CN109200272B

CN109200272B - Oral exenatide nanoparticle preparation and preparation method and application thereof

Info

Publication number: CN109200272B
Application number: CN201811063975.3A
Authority: CN
Inventors: 刘志佳; 田厚宽; 陈永明; 刘利新; 梁锦荣; 毛海泉
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2018-09-12
Filing date: 2018-09-12
Publication date: 2021-11-26
Anticipated expiration: 2038-09-12
Also published as: CN109200272A

Abstract

The invention discloses an exenatide-loaded nanoparticle and a preparation thereof. By adopting a rapid nano-composite technology, leading a tannic acid solution to pass through the channels 1 and 2, leading an exenatide solution to pass through the channels 3 and 4, and reaching a vortex mixing area for mixing to obtain a nano-composite core mixed solution consisting of tannic acid and exenatide; and then the mixed solution passes through the channels 1 and 2, and the chitosan solution passes through the channels 3 and 4 to reach a vortex mixing area for mixing, so that the nano-particles with the surfaces coated with chitosan are obtained. The nanoparticle can be further coated with an enteric Eudragit material on the surface of the nanoparticle, so as to prepare an enteric microcapsule preparation loaded with exenatide. The nano-particle and microcapsule preparation can be effectively absorbed by small intestine through oral administration, has high oral administration utilization degree and has larger clinical application prospect.

Description

Oral exenatide nanoparticle preparation and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an oral exenatide nanoparticle preparation as well as a preparation method and application thereof.

Background

Diabetes is a metabolic disease characterized by hyperglycemia. Hyperglycemia is caused by a defect in insulin secretion or an impaired biological action, or both. The hyperglycemia that is present in diabetes for a long time can lead to chronic damage and dysfunction of various tissues, particularly eyes, kidneys, heart, blood vessels and nerves. Diabetes has become the leading chronic non-infectious disease that seriously harms human health following cardiovascular and cerebrovascular diseases; at present, the number of diabetic patients in China is more than 9400 ten thousand, wherein the type 2 diabetes (T2 DM) is the main body of diabetes and accounts for about 90 percent of the diabetic patients. T2DM is defective in insulin resistance or secretion caused by the interaction of genetic and various environmental factors. It has a high prevalence rate, especially for the middle-aged and the elderly, and shows a tendency of youthfulness. The reduction in the number of islet beta cells or dysfunction in secretion is a central link leading to the pathogenesis of T2 DM. Treatment generally follows lifestyle interventions (e.g. diet control, exercise therapy, etc.), oral hypoglycemic agents and combined use of insulin. The traditional medicine for treating T2DM has large side effects, the long-term use of the traditional medicine can cause the function failure of islet cells due to the over-secretion of insulin, and the long-term use of insulin injection can cause the increase of insulin resistance (insulin resistance).

Glucagon-like peptide-1 (GLP-1) is a 30 amino acid long peptide hormone derived from tissue-specific post-translational processing of the human glucagon protogene. GLP-1 produces weight loss effects through a variety of pathways, including inhibition of gastrointestinal motility and gastric secretion, inhibition of appetite and feeding, and delay of gastric emptying. In addition, GLP-1 acts on the central nervous system (especially hypothalamus), thereby causing satiety and appetite reduction in humans. In addition, GLP-1 has many other biological properties and functions, for example, GLP-1 may play a role in lowering blood lipid and blood pressure, thereby protecting the cardiovascular system; it can also act on the central nervous system to enhance learning and memory functions and protect nerves.

GLP-1 can stimulate insulin secretion by binding to GLP-1 receptor at higher blood glucose, thereby lowering blood glucose. However, GLP-1 is rapidly degraded and inactivated by dipeptidyl peptidase IV (DPP-IV) in vivo, and the half-life is less than 2 minutes, so that the clinical application of the GLP-1 is limited. To overcome this problem, researchers have developed GLP-1 receptor agonists and inhibitors of DPP-IV to address the above-mentioned challenges.

