WO2019148811A1 - 一种负载胰岛素的肠溶性纳微颗粒及其制备方法和应用 - Google Patents

一种负载胰岛素的肠溶性纳微颗粒及其制备方法和应用 Download PDF

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WO2019148811A1
WO2019148811A1 PCT/CN2018/101177 CN2018101177W WO2019148811A1 WO 2019148811 A1 WO2019148811 A1 WO 2019148811A1 CN 2018101177 W CN2018101177 W CN 2018101177W WO 2019148811 A1 WO2019148811 A1 WO 2019148811A1
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insulin
eudragit
solution
particle
enteric
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PCT/CN2018/101177
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French (fr)
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刘志佳
孙立泷
陈永明
刘利新
毛海泉
梁锦荣
孙成新
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中山大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—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
    • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
    • 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
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • 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
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • 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
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the invention relates to the field of biomedical technology, and more particularly to an insulin-loaded enteric nano-particle and a preparation method and application thereof.
  • Diabetes is a chronic metabolic disorder, which is mainly divided into two types, type 1 and type 2.
  • Insulin has always been an important treatment for diabetic patients, but its traditional subcutaneous injection method has brought great physical pain or poor compliance to patients, so people have been working on the development of non-injectable insulin preparations.
  • oral administration of insulin is more popular among the public. Compared with other routes of administration, oral insulin has the following advantages: 1. It is convenient and non-invasive, and improves patient compliance; 2. Prevents high blood insulin symptoms in peripheral blood and reduces adverse reactions caused by traditional diabetes treatment; After oral administration of insulin, intestinal absorption into the liver from the portal vein to exert hypoglycemic effects mimics the normal physiological pathway of autologous insulin secretion.
  • insulin has a relatively high molecular weight (molecular weight of 5,850) and is highly hydrophilic, which results in poor intestinal osmotic absorption after oral administration.
  • protein drugs such as insulin are unstable compared to small molecule drugs, and are easily degraded by gastrointestinal pH and proteases, resulting in extremely low oral bioavailability.
  • Nanomedicine and nanotechnology bring new expectations for oral insulin.
  • the use of nano-micron-loaded insulin can greatly improve its oral bioavailability and exhibit good hypoglycemic effects in vivo.
  • some acid-insoluble, neutral and alkaline soluble enteric polymer materials have been developed, such as Eudragit L100-55 (pH> 5.5 Dissolution), Eudragit L100 (pH > 6.0 dissolved), Eudragit S100 (pH > 7.0 dissolved), and the like.
  • the prior art mainly produces an enteric capsule by coating a layer of an enteric polymer on the outer shell of the medicinal capsule, and then drying, so that the enteric capsule is prepared after repeated operations, and then the nano pharmaceutical preparation is filled therein.
  • the method specifically relates to the use of an organic solvent, and the operation process is complicated and time consuming.
  • the technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art mentioned above, and provide a new method for preparing enteric nano-particles in a fast, efficient and in-situ manner, and the prepared nano-particles can solve the existing insulin nano-preparation.
  • the surface-positively charged nanoparticles were prepared by electrostatic compounding between quaternized chitosan, insulin and sodium tripolyphosphate, and then Utech polymers were coated on the surface to obtain particle sizes ranging from nanometers to micrometers. Enteric nano-particles inside.
  • the granule has the characteristics of slowing drug release and avoiding drug degradation in the acidic environment of the stomach, improving the oral absorption efficiency of the insulin preparation in the small intestine, showing good blood sugar lowering effect and high bioavailability, and can be given 1 Type 2 diabetes patients provide a safe, convenient and compliant method of administration.
  • a first object of the present invention is to provide an insulin-loading enteric nanoparticle.
  • a second object of the present invention is to provide a process for preparing the insulin-loaded enteric nanoparticle.
  • a third object of the present invention is to provide the use of the insulin-loaded enteric nanoparticle.
  • An insulin-loading enteric nanoparticle granule obtained by electrostatically compounding quaternized chitosan, insulin and sodium tripolyphosphate and Eudragit coated on the surface of the nanoparticle )composition.
  • the quaternized chitosan used in the particles of the present invention has water solubility and good biocompatibility, and can reversibly open the tight junction between small intestinal epithelial cells; sodium tripolyphosphate is a cross-linking agent; Eudragit Odd) is a class of acid-insoluble, neutral and alkaline soluble enteric polymers that protect protein proteins from excessive release or degradation by acids and proteases in the acidic environment of the stomach, while Eudragit (especially) In addition, it can be quickly dissolved in a specific position in the intestine, thereby releasing the loaded drug and improving its oral absorption efficiency.
  • the quaternized chitosan has a molecular weight of from 50 kDa to 200 kDa.
  • the Eudragit is Eudragit L100-55 (pH > 5.5 dissolved, duodenal site), Eudragit L100 (pH > 6.0 dissolved, jejunal site) or Eudragit S100 (pH > 7.0 dissolved, colon site).
  • the Eudragit is Eudragit L100-55.
  • the particles have a particle diameter of 50 nm to 2 ⁇ m.
  • the particles have a PDI of from 0.1 to 0.5.
  • the potential of the particles is from -5 mV to -20 mV.
  • the granules are loaded in an amount of from 20% to 40%.
  • the particles have an encapsulation efficiency of from 70% to 95%.
  • the insulin-loaded enteric nano-particles of the invention have the characteristics of slowing drug release and avoiding drug degradation in an acidic environment of the stomach, and rapidly dissolving at a specific position in the intestine, thereby releasing the loaded drug and improving the absorption efficiency thereof.
  • An oral insulin pharmaceutical preparation comprising the above-mentioned insulin-loaded nanoparticle.
  • the pharmaceutical formulation further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutical preparation is a lyophilized preparation
  • the pharmaceutical preparation is a capsule.
  • the present invention also claims a method for preparing an insulin-loaded enteric nanoparticle, comprising the following steps:
  • the Utech solution is introduced into the third and fourth channels, and the solution of each channel simultaneously reaches the vortex mixing region for rapid mixing, thereby obtaining the surface coated Utech
  • the soluble nanoparticles, wherein the flow rate of the four channels is controlled from 1 mL/min to 50 mL/min (preferably from 10 mL/min to 50 mL/min, more preferably 40 mL/min).
  • the present invention prepares insulin-loaded enteric nanoparticle by a multi-channel vortex mixing technique and a rapid nanocomposite (FNC) method.
  • the multi-channel vortex mixing device is shown in FIGS. 1A and 1B, and FIG. 1A shows the overall configuration of the device, which is composed of three identical cylindrical metal bodies; FIG. 1B shows the difference of the three identical cylindrical metal bodies.
  • the structural diagram where 1 is the uppermost metal body, which contains 4 channels and is directly connected to the outer plastic pipe; 2 is an intermediate metal body, which mainly vortexes and introduces the solution introduced by 4 channels of 1 to the center thereof. 3 is the lowermost metal body, and the mixed solution introduced into the center portion of 2 is externally collected through its pores.
  • This FNC process can be operated continuously and efficiently in aqueous solution, featuring high throughput and high controllability to produce particles. Further, the granules prepared therefrom have the advantages of small particle size, uniform dispersion, high batch-to-batch reproducibility, and the like.
  • the above-mentioned technique and apparatus are described in the inventor's patent application No. PCT/US2017/014080.
