Exosome for treating fatty liver disease and cognitive disorder related to metabolic dysfunction
By transfecting mesenchymal stem cells with miRNA mimics and subjecting them to hypoxia stimulation, engineered exosomes were prepared, solving the problem of the lack of target cell specificity in exosomes and achieving effective treatment of fatty liver disease and cognitive impairment related to metabolic dysfunction, as well as delaying dementia.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BOYALIFE
- Filing Date
- 2025-12-18
- Publication Date
- 2026-04-17
AI Technical Summary
Existing mesenchymal stem cell exosomes lack target cell specificity, which limits their clinical application in the treatment of fatty liver disease and cognitive impairment related to metabolic dysfunction.
By transfecting mesenchymal stem cells with miRNA mimics, stimulating them to secrete exosomes under hypoxic conditions, and purifying them through methods such as differential centrifugation, engineered exosomes were prepared to specifically target liver cells and brain neurons.
It has achieved effective treatment of fatty liver disease and cognitive impairment associated with metabolic dysfunction, while slowing the progression of Alzheimer's disease, thus improving the therapeutic efficacy and targeting of exosomes.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine, specifically relating to the preparation of engineered exosomes derived from mesenchymal stem cells, and their application in treating metabolic dysfunction-associated fatty liver disease (MAFLD), cognitive impairment, and delaying dementia. This invention relates to a method for preparing engineered exosomes from mesenchymal stem cells, which can serve as a technological platform. The invention provides a safe and efficient method for preparing engineered mesenchymal stem cell exosomes, which can be used to treat MAFLD and delay cognitive impairment and dementia. Background Technology
[0002] In 2020, liver disease experts proposed a new definition for metabolic dysfunction-related fatty liver disease (MAFLD) (Associations of metabolic dysfunction-related fatty liver disease and dementia risk: A prospective study based on the UK biobank. Chaofan Geng, Peiyang Gao, Yi Tang. Arch Gerontol Geriatr. 2025,135:105845; Associations between metabolic dysfunction-associated fatty liver disease and atherosclerotic cardiovascular disease. Wen Wen, Hua Fan, Shenghui Zhang, et al. Am J Med Sci. 2024, 368(6):557-568). Metabolic dysfunction-related fatty liver disease (MAFLD) is the most common chronic liver disease worldwide, primarily caused by rising obesity rates, metabolic syndrome, and type 2 diabetes mellitus (T2DM). It is currently estimated to affect 38% of the global population. In 2021, approximately 1.27 billion cases of MAFLD were reported globally, with an age-standardized prevalence of 15,018 cases per 100,000 people. A study involving 2,667,052 participants reported an estimated global prevalence of MAFLD (fatty liver disease) of up to 50.7% among overweight or obese adults. With my country's aging population, the burden of metabolic dysfunction-related fatty liver disease is increasing. There is an urgent need for preventative and early intervention measures to mitigate future impacts.
[0003] Studies have shown a significant association between metabolic dysfunction-related fatty liver disease (MAFLD) and dementia risk. As a key metabolic organ, liver metabolic dysfunction can lead to dementia through altered communication along the liver-brain axis. A robust cohort study based on 415,116 participants from the UK Biobank showed that MAFLD significantly increases the risk of dementia, including Alzheimer's disease and vascular dementia. Research indicates that MAFLD shares common pathological pathways with cognitive impairment, such as systemic inflammation, insulin resistance, and changes in brain structure. Early intervention in MAFLD may reduce dementia risk. Animal studies have shown that improving liver function can reduce Aβ deposition in the brains of aging mice.
[0004] Mesenchymal stem cell exosomes, ranging from 20 to 300 nm in diameter, contain abundant microRNAs (miRNAs), which are non-coding RNA molecules composed of 22 nucleotides. miRNAs bind to the 3′-untranslated region (UTR) of mRNA, leading to reduced protein synthesis or mRNA degradation, and are key factors in the post-transcriptional regulation of gene expression. Mesenchymal stem cell exosomes possess the advantages of low immunogenicity and the ability to cross the blood-brain barrier; however, the lack of target cell specificity in natural exosomes limits their clinical application.
[0005] This invention patent describes the development of a novel type of engineered exosomes from mesenchymal stem cells. Through engineered design, these exosomes are constructed and targeted with specific miRNAs to precisely target liver cells and brain neurons. Prepared via intelligent drug delivery and large-scale production, these novel engineered exosomes from mesenchymal stem cells can simultaneously target metabolic dysfunction-related fatty liver disease (MAFLD), Alzheimer's disease (AD), and cognitive impairment. They improve liver metabolic function while delaying cognitive decline and dementia. Summary of the Invention
[0006] The purpose of this invention is to provide a novel treatment for improving metabolic dysfunction-related fatty liver disease (MAFLD), which is significantly associated with cognitive impairment and dementia risk. The technical problem this invention aims to solve is to develop a method for preparing mesenchymal stem cell-engineered exosomes. The resulting mesenchymal stem cell-engineered exosomes can be used to treat metabolic dysfunction-related fatty liver disease while simultaneously delaying cognitive impairment or dementia.
[0007] Therefore, in a first aspect, the present invention provides the use of mesenchymal stem cell-engineered exosomes in the preparation of medicaments for the treatment of fatty liver disease and cognitive impairment associated with metabolic dysfunction.
[0008] According to the first aspect of the invention, the mesenchymal stem cell-engineered exosomes are prepared from mesenchymal stem cells transfected with miRNA mimics.
[0009] According to the first aspect of the invention, the miRNA mimic is selected from any one or a combination of several of miR-100-5p, miR-99a-5p, let-7e-5p, miR-146a-5p, miR-29b-3p, miR-133b, miR-191-5p, miR-199b-3p, and miR-199a-3p. These miRNA mimics are well known to those skilled in the art; for example, their base sequences can be found on the public website https: / / www.mirbase.org / . For instance, the miRBase accession number for miRNA miR-100-5p is MIMAT0000098, for miRNA miR-99a-5p it is MIMAT0000099, for miRNA miRNA miR-let-7e-5p it is MIMAT0000065, and for miRNA miRNA miR-146a-5p it is... The miRBase accession number for miRNA miR-29b-3p is MIMAT0000100, for iRNA miR-133b it is MIMAT0000427, for miRNA miR-191-5p it is MIMAT0000465, for miRNA miR-199b-3p it is MIMAT0004563, and for miRNA miR-199a-3p it is MIMAT0000232.
