WO2021212678A1 - 一种治疗炎症性肠病及其他肠道疾病的口服MSCs充液胶囊 - Google Patents

一种治疗炎症性肠病及其他肠道疾病的口服MSCs充液胶囊 Download PDF

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WO2021212678A1
WO2021212678A1 PCT/CN2020/103455 CN2020103455W WO2021212678A1 WO 2021212678 A1 WO2021212678 A1 WO 2021212678A1 CN 2020103455 W CN2020103455 W CN 2020103455W WO 2021212678 A1 WO2021212678 A1 WO 2021212678A1
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mscs
oral
liquid
capsule
sodium alginate
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French (fr)
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孙凌云
甘璟璟
赵远锦
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南京鼓楼医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/501—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5026—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5052—Proteins, e.g. albumin
    • A61K9/5057—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5073—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the invention relates to the field of therapeutic drugs for intestinal diseases, in particular to an oral MSCs liquid-filled capsule for the treatment of inflammatory bowel diseases and other intestinal diseases.
  • MSCs Mesenchymal stem cells
  • MSCs mesenchymal stem cells
  • the main obstacle of traditional intravenous injection of MSCs is that the first pass effect of the lung leads to low homing efficiency, large dose requirements and treatment differences. Rectal administration is not only complicated, but also has a large amount of intestinal contents. Substances can reduce the survival rate of cells.
  • the delivery site is limited to the rectum and descending colon, and cannot reach the major ulcer areas such as the transverse colon and ascending colon, which affects the efficacy of MSCs.
  • Oral colon-targeted administration can protect the targeted release of the drug in the colon, fully improve the efficacy of the drug, reduce the dose, and reduce the negative effect of the drug on the human body. It is an ideal route of administration. At present, most of the drugs that have been on the market or entered clinical research are packaged in capsules of medicinal gelatin and coating materials, such as sulfasalazine colon-coated capsules and Changweining capsules.
  • MSCs microspheres in a hydrogel culture solution, and encapsulate them in colon-soluble soft capsules with cytoprotective function through traditional soft capsule technology or 3D printing technology.
  • MSCs can reach the colon. It starts to release after the site, which plays a dual role of slow release and colon positioning.
  • MSCs-filled oral capsules are easy to administer, safe and effective, and improve treatment efficiency.
  • the successful application of oral MSCs-filled capsules in pre-clinical studies such as autoimmunity or inflammation has opened the way for clinical trials. For the development of oral safety, feasibility and effectiveness. Therefore, the treatment research for oral MSCs has more application prospects and feasibility, which is worthy of attention.
  • the present invention provides an oral MSCs-filled bionic capsule and its application in the treatment of colitis.
  • An oral MSCs liquid-filled capsule comprising a three-layered capsule shell and a capsule content.
  • the capsule shell is sequentially coated with calcium carbonate to neutralize gastric acid and water-soluble gelatin-hydroxypropyl methyl from the inside to the outside.
  • Cellulose gel and Eudragit S100 coating coating the capsule content is MSCs microspheres suspended in sodium alginate hydrogel culture solution; the preparation method includes the following steps:
  • Preparation of calcium carbonate solution Weigh a certain amount of calcium carbonate and add it to double distilled water to disperse, and fully dissolve at room temperature;
  • Gelatin-Hydroxypropyl Methyl Cellulose Gel Preparation Weigh a certain amount of gelatin and hydroxypropyl methyl cellulose respectively, add an appropriate amount of double-distilled water to fully dissolve them; after cooling to room temperature, they are Mix in proportions, stir well until uniform, put in the refrigerator to form a uniform mixed gel; the total concentration of gelatin and hydroxypropyl methylcellulose in the final mixed gel is 0.5-10%, and the gelatin concentration is 0.05 ⁇ 5.0%;
  • the umbilical cord mesenchymal stem cell MSCs are uniformly mixed in sodium alginate hydrogel at a certain density, and then the mixed solution is sprayed into the calcium chloride solution under the action of high voltage through the electrospray microfluidic technology to obtain wrapped cells
  • the size of the microgel droplets, the size of the microspheres can be adjusted by voltage and flow rate;
  • the calcium carbonate in step (1) is dissolved at room temperature for 20 minutes to 40 minutes, and the concentration of calcium carbonate is 5 to 30% w/v; gelatin and hydroxypropyl methyl cellulose are each added to an appropriate amount of double distilled water, and the reaction temperature is 80° C., and stirring It takes 1 to 4 hours to fully dissolve, and the two aqueous solutions are mixed and placed in a refrigerator at 4°C for more than 24 hours to form a uniform mixed gel.
  • the umbilical cord mesenchymal stem cells MSCs charge selected from human umbilical cord mesenchymal stem cells 2-4 generations, cell seeding density of 106 cells / ml.
  • the electrospray parameters of the electrospray microfluidic technology are set as follows: the voltage is 5kV, and the flow rate is 30 ⁇ l/min.
  • the sodium alginate hydrogel culture solution is prepared by using F12/DMEM complete culture solution.
  • the MSCs-loaded sodium alginate microspheres prepared by the microfluidic electrospray technology have a size of 143 ⁇ 5.2 ⁇ m and are suspended in 1% w/v sodium alginate hydrogel.
  • Step (3) Print with a 3D printer through repeater hostV1.0.5 software; the bionic culture medium is MSCs medium containing F12/DMEM+10% fetal bovine serum+antibiotics.
  • the invention also protects the application of the oral MSCs liquid-filled capsule as a medicine for treating intestinal diseases.
  • the intestinal disease includes inflammatory bowel disease.
  • the present invention first analyzes the characteristics of the colon oral drug delivery system, combines the properties of biological materials, and constructs liquid-filled oral microcapsules loaded with MSCs through high-precision 3D printing technology to achieve oral delivery of MSCs to the colon and alleviate intestinal diseases The role of.
