Application of mesenchymal stem cell-derived exosome subsets in the treatment of chronic obstructive pulmonary disease

Abstract

This invention relates to the field of stem cell biomedical technology, and provides an application of a subpopulation of mesenchymal stem cell exosomes in the treatment of COPD. This invention develops a mesenchymal stem cell-derived exosome subpopulation S1, and discovers and verifies that exosomes isolated from this subpopulation have better therapeutic effects on chronic obstructive pulmonary disease (COPD), which will provide a new therapeutic agent for COPD.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a mesenchymal stem cell-derived exosome subset S1 in the treatment of chronic obstructive pulmonary disease. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a progressive, irreversible respiratory disease characterized primarily by persistent airflow limitation. With the aging of the global population, the incidence and mortality rates of COPD continue to rise, making it one of the leading causes of death worldwide. Although current drug treatments such as bronchodilators, corticosteroids, and antibiotics are available, these therapies mainly aim to relieve symptoms and control acute exacerbations, and cannot effectively reverse the disease progression. COPD treatment faces significant clinical challenges, particularly the lack of fundamental treatments that can improve lung function and prevent disease progression. As the limitations of traditional treatments become increasingly apparent, there is an urgent need to develop new treatment strategies to more effectively address the long-term pathological damage and functional decline in COPD patients. Therefore, exploring innovative therapies based on immune modulation and tissue repair has become a focus of current COPD treatment research.

[0003] Exosomes, as nanoscale vesicles secreted by cells, possess natural immunomodulatory and repair capabilities due to their ability to carry biomolecules such as proteins, lipids, and RNA, and have gradually become a research hotspot in the field of COPD treatment in recent years. In particular, exosomes derived from mesenchymal stem cells (MSC-Exo) have demonstrated promising therapeutic potential in animal models due to their ability to effectively regulate immune responses, reduce inflammation, and promote tissue repair. However, exosome therapy faces bottlenecks such as the complexity of preparation processes, product consistency, and variability in efficacy among different patients, which require breakthroughs and solutions. Summary of the Invention

[0004] This invention provides the use of the subpopulation S1 of mesenchymal stem cell exosomes in COPD treatment. By subdividing stem cell exosomes into subpopulations, the homogeneity of the product is improved, while achieving a more efficient and stable COPD treatment effect.

[0005] This invention provides the application of mesenchymal stem cell exosome subpopulation S1 in the preparation of a formulation for treating COPD, wherein the formulation uses exosome subpopulation S1 as the active ingredient; the exosome subpopulation S1 has a peak particle size range of 105-135 nm and an average particle size of 115-145 nm, and its surface markers include HRS, HSPA4, RAS1B, A2M, Syntenin-1, CD9, GPC1, DPP4, HSP90AA1, HSP90AB1, ALIX, CD81, TSG101, and Filamin-A.

[0006] As an optional option, the COPD includes stable COPD and acute exacerbation COPD.

[0007] Alternatively, the formulation may comprise a combination of a subgroup S1 of mesenchymal stem cell exosomes and a drug for treating COPD; the drug for treating COPD may include an inhaled bronchodilator, an inhaled corticosteroid, a phosphodiesterase 4 inhibitor, a corticosteroid, or an antibiotic.

[0008] As an optional feature, the surface markers of the exosome subgroup S1 also include CD63, wherein CD63 is expressed at low levels, and CD9 and ALIX are expressed at high levels, and the exosome purity is not less than 1×10⁻⁶. 8 Particle count / μg protein.

[0009] As an optional method, the preparation method of the exosome subpopulation S1 includes: isolating and culturing mesenchymal stem cells derived from human umbilical cord, collecting their culture supernatant; filtering; using a tangential flow filtration system to obtain a concentrate from the filtered supernatant, and separating and purifying the concentrate by size exclusion chromatography and / or reverse affinity chromatography to obtain the exosome subpopulation S1.

[0010] As an alternative, when size exclusion chromatography is used to separate and purify the concentrate, the component corresponding to the first ultraviolet absorption peak in the eluent is collected as the exosome subgroup S1.

[0011] As an optional method, the preparation of the culture supernatant includes: selecting and isolating mesenchymal stem cells derived from umbilical cords from healthy mothers who are in the logarithmic growth phase and within the seventh generation; seeding the mesenchymal stem cells in a serum-free, well-defined mesenchymal stem cell-specific culture medium and culturing them to the 5th to 7th generation until the cell confluence reaches 70%; discarding the culture medium, washing the cells three times with PBS, replacing with fresh culture medium, continuing the culture, and collecting the cell culture supernatant containing exosomes.

