CN111317824A

CN111317824A - Oral nano preparation carrying polypeptide medicine and preparation method thereof

Info

Publication number: CN111317824A
Application number: CN202010131850.0A
Authority: CN
Inventors: 姚萍; 包晓燕
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2020-02-29
Filing date: 2020-02-29
Publication date: 2020-06-23
Anticipated expiration: 2040-02-29
Also published as: CN111317824B

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an oral nano preparation carrying a polypeptide medicine and a preparation method thereof. The oral nano preparation carrying the polypeptide medicament takes alcohol-soluble corn protein as a main carrier, casein-glucan or casein-PEG covalent graft as a stabilizing agent, a protective agent and a lymphatic transport promoter, cholic acid and derivatives thereof as intestinal absorption promoters, and a nano particle system carrying the polypeptide medicament; the nano particle system is used as an oral preparation of the polypeptide medicament, and can effectively improve the oral treatment effect of the polypeptide medicament.

Description

Oral nano preparation carrying polypeptide medicine and preparation method thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an oral nano preparation carrying a polypeptide medicine and a preparation method thereof.

Background

The oral administration of polypeptide drugs has been a difficult problem in the field of drug research [ Advanced drug delivery reviews,2016,106:196-222 ]. For oral delivery systems of polypeptide drugs, three problems need to be solved first [ Advanced drug delivery reviews,2016,106:256-276 ]: firstly, the polypeptide medicament is protected from denaturation and decomposition in the severe pH change process from gastric juice to intestinal juice in the digestive tract; secondly, the polypeptide drug is protected from being degraded by protease which exists in the digestive tract in a large amount; the third problem to be solved is to promote the absorption of the polypeptide drug in the digestive tract, because the polypeptide has a large molecular weight and strong surface hydrophilicity, so that the polypeptide drug is difficult to pass through the intestinal mucus layer and the intestinal wall cell membrane to enter the blood circulation; there is also a need to address the issues of biocompatibility and safety of oral delivery systems [ Journal of controlled release,2013,166(2): 182-. Due to these problems, although oral formulations of polypeptide drugs have been studied for many years, their use is very limited.

Polypeptide drugs and polypeptide-loaded nanoparticles are absorbed in the intestinal tract mainly through two ways of transcellular transport and paracellular transport [ Advanced drug delivery reviews,2016,106:256-276 ]. Since tight junctions between enteroepithelial cells need to be reversibly opened for paracellular transport, there is a potential to increase the risk of diarrhea and autoimmune diseases [ Medical Electron Microscopy, 2003; 36: 147-56; autoimmiture Reviews, 2015; 479-89 ]. For the treatment and control of some diseases that require long-term or even lifelong treatment, such as diabetes, the safety, effectiveness, and compliance of pharmaceutical preparations are critical [ Advanced drug delivery reviews,2016,106: 196: 222 ]. Thus, transcellular transport may be a safer means of drug absorption. The nanoparticles and drugs are absorbed and transported by intestinal epithelial cells and enter the capillaries and lymphatic capillaries [ Journal of pharmacological-biological-dynamics, 1984,7(1):1-6 ]. The efficiency of nanoparticles and drugs entering the circulatory system through capillaries [ Science,1957,126(3284): 1176-.

Cholic acid is an amphiphilic small molecule produced by the liver, and common derivatives thereof include deoxycholic acid, taurocholic acid, glycocholic acid and the like. Cholic acid and its derivatives are widely used in the formulation of orally delivered polypeptides as intestinal absorption enhancers [ Advanced drug delivery reviews,2016,106:277- ] U.S. patent publication No. US 7196059. Studies have shown that the addition of cholic acid or cholic acid derivatives to insulin-loaded liposomes increases the resistance of insulin to enzymatic digestion and increases the endocytosis of the liposomes by epithelial cells [ European Journal of pharmaceuticals and biopharmaceutical therapeutics, 2012,81(2):265- ]. Bile acid transport receptors in the small intestine are capable of transporting nanoparticles with surface-bound bile acid groups, facilitating the nanoparticles to cross the cell membrane of the small intestine into the circulatory system [ Drug Delivery 2018,25(1): 1224-123 ]. The insulin-loaded nanoparticles have lysosome escape capacity after being transported into cells through bile acid receptors, and can avoid the degradation of insulin in cells [ Biomaterials,2018,151:13-23 ].

Glucan is a polysaccharide produced by microbial fermentation and mainly formed by linear polymerization of D-glucose through α - (1 → 6) glycosidic linkages [ Expert Opinion on Drug Delivery,2012,9(5): 509-.

