Mechanical response type engineered mesenchymal stem cell exosome preparation for treating diseases caused by fibrosis inflammation as well as preparation method and application of mechanical response type engineered mesenchymal stem cell exosome preparation

Abstract

The invention discloses a mechanical response type engineered mesenchymal stem cell exosome preparation for treating diseases caused by fibrosis inflammation, a preparation method and application, and the exosome preparation is an exosome which is secreted by mesenchymal stem cells and has high cholesterol and high-efficacy protein. The exosome is prepared by culturing mesenchymal stem cells on a hydrogel matrix in a modulus hardness range of 1-50KPa, the exosome secretion amount of a single cell is (3-5) * 10 < 4 > / cell, the cholesterol content is (1-10) nM / 5mg exosome, and the size of the exosome is 110-140nm; the high-efficacy protein is a key functional protein for regulating and controlling the biological activity of the exosome and the cell action efficiency. The cholesterol content in the exosome is precisely regulated and controlled, the cytoplasm delivery efficiency and tissue penetrability of the exosome are improved, a whole-process control system of hydrogel preparation, stem cell culture and exosome purification is constructed, and the stability of the preparation and the yield of the exosome are improved.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a mechanically responsive engineered mesenchymal stem cell exosome preparation, its preparation method, and its application for treating fibrotic inflammatory diseases. Background Technology

[0002] Fibrotic diseases are a spectrum of cross-organ diseases characterized by fibroblast activation and proliferation, excessive deposition of extracellular matrix replacing normal tissue, and have become a major public health challenge with high morbidity and mortality worldwide. Although significant progress has been made in understanding their pathobiological mechanisms, effective interventions to control disease progression remain lacking in clinical practice. These diseases were once widely considered irreversible, but recent studies have confirmed their high pathological dynamism and reversal potential, providing a key technological entry point for treatment strategy development. Pulmonary fibrosis, liver fibrosis, and ocular fibrosis are the most representative subtypes of these diseases, and all three share a common clinical challenge: existing treatments have significantly limited effectiveness in controlling disease progression, failing to meet clinical needs. Therefore, developing therapeutic agents that can effectively intervene in fibrotic diseases has extremely high clinical value and is urgently needed.

[0003] Mesenchymal stem cell therapy, as an emerging technology in regenerative medicine, has become a research hotspot in this field due to its core functional advantages such as tissue repair, immune regulation, and angiogenesis. Mesenchymal stem cells can secrete exosomes, which are loaded with various bioactive molecules such as microRNAs, proteins, and lipids, and can play a role in regulating the cellular microenvironment and promoting tissue regeneration. However, current research mainly focuses on the application of exosomes derived from mesenchymal stem cells in wound repair and immune regulation. Furthermore, natural mesenchymal stem cell-derived exosomes face significant technical bottlenecks, specifically low effector cell uptake efficiency, limited production yield, and insufficient bioactivity. These deficiencies severely restrict the clinical translation and application of natural exosome preparations.

[0004] CN 120549978 A discloses the application of enhanced mesenchymal stem cells and exosomes in chronic nephritis and renal failure, belonging to the field of biopharmaceutical manufacturing technology. This invention significantly enhances the therapeutic potential of mesenchymal stem cells in secreting exosomes through a compound pretreatment with rhodioloside and astragalus polysaccharide, providing a new approach to the intervention of chronic kidney disease. It covers the application of multicellular secretions in chronic kidney disease and the composition in pulmonary fibrosis. The technical solution has been verified through systematic pharmacodynamic experiments. The composition can significantly reduce the content of the key pro-fibrotic factor TGF-β1 in the blood of rats with pulmonary fibrosis, while effectively improving blood gas exchange function, manifested as a significant increase in blood oxygen saturation (SaO2) and a significant decrease in carbon dioxide partial pressure (pCO2). The improvement of these key indicators confirms that the composition has a clear anti-fibrotic effect and lung function protection effect, providing a novel treatment strategy for pulmonary fibrosis, which currently lacks specific treatments in clinical practice. This technology not only has significant clinical translational value, but its mechanism of action based on stem cell secretions has also opened up innovative research ideas for the treatment of lung diseases, which is of great scientific significance. However, the specific parameters (such as sequence information and loading concentration) and functional verification data of the target microRNA (such as miR-29 family members) have not yet been disclosed. In addition, in the preparation of mesenchymal stem cell secretions, the yield of exosomes obtained by conventional plate culture is extremely low, which is difficult to meet the needs of industrial-scale production, thus restricting its market development prospects.

