An oral drug delivery system with double emulsion structure and its use in the preparation of a medicament for treating premature ovarian failure

Abstract

The application relates to an oral drug delivery system with a double-emulsion structure and application thereof in preparation of a medicine for treating premature ovarian insufficiency, and belongs to the technical field of biological medicines. The oral drug delivery system comprises an oil-phase solvent, MSCs conditioned medium is wrapped in the inside to form a droplet particle, and exosomes are uniformly distributed on the outer surface of the droplet particle, thereby forming a double-emulsion structure. The water-soluble MSC-CM is wrapped by a water-in-oil-in-water method to form a double-emulsion structure, so that the water-soluble protein is absorbed into the human body in an oral mode. The application has good structural stability and viscoelasticity, the particle size range is about 10-50 mu m, the contact with the intestinal epithelial cells is facilitated, meanwhile, the skeleton of the intestinal epithelial cells can be changed, the expression of the tight junction protein of the intestinal epithelial cells is lowered, the tight junction barrier of the intestinal epithelial cells is opened, and therefore the bioavailability of the MSC-CM is improved, and the role of treating POI is played.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an oral drug delivery system with a double emulsion structure and its application in the preparation of drugs for treating early-onset ovarian insufficiency. Background Technology

[0002] Premature ovarian insufficiency (POI) is a common disease in the field of gynecological endocrinology, referring to decreased ovarian function in women before the age of 40, characterized by high gonadotropin levels, low estrogen levels, oligomenorrhea, and amenorrhea. POI not only affects female fertility, leading to infertility, but can also cause dysfunction in multiple systems, including the neurocognitive, urogenital, skeletal, cardiovascular, and endocrine metabolic systems, seriously endangering women's physical and mental health and quality of life. POI is caused by a variety of etiologies and complex mechanisms. Currently known causes include genetics, infection, immunity, and iatrogenic injury. Among these, POI caused by immune factors (autoimmune POI) is the most common type, accounting for approximately 5%-30% of POI cases. Current treatments, including hormone therapy, immunosuppressive therapy, and assisted reproductive technologies, have not achieved a complete cure for autoimmune POI. Therefore, exploring new therapeutic targets and developing innovative therapies to enhance treatment efficacy and promote women's health has become a key issue that urgently needs to be addressed in scientific research and clinical practice.

[0003] In the pathogenesis of autoimmune premature ovarian insufficiency (aPOI), under the influence of various immune factors, immune cells are overactivated, producing large amounts of reactive oxygen species (ROS). ROS are byproducts of metabolism that determine the balance between free radicals and antioxidants, regulating cellular function. In ovarian tissue, normal levels of ROS are widely involved in the activation of transcription factors, thereby promoting cell proliferation and differentiation. They play an important regulatory role in follicle growth, angiogenesis, and sex hormone synthesis, and are crucial for the development of follicles at all stages in the ovary. However, excessively high ROS levels induce oxidative stress in ovarian granulosa cells, leading to impaired or abnormally depleted follicle development. Imbalances in oxidative stress can cause damage to cellular molecules such as DNA, mitochondria, proteins, and lipids. Many factors affecting POI ultimately result in oxidative stress; therefore, reducing ovarian oxidative stress is an important means of 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 increased the risk of diminished ovarian reserve (DOR) (Environmental Health Perspectives, 2023, 131(6):067005.). In contrast, natural antioxidants are not only more potent and safer, but also more readily accepted, making them a better choice for aPOI antioxidant therapy.

[0004] Mesenchymal stem cells (MSCs) hold great promise for treating poikiloma (POI) due to their self-renewal and induced differentiation capabilities. Previous studies have shown that MSCs can reduce ROS levels in granulosa cells, inhibit oxidative stress, and have a repairing effect on POI. However, ethical controversies, immune rejection, and post-transplant apoptosis during stem cell therapy cannot be ignored. Using MSC-conditioned medium (MSC-CM) can effectively mitigate these risks. The repair function of MSCs is closely related to paracrine function. MSCs can secrete various paracrine factors, including extracellular matrix, proteins, growth factors, cytokines, and chemokines, exerting a therapeutic effect on diseases through the paracrine pathway. Protein detection analysis has confirmed that MSC-CM contains various factors secreted by MSCs, including vascular endothelial growth factor, epidermal growth factor, and insulin-like growth factor I and II, achieving therapeutic effects equivalent to MSCs. Nevertheless, MSC-CM is a protein composed of various secreted factors, and its administration method is a major issue limiting its clinical application. Currently, oral administration is the most common clinical administration method, with high patient compliance and fewer side effects. However, proteins are easily degraded in the gastrointestinal tract, resulting in low bioavailability, which limits the efficacy of MSC-CM. Therefore, there is an urgent need to develop oral delivery systems for MSC-CM that improve its stability and bioavailability in the gastrointestinal tract, thereby benefiting the improvement of premature ovarian failure.

[0005] In recent years, exosomes (also known as extracellular vesicles, EVs) have attracted widespread attention as drug delivery carriers. These are nanoscale natural vesicles with a phospholipid bilayer structure secreted by cells, which can be used to encapsulate various drugs. Compared with other traditional drug delivery systems, they have significant advantages, such as good biocompatibility, high stability under physiological conditions, and good ability to penetrate biological barriers. However, the small size and particle size of exosomes limit their drug loading capacity, restricting their further development as drug carriers. Based on this, leveraging the excellent lipid bilayer properties of exosomes, we designed an oil-in-water (OI-water) oral drug delivery system. The system consists of exosomes stabilized at the oil-water interface, with a water-soluble protein as the inner aqueous phase, further encapsulated in the oil phase. This system, stable by exosomes and with a water-soluble protein as the inner aqueous phase, is used for in vivo delivery to MSC-CM. This system achieves high drug loading and exhibits good structural stability. A 2023 study used fruit-derived exosomes (EVs) as stabilizing particles to dissolve the hydrophobic chemotherapy drug doxorubicin (DOX) in squalene, preparing extracellular vesicle-stabilized oil-in-water structured droplet drug (Advanced Materials, 2023:2304187). This system can achieve the encapsulation of hydrophobic drugs, producing particles with a size of around nanometers, and is intended for the development of intravenous drug delivery for glioma treatment. However, this drug delivery system mainly targets the development of hydrophobic drugs, and an oral delivery system suitable for water-soluble drugs (especially protein drugs) has not yet been established for disease treatment.

[0006] This invention addresses the challenge of constructing oral drug delivery systems for water-soluble proteins by developing a water-in-oil-in-water double emulsion droplet structure. The system's surface is stabilized with viscoelastic natural exosomes, while a flexible liquid serves as the core, thus giving the entire system a degree of viscoelasticity. The prepared oral drug delivery system has a particle size range of approximately 10-50 μm, reaching the size of a single cell, which facilitates greater contact with intestinal cells. This drug delivery system maintains structural stability under the harsh physiological conditions of the gastrointestinal tract. Furthermore, after interacting with intestinal epithelial cells, the system influences the intestinal epithelial cell cytoskeleton, further opening the tight junction barrier of intestinal epithelial cells, promoting the absorption of MSC-CM into the bloodstream, and improving the oral bioavailability of MSC-CM, thereby achieving effective treatment against aPOIs. Summary of the Invention

[0007] To address the problems existing in the prior art, the present invention aims to design and provide an oral drug delivery system and its preparation method for a conditioned medium containing mesenchymal stem cells used to treat early-onset ovarian insufficiency. The E@MSC-CM of the present invention uses exosomes (EVs) as a particle stabilizer, and through a two-step emulsification method, MSC-CM is encapsulated in an oil phase solvent. EVs are uniformly 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 using various oral and topical natural oils such as squalene, squalane, rapeseed oil, peanut oil, perilla seed oil, and sweet almond oil.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

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

[0010] The oral drug delivery system having a dual emulsion structure, wherein the oil phase solvent is a food-grade oil phase solvent.

[0011] The oral drug delivery system having a dual emulsion structure, wherein the oil phase solvent is squalene, rapeseed oil, peanut oil, perilla seed oil, or sweet almond oil.

[0012] Secondly, the present invention provides a method for preparing an oral drug delivery system having a dual emulsion structure as described in any one of the claims, comprising the following steps:

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

[0014] (2) Take the oil phase solvent, mix it with MSCs conditioned medium, and perform ultrasonic emulsification under ice bath conditions. Add PBS solution and exosomes, and perform secondary emulsification using a high-speed homogenizer under ice bath conditions. Let it stand, and aspirate the upper emulsion liquid to obtain an oral drug delivery system (E@MSC-CM) loaded with MSCs conditioned medium.

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

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

[0017] Thirdly, the present invention provides the use of any of the oral drug delivery systems having a dual emulsion structure in the preparation of a medicament for treating early-onset ovarian insufficiency.

[0018] As described above, the oral drug delivery system can reduce ROS levels.

[0019] As described above, the oral delivery system can restore ovarian structure and improve the levels of estradiol and follicle-stimulating hormone in ovarian cells.

[0020] Fourthly, the present invention provides a pharmaceutical composition for treating early-onset ovarian insufficiency, comprising an oral delivery system having a double emulsion structure as described in any one of the claims and pharmaceutically acceptable excipients.

[0021] Fifthly, the present invention provides the use of the oral delivery system having a dual emulsion structure as described in any one of the claims in the preparation of formulations that reduce 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 this invention uses exosomes as solid stabilizers. Using ultrasonic emulsification, water-soluble MSC-CM is mixed with an oil-phase solvent and water-in-water method to form a double emulsion core, thereby enabling the water-soluble protein to be absorbed into the human body via oral administration.

[0024] This oral drug delivery system not only possesses excellent structural stability, but the structure of the exosomes and emulsion also endows it with good viscoelasticity. The particle size range of the oral drug delivery system can reach approximately 10μm-50μm. This facilitates increased contact with intestinal epithelial cells, while simultaneously altering the cytoskeleton of intestinal epithelial cells, downregulating tight junction proteins, opening the tight junction barrier of intestinal epithelial cells, and promoting the absorption of released MSC-CM into the bloodstream, thereby improving the bioavailability of MSC-CM and exerting its therapeutic effect on POI. Attached Figure Description

[0025] Figure 1 The distribution and localization of each component in the E@MSC-CM delivery system are observed by laser confocal microscopy; where a is the morphology of E@MSC-CM observed by laser confocal microscopy with PKH67-labeled EV; b is the morphology of MSC-CM observed by laser confocal microscopy with FITC-labeled MSC-CM.

[0026] Figure 2 This is a light microscopic view of the structure of E@MSC-CM;

[0027] Figure 3 The image shows the particle size results of E@MSC-CM observed under an optical microscope at different time points in simulated gastric fluid, simulated intestinal fluid, and PBS solution.

[0028] Figure 4The graph shows the change in cell transmembrane resistance over time after the addition of E@MSC-CM.

[0029] Figure 5 To observe changes in the cytoskeleton after E@MSC-CM was applied to human colon adenocarcinoma cells Caco-2 using laser confocal microscopy, red fluorescent phalloidin was used to label cell actin and observe the changes in the cytoskeleton.

[0030] Figure 6 To observe the changes in the cell tight junction protein ZO-1 (green fluorescence) in a simulated intestinal epithelial monolayer barrier constructed by E@MSC-CM acting on human colon adenocarcinoma cells Caco-2 using laser confocal microscopy;

[0031] Figure 7 Cell viability assays of IFN-γ-induced granulocytes (POI cells) after treatment with MSC-CM, EV and E@MSC-CM;

[0032] Figure 8 The levels of reactive oxygen species (ROS) in IFN-γ-induced granulosa cells (POI cells) were measured by MSC-CM, EV, and E@MSC-CM, with normal granulosa cells serving as a control.

[0033] Figure 9 The image shows the results of ELISA detection of estradiol (E2) and follicle-stimulating hormone (FSH) levels in mouse serum.

[0034] Figure 10 This is a comparison of the number of ovaries in mice at different stages after drug treatment, observed using HE staining. Detailed Implementation

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

[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 juicing fresh strawberries, large particles of impurities are removed by differential centrifugation, followed by ultracentrifugation. The resulting precipitate is then purified by sucrose density gradient centrifugation to obtain strawberry-derived exosomes (ST-EV).

[0040] (2) Preparation of MSC-CM

[0041] Once the confluence of umbilical cord mesenchymal stem cells (UC-MSCs) reaches 80%, the DMEM / F12 medium containing 10% serum is replaced with DMEM / F12 serum-free medium. After 96 hours, the supernatant is collected, cell debris and apoptotic bodies are removed by centrifugation, ultrafiltration is performed to concentrate the MSCs, and they are freeze-dried and stored at -80℃.

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

[0043] Add 1 mL of squalene to a centrifuge tube, then add 250 μL of MSC-CM. Sonicate the mixture using an ultrasonic cell disruptor while on ice. Add 3 mL of PBS solution and 300 μg of ST-EV to the product. Perform a secondary emulsification using a high-speed homogenizer while on ice. After this process, allow the mixture to stand briefly; clear stratification will be observed in the centrifuge tube. Carefully aspirate the upper emulsion layer. The resulting product is the strawberry exosome structured delivery system (E@MSC-CM) loaded with MSC-CM.

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

[0045] To determine the specific distribution of the components EV, MSC-CM, and squalene in the E@MSC-CM delivery system, confocal fluorescence microscopy was used to observe the relative positioning of E@MSC-CM synthesized from PKH67-labeled EV, squalene-labeled with Nile Red, and FITC-MSC-CM. The results are as follows: Figure 1 As shown in Figure a, the green fluorescence is distributed around the droplet, indicating that EV is uniformly distributed on the outermost surface of the emulsion particles. Figure 1 As shown in Figure b, when squalene is labeled with Nile Red, small green bubbles can be observed inside the droplet. The red oil phase is located inside the droplet, and the outer green ring is due to a reflective effect. MSC-CM is located inside the squalene, and MSC-CM together with squalene constitute the core structure of the emulsion. Figure 2 Under light microscopy, E@MSC-CM can be observed as granular droplets, with smaller water bubbles visible in the center of the droplets, indicating the presence of an internal aqueous phase. This suggests that the prepared drug delivery system has a water-in-oil-in-water structural characteristic.

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

[0047] The prepared E@MSC-CM was dissolved in simulated gastric fluid, simulated intestinal fluid, and PBS solution at room temperature. The morphological changes of E@MSC-CM were observed using an optical microscope, and the particle size of E@MSC-CM at different time points was recorded to assess its stability. The results are as follows: Figure 3As shown, E@MSC-CM maintained good stability in gastric juice for the first 6 hours, but demulsification gradually began to occur after 12 hours. It maintained good stability in artificial intestinal fluid and PBS solution. In artificial intestinal fluid, it retained its original morphology after 48 hours, and after 2 weeks of storage in PBS solution, no significant changes were observed under the naked eye or optical microscope. This indicates that the constructed E@MSC-CM can maintain a stable structure in PBS solution and artificial gastrointestinal fluid, and can effectively protect 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 established, E@MSC-CM prepared in Example 1 was added, and the decrease in transmembrane electrical resistance (TEER) was detected at different time points. Figure 4 After the addition of E@MSC-CM, the transmembrane resistance decreased significantly, indicating a decrease in barrier integrity and an opening of the intestinal epithelial barrier, allowing MSC-CM in E@MSC-CM to cross the intestinal barrier and enter the bloodstream. Subsequently, starting from hour 6, the transmembrane resistance gradually recovered, reaching a level comparable to the original level by hour 24. This indicates that the oral drug delivery system can alter the tight junctions of the simulated intestinal epithelial barrier, but recovers after a certain period of action, protecting the epithelial barrier from damage. Further immunofluorescence was 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 cytoskeleton was altered, 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 granulocytes (KGN cells)

[0051] After digesting KGN cells in the logarithmic growth phase into a cell suspension, count the cells at a ratio of 1 × 10⁶ cells per well. 3Cells were seeded into 96-well plates and cultured overnight in a constant temperature incubator. After cell adhesion, an immune-induced early-onset ovarian insufficiency model was constructed using IFN-γ. The cell culture medium was replaced with 200 μL of fresh complete medium or complete medium containing E@MSC-CM, ST-EV, or MSC-CM, serving as the drug group. Cells without any drugs served as the control group, and a zero-adjustment well without cells or drugs was set up as the zero-adjustment group. After culturing at 37°C for 48 h, the medium was replaced with 100 μL of basal medium containing 10% CCK-8 and cultured for another 1 h. The 96-well plates were then removed from the cell culture incubator. The multi-plate reader was preheated to 37°C, and the absorbance at 450 nm was measured using the multi-plate reader. Cell viability was counted according to the following formula:

[0052] Cell viability = (Absorbance of drug group - Absorbance of zeroing group) / (Absorbance of control group - Absorbance of zeroing group)

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

[0054] Human ovarian granulosa cells (KGN cells) in the logarithmic growth phase were harvested, digested, centrifuged, resuspended, and counted. Cell concentration was adjusted with complete culture medium. A sterile cell slide was placed in a 6-well plate, and the adjusted cell suspension was carefully added dropwise onto the slide. The plate was then carefully transferred to a cell culture incubator and incubated overnight. The next day, after cell attachment, an immune-induced early-onset ovarian insufficiency model was constructed using IFN-γ. After the immune POI model was established, ST-EV, MSC-CM, and E@MSC-CM were added. After incubation for 24 hours, the supernatant was discarded, and the cells were gently washed three times with sterile PBS buffer. DCFH-DA was diluted with serum-free culture medium to a final concentration of 10 μmol / L. 1 mL of this solution was added to each well, and the 6-well plate was incubated for 20 minutes. After incubation, the cells were gently washed three times with serum-free culture medium to thoroughly remove any unadsorbed DCFH-DA. Gently wash the cells three times with PBS solution, 5 min each time. After each wash, add 4% DAPI and fix at room temperature for 20 min. Then wash off the fixative with PBS three times, 5 min each time. Prepare clean glass slides, add 10 μL of anti-fluorescence quencher (containing DAPI) to each slide, carefully remove the cell smears, and invert them onto clean glass slides. Observe the samples using an inverted laser confocal microscope and take images to record 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 ROS levels of cells after droplet treatment were studied using the green fluorescent probe DCFH-DA, and cell nuclei were labeled with DAPI. Merged plots were then analyzed. The results showed that the green fluorescence intensity of POI cells increased, indicating that POI could induce elevated ROS levels and severe oxidative stress. The addition of 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] An immune POI animal model was established in mice by subcutaneous injection of the immunomodulator ZP3. After confirming model establishment by monitoring the estrous cycle, mice were administered E@MSC-CM and MSC-CM (protein quantification 500 μg) orally by gavage for 5 consecutive days. Four weeks after administration, mice were sacrificed, and serum and ovarian samples were collected. Serum levels of estradiol (E2) and follicle-stimulating hormone (FSH) were detected using ELISA. Figure 9 The results showed that after E@MSC-CM treatment, serum E2 expression increased and FSH expression decreased in POI mice, indicating that direct oral administration of MSC-CM could not improve premature ovarian failure in the affected mice. Oral administration of E@MSC-CM effectively improved serum estradiol and follicle-stimulating hormone levels in POI mice. Additionally, HE staining was used to observe the ovarian condition of the mice after drug action. Figure 10 The results showed that oral administration of E@MSC-CM restored ovarian structure, promoted the development of primordial, primary, 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 having a dual emulsion structure, characterized in that, The droplet particles, which contain an oil phase solvent that encapsulates the conditioned medium for MSCs, and the exosomes that are uniformly distributed on the outer surface of the droplet particles, constitute a biemulsion structure. The preparation method of the oral drug delivery system with a dual emulsion structure includes the following steps: (1) Umbilical cord mesenchymal stem cells were cultured in serum-free medium. After the culture was completed, the supernatant was collected, centrifuged to remove cell debris and apoptotic bodies, concentrated by ultrafiltration, and then lyophilized and reconstituted to obtain MSCs conditioned medium. (2) Take the oil phase solvent, mix it with MSCs conditioned medium, perform ultrasonic emulsification under ice bath conditions, add PBS solution and exosomes, perform secondary emulsification using a high-speed homogenizer under ice bath conditions, let stand, and aspirate the upper emulsion liquid to obtain an oral drug delivery system loaded with MSCs conditioned medium.

2. The oral drug delivery system with a dual emulsion structure as described in claim 1, characterized in that, The oil phase solvent is a food-grade oil phase solvent.

3. An oral drug delivery system with a dual emulsion structure as described in claim 1, characterized in that, The oil phase solvent is squalene, rapeseed oil, peanut oil, perilla seed oil, or sweet almond oil.

4. An oral drug delivery system with a dual emulsion structure as described in claim 1, characterized in that, The culture time is 48-96 h; The concentration of the conditioned medium for the MSCs was 8-15 mg / mL.

5. Use of the oral delivery system having a dual emulsion structure as described in any one of claims 1-4 in the preparation of a medicament for treating early-onset ovarian insufficiency.

6. The use as described in claim 5, characterized in that, The oral drug delivery system can reduce ROS levels.

7. The use as described in claim 5, characterized in that, The oral delivery system can restore ovarian structure and improve the levels of estradiol and follicle-stimulating hormone in the serum of POI mice.

8. A pharmaceutical composition for treating early-onset ovarian insufficiency, characterized in that, It includes an oral delivery system having a dual emulsion structure as described in any one of claims 1-4 and pharmaceutically acceptable excipients.

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