CN100374477C - A kind of mucoadhesive nanoparticle and its preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of adhesive nano-materials, and specifically relates to a mucoadhesive nano-particle and a preparation method and application thereof. The mucoadhesive nanoparticles of the present invention use polyalkyl acrylate, polyalkyl methacrylate or polycyanoacrylate as the skeleton, and mucoadhesive polymers as the coating material. It is prepared according to the following steps: after dissolving the mucoadhesive polymer, adding the framework material, adding the initiator, heating, continuing the reaction, and purifying. It can be used in the production of medicines, nutrition and cosmetics. Compared with the prior art, it has the characteristics of better stability and prolonged action time of active ingredients.

Description

A kind of mucoadhesive nanoparticle and its preparation method and application

technical field

The invention belongs to the technical field of adhesive nanomaterials, and in particular relates to a mucoadhesive nanoparticle and a preparation method and application thereof.

Background technique

Protein and polypeptide drugs have short biological half-lives, poor biofilm permeability, and are easily degraded by gastrointestinal enzymes. Improving their oral bioavailability has become one of the current research hotspots in pharmacy. Mucoadhesive nanoparticles, which have been researched in recent years, can prolong the residence time of drugs at the absorption site to promote drug absorption; some preparation materials also have enzyme inhibition and permeation promotion effects, which can prevent enzymatic degradation of drugs and promote transmembrane transport of drugs. Thereby improving the bioavailability of protein peptide drugs. At present, there are relatively few studies on mucoadhesive nanoparticles at home and abroad.

Takeucki et al improved the oral bioavailability of calcitonin by coating polylactide copolymer nanoparticles with chitosan (Mucoahesive DL-Lactide/glycolide copolymer nanoparticles coated with chitosan to improve oraldelivery of elcatonin, Pharm.Dev. Tech.2000 (5): 77-85), in the preparation process of the above-mentioned nanoparticles, organic solvents such as methanol and chloroform were used and ultrasound was carried out. These methods easily lead to the inactivation of protein and polypeptide drugs. Studies have shown that chitosan cannot improve the oral bioavailability of protein and peptide drugs under high pH conditions. Xu Yongmei et al. formed adhesive nanoparticles by ionically cross-linking chitosan and chitosan quaternary ammonium salt with sodium tripolyphosphate (Effect of molecular structure of chitosan nanoparticles Int.J.Pharm.2003, 25 (1) : 215-226), although the destruction of the active ingredient by organic solvents has been avoided, the nanoparticle has pH instability, which limits its application as an oral delivery carrier for protein and polypeptide drugs.

Contents of the invention

The object of the present invention is to provide a mucoadhesive nanoparticle with stable performance and wide application and its preparation method, and to provide the application of the mucoadhesive nanoparticle to overcome the deficiencies and defects of the prior art.

Design of the present invention is as follows:

In the gastrointestinal tract, alkyl acrylates, alkyl methacrylates, or alkyl cyanoacrylates are not easily degraded and can protect the drug; in aqueous solution, alkyl acrylates, alkyl methacrylates, or cyanoacrylates Alkyl esters are easier to form nanoparticles, avoid the use of various organic solvents, and are beneficial to improve the stability of protein and polypeptide drugs.

Chitosan derivatives with different degrees of substitution-chitosan quaternary ammonium salt, carboxylated chitosan and carboxymethyl chitosan undergo graft polymerization with alkyl acrylate or methacrylate to form mucoadhesive Sexual nanoparticles, and then mixed with protein peptide drugs for encapsulation, not only avoided the use of organic solvents in the preparation process, but also chitosan derivatives will not be precipitated due to insolubility under high pH conditions, And chitosan derivatives have the functions of promoting penetration, inhibiting enzymes, and stabilizing proteins. Sodium alginate can prevent the diffusion of drugs or enzymes to a certain extent through the steric hindrance and viscosity of the polymer, and protect the drugs from being degraded by enzymes. Nanoparticles coated with PEG can increase the absorption of protein and polypeptide drugs loaded in nanoparticles through the mucosa, and PEG-coated nanoparticles are a better carrier for vaccine delivery through the mucosa. In the present invention, when the alkyl acrylate, the alkyl methacrylate or the alkyl cyanoacrylate forms ordinary nanoparticles, the mucoadhesive material is coated on the nanoparticles by grafting or physical adsorption. The properties of mucoadhesive nanoparticles and drug efficacy after drug loading were investigated.