Zein is a prolamin extracted from corn grain [ Industrial crops and products,2001,13(3): 171-. Zein is rich in hydrophobic amino acid residues and is deficient in basic and acidic amino acid residues, so zein can be dissolved in 40-95% ethanol solution and is insoluble in water [ Soft Matter,2013,9(25): 5933-. The zein has certain resistance to pepsin, and can increase the stability of the loaded drug in the stomach [ Current opinion in Colloid & Interface Science 2014,19(5): 450-. Since hydrophobic zein nanoparticles aggregate easily in aqueous solution [ Food Hydrocolloids,2015,49: 127-.

Casein is the major protein in mammalian milk casein in milk consists of 4 components α s1-, α s2-, β -, kappa-casein in a ratio of 4:1:4: 1. casein solubility in aqueous solution is related to pH and solubility is minimal near isoelectric point the isoelectric points of milk casein are pH 4.6, α -casein, β -casein, kappa-casein are pH 4.2, pH 4.7 and pH 5.3-5.8 respectively [ Food research international,2000,33(8):637 647 ] casein is generally considered to have a secondary structure, no tertiary structure, its behavior in aqueous solution is similar to that of amphiphilic block polymers, with hydrophobic and hydrophilic interactions [ casein & lacton & loc & Science 2002,7 (5-456): 5-6 ] casein is covalently grafted with polysaccharide via covalent reaction, covalent reaction with polysaccharide, covalent reaction, and covalent reaction, and covalent reaction, wherein casein is covalently grafted on water, polysaccharide is covalently grafted with polysaccharide, polysaccharide is covalently grafted on polysaccharide, polysaccharide is covalently grafted, polysaccharide is grafted, polysaccharide is grafted, polysaccharide is grafted, polysaccharide is grafted, polysaccharide.

Disclosure of Invention

The invention aims to provide an oral nano preparation carrying polypeptide medicine and capable of effectively improving the treatment effect of the medicine and a preparation method thereof.

The oral nano preparation for carrying the polypeptide drug is a nano particle which takes alcohol-soluble corn protein as a main carrier, casein-glucan or casein-PEG covalent graft as a stabilizing agent, a protective agent and a lymphatic transport promoter, and cholic acid and derivatives thereof as intestinal absorption promoters and loads the polypeptide drug.

In the casein-glucan covalent graft, the molecular weight of glucan in the casein-glucan covalent graft is 2-200kDa, preferably 5-70 kDa; the mass ratio of casein to dextran is 1:10 to 10:1, preferably 1:5 to 4: 1.

In the casein-PEG covalent graft, the molecular weight of PEG is 0.4-20kDa, preferably 1-5 kDa; the mass ratio of casein to PEG is between 1:10 and 10:1, preferably between 1:5 and 4: 1.

In the present invention, the mass ratio of the zein to the casein-dextran covalent graft, or the zein to the casein-PEG covalent graft is 1:10 to 10:1, preferably 1:1 to 5: 1.

In the invention, the mass ratio of the zein to the loaded polypeptide medicament is 10:1 to 500:1, and preferably 20:1 to 200: 1.

In the present invention, the mass ratio of zein to cholic acid or a derivative thereof is in the range of 5:1 to 200:1, preferably 10:1 to 50: 1.

In the invention, the polypeptide drug is a therapeutic polypeptide such as insulin, insulin glargine, insulin detemir, liraglutide, exenatide, somaglutide, lixivide, tasaglutide, albiglutide or dolaglutide.

In the invention, the intestinal absorption enhancer is cholic acid or derivatives thereof, including cholic acid, deoxycholic acid, glycocholic acid or taurocholic acid and the like.

The invention provides a preparation method of the oral nano preparation loaded with the polypeptide drug, which comprises the following specific steps:

(1) covalently grafting glucan to casein through Maillard reaction to obtain a casein-glucan covalent graft; wherein the molecular weight of dextran is 2-200kDa, preferably 5-70 kDa; the mass ratio of casein to glucan in the grafting reaction is 1:10-10:1, preferably 1:5-4: 1;

(2) covalently grafting PEG to casein through coupling reaction to obtain a casein-PEG covalent graft; wherein the molecular weight of PEG is 0.4-20kDa, preferably 1-5 kDa; the mass ratio of casein to PEG in the grafting reaction is 1:10-10:1, preferably 1:5-4: 1;

(3) respectively dissolving the polypeptide and the intestinal absorption enhancer in an aqueous solution, and then mixing the solutions to obtain a polypeptide/absorption enhancer mixed solution, wherein the concentration of the polypeptide is 0.1-100mg/mL, preferably 0.5-30 mg/mL; the concentration of the absorption enhancer is 1-1000mg/mL, preferably 5-80 mg/mL;