[0010] According to the first aspect of the invention, the mesenchymal stem cell-engineered exosomes are prepared by a method comprising the steps of: (1) miRNA-Mimics transfection of mesenchymal stem cells; (2) Transfected mesenchymal stem cells were cultured under hypoxic conditions to induce them to secrete exosomes; (3) Separate and purify the exosomes from the cell culture medium obtained in step (2).
[0011] According to the first aspect of the invention, the transfection of mesenchymal stem cells with miRNA-Mimics is performed as follows: i) The miRNA mimic was briefly centrifuged, and the precipitate was reconstituted with water without nuclease to obtain a miRNA mimic stock solution with a concentration of 20 μM. ii) Add 25 μL of transfection mixture A and 25 μL of transfection mixture B to each well of a 96-well plate, mix gently to obtain transfection mixture (A+B), and incubate at room temperature for 15-20 minutes. iii) Resuspend P3-8 generation human (umbilical cord) mesenchymal stem cells in MSC complete medium, seed them into culture flasks, supplement with MSC complete medium, and culture at 37°C and 5% CO2 until the cell confluence reaches more than 65%; iv) Remove the culture medium from the cell culture medium in step iii), wash with PBS, add 50 μL of serum-free culture medium to the cells, then add 50 μL of transfection mixture (A+B), mix well, and then incubate the cells at 37°C to complete the transfection.
[0012] According to the first aspect of the invention, the transfection mixture A is a mixture of 24.75 μL of serum-free culture medium and 0.25 μL of miRNA mimic stock solution.
[0013] According to the first aspect of the invention, the transfection mixture B is a mixture of 24.5 μL of serum-free culture medium and 0.5 μL of siRNA / miRNA transfection reagent.
[0014] According to the first aspect of the invention, 50 μL of the transfection mixture (A+B) is added and mixed well, and the cells are incubated at 37°C for 48 h to complete the transfection.
[0015] According to the first aspect of the invention, the culture of transfected mesenchymal stem cells is carried out in the following manner: a) Take miRNA-Mimics transfected mesenchymal stem cells, digest them with recombinant trypsin, and then use 1×10 6 Cells were seeded into 10cm culture dishes, 8ml of mesenchymal stem cell complete culture medium was added, and the dishes were incubated at 37℃ in a 5% CO2 incubator to allow the cells to adhere to the culture dish. b) Continue culturing the cells at 37°C and 5% CO2 until the cell confluence is not less than 80%, and collect the culture supernatant; c) Wash three times with PBS, add 8 mL of FBS-free DMEM-high glucose basal medium, and culture in a hypoxic environment at 37°C in an incubator containing 2% O2 and 5% CO2 to allow the transfected mesenchymal stem cells to fully release exosomes.
[0016] According to the first aspect of the invention, the DMEM-high glucose basal medium is supplemented with 12-20 µg / ml estrogen E2, 0.3-0.7 mg / ml inositol and 0.1-0.2 mg / ml zinc sulfate.
[0017] According to the first aspect of the invention, the DMEM-high glucose basal medium is supplemented with 16 µg / ml estrogen E2, 0.5 mg / ml inositol and 0.15 mg / ml zinc sulfate.
[0018] According to the first aspect of the invention, the DMEM-high glucose basal medium is supplemented with 12 µg / ml estrogen E2, 0.7 mg / ml inositol and 0.2 mg / ml zinc sulfate.
[0019] According to the first aspect of the invention, the DMEM-high glucose basal medium is supplemented with 20 µg / ml estrogen E2, 0.3 mg / ml inositol and 0.1 mg / ml zinc sulfate.
[0020] According to the first aspect of the invention, the DMEM-high glucose basal medium is supplemented with 16 µg / ml estrogen E2, 0.3 mg / ml inositol and 0.1 mg / ml zinc sulfate.
[0021] According to the first aspect of the invention, the DMEM-high glucose basal medium is supplemented with 17 µg / ml estrogen E2, 0.7 mg / ml inositol and 0.2 mg / ml zinc sulfate.
[0022] According to the first aspect of the invention, the culture is carried out for 48 hours under hypoxic conditions stimulating a 2% O2 environment.
[0023] According to the first aspect of the invention, step (3) of separating and purifying exosomes from the cell culture medium obtained in step (2) is carried out by differential centrifugation.
[0024] According to the first aspect of the invention, step (3) of separating and purifying exosomes from the cell culture medium obtained in step (2) is carried out as follows: The cell supernatant obtained in step (2) was placed in a centrifuge tube and centrifuged as follows: Centrifuge at 300g, 4℃ for 10 minutes to remove dead cells and larger cell debris, and transfer the supernatant to another centrifuge tube; Centrifuge at 2000g, 4℃ for 20 minutes to further remove cell debris and other impurities, and transfer the supernatant to another high-speed centrifuge tube; Centrifuge at 12000g, 4℃ for 30 minutes to further remove smaller cell debris and impurities. Filter the supernatant through a 0.22μm sterile filter and place it in another ultracentrifuge tube. Centrifuge at 100,000g, 4℃ for 90 minutes, and discard the supernatant; Add sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 100,000g and 4°C for 90 minutes, discard the supernatant, add sterile PBS to resuspend the exosome, and obtain the exosome suspension.
[0025] According to the first aspect of the invention, the MSC complete culture medium is a serum-free culture medium containing 2% human platelet lysate.
[0026] Furthermore, a second aspect of the present invention provides a mesenchymal stem cell-engineered exosome, which is prepared from mesenchymal stem cells transfected with miRNA mimics.
[0027] According to a second aspect of the present invention, the mesenchymal stem cell-engineered exosomes wherein the miRNA mimic is selected from any one or a combination of several of miR-100-5p, miR-99a-5p, let-7e-5p, miR-146a-5p, miR-29b-3p, miR-133b, miR-191-5p, miR-199b-3p, and miR-199a-3p.