Technical scheme of the present invention is as follows:

The mucoadhesive nanoparticles provided by the present invention use polyalkyl acrylate, polyalkyl methacrylate or polycyanoacrylate as the skeleton, and mucoadhesive polymers as the coating material.

Wherein the weight ratio of the coating material to the framework material is in the range of 1:0.05 to 1:20.

Said alkyl methacrylate is one of methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, isobutyl methacrylate and hexyl methacrylate or more than one.

Said alkyl acrylate is one or more of methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, isobutyl acrylate and hexyl acrylate.

Said alkyl cyanoacrylate is one of methyl cyanoacrylate, ethyl cyanoacrylate, propyl cyanoacrylate, butyl cyanoacrylate, isobutyl cyanoacrylate and hexyl cyanoacrylate or more than one.

The mucoadhesive polymer is one of chitosan derivatives, sodium alginate, polyethylene glycol, polyacrylic acid and carbopol.

Said chitosan derivatives are carboxymethyl chitosan, carboxylated chitosan, chitosan quaternary ammonium salt, chitosan hydrochloride, chitosan glutamate and chitosan lactate One of.

The molecular weight of said polyethylene glycol is one of 400, 1500, 6000, 20,000.

Said Carbopol class is one of Carbopol 934PNF, 974PNF, 971PNF.

The mucoadhesive nanoparticles of the present invention have a particle size of 100-300nm.

The preparation method of the mucoadhesive nanoparticles of the present invention is as follows:

Dissolve the mucoadhesive polymer as the coating material in a solvent, heat to 25-60°C, add alkyl acrylate, alkyl methacrylate or alkyl cyanoacrylate as the skeleton material, and react for 10-30 Minutes, then add the initiator, heat to 70-85°C, continue the reaction for 10-36 hours, and obtain the product after purification.

Wherein the weight ratio of the coating material to the framework material is in the range of 1:0.05 to 1:20.

Said alkyl methacrylate is one of methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, isobutyl methacrylate and hexyl methacrylate or more than one.

Said alkyl acrylate is one or more of methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, isobutyl acrylate and hexyl acrylate.

Said alkyl cyanoacrylate is one of methyl cyanoacrylate, ethyl cyanoacrylate, propyl cyanoacrylate, butyl cyanoacrylate, isobutyl cyanoacrylate and hexyl cyanoacrylate or more than one.

The mucoadhesive polymer is one of chitosan derivatives, sodium alginate, polyethylene glycol, polyacrylic acid and carbopol.

Said chitosan derivatives are carboxymethyl chitosan, carboxylated chitosan, chitosan quaternary ammonium salt, chitosan hydrochloride, chitosan glutamate and chitosan lactate One of. Its dosage is 0-1%, calculated by mass percentage.

The molecular weight of said polyethylene glycol is one of 400, 1500, 6000, 20,000.

Said Carbopol class is one of Carbopol 934PNF, 974PNF, 971PNF.

The solvent for dissolving the mucoadhesive polymer is 1% acetic acid or water.

Said initiator is ammonium persulfate or persulfuric acid. Its dosage is 0.01-0.2% g/ml of the reaction system.

In the present invention, a semipermeable membrane is used for dialysis for purification, and the specification of the semipermeable membrane used is 12,000 Daltons.

In the present invention, the suspension obtained by dialysis is mixed with protein polypeptide drugs or other macromolecular drugs, warmed for 2 to 4 hours, then centrifuged at high speed, the supernatant is taken out, and the protein polypeptide drugs or other macromolecular drugs in the supernatant are determined. The concentration of the drug was calculated, the encapsulation efficiency was calculated, and the release test and in vivo test were performed on the mucoadhesive drug-loaded nanoparticles obtained by centrifugation.

