Stem cell exosome preparation absorbed through nose and preparation method thereof

Abstract

The invention provides a nasal-absorbed stem cell exosome preparation and a preparation method thereof, the nasal-absorbed stem cell exosome preparation comprises an exosome and a matrix solution, and the matrix solution comprises 0.9% of sodium chloride injection, 5-10% of human serum albumin, 10-20% of dextran 40 glucose injection, 20-30% of glycerin fructose sodium chloride injection, 20-50 [mu] g / ml of ganglioside sodium and 4-8 [mu] g / ml of edaravone injection. The stem cell exosome preparation prepared from the stem cell exosome and the matrix solution can be preserved at-20 DEG C for 12 months, and can be preserved at 2-8 DEG C for 15 days after thawing, the number of the exosomes, surface protein expression and the form of vesicles can be well maintained, and the stem cell exosome preparation is clear and safe in component. Exosome administration is carried out through nasal mucosa to enter brain tissue parts, endogenous neural stem cells of organisms are activated and nourished, a regeneration microenvironment is established, nerve injury repair is promoted, and the exosome is used for preventing, treating and improving cognitive impairment and traumatic brain injury of senile dementia. The preparation is simple and convenient to prepare, small in toxic and side effects and convenient to dose.

Description

Technical Field

[0001] The present invention relates to a stem cell exosome preparation for nasal absorption and a preparation method thereof, and belongs to the technical field of biomedicine. Background Art

[0002] Alzheimer's disease (AD) is a common neurodegenerative disorder that places a heavy mental and economic burden on patients, their families, and society. The etiology of the disease is complex, and its pathogenesis remains unclear. Hypotheses include abnormal deposition of β-amyloid protein, abnormal tau protein phosphorylation, neuroinflammation, metabolic reprogramming of APOE, and vascular disease. Due to these factors, treatment options are relatively limited, making it considered a "blind spot" in modern medicine. Statistics indicate that in 2020, there were approximately 50 million AD patients worldwide, and the number is expected to reach 152 million by 2050. However, there is still no effective cure. Currently, only five drugs are approved for the treatment of the disease, and these drugs only provide short-term symptom control, failing to target the clear etiology and slow disease progression. After diagnosis, the majority of patients receive medication, but nearly half of those surveyed report being dissatisfied with the effectiveness of existing treatments, highlighting the limitations of current treatment approaches. In recent years, with the rise of regenerative medicine, stem cell and extracellular vesicle (EV)-derived therapies have brought new hope to the treatment of AD. Stem cells and exosomes exert their therapeutic effects on AD through neuroprotection, anti-inflammatory, immunomodulatory, and tissue repair. For example, mesenchymal stem cell exosomes contain a variety of anti-inflammatory and neurotrophic factors, which can improve cognitive function in AD model animals. Furthermore, mesenchymal stem cell exosomes possess neuroprotective properties, mitigating the effects of dementia-related neuroinflammation and cognitive decline, opening up new avenues for AD treatment.

[0003] Maintaining the integrity, bioactivity, and stability of exosomes over a long period of time remains a major challenge in clinical exosome therapy. Cryoprotectants, such as culture medium, bovine serum, and trehalose, are often added to preserve exosome activity. However, exosome activity decreases dramatically within three months of cryopreservation in culture medium. Exosomes stored in bovine serum also harbor potential pathogenic factors, leading to batch-to-batch instability. Lyophilized formulations designed for exosomes have also been developed. While these agents can maintain exosome activity to a certain extent, their complex composition and cumbersome preparation hinder the quality control of exosome preparations. Currently, exosomes are stable for only three days at 2-8°C and one month at -20°C. Long-term storage requires temperatures as low as -80°C and requires no freeze-thaw cycles, resulting in demanding storage conditions that hinder clinical research. Therefore, developing a formulation with a defined composition, safe for clinical use, and effective long-term maintenance of exosome integrity, bioactivity, and stability has become a pressing clinical challenge. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide an exosome preparation that can maintain the number of exosomes, surface protein expression and morphological integrity of vesicles, uniformity, prevent exosome aggregation, and has clear ingredients and safety.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: a stem cell exosome preparation for nasal absorption, comprising exosomes and a matrix solution, wherein the matrix solution comprises 0.9% sodium chloride injection, 5-10% human serum albumin (V / V), 10-20% dextran 40 glucose injection (V / V), 20-30% glycerol fructose sodium chloride injection (V / V), 20-50 ug / ml ganglioside sodium, and 4-8 ug / ml edaravone injection; The preparation method of stem cell exosomes is as follows: the supernatant of mesenchymal stem cells cultured in serum-free medium under hypoxic conditions on P3-P10 surrogate 3D microcarriers is collected; after filtering through 0.45 um filter membrane and 0.22 um filter membrane successively, the supernatant is passed through a nano-ultrafiltration chip; the free nucleotides and protein impurities in the supernatant are removed using a negative pressure oscillation and a dual-coupled ultrasonic oscillation system, and the 30-200 nm exosome vesicles are intercepted and purified.

