Microcapsule with intestinal mucosa adsorption effect and preparation method thereof

Abstract

The invention relates to the technical field of medical, dental or dressing preparations, in particular to a microcapsule with an intestinal mucosa adsorption effect and a preparation method of the microcapsule. Compared with the prior art, the chitosan is modified, so that free thiol groups are introduced to the surface of the chitosan, the coating material is obtained through crosslinking, then the coating material and the active ingredients are embedded to obtain the microcapsule, and the microcapsule can improve mucous membrane adhesion and prolong retention time, so that the retention time of the medicine in the digestive tract is prolonged, and the bioavailability of the medicine is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical, dental or cosmetic preparations, and in particular to a microcapsule capable of adsorbing intestinal mucosa and a preparation method thereof. Background Art

[0002] Bioadhesion technology is a special drug delivery mechanism that relies on the interaction between certain polymer materials and biological mucosal surfaces to generate adhesion. This technology has achieved remarkable results in drug delivery systems in the oral cavity, nasal cavity, uterus and vagina, especially in improving drug efficacy, prolonging drug action time, controlling drug release rate, reducing toxic side effects and improving bioavailability. Gastrointestinal bioadhesive preparations are a form of oral drugs that prolong the residence time of drugs in the gastrointestinal tract through the adhesion between polymer materials and the gastric and intestinal (mainly small intestine) mucosa. This preparation does not cover oral mucosal adhesion, colon-localized drug delivery systems, rectal administration preparations and other mucosal adhesive preparations. The benefits of this preparation include: after oral administration, it adheres to the digestive tract mucosa, prolongs the residence of the preparation in the gastrointestinal tract, helps to develop long-acting drugs, and reduces the frequency of administration. For drugs with low solubility or absorption in specific parts, delaying the residence time of the preparation can improve its absorption effect, thereby improving bioavailability. The close contact between the preparation and the mucosa not only increases the amount of drug absorbed, but also accelerates the absorption rate. It can be targeted to the gastrointestinal lesion area to achieve the purpose of local treatment.

[0003] CN108601846B relates to a mucoadhesive pharmaceutical composition comprising a poly-ionic complex of chitosan and a polyacrylic acid polymer. When the composition of the invention is used as an oral preparation, the retention time of the preparation is prolonged by virtue of its mucosal adhesion and swelling properties in the gastrointestinal tract, thereby contributing to improving the absorption of sustained-release preparations for oral administration, retention preparations of active ingredients with limited absorption sites, and active ingredients whose solubility changes with pH.

[0004] CN116271095A relates to an intestinal adhesive anti-enzymatic hydrolysis gel microcapsule oral delivery system for bioactive macromolecular drugs, which comprises: sodium alginate drug-loaded gel microcapsules and chitosan grafted with digestive enzyme inhibitors adsorbed on the surface of the sodium alginate drug-loaded gel microcapsules; the sodium alginate drug-loaded gel microcapsules are composed of sodium alginate wall materials and bioactive macromolecular drugs embedded in the sodium alginate wall materials. The oral delivery system can resist the effects of gastric acid and digestive enzyme degradation, and has intestinal adhesiveness, and can maintain protease activity in the digestive tract for a long time. The invention also relates to a preparation method for an intestinal adhesive anti-enzymatic hydrolysis gel microcapsule oral delivery system for bioactive macromolecular drugs, wherein no organic solvent is introduced during the preparation process, the material has low biological toxicity, and the product is safe and reliable.

[0005] A large number of studies have reported that chitosan can be used as tablet excipients, disintegrants, adhesives, etc. for colon-targeted drug delivery. In addition, chitosan can be degraded by special enzymes in the colon and is also used as a coating material for colon-targeted drug delivery systems. Microparticles or microcapsules are considered to be one of the best ways to release drugs in the intestinal tract. They can adhere to the surface of the diseased colon mucosa, slowly release drugs, and increase the local drug concentration in the lesion site, thereby better treating intestinal diseases. Chitosan, as a cationic polymer, also has unique properties. The mucin surface in the intestinal mucus layer carries a negative charge, so the positively charged chitosan surface can bind to mucin under the action of electrostatic force, thereby adhering to the intestine and remaining in the digestive tract for a longer time. However, the electrostatic effect of chitosan binding to mucin is weak, so this adhesion cannot be maintained for too long and the adhesion is also poor. Summary of the invention

[0006] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a microcapsule having the function of adsorbing intestinal mucosa and a preparation method thereof.