The mucoadhesive nanoparticles of the present invention can be used as carriers for carrying proteins, polypeptides, nucleic acids and vaccines.

The nanoparticles according to the invention can also be used for the production of special products for nutrition, plant protection or cosmetics. Cosmetic applications such as the combination of nanoparticles and active ingredients can be used transdermally.

The advantages of the present invention are:

(1) The particle size of the mucoadhesive nanoparticles is uniform, and the size can be controlled, and the dispersion is uniform. Stable under room temperature storage and in vivo conditions.

(2) It has mucoadhesive properties, which can prolong the residence time of the active ingredient on the mucous membrane and improve the effect of the active ingredient.

(3) The preparation is convenient and easy, and no organic solvent is required.

(4) Under neutral and alkaline conditions, the chitosan quaternary ammonium salt is positively charged, and the formed mucoadhesive nanoparticles are still positively charged. Therefore, when the active ingredient is administered orally with it as a carrier, the pH The tight junctions of the mucosal epithelium are also opened in the higher intestinal segments, increasing the penetration of active ingredients through the intestinal mucosa.

(5) Under strong acid conditions, the mucoadhesive nanoparticles of carboxylated chitosan and carboxymethyl chitosan coagulate, and then disperse into nanoparticles under weak acid, neutral and alkaline conditions, which is beneficial to the active ingredients through Oral administration.

(6) Carboxylated chitosan and carboxymethyl chitosan contain carboxyl groups, which can inhibit enzymes.

(7) Sodium alginate is used as a coating material, which improves the stability of the nanoparticles and has mucoadhesive properties. Sodium alginate is a hydrophilic polysaccharide, which is easy to contain hydrophilic active ingredients. Sodium alginate provides controlled release of active ingredients.

(8) Polyethylene glycol is used as a coating material to improve the stability of nanoparticles and have mucoadhesive properties. Polyethylene glycol prolongs the action of the active ingredient on the mucous membranes and stabilizes the protein.

(9) Hydrophilic drugs such as protein can be adsorbed on mucoadhesive nanoparticles, and the encapsulation efficiency is high.

(10) The combination and release of the active ingredient can be controlled by changing the type and amount of the adhesive polymer.

Description of drawings

Figure 1 Effect of chitosan concentration on particle size and zeta potential of mucoadhesive nanoparticles (n=3)

Figure 2 Effect of polyacrylic acid concentration on particle size and zeta potential of mucoadhesive nanoparticles (n=3)

Figure 3 Effect of initiator concentration on particle size of mucoadhesive nanoparticles (n=3)

Fig. 4 is an electron micrograph of several nanoparticles of the present invention. Among them, (a) chitosan coated nanoparticles (b) polyacrylic acid coated nanoparticles (c) Carbopol 934PNF coated nanoparticles (d) Carbopol974PNF coated nanoparticles

The hypoglycemic effect (100IU/Kg) of the carboxymethyl chitosan coating nanoparticle loaded with insulin of Fig. 5

Detailed ways

Embodiment 1: N, N, the synthesis of N-trimethyl chitosan quaternary ammonium salt

Disperse 2g of chitosan (30,000, 100,000-300,000, greater than 300,000) in 50ml of dimethyl sulfoxide in an eggplant-shaped bottle, soak and stir at room temperature for 24 hours, then add 4.8g of potassium iodide, 2g of sodium hydroxide and A solution made of 6ml of double distilled water was stirred slowly at 36°C for 16h. Then use four times the volume of the mixed solution of absolute ethanol and absolute ether to precipitate the crude product of the reaction, i.e. iodide N, N, N-trimethyl chitosan quaternary ammonium salt. Due to the poor stability of the iodized salt, it was dissolved in water, converted into N, N, N-trimethyl chitosan quaternary ammonium chloride by ion exchange, and then precipitated with absolute ethanol and anhydrous ether, and then After filtration and vacuum drying, the pure product was obtained, which was used for the following synthesis of mucoadhesive nanoparticles.

