WO2020088306A1 - 一种用于胰岛素口服递送的聚电解质复合物 - Google Patents

一种用于胰岛素口服递送的聚电解质复合物 Download PDF

Info

Publication number
WO2020088306A1
WO2020088306A1 PCT/CN2019/112522 CN2019112522W WO2020088306A1 WO 2020088306 A1 WO2020088306 A1 WO 2020088306A1 CN 2019112522 W CN2019112522 W CN 2019112522W WO 2020088306 A1 WO2020088306 A1 WO 2020088306A1
Authority
WO
WIPO (PCT)
Prior art keywords
insulin
nanoparticles
solution
mpeg
copolymer nanoparticles
Prior art date
Application number
PCT/CN2019/112522
Other languages
English (en)
French (fr)
Inventor
曾庆冰
陈婷婷
李顺英
朱雯婷
王俊
秦婷婷
Original Assignee
南方医科大学
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 南方医科大学 filed Critical 南方医科大学
Publication of WO2020088306A1 publication Critical patent/WO2020088306A1/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
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—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
    • 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
    • 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 present invention relates to a polyelectrolyte complex for oral administration of insulin and a preparation method thereof, and more specifically, to two types of insulin controlled-release nanoparticles with opposite charges, which are further cross-linked to each other to form Polyelectrolyte complex.
  • Diabetes is an endocrine disease caused by the pancreas can not secrete enough insulin, or the body can not effectively use insulin.
  • Insulin subcutaneous injection is currently the most commonly used and effective treatment, but due to its difficulty in maintaining glucose homeostasis, low compliance, and strong side effects in patients, it has been an attempt to develop a safe and effective non-invasive drug delivery route. Research hotspots.
  • Oral administration is the most ideal route of administration because it not only avoids the side effects of subcutaneous injection, but also greatly improves patient compliance.
  • macromolecules of insulin are difficult to pass through intestinal epithelial cells, they are degraded in the gastrointestinal tract before reaching the intestinal absorption site, so the oral bioavailability of insulin is extremely low.
  • Nanocarriers such as polymer nanoparticles and micelles can be used for oral administration of insulin. These nanocarriers can protect insulin from degradation and promote absorption during transmembrane and paracellular pathways. Therefore, insulin oral preparations should prolong the residence time of insulin in the intestine under the premise of protecting insulin from being destroyed before release to provide more opportunities for its absorption, and accelerate the release of insulin in the preparation at its main absorption site, thereby improving Insulin oral bioavailability.
  • the ideal polymer carrier should be biodegradable, biocompatible, and able to prolong the intestinal residence time under the action of resisting gastric pH gradient and enzymes.
  • Polyethylene glycol-polylactic acid-glycolic acid copolymer (mPEG-b-PLGA) is a block polymer formed by polymerizing PLGA and methoxy-terminated polyethylene glycol (mPEG).
  • the strong mPEG molecule makes mPEG-b-PLGA a hydrophilic amphiphilic molecule at one end and hydrophobic at the other end.
  • the prepared nanoparticles can avoid the rapid clearance of macrophages, thereby prolonging the residence time of the loaded drug in the systemic circulation and reducing the drug Released prematurely.
  • Chitosan is a natural cationic polysaccharide. Its side chain contains amino groups, and the amino groups are protonated under acidic conditions, making it a positively charged polymer with pH sensitivity. In the intestinal environment, the positively charged shell The glycan and the negatively charged sialic acid groups on mucin attract each other and have membrane adhesion.
  • Alginic acid is another water-soluble natural linear polysaccharide, which can shrink at a lower pH, so that the encapsulated drug can stay in the stomach, and at the same time protect the drug from the enzyme and inactivate it. Therefore, it is obtained in pH-responsive polymers. Has been widely used.
  • the present invention prepares a polyelectrolyte complex that can be used for oral delivery of insulin.
  • the invention relates to a polyelectrolyte complex formed by cross-linking insulin controlled release nanoparticles with opposite charges, which can be used for oral administration.
  • the components of the polyelectrolyte composite are non-toxic, biocompatible, biodegradable synthetic or natural polymer.
  • the present invention designs two kinds of nanoparticles with opposite charges, one of which has a positive charge on the surface, and the other has a negative charge on the surface, and its interaction can be cross-linked to form a polyelectrolyte for oral administration
  • the compound has pH responsiveness. It has a protective effect on the drug contained in the acidic environment of the stomach under the oral absorption route, and releases the drug slowly in a neutral environment of the intestine. It has a good sustained-release effect and relative bioavailability.
  • One aspect of the present invention relates to a polyelectrolyte composite made of a mixture of positively charged copolymer nanoparticles and negatively charged copolymer nanoparticles;
  • the copolymer nanoparticles are water-in-oil (W / O) copolymer nanoparticle colostrums prepared by using an amphiphilic block copolymer as the oil phase and an aqueous solution of the active drug as the internal water phase. ;
  • the external water phase of the positively charged copolymer nanoparticles is a solution of a cationic biopolymer or its modified biopolymer.
  • a water-in-oil-in-water (W / O / W) re-emulsion is prepared and The emulsion is prepared by dispersing in the emulsifier diluent;
  • the external water phase of the negatively charged copolymer nanoparticles is a solution of an anionic biopolymer or its modified biopolymer.
  • a water-in-oil-in-water (W / O / W) re-emulsion is prepared and The emulsion is prepared by dispersing in the emulsifier diluent.
  • the amphiphilic block polymer is selected from polyethylene glycol-polylactic acid-glycolic acid (mPEG-b-PLGA), polyethylene glycol-polylactic acid (PEG-b- PLA), polyethylene glycol-polyacetone (PEG-b-PCL), polyethylene glycol-polylactide-caprolactone (PEG-b-PLCL), preferably mPEG-b-PLGA
  • Oxygen-terminated polyethylene glycol (mPEG) is mPEG 5000 ; more preferably, the mPEG-b-PLGA is synthesized from D, L-lactide and glycolide by ring-opening polymerization method, which is invested by mPEG 5000 The quality score is 10%.
  • the outer aqueous phase of the positively charged copolymer nanoparticles is selected from a solution of a cationic biopolymer or its modified biopolymer, is a hydrogen bond acceptor, and is selected from chitosan , N-succinyl chitosan, carboxymethyl chitosan, polyethylene imine (PEI), polyallylamine (PAH), poly (L-lysine), poly (L-arginine), poly Vinylamine.
  • the outer aqueous phase of the negatively charged copolymer nanoparticles is selected from the solution of an anionic biopolymer or its modified biopolymer, is a hydrogen bond donor, and is selected from alginate , Polyglutamic acid, hyaluronic acid, pectin, glycosaminoglycans, polyaspartic acid, carboxymethyl cellulose, carboxymethyl dextran.
  • the emulsifier diluent is selected from different concentrations of poloxamer 188 (F68), polyvinyl alcohol (PVA), polyoxyethylene alkyl ether, sodium lauryl sulfate ( SDS) solution.
  • the active drug is selected from insulin, insulin analogs, diabetes drugs, protein drugs, and polypeptide drugs.
  • the above-mentioned positively charged copolymer nanoparticles or negatively charged copolymer nanoparticles are prepared by the double emulsion volatilization method; preferably, the following specific steps are included:
  • step (3) Slowly drop the active ingredient solution prepared in step (1) into the organic solution of the amphiphilic block copolymer prepared in step (2), ultrasonically emulsify to prepare a colostrum;
  • the preparation method of the polyelectrolyte composite is to mix positively charged copolymer nanoparticles or negatively charged copolymer nanoparticles, preferably, positively charged copolymer nanoparticles or The negatively charged copolymer nanoparticles are dispersed in an aqueous solution and mixed with positively charged copolymer nanoparticles or negatively charged copolymer nanoparticles.
  • Another aspect of the present invention provides a method for preparing a polyelectrolyte composite, which includes the following steps:
  • step i) is made by the following steps,
  • step (3) Slowly drop the active ingredient solution prepared in step (1) into the organic solution of the amphiphilic block copolymer prepared in step (2), ultrasonically emulsify to prepare a colostrum;
  • the colostrum emulsification condition is ultrasonic time 5 s, interval time 5 s, ultrasound time 1 min, working cycle time is 60%; double emulsion emulsification condition is ultrasonic time 10 s, interval time 10 s, total ultrasound time 2 min, The working cycle time is 60%.
  • the specific prescription process in the step is 1: 10: 40: 100, and the ratio of the organic solvent methylene chloride and acetone as the oil phase is 3: 2.
  • the preparation method of mPEG-b-PLGA nanoparticles coated with positively charged chitosan is that the external water phase is 8 ml of 0.5% chitosan acetic acid solution (1% acetic acid solution) and 12ml mixed solution of 2% polyvinyl alcohol; the diluted solution is 50ml 0.5% polyvinyl alcohol solution; the active ingredient contained is insulin, and the mass feeding ratio with the copolymer mPEG-PLGA carrier material is preferably 5-20%, more preferably 10%.
  • the external aqueous phase is 20 times the volume of 0.5% sodium alginate solution; the diluted phase is 50 times 2% by volume of poloxamer 188 solution; the active ingredient contained is insulin, and the mass feeding ratio with the copolymer mPEG-PLGA carrier material is preferably 5-20%, more preferably 15%.
  • the two oppositely charged nanoparticles are modified by adding the surface modifiers chitosan and sodium alginate as the organic phase by dissolving the copolymer mPEG-b-PLGA in an organic solvent,
  • the nanoparticles are prepared by the emulsion evaporation method. The specific steps are as follows: the prepared chitosan-coated mPEG-PLGA nanoparticle solution and the sodium alginate-coated mPEG-PLGA nanoparticle solution are mixed at a volume ratio of 1: 1 (w / w) under ultrasonic conditions, The ultrasonic time is 2min, and the result is the polyelectrolyte complex.
  • the polyelectrolyte complex is pH-responsive. In an acidic environment, the charge intensity on the surface of the nanoparticles is obvious, forming a tight network structure can protect the drug. In a neutral environment, the polyelectrolyte complex The interaction force of the compound is weakened, and it becomes a loose structure to facilitate drug release.
  • Another aspect of the present invention provides the use of the above polyelectrolyte complex in the preparation of a pharmaceutical carrier.
  • Another aspect of the present invention relates to the use of the aforementioned polyelectrolyte complex in preparing an oral preparation for treating metabolic diseases.
  • the metabolic disease is selected from diabetes and hyperglycemia, more preferably type I diabetes or type II diabetes, and the drug is insulin or insulin analog.
  • the formulation is administered to maintain normal blood glucose levels.
  • the oral administration system of the present invention by adding a hydrophilic segment, the synthetic mPEG-PLGA polymer has improved hydrophilicity, and as a material matrix for preparing nanoparticles, it is better to contain water soluble Sexual drugs (such as insulin and protein drugs).
  • the oral administration system of the present invention modifies drug-loaded sustained-release polymer nanoparticles by adding surface modifiers, which are chitosan with positively charged amino groups and sodium alginate with negatively charged carboxyl groups, which intersect Combined to form a polyelectrolyte complex to increase the total drug loading.
  • surface modifiers which are chitosan with positively charged amino groups and sodium alginate with negatively charged carboxyl groups, which intersect Combined to form a polyelectrolyte complex to increase the total drug loading.
  • the intensity of the positive and negative charges on the surface of the nanoparticles changes with the pH environment. It has pH sensitivity, which can make the drug reach the intestinal absorption site smoothly through the oral absorption path. It is an ideal oral preparation with slow and controlled release.
  • Figure 1 depicts the preparation of insulin-coated chitosan-coated mPEG-PLGA nanoparticles and sodium alginate-coated mPEG-PLGA nanoparticles by the double emulsion method. After mixing the two nanoparticles, the insulin-loaded polyelectrolyte composite The formation of things.
  • Figure 2 is a schematic diagram of the synthesis of the diblock copolymer mPEG-PLGA.
  • Figure 3 is the FT-IR spectrum of the diblock copolymer mPEG-PLGA.
  • Fig. 4 is a 1 H-NMR spectrum of the diblock copolymer mPEG-PLGA.
  • Fig. 5 is a graph showing the particle size distribution of insulin-coated chitosan-coated mPEG-PLGA nanoparticles (CSNPs) and sodium alginate-coated mPEG-PLGA nanoparticles (ALNPs).