[0028] According to a second aspect of the present invention, mesenchymal stem cell-engineered exosomes are prepared by a method comprising the following steps: (1) miRNA-Mimics transfection of mesenchymal stem cells; (2) Transfected mesenchymal stem cells were cultured under hypoxic conditions to induce them to secrete exosomes; (3) Separate and purify the exosomes from the cell culture medium obtained in step (2).
[0029] According to the second aspect of the present invention, mesenchymal stem cell-engineered exosomes, wherein miRNA-Mimics transfection of mesenchymal stem cells is performed as follows: i) The miRNA mimic was briefly centrifuged, and the precipitate was reconstituted with water without nuclease to obtain a miRNA mimic stock solution with a concentration of 20 μM. ii) Add 25 μL of transfection mixture A and 25 μL of transfection mixture B to each well of a 96-well plate, mix gently to obtain transfection mixture (A+B), and incubate at room temperature for 15-20 minutes. iii) Resuspend P3-8 generation human (umbilical cord) mesenchymal stem cells in MSC complete medium, seed them into culture flasks, supplement with MSC complete medium, and culture at 37°C and 5% CO2 until the cell confluence reaches more than 65%; iv) Remove the culture medium from the cell culture medium in step iii), wash with PBS, add 50 μL of serum-free culture medium to the cells, then add 50 μL of transfection mixture (A+B), mix well, and then incubate the cells at 37°C to complete the transfection.
[0030] According to a second aspect of the invention, mesenchymal stem cell-engineered exosomes are provided, wherein the transfection mixture A is a mixture of 24.75 μL of serum-free culture medium and 0.25 μL of miRNA mimic stock solution.
[0031] According to a second aspect of the present invention, mesenchymal stem cell-engineered exosomes are provided, wherein the transfection mixture B is a mixture of 24.5 μL of serum-free culture medium and 0.5 μL of siRNA / miRNA transfection reagent.
[0032] According to the second aspect of the present invention, mesenchymal stem cell engineered exosomes are prepared by adding 50 μL of transfection mixture (A+B) and mixing well, and then incubating the cells at 37°C for 48 h to complete the transfection.
[0033] According to the second aspect of the present invention, mesenchymal stem cell-engineered exosomes are used to culture transfected mesenchymal stem cells in the following manner: a) Take miRNA-Mimics transfected mesenchymal stem cells, digest them with recombinant trypsin, and then use 1×10 6 Cells were seeded into 10cm culture dishes, 8ml of mesenchymal stem cell complete culture medium was added, and the dishes were incubated at 37℃ in a 5% CO2 incubator to allow the cells to adhere to the culture dish. b) Continue culturing the cells at 37°C and 5% CO2 until the cell confluence is not less than 80%, and collect the culture supernatant; c) Wash three times with PBS, add 8 mL of FBS-free DMEM-high glucose basal medium, and culture in a hypoxic environment at 37°C in an incubator containing 2% O2 and 5% CO2 to allow the transfected mesenchymal stem cells to fully release exosomes.
[0034] According to the second aspect of the present invention, mesenchymal stem cell engineered exosomes are wherein the DMEM-high glucose basal medium is supplemented with 12-20 µg / ml estrogen E2, 0.3-0.7 mg / ml inositol and 0.1-0.2 mg / ml zinc sulfate.
[0035] According to a second aspect of the present invention, mesenchymal stem cell-engineered exosomes are wherein the DMEM-high glucose basal medium is supplemented with 16 µg / ml estrogen E2, 0.5 mg / ml inositol and 0.15 mg / ml zinc sulfate.
[0036] According to a second aspect of the present invention, mesenchymal stem cell-engineered exosomes are wherein the DMEM-high glucose basal medium is supplemented with 12 µg / ml estrogen E2, 0.7 mg / ml inositol and 0.2 mg / ml zinc sulfate.
[0037] According to a second aspect of the present invention, mesenchymal stem cell-engineered exosomes are wherein the DMEM-high glucose basal medium is supplemented with 20 µg / ml estrogen E2, 0.3 mg / ml inositol and 0.1 mg / ml zinc sulfate.
[0038] According to a second aspect of the present invention, mesenchymal stem cell-engineered exosomes are wherein the DMEM-high glucose basal medium is supplemented with 16 µg / ml estrogen E2, 0.3 mg / ml inositol and 0.1 mg / ml zinc sulfate.
[0039] According to a second aspect of the present invention, mesenchymal stem cell-engineered exosomes are wherein the DMEM-high glucose basal medium is supplemented with 17 µg / ml estrogen E2, 0.7 mg / ml inositol and 0.2 mg / ml zinc sulfate.
[0040] According to a second aspect of the invention, mesenchymal stem cell-engineered exosomes are cultured for 48 hours under hypoxic conditions stimulating a 2% O2 environment.
[0041] According to the second aspect of the present invention, the mesenchymal stem cell engineered exosomes, wherein step (3) involves separating and purifying the exosomes from the cell culture medium obtained in step (2) by differential centrifugation.
[0042] According to the second aspect of the present invention, mesenchymal stem cell-engineered exosomes, wherein step (3) of separating and purifying the exosomes from the cell culture medium obtained in step (2) is performed as follows: The cell supernatant obtained in step (2) was placed in a centrifuge tube and centrifuged as follows: Centrifuge at 300g, 4℃ for 10 minutes to remove dead cells and larger cell debris, and transfer the supernatant to another centrifuge tube; Centrifuge at 2000g, 4℃ for 20 minutes to further remove cell debris and other impurities, and transfer the supernatant to another high-speed centrifuge tube; Centrifuge at 12000g, 4℃ for 30 minutes to further remove smaller cell debris and impurities. Filter the supernatant through a 0.22μm sterile filter and place it in another ultracentrifuge tube. Centrifuge at 100,000g, 4℃ for 90 minutes, and discard the supernatant; Add sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 100,000g and 4°C for 90 minutes, discard the supernatant, add sterile PBS to resuspend the exosome, and obtain the exosome suspension.
[0043] According to a second aspect of the present invention, mesenchymal stem cell engineered exosomes are wherein the MSC complete culture medium is a serum-free culture medium containing 2% human platelet lysate.
