、泡腾剂、乳化剂、絮凝剂、反絮凝剂、抑菌剂、增溶剂。在某些实施方案中，所述药用载体选自:离子交换剂、氧化铝、硬脂酸铝、卵磷脂、血清蛋白(例如人血清蛋白)、甘油、山梨酸、山梨酸钾、水、硫酸鱼精蛋白、磷酸氢二钠、磷酸氢钾、氯化钠、锌盐、胶态氧化硅、三硅酸镁、聚乙烯吡咯烷酮、纤维素物质、聚乙二醇、羧甲基纤维素钠、聚丙烯酸酯、蜂蜡、聚乙烯-聚氧丙烯嵌段聚合物、羊毛脂及其任意组合。
在某些实施方案中，所述药物组合物包含冻干保护剂。在某些实施方案中，所述冻干保护剂为醇类冻干保护剂，例如木糖醇、甘露醇或山梨醇。
本发明的药物组合物可被制成各种合适的剂型，包括但不限于：口服剂型、注射剂型(例如适于皮下注射、肌肉注射或静脉注射的剂型)、吸入剂型、粘膜给药剂型或者局部给药剂型。在某些实施方案中，所述药物组合物被制成口服剂型，例如片剂、胶囊剂、颗粒剂、口服溶液、口服混悬液、微丸剂或微片剂。
在一个方面，本申请提供了本发明的纳米粒或微胶囊用于制备药物组合物的用途，所述药物组合物用于预防或治疗所述纳米粒或微胶囊中包含的治疗性蛋白所能够预防或治疗的疾病。在某些实施方案中，所述治疗性蛋白为胰岛素，所述疾病为高血糖症。在某些实施方案中，所述高血糖症包括应激诱发高血糖症、糖尿病(包括1型糖尿病和2型糖尿病)和葡萄糖耐量受损。
在一个方面，本申请提供了一种预防或治疗疾病的方法，包括给有此需要的受试者施用本发明的纳米粒、微胶囊或药物组合物，所述疾病为所述纳米粒、微胶囊或药物组合物中包含的治疗性蛋白所能够预防或治疗的疾病。在某些实施方案中，所述治疗性蛋白为胰岛素，所述疾病为高血糖症。在某些实施方案中，所述高血糖症包括应激诱发高血糖症、糖尿病(包括1型糖尿病和2型糖尿病)和葡萄糖耐量受损。
在本申请的实施方案中，所述受试者优选为哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物；例如，所述受试者为人。
在一个方面，本申请提供了制备本发明纳米粒的方法，所述方法包括以下步骤：
步骤1：制备纳米粒A，所述纳米粒A为包含治疗性蛋白和CPP的纳米粒；
步骤2：使用聚阴离子对步骤1得到的纳米粒A进行包被。
在某些实施方案中，所述步骤1包括：使包含治疗性蛋白的溶液与包含CPP的溶液进行混合。
在某些实施方案中，所述步骤1进一步包括以下步骤：
步骤1-1：提供包含治疗性蛋白的溶液和包含CPP的溶液；
步骤1-2：提供包含涡流混合区域和多个可供流体流向涡流混合区域的通道的装置；
步骤1-3：使包含治疗性蛋白的溶液和包含CPP的溶液通过不同的通道到达涡流混合区域中，进行混合，得到包含纳米粒A的混悬液。
在某些实施方案中，步骤1-1的包含CPP的溶液具有0.1-0.5mg/mL(例如0.1mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL或0.5mg/mL)的质量浓度。
在某些实施方案中，步骤1-1中，包含CPP的溶液：包含治疗性蛋白的溶液的质量浓度比为0.1-0.5：1(例如0.1：1、0.2：1、0.3：1、0.4：1或0.5：1)。
在某些实施方案中，所述包含治疗性蛋白的溶液为水溶液。
在某些实施方案中，所述包含CPP的溶液为水溶液。
本发明中，包含治疗性蛋白的溶液的质量浓度是指所述溶液中的治疗性蛋白的质量浓度。
本发明中，包含CPP的溶液的质量浓度是指所述溶液中的CPP的质量浓度。
在某些实施方案中，所述步骤1-1还包括：使用酸性溶液(例如盐酸)或碱性溶液(例如氢氧化钠溶液)将所述包含治疗性蛋白的溶液的pH调整至6.5-7.0(例如6.5、6.6、6.7、6.8、6.9或7.0)。
在某些实施方案中，所述步骤1-2中的装置为多入口涡流混合器(multi-inlet vortex mixer(MIVM))。
在某些实施方案中，所述步骤1-3中，包含治疗性蛋白的溶液，以及包含CPP的溶液在通道中以相同的流速匀速流动。在某些实施方案中，所述流速为1-50mL/min(例如1-15mL/min、15-25mL/min或25-50mL/min)。
在某些实施方案中，步骤1制备的纳米粒A具有窄的粒径和/或均一的粒径分布。在某些实施方案中，所述纳米粒A的粒径为30nm-500nm(例如30nm-100nm、100-200nm、200-300nm、300-400nm或400-500nm)。在某些实施方案中，所述纳米 粒A的粒径的多分散指数(PDI)为0.1-0.5(例如0.1-0.2、0.2-0.3、0.3-0.4或0.4-0.5)。
在某些实施方案中，步骤1制备的纳米粒A呈电正性。在某些实施方案中，所述纳米粒A的Zeta电位为+10mV至+50mV(例如+10mV至+20mV、+20mV至+30mV、+30mV至+40mV或+40mV至+50mV)。
在某些实施方案中，步骤1制备的纳米粒A具有较高的包封率和/或载药量。在某些实施方案中，所述纳米粒A的包封率为90％-99％(例如90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)。在某些实施方案中，所述纳米粒A的载药量为60％-90％(例如60％-65％、65％-70％、70％-75％、75％-80％、80％-85％或85％-90％)。
本发明中，纳米粒A的包封率(EE)和载药量(LC)可以通过包含以下步骤的方法测得：1)使用荧光标记的治疗性蛋白制得纳米粒A；2)将包含所述纳米粒A的混悬液置于超滤管中进行离心，得到滤液；3)测定滤液中治疗性蛋白的荧光强度，进而计算滤液中的治疗性蛋白的浓度，以确定游离治疗性蛋白的量。按下式计算包封率(EE)和载药量(LC)：
Figure PCTCN2018101106-appb-000004
Figure PCTCN2018101106-appb-000005
在某些实施方案中，所述步骤2包括：使包含聚阴离子的溶液与包含纳米粒A的混悬液进行混合。
在某些实施方案中，所述步骤2进一步包括以下步骤：
步骤2-1：提供包含聚阴离子的溶液和包含纳米粒A的混悬液；
步骤2-2：提供包含涡流混合区域和多个可供流体流向涡流混合区域的通道的装置；
步骤2-3：使包含聚阴离子的溶液和包含纳米粒A的混悬液通过不同的通道到达涡流混合区域中，进行混合，得到包含本发明纳米粒的混悬液。
在某些实施方案中，步骤2-1中的包含纳米粒A的混悬液由包含步骤1-1、步骤1-2和步骤1-3的方法得到。
在某些实施方案中，步骤2-1的包含聚阴离子的溶液具有0.5-0.9mg/mL(例如0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL或0.9mg/mL)的质量浓度。
在某些实施方案中，步骤2-1中，包含聚阴离子的溶液：包含纳米粒A的混悬液的质量浓度比为0.5-0.9：1(例如0.5：1、0.6：1、0.7：1、0.8：1或0.9：1)。
在某些实施方案中，所述包含聚阴离子的溶液为水溶液。
在某些实施方案中，所述包含纳米粒A的混悬液中，分散介质为水。
本发明中，包含聚阴离子的溶液的质量浓度是指所述溶液中的聚阴离子的质量浓度。
本发明中，包含纳米粒A的混悬液的质量浓度是指所述混悬液中的纳米粒A所含有的治疗性蛋白的质量浓度。
在某些实施方案中，所述步骤2-2中的装置为多入口涡流混合器。
在某些实施方案中，所述步骤2-3中，包含聚阴离子的溶液，以及包含纳米粒A的混悬液在通道中以相同的流速匀速流动、
。在某些实施方案中，所述流速为1-50mL/min(例如1-15mL/min、15-25mL/min或25-50mL/min)。
本申请还提供了制备本发明微胶囊的方法，所述方法包括以下步骤：
步骤1’：制备本发明的纳米粒；
步骤2’：使用肠溶材料对步骤1’得到的纳米粒进行包被。
在某些实施方案中，步骤1’包括：以如上所述的制备方法制备本发明的纳米粒。
在某些实施方案中，所述步骤2’进一步包括以下步骤：
步骤2’-1：提供包含本发明纳米粒的混悬液和包含肠溶材料的溶液；
步骤2’-2：提供包含涡流混合区域和多个可供流体流向涡流混合区域的通道的装置；
步骤2’-3：使包含本发明纳米粒的混悬液、包含肠溶材料的溶液和任选的酸性溶液(例如盐酸)通过不同的通道到达涡流混合区域中，进行混合，得到包含本发明微胶囊的溶液。
在某些实施方案中，步骤2’-1的包含肠溶材料的溶液具有0.25-1mg·mL -1(例如0.25mg/mL、0.3mg/mL、0.35mg/mL、0.4mg/mL、0.45mg/mL、0.5mg/mL、0.55mg/mL、0.6mg/mL、0.65mg/mL、0.7mg/mL、0.75mg/mL、0.8mg/mL、0.85mg/mL、0.9mg/mL、0.95mg/mL或1mg/mL)的质量浓度。
在某些实施方案中，步骤2’-1中，包含肠溶材料的溶液：包含本发明纳米粒的混悬液的质量浓度比为0.25-1：1(例如0.25:1、0.3:1、0.35:1、0.4:1、0.45:1、0.5:1、0.55:1、0.6:1、0.65:1、0.7:1、0.75:1、0.8:1、0.85:1、0.9:1、0.95:1或1:1)。
在某些实施方案中，所述包含肠溶材料的溶液为水溶液。
在某些实施方案中，所述包含本发明纳米粒的混悬液中，分散介质为水。
本发明中，包含肠溶材料的溶液的质量浓度是指所述溶液中的肠溶材料的质量浓度。
本发明中，包含本发明纳米粒的混悬液的质量浓度是指所述混悬液中的本发明的纳米粒所含有的治疗性蛋白的质量浓度。
在某些实施方案中，所述步骤2’-2中的装置为多入口涡流混合器。
在某些实施方案中，所述步骤2’-3中，包含肠溶材料的溶液，以及包含本发明纳米粒的混悬液在通道中以相同的流速匀速流动。在某些实施方案中，所述流速为1-50mL/min(例如1-15mL/min、15-25mL/min或25-50mL/min)。
图1示例性地显示了制备本发明纳米粒和微胶囊的过程，其中，用于制备纳米粒的治疗性蛋白为胰岛素，聚阴离子为透明质酸(HA)，用于制备微胶囊的肠溶材料为HPMCP。制备过程如下：第一步，将CPP溶液与胰岛素溶液在多入口涡流混合器中进行混合，形成表面带正电的纳米粒A(NP-A)；第二步，将包含NP-A的混悬液与包含HA的溶液进行混合，形成以NP-A为核心，表面包被HA的纳米粒(NP-B)；第三步，将包含NP-B的混悬液、包含HPMCP的溶液和pH为2.5的稀盐酸混合，使HPMCP包被在纳米粒上，得到肠溶微胶囊。各步骤中，使用多入口涡流混合器对液体进行快速混合。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所涉及的实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中使用的，术语“治疗性蛋白”是指能够用于预防或治疗疾病的蛋白，包括但不限于激素、激素类似物、酶、酶抑制剂和抗体。