[0006] Furthermore, the concentration of the exosomes in the preparation is 1-10×10 9 / ml, the exosome preparation was stored at -20℃ for 12 months and stored at 2-8℃ for 15 days after thawing, and the number of exosomes, surface protein expression and vesicle morphology were well maintained.

[0007] Furthermore, the optimal concentration of the exosome preparation is: 7.5% human serum albumin (V / V), 15% dextran 40 glucose injection (V / V), 25% glycerol fructose sodium chloride injection (V / V), 35 ug / ml ganglioside sodium, 6 ug / ml edaravone injection, and the exosome concentration is 5×10 9 / ml.

[0008] Furthermore, the exosomes can be obtained from the culture supernatant of mesenchymal stem cells separated from tissues such as umbilical cord, placenta, fat, amniotic membrane, dental pulp, and endometrium.

[0009] Furthermore, the exosomes have a diameter of 30-200 nm, and protein detection results show that the exosome markers CD9, CD63 and Tsg101 are positively expressed, the negative markers GM130 or Calnexin are not detected, and the ratio of the number of exosome particles to the amount of protein is higher than 1×10 9particles / μg protein, with saucer-shaped or cup-shaped structures with clear membrane structures and clear edges under the electron microscope.

[0010] Furthermore, a method for preparing a stem cell exosome preparation for nasal absorption comprises the following steps: Step 1: Preparation of the matrix solution: 5-10 ml human serum albumin (V / V), 10-20 ml dextran 40 glucose injection (V / V), 20-30 ml glycerol fructose sodium chloride injection (V / V), 40-100 ul ganglioside sodium 100 mg / 2 ml, 267-533 ul edaravone injection 15 mg / 10 ml; Step 2: Add 0.9% sodium chloride injection to 100 ml, shake thoroughly on a micro-oscillator at 180 rpm for 30 seconds to 1 minute to mix, and store at 2-8°C. Step 3: Add exosomes to the prepared matrix solution to make the exosome concentration in the preparation 1-10×10 9 / ml.

[0011] Beneficial effects of the present invention: Compared with existing technologies, the present invention effectively extracts mesenchymal stem cell culture supernatant through a nanofiltration chip. Using a negative pressure oscillation and dual-coupled ultrasonic oscillation system, free nucleotides and protein impurities are removed from the supernatant, trapping and purifying vesicles sized 30-200 nm, yielding a large number of highly pure exosomes. The exosome preparation, prepared from exosomes and a matrix solution, maintains exosome quantity, surface protein expression, and vesicle morphological integrity, ensuring uniformity, preventing exosome aggregation, and maintaining a clear composition and safety profile. The preparation maintains good exosome quantity, surface protein expression, and vesicle morphology when stored at -20°C for 12 months or at 2-8°C for 15 days after thawing. Exosomes are administered through the nasal mucosa into brain tissue, activating and nourishing endogenous neural stem cells, establishing a regenerative microenvironment, and promoting neural repair. It is used to prevent and improve cognitive impairment associated with Alzheimer's disease and traumatic brain injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings: Figure 1 This is a fluorescence staining image of the cells of the present invention growing on microcarriers; Figure 2 This is the particle size distribution diagram of the exosomes of the present invention; Figure 3 This is a diagram of the exosome membrane structure of the present invention; Figure 4This is a graph showing positive expression of the exosome surface markers CD9, CD63 and the internal marker Tsg101; Figure 5 The exosome particle size distribution diagram of the exosome preparation of the present invention after 1, 4, 8, and 12 months of storage; Figure 6 These are the morphological diagrams of exosomes stored in the exosome preparation of the present invention for 1, 4, 8, and 12 months. DETAILED DESCRIPTION

[0013] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below in conjunction with specific implementation methods.

