CN111195238A

CN111195238A - Polyelectrolyte complex for oral delivery of insulin

Info

Publication number: CN111195238A
Application number: CN201811286230.3A
Authority: CN
Inventors: 曾庆冰; 陈婷婷; 李顺英; 朱雯婷; 王俊; 秦婷婷
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2018-10-31
Filing date: 2018-10-31
Publication date: 2020-05-26
Also published as: WO2020088306A1

Abstract

The invention relates to a polyelectrolyte compound for oral delivery of insulin, which is characterized in that two block copolymers MePEG-PLGA are used as matrixes to respectively prepare two types of nanoparticles with positive charges and negative charges, and the two types of nanoparticles are mixed and then cross-linked with each other to form the polyelectrolyte compound. After the insulin polyelectrolyte compound is orally taken, the damage of gastric acid, enzyme and other environments to the activity of insulin when the medicine passes through the stomach can be avoided, so that the medicine can smoothly reach the absorption part of the intestinal tract and has pH sensitivity. The oral preparation has good blood sugar lowering effect and sustained and controlled release effect, and can be used for oral delivery of insulin and protein drugs.

Description

Polyelectrolyte complex for oral delivery of insulin

Technical Field

The present invention relates to a polyelectrolyte complex for oral administration of insulin and a preparation method thereof, and more particularly, to two types of insulin sustained-release nanoparticles having opposite charges, which are further cross-linked with each other to form a polyelectrolyte complex.

Background

Diabetes is an endocrine disease caused by the inability of the pancreas to secrete sufficient insulin, or the body to utilize insulin effectively. Subcutaneous insulin injection is the most common and effective treatment method at present, but because of the problems of difficulty in maintaining glucose homeostasis in patients, low compliance, strong side effects and the like, the development of a safe and effective noninvasive administration route is a research hotspot. Oral administration is the most desirable route of administration because it not only avoids the side effects associated with subcutaneous injection, but also greatly increases patient compliance. However, since insulin macromolecules are difficult to pass through the intestinal epithelium and are degraded in the gastrointestinal tract before reaching the site of intestinal absorption, the oral bioavailability of insulin is extremely low. The main barriers of the current oral insulin formulations are three points: the enzyme barrier, the physiological barrier of the gastrointestinal tract and the biochemical nature of insulin itself. Nanocarriers such as polymeric nanoparticles and micelles can be used for oral administration of insulin, and these nanocarriers can protect insulin from degradation and promote absorption during transport through transmembrane and paracellular pathways. Therefore, the insulin oral preparation should prolong the retention time of insulin in intestinal tract to provide more opportunity for its absorption and accelerate the release of insulin in the preparation at its main absorption site to improve the oral bioavailability of insulin on the premise of protecting insulin from being destroyed before being released.

The ideal polymeric carrier should be biodegradable, biocompatible, and capable of extending intestinal residence time against the action of gastric pH gradients and enzymes. The polyethylene glycol-polylactic acid-glycolic acid copolymer (mPEG-b-PLGA) is a block polymer formed by polymerizing PLGA and methoxy-terminated polyethylene glycol (mPEG), and due to the addition of the mPEG molecules with strong hydrophilicity, the mPEG-b-PLGA becomes amphiphilic molecules with one hydrophilic end and the other hydrophobic end, and the prepared nanoparticles can avoid the rapid removal of macrophages, so that the retention time of the carried medicine in the systemic circulation is prolonged, and the premature release of the medicine is reduced. The chitosan is natural cationic polysaccharide, the side chain of the chitosan contains amino, the amino is protonated under acidic conditions, so that the chitosan becomes an electropositive polymer, has pH sensitivity, and has membrane adhesion property because the chitosan with positive charge and sialic acid groups with negative charge on mucin attract each other in intestinal environment. Alginic acid is another water-soluble natural linear polysaccharide that shrinks at a lower pH, retaining the encapsulated drug in the stomach, while protecting the drug from inactivation by enzymes, and is therefore widely used in pH-responsive polymers.

Therefore, the present invention prepares a polyelectrolyte complex that can be used for oral delivery of insulin based on the electrostatic force interaction of two oppositely charged nanoparticles.

Disclosure of Invention

The invention relates to a polyelectrolyte compound which is formed by mutually crosslinking insulin sustained and controlled release nanoparticles with opposite charges and can be used for oral administration. The components of the polyelectrolyte compound are synthetic or natural high molecular polymers with no toxicity, biocompatibility and biodegradability.

The invention designs two types of nanoparticles with opposite charges, wherein the surface of one type of nanoparticle has positive charges, the surface of the other type of nanoparticle has opposite negative charges, the interaction of the two types of nanoparticles can form a polyelectrolyte compound for oral administration through crosslinking, the polyelectrolyte compound has pH responsiveness, has a protection effect on a carried medicine in a gastric acid environment under an oral administration absorption path, slowly releases the medicine in an intestinal neutral environment, and has good slow release effect and relative bioavailability.

One aspect of the present invention relates to a polyelectrolyte complex, which is prepared by mixing positively charged copolymer nanoparticles and negatively charged copolymer nanoparticles;

wherein the copolymer nanoparticles are water-in-oil (W/O) copolymer nanoparticle primary emulsion prepared by taking an amphiphilic block copolymer as an oil phase and taking an aqueous solution of an active medicament as an internal water phase;

the external water phase of the copolymer nanoparticle with positive charge is a solution of a cationic biopolymer or a modified biopolymer thereof, a double emulsion of water-in-oil-in-water (W/O/W) is prepared, and the double emulsion is dispersed in an emulsifier diluent to prepare the composite emulsion;

the external water phase of the copolymer nanoparticle with negative charge is a solution of an anionic biopolymer or a modified biopolymer thereof, a double emulsion of water-in-oil-in-water (W/O/W) is prepared, and the double emulsion is dispersed in an emulsifier diluent.