  • CSNPs insulin-coated chitosan-coated mPEG-PLGA nanoparticles
  • ANPs sodium alginate-coated mPEG-PLGA nanoparticles
  • CSNPs insulin-coated chitosan-coated mPEG-PLGA nanoparticles
  • ANPs sodium alginate-coated mPEG-PLGA nanoparticles
  • 7A is a scanning transmission electron microscope image of insulin-loaded chitosan-coated mPEG-PLGA nanoparticles.
  • 7B is a scanning transmission electron microscope image of insulin-loaded sodium alginate-coated mPEG-PLGA nanoparticles.
  • FIG. 8A is a scanning transmission electron micrograph of insulin-loaded polyelectrolyte complex at pH 1.2.
  • FIG. 8B is a scanning transmission electron micrograph of insulin-loaded polyelectrolyte complex at pH 6.8.
  • FIG. 8C is a scanning transmission electron micrograph of insulin-loaded polyelectrolyte complex at pH 7.4.
  • Fig. 9 is a graph showing the change in cell viability (percentage) with concentration (mg / mL) of mPEG-PLGA nanoparticles coated with chitosan or sodium alginate after 48 hours of culture with Caco-2 cells.
  • FIG. 10 is a graph showing the change in cell viability (percentage) of chitosan or sodium alginate-coated blank mPEG-PLGA nanoparticles with concentration (mg / mL) after 72 hours of culture with Caco-2 cells.
  • FIG. 11 is an in vitro cumulative release curve of insulin-loaded polyelectrolyte complexes under different pH 1.2 and pH 6.8 solution media.
  • Figure 12 is a circular dichroism diagram of insulin and standard insulin solution released from the polyelectrolyte complex.
  • the synthesized product has a strong absorption peak at 1760 cm -1 , which is a typical carbonyl stretching vibration peak, indicating that the ring-opening polymerization of caprolactone and propiolactone forms a linear ester bond.
  • the CH stretching vibration peak in the PLGA segment overlaps with the CH stretching vibration peak in the mPEG in the region from 2900cm -1 to 3000cm -1 , and the bending vibration peak in the PEG segment also exists, indicating that the mPEG has formed a double embedding with the PLGA Segment copolymer.
  • TMS tetramethyl silicon
  • mPEG-PLGA polyethylene glycol-polylactic acid-glycolic acid copolymer
  • CSNPs Chitosan-coated insulin-carrying mPEG-PLGA nanoparticles
  • ALNPs insulin alginate mPEG-PLGA nanoparticles coated with sodium alginate
  • Two kinds of insulin nanoparticles with opposite charges were prepared by double emulsion method (water-in-oil-in-water). Precisely weigh a certain amount of porcine insulin raw material, dissolve it in 0.1M HCl solution, and configure it as an insulin solution. Take 500 ⁇ l of insulin solution as the internal water phase and add it to 5 ml of oil phase (dichloromethane / acetone (3: 2) solution containing polymer). The ultrasonic probe is emulsified. The total ultrasonic time is 1 min (working cycle time is 60%). colostrum.
  • the colostrum was transferred to the external aqueous phase of 20 ml of chitosan / PVA solution, and the ultrasonic probe was emulsified for 2 min (working cycle time was 60%).
  • the obtained double milk was slowly added dropwise to the diluted phase of 50 ml of 0.5% PVA solution, stirred and evaporated at room temperature to eliminate the organic solvent. After stirring overnight, it was centrifuged at 12000 ⁇ g at 4 ° C for 30 min, resuspended in distilled water and centrifuged to precipitate, and washed three times. The resulting positively charged chitosan coated nanoparticles.
  • the sodium alginate solution was used as the external aqueous phase, and the 2% poloxamer 188 solution was used as the diluted phase.
  • the other preparation conditions were the same, that is, the negatively charged sodium alginate coated nanoparticles were obtained. Freeze-dried to obtain two kinds of insulin-loaded nanoparticles with opposite charges.
  • Example 3-2 Particle size and potential characterization of drug-loaded nanoparticles
  • the two kinds of nanoparticles prepared were characterized by dynamic light scattering laser particle size and Zeta potential analyzer.
  • the nanoparticle solution to be tested was diluted with deionized water to make them evenly dispersed, and the particle size and dispersion coefficient (PDI) were measured. And Zeta potential.
  • the results are shown in Table 1.
  • Example 3-3 Determination of encapsulation rate and drug loading rate of drug-loaded nanoparticles
  • Encapsulation rate (EE%) (total quality of drug administration-quality of supernatant drug) / total quality of drug administration ⁇ 100%
  • Drug loading rate (DL%) (total mass of drug administered-mass of supernatant drug) / total mass of nano-formulation ⁇ 100%
  • Protein-like peptide substances such as insulin are prone to change in the secondary structure and lead to inactivation under acidic environment.
  • the organic solution of mPEG-PLGA is selected as the oil phase, and the insulin solution as the internal water phase.
  • the Zeta value of the nanoparticles was measured. The results are shown in Figure 6. Under acidic conditions, the amino group in the chitosan molecule is protonated with a positive charge, so 1% acetic acid is used as a solvent to dissolve the chitosan. However, due to the poor water solubility of chitosan, the pH of the solution increased after a large volume of dilution phase, which reduced the solubility of chitosan and weakened the protonation strength of amino groups. Therefore, the final measured Zeta potential value of chitosan nanoparticles The absolute value is lower than the Zeta value of sodium alginate nanoparticles. Sodium alginate has strong hydrophilicity, high solubility in aqueous solution, and high encapsulation rate of hydrophilic insulin; the measured Zeta value is high, the charge repulsion between particles is large, and the system is stable.
  • Dynamic light scattering (DSL) results show that the particle size of insulin-loaded nanoparticles coated with chitosan is (227.9 ⁇ 0.7) nm, Zeta potential is (+ 12.55 ⁇ 1.35) mV, and the encapsulation rate is (33.27 ⁇ 6.84) wt%, drug loading rate is (3.18 ⁇ 0.60) wt%.
  • the particle size of insulin-loaded nanoparticles coated with sodium alginate is (271.8 ⁇ 3.5) nm, the Zeta potential is (-54.27 ⁇ 2.75) mV, the encapsulation rate is (75.91 ⁇ 5.11) wt%, and the drug loading rate is ( 9.77 ⁇ 0.71) wt%.
  • the two nanoparticles have a particle size ranging from 200 to 300 nm, and both have good dispersion coefficients and good dispersibility.
  • TEM Transmission electron microscopy
  • TEM observed the morphological changes of the polyelectrolyte complex under different pH environments.
  • pH1.2, pH6.8 and pH7.4 simulate the acidic environment of gastric juice, intestinal juice environment and physiological environment respectively.
  • the two nanoparticles cross-link with each other to form a flocculent and network structure, which is relatively tightly agglomerated, and the shape of the nanoparticles is still spherical or elliptical. This is because the pKa value of chitosan is about 5.6.
  • the structure-like polyelectrolyte complex has a certain protective effect on the encapsulated insulin, avoids the release of drugs in the stomach, and reduces the destruction of the structure of insulin by gastric acid and pepsin.
  • the pH value of the solution medium is greater than the pKa value of chitosan, the electrostatic interaction between chitosan and sodium alginate is greatly reduced, and the redispersion of polyelectrolyte complex is observed under TEM It is a single nanoparticle, and the shape of the nanoparticle collapsed.
  • simulating oral administration of insulin-loaded polyelectrolyte complex it first passes through the stomach and forms a tight network structure under the acidic pH gastric juice environment, which can protect the drug, and then enters the intestine, and the interaction force of the polyelectrolyte complex weakens.
  • insulin can be released from a single nanoparticle, allowing the drug to absorb in the intestine and play a role in lowering blood sugar.
  • the mixed two nanoparticles are ellipsoidal or spherical in shape and uniformly dispersed.
  • the process of simulating insulin traversing the gastrointestinal tract needs to undergo a change from a strong acid (intragastric pH 1.2 to 3.0) to a weak alkaline environment (intestinal pH 6.5 to 8.0).
  • the simulated gastric juice environment and intestinal juice environment were selected, that is, pH 1.2 and pH 6.8 solution media were used for in vitro release test.
  • Circular dichroism spectroscopy was used to determine the secondary structure of the insulin released in the polyelectrolyte complex, and the stability of the released insulin was investigated. Dilute the collected solution after the in vitro release experiment to a certain concentration for circular dichroism determination. At the same time, dissolve the porcine insulin drug substance in 0.1 mol / L HCl and configure it as a 100 ⁇ g / mL porcine insulin solution. Conformation determination was used as a control.
  • the detection conditions are: scanning range 190 ⁇ 250nm, precision 0.2nm, bandwidth 1.0nm, scanning speed 100nm / min, response time 0.25 seconds, sample cell 0.1cm, room temperature 25 ° C, repeated measurement 3 times in parallel.
  • Porcine insulin has two negative peaks at 208nm and 223nm in the far ultraviolet region, which are characteristic peaks of ⁇ -helix and ⁇ -sheet, respectively.
  • the intensity ratio of the negative peaks at 208nm and 223nm ([ ⁇ ] 208 / [ ⁇ ] 223) quantitatively reflects the overall structure of insulin.
  • the [ ⁇ ] 208 / [ ⁇ ] 223 values of porcine insulin and released insulin are 1.29 and 1.33, respectively. It can fully show that the secondary conformation of released insulin is not significantly different from that of porcine insulin. In the process of drug loading and release of polyelectrolyte complex, insulin maintains its conformational stability.
  • Group A is a positive control group: subcutaneous injection of insulin solution (5IU / kg), the other three groups are all dosing experimental groups: group B orally administered AL NPs (60IU / kg); group C orally administered CS NPs ( 60IU / kg); Group D was administered orally with insulin-containing polyelectrolyte complex (60IU / kg). After administration, blood glucose was measured and blood samples were collected at each set time point.
  • Diabetic rats were injected subcutaneously with insulin solution as a positive control group.
  • the insulin-containing CS, NPs, insulin-containing NPs, and insulin-containing PEC were orally administered as the experimental group to evaluate the hypoglycemic effect of oral PEC in vivo.
  • hypoglycemic effect of oral insulin-loaded PEC also appears at the 4h time point, but the hypoglycemic effect is more obvious than that of ALNPs, and the steady-state decline continues from 4-8h.
  • the optimal hypoglycemic effect reaches the initial level of 28.29 at 8h %, Within the fasting normal blood glucose value range of SD rats, and then keep the overall blood sugar level fluctuate not much, it is still 35.75% of the initial blood glucose value at 12h, and has a good sustained release and hypoglycemic effect.
  • Insulin-containing CS NPs group had a small peak at 1h, and then decreased, and the plasma insulin concentration was not high, the fluctuation range was not large; insulin-containing AL NPs group also had a small peak at 1h, 2h-8h At this time, the blood drug concentration slowly increased, and after 8 hours reached the second small peak, it gradually decreased and returned to the pre-dose level, but the plasma insulin concentration detected after the overall drug administration was higher than that of the CSNPs group.
  • the first small peak after administration in the PEC group appeared at 2h.
  • CS NPs and AL NPs are administered alone as a carrier, and the hypoglycemic effect is not ideal.
  • the drug loading of CS NPs is low. Although it has bioadhesion, it can prolong the action time of the drug on the small intestinal mucosa, but it is used as a carrier alone During administration, when passing through the stomach, the acidic environment and enzymes will affect the stability of the drug and the nanoparticle carrier, so that only a small amount of insulin can reach the intestinal absorption site and exert the drug effect.
  • the drug loading of ALNPs can reach more than 70%, so after oral administration as a carrier alone, its hypoglycemic effect and duration are better than CSNPs, which is consistent with the experimental results.
  • sodium alginate does not have the functions of chitosan bioadhesion and opening the tight junction between small intestinal epithelial cells. Therefore, in the oral absorption route, the absorption of insulin at the intestinal site is limited.
  • the polyelectrolyte complex synergizes the advantages of the two types of nanoparticles.
  • the PEC In the acidic environment of the stomach, the PEC forms a tight network cross-linked structure that has a protective effect and provides a physical protective layer for insulin to protect it from damage by strong acids and enzymes before release. In the neutral environment of intestinal juice, it can slowly and steadily release insulin, and has a slow-release effect of lowering blood sugar. Therefore, PEC is used as a carrier for administration, synergistically with the high drug loading of AL NPs and the advantages of CS NPs in the intestinal absorption enhancer and intestinal mucosal adhesive agent. It has a good hypoglycemic effect, slow release effect and safety. Ideal drug oral carrier.

