Oral drug delivery system with double-emulsion structure and application of oral drug delivery system in preparation of drugs for treating premature ovarian insufficiency

Abstract

The invention discloses an oral drug delivery system with a double-emulsion structure and application of the oral drug delivery system in preparation of drugs for treating premature ovarian insufficiency, and belongs to the technical field of biological medicines. The oral drug delivery system comprises droplet particles formed by wrapping an MSCs conditioned culture medium with an oil-phase solvent, and exosomes uniformly distributed on the outer surfaces of the droplet particles, so that a double-emulsion structure is formed. Water-soluble MSC-CM is wrapped by a water-in-oil-in-water method to form a double-emulsion structure, so that water-soluble protein enters a human body to be absorbed in an oral manner. The MSC-CM composite has good structural stability and viscoelasticity, the particle size range is about 10-50 microns, contact with intestinal epithelial cells can be increased, meanwhile, the skeleton of the intestinal epithelial cells can be changed, expression of tight junction protein of the intestinal epithelial cells can be lowered, tight junction barriers of the intestinal epithelial cells can be opened, and therefore the bioavailability of MSC-CM is improved, and the MSC-CM composite has a good application prospect. The effect of treating the POI is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to an oral drug delivery system with a double emulsion structure and application thereof in preparing a drug for treating premature ovarian insufficiency. Background Art

[0002] Premature ovarian insufficiency (POI) is a common condition in gynecological endocrinology. It refers to ovarian dysfunction that occurs before the age of 40, characterized by high gonadotropin levels, low estrogen levels, oligomenorrhea, and amenorrhea. POI not only affects female fertility and leads to infertility, but can also cause dysfunction in multiple systems, including neurocognitive, urogenital, skeletal, cardiovascular, and endocrine and metabolic systems, severely impacting women's physical and mental health and quality of life. POI is a disease caused by multiple etiologies and complex mechanisms. Currently known causes include genetics, infections, immunity, and iatrogenic injury. Among them, immune-related POI (autoimmune POI) is the most common type, accounting for approximately 5%-30% of POI cases. Current treatment options, including hormone therapy, immunosuppressive therapy, and assisted reproductive technology, have failed to achieve a definitive cure for autoimmune POI. Therefore, exploring new therapeutic targets and developing innovative therapies to enhance treatment efficacy and promote women's health have become critical research and clinical challenges.

[0003] During the pathogenesis of autoimmune premature ovarian insufficiency (aPOI), various immune factors trigger overactivation of immune cells, leading to the production of large amounts of reactive oxygen species (ROS). ROS, a metabolic byproduct, dictate the balance between free radicals and antioxidants, regulating cellular function. In ovarian tissue, normal levels of ROS are widely involved in activating transcription factors, promoting cell proliferation and differentiation. They play a crucial role in regulating follicular growth, angiogenesis, and sex hormone synthesis, and are crucial for follicular development at all stages of the ovary. However, excessive ROS levels induce oxidative stress in granulosa cells, leading to impaired follicular development or abnormal exhaustion. Imbalanced oxidative stress can damage cellular molecules such as DNA, mitochondria, proteins, and lipids. Many factors that influence POI ultimately contribute to this by activating oxidative stress. Therefore, alleviating ovarian oxidative stress is an important approach to treating POI. Antioxidants can be divided into synthetic antioxidants and natural antioxidants. Synthetic antioxidants are economical and have high yields, but studies have reported that many synthetic antioxidants have potential toxicity and carcinogenic effects. In 2023, a study published in Environmental Health Perspectives found that high exposure to synthetic phenolic antioxidants and their metabolites increases the risk of diminished ovarian reserve (DOR) (Environmental Health Perspectives, 2023, 131(6):067005.). Relatively speaking, natural product antioxidants are not only more effective, but also safer and more easily accepted, making them a better choice for antioxidant treatment of aPOI.