[0005] CN 120555364 A discloses a method for preparing engineered exosomes for pulmonary fibrosis and the product thereof. This invention belongs to the field of biopharmaceutical manufacturing technology. Specific embodiments provide a method for preparing engineered exosomes capable of loading mitochondria for the treatment of pulmonary fibrosis, and the engineered exosomes themselves. The method includes constructing a fusion plasmid expressing a mitochondrial-targeting peptide-therapeutic protein gene, dissolving it in enzyme-free ultrapure water, adding it to a lipid nanoparticle formulation, and thoroughly mixing to obtain a nanoparticle formulation containing the therapeutic protein gene; delivering this nanoparticle formulation into alveolar epithelial cells and efficiently transfecting it, effectively regulating the homeostasis of damaged alveolar epithelial cells during the development of pulmonary fibrosis, promoting the secretion of exosomes with therapeutic effects, and further modifying it with mannose to obtain engineered exosomes. These exosomes can effectively load mitochondria, and based on the mannose target and the mitochondrial-targeting peptide, they sequentially and precisely target macrophage mitochondria, thereby effectively regulating the mitochondrial homeostasis of macrophages and achieving the goal of reversing pulmonary fibrosis, showing high clinical application value. This technical solution does not disclose the pharmacokinetic parameters and long-term toxicological data of engineered exosomes in vivo, nor does it disclose the influence of different preparation parameters on the function of engineered exosomes, or the actual expression level and activity maintenance period of SIRT3 protein in engineered exosomes. The engineered exosomes obtained rely on ultracentrifugation extraction, resulting in low production efficiency and long processing time, making it difficult to meet the needs of industrial-scale production. Furthermore, the ultrasonic treatment parameters during mitochondrial loading are not clearly optimized, which can easily cause fluctuations in mitochondrial activity between different batches, affecting the consistency of efficacy. At the same time, it does not mention long-term storage conditions and stability data, which may lead to activity decay during clinical transportation and storage, thus restricting practical application.

[0006] CN 120324468 A discloses a stem cell preparation for scalp hair follicle regeneration and its preparation method, the raw materials of which include: exosomes, physiological saline, galangal extract, and hydrogel. This stem cell preparation for scalp hair follicle regeneration and its preparation method utilize mRNA, miRNA, and proteins carried by exosomes to target hair follicle stem cells, activate cell cycle-related genes, and promote cell transition from the G0 phase to the proliferative phase; simultaneously, by delivering signaling molecules such as BMP and Notch, it inhibits the differentiation of hair follicle stem cells into epidermal cells and induces their differentiation into hair follicle-specific cells, promoting the reconstruction of hair follicle structure; the anti-inflammatory factors in the exosomes can inhibit the excessive activation of fibroblasts around the hair follicle, reduce collagen fiber deposition, and relieve the inhibition of hair follicle regeneration by fibrosis; at the same time, it reduces the levels of TNF-α, IL-6, and other pro-inflammatory factors. The inflammatory factor level provides a suitable growth environment for hair follicle stem cells; however, this technical solution does not disclose key quality control parameters of exosomes, quantitative data of active ingredients, specific centrifugation parameters for exosome extraction, batch yield stability, or data on the purity, extraction rate, and synergistic mechanism of galangin extract with exosomes. The obtained exosomes have the risk of immunogenicity due to allogeneic fat, and due to the lack of quality control standards, the consistency of activity and purity of different batches of exosomes is difficult to guarantee. At the same time, the release kinetics of hydrogel and exosomes are not matched, which may lead to premature or slow release of exosomes, affecting the sustained efficacy of hair follicle regeneration.