Landscapes

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

Abstract

一种用于胰岛素口服递送的复合载体，其由两嵌段共聚物MePEG-PLGA为基质，分别制备成带有正带电荷和带有负电荷的两种纳米粒，混合后相互交联形成聚电解质复合物。该胰岛素聚电解质复合物口服给药后，能避免药物通过胃时胃酸及酶等环境对胰岛素活性的破坏，使药物顺利到达肠道吸收部位，具有pH敏感性。这一口服制剂具有良好的降血糖效果及缓控释作用，可以用于胰岛素和蛋白类的药物的口服递送。

Description

一种用于胰岛素口服递送的聚电解质复合物 技术领域
本发明涉及一种用于胰岛素口服给药的聚电解质复合物和其制备方法，并且更具体地，涉及两种带有相反电荷的胰岛素缓控释纳米粒，所述纳米粒进一步相互交联形成聚电解质复合物。
背景技术
糖尿病是一种由于胰腺不能分泌足够的胰岛素，或者机体不能有效地利用胰岛素所导致的内分泌疾病。胰岛素皮下注射是目前最常用和有效的治疗方法，但由于其难在病人体内保持葡萄糖的稳态、顺应性低及副作用强等问题，因此尝试开发一种安全有效的无创给药途径是一直以来的研究热点。口服给药是最为理想的给药途径，因为其不仅避免了皮下注射带来的副作用，而且大大提高了病人的顺应性。但由于胰岛素大分子很难通过肠道上皮细胞，其在到达肠道吸收部位前就在胃肠道中被降解，因此胰岛素的口服生物利用度极低。目前胰岛素口服制剂主要的屏障在于三点：酶屏障，胃肠道生理屏障和胰岛素自身的生化性质。纳米载体如聚合物纳米粒和胶束可以用于胰岛素的口服,这些纳米载体可以保护胰岛素免于降解，并在在跨膜和旁细胞途径转运时促进吸收。因此，胰岛素口服制剂应在保护胰岛素在释放前不被破坏的前提下，延长胰岛素在肠道内的滞留时间从而为其吸收提供更多机会，加快制剂中的胰岛素在其主要吸收部位释放，从而提高胰岛素口服生物利用度。
理想的聚合物载体应该是可生物降解的，生物相容性的，能够在抵抗胃pH梯度和酶的作用下延长肠道停留时间。聚乙二醇-聚乳酸-羟基乙酸共聚物(mPEG-b-PLGA)是由PLGA与甲氧基封端的聚乙二醇(mPEG)聚合而成的嵌段聚合物,由于加入了亲水性强的mPEG分子,使mPEG-b-PLGA成为一端亲水另一端疏水的两性分子，所制备的纳米粒能够避免巨噬细胞的快速清除，从而延长所载药物在体循环中的滞留时间，减少药物过早释放。壳聚糖为天然的阳离子多糖，其侧链含有氨基，酸性条件下氨基质子化，使其成为一种正电性的聚合物， 具有pH敏感性，在肠道环境中，带正电荷的壳聚糖与粘蛋白上带负电荷的唾液酸基团相互吸引，具有膜粘附性。海藻酸是另一种水溶性的天然线性多糖，能在较低的pH收缩，使包封的药物滞留在胃，同时保护药物免受酶的作用而失活，因此pH响应性聚合物中得到了广泛的应用。
因此，本发明基于两种带相反电荷的纳米粒的静电力相互作用，制备了可以用于胰岛素口服递送的聚电解质复合物。
发明内容
本发明涉及一种由带相反电荷的胰岛素缓控释纳米粒相互交联形成的，可用于口服给药的聚电解质复合物。该聚电解质复合物的组份均为无毒性、生物相容性、生物可降解性的合成或天然高分子聚合物。
本发明设计两种带有相反电荷的纳米粒，其中一种纳米粒的表面具有正电荷，另一种纳米粒的表面具有相反的负电荷，其相互作用可以交联形成可用于口服的聚电解质复合物，具有pH响应性，在口服吸收路径下，在胃酸性环境下对所载药物具有保护作用，在肠中性环境下缓慢释放药物，具有良好的缓释作用和相对生物利用度。
本发明一个方面涉及一种聚电解质复合物，由带正电荷的共聚物纳米粒和带负电荷的共聚物纳米粒混合制成；
其中，所述的共聚物纳米粒为以两亲性嵌段共聚物作为油相，以活性药物的水溶液为内水相，制备得到的油包水(W/O)的共聚物纳米粒初乳剂；
带正电荷的共聚物纳米粒的外水相为阳离子生物聚合物或其修饰后生物聚合物的溶液，制备得到水包油包水(W/O/W)的复乳剂，并通过将该复乳剂分散在乳化剂稀释液中制得；
带负电荷的共聚物纳米粒的外水相为阴离子生物聚合物或其修饰后生物聚合物的溶液，制备得到水包油包水(W/O/W)的复乳剂，并通过将该复乳剂分散在乳化剂稀释液中制得。
在本发明的技术方案中，所述的两亲性嵌段聚合物选自聚乙二醇-聚乳酸-羟基乙酸(mPEG-b-PLGA)、聚乙二醇-聚乳酸(PEG-b-PLA)、聚乙二醇-聚乙酸内酯(PEG-b-PCL)、聚乙二醇-聚丙交酯-己内酯(PEG-b-PLCL)，优选地，mPEG-b-PLGA中甲氧基封端的聚乙二醇(mPEG)为mPEG 5000；更优选地，所 述mPEG-b-PLGA由D,L-丙交酯和乙交酯通过开环聚合法合成，mPEG 5000所投入的质量分数为10％。
在本发明的技术方案中，所述的带正电荷的共聚物纳米粒的外水相选自阳离子生物聚合物或其修饰后生物聚合物的溶液，为氢键受体，选自壳聚糖、N-琥珀酰壳聚糖、羧甲基壳聚糖、聚乙烯亚胺(PEI)、聚烯丙胺(PAH)、聚(L-赖氨酸)、聚(L-精氨酸)、聚乙烯胺。
在本发明的技术方案中，所述的带负电荷的共聚物纳米粒的外水相选自阴离子生物聚合物或其修饰后生物聚合物的溶液，为氢键供体，选自海藻酸盐、聚谷氨酸、透明质酸、果胶、糖胺聚糖、聚天冬氨酸、羧甲基纤维素、羧甲基葡聚糖。
在本发明的技术方案中，所述的乳化剂稀释液选自不同浓度的泊洛沙姆188(F68)、聚乙烯醇(PVA)、聚氧乙烯烷基醚、十二烷基硫酸钠(SDS)的溶液。
在本发明的技术方案中，所述活性药物选自胰岛素、胰岛素类似物、糖尿病药物、蛋白质类药物、多肽类药物。
在本发明的技术方案中，上述带正电荷的共聚物纳米粒或带负电荷的共聚物纳米粒通过双乳剂挥发法制备而成；优选地，包括如下具体步骤：
(1)制备活性成分溶液，作为内水相；
(2)将所述的两亲性嵌段共聚物溶解于有机溶剂中，作为油相；
(3)将步骤(1)制得的活性成分溶液缓慢滴加入步骤(2)制得的两亲性嵌段共聚物有机溶液中，超声乳化，制备初乳剂；
(4)将初乳缓慢滴加入相应的外水相中，超声乳化，制备复乳剂；
(5)制备相应的乳化剂稀释液，将复乳剂和稀释液混合，去除有机溶剂，离心并收集沉淀，即得到载药共聚物纳米粒。
在本发明的技术方案中，上述聚电解质复合物的制备方法为将带正电荷的共聚物纳米粒或带负电荷的共聚物纳米粒混合，优选地，将带正电荷的共聚物纳米粒或带负电荷的共聚物纳米粒在水溶液中分散，并以带正电荷的共聚物纳米粒或带负电荷的共聚物纳米粒等质量混合。
本发明另一个方面提供了聚电解质复合物的制备方法，其包括如下步骤：
i)制备带正电荷的共聚物纳米粒或带负电荷的共聚物纳米粒；
ii)将带正电荷的共聚物纳米粒或带负电荷的共聚物纳米粒混合，得到聚电解质复合物。
其中，所述步骤i)通过以下步骤制成，
(1)制备活性成分溶液，作为内水相；
(2)将所述的两亲性嵌段共聚物溶解于有机溶剂中，作为油相；
(3)将步骤(1)制得的活性成分溶液缓慢滴加入步骤(2)制得的两亲性嵌段共聚物有机溶液中，超声乳化，制备初乳剂；
(4)将初乳缓慢滴加入相应的外水相中，超声乳化，制备复乳剂；
(5)制备相应的稀释液，将复乳剂和稀释液混合，去除有机溶剂，离心并收集沉淀，即得到载药共聚物纳米粒。
进一步地，所述步骤中：初乳乳化条件为超声时间5s，间隔时间5s，超声时间1min，工作循环时间为60％；复乳乳化条件为超声时间10s，间隔时间10s，总超声时间2min，工作循环时间为60％。
进一步地，所述步骤中的具体处方工艺：内水相、油相、外水相、稀释液的体积比为1:10:40:100，作为油相的有机溶剂二氯甲烷与丙酮比例为3:2。