[0004] Mesenchymal stem cells (MSCs) hold great promise in the treatment of POI due to their self-renewal and differentiation-inducing properties. Previous studies have demonstrated that MSCs can reduce ROS levels in granulosa cells, inhibit oxidative stress, and have a reparative effect on POI. However, ethical concerns, immune rejection, and post-transplant apoptosis remain important issues in stem cell therapy. The use of MSC-conditioned medium (MSC-CM) can effectively mitigate these risks. The reparative effects of MSCs are closely related to their paracrine function. MSCs secrete a variety of paracrine factors, including extracellular matrix, proteins, growth factors, cytokines, and chemokines, which exert therapeutic effects through paracrine pathways. Protein analysis has confirmed that MSC-CM contains various MSC-secreted factors, including vascular endothelial growth factor, epidermal growth factor, and insulin-like growth factors I and II, achieving therapeutic effects comparable to those of MSCs. However, MSC-CM is a protein composed of a variety of secreted factors, and its delivery method is a major limitation to its clinical application. Currently, oral administration is the most common route of administration in clinical practice, with high patient compliance and minimal side effects. However, proteins are easily degraded in the gastrointestinal tract, resulting in low bioavailability, which limits the therapeutic efficacy of MSC-CM. Therefore, there is an urgent need to develop an oral delivery system for MSC-CM that is highly stable in the gastrointestinal tract and has good bioavailability, thereby improving premature ovarian failure.

[0005] In recent years, exosomes (also known as extracellular vesicles, EVs) have garnered widespread attention as drug delivery vehicles. These natural, nanoscale vesicles secreted by cells and possessing a phospholipid bilayer structure can be used to encapsulate a variety of drugs. They offer significant advantages over other traditional drug delivery systems, including excellent biocompatibility, high stability under physiological conditions, and the ability to penetrate biological barriers. However, their nanoparticle size and small size limit their drug-carrying capacity, hindering their further development as drug carriers. Leveraging the well-defined lipid bilayer properties of exosomes, we designed an exosome-stabilized, water-soluble protein-encapsulated, oil-in-water oral drug delivery system for in vivo delivery of MSC-CM. This system achieves high drug loading and exhibits excellent structural stability. A 2023 study used fruit-derived exosomes (EVs) as stable particles and dissolved the hydrophobic chemotherapy drug doxorubicin (DOX) in squalene to create extracellular vesicle-stabilized oil-in-water structured drug droplets (Advanced Materials, 2023: 2304187). This system can encapsulate hydrophobic drugs, producing nanoparticles with a size of approximately 200 nanometers, for the development of intravenous treatments for gliomas. However, this drug delivery system is primarily targeted at the development of hydrophobic drugs, and an oral delivery system suitable for water-soluble drugs (especially protein drugs) for disease treatment has not yet been established.

[0006] The present invention solves the problem of difficulty in constructing an oral drug delivery system for water-soluble proteins, and constructs a water-in-oil-in-water double emulsion droplet structure. The surface of the system is stabilized by natural exosomes with a certain viscoelasticity, and a flexible liquid serves as the core, so the entire system has a certain viscoelasticity. The particle size range of the oral drug delivery system prepared at the same time is about 10μm-50μm, which can reach the size of a single cell, which is conducive to contact with a larger area of cells in the intestine. The drug delivery system can maintain structural stability under the harsh physiological conditions of the gastrointestinal tract; in addition, after the drug delivery system interacts with intestinal epithelial cells, it can affect the intestinal epithelial cell skeleton, thereby further opening the tight junction barrier of the intestinal epithelial cells, facilitating the absorption of MSC-CM into the blood, and improving the oral utilization of MSC-CM, thereby exerting an effective treatment for aPOI. Summary of the Invention

[0007] In response to the problems existing in the above-mentioned prior art, the object of the present invention is to design and provide an oral drug delivery system carrying mesenchymal stem cell conditioned medium for the treatment of premature ovarian insufficiency and its preparation method. The E@MSC-CM described in the present invention uses exosomes (EVs) as a fixed particle stabilizer, and through a two-step emulsification method, MSC-CM is encapsulated in an oil phase solvent, and EVs are evenly distributed on the outermost surface of the droplet particles composed of the oil phase and MSC-CM, forming a water-in-oil-in-water oral delivery system. The oil phase of the present invention can be prepared with various oral and topical natural source oils such as squalene, squalane, rapeseed oil, peanut oil, perilla seed oil, and sweet almond oil.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] On the one hand, the present invention provides an oral drug delivery system with a double emulsion structure, comprising droplet particles composed of MSCs-conditioned medium encapsulated by an oil phase solvent, and exosomes uniformly distributed on the outer surface of the droplet particles, forming a double emulsion structure.