In the technical scheme of the invention, 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-polyacetic acid lactone (PEG-b-PCL), polyethylene glycol-polylactide-caprolactone (PEG-b-PLCL), preferably, the methoxy-terminated polyethylene glycol (mPEG) in the mPEG-b-PLGA is mPEG₅₀₀₀(ii) a More preferably, the mPEG-b-PLGA is synthesized by D, L-lactide and glycolide through a ring-opening polymerization method, and the mPEG₅₀₀₀The mass fraction charged was 10%.

In the technical scheme of the invention, the external water phase of the copolymer nanoparticle with positive charge is selected from a solution of a cationic biopolymer or a modified biopolymer thereof, is a hydrogen bond receptor, and is selected from chitosan, N-succinyl chitosan, carboxymethyl chitosan, Polyethyleneimine (PEI), Polyallylamine (PAH), poly (L-lysine), poly (L-arginine) and polyvinylamine.

In the technical scheme of the invention, the external water phase of the copolymer nanoparticle with negative charge is selected from a solution of an anionic biopolymer or a modified biopolymer thereof, is a hydrogen bond donor, and is selected from alginate, polyglutamic acid, hyaluronic acid, pectin, glycosaminoglycan, polyaspartic acid, carboxymethyl cellulose and carboxymethyl dextran.

In the technical scheme of the invention, the emulsifier diluent is selected from solutions of poloxamer 188(F68), polyvinyl alcohol (PVA), polyoxyethylene alkyl ether and Sodium Dodecyl Sulfate (SDS) with different concentrations.

In the technical scheme of the invention, the active medicament is selected from insulin, insulin analogues, diabetes drugs, protein drugs and polypeptide drugs.

In the technical scheme of the invention, the copolymer nanoparticles with positive charges or the copolymer nanoparticles with negative charges are prepared by a double-emulsion volatilization method; preferably, the method comprises the following specific steps:

(1) preparing an active ingredient solution as an internal aqueous phase;

(2) dissolving the amphiphilic block copolymer in an organic solvent to form an oil phase;

(3) slowly dripping the active component solution prepared in the step (1) into the amphiphilic block copolymer organic solution prepared in the step (2), and ultrasonically emulsifying to prepare a primary emulsion;

(4) slowly dripping the primary emulsion into corresponding external water phase, and ultrasonically emulsifying to prepare a double emulsion;

(5) preparing corresponding emulsifier diluent, mixing the double emulsion and the diluent, removing the organic solvent, centrifuging and collecting the precipitate to obtain the drug-loaded copolymer nanoparticles.

In the technical scheme of the invention, the preparation method of the polyelectrolyte complex comprises the steps of mixing the copolymer nanoparticles with positive charges or the copolymer nanoparticles with negative charges, preferably, dispersing the copolymer nanoparticles with positive charges or the copolymer nanoparticles with negative charges in an aqueous solution, and mixing the copolymer nanoparticles with positive charges or the copolymer nanoparticles with negative charges in equal mass.

In another aspect, the present invention provides a method for preparing a polyelectrolyte complex, comprising the steps of:

i) preparing copolymer nanoparticles with positive charges or copolymer nanoparticles with negative charges;

ii) mixing the copolymer nanoparticles with positive charges or the copolymer nanoparticles with negative charges to obtain the polyelectrolyte complex.

Wherein the step i) is made by the steps of,

(1) preparing an active ingredient solution as an internal aqueous phase;

(5) preparing corresponding diluent, mixing the double emulsion and the diluent, removing the organic solvent, centrifuging and collecting the precipitate to obtain the drug-loaded copolymer nano-particles.

Further, in the step: the colostrum emulsification condition is ultrasonic time 5s, interval time 5s, ultrasonic time 1min, and work cycle time 60%; the multiple emulsion emulsification condition is ultrasonic time 10s, interval time 10s, total ultrasonic time 2min, and work cycle time 60%.

Further, the specific prescription process in the step: the volume ratio of the internal water phase, the oil phase, the external water phase and the diluent is 1:10:40:100, and the ratio of the organic solvent dichloromethane and acetone as the oil phase is 3: 2.

In the technical scheme of the invention, the preparation method of the mPEG-b-PLGA nanoparticles of the chitosan coating with positive charge comprises the following steps of mixing 8ml of 0.5% chitosan acetic acid solution (1% acetic acid solution) and 12ml of 2% polyvinyl alcohol as external water phase; the diluent is 50ml of 0.5% polyvinyl alcohol solution; the loaded active ingredient is insulin, and the mass feeding ratio of the loaded active ingredient to the copolymer mPEG-PLGA carrier material is preferably 5-20%, and more preferably 10%.

In the technical scheme of the invention, the preparation method of the mPEG-b-PLGA nano-particle coated with the negatively charged sodium alginate comprises the steps that the external water phase is a 0.5% sodium alginate solution with the volume 20 times that of the external water phase; the diluted phase was a 50-fold volume of 2% poloxamer 188 solution; the loaded active ingredient is insulin, and the mass feeding ratio of the loaded active ingredient to the copolymer mPEG-PLGA carrier material is preferably 5-20%, and more preferably 15%.

Furthermore, the two types of nanoparticles with opposite charges are prepared by dissolving the copolymer mPEG-b-PLGA in an organic solvent to serve as an organic phase, adding surface modifiers chitosan and sodium alginate for modification, and performing a double-emulsion volatilization method. The method comprises the following specific steps: mixing the prepared mPEG-PLGA nanoparticle solution coated with the chitosan and the mPEG-PLGA nanoparticle solution coated with the sodium alginate according to the volume ratio of 1:1(w/w) under the ultrasonic condition, and carrying out ultrasonic treatment for 2min to obtain the polyelectrolyte compound.