在本发明的技术方案中，带正电荷的壳聚糖覆膜的mPEG-b-PLGA纳米粒的制备方法为，其外水相为8ml 0.5％壳聚糖醋酸溶液(1％醋酸溶液)与12ml 2％聚乙烯醇的混合溶液；稀释液为50ml 0.5％聚乙烯醇溶液；所载活性成分为胰岛素，且与共聚物mPEG-PLGA载体材料的质量投料比优选为5-20％，更优选为10％。
在本发明的技术方案中，带负电荷的海藻酸钠覆膜的mPEG-b-PLGA纳米粒的制备方法，其外水相为20倍体积的0.5％海藻酸钠溶液；稀释相为50倍体积的2％泊洛沙姆188溶液；所载活性成分为胰岛素，且与共聚物mPEG-PLGA载体材料的质量投料比优选为5-20％，更优选为15％。
进一步地，两种带相反电荷的纳米粒是通过将所述共聚物mPEG-b-PLGA溶解在有机溶剂中，作为有机相，通过加入表面改性剂壳聚糖和海藻酸钠修饰，通过双乳剂挥发法制备成纳米粒。具体步骤为：将制得的壳聚糖覆膜的mPEG-PLGA纳米粒溶液与海藻酸钠覆膜的mPEG-PLGA纳米粒溶液，超声条件下按1:1(w/w)体积比混合，超声时间为2min，所得即为聚电解质复合物。
在一些实施例中，所述聚电解质复合物具有pH响应性，酸性环境下，纳米粒表面的电荷强度明显，形成紧密的网状结构可以对药物起保护作用，在中性环境下，聚电解质复合物相互作用力减弱，变为疏松结构利于药物释放。
本发明另一个方面提供了上述聚电解质复合物在制备药物载体中的用途。
本发明另一个方面涉及前述聚电解质复合物在制备治疗代谢性疾病的口服制剂中的应用。优选地，所述代谢性疾病选自糖尿病、高糖血症，更优选为I型糖尿病或II型糖尿病，所述的药物是胰岛素或胰岛素类似物。在一些实施例中，投与所述制剂以维持正常血糖水平。
本发明所述的口服给药体系，通过加入了亲水性链段，合成的mPEG-PLGA高分子聚合物的提高了亲水性，以此作为材料基质制备纳米粒，更好的包载水溶性药物(如胰岛素和蛋白类药物)。
本发明所述的口服给药体系，通过加入表面改性剂修饰载药缓释聚合物纳米粒，分别是带有正电荷氨基的壳聚糖和带有负电荷羧基的海藻酸钠，相互交联形成聚电解质复合物，提高总载药量。纳米粒表面正负电荷的强度随pH环境变化而变化，具有pH敏感性，可以使药物顺利通过口服吸收路径到达肠道吸收部位，为一种理想的具有缓控释作用的口服制剂。
附图说明
图1描绘了通过双乳剂法分别制备了包载了胰岛素的壳聚糖覆膜的mPEG-PLGA纳米粒和海藻酸钠覆膜mPEG-PLGA纳米粒，两种纳米粒混合后载胰岛素聚电解质复合物的形成。
图2为二嵌段共聚物mPEG-PLGA的合成示意图。
图3为二嵌段共聚物mPEG-PLGA的FT-IR谱图。
图4为二嵌段共聚物mPEG-PLGA的 1H-NMR谱图。
图5为包载了胰岛素的壳聚糖覆膜的mPEG-PLGA纳米粒(CS NPs)和海藻酸钠覆膜mPEG-PLGA纳米粒(AL NPs)的粒径分布图。
图6为包载了胰岛素的壳聚糖覆膜的mPEG-PLGA纳米粒(CS NPs)和海藻酸钠覆膜mPEG-PLGA纳米粒(AL NPs)的Zeta电位图。
图7A为载胰岛素的壳聚糖覆膜的mPEG-PLGA纳米粒的扫描透射电镜图。
图7B为载胰岛素的海藻酸钠覆膜的mPEG-PLGA纳米粒的扫描透射电镜图。
图8A为pH1.2下，载胰岛素的聚电解质复合物的扫描透射电镜图。
图8B为pH6.8下，载胰岛素的聚电解质复合物的扫描透射电镜图。
图8C为pH7.4下，载胰岛素的聚电解质复合物的扫描透射电镜图。
图9是在用Caco-2细胞培养48小时后壳聚糖或海藻酸钠覆膜空白 mPEG-PLGA纳米粒的细胞活力(百分比)随浓度(mg/mL)变化的图。
图10是在用Caco-2细胞培养72小时后壳聚糖或海藻酸钠覆膜空白mPEG-PLGA纳米粒的细胞活力(百分比)随浓度(mg/mL)变化的图。
图11为载胰岛素聚电解质复合物在不同pH1.2和pH6.8溶液介质下的体外累积释放曲线图。
图12为从聚电解质复合物中释放的胰岛素和标准胰岛素溶液的圆二色谱图。
图13为皮下注射纯胰岛素溶液、口服载胰岛素壳聚糖覆膜mPEG-PLGA纳米粒(CS NPs)、口服载胰岛素海藻酸钠覆膜mPEG-PLGA纳米粒(AL NPs)和口服载胰岛素聚电解质复合物(PEC)后，在STZ诱发的SD糖尿病大鼠中的降血糖效果图。数据点表示平均值±SD(n＝5)。
图14为皮下注射纯胰岛素溶液、口服载胰岛素壳聚糖覆膜mPEG-PLGA纳米粒(CS NPs)、口服载胰岛素海藻酸钠覆膜mPEG-PLGA纳米粒(AL NPs)和口服载胰岛素聚电解质复合物(PEC)后，在STZ诱发的SD糖尿病大鼠血浆中的猪胰岛素浓度。数据点表示平均值±SD(n＝3)。
具体实施方式
实施例1 载体材料二嵌段聚合物10％mPEG 5000-PLGA的合成
采用开环聚合法合成。精密称量mPEG 5000(10％质量分数)于圆底烧瓶中作为引发剂，随后加入按50:50投料比加入D,L-丙交酯和乙交酯，加入0.1％单体量的辛酸亚锡作为催化剂，三次抽真空，于120℃下氮气环境油浴反应24h。移出油浴锅停止反应。用适量二氯甲烷溶解反应物，在过量冷甲醇溶液中沉淀，重复三次，40℃下真空干燥至恒重。反应式如图2所示。
实施例2 合成产物的表征
实施例2-1 mPEG-PLGA红外光谱表征分析
取适量的待测产物与干燥的溴化钾混合，于室温下研磨成粉末，取适量粉末样品压片，在350～7800cm -1波数扫描，测定红外吸收光谱。
如下图3所示，合成产物在1760cm -1处有强吸收峰，为典型的羰基伸缩振动峰，表明己内酯和丙内酯发生开环聚合反应形成了直链酯键。在2900cm -1～3000cm -1区域中出现PLGA链段中CH伸缩振动峰与mPEG中的CH伸缩振动峰发生重叠，而PEG链段中的弯曲振动峰同时存在，表明mPEG已与PLGA形成二嵌段 共聚物。在3500cm -1处有宽吸收峰，为mPEG和PLGA中的羟基的伸缩振动峰，红外图谱分析结果与聚合物预计结构一致，表明合成物为所需二嵌段共聚物mPEG-PLGA。
实施例2-2 mPEG-PLGA  1H-NMR表征分析
氘代氯仿(CDCl 3)为溶剂，四甲基硅(TMS)为内标，25℃下测定待测样品的核磁氢谱图。依据谱图中相关峰的位移位置及面积，确定聚合物的结构组成。
如下图4所示，图中δ＝5.24和δ＝4.85的吸收峰分别为PLGA链段中乙交酯LA的次甲基质子峰(–O–CH*(CH 3)–CO–)和丙交酯GA的亚甲基质子峰(–O–CH 2*–CO–)；δ＝3.66处出现较强的吸收峰，此为mPEG 5000中的重复单元的亚甲基质子特征峰(–O–CH 2*–CH 2*–)；δ＝1.60处的吸收峰为乙交酯LA中的重复单元的甲基质子峰(–CH 3*)。由此可见聚乙二醇-聚乳酸-羟基乙酸共聚物(mPEG-PLGA)已成功合成。
实施例3 载药聚合物纳米粒的制备及性能表征
实施例3-1 纳米粒的制备
CS NPs:壳聚糖覆膜的载胰岛素mPEG-PLGA纳米粒
AL NPs:海藻酸钠覆膜的载胰岛素mPEG-PLGA纳米粒
采用双乳剂法(水包油包水)制备两种带有相反电荷的胰岛素纳米粒。精密称取一定量的猪胰岛素原料药，溶解于0.1M HCl溶液中，配置成胰岛素溶液。取500μl胰岛素溶液作为内水相加入到5ml油相(含聚合物的二氯甲烷/丙酮(3:2)溶液)中,超声探头乳化，总超声时间1min(工作循环时间为60％),得到初乳。随后将初乳转移到20ml壳聚糖/PVA溶液的外水相中，超声探头乳化2min(工作循环时间为60％)。将所得到的复乳缓慢滴加到50ml 0.5％PVA溶液的稀释相中，室温下搅拌蒸发消除有机溶剂。搅拌一晚后，12000×g，4℃下离心30min，再悬浮在蒸馏水中离心沉淀，重复三次洗净，所得即带正电荷的壳聚糖覆膜纳米粒。