Embodiment 2: chitosan coated nanoparticles

Weigh 1g of chitosan (greater than 300,000), place it in an Erlenmeyer flask, add 98ml of 1% acetic acid solution, stir, heat to 25°C, dissolve, then add 1.6ml of methyl methacrylate to it, after 20min Ammonium persulfate was added, the temperature rose to 75°C, and the reaction was continued for 12 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons. After the suspension was diluted 100 times with water, the particle size and Zeta potential under different conditions were measured with a particle size analyzer (Nicomp 380/ZLS, Santa Barbara, Calif., USA), see accompanying drawings 1 and 3.

Embodiment 3: chitosan coated nanoparticles

Weigh 0.5g chitosan (greater than 300,000), place it in an Erlenmeyer flask, add 98ml 1% acetic acid solution, stir, heat to 40°C, dissolve, then add 1.1ml methyl methacrylate to it for 10min Then ammonium persulfate was added, the temperature rose to 80°C, and the reaction was continued for 14 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons. After the suspension was diluted 100 times with water, the particle size and Zeta potential under different conditions were measured with a particle size analyzer (Nicomp 380/ZLS, Santa Barbara, Calif., USA), see accompanying drawings 1 and 3.

Embodiment 4: chitosan coated nanoparticles

Weigh 0.2g of chitosan (greater than 300,000), put it in a Erlenmeyer flask, add 98ml of 1% acetic acid solution, stir, heat to 30°C, dissolve, then add 1.9ml of methyl methacrylate to it, and add it after 30min ammonium persulfate, the temperature was raised to 70°C, and the reaction was continued for 16 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons. After the suspension was diluted 100 times with water, the particle size and Zeta potential under different conditions were measured with a particle size analyzer (Nicomp 380/ZLS, Santa Barbara, Calif., USA), see accompanying drawings 1 and 3.

Embodiment 5: carboxylated chitosan coated nanoparticles

Weigh 1g of carboxylated chitosan, place it in an Erlenmeyer flask, add 98ml of double-distilled water, stir, heat to 60°C, dissolve, then add 1.1ml of methyl methacrylate to it, add ammonium persulfate after 20min, the temperature Raise to 80°C and continue the reaction for 24 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Embodiment 6: carboxylated chitosan coated nanoparticles

Weigh 0.5g carboxylated chitosan, place it in a Erlenmeyer flask, add 98ml double distilled water, stir, heat to 50°C, dissolve, then add 1.1ml methyl methacrylate to it, and add ammonium persulfate after 30min, The temperature was raised to 80°C and the reaction was continued for 18 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Embodiment 7: carboxylated chitosan coated nanoparticles

Weigh 0.2g carboxylated chitosan, place it in a Erlenmeyer flask, add 98ml double distilled water, stir, heat to 60°C, dissolve, then add 1.1ml methyl methacrylate to it, and add ammonium persulfate after 10min, The temperature was raised to 80°C, and the reaction was continued for 20 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Embodiment 8: carboxymethyl chitosan coating nanoparticle

Weigh 1g of carboxymethyl chitosan, place it in an Erlenmeyer flask, add 98ml of double distilled water, stir, heat to 35°C, dissolve, then add 1.1ml of methyl methacrylate to it, add ammonium persulfate after 20min, The temperature was raised to 78°C and the reaction was continued for 28 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Embodiment 9: carboxymethyl chitosan coating nanoparticle

Weigh 0.5g carboxymethyl chitosan, put it in a Erlenmeyer flask, add 98ml double distilled water, stir, heat to 50°C, dissolve, then add 1.1ml methyl methacrylate to it, and add ammonium persulfate after 20min , the temperature rose to 75°C, and the reaction was continued for 30 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Embodiment 10: carboxymethyl chitosan coating nanoparticle

Weigh 0.2g carboxymethyl chitosan, put it in a Erlenmeyer flask, add 98ml double distilled water, stir, heat to 45°C, dissolve, then add 1.1ml methyl methacrylate to it, and add ammonium persulfate after 30min , the temperature rose to 75°C, and the reaction was continued for 36 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Embodiment 11: Chitosan quaternary ammonium salt coating nanoparticle