(4) dissolving zein in an ethanol-water mixed solvent, wherein the concentration of the zein is 5-500mg/mL, preferably 10-200 mg/mL;

(5) dissolving the casein covalent graft prepared in the step (1) or the step (2) in water, wherein the concentration is 1-600mg/mL, and preferably 1-30 mg/mL;

(6) mixing the solution prepared in the step (3) with the solution prepared in the step (4), wherein the volume ratio of ethanol to water in the mixed solution is 1:2-9:1, preferably 1:1-9: 1;

(7) and (3) mixing the solution prepared in the step (6) and the solution prepared in the step (5) according to the volume ratio of 2:1-1:20 (preferably 1:1-1: 10), and stirring to obtain the polypeptide-loaded nanoparticle solution.

The nano preparation can be used as an oral preparation of a polypeptide-carrying medicine.

In the invention, the zein nanoparticles loaded with the polypeptide drug, the protective agent, the stabilizing agent and the absorption enhancer are prepared by using the alcohol-soluble zein as a main carrier. The protective agent loaded by the corn protein nano particles can protect the loaded polypeptide drug from being degraded in the digestive tract, and the loaded absorption enhancer can increase the absorption of the polypeptide drug in the intestinal tract and the transport of the polypeptide drug in the lymphatic capillaries.

In the invention, the glucan is covalently grafted to the casein through Maillard reaction to obtain a casein-glucan covalent graft. The grafted glucan not only has the function of promoting lymphatic transport [ Journal of pharmacological-biological, 1984,7(1):1-6 ], but also has the function of inhibiting degradation of digestive tract protease [ Journal of aggregative and food chemistry,2014,62(35):8900-8907 ], so that the loaded polypeptide drug can be protected from degradation in the digestive tract; in addition, the nano particles also have the effects of increasing the dispersibility of the nano particles in an aqueous solution and inhibiting the aggregation of the nano particles in the aqueous solution. Maillard reaction conditions of casein and glucan can be realized by the prior experimental technology. The number and chain length of the polysaccharides grafted in the casein-dextran covalent grafts can be adjusted by controlling the dosage ratio of casein and dextran and the molecular weight of dextran [ Critical reviews in food science and nutrition,2016,56(7):1108-1125 ].

Experiments show that the lymphatic transport promoter glucan or PEG is introduced into the polypeptide drug-loaded nanoparticles, so that the oral treatment effect of the loaded polypeptide drug can be remarkably improved.

Drawings

Fig. 1 is a graph showing the relative blood glucose concentration versus time for diabetic mice injected with insulin subcutaneously, with normal saline orally, with nanoparticles orally, with cyclimide (a lymphatic transport inhibitor) injected intraperitoneally first followed by nanoparticles orally, with cholic acid (a bile acid channel transport inhibitor) first followed by nanoparticles orally.

Detailed Description

Example 1 preparation of Casein-dextran covalent grafts by Maillard reaction

Adding Casein (CN) into deionized water, wherein the concentration is 20mg/mL, and adding NaOH to adjust the pH of the solution to 7.4; adding Dextran (DEX) with molecular weight of 1 ten thousand (10kDa) or 3.5 ten thousand (35kDa) into deionized water, and the concentration is 20 mg/mL; uniformly mixing a casein aqueous solution and a glucan aqueous solution, wherein the mass ratio of casein to glucan is 1:1 or 1:2, freezing and drying the mixed solution, placing the freeze-dried powder in a beaker, placing the beaker in a closed container filled with a saturated KBr solution (the relative humidity of the beaker is 79%), and reacting for 24 hours at 60 ℃ to obtain a casein-glucan covalent graft, wherein the sample name is shown in Table 1.

Table 1. casein-dextran covalent grafts prepared by Maillard reaction.