[0044] Furthermore, a third aspect of the present invention provides a method for preparing engineered exosomes of mesenchymal stem cells, comprising the steps of culturing mesenchymal stem cells transfected with miRNA mimics under hypoxic stimulation, and then isolating and purifying them from the cell culture medium.
[0045] According to the method of the third aspect of the present invention, the miRNA mimic is selected from any one or a combination of several of miR-100-5p, miR-99a-5p, let-7e-5p, miR-146a-5p, miR-29b-3p, miR-133b, miR-191-5p, miR-199b-3p, and miR-199a-3p.
[0046] According to a third aspect of the present invention, the method comprises the following steps: (1) miRNA-Mimics transfection of mesenchymal stem cells; (2) Transfected mesenchymal stem cells were cultured under hypoxic conditions to induce them to secrete exosomes; (3) Separate and purify the exosomes from the cell culture medium obtained in step (2).
[0047] According to the method of the third aspect of the present invention, the transfection of mesenchymal stem cells with miRNA-Mimics is performed as follows: i) The miRNA mimic was briefly centrifuged, and the precipitate was reconstituted with water without nuclease to obtain a miRNA mimic stock solution with a concentration of 20 μM. ii) Add 25 μL of transfection mixture A and 25 μL of transfection mixture B to each well of a 96-well plate, mix gently to obtain transfection mixture (A+B), and incubate at room temperature for 15-20 minutes. iii) Resuspend P3-8 generation human (umbilical cord) mesenchymal stem cells in MSC complete medium, seed them into culture flasks, supplement with MSC complete medium, and culture at 37°C and 5% CO2 until the cell confluence reaches more than 65%; iv) Remove the culture medium from the cell culture medium in step iii), wash with PBS, add 50 μL of serum-free culture medium to the cells, then add 50 μL of transfection mixture (A+B), mix well, and then incubate the cells at 37°C to complete the transfection.
[0048] According to the method of a third aspect of the invention, the transfection mixture A is a mixture of 24.75 μL of serum-free culture medium and 0.25 μL of miRNA mimic stock solution.
[0049] According to the method of the third aspect of the present invention, the transfection mixture B is a mixture of 24.5 μL of serum-free culture medium and 0.5 μL of siRNA / miRNA transfection reagent.
[0050] According to the method of the third aspect of the present invention, 50 μL of transfection mixture (A+B) is added and mixed well, and the cells are incubated at 37°C for 48 h to complete the transfection.
[0051] According to the method of a third aspect of the present invention, the culture of transfected mesenchymal stem cells is performed in the following manner: a) Take miRNA-Mimics transfected mesenchymal stem cells, digest them with recombinant trypsin, and then use 1×10 6 Cells were seeded into 10cm culture dishes, 8ml of mesenchymal stem cell complete culture medium was added, and the dishes were incubated at 37℃ in a 5% CO2 incubator to allow the cells to adhere to the culture dish. b) Continue culturing the cells at 37°C and 5% CO2 until the cell confluence is not less than 80%, and collect the culture supernatant; c) Wash three times with PBS, add 8 mL of FBS-free DMEM-high glucose basal medium, and culture in a hypoxic environment at 37°C in an incubator containing 2% O2 and 5% CO2 to allow the transfected mesenchymal stem cells to fully release exosomes.
[0052] According to the method of the third aspect of the present invention, the DMEM-high glucose basal medium is supplemented with 12-20 µg / ml estrogen E2, 0.3-0.7 mg / ml inositol and 0.1-0.2 mg / ml zinc sulfate.
[0053] According to the method of the third aspect of the present invention, the DMEM-high glucose basal medium is supplemented with 16 µg / ml estrogen E2, 0.5 mg / ml inositol and 0.15 mg / ml zinc sulfate.
[0054] According to the method of the third aspect of the present invention, the DMEM-high glucose basal medium is supplemented with 12 µg / ml estrogen E2, 0.7 mg / ml inositol and 0.2 mg / ml zinc sulfate.
[0055] According to the method of the third aspect of the present invention, the DMEM-high glucose basal medium is supplemented with 20 µg / ml estrogen E2, 0.3 mg / ml inositol and 0.1 mg / ml zinc sulfate.
[0056] According to the method of the third aspect of the present invention, the DMEM-high glucose basal medium is supplemented with 16 µg / ml estrogen E2, 0.3 mg / ml inositol and 0.1 mg / ml zinc sulfate.
[0057] According to the method of the third aspect of the present invention, the DMEM-high glucose basal medium is supplemented with 17 µg / ml estrogen E2, 0.7 mg / ml inositol and 0.2 mg / ml zinc sulfate.
[0058] According to the method of the third aspect of the invention, the culture is carried out for 48 hours under hypoxic conditions stimulating the environment with 2% O2.
[0059] According to the method of the third aspect of the present invention, step (3) of separating and purifying exosomes from the cell culture medium obtained in step (2) is carried out by differential centrifugation.
[0060] According to the method of the third aspect of the present invention, step (3) of separating and purifying exosomes from the cell culture medium obtained in step (2) is performed as follows: The cell supernatant obtained in step (2) was placed in a centrifuge tube and centrifuged as follows: Centrifuge at 300g, 4℃ for 10 minutes to remove dead cells and larger cell debris, and transfer the supernatant to another centrifuge tube; Centrifuge at 2000g, 4℃ for 20 minutes to further remove cell debris and other impurities, and transfer the supernatant to another high-speed centrifuge tube; Centrifuge at 12000g, 4℃ for 30 minutes to further remove smaller cell debris and impurities. Filter the supernatant through a 0.22μm sterile filter and place it in another ultracentrifuge tube. Centrifuge at 100,000g, 4℃ for 90 minutes, and discard the supernatant; Add sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 100,000g and 4°C for 90 minutes, discard the supernatant, add sterile PBS to resuspend the exosome, and obtain the exosome suspension.
[0061] According to a third aspect of the invention, the MSC complete culture medium is a serum-free culture medium containing 2% human platelet lysate.
[0062] The exosomes prepared by this invention exhibit the excellent effects described herein. Attached Figure Description
[0063] Figure 1 Transmission electron microscopy image of mesenchymal stem cell-engineered exosomes (overexpressing miR-99a-5p).