  • the method is simple and easy to operate.
  • the present invention precisely controls the parameters of each layer structure of the liquid-filled capsule through the precise 3D printing technology of the four-layer structure, highly optimizes the structural characteristics of each layer of the soft capsule shell, highlights the functions of each layer, and solves the problem of the traditional soft capsule process.
  • the present invention proposes a multilayer structure printed soft capsule to wrap the content of MSCs to form an oral liquid-filled capsule with anti-acid, anti-digestive functions (prevent gastric acid and digestive juice from penetrating into the cell layer) and colon-positioned release.
  • the present invention solves the problem of long-term survival of oral MSCs in the digestive tract. Since the outermost layer of the soft capsule shell is a colon-coated material, after oral administration, the outer enteric-coated capsule does not dissolve in the stomach and small intestine, which protects the capsule from degradation by gastrointestinal proteases.
  • the middle layer is a water-soluble capsule (water-soluble gelatin-hydroxypropyl methyl cellulose gel), which plays the role of isolating the contents from contact with the digestive tract.
  • the innermost layer is the calcium carbonate layer, which neutralizes the infiltrated hydrogen ions and acts as a buffer for gastric acid to affect cell activity.
  • the outer layer of Eudragit S100 coating is first dissolved, and then the outer layer of water-soluble capsule (water-soluble gelatin-hydroxypropyl methylcellulose gel) bursts and releases. , Release the MSCs microspheres and sodium alginate hydrogel. Under the protection of the hydrogel that easily adheres to the intestinal tract, the MSCs in the microspheres can play anti-inflammatory and immunomodulatory effects through paracrine for a long time, alleviate the symptoms of colon-related diseases, and have strong practicability.
  • water-soluble capsule water-soluble gelatin-hydroxypropyl methylcellulose gel
  • the present invention provides a route of oral administration of MSCs and related medical applications, which alleviate the problem of cell-based oral capsules being destroyed by proteases and acids in the digestive tract, while ensuring long-term survival and stable performance of MSCs in the colon and intestines.
  • Biological function so that oral capsules of MSCs and other cells can reach the minimum cell mass for treatment in the body.
  • Figure 1 is a schematic diagram of the structure of the MSCs oral liquid-filled capsule of the present invention; where 1 is the Eudragit S100 coating material layer, 2 is the calcium carbonate coating, and 3 is the water-soluble gelatin-hydroxypropyl methylcellulose gel, 4 is sodium alginate microspheres, 5 is MSCs, and 6 is alginate hydrogel.
  • Figure 2 is a photo of the colon of MSCs oral liquid-filled capsules for the treatment of DSS-induced inflammatory diseases in mice.
  • Figure 3 is a pathological picture of MSCs oral liquid-filled capsules for the treatment of DSS-induced H&E in the colon of mice.
  • Figures 4A and 4B show the curative effect assessment of MSCs oral liquid-filled capsules on TNBS-induced inflammatory diseases in mice.
  • Figure 4A shows the change of MSCs oral liquid-filled capsules on TNBS-induced mouse body weight curve
  • Figure 4B shows MSCs oral liquid-filled capsules. The effect of capsules on TNBS-induced disease activity index.
  • An oral MSCs liquid-filled capsule comprising a three-layered capsule shell and a capsule content.
  • the capsule shell is sequentially coated with calcium carbonate to neutralize gastric acid and water-soluble gelatin-hydroxypropyl methyl from the inside to the outside.
  • Cellulose gel and Eudragit S100 coating coating the capsule content is MSCs microspheres suspended in sodium alginate hydrogel culture solution; the preparation method includes the following steps:
  • Preparation of calcium carbonate solution Weigh a certain amount of calcium carbonate and add it to double distilled water to disperse, and fully dissolve at room temperature;
  • Gelatin-Hydroxypropyl Methyl Cellulose Gel Preparation Weigh a certain amount of gelatin and hydroxypropyl methyl cellulose respectively, add an appropriate amount of double-distilled water to fully dissolve them; after cooling to room temperature, they are Mix in proportions, stir well until uniform, put it in the refrigerator to form a uniform mixed gel; the total concentration of gelatin and hydroxypropyl methylcellulose in the final mixed gel is 2%, of which the gelatin concentration is 4%;
  • the MSCs of umbilical cord mesenchymal stem cells are uniformly mixed in sodium alginate solution at a certain density, and then the mixed solution is sprayed into the calcium chloride solution under the action of high voltage through the electrospray microfluidic technology to obtain the microstructure of the encapsulated cells.
  • Gel droplets, the size of microspheres can be adjusted by voltage and flow rate; MSCs-encapsulated microspheres are suspended in sodium alginate hydrogel;
  • Collect the biological information of the soft capsule input it into the computer for bionic modeling, load the pre-adjusted three-layer soft capsule shell and the cell microspheres into the prepared extrusion 3D printing silo, set the printing parameters, and then perform 3D printing.
  • the printed and formed liquid-filled capsules are continuously circulated and perfused cultured in the bionic culture medium to realize the pre-forming of the printed liquid-filled capsules; printed with a 3D printer through repetier hostV1.0.5 software.
  • step (1) calcium carbonate is dissolved at room temperature for 40 minutes, and the concentration of calcium carbonate is 10% w/v; gelatin and hydroxypropyl methyl cellulose are each added to an appropriate amount of double distilled water, the reaction temperature is 80 °C, and the stirring time is 1 to 4 hours. It is fully dissolved and placed in a refrigerator at 4°C for more than 24 hours after mixing to form a uniform mixed gel.
  • the umbilical cord mesenchymal stem cells MSCs charge selected from human umbilical cord mesenchymal stem cells 2-4 generations, cell seeding density of 106 cells / ml.