如本文中使用的，术语“细胞穿透肽”(cell penetrating peptide,CPP)是指由一般不超过30个氨基酸构建的短肽，其可以穿过细胞膜进入细胞，可以用来将外源性分子携带进入细胞。细胞穿透肽可以是天然存在的肽或人工合成的肽。常见的细胞穿透肽包括：阳离子CPP，例如：TAT(48-60)、Penetratin、聚精氨酸、Oct4、WT1-pTj、DPV3；两亲性CPP，例如：Transportan、MAP、VP22、Pep1、KW；疏水性CPP，例如：KFGF、FGF12、Integrinβ3Peptide、C105Y、TP2。常见的细胞穿透肽的来源和序列可参见，例如Joshua D.Ramsey,Nicholas H.Flynn.Cell-penetrating peptides transport therapeutics into cells,Pharmacology&Therapeutics 154(2015)78–86。可以对细胞穿透肽进行修饰，例如，在CPP的C端或N端进行修饰(例如烷基化修饰)。
如本文中使用的，术语“饱和脂肪酸”是指至少一端含有羧基的饱和碳氢链，其中， 所述饱和碳氢链多为直链，碳原子数可以是小于6个、6-12个或大于12个(例如12-18个)。饱和脂肪酸的实例包括但不限于：己酸、辛酸、癸酸、月桂酸(十二烷酸)、豆蔻酸(十四烷酸)、软脂酸(十六烷酸)、硬脂酸(十八烷酸)、花生酸(二十烷酸)。
如本文中使用的，术语“纳米粒”是指尺寸(即颗粒的最长维度中的直径)在纳米级的颗粒，例如尺寸为1-100nm、100-500nm、500-1000nm或1000-2000nm的颗粒。
如本文中使用的，术语“粒径”即“等效粒径”，是指当被测颗粒的某种物理特性或物理行为与某一直径的同质球体(或组合)最相近时，就把该球体的直径(或组合)作为被测颗粒的等效粒径(或粒度分布)。
如本文中使用的，术语“平均粒径”是指，对于一个由大小和形状不相同的粒子组成的实际粒子群，与一个由均一的球形粒子组成的假想粒子群相比，如果两者的粒径全长相同，则称此球形粒子的直径为实际粒子群的平均粒径。平均粒径的测量方法是本领域技术人员已知的，例如光散射法；平均粒径的测量仪器包括但不限于马尔文粒径仪。
如本文中使用的，术语“微胶囊”是指一种固体微粒，其具有壁层，以及包埋在壁层中的内容物。构成壁层的物质通常为高分子。微胶囊可以是各种形状，例如球形，其直径通常在微米级或毫米级。
如本文中使用的，术语“肠溶微胶囊”是指以肠溶材料作为壁层的主要材料制得的微胶囊，其能够耐受胃酸，在进入肠道后能够崩解并释放出内容物。
如本文中使用的，术语“肠溶材料”是指在胃液中不溶或几乎不溶，而在肠液中能崩解或溶解的材料。肠溶材料的溶解度随pH不同而不同。可用于本发明的肠溶材料包括但不限于纤维素及其衍生物，例如羟丙甲纤维素邻苯二甲酸酯(HPMCP)、邻苯二甲酸乙酸纤维素(CAP)、1,2,4-苯三甲酸乙酸纤维素(CAT)等。
如本文中使用的，术语“混悬液”是指固体微粒分散于液体分散介质中形成的液态分散体系，所述液体分散介质包括但不限于水。
如本文中使用的，术语“约”应该被本领域技术人员理解，并将随其所用之处的上下文而有一定程度的变化。如果根据术语应用的上下文，对于本领域技术人员而言，其含义不是清楚的，那么“约”的意思是偏差不超过所述特定数值或范围的正负10％。
如本文中使用的，术语“预防”是指阻止或延迟疾病的发生。
如本文中使用的，术语“治疗”是指治愈或至少部分阻止疾病的进展，或缓解疾病的症状。
发明的有益效果
本发明得到了一种负载治疗性蛋白的纳米粒，其具有核壳结构，其中，核心包含负载治疗性蛋白的CPP，壳层包含聚阴离子。本发明的纳米粒实现了较高的黏液层渗透率和肠上皮细胞转运效率，最终提高了治疗性蛋白的口服生物利用度和药效。进一步地，包含本发明纳米粒的微胶囊可在胃酸中保护纳米粒、而在肠道中快速释药，药效持续平稳。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是，本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1示例性地显示了制备本发明纳米粒和微胶囊的过程，其中，用于制备纳米粒的治疗性蛋白为胰岛素，聚阴离子为透明质酸(HA)，用于制备微胶囊的肠溶材料为HPMCP。制备过程如下：第一步，将CPP溶液与胰岛素溶液在多入口涡流混合器中进行混合，形成表面带正电的纳米粒A(NP-A)；第二步，将包含NP-A的混悬液与包含HA的溶液进行混合，形成以NP-A为核心，表面包被HA的纳米粒(NP-B)；第三步，将包含NP-B的混悬液、包含HPMCP的溶液和pH为2.5的稀盐酸混合，使HPMCP包被在纳米粒上，得到肠溶微胶囊。各步骤中，使用多入口涡流混合器对液体进行快速混合。
图2显示了实施例1的制备纳米粒的过程中，流速1以及CPP溶液的初始pH值对NP-A粒径大小、分散度和/或表面电位的影响，图中数据为平均值±SD(n＝6)。
图2A)显示了流速1为50mL·min -1的条件下，不同初始pH值的CPP溶液制得的NP-A的粒径大小(■)、分散度(▲)及表面电位
Figure PCTCN2018101106-appb-000006
结果表明，在pH为5.0～8.0的范围内，随着CPP溶液初始pH值的升高，NP-A的粒径和表面电位均降低。
图2B)显示了在CPP溶液初始pH值为8.0的条件下，不同流速1下制得的NP-A的粒径(■)和分散度(▲)。如图所示，在流速1为1～50mL·min -1范围内，通过调控流速，可将NP-A的粒径控制在约75nm至约480nm范围内，分散度控制在0.12～0.45范围内；且随着流速的增加，NP-A的粒径和分散度急剧减小，并在流速增至30 mL·min -1后达到一个平台期。
图3显示了不同流速2下制得的NP-B3的粒径(■)和分散度(▲)，所使用的NP-A是在CPP溶液的初始pH为8.0，流速1为50mL·min -1的条件下制得的。如图所示，随着流速的提高，NP-B的粒径从约219nm降至约105nm，分散度从0.46降至0.067。
图4显示了表1中NP-A、NP-B1、NP-B2、NP-B3的形貌。图中，所有纳米粒均为近似球形，粒径均一且分散均匀。由于使用磷钨酸染色，显深色部位应为带正电的细胞穿透肽的富集区域。图A1-A3为NP-A的图像，标尺分别为1μm、200nm、100nm。可以清楚地看到，绝大多数的细胞穿透肽位于NP-A的表面，形成蓬松的薄层。图A4-A6分别为NP-B1、NP-B2、NP-B3的图像，标尺均为200nm。如图所示，经HA包被后，在纳米核心表面出现可见的浅灰色层，表明纳米核心表面成功地包被上了HA，形成了壳核结构。此外，HA对纳米核心表面进行包被后，内部的纳米核心结构变得更加致密，说明对纳米核心的表面进行包被，可以压实其内部结构，使整个纳米粒的结构更加稳定。
图5显示了FRET实验的测试结果。单荧光标记的纳米粒(Rho123-HA/(CPP/Insulin)和HA/(CPP/RITC-Insulin))在450nm激发光激发后，可分别在530nm、580nm处观察到各自的发射峰。然而，对于双荧光标记制备的纳米粒(Rho123-HA/(CPP/RITC-Insulin))，经450nm的激发光激发后，Rho123-HA的发射峰(530nm)明显降低，而RITC-Insulin的发射峰(580nm)显著增高，意味着能量从供体转移到受体上，表明HA成功包被在载有胰岛素的纳米核心上。
图6显示了表1中的NP-A和NP-B3在低温(4℃)或常温(25℃)，下的粒径和粒径分布。图中数据为平均值±SD(n＝6)。NP-A在25℃条件下可稳定存在9h，9h后粒径、分散度明显增加。经HA包被后，纳米粒的稳定性显著提高，粒径和分散度在48h内几乎不变。低温条件下，NP-A和NP-B3的纳米粒的稳定性较高，粒径、PDI值无显著变化。以上结果表明，纳米核心经亲水性的HA包被后，稳定性显著提高。
图7显示了NP-A、NP-B1、NP-B2、NP-B3在模拟胃液(pH2.5)和模拟肠液(pH7.0)中的胰岛素损失百分率。如图所示，NP-A在模拟胃液(1h)及模拟小肠液(2h)中，胰岛素损失量分别高达24％和21％。经HA的包被后，可显著减少胰岛素损失。上述结果表明，HA的包被可提高纳米粒的稳定性，减少胃肠道消化液对纳米粒的破坏，且HA分子量越高，保护效果越好。
图8显示了NP-A和NP-B3在pH值为7.4的含有透明质酸酶(0.01mg/mL)的PBS中的胰岛素释放曲线，以及NP-A和NP-B3在pH值为7.4的不含透明质酸酶的PBS中的胰岛素释放曲线。如图所示，无论有无透明质酸酶的存在，NP-A释放胰岛素的速率始终是最快，在最初2h内存在明显的突释现象(累积释放率为45％)。经HA表面包被后，释放胰岛素的速率减缓，而且释放胰岛素的速率随着表面HA分子量的增加而降低。
图9显示了实施例9中，纳米粒与不同浓度的粘蛋白溶液在37℃下共同孵育1h后，形成的聚集物沉淀中的胰岛素含量。将NP-A与0.5％的粘蛋白得到的聚集物沉淀的荧光强度作为对照并归一化；#表示与其他0.5％粘蛋白组相比，p<0.01；*表示与其他1.0％粘蛋白组相比，p<0.001。图中数据为平均值±SD(n＝6)。在无粘蛋白存在时，各纳米粒孵育后所得沉淀中的胰岛素含量极低。然而，与浓度为0.5％或1.0％的粘蛋白在37℃孵育1h后，NP-A与粘蛋白发生了严重聚集，离心所得沉淀中的胰岛素含量高，表明表面带强正电的NP-A易通过静电作用与带负电的粘蛋白络合成聚集体；经HA表面包被后，纳米粒与粘蛋白的相互作用明显减弱，聚集体形成的量显著减少，随着HA分子量的增加，抗粘蛋白吸附效果越显著。NP-B3分别与浓度为0.5％和1.0％的粘蛋白共孵育后，沉淀中的胰岛素量仅是NP-A组的16.5％和22.9％。
图10显示了NP-A、NP-B1、NP-B2、NP-B3对HT29-MTX细胞的毒性，如图所示，纳米粒对HT29-MTX均没有明显的细胞毒性，安全性较好。