[0064] Figure 2 Nanoparticle tracking analysis (NTA) was used to determine the particle size of mesenchymal stem cell-engineered exosomes (overexpressing miR-99a-5p).
[0065] Figure 3 Effects of engineered exosomes on liver function (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) in mice. The groups in the figure are: model group (G1), low-dose MSC-EXO group (G2), high-dose MSC-EXO group (G3), positive control group (G4), and blank control group (G5).
[0066] Figure 4 Effects of mesenchymal stem cell-engineered exosomes on mouse liver and brain. The groups in the figure are: model group (G1), low-dose MSC-EXO group (G2), high-dose MSC-EXO group (G3), positive control group (G4), and blank control group (G5).
[0067] Figure 5 Effect of mesenchymal stem cell exosomes on oil red staining of mouse liver. The groups in the figure are: model group (G1), low-dose MSC-EXO group (G2), high-dose MSC-EXO group (G3), positive control group (G4), and blank control group (G5).
[0068] Figure 6 Effects of mesenchymal stem cell-engineered exosomes on inflammatory factors in mice. The groups in the figure are: model group (G1), low-dose MSC-EXO group (G2), high-dose MSC-EXO group (G3), positive control group (G4), and blank control group (G5).
[0069] Figure 7 Effects of mesenchymal stem cell exosomes on mouse behavior. The groups in the figure are: model group (G1), low-dose MSC-EXO group (G2), high-dose MSC-EXO group (G3), positive control group (G4), and blank control group (G5).
[0070] Figure 8 Effects of mesenchymal stem cell exosomes on mouse brain galactosidase. The groups in the figure are: model group (G1), low-dose MSC-EXO group (G2), high-dose MSC-EXO group (G3), positive control group (G4), and blank control group (G5). Detailed Implementation
[0071] The invention can be further described through the following embodiments; however, the scope of the invention is not limited to the embodiments described below. Those skilled in the art will understand that various changes and modifications can be made to the invention without departing from its spirit and scope. The invention provides a general and / or specific description of the materials and methods used in the experiments. Although many materials and methods of operation used to achieve the objectives of the invention are well known in the art, the invention is still described herein in as much detail as possible.
[0072] Unless otherwise stated, some of the reagents used in this invention are conventional in the art or readily available from commercial sources. For example, serum-free culture medium (Gibco) was purchased from Thermo Fisher Scientific; human platelet lysate was purchased from Precicion BioMedicals; phosphate-buffered saline (PBS) was prepared by taking 250 ml of 0.2 mol / L potassium dihydrogen phosphate solution, adding 118 ml of 0.2 mol / L sodium hydroxide solution, diluting with water to 1000 ml, shaking well, and sterilizing at 121°C for 15 min; the MSC complete culture medium described herein, i.e., mesenchymal stem cell complete culture medium, is a serum-free culture medium containing 2% human platelet lysate. Recombinant trypsin was purchased from Merck (EMS0006) and used according to the instructions for use.
[0073] Example 1: miRNA-mimics transfection of MSC cells Chemically synthesized miRNA mimics are double-stranded RNA molecules that mimic endogenous miRNAs after transfection into cells. In this example, a commercially available synthetic miRNA mimic, miR-99a-5p mimic (purchased from MedChemExpres, USA, catalog number HY-R02554), was used to transfect MSC cells.
[0074] i) Briefly centrifuge the miRNA mimic to ensure that the miRNA is at the bottom of the test tube, discard the supernatant, and reconstitute the precipitate with nuclease-free water to obtain a stock solution with a miRNA concentration of 20 μM. If necessary, aliquot the stock solution into one or more tubes to reduce the number of freeze-thaw cycles and store it at -20°C or -80°C for later use. ii) Preparation of transfection mixture: In a 96-well plate, add 25 μL of transfection mixture A (24.75 μL serum-free medium + 0.25 μL miRNA mimic stock solution) and 25 μL of transfection mixture B (24.5 μL serum-free medium + 0.5 μL siRNA / miRNA transfection reagent (purchased from MedChemExpres, USA, catalog number HY-K2017)) to each well, mix gently to obtain transfection mixture (A+B), and incubate at room temperature for 15-20 minutes; iii) Resuspend P3-8 generation human (umbilical cord) mesenchymal stem cells in MSC complete medium, seed them into culture flasks, supplement with MSC complete medium, and culture at 37°C and 5% CO2 until the cell confluence reaches more than 65%; iv) Remove the culture medium from the cell culture medium in step iii), wash with PBS, add 50 μL of serum-free culture medium to the cells, then add 50 μL of transfection mixture (A+B), mix well, and then incubate the cells at 37°C for 48 h to complete the transfection.
[0075] Example 2: Culture of MSC cells a) Mesenchymal stem cells transfected with the miRNA-Mimics obtained in Example 1 for 48 hours were enzymatically digested with recombinant trypsin (Merck, EMS0006), and then 1×10 6 Cells were seeded into 10cm culture dishes, 8ml of mesenchymal stem cell complete culture medium was added, and the dishes were incubated at 37℃ in a 5% CO2 incubator to allow the cells to adhere to the culture dish. b) Continue culturing the cells at 37°C and 5% CO2 until the cell confluence is not less than 80%, and collect the culture supernatant; c) Wash three times with PBS, add 8 mL of FBS-free DMEM-high glucose basal medium (Sigma-Aldrich, catalog number D7596), and incubate at 37°C in an anaerobic environment containing 2% O2 and 5% CO2 for 48 hours to allow the transfected mesenchymal stem cells to fully release exosomes. The DMEM-high glucose basal medium was supplemented with 16 µg / ml estrogen E2, 0.5 mg / ml inositol, and 0.15 mg / ml zinc sulfate.
[0076] Example 3: Purification of exosomes The cell culture medium obtained in Example 2 was placed in a centrifuge tube (10 ml, the same below) and centrifuged as follows: Centrifuge at 300g, 4℃ for 10 minutes to remove dead cells and larger cell debris, and transfer the supernatant to another centrifuge tube; Centrifuge at 2000g, 4℃ for 20 minutes to further remove cell debris and other impurities, and transfer the supernatant to another high-speed centrifuge tube; Centrifuge at 12000g, 4℃ for 30 minutes to further remove smaller cell debris and impurities. Filter the supernatant through a 0.22μm sterile filter (Millipore) and place it in another ultracentrifuge tube. Centrifuge at 100,000g, 4℃ for 90 minutes, and discard the supernatant; Add 8 ml of sterile PBS to the centrifuge tube to resuspend the exosome precipitate. Centrifuge at 100,000 g and 4 °C for 90 minutes. Discard the supernatant and add 1 ml of sterile PBS to resuspend the exosome, obtaining 1 ml of exosome suspension.