[0007] CN 119040258 A discloses the preparation and application of adipose-derived mesenchymal stem cell exosomes with SphK1 inhibitory function. Through a specific preparation method, the obtained adipose-derived mesenchymal stem cell exosomes are identified and verified as a biological product capable of inhibiting Sphk1, a key target gene in liver fibrosis. Compared to other chemically synthesized Sphk1 inhibitors, because it originates from adipose-derived stem cells (ADSCs), it possesses biological functions similar to ADSCs, such as tissue repair and regulation of the immune microenvironment, while also inhibiting Sphk1 expression. This makes it applicable to the development and application of new drugs for liver fibrosis. However, this technical solution does not disclose the specific mechanism of action of exosomes in inhibiting SphK1, the key quality control parameters of exosomes (and batch-to-batch consistency data on SphK1 inhibitory activity of exosomes prepared from different donor adipose tissues); the obtained engineered exosomes rely on ultracentrifugation for preparation, resulting in low yield and time-consuming operation, making it difficult to meet the needs of industrial-scale production; and the immunogenicity of allogeneic exosomes has not been verified, which may affect the safety of clinical applications. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing engineered mesenchymal stem cell exosomes and the engineered mesenchymal stem cell exosomes obtained by this method. The engineered exosomes of this invention are based on the regulatory mechanisms of cholesterol (a key determinant of exosome cytoplasmic delivery) and ABCA family genes (key proteins that regulate cholesterol content in stem cell exosomes and are matrix stiffness-dependent). By activating stem cell ABCA family genes through a soft matrix culture system, the invention achieves "three-high" engineered modification (high secretion yield, high bioactivity, and high effector cell internalization capacity). Furthermore, it exhibits superior tissue penetration and therapeutic efficacy in mouse models of fibrosis-related diseases, contributing to the reversal of the pathological process of fibrosis at the levels of vital signs and extracellular matrix microenvironment.

[0009] According to a first aspect of the present invention, an engineered mesenchymal stem cell exosome preparation is provided for treating the mechanical response of fibrotic inflammatory diseases. The exosome preparation is an exosome derived from mesenchymal stem cells and contains high cholesterol and high-efficacy proteins. The exosomes are prepared by culturing mesenchymal stem cells on a hydrogel matrix with a modulus hardness range of 1-50 kPa. The secretion amount of the exosomes is (3-5) × 10⁻⁶. 4 The exosomes contain (1-10) nM cholesterol per 5mg cell, and the exosome size is 110-140nm; the high-efficacy protein is a key functional protein that regulates the biological activity of exosomes and the efficacy of cell action. Based on the above technical solutions, the fibrotic diseases include pulmonary fibrosis, liver fibrosis, and ocular fibrosis; The key functional proteins are selected from the ABC superfamily, namely the ATP-binding cassette protein gene family, preferably ABCA family genes. The cholesterol content is (3-5) nM / 5mg.

[0010] Based on the above technical solution, the base sequence of the key functional protein is ABCA1: ENSG00000165029, ABCA4: ENSG00000198691, ABCA13: ENSG00000179869, ABCA12: ENSG00000144452, ABCA3: ENSG0000016797, ABCA5: ENSG00000154265, ABCA7: ENSG00000064687, ABCA6: ENSG00000154262, ABCA8: ENSG00000141338, ABCA2: ENSG00000107331, ABCA9: ENSG00000154258, ABCA10: ENSG00000154263.

