CN113171355B

CN113171355B - M cell targeted self-assembly nano-particle of colistin sulfate, preparation method and application

Info

Publication number: CN113171355B
Application number: CN202110445849.XA
Authority: CN
Inventors: 谢书宇; 陈冬梅; 李超; 潘源虎; 瞿玮
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-04-25
Filing date: 2021-04-25
Publication date: 2022-07-19
Anticipated expiration: 2041-04-25
Also published as: CN113171355A

Abstract

The invention discloses a self-assembled mucositin sulfate nanoparticle with M cell targeting, a preparation method and application, and relates to the field of pharmaceutical preparations; the self-assembled mucobactin sulfate nanoparticle disclosed by the invention is composed of mucobactin sulfate, a polyanion compound, a polycation compound and a coating material, and has the advantages of reasonable formula, simple process and safe quality; the self-assembled nano-particles of the colistin sulfate are resistant to gastric acid, have slow release property, can obviously reduce the release of the colistin sulfate in the stomach, improve the ability of the colistin sulfate to penetrate through a mucus barrier and be absorbed at a target site of an M cell in an intestinal section, and ensure that the clinical use of the colistin sulfate is more economical and effective; meanwhile, the complete nano shape can be kept in simulated gastric juice, intestinal tight connection can be effectively and transiently opened, and the oral bioavailability of the mouse is improved by more than 3 times.

Description

M cell targeted colistin sulfate self-assembly nanoparticles, preparation method and application

Technical Field

The invention relates to the field of veterinary drug preparations, in particular to a mucositin sulfate self-assembly nanoparticle with M cell targeting, a preparation method and an application thereof.

Background

In recent years, due to the progress of biotechnology and genetic engineering technology, a series of polypeptide drugs based on biotechnology, such as mucobactin sulfate, bacitracin, and the like, have been rapidly developed. Although many polypeptide drugs have good pharmacological activity in vitro, the stability is poor, the transmembrane transport capacity is low, and the drugs are required to be continuously and repeatedly taken for a long time during the treatment period, so that the clinical application of the polypeptide drugs is greatly limited, and a new preparation is required to be developed for the application of the polypeptide drugs so as to overcome the scientific problem faced by the clinical application.

The oral administration has the advantages of no pain, convenience, good compliance and the like, is the most widely applied administration route at present, and is the hotspot and focus of the development of a drug delivery system. The orally administered drug molecule must overcome three major physiological barriers of the gastrointestinal tract to be effectively absorbed by the gastrointestinal tract: firstly, the degradation of a super acidic (pH 1-3) environment and a large amount of functional enzymes in the stomach is avoided, and the protein drug is easy to inactivate in an adverse physiological environment in the stomach; secondly, a mucus layer on the surface of the intestinal epithelium, and the highly viscoelastic and adhesive mucus can lead the medicament to be captured rapidly and removed from epithelial cells, thus severely limiting the transmission of medicament molecules to the mucosal epithelium; thirdly, intestinal mucosal barriers prevent efficient transmembrane transport of polypeptide drugs. The strong acidity and complex enzyme in stomach, the block of gastrointestinal tract mucus and the physiological barrier of mucosa and other factors seriously limit the oral absorption of polypeptide drugs, so that at present, almost no oral preparation of polypeptide drugs is approved at home and abroad to be put on the market. Therefore, the development of oral polypeptide drug preparations is always a critical scientific problem and a technical problem to be broken through in the process of creating new drugs, and a new strategy is urgently needed to improve the capability of the drugs for penetrating through the gastrointestinal biomembrane barrier and the stability of the drugs in the gastrointestinal physiological environment so as to improve the oral absorption of the drugs.

In order to overcome the three limiting factors of oral absorption of polypeptide drugs, researchers try to solve the problems of oral absorption of the polypeptide drugs by different modes such as chemical modification, novel drug delivery systems and the like. The functions of the nano-drug delivery system, such as high efficiency (taking a small amount of drug to play a role and efficacy), low toxicity and convenient use, are undoubtedly achieved, and a new technology is provided for the efficient and reasonable utilization of polypeptide drugs for human beings. With the increasingly deepened understanding of the action mechanism and the safety of the nano-drugs, the nano-drugs will meet the development stage from basic research to practical application, and a new milestone is created for improving the treatment level of human diseases.

One of the most potential methods for enhancing drug absorption is to wrap the drug into a nano-carrier system, and improve the internal absorption efficiency of the polypeptide drug by utilizing the efficient nano-transmucus and membrane transport performance. In order to overcome the degradation of the nanoparticles and the drug loaded therein in the stomach, enteric coating can be performed on the nanoparticles to overcome the degradation in the stomach, so that the nanoparticles reach the intestinal tract in an intact form. For example, it has recently been reported that freeze-dried powder of zwitterionic betaine micelles can effectively allow insulin to penetrate mucus and effectively promote epithelial absorption by encapsulating it in enteric capsules.