[0077] The exosome suspension can be stored at -80°C and / or aliquoted for performance testing. Preliminary measurements show that the purified engineered exosomes have a diameter of 50-200 nm and a vesicle purity >95%.
[0078] Example 4: Detection of exosome protein content The determination of exosomal protein content was performed using the Pierce™ Protein Quantification Kit (catalog number: 23225, Thermo Scientific). The specific procedures are as follows: Take 10 µl of 5 mg / mL BCA (2,2-biquinoline-4,4-dicarboxylate disodium salt) standard, dilute with PBS to a final concentration of 0.5 mg / mL, and use this as the BCA standard solution. Add 0, 2, 4, 6, 8, 12, 16, and 20 µL of this standard solution to the protein standard wells of a 96-well plate, and add PBS to bring the total to 20 µL. Dilute the exosome sample appropriately and add 20 µL to the sample wells of the 96-well plate. Since the pipette has a large error when taking small amounts of sample, the points before the standard line may not be very accurate, so try to make the sample concentration point fall within 1 / 2 of the standard line. Add 200 µL of BCA working solution to each well, incubate at 37°C for 15-30 minutes, and measure the OD value at A562 nm using a microplate reader. Calculate the protein concentration based on the standard curve.
[0079] The exosomes (suspension) obtained in Example 3 of the present invention were tested using the above method, and the exosome protein content obtained from 1×10^6 cells was calculated, with a result of 358.4µg.
[0080] In some exploratory experiments, the inventors discovered that during the culture process of stimulating cells to produce exosomes in a hypoxic environment as described in step c) of Example 2, it is necessary to supplement the DMEM-high glucose basal medium with estrogen E2, as well as simultaneously supplement with inositol and zinc sulfate. Otherwise, the protein content of the exosomes obtained in Example 3 is significantly reduced, which was completely unexpected. Specific details are shown in the following supplementary examples. Supplementary Example 231: Refer to Examples 2 and 3, except that inositol is not added in step c) of Example 2, and an exosome suspension is finally obtained; Supplementary Example 232: Refer to Examples 2 and 3, except that zinc sulfate is not added in step c) of Example 2, and an exosome suspension is finally obtained; Supplementary Example 233: Refer to Examples 2 and 3, except that neither inositol nor zinc sulfate is added in step c) of Example 2, and an exosome suspension is finally obtained; Supplementary Example 234: Refer to Examples 2 and 3, except that 12 µg / ml estrogen E2, 0.7 mg / ml inositol, and 0.2 mg / ml zinc sulfate are added to all DMEM-high glucose basal media in step c) of Example 2; Supplementary Example 235: Refer to Examples 2 and 3 differ only in that step c) of Example 2 is supplemented with 20 µg / ml estrogen E2, 0.3 mg / ml inositol, and 0.1 mg / ml zinc sulfate. Supplementary Example 236: Refer to Examples 2 and 3, except that step c) of Example 2 is supplemented with 16 µg / ml estrogen E2, 0.3 mg / ml inositol, and 0.1 mg / ml zinc sulfate. Supplementary Example 237: Refer to Examples 2 and 3, except that step c) of Example 2 is supplemented with 17 µg / ml estrogen E2, 0.7 mg / ml inositol, and 0.2 mg / ml zinc sulfate. The protein content in the exosomes obtained in Supplement Examples 231-237 was determined using the method of Example 4, and the results were 183.6 µg, 156.4 µg, 112.7 µg, 327.6 µg, 382.1 µg, 367.6 µg, and 342.7 µg, respectively. These results demonstrate the benefit of simultaneously supplementing the culture medium with inositol and zinc sulfate when culturing cells under hypoxic conditions.
[0081] Example 5: Characterization of dual-targeted engineered exosomes of mesenchymal stem cells This embodiment uses the exosomes obtained by the method in Example 3 for performance characterization.
[0082] 5.1) Morphological identification of dual-target engineered exosomes using transmission electron microscopy The exosomes extracted in Example 3 were observed using transmission electron microscopy. The specific procedure was as follows: 1) Take 50µl of the separated and purified exosome precipitate, add an equal volume of 2.5% glutaraldehyde, and fix it in a 4℃ refrigerator for 1 hour; 2) Add 20 µl of the fixed exosome suspension to the front side of the copper mesh and let it stand for 20 min; 3) Carefully absorb the excess solution with absorbent filter paper; then rinse the copper mesh with ultrapure water 5 times, 30 seconds each time, and blot dry with filter paper. 4) Add 1 drop of 2% uranium acetate staining solution to the front of the copper mesh, stain for 1 minute, and then use filter paper to blot away the excess staining solution along the edge of the copper mesh. 5) Place the copper mesh in room temperature air to dry naturally in the dark. After it is dry, observe it on the machine and calculate and count the particle size and distribution of exosomes.
[0083] 6) Finally, exosomes were observed using transmission electron microscopy.
[0084] Typical observation results are as follows Figure 1 As shown.
[0085] 5.2) Particle size and exosome concentration were determined by nanoparticle tracking analysis (NTA). The particle size and concentration of exosomes were determined using a nanoparticle tracking and analysis system (NTA, NanoSight Pro). A typical result is as follows: Figure 2 As shown, the results of the exosomes showed that the particle size was mainly distributed in the range of 60~280nm.
[0086] Example 6: Transplantation of engineered exosomes into aging mice to treat metabolic and cognitive dysfunction. This experiment used exosomes obtained by the method in Example 3 to investigate whether mesenchymal stem cell-derived engineered exosomes (MSC-EXO) have a slowing effect on the weight, general condition, inflammation, liver function and metabolic function, neurocognitive function and overall aging process of rapidly aging mice. Through in vivo pharmacodynamic studies, the effects of dual-target engineered exosomes on metabolic dysfunction and Alzheimer's disease were clarified.