  • the EFI parameters of the EFI microfluidic technology are set as follows: the voltage is 5kV, and the flow rate is 30 ⁇ l/min.
  • the sodium alginate hydrogel culture solution is prepared by using F12/DMEM complete culture solution.
  • the MSCs-loaded sodium alginate microspheres prepared by the microfluidic electrospray technology have a size of 143 ⁇ 5.2 ⁇ m and are suspended in 1% w/v sodium alginate hydrogel.
  • An oral MSCs liquid-filled capsule comprising a three-layered capsule shell and a capsule content.
  • the capsule shell is sequentially coated with calcium carbonate to neutralize gastric acid and water-soluble gelatin-hydroxypropyl methyl from the inside to the outside.
  • Cellulose gel and Eudragit S100 coating coating the capsule content is MSCs microspheres suspended in sodium alginate hydrogel culture solution; the preparation method includes the following steps:
  • Preparation of calcium carbonate solution Weigh a certain amount of calcium carbonate and add it to double distilled water to disperse, and fully dissolve at room temperature;
  • Gelatin-Hydroxypropyl Methyl Cellulose Gel Preparation Weigh a certain amount of gelatin and hydroxypropyl methyl cellulose respectively, add an appropriate amount of double-distilled water to fully dissolve them; after cooling to room temperature, they are Mix in the proportions, stir thoroughly until uniform, put it in the refrigerator to form a uniform mixed gel; the total concentration of gelatin and hydroxypropyl methylcellulose in the final mixed gel is 5%, of which the concentration of gelatin is 2%;
  • the MSCs of umbilical cord mesenchymal stem cells are uniformly mixed in sodium alginate solution at a certain density, and then the mixed solution is sprayed into the calcium chloride solution under the action of high voltage through the electrospray microfluidic technology to obtain the microstructure of the encapsulated cells.
  • Gel droplets, the size of microspheres can be adjusted by voltage and flow rate; MSCs-encapsulated microspheres are suspended in sodium alginate hydrogel;
  • Collect the biological information of the soft capsule input it into the computer for bionic modeling, load the pre-adjusted three-layer soft capsule shell and the cell microspheres into the prepared extrusion 3D printing silo, set the printing parameters, and then perform 3D printing.
  • the printed and formed liquid-filled capsules are continuously circulated and perfused cultured in the bionic culture solution to realize the pre-forming of the printed liquid-filled capsules; printed with a 3D printer through the repeater hostV1.0.5 software.
  • the calcium carbonate in step (1) is dissolved at room temperature for 20 minutes to 40 minutes, and the concentration of calcium carbonate is 5 to 30% w/v; gelatin and hydroxypropyl methyl cellulose are each added to an appropriate amount of double distilled water, and the reaction temperature is 80° C., and stirring It takes 1 to 4 hours to fully dissolve it. After mixing, put it in a refrigerator at 4°C for more than 24 hours to form a uniform mixed gel.
  • the umbilical cord mesenchymal stem cells MSCs charge selected from human umbilical cord mesenchymal stem cells 2-4 generations, cell seeding density of 106 cells / ml.
  • the EFI parameters of the EFI microfluidic technology are set as follows: the voltage is 6kV, and the flow rate is 50 ⁇ l/min.
  • the sodium alginate hydrogel culture solution is prepared by using F12/DMEM complete culture solution.
  • the MSCs-loaded sodium alginate microspheres prepared by the microfluidic electrospray technology have a size of 123 ⁇ 6.1 ⁇ m and are suspended in 1% w/v sodium alginate hydrogel.
  • An oral MSCs liquid-filled capsule comprising a three-layered capsule shell and a capsule content.
  • the capsule shell is sequentially coated with calcium carbonate to neutralize gastric acid and water-soluble gelatin-hydroxypropyl methyl from the inside to the outside.
  • Cellulose gel and Eudragit S100 coating coating the capsule content is MSCs microspheres suspended in sodium alginate hydrogel culture solution; the preparation method includes the following steps:
  • Preparation of calcium carbonate solution Weigh a certain amount of calcium carbonate and add it to double distilled water to disperse, and fully dissolve at room temperature;
  • Gelatin-Hydroxypropyl Methyl Cellulose Gel Preparation Weigh a certain amount of gelatin and hydroxypropyl methyl cellulose respectively, add an appropriate amount of double-distilled water to fully dissolve them; after cooling to room temperature, they are Mix in the proportions, stir thoroughly until uniform, put in the refrigerator to form a uniform mixed gel; the total concentration of gelatin and hydroxypropyl methylcellulose in the final mixed gel is 6%, of which the concentration of gelatin is 2%;
  • the MSCs of umbilical cord mesenchymal stem cells are uniformly mixed in sodium alginate solution at a certain density, and then the mixed solution is sprayed into the calcium chloride solution under the action of high voltage through the electrospray microfluidic technology to obtain the microstructure of the encapsulated cells.
  • Gel droplets, the size of microspheres can be adjusted by voltage and flow rate; MSCs-encapsulated microspheres are suspended in sodium alginate hydrogel;
  • Collect the biological information of the soft capsule input it into the computer for bionic modeling, load the pre-adjusted three-layer soft capsule shell and the cell microspheres into the prepared extrusion 3D printing silo, set the printing parameters, and then perform 3D printing.
  • the printed and formed liquid-filled capsules are continuously circulated and perfused cultured in the bionic culture medium to realize the pre-forming of the printed liquid-filled capsules; printed with a 3D printer through repetier hostV1.0.5 software.
  • the calcium carbonate in step (1) was dissolved at room temperature for 40 minutes, and the concentration of calcium carbonate was 20% w/v; gelatin and hydroxypropyl methylcellulose were each added to an appropriate amount of double distilled water, the reaction temperature was 80°C, and the stirring time was 1 to 4 hours Make it fully dissolved, put it in a refrigerator at 4°C for more than 24 hours after mixing, to form a uniform mixed gel.