In addition to intragastric degradation, nanoparticles, like polypeptide drug molecules, are also captured and eliminated by the intestinal mucus layer due to steric hindrance or electrostatic interactions, hydrophobic forces, hydrogen bonding, and mucus interactions. Therefore, how to improve the mucus permeability of the nano-carrier, avoid the capture of mucus and quickly clear the mucus has important significance for promoting the oral absorption of the medicine. To address the issue of mucus barrier, one uses functional modifications to the nano-meter to prevent mucus adhesion and rapid clearance. For example, researchers have functionally modified nanoparticles with hydrophilic and neutral polymers (e.g., polyethylene glycol (PEG), poly (2-ethyl-2-oxazoline)) to effectively reduce the adsorption of mucus on nanoparticles by reducing the interaction with highly viscous mucin (the major structural component of mucus), and to promote the rate of nanoparticle transport in mucus and the ability to penetrate mucus.

To enhance the transport of nanoparticles across the intestinal mucosal barrier, a number of absorption enhancers (such as surfactant molecules and ionic liquids) have been used to open tight junctions between intestinal epithelial cells to enhance their ability to transport by the cellular pathway. Some cellular receptors (e.g., Fc receptors)/transporters (bile acid transporters and proton-assisted amino acid transporter 1) have also been used to enhance the ability of the nano-scale transport across intestinal epithelial cells.

Despite the great progress made by oral carrier systems, multifunctional nano drug delivery systems that overcome three major physiological barriers of the gastrointestinal tract of the body at the same time have not been successfully developed, resulting in the oral administration of polypeptide drugs still facing great challenges.

Disclosure of Invention

The present invention aims to provide a self-assembled mucositin sulfate nanoparticle with M cell targeting, a preparation method and an application thereof, so as to solve the problems of the prior art.

In order to achieve the above objects, the present invention provides a mucobactin sulfate self-assembled nanoparticle with M-cell targeting, which is composed of mucobactin sulfate, a polyanion compound, a polycation compound and a coating material;

wherein the polyanionic compound is selected from one or more of carrageenan, hyaluronic acid, peach gum, carrageenan and sodium alginate; the polycationic compound is selected from one or more of guar gum, xanthan gum, carob gum, agglutinin and mannosamine; the coating material is selected from one or more of heparin, citric acid, sodium carboxymethyl cellulose, cellulose acetate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, and polyvinyl alcohol ester.

Further, the mass ratio of the mucobactin sulfate, the polyanion compound, the polycation compound and the coating material is 6-12:7-30:7-15: 50-64.

Further, the mass ratio of the mucobactin sulfate to the polyanion compound to the polycation compound to the coating material is 10:25:15: 50.

Further, the preparation method of the self-assembled mucomycin sulfate nanoparticle with M cell targeting comprises the following steps:

adding polyanion compound into the aqueous solution of the mucobactin sulfate, mixing, and adding polycation compound to prepare nano particle solution; and dissolving the coating material, and then adding the dissolved coating material into the nano-particle solution to be continuously stirred to prepare the self-assembled nano-particles.

Further, the preparation method of the self-assembled mucomycin sulfate nanoparticle with the M cell targeting function specifically comprises the following steps:

(1) dissolving polyanion compound in water, adding aqueous solution of colistin sulfate, and mixing to obtain solution A;

(2) adding a dissolved polycation compound into the solution A obtained in the step (1) to prepare a nanoparticle solution, wherein the specific conditions are that the titration speed is 0.2-1mL/min, the stirring speed is 500-2000r/min, and the time is 2-12 h;

(3) and (3) adding the dissolved coating material into the nanoparticle solution prepared in the step (2) to prepare the M cell targeted self-assembled mucomycin sulfate nanoparticles under the conditions of stirring speed of 500-2000r/min and time of 2-12 h.

The invention also provides a preparation method of the self-assembled mucositin sulfate nanoparticles with M cell targeting, which comprises the following steps: adding a polyanion compound into a mucomycin sulfate aqueous solution, mixing, adding a polycation compound, and preparing a nanoparticle solution; and dissolving the coating material, and then adding the dissolved coating material into the nano-particle solution to be continuously stirred to prepare the self-assembled nano-particles.

Further, the preparation method specifically comprises the following steps:

The invention also provides an application of the self-assembled M cell targeted colistin sulfate nanoparticle in preparation of a colistin sulfate product.

The invention also provides application of the self-assembled mucositin sulfate nanoparticles with the M cell targeting function in preparation of a mucositin sulfate product with the M cell targeting function.

The invention discloses the following technical effects:

the colistin sulfate self-assembly nanoparticles with M cell targeting function prepared by the invention are innovatively synthesized to have the functions of M cell targeting function, high-efficiency trans-enterocyte and opening the precise connection between enterocyte, and the self-assembly nanoparticles are functionally modified by using a hydrophilic material with mucus penetrating power and gastric acid resistance, so that the oral bioavailability of the colistin sulfate can be remarkably improved, and the oral bioavailability of the colistin sulfate nanoparticles prepared by the invention is improved by more than 3 times. On one hand, the problems of drug degradation and nanoparticle damage caused by strong acidity of gastric juice are solved, on the other hand, the release of the mucositin sulfate in the stomach is reduced, so that the drug can smoothly enter the intestinal tract to penetrate a mucus barrier, and the targeted drug release performance of the mucositin sulfate in the intestinal tract is endowed. Meanwhile, the nanoparticle combines a functional material capable of reversibly opening tight connection, and the transmembrane transport efficiency of the drug carried by the nanoparticle is further promoted, so that the oral bioavailability of the myxobacter sulfatus is improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.