[0087] 6.1) Establishment and grouping of aging mouse models Twenty-four male SAMP8 mice and six SAMR1 mice, aged 6-8 weeks and classified as SPF, were purchased. All mice underwent 7 days of acclimatization. All mice were then randomly divided into five groups: model group (G1), low-dose MSC-EXO group (G2), high-dose MSC-EXO group (G3), positive control group (G4), and blank control group (G5).
[0088] 6.2) Engineered exosome delivery Route of administration: The drug was administered via tail vein injection in mice, mimicking the clinical administration route. Administration volume: 0.3 ml / mouse.
[0089] Treatment: Group G1 was given sodium chloride injection, Group G2 was given MSC-EXO (2.5×10e8 / mouse), Group G3 was given MSC-EXO (1.5×10e9 / mouse), Group G4 was given metformin, and Group G5 was given sodium chloride injection. All treatments were administered via tail vein injection once a week for 16 weeks. Mice were euthanized 24 hours after the last exosome administration.
[0090] 6.3) Mouse body weight and general condition Weight: Mice were weighed and recorded weekly during the first month and bi-weekly thereafter, and weight changes were plotted. Results: Throughout the experimental period, the weight of the mice in the experimental group remained stable. The engineered exosomes exerted their therapeutic effect without causing weight loss due to adverse drug reactions.
[0091] General condition: Observe physical appearance, hair, behavior, mental state, respiratory status, fecal characteristics, and drug administration site. No abnormalities were observed in the general condition of the mice throughout the experimental period. Back skin: New hair growth was slow in the model group mice. In other groups, the hair growth was normal, with continuous renewal and vigorous growth and a good appearance.
[0092] 6.4) Complete blood count After the experiment, blood was collected from the mice for routine blood tests. The results showed that all blood count indicators, including white blood cell count, red blood cell count, hemoglobin content, and platelet count, were within the normal reference range.
[0093] 6.5) Serum alanine aminotransferase (ALT) / aspartate aminotransferase (AST) measurement Blood was collected from mice immediately after euthanasia and before liver separation, and stored at room temperature for 4 hours. Serum was separated by centrifugation (2500 g, 10 min). Serum ALT and AST levels were detected using either an aspartate aminotransferase (AST) kit or an alanine aminotransferase (ALT) kit. 10 μL of serum diluted in PBS was mixed with the substrate at 37°C for 30 min, followed by the addition of kit reagents at 37°C for 20 min. Measurements were taken at 505 nm using a microplate reader. Results are as follows: Figure 3 As shown.
[0094] The results showed that, compared with the G1 model group, the serum ALT / AST index of the engineered exosome group was significantly reduced, and the exosomes could significantly reduce the abnormally elevated liver and kidney function-related biochemical indicators in the model group mice, thus playing a protective role.
[0095] 6.6) H&E staining After the experiment, samples of liver, spleen, brain, and other tissues and organs were collected, paraffin sections were prepared, and hematoxylin and eosin (HE) staining was performed. Mouse liver sections were deparaffinized in xylene and hydrated via an ethanol gradient. After rehydration in distilled water, the sections were immersed in hematoxylin for 1 minute, rinsed in tap water, and then fractionated in Scott's tap water for 3 minutes. After rinsing in tap water, these sites were stained with Eosin-γ for 3 minutes and dehydrated with absolute ethanol. Results are as follows: Figure 4 As shown.
[0096] The results showed that HE staining revealed normal cell morphology and structure in mouse liver and brain tissues, with no pathological damage such as cell edema, fatty degeneration, necrosis, inflammatory cell infiltration, congestion, hemorrhage, abnormal cell proliferation or apoptosis, neuronal degeneration, glial cell proliferation, or vascular lesions. Engineered exosomes improved the pathological damage in mouse liver, brain, and other tissues and organs.
[0097] 6.7) Oil Red O staining The study examined the extent of fat accumulation in the liver. Frozen liver sections were fixed, stained, and differentiated. They were then washed with 60% isopropanol, counterstained with hematoxylin, and finally observed under a microscope to determine the degree of fat accumulation by observing the oil red O staining of lipid droplets within the liver cells. Results are as follows: Figure 5 As shown.
[0098] The results showed that there was no significant lipid droplet aggregation in any group, indicating that there were no significant hepatic fat accumulation lesions in any group.
[0099] 6.8) Detection of inflammatory factors After the entire drug administration process was completed, blood samples were collected from the mice, and ELISA was used to detect interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), phosphorylated histone H2AX (H2AX), and immunoglobulin G (IgG). Results are as follows: Figure 6 As shown, the results fully demonstrate that engineered exosomes can significantly suppress inflammation in a dose-dependent manner.
[0100] ELISA results showed that, compared with the control group, the two key inflammatory markers (IL-6 and TNF-α) in the G1 model group were significantly increased, and the differences were statistically significant (P < 0.05). This indicates a significant increase in inflammation levels in aging mice. In contrast to the G1 model group, both the low-dose and high-dose exosome groups showed significant decreases in IL-6 and TNF-α, with the high-dose group showing a more pronounced reduction (P < 0.05).
[0101] 6.9) New Object Recognition Experiment Measurement methods: A mouse behavioral observation box was used for testing. VisuTrack was used to record and analyze data such as exploration time, number of explorations, and approach distance to assess cognitive flexibility, memory retention, and attention. Measurement frequency: Measurements were performed once after the last administration. Results are as follows: Figure 7 As shown.
[0102] Results: Behavioral observation tests in mice revealed significant increases in cognitive indices in both the low-dose and high-dose exosome groups compared to the G1 model group (P < 0.05). This demonstrates that exosome intervention enhances cognitive flexibility, memory retention, and attention in mice, providing important clues for further investigation into the mechanisms by which exosomes regulate the nervous system and improve cognitive function.
[0103] 6.10) Staining with SA-β-gal, a marker of aging Senescence-associated beta-galactosidase (SA-β-gal) is widely recognized as a classic and reliable biomarker of cellular senescence. It is an enzyme that accumulates in the lysosomes of senescent cells and hydrolyzes β-galactoside substrates under specific conditions, allowing it to be detected through colorimetric or fluorescent reactions. SA-β-gal, along with molecules such as p16, constitutes an important tool for assessing cellular senescence status in vivo and in vitro.