In recent years, glucagon-like peptide (GLP-1) receptor agonists have become hot spots for diabetes treatment studies, and represent drugs such as exenatide (exenatide) and liraglutide (liraglutide); wherein the exenatide is an artificial synthetic product of GLP-1 analogue Exendin-4 separated from the salivary gland of Eremias gigantea, consists of 39 amino acids, has a molecular weight of about 4.186kD, has 53 percent of sequence homology with human GLP-1, is the first GLP-1 analogue approved by the United states Food and Drug Administration (FDA), and is the only drug which can help type II diabetics to control blood sugar and weight at the same time at present. The exenatide can promote the secretion of blood sugar-dependent insulinotropic hormone, rebuild the ratio of insulin/glucagon, increase the number of islet beta cells, delay gastric emptying, inhibit appetite and increase insulin sensitivity, can well control the weight of a patient while realizing the control of blood sugar level, has the effect of reducing blood sugar only for a hyperglycemic person, and can disappear when the blood sugar of the patient is reduced to a normal value, thereby greatly reducing the risk of hypoglycemia for a user.

The exenatide is a polypeptide drug, and the current clinical administration modes are injection administration. The long-term injection administration brings great pain to patients and has poor patient compliance, and the oral administration route is the most widely applied and more convenient administration mode at present. The development of exenatide oral dosage forms presents the following technical difficulties: (1) the exenatide has large molecular weight and poor fat solubility, and is difficult to be absorbed into blood through a biological barrier of the gastrointestinal tract; (2) the exenatide is a polypeptide drug and is easily degraded by various proteases in the gastrointestinal tract to lose activity; (3) after being absorbed, the medicine is easy to be eliminated by liver, so that the oral bioavailability is extremely low.

Therefore, in order to expand the clinical applications of exenatide formulations, it is required to develop an exenatide oral formulation that is more convenient to administer.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the existing exenatide preparation and provide exenatide nanoparticles capable of being effectively absorbed by small intestine through oral administration and a preparation thereof.

A first object of the present invention is to provide an exenatide-loaded nanoparticle.

The second purpose of the invention is to provide a preparation method of the exenatide-loaded nanoparticle.

The third purpose of the invention is to provide an enteric microcapsule preparation loaded with exenatide.

The fourth purpose of the invention is to provide a preparation method of the enteric microcapsule preparation.

The above object of the present invention is achieved by the following technical solutions:

the exenatide-loaded nanoparticle is characterized in that the nanoparticle is of a core-shell structure, the core is a nano composite core consisting of tannic acid and exenatide, and the shell is chitosan coated on the surface of the core.

According to the invention, the nano preparation is carried out on the exenatide through the hydrogen bond complexing action by adopting the tannic acid as a hydrogen bond donor and the exenatide as a hydrogen bond acceptor, so as to obtain the nano particles loaded with the exenatide. The nanoparticle has extremely high encapsulation efficiency and drug-loading rate, and can be effectively absorbed by small intestine through oral administration. Further, coating chitosan on the surface of the nano-particles through electrostatic interaction to obtain surface functionalized modified nano-particles; the electropositive chitosan can enhance the interaction between the particles and the mucus layer, improve the affinity of the particles and the epithelial cells of the small intestine and promote the permeability of the EXE in the epithelial cell layer of the small intestine; meanwhile, the coated chitosan layer also has the capability of reversibly opening the epithelial tight junction of the small intestine, the EXE is further promoted to be transported through the wall of the small intestine through a cell bypass way, and the oral availability of the nanoparticles is improved.

The preparation method of the exenatide-loaded nanoparticle adopts a rapid nano composite technology, and comprises the following steps:

s1, enabling a Tannic Acid (TA) solution to pass through

channels

1 and 2, enabling an exenatide (EXE) solution to pass through channels 3 and 4, and reaching a vortex mixing area for mixing to obtain a nano composite core consisting of tannic acid and exenatide; the liquid in the four channels flows at constant speed, and the flow rate is 5mL/min to 50mL/min (preferably 30 mL/min);

s2, enabling the mixed liquor obtained in the step S1 to pass through

channels

1 and 2, enabling the chitosan solution to pass through channels 3 and 4, and enabling the chitosan solution to reach a vortex mixing area for mixing to obtain nanoparticles with surfaces coated with chitosan; the liquid in the four channels flows at constant speed, and the flow rate is 5mL/min to 50mL/min (preferably 30 mL/min).