[0007] The intestinal mucosa wraps around the intestinal epithelial cells, and its main components include water, carbohydrates, inorganic salts, fatty acids, lipids and glycoproteins. Depending on the physiological position and gene expression, the glycoproteins in the mucus matrix have different hydrophilic / lipophilic properties and charges. Therefore, chitosan with a positive charge on the surface can bind to mucin under the action of electrostatic force. In addition, the amino bond and hydrophobic interaction between chitosan and the mucosa may also be one of the reasons for its mucosal adhesion. However, these ionic interactions between cationic polymers such as chitosan and mucus are weak, making it difficult to achieve long-term and stable adhesion. The present invention modifies chitosan, thereby introducing free thiol groups on its surface, and then cross-linking to obtain a packaging material, and then embedding with active ingredients to obtain microcapsules. The present invention modifies chitosan, thereby introducing free thiol groups on its surface, cross-linking to obtain a packaging material, and then embedding with active ingredients to obtain microcapsules, which can improve mucosal adhesion and prolong retention time, so that the retention time of the drug in the digestive tract is prolonged, and the bioavailability of the drug is improved.

[0008] To achieve the above object, the present invention provides a method for preparing microcapsules having the function of adsorbing intestinal mucosa, comprising the following steps:

[0009] S1. Add 2-(mercaptomethyl)-cysteine ​​to dilute hydrochloric acid, then add N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, stir for 1 to 2 hours and set aside;

[0010] S2, adding chitosan to dilute hydrochloric acid, stirring until completely dissolved, adding the mixed solution of 2-(mercaptomethyl)-cysteine ​​in S1, adding 1 mol / L sodium hydroxide solution to adjust the pH to 5-6, continuing to stir for 4-6 hours under an inert atmosphere, dialyzing, and freeze-drying to obtain modified chitosan;

[0011] S3. Disperse the modified chitosan in 1% acetic acid solution, add the active drug, let it stand for use to obtain a mixture A, mix the liquid paraffin and Span 80, heat and stir evenly, then add it to the mixture A, stir and disperse, add 5% sodium tripolyphosphate for cross-linking for 2 to 3 hours, centrifuge and separate, then freeze-dry the lower precipitate to obtain the mixture.

[0012] Furthermore, the solid-liquid ratio of the 2-(mercaptomethyl)-cysteine ​​to dilute hydrochloric acid is 0.4 to 1:10 g / mL.

[0013] Furthermore, the molar ratio of the 2-(mercaptomethyl)-cysteine ​​to N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1-2:1-2:2-2.5.

[0014] Furthermore, the solid-liquid ratio of the chitosan to the dilute hydrochloric acid is 1:80-100 g / mL.

[0015] Furthermore, the mass ratio of chitosan to 2-(mercaptomethyl)-cysteine ​​is 1:1-6.

[0016] Furthermore, the mass concentration of the modified chitosan in the acetic acid solution is 1-2%.

[0017] Furthermore, the active drug is one of nucleic acid, polypeptide, mesalazine or other active ingredients.

[0018] Furthermore, the mass concentration of the active drug in step S3 is 0.001-10%.

[0019] Furthermore, the volume fraction of the liquid paraffin in the total volume is 40-50%, and the volume ratio of Span 80 to the liquid paraffin is 1:8-10.

[0020] Preferably, the method for preparing the microcapsules having the function of adsorbing intestinal mucosa comprises the following steps:

[0021] S1. Add 2-(mercaptomethyl)-cysteine ​​to 1wt% dilute hydrochloric acid at a solid-liquid ratio of 0.4-1:10 g / mL, then add N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, the molar ratio of 2-(mercaptomethyl)-cysteine ​​to N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1-2:1-2:2-2.5, stir for 1-2h and set aside;