Sample name	Casein to dextran mass ratio	Dextran molecular weight (Da)
			C1-D1-10k	1:1	10k
C1-D2-10k	1:2	10k
			C1-D1-35k	1:1	35k

。

Example 2 preparation of nanoparticles containing 4 Components by direct Dispersion-aqueous solution Medium pH7.4 buffer

Preparing an organic phase solution: dissolving insulin in 1% citric acid aqueous solution with the concentration of 8 mg/mL; adding cholic acid into deionized water, wherein the concentration is 20mg/mL, and adding NaOH to adjust the pH of the solution to 7.4; dissolving zein in 90% ethanol, wherein the concentration is 50-100 mg/mL; uniformly mixing an insulin solution, a cholic acid solution, absolute ethyl alcohol and a zein solution according to the volume ratio of 1:1:2:4 to obtain an organic phase solution. The following aqueous solutions were prepared: (1) dissolving casein in 20mmol/LpH7.4 phosphate buffer solution, wherein the concentration of casein is 2.5 mg/mL; (2) the casein-dextran covalent grafts were dissolved in 20mmol/LpH7.4 phosphate buffer, the concentration of the grafts was 5 or 7.5mg/mL respectively depending on the sample, but 2.5mg/mL was calculated as the casein concentration. And (3) adding 4mL of aqueous phase solution into 1mL of the organic phase solution, placing the mixed solution at room temperature, stirring for 3 hours under an open condition to volatilize ethanol, and then adding deionized water to reach a constant volume of 5mL to obtain the insulin-loaded nanoparticles shown in Table 2. The particle size (hydration diameter D) of the nanoparticles was determined by means of a light scattering particle size analyser (Zetasizer Nano ZS90, Malvern)_h) Polydispersity (PDI) and zeta potential.

According to the literature, a fluorescent probe Fluorescein Isothiocyanate (FITC) labeled insulin (F-INS) [ molecular conjugates, 2016,13:2433-42 ] is prepared, F-INS loaded nanoparticles are prepared by the method, then F-INS which is not embedded in the solution is separated by an ultrafiltration method (ultrafiltration membrane molecular weight cutoff is 50kDa), the concentration of the F-INS in the ultrafiltrate is determined by a multifunctional microplate tester (catalysis 3, BioTek), and the insulin embedding efficiency of the nanoparticles is calculated by the following formula:

encapsulation efficiency (%) - ((total insulin mass-non-encapsulated insulin mass)/total insulin mass) × 100%.

Table 2 composition, properties and encapsulation efficiency of insulin-loaded nanoparticles prepared according to the method of example 2 (n-3).

a:C1-D1-10k；b:CN；c:C1-D1-35k；d:C1-D2-10k。

Example 3 preparation of nanoparticles containing 6 components by direct Dispersion method-aqueous phase solution medium organic phase solution was prepared with pH7.4 buffer: dissolving decanoic acid in absolute ethyl alcohol, wherein the concentration is 20 mg/mL; dissolving egg yolk lecithin in anhydrous ethanol, wherein the concentration is 20 mg/mL; dissolving insulin in 1% citric acid aqueous solution with the concentration of 8 mg/mL; adding cholic acid into deionized water, wherein the concentration is 20mg/mL, and adding NaOH to adjust the pH of the solution to 7.4; dissolving zein in 90% ethanol, wherein the concentration is 50-100 mg/mL; uniformly mixing an insulin solution, a cholic acid solution, a capric acid solution, a phospholipid solution and a zein solution according to the volume ratio of 1:1:1:1:4 to obtain an organic phase solution. The following aqueous solutions were prepared: (1)20mmol/LpH7.4 phosphate buffer; (2) dissolving casein in 20mmol/LpH7.4 phosphate buffer solution, wherein the concentration of casein is 2.5 mg/mL; (3) the casein-dextran covalent grafts were dissolved in 20mmol/LpH7.4 phosphate buffer, the concentration of the grafts was 5 or 7.5mg/mL respectively depending on the sample, but 2.5mg/mL was calculated as the casein concentration. And (3) adding 4mL of aqueous phase solution into 1mL of the organic phase solution, placing the mixed solution at room temperature, stirring for 3 hours under an open condition to volatilize ethanol, and then adding deionized water to reach a constant volume of 5mL to obtain the insulin-loaded nanoparticles shown in Table 3. The nanoparticles were analyzed for particle size, zeta potential and insulin encapsulation efficiency as per example 2. As can be seen from the data in Table 3, the insulin embedding efficiency of the nanoparticles is greatly improved when the zein content is increased from 5mg/mL to 10 mg/mL.