图11为实施例11中的共聚焦显微镜照片，显示了游离胰岛素(图11A)、NP-A(图11B)、NP-B1(图11C)、NP-B2(图11D)、NP-B3(图11E)在黏液层中的渗透情况，以及在穿透黏液层的过程中核心结构的完整性。图中的标尺为10μm，绿色为FITC-CPP，红色为Cy-5-胰岛素，蓝色为DIPA染色的细胞核。
如图所示，对于游离胰岛素，将其溶液与细胞孵育3h后，胰岛素总体荧光强度明显小于各纳米粒组，且多数滞留在黏液层中(参见15μm处的照片)，最终能够穿过黏液层到达细胞表面(参见30μm处的照片)的量很少。对于NP-A组，纳米粒在黏液层中渗透的初期就形成大量的粘蛋白-纳米粒聚集体，难以向下穿行而在原位滞留(参见0μm处的照片)，但与胰岛素溶液相比，在到达细胞层面的荧光强度有所提高。对于NP-B，随着HA分子量的增大，纳米粒在黏液层中的滞留量逐渐减少，到达细胞表面的信号逐渐增强。
此外，从图中可以清晰地看到，NP-A及NP-B1～3在穿过黏液层到达上皮细胞顶 端时，纳米核心中的细胞穿透肽(绿色)和胰岛素(红色)信号几乎完全共定位，说明在纳米粒自上而下的渗透过程中，黏液层的存在对纳米核心的结构完整性没有明显影响。
图12显示了实施例12中，HT29-MTX细胞对游离胰岛素和各组纳米粒的相对摄取量，图中，数据为平均值±SD(n＝6)。如图所示，各纳米粒组的摄取量显著高于游离胰岛素组。纳米粒按照摄取量由低到高的排序为：NP-A<NP-B1<NP-B2<NP-B3。NP-B3的细胞摄取量最高，分别是游离胰岛素和NP-A组的11倍、1.9倍。
图13显示了实施例13中，游离胰岛素组和纳米粒在Caco-2/mucin模型中的表观渗透常数(Papp)。Δ代表p<0.01；*代表p<0.001。图中数据为平均值±SD(n＝6)。与游离胰岛素溶液相比，各纳米粒组的Papp值均明显更高，NP-B的Papp值较NP-A有所增加，且随着HA分子量的增加，Papp增加，说明随着HA分子量越高，转运量越高。
图14为实施例14中，各组小鼠空肠组织横截面的荧光共聚焦显微照片，图中，游离胰岛素和各纳米粒呈红色(Cy-5的荧光颜色)，组织中的黏液层呈绿色(WGA-647的荧光颜色)，细胞核呈蓝色(DAPI的颜色)。图中的标尺为50μm。
如图所示，对于口服游离胰岛素溶液组，仅在小肠绒毛及上方的黏液层中观察到少量的胰岛素荧光(红色信号)，证明口服胰岛素溶液很难达到有效的药物吸收。对于口服NP-A组，观察到位于黏液层中的胰岛素荧光很强，而下方的小肠绒毛内的荧光很弱，说明多数NP-A在黏液层滞留，能够渗透过黏液层、进而被肠上皮细胞吸收的量较少。与之相比，NP-B1、NP-B2、NP-B3的黏液穿透能力及被小肠上皮细胞吸收转运的效率明显提高。其中，NP-B3在小肠绒毛中的信号最强，且荧光多位于绒毛毛细血管存在的部位，表明NP-B3可被上皮细胞有效吸收并转运到血液循环。上述结果证明，相对于表面带正电且未经修饰的纳米核心，经HA包被后的纳米粒的抗黏液层滞留并快速向下渗透转运的能力显著增强，进而使体内胰岛素的小肠吸收效率提高。
图15显示了实施例15-试验1中，各组大鼠给药后的血糖水平随时间的变化。图中，#代表相对于NP-B1组，p<0.05；*代表相对于NP-B2组，p<0.05。图中数据为平均值±SD(n＝8)。如图所示，给药八小时内，口服去离子水的大鼠的血糖水平稍有降低。皮下注射组的血糖水平在1h内急剧下降至初始水平的25％，在接下来的4h内血糖都一直保持在较低水平。相比之下，给予纳米粒的大鼠的血糖水平在给药后的8h内均显著但缓慢降低。NP-A、NP-B1和NP-B2纳米粒在口服给药后，在给药初期(0～5 h)的降血糖效果并无明显差异。在5～8h期间，NP-B2的降血糖效果比NP-A和NP-B1更为明显，可将血糖值降低至初始值的45％。NP-B3组的体内降血糖效果最为显著和持久，8h内可将血糖水平缓慢平稳地降低至初始值的40％。
图16显示了实施例15-试验2中，各组大鼠给药后的血糖水平随时间的变化。图中，*代表相对于NP-B1微胶囊组，p<0.05；#代表相对于NP-B2微胶囊组，p<0.05。图中数据为平均值±SD(n＝6)。
如图所示，经口服给药后，NP-B1和NP-B2微胶囊组的降血糖效果明显。这两组大鼠在口服给药后，血糖水平一直持续平稳地下降，大鼠的血糖值在给药8h后分别降低至初始水平的50％和40％。NP-B3微胶囊组的体内降血糖效果极其显著，可以使大鼠血糖在8h内平缓持久地降低至初始值的20％，此时，糖尿病大鼠的血糖水平达到正常范围(3.5～5mM)。
图17显示了实施例16中，各组大鼠体内血清胰岛素浓度随时间的变化曲线。图中数据为平均值±SD(n＝6)。如图所示，皮下注射给予胰岛素溶液(5IU·kg -1)后，大鼠血清中胰岛素浓度急剧增加，并于1h达到峰值(～120mIU·L -1)。NP-B2微胶囊和NP-B3微胶囊经口服给药后，大鼠血清胰岛素水平缓慢上升，达峰时间为4h。经计算，NP-B1微胶囊、NP-B2微胶囊和NP-B3微胶囊的相对生物利用度分别为5.3％、7.4％和11％。
图18显示了实施例17中，各组大鼠血清中的ALP、AST、ALT、γ-GT含量。图中数据为平均值±SD(n＝6)。与阴性对照组(口服PBS的正常大鼠)和阳性对照组(口服PBS的模型大鼠)相比，口服胰岛素纳米粒或胰岛素纳米粒肠溶微胶囊后，模型动物体内的四种肝药酶(ALP、AST、ALT、γ-GT)的活性没有明显升高，表明口服递送本发明的纳米粒及其肠溶微胶囊在动物体内没有明显毒性。
图19显示了实施例17中，各组大鼠肝脏组织HE染色切片的显微照片，A1：口服PBS的正常大鼠；A2：口服PBS的模型大鼠；A3：口服NP-B3的模型大鼠；A4：口服HPMCP包被的NP-B3的模型大鼠。与对照组大鼠的情况相似，实验组大鼠的肝脏结构完整，肝细胞排列较整齐，说明本发明的纳米粒及其肠溶微胶囊在多剂量重复口服给药后，未引起明显的肝损伤。
序列信息
本发明涉及的序列的信息提供于下表中：
序列号(SEQ ID NO:) 描述
1 Penetratin的氨基酸序列
序列信息
序列1(SEQ ID NO:1):16aa
Figure PCTCN2018101106-appb-000007
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
材料
异硫氰酸荧光素(FITC)、罗丹明异硫氰酸酯(RITC)、罗丹明123(Rho123)购自上海阿拉丁生化科技有限公司；Cy5购自武汉Little-PA Sciences有限公司；(3-二甲氨基丙基)碳化二亚胺盐酸盐(EDC·HCl)、N-羟基琥珀酰亚胺(NHS)购自上海阿拉丁试剂有限公司；CPP(Ste-RQIKIWFQNRRMKWKK，N端修饰正十八烷基的Penetratin)购自南京肽业生物科技有限公司，货号：NJP12879；粘蛋白、透明质酸酶(HAase)购自上海原叶生物有限公司；透明质酸钠(Mw分别为4.7KDa、35KDa、190KDa)购自山东华熙福瑞达生物医药有限公司；猪胰岛素(27.4IU/mg)购自江苏徐州万邦生物制药有限责任公司；DAPI、MTT以及Alexa
Figure PCTCN2018101106-appb-000008
647小麦胚芽凝集素配合物(AF-647)购自英国Abcam公司；猪胰岛素ELISA试剂盒购自瑞典Mercodia公司；碱性磷酸酶(alkaline phosphatase,ALP)试剂盒、天冬氨酸转氨酶(aspartate transaminase,AST)试剂盒、丙氨酸氨基转移酶(alanine aminotransferase,ALT)试剂盒、γ-谷氨酰转肽酶(glutamyl transpeptidase,γ-GT)试剂盒购自江苏南京建成生物科技有限公司；HPMCP(密度＝42.0cP·s)购自浙江绍兴市康可胶囊有限公司；其余所有试剂均为商购的分析纯试剂。
仪器
马尔文激光粒度测定仪(Malvern Zetasizer Nano ZS，英国Malvern公司)；冷冻干燥机(Alpha 1-2LD Plus，德国Christ公司)；pH计(Seven Compact pH Meter，瑞士Mettler公司)；自动双重纯水蒸馏器(
Figure PCTCN2018101106-appb-000009
美国Millipore公司)；高速 离心机(5810R，德国Eppendorf公司)；紫外可见分光光度计(Evo lution，美国Thermo公司)；荧光分光光谱仪(FS-980,英国Edinburgh公司)；激光共聚焦显微镜(SP8，德国Leica公司)；TEM(JEOL-1400，日本JEOL公司)；多功能酶标仪(Synergy 2，美国Biotek公司)；细胞培养箱(Forma TMSteri-Cycle TM，美国Thermo公司)；血糖仪(
Figure PCTCN2018101106-appb-000010
UltraVue,美国Johnson公司)。
统计分析
所有数值均表示为平均数±标准差。采用GraphPad Prism version 5.0(GraphPad Software，USA)进行单因素ANOVA或t检验评价，p<0.05表示统计学显著差异。
实施例1 HA包被的CPP-胰岛素纳米粒及肠溶微胶囊的制备
1、配制溶液：将细胞穿透肽粉末溶于双蒸水中，配制成浓度为0.3mg·mL -1的溶液，并用3M的氢氧化钠溶液调至所需pH值(5.0、5.5、6.0、6.5、7.0、7.5或8.0)。胰岛素粉末用0.01M的稀盐酸溶液溶解，配制成浓度为1mg·mL -1的溶液，并用3M的氢氧化钠溶液将pH值调过其pI，并缓慢调至6.8，使其再次形成透明澄清的均一溶液。三种不同分子量的透明质酸钠(Mw＝4.7KDa、35KDa或190KDa)粉末用双蒸水制备成浓度分别为0.28、0.30、0.70mg·mL -1的溶液。
2、制备CPP-胰岛素纳米核心(纳米粒A)：将细胞穿透肽溶液从多入口涡流混合器的入口2、3注入，胰岛素溶液从入口1、4注入，保持各溶液的流速一致(流速1)，得到包含纳米粒A(NP-A)的混悬液。
制备HA包被的CPP-胰岛素纳米粒：将得到的含NP-A的混悬液从入口1、2、3注入，HA溶液从入口4注入，保持各溶液的流速一致(流速2)，得到混悬液，其中含有表面包被HA的CPP-胰岛素纳米粒(NP-B)。使用三种不同分子量的HA(Mw＝4.7KDa、35KDa、190KDa)，制得的纳米粒分别命名为NP-B1、NP-B2、NP-B3。
