Self-releasing microcapsules for intestinal biopsy and uses thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a self-releasing microcapsule for intestinal sampling and application thereof. The self-releasing microcapsule comprises a capsule cavity, a capsule shell, a sealing member and a coating. The capsule shell is provided with a sampling hole, the capsule cavity is internally provided with sampling hydrogel for absorbing sample fluid, the sealing member is arranged in the capsule cavity between the sampling hydrogel and the sampling hole, wherein the expansion of the sampling hydrogel in the capsule cavity presses the sealing member to engage with the sampling hole to seal the capsule cavity, and the coating is arranged on the surface of the capsule shell. The coating comprises the following coating materials in parts by weight: 15-20 parts of hydroxypropyl methyl cellulose phthalate, 25-30 parts of Eudragit L100, 8-12 parts of algal extract, 1-3 parts of polyethylene glycol and 1-2 parts of talc. The self-releasing microcapsule can be used for precise sampling of the colon.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and specifically relates to a self-releasing microcapsule for intestinal sampling and application thereof. BACKGROUND

[0002] As an important part of human health, the state of intestinal microecology is closely related to the occurrence and development of various diseases, especially in the field of tumors and immune system-related diseases. Studies have shown that whether it is the development of tumors or the functional changes of the immune system, the intestinal ecology will show corresponding signs very early. For example, in the early stages of tumor occurrence, changes in the structure of intestinal microbial communities may indicate potential pathological processes, and these changes often precede the appearance of clinical symptoms. Similarly, when the body's immune system is abnormal, the composition and function of the intestinal flora will also change, which can be regarded as an important indicator of immune status.

[0003] However, despite the recognition of the importance of intestinal microecology, the lack of precise sampling technology has seriously hampered the development of precision medicine. Precision sampling refers to the ability to accurately and timely obtain key biomarkers or data that reflect the individual's health status. Only in this way can we truly achieve the goal of developing personalized treatment plans based on the unique intestinal microecological environment of each patient.

[0004] Colon cancer is a malignant tumor that originates in the mucosal epithelium of the colon and is one of the most common malignant tumors in the digestive tract. It usually occurs in the inner layer of cells in the colon and can invade deeper tissues or spread to other parts of the body over time. Early colon cancer may have no obvious symptoms. Chinese patent CN119325357A discloses a gastrointestinal sampling capsule, but the coating layer disclosed in the method has a long dissolution time in the intestinal tract, which cannot meet the increasingly high market requirements.

[0005] Therefore, there is an urgent need for a self-releasing microcapsule for intestinal sampling and application thereof for colon sampling. SUMMARY

[0006] The purpose of the present application is to provide a self-releasing microcapsule for intestinal sampling and application thereof.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] A self-releasing microcapsule for intestinal sampling, comprising a capsule cavity, a capsule shell, a sealing member and a coating; the capsule shell is provided with a sampling hole, the capsule cavity is filled with a sampling hydrogel for absorbing sample fluid; the sealing member is arranged in the capsule cavity between the sampling hydrogel and the sampling hole, wherein the expansion of the sampling hydrogel in the capsule cavity presses the sealing member to engage with the sampling hole to seal the capsule cavity; the coating is arranged on the surface of the capsule shell to cover the sampling hole, and the degradation of the coating layer exposes the sampling hole to allow fluid to flow into the capsule cavity.

[0009] The coating comprises the following weight proportions of coating materials: 15-20 parts of hypromellose phthalate, 25-30 parts of Eudragit L100, 8-12 parts of algal extract, 1-3 parts of polyethylene glycol, and 1-2 parts of talc.

[0010] The present application balances environmental protection and performance requirements by compounding various coating materials, and balances the physical barrier effect provided by synthetic polymer materials and the biological response characteristics of natural products, so as to achieve the goal of keeping intact in gastric juice and high-efficiency release in colon, and realize precise positioning sampling in colon.

[0011] Further, the coating comprises the following weight proportions of coating materials: 17 parts of hypromellose phthalate, 26 parts of Eudragit L100, 10 parts of algal extract, 2 parts of polyethylene glycol, and 1.5 parts of talc.

[0012] Further, the preparation method of the algal extract comprises the following steps:

[0013] (1) Gracilaria (Gracilaria) is soaked with water, washed with sodium hydroxide aqueous solution, filtered, washed to neutral, dried, crushed, and the powder raw material is obtained; Gracilaria lemaneiformis

[0014] (2) The powder raw material is added to water to obtain a fermentation raw material, Bacillus licheniformis is added to the fermentation raw material, and fermentation is carried out, and after sterilization, a fermentation product is obtained;

[0015] (3) The fermentation product is added with an ethanol aqueous solution, stirred, cooled, placed, filtered with a filter membrane, and an alcohol precipitation liquid is obtained;

[0016] (4) The alcohol precipitation liquid is filtered with an ultrafiltration membrane, freeze-dried, and an algal extract is obtained.