Table 3 composition, properties and encapsulation efficiency of insulin-loaded nanoparticles prepared according to the method of example 3 (n-3).

a:C1-D1-10；b:CN；c:C1-D1-35k；d:C1-D2-10k。

EXAMPLE 4 preparation of nanoparticles containing 6 Components by direct Dispersion deionized Water as the aqueous solution Medium

An organic phase solution was prepared as in example 3. The following aqueous solutions were prepared: (1) deionized water; (2) dissolving casein in deionized water, wherein the concentration of the casein is 2.5 mg/mL; (3) the casein-dextran covalent grafts were dissolved in deionized water at a concentration of 5, 7.5 or 10mg/mL, respectively. And (3) adding 4mL of aqueous phase solution into 1mL of organic phase solution, placing the mixed solution at room temperature, stirring for 3 hours under an open condition to volatilize ethanol, and then adding deionized water to a constant volume of 5mL to obtain the insulin-loaded nanoparticles shown in Table 4. The nanoparticles were analyzed for particle size, zeta potential and insulin encapsulation efficiency as per example 2. Comparing the data in tables 4 and 3, it can be seen that the nanoparticles prepared in the aqueous solution medium of deionized water and 20mmol/L of phosphate buffer solution with pH7.4 have similar insulin embedding efficiency. The data in table 4 also show that casein without grafted dextran cannot produce nanoparticles under the condition that the aqueous medium is deionized water; increasing the concentration of graft C1-D1-10k from 4mg/mL to 8mg/mL reduced the insulin-embedding efficiency of the nanoparticles.

Table 4 composition, properties and encapsulation efficiency of insulin-loaded nanoparticles prepared according to the method of example 4 (n-3).

a:C1-D1-10k；b:C1-D1-35k；c:C1-D2-10k；d:CN。

EXAMPLE 5 preparation of nanoparticles comprising 6 Components by Complex method-aqueous solution Medium is pH7.4 buffer

Dissolving insulin in a methanol solution containing 1% citric acid, wherein the concentration is 4 mg/mL; dissolving cholic acid in methanol at a concentration of 5 mg/mL; dissolving egg yolk lecithin in methanol at a concentration of 10 mg/mL; capric acid was dissolved in methanol at a concentration of 10 mg/mL. Cholic acid was dissolved in ethanol at a concentration of 2.5 mg/mL. Uniformly mixing the insulin methanol solution, the cholic acid methanol solution, the decanoic acid methanol solution and the phospholipid methanol solution according to the volume ratio of 1:2:2:2, removing the solvent by vacuum rotary evaporation at the temperature of 30 ℃, and drying by using nitrogen flow. Adding cholic acid ethanol solution into dried insulin/phospholipid/capric acid/cholic acid complex, and performing ultrasonic treatment for 1min to obtain insulin/phospholipid/capric acid/cholic acid complex solution.

Zein was dissolved in 90% ethanol at a concentration of 100 mg/mL. And uniformly mixing the compound solution and the zein solution according to the volume ratio of 1:1 to obtain an organic phase solution. The following aqueous solutions were prepared: (1)20mmol/LpH7.4 phosphate buffer; (2) dissolving casein in 20mmol/LpH7.4 phosphate buffer solution, wherein the concentration of casein is 2.5 mg/mL; (3) the casein-dextran covalent grafts were dissolved in 20mmol/LpH7.4 phosphate buffer, the concentration of the grafts was 5 or 7.5mg/mL respectively depending on the sample, but 2.5mg/mL was calculated as the casein concentration. And (3) adding 4mL of aqueous phase solution into 1mL of the organic phase solution, placing the mixed solution at room temperature, stirring for 3 hours under an open condition to volatilize ethanol, and then adding deionized water to fix the volume to 5mL to obtain the nanoparticles carrying the insulin/phospholipid/decanoic acid/cholic acid compound shown in the table 5, wherein the pH value of the nanoparticle solution is approximately equal to 6. The nanoparticles were analyzed for particle size, zeta potential and insulin encapsulation efficiency as per example 2. Comparing the data in tables 5 and 3, it was found that the nanoparticle loaded with the complex can improve the insulin embedding efficiency, but the improvement is not large.

Table 5 composition, properties and encapsulation efficiency of nanoparticles loaded with insulin/phospholipid/decanoic acid/cholic acid complexes prepared according to the method of example 5 (n ═ 3).

a:C1-D1-10k；b:C1-D1-35k；c:C1-D2-10k；d:CN。

EXAMPLE 6 preparation of nanoparticles comprising 6 Components by Complex method-aqueous solution Medium is deionized Water