  • the umbilical cord mesenchymal stem cells MSCs charge selected from human umbilical cord mesenchymal stem cells 2-4 generations, cell seeding density of 106 cells / ml.
  • the EFI parameters of the EFI microfluidic technology are set as follows: the voltage is 7kV, and the flow rate is 50 ⁇ l/min.
  • the sodium alginate hydrogel culture solution is prepared by using F12/DMEM complete culture solution.
  • the MSCs-loaded sodium alginate microspheres prepared by the microfluidic electrospray technology have a size of 101 ⁇ 3.7 ⁇ m and are suspended in 1% w/v sodium alginate hydrogel.
  • the present invention adopts oral MSCs liquid-filled capsules to give a proper dose by gavage.
  • the effective amount of the capsule is the amount that can significantly reduce the symptoms of DSS enteritis.
  • mice were given 3% DSS drinking water, and the treatment group was given different doses of pre-prepared MSCs-filled capsules and infused into the mouse stomach by gavage.
  • the drug was administered continuously for one week. After intragastric administration, the hair condition and fecal characteristics of the mice were observed every day, and the mice were weighed and recorded in detail.
  • mice of each group were sacrificed. Take the colon tissue, observe and take pictures, and fix and embed part of the colon for H&E staining and other pathological analysis.
  • Fig. 2 A normal control group was established, the enteritis model group was given DSS free drinking water, and the treatment group was given capsules on the first 1, 3, and 5 days after modeling. Eight days later, the mice were sacrificed and their colons were taken. The photographed results are shown in Fig. 2. As can be seen from Fig. 2, the control mice have longer colons, no redness and swelling, and no obvious other inflammatory symptoms. In the DSS group, the symptoms of inflammation were obvious, with redness and swelling, and the colon became shorter. The inflammatory symptoms in the MSCs suspension group were not significantly different from the model group, which may be due to the inactivation of unprotected MSCs in gastric acid and digestive juices. The MSCs capsule group was similar to normal, and there was no obvious redness and swelling.
  • MSCs capsules does alleviate the DSS-induced colonic inflammation in mice.
  • the H&E staining picture of the MSCs-filled capsule is shown in Figure 3.
  • MSCs liquid-filled capsules are used for intragastric administration to give an appropriate dose.