以海藻酸钠溶液作为外水相，2％泊洛沙姆188溶液作为稀释相，其他制备条件相同，即得到带负电荷的海藻酸钠覆膜纳米粒。冷冻干燥，得到两种带相反电荷的载胰岛素纳米粒。
实施例3-2 载药纳米粒的粒径及电位表征
采用动态光散射激光粒度和Zeta电位分析仪对制备所得的两种纳米粒进行表征，分别取待测纳米粒溶液用去离子水稀释使其均匀分散，测定其粒径大小、分 散系数(PDI)及Zeta电位。结果见表1。
实施例3-3 载药纳米粒的包封率及载药率的测定
分别制备所得载胰岛素聚合物纳米粒溶液在4℃，12000×g条件下离心30min后收集上清液，用去离子水洗涤沉淀2～3次，将洗涤液合并至上清液记录体积，摇匀混悬后随机取3次2ml溶液到2.5ml离心管中，10000×g离心8min后取上清液为待测样品溶液，采用BCA法测定上清中胰岛素含量，再根据下式计算载胰岛素聚合物纳米粒的包封率和载药量，结果见表1。
包封率(EE％)＝(投药总质量－上清药物的质量)/投药总质量×100％
载药率(DL％)＝(投药总质量－上清药物的质量)/纳米制剂总质量×100％
表1 CS NPs和AL NPs的表征
Figure PCTCN2019112522-appb-000001
Note:Data are shown as Mean±SD(n≥5)
为了获得两种带有相反电荷的纳米粒，分别加入两种带有相反电荷的天然高分子聚合物壳聚糖和海藻酸钠，在mPEG-PLGA纳米粒表面引入正电荷和负电荷。
为了通过双乳剂法制备负载胰岛素的壳聚糖覆膜纳米粒(+)和海藻酸钠覆膜纳米粒(-)，对乳化剂的选择、投料比、油水相比例等一系列因素的考察，以粒径大小和分散系数PDI为指标，筛选出粒径均匀分散的纳米粒制备工艺。
蛋白类多肽物质如胰岛素在酸性环境下二级结构易发生改变导致失活，为了保证包载药物的活性及稳定性，选择先以mPEG-PLGA的有机溶液作为油相，胰岛素溶液作为内水相，先制备成纳米粒初乳，包载胰岛素，具有保护作用。再以壳聚糖醋酸溶液作为外水相，并加入一定浓度的乳化剂提高纳米粒的稳定性和分散性，超声乳化使其覆膜在纳米粒表面，得到带有正电荷的载胰岛素纳米粒。
为了验证共聚物纳米粒表面是否成功带上电荷，测定了纳米粒的Zeta值，结果如图6。酸性条件下，壳聚糖分子中的氨基被质子化带正电荷，因此用1％的醋酸作为溶剂溶解壳聚糖。但由于壳聚糖的水溶性差，在大体积的稀释相后，溶液pH增大，使得壳聚糖溶解度降低和氨基质子化强度减弱，因此最终测得的壳聚糖 纳米粒的Zeta电位值的绝对值比海藻酸钠纳米粒的Zeta值低。海藻酸钠的亲水性较强，在水溶液中的溶解度较大，对亲水性的胰岛素的包封率较高；测得的Zeta值较高，粒子间电荷斥力较大，体系稳定。
动态光散射(DSL)结果显示，壳聚糖覆膜的载胰岛素纳米粒粒径大小为(227.9±0.7)nm，Zeta电位为(+12.55±1.35)mV，包封率为(33.27±6.84)wt％，载药率为(3.18±0.60)wt％。海藻酸钠覆膜的载胰岛素纳米粒的粒径大小为(271.8±3.5)nm，Zeta电位为(-54.27±2.75)mV，包封率为(75.91±5.11)wt％，载药率为(9.77±0.71)wt％。两种纳米粒的粒径范围为200～300nm，均具有较好的分散系数，分散性好。
实施例4 载药聚电解质复合物的制备
为了获得载药聚电解质复合物，将制备所得壳聚糖或海藻酸钠覆膜的载药纳米粒单独地分散在10ml去离子水中，超声下混合两种纳米粒溶液(w/w＝1/1)，在3000rpm，4℃下离心3分钟，所得为载药聚电解质复合物，储存在4℃下。
实施例5 载药聚合物纳米粒及载药电解质复合物的透射电镜图
采用透射电子显微镜(TEM)观察两种载药纳米粒分散在水溶液中的大小和形态，以及聚电解质复合物在pH1.2，pH6.8和pH7.4下的形态。取适量待测样品溶液，充分稀释使其均匀分散，滴于碳膜铜网上，停留2min，用滤纸吸去多余的溶液，滴加2％磷钨酸溶液负染30s，室温下干燥，透射电镜观察，拍照，结果如图7和图8。
为了进一步观测两种载胰岛素共聚物纳米粒的形态。TEM结果如图7A和图7B，两种纳米粒在水溶液中均呈球形形态，且分散均一。
为了进一步验证带相反电荷的纳米粒之间的相互作用形成聚电解质复合物，TEM观测聚电解质复合物在不同pH环境下的形态变化，结果如图8A，8B和8C。pH1.2、pH6.8和pH7.4分别模拟胃液的酸性环境、肠液环境和生理环境。pH1.2溶液介质下，两种纳米粒相互交联形成絮状和网状的结构，较为紧密团聚，纳米粒形态仍呈类球形或椭圆形。这是由于壳聚糖的pKa值约为5.6，在pH1.2酸性条件下，质子化强度增大，与带有负电荷的海藻酸钠纳米粒的静电作用强，相互交联形成了纳米网状结构的聚电解质复合物，对包载的胰岛素起到了一定的保护作用，避免药物在胃中释放，降低胃酸及胃蛋白酶等对胰岛素的结构的破坏。
可以进一步观测到，在pH6.8的模拟肠液中，溶液介质的pH值大于壳聚糖的 pKa值，壳聚糖与海藻酸钠的静电作用大大降低，TEM下观测到聚电解质复合物重新分散为单个的纳米粒，且纳米粒的形态有所崩塌。模拟口服载胰岛素的聚电解复合物后，首先通过胃，在酸性pH胃液环境下，形成紧密的网状结构可以对药物起保护作用，随后进入到肠道，聚电解质复合物相互作用力减弱，变为疏松结构，胰岛素可以从单个的纳米粒中释放出来，使药物在肠道内吸收发挥降血糖作用。此外，在生理环境pH7.4下，可以看到混合的两种纳米粒形态均呈椭圆形或类球形，均匀分散。
实施例6 载胰岛素聚电解质复合物的体外释放行为考察
精密称取载胰岛素聚电解质复合物5mg置于离心管中，加入5ml事先预热好至37℃的溶液介质溶解，配制成载胰岛素聚电解质复合物溶液。将载待测溶液装入透析袋(MWCO 30,000)中，用封口夹密封好，分别浸泡在50ml pH1.2和pH6.8溶液介质中，37±1℃恒温水浴中，100rpm匀速搅拌。按设定的时间点即1,2,3,4,5,6,7,8,9,10,25,35,50,60h取出0.5ml样品后，补充等温等量与释放介质相同的新鲜介质。样品在4℃离心机中，10000rpm离心10min后，取出上清液BCA法测定胰岛素含量。每个样品平行操作3次，以释放时间为横坐标，以累积释放率的平均值为纵坐标，绘制体外累计释放曲线，结果如图9。
为了进一步评价载药电解质的体外释放效果，模拟胰岛素穿越胃肠道的过程中需要经历从强酸(胃内pH1.2～3.0)到弱碱环境(肠道pH 6.5～8.0)的变化，因此分别选择了模拟胃液环境和肠液环境下，即pH1.2和pH6.8溶液介质中进行体外释放性试验。
结果如图9，pH1.2条件下，在前4h为快速释放期，胰岛素在此阶段的累计释放量为13.91％，前10h累积释放量仅为20.69％，且释放曲线呈现缓慢上升状态，60h后的累积释放量仅为47.66％。相比之下，在pH7.4条件下，药物的累积释放量前4h即达到38.03％，随后持续缓慢释放胰岛素，10h累积释放量达到51.57％，但释放曲线仍为平滑曲线，表明以聚电解质复合物作为载体具有控释的作用，不会发生药物的突释。此后药物释放速率逐步趋于平缓，60h的累计释放量达80.54％。
进一步论证了前期实验结果，酸性条件下，覆膜在纳米粒表面的壳聚糖质子化，带有相反电荷的两种载药纳米粒之间发生静电作用，形成了紧密交联的聚电解质复合物，使包载的药物难以释放出来，具有保护作用。在生理条件下，壳聚糖所带的正电荷强度大大减弱，纳米粒分散在介质中，并随着时间延长缓慢释放 出胰岛素，具有缓释作用，可以延长药物的作用时间。
实施例7 圆二色光谱