  • the quaternized chitosan has a degree of quaternization of from 5% to 30%; and the quaternized chitosan concentration is from 0.5 to 3 mg/mL (preferably 1.5 mg/mL).
  • the pH of the insulin solution is 7 to 8.5 (preferably 8), the concentration is 0.1 to 4 mg/mL (preferably 2 mg/mL), and the insulin solution is mixed with 0.1 to 1 mg/mL sodium tripolyphosphate (preferably 0.1 mg). /mL).
  • the pH of the composite nanoparticle core preparation system is 6.5 to 7.3 (preferably 7.3).
  • the concentration of Eudragit is 0.1 to 2 mg/mL (preferably 0.5 mg/mL).
  • the present invention has the following beneficial effects:
  • the invention coats the enteric characteristic material-Eudragit on the surface of the positively charged insulin-loaded composite nanoparticles by a two-step method of rapid nanocomposite (FNC) technology, so that when passing through the acidic environment of the stomach , slows the release of the drug, and avoids the enzyme or acid degradation of the protein protein in the stomach; when the particle reaches a specific part of the intestine, due to the rapid dissolution of the coated Eudragit, it can be quickly positively
  • the internal composite nanoparticles of charge are exposed, and the oral absorption of the small intestinal epithelium is enhanced to enhance the oral absorption efficiency of the drug, and the insulin is allowed to enter the blood to exert a stable hypoglycemic effect.
  • the insulin-loaded enteric nano-particles prepared by the invention not only have the characteristics of high encapsulation rate and drug loading amount, but also have good blood sugar lowering effect, and also have high oral bioavailability, which can be very To a large extent, the patient's symptoms of diabetes are alleviated, and the mode of administration is simple and convenient and the compliance is high.
  • FIG. 1 is a exemplarily depicted multi-inlet vortex mixer for preparing nanoparticles of the present invention
  • FIG. 1A is a state in which the first member, the second member, and the third member are assembled and connected to an external pipe
  • FIG. 1 is a bottom view of the first component
  • FIG. 1B-2 is a top view of the second component
  • FIG. 1B-3 is a top view of the third component
  • FIG. 1C shows a device for preparing nanoparticles
  • FIG. 1C-1 shows a syringe High pressure pump, plastic tube and multi-inlet vortex mixer
  • Figure 1C-2 is an enlarged view of a multi-inlet vortex mixer connected to a plastic tube.
  • Figure 2 shows composite nanoparticles prepared at different flow rates.
  • concentration of quaternized chitosan (HTCC) was 1.5 mg/mL
  • concentration of insulin was 2 mg/mL
  • concentration of sodium tripolyphosphate was 0.1 mg/mL
  • pH was 7.3.
  • Figure 3 shows the particle size and zeta potential of composite nanoparticles prepared at different pH values.
  • the quaternized chitosan (HTCC) concentration was 1.5 mg/mL
  • the insulin concentration was 2 mg/mL
  • the sodium tripolyphosphate concentration was 0.1 mg/mL
  • the flow rate was 40 mg/mL.
  • Figure 4 shows the insulin encapsulation efficiency and drug loading of composite nanoparticles prepared under different pH conditions.
  • the quaternized chitosan (HTCC) concentration was 1.5 mg/mL
  • the insulin concentration was 2 mg/mL
  • the sodium tripolyphosphate concentration was 0.1 mg/mL
  • the flow rate was 40 mg/mL.
  • Figure 5 shows the stability of the prepared composite nanoparticles (particle-a).
  • Figure 6 is a graph showing the particle size distribution of composite nanoparticles obtained by comparing different preparation methods.
  • Figure 7 shows the surface coated Eudragit L100-55 nanoparticles (particle-b1) prepared at different flow rates.
  • the concentration of Eudragit L100-55 was 0.5 mg/mL and the pH was 6.8.
  • Figure 8 shows the preparation of enteric particles by coating the composite nanoparticles with different concentrations of Eudragit L100-55, wherein the flow rate is 40 mL/min, and ⁇ represents the phenomenon of coagulation of the particles obtained under the conditions.
  • Figure 9 shows the preparation of enteric particles of different particle sizes by varying the pH of Eudragit L100-55.
  • Figure 10 is a graph showing the fluorescence resonance energy transfer spectrum of the enteric particle-b1 after fluorescent labeling.
  • Figure 11 is a transmission electron micrograph of composite nanoparticles (particle-a), enteric particles-b1, particles-b2, and particles-b3.
  • Figure 12 shows the in vitro release profile of insulin loaded with insulin-loaded granules or insulin solution at simulated gastrointestinal pH conditions.
  • Figure 13 shows the cytotoxicity of pellet-a, granule-b1 or insulin solution samples after co-incubation with E12 or Caco-2 cells.
  • Figure 14 shows the change in transmembrane resistance of Caco-2 monolayers with different particle or insulin solutions on the surface without covering the mucus layer.
  • Figure 15 shows the change in transmembrane resistance of Caco-2 monolayers with different particle or insulin solutions covering the mucus layer.
  • Figure 16 shows the apparent permeability coefficient (cm/s) of insulin penetrating cell layers in Caco-2 monolayer cells with different particle or insulin solutions that do not cover or cover the mucus layer.
  • Figure 17 shows the treatment of monolayer Caco-2 cells by enteric granule-b1 and observation of changes in tight junctions between cells by immunofluorescence labeling.
  • Figure 18 shows the changes in blood glucose by oral administration of different granules or insulin solutions and subcutaneous injection of insulin solution.
  • Figure 19 shows the changes in serum insulin content over time in oral granule-b1 or insulin solution and subcutaneous injection of insulin solution.
  • Figure 20 shows the biosafety of oral granule-a or granule-b1.
  • Chitosan (2g) was dissolved in 100mL aqueous solution containing 2wt% acetic acid, then heated to 80 ° C, then slowly added 5mL aqueous solution of glycidyl trimethylammonium chloride (GTMAC) to the solution, further reaction for 24h, the resulting solution was cooled Thereafter, it was precipitated 3 times in 10 volumes of acetone, then dialyzed against water for 3 days, and lyophilized to obtain the final product HTCC.
  • the degree of quaternization of HTCC is 43%.
  • FIG. 1A is an overall structural view of the device, which is composed of three identical cylindrical metal bodies; FIG.
  • 1B is a top view of the respective structures of three identical cylindrical metal bodies.
  • 1 is the uppermost metal body, which has 4 channels and can be directly connected to the plastic pipe.
  • 2 is the intermediate metal body, which is the liquid introduced by the 4 channels of 1 through the rapid eddy current mixing to reach the center of the channel, 3
  • the HTCC at a concentration of 1.5 mg/mL was dissolved in deionized water, and the pH of the 2 mg/mL insulin solution was adjusted to 8.0 with HCl and NaOH solution, and the solution contained 0.1 mg/mL sodium tripolyphosphate.
  • the above two solutions were respectively loaded into four 20 mL syringes and connected to four plastic connecting tubes, respectively, and the other end of the plastic tube was respectively connected and fixed to the multi-channel vortex mixer integral device, in the multi-channel vortex mixer.
  • a container for collecting the prepared particles is placed under the unitary device, and an overall schematic view of the process is shown in Fig. 1C. By simultaneously opening the high pressure pump, the four syringes carrying the aqueous sample were run at the same flow rate through four plastic tubes into a multi-channel vortex mixer for rapid mixing to prepare the desired composite nanoparticles.