[0014] Example 1: Mesenchymal stem cell culture. After thawing the frozen P3 cells in the working bank in a 37±2°C water bath, each tube of cells was added to a 50 ml centrifuge tube containing 10 ml of normal saline and centrifuged at 1400 rpm for 5-8 min. The cells at the bottom were collected and serum-free medium (DMEM / F12 + 10% serum replacement + 20 ng / ml PDGF-BB + 10 ng / ml EGF + 1×NEAA) was added, and the cells were resuspended and counted. Serum-free medium was added to the spinner flask, and fully swollen gelatin microcarriers were added at a ratio of 1.3-1.5 mg / ml. The cells were plated at a density of 0.3-0.5×10 5 The cells were inoculated at a density of 100 μg / ml into a bioreactor containing fully swollen microcarriers. The bioreactor process control parameters were set, including temperature at 37°C, pH 7.2-7.4, and dissolved oxygen concentration of 2-5%. On Day 0, intermittent stirring was used: stirring at 35-45 rpm for 5 minutes, then at 0 rpm for 25 minutes, and amplification culture was repeated for at least 48 times. On Day 1, the cells were adjusted to a constant speed of 40 rpm. When the cells grew to 80-90% confluence on the microcarriers, samples were taken for AMPI staining and photographed under a fluorescence microscope. The results were as follows: Figure 1 Green fluorescently stained stem cells were grown on the carrier. Cell supernatant was collected for exosome extraction.

[0015] Enzymatic cell dissociation and harvesting: Once cells have grown to a sufficient number, remove the spinner flask, stop stirring, and let it stand for approximately 2-5 minutes. After the microcarriers have settled, aspirate approximately 80% of the supernatant from the culture medium. Add 5× lysis buffer at a 1:5 mass ratio of lysis enzyme to microcarriers, and adjust the volume to approximately 40% of the original culture volume with physiological saline, so that the lysis enzyme concentration is approximately 1 mg / ml. Lyse the cells at 37°C for 30-40 minutes. Once the microcarriers are fully lysed, collect the cell suspension. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, wash with physiological saline, resuspend by centrifugation 2-3 times, and count the cells. Serial passage or cryopreservation is performed, and cell samples are collected for phenotypic analysis. The results are shown in Table 1. Flow cytometry analysis shows that the positive rates for CD73, CD90, and CD105 are greater than 95%, while those for CD34 and HLA-DR are less than 2%. This indicates that the cells obtained are mesenchymal stem cells.

[0016] Lysis enzyme preparation (5×): Take 0.5 g of lysase and add 100 ml of normal saline to prepare 5 mg / ml 5× lysis solution. After filtering through a 0.22 μm filter, aliquot and store at -20°C.

[0017] Table 1 shows the phenotypic detection of 3D cultured cells; Example 2: Exosome extraction and identification. The culture supernatant of human umbilical cord mesenchymal stem cells cultured in serum-free medium from generations P3 to P10 was collected and filtered continuously through a 0.45 μm filter membrane and a 0.22 μm filter membrane. The supernatant was then passed through a nano-ultrafiltration chip. Free nucleotides and protein impurities in the supernatant were removed using a negative pressure oscillation and a dual-coupled ultrasonic oscillation system. Vesicles of 30-200 nm were trapped and purified. The trapped vesicles were eluted with a matrix solution and frozen or detected.

[0018] NTA identification: Take the exosome stock solution, dilute it 1000 times and use the nanoparticle tracking analyzer to detect the particle size. The results show that Figure 2 The diameter of exosomes is 30-150 nm, the peak particle size is at 76 nm, and the concentration of original exosomes is 1.87E+11+ / -1.04E+08 / ml.

[0019] Transmission electron microscopy: Fix the sample with 2.5% glutaraldehyde solution at 4°C overnight, take 5-10 ul of sample and drop it onto a copper grid, let it stand at room temperature for 5 minutes, and absorb the liquid with filter paper. Add staining solution (saturated uranyl acetate solution) to the copper grid and stain for 1 minute, and absorb the liquid with filter paper. Add ddH2O to the copper grid and let it stand at room temperature for 5 minutes, and absorb the liquid with filter paper; repeat once. Dry at room temperature, observe and photograph on the microscope. The results show that exosomes have a clear membrane structure, a saucer-shaped or cup-shaped structure, and clear edges, such as Figure 3 .