  • the effective amount of the agent is an amount that can significantly reduce the symptoms of inflammatory bowel disease.
  • mice were fasted for 24 hours, and TNBS solution was administered by gavage.
  • the pre-prepared MSCs liquid-filled capsules were injected into the mouse colon by gavage.
  • MSCs capsules The effect of MSCs capsules on TNBS colitis is shown in Figure 4.
  • MSCs capsules By statistically calculating the daily weight of mice and analyzing the weight change curve (see Figure 4A), MSCs capsules can effectively slow down the trend of TNBS-induced weight loss in mice.
  • TNBS enema After TNBS enema, the model mice developed diarrhea, their body weight was significantly reduced, and there were symptoms such as blood in the stool.
  • the disease activity score can be used to evaluate the severity of colitis in mice based on the above indicators.
  • MSCs-filled liquid capsules have a good protective effect on the mouse TNBS model at the used dose. Compared with the blank control group, it has a better inhibition rate on disease activity, and the DAI index is significantly lower than that of the model control group.
  • the scoring results are shown in Figure 4B.

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Abstract

一种口服间充质干细胞（MSCs）充液胶囊，包括三层结构的胶囊外壳和胶囊内容物。所述的胶囊外壳由内至外分别为碳酸钙涂层、明胶-羟丙甲基纤维素凝胶和尤特奇S100涂层，所述的胶囊内容物为混悬于海藻酸钠水凝胶培养液中的MSCs微球。所述充液胶囊采用3D打印技术制备，能够将MSCs口服递送至结肠部位，用于治疗肠道疾病。

Description

一种治疗炎症性肠病及其他肠道疾病的口服MSCs充液胶囊 技术领域
本发明涉及肠道疾病治疗药物领域，具体涉及一种治疗炎症性肠病及其他肠道疾病的口服MSCs充液胶囊。
背景技术
间充质干细胞MSCs是一种多能性干细胞，具有免疫调节和抗炎功能。近年来，大量的研究表明，间充质干细胞(MSCs)具有治疗多种炎症性疾病的临床潜能，如复杂性肛瘘和炎症性肠病等。据报道，MSCs移植后可归巢到受损的炎症肠道部位，可有效缓解结肠炎症的病程。因此MSCs移植的方式是决定其治疗效果的首要因素。
传统的静脉注射MSCs的给药方式主要障碍是肺部的首过效应导致归巢效率低，存在剂量需求量大及治疗差异性等问题；直肠给药不仅操作较为复杂，且大量的肠道内容物会降低细胞的存活率，此外运送部位局限于直肠和降结肠，无法到达横结肠、升结肠等主要溃疡病灶区，影响MSCs疗效的发挥。口服结肠靶向给药可保护药物在结肠部分靶向释放，充分提高药物疗效，降低剂量和减少药物对人体的负作用等功效，是较为理想的给药途径。目前已上市或进入临床研究的药物多以药用明胶和包衣材料的胶囊包裹，例如柳氮磺吡啶结肠溶胶囊和肠胃宁胶囊等。
因此，在本发明中，我们将MSCs微球混合在水凝胶培养液中，通过传统软胶囊工艺或3D打印技术将其封装于具有细胞保护功能的结肠溶软胶囊，口服后使MSCs到达结肠部位后开始释放，起到缓释和结肠定位的双重作用。MSCs充液口服胶囊给药简便易行，安全有效，提高了治疗效率，口服MSCs充液胶囊在自身免疫或炎症等临床前期研究中的成功应用为临床试验开辟了道路。对于开发口服安全性、可行性和有效性。因此针对口服MSCs的治疗研究更有应用前景和可行性，值得关注。
发明内容
为了解决传统MSCs治疗慢性肠道疾病归巢率低、输注剂量大、治疗效果不稳定等缺点，本发明提供了一种口服MSCs充液仿生胶囊及其在结肠炎治疗中的应用。
为实现上述目的，本发明提供的技术方案是：
一种口服MSCs充液胶囊，包括三层结构的胶囊外壳和胶囊内容物，所述的胶囊外壳从内到外依次为中和胃酸的碳酸钙涂层、水溶性的明胶-羟丙基甲基纤维素凝胶和尤特奇S100包衣涂层，所述的胶囊内容物为混悬于海藻酸钠水凝胶培养液中的MSCs微球；其制备方法包括以下步骤：
(1)软胶囊外壳的制备：
碳酸钙溶液的配制：称取一定量的碳酸钙加入到双蒸水中分散，室温充分溶解；
明胶-羟丙基甲基纤维素凝胶的配制：分别称取一定量的明胶和羟丙基甲基纤维素，各自加入适量的双蒸水中，充分溶解；冷却至室温后将二者按照一定比例混合，充分搅拌直至均匀，放入冰箱冷藏，使其形成均一的混合凝胶；最终混合凝胶中明胶和羟丙基甲基纤维素的总浓度为0.5～10％，其中明胶浓度为0.05～5.0％；