  • different sizes of insulin/sodium tripolyphosphate can be obtained by adjusting the fluid flow rates of the four channels to 5 mL/min, 10 mL/min, 20 mL/min, 30 mL/min, 40 mL/min, and 50 mL/min, respectively.
  • /Quaternized chitosan composite nanoparticles When the flow rate was increased from 5 mL/min to 50 mL/min, the particle size of the composite nanoparticle decreased from about 193 nm to about 80 nm, and the PDI decreased from 0.29 to about 0.16.
  • the flow rate is 40 mL/min, the obtained composite particles have a small particle size and the best dispersibility.
  • Glycan composite nanoparticles (particle-a).
  • Figure 5 shows the storage stability study of the particle-a. It can be seen that the particle size and polydispersity index (PDI) of the particle remain substantially unchanged over a period of 3 days, indicating that the particle has good stability.
  • PDI polydispersity index
  • Figure 6 shows the size distribution of nanoparticles prepared by bulk mixing, stepwise dropping or FNC method. It can be concluded that our FNC technology produces nanoparticles with smaller size than traditional bulk mixing or stepwise dropping. Dimensions and more uniform dimensional dispersion.
  • Figure 7 shows the effect of changing the four-channel flow rate from 5 mL/min to 50 mL/min on the enteric particle size and polydispersity index at pH 6.8 and Eudragit L100-55 at a concentration of 0.5 mg/mL. It can be seen that the particle size and PDI prepared by different flow rates are significantly different. The particle size of the particles will gradually decrease with the increase of the flow rate, and the particle size of the particles will be smaller and more dispersed when the flow rate is 40 mL/min. The sex index was the smallest, so the flow rate of 40 mL/min was chosen as the optimization condition.
  • Figure 8 shows the preparation of enteric particles using different concentrations of Eudragit L100-55.
  • concentration is lower than 0.5mg/mL, the prepared particles will accumulate; when the concentration is higher than 0.5mg/mL, the particle size will increase, so the optimum concentration of Eudragit L100-55 is 0.5mg. /mL.
  • Figure 9 shows a guaranteed flow rate of 40 mL/min and an Eudragit L100-55 concentration of 0.5 mg/mL.
  • pH of Eudragit L100-55 solution 6.8, 6.5 or 6.0
  • three enteric properties with different particle sizes were prepared.
  • the particles are particles - b1, particles - b2, and particles - b3, and their various physical and chemical properties are characterized as shown in Table 1.
  • Figure 10 shows that the fluorescence of the fluorescein isothiocyanate (FITC)-labeled Eudragit L100-55 in the enteric particle-b1 is reduced and the rhodamine isothiocyanate (RITC)-labeled insulin should be enhanced, indicating two fluorescent molecules in the particle. It has the property of fluorescence resonance energy transfer, which proves the particle integrity of the composite nanoparticles coated with Eudragit L100-55.
  • FITC fluorescein isothiocyanate
  • RITC rhodamine isothiocyanate
  • Nanoparticle Particle size (nm) Polydispersity index ⁇ -potential (mV) Encapsulation rate (%) Drug loading(%) Particle-a 87 ⁇ 3 0.16 ⁇ 0.01 23.8 ⁇ 0.50 95.3 ⁇ 0.3 52.9 ⁇ 0.2 Particle-b1 115 ⁇ 5 0.13 ⁇ 0.01 -18.7 ⁇ 1.4 81.9 ⁇ 1.1 35.6 ⁇ 0.5 Particle-b2 354 ⁇ 11 0.11 ⁇ 0.06 -13.0 ⁇ 1.2 76.1 ⁇ 0.2 33.1 ⁇ 0.1 Particle-b3 1431 ⁇ 35 0.30 ⁇ 0.06 -5.5 ⁇ 0.30 70.2 ⁇ 0.3 30.5 ⁇ 0.2
  • Preparation of insulin/trimeric phosphate coated with Eudragit L100 or Eudragit S100 by rapid mixing of insulin/sodium tripolyphosphate/quaternized chitosan particles (particle-a) and Eudragit L100 or Eudragit S100 in a multi-channel vortex mixer
  • the sodium/quaternized chitosan particles (particle-c1 and particle-d1), the particle size, polydispersity index, surface potential, encapsulation efficiency, and drug loading amount are shown in Table 2.
  • Table 2 Enteric granules prepared using Eudragit L100 or Eudragit S100
  • Nanoparticle Particle size (nm) Polydispersity index ⁇ -potential (mV) Encapsulation rate (%) Drug loading(%) Particle-c1 103 ⁇ 6 0.19 ⁇ 0.01 -20.3 ⁇ 0.8 83.3 ⁇ 0.4 34.5 ⁇ 0.7 Particle-d1 105 ⁇ 2 0.15 ⁇ 0.01 -25.0 ⁇ 0.9 82.3 ⁇ 0.6 31.7 ⁇ 0.4
  • the particle size, polydispersity index and surface potential of the particle samples of Example 2 were measured by a Malvern particle size meter, and the structural morphology of various particles was characterized by transmission electron microscopy.
  • Figure 11 shows a transmission electron micrograph of composite nanoparticles (particle-a) and enteric particles (particle-b1, particle-b2, particle-b3). Electron micrographs show that the particle-a, particle-b1, particle-b2, and particle-b3 sizes are consistent with the particle size measurements measured by the Malvern particle size analyzer.
  • the medium solution was added with an equal volume of fresh medium solution, and the concentration of insulin in the release medium was measured by BCA protein concentration detection method to calculate the cumulative release content of insulin.
  • the safety of insulin solution, granule-a and granule-b1 for E12 and Caco-2 cells was examined by the MTT method.
  • 1.0 ⁇ 10 4 /well Caco-2 or E12 cells were cultured in 96-well plates, and added to 200 ⁇ L of medium for 24 h, then replaced with 200 ⁇ L of fresh medium and containing different concentrations of insulin solution, granule-a or granules- B1.
  • DMSO dimethyl sulfoxide
  • the insulin solution, granule-a or granule-b1 did not affect the proliferation of E12 and Caco-2 cells, indicating that it was not toxic to E12, Caco-2 cells.
  • Caco-2 single cell layer model to simulate small intestinal epithelial cells.
  • Caco-2 cells were cultured on a 12-well plate Transwell polyester membrane, cultured in a 37 ° C, 5% CO 2 cell culture incubator, and the medium was changed every 2 days and the resistance value was measured. The culture period is about 2 weeks.
  • the transmembrane resistance of Caco-2 cells is stable and higher than 750 ⁇ , it can be used in subsequent experimental studies.
  • the upper and lower media were replaced with Hank's Balanced Salt Solution (HBSS) or HBSS containing 1% mucin before the experiment.
  • HBSS Hank's Balanced Salt Solution
  • Transmembrane resistance (TEER) tracking 200 ⁇ L of HBSS insulin-containing solution, particle-a, particle-b1, particle-b2, particle-b3 and Caco-2 single cell layer or Caco- covering 1% mucin, respectively 2 The single cell layer was incubated for 2 h, then the sample solution was removed and washed, and incubation was continued for 24 h, and the transmembrane resistance (TEER) was measured within a preset time.
  • TEER Transmembrane resistance
  • the transmembrane resistance value remained substantially unchanged for the insulin solution group; for the particle-a, particle-b1, particle-b2 or particle-b3, the cell transmembrane resistance value Both decreased, but the decrease in the transmembrane resistance of the particle-a group was more pronounced (down to about 40%), mainly due to the positive charge on the surface of the particle-a, which is more conducive to interaction with the cell and the season.