The particle size of the nanoparticle prepared by the method is 40-500 nm, the polydispersity index (PDI) is 0.1-0.4, the encapsulation efficiency is 70-99%, and the drug loading rate is 10-60%.

Preferably, the pH of the exenatide solution in S1 is 5.5 to 6.8 (preferably 6.2), specifically, the pH is adjusted to 5.5 to 6.8 (preferably 6.2) by dissolving exenatide in 0.2% acetic acid (HAc) solution and then using NaOH solution; the concentration of the exenatide solution is 0.1-0.9 mg/mL (preferably 0.3 mg/mL).

Preferably, the concentration of the Tannic Acid (TA) solution of S1 is 0.1-0.9 mg/mL (preferably 0.3 mg/mL).

Preferably, the chitosan solution of S2 has a pH value of 5.0-6.4 (preferably 6.2) and a concentration of 0.1-1 mg/mL (preferably 0.5 mg/mL).

Preferably, the mass ratio of the tannic acid to the exenatide is 0.3-3: 1.

the invention also claims the application of the nano-particles in preparing exenatide oral nano-preparations.

An oral exenatide pharmaceutical preparation, comprising the exenatide-loaded nanoparticle.

Preferably, the pharmaceutical formulation further comprises a pharmaceutically acceptable excipient, a lyoprotectant.

An enteric microcapsule preparation loaded with exenatide is prepared by coating enteric Eudragit material on the surface of the exenatide loaded nanoparticles; the enteric microcapsule preparation obtained by the Eudragit coating can be rapidly disintegrated in the small intestine environment, and rapidly release the exenatide-loaded nanoparticles.

Preferably, the Eudragit is Eudragit L100-55.

The invention also claims a preparation method of the enteric microcapsule preparation loaded with exenatide, which comprises the steps of adopting a rapid nano composite technology, enabling the nanoparticle solution loaded with exenatide to pass through

channels

1 and 2, enabling the ewing solution to pass through a channel 3, enabling the acetic acid aqueous solution to reach a vortex mixing area through a channel 4, and mixing to obtain the enteric microcapsule preparation; the liquid in the four channels flows at constant speed, and the flow rate is 30 mL/min.

Preferably, the Eudragit concentration is 0.3-1.5 mg/mL (preferably 0.9-1.5 mg/mL, most preferably 1.5 mg/mL).

Preferably, the pH value of the reaction system is 3.5-5.2 (preferably, pH = 5.2).

The exenatide-loaded enteric microcapsule preparation disclosed by the invention has the advantages that the blood concentration is gradually increased and tends to be stable after oral administration, the blood sugar can be effectively controlled, the blood sugar change is more stable compared with subcutaneous injection, and the exenatide-loaded enteric microcapsule preparation has a good blood sugar reduction effect and high bioavailability. The preparation can effectively control blood sugar, greatly relieve unnecessary pain of diabetic patients, and is safe and convenient.

Compared with the prior art, the invention has the following beneficial effects:

the exenatide-loaded nanoparticle is prepared through hydrogen bond complexation between tannic acid and exenatide, the nanoparticle has extremely high encapsulation efficiency and drug loading capacity, and the active function of a drug can be efficiently maintained as the processing process of the nanoparticle is carried out in a pure water phase. The nano-particle and microcapsule preparation loaded with the exenatide can be effectively absorbed by small intestine through an oral way, the blood concentration is gradually increased and tends to be stable after oral administration, the blood sugar can be effectively controlled, the change of the blood sugar is more stable compared with subcutaneous injection, the nano-particle and microcapsule preparation has good blood sugar reduction effect and high bioavailability, the unnecessary pain of a diabetic patient is relieved, and the application prospect is wide.

Drawings

Fig. 1 is a schematic view of a multi-inlet vortex mixer.

FIG. 2 shows the effect of flow rate on NC particle size and PDI.

FIG. 3 shows the effect of pH on the NC particle size PDI.

FIG. 4 shows the effect of TA concentration on NC particle size, PDI and surface potential.

Figure 5 shows the effect of TA concentration on NC particle encapsulation efficiency and drug loading.

FIG. 6 shows the effect of CS concentration on NP particle size, PDI, and surface potential.

FIG. 7 shows the effect of pH on NP particle size, PDI, and surface potential.