3、制备肠溶微胶囊：从多入口涡流混合器的入口1注入包含NP-B的混悬液，入口2注入HPMCP的乙醇溶液(浓度为0.75mg·mL -1)，入口3、4注入稀盐酸(pH 2.5)，得到包含肠溶微胶囊的混悬液。
图1示例性地显示了上述制备过程。
实施例2 荧光标记的纳米粒的制备
1、荧光标记组分的合成
(1)异硫氰酸荧光素标记的细胞穿透肽(FITC-CPP)的制备
将400mg细胞穿透肽溶于40mL 0.05M碳酸盐缓冲液(配制：1.59g Na 2CO 3、2.93g NaHCO 3加至1L双蒸水中，pH 9.6)中，10mg FITC溶于1.5mL的甲醇，将FITC的甲醇溶液与细胞穿透肽溶液混合后，于4℃、温和搅拌下避光反应24h。反应完毕后，用截留分子量为1.0KDa的透析袋在去离子水中对样品进行透析，全程避光，吸取透析袋外侧的透析介质，监测其荧光值，直至游离染料透析完毕。冻干后避光保存备用。
(2)罗丹明异硫氰酸酯标记的胰岛素(RITC-胰岛素)及Cy5标记的胰岛素(Cy5-胰岛素)的制备
称取400mg胰岛素，以0.05M碳酸盐缓冲液(1.59g Na 2CO 3、2.93g NaHCO 3加至1L双蒸水中，pH 9.6)中配制5mg·mL -1的胰岛素溶液，加入5mL浓度为2mg·mL -1的RITC甲醇溶液，于4℃、避光反应24h。反应液于4℃透析(透析袋的截留分子量为3.5KDa，透析介质为pH 3.0的盐酸溶液)。监测到透析介质中无RITC荧光信号，即可认为透析彻底，冻干即得RITC-胰岛素粉末。Cy5-胰岛素的标记方法同上，除了将RITC的甲醇溶液换成Cy5的水溶液。
(3)罗丹明123标记的透明质酸(Rho123-HA)的制备
称取200mg 190KDa的透明质酸钠，溶于20mL甲醇与水(1:1，v/v)的混合溶剂中，充分溶解后，加入95.6mg EDC·HCl，57.4mg NHS，调pH至5.0，反应2h后，加入40mg罗丹明123，调pH至6.0，过夜反应24h，用截留分子量为14KDa的透析袋在去离子水中进行透析，定时吸取透析袋外侧的透析介质，监测荧光值，判断游离染料是否透析完毕。透析结束后，冻干成粉末，避光保存，备用。
(4)罗丹明异硫氰酸酯标记的透明质酸(RITC-HA)的制备
称取200mg 190KDa的透明质酸钠，溶于20mL甲醇与水(1:1，v/v)混合溶剂中，充分溶解后，加入40mg RITC的甲醇溶液，过夜反应24h，用截留分子量为14KDa的透析袋在去离子水中进行透析，定时吸取透析袋外侧的透析介质，监测荧光值，判断游离染料是否透析完毕。透析结束后，冻干成粉末，避光保存，备用。
2、按照实施例1描述的过程，使用荧光标记的原料制备纳米粒，制备条件：细胞穿透肽溶液初始pH值为8.0，流速1和流速2均为50mL·min -1。
实施例3 粒径、粒子分散性和表面电位的测定
采用马尔文激光粒度测定仪测定纳米粒的粒径、分散度和表面电位。测定方法如下：1)直接对制备好的包含纳米粒的混悬液进行测定；2)将混悬液用截留分子量为100KDa的超滤管于300×g的转速下进行超滤，浓缩液用0.01M的磷酸盐缓冲液(PBS，pH 6.6)重悬，再进行后续测定。将样品放入样品池中，于25℃平衡120s后，进行测定。每一样品平行测定三次。
图2A)显示了流速1为50mL·min -1的条件下，不同初始pH值的CPP溶液制得的NP-A的粒径大小(■)、分散度(▲)及表面电位
Figure PCTCN2018101106-appb-000011
结果表明，在pH为5.0～8.0的范围内，随着CPP溶液初始pH值的升高，NP-A的粒径和表面电位均降低。上述结果表明，本发明的方法可以通过调节CPP溶液的初始pH值得到不同粒径和表面电位的NP-A。
图2B)显示了在CPP溶液初始pH值为8.0的条件下，不同流速1下制得的NP-A的粒径(■)和分散度(▲)。如图所示，在流速1为1～50mL·min -1范围内，通过调控流速，可将NP-A的粒径控制在约75nm至约480nm范围内，分散度控制在0.12～0.45范围内；且随着流速的增加，NP-A的粒径和分散度急剧减小，并在流速增至30mL·min -1后达到一个平台期。上述结果表明，本发明的方法可以通过调节流速得到不同粒径和分散度的NP-A。
图3显示了不同流速2下制得的NP-B3的粒径(■)和分散度(▲)，所使用的NP-A是在CPP溶液的初始pH为8.0，流速1为50mL·min -1的条件下制得的。如图所示，随着流速的提高，NP-B的粒径从约219nm降至约105nm，分散度从0.46降至0.067。上述结果表明，本发明的方法可以通过调节流速得到不同粒径和分散度的NP-B。
在CPP溶液的初始pH为8.0，流速1和2均为50mL·min -1的条件下，制备NP-A和NP-B，其粒径、粒子分散性和表面电位如表1所示。
表1
Figure PCTCN2018101106-appb-000012
如表1所示，在CPP溶液的初始pH为8.0，流速1和2均为50mL·min-1的条件下，制得的NP-A的粒径小(～75nm)，分布窄(PDI:～0.12)，表面带正电(～+22mV)。经不同分子量的HA包被后，粒径增大(～100nm)，表面呈现负电性(～-20mV)。电荷的反转表明，HA成功包被于NP-A表面。经HA包被后，纳米粒的粒径分布仍较为均匀(PDI:～0.1)。
实施例4 包封率和载药量的测定
按照实施例2的方法，使用RITC-胰岛素制得NP-A，并进一步得到包含NP-B的混悬液。将包含NP-B的混悬液置于超滤管(截留分子量为100KDa)中，于4℃、300×g离心20min。用多功能酶标仪测定滤液中胰岛素的荧光强度，进而计算滤液中的胰岛素浓度，以确定游离胰岛素量。按下式计算纳米粒的包封率(Encapsulation efficiency，EE)和载药量(Loading content，LC)：
Figure PCTCN2018101106-appb-000013
Figure PCTCN2018101106-appb-000014
测定结果如表2所示。
表2
样品 HA(M w) EE(％) LC(％)
NP-A — 93.9±1.6 75.8±0.3
NP-B1 4.7KDa 95.6±2.4 55.5±0.6
NP-B2 35KDa 97.3±1.8 66.1±0.4
NP-B3 190KDa 96.6±1.7 66.7±0.5
如图所示，各纳米粒的包封率均在90％以上，载药量在55％以上。此外，NP-B3纳米粒的总产量可达到6.6g·h -1，相当于约4.5g·h -1的胰岛素产量(由纳米粒的总产量乘以载药量算得)，表明本发明的方法可应用于胰岛素纳米粒的连续和规模化的生产。
实施例5 纳米粒微观形态的观察
采用透射电镜观察纳米粒的形貌。取含有纳米粒的混悬液，滴在覆有碳支持膜的200目铜网上，停留20min，用滤纸吸去多余的液体，滴加2％的磷钨酸溶液染色2min，再用滤纸吸去多余的磷钨酸溶液，自然晾干，用透射电镜观察并拍照。
图4显示了表1中NP-A、NP-B1、NP-B2、NP-B3的形貌。图中，所有纳米粒均为近似球形，粒径均一且分散均匀。由于使用磷钨酸染色，显深色部位应为带正电的细胞穿透肽的富集区域。
图A1-A3为NP-A的图像，标尺分别为1μm、200nm、100nm。可以清楚地看到，绝大多数的细胞穿透肽位于NP-A的表面，形成蓬松的薄层。
图A4-A6分别为NP-B1、NP-B2、NP-B3的图像，标尺均为200nm。如图所示，经HA包被后，在纳米核心表面出现可见的浅灰色层，表明纳米核心表面成功地包被上了HA，形成了壳核结构。此外，HA对纳米核心表面进行包被后，内部的纳米核心结构变得更加致密，说明对纳米核心的表面进行包被，可以压实其内部结构，使整个纳米粒的结构更加稳定。
实施例6 FRET实验考察胰岛素纳米粒的结构
本实施例采用荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术，确认透明质酸与胰岛素的相互作用。
根据实施例2的方法，使用Rho 123(ex：490nm，em：530nm)标记的HA，和RITC(ex：540nm，em：580nm)标记的胰岛素，制备双荧光标记的纳米粒NP-B3，形成荧光共振能量对。以单独标记Rho123的透明质酸或单独标记RITC的胰岛素制备而成的纳米粒作为对照，在450nm的激发波长下测定各样品荧光光谱的变化。测试结果如图5所示。单荧光标记的纳米粒(Rho123-HA/(CPP/Insulin)和HA/(CPP/RITC-Insulin))在450nm激发光激发后，可分别在530nm、580nm处观察到各自的发射峰。然而，对于双荧光标记制备的纳米粒(Rho123-HA/(CPP/RITC-Insulin))，经450nm的激发光激发后，Rho123-HA的发射峰(530nm)明显降低，而RITC-Insulin的发射峰(580nm)显著增高，意味着能量从供体转移到受体上，表明HA成功包被在载有胰岛素的纳米核心上。
实施例7 纳米粒的贮存稳定性及在模拟胃肠液中的稳定性
将表1中的NP-A和NP-B3放置于低温(4℃)或常温(25℃)，在特定的时间 点(1、3、6、9、12、18、24、48h)监测纳米粒的粒径和粒径分布，初步评价纳米粒在短期贮存过程中的稳定性。
如图6所示，NP-A在25℃条件下可稳定存在9h，9h后粒径、分散度明显增加。经HA包被后，纳米粒的稳定性显著提高，粒径和分散度在48h内几乎不变。低温条件下，NP-A和NP-B3的纳米粒的稳定性较高，粒径、PDI值无显著变化。以上结果表明，纳米核心经亲水性的HA包被后，稳定性显著提高。
按照实施例2的方法，使用RITC-胰岛素制备NP-A、NP-B1、NP-B2和NP-B3，分散于模拟胃液(稀盐酸溶液，pH 2.5)和模拟肠液(0.01M PBS，pH 7.0，含有透明质酸酶0.01mg·mL -1)中，分别于1h、2h后用超滤管(截留分子量为100KDa)超滤，用多功能酶标仪测定滤液中的荧光强度(ex：540nm，em：580nm)，从而计算胰岛素含量，得出胰岛素损失百分率，考察纳米粒在模拟胃液及肠液中的药物损失情况。
图7显示了NP-A、NP-B1、NP-B2、NP-B3在模拟胃液(pH2.5)和模拟肠液(pH7.0)中的胰岛素损失百分率。如图所示，NP-A在模拟胃液(1h)及模拟小肠液(2h)中，胰岛素损失量分别高达24％和21％。经HA的包被后，可显著减少胰岛素损失。上述结果表明，HA的包被可提高纳米粒的稳定性，减少胃肠道消化液对纳米粒的破坏，且HA分子量越高，保护效果越好。