Take by weighing the chitosan quaternary ammonium salt that 1g embodiment 1 obtains, place in Erlenmeyer flask, add the acetic acid solution of 98ml 1%, stir, be heated to 55 ℃, dissolve, then add 1.6ml methyl methacrylate wherein, After 25 minutes, potassium persulfate was added, the temperature rose to 75°C, and the reaction was continued for 18 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Embodiment 12: Chitosan quaternary ammonium salt coating nanoparticle

Take by weighing the chitosan quaternary ammonium salt that 0.5g embodiment 1 obtains, place in conical flask, add the acetic acid solution of 98ml 1%, stir, be heated to 25 ℃, dissolve, then add 1.6ml methyl methacrylate wherein After 15 minutes, ammonium persulfate was added, the temperature rose to 70°C, and the reaction was continued for 36 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Embodiment 13: Chitosan quaternary ammonium salt coating nanoparticle

Take by weighing the chitosan quaternary ammonium salt that 0.2g embodiment 1 obtains, place in Erlenmeyer flask, add 98ml 1% acetic acid solution, stir, be heated to 35 ℃, dissolve, then add 1.6ml methyl methacrylate wherein After 20 minutes, potassium persulfate was added, the temperature rose to 75°C, and the reaction was continued for 10 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Example 14: Sodium Alginate Coated Nanoparticles

Weigh 1g of sodium alginate, put it in a Erlenmeyer flask, add 98ml of double distilled water, stir, heat to 60°C, dissolve, then add 1.1ml of methyl methacrylate to it, add ammonium persulfate after 30min, and the temperature rises to 75°C, the reaction was continued for 10 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Example 15: Sodium Alginate Coated Nanoparticles

Weigh 0.5g of sodium alginate, put it in a Erlenmeyer flask, add 98ml of double distilled water, stir, heat to 30°C, dissolve, then add 1.1ml of methyl methacrylate to it, add ammonium persulfate after 30min, the temperature rises to 80° C., and the reaction was continued for 36 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Embodiment 16: Sodium alginate coated nanoparticles

Weigh 0.2g of sodium alginate, put it in a Erlenmeyer flask, add 98ml of double distilled water, stir, heat to 30°C, dissolve, then add 1.1ml of methyl methacrylate to it, add ammonium persulfate after 20min, the temperature rises to 80°C and continue the reaction for 28 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Example 17: PEG-coated nanoparticles

Weigh 1g of PEG, put it in a Erlenmeyer flask, add 98ml of double distilled water, stir, heat to 30°C, dissolve, then add 1.1ml of methyl methacrylate to it, add ammonium persulfate after 30min, and the temperature rises to 70°C , continue to react for 24 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Example 18: PEG-coated nanoparticles

Weigh 0.5g of PEG, put it in a Erlenmeyer flask, add 98ml of double-distilled water, stir, heat to 45°C, dissolve, then add 1.1ml of methyl methacrylate to it, add ammonium persulfate after 10min, and the temperature rises to 85°C °C, the reaction was continued for 26 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Embodiment 19: PEG-coated nanoparticles

Weigh 0.2g of PEG, put it in a Erlenmeyer flask, add 98ml of double-distilled water, stir, heat to 35°C, dissolve, then add 1.1ml of methyl methacrylate to it, add ammonium persulfate after 30min, and the temperature rises to 80 °C, the reaction was continued for 30 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons.

Embodiment 20: Polyacrylic acid coated nanoparticles

Weigh 1g of polyacrylic acid, place it in a Erlenmeyer flask, add 98ml of double distilled water, stir, heat to 40°C, dissolve, then add 1.1ml of methyl methacrylate to it, add ammonium persulfate after 20min, and the temperature rises to 80°C °C, the reaction was continued for 24 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons. Determination of the impact of different amounts of polyacrylic acid and initiator on the size and Zeta potential of the obtained nanoparticles, see accompanying drawings 2 and 3.