[0022] S2, adding chitosan to 1wt% dilute hydrochloric acid with a solid-liquid ratio of 1:80-100g / mL, stirring until completely dissolved, adding the mixed solution of 2-(mercaptomethyl)-cysteine ​​in S1, so that the mass ratio of chitosan to 2-(mercaptomethyl)-cysteine ​​is 1:1-6, adding 1mol / L sodium hydroxide solution to adjust the pH to 5-6, continuing to stir for 4-6h under an inert atmosphere, dialyzing, and freeze-drying to obtain modified chitosan;

[0023] S3. Disperse 1-2% (w / v) modified chitosan in 1% (v / v) acetic acid solution, add 0.001-10% (w / v) active drug, let stand for use to obtain mixture A, mix liquid paraffin and Span 80, heat and stir evenly, and then add to mixture A, the volume fraction of liquid paraffin in the total volume is 40-50%, and the volume ratio of Span 80 to liquid paraffin is 1:8-10. After stirring and dispersing, add 5% (w / v) sodium tripolyphosphate for cross-linking for 2-3 hours, centrifuge and separate, and freeze-dry the lower precipitate to obtain the mixture.

[0024] The present invention also provides a microcapsule with the function of adsorbing intestinal mucosa, which is prepared by the above method.

[0025] Beneficial effects of the present invention:

[0026] The present invention modifies chitosan to introduce free thiol groups on its surface, obtains a packaging material through cross-linking, and then embeds the active ingredients to obtain microcapsules. The microcapsules can improve mucosal adhesion and prolong retention time, thereby prolonging the retention time of the drug in the digestive tract and improving the bioavailability of the drug. DETAILED DESCRIPTION

[0027] 2-(Mercaptomethyl)-cysteine, Cysteine, 2-(mercaptomethyl)-, CAS No.: 501950-56-7.

[0028] Example 1

[0029] A method for preparing microcapsules with intestinal mucosal adsorption function comprises the following steps, measured in parts by weight:

[0030] S1. Add 4.75 parts of 2-(mercaptomethyl)-cysteine ​​to 100 parts of 1 wt% dilute hydrochloric acid, then add 6.5 parts of N-hydroxysuccinimide and 8.5 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, stir for 2 hours and set aside;

[0031] S2, adding 5 parts of chitosan to 400 parts of 1wt% dilute hydrochloric acid, stirring until completely dissolved, adding the mixed solution of 2-(mercaptomethyl)-cysteine ​​in S1, so that the mass ratio of chitosan to 2-(mercaptomethyl)-cysteine ​​is 1:1, adding 1 mol / L sodium hydroxide solution to adjust the pH to 5.5, continuing to stir for 6 hours under an inert atmosphere, dialyzing, and freeze-drying to obtain modified chitosan;

[0032] S3. Disperse 1.5% (w / v) modified chitosan in 1% (v / v) acetic acid solution, add 0.005% (w / v) mesalazine, let stand and set aside to obtain mixture A, mix liquid paraffin and Span 80, heat and stir evenly, and then add to mixture A, the volume fractions of liquid paraffin and Span 80 are 45% and 5% of the total volume, stir and disperse, add an equal volume of 5% (w / v) sodium tripolyphosphate, cross-link for 3 hours, centrifuge and separate, and freeze-dry the lower precipitate to obtain the mixture.

[0033] Example 2

[0034] The method is basically the same as Example 1, except that the mass ratio of chitosan to 2-(mercaptomethyl)-cysteine ​​in step S2 is 1:2.

[0035] Example 3

[0036] The method is basically the same as Example 1, except that the mass ratio of chitosan to 2-(mercaptomethyl)-cysteine ​​in step S2 is 1:4.

[0037] Example 4

[0038] The method is basically the same as Example 1, except that the mass ratio of chitosan to 2-(mercaptomethyl)-cysteine ​​in step S2 is 1:6.

[0039] Comparative Example 1

[0040] A method for preparing microcapsules with intestinal mucosal adsorption function comprises the following steps:

[0041] 1.5% (w / v) chitosan is dispersed in 1% (v / v) acetic acid solution, and then 0.005% (w / v) mesalazine is added, and the mixture is allowed to stand for use to obtain a mixture A. Liquid paraffin and Span 80 are mixed, heated and stirred evenly, and then added to the mixture A. The volume fractions of liquid paraffin and Span 80 are 45% and 5% of the total volume. After stirring and dispersing, an equal volume of 5% (w / v) sodium tripolyphosphate is added, cross-linked for 3 hours, and the lower precipitate is freeze-dried after centrifugation to obtain the mixture.