  • Ammonium chitosan has the effect of opening tight junctions between cells.
  • Figure 15 shows the change in transmembrane resistance of Caco-2 monolayer cells covered with 1% mucin on different sample treatment surfaces.
  • the transmembrane resistance of the insulin solution group remained unchanged within 2 hours of sample treatment; a.
  • the transmembrane resistance of the particle-b1, granule-b2 or granule-b3 group decreased, but the transmembrane resistance of these granules after adding 1% mucin was higher than that of non-mucin.
  • the transmembrane resistance of the particle-a group decreased to about 60%
  • the transmembrane resistance of the particle-b1, particle-b2 or particle-b3 group decreased to about 65% to 80%.
  • the sample solution was removed, and the transmembrane resistance values of each group were restored within 24 hours, indicating that the tight junction between the Caco-2 single cell layers was restored after opening.
  • Papp is the apparent permeability coefficient
  • dQ/dt is the amount that permeates from the Transwell plate to the lower layer within a certain period of time
  • C o is the initial concentration of the upper drug
  • A is the area of the polyester film of the transewell plate.
  • Figure 16 shows the apparent permeability coefficient of insulin solution, particle-a, particle-b1, particle-b2 or particle-b3 through monolayer cells in Caco-2 monolayer cells with or without 1% mucin.
  • the particle-a has a higher apparent permeability coefficient relative to the insulin solution or other particle group.
  • the apparent permeability coefficient of all groups of Caco2 monolayer cells covered with 1% mucin decreased, indicating that the mucus layer will affect the penetration efficiency of the drug to some extent.
  • Figure 17 shows the changes in intercellular tight junctions of Caco-2 monolayers over different time periods.
  • the initial Caco-2 monolayer cells showed a clear and tight tight junctional structure; after adding granule-b1 and Caco-2 cells for 2 h (2 hours), It can be seen that the tightly connected ring structure disappears substantially, indicating that the tight junction has been opened; after the particle-b1 is removed and culture is continued for 10 hours (12 hours chart), it can be seen that the tightly connected ring structure reappears, indicating that the compact The connection is recoverable after it is opened.
  • SD rats weighing 200 g were intraperitoneally injected with 80 mg/kg of streptozotocin, and the blood glucose level of the rats was monitored regularly.
  • the blood glucose level was stable at 16.6 mmol/L, it was considered to be a type I diabetic model rat.
  • the model mice were divided into 7 groups, 6 in each group, and the rats were also fasted for about 12 hours.
  • the first group was given oral saline
  • the second group was given oral insulin solution (80 IU/kg)
  • the third, fourth, fifth, and sixth groups were given oral granule-a, granule-b1, granule-b2 or granule-b3
  • group 7 a subcutaneous injection of insulin solution (5 IU/kg) was administered, and the blood glucose level of the rats was measured every 1 hour using a blood glucose meter and a blood glucose test strip.