实施例8 体外释放实验
按照实施例2的方法，使用RITC-胰岛素制备NP-A和NP-B3。将纳米粒分散于含有透明质酸酶(0.01mg·mL -1)的pH 7.4的PBS中，37℃，100rpm恒温振荡。定时取样，超滤(截留分子量为100KDa)，采用多功能酶标仪测定滤液的荧光强度(ex：540nm，em：580nm)，进而计算出胰岛素含量，得出累积药物释放百分率。同时，将纳米粒分散于不含有透明质酸酶的分散介质中进行体外释放研究，用作对照实验。
图8显示了NP-A和NP-B3在pH值为7.4的含有透明质酸酶(0.01mg/mL)的PBS中的胰岛素释放曲线，以及NP-A和NP-B3在pH值为7.4的不含透明质酸酶的PBS中的胰岛素释放曲线。如图所示，无论有无透明质酸酶的存在，NP-A释放胰岛素的速率始终是最快，在最初2h内存在明显的突释现象(累积释放率为45％)。经HA表面包被后，释放胰岛素的速率减缓，而且释放胰岛素的速率随着表面HA分子 量的增加而降低。
实施例9 体外模拟粘蛋白-纳米粒吸附
本实施例在体外考察纳米粒与粘蛋白的相互作用。按照实施例2的方法，使用RITC-胰岛素制备荧光标记的纳米粒。将不同浓度的粘蛋白溶液和纳米粒共孵育一定时间，通过监测最终形成的粘蛋白-纳米粒聚集物沉淀中的胰岛素含量，来考察纳米粒与粘蛋白的相互作用情况。
实验过程：将荧光标记的纳米粒分别分散于0.5％或1.0％(m/v)的粘蛋白水溶液中，用不含粘蛋白的空白溶液作对比，在37℃震荡孵育1h。混合物于3000rpm离心10min，所得沉淀用PBS洗涤2次，并用200μL的氢氧化钠溶液(3M)充分破坏，涡旋振荡，形成均一溶液后，用多功能酶标仪测定溶液的荧光强度(ex：540nm，em：580nm)。
实验结果如图9所示。在无粘蛋白存在时，各纳米粒孵育后所得沉淀中的胰岛素含量极低。然而，与浓度为0.5％或1.0％的粘蛋白在37℃孵育1h后，NP-A与粘蛋白发生了严重聚集，离心所得沉淀中的胰岛素含量高，表明表面带强正电的NP-A易通过静电作用与带负电的粘蛋白络合成聚集体；经HA表面包被后，纳米粒与粘蛋白的相互作用明显减弱，聚集体形成的量显著减少，随着HA分子量的增加，抗粘蛋白吸附效果越显著。NP-B3分别与浓度为0.5％和1.0％的粘蛋白共孵育后，沉淀中的胰岛素量仅是NP-A组的16.5％和22.9％。
上述结果表明，HA的表面包被使纳米核心的表面由强正电反转至呈现负电，且使其亲水性增加，进而减少了纳米粒与粘蛋白之间的静电和疏水相互作用，有望增加纳米粒在黏液层中自上而下的渗透效率，提高载药纳米粒到达上皮细胞表面并进一步跨膜转运的几率。
实施例10 细胞毒性实验
本实施例采用MTT检测方法评价各纳米粒对HT29-MTX细胞的安全性。
取对数生长期的HT29-MTX细胞，以1.0×10 4个/孔的密度接种于96孔板，加入200μL培养基培养24h后吸去培养液，加入200μL含游离胰岛素、NP-A、NP-B1、NP-B2或NP-B3的培养基，于37℃孵育24h。随后加入20μL MTT溶液(5mg·mL -1，pH 7.4PBS)继续孵育4h。移除上清液后加入100μL DMSO，震荡混匀，接着用酶 标仪于570nm测定吸光度。以空白培养液孵育细胞，相同处理后作为对照；以无细胞的培养基相同操作处理后作为本底。每个样品设置三个复孔，根据所测得吸光度按照下述公式计算细胞存活率：
Figure PCTCN2018101106-appb-000015
其中，OD 样本是实验组吸光度，OD 对照是含细胞的空白培养液的吸光度，OD 0是本底的吸光度。
如图10所示，各纳米粒对HT29-MTX细胞的生长均没有表现出明显的毒性，安全性较好。
实施例11 纳米粒在黏液层中的渗透能力实验
本实施例考察纳米粒在黏液层中的渗透能力，以及在穿透黏液层的过程中纳米核心结构的完整性。按照实施例2的方法，使用FITC-CPP和Cy5-胰岛素制备双荧光标记的纳米粒，并与可分泌黏液的HT29-MTX细胞共孵育，用共聚焦显微镜进行观察。
实验过程：将HT29-MTX细胞以2×10 4个/孔的密度接种于专用的共聚焦小皿(1×1cm)，培养2～3天。加入双荧光标记的纳米粒，孵育3h后，移除培养基，并用PBS温和洗涤2次，去除黏液层表面吸附的样品，用4％多聚甲醛固定细胞10min，用PBS洗涤2次。随后在共聚焦显微镜下逐层扫描。以Cy-5标记的游离胰岛素作为对照组。
实验结果:图11显示了游离胰岛素(图11A)、NP-A(图11B)、NP-B1(图11C)、NP-B2(图11D)、NP-B3(图11E)在黏液层的渗透情况和核心结构的完整性。图中的标尺为10μm，绿色为FITC-CPP，红色为Cy-5-胰岛素，蓝色为DIPA染色的细胞核。
如图所示，对于游离胰岛素，将其溶液与细胞孵育3h后，胰岛素总体荧光强度明显小于各纳米粒组，且多数滞留在黏液层中(参见15μm处的照片)，最终能够穿过黏液层到达细胞表面(参见30μm处的照片)的量很少。原因可能在于，胰岛素分子直接暴露于黏液环境，易被黏液中的酶降解破坏，因而表现出的总体荧光强度较低。此外，胰岛素为亲水性大分子，难以渗透通过黏液层并被肠上皮细胞摄取。
对于NP-A组，纳米粒在黏液层中渗透的初期就形成大量的粘蛋白-纳米粒聚集体，难以向下穿行而在原位滞留(参见0μm处的照片)，可能的原因在于NP-A表面带正电荷，易与黏液层中的多种粘蛋白发生静电作用。但与胰岛素溶液相比，在到达细 胞层面的荧光强度有所提高，原因可能在于NP-A对其内部的胰岛素还是具有一定的保护效果，且易与带负电的细胞膜作用而入胞。
对于NP-B，随着HA分子量的增大，纳米粒在黏液层中的滞留量逐渐减少，到达细胞表面的信号逐渐增强，说明亲水性强、带负电的HA的包被的确提高了纳米粒的稳定性，降低了与黏液层中粘蛋白的亲和性，并且保护效果与HA的分子量有关。
此外，从图中可以清晰地看到，NP-A及NP-B1～3在穿过黏液层到达上皮细胞顶端时，纳米核心中的细胞穿透肽(绿色)和胰岛素(红色)信号几乎完全共定位，说明在纳米粒自上而下的渗透过程中，黏液层的存在对纳米核心的结构完整性没有明显影响。
实施例12 HT29-MTX细胞对纳米粒的摄取实验
实验过程：HT29-MTX细胞以5×10 4个/孔的密度接种于24孔板，加入使用RITC-胰岛素制得荧光标记的纳米粒。孵育3h后，移除培养基，并用PBS温和洗涤3次，去除残留黏液及细胞表面吸附的样品。采用Scepter TM2.0手持自动细胞计数器测定细胞数目。随后用细胞裂解液对细胞进行充分裂解破坏后，采用多功能酶标仪测定细胞裂解液的荧光强度。细胞摄取的相对定量统一为每5.0×10 3个细胞所对应的荧光强度值。以RITC标记的游离胰岛素作为对照组。
实验结果：图12显示了HT29-MTX细胞对游离胰岛素和各组纳米粒的相对摄取量，图中数据为平均值±SD(n＝6)。如图所示，各纳米粒组的摄取量显著高于游离胰岛素组。纳米粒按照摄取量由低到高的排序为：NP-A<NP-B1<NP-B2<NP-B3。NP-B3的细胞摄取量最高，分别是游离胰岛素和NP-A组的11倍、1.9倍。
在含有黏液层的细胞模型(HT29-MTX)中，纳米核心NP-A的摄取量较低，可能是由于带正电的纳米核心NP-A易与负电性粘蛋白聚集成较大颗粒，在黏液层中的渗透受到阻碍，到达细胞表面的量显著减少，进而细胞摄取效率降低；而HA包被的纳米粒可以高效地透过粘液层，容易被细胞摄取。
实施例13 纳米粒的跨上皮细胞转运
按照实施例2的方法，用RITC-胰岛素制备荧光标记的纳米粒。采用表面人为添加粘蛋白的Caco-2细胞模型，以胰岛素的Papp值为指标，考察纳米粒的跨细胞转运能力以及黏液层对纳米粒转运效率的影响。
实验过程：
细胞培养：将Caco-2置于37℃、5％CO 2的培养箱中，分别用含10％胎牛血清、1％L-谷氨酰胺、1％青-链霉素(100IU·mL -1)、1％非必需氨基酸、10mg·mL -1黏蛋白的DMEM高糖培养基培养。
Caco-2细胞单层模型的构建：将Caco-2细胞接种在Costar Transwell 12孔板的聚酯膜上(聚酯膜孔径0.4μm，直径12mm，细胞生长区域1.1cm 2)，每隔2天采用Millicell ERS-2型电阻仪监测其跨膜电阻(Trans-epithellal Electric Resistance，TEER)值；并且每两天更换一次培养液。培养2～3周后，当TEER值高于700Ω·cm 2，即可用于后续实验。
实验前，将Transwell上、下室的培养液换成Hanks平衡盐溶液(HBSS)，于37℃平衡30min。上室换成200μL含荧光标记的纳米粒样品(其中胰岛素被标记，浓度为0.1mg·mL -1)的HBSS。孵育4h后，取适量下室溶液，用多功能酶标仪检测溶液中的荧光强度，从而测定胰岛素浓度，计算表观渗透常数(Papp)。以荧光标记的游离胰岛素作为对照组。
Figure PCTCN2018101106-appb-000016
Q为渗透的药物总量(ng)；c是上室药物的初始浓度(ng·cm -3)；A是膜面积(cm 2)；t是试验时间(s)。
实验结果：图13显示了游离胰岛素组和纳米粒在Caco-2/mucin模型中的表观渗透常数(Papp)。Δ代表p<0.01；*代表p<0.001。图中数据为平均值±SD(n＝6)。与游离胰岛素溶液相比，各纳米粒组的Papp值均明显更高，NP-B的Papp值较NP-A有所增加，且随着HA分子量的增加，Papp增加，说明随着HA分子量越高，转运量越高。
实施例14 大鼠体内的吸收研究
按照实施例2的方法，用Cy5-胰岛素制备荧光标记的纳米粒，进而示踪胰岛素在大鼠小肠绒毛中的吸收行为。
实验过程：
取若干只正常雄性SD大鼠(230～250g)，分为5组：胰岛素溶液组、NP-A组、NP-B1组、NP-B2组、NP-B3组(胰岛素剂量：1.5mg·kg -1)。对胰岛素溶液组的大鼠，给予游离胰岛素溶液；对各纳米粒组，将实施例1制得的包含纳米粒的混悬液离 心(4000rpm)冻干，将得到的固体用双蒸水重悬，将得到的混悬液给予大鼠。给药前，动物禁食过夜，可自由饮水。经口服给药2h后，大鼠用戊巴比妥钠(0.04mg·kg -1)进行麻醉，沿腹中线打开大鼠腹腔，轻柔地取出肠组织，并截取靠近十二指肠端的空肠段(～10cm)，沿着肠管腔方向纵向切割，用PBS轻柔冲洗表面粘附的纳米粒及肉眼可见的杂质，然后轻轻地将其卷曲，快速放入液氮冷却的O.C.T.(optimum cutting temperature)化合物中固定。用冷冻切片机进行组织切片(8μm)，用Alexa Fluor 647标记的小麦胚芽凝集素(WGA)和DAPI染料分别对组织横截面的黏液层中的粘蛋白和细胞核进行染色，并用共聚焦显微镜观察。