Example 21: Polyacrylic acid coated nanoparticles

Weigh 0.5g of polyacrylic acid, place it in a Erlenmeyer flask, add 98ml of double distilled water, stir, heat to 50°C, dissolve, then add 1.1ml of methyl methacrylate to it, add ammonium persulfate after 20min, and the temperature rises to 75°C, continue to react for 24 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons. Determination of the impact of different amounts of polyacrylic acid and initiator on the size and Zeta potential of the obtained nanoparticles, see accompanying drawings 2 and 3.

Example 22: Polyacrylic acid coated nanoparticles

Weigh 0.2g of polyacrylic acid, place it in a Erlenmeyer flask, add 98ml of double distilled water, stir, heat to 45°C, dissolve, then add 1.1ml of methyl methacrylate to it, add ammonium persulfate after 30min, and the temperature rises to 80°C, continue to react for 30 hours. The resulting suspension was dialyzed against a semipermeable membrane with a molecular size of 12,000 Daltons. Determination of the impact of different amounts of polyacrylic acid and initiator on the size and Zeta potential of the obtained nanoparticles, see accompanying drawings 2 and 3.

Example 23: Carbopol-coated nanoparticles

Weigh 0.1g of Carbopol, place it in a Erlenmeyer flask, add 98ml of double-distilled water, stir, heat to 35°C, dissolve, then add 2ml of methyl methacrylate to it, add ammonium persulfate after 15min, and the temperature rises to 85°C, continue to react for 20 hours. The particle size and Zeta potential of mucoadhesive nanoparticles obtained from different kinds of Carbopol are shown in Table 1 below.

Table 1 Nanoparticle size and Zeta potential determination

type Nanoparticle size (nm) Zeta potential (mv) Carbopol934PNF-PMMA 174.7±38.5 -18 Carbopol974PNF-PMMA 103.4±47.067 ND Carbopol971PNF-PMMA 197.5±23.305 -16.56

Example 24: Transmission electron microscopy confirms the morphology of nanoparticles

The nanoparticle suspension obtained in Examples 1-10 was negatively stained with 3% sodium phosphomolybdate to prepare a sample, and observed with a transmission electron microscope, see Figure 4. It can be seen from the figure that the mucoadhesive nanoparticles are relatively uniform in size and dispersed evenly.

Example 25: Binding test of nanoparticles and insulin

Encapsulation of carboxylated chitosan and carboxymethyl chitosan nanoparticles: take insulin, add 0.01N HCl, dissolve, mix with the above nanoparticles, incubate at room temperature for 3 hours, and centrifuge at high speed (15,000×g, 50min) to dissociate separation of insulin and nanoparticles. The concentration of free insulin was determined using the Lowry method.

Encapsulation of several other mucoadhesive nanoparticles: Take insulin, add isotonic phosphate buffer (pH7.4), dissolve, and prepare 1mg/ml (28IU/ml) insulin solution. Disperse the suspensions of the above several nanoparticles into the insulin solution, incubate at room temperature for 3 hours, and then centrifuge at a high speed (15,000×g, 50 min) to separate the free insulin from the nanoparticles. The concentration of free insulin was determined using the Lowry method.

Embodiment 26: Release test of nanoparticles

The drug-loaded nanoparticles in Example 12 were redispersed in hydrochloric acid at pH 1.2, shaken at 37°C, and two hours later, the drug-loaded nanoparticles were taken out, centrifuged at high speed, and the supernatant was taken, and the concentration of insulin was measured by the Lowry method. The results are shown in Table 2 below.

Table 2 Encapsulation efficiency of mucoadhesive nanoparticles and release at pH 1.2

type Chitosan-PMMA CMC-PMMA TMC-PMMA Alginate-PMMA encapsulation rate 80.3% 75.4% 71.0% 76.7% release percentage 38.9% 19.1% 20.1% 33.4%

It can be seen from Table 2 that under acidic conditions, the release of carboxymethyl chitosan-coated nanoparticles is relatively small, which is more suitable for oral administration of active ingredients.