Fig. 1 is a physical diagram and a schematic diagram of a mucobactin sulfate nanoparticle with M cell targeting, wherein a is a physical diagram of a mucobactin sulfate nanoparticle lyophilized powder with M cell targeting; b is a real picture of the M cell targeted colistin sulfate nanoparticle aqueous solution; c is a schematic diagram of the mucobactin sulfate nanoparticle freeze-dried powder with M cell targeting;

FIG. 2 is an electron micrograph of M-cell targeted colistin sulfate nanoparticles prepared in example 5;

FIG. 3 is a time course of colistin sulfate nanoparticles and colistin sulfate in plasma;

fig. 4 is an SEM of colistin sulfate nanoparticles at pH 2;

FIG. 5 is the transport of colistin sulfate nanoparticles in recombinant porcine intestinal mucus, where A is the mean square displacement curve of the nanoparticles at different times; b is a two-photon confocal microscopic result picture;

figure 6 is an in vivo imaging of colistin sulfate nanoparticles.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.

The invention takes mucobactin sulfate as a model polypeptide medicament, and takes one or more of carrageenan, hyaluronic acid, peach gum, carrageenan and sodium alginate as polyanion matrix; one or more of guar gum, xanthan gum, carob bean gum, agglutinin and mannosamine are used as polycation matrix; one or other of heparin, citric acid, sodium carboxymethyl cellulose, cellulose acetate process ester, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate and polyvinyl alcohol ester is used as a coating material. The self-assembled mucositin sulfate nanoparticles are prepared by a classical solution method, and then the mucositin sulfate nanoparticles are added into a coating material to prepare the M cell targeted self-assembled mucositin sulfate nanoparticles which can overcome gastrointestinal tract barriers and realize transmembrane transport. The invention synthesizes a functional self-assembly nano-carrier system which has the M cell targeting function, high efficiency and can cross the intestinal epithelial cells and open the precise connection between the intestinal epithelial cells. Then, the coating material is utilized to modify the self-assembled nanoparticles, three physiological barriers which can overcome gastrointestinal transport of the drug are developed, a high-efficiency polypeptide drug oral administration delivery system is established, the development of a polypeptide drug oral preparation is promoted, and the clinical application of the polypeptide drug is promoted.

The materials used in the present invention are commercially available, unless otherwise specified; the experimental methods used are all routine experimental methods in the field unless otherwise specified.

Example 1 self-assembled nanoparticles of colistin sulfate formula 1 and its in vitro cumulative release

1.1 self-assembled nano-particles of mucobactin sulfate formula 1 and preparation

Formula 1 of self-assembled M cell-targeted colistin sulfate nanoparticles is shown in table 1.

Table 1 formula 1 with M cell targeted self-assembled mucobactin sulfate nanoparticles

The preparation method comprises the following steps:

(1) heating and dissolving 0.2g carrageenan in 25ml water, adding 10ml of 0.16g mucobacteriocin sulfate aqueous solution, and stirring to fully mix the two solutions to obtain a solution A;

(2) heating and dissolving 35mL of 0.714% guar gum solution, adding the solution A in the step (1), and continuously stirring for 2 hours under the conditions of a titration speed of 0.2mL/min and a stirring speed of 500r/min to prepare 100nm nanoparticle solution B, namely obtaining inner core NPs;

(3) respectively dissolving 5ml of 2.8% heparin and 20ml of 1.25% hydroxypropyl methyl cellulose phthalate, uniformly mixing, and continuously stirring for 2 hours at the stirring speed of 500r/min and the temperature of 37 ℃ to prepare a solution C;

(4) adding the solution C in the step (3) into the solution B in the step (2), continuously stirring for 2h under the condition of stirring speed of 500-2000r/min, preparing the self-assembled nano particles and freeze-drying.

1.2 measurement of sustained-release property of self-assembled mucobactin sulfate nanoparticles in simulated gastric fluid environment (pH 2) and results thereof

The preparation method comprises the steps of releasing a mucobactin sulfate bulk drug, mucobactin sulfate core nanoparticles (namely core nanoparticles obtained in the step (2) of 1.1) and mucobactin sulfate coated nanoparticles in an environment simulating gastric juice, sampling 1ml from corresponding preparation and bulk drug bottles for 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h and 12h and time periods, storing the samples in 2ml centrifuge tubes for later use, and adding the volume of a 1ml simulated gastric juice retention system. And detecting the sample by a high performance liquid chromatograph to obtain accumulated release data, and analyzing to determine the final coating material. And (3) carrying out release sampling and data analysis on the screened coating material-coated colistin sulfate nano granules in the condition of pH 8 simulated intestinal fluid, and inspecting the enteric-coated effect.

The in vitro cumulative release test results of the prepared self-assembled myxobactin sulfate nanoparticles of formula 1, the myxobactin sulfate and the inner core myxobactin sulfate nanoparticles are shown in table 2.

Table 2 in vitro release of nanoparticles at different pH values in example 1

From table 2 it can be seen that in the environment of simulated gastric fluid (pH 2), the cumulative release of both the mucobactin sulfate drug substance and the uncoated inner core nanosuspension is faster, releasing over 70% at 4h of the average pig stomach emptying time, with the drug substance releasing as fast as 90%. It can be seen that the nanoparticles alone in simulated gastric fluid do not have significant sustained release properties.