采用圆二色光谱法对聚电解质复合物中释放出的胰岛素进行二级结构的测定，考察所释放出的胰岛素的稳定性。将体外释放实验后的收集液稀释至一定浓度，进行圆二色谱测定，同时，将猪胰岛素原料药溶解在0.1mol/L HCl中，配置成100μg/mL猪胰岛素溶液，用同样的方法进行二级构象测定作为对照。检测条件为：扫描范围190～250nm，精密度0.2nm，带宽1.0nm，扫描速度100nm/min,响应时间0.25秒，样品池0.1cm，室温25℃,平行重复测定3次。
蛋白质的折叠和构象的变化均会引起其活性发生改变，包括超声、温度、有机溶剂、酸碱环境等一系列因素均可能使胰岛素构象变化而导致变性失活。
进一步通过圆二色谱对照从PEC中释放的胰岛素与胰岛素溶液的二级构象，评价聚电解质复合物(PEC)的制备过程对胰岛素的构象稳定性的影响。结果如图10所示，猪胰岛素分别在远紫外区的208nm和223nm处有两个负峰，分别为α-螺旋和β-折叠的特征峰。208nm和223nm负峰的强度比值([Ф]208/[Ф]223)定量地反映了胰岛素的整体结构，猪胰岛素和释放的胰岛素的[Ф]208/[Ф]223值分别为1.29和1.33，可以充分表明释放出来的胰岛素的二级构象与猪胰岛素的二级构象无明显差异，在聚电解质复合物的载药过程和释放过程中，胰岛素保持其构象的稳定性。
实施例8 细胞毒性实验
取对数生长期的Caco-2细胞进行实验。首先弃去培养瓶中原培养基，用无菌PBS漂洗细胞后吸弃，加入1ml 0.25％柠檬酸胰蛋白酶消化细胞后，加入3ml新鲜全培轻轻吹打细胞，使之分散成单细胞悬液后计数。根据实验所需每孔接种的细胞个数(5×10 3个)配制细胞悬液浓度,浓度为5×10 4/mL，然后向96孔板中每孔加入100μl接种细胞，37℃、5％CO 2细胞培养箱饱和湿度下培养24h。更换介质为不同浓度的空白AL NPs溶液和空白CS NPs纳米粒(400μg/ml,200μg/ml,100μg/ml,50μg/ml,25μg/ml,12.5μg/ml)，每组每个时间点设置5个复孔，放入37℃、5％CO 2饱和湿度下培养48h和96h。每孔加入100μl MTT(0.5mg/ml)的完全培养基，在37℃、5％CO 2细胞培养箱饱和湿度下培养4h后小心吸净孔上清培养液，每孔加入150μl DMSO。置低速震荡仪上震荡10min待甲臢完全溶解于DMSO呈均匀紫色溶液时，用酶标仪测定各孔在波长570nm处的吸光度OD值，将各孔吸 光值减去调零孔吸光值，同时设置空白对照组(1％DMSO溶液)的细胞活性100％,得到样品孔校正后的吸光度平均值average(OD570’),对照孔校正后的吸光度平均值average(OD570C’)。根据校正后的平均OD值计算样品孔细胞相对活力:
细胞存活率％＝(avg(OD570’)/avg(OD570C’))×100％
为了进一步验证该聚电解质复合物的安全性和生物相容性，采用体外细胞毒性试验对空白的海藻酸钠覆膜的mPEG-PLGA纳米粒和壳聚糖覆膜mPEG-PLGA纳米粒的生物相容性和毒性进行初步评价。
考察了不同浓度(0～400μg/mL)的两种纳米粒溶液48h和72h下对Caco-2细胞生长的影响。结果如图11和图12，空白纳米粒溶液与细胞孵育48h后，AL NPs组和CS NPs细胞存活率均在95％以上，没有明显的细胞毒性作用。孵育72h后，AL NPs组的细胞存活率均大于90％，CS NPs组的细胞存活率均大于100％。以上结果均表明，在实验采用的浓度范围内，两种聚合物纳米粒对Caco-2细胞均没有毒性作用，对Caco-2细胞的增殖无抑制作用，具有良好的生物相容性。
实施例9 体内药效考察
选择200±20g的雄性SD大鼠，采取大剂量、一次腹腔注射给药的方法构建STZ诱导的糖尿病大鼠模型。造模前禁食过夜，第二天空腹腹腔注射链脲佐菌素(65mg/kg，柠檬酸盐缓冲液，pH 4.2)，一周后开始监测血糖。当模型大鼠的血糖持续高于16.7mmol/L，可视为糖尿病大鼠模型造模成功。将糖尿病模型大鼠随机分为4组，每组5只，给药前禁食过夜，保持充足饮水量。A组为阳性对照组：皮下注射胰岛素溶液(5IU/kg)，其它三组均为给药实验组：B组口服给药AL NPs(60IU/kg)；C组口服给药载药CS NPs(60IU/kg)；D组口服给药载胰岛素聚电解质复合物(60IU/kg)。给药后，在各设定时间点分别测量血糖值和尾静脉采集血样。绘制体内血糖浓度与时间曲线图；用含肝素钠的取血管采集血样后4℃，3000×g离心10min，取上清血浆保存于-80℃，用猪胰岛素ELISA试剂盒测定血浆中猪胰岛素的含量，绘制药时曲线，计算药时曲线下面积(AUC)和相对生物利用度(F％)。
糖尿病大鼠皮下注射胰岛素溶液作为阳性对照组，载胰岛素CS NPs、载胰岛素AL NPs和载胰岛素PEC口服给药为实验组，评估口服PEC的体内降糖效果。
结果如图13所示，胰岛素溶液皮下注射组血糖迅速下降，并且在3h时达到最低，随后逐渐回升，8-10h即恢复到最初的高血糖水平。载胰岛素CS NPs口服 给药后，在前8h没有降血糖效果，8h后血糖开始降低，并持续下降，在12h时血糖降至初始水平的43％；载胰岛素AL NPs口服给药后，4h即开始出现降血糖效果，但并不明显，并在之后4h-12h的测定时间内，持续保持血糖稳定下降的趋势，最终在12h时血糖降至初水平的36％。对比之下，口服载胰岛素PEC的降血糖效果也是出现在4h时间点，但降血糖效果比AL NPs明显，4-8h持续保持稳态下降，在8h时降血糖效果最佳达到初始水平的28.29％，在SD大鼠空腹正常血糖值范围内，随后保持血糖水平总体波动不大，12h时仍为初始血糖值的35.75％，具有良好的缓释和降血糖效果。
与之相对应的药时曲线，如图14所示，糖尿病大鼠皮下注射胰岛素溶液后，血浆中胰岛素浓度迅速上升。载胰岛素CS NPs组在1h时有一个小的峰值，随后即降低，并血浆中胰岛素浓度始终不高，波动范围不大；载胰岛素AL NPs组同样在1h时有一个小的峰值，2h-8h时血药浓度缓慢上升，8h达到第二个小峰值后开始逐渐降低恢复到给药前水平，但整体给药后所检测出血浆中的胰岛素浓度比CS NPs组高。PEC组给药后的第一个小峰值出现在2h，血浆中胰岛素浓度降低后恢复后，4h-10h开始稳定持续上升，并且在10h时血药浓度达到最高44.19μIU/mL，在12h时仍测得35.75μIU/mL，相对生物利用度为7.51％，均高于另外三组的血药浓度值。
与游离胰岛素相比，以聚合物纳米粒和聚电解质复合物作为载体呈递胰岛素，明显具有缓释作用，更好地发挥降血糖作用。但CS NPs和AL NPs单独作为载体给药，降血糖效果均不理想，CS NPs的载药量低，虽然其具有生物粘附性，可以延长在小肠粘膜上药物作用时间，但单独作为载体口服给药时，在通过胃时，酸性环境和酶的作用均会对药物和纳米粒载体的稳定性造成影响，从而使得能顺利到达肠道吸收部位并发挥药效的胰岛素仅为极少量。相比之下，AL NPs的载药量能达到70％以上，因此单独作为载体口服给药后，其降血糖效果和持续时间均比CS NPs好，与实验结果一致。但是海藻酸钠并不具有壳聚糖生物粘附性和打开小肠上皮细胞之间的紧密连接的功能，因此在口服吸收途径中，胰岛素在肠道作用部位的吸收是有限的。
聚电解质复合物协同两种纳米粒的优点，胃酸性环境下，PEC的形成紧密的网状交联结构具有保护作用，为胰岛素提供一道物理防护层，在释放之前保护其不被强酸和酶破坏，而在肠液中性环境下，能缓慢稳定释放胰岛素，具有缓释的 降血糖作用。因此，PEC作为载体给药，协同AL NPs高载药量和CS NPs在肠道吸收促进剂和肠粘膜粘附剂的优点，具有良好的降血糖效果，缓释作用和安全性，是一种理想的药物口服载体。