An insulin/phospholipid/decanoic acid/cholic acid complex solution was prepared by the method of example 5. Zein was dissolved in 90% ethanol at a concentration of 100 mg/mL. And uniformly mixing the compound solution and the zein solution according to the volume ratio of 1:1 to obtain an organic phase solution. The following aqueous solutions were prepared: (1) deionized water; (2) dissolving casein in deionized water, wherein the concentration of the casein is 2.5 mg/mL; (3) the casein-dextran covalent grafts were dissolved in deionized water at a concentration of 5, 7.5 or 10mg/mL, respectively. Taking 1mL of organic phase solution, adding 4mL of aqueous phase solution, placing the mixed solution at room temperature, stirring for 3 hours under an open condition to volatilize ethanol, then adding deionized water to fix the volume to 5mL to obtain the nano particles loaded with the insulin/phospholipid/decanoic acid/cholic acid compound shown in the table 6, wherein the pH value of the nano particle solution is approximately equal to 4.5. The nanoparticles were analyzed for particle size, zeta potential and insulin encapsulation efficiency as per example 2. Comparing the data in tables 5 and 6, it can be seen that the nanoparticles prepared by the method of example 5 have higher insulin embedding efficiency for the nanoparticles loaded with the complexes. The data in table 6 also show that casein without grafted dextran cannot produce nanoparticles under the condition that the aqueous medium is deionized water.

Table 6 composition, properties and entrapment efficiency of nanoparticles loaded with insulin/phospholipid/decanoic acid/cholic acid complexes prepared according to the method of example 6 (n ═ 3).

a:C1-D1-10k；b:C1-D1-35k；c:C1-D2-10k；d:CN。

EXAMPLE 7 preparation of a Casein-Casein covalent graft containing 6 Components by Complex Process to Casein-PEG

Dissolving casein in deionized water, wherein the concentration of the casein is 0.2mg/mL, and adjusting the pH of the solution to 8.0 by using NaOH; dissolving polyethylene glycol-ester (PEG-ester, mPEG-NHS-2k, molecular weight 2kDa) with the end group modified with succinimide group in deionized water, wherein the concentration is 2 mg/mL; mixing the two aqueous solutions according to the mass ratio of casein to PEG-ester of 1:1, stirring at room temperature for 4 hours, dialyzing to remove unreacted PEG-ester, and lyophilizing to obtain casein-PEG covalent graft (CN-PEG). The covalent grafts were dissolved in 20mmol/L phosphate buffer pH7.4 at a concentration of 5mg/mL as aqueous phase. The organic phase was prepared as in example 5. And (3) adding 4mL of aqueous phase solution into 1mL of organic phase solution, placing the mixed solution at room temperature, stirring for 3 hours under an open condition to volatilize ethanol, and then adding deionized water to reach a constant volume of 5mL to obtain the nanoparticles carrying the insulin/phospholipid/decanoic acid/cholic acid compound shown in the table 7. The nanoparticles were analyzed for particle size, zeta potential and insulin encapsulation efficiency as per example 2.

Table 7 composition, properties and entrapment efficiency of nanoparticles loaded with insulin/phospholipid/decanoic acid/cholic acid complexes prepared according to the method of example 7 (n ═ 3).

Example 8 Effect of nanoparticles on Caco-2 cell Activity

At 37 deg.C, 5% CO₂In a constant temperature incubator in a humid environment, Caco-2 cells are cultured by using a complete culture medium, and the cells are cultured in a 96-well plate according to the density of 5000 cells/well; after 24 hours, replacing the culture solution with an incomplete culture medium solution containing nanoparticles with different concentrations; after 48 hours of culture, the cells were replaced with 120. mu.L of an incomplete medium solution containing 16.7% MTS reagent, and after further 2 hours of incubation in the dark, the absorbance A of the sample at 490nm was measured using a multi-function microplate reader, and the cell survival rate was calculated using the following formula:

the data in Table 8 show that the nanoparticle sample containing capric acid and phospholipid (B1-1) has low cytotoxicity, and the cell survival rate is about 70% when the nanoparticle concentration is 0.5 mg/mL. The samples of nanoparticles (A1, A3, A4, A5) without decanoic acid and phospholipid, prepared according to the method of example 2, had very good cell compatibility, and the cell viability of each sample was greater than 90% at a nanoparticle concentration of 1 mg/mL.

Table 8 survival of Caco-2 cells after 48 hours co-incubation with different concentrations of nanoparticles (n-3).