The prepared colistin sulfate nanoparticles can be slowly released for 12 hours in simulated gastric fluid, only 54 percent of the colistin sulfate nanoparticles are released when the average emptying time of the pig stomach is 4 hours, the slow release can be maintained in the environment simulating intestinal fluid, and the drug release characteristic is undoubtedly favorable for more nanoparticles carrying drugs to be absorbed in intestinal segments and the maximum drug effect of the colistin sulfate.

Example 2 formulation 2 of self-assembled nanoparticles of colistin sulfate and its in vitro cumulative release

2.1 self-assembled Nanoparticulate Mucosus sulfate formula 1 and preparation

Formula 2 of the self-assembled mucobactin sulfate nanoparticles is shown in table 3,

table 3 formula 2 of self-assembled nanoparticles of colistin sulfate

The preparation steps are as follows:

(1) heating and dissolving 0.15g of hyaluronic acid in 25ml of water, adding 10ml of a 0.17 g-containing aqueous solution of colistin sulfate, and stirring to fully mix the two solutions to obtain a solution A;

(2) heating 35mL of 0.714% lectin solution for dissolving, adding into the solution A obtained in the step (1), continuously stirring (technical conditions: titration speed of 0.4mL/min, stirring speed of 600r/min, and time of 4h), and preparing into 300nm nanoparticle solution B to obtain kernel NPs;

(3) respectively dissolving 5ml of 2.6% citric acid and 20ml of 1.5% hydroxypropyl methylcellulose acetate succinate, mixing, and stirring (at a stirring speed of 700r/min, time of 6h, and temperature of 50 deg.C) to obtain solution C;

(4) adding the solution C in the step (3) into the solution B in the step (2) (technical conditions: stirring speed 800r/min, time 5 h); continuously stirring to prepare the self-assembled nano particles, and freeze-drying.

2.2 determination of sustained-release property of self-assembled nano-particles of mucobactin sulfate in environment simulating gastric juice (pH 2) and results thereof

The sustained release performance of the self-assembled mucobactin sulfate nanoparticles in simulated gastric fluid environment (pH 2) was determined as in example 1. The in vitro cumulative release test results of the prepared self-assembled myxobactin sulfate nanoparticles of formula 2 and the myxobactin sulfate and uncoated myxobactin sulfate nanoparticles are shown in table 4.

Table 4 in vitro release of nanoparticles at different pH values in example 2

From table 4 it can be seen that in the environment of simulated gastric fluid (pH 2), the cumulative release of both the mucobactin sulfate drug substance and the uncoated inner core nanosuspension is faster, releasing over 77% at 4h of the average gastric emptying time of pigs, with the drug substance releasing as fast as 90%. It can be seen that the nanoparticles alone in simulated gastric fluid do not have significant sustained release properties.

The prepared colistin sulfate nanoparticles can be slowly released for 12 hours in simulated gastric juice, only 28% of the colistin sulfate nanoparticles are released when the average emptying time of the pig stomach is 4 hours, and the slow release is maintained to be 34% in the environment simulating intestinal juice, so that transmembrane transport of the colistin sulfate nanoparticles can be facilitated, and the drug effect can be maximally exerted.

Example 3 formulation 3 of self-assembled nanoparticles of colistin sulfate and its in vitro cumulative release

3.1 formula 3 and preparation of self-assembled nanoparticles of colistin sulfate

Formula 3 of the self-assembled mucobactin sulfate nanoparticles is shown in table 5.

Table 5 formula 3 of self-assembled nanoparticles of colistin sulfate

The preparation steps are as follows:

(1) heating and dissolving 0.25g of peach gum in 15ml of water, adding 15ml of a mucositis sulfate aqueous solution containing 0.10g of peach gum, and stirring to fully mix the two solutions to obtain a solution A;

(2) uniformly stirring 7.5mL of 2.0% carob bean gum solution and 7.5mL of 2.0% xanthan gum solution, adding the mixture into the solution A obtained in the step (1), and continuously stirring (the technical conditions are that the titration speed is 0.7mL/min, the stirring speed is 1000r/min, and the time is 7 hours) to prepare 600nm nanoparticle solution B, so as to obtain kernel NPs;

(3) respectively dissolving 10ml of 1.0% citric acid, 20ml of 0.25% heparin and 25ml of 0.8% hydroxypropyl methylcellulose acetate succinate, uniformly mixing, and continuously stirring (the technical conditions are that the stirring speed is 1000r/min, the stirring time is 6h, and the temperature is 57 ℃), thus obtaining a solution C;

(4) adding the solution C in the step (3) into the solution B in the step (2) (technical conditions: stirring speed 1800r/min, time 2 h); continuously stirring to prepare the self-assembled nano particles and freeze-drying.

3.2 determination of sustained-release property of self-assembled myxobactin sulfate nanoparticles in simulated gastric fluid environment (pH 2) and results

The sustained release performance of the self-assembled mucobactin sulfate nanoparticles in simulated gastric fluid environment (pH 2) was determined as in example 1. The in vitro cumulative release test results of the prepared self-assembled myxobactin sulfate nanoparticles of formula 3 with myxobactin sulfate and uncoated myxobactin sulfate nanoparticles are shown in table 6.

Table 6 in vitro release of nanoparticles at different pH values in example 3

From table 6 it can be seen that in the environment of simulated gastric fluid (pH 2), the cumulative release of both the mucositin sulfate drug substance and the uncoated inner core nanosuspension is faster, releasing over 80% at 4h of the average pig stomach emptying time, with the drug substance releasing as fast as 92%. It can be seen that the nanoparticles alone in simulated gastric fluid do not have significant sustained release properties.