[0010] In the oral drug delivery system with a double emulsion structure, the oil phase solvent is a food-grade oil phase solvent.

[0011] In the oral drug delivery system with a double emulsion structure, the oil phase solvent is squalene, rapeseed oil, peanut oil, perilla seed oil or sweet almond oil.

[0012] In a second aspect, the present invention provides a method for preparing an oral drug delivery system having a double emulsion structure as described in any one of the above, comprising the following steps:

[0013] (1) Umbilical cord mesenchymal stem cells were cultured in serum-free medium. After the culture, the supernatant was collected, centrifuged to remove cell debris and apoptotic bodies, concentrated by ultrafiltration, and freeze-dried and reconstituted to obtain MSCs-conditioned medium;

[0014] (2) The oil phase solvent was mixed with the MSCs conditioned medium, and ultrasonic emulsification was performed under ice bath conditions. PBS solution and exosomes were added, and secondary emulsification was performed using a high-speed homogenizer under ice bath conditions. The mixture was allowed to stand, and the upper emulsion-like liquid was aspirated to obtain an oral drug delivery system loaded with MSCs conditioned medium (E@MSC-CM).

[0015] The preparation method is characterized in that the culturing time is 48-96 hours;

[0016] The concentration of the MSCs conditioned medium is 8-15 mg / mL.

[0017] In a third aspect, the present invention provides use of any one of the oral drug delivery systems having a double emulsion structure in the preparation of a drug for treating premature ovarian insufficiency.

[0018] The use described above, the oral drug delivery system can reduce the level of ROS.

[0019] The oral drug delivery system can restore the ovarian structure and improve the levels of estradiol and follicle-stimulating hormone in ovarian cells.

[0020] In a fourth aspect, the present invention provides a pharmaceutical composition for treating premature ovarian insufficiency, comprising any one of the oral drug delivery systems with a double emulsion structure and pharmaceutically acceptable excipients.

[0021] In a fifth aspect, the present invention provides use of any one of the oral drug delivery systems having a double emulsion structure in preparing a preparation for reducing the tightness of the intestinal epithelial barrier.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The oral drug delivery system prepared by the method of the present invention uses exosomes as a solid stabilizer. By using ultrasonic emulsification, an oil phase solvent and water are mixed, and then the water-soluble MSC-CM is wrapped by a water-in-oil-in-water method to form the core of a double emulsion, thereby achieving oral absorption of water-soluble proteins into the human body.

[0024] The oral drug delivery system of the present invention not only has excellent structural stability, but also possesses excellent viscoelasticity due to the structure of the exosomes and emulsion. The particle size range of the oral drug delivery system can reach approximately 10-50 μm. This facilitates increased contact with intestinal epithelial cells, while also altering the intestinal epithelial cell skeleton, downregulating tight junction proteins in intestinal epithelial cells, and opening the tight junction barrier of intestinal epithelial cells, promoting the absorption of released MSC-CM into the bloodstream, thereby increasing the bioavailability of MSC-CM and exerting its therapeutic effect on POI. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The distribution and localization diagram of each component in the E@MSC-CM delivery system observed by laser confocal microscopy; a shows the morphology of E@MSC-CM observed by laser confocal microscopy using PKH67-labeled EVs; b shows the morphology of MSC-CM observed by laser confocal microscopy using FITC-labeled MSCs;

[0026] Figure 2 This is the structure diagram of E@MSC-CM observed by light microscopy;

[0027] Figure 3 The results of optical microscope observation of the particle size of E@MSC-CM at different times in artificial gastric fluid, artificial intestinal fluid and PBS solution;

[0028] Figure 4This is a graph showing the changes in cell transmembrane resistance over time after the addition of E@MSC-CM.

[0029] Figure 5 Laser confocal microscopy was used to observe the changes in the cytoskeleton after E@MSC-CM acted on human colon adenocarcinoma cells Caco-2. Red fluorescent phalloidin was used to label cell actin.