[0011] According to a second aspect of the present invention, a method for preparing an engineered mesenchymal stem cell exosome formulation is provided, the method comprising the following steps: Step 1, hydrogel modification treatment: After filtering the hydrogel prepolymer solution through a 0.22um filter membrane, a hydrogel matrix with a mechanical strength of 1-50KPa is obtained by cross-linking polymerization. Then, the surface of the hydrogel matrix is ​​modified to obtain the modified hydrogel. Step 2, stem cell culture: Mesenchymal stem cells were seeded onto the modified hydrogel obtained in Step 1 and cultured to passage 3-6 in αMEM medium containing 10% exosome-free serum and 1% penicillin-streptomycin. After washing with PBS, the cells were cultured in αMEM medium containing 1% penicillin-streptomycin and 100-1000 nM 24(S), 25-Epoxycholesterol for 12-48 hours. 100-300 ml of supernatant was collected and stored. Step 3, exosome purification: Centrifuge the supernatant obtained in Step 2, add exosome precipitation reagent, let stand and centrifuge, discard the supernatant, then add PBS at a volume of 1:5-1:10 of the supernatant obtained in Step 2 to resuspend the precipitate and exosome precipitation reagent, let stand and centrifuge, discard the supernatant, and again add PBS at a volume of 1:50-1:100 of the supernatant obtained in Step 2 to resuspend the precipitate to obtain the exosome preparation; The hydrogel matrix is ​​in the form of a block hydrogel or a microcarrier hydrogel.

[0012] Based on the above technical solution, the concentration of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone in the polyacrylamide hydrogel prepolymer is 0.1-1 w / v, the concentration of acrylamide is 5-10 w / v, the concentration of N,N'-methylenebisacrylamide is 0.01-1 w / v, and the concentration of N-acryloyloxysuccinimide is 0.01-1 w / v.

[0013] Based on the above technical solutions, the modified hydrogels include polyacrylamide hydrogels, gelatin hydrogels, methacrylamide gelatin hydrogels, sodium alginate hydrogels, methacrylamide sodium alginate hydrogels, hyaluronic acid hydrogels, and methacrylamide hyaluronic acid hydrogels. When the modified hydrogel is a polyacrylamide hydrogel, the composition of the hydrogel prepolymer is as follows: acrylamide concentration of 6-9 w / v%, N,N'-methylenebisacrylamide concentration of 0.05-0.8 w / v%, N-acryloyloxysuccinimide concentration of 0.05-0.8 w / v%, and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone concentration of 0.3-0.8 w / v%. When the modified hydrogel is a gelatin hydrogel, the concentration of gelatin in the hydrogel prepolymer solution is 5-20 w / v%. When the modified hydrogel is a methacrylamide gelatin hydrogel, the composition of the hydrogel prepolymer is as follows: the concentration of methacrylamide gelatin is 5-20 w / v, and the concentration of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone is 0.1-1 w / v. When the modified hydrogel is a sodium alginate hydrogel, the concentration of sodium alginate in the hydrogel prepolymer solution is 1-8 w / v%. When the modified hydrogel is a sodium alginate hydrogel with methacrylamide, the composition of the hydrogel prepolymer is as follows: the concentration of sodium alginate with methacrylamide is 1-8 w / v, and the concentration of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone is 0.1-1 w / v. When the modified hydrogel is a hyaluronic acid hydrogel, the concentration of hyaluronic acid in the hydrogel prepolymer solution is 1-8 w / v%. When the modified hydrogel is a methacrylamide hyaluronic acid hydrogel, the composition of the hydrogel prepolymer is as follows: the concentration of methacrylamide hyaluronic acid is 1-8 w / v, and the concentration of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone is 0.1-1 w / v.

[0014] Based on the above technical solution, step 1 is selected from at least one of chemical crosslinking, photocrosslinking, thermal crosslinking, enzyme crosslinking, and physical crosslinking; The surface modification agent used in step 1 is selected from at least one of gelatin, collagen, fibrinogen and polylysine, with a concentration of 0.1-1 mg / ml; The modified hydrogel described in step 1 has a coating containing cell adhesion sites, the coating being selected from at least one of gelatin, collagen, fibrinogen, and polylysine.