  • Figure 18 shows the in vivo hypoglycemic effect of rats after oral administration of different insulin preparations. It can be seen that oral insulin solution and oral water did not cause a decrease in blood glucose level; subcutaneous injection of insulin solution (5 IU/kg) rapidly decreased blood glucose levels to 20% at 2 h; and oral granule-a, granule-b1, granules After -b2 or granule-b3, the blood glucose of rats showed a steady downward trend, and it can be seen that granule-b1, granule-b2 or granule-b3 has more obvious hypoglycemic effect than granule-a, and small-sized granules- B1 showed the best hypoglycemic effect.
  • Rats with type 1 diabetes were fasted for 12 hours, and the rats were divided into 3 groups of 5 rats each.
  • Group 1 was given a subcutaneous injection of insulin solution (5 IU/kg);
  • Group 2 was given an oral insulin solution (80 IU/kg);
  • Group 3 was given an oral granule-b1 (80 IU/kg), blood samples were taken every 1 hour and passed through a centrifuge.
  • the serum insulin content in rats was tested by ELISA kit.
  • Figure 19 is a graph showing serum insulin concentration versus time.
  • the serum insulin content of the oral insulin solution group was extremely low, and the bioavailability of oral granule-b1 was calculated to be about 11.6% relative to the subcutaneous injection of insulin.
  • Rats were first divided into 4 groups: the first group was normal mice, the second group was diabetic model rats, the third group was diabetic model rats oral granule-a group, and the fourth group was diabetic model rats oral granule-b1 group.

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Abstract

一种负载胰岛素的肠溶性纳微颗粒及其制备方法和应用，所述纳微颗粒由季铵化壳聚糖、胰岛素和三聚磷酸钠通过静电作用复合得到的纳米粒及涂覆在纳米粒表面的尤特奇组成，所述制备方法包括利用FNC技术先制备负载胰岛素的表面带正电荷的复合纳米粒，再在复合颗粒表面涂覆尤特奇。

Description

一种负载胰岛素的肠溶性纳微颗粒及其制备方法和应用 技术领域
本发明涉及生物医药技术领域，更具体地，涉及一种负载胰岛素的肠溶性纳微颗粒及其制备方法和应用。
背景技术
糖尿病是一种慢性代谢障碍性疾病，主要分为1型和2型两大类。胰岛素一直都是糖尿病患者的重要治疗药物，但是其传统的皮下注射给药方式给患者带来了极大身体痛苦或差的依顺性，因而人们一直在致力于非注射型胰岛素制剂的开发，这其中以胰岛素口服给药方式更为大众所青睐。口服胰岛素较之其它给药途径而言具有如下优势：1.具有方便和无创性，提高了病人依顺性；2.防止外周血液高胰岛素症状，减少传统糖尿病治疗所带来的不良反应；3.胰岛素口服给药后经肠道吸收由肝门静脉进入肝脏从而发挥降糖作用，这模拟了自体胰岛素分泌的正常生理途径。然而，胰岛素的口服递送一直面临诸多问题，主要是因为胰岛素的分子量较大(分子量为5850)且亲水性强，这导致其口服后小肠渗透吸收性差。更重要的是，相较之小分子药物而言，胰岛素等蛋白类药物不稳定，容易受胃肠道pH和蛋白酶影响而降解，这导致其口服生物利用度极低。
纳米医学和纳米技术给口服胰岛素带来了新期待。利用纳微米颗粒负载胰岛素可以大幅度提高其口服生物利用度且表现出良好体内降血糖效果。为了避免胰岛素纳米制剂口服给药后受到胃酸和蛋白酶的降解，人们开发了一些酸性不溶、偏中性及碱性溶解的肠溶性聚合物材料，例如Eudragit(尤特奇)L100-55(pH>5.5溶解)，Eudragit L100(pH>6.0溶解)，Eudragit S100(pH>7.0溶解)等。现有技术主要通过在药用胶囊外壳涂覆一层肠溶性聚合物，然后干燥，如此经过反复多次操作后制得肠溶性胶囊，然后在它内部填装纳米药物制剂。该方法具体涉及有机溶剂的使用，且操作过程复杂，耗时长。当前仍然缺乏一种快速、高效、原位制备肠溶性纳微颗粒的新技术，从而改善胰岛素纳米制剂的口服递送效率，提高其口服生物利用度，增强其体内降血糖效果。
发明内容
本发明所需解决的技术问题是克服上述现有技术的缺陷和不足，提供一种快 速、高效、原位制备肠溶性纳微颗粒的新方法，制备得到的纳微颗粒解决现有胰岛素纳米制剂存在低口服生物利用度的问题。首先利用季铵化壳聚糖、胰岛素和三聚磷酸钠之间的静电复合制备了表面带正电荷的纳米粒，然后在其表面涂覆尤特奇聚合物得到了颗粒尺寸从纳米到微米范围内的肠溶性纳微颗粒。所述颗粒在胃部酸性环境下具有减缓药物释放和避免药物降解的特点，提高了胰岛素制剂在小肠部位的口服吸收效率，表现出良好的降血糖效果和较高的生物利用度，可以给1型糖尿病患者提供一种安全便利且依顺性好的给药方式。
本发明的第一个目的是提供一种负载胰岛素肠溶性纳微颗粒。
本发明的第二个目的是提供所述负载胰岛素肠溶性纳微颗粒的制备方法。
本发明的第三个目的是提供所述负载胰岛素肠溶性纳微颗粒的应用。
本发明的上述目的是通过以下技术方案给予实现的：
一种负载胰岛素的肠溶性纳微颗粒，所述纳微颗粒由季铵化壳聚糖、胰岛素和三聚磷酸钠通过静电作用复合得到的纳米粒及涂覆在纳米粒表面的Eudragit(尤特奇)组成。
本发明中所述颗粒使用的季铵化壳聚糖具有水溶性和良好生物相容性，它可以可逆瞬时地打开小肠上皮细胞间紧密连接；三聚磷酸钠为交联剂；Eudragit(尤特奇)是一类酸性不溶、偏中性和碱性溶解的肠溶特性聚合物，它可以在胃部酸性环境中保护蛋白类药物防止其过快释放或被酸和蛋白酶降解，同时Eudragit(尤特奇)又可以在肠部特定位置快速溶解，从而释放出所负载的药物，提高其口服吸收效率。
优选地，所述季铵化壳聚糖的分子量为50kDa～200kDa。
优选地，所述尤特奇为EudragitL100-55(pH>5.5溶解，十二指肠部位)、EudragitL100(pH>6.0溶解，空肠部位)或EudragitS100(pH>7.0溶解，结肠部位)。
最优选地，所述尤特奇为EudragitL100-55。