Example 9 uptake of nanoparticles by Caco-2 cells

Preparation of fluorescent Probe Fluorescein Isothiocyanate (FITC) -labeled insulin (F-INS) according to example 2, preparation of rhodamine B isothiocyanate (RITC) -labeled Zein (R-Zein) according to the literature [ Journal of Materials Chemistry,2011,21(45):18227-⁵The cells were cultured in a 24-well plate in a constant temperature incubator for 24 hours. The nanoparticle solutions were diluted with cell culture medium to a final zein concentration of 50. mu.g/mL in each nanoparticle solution. 0.5mL of the solution was added to each well, after incubation for 2 hours, the medium was discarded, the uningested nanoparticles were removed by washing three times with PBS, the cells were trypsinized and then resuspended with PBS, and the fluorescence intensity after nanoparticle uptake by the cells was measured and counted using a flow cytometer (Gallios, Beckman Coulter). From the cell geometric mean fluorescence intensities listed in table 9, the following conclusions can be drawn: (1) the nanoparticle samples A1, B1-1 and C1-1 prepared by the methods of example 2, example 3 and example 5 had similar cellular uptake effects; (2) the efficiency of cellular uptake of cholic acid-free nanoparticle sample a2 was reduced by about 47% compared to cholic acid-containing nanoparticle sample a 1; (3) the samples B1-4 prepared using Casein (CN) without grafted dextran, and the samples A4 and A5 prepared using C1-D1-35k and C1-D2-10k, were not taken up by cells as well as the samples B1-1 and A1 prepared using C1-D1-10 k. Number of Table 9The cholic acid in the nanoparticles can promote the Caco-2 cells to take up the nanoparticles; the casein-glucan covalent graft in the nanoparticle is C1-D1-10k, which is beneficial to the uptake of Caco-2 cells.

Table 9 geometric mean fluorescence intensity of Caco-2 cells after 2 hours co-incubation with various nanoparticles labeled with dual fluorescent probes (n ═ 3).

Example 10 diabetic mouse model establishment and evaluation of in vivo hypoglycemic Effect

Healthy female ICR mice (25 +/-2 g) are fasted for 12 hours (can freely drink water), and then are intraperitoneally injected with a tetraoxypyrimidine physiological saline solution with the concentration of 24mg/mL at the dose of 180-190mg/kg, and the mice can freely feed and feed water after 1 hour of injection; after 3 days, the fasting blood glucose of the mice is measured by a glucometer (activity type, Roche) and blood glucose test paper, and the mice with the fasting blood glucose concentration higher than 16mmol/L are selected as the mice with type I diabetes which are successfully modeled.

Diabetic mice were randomly grouped into 5 mice each. The group orally took normal saline as a negative control group; the insulin solution injected subcutaneously in the second group is used as a positive control group, and the other groups are orally taken with the nano particle solution; measuring the blood glucose value by tail vein blood sampling at a predetermined time point; mice were free to feed for 1 hour at 12, 24 and 36 hour time points post-dose, and were fasted (freely accessible to water) for the remainder of the time. Area above blood glucose reduction curve (AAC) in interval 0-48h after administration according to administration group_0-48) The pharmacodynamic bioavailability (PA) of the oral group was calculated by the following formula, Dose being insulin:

the results of the batch 1 experiment in Table 10 show that the oral pharmacodynamic availability of insulin is 3 times greater for the C1-8 sample prepared with C1-D1-10k than for the C1-7 sample prepared with Casein (CN) using the method of example 5, indicating that dextran covalently grafted to casein has the effect of protecting insulin and promoting the oral pharmacodynamic action of insulin.

The experimental results of batch 2 showed that for the same composition of nanoparticles, the B1-1 sample prepared by the direct dispersion method of example 3 and the C1-1 sample of the loaded complex prepared by the method of example 5 had similar oral pharmacodynamic availability of insulin; the blood sugar reducing effect of the C1-1 sample with the zein concentration of 10mg/mL is better than that of the C1-2 sample with the zein concentration of 5 mg/mL.

The experimental results of batch 3 showed that the sample of B1-1 prepared by the method of example 3, the sample of B2-1 prepared by the method of example 4, and the sample of C1-1 prepared by the method of example 5 had similar oral pharmacodynamic availability of insulin for nanoparticles of the same composition.

The results of the batch 4 experiment show that 3 different samples B1-1(C1-D1-10k), B1-6(C1-D1-35k), B1-7(C1-D2-10k) prepared using the method of example 3 and 3 casein-dextran covalent grafts have similar oral pharmacodynamic availability of insulin.

Table 10 analysis of pharmacodynamic data of diabetic mice orally administered with normal saline, subcutaneously injected with insulin, and orally administered with various nanoparticles (n ═ 5).

^aΔ BGL is the difference in percent blood glucose concentration between the saline group and the administered group.