[0015] Based on the above technical solution, in step 2, the culture time in αMEM medium containing 1% penicillin-streptomycin and 100-1000 nM24(S), 25-Epoxycholesterol is 24-48 h. The supernatant is collected, stored at -80-4℃, and then centrifuged at 4000-10000 rpm for 15-60 minutes to remove cells, apoptotic bodies and cell debris. Preferably, the concentration of 24(S),25-Epoxycholesterol is 300-500 nM; The exosome precipitation reagent in step 2 consists of 5-10 w / v% polyethylene glycol phase and 0.1-0.5 mM salt ions, wherein the polyethylene glycol phase is selected from at least one of PEG6000, PEG8000, and PEG10000, and the salt ions are selected from at least one of magnesium chloride, calcium chloride, and sodium chloride. The conditions for centrifugation in step 2 are as follows: The settling time in the centrifugation process is 8-12 hours. The centrifugation speed during static centrifugation is 8000-12000 rpm; The centrifugation time in the static centrifugation is 30-60 minutes.

[0016] According to a third aspect of the present invention, an engineered mesenchymal stem cell exosome preparation is provided for use in the preparation of a fibrotic disease treatment drug, wherein the fibrotic disease treatment drug is administered via intravenous injection, intratracheal injection, or local injection; the local injection includes ocular injection and local liver injection.

[0017] Based on the above technical solution, the fibrotic disease treatment drug reduces the content of pro-fibrotic factor TGF-β1 in the blood of fibrotic model animals to within the range of ±2SD of the mean of the healthy control group, or decreases by more than 25% compared with before treatment.

[0018] Based on the above technical solution, the fibrotic disease treatment drug regulates the polarization of macrophages towards the M2 anti-inflammatory phenotype, thereby increasing the expression level of the M2 marker arginase by 65.4-68.9%, decreasing the levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α by 30-50%, and increasing the levels of anti-inflammatory factors IL-10 and TGF-β1 by 40-60%.

[0019] Beneficial effects (1) The hydrogel prepared by the technical solution disclosed in this invention is precise and controllable and functionally adapted, which is superior to the existing matrix design. The existing hydrogels used for stem cell culture do not emphasize mechanical properties and are difficult to adapt to the regulatory needs of stem cell secretion of exosomes.

[0020] (2) The technical solution disclosed in this invention can increase exosome production, achieve high yield through mechanistic regulation, and break through the existing production bottleneck. In the existing technology, exosomes derived from natural mesenchymal stem cells generally have low secretion yield (no clear high-yield parameters), which makes it difficult to meet the supply needs of clinical treatment formulations. This invention, through a matrix stiffness-dependent regulation mechanism, combined with the soft matrix culture system and the specific activation of 24(S),25-Epoxycholesterol (optimized concentration 300nM-500nM), enables the exosome secretion of a single mesenchymal stem cell to stably reach (3-5)×10⁻⁶. 4This technology significantly increases the yield of natural exosomes compared to existing technologies, addressing the production constraints of exosome preparations for clinical translation from a supply perspective and ensuring sufficient preparations during treatment.

[0021] (3) Through the technical solution disclosed in this invention, the cholesterol content of exosomes is precisely regulated, overcoming the existing delivery defects. Most existing technologies do not focus on the regulation of cholesterol content in exosomes or lack precise control methods, resulting in low cytoplasmic delivery efficiency and poor tissue penetration of exosomes. This invention clearly identifies cholesterol as the core determinant of exosome cytoplasmic delivery. Through the matrix stiffness-dependent regulation mechanism of the ABCA family (key proteins, key molecules that regulate the cholesterol content of stem cell exosomes), combined with the targeted activation of 24(S),25-Epoxycholesterol, the cholesterol content of exosomes is precisely controlled at (3-5 nM) / 5 mg of exosomes. This precise regulation enables exosomes to not only have high cytoplasmic delivery efficiency, but also to achieve excellent penetration in fibrotic lesions, thereby playing a role in reversing fibrosis from the perspectives of vital signs and extracellular matrix microenvironment, breaking through the bottleneck of "insufficient therapeutic efficacy due to uncontrolled cholesterol content" of existing exosomes.