优选地，所述颗粒的粒径为50nm～2μm。
优选地，所述颗粒的PDI为0.1～0.5。
优选地，所述颗粒的电位为-5mV～-20mV。
优选地，所述颗粒的载药量为20％～40％。
优选地，所述颗粒的包封效率为70％～95％。
本发明的负载胰岛素的肠溶性纳微颗粒具有在胃部酸性环境下减缓药物释放和避免药物降解特点，同时在肠部特定位置快速溶解，从而释放出所负载的药物，提高其吸收效率，具有高的口服生物利用度；因此本发明请求保护上述肠溶性纳微颗粒在制备口服胰岛素制剂中的应用。
一种口服胰岛素药物制剂，包含上述负载胰岛素的纳微颗粒。
优选地，所述药物制剂还包括药学上可接受的赋形剂。
优选地，所述药物制剂为冻干制剂
优选地，所述药物制剂为胶囊。
同时，本发明还请求保护一种负载胰岛素的肠溶性纳微颗粒的制备方法，包括如下步骤：
S1.将季铵化壳聚糖溶液引入第1和2通道，将胰岛素和三聚磷酸钠混合溶液引入第3和4通道，各通道溶液同时到达涡流混合区域内进行快速混合，得到表面带正电荷的复合纳米粒；其中四个通道的流速控制为1mL/min～50mL/min(优选为10mL/min～50mL/min，更优选为40mL/min)；
S2.将S1得到复合纳米粒溶液引入第1和2通道，尤特奇溶液引入第3和4通道，各通道溶液同时到达涡流混合区域内进行快速混合，从而得到表面涂覆尤特奇的肠溶性纳微颗粒，其中四个通道的流速控制为1mL/min～50mL/min(优选为10mL/min～50mL/min，更优选为40mL/min)。
优选地，本发明通过一种多通道涡流混合技术并采用快速纳米复合(FNC)方法制备了负载胰岛素的肠溶性纳微颗粒。其中多通道涡流混合装置如图1A和1B所示，图1A显示了该装置的整体构造图，它由3个相同圆柱形金属体构成；图1B显示了该3个相同圆柱形金属体的分别构造图，其中1为最上层金属体，它含有4个通道且直接与外部塑料管道相连；2为中间金属体，它主要将1中4个通道引入的溶液进行涡流混合并到达其中心部位，3为最下层金属体，它可将2中引入到中心部位的混合溶液通过其孔道被外部收集。这种FNC方法可以在水溶液中连续、高效地进行操作，具有高通量和高可控性生产颗粒的特点。此外由它制备的颗粒还具有粒径小，分散均匀、批次间重复性高等优点；上述技术及装置记载在本发明人前期申请号为PCT/US2017/014080的专利中。
优选地，所述季铵化壳聚糖的季铵化程度为5％～30％；季铵化壳聚糖浓度 为0.5～3mg/mL(优选1.5mg/mL)。
优化地，所述胰岛素溶液的pH为7～8.5(优选8)，浓度为0.1～4mg/mL(优选2mg/mL)，胰岛素溶液中混合有0.1～1mg/mL三聚磷酸钠(优选0.1mg/mL)。
优化地，复合纳米粒核心制备体系的pH值为6.5～7.3(优选7.3)。
优化地，所述Eudragit(尤特奇)的浓度为0.1～2mg/mL(优选0.5mg/mL)。
与现有技术相比，本发明具有以下有益效果：
本发明通过快速纳米复合(FNC)技术两步法将肠溶特性材料-Eudragit(尤特奇)涂覆在表面带正电荷的负载胰岛素的复合纳米粒上，使其在通过胃部酸性环境时，减缓药物的释放，并避免胃部酶或酸降解负载的蛋白类药物；当颗粒到达肠部特定部位时，由于涂覆的Eudragit(尤特奇)可以快速溶解的特点，可以快速将带正电荷的内部复合纳米粒暴露出来，进而通过打开小肠上皮紧密连接增强药物的口服吸收效率，促使胰岛素进入血液后发挥稳定的降血糖效果。本发明制备的负载胰岛素的肠溶性纳微颗粒，不但具有较高的包封率和载药量的特点，同时还具有良好的降血糖效果，也具有较高的口服生物利用度，可以在很大程度上对病人的糖尿病症状得到缓解，且给药方式简单方便和依顺性高。
附图说明
图1为示例性地描述了用于制备本发明的纳米粒的多入口涡流混合器；图1A为第一部件、第二部件和第三部件组装后并连接了外部管道的状态；图1B-1为第一部件的仰视图；图1B-2为第二部件的俯视图；图1B-3为第三部件的俯视图；图1C显示了用于制备纳米粒的装置，图1C-1显示了注射器、高压泵、塑料管和多入口涡流混合器，图1C-2为连接了塑料管的多入口涡流混合器的放大图。
图2所示为在不同流速下制备的复合纳米颗粒。其中季铵化壳聚糖(HTCC)浓度为1.5mg/mL，胰岛素浓度为2mg/mL，三聚磷酸钠浓度为0.1mg/mL，pH值为7.3。
图3所示为不同pH值条件下制备的复合纳米颗粒的粒径大小和Zeta电位。季铵化壳聚糖(HTCC)浓度为1.5mg/mL，胰岛素浓度为2mg/mL，三聚磷酸钠浓度为0.1mg/mL，流速为40mg/mL。
图4所示为不同pH值条件下制备的复合纳米颗粒的胰岛素包封率和载药量。 季铵化壳聚糖(HTCC)浓度为1.5mg/mL，胰岛素浓度为2mg/mL，三聚磷酸钠浓度为0.1mg/mL，流速为40mg/mL。
图5所示为制备的复合纳米颗粒(颗粒-a)的稳定性情况。
图6所示为比较不同制备方法所得到的复合纳米粒的粒径分布图。
图7所示为不同流速下制备的表面涂覆Eudragit L100-55的纳米粒(颗粒-b1)。其中Eudragit L100-55浓度为0.5mg/mL，pH 6.8。
图8所示为利用不同浓度Eudragit L100-55涂覆复合纳米粒制备肠溶性颗粒，其中流速为40mL/min，Δ代表该条件下所得颗粒发生聚沉现象。
图9所示为通过改变Eudragit L100-55的pH值制备得到不同粒径的肠溶性颗粒。
图10所示为荧光标记后肠溶性颗粒-b1的荧光共振能量转移谱图。
图11所示为复合纳米颗粒(颗粒-a)、肠溶性颗粒-b1、颗粒-b2、颗粒-b3的透射电镜图。
图12所示为负载胰岛素的颗粒或胰岛素溶液在模拟胃肠道pH条件下的胰岛素体外释放曲线。
图13所示为颗粒-a、颗粒-b1或胰岛素溶液样品与E12或Caco-2细胞共孵育后的细胞毒性情况。
图14所示为不同颗粒或胰岛素溶液在表面不覆盖黏液层的Caco-2单层细胞中跨膜电阻值变化情况。
图15所示为不同颗粒或胰岛素溶液在表面覆盖黏液层的Caco-2单层细胞中跨膜电阻值变化情况。
图16所示为不同颗粒或胰岛素溶液在表面不覆盖或覆盖黏液层的Caco-2单层细胞中胰岛素穿透细胞层的表观渗透系数(cm/s)。
图17所示为肠溶性颗粒-b1处理单层Caco-2细胞并通过免疫荧光标记后观察细胞间紧密连接变化的情况。
图18所示为口服不同颗粒或胰岛素溶液及皮下注射胰岛素溶液的血糖变化情况。
图19所示为口服颗粒-b1或胰岛素溶液及皮下注射胰岛素溶液的血清胰岛素含量随时间变化情况。
图20所示为口服颗粒-a或颗粒-b1的生物安全性情况。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1季铵化壳聚糖(HTCC)的合成
壳聚糖(2g)溶解于100mL水溶液中含2wt％醋酸，随后加热至80℃后向溶液内缓慢滴加5mL氯化缩水甘油基三甲基铵(GTMAC)水溶液，进一步反应24h，所得溶液冷却后在10倍体积丙酮中沉淀3次，然后对水透析3天，冻干，得到最终产物HTCC。HTCC的季铵化程度是43％。
实施例2负载胰岛素的肠溶性纳微颗粒制备
快速纳米复合(FNC)技术是一种利用多通道涡流混合器快速混合聚电解质水溶液高效、连续、可控制备载药颗粒的方法(记载在本发明人前期申请号为PCT/US2017/014080的专利中)。该方法制备的纳米颗粒具有粒径小、尺寸分布均匀、批次重现性高等优点，而且制备过程中不涉及使用有机溶剂，非常适合于蛋白质、多肽、核酸等生物制剂的纳米制剂化。多通道涡流混合器装置图如图1A和1B所示，图1A为装置的整体构造图，是由3个相同圆柱形金属体构成；图1B为3个相同圆柱形金属体的分别的构造俯视图，其中1为最上层金属体，它有4个通道且可以直接与塑料管道相连，2为中间金属体，它是将1中4个通道引入的液体通过快速涡流混合到达其通道中心部位，3为最下层金属体，它可将2中引入到涡流中心部位的混合溶液通过其孔道被外部收集。