染色步骤：
1)配制黏液层染色液：将1.0mg·mL -1的WGA储备液用HBSS稀释，WGA溶液的推荐浓度为5.0μg·mL -1；
2)组织切片用染色液室温染色10min后移除染色液，用HBSS冲洗细胞2次；
3)组织切片用细胞核DAPI染液避光染色5min后，冲洗细胞2次。
实验结果：
图14为各组小鼠空肠组织横截面的荧光共聚焦显微照片，图中，游离胰岛素和各纳米粒呈红色(Cy-5的荧光颜色)，组织中的黏液层呈绿色(WGA-647的荧光颜色)，细胞核呈蓝色(DAPI的颜色)。图中的标尺为50μm。
如图所示，对于口服游离胰岛素溶液组，仅在小肠绒毛及上方的黏液层中观察到少量的胰岛素荧光(红色信号)，证明口服胰岛素溶液很难达到有效的药物吸收。对于口服NP-A组，观察到位于黏液层中的胰岛素荧光很强，而下方的小肠绒毛内的荧光很弱，说明多数NP-A在黏液层滞留，能够渗透过黏液层、进而被肠上皮细胞吸收的量较少。这一现象与体外模拟粘蛋白-纳米粒吸附实验(实施例9)、纳米粒在黏液层中的渗透能力实验(实施例11)、及纳米粒在含有黏液层的细胞模型(HT29-MTX)中的摄取实验(实施例12)的结果相吻合。与之相比，NP-B1、NP-B2、NP-B3的黏液穿透能力及被小肠上皮细胞吸收转运的效率明显提高。其中，NP-B3在小肠绒毛中的信号最强，且荧光多位于绒毛毛细血管存在的部位，表明NP-B3可被上皮细胞有效吸收并转运到血液循环。上述结果证明，相对于表面带正电且未经修饰的纳米核心，经HA包被后的纳米粒的抗黏液层滞留并快速向下渗透转运的能力显著增强，进而使体内胰岛素的小肠吸收效率提高。
实施例15 I型糖尿病大鼠模型的建立及体内降血糖试验
I型糖尿病大鼠模型的建立
取若干只雄性SD大鼠，腹腔注射链脲霉素(STZ,70mg·kg -1)，注射部位靠近大鼠仰卧位的腹部左上方。造模前需禁食6h以上，造模后4h后再重新恢复给予进食。一周后用血糖仪测定血糖水平，若空腹血糖水平值高于16.0mM，则视为I型糖尿病模型造模成功。
试验1 口服纳米粒的降血糖实验
取56只I型糖尿病模型大鼠，分为7组：阴性对照组(口服去离子水)，胰岛素水溶液(胰岛素浓度1mg·mL -1)皮下(s.c.)注射组(5IU·kg -1)，以及胰岛素水溶液、NP-A、NP-B1、NP-B2、NP-B3混悬液(胰岛素浓度1mg·mL -1)口服给药组(80IU·kg -1)。各纳米粒混悬液通过以下方法得到：将实施例1制得的包含纳米粒的混悬液离心(4000rpm)冻干，将得到的固体用双蒸水重悬。给药前，动物禁食过夜，可自由饮水。分别于给药前及给药后1、2、3、4、5、6、7和8h尾静脉收集血液样品，用血糖仪测定其血糖水平。
图15显示了各组大鼠给药后的血糖水平随时间的变化。图中，#代表相对于NP-B1组，p<0.05；*代表相对于NP-B2组，p<0.05。图中数据为平均值±SD(n＝8)。
如图所示，给药八小时内，口服去离子水的大鼠的血糖水平稍有降低，可能是由于糖尿病大鼠在给药期间处于禁食状态，存在一个基础降低值。口服胰岛素溶液的大鼠的血糖水平与阴性对照组相差不明显。
皮下注射组的血糖水平在1h内急剧下降至初始水平的25％，在接下来的4h内血糖都一直保持在较低水平。但是，血糖水平在较短时间内从较高水平到达较低水平的骤降效应，对于糖尿病患者来说具有高风险性，严重的会引起低血糖、昏迷、休克，甚至死亡。
相比之下，给予纳米粒的大鼠的血糖水平在给药后的8h内均显著但缓慢降低。NP-A、NP-B1和NP-B2纳米粒在口服给药后，在给药初期(0～5h)的降血糖效果并无明显差异。在5～8h期间，NP-B2的降血糖效果比NP-A和NP-B1更为明显，可将血糖值降低至初始值的45％。NP-B3组的体内降血糖效果最为显著和持久，8h内可将血糖水平缓慢平稳地降低至初始值的40％。
上述结果说明，本发明的纳米粒可以平稳地起到降低血糖的作用，药效明显，安全性高。
试验2 口服肠溶微胶囊的降血糖实验
取18只I型糖尿病模型大鼠，分为3组：HPMCP包被的NP-B1微胶囊组、NP-B2微胶囊组、NP-B3微胶囊组，(胰岛素浓度1mg·mL -1)微胶囊用双蒸水重悬，均口服给药(80IU·kg -1)。
微胶囊的制备是通过将制备得到的NP-B1、NP-B2、NP-B3纳米粒子注入FNC入口1，制备好的HPMCP的乙醇溶液注入入口2，入口3、4注入稀盐酸溶液(pH＝2.5)，最终得到相应纳米粒子的微胶囊。
给药前，动物禁食过夜，可自由饮水。分别于给药前及给药后1、2、3、4、5、6、7和8h尾静脉收集血液样品，用血糖仪测定血糖水平。
图16显示了各组大鼠口服微胶囊后的血糖水平随时间的变化。图中，*代表相对于NP-B1微胶囊组，p<0.05；#代表相对于NP-B2微胶囊组，p<0.05。图中数据为平均值±SD(n＝6)。
如图所示，经口服给药后，NP-B1和NP-B2微胶囊组的降血糖效果明显。这两组大鼠在口服给药后，血糖水平一直持续平稳地下降，大鼠的血糖值在给药8h后分别降低至初始水平的50％和40％。NP-B3微胶囊组的体内降血糖效果极其显著，可以使大鼠血糖在8h内平缓持久地降低至初始值的20％，此时，糖尿病大鼠的血糖水平达到正常范围(3.5～5mM)。相对于直接皮下注射胰岛素，本发明的微胶囊经过口服，可以缓慢、平稳、持久地起到降血糖作用，安全性高，避免了产生血糖骤降等风险，更能为广大糖尿病患者所接受。
实施例16 体内生物利用度研究
取24只I型糖尿病大鼠，分为4组：胰岛素水溶液注射组(s.c.5IU·kg -1)，NP-B1微胶囊组、NP-B2微胶囊组、NP-B3微胶囊组(胰岛素浓度1mg·mL -1)。微胶囊用双蒸水重悬，口服给药(80IU·kg -1)。微胶囊的制备是通过将制备得到的NP-B1、NP-B2、NP-B3纳米粒子注入FNC入口1，制备好的HPMCP的乙醇溶液注入入口2，入口3、4注入稀盐酸溶液(pH＝2.5)，最终得到相应纳米粒子的微胶囊。
分别于给药后0.3、0.6、1、2、3、4、5、6、7和8h尾静脉收集血液样品，置于1.5mL采集管中，凝血后于3000rpm离心15min，小心收集上层血清，-80℃保存备用，避免反复冻融。使用猪胰岛素ELISA试剂盒，采用双固相酶免疫测定法，测定血清中的胰岛素浓度。具体操作如下：
测试前，先将所有试剂和样品恢复至室温。
1)配制酶结合物溶液1×和清洗液1×：将酶结合物溶液(Enzyme Conjugate 11×)按1:10用酶结合物缓冲液(Enzyme Conjugate Buffer)稀释混匀；清洗液(Wash Buffer21×)按1:20用去离子水稀释混匀；
2)取一块96孔的酶标板，精密吸取25μL的标定液和血清样品置于96孔板中，一式三份；
3)每孔加入100μL的酶结合物溶液1×；
4)室温(18～25℃)下振荡(700～900rpm)孵育2h；
5)洗板：翻转酶标板弃去反应液，每孔加入350μL清洗液，轻摇数秒后弃去清洗液，在吸附纸上用力拍打脱干清洗液，重复洗板5次；
6)每孔加入酶结合底物(Substrate TMB)200μL，室温(18～25℃)下孵育15min；
7)每孔加入50μL反应终止液，轻摇5s，充分混匀。用酶标仪于450nm波长下测定吸光度值，读取在30min内完成。
按照下式计算口服胰岛素肠溶微胶囊的相对生物利用度(BA)：
Figure PCTCN2018101106-appb-000017
其中，AUC 口服灌胃和AUC 皮下注射分别代表口服灌胃和皮下注射后血清胰岛素浓度对时间曲线下的总面积。
图17显示了各组大鼠体内血清胰岛素浓度随时间的变化曲线。图中数据为平均值±SD(n＝6)。皮下注射给予胰岛素溶液(5IU·kg -1)后，大鼠血清中胰岛素浓度急剧增加，并于1h达到峰值(～120mIU·L -1)，这与实施例15中，在糖尿病大鼠模型中皮下注射等剂量的胰岛素后，血糖水平在1h内迅速降低至初始值的25％的结果互相吻合(参见图15)。NP-B2微胶囊和NP-B3微胶囊经口服给药后，大鼠血清胰岛素水平缓慢上升，达峰时间为4h。经计算，NP-B1微胶囊、NP-B2微胶囊和NP-B3微胶囊的相对生物利用度分别为5.3％、7.4％和11％。
实施例17 大鼠体内安全性研究
实验过程：取6只正常大鼠用作阴性对照组，18只I型糖尿病模型大鼠，分为3组：口服PBS组、口服NP-B3组、口服HPMCP包被的NP-B3组(胰岛素浓度1mg·mL -1)， 纳米粒和微胶囊均用双蒸水重悬(80IU·kg -1)。连续口服给药1周后，尾静脉收集血液样品，置于1.5mL采集管中，凝血后于3000rpm离心15min，小心收集上层血清，置于-80℃冻存，避免反复冻融。按照试剂盒说明书中的方法，测定血清中的碱性磷酸酶(ALP)、天冬氨酸转氨酶(AST)、丙氨酸氨基转移酶(ALT)和γ-谷氨酰转肽酶(γ-GT)的含量。
动物用戊巴比妥钠(0.04mg·kg -1)进行麻醉，沿腹中线打开腹腔，用PBS进行心脏灌流冲洗体内残余血液后，轻柔地取出肝脏组织，用4％多聚甲醛固定后，经石蜡包埋，用冷冻切片机进行组织切片(8μm)，进行HE染色。
实验结果：图18显示了各组大鼠血清中的ALP、AST、ALT、γ-GT含量。图中数据为平均值±SD(n＝6)。与阴性对照组(口服PBS的正常大鼠)和阳性对照组(口服PBS的模型大鼠)相比，口服胰岛素纳米粒或胰岛素纳米粒肠溶微胶囊后，模型动物体内的四种肝药酶(ALP、AST、ALT、γ-GT)的活性没有明显升高，表明口服递送本发明的纳米粒及其肠溶微胶囊在动物体内没有明显毒性。
图19显示了各组大鼠肝脏组织HE染色切片的显微照片，A1：口服PBS的正常大鼠；A2：口服PBS的模型大鼠；A3：口服NP-B3的模型大鼠；A4：口服HPMCP包被的NP-B3的模型大鼠。与对照组大鼠的情况相似，实验组大鼠的肝脏结构完整，肝细胞排列较整齐，说明本发明的纳米粒及其肠溶微胶囊在多剂量重复口服给药后，未引起明显的肝损伤。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (9)