FIG. 8 shows the effect of pH on the encapsulation efficiency of E-NP.

FIG. 9 shows the effect of Ewing concentration on the encapsulation efficiency of E-NP.

Fig. 10 shows the release behavior of E-NP in a pH =2.5 environment.

FIG. 11 shows the release behavior of NC, NP and E-NP under optimal conditions under physiological conditions.

Fig. 12 shows the small intestine epithelial penetration behavior of drug-loaded nanoparticles of the invention.

FIG. 13 is a graph showing the serum pharmacokinetic profiles of exenatide in oral NP and E-NP of the present invention.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 preparation of Exenatide-loaded nanoparticle and enteric microcapsule formulations by FNC technique

1. Preparation of Tannin (TA) and Exenatide (EXE) nanocomposite cores

TA is dissolved in deionized water, and the concentration is 0.1-0.9 mg/mL. EXE is dissolved in 0.2% acetic acid solution (HAc), and then the pH value is adjusted to different pH values (5.5-6.8) by NaOH solution, and the concentration of the EXE solution is 0.3 mg/mL. Distributing TA solution in the first channel and the second channel and EXE solution in the third channel and the fourth channel, as shown in figure 1, the flow rates of the four channels are consistent, adjusting the flow rates of the channels to 5mL/min, 10 mL/min, 20 mL/min, 30mL/min, 40 mL/min and 50mL/min, and enabling the two solutions to reach a vortex mixing area through the four channels for mixing to obtain a nano composite core (NC) of TA and EXE.

FIG. 2 shows the effect of flow rate on NC particle size and polydispersity index (PDI). TA concentration was 0.3mg/mL and EXE concentration was 0.3 mg/mL. When the flow rate is 5-30 mL/min, the particle size of the particles is reduced along with the increase of the flow rate, and when the flow rate is 30-50 mL/min, the particle size of the particles is increased along with the increase of the flow rate. In the experimental flow rate range, PDI decreased with increasing flow rate. At a flow rate of 30mL/min, the resulting particles were minimal and relatively uniform. The preferred flow rate of 30mL/min was chosen.

FIG. 3 shows the effect of pH on NC particle size and polydispersity index. As the pH decreases, the particle size gradually increases, and at pH 5.5, stable suspended particles are not formed, but rapid precipitation occurs. At pH =6.2, there is a smaller particle size and a minimum PDI, so the preferred condition of pH =6.2 is chosen.

FIG. 4 shows the effect of TA/EXE ratio on NC particle size, PDI and surface potential. The EXE concentration was maintained at 0.3mg/mL and the flow rate was 30 mL/min. When the TA concentration is 0.3-0.9 mg/mL, the particle size and PDI are basically unchanged and are respectively-45 nm and-0.25; the potential increases slightly with increasing TA concentration, from 25 mV to 30 mV.

FIG. 5 shows the encapsulation efficiency and drug loading of NC at different ratios. In the experimental range, the TA concentration basically has no influence on PDI, particle size and surface potential, and nanoparticles with small particle size, uniform distribution and negatively charged surface can be obtained. FIG. 5 shows the preferred conditions for the encapsidation rate and drug loading of NC at different TA concentrations, with the encapsidation rate of NC being substantially higher than 95% and the drug loading being increased from 24% to 48% as the TA concentration is decreased from 0.9mg/mL to 0.3mg/mL, and the EXE concentration being selected to be 0.3mg/mL for higher drug loadings.

2. Coating a layer of Chitosan (CS) on the NC surface by using FNC technology

CS was dissolved in 0.2% aqueous acetic acid (HAc) at concentrations of 0.1mg/mL, 0.2m g/mL, 0.3mg/mL, 0.4 mg/mL, 0.5 mg/mL. The NC suspension prepared in the first step is distributed in the first channel and the second channel, the CS aqueous solution is distributed in the third channel and the fourth channel, as shown in figure 1, the flow rates of the four channels are consistent, the flow rate of the channels is adjusted to be 30mL/min, and the two liquids reach a vortex mixing area through the four channels to be mixed, so that the Nanoparticles (NP) coated with CS on the surface are obtained.