The prepared colistin sulfate nanoparticles can be released for 8 hours in simulated gastric juice, and 77% of the colistin sulfate nanoparticles are released for 4 hours, which indicates that the acid resistance effect of the colistin sulfate nanoparticles cannot be achieved. And the release is also quick in the environment simulating intestinal juice, and the release characteristic is undoubtedly not beneficial to more colistin sulfate being absorbed in the intestinal segment.

Example 4 formulation 4 of self-assembled nanoparticles of colistin sulfate and its in vitro cumulative release

4.1 formula 4 and preparation of self-assembled nanoparticles of colistin sulfate

Formula 4 of the self-assembled mucobactin sulfate nanoparticles is shown in table 7.

Table 7 formula 4 of self-assembled nanoparticles of mucobacteriocin sulfate

The preparation method comprises the following steps:

(1) heating and dissolving 0.15g of carrageenan in 15ml of water, adding 15ml of a 0.10 g-containing aqueous solution of colistin sulfate, and stirring to fully mix the two solutions to obtain a solution A;

(2) adding 27mL of 3.7% lectin solution into the solution A in the step (1), continuously stirring (technical conditions: titration speed of 1mL/min, stirring speed of 2000r/min, time of 12h), and preparing into 900nm nanoparticle solution B to obtain kernel NPs;

(3) respectively dissolving 10ml of 2.3% citric acid, 10ml of 1.2% sodium carboxymethylcellulose, 20ml of 1.9% heparin and 10ml of 0.5% hydroxypropyl methylcellulose acetate succinate, uniformly mixing, and continuously stirring (the technical conditions are that the stirring speed is 2000r/min, the time is 12 hours, and the temperature is 78 ℃) to prepare a solution C;

(4) adding the solution C in the step (3) into the solution B in the step (2) (technical conditions: stirring speed 2000r/min, time 12 h); continuously stirring to prepare the self-assembled nano particles and freeze-drying.

4.2 determination of sustained-release property of self-assembled myxobactin sulfate nanoparticles in simulated gastric fluid environment (pH 2) and results thereof

The sustained release performance of the self-assembled mucobactin sulfate nanoparticles in an environment simulating gastric juice (pH 2) was determined as in example 1. The in vitro cumulative release test results of the prepared self-assembled myxobactin sulfate nanoparticles of formula 4 with myxobactin sulfate and uncoated myxobactin sulfate nanoparticles are shown in table 8.

Table 8 in vitro release of nanoparticles at different pH values in example 4

From table 8 it can be seen that in the environment of simulated gastric fluid (pH 2), the cumulative release of both the mucositin sulfate drug substance and the uncoated inner core nanosuspension is faster, releasing over 80% at 4h of the average pig stomach emptying time, with the drug substance releasing as fast as 92%. It can be seen that the nanoparticles alone in simulated gastric fluid do not have significant sustained release properties.

The release time of the prepared colistin sulfate nanoparticles in the pig stomach is only 35% within 4h, and the slow release of the prepared colistin sulfate nanoparticles in the simulated intestinal fluid environment is 41%, so that transmembrane transport of the colistin sulfate nanoparticles is possibly facilitated, and the drug effect is maximized.

Example 5 formulation 5 of self-assembled nanoparticles of colistin sulfate and its in vitro cumulative release

5.1 formula 5 and preparation of self-assembled nano-particles of mucobacteriocin sulfate

Formula 5 of the self-assembled mucobactin sulfate nanoparticles is shown in table 9.

TABLE 9 self-assembled nanoparticles of colistin sulfate formula 5

The preparation steps are as follows:

(1) heating and dissolving 0.11g of hyaluronic acid in 10ml of water, adding 10ml of a 0.11 g-containing aqueous solution of colistin sulfate, and stirring to fully mix the two solutions to obtain a solution A;

(2) adding 10mL of 1.1% mannosamine solution into the solution A obtained in the step (1), continuously stirring (the technical conditions are that the titration speed is 0.9mL/min, the stirring speed is 1600r/min, and the time is 10 hours), and preparing into 800nm nano-particle solution B, namely obtaining kernel NPs;

(3) respectively dissolving 15ml of 2.3% citric acid and 15ml of 2.3% sodium carboxymethylcellulose, mixing, and stirring (with stirring speed of 2000r/min, time of 10h, and temperature of 66 deg.C) to obtain solution C;

(4) adding the solution C in the step (3) into the solution B in the step (2) (technical conditions: stirring speed 2000r/min, time 11 h); continuously stirring to prepare the self-assembled nano particles and freeze-drying.

5.2 measurement of sustained-release property of self-assembled myxobactin sulfate nanoparticles in simulated gastric fluid environment (pH 2) and results thereof

The sustained release performance of the self-assembled mucobactin sulfate nanoparticles in an environment simulating gastric juice (pH 2) was determined as in example 1. The in vitro cumulative release test results of the prepared self-assembled myxobactin sulfate nanoparticles of formula 5 with myxobactin sulfate and uncoated myxobactin sulfate nanoparticles are shown in table 10.