  1. 一种纳米粒，其包含核心和包被在核心上的聚阴离子；所述核心包含治疗性蛋白，还包含细胞穿透肽(CPP)；
    优选地，所述聚阴离子选自三聚磷酸钠、海藻酸、肝素、透明质酸(HA)、透明质酸盐、硫酸软骨素、聚丙烯酸类聚合物、聚苯乙烯磺酸类聚合物或其任意组合；
    优选地，所述聚阴离子选自透明质酸、透明质酸盐(例如透明质酸钠)或其组合；
    优选地，所述聚阴离子的重均分子量为4kDa-200kDa(例如4kDa-10kDa、10kDa-50kDa、50kDa-100kDa、100kDa-150kDa或150kDa-200kDa)；
    优选地，所述核心包含的治疗性蛋白选自激素、激素类似物、酶、酶抑制剂或抗体；
    优选地，所述治疗性蛋白为胰岛素；
    优选地，所述CPP包含精氨酸残基；
    优选地，所述CPP的N端为精氨酸残基；
    优选地，所述CPP为Penetratin；
    优选地，所述CPP被烷基修饰；
    优选地，所述CPP的N端被烷基修饰；
    优选地，所述烷基是C 12-C 18烷基(例如C 12烷基、C 14烷基、C 16烷基或C 18烷基)；
    优选地，所述烷基是直链烷基；
    优选地，所述CPP为N端被正十八烷基修饰的Penetratin。
  2. 权利要求1的纳米粒，其还包含荧光化合物或荧光化合物的生色团；
    优选地，所述荧光化合物选自异硫氰酸荧光素(FITC)、罗丹明异硫氰酸酯(RITC)、3H-吲哚菁类染料(例如Cy3、Cy5)或罗丹明(例如罗丹明6G、罗丹明123、罗丹明B)；
    优选地，所述荧光化合物标记在聚阴离子、治疗性蛋白和/或CPP上。
  3. 权利要求1或2的纳米粒，其为球形；
    优选地，所述纳米粒包含的核心为球形；
    优选地，所述纳米粒的粒径为100nm-900nm(例如100nm-200nm、200-300nm、 300-400nm、400-500nm、500-600nm、600-700nm、700-800nm或800-900nm)；
    优选地，所述纳米粒粒径的多分散指数(PDI)为0.05-0.5(例如0.05-0.1、0.1-0.2、0.2-0.3、0.3-0.4或0.4-0.5)；
    优选地，所述核心的粒径为30nm-500nm(例如30nm-100nm、100-200nm、200-300nm、300-400nm或400-500nm)；
    优选地，所述核心的粒径的多分散指数(PDI)为0.1-0.5(例如0.1-0.2、0.2-0.3、0.3-0.4或0.4-0.5)；
    优选地，所述纳米粒的Zeta电位为-10mV至-50mV(例如-10mV至-20mV、-20mV至-30mV、-30mV至-40mV或-40mV至-50mV)；
    优选地，所述纳米粒的包封率为90％-99％(例如90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)；
    优选地，所述纳米粒的载药量为50％-90％(例如50％-55％、55％-60％、60％-65％、65％-70％、70％-75％、75％-80％、80％-85％或85％-90％)。
    优选地，所述纳米粒中，治疗性蛋白：聚阴离子：CPP的质量比为1：0.5-0.9：0.1-0.5(例如1：0.5：0.1-0.5、1：0.6：0.1-0.5、1：0.7：0.1-0.5、1：0.8：0.1-0.5或1：0.9：0.1-0.5，例如1：0.5-0.9：0.1、1：0.5-0.9：0.2、1：0.5-0.9：0.3、1：0.5-0.9：0.4或1：0.5-0.9：0.5)。
  4. 一种微胶囊，所述微胶囊包含壁层，以及包埋在壁层中的权利要求1-3任一项的纳米粒，所述壁层包含肠溶材料或主要由肠溶材料组成；
    优选地，所述肠溶材料选自纤维素及其衍生物，例如羟丙甲纤维素邻苯二甲酸酯(HPMCP)、邻苯二甲酸乙酸纤维素(CAP)、1,2,4-苯三甲酸乙酸纤维素(CAT)；
    优选地，所述肠溶材料为HPMCP；
    优选地，所述微胶囊的粒径为1-10μm(例如1μm-2μm、2μm-3μm、3μm-4μm、4μm-5μm、5μm-6μm、6μm-7μm、7μm-8μm、8μm-9μm或9μm-10μm)；
    优选地，所述微胶囊的形状为球形；
    优选地，所述微胶囊的包封率为30％-95％(例如30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％或95％)。
  5. 一种药物组合物，其包含权利要求1-3任一项的纳米粒或权利要求4的微胶囊；
    优选地，所述药物组合物用于预防或治疗所述纳米粒或微胶囊中包含的治疗性蛋白所能够预防或治疗的疾病；
    优选地，所述治疗性蛋白为胰岛素，所述药物组合物用于预防或治疗受试者中的高血糖症；
    优选地，所述高血糖症包括应激诱发高血糖症、糖尿病(包括1型糖尿病和2型糖尿病)和葡萄糖耐量受损；
    优选地，所述药物组合物包含预防或治疗有效剂量的纳米粒或微胶囊；
    优选地，所述药物组合物包含一种或多种药用载体；
    优选地，所述药物组合物包含冻干保护剂；
    优选地，所述冻干保护剂为醇类冻干保护剂，例如木糖醇、甘露醇或山梨醇；
    优选地，所述受试者为哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物；例如，所述受试者为人。
  6. 权利要求1-3任一项的纳米粒或权利要求4的微胶囊用于制备药物组合物的用途，所述药物组合物用于预防或治疗所述纳米粒或微胶囊中包含的治疗性蛋白所能够预防或治疗的疾病；
    优选地，所述治疗性蛋白为胰岛素，所述疾病为受试者中的高血糖症；
    优选地，所述高血糖症包括应激诱发高血糖症、糖尿病(包括1型糖尿病和2型糖尿病)和葡萄糖耐量受损；
    优选地，所述受试者为哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物；例如，所述受试者为人。
  7. 一种预防或治疗疾病的方法，包括给有此需要的受试者施用权利要求1-3任一项的纳米粒、权利要求4的微胶囊或权利要求5的药物组合物，所述疾病为所述纳米粒、微胶囊或药物组合物中包含的治疗性蛋白所能够预防或治疗的疾病；
    优选地，所述治疗性蛋白为胰岛素，所述疾病为高血糖症；
    优选地，所述高血糖症包括应激诱发高血糖症、糖尿病(包括1型糖尿病和2型糖尿病)和葡萄糖耐量受损；
    优选地，所述受试者为哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物；例如，所述受试者为人。
  8. 制备权利要求1-3任一项的纳米粒的方法，所述方法包括以下步骤：
    步骤1：制备纳米粒A，所述纳米粒A为包含治疗性蛋白和CPP的纳米粒；
    步骤2：使用聚阴离子对步骤1得到的纳米粒A进行包被；
    优选地，所述步骤1包括：使包含治疗性蛋白的溶液与包含CPP的溶液进行混合；
    优选地，所述步骤1进一步包括以下步骤：
    步骤1-1：提供包含治疗性蛋白的溶液和包含CPP的溶液；
    步骤1-2：提供包含涡流混合区域和多个可供流体流向涡流混合区域的通道的装置；
    步骤1-3：使包含治疗性蛋白的溶液和包含CPP的溶液通过不同的通道到达涡流混合区域中，进行混合，得到包含纳米粒A的混悬液；
    优选地，步骤1制备的纳米粒A的粒径为30nm-500nm(例如30nm-100nm、100-200nm、200-300nm、300-400nm或400-500nm)；
    优选地，步骤1制备的纳米粒A的粒径的多分散指数(PDI)为0.1-0.5(例如0.1-0.2、0.2-0.3、0.3-0.4或0.4-0.5)；
    优选地，步骤1制备的纳米粒A的Zeta电位为+10mV至+50mV(例如+10mV至+20mV、+20mV至+30mV、+30mV至+40mV或+40mV至+50mV)；
    优选地，步骤1制备的纳米粒A的包封率为90％-99％(例如90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)；
    优选地，步骤1制备的纳米粒A的载药量为60％-90％(例如60％-65％、65％-70％、70％-75％、75％-80％、80％-85％或85％-90％)；
    优选地，所述步骤2包括：使包含聚阴离子的溶液与包含纳米粒A的混悬液进行混合；
    优选地，所述步骤2进一步包括以下步骤：
    步骤2-1：提供包含聚阴离子的溶液和包含纳米粒A的混悬液；
    步骤2-2：提供包含涡流混合区域和多个可供流体流向涡流混合区域的通道的装置；
    步骤2-3：使包含聚阴离子的溶液和包含纳米粒A的混悬液通过不同的通道到达涡流混合区域中，进行混合，得到包含所述纳米粒的混悬液；
    优选地，步骤2-1中的包含纳米粒A的混悬液由包含步骤1-1、步骤1-2和步骤1-3的方法得到。
  9. 制备权利要求4的微胶囊的方法，所述方法包括以下步骤：
    步骤1’：制备权利要求1-3任一项的纳米粒；
    步骤2’：使用肠溶材料对步骤1’得到的纳米粒进行包被；
    优选地，步骤1’包括：以权利要求8的方法权利要求1-3任一项的纳米粒；
    优选地，所述步骤2’进一步包括以下步骤：
    步骤2’-1：提供包含权利要求1-3任一项的纳米粒的混悬液和包含肠溶材料的溶液；
    步骤2’-2：提供包含涡流混合区域和多个可供流体流向涡流混合区域的通道的装置；
    步骤2’-3：使包含权利要求1-3任一项的纳米粒的混悬液、包含肠溶材料的溶液和任选的酸性溶液(例如盐酸)通过不同的通道到达涡流混合区域中，进行混合，得到包含所述微胶囊的溶液。
PCT/CN2018/101106 2017-10-20 2018-08-17 负载治疗性蛋白的纳米粒及其微胶囊 WO2019076125A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710983761.7 2017-10-20
CN201710983761.7A CN108272768B (zh) 2017-10-20 2017-10-20 负载治疗性蛋白的纳米粒及其微胶囊