In some embodiments, the polyelectrolyte complex has pH responsiveness, the charge intensity on the surface of the nanoparticle is obvious in an acidic environment, a compact network structure is formed to protect the drug, and the interaction force of the polyelectrolyte complex is weakened in a neutral environment and is changed into a loose structure to facilitate the drug release.

In another aspect, the present invention provides the use of the polyelectrolyte complex described above in the preparation of a pharmaceutical carrier.

Another aspect of the present invention relates to the use of the aforementioned polyelectrolyte complexes for the preparation of an oral formulation for the treatment of metabolic disorders. Preferably, the metabolic disease is selected from diabetes, hyperglycaemia, more preferably type I diabetes or type II diabetes, and the drug is insulin or an insulin analogue. In some embodiments, the formulation is administered to maintain normal blood glucose levels.

According to the oral administration system, the hydrophilicity of the synthesized mPEG-PLGA high molecular polymer is improved by adding the hydrophilic chain segment, and the mPEG-PLGA high molecular polymer is used as a material matrix to prepare the nanoparticles to better entrap water-soluble drugs (such as insulin and protein drugs).

According to the oral administration system, the surface modifier is added to modify the drug-loaded sustained-release polymer nanoparticles, namely chitosan with positive charge amino and sodium alginate with negative charge carboxyl, and the chitosan and the sodium alginate are mutually crosslinked to form a polyelectrolyte compound, so that the total drug-loaded amount is increased. The strength of the positive and negative charges on the surface of the nanoparticle changes along with the change of a pH environment, has pH sensitivity, can ensure that the medicament can smoothly reach the intestinal absorption part through an oral absorption path, and is an ideal oral preparation with a sustained and controlled release effect.

Drawings

FIG. 1 depicts the formation of insulin-loaded polyelectrolyte complex by the double emulsion method of separately preparing mPEG-PLGA nanoparticles coated with chitosan and mPEG-PLGA nanoparticles coated with sodium alginate.

FIG. 2 is a schematic diagram of the synthesis of diblock copolymer mPEG-PLGA.

FIG. 3 is a FT-IR spectrum of the diblock copolymer mPEG-PLGA.

FIG. 4 shows the preparation of diblock copolymer mPEG-PLGA¹H-NMR spectrum.

FIG. 5 is a graph showing the distribution of the particle size of mPEG-PLGA nanoparticles (CS NPs) coated with chitosan loaded with insulin and mPEG-PLGA nanoparticles (AL NPs) coated with sodium alginate.

FIG. 6 is Zeta potential diagram of mPEG-PLGA nanoparticles (CS NPs) coated with chitosan of insulin and mPEG-PLGA nanoparticles (AL NPs) coated with sodium alginate.

FIG. 7A is a scanning transmission electron microscope image of mPEG-PLGA nanoparticles coated with chitosan loaded with insulin.

FIG. 7B is a scanning transmission electron microscope image of mPEG-PLGA nanoparticles coated with insulin-loaded sodium alginate.

FIG. 8A is a scanning transmission electron micrograph of an insulin-loaded polyelectrolyte complex at pH 1.2.

FIG. 8B is a scanning transmission electron micrograph of the insulin loaded polyelectrolyte complex at pH 6.8.

FIG. 8C is a scanning transmission electron micrograph of the insulin-loaded polyelectrolyte complex at pH 7.4.

FIG. 9 is a graph of cell viability (percentage) of chitosan or sodium alginate coated blank mPEG-PLGA nanoparticles as a function of concentration (mg/mL) after 48 hours of culture with Caco-2 cells.

FIG. 10 is a graph of cell viability (percentage) of chitosan or sodium alginate coated blank mPEG-PLGA nanoparticles as a function of concentration (mg/mL) after 72 hours of culture with Caco-2 cells.

FIG. 11 is a graph of in vitro cumulative release of insulin-loaded polyelectrolyte complexes in solution media at different pH1.2 and pH6.8.

FIG. 12 is a circular dichroism spectrum of insulin released from polyelectrolyte complexes and a standard insulin solution.

FIG. 13 is a graph showing the effect of blood glucose lowering in STZ-induced SD diabetic rats after subcutaneous injection of pure insulin solution, oral insulin-loaded chitosan-coated mPEG-PLGA nanoparticles (CSNPs), oral insulin-loaded sodium alginate-coated mPEG-PLGA nanoparticles (AL NPs) and oral insulin-loaded polyelectrolyte complex (PEC). Data points represent mean ± SD (n ═ 5).

FIG. 14 shows the porcine insulin concentrations in the plasma of STZ-induced SD diabetic rats after subcutaneous injection of pure insulin solution, oral insulin-loaded chitosan-coated mPEG-PLGA nanoparticles (CSNPs), oral insulin-loaded sodium alginate-coated mPEG-PLGA nanoparticles (AL NPs) and oral insulin-loaded polyelectrolyte complexes (PEC). Data points represent mean ± SD (n-3).

Detailed Description

Example 1 support Material diblock Polymer 10% mPEG₅₀₀₀Synthesis of PLGA

Synthesized by ring-opening polymerization. Precision weighing mPEG₅₀₀₀(10 mass percent) is used as an initiator in a round-bottom flask, then D, L-lactide and glycolide are added according to a 50:50 feeding ratio, stannous octoate with 0.1 percent of monomer amount is added as a catalyst, vacuum pumping is carried out for three times, and the reaction is carried out for 24 hours in a nitrogen environment oil bath at the temperature of 120 ℃. The reaction was stopped by removing the oil bath pan. The reaction was dissolved in an appropriate amount of dichloromethane, precipitated in an excess of cold methanol solution, repeated three times and dried under vacuum at 40 ℃ to constant weight. The reaction formula is shown in FIG. 2.

Example 2 characterization of the synthesized product

Example 2-1 mPEG-PLGA Infrared Spectroscopy

Mixing a proper amount of product to be detected with dry potassium bromide, grinding the mixture into powder at room temperature, taking a proper amount of powder sample, tabletting the powder sample at 350-7800 cm^-1Wave number scanning and measuring infrared absorption spectrum.