FIG. 6 shows the particle size, polydispersity index and surface potential of NPs prepared at different CS concentrations. As the CS concentration was decreased from 0.5mg/mL to 0.2 mg/mL, the particle size increased from 101 nm to 140 nm, the potential decreased from 18mV to 12mV, and precipitation occurred when the CS concentration was decreased to 0.1 mg/mL. When the CS concentration is 0.5mg/mL, the preferable condition that the CS concentration is 0.5mg/mL is finally selected, with the smallest particle size and smaller PDI.

The effect of system pH on NP was investigated. Figure 7 shows NPs prepared at different pH values. The particle size increased from 103 nm to 131 nm and the PDI increased from 0.27 to 0.526 as the pH decreased from 6.2 to 5.0, since the binding ability of TA to EXE increased with decreasing pH, resulting in aggregation during CS coating, increased particle size, and decreased dispersion uniformity; the surface sites were elevated from 18.3 mV to 26.4 mV due to the increased dissociation of CS and increased electropositivity as the pH was lowered, resulting in an increase in the surface sites of the particles. As the pH increased from 6.2 to 6.7, the particle size decreased to 83 nm and the PDI increased to 0.38; the reason is that the interaction force between TA and EXE is weakened due to the increase of pH, and the TA/EXE core is partially dissociated in the process of coating CS, so that the particle size is reduced, and the particle size and the dispersion uniformity are reduced; the surface potential drops to 12mV because, as the pH increases, the CS dissociation decreases and the electropositivity decreases, resulting in a decrease in NP surface potential. Since changes in pH during coating lead to changes in the core properties of TA/EXE and further affect the particle size and PDI of the NP after coating, pH =6.2 (pH in NC preparation) was chosen as the preferred pH for the coating process.

3. Preparation of enteric microcapsule preparation

Eugradit L100-55 is dissolved in NaOH aqueous solution with pH =11, the concentration is 0.3-1.5 mg/mL, and after complete dissolution, the pH value is adjusted to 7.4 by using HCl solution. Placing the NP particle suspension in a channel 1 and a channel 3, placing Eugradit L100-55 solution in a channel 2, placing acetic acid (HAc) aqueous solution in a channel 4 to regulate and control the pH value of the system, setting the flow rate of the channel to be 30mL/min, enabling the three liquids to reach a vortex mixing region through four channels for mixing to obtain an enteric microcapsule (E-NP) wrapped by Eudragit L100-55, and finally setting the pH value of the system to be 3.5-5.2.

At pH values above 5.5, Eudragit L100-55 was in solution and after complexing with NP, it was not possible to form microencapsulated particles. Micron-sized particles or precipitates of encapsulated NPs can be obtained when the pH is less than 5.5. FIG. 8 shows the encapsulation efficiency of E-NP to NP obtained from different pH values of the system. The pH value is reduced from 5.2 to 3.5, and the encapsulation efficiency of the E-NP is reduced from 97.7 percent to 57.3 percent. This is because the ionization degree of Eudragit L100-55 decreases with the decrease of pH, and the negative charge carried by it decreases, and the binding degree to the electropositive NP decreases, resulting in the increase of NP not bound to Eudragit L100-55 during precipitation and the decrease of encapsulation efficiency. When pH =5.2, the highest encapsulation efficiency is exhibited, and therefore system pH =5.2 is selected as the preferred condition for preparing E-NP.

FIG. 9 shows the encapsulation efficiency of E-NP versus NP at different Eudragit L100-55 concentrations. When the concentration of Eudragit L100-55 is higher than 0.9mg/mL, the E-NP has higher encapsulation efficiency (97%); when the concentration of the Eudragit L100-55 is lower than 0.9mg/mL, the E-NP encapsulation efficiency is reduced from 97% to 75% along with the reduction of the concentration of the Eudragit L100-55 to 0.3 mg/mL. This is because Eudragit L100-55 at higher concentrations can efficiently wrap NPs in excess of NPs; at lower concentrations of Eudragit L100-55, insufficient binding to all NPs relative to NPs resulted in a decrease in encapsulation efficiency. Therefore, the concentration of Eudragit L100-55 in the preparation process should be more than 0.9 mg/mL.