Publications (1)

Publication Number Publication Date
WO2019076125A1 true WO2019076125A1 (zh) 2019-04-25

Family

ID=62801306

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/101106 WO2019076125A1 (zh) 2017-10-20 2018-08-17 负载治疗性蛋白的纳米粒及其微胶囊

Country Status (2)

Country Link
CN (1) CN108272768B (zh)
WO (1) WO2019076125A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021011398A1 (en) * 2019-07-12 2021-01-21 The Board Of Trustees Of The Leland Stanford Junior University Functionalized nanoparticles and their use in treating bacterial infections
WO2022177980A1 (en) * 2021-02-16 2022-08-25 The Johns Hopkins University Methods for preparation of shelf-stable plasmid dna/polycation particles with defined sizes for cell transfection
US11998615B2 (en) 2021-04-14 2024-06-04 The Board Of Trustees Of The Leland Stanford Junior University Functionalized nanoparticles and their use in treating bacterial infections

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108272768B (zh) * 2017-10-20 2019-07-26 中山大学 负载治疗性蛋白的纳米粒及其微胶囊
CN111265493A (zh) * 2018-12-05 2020-06-12 沈阳药科大学 磷脂包覆的聚丙烯酸/磷酸锌纳米粒的制备方法及应用
CN117562869A (zh) * 2023-05-05 2024-02-20 中南大学湘雅医院 一种治疗关节疼痛的氢氧化镁纳米粒、其制备方法及应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102988295A (zh) * 2011-09-09 2013-03-27 复旦大学 一种穿膜肽修饰的纳米粒及其制备方法
CN104353062A (zh) * 2014-11-21 2015-02-18 中国人民解放军南京军区福州总医院 一种胰岛素口服纳米制剂及其制备方法
CN105056212A (zh) * 2015-07-14 2015-11-18 江西省药物研究所 一种提高胰岛素口服结肠吸收的壳聚糖纳米粒及制备方法
CN108272768A (zh) * 2017-10-20 2018-07-13 中山大学 负载治疗性蛋白的纳米粒及其微胶囊

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2014112958A (ru) * 2011-09-21 2015-10-27 Йиссум Ресерч Девелопмент Компани оф де Хебрю Юниверсити оф Джерусалем Лтд. Наносистемы доставки для мирнк

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102988295A (zh) * 2011-09-09 2013-03-27 复旦大学 一种穿膜肽修饰的纳米粒及其制备方法
CN104353062A (zh) * 2014-11-21 2015-02-18 中国人民解放军南京军区福州总医院 一种胰岛素口服纳米制剂及其制备方法
CN105056212A (zh) * 2015-07-14 2015-11-18 江西省药物研究所 一种提高胰岛素口服结肠吸收的壳聚糖纳米粒及制备方法
CN108272768A (zh) * 2017-10-20 2018-07-13 中山大学 负载治疗性蛋白的纳米粒及其微胶囊

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HAN, LINA ET AL.: "Insulin-Loaded pH-Sensitive Hyaluronic Acid Nanoparticles Enhance Transcellular Delivery", AAPS PHARMSCITECH, vol. 13, no. 3, 30 May 2012 (2012-05-30), pages 836 - 845, XP035104455, DOI: doi:10.1208/s12249-012-9807-2 *
HE, ZHIYU ET AL.: "Scalable Fabrication of Size-Controlled Chitosan Nanoparticles for Oral Delivery of Insulin", BIOMATERIALS, vol. 130, 22 March 2017 (2017-03-22), pages 28 - 41, XP029968827, DOI: doi:10.1016/j.biomaterials.2017.03.028 *
HU , CHULING ET AL.: "Application of Cell Penetrating Peptides in the Field of Biomedicine", PHARMACEUTICAL JOURNAL OF CHINESE PEOPLE'S LIBERATION ARMY, vol. 32, no. 4, 20 August 2016 (2016-08-20), pages 351 - 356 *
WANG, ZHAOYANG ET AL.: "Application of Cell Penetrating Peptides in Oral Drug Delivery Systems", THE 12TH NATIONAL YOUTH PHARMACY WORKING STUFF SYMPOSIUM ON LATEST SCIENTIFIC RESEARCH ACHIEVEMENTS, 10 June 2014 (2014-06-10), pages 402 - 406 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021011398A1 (en) * 2019-07-12 2021-01-21 The Board Of Trustees Of The Leland Stanford Junior University Functionalized nanoparticles and their use in treating bacterial infections
WO2022177980A1 (en) * 2021-02-16 2022-08-25 The Johns Hopkins University Methods for preparation of shelf-stable plasmid dna/polycation particles with defined sizes for cell transfection
US11998615B2 (en) 2021-04-14 2024-06-04 The Board Of Trustees Of The Leland Stanford Junior University Functionalized nanoparticles and their use in treating bacterial infections

Also Published As

Publication number Publication date
CN108272768A (zh) 2018-07-13
CN108272768B (zh) 2019-07-26

Similar Documents

Publication Publication Date Title
WO2019076125A1 (zh) 负载治疗性蛋白的纳米粒及其微胶囊
Yu et al. Mannan-modified solid lipid nanoparticles for targeted gene delivery to alveolar macrophages
Wu et al. A novel small Odorranalectin-bearing cubosomes: preparation, brain delivery and pharmacodynamic study on amyloid-β25–35-treated rats following intranasal administration
Wang et al. A cooperative dimensional strategy for enhanced nucleus‐targeted delivery of anticancer drugs
Woitiski et al. Pharmacological effect of orally delivered insulin facilitated by multilayered stable nanoparticles
Chen et al. Eudragit S100-coated chitosan nanoparticles co-loading tat for enhanced oral colon absorption of insulin
Regier et al. Fabrication and characterization of DNA-loaded zein nanospheres
Inchaurraga et al. Zein-based nanoparticles for the oral delivery of insulin
Zhang et al. The use of low molecular weight protamine to enhance oral absorption of exenatide
Chen et al. Glucosamine derivative modified nanostructured lipid carriers for targeted tumor delivery
Chen et al. Cp1-11 peptide/insulin complex loaded pH-responsive nanoparticles with enhanced oral bioactivity
WO2019148811A1 (zh) 一种负载胰岛素的肠溶性纳微颗粒及其制备方法和应用
Gong et al. Hyaluronic acid modified doxorubicin loaded Fe 3 O 4 nanoparticles effectively inhibit breast cancer metastasis
US10525013B2 (en) Cationic materials and formulations for drug delivery
Zhang et al. Enhanced oral bioavailability from food protein nanoparticles: A mini review
Sharma et al. Hyaluronic acid anchored paclitaxel nanocrystals improves chemotherapeutic efficacy and inhibits lung metastasis in tumor-bearing rat model
Lv et al. Biological and intracellular fates of drug nanocrystals through different delivery routes: Recent development enabled by bioimaging and PK modeling
Zhang et al. Novel solid lipid nanoparticles as carriers for oral administration of insulin
Wang et al. Nano-porous silica aerogels as promising biomaterials for oral drug delivery of paclitaxel
Esparza et al. Phospholipid micelles for peptide drug delivery
Wang et al. Mitochondria-targeting folic acid-modified nanoplatform based on mesoporous carbon and a bioactive peptide for improved colorectal cancer treatment
Qi et al. Gastrointestinal absorption enhancement of insulin by administration of enteric microspheres and SNAC to rats
CN114588275A (zh) 一种具有脑靶向性的细胞膜仿生修饰药物纳米晶及其制备方法与应用
Shan et al. RETRACTED ARTICLE: In vitro and in vivo protein release and anti-ischemia/reperfusion injury properties of bone morphogenetic protein-2-loaded glycyrrhetinic acid-poly (ethylene glycol)-b-poly (l-lysine) nanoparticles
Arpaç et al. Design and in vitro/in vivo evaluation of polyelectrolyte complex nanoparticles filled in enteric-coated capsules for oral delivery of insulin

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18868538

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18868538

Country of ref document: EP

Kind code of ref document: A1