Claims (10)

  1. 一种聚电解质复合物，由带正电荷的共聚物纳米粒和带负电荷的共聚物纳米粒混合制成；
    其中，所述的共聚物纳米粒为以两亲性嵌段共聚物作为油相，以活性药物的水溶液为内水相，制备得到的油包水(W/O)的共聚物纳米粒初乳剂；然后与外水相制备得到水包油包水(W/O/W)的复乳剂，并通过将该复乳剂分散在乳化剂稀释液中制得；
    其中，带正电荷的共聚物纳米粒的外水相为阳离子生物聚合物或其修饰后生物聚合物的溶液；
    带负电荷的共聚物纳米粒的外水相为阴离子生物聚合物或其修饰后生物聚合物的溶液。
  2. 根据权利要求1所述的聚电解质复合物，其中，两亲性嵌段聚合物选自聚乙二醇-聚乳酸-羟基乙酸(mPEG-b-PLGA)、聚乙二醇-聚乳酸(PEG-b-PLA)、聚乙二醇-聚乙酸内酯(PEG-b-PCL)、聚乙二醇-聚丙交酯-己内酯(PEG-b-PLCL)，优选地，mPEG-b-PLGA中甲氧基封端的聚乙二醇(mPEG)为mPEG 5000；更优选地，所述mPEG-b-PLGA由D,L-丙交酯和乙交酯通过开环聚合法合成，mPEG 5000所投入的质量分数为10％。
  3. 根据权利要求1所述的聚电解质复合物，其中，带正电荷的共聚物纳米粒的外水相选自壳聚糖、N-琥珀酰壳聚糖、羧甲基壳聚糖、聚乙烯亚胺(PEI)、聚烯丙胺(PAH)、聚(L-赖氨酸)、聚(L-精氨酸)、聚乙烯胺。
  4. 根据权利要求1所述的聚电解质复合物，其中，所述的带负电荷的共聚物纳米粒的外水相选自海藻酸盐、聚谷氨酸、透明质酸、果胶、糖胺聚糖、聚天冬氨酸、羧甲基纤维素、羧甲基葡聚糖。
  5. 根据权利要求1所述的聚电解质复合物，其中，所述的乳化剂稀释液选自泊洛沙姆188(F68)、聚乙烯醇(PVA)、聚氧乙烯烷基醚、十二烷基硫酸钠(SDS) 的溶液。
  6. 根据权利要求1所述的聚电解质复合物，其中，所述活性药物选自胰岛素、胰岛素类似物、糖尿病药物、蛋白质类药物、多肽类药物。
  7. 一种聚电解质复合物的制备方法，其包括如下步骤：
    i)制备带正电荷的共聚物纳米粒或带负电荷的共聚物纳米粒；
    ii)将带正电荷的共聚物纳米粒或带负电荷的共聚物纳米粒混合，得到聚电解质复合物；
    其中，所述步骤i)中带正电荷的共聚物纳米粒或带负电荷的共聚物纳米粒通过以下步骤制成，
    (1)制备活性成分溶液，作为内水相；
    (2)将所述的两亲性嵌段共聚物溶解于有机溶剂中，作为油相；
    (3)将步骤(1)制得的活性成分溶液缓慢滴加入步骤(2)制得的两亲性嵌段共聚物有机溶液中，超声乳化，制备初乳剂；
    (4)将初乳缓慢滴加入相应的外水相中，超声乳化，制备复乳剂；
    (5)制备相应的稀释液，将复乳剂和稀释液混合，去除有机溶剂，离心并收集沉淀，即得到带正电荷的共聚物纳米粒或带负电荷的共聚物纳米粒；
    其中，带正电荷的共聚物纳米粒的外水相为阳离子生物聚合物或其修饰后生物聚合物的溶液；
    带负电荷的共聚物纳米粒的外水相为阴离子生物聚合物或其修饰后生物聚合物的溶液。
  8. 根据权利要求7所述的制备方法，其中，在制备过程中，内水相、油相、外水相、稀释液的体积比为1:10:40:100。
  9. 根据权利要求7所述的制备方法，其中，所载活性成分为胰岛素，且与共聚物mPEG-PLGA载体材料的质量投料比优选为5-20％，更优选为10％。
  10. 根据权利要求1-5所述的聚电解质复合物在制备治疗代谢性疾病的口服制剂中的应用；优选地，所述代谢性疾病选自糖尿病、高糖血症，更优选为I型糖 尿病或II型糖尿病，所述的药物是胰岛素或胰岛素类似物。
PCT/CN2019/112522 2018-10-31 2019-10-22 一种用于胰岛素口服递送的聚电解质复合物 WO2020088306A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811286230.3A CN111195238A (zh) 2018-10-31 2018-10-31 一种用于胰岛素口服递送的聚电解质复合物
CN201811286230.3 2018-10-31