1、制备胰岛素/三聚磷酸钠/季铵化壳聚糖(HTCC)复合纳米粒
浓度为1.5mg/mL的HTCC溶解在去离子水中，利用HCl和NaOH溶液调节2mg/mL胰岛素溶液的pH值为8.0，且该溶液中含有0.1mg/mL三聚磷酸钠。将以上两种溶液分别两两装入4支20mL注射器并分别与4支塑料连通管相连，同时将塑料管的另一端分别与多通道涡流混合器整体装置相连并固定，在多通道涡流混合器整体装置下方放置收集制备的颗粒的容器，图1C所示为该过程的整体示意图。通过同时打开高压泵，使4支装载水溶液样品的注射器以相同流速运行通过4支塑料管进入多通道涡流混合器进行快速混合制备所需复合纳米 粒。
如图2所示，通过调节四通道的流体流速分别为5mL/min，10mL/min，20mL/min，30mL/min，40mL/min，50mL/min，可得到不同尺寸的胰岛素/三聚磷酸钠/季铵化壳聚糖复合纳米粒。当流速从5mL/min升至50mL/min，复合纳米颗的粒径由约193nm减小至约80nm，PDI由0.29降至0.16左右。当流速在40mL/min时，所制得的复合颗粒粒径小且分散性最好。
图3和图4所示为调节HTCC溶液体系pH值对复合纳米颗粒的粒径、表面电位、包封率和载药量的影响，其中通过调节pH值由6.5至7.3，颗粒粒径由137nm降至87nm左右；当pH值为7.3时，复合颗粒具有较小的粒径和最高的包封率和载药量。因此我们在pH＝7.3条件下，HTCC浓度为1.5mg/mL，胰岛素浓度为2mg/mL(含0.1mg/mL三聚磷酸钠)，得到了优化的胰岛素/三聚磷酸钠/季铵化壳聚糖复合纳米粒(颗粒-a)。
图5所示为颗粒-a的储存稳定性研究，从中可看出该颗粒在3天时间内其粒径及多分散指数(PDI)基本保持不变，表明颗粒具有较好的稳定性。
图6为利用本体混合、逐步滴加或FNC方法制备纳米颗粒的尺寸分布情况，从中可以得出，相对于传统的本体混合或逐步滴加而言，我们的FNC技术制备的纳米颗粒具有更小的尺寸及更均匀的尺寸分散性。
2、制备表面涂覆Eudragit L100-55的胰岛素/三聚磷酸钠/季铵化壳聚糖颗粒
将浓度0.2～0.6mg/mL的Eudragit L100-55溶解在水中(pH＝11)，然后再分别调节其pH值为6.0、6.5和6.8。利用多通道涡流混合器快速混合胰岛素/三聚磷酸钠/季铵化壳聚糖颗粒(颗粒-a)和Eudragit L100-55水溶液制备表面涂覆Eudragit L100-55的胰岛素/三聚磷酸钠/季铵化壳聚糖颗粒。
如图7显示了在pH6.8和Eudragit L100-55浓度为0.5mg/mL条件下，改变四通道流速从5mL/min到50mL/min对肠溶性颗粒粒径和多分散性指数的影响。从中可以看出不同流速制备的颗粒粒径和PDI都有明显的不同，随着流速的增加颗粒的粒径会逐渐变小，而当流速在40mL/min时的颗粒粒径较小与多分散性指数最小，因而选择流速40mL/min作为优化条件。
图8显示了利用不同浓度Eudragit L100-55制备肠溶性颗粒的情况。当其浓度低于0.5mg/mL时，制备的颗粒会发生聚沉；而当浓度高于0.5mg/mL时，颗粒粒径会升高，因而选择Eudragit L100-55的最佳浓度为0.5mg/mL。
图9所示为保证流速为40mL/min，Eudragit L100-55浓度为0.5mg/mL，通过调整Eudragit L100-55溶液pH值为6.8，6.5或6.0，分别制备了三种粒径不同的肠溶性颗粒即颗粒-b1，颗粒-b2，颗粒-b3，它们各种理化性质的表征如表1所示。
图10显示了肠溶性颗粒-b1中异硫氰酸荧光素(FITC)标记Eudragit L100-55的荧光降低而异硫氰酸罗丹明(RITC)标记胰岛素应该增强，表明颗粒中的两种荧光分子具有荧光共振能量转移的性质，从而证明了表面涂有Eudragit L100-55的复合纳米颗粒的颗粒完整性。
表1优化制备的胰岛素/三聚磷酸钠/季铵化壳聚糖颗粒(颗粒-a)和不同尺寸表面涂覆Eudragit L100-55的肠溶颗粒(颗粒-b1，颗粒-b2，颗粒-b3)的粒径、多分散性指数、表面电位、包封率和载药量
纳米颗粒 粒径(nm) 多分散性指数 ζ-电位(mV) 包封率(％) 载药量(％)
颗粒-a 87±3 0.16±0.01 23.8±0.50 95.3±0.3 52.9±0.2
颗粒-b1 115±5 0.13±0.01 -18.7±1.4 81.9±1.1 35.6±0.5
颗粒-b2 354±11 0.11±0.06 -13.0±1.2 76.1±0.2 33.1±0.1
颗粒-b3 1431±35 0.30±0.06 -5.5±0.30 70.2±0.3 30.5±0.2
3、制备表面涂覆Eudragit L100或Eudragit S100的胰岛素/三聚磷酸钠/季铵化壳聚糖颗粒
将浓度为0.5mg/mL的Eudragit L100或Eudragit S100溶解在水中(pH＝11)，然后再分别调节其pH值为7.4。利用多通道涡流混合器快速混合胰岛素/三聚磷酸钠/季铵化壳聚糖颗粒(颗粒-a)和Eudragit L100或Eudragit S100水溶液分别制备表面涂覆Eudragit L100或Eudragit S100的胰岛素/三聚磷酸钠/季铵化壳聚糖颗粒(颗粒-c1和颗粒-d1)，其粒径、多分散性指数、表面电位、包封率和载药量等如表2所示。
表2利用Eudragit L100或Eudragit S100制备的肠溶性颗粒
纳米颗粒 粒径(nm) 多分散性指数 ζ-电位(mV) 包封率(％) 载药量(％)
颗粒-c1 103±6 0.19±0.01 -20.3±0.8 83.3±0.4 34.5±0.7
颗粒-d1 105±2 0.15±0.01 -25.0±0.9 82.3±0.6 31.7±0.4
实施例3粒径、电位和形态表征
利用马尔文粒径仪测量了实施例2中颗粒样品的粒径、多分散性指数以及表面电位，利用透射电镜显微镜表征了各种颗粒的结构形貌。
图11显示了复合纳米粒(颗粒-a)、肠溶性颗粒(颗粒-b1，颗粒-b2，颗粒-b3)的透射电镜图。电镜图可以看出颗粒-a、颗粒-b1，颗粒-b2，颗粒-b3的尺寸与马尔文粒径仪测出的粒径结果是相一致的。
实施例4体外药物释放实验
将胰岛素溶液、颗粒-a、颗粒-b1、颗粒-b2、颗粒-b3分别移入特定1mL体积的透析管中。将其放在不同pH值(pH＝2.5、pH＝6.8、pH＝7.4)的释放介质中并置于37℃，120rpm的摇床中进行释放实验，每隔一定时间从释放介质中取出1mL释放介质溶液并加入等体积的新鲜介质溶液，通过BCA蛋白浓度检测法来检测释放介质的胰岛素浓度，从而计算得到胰岛素的累积释放含量百分比。
如图12所示，从胰岛素溶液或不同颗粒溶液在不同释放介质中的释放结果可看出，在pH＝2.5的胃部模拟环境中，胰岛素溶液在2小时内释放了约80％，颗粒-a释放了约40％的胰岛素，但是颗粒-b1、颗粒-b2、颗粒-b3仅有约14％的胰岛素释放出来，表明颗粒表面涂覆肠溶性Eudragit L100-55在胃部环境下可以减缓胰岛素释放，同时间接地避免了胰岛素被酸或酶降解；而在pH＝6.8或7.4的小肠模拟环境下24小时内，颗粒-b1、颗粒-b2、颗粒-b3可以释放约80％胰岛素。
实施例5颗粒的体外细胞毒性
通过MTT方法检测了胰岛素溶液、颗粒-a以及颗粒-b1对于E12和Caco-2细胞的安全性。1.0×10 4个/孔Caco-2或E12细胞培养于96孔板中，加入200μL培养基培养24h后，分别替换成200μL新鲜的培养基并含有不同浓度的胰岛素溶液、颗粒-a或颗粒-b1。继续培养24h后，加入一定量MTT溶液共孵育4h；最后将溶液移除，加入二甲基亚砜(DMSO)溶液进行溶解，并用酶标仪在570nm测定吸光度得到细胞的活力。