[0042] Comparative Example 2

[0043] A method for preparing microcapsules with intestinal mucosal adsorption function comprises the following steps, measured in parts by weight:

[0044] S1. Add 3.5 parts of cysteine ​​to 100 parts of 1 wt% dilute hydrochloric acid, then add 6.5 parts of N-hydroxysuccinimide and 8.5 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, stir for 2 hours and set aside;

[0045] S2, adding 5 parts of chitosan to 400 parts of 1wt% dilute hydrochloric acid, stirring until completely dissolved, adding the mixed solution of cysteine ​​in S1, so that the mass ratio of chitosan to cysteine ​​is 1:1, adding 1 mol / L sodium hydroxide solution to adjust the pH to 5.5, continuing to stir for 6 hours under an inert atmosphere, dialyzing, and freeze-drying to obtain modified chitosan;

[0046] S3. Disperse 1.5% (w / v) modified chitosan in 1% (v / v) acetic acid solution, add 0.005% (w / v) mesalazine, let stand and set aside to obtain mixture A, mix liquid paraffin and Span 80, heat and stir evenly, and then add to mixture A, the volume fractions of liquid paraffin and Span 80 are 45% and 5% of the total volume, stir and disperse, add an equal volume of 5% (w / v) sodium tripolyphosphate, cross-link for 3 hours, centrifuge and separate, and freeze-dry the lower precipitate to obtain the mixture.

[0047] Test Example 1

[0048] The microcapsules in the control examples and the examples were tested for intestinal mucosal adhesion. First, male C57BL / 6 mice were fasted for 24 hours, but free drinking water was allowed. The next day, anesthesia was performed by intraperitoneal injection of chloral hydrate, and then the abdomen was opened along the midline of the abdomen to remove the duodenum and colon. These intestinal sections were washed with normal saline, the contents were removed, and then 6 cm duodenum and colon segments were separated and accurately weighed. The ulcerative colitis rat model was established using the TNBS induction method, and a 6 cm inflamed colon segment was obtained and weighed in the same way. After these intestinal segments were turned outward into a sac shape, they were placed in 10 mL of PBS solution containing 10 mg of microcapsules, and the pH values ​​of the duodenum and colon buffers were 5.5 and 7.4, respectively. At 37°C, the intestinal sac was slowly shaken for 30 minutes, and then the intestinal sac was removed, the microcapsules were centrifuged, freeze-dried and weighed. Mucosal adhesion = (initial microcapsule weight-unadherent microcapsule weight) / initial microcapsule weight*100%.

[0049] Table 1 Microcapsule mucosal adhesion test results

[0050]

[0051] The surface of the colon mucosa is covered with abundant villi, which helps the mucosa and epithelial cells adhere to the microparticles more effectively. In addition, the number of goblet cells in the colon area is significantly greater than that in the duodenum, which leads to higher mucin levels in the colon mucosa than in the duodenum, thus increasing the adhesion of microcapsules to the colon mucosa. The inflamed colon has significantly enhanced mucosal adhesion to microcapsules compared to the normal colon. This is mainly because the colon in an inflammatory state can produce more mucus glycoproteins, which can react with the thiol groups in the composite microparticles, thereby enhancing the adhesion of the inflamed colon to these microcapsules. From the comparison between Example 1 and Comparative Example 1, it can be seen that the mucosal adhesion of the microcapsules prepared by modified chitosan is significantly higher than that of chitosan. This may be because the chitosan with positive surface charge in Comparative Example 1 binds to mucin under the action of electrostatic force. In addition, the amino bond and hydrophobic interaction between chitosan and mucosa may also be one of the reasons for its mucosal adhesion. However, in addition to these adhesions with low interaction forces in Example 1, the thiol groups on the surface of chitosan after modification can also bind to glycoproteins to form disulfide bonds. The covalent action is stronger, so the binding is tighter, resulting in improved adhesion of the microcapsules to the mucosa. The thiol groups in Example 1 are more than those in Comparative Example 2, so the adhesion is also better. Compared with Examples 2 to 4, Example 1 has a different mass ratio of chitosan to 2-(mercaptomethyl)-cysteine. In Examples 1 to 4, the mass ratio of the two gradually increases, but the adhesion does not increase accordingly. This shows that the increase of 2-(mercaptomethyl)-cysteine ​​does not help the adhesion, but reduces the adhesion. Therefore, the dosage ratio in Example 1 is the best.