尤特奇S100包衣液的配制：配制1～3％w/v的Eudragit S100的乙醇溶液；
(2)负载MSCs微球海藻酸钠水凝胶的制备：
将脐带间充质干细胞MSCs按一定密度均匀混合于海藻酸钠水凝胶中，再通过电喷微流控技术，在高电压的作用下将混合溶液喷射到氯化钙溶液中，获得包裹细胞的微凝胶液滴，微球大小可通过电压和流速进行调控；
(3)3D打印制备MSCs口服胶囊：
采集软胶囊的生物信息，输入计算机进行仿生建模，将预调量的三层软胶囊外壳与负载MSCs微球海藻酸钠水凝胶分别加载于准备好的挤压式3D打印料仓，设置打印参数后进行3D打印，打印成型的充液胶囊在仿生培养液中进行连续循环灌注培养，实现打印充液胶囊的预成型。
步骤(1)中的碳酸钙室温溶解20min～40min，碳酸钙的浓度为5～30％w/v；明胶和羟丙基甲基纤维素各自加入适量的双蒸水中，反应温度80℃，搅拌时间1～4h充分溶解，二者的水溶液混合后放入4℃冰箱保持24小时以上，使形成均一的混合凝胶。
步骤(1)中，所述的羟丙基甲基纤维素的粘度为100～10000mPa.s；所述的Eudragit S100成分为甲基丙烯酸酯：甲基丙烯酸质量比＝1:2。
所述脐带间充质干细胞MSCs选自人脐带间充质干细胞第2～4代，接种细胞密度为10 6个/ml。
电喷微流控技术的电喷参数设置为：电压为5kV，流速为30μl/min。
所述海藻酸钠水凝胶培养液使用F12/DMEM完全培养液配制而成。
所述微流控电喷技术制备的负载MSCs的海藻酸钠微球尺寸为143±5.2μm，且混悬于1％w/v的海藻酸钠水凝胶中。
步骤(3)通过repetier hostV1.0.5软件用3D打印机打印；仿生培养液为含有F12/DMEM+10％胎牛血清+抗生素的MSCs培养基。
本发明还保护所述的口服MSCs充液胶囊在作为治疗肠道疾病的药物中的应用。
所述肠道疾病包括炎症性肠病。
与现有技术相比，本发明的有益效果是：
1)本发明以首先分析结肠口服给药系统特征，结合生物材料性质，通过高精度生物3D打印技术，构建出负载MSCs的充液口服微胶囊，实现MSCs口服递送到结肠部位，缓解肠道疾病的作用。方法简单，操作方便。
2)本发明通过四层结构精确3D打印技术，精准调控充液胶囊各层结构的参数，高度优化了软胶囊外壳各层的结构特征，突出各层的功能，解决了传统软胶囊工艺中涂层厚度不均一、结构简单、差异性较大的问题。本发明提出了多层结构打印软胶囊包裹MSCs内容物，形成具有抗酸、抗消化功能(防胃酸和消化液渗透到细胞层)以及结肠定位释放的口服充液胶囊。
3)本发明解决了口服MSCs在消化道长期存活的问题。由于软胶囊壳的最外层为结肠溶材料，口服后因外层肠溶胶囊不在胃和小肠内溶解，保护胶囊不被胃肠蛋白酶降解。中间层为水溶性胶囊(水溶性的明胶-羟丙基甲基纤维素凝胶)，起到隔离内容物与消化道接触的作用。最内层为碳酸钙层，中和渗入的氢离子，起到缓冲胃酸影响细胞活性的作用。胶囊完整通过胃和小肠，进入结肠肠道后，外层尤特奇S100包衣首先溶解，然后依次次外层水溶性胶囊(水溶性的明胶-羟丙基甲基纤维素凝胶)崩裂释放，释放出含MSCs微球和海藻酸钠水凝胶。在易黏附于肠道的水凝胶保护下，实现了微球中的MSCs长时间通过旁分泌发挥抗炎和免疫调节作用，缓解结肠相关疾病症状，实用性强。
4)本发明提供了一种MSCs口服给药途径及其相关医学应用，缓解了细胞类口服胶囊在消化道被蛋白酶和酸破坏的问题，同时确保了MSCs在结肠肠道内长时间存活并稳定发挥生物功能，使MSCs及其他细胞口服胶囊可以在体内达到治疗最小细胞量。
附图说明
图1为本发明的MSCs口服充液胶囊的结构示意图；其中1为Eudragit S100包衣材料层，，2为碳酸钙涂层，3为水溶性的明胶-羟丙基甲基纤维素凝胶，4为海藻酸钠微球，5为MSCs,6为海藻酸盐水凝胶。
图2为MSCs口服充液胶囊治疗DSS诱导的小鼠炎症性疾病的结肠照片。
图3为MSCs口服充液胶囊治疗DSS诱导的小鼠结肠H&E病理图片。
图4A和4B为MSCs口服充液胶囊对TNBS诱导的小鼠炎症性疾病的疗效考核，其中，图4A为MSCs口服充液胶囊对TNBS诱导的小鼠体重曲线变化，图4B为MSCs口服充液胶囊对TNBS 诱导的疾病活动指数的影响。
具体实施方式
下面结合具体实施例对本发明作进一步说明。
实施例1
一种口服MSCs充液胶囊，包括三层结构的胶囊外壳和胶囊内容物，所述的胶囊外壳从内到外依次是中和胃酸的碳酸钙涂层、水溶性的明胶-羟丙基甲基纤维素凝胶和尤特奇S100包衣涂层，所述的胶囊内容物为混悬于海藻酸钠水凝胶培养液中的MSCs微球；其制备方法包括以下步骤：
(1)软胶囊外壳的制备：
碳酸钙溶液的配制：称取一定量的碳酸钙加入到双蒸水中分散，室温充分溶解；
明胶-羟丙基甲基纤维素凝胶的配制：分别称取一定量的明胶和羟丙基甲基纤维素，各自加入适量的双蒸水中，充分溶解；冷却至室温后将二者按照一定比例混合，充分搅拌直至均匀，放入冰箱冷藏，使其形成均一的混合凝胶；最终混合凝胶中明胶和羟丙基甲基纤维素的总浓度为2％，其中明胶浓度为4％；
尤特奇S100包衣液的配制：配制2％w/v的Eudragit S100的乙醇溶液；
(2)负载MSCs微球海藻酸钠水凝胶的制备：
将脐带间充质干细胞MSCs按一定密度均匀混合于海藻酸钠溶液中，再通过电喷微流控技术，在高电压的作用下将混合溶液喷射到氯化钙溶液中，获得包裹细胞的微凝胶液滴，微球大小可通过电压和流速进行调控；将包裹MSCs的微球混悬于海藻酸钠水凝胶中；
(3)3D打印制备MSCs口服胶囊
采集软胶囊的生物信息，输入计算机进行仿生建模，将预调量的三层软胶囊外壳与细胞微球分别加载于准备好的挤压式3D打印料仓，设置打印参数后进行3D打印，打印成型的充液胶囊在仿生培养液中进行连续循环灌注培养，实现打印充液胶囊的预成型；通过repetier hostV1.0.5软件用3D打印机打印。