Example 2 in vitro drug Release assay

Three preparations, NC, NP and E-NP, were prepared under optimal conditions using RITC labeled EXE. Placing the E-NP formulation at pH =2.5In HCl solution of (2) at 37^oC, incubation in a shaker at 150rpm, centrifugation at 10000 rpm for 10 min at 2h intervals, and taking the supernatant to measure the EXE concentration. FIG. 10 is an EXE release curve of E-NP prepared at different concentrations of Ewing at pH =2.5, from which it can be seen that at the concentration of Ewing of 0.9mg/mL, 1.2mg/mL, 1.5mg/mL, 50%, 18% and 7% EXE is released within 2h, respectively, thus indicating that at lower concentrations of Ewing, a large amount of EXE is released, which is not effective in protecting EXE from the strong acid environment in the stomach, and that when the concentration of Ewing is increased to 1.5mg/mL, EXE is effectively protected from the rapid release of the acid environment in the tail, and finally the concentration of Ewing of 1.5mg/mL is selected as the preferred condition.

1mL of the suspension of NC, NP, and E-NP particles was taken and placed in a 1mL dialysis tube (M)_w= 50 kDa), sealed. The dialysis tube was placed in 20 mL of 10 mM PBS buffer (pH = 7.4), incubated at 37 ℃ in a shaker at 150rpm, 1mL of buffer was removed at specified intervals (subsequently supplemented with 1mL of blank buffer, keeping the volume of the system constant), and the concentration of the liberated EXE was measured by fluorescence intensity. Figure 11 shows the release profiles under physiological conditions for three different formulations. NC has the fastest release rate, NP has the protection effect of a CS coating layer, EXE release rate is less than that of NC, E-NP shows the release rate similar to that of NP, and after 24h, more than 80% of EXE release amount is obtained, thereby showing that the enteric microcapsule preparation E-NP obtained by the Ewing coating can be rapidly disintegrated in the small intestine environment and rapidly release NP.

Example 3 intestinal permeability test with Exenatide nanoparticles

Both NC and NP formulations were prepared under preferred conditions using RITC-labeled EXE for use. SD rats were fasted for 12h, anesthetized, and then the duodenum, jejunum, and ileum portions of the small intestine of the rats were surgically removed for 5cm lengths, after which the rats were sacrificed. The two ends of the small intestine at different sites were ligated, and 1mL of EXE solution, NC particle suspension, NP particle suspension was injected, after which the small intestine was placed in 10mL of krebs ringer buffer, 37^oC, dialyzing in a shaking table at 150rpm, taking out 1mL of dialysate every 0.5h, measuring the concentration of EXE in the dialysate, and calculating according to the formula (3-3)Apparent permeability coefficient (PAPP value) with EXE formulation.

Wherein,

is the flux of fluorescently labeled nanoparticles or EXEs permeating from the inside of the small intestine through the wall of the small intestine to the outside of the small intestine,

is the initial fluorescence intensity of the nanoparticles or EXE inside the small intestine, and A is the area of the small intestine wall.

FIG. 12 shows the apparent permeability coefficient (Papp) values of NP, NC, and EXE solutions in the small intestine, duodenum, jejunum, and ileum. NP has a significantly higher apparent permeability coefficient in the duodenum, jejunum, and ileum than NC and EXE solutions. The NP has the highest small intestine penetration efficiency, because the surface of the NP is coated with a layer of CS, the electropositive CS enhances the interaction between particles and a mucus layer, improves the affinity between the particles and small intestine epithelial cells and promotes the absorption of EXE by the small intestine epithelial cells; at the same time, this layer of CS also has the ability to reversibly open the tight junctions of the small intestine epithelium, further facilitating the transport of EXE through the small intestine via the paracellular pathway. Comparing the apparent permeability coefficients of the same drug-loaded nanoparticles at different parts of the small intestine, it can be seen that the penetration efficiency of EXE also depends on the part of the small intestine. The same drug-loaded particles had higher penetration efficiency in the duodenum and jejunum than in the ileum, probably because the duodenum and jejunum have a thinner mucus layer (< 300 μm) and the particles can reach the small intestine epithelium more easily and be absorbed, while the ileum has a thicker mucus layer (-500 nm), about 2 times that in the duodenum and jejunum, and the drug-loaded particles are harder to reach the small intestine epithelium.