Table 10 in vitro release of nanoparticles at different pH values in example 5

From table 10, it can be seen that the self-assembled myxobactin sulfate nanoparticles have obvious sustained release effects in simulated gastric fluid and intestinal fluid, and such a drug release characteristic will undoubtedly facilitate more absorption of myxobactin sulfate in intestinal segment and exertion of the maximum drug effect of myxobactin sulfate.

As a control, the cumulative release of the colistin sulfate drug substance and the uncoated inner core nanosuspension was faster, and the release exceeded 69% at 4h of the average emptying time of the pig stomach, with the drug substance being released as fast as 92%. It can be seen that the nanoparticles alone in simulated gastric fluid do not have significant sustained release properties.

Example 6 formulation 6 of self-assembled nanoparticles of colistin sulfate and its in vitro cumulative release

6.1 formula 6 and preparation of self-assembled nano-particles of colistin sulfate

Formula 6 of the self-assembled mucobactin sulfate nanoparticles is shown in table 11.

TABLE 11 self-assembled nanoparticulate colistin sulfate formulation 6

The preparation method comprises the following steps:

(1) heating and dissolving 0.15g of hyaluronic acid in 12ml of water, adding 12ml of a 0.10 g-containing aqueous solution of colistin sulfate, and stirring to fully mix the two solutions to obtain a solution A;

(2) adding 12mL of 1.25% xanthan gum solution into the solution A in the step (1), continuously stirring (the technical conditions are that the titration speed is 0.2mL/min, the stirring speed is 2000r/min, and the time is 27h), and preparing a nanoparticle solution B with the particle size of 900nm to obtain kernel NPs;

(3) respectively dissolving 18ml of 1.6% citric acid and 18ml of 1.6% hydroxypropyl methylcellulose acetate succinate, mixing, and stirring (with stirring speed of 2000r/min, time of 9h, and temperature of 90 deg.C) to obtain solution C;

(4) adding the solution C in the step (3) into the solution B in the step (2) (the technical conditions are that the stirring speed is 2000r/min, and the time is 2-12 h); continuously stirring to prepare the self-assembled nano particles and freeze-drying.

6.2 measurement of sustained-release property of self-assembled myxobactin sulfate nanoparticles in simulated gastric fluid environment (pH 2) and results thereof

The sustained release performance of the self-assembled mucobactin sulfate nanoparticles in an environment simulating gastric juice (pH 2) was determined as in example 1. The in vitro cumulative release test results of the prepared self-assembled myxobactin sulfate nanoparticles of formula 6 with myxobactin sulfate and uncoated myxobactin sulfate nanoparticles are shown in table 12.

Table 12 in vitro release of nanoparticles at different pH values in example 6

From table 12, it can be seen that the self-assembled myxobactin sulfate nanoparticles have obvious sustained release effects in simulated gastric fluid and intestinal fluid, and such a drug release characteristic will undoubtedly facilitate more absorption of myxobactin sulfate in intestinal segment and exertion of the maximum drug effect of myxobactin sulfate.

At PH 2, the mucositin sulfate bulk drug and the uncoated inner core nanosuspension are both rapidly released, and it can be seen that simple nanoparticles in simulated gastric juice do not have an obvious sustained release property, which is mainly that under the strong acid environment of gastric juice, part of nanoparticles are damaged, resulting in rapid release of the drug. The self-assembled nanoparticles were released at pH 2 in the order of example 3, example 1, example 4, example 5, example 6, and example 2, and although the nanoparticles in example 2 released the slowest drug, the coating material in example 5 was used as the coating material of the mucositin sulfate nanoparticles and the nanoparticles were prepared for the cross-mucodynamic study in consideration of the cost, the preparation process, and other comprehensive conditions.

Test example 1 preparation of mucositin sulfate nanoparticles containing different surface charges and hydrophilicity and Trans-mucus test

1.1 preparation of mucobactin sulfate nanoparticles of different surface charges and hydrophilicity and hydrophobicity

According to tables 13 and 14, two test-group preparations containing mucositin sulfate nanoparticles of different surface charges and hydrophobicity were prepared.

1.1.1 self-assembling nanoparticles of colistin sulfate test group 1

TABLE 13 preparation of mucobactin sulfate self-assembled nanoparticles test 1

Preparation of mucobactin sulfate nanoparticles assay 1 group:

(1) heating and dissolving 0.25g of hyaluronic acid in 15ml of water, adding 15ml of a 0.10 g-containing aqueous solution of colistin sulfate, and stirring to fully mix the two solutions to obtain a solution 1;

(2) adding 15mL of 0.5% mannosamine solution into the solution 1 in the step (1), and continuously stirring (technical conditions: titration speed 0.9mL/min, stirring speed 1600r/min, time 10h) to prepare 800nm nanoparticle solution 2;

(3) respectively dissolving 15ml of 2% citric acid and 15ml of 2% carboxymethyl cellulose, mixing, and stirring (with stirring speed of 2000r/min, time of 10 hr, and temperature of 66 deg.C) to obtain solution 3;

(4) adding the solution 2 in the step (3) into the solution 2 in the step (2) (technical conditions: stirring speed 2000r/min, time 11 h); continuously stirring to prepare the self-assembled nano particles and freeze-drying.