步骤(1)中碳酸钙室温溶解40min，碳酸钙的浓度为10％w/v；明胶和羟丙基甲基纤维素各自加入适量的双蒸水中，反应温度80℃，搅拌时间1～4h使其充分溶解，混合后放入4℃冰箱保持24小时以上，形成均一的混合凝胶。所述的羟丙基甲基纤维素的粘度为4000mPa.s；所述的Eudragit S100成分为甲基丙烯酸酯：甲基丙烯酸＝1:2。
所述脐带间充质干细胞MSCs选自人脐带间充质干细胞第2～4代，接种细胞密度为10 6个 /ml。电喷微流控技术的电喷参数设置为：电压为5kV，流速为30μl/min。所述海藻酸钠水凝胶培养液使用F12/DMEM完全培养液配制而成。所述微流控电喷技术制备的负载MSCs的海藻酸钠微球尺寸为143±5.2μm，且混悬于1％w/v海藻酸钠水凝胶中。
实施例2
一种口服MSCs充液胶囊，包括三层结构的胶囊外壳和胶囊内容物，所述的胶囊外壳从内到外依次是中和胃酸的碳酸钙涂层、水溶性的明胶-羟丙基甲基纤维素凝胶和尤特奇S100包衣涂层，所述的胶囊内容物为混悬于海藻酸钠水凝胶培养液中的MSCs微球；其制备方法包括以下步骤：
(1)软胶囊外壳的制备步骤：
碳酸钙溶液的配制：称取一定量的碳酸钙加入到双蒸水中分散，室温充分溶解；
明胶-羟丙基甲基纤维素凝胶的配制：分别称取一定量的明胶和羟丙基甲基纤维素，各自加入适量的双蒸水中，充分溶解；冷却至室温后将二者按照一定比例混合，充分搅拌直至均匀，放入冰箱冷藏，使其形成均一的混合凝胶；最终混合凝胶中明胶和羟丙基甲基纤维素的总浓度为5％，其中明胶浓度为2％；
尤特奇S100包衣液的配制：配制2％w/v的Eudragit S100的乙醇溶液；
(2)负载MSCs微球海藻酸钠水凝胶的制备：
将脐带间充质干细胞MSCs按一定密度均匀混合于海藻酸钠溶液中，再通过电喷微流控技术，在高电压的作用下将混合溶液喷射到氯化钙溶液中，获得包裹细胞的微凝胶液滴，微球大小可通过电压和流速进行调控；将包裹MSCs的微球混悬于海藻酸钠水凝胶中；
(3)3D打印制备MSCs口服胶囊
采集软胶囊的生物信息，输入计算机进行仿生建模，将预调量的三层软胶囊外壳与细胞微球分别加载于准备好的挤压式3D打印料仓，设置打印参数后进行3D打印，打印成型的充液胶囊在仿生培养液中进行连续循环灌注培养，实现打印充液胶囊的预成型；通过repetier hostV1.0.5软件用3D打印机打印。
步骤(1)中的碳酸钙室温溶解20min～40min，碳酸钙的浓度为5～30％w/v；明胶和羟丙基甲基纤维素各自加入适量的双蒸水中，反应温度80℃，搅拌时间1～4h使其充分溶解，混合后放入4℃冰箱保持24小时以上，形成均一的混合凝胶。所述的羟丙基甲基纤维素的粘度为1000mPa.s；所述的Eudragit S100成分为甲基丙烯酸酯：甲基丙烯酸＝1:2。
所述脐带间充质干细胞MSCs选自人脐带间充质干细胞第2～4代，接种细胞密度为10 6个 /ml。电喷微流控技术的电喷参数设置为：电压为6kV，流速为50μl/min。所述海藻酸钠水凝胶培养液使用F12/DMEM完全培养液配制而成。所述微流控电喷技术制备的负载MSCs的海藻酸钠微球尺寸为123±6.1μm，且混悬于1％w/v海藻酸钠水凝胶中。
实施例3
一种口服MSCs充液胶囊，包括三层结构的胶囊外壳和胶囊内容物，所述的胶囊外壳从内到外依次是中和胃酸的碳酸钙涂层、水溶性的明胶-羟丙基甲基纤维素凝胶和尤特奇S100包衣涂层，所述的胶囊内容物为混悬于海藻酸钠水凝胶培养液中的MSCs微球；其制备方法包括以下步骤：
(1)软胶囊外壳的制备步骤：
碳酸钙溶液的配制：称取一定量的碳酸钙加入到双蒸水中分散，室温充分溶解；
明胶-羟丙基甲基纤维素凝胶的配制：分别称取一定量的明胶和羟丙基甲基纤维素，各自加入适量的双蒸水中，充分溶解；冷却至室温后将二者按照一定比例混合，充分搅拌直至均匀，放入冰箱冷藏，使其形成均一的混合凝胶；最终混合凝胶中明胶和羟丙基甲基纤维素的总浓度为6％，其中明胶浓度为2％；
尤特奇S100包衣液的配制：配制3％w/v的Eudragit S100的乙醇溶液；
(2)负载MSCs微球海藻酸钠水凝胶的制备：
将脐带间充质干细胞MSCs按一定密度均匀混合于海藻酸钠溶液中，再通过电喷微流控技术，在高电压的作用下将混合溶液喷射到氯化钙溶液中，获得包裹细胞的微凝胶液滴，微球大小可通过电压和流速进行调控；将包裹MSCs的微球混悬于海藻酸钠水凝胶中；
(3)3D打印制备MSCs口服胶囊
采集软胶囊的生物信息，输入计算机进行仿生建模，将预调量的三层软胶囊外壳与细胞微球分别加载于准备好的挤压式3D打印料仓，设置打印参数后进行3D打印，打印成型的充液胶囊在仿生培养液中进行连续循环灌注培养，实现打印充液胶囊的预成型；通过repetier hostV1.0.5软件用3D打印机打印。
步骤(1)中的碳酸钙室温溶解40min，碳酸钙的浓度为20％w/v；明胶和羟丙基甲基纤维素各自加入适量的双蒸水中，反应温度80℃，搅拌时间1～4h使其充分溶解，混合后放入4℃冰箱保持24小时以上，形成均一的混合凝胶。所述的羟丙基甲基纤维素的粘度为1000mPa.s；所述的Eudragit S100成分为甲基丙烯酸酯：甲基丙烯酸＝1:2。
所述脐带间充质干细胞MSCs选自人脐带间充质干细胞第2～4代，接种细胞密度为10 6个 /ml。电喷微流控技术的电喷参数设置为：电压为7kV，流速为50μl/min。所述海藻酸钠水凝胶培养液使用F12/DMEM完全培养液配制而成。所述微流控电喷技术制备的负载MSCs的海藻酸钠微球尺寸为101±3.7μm，且混悬于1％w/v海藻酸钠水凝胶中。
实施例4
口服MSCs充液胶囊在DSS诱导的肠炎模型的治疗应用：
本项发明采取口服MSCs充液胶囊灌胃给药方式给予合适的剂量。该胶囊的有效量是能导致DSS肠炎疾病症状明显降低的量。
C57BL/6雄性小鼠给予3％的DSS饮用水，治疗组给予不同剂量的预先制备好的MSCs充液胶囊通过灌胃给药的方式输入小鼠胃中。
DSS模型中均为一周连续给药，灌胃后每天观察小鼠毛发状况，粪便性状，称量小鼠体重，并详细记录。