[0012] As an optional approach, the filtration, concentration, and separation and purification steps in the preparation method include: deep filtration of the culture supernatant to remove large vesicles and apoptotic bodies to obtain a pretreatment solution; concentration of the pretreatment solution by a tangential flow ultrafiltration system, separation and purification by size exclusion chromatography, collection of the component corresponding to the first ultraviolet absorption peak in the eluent, and correspondingly obtaining the exosome subgroup S1.

[0013] Alternatively, the concentration of exosome subpopulation S1 in the formulation is 1×10⁻⁶. 8 ~ 1×10¹¹ particles / mL.

[0014] Alternatively, the dosage form of the formulation may include lyophilized powder, hydrogel, or sterile solution; the method of administration of the formulation may include intravenous injection or nebulized inhalation.

[0015] This invention provides a novel application for exosome subset S1 derived from mesenchymal stem cells, which is of great significance for improving the efficacy of exosome therapy for chronic obstructive pulmonary disease (COPD). Compared to undifferentiated exosome TFF and the positive control drugs LABA+LAMA+ICS, the S1 subpopulation of this invention significantly improves the state of lung epithelial cells, lung function, and lung tissue structure in COPD rats. It also boasts high purity, can be mass-produced, and can be formulated into effective pharmaceutical products. This invention offers a new treatment option for COPD and has promising application prospects. Attached Figure Description

[0016] Figure 1 This image shows the preparation and identification of the S1 subpopulation of exosomes derived from mesenchymal stem cells.

[0017] Figure 2 The figure shows the effect of exosome subpopulation S1 and undifferentiated exosome TFF derived from mesenchymal stem cells on BEAS-2B cells damaged by smoke extract.

[0018] Figure 3 The results of efficacy evaluation of endotracheal infusion of mesenchymal stem cell-derived exosome subset S1 and endotracheal infusion of positive drugs in the treatment of COPD rats. Detailed Implementation

[0019] The function of exosomes depends on the cellular origin and molecular composition of their subpopulations. Further subdividing exosomes into different subpopulations can enhance their therapeutic effects. For example, specific subpopulations of exosomes may more effectively modulate immune responses and reduce chronic inflammation in COPD patients, thus providing more precise treatment options for COPD. Therefore, developing exosome-based therapeutic strategies based on subpopulations holds promise for overcoming existing limitations and providing more personalized and effective treatments.

[0020] This invention provides a novel use of mesenchymal stem cell-derived exosome subset S1 in the treatment of chronic obstructive pulmonary disease (COPD). The mesenchymal stem cell-derived exosome subset S1 is defined by its surface markers, particle size, or preparation method. The particle size of the exosome subset S1 has a peak value of 105–135 nm and an average particle size of 115–145 nm. Surface markers include HRS, HSPA4, RAS1B, A2M, Syntenin-1, CD9, GPC1, DPP4, HSP90AA1, HSP90AB1, ALIX, CD81, TSG101, and Filamin-A.

[0021] More specifically, regarding novel applications in COPD treatment, this invention experimentally validates that the S1 subset of exosomes derived from mesenchymal stem cells can serve as an effective active ingredient in COPD treatment formulations. It can also be used in combination with other types of COPD treatment drugs, such as inhaled bronchodilators, inhaled corticosteroids, phosphodiesterase 4 inhibitors, corticosteroids, or antibiotics. The COPD includes stable COPD and acute exacerbations of COPD. Example 1

[0022] This embodiment describes the preparation and identification of the S1 subset of exosomes derived from mesenchymal stem cells, including the following steps: (1) Preparation of mesenchymal stem cell culture supernatant: Mesenchymal stem cells were cultured in vitro in exosome-specific culture medium and passaged to a predetermined number of passages under carbon dioxide culture conditions. After the cell viability reached the set standard, the cell culture medium was collected. The culture medium was clarified and filtered to remove cells and cell debris to obtain mesenchymal stem cell culture supernatant. The mesenchymal stem cell culture supernatant was concentrated 20 times using a tangential flow filtration concentration system to obtain a concentrated solution. The tangential flow concentration system used a 300kDa hollow fiber filter, washed with PBS 16-25 times, with a transmembrane pressure (TMP) of 0.5 bar and a flow rate of 385 LMH.

[0023] (2) The eluent obtained from the first step of chromatography purification was used as the sample for the second step of chromatography. Gel filtration chromatography (size exclusion chromatography) was used to further separate and purify the exosomes. The gel filtration chromatography packing material was a porous gel medium suitable for separating exosomes within a certain particle size range. The chromatography column was equilibrated with buffer before loading the sample.

[0024] The equilibrium volume of the chromatography column is 2 column volumes (CV), and the equilibration and elution buffer used is phosphate buffer. The sample is loaded into the chromatography column at a loading rate of 0.5–30 mg / mL, preferably 1–10 mg / mL. After loading, isocratic elution is performed using the buffer, with continuous monitoring of the UV absorption signal during the elution process.