[0022] (4) The technical solution disclosed in this invention enables the controllable preparation system and the standardization of the entire process, ensuring the stability and purity of the formulation, which is superior to the existing process. The existing exosome preparation often lacks a systematic process design, and often results in low purity (containing cell debris and apoptotic bodies), large batch differences, and poor stability due to residual cell impurities, crude purification steps, or inconsistent batch parameters. This invention constructs a fully controllable system for "hydrogel preparation - stem cell culture - exosome purification": stem cell culture uses 3rd-6th generation mesenchymal stem cells, combined with a culture medium containing 10% exosome-free serum to avoid exogenous interference; in the purification stage, cell debris is removed by centrifugation at 4000-10000 rpm for 15-60 minutes, followed by gradient centrifugation (8000-12000 rpm) using a two-step precipitation reagent containing 5-10 w / v% PEG phase and 0.1-0.5 mM salt ions, combined with aseptic control using a 0.22 μm filter membrane to effectively remove impurities and maintain purity; at the same time, the concentration, time, speed and other parameters of each step are clearly defined to achieve batch standardization, significantly improve the stability of the formulation, and overcome the defects of existing processes such as "high randomness and poor purity and stability". Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the hydrogel preparation process for culturing mesenchymal stem cells in Example 1 of the present invention; Figure 2 The results of exosome secretion after culturing hydrogels with different compositions prepared in Examples 1-4 of this invention; Figure 3Transmission electron micrographs of exosomes secreted by hydrogels with different compositions prepared in Examples 1-4 of this invention after culture; Figure 4 These are immunofluorescence images of mesenchymal stem cells treated with and without 24(S),25-Epoxycholesterol in Test Example 2 of this invention, after being cultured in hydrogels of different compositions, and then co-cultured with secreted exosomes and macrophages. Figure 5 Immunofluorescence micrographs of mesenchymal stem cells treated with and without 24(S),25-Epoxycholesterol in Test Example 4 of this invention, after being cultured in hydrogels of different compositions, and their secreted exosomes co-cultured with corneal epithelial cells; Figure 6 This image shows the exosome penetration and retention in lung tissue of an animal model of pulmonary fibrosis after mesenchymal stem cells treated with and without 24(S),25-Epoxycholesterol in Comparative Example 1 of this invention were cultured in hydrogels with different compositions and secreted exosomes. Detailed Implementation

[0024] To make the objectives and technical solutions of this invention clearer, the following embodiments are provided for further explanation. However, the scope of protection of this invention is not limited to these embodiments; the embodiments are merely for illustrative purposes. Those skilled in the art should understand that any changes or equivalent substitutions that do not depart from the concept of this invention are included within the scope of protection of this invention.

[0025] Unless otherwise specified, all reagents and raw materials used in this invention are obtained through purchase.

[0026] Preparation Example 1 Step 1, composition of the hydrogel prepolymer: acrylamide concentration of 8 w / v%, N,N'-methylenebisacrylamide concentration of 0.05 w / v%, N-acryloyloxysuccinimide concentration of 0.5 w / v%, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone concentration of 0.2 w / v (dissolved in 1 ml of pure water to form a 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone) solution, and then the above solutions are mixed to form a homogeneous hydrogel prepolymer.

[0027] Step 2, preparation of hydrogel: The hydrogel prepolymer obtained in Step 1 was sterilized by passing it through a 0.22 μm aqueous filter membrane and then placed at 365 nm and 1200 mW / cm. 2 After irradiation under a UV lamp for 5 minutes, cross-linking occurs to form a hydrogel, the mechanical strength of which is 2 kPa.

[0028] Step 3, Surface Modification: 1 ml of a 0.2 mg / ml type I collagen solution was added dropwise to the surface of 2 g of the hydrogel obtained in Step 2. The surface was then allowed to stand overnight at 4°C to complete the surface modification, resulting in the modified hydrogel. The procedure is as follows: Figure 1 As shown.

[0029] Step 4, preparation of exosomes: The modified hydrogel obtained in Step 3 was washed with PBS to remove residual collagen on the surface. Mesenchymal stem cells were then seeded onto the hydrogel surface and cultured in αMEM medium containing 10% exosome-free serum and 1% penicillin-streptomycin for 24 hours until complete cell attachment. The medium was discarded, and the cells were washed three times with PBS. The medium was then replaced with αMEM medium containing 1% penicillin-streptomycin and 500 nM 24(S), 25-Epoxycholesterol. After culturing for another 48 hours, 100 ml of the supernatant was collected.