As shown in FIG. 3 below, the synthesized product was found to be 1760cm^-1The peak has a strong absorption peak, which is a typical carbonyl stretching vibration peak, and indicates that caprolactone and propiolactone undergo ring-opening polymerization to form a linear ester bond. At 2900cm^-1～3000cm^-1The presence of PLGA chains in the regionThe CH stretching vibration peak in the segment is overlapped with the CH stretching vibration peak in the mPEG, and the bending vibration peak in the PEG segment exists at the same time, which indicates that the mPEG and the PLGA form a diblock copolymer. At 3500cm^-1The compound has a wide absorption peak which is a stretching vibration peak of hydroxyl groups in mPEG and PLGA, and an infrared spectrum analysis result is consistent with a predicted structure of the polymer, which indicates that the compound is the required diblock copolymer mPEG-PLGA.

Example 2-2 mPEG-PLGA¹H-NMR characterization analysis

Deuterated chloroform (CDCl)₃) Taking Tetramethylsilicon (TMS) as an internal standard as a solvent, and measuring the nuclear magnetic hydrogen spectrum of the sample to be measured at 25 ℃. And determining the structural composition of the polymer according to the displacement position and the area of the related peak in the spectrogram.

As shown in fig. 4 below, absorption peaks at δ ═ 5.24 and δ ═ 4.85 in the graph are methine proton peaks (-O — CH — (CH) of glycolide LA in the PLGA segment, respectively₃) -CO-) and methylene proton peak (-O-CH) of lactide GA₂-CO-); a strong absorption peak appears at δ of 3.66, mPEG₅₀₀₀Characteristic peak of methylene proton of repeating unit (-O-CH)₂*–CH₂X-); the absorption peak at δ ═ 1.60 is the methyl proton peak (-CH) of the repeating unit in glycolide LA₃*). Therefore, the polyethylene glycol-polylactic acid-glycolic acid copolymer (mPEG-PLGA) has been successfully synthesized.

Example 3 preparation and Performance characterization of drug-loaded Polymer nanoparticles

EXAMPLE 3-1 preparation of nanoparticles

CS NPs (chitosan-chitosan coated insulin-loaded mPEG-PLGA (methoxy polyethylene glycol-poly (lactic-co-glycolic acid)) nanoparticle

AL NPs (sodium alginate coated) insulin-loaded mPEG-PLGA (methoxy polyethylene glycol-poly (lactic-co-glycolic acid)) nanoparticle

Two types of insulin nanoparticles with opposite charges are prepared by a double-emulsion method (water-in-oil-in-water). Precisely weighing a certain amount of pig insulin raw material medicine, dissolving the pig insulin raw material medicine in 0.1M HCl solution, and preparing the pig insulin solution. Adding 500 μ l insulin solution as internal water phase into 5ml oil phase (dichloromethane/acetone (3:2) solution containing polymer), and emulsifying with ultrasonic probe for total ultrasonic time of 1min (working cycle time of 60%) to obtain colostrum. The colostrum was subsequently transferred to the outer aqueous phase of 20ml chitosan/PVA solution and emulsified with an ultrasonic probe for 2min (duty cycle time 60%). The resulting multiple emulsion was slowly added dropwise to a diluted phase of 50ml of 0.5% PVA solution, and the organic solvent was evaporated off by stirring at room temperature. Stirring overnight, centrifuging at 12000 Xg at 4 deg.C for 30min, suspending in distilled water, centrifuging, precipitating, and washing for three times to obtain chitosan coated nanoparticles with positive charge.

And (3) taking a sodium alginate solution as an external water phase, taking a 2% poloxamer 188 solution as a diluent phase, and obtaining the sodium alginate coated nanoparticles with negative charges under the same other preparation conditions. Freeze drying to obtain two kinds of insulin carrying nanometer particle with opposite charge.

Example 3-2 characterization of particle size and potential of drug-loaded nanoparticles

The two prepared nanoparticles are characterized by adopting a dynamic light scattering laser particle size and Zeta potential analyzer, the solution of the nanoparticles to be detected is respectively diluted by deionized water to be uniformly dispersed, and the particle size, the dispersion coefficient (PDI) and the Zeta potential of the solution are measured. The results are shown in Table 1.

Example 3-3 measurement of encapsulation efficiency and drug-loading efficiency of drug-loaded nanoparticles

Centrifuging the prepared insulin-loaded polymer nanoparticle solution for 30min at 4 ℃ under 12000 Xg, collecting supernatant, washing the supernatant with deionized water for 2-3 times, combining washing solutions to record the volume of the supernatant, shaking up and suspending, randomly taking 2ml of solution 3 times to a 2.5ml centrifuge tube, centrifuging for 8min at 10000 Xg, taking the supernatant as a sample solution to be tested, measuring the content of insulin in the supernatant by using a BCA method, and calculating the encapsulation rate and the drug loading rate of the insulin-loaded polymer nanoparticles according to the following formula, wherein the results are shown in Table 1.

The entrapment efficiency (EE%) (total administered mass-mass of supernatant drug)/total administered mass × 100%

The drug loading rate (DL%) (total weight of drug administration-weight of supernatant drug)/total weight of nanometer preparation × 100%

TABLE 1 characterization of CS NPs and AL NPs

Note:Data are shown as Mean±SD(n≥5)

In order to obtain two types of nanoparticles with opposite charges, two types of natural high molecular polymers chitosan and sodium alginate with opposite charges are respectively added, and positive charges and negative charges are introduced on the surfaces of the mPEG-PLGA nanoparticles.