[0052] Test Example 2

[0053] 1 mL of 10 mg / mL microcapsule saline dispersion was added to simulated gastric fluid (9 mL, pH = 1.2) and incubated at 37 ° C with gentle stirring for 2 h. Then, the simulated gastric fluid was replaced with 9 mL of simulated intestinal fluid (pH 6.8) and left for 6 h, and then replaced with 9 mL of simulated intestinal fluid (pH 7.4) and left for another 24 h. The drug content was measured at different time periods to obtain the drug release rate.

[0054] Table 2 Results of in vitro release experiment of microcapsules

[0055]

[0056]

[0057] From the results of in vitro release, it can be seen that the microcapsules prepared by the present invention basically do not release in the simulated gastric fluid environment, and only start to release when replaced with a simulated intestinal fluid environment. When the chitosan is modified and cross-linked to improve its stability, and then the active drug is encapsulated, it can achieve the goal of basically no release under gastric acid conditions for a short time. As time goes by, after the drug begins to enter the intestine, the drug begins to be released in large quantities, thereby achieving the result of directional release of the drug.

[0058] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for preparing microcapsules with intestinal mucosal adsorption function, characterized in that: The steps include: S1. Add 2-(mercaptomethyl)-cysteine ​​to dilute hydrochloric acid, then add N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, stir for 1 to 2 hours and set aside; S2, adding chitosan to dilute hydrochloric acid, stirring until completely dissolved, adding the mixed solution of 2-(mercaptomethyl)-cysteine ​​in S1, adding 1 mol / L sodium hydroxide solution to adjust the pH to 5-6, continuing to stir for 4-6 hours under an inert atmosphere, dialyzing, and freeze-drying to obtain modified chitosan; S3. Disperse the modified chitosan in 1% acetic acid solution, add the active drug, let it stand for use to obtain a mixture A, mix the liquid paraffin and Span 80, heat and stir to mix, then add to the mixture A, stir and disperse, add 5% sodium tripolyphosphate to cross-link for 2 to 3 hours, centrifuge and separate, and freeze-dry the lower precipitate to obtain the mixture.

2. The method for preparing microcapsules having the function of adsorbing intestinal mucosa according to claim 1, characterized in that: The solid-liquid ratio of the 2-(mercaptomethyl)-cysteine ​​to the dilute hydrochloric acid is 0.4 to 1:10 g / mL.

3. The method for preparing microcapsules having the function of adsorbing intestinal mucosa according to claim 1, characterized in that: The molar ratio of the 2-(mercaptomethyl)-cysteine ​​to N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1-2:1-2:2-2.

5.

4. The method for preparing microcapsules having the function of adsorbing intestinal mucosa according to claim 1, characterized in that: The solid-liquid ratio of the chitosan to the dilute hydrochloric acid is 1:80-100 g / mL.

5. The method for preparing microcapsules having the function of adsorbing intestinal mucosa according to claim 1, characterized in that: The mass ratio of the chitosan to 2-(mercaptomethyl)-cysteine ​​is 1:1-6.

6. The method for preparing microcapsules having the function of adsorbing intestinal mucosa according to claim 1, characterized in that: The mass concentration of the modified chitosan in the acetic acid solution is 1-2%.

7. The method for preparing microcapsules having the function of adsorbing intestinal mucosa according to claim 1, characterized in that: The active drug is one of nucleic acid, polypeptide, mesalazine or other active ingredients.

8. The method for preparing microcapsules having the function of adsorbing intestinal mucosa according to claim 1, characterized in that: The mass concentration of the active drug in step S3 is 0.001-10%.

9. The method for preparing microcapsules having the function of adsorbing intestinal mucosa according to claim 1, characterized in that: The volume fraction of the liquid paraffin in the total volume is 40-50%, and the volume ratio of Span 80 to the liquid paraffin is 1:8-10.

10. A microcapsule having the function of adsorbing intestinal mucosa, characterized in that: Prepared by the method according to any one of claims 1 to 9.

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