1.1.2 self-assembling nanoparticles of Muslim sulphated bacitracin assay 2 groups

Table 14 test set 2 formulation of self-assembled nanoparticles of colistin sulfate

Preparation of mucobactin sulfate nanoparticles assay group 2:

(1) heating and dissolving 0.10g of hyaluronic acid in 10ml of water, adding 10ml of a 0.10 g-containing aqueous solution of colistin sulfate, and stirring to fully mix the two solutions to obtain a solution 1;

(2) adding 10mL of 2% mannosamine solution into the solution 1 in the step (1), and continuously stirring (technical conditions: titration speed of 0.9mL/min, stirring speed of 1600r/min, time of 10h) to prepare 800nm nanoparticle solution 2;

(4) adding the solution 2 in the step (3) into the solution 2 in the step (2) (technical conditions: stirring speed 2000r/min, time 11 h); continuously stirring to prepare the self-assembled nano particles, and freeze-drying.

1.2 Transmyxoid assay containing differently surface charged and hydrophobic colistin sulfate nanoparticles

1.2.1 materials and methods

(1) Medicine preparation: 1.1 two groups of colistin sulfate nanoparticles prepared in

(2) Test reagent porcine gastric mucin.

(3) Instrument two-photon confocal microscope, glass dish.

(4) The test method comprises the following steps:

preparation of simulated gastric fluid: the mucus was constructed to dissolve porcine pepsin in PBS at a concentration of 30 mg/ml. Nanoparticles were added to 200. mu.l mucus before observation to 4% v/v (final particle concentration 8.25X 10)^-7w/v)。

The testing steps are as follows: the dynamic process of the nanoparticles in the mucus is followed. The nanoparticles were labeled with FITC or RBITC by covalent cross-linking, then dispersed in 200ul of fresh pig gastric mucus solution (4% v/v) at a concentration of 30mg/ml (bottom thickness 0.17mm) and incubated at 37 ℃ for 30 minutes. Video was captured with 100 ms temporal resolution using x 64 oil immersion objective in a spinning disk confocal microscope. For different types of NPs, traces of n-50 NPs were analyzed and three replicates were performed. The model is calculated by the following equation:

where x and y represent the coordinates of the nanoparticle at a given time scale (t) and τ represents the time interval or exposure time.

1.2.2 results

The results of the experiments with mucositin sulfate nanoparticles of different surface charges and hydrophobicity are shown in table 15, fig. 5.

TABLE 15 mean square displacement of nanoparticles at different times

Group of	0.3s	0.6s	0.9s	1.2s	1.5s
						Group 1	0.03	0.045	0.081	0.11	0.13
2 groups of	0.1	0.35	0.6	0.95	1.4

Two groups of nanoparticles were incubated with the constructed mucus, and the diffusion traces of these particles were traced through a two-photon confocal microscope. From the test results in table 15, it can be seen that the diffusion speed of the mucositin sulfate nanoparticles with different surface charges and hydrophobicity in mucus is greatly different, and we found that the overall average geometric mean square shift of the nanoparticle 2 group is about 8.5 times that of the nanoparticle 1 group. Compared with the reported and accepted movement rate of the PEG modified nanoparticles, the movement rate of the nanoparticles in the 2 group is 11.3 times faster, and the movement rate of the nanoparticles in the 1 group is 1.3 times faster. We can conclude from this that both sets of nanoparticles of mucobactin sulphate can move rapidly in the mucus, thereby penetrating the mucus barrier and promoting the entry of the drug into the epithelial layer.

The optimum amount of the formulation of the present invention of the mucobactin sulphate nanoparticles was therefore determined as the amount of the nanoparticle test group 2.

Test example 2M cell targeting test of colistin sulfate nanoparticles

The M cell is a special intestinal epithelial cell, is mainly covered on a lymph follicle at the tail end of an ileum, has the capacity of efficient transcellular transport, can effectively transport various substances, and can avoid the first-pass effect of a medicament. Therefore, the ability of the M cell targeted transport is enhanced through the surface modification of the nanometer, and the method is an important way for further improving the absorption of the nanometer loading drug.

2.1 test methods

Firstly, covalently labeling the colistin sulfate by CY5.5, and comprises the following specific steps: 5mg of CY5.5 dye are weighed into a 1% mucositin sulfate solution and stirred for 5h for sufficient binding. Unbound CY5.5 was then removed using dialysis bag purification and nanoparticle preparation was carried out according to the ratio and preparation method of 1.1.2 in test example 1.

Then, the absorption sites of the self-assembled mucositin sulfate nanoparticles are verified through a mouse in vivo test, and the method comprises the following specific steps: fasting was performed for 12 hours before oral administration, and the two groups were randomly divided into three groups (drug-only group, nanoparticle group). Subsequently, mice were euthanized at fixed times of 1 hour, 4 hours, and 8 hours, and the entire digestive organ (from stomach to rectum) was collected into a glass dish. These glass disks were used to study nanoparticle absorption sites and M cell targeting by using an ivis spectroscopy system.

2.2 results

The results for the mucobactin sulfate nanoparticles are shown in figure 6. The significant difference between the two groups of samples can be seen from fig. 6, the drug of the simple raw material drug group is hardly absorbed, and is basically in the duodenum after about 1h, but is almost eliminated from the body later. As a contrast, the nanoparticle group had a certain sustained release effect, and an obvious fluorescence signal could still be seen in the 8h intestinal tract. Importantly, nanoparticles were observed to be mainly concentrated in the ileum stage enriched for M cells at the 4h and 8h time points, from which it could be preliminarily verified that the mucobactin sulfate nanoparticles have a certain M cell targeting. Subsequently, to verify this hypothesis, we took off the peyer's patches and observed the change in the number of nanoparticles and the increase and decrease of M cells at different times by confocal microscopy, from which we can conclude that the mucobactin sulfate nanoparticles are co-absorbed by M cells.