[0017] ​The algal extract prepared by using the Gracilaria dura using specific bacteria for fermentation, and the remaining coating material can improve the release speed in the colon, and at the same time improve the storage stability. The Gracilaria dura can further change its chemical structure and physical properties by specific microbial fermentation, and generate metabolites with higher functionality, which have low solubility in gastric juice and high solubility in colon, and these substances can be quickly recognized and degraded by intestinal flora in the colon environment, thereby promoting the disintegration of the coating layer and accelerating the drug release. At the same time, fermentation can effectively remove or transform some components that may affect the storage stability, not only improving the safety of the final product, but also enhancing its long-term storage capability.

[0018] Further, the concentration of the sodium hydroxide aqueous solution in step (1) is 0.1-0.12 mol / L.

[0019] Further, in step (1), the sodium hydroxide aqueous solution is soaked at 50-55℃ for 15-20h.

[0020] Further, in step (1), the crushing is to less than 100 mesh.

[0021] Further, in step (2), the fermentation conditions are 25~30℃ for 30-35h, and the aeration amount is 7.0-7.5L / min.

[0022] Further, in step (3), the fermentation product is added with a volume fraction of 60-70% ethanol aqueous solution, stirred at 60-70℃ for 1-2h, cooled to 15-20℃, and stood for 2-4h, and then filtered using a 0.45μm filter membrane to obtain the alcohol precipitation liquid.

[0023] Further, in step (4), the molecular weight cut-off of the ultrafiltration membrane is 10000-20000 daltons.

[0024] The application provides the use of the self-releasing microcapsule for intestinal sampling in intestinal sampling.

[0025] Compared with the prior art, the application has the following advantages and beneficial effects:

[0026] 1. The application can balance environmental protection and performance requirements by compounding a variety of coating materials and compounding natural and synthetic materials, and realize precise positioning sampling in the colon.

[0027] 2. The algal extract prepared by using the Gracilaria dura using specific bacteria for fermentation, and the remaining coating material can improve the release speed in the colon, and at the same time improve the storage stability. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all of them. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present application.

[0029] The raw materials used in the following embodiments of the present application are all commercially available goods:

[0030] Bacillus licheniformis is a commercially available strain, numbered: BNCC186189, Beinaelieli Biotechnology Co., Ltd.

[0031] Bacillus subtilis is a commercially available strain, numbered: BNCC361064, Beinaelieli Biotechnology Co., Ltd.

[0032] Hydroxypropyl methyl cellulose phthalate, Xi'an Jinxing Pharmaceutical Auxiliary Co., Ltd.

[0033] Eudragit L100, company: Germany Rom, Eudragit series.

[0034] Example 1

[0035] The present embodiment provides a self-releasing microcapsule for intestinal sampling, comprising a capsule cavity, a capsule shell, a sealing member and a coating; the capsule shell is provided with a sampling hole, the capsule cavity is filled with a sampling hydrogel for absorbing sample fluid; the sealing member is arranged in the capsule cavity between the sampling hydrogel and the sampling hole, wherein the expansion of the sampling hydrogel in the capsule cavity presses the sealing member to engage with the sampling hole to seal the capsule cavity; the coating is arranged on the surface of the capsule shell to cover the sampling hole, and the degradation of the coating layer exposes the sampling hole to allow fluid to flow into the capsule cavity.

[0036] The coating comprises the following weight proportions of coating materials: 17 parts of hydroxypropyl methyl cellulose phthalate, 26 parts of Eudragit L100, 10 parts of algal extract, 2 parts of polyethylene glycol, and 1.5 parts of talc.

[0037] The preparation method of the algal extract comprises the following steps:

[0038] (1) Soak the Gracilaria dura with water, wash it clean, soak it with a sodium hydroxide aqueous solution, soak it at 52℃ for 18h, the concentration of the sodium hydroxide aqueous solution is 0.1mol / L, filter after soaking, wash it with water until it is neutral, dry it, crush it to less than 100 mesh to obtain a powder raw material;

[0039] (2) The powder raw material is added to water, and the weight ratio of the powder raw material to water is 1:6 to obtain fermentation raw material. Bacillus licheniformis is added to the fermentation raw material to perform fermentation. The fermentation conditions are 28℃ for 32h, and the aeration amount is 7.0L / min. After sterilization, the fermentation product is obtained. The dosage of Bacillus licheniformis is 10 8 CFU / mL.