Example 27: Sensitivity of nanoparticles to different pH

Prepare a series of buffers at different pHs in which to disperse the mucoadhesive nanoparticles. The particle size change of mucoadhesive nanoparticles was measured at different pH, and the results are shown in Table 3 below.

Table 3 Changes in particle size of mucoadhesive nanoparticles under different pH conditions

As can be seen from Table 3, under neutral and alkaline conditions, the nanoparticles coated with chitosan are easy to aggregate; under strong acid conditions, the nanoparticles coated with carboxymethyl chitosan are easy to aggregate.

Embodiment 28: Nanoparticle hypoglycemic test

After the SD rats were fasted for 12 hours, insulin-loaded nanoparticles were administered intragastrically, and blood was collected from the tail vein of the rats at regular intervals. After the blood coagulated, it was centrifuged, and the serum was collected, and the blood sugar content was measured by the glucose oxidase method. The results are shown in Figure 5. It can be seen from accompanying drawing 5 that after intragastric administration of insulin-loaded carboxymethyl chitosan coated nanoparticles to rats, a significant hypoglycemic effect is produced.

Claims (9)

1. nano granules adhesive to mucous membrane preparation method is characterized in that concrete steps are as follows:

Will be as the mucosa adhesion polymkeric substance dissolution with solvents of coating material, be heated to 25～60 ℃, adding is as alkyl acrylate, alkyl methacrylate or the alkyl cyanoacrylate of framework material, reacted 10～30 minutes, add initiator again, be heated to 70～85 ℃, continue reaction 10～36 hours, purifying promptly; Wherein:

The weight ratio of coating material and framework material is 1: 0.05～1: 20;

Said alkyl methacrylate is one or more in methyl methacrylate, Jia Jibingxisuanyizhi, propyl methacrylate, butyl methacrylate, Propenoic acid, 2-methyl, isobutyl ester and the N-Hexyl methacrylate; Said alkyl acrylate is one or more in methyl acrylate, ethyl propenoate, propyl acrylate, butyl acrylate, isobutyl acrylate and the Ethyl acrylate; Said alkyl cyanoacrylate is one or more in Methyl 2-cyanoacrylate, cyanacrylate, alpha-cyanoacrylate propyl ester, Tisuacryl, isobutylcyanoacrylate and the own ester of alpha-cyanoacrylate;

Said mucosa adhesion polymkeric substance is a kind of in chitosan derivatives, sodium alginate, polyoxyethylene glycol, polyacrylic acid and the carbopol.

2. nano granules adhesive to mucous membrane preparation method according to claim 1, the molecular weight that it is characterized in that said polyoxyethylene glycol is 400,1500,6000 or 20,000.

3. nano granules adhesive to mucous membrane preparation method according to claim 1 is characterized in that said carbopol class is carbopol 934PNF, 974PNF or 971PNF.

4. nano granules adhesive to mucous membrane preparation method according to claim 1 is characterized in that said chitosan derivatives is a kind of in cm-chitosan, using carboxyl chitosan, chitosan quaternary ammonium salt, chitosan hydrochloride, chitosan glutamate salt and the chitosan lactate.

5. nano granules adhesive to mucous membrane preparation method according to claim 1 is characterized in that said initiator is ammonium persulphate or Potassium Persulphate, and consumption is reaction system 0.01～0.2%g/ml.

6. nano granules adhesive to mucous membrane by the preparation of the described method of one of claim 1-5, it is characterized in that with polyalkyl acrylate, polyalkyl methacrylate or polybutylcyanoacrylate be skeleton, with the mucosa adhesion polymkeric substance is coating material, and the weight ratio of coating material and framework material is 1: 0.05～1: 20.

7. nano granules adhesive to mucous membrane according to claim 6 is characterized in that particle diameter is 100～300nm.

8. a nano granules adhesive to mucous membrane as claimed in claim 6 is as the application of the carrier of protein, polypeptide, nucleic acid and vaccine.

9. application according to claim 8, wherein protein is Regular Insulin, tethelin or Thymosin alpha 1.

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