Test example 3 pharmacokinetics of colistin sulfate nanoparticles

3.1 materials and methods

A medicine mucomycin sulfate bulk drug, a mucomycin sulfate nanoparticle.

(1) The experimental animals, 12 mice and 200g, were raised in cages in the SPF animal house of national veterinary drug residue benchmark laboratory of Huazhong university of agriculture.

(2) High speed centrifuge, liquid chromatography.

(3) Test method

The experiment is carried out on the premise of strictly complying with the animal welfare principle, and the conditions of proper temperature, sufficient feed and free drinking water are ensured in the period. 12 rats weighing 200g are randomly divided into a mucositin sulfate bulk drug group and a mucositin sulfate nanoparticle group. The administration is carried out by intragastric administration according to the recommended dosage of 15mg/kg body weight. Collecting blood samples of mice at 0.125, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after administration, centrifuging, taking supernatant, detecting the content of the colistin sulfate in the blood by a liquid chromatography mass spectrometer after pretreatment, and drawing a pharmaceutical time curve.

3.2 results

The pharmacokinetic data of the colistin sulfate drug and the colistin sulfate nanoparticles prepared by the present invention are shown in table 16.

TABLE 16 pharmacokinetics of mucobacteriocin sulfate nanoparticles

As can be seen from Table 16, the area under the curve of the granules is 3.01 times that of the bulk drug, i.e., the relative bioavailability is 301.21%, and C is_maxCompared with the bulk drug, the nano-particle is obviously improved by 2.77 times, which is probably that after the nano-particle is orally taken, less drug is metabolized by gastric acid, more drug is transported to intestinal segment to be absorbed, and the mucomycin sulfate bulk drug is less absorbed by oral administration due to too strong polarity. After entering the intestinal tract, the nanoparticles penetrate through a mucus layer and are absorbed by M cells in a targeted manner, so that the in-vivo drug release time of the colistin sulfate is prolonged. In conclusion, the present invention successfully prepares a mucositin sulfate nanoparticle that can overcome gastrointestinal tract barriers and achieve M cell targeting, and has a high maximum blood concentration while having a strong sustained release capability, which undoubtedly will be beneficial to the exertion of the maximum drug effect of mucositin sulfate, and will also be beneficial to the clinical use of mucositin sulfate in veterinary medicine for disease prevention and control.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A mucobactin sulfate self-assembled nanoparticle having M-cell targeting, wherein the mucobactin sulfate self-assembled nanoparticle comprises a mucobactin sulfate, a polyanionic compound, a polycationic compound, and a coating material;

wherein the polyanionic compound is selected from one or more of carrageenan, hyaluronic acid, peach gum, carrageenan and sodium alginate; the polycationic compound is selected from one or more of guar gum, xanthan gum, carob bean gum, agglutinin and mannosamine; the coating material is selected from one or more of heparin, citric acid, sodium carboxymethyl cellulose, cellulose acetate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate and polyvinyl alcohol ester;

the mass ratio of the mucobactin sulfate to the polyanion compound to the polycation compound to the coating material is 6-17:5-30:5-20: 40-80;

the preparation method of the self-assembled mucobacteriocin sulfate nanoparticle comprises the following steps:

adding polyanion compound into the aqueous solution of the colistin sulfate, mixing, adding polycation compound, and preparing into a nano-particle solution; and dissolving the coating material, and then adding the dissolved coating material into the nano-particle solution to be continuously stirred to prepare the self-assembled nano-particles.

2. The self-assembled mucobactin sulfate nanoparticle with M-cell targeting according to claim 1, wherein the mass ratio of the mucobactin sulfate, the polyanionic compound, the polycationic compound and the coating material is 10:25:15: 50.

3. The M-cell targeted self-assembled mucobactin sulfate nanoparticle according to claim 1, wherein the method for preparing the M-cell targeted self-assembled mucobactin sulfate nanoparticle comprises:

(3) and (3) adding the dissolved coating material into the nanoparticle solution prepared in the step (2) to prepare the M cell targeted self-assembled mucomycin sulfate nanoparticles under the conditions of stirring speed of 500-.

4. A method for preparing M-cell targeted self-assembled mucobactin sulfate nanoparticles according to any one of claims 1 to 3, wherein the method comprises: adding a polyanion compound into a mucomycin sulfate aqueous solution, mixing, adding a polycation compound, and preparing a nanoparticle solution; and dissolving the coating material, and then adding the dissolved coating material into the nano-particle solution to be continuously stirred to prepare the self-assembled nano-particles.

5. The preparation method according to claim 4, wherein the preparation method specifically comprises:

6. Use of the self-assembled M cell-targeted colistin sulfate nanoparticles of any one of claims 1-3 for the preparation of colistin sulfate products.

7. Use of the self-assembled mucositin sulfate nanoparticles with M cell targeting according to any one of claims 1 to 3 for the preparation of a mucositin sulfate product with M cell targeting function.