如图13所示，胰岛素溶液、颗粒-a或颗粒-b1不会影响E12和Caco-2细胞的增殖，表明其对E12、Caco-2细胞无毒性。
实施例6细胞穿透实验
利用Caco-2单细胞层模型模拟小肠上皮细胞研究了颗粒样品的细胞穿透情况。将Caco-2细胞培养于12孔板Transwell聚酯膜上，通过在37℃、5％CO 2的细胞培养箱中进行培养，同时每2天换一次培养基且测定其电阻值变化情况，一般培养周期为2周左右，当Caco-2细胞跨膜电阻值稳定且高于750Ω，可用于后续实验研究。实验前将上下层培养基换成Hank's平衡盐溶液(HBSS)或含有1％粘蛋白的HBSS。
(1)跨膜电阻(TEER)跟踪：200μL的HBSS含胰岛素溶液、颗粒-a、颗粒-b1、颗粒-b2、颗粒-b3分别与Caco-2单细胞层或覆盖1％粘蛋白的Caco-2单细胞层共同孵育2h，然后将样品溶液移除并清洗，继续孵育至24h，在预设时间内测定跨膜电阻(TEER)。
如图14所示，样品处理2小时内，对于胰岛素溶液组，跨膜电阻值基本保持不变；对于颗粒-a、颗粒-b1、颗粒-b2或颗粒-b3而言，细胞跨膜电阻值均有所下降，但是颗粒-a组的跨膜电阻值下降更为明显(下降至约40％)，主要是由于颗粒-a的表面带正电荷，这更有利于与细胞的相互作用且季铵化壳聚糖具有打开细胞间紧密连接的作用。
图15显示了不同样品处理表面覆盖了1％粘蛋白的Caco-2单层细胞的跨膜电阻变化情况，样品处理2小时内，胰岛素溶液组的跨膜电阻值仍保持不变；对于颗粒-a、颗粒-b1、颗粒-b2或颗粒-b3组的跨膜电阻值均有所下降，但是加入1％粘蛋白后这些颗粒组的跨膜电阻较之无粘蛋白加入时跨膜电阻值均有所上升，其中颗粒-a组跨膜电阻下降至约60％，颗粒-b1、颗粒-b2或颗粒-b3组的跨膜电阻值下降至约65％～80％。2小时后移除样品溶液，各组的跨膜电阻值在24小时内均得以恢复，表明Caco-2单细胞层间的紧密连接打开后是可以恢复的。
(2)表观渗透系数(Papp)测量：将异硫氰酸罗丹明(RITC)荧光标记的胰岛素溶液、颗粒-a、颗粒-b1、颗粒-b2或颗粒-b3分别与Caco-2单层细胞或表面覆盖1％粘蛋白的Caco-2单层细胞共孵育4h，在预设时间内从下室取出100μL体积并补充新溶液，同时将取出的溶液进行荧光强度的测量，并通过下述公式计算其Papp值
Figure PCTCN2018101177-appb-000001
其中Papp为表观渗透系数，dQ/dt为代表一定时间内从Transwell板上层渗 透至下层的量，C o为上层药物起始浓度，A为transewell板聚酯膜面积。
图16显示了在表面覆盖或不覆盖1％粘蛋白的Caco-2单层细胞中胰岛素溶液、颗粒-a、颗粒-b1、颗粒-b2或颗粒-b3穿过单层细胞的表观渗透系数情况。对于无粘蛋白加入的Caco2单层细胞，相对于胰岛素溶液或其他颗粒组而言，颗粒-a具有较高的表观渗透系数。然而Caco2单层细胞表面覆盖1％粘蛋白后所有组的表观渗透系数均有所下降，表明黏液层在一定程度上会影响药物的渗透效率。
(3)紧密连接打开与闭合：将颗粒-b1与Caco-2单层细胞孵育2小时后移除并清洗，然后继续培养至24小时，在0、2和24小时分别取样然后用4％多聚甲醛固定30min，并用PBS清洗3次；再用10ug/mL闭合蛋白抗体一抗孵育1h，并用PBS清洗3次；最后20ug/mLAF488荧光标记的二抗进行孵育1h，并用PBS清洗3次。
图17显示了不同时间段内Caco-2单层细胞的细胞间紧密连接变化情况。加入颗粒-b1之前(0小时图)，初始Caco-2单层细胞可看到明显、清晰的紧密连接环状结构；加入颗粒-b1与Caco-2细胞共孵育2h后(2小时图)，可看到紧密连接环状结构基本消失，表明紧密连接已打开；随后将颗粒-b1移除并继续培养10h后(12小时图)，可看到紧密连接环状结构又重新出现，表明该紧密连接打开之后是可恢复的。
实施例7口服降血糖效果
将体重200g的SD大鼠腹腔注射80mg/kg的链脲佐菌素，同时定期监测大鼠血糖情况，当血糖值稳定在16.6mmol/L可认为是I型糖尿病模型鼠。随后将模型鼠分为7组，每组6只，同时给予鼠禁食12h左右。第1组给予口服生理盐水，第2组组给予口服胰岛素溶液(80IU/kg),第3、4、5、6组分别给予口服颗粒-a、颗粒-b1、颗粒-b2或颗粒-b3(80IU/kg)，第7组给予皮下注射胰岛素溶液(5IU/kg)，每隔1h利用血糖仪及血糖试纸测试大鼠的血糖值变化情况。
图18显示了大鼠口服不同胰岛素制剂后的体内降血糖效果。从中可看出口服胰岛素溶液、口服水都未造成血糖值下降；皮下注射胰岛素溶液(5IU/kg)在2h时可使血糖值迅速下降至20％；而口服颗粒-a、颗粒-b1、颗粒-b2或颗粒-b3后大鼠的血糖出现平稳的下降趋势，同时可看出颗粒-b1、颗粒-b2或颗粒-b3较之颗粒-a的降血糖效果更明显，且小尺寸的颗粒-b1表现出最佳的降血糖效果。
实施例8药代动力学评价
I型糖尿病模型鼠禁食12h后，将大鼠分为3组，每组5只。第1组给予皮下注射胰岛素溶液(5IU/kg)；第2组给予口服胰岛素溶液(80IU/kg)；第3组给予口服颗粒-b1(80IU/kg)，每隔1h取血样并离心后通过ELISA试剂盒测试大鼠体内血清胰岛素含量。
图19所示是血清胰岛素浓度与时间的曲线图。口服胰岛素溶液组的血清胰岛素含量极低，相对于皮下注射胰岛素组，计算得到口服颗粒-b1的生物利用度约为11.6％。
实施例9体内生物安全性
首先将大鼠分为4组，第一组为正常鼠、第二组为糖尿病模型鼠、第三组为糖尿病模型鼠口服颗粒-a组、第四组为糖尿病模型鼠口服颗粒-b1组。
如图20所示，通过对比γ-谷氨酰转移酶(γ-GT)、谷丙转氨酶(ALT)、谷草转氨酶(AST)和碱性磷酸酶(ALP)四种反应肝毒性酶的活力评价了不同制剂的体内安全性情况。结果表明颗粒-a或颗粒-b1具有良好的生物安全性，对肝不明显毒性。

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  1. 一种负载胰岛素的肠溶性纳微颗粒，其特征在于，所述纳微颗粒由季铵化壳聚糖、胰岛素和三聚磷酸钠通过静电作用复合得到的纳米粒及涂覆在纳米粒表面的尤特奇组成。
  2. 根据权利要求1所述的纳微颗粒，其特征在于，所述季铵化壳聚糖的分子量为50kDa～200kDa。
  3. 根据权利要求1所述的纳微颗粒，其特征在于，所述尤特奇为尤特奇L100-55、尤特奇L100或尤特奇S100。
  4. 根据权利要求1所述的颗粒，其特征在于，所述肠溶性微纳颗粒粒径为50nm～2μm。
  5. 权利要求1～4任一项所述的肠溶性纳微颗粒在制备口服胰岛素制剂方面的应用。
  6. 一种负载胰岛素的肠溶性纳微颗粒的制备方法，其特征在于，包括如下步骤：
    S1.将季铵化壳聚糖溶液引入第1和2通道，将胰岛素和三聚磷酸钠混合溶液引入第3和4通道，各通道溶液同时到达涡流混合区域内进行快速混合，得到表面带正电荷的复合纳米粒；其中四个通道的流速控制为1mL/min～50mL/min；
    S2.将S1得到复合纳米粒溶液引入第1和2通道，尤特奇溶液引入第3和4通道，各通道溶液同时到达涡流混合区域内进行快速混合，从而得到表面涂覆尤特奇的肠溶性纳微颗粒，其中四个通道的流速控制为1mL/min～50mL/min。
  7. 根据权利要求6所述的制备方法，其特征在于，所述季铵化壳聚糖的季铵化程度为5％～30％；季铵化壳聚糖浓度为0.5～3mg/mL。
  8. 根据权利要求6所述的制备方法，其特征在于，所述胰岛素浓度为0.1～4mg/mL；三聚磷酸钠浓度为0.1～1mg/mL。
  9. 根据权利要求6所述的制备方法，其特征在于，所述尤特奇的浓度为0.1～2mg/mL。
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