当结肠炎症最严重时，处死各组小鼠。取结肠组织，观察拍照后，部分结肠固定包埋用于H&E染色等病理学分析。
设立正常对照组，肠炎模型组给予DSS自由饮水，治疗组在造模第1、3、5天灌胃胶囊。8天后处死小鼠取结肠，拍照结果如图2，从图2可以看出，对照小鼠结肠长度较长，无红肿现象，无明显其他炎症症状。DSS组炎症症状明显，有红肿，结肠变短的现象。MSCs悬液组的炎症症状存在，与模型组无明显差异，可能是由于未受保护MSCs在胃酸和消化液里失活。MSCs胶囊组与正常相似，无明显红肿现象。由此可见MSCs胶囊经口服确有缓解DSS诱导的小鼠结肠炎症的作用。MSCs充液胶囊的治疗H&E染色图片见图3。我们可以发现，相比于DSS模型组和MSCs组，胶囊治疗组中仅见少量炎症细胞浸润，结肠结构较为完整，由此可见MSCs充液胶囊的治疗可以有效缓解DSS诱导的肠道炎症。
实施例5
口服MSCs充液胶囊在小鼠TNBS诱导溃疡性炎结肠炎的治疗应用：
本项发明采取MSCs充液胶囊灌胃给药方式给予合适的剂量。该试剂的有效量是能导致炎症性肠病症状明显降低的量。
本发明所述的MSCs充液胶囊在TNBS肠炎模型治疗中的应用：
a.BALB/C小鼠禁食24小时，灌胃给药TNBS溶液。
b.TNBS小鼠模型建立12小时后，将预先制备好的MSCs充液胶囊通过灌胃给药的方式注入小鼠结肠内。
c.造模后每天称量小鼠体重，观察小鼠是否腹泻，便血，粪便性状等，记录疾病活动度评分(DAI)，DAI评分＝(体重丢失积分+粪便连续积分+隐血肉眼血积分)/3。
MSCs胶囊治疗TNBS结肠炎的效果见图4。通过对小鼠每日体重进行统计，分析体重变化曲线(见图4A)，MSCs胶囊能够有效减缓TNBS诱导小鼠体重下降的趋势。TNBS灌肠后，模型小鼠出现腹泻，体重明显降低，存在便血等症状，疾病活动度评分可以综合上述指标评价小鼠结肠炎的疾病严重程度。MSCs充液胶囊在所用剂量下对小鼠TNBS模型有良好的保护作用，与空白对照组比较对疾病活动度有较好的抑制率，DAI指数明显低于模型对照组。评分结果见图4B。
以上所述，仅是本发明的较佳实施例，并非对本发明作任何形式上的限制，任何熟悉本专业的技术人员，在不脱离本发明技术方案范围内，依据本发明的技术实质，对以上实施例所作的任何简单的修改、等同替换与改进等，均仍属于本发明技术方案的保护范围之内。

Claims (10)

  1. 一种口服MSCs充液胶囊，其特征在于：包括三层结构的胶囊外壳和胶囊内容物，所述的胶囊外壳从内到外依次为中和胃酸的碳酸钙涂层、水溶性的明胶-羟丙基甲基纤维素凝胶和尤特奇S100包衣涂层，所述的胶囊内容物为混悬于海藻酸钠水凝胶培养液中的MSCs微球；其制备方法包括以下步骤：
    (1)软胶囊外壳的制备：
    碳酸钙溶液的配制：称取一定量的碳酸钙加入到双蒸水中分散，室温充分溶解；
    明胶-羟丙基甲基纤维素凝胶的配制：分别称取一定量的明胶和羟丙基甲基纤维素，各自加入适量的双蒸水中，充分溶解；冷却至室温后将二者按照一定比例混合，充分搅拌直至均匀，放入冰箱冷藏，使其形成均一的混合凝胶；最终混合凝胶中明胶和羟丙基甲基纤维素的总浓度为0.5～10％，其中明胶浓度为0.05～5.0％；
    尤特奇S100包衣液的配制：配制1～3％w/v的Eudragit S100的乙醇溶液；
    (2)负载MSCs微球海藻酸钠水凝胶的制备：
    将脐带间充质干细胞MSCs按一定密度均匀混合于海藻酸钠水凝胶中，再通过电喷微流控技术，在高电压的作用下将混合溶液喷射到氯化钙溶液中，获得包裹细胞的微凝胶液滴，微球大小可通过电压和流速进行调控；
    (3)3D打印制备MSCs口服胶囊：
    采集软胶囊的生物信息，输入计算机进行仿生建模，将预调量的三层软胶囊外壳与负载MSCs微球海藻酸钠水凝胶分别加载于准备好的挤压式3D打印料仓，设置打印参数后进行3D打印，打印成型的充液胶囊在仿生培养液中进行连续循环灌注培养，实现打印充液胶囊的预成型。
  2. 根据权利要求1所述的口服MSCs充液胶囊，其特征在于：步骤(1)中的碳酸钙室温溶解20min～40min，碳酸钙的浓度为5～30％w/v；明胶和羟丙基甲基纤维素各自加入适量的双蒸水中，反应温度80℃，搅拌时间1～4h充分溶解，二者的水溶液混合后放入4℃冰箱保持24小时以上，使形成均一的混合凝胶。
  3. 根据权利要求1所述的口服MSCs充液胶囊，其特征在于：步骤(1)中，所述的羟丙基甲基纤维素的粘度为100～10000mPa.s；所述的Eudragit S100成分为甲基丙烯酸酯：甲基丙烯酸质量比＝1:2。
  4. 根据权利要求1所述的口服MSCs充液胶囊，其特征在于：所述脐带间充质干细胞MSCs选自人脐带间充质干细胞第2～4代，接种细胞密度为10 6个/ml。
  5. 根据权利要求1所述的口服MSCs充液胶囊，其特征在于：电喷微流控技术的电喷参数设置为：电压为5kV，流速为30μl/min。
  6. 根据权利要求1所述的口服MSCs充液胶囊，其特征在于：所述海藻酸钠水凝胶培养液使用F12/DMEM完全培养液配制而成。
  7. 根据权利要求1所述的口服MSCs充液胶囊，其特征在于：所述微流控电喷技术制备的负载MSCs的海藻酸钠微球尺寸为143±5.2μm，且混悬于1％w/v的海藻酸钠水凝胶中。
  8. 根据权利要求1所述的口服MSCs充液胶囊，其特征在于：步骤(3)通过repetier hostV1.0.5软件用3D打印机打印；仿生培养液为含有F12/DMEM+10％胎牛血清+抗生素的MSCs培养基。
  9. 权利要求1-8任一项所述的口服MSCs充液胶囊在作为治疗肠道疾病的药物中的应用。
  10. 根据权利要求9所述的口服MSCs充液胶囊在作为治疗肠道疾病的药物中的应用，其特征在于：所述肠道疾病包括炎症性肠病。
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