[0020] Example 3: Western blot experiment. After the protein sample concentration was tested, the total protein concentration was 143.8 ug / ml. 20 μg of protein was taken from each sample (an equal amount of culture supernatant was used as a control) and added to 5× SDS-PAGE Loading Buffer, mixed evenly, and heated at 95°C for 5 minutes. Electrophoresis was performed, followed by transfer to a membrane, blocking, incubation with primary and secondary antibodies, and imaging using ELC luminescent solution. The results showed that the exosome surface markers CD9 and CD63 and the internal marker Tsg101 were positively expressed, while the negative markers GM130 or Calnexin were not detected. Figure 4 , indicating that the vesicles we isolated have typical characteristics of exosomes, with high concentration and good integrity. The ratio of exosome particle number to protein amount is 1.3×10 9 particles / μg protein, high purity.

[0021] Example 4: Preparation of exosome preparation, preparation of 100 ml matrix solution: 5 ml human serum albumin (V / V), 10 ml dextran 40 glucose injection (V / V), 20 ml glycerol fructose sodium chloride injection (V / V), 40 ul ganglioside sodium 100 mg / 2 ml, 267 ul edaravone injection 15 mg / 10 ml, add 0.9% sodium chloride injection to 100 ml, place in a micro-oscillator (VORTEX GENIE) and shake thoroughly at 180 rpm for 30s-1 min to mix, and store at 2-8°C. Add exosomes to the prepared matrix solution to make the exosome concentration in the preparation 1×10 9 / ml.

[0022] Example 5: Preparation of exosome preparation, preparation of 100 ml matrix solution: 7.5 ml human serum albumin (V / V), 15 ml dextran 40 glucose injection (V / V), 25 ml glycerol fructose sodium chloride injection (V / V), 70 ul ganglioside sodium 100 mg / 2 ml, 400 ul edaravone injection 15 mg / 10 ml, add 0.9% sodium chloride injection to 100 ml, place in a micro-oscillator (VORTEX GENIE) and shake thoroughly at 180 rpm for 30s-1 min to mix, and store at 2-8°C. Add exosomes to the prepared matrix solution to make the exosome concentration in the preparation 5×10 9 / ml.

[0023] Example 6: Preparation of exosome preparation: Preparation of 100 ml matrix solution: 10 ml human serum albumin (V / V), 20 ml dextran 40 glucose injection (V / V), 30 ml glycerol fructose sodium chloride injection (V / V), 100 ul ganglioside sodium 100 mg / 2 ml, 533 ul edaravone injection 15 mg / 10 ml, add 0.9% sodium chloride injection to 100 ml, place in a micro-oscillator (VORTEX GENIE) and shake thoroughly at 180 rpm for 30 s-1 min to mix, and store at 2-8°C. Add exosomes to the prepared matrix solution to make the exosome concentration in the preparation 10×10 9 / ml.

[0024] Example 7: Normal saline plus 10% human serum albumin was used as a control, and the exosome concentration in the preparation was 5×10 9 / ml, and the exosome particle size, concentration, protein, and integrity were stored at -20°C for different time periods. The results are shown in Table 2; As can be seen from Table 2, our exosome preparation can be stored at -20°C for 12 months, and the number of exosomes, surface protein expression and vesicle morphology are well maintained, which is significantly better than the control group. Example 5 has the best effect.

[0025] The exosome preparation stored at -20°C for 8 months was thawed and stored at 2-8°C for 15 days. The particle size, concentration, protein content, and integrity of the exosomes were detected at different times. The results are shown in Table 3.

[0026] As can be seen from Table 3, our exosome preparations can be stored at 4-8°C for 15 days after thawing, and the number of exosomes, surface protein expression, and vesicle morphology are well maintained, providing quality assurance for subsequent clinical use or treatment.