Example 11 Effect of inhibitors on the hypoglycemic Effect of nanoparticles

A sample of nanoparticle a1 was prepared using the method of example 2; type I diabetic mice were molded, screened and the A1 samples were evaluated for hypoglycemic effects according to the method of example 10. Free cholic acid molecules can inhibit the absorption of nanoparticles through cholic acid channels [ Journal of Controlled Release,2014,177:64-73 ]; cyclo-imides are inhibitors of uptake by lymphatic transport [ European journal of pharmaceutical sciences,2005,24(4):381-388 ]. For the cholic acid inhibitor group (A1+ cholic acid), mice were orally administered cholic acid solution 30 minutes prior to oral administration of the A1 sample at a dose of 25 mg/kg; for the lymphokinesis inhibitor cyclo-succinimide group (A1+ cyclo-succinimide), mice were injected intraperitoneally with a cyclo-imide solution at a dose of 6mg/kg prior to oral administration of the A1 sample. The results in table 11 show that cyclic succinimide has a significant inhibitory effect on the drug effect of a1, indicating that a part of a1 nanoparticles are absorbed through the lymphatic system; cholic acid also has inhibitory effects on the drug effect of a1, indicating that a portion of a1 nanoparticles may also be absorbed through cholic acid channels.

Table 11. analysis of pharmacodynamic data of diabetic mice orally administered physiological saline, subcutaneously injected insulin, and orally administered a1 nanoparticles, and effects of cholic acid and cyclic imide on hypoglycemic effect of a1 nanoparticles (n ═ 5).

Claims

1. An oral nano preparation carrying polypeptide drugs is characterized in that the oral nano preparation is a nano particle system which takes alcohol-soluble corn protein as a main carrier, casein-dextran or casein-PEG covalent grafts as a stabilizing agent, a protective agent and a lymphatic transport promoter, and cholic acid and derivatives thereof as intestinal absorption promoters and carries the polypeptide drugs.

2. The oral nanoformulation of polypeptide-loaded drugs according to claim 1, wherein in the casein-dextran covalent grafts, dextran has a molecular weight of 2-100 kDa; the mass ratio of casein to dextran is 1:10 to 10: 1.

3. The oral nanoformulation of polypeptide-loaded drugs according to claim 1, wherein, in the casein-PEG covalent grafts, the PEG has a molecular weight of 0.4-20 kDa; the mass ratio of casein to PEG is 1:10 to 10: 1.

4. The oral nanoformulation for carrying polypeptide drugs according to claim 1, wherein the mass ratio of zein to casein-dextran covalent grafts is 1:10 to 10: 1; the mass ratio of zein to casein-PEG covalent grafts ranges from 1:10 to 10: 1.

5. The oral nanoformulation of polypeptide-loaded drug according to claim 1, wherein the mass ratio of zein to loaded polypeptide drug is in the range of 10:1 to 500: 1.

6. The oral nano-preparation of the polypeptide-loaded drug as claimed in claim 1, wherein the mass ratio of zein to cholic acid or its derivatives is in the range of 5:1 to 200: 1.

7. The polypeptide-drug-loaded oral nanoformulation of claim 1, wherein the polypeptide drug is an insulin, insulin glargine, insulin detemir, liraglutide, exenatide, somaglutide, lixivide, tasaglutide, albiglutide, or dolaglutide therapeutic polypeptide.

8. The oral nanoformulation according to claim 1, wherein the cholic acid or derivative is cholic acid, deoxycholic acid, glycocholic acid or taurocholic acid.

9. A method for preparing the oral nano preparation of the polypeptide-carrying drug according to any one of claims 1 to 8, which comprises the following steps:

(1) covalently grafting glucan to casein through Maillard reaction to obtain a casein-glucan covalent graft; wherein the molecular weight of the glucan is 2-200 kDa; the mass ratio of casein to glucan in the grafting reaction is 1:10-10: 1;

(2) covalently grafting PEG to casein through coupling reaction to obtain a casein-PEG covalent graft; wherein the molecular weight of PEG is 0.4-20 kDa; the mass ratio of casein to PEG in the grafting reaction is 1:10-10: 1;

(3) respectively dissolving the polypeptide and the intestinal absorption enhancer in an aqueous solution, and then mixing the solutions to obtain a polypeptide/absorption enhancer mixed solution, wherein the concentration of the polypeptide is 0.1-100 mg/mL; the concentration of the absorption enhancer is 1-1000 mg/mL;

(4) dissolving zein in an ethanol-water mixed solvent, wherein the concentration of the zein is 5-500 mg/mL;

(5) dissolving the casein covalent graft prepared in the step (1) or the step (2) in water, wherein the concentration is 1-600 mg/mL;

(6) mixing the solution prepared in the step (3) with the solution prepared in the step (4), wherein the volume ratio of ethanol to water in the mixed solution is 1:2-9: 1;

(7) and (3) mixing the solution prepared in the step (6) and the solution prepared in the step (5) according to the volume ratio of 2:1-1:20, and stirring to obtain the polypeptide-loaded nanoparticle solution.