[0025] During the elution process, at least two separate elution peaks are formed based on the changes in the ultraviolet absorption signal. The eluents corresponding to different elution peaks are collected sequentially according to the elution order, and the eluent corresponding to the first elution peak is defined as exosome subgroup S1.

[0026] After the chromatography separation is completed, the chromatography column is rinsed with buffer solution and then cleaned in situ with alkaline cleaning solution to restore the performance of the chromatography column for subsequent reuse.

[0027] (3) Exosome identification experiment The extracted exosomes were identified using different detection indicators, including observing the morphology of exosomes by transmission electron microscopy (TEM) and analyzing the particle size distribution and particle concentration in exosome subpopulations by nanoparticle tracking technology (NTA).

[0028] The results showed that, through clarification filtration, concentration, and the first-step purification, exosomes were purified as follows during the second-step purification process: Figure 1 As shown in Figure A, two ultraviolet absorption peaks appear. The first absorption peak is collected, named S1, and further identified. The results are as follows: Figure 1 As shown in B and 1C, the average particle size of S1 is 136 nm, which is within the range of 30-150 nm of exosomes. In addition, transmission electron microscopy results show that S1 exhibits the classic characteristics of exosomes, namely the saucer-like morphology.

[0029] Furthermore, omics-level analysis was conducted on the S1 exosome subsets to compare the differences in molecular composition among the different subsets. The omics analysis included, but was not limited to, at least one of proteomics, nucleomics, and lipidomics. High-throughput detection methods were used to analyze the protein, nucleic acid, or lipid components contained in the exosome subsets. The results showed that S1 highly expressed miRNAs (miR-155, miR-146, etc.) that regulate Th2 inflammation-related pathways. Figure 1 D), and the Th2 pathway-mediated target is most widely recognized as the most promising target for COPD treatment, indicating that S1 has good potential for treating COPD.

[0030] In summary, through morphological analysis, particle size distribution detection, and molecular composition analysis at the omics level, it can be confirmed that the S1 obtained by the preparation method of this invention conforms to the basic structure and physicochemical properties of exosomes, and provides a mechanistic basis for subsequent pharmacodynamic studies. Example 2

[0031] One of the pathological features of chronic obstructive pulmonary disease is lung epithelial cell damage caused by cigarette smoke and inflammation. Therefore, this invention uses an in vitro lung epithelial cell damage experiment to detect the difference in the effects of S1 subset and undifferentiated subset of exosome TFF in protecting cell viability and controlling inflammatory response.

[0032] The in vitro lung epithelial cell injury experiment includes the following steps: (1) adding cigarette extract and lipopolysaccharide to the lung epithelial cell culture medium to establish a COPD injury model; (2) adding exosome subsets to the culture medium; (3) evaluating the lung epithelial cell status of the COPD in vitro model.

[0033] Specifically, the steps include the following: (1) Day 1: Human lung epithelial cells BEAS-2B were placed in 96-well plates at a density of 6.0E+04 cells / well and cultured overnight to allow them to adhere to the plate. (2) Day 2: Remove the old culture medium and add a culture medium containing 1, 2, 3, 4% smoke extract (CSE) and 0.1 μg lipopolysaccharide (LPS) and continue culturing for 24 h. For the exosome group, add 1.0E+07 particle number S1 or TFF and 0.5 μM dexamethasone (DEX) as a positive control and continue culturing for 24 h. The method for preparing the cigarette smoke extract is as follows: one end of a Boucher tube is connected to a lit cigarette, and the other end is connected to a 50 mL syringe. The tube contains 20 mL of DMEM culture medium. After burning four cigarettes, 100% cigarette smoke extract is obtained.

[0034] (3) Day 3: IL-6 content in cell supernatant was detected by ELISA; cell viability was detected by CCK-8 assay.

[0035] The results are as follows Figure 2 As shown in Figure A, 1% CSE + 0.1 μg / mL LPS significantly reduced BEAS-2B cell viability, which was determined as the modeling concentration for subsequent studies. Figure 2 As shown in B and 2C, S1, TFF, and DEX all have significant effects on restoring cell vitality and inhibiting inflammatory factors, with S1 showing better efficacy than TFF and DEX. Example 3

[0036] A rat model of COPD was established as follows: Day 1 of modeling was designated as D1. On D1 and D15, rats in the model group (SD rats, weighing 200-220g) received intratracheal inhalation of LPS (200μg / 200μL). From D2 to D14 and D16 to D49, rats in the model group were continuously inhaled with cigarette smoke for 30 minutes daily in a self-made sealed acrylic glass fumigation chamber (160cm×60cm×50cm), smoking 15 cigarettes at a time. After the cigarettes were finished, the diaphragm vacuum pump was turned off, and the rats remained in the fumigation chamber for the remaining time. On D49, intratracheal inhalation of the drug was initiated at a dose of 5.0E+10 particles for seven consecutive days. Pharmacodynamic evaluation was performed on the eighth day. Symptom indicators included, but were not limited to, pulmonary respiratory function tests, HE staining, Masson staining, and AB-PAS staining.