[0027] Example 8: Recruit 5 eligible participants, 3 males and 2 females, aged 70-75 years. Five subjects were diagnosed with mild to moderate Alzheimer's disease (AD). The exosome preparation of Example 5 was administered intranasally to the MSCs-Exos. Each participant received 5×10 9Participants were administered 0.5 ml / ml twice daily via a nasal spray device (20 treatments total). Four weeks after treatment, efficacy assessments were conducted, including assessments of cognitive function and daily activities. Outcome measures included the Alzheimer's Disease Assessment Scale-Cognitive (ADAS-cog, scores range from 0 to 70), the Mini-Mental State Examination (MMSE, scores range from 0 to 30), and the Montreal Cognitive Assessment-Basic (MoCA-B, scores range from 0 to 30). Higher ADAS-cog scores indicate worse cognition, while decreased ADAS-cog scores indicate improved cognition. Conversely, increases in MMSE or MoCA-B scores indicate improved cognition. The results are shown in Table 4.

[0028] Table 4 shows that the ADAS-cog scores showed a consistent trend of decrease compared to pre-treatment levels. MoCA-B scores and MoCA-B scores also showed an increasing trend of improvement. Participants did not experience any physical discomfort during treatment, and their blood pressure, electrocardiogram, liver, and kidney functions were normal. This indicates that our exosome preparation is safe and effective.

[0029] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, from all points of view, the embodiments should be regarded as illustrative and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and range of equivalents of the claims are included in the present invention. Any reference signs in the claims should not be construed as limiting the claim to which they relate.

[0030] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A stem cell exosome preparation for nasal absorption, consisting of exosomes and a matrix solution. The matrix solution consists of 0.9% sodium chloride injection, 5-10% human albumin (v / v), 10-20% dextran 40 glucose injection (v / v), 20-30% glycerol fructose sodium chloride injection (v / v), 20-50 μg / ml ganglioside sodium, and 4-8 μg / ml edaravone injection. The preparation method of stem cell exosomes is as follows: the supernatant of mesenchymal stem cells cultured in serum-free medium under hypoxic conditions on P3-P10 surrogate 3D microcarriers is collected; after filtering through 0.45 um filter membrane and 0.22 um filter membrane successively, the supernatant is passed through a nano-ultrafiltration chip; the free nucleotides and protein impurities in the supernatant are removed using a negative pressure oscillation and a dual-coupled ultrasonic oscillation system, and the 30-200 nm exosome vesicles are intercepted and purified.

2. The stem cell exosome preparation for nasal absorption according to claim 1, characterized in that: The concentration of the exosomes in the preparation is 1-10×10 9 / ml, the exosome preparation was stored at -20℃ for 12 months and stored at 2-8℃ for 15 days after thawing, and the number of exosomes, surface protein expression and vesicle morphology were well maintained.

3. The stem cell exosome preparation for nasal absorption according to claim 1, characterized in that: The optimal concentrations of the exosome preparation are: 7.5% human serum albumin (V / V), 15% dextran 40 glucose injection (V / V), 25% glycerol fructose sodium chloride injection (V / V), 35 ug / ml ganglioside sodium, 6 ug / ml edaravone injection, and the exosome concentration is 5×10 9 / ml.

4. The stem cell exosome preparation for nasal absorption according to claim 1, characterized in that: The exosomes can be obtained from the culture supernatant of mesenchymal stem cells separated from tissues such as umbilical cord, placenta, fat, amniotic membrane, dental pulp, and endometrium.

5. The stem cell exosome preparation for nasal absorption according to claim 1, characterized in that: The exosomes were 30-200 nm in diameter, and protein detection results showed that the exosome markers CD9, CD63, and Tsg101 were positively expressed, while the negative markers GM130 or Calnexin were not detected. The ratio of the number of exosome particles to the amount of protein was higher than 1×10 9 particles / μg protein, with saucer-shaped or cup-shaped structures with clear membrane structures and clear edges under the electron microscope.

6. A method for preparing a stem cell exosome preparation for nasal absorption, characterized in that: The following steps are involved: Step 1: Preparation of the matrix solution: 5-10 ml human serum albumin (V / V), 10-20 ml dextran 40 glucose injection (V / V), 20-30 ml glycerol fructose sodium chloride injection (V / V), 40-100 ul ganglioside sodium 100 mg / 2 ml, 267-533 ul edaravone injection 15 mg / 10 ml; Step 2: Add 0.9% sodium chloride injection to 100 ml, shake thoroughly on a micro-oscillator at 180 rpm for 30 seconds to 1 minute to mix, and store at 2-8°C. Step 3: Add exosomes to the prepared matrix solution to make the exosome concentration in the preparation 1-10×10 9 / ml.

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