Example 4 oral bioavailability of Exenatide-loaded nanoparticles

200-250 g of SD mice are divided into 4 groups, and all experimental groups are fasted for 10-12 h before the experiment. Group 1 was injected subcutaneously with EXE solution (50. mu.g/kg), group 2 with intragastric NP particle suspension (500. mu.g/kg), group 3 with intragastric E-NP particle suspension (500. mu.g/kg), and group 4 with intragastric EXE solution (500. mu.g/kg). At specific time intervals, blood was collected from the orbit, and the EXE concentration in the blood was measured by using an exenatide ELISA kit to obtain a curve of EXE blood concentration versus time, as shown in fig. 13. The subcutaneous EXE solution group served as a 100% control, and bioavailability was obtained by comparing the area under the curve of the oral granule group and the subcutaneous EXE solution group. Oral bioavailability of NP was: 13.8%, oral bioavailability of E-NP was: 15.1 percent. The EXE solution group is taken as a negative control, and the EXE blood concentration is close to zero.

After the nano-particles loaded with the exenatide are taken orally, the blood concentration gradually rises and tends to be stable, the blood sugar can be effectively controlled, the change of the blood sugar is more stable compared with subcutaneous injection, and the nano-particles have a good blood sugar reducing effect and high bioavailability. The preparation can effectively control blood sugar, greatly relieve unnecessary pain of diabetic patients, and is safe and convenient.

Claims

1. The exenatide-loaded nanoparticle is characterized in that the nanoparticle is of a core-shell structure, the core is a nano composite core consisting of tannic acid and exenatide, and the shell is chitosan coated on the surface of the core; the nano preparation is carried out on the exenatide through the hydrogen bond complexing action by adopting tannic acid as a hydrogen bond donor and the exenatide as a hydrogen bond acceptor, so as to obtain nano particles loaded with the exenatide; and coating chitosan on the surface of the exenatide-loaded nanoparticle through electrostatic interaction to obtain the surface functionalized chitosan-modified nanoparticle.

2. The method of claim 1, wherein the method of preparing nanoparticles is performed by a rapid nanocomposite technique, comprising the steps of:

s1, leading a tannic acid solution to pass through channels 1 and 2, leading an exenatide solution to pass through channels 3 and 4, and reaching a vortex mixing area for mixing to obtain a nano composite core consisting of tannic acid and exenatide; the liquid in the four channels flows at constant speed, and the flow rate is 5mL/min to 50 mL/min;

s2, enabling the mixed liquor obtained in the step S1 to pass through channels 1 and 2, enabling the chitosan solution to pass through channels 3 and 4, and enabling the chitosan solution to reach a vortex mixing area for mixing to obtain nanoparticles with surfaces coated with chitosan; the liquid in the four channels flows at constant speed with the flow rate of 5 mL/min-50

mL/min。

3. The method according to claim 2, wherein the Exenatide solution of S1 has a pH of 5.5 to 6.8 and a concentration of 0.1 to 0.9 mg/mL.

4. The method as set forth in claim 2, wherein the concentration of the tannic acid solution S1 is 0.1 to 0.9

mg/mL。

5. The method according to claim 2, wherein the chitosan solution of S2 has a pH of 5.0-6.4 and a concentration of 0.1-1 mg/mL.

6. Use of the nanoparticle of claim 1 for the preparation of exenatide oral nano formulation.

7. An exenatide-loaded enteric microcapsule formulation, wherein an enteric ewing's material is coated on the surface of the exenatide-loaded nanoparticle of claim 1.

8. The method for preparing an exenatide-loaded enteric microcapsule formulation as claimed in claim 7, wherein the enteric microcapsule formulation is obtained by mixing the nanoparticle solution of claim 1 through channels 1 and 2, the ewing's aqueous solution through channel 3, and the acetic acid aqueous solution through channel 4 into a vortex mixing region using a rapid nano-compounding technique; the liquid in the four channels flows at constant speed, and the flow rate is 30 mL/min.

9. The method of claim 8, wherein the Ewing concentration is 0.3-1.5 mg/mL.

10. The preparation method according to claim 8, wherein the reaction system has a pH of 3.5 to 5.2.