In order to prepare chitosan coated nanoparticles (+) and sodium alginate coated nanoparticles (-) loaded with insulin by a double-emulsion method, a nanoparticle preparation process with uniformly dispersed particle sizes is screened by taking the particle size and a dispersion coefficient PDI as indexes through investigating a series of factors such as selection of an emulsifier, a feed ratio, an oil-water phase ratio and the like.

In order to ensure the activity and stability of the encapsulated drug, an organic solution of mPEG-PLGA is selected as an oil phase, an insulin solution is selected as an inner water phase, and the nano-particle colostrum is prepared and encapsulated with the insulin, so that the protective effect is achieved. And then, taking a chitosan acetic acid solution as an external water phase, adding an emulsifier with a certain concentration to improve the stability and the dispersibility of the nanoparticles, and carrying out ultrasonic emulsification to coat the nanoparticles on the surfaces of the nanoparticles to obtain the insulin-loaded nanoparticles with positive charges.

To verify whether the copolymer nanoparticle surface was successfully charged, the Zeta value of the nanoparticles was determined and the results are shown in fig. 6. Under acidic conditions, amino groups in chitosan molecules are protonated and positively charged, so that 1% acetic acid is used as a solvent to dissolve chitosan. However, due to poor water solubility of chitosan, after a large-volume diluted phase, the pH value of the solution is increased, so that the solubility of chitosan is reduced and the protonation strength of amino groups is weakened, and the finally measured Zeta potential value of the chitosan nanoparticles is lower than that of the sodium alginate nanoparticles. The sodium alginate has stronger hydrophilicity, larger solubility in aqueous solution and higher encapsulation efficiency on hydrophilic insulin; the Zeta value is high, the charge repulsion between particles is large, and the system is stable.

The dynamic light scattering (DSL) result shows that the particle size of the insulin-loaded nanoparticle of the chitosan coating film is (227.9 +/-0.7) nm, the Zeta potential is (+12.55 +/-1.35) mV, the encapsulation efficiency is (33.27 +/-6.84) wt%, and the drug loading efficiency is (3.18 +/-0.60) wt%. The particle size of the sodium alginate coated insulin-loaded nano-particles is (271.8 +/-3.5) nm, the Zeta potential is (-54.27 +/-2.75) mV, the encapsulation efficiency is (75.91 +/-5.11) wt%, and the drug loading efficiency is (9.77 +/-0.71) wt%. The particle size range of the two types of nanoparticles is 200-300 nm, and both nanoparticles have good dispersion coefficients and good dispersibility.

Example 4 preparation of drug-loaded polyelectrolyte complexes

In order to obtain the drug-loaded polyelectrolyte complex, the drug-loaded nanoparticles coated with chitosan or sodium alginate prepared by the method are separately dispersed in 10ml of deionized water, two nanoparticle solutions (w/w: 1/1) are mixed under ultrasound, and the mixture is centrifuged at 3000rpm and 4 ℃ for 3 minutes to obtain the drug-loaded polyelectrolyte complex which is stored at 4 ℃.

Example 5 Transmission Electron microscopy of drug-loaded Polymer nanoparticles and drug-loaded electrolyte complexes

The size and the form of the dispersion of the two drug-loaded nanoparticles in the aqueous solution and the form of the polyelectrolyte complex at pH1.2, pH6.8 and pH7.4 are observed by a Transmission Electron Microscope (TEM). Taking a proper amount of sample solution to be detected, fully diluting to uniformly disperse the sample solution, dripping the sample solution on a carbon film copper net, staying for 2min, absorbing excess solution by using filter paper, dripping 2% phosphotungstic acid solution for negative dyeing for 30s, drying at room temperature, observing by using a transmission electron microscope, and taking a picture, wherein the result is shown in a figure 7 and a figure 8.

To further observe the morphology of the two insulin-loaded copolymer nanoparticles. TEM results are shown in FIG. 7A and FIG. 7B, and both nanoparticles are spherical in shape and uniformly dispersed in the aqueous solution.

To further verify that the interaction between the oppositely charged nanoparticles forms polyelectrolyte complexes, TEM observed the morphology change of the polyelectrolyte complexes in different pH environments, and the results are shown in FIGS. 8A, 8B and 8C. pH1.2, pH6.8 and pH7.4 respectively simulate the acidic environment, intestinal juice environment and physiological environment of gastric juice. Under the medium of a solution with the pH value of 1.2, the two types of nanoparticles are mutually crosslinked to form a flocculent and reticular structure, the flocculent and reticular structures are tightly agglomerated, and the nanoparticle forms are still in a similar spherical or elliptical shape. The pKa value of the chitosan is about 5.6, the protonation strength is increased under the acidic condition of pH1.2, the electrostatic interaction with sodium alginate nanoparticles with negative charges is strong, and the chitosan and the sodium alginate nanoparticles are mutually crosslinked to form a polyelectrolyte compound with a nano-network structure, so that a certain protection effect on the entrapped insulin is achieved, the release of drugs in the stomach is avoided, and the damage of gastric acid, pepsin and the like to the structure of the insulin is reduced.

It can be further observed that in the simulated intestinal fluid with the pH value of 6.8, the pH value of the solution medium is greater than the pKa value of chitosan, the electrostatic interaction of chitosan and sodium alginate is greatly reduced, and under TEM, the polyelectrolyte complex is observed to be re-dispersed into single nanoparticles, and the morphology of the nanoparticles is collapsed to some extent. After the oral insulin-loaded polyelectrolyte compound is simulated, the polyelectrolyte compound firstly passes through the stomach and forms a compact reticular structure in an acid pH gastric juice environment to protect the drug, then enters the intestinal tract, the interaction force of the polyelectrolyte compound is weakened and the polyelectrolyte compound is changed into a loose structure, and the insulin can be released from single nanoparticles, so that the drug can be absorbed in the intestinal tract to play a role in reducing blood sugar. In addition, under the physiological environment of pH7.4, the two mixed nanoparticles are in an elliptical or sphere-like shape and are uniformly dispersed.