[0040] (3) The fermentation product is added with 65% ethanol aqueous solution. The volume ratio of the fermentation product to the ethanol aqueous solution is 1:2. The mixture is stirred at 65℃ for 1.5h, cooled to 18℃, and then left to stand for 2-4h. The mixture is filtered by using a 0.45μm filter membrane to obtain an alcohol precipitation solution.

[0041] (4) The alcohol precipitation solution is filtered by using an ultrafiltration membrane. The component with a molecular weight cut-off of 10000-20000 Dalton is obtained by using the ultrafiltration membrane. The component is freeze-dried to obtain the algal extract.

[0042] Example 2

[0043] The present embodiment provides a self-releasing microcapsule for intestinal sampling, which comprises a capsule cavity, a capsule shell, a sealing member and a coating. The capsule shell is provided with a sampling hole. The capsule cavity is filled with a sampling hydrogel for absorbing sample fluid. The sealing member is arranged in the capsule cavity between the sampling hydrogel and the sampling hole. The expansion of the sampling hydrogel in the capsule cavity presses the sealing member to engage with the sampling hole to seal the capsule cavity. The coating is arranged on the surface of the capsule shell to cover the sampling hole. The degradation of the coating layer exposes the sampling hole to allow fluid to flow into the capsule cavity.

[0044] The coating comprises the following coating materials in parts by weight: 20 parts of hydroxypropyl methyl cellulose phthalate, 25 parts of Eudragit L100, 12 parts of algal extract, 3 parts of polyethylene glycol, and 1 part of talc.

[0045] The preparation method of the algal extract comprises the following steps:

[0046] (1) The Gracilaria dura is soaked with water and then soaked with a sodium hydroxide aqueous solution. The soaking is performed at 55℃ for 15h. The concentration of the sodium hydroxide aqueous solution is 0.12mol / L. After the soaking is completed, the Gracilaria dura is filtered, washed with water until neutral, dried, and pulverized to less than 100 mesh to obtain a powder raw material.

[0047] (2) The powder raw material is added to water, and the weight ratio of the powder raw material to water is 1:6 to obtain fermentation raw material. Bacillus licheniformis is added to the fermentation raw material to perform fermentation. The fermentation conditions are 30℃ for 30h, and the aeration amount is 7.5L / min. After sterilization, the fermentation product is obtained. The dosage of Bacillus licheniformis is 10 8 CFU / mL.

[0048] (3) The fermentation product is added with 70% ethanol aqueous solution by volume fraction, the volume ratio of fermentation product and ethanol aqueous solution is 1:2; stirring at 70℃ for 1h, cooling to 20℃, standing for 2h, filtering with 0.45μm filter membrane, to obtain the alcohol precipitation liquid.

[0049] (4) The alcohol precipitation liquid is filtered with ultrafiltration membrane, the component with molecular weight cut-off of 10000-20000 Dalton is used, the component is freeze-dried to obtain the algal extract.

[0050] Comparative Example 1

[0051] The difference between this comparative example and Example 1 is that the coating includes the following weight fractions of coating materials: 24 parts of hydroxypropyl methyl cellulose phthalate, 23 parts of Eudragit L100, 6 parts of algal extract, 2 parts of polyethylene glycol, 1.5 parts of talc.

[0052] Comparative Example 2

[0053] The difference between this comparative example and Example 1 is that the Gracilaria lemaneiformis is replaced by Gelidium amansii. Gelidium amansii (Lamouroux Lamouroux ,1813)

[0054] Comparative Example 3

[0055] The difference between this comparative example and Example 1 is that the Bacillus licheniformis is replaced by Bacillus subtilis, the number of Bacillus subtilis is BNCC361064, and it is from Beinaeliang Biotechnology Co., Ltd.

[0056] Comparative Example 4

[0057] The difference between this comparative example and Example 1 is that no algal extract is added.

[0058] The coating includes the following weight fractions of coating materials: 22 parts of hydroxypropyl methyl cellulose phthalate, 31 parts of Eudragit L100, 2 parts of polyethylene glycol, 1.5 parts of talc.

[0059] Performance test.

[0060] Referring to Chinese Patent CN119325357A, the solubility of the coating materials prepared in Examples 1-2 and Comparative Examples 1-4 of the gastrointestinal sampling capsule method is determined. Specifically as follows:

[0061] Methylene blue and deep orange food coloring dyes were added to the solution of the coating material, respectively. These dyes allowed the polymer to reach the peak absorption during the UV-Vis spectrophotometry. The solution was then cast on an acrylic plate using a doctor blade to obtain a uniform film thickness. After casting each layer, to avoid solvent retention in the film composition, the film was completely dried using NIR (Adphos NIR-126-250 Modul, line speed of the conveyor 3.6 m.min-1, lamp power 3 kW). A computer-controlled CO2 laser cutting and engraving system was used to cut identical discs of 10 mm in diameter at a wavelength of 10.6 μm.