[0030] Figure 6 Laser confocal microscopy was used to observe the effect of E@MSC-CM on the simulated intestinal epithelial monolayer barrier constructed by human colon adenocarcinoma cells Caco-2, and to observe the changes in the cell tight junction protein ZO-1 (green fluorescence);

[0031] Figure 7 This is a cell viability experiment after MSC-CM, EV and E@MSC-CM acted on IFN-γ-induced granulosa cells (POI cells);

[0032] Figure 8 The ROS levels of MSC-CM, EV and E@MSC-CM in IFN-γ-induced granulosa cells (POI cells) were compared with normal granulosa cells as the control.

[0033] Figure 9 This is the result of ELISA detection of the levels of sex hormones estradiol (E2) and follicle-stimulating hormone (FSH) in mouse serum;

[0034] Figure 10 This is a comparison chart of the number of ovaries in mice at different stages after drug action observed using HE staining. DETAILED DESCRIPTION

[0035] The present invention will be further described below with reference to the accompanying drawings and examples.

[0036] Example 1: Preparation of E@MSC-CM

[0037] 1. Preparation of E@MSC-CM

[0038] (1) Extraction and purification of extracellular vesicles:

[0039] After fresh strawberry juice is squeezed, large particle impurities are removed by differential centrifugation, and then ultracentrifugation is performed using an ultracentrifuge. The resulting precipitate is purified by sucrose density gradient centrifugation to obtain strawberry-derived exosomes (ST-EV).

[0040] (2) Preparation of MSC-CM

[0041] When the confluence of umbilical cord mesenchymal stem cells (UC-MSC) reached 80%, the DMEM / F12 medium containing 10% serum was replaced with DMEM / F12 serum-free medium. After 96 h, the supernatant was collected, and cell debris and apoptotic bodies were removed by centrifugation. The supernatant was concentrated by ultrafiltration and freeze-dried to obtain MSC-CM, which was stored at -80°C.

[0042] (3) Preparation of E@MSC-CM

[0043] Place 1 mL of squalene in a centrifuge tube, add 250 μL of MSC-CM, and perform ultrasonic emulsification using an ultrasonic cell disruptor in an ice bath. Add 3 mL of PBS and 300 μg of ST-EV to the product. Perform a secondary emulsification using a high-speed homogenizer in an ice bath. After the mixture is allowed to stand for a while, distinct layers of liquid will be visible in the centrifuge tube. Carefully aspirate the upper emulsion-like liquid. The resulting product is the MSC-CM-loaded strawberry exosome structured delivery system (E@MSC-CM).

[0044] 2. Characterization and Identification of E@MSC-CM

[0045] In order to determine the specific distribution of components EV, MSC-CM, and squalene in the E@MSC-CM delivery system, confocal fluorescence microscopy was used to observe the relative localization of E@MSC-CM synthesized by EV labeled with PKH67, squalene labeled with Nile red, and E@MSC-CM synthesized by FITC-MSC-CM. Figure 1 As shown in Figure a, green fluorescence is distributed around the periphery of the droplet, indicating that EVs are evenly distributed on the outermost surface of the emulsion particles. Figure 1 As shown in Figure b, using Nile red to label squalene, we can see green bubbles inside the droplet. The red oil phase is inside the droplet, and the green outer circle is a certain reflection effect. MSC-CM is inside the squalene, and MSC-CM and squalene together form the core structure of the emulsion. Figure 2 Under light microscopy, E@MSC-CM can be observed as granular droplets, and smaller bubbles can be observed in the middle of the droplets, indicating the existence of an internal water phase, indicating that the prepared drug delivery system has the structural characteristics of water-in-oil-in-water.

[0046] 3. Stability testing of E@MSC-CM

[0047] The prepared E@MSC-CM was dissolved in artificial gastric juice, artificial intestinal juice and PBS solution at room temperature. The morphological changes of E@MSC-CM were observed under an optical microscope, and the particle size of E@MSC-CM at different times was counted to detect the stability of E@MSC-CM. Figure 3As shown, E@MSC-CM maintained good stability in gastric fluid for the first 6 hours, but gradually began to demulsify after 12 hours. It also remained stable in artificial intestinal fluid and PBS solution, retaining its original morphology after 48 hours in artificial intestinal fluid. After two weeks in PBS solution, no significant changes were observed under both the naked eye and an optical microscope. This demonstrates that the constructed E@MSC-CM maintains a stable structure in PBS solution and artificial gastrointestinal fluid, effectively protecting the in vivo delivery of MSC-CM as a drug carrier.