[0030] The collected 100 ml supernatant was centrifuged at 10,000 rpm for 60 minutes. The supernatant was then mixed with exosome precipitant 1 (working concentration: 8 w / v% PEG1000 + 0.2 M sodium chloride), allowed to stand overnight, and then centrifuged at 12,000 rpm for 60 minutes. The precipitate was collected and the supernatant was discarded. The precipitate was resuspended in 1 / 5 volume of PBS with the collected supernatant (100 ml). The resuspended precipitate was mixed with exosome precipitant 2, allowed to stand for 120 minutes, and then centrifuged at 12,000 rpm for 60 minutes to obtain exosomes. Western blotting (WB) was used to detect the characteristic surface proteins of the obtained exosomes. The results showed that CD9, CD81, TSG101, Flotilin-2, and CD63 were all strongly positively expressed, and the endoplasmic reticulum marker Calnexin was not expressed, confirming that the extracted product was exosomes. Analysis of exosome charge revealed that the exosomes secreted by mesenchymal stem cells cultured in substrates of varying hardness had a similar charge, ranging from -3.2 mV. Particle size analysis showed that exosome size increased with increasing substrate hardness, ranging from 110 to 140 nm. Secretion volume analysis showed that exosome secretion decreased with increasing substrate hardness, with a single cell secreting approximately 4 × 10⁻⁶ exosomes. 4 Cells Figure 2 (i.e., 8 + 0.05). Transmission electron microscopy images of exosomes secreted by cells under a hardness of 1.2 kPa are shown below. Figure 3 As shown (i.e., 8+0.05).

[0031] Preparation Example 2 The difference from Preparation Example 1 lies in the composition of the hydrogel; specifically, the concentration of N,N'-methylenebisacrylamide in step 1 is 0.1 w / v%, and the mechanical strength of the hydrogel is 4.5 kPa. The remaining steps are consistent with Preparation Example 1. The resulting extract is exosomes with a charge of -4.3 mV, a particle size of 110 nm, and a secretion rate of 3 × 10⁻⁶ exosomes per cell. 4 Transmission electron microscopy image of exosomes per cell, as shown below. Figure 3 As shown (i.e., 8+0.1).

[0032] Preparation Example 3 The difference from Preparation Example 1 lies in the composition of the hydrogel. Specifically, the concentration of N,N'-methylenebisacrylamide in step 1 is 0.2 w / v%, and the mechanical strength of the hydrogel is 15 kPa. The remaining steps are consistent with Preparation Example 1. The extracted product is exosomes with a charge of -5.1 mV, a particle size of 130 nm, and a secretion rate of 2.1 × 10⁻⁶ cells per cell. 4 cells / cell Figure 2 (i.e., 8+0.2), transmission electron microscopy image of exosomes as shown below. Figure 3 As shown (i.e., 8+0.2).

[0033] Preparation Example 4 The difference from Preparation Example 1 lies in the composition of the hydrogel; specifically, the concentration of N,N'-methylenebisacrylamide in step 1 is 0.8 w / v%, and the mechanical strength of the hydrogel is 46 kPa. The remaining steps are consistent with Preparation Example 1. The resulting extract is exosomes with a charge of -5 mV, a particle size of 130 nm, and a secretion rate of 1.5 × 10⁴ exosomes per cell. Figure 2 Transmission electron microscopy (TEM) images of exosomes (8+0.8) are shown below. Figure 3 As shown (i.e., 8+0.8).

[0034] Test Example 1 Detection of cholesterol content in exosomes under different matrix stiffness and 24(S),25-Epoxycholesterol treatment To investigate the effects of matrix stiffness and 24(S),25-Epoxycholesterol on cholesterol content in exosomes secreted by mesenchymal stem cells, this example quantitatively detected cholesterol content in exosome samples prepared in Examples 1-4. The specific process and results are as follows: 1. Experimental Methods Exosomes secreted by mesenchymal stem