[0062] Dissolution testing of the samples was performed in 900 mL of various dissolution media at 37°C using a USP type II apparatus (PTWS apparatus, Pharma Test, Hainburg, Germany) at a paddle speed of 100 rpm. The samples were removed from the dissolution media after 1 h and analyzed by UV-Vis using a BMG Clariostar microplate reader (BMG Labtech, Germany) before returning to the dissolution media. The percentage of dye released from the formulation was measured using UV-Vis technique at the wavelength corresponding to the peak wavelength of the appropriate dye. All tests were repeated three times.

[0063] Dissolution = amount of dye released ÷ total amount of dye * 100%.

[0064] Table 1 dissolution test results (%)

[0065]

[0066] From Table 1, it can be seen that the coatings of Examples 1-2 have good solubility in the colon, and can achieve complete dissolution in 1 h. Meanwhile, the dissolution state remains stable after long-term storage.

[0067] In Comparative Example 1, the ratio of the coating material was changed, the dissolution time of the coating in the colon was prolonged, and the storage stability decreased, indicating that the coating materials of the present application achieve a synergistic effect.

[0068] In Comparative Example 2, Gracilaria dura was used, and in Comparative Example 3, different strains were used, the solubility of the coating in the gastric juice was increased, the dissolution time in the colon was prolonged, and the storage stability decreased, indicating that only the seaweed extract prepared by fermenting Gracilaria dura with a specific strain can have a synergistic effect with the remaining components, which can have low solubility in the gastric juice and at the same time achieve rapid dissolution in the colon.

[0069] In Comparative Example 4, no seaweed extract was added, the dissolution time of the coating in the colon was prolonged, and the storage stability was reduced. Ideally, the intestinal sampling microcapsule should be able to quickly open the sampling device after reaching the target area, so as to accurately collect samples from a specific location. If the coating dissolves too slowly, the capsule may have moved to a non-target area before starting to work, which causes the sampling point to deviate from the expected location, reducing the effectiveness and accuracy of the diagnosis. Especially in cases where accurate sampling of the lesion site is required, such errors are unacceptable.

[0070] The above describes the preferred embodiments of the present application. It should be noted that, for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. A self-releasing microcapsule for intestinal biopsy, characterized in that, The capsule comprises a cavity, a capsule shell, a seal, and a coating. The capsule shell has a sampling port, and the cavity contains a sampling hydrogel for absorbing sample fluid. The seal is disposed within the cavity between the sampling hydrogel and the sampling port, wherein the expansion of the sampling hydrogel within the cavity presses the sealing member to engage with the sampling port to seal the cavity. The coating is disposed on the surface of the capsule shell to cover the sampling port, and the degradation of the coating layer exposes the sampling port to allow fluid to flow into the cavity. The coating comprises the following coating materials in parts by weight: 15-20 parts hydroxypropyl methylcellulose phthalate, 25-30 parts Eudragit L100, 8-12 parts algae extract, 1-3 parts polyethylene glycol, and 1-2 parts talc. Methods for preparing algal extracts include: (1) Soak and wash the dragon beard vegetable with water, soak it in a sodium hydroxide aqueous solution with a concentration of 0.1-0.12 mol / L at 50-55℃ for 15-20 hours, filter it, wash it with water until it is neutral, dry it, and pulverize it to less than 100 mesh to obtain powder raw material; (2) Add the powdered raw material to water to obtain the fermentation raw material. Add Bacillus licheniformis to the fermentation raw material, ferment at 25~30℃ for 30-35h, with an aeration rate of 7.0-7.5L / min. After sterilization, the fermentation product is obtained. (3) Add an aqueous ethanol solution to the fermentation product, stir, cool, let stand, filter through a filter membrane to obtain an alcohol precipitate; (4) The alcohol precipitate was filtered through an ultrafiltration membrane with a molecular weight cutoff of 10,000-20,000 Daltons and freeze-dried to obtain an algal extract.

2. The self-releasing mini-capsule for intestinal biopsy according to claim 1, wherein, The coating comprises the following parts by weight of coating material: 17 parts hydroxypropyl methylcellulose phthalate, 26 parts Eudragit L100, 10 parts algae extract, 2 parts polyethylene glycol, and 1.5 parts talc.

3. The self-releasing microcapsule for intestinal biopsy according to claim 1, wherein In step (3), 60-70% ethanol aqueous solution by volume is added to the fermentation product, stirred at 60-70℃ for 1-2 hours, cooled to 15-20℃, allowed to stand for 2-4 hours, and filtered using a 0.45μm filter membrane to obtain the alcohol precipitate.

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