[0048] Example 2: Effects of E@MSC-CM on the simulated intestinal epithelial cell barrier

[0049] Caco-2 cells were cultured in Transwell chambers to simulate the intestinal epithelial barrier in vitro. After the barrier was intact, the E@MSC-CM prepared in Example 1 was added and the reduction in cell transmembrane electrical resistance (TEER) was measured at different times. Figure 4 After the addition of E@MSC-CM, the resistance dropped significantly, indicating that the barrier integrity decreased, the intestinal epithelial barrier was opened, and the MSC-CM in E@MSC-CM passed through the intestinal barrier into the blood circulation. Afterwards, starting from the 6th hour, the cell transmembrane resistance gradually recovered, and at the 24th hour it reached a level equivalent to the original level, indicating that the oral drug delivery system can change the tight junctions of the simulated intestinal epithelial barrier, but it can recover after a certain period of action, protecting the epithelial barrier from damage. Immunofluorescence was further used to observe the effects of E@MSC-CM on the Caco-2 cytoskeleton and the tight junction protein ZO-1. Figure 5 and 6 After 6 hours of treatment with E@MSC-CM, the actin (F-actin) of the Caco-2 cell skeleton was changed, and the tight junction protein ZO-1 was downregulated, indicating that the prepared oral drug delivery system can reduce the tightness of the intestinal epithelial barrier.

[0050] Example 3: E@MSC-CM repairs IFN-γ-induced immune damage in KGN cells

[0051] KGN in the logarithmic growth phase was digested into cell suspension and counted, with 1×10 3Inoculate into 96-well plates and culture in a constant temperature incubator overnight. After the cells adhered, IFN-γ was used to construct an immune damage premature ovarian insufficiency model, and the cell culture medium was replaced with 200 μL of fresh complete culture medium or complete culture medium containing E@MSC-CM, ST-EV, and MSC-CM as the drug group. Cells without any drugs were used as the control group, and zero-adjustment wells without cells and drugs were set as the zero-adjustment group. After culturing at 37°C for 48 hours, the culture was replaced with 100 μL of basal culture medium containing 10% CCK-8 and continued to be cultured for 1 hour. Remove the 96-well plate from the cell culture incubator. Turn on the multifunctional microplate reader in advance for preheating. After the temperature reaches 37°C, use the multifunctional microplate reader to measure the absorbance at 450 nm. Count cell viability according to the following formula:

[0052] Cell viability = (absorbance value of drug group - absorbance value of zero adjustment group) / (absorbance value of control group - absorbance value of zero adjustment group)

[0053] The results are as follows Figure 7 As shown in the results, MSC-CM itself can rescue the negative effects of IFN-γ and promote the proliferation of granulocytes, but at lower concentrations, E@MSC-CM has a better proliferation-promoting effect than ST-EV and MSC-CM at the same concentration.

[0054] Take human ovarian granulosa cells (KGN cells for short) in the logarithmic growth phase, digest, centrifuge, resuspend and count, and adjust the cell concentration with complete culture medium. Place a sterile and clean cell slide in a 6-well plate, carefully drop the cell suspension with adjusted density on the slide, carefully transfer to a cell culture incubator, and culture overnight. Observe the next day, and after the cells adhere to the wall, use IFN-γ to construct an immune-damaged premature ovarian insufficiency model. After the immune POI model is constructed, add ST-EV, MSC-CM and E@MSC-CM, incubate for 24 hours, discard the supernatant, gently wash three times with sterile PBS buffer, and dilute DCFH-DA with serum-free medium to a final concentration of 10μmol / L. Add 1mL to each well and incubate the 6-well plate in an incubator for 20 minutes. After incubation, gently wash the cells three times with serum-free medium to completely remove DCFH-DA that has not entered the cells. Gently shake the cells three times with PBS, each for 5 minutes. Add 4% polyacrylamide and fix for 20 minutes at room temperature. Remove the fixative and wash three times with PBS, each for 5 minutes. Prepare clean slides and add 10 μL of anti-fluorescence quencher (containing DAPI) to each slide. Carefully remove the cells from the slide and invert them onto a clean slide. Observe the samples using an inverted laser confocal microscope and capture images to document the experimental results.