Example 6 in vitro Release behavior Studies of insulin-loaded polyelectrolyte complexes

Accurately weighing 5mg of the insulin-loaded polyelectrolyte complex, placing the insulin-loaded polyelectrolyte complex into a centrifuge tube, adding 5ml of solution medium preheated to 37 ℃ in advance, and dissolving to prepare the insulin-loaded polyelectrolyte complex solution. The solution to be tested is put into a dialysis bag (MWCO30,000), sealed by a sealing clip, and respectively soaked in 50ml of solution media with pH value of 1.2 and pH value of 6.8, and stirred in a constant-temperature water bath at 37 +/-1 ℃ at a constant speed of 100 rpm. After 0.5ml of sample was taken at the set time points, i.e. 1,2,3,4,5,6,7,8,9,10,25,35,50,60h, isothermal equivalent amounts of fresh medium identical to the release medium were replenished. The sample is centrifuged at 10000rpm for 10min in a 4 ℃ centrifuge, and then the supernatant is taken out for measuring the content of the insulin by a BCA method. Each sample was run in parallel 3 times, with release time as the abscissa and the average of the cumulative release rate as the ordinate, to plot the cumulative release profile in vitro, with the results shown in figure 9.

In order to further evaluate the in-vitro release effect of the drug-loaded electrolyte, the simulated insulin needs to undergo the change from strong acid (pH 1.2-3.0 in the stomach) to weak base (pH 6.5-8.0 in the intestinal tract) in the process of passing through the gastrointestinal tract, so that the simulated gastric juice environment and the intestinal juice environment, namely the pH1.2 and the pH6.8 solution media, are respectively selected for carrying out in-vitro release tests.

The results are shown in FIG. 9, where the release period in the first 4h is a fast release period, the cumulative release of insulin in this period is 13.91%, the cumulative release in the first 10h is only 20.69%, the release curve shows a slow-rising state, and the cumulative release after 60h is only 47.66%, under the condition of pH1.2. In contrast, the cumulative release amount of the drug reached 38.03% 4h before and then insulin was continuously and slowly released at pH7.4, and the cumulative release amount reached 51.57% 10h, but the release curve was still smooth, indicating that the polyelectrolyte complex as a carrier had a controlled release effect and no burst release of the drug occurred. The drug release rate gradually becomes flat after that, and the cumulative release amount of 60h reaches 80.54%.

Further argumenting the earlier stage experimental results, under the acidic condition, the chitosan of the coating film on the surface of the nanoparticles is protonated, and the two drug-loaded nanoparticles with opposite charges are subjected to electrostatic interaction to form a closely cross-linked polyelectrolyte compound, so that the encapsulated drug is difficult to release, and the protective effect is achieved. Under physiological conditions, the strength of positive charges carried by chitosan is greatly reduced, and the nanoparticles are dispersed in a medium and slowly release insulin along with the time extension, so that the chitosan nanoparticle sustained release drug has a sustained release effect and can prolong the action time of the drug.

Example 7 circular dichroism Spectroscopy

And (3) measuring the secondary structure of the insulin released from the polyelectrolyte complex by adopting circular dichroism spectroscopy, and inspecting the stability of the released insulin. And (3) diluting the collected liquid after the in vitro release experiment to a certain concentration, performing circular dichroism chromatogram determination, simultaneously dissolving the porcine insulin bulk drug in 0.1mol/L HCl to prepare a porcine insulin solution with the concentration of 100 mu g/mL, and performing secondary conformation determination by the same method to serve as a control. The detection conditions are as follows: the scanning range is 190-250 nm, the precision is 0.2nm, the bandwidth is 1.0nm, the scanning speed is 100nm/min, the response time is 0.25 seconds, the sample cell is 0.1cm, the room temperature is 25 ℃, and the parallel repeated determination is carried out for 3 times.

The folding and conformational changes of the protein can cause the change of the activity of the protein, and a series of factors including ultrasound, temperature, organic solvents, acid-base environment and the like can cause the conformational change of the insulin to cause denaturation and inactivation.

The results are shown in FIG. 10, pig insulin has two negative peaks at 208nm and 223nm of far ultraviolet region, α -helix and β -fold characteristic peaks, the intensity ratio ([ Phi ]208/[ Phi ]223) of the negative peaks at 208nm and 223nm quantitatively reflects the whole structure of the insulin, the values of [ Phi ]208/[ Phi ]223 of the pig insulin and the released insulin are 1.29 and 1.33, respectively, which can fully indicate that the secondary conformation of the released insulin has no obvious difference from the secondary conformation of the pig insulin, and the insulin keeps the conformational stability during the drug loading process and the release process of the polyelectrolyte complex.

Example 8 cytotoxicity assay

Caco-2 cells were taken for experiments in logarithmic growth phase. Firstly, the original culture medium in a culture bottle is discarded, the cells are rinsed by sterile PBS and then are discarded by suction, 1ml of 0.25% citric acid trypsin is added to digest the cells, 3ml of fresh full culture is added to gently blow and beat the cells, and the cells are dispersed into single cell suspension and counted. According to the number of cells seeded per well (5X 10) required for the experiment³One) to prepare a cell suspension concentration of 5X 10⁴mL, then 100. mu.l of seeded cells were added to each well of a 96-well plate at 37 ℃ with 5% CO₂The cells are cultured for 24h in a cell culture box under saturated humidity. The medium was replaced with blank AL NPs solution and blank CS NPs nanoparticles (400. mu.g/ml, 200. mu.g/ml, 100. mu.g/ml, 50. mu.g/ml, 25. mu.g/ml, 12.5. mu.g/ml) of different concentrations, 5 multiple wells per set per time point were placed, and 37 ℃ and 5% CO were placed₂Culturing for 48h and 96h under saturated humidity. Mu.l MTT (0.5mg/ml) complete medium was added to each well at 37 ℃ with 5% CO₂Carefully sucking and cleaning the supernatant culture solution of the holes after culturing for 4 hours in a cell culture box under saturated humidityMu.l DMSO was added to each well. And (3) placing the mixture on a low-speed oscillator to oscillate for 10min until the formazan is completely dissolved in DMSO and presents a uniform purple solution, measuring the absorbance OD value of each well at the position of 570nm by using an enzyme labeling instrument, subtracting the absorbance value of each well from the absorbance value of a zero-setting well, setting the cell activity of a blank control group (1% DMSO solution) to be 100%, and obtaining the corrected absorbance average value average (OD570 ') of the sample wells and the corrected absorbance average (OD 570C') of the control wells. Calculating the relative activity of the cells in the sample wells according to the corrected average OD value:

percent cell viability ═ avg (OD570 ')/avg (OD 570C')) x 100%

In order to further verify the safety and biocompatibility of the polyelectrolyte compound, the biocompatibility and toxicity of the blank sodium alginate-coated mPEG-PLGA nanoparticles and chitosan-coated mPEG-PLGA nanoparticles are preliminarily evaluated by an in vitro cytotoxicity test.

The influence of two nanoparticle solutions with different concentrations (0-400 mu g/mL) on the growth of Caco-2 cells under 48h and 72h is examined. The results are shown in fig. 11 and fig. 12, after the blank nanoparticle solution is incubated with the cells for 48 hours, the cell survival rates of the AL NPs group and the CS NPs group are both above 95%, and no obvious cytotoxicity effect is caused. After incubation for 72h, the cell survival rates of the AL NPs are all more than 90%, and the cell survival rates of the CS NPs are all more than 100%. The results show that in the concentration range adopted in the experiment, the two polymer nanoparticles have no toxic effect on Caco-2 cells, have no inhibition effect on the proliferation of the Caco-2 cells, and have good biocompatibility.

Example 9 in vivo drug efficacy examination

Male SD rats of 200 ± 20g were selected, and a large dose, one-time intraperitoneal injection administration method was employed to construct STZ-induced diabetic rat models. Fasting was overnight before molding, streptozotocin (65mg/kg, citrate buffer, pH 4.2) was intraperitoneally injected second sky, and blood glucose monitoring was started after one week. When the blood sugar of the model rat is continuously higher than 16.7mmol/L, the model rat model with diabetes is considered to be successfully modeled. Diabetic model rats were randomly divided into 4 groups of 5 rats each, fasted overnight before dosing, and kept at adequate water intake. Group a is a positive control group: subcutaneous insulin solution (5IU/kg) was injected, and the other three groups were all administration experimental groups: group B oral administration of AL NPs (60 IU/kg); group C oral drug-loaded CS NPs (60 IU/kg); group D was orally administered with insulin-loaded polyelectrolyte complex (60 IU/kg). After administration, blood glucose values were measured at each set time point and blood samples were collected from the tail vein. Drawing a graph of blood glucose concentration in vivo and time; collecting blood sample with blood vessel containing heparin sodium, centrifuging at 4 deg.C and 3000 Xg for 10min, collecting supernatant, storing at-80 deg.C, measuring porcine insulin content in plasma with porcine insulin ELISA kit, drawing pharmaceutical time curve, and calculating area under the curve (AUC) and relative bioavailability (F%).

The diabetic rats injected with insulin solution subcutaneously as a positive control group, and orally administered with insulin-loaded CS NPs, insulin-loaded ALNPs and insulin-loaded PEC as experimental groups to evaluate the in vivo blood sugar lowering effect of the oral PEC.

As shown in FIG. 13, the blood glucose level of the group injected subcutaneously with insulin solution rapidly decreased and reached the lowest level at 3 hours, and then gradually increased back to the original high blood glucose level after 8-10 hours. After oral administration of the insulin-loaded CS NPs, the blood sugar is not reduced in the first 8h, the blood sugar is reduced after 8h and is continuously reduced, and the blood sugar is reduced to 43 percent of the initial level in 12 h; after oral administration of the insulin-loaded AL NPs, the hypoglycemic effect begins to appear at 4h, but is not obvious, and the trend of stable decline of blood sugar is continuously maintained within the measuring time of 4h-12h, and finally the blood sugar is reduced to 36% of the original level at 12 h. In contrast, the oral insulin-loaded PEC also has the hypoglycemic effect at the time point of 4h, but the hypoglycemic effect is obvious compared with the AL NPs, the steady decline is continuously maintained for 4-8h, the hypoglycemic effect reaches 28.29 percent of the initial level optimally at 8h, the total fluctuation of the blood sugar level is kept small in the range of normal fasting blood sugar level of SD rats, and the blood sugar level still reaches 35.75 percent of the initial blood sugar level at 12h, so that the oral insulin-loaded PEC has good slow release and hypoglycemic effects.

The corresponding time curve is shown in FIG. 14, and the insulin concentration in plasma rapidly increases after the subcutaneous injection of insulin solution in diabetic rats. The group of the insulin-loaded CS NPs has a small peak value within 1h, and then the peak value is reduced, and the concentration of the insulin in blood plasma is not high all the time, and the fluctuation range is not large; the group of insulin-loaded AL NPs also had a small peak at 1h, the blood concentration slowly increased from 2h to 8h, and after 8h reached the second small peak, the blood concentration began to gradually decrease and return to the level before the administration, but the plasma insulin concentration detected after the total administration was higher than that of the group of CSNPs. The first small peak after PEC group administration occurred at 2h, and after recovery after a decrease in plasma insulin concentration, a steady continuous increase began between 4h and 10h, and plasma concentrations reached a maximum of 44.19 μ IU/mL at 10h, and 35.75 μ IU/mL was still measured at 12h, with a relative bioavailability of 7.51%, all higher than those of the other three groups.