[0037] Figure 3 A represents the group assignments and drug administration details for this trial. The results are as follows: Figure 3 As shown in Figure B, compared with group G1, group G2 showed significantly worse pulmonary function indicators, manifested by a decreased FEV1 / FVC ratio, consistent with typical COPD pulmonary function characteristics. Group G3 showed significant improvement in pulmonary function parameters compared to group G2, indicating that S1 effectively relieves airway obstruction. Group G4 showed limited improvement in pulmonary function. Regarding the improvement in pulmonary pathology (… Figure 3S1 showed superior effects compared to LABA+LAMA+ICS in terms of lung tissue structure repair, inhibition of goblet cell metaplasia, and inhibition of pulmonary fibrosis development, and was comparable to S1+LABA+LAMA. This indicates that compared to the use of conventional bronchodilators combined with hormones, S1's effect lies in directly targeting the repair of lung tissue function, thereby inhibiting the progression of COPD.

[0038] In summary, the exosome subgroup S1, compared with existing drugs in the field of COPD treatment, is not limited to the suppression of lung inflammation and the temporary dilation of airways, but has the effect of delaying or even stopping the progression of COPD, and has greater potential for future market application.

[0039] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the present invention, and all such modifications and improvements fall within the scope of the present invention.

Claims

1. The application of mesenchymal stem cell exosome subpopulation S1 in the preparation of formulations for treating COPD, characterized in that, The formulation uses exosome subgroup S1 as its active ingredient; the peak particle size range of exosome subgroup S1 is 105-135 nm, the average particle size is 115-145 nm, and the surface markers include HRS, HSPA4, RAS1B, A2M, Syntenin-1, CD9, GPC1, DPP4, HSP90AA1, HSP90AB1, ALIX, CD81, TSG101, and Filamin-A.

2. The application according to claim 1, characterized in that, The COPD mentioned includes stable COPD and acute exacerbation COPD.

3. The application according to claim 1, characterized in that, The formulation comprises a combination of a subgroup S1 of mesenchymal stem cell exosomes and a drug for treating COPD; the drug for treating COPD includes an inhaled bronchodilator, an inhaled corticosteroid, a phosphodiesterase 4 inhibitor, a corticosteroid, or an antibiotic.

4. The application according to claim 1, characterized in that, The surface markers of the exosome subpopulation S1 also include CD63, with low expression of CD63 and high expression of CD9 and ALIX, and the exosome purity is not less than 1×10⁻⁶. 8 Particle count / μg protein.

5. The application according to claim 1, characterized in that, The preparation method of the exosome subpopulation S1 includes: isolating and culturing mesenchymal stem cells derived from human umbilical cord, collecting their culture supernatant; filtering; using a tangential flow filtration system to obtain a concentrate from the filtered supernatant, and separating and purifying the concentrate by size exclusion chromatography and / or reverse affinity chromatography to obtain the exosome subpopulation S1.

6. The application according to claim 5, characterized in that, When size exclusion chromatography is used to separate and purify the concentrate, the component corresponding to the first ultraviolet absorption peak in the eluent is collected as the exosome subgroup S1.

7. The application according to claim 5, characterized in that, The method for preparing the culture supernatant includes: selecting and isolating mesenchymal stem cells derived from umbilical cords from healthy mothers who are in the logarithmic growth phase and within the seventh generation; seeding the mesenchymal stem cells in a serum-free, well-defined mesenchymal stem cell-specific culture medium and culturing them to the 5th to 7th generation until the cell confluence reaches 70%; discarding the culture medium, washing the cells three times with PBS, replacing with fresh culture medium, continuing the culture, and collecting the cell culture supernatant containing exosomes.

8. The application according to claim 5 or 7, characterized in that, The filtration, concentration, and separation and purification steps in the preparation method include: deep filtration of the culture supernatant to remove large vesicles and apoptotic bodies to obtain a pretreatment solution; concentration of the pretreatment solution by a tangential flow ultrafiltration system, separation and purification by size exclusion chromatography, collection of the component corresponding to the first ultraviolet absorption peak in the eluent, and correspondingly obtaining the exosome subgroup S1.

9. The application according to claim 1, characterized in that, The concentration of exosome subpopulation S1 in the formulation is 1×10⁻⁶. 8 ~ 1×10¹¹ particles / mL.

10. The application according to claim 1, characterized in that, The dosage form of the formulation includes lyophilized powder, hydrogel, or sterile solution; the method of administration of the formulation includes intravenous injection or nebulized inhalation.

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