[0055] like Figure 8Using normal cells (CON) as a control and IFN-γ-induced immune-damaged KGN cells (POI) as a positive control, the green fluorescent probe DCFH-DA was used to investigate ROS levels in cells after droplet treatment. Cell nuclei were labeled with DAPI, and the images were merged for analysis. The results showed an increase in green fluorescence intensity in POI cells, indicating that POI can elevate ROS levels and lead to severe oxidative stress. Adding MSC-CM significantly reduced ROS levels, and E@MSC-CM further alleviated oxidative stress in POI cells.

[0056] Example 4: E@MSC-CM improves endocrine function in autoimmune POI mice

[0057] The immune POI animal model was established by subcutaneously injecting mice with the immune agent ZP3. After confirming the model establishment by monitoring the estrous cycle, E@MSC-CM and MSC-CM with a protein content of 500 μg were administered orally for 5 consecutive days. Four weeks after administration, the mice were killed and serum and ovarian samples were collected. The levels of sex hormones estradiol (E2) and follicle-stimulating hormone (FSH) in mouse serum were measured by ELISA. Figure 9 The results showed that after POI mice were treated with E@MSC-CM, the expression of E2 in the serum increased and the expression level of FSH decreased, indicating that direct oral administration of MSC-CM could not improve the premature ovarian failure of the mice. Oral administration of E@MSC-CM could effectively improve the levels of estradiol and follicle-stimulating hormone in the serum of POI mice. In addition, HE staining was used to observe the ovarian condition of the mice after the drug effect ( Figure 10 The results showed that oral administration of E@MSC-CM restored ovarian structure, promoted the development of primordial follicles, primary follicles, and secondary follicles, and reduced atretic follicles. This indicates that oral administration of E@MSC-CM can effectively improve the levels of estradiol and follicle-stimulating hormone in the serum of POI mice, promote ovarian structural recovery and follicle development, and demonstrate that this oral drug delivery system enhances the bioavailability of MSC-CM.

Claims

1. An oral drug delivery system with a double emulsion structure, characterized in that: The double emulsion structure is composed of droplet particles composed of an oil phase solvent encapsulating MSCs conditioned medium inside and exosomes uniformly distributed on the outer surface of the droplet particles.

2. The oral drug delivery system with a double emulsion structure according to claim 1, characterized in that: The oil phase solvent is a food grade oil phase solvent.

3. The oral drug delivery system with a double emulsion structure according to claim 1, wherein: The oil phase solvent is squalene, rapeseed oil, peanut oil, perilla seed oil or sweet almond oil.

4. A method for preparing an oral drug delivery system having a double emulsion structure according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Umbilical cord mesenchymal stem cells were cultured in serum-free medium. After the culture, the supernatant was collected, centrifuged to remove cell debris and apoptotic bodies, concentrated by ultrafiltration, and freeze-dried and reconstituted to obtain MSCs-conditioned medium; (2) The oil phase solvent was mixed with the MSCs conditioned medium, and ultrasonic emulsification was performed under ice bath conditions. PBS solution and exosomes were added, and secondary emulsification was performed using a high-speed homogenizer under ice bath conditions. The mixture was allowed to stand, and the upper emulsion-like liquid was aspirated to obtain an oral drug delivery system loaded with MSCs conditioned medium.

5. The preparation method according to claim 4, wherein The culture time is 48-96h; The concentration of the MSCs conditioned medium is 8-15 mg / mL.

6. Use of the oral drug delivery system with a double emulsion structure according to any one of claims 1 to 3 in the preparation of a drug for treating premature ovarian insufficiency.

7. The use according to claim 6, characterized in that The oral drug delivery system can reduce ROS levels.

8. The use according to claim 6, characterized in that The oral drug delivery system can restore the ovarian structure and improve the levels of estradiol and follicle-stimulating hormone in the serum of POI mice.

9. A pharmaceutical composition for treating premature ovarian insufficiency, characterized in that: The oral drug delivery system having a double emulsion structure according to any one of claims 1 to 4 and pharmaceutically acceptable excipients.

10. Use of the oral drug delivery system having a double emulsion structure according to any one of claims 1 to 4 in the preparation of a preparation for reducing the tightness of the intestinal epithelial barrier.

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