Compared with free insulin, the polymer nanoparticles and the polyelectrolyte compound are used as carriers to present insulin, so that the slow-release effect is obvious, and the hypoglycemic effect is better played. However, when the CS NPs and the AL NPs are taken as carriers alone for administration, the effect of reducing blood sugar is not ideal, the drug loading capacity of the CS NPs is low, the CS NPs have biological adhesion and can prolong the action time of the drugs on the mucosa of the small intestine, but when the CS NPs are taken as carriers alone for oral administration, the acidic environment and the action of enzyme can influence the stability of the drugs and the nanoparticle carriers when the CS NPs and the AL NPs pass through the stomach, so that the insulin which can successfully reach the absorption part of the intestinal tract and exert the drug effect is only a very small amount. Compared with the prior art, the drug loading rate of the AL NPs can reach more than 70 percent, so that the effect and the duration of the blood sugar reduction are better than those of the CS NPs after the AL NPs are taken as a carrier for oral administration, and the results are consistent with the experimental results. However, sodium alginate does not have the functions of chitosan bioadhesive and opening tight junctions between small intestinal epithelial cells, and thus insulin absorption at the site of action in the intestine is limited in the oral absorption route.

The polyelectrolyte compound has the advantage of cooperating with the two nanoparticles, the tight reticular cross-linked structure formed by the PEC has a protective effect in a gastric acid environment, provides a physical protective layer for insulin, protects the insulin from being damaged by strong acid and enzyme before release, can slowly and stably release the insulin in an intestinal juice neutral environment, and has a sustained-release hypoglycemic effect. Therefore, the PEC is taken as a carrier for administration, has the advantages of good hypoglycemic effect, slow release effect and safety in cooperation with the high drug-loading rate of the AL NPs and the advantages of the CS NPs in intestinal absorption promoters and intestinal mucoadhesives, and is an ideal oral drug carrier.

Claims

1. A polyelectrolyte complex is prepared by mixing copolymer nanoparticles with positive charges and copolymer nanoparticles with negative charges;

wherein the copolymer nanoparticles are water-in-oil (W/O) copolymer nanoparticle primary emulsion prepared by taking an amphiphilic block copolymer as an oil phase and taking an aqueous solution of an active medicament as an internal water phase; then preparing a double emulsion of water-in-oil-in-water (W/O/W) with an external aqueous phase, and dispersing the double emulsion in an emulsifier diluent;

wherein, the outer water phase of the copolymer nanoparticle with positive charge is a solution of a cationic biopolymer or a modified biopolymer thereof;

the outer water phase of the copolymer nanoparticle with negative charge is a solution of an anionic biopolymer or a biopolymer modified by the anionic biopolymer.

2. The polyelectrolyte complex according to claim 1, wherein the amphiphilic block polymer is selected from the group consisting of polyethylene glycol-polylactic acid-glycolic acid (mPEG-b-PLGA), polyethylene glycol-polylactic acid (PEG-b-PLA), polyethylene glycol-polyacetolactone (PEG-b-PCL), polyethylene glycol-polylactide-caprolactone (PEG-b-PLCL), preferably, the methoxy-terminated polyethylene glycol (mPEG) in mPEG-b-PLGA is mPEG₅₀₀₀(ii) a More preferably, the mPEG-b-PLGA is synthesized by D, L-lactide and glycolide through a ring-opening polymerization method, and the mPEG₅₀₀₀The mass fraction charged was 10%.

3. The polyelectrolyte complex according to claim 1, wherein the outer aqueous phase of the positively charged copolymer nanoparticles is selected from the group consisting of chitosan, N-succinylchitosan, carboxymethyl chitosan, Polyethyleneimine (PEI), Polyallylamine (PAH), poly (L-lysine), poly (L-arginine), polyvinylamine.

4. The polyelectrolyte complex according to claim 1, wherein the outer aqueous phase of the negatively charged copolymer nanoparticles is selected from the group consisting of alginate, polyglutamic acid, hyaluronic acid, pectin, glycosaminoglycan, polyaspartic acid, carboxymethylcellulose, and carboxymethylglucan.

5. The polyelectrolyte complex according to claim 1, wherein the emulsifier diluent is selected from poloxamer 188(F68), polyvinyl alcohol (PVA), polyoxyethylene alkyl ether, Sodium Dodecyl Sulfate (SDS) solution.

6. The polyelectrolyte complex according to claim 1, wherein the active drug is selected from the group consisting of insulin, insulin analogs, diabetic drugs, proteinaceous drugs, polypeptide drugs.

7. A method for preparing a polyelectrolyte complex, comprising the steps of:

ii) mixing the copolymer nanoparticles with positive charges or the copolymer nanoparticles with negative charges to obtain a polyelectrolyte compound;

wherein the positively charged copolymer nanoparticles or negatively charged copolymer nanoparticles of step i) are prepared by the following steps,

(1) preparing an active ingredient solution as an internal aqueous phase;

(5) preparing corresponding diluent, mixing the double emulsion and the diluent, removing the organic solvent, centrifuging and collecting the precipitate to obtain copolymer nanoparticles with positive charges or copolymer nanoparticles with negative charges;

8. The preparation method according to claim 7, wherein the volume ratio of the internal aqueous phase, the oil phase, the external aqueous phase and the diluent is 1:10:40:100 during the preparation process.

9. The preparation method according to claim 7, wherein the active ingredient is insulin, and the mass ratio of the loaded active ingredient to the copolymer mPEG-PLGA carrier material is preferably 5-20%, more preferably 10%.

10. Use of a polyelectrolyte complex according to claims 1-5 for the preparation of an oral formulation for the treatment of metabolic disorders; preferably, the metabolic disease is selected from diabetes, hyperglycaemia, more preferably type I diabetes or type II diabetes, and the drug is insulin or an insulin analogue.