Publications (1)

Publication Number Publication Date
WO2020088306A1 true WO2020088306A1 (zh) 2020-05-07

Family

ID=70462529

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/112522 WO2020088306A1 (zh) 2018-10-31 2019-10-22 一种用于胰岛素口服递送的聚电解质复合物

Country Status (2)

Country Link
CN (1) CN111195238A (zh)
WO (1) WO2020088306A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113893333B (zh) * 2021-10-18 2024-04-09 中国药科大学 一种胰岛素缓释口腔贴片及其制备方法、应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102380103A (zh) * 2011-10-28 2012-03-21 复旦大学 甘露糖修饰巯基化壳聚糖季铵盐纳米粒及其制备方法和应用
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
CN105813652A (zh) * 2013-04-30 2016-07-27 麻省理工学院 用于糖尿病治疗的可注射纳米网络凝胶
CN108379560A (zh) * 2018-02-02 2018-08-10 中山大学 一种负载胰岛素的肠溶性纳微颗粒及其制备方法和应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101053811A (zh) * 2007-03-08 2007-10-17 上海交通大学 兼具表面官能团和可控功能性的复合微球的制备方法
CN101675996B (zh) * 2008-09-19 2011-12-14 中国科学院过程工程研究所 壳聚糖纳微球产品及其制备方法
CN102813937A (zh) * 2012-06-12 2012-12-12 天津大学 含有疏水性药物的聚电解质复合物及其制备方法和应用
CN103255174B (zh) * 2013-05-02 2015-10-28 天津大学 以聚乙二醇接枝的透明质酸为外层的三元复合物及三元复合物的液体与应用
CN104922694B (zh) * 2015-07-20 2018-09-21 南方医科大学 一种胰岛素缓释纳米颗粒及其制备方法

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
CN102380103A (zh) * 2011-10-28 2012-03-21 复旦大学 甘露糖修饰巯基化壳聚糖季铵盐纳米粒及其制备方法和应用
CN105813652A (zh) * 2013-04-30 2016-07-27 麻省理工学院 用于糖尿病治疗的可注射纳米网络凝胶
CN108379560A (zh) * 2018-02-02 2018-08-10 中山大学 一种负载胰岛素的肠溶性纳微颗粒及其制备方法和应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PIYASI MUKHOPADHYAY. ET AL: "Formulation of pH-Responsive Carboxymethyl Chitosan and Alginate Beads for the Oral Delivery of Insulin", JOURNAL OF APPLIED POLYMER SCIENCE, vol. 129, no. 2, 18 December 2012 (2012-12-18), pages 835 - 845, XP055698396, ISSN: 0021-8995, DOI: 10.1002/app.38814 *
SARMENTO, B. ET AL.: "Insulin-Loaded Nanoparticles are Prepared by Alginate Ionotropic Pre-Gelation Followed by Chitosan Polyelectrolyte Complexation", JOURNAL OF NANOSCIENCE AND NANOTECHNOLOGY, vol. 7, no. 8, 1 August 2007 (2007-08-01), pages 2833 - 2841, XP009520611, ISSN: 1533-4880, DOI: 10.1166/jnn.2007.609 *
ZHANG, XIAOYAN ET AL.: "Preparation and characterization of insulin-loaded bioadhesive PLGA nanoparticles for oral administration,", EUROPEAN JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 45, no. 5, 11 April 2012 (2012-04-11), pages 632 - 638, XP028464586, ISSN: 0928-0987, DOI: 10.1016/j.ejps.2012.01.002 *

Also Published As

Publication number Publication date
CN111195238A (zh) 2020-05-26

Similar Documents

Publication Publication Date Title
Wong et al. Microparticles, microcapsules and microspheres: a review of recent developments and prospects for oral delivery of insulin
Chen et al. Self-assembly pH-sensitive chitosan/alginate coated polyelectrolyte complexes for oral delivery of insulin
Xiong et al. Vesicles from Pluronic/poly (lactic acid) block copolymers as new carriers for oral insulin delivery
Zhang et al. Sustained intravitreal delivery of dexamethasone using an injectable and biodegradable thermogel
Kanwar et al. In situ forming depot as sustained-release drug delivery systems
US20100226985A1 (en) Viscoelastic aqueous gels comprising microspheres
Bongiovì et al. Hyaluronic Acid‐Based Micelles as Ocular Platform to Modulate the Loading, Release, and Corneal Permeation of Corticosteroids
Cheng et al. Mucoadhesive versus mucopenetrating nanoparticles for oral delivery of insulin
Hu et al. Smart pH-responsive polymeric micelles for programmed oral delivery of insulin
Bongiovì et al. Imatinib-loaded micelles of hyaluronic acid derivatives for potential treatment of neovascular ocular diseases
US8859004B2 (en) pH-sensitive nanoparticles for oral insulin delivery
CN115192542B (zh) 一种利用小分子营养物质介导的口服纳米递药系统
CN108926531B (zh) 一种还原及pH双重响应性的纳米胶束及其制备方法与应用
Zhang et al. pH-sensitive O-carboxymethyl chitosan/sodium alginate nanohydrogel for enhanced oral delivery of insulin
KR20090101758A (ko) 고분자 마이셀 약물 조성물 및 이의 제조방법
CN107638388B (zh) 一种积雪草酸壳聚糖去氧胆酸嫁接物胶束及制备方法
Fang et al. Gastrointestinal responsive polymeric nanoparticles for oral delivery of insulin: optimized preparation, characterization, and in vivo evaluation
Han et al. Oral Delivery of Protein Drugs via Lysine Polymer‐Based Nanoparticle Platforms
WO2020088306A1 (zh) 一种用于胰岛素口服递送的聚电解质复合物
EP2042166A1 (en) Nanocapsules for oral delivery of proteins
Arpaç et al. Design and in vitro/in vivo evaluation of polyelectrolyte complex nanoparticles filled in enteric-coated capsules for oral delivery of insulin
CN102908332A (zh) 用于口服胰岛素递送的包含阳离子纳米粒子的肠溶包衣胶囊
CN106581647A (zh) 一种pH响应胰岛素缓释纳米粒及其制备方法和应用
Thanos et al. Enhancing the oral bioavailability of the poorly soluble drug dicumarol with a bioadhesive polymer
Liu et al. Development of controlled-release soy β-conglycinin nanoparticles containing curcumin using genipin as crosslinker

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: 19877970

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: 19877970

Country of ref document: EP

Kind code of ref document: A1