[0104] This invention uses a method described in the literature (Florence Debacq-Chainiaux, et al. Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nature protocols. 2009:191) to detect the content of the aging marker SA-β-gal in brain tissue. The results are as follows... Figure 8 As shown, staining results were significantly improved in the brains of mice treated with exosomes. Exosomes have an inhibitory effect on the accumulation of senescent cells.
[0105] Overall, the present invention has demonstrated through experiments that the engineered exosomes prepared by the present invention have significant and excellent effects in regulating inflammation and improving multiple important tissues and organs such as the liver and brain, showing good prospects for industrial application.
[0106] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.
Claims
1. Use of mesenchymal stem cell-engineered exosomes in the preparation of drugs for the treatment of fatty liver disease and cognitive impairment associated with metabolic dysfunction.
2. The use according to claim 1, wherein the mesenchymal stem cell-engineered exosomes are prepared from mesenchymal stem cells transfected with miRNA mimics.
3. According to the use of claim 1, wherein the miRNA mimic is selected from any one or a combination of several of miR-100-5p, miR-99a-5p, let-7e-5p, miR-146a-5p, miR-29b-3p, miR-133b, miR-191-5p, miR-199b-3p, and miR-199a-3p.
4. The use according to claim 1, wherein the mesenchymal stem cell-engineered exosomes are prepared by a method comprising the following steps: (1) miRNA-Mimics transfection of mesenchymal stem cells; (2) Transfected mesenchymal stem cells were cultured under hypoxic conditions to induce them to secrete exosomes; (3) Separate and purify the exosomes from the cell culture medium obtained in step (2).
5. The use according to claim 1, wherein the miRNA-Mimics transfection of mesenchymal stem cells is performed as follows: i) The miRNA mimic was briefly centrifuged, and the precipitate was reconstituted with water without nuclease to obtain a miRNA mimic stock solution with a concentration of 20 μM. ii) Add 25 μL of transfection mixture A and 25 μL of transfection mixture B to each well of a 96-well plate, mix gently to obtain transfection mixture (A+B), and incubate at room temperature for 15-20 minutes. iii) Resuspend P3-8 generation human (umbilical cord) mesenchymal stem cells in MSC complete medium, seed them into culture flasks, supplement with MSC complete medium, and culture at 37°C and 5% CO2 until the cell confluence reaches more than 65%; iv) Remove the culture medium from the cell culture medium in step iii), wash with PBS, add 50 μL of serum-free culture medium to the cells, then add 50 μL of transfection mixture (A+B), mix well, and then incubate the cells at 37°C to complete the transfection.
6. The use according to claim 1, wherein the culture of the transfected mesenchymal stem cells is performed in the following manner: a) Take the mesenchymal stem cells transfected with miRNA-Mimics, digest them with recombinant trypsin, then seed 1×106 cells into a 10cm culture dish, add 8ml of complete mesenchymal stem cell culture medium, and incubate at 37℃ and 5% CO2 to allow the cells to adhere to the culture dish. b) Continue culturing the cells at 37°C and 5% CO2 until the cell confluence is not less than 80%, and collect the culture supernatant; c) Wash three times with PBS, add 8 mL of FBS-free DMEM-high glucose basal medium, and culture in a hypoxic environment at 37°C in an incubator containing 2% O2 and 5% CO2 to allow the transfected mesenchymal stem cells to fully release exosomes.
7. The use according to claim 1, wherein step (3) of separating and purifying exosomes from the cell culture medium obtained in step (2) is performed by differential centrifugation; for example, step (3) of separating and purifying exosomes from the cell culture medium obtained in step (2) is performed as follows: The cell supernatant obtained in step (2) was placed in a centrifuge tube and centrifuged as follows: Centrifuge at 300g, 4℃ for 10 minutes to remove dead cells and larger cell debris, and transfer the supernatant to another centrifuge tube; Centrifuge at 2000g, 4℃ for 20 minutes to further remove cell debris and other impurities, and transfer the supernatant to another high-speed centrifuge tube; Centrifuge at 12000g, 4℃ for 30 minutes to further remove smaller cell debris and impurities. Filter the supernatant through a 0.22μm sterile filter and place it in another ultracentrifuge tube. Centrifuge at 100,000g, 4℃ for 90 minutes, and discard the supernatant; Add sterile PBS to the centrifuge tube to resuspend the exosome precipitate, centrifuge at 100,000g and 4°C for 90 minutes, discard the supernatant, add sterile PBS to resuspend the exosome, and obtain the exosome suspension.
8. A mesenchymal stem cell-engineered exosome, prepared from mesenchymal stem cells transfected with a miRNA mimic; for example, the miRNA mimic is selected from any one or a combination of miR-100-5p, miR-99a-5p, let-7e-5p, miR-146a-5p, miR-29b-3p, miR-133b, miR-191-5p, miR-199b-3p, and miR-199a-3p.
9. The mesenchymal stem cell-engineered exosomes according to claim 8, wherein the mesenchymal stem cell-engineered exosomes are prepared by a method comprising the following steps: (1) miRNA-Mimics transfection of mesenchymal stem cells; (2) Transfected mesenchymal stem cells were cultured under hypoxic conditions to induce them to secrete exosomes; (3) Separate and purify the exosomes from the cell culture medium obtained in step (2).
10. A method for preparing engineered exosomes of mesenchymal stem cells, comprising the steps of culturing mesenchymal stem cells transfected with miRNA mimics under hypoxic stimulation, and then isolating and purifying them from the cell culture medium; for example, the miRNA mimics are selected from any one or a combination of miR-100-5p, miR-99a-5p, let-7e-5p, miR-146a-5p, miR-29b-3p, miR-133b, miR-191-5p, miR-199b-3p, and miR-199a-3p; for example, the method includes the following steps: (1) miRNA-Mimics transfection of mesenchymal stem cells; (2) Transfected mesenchymal stem cells were cultured under hypoxic conditions to induce them to secrete exosomes; (3) Separate and purify the exosomes from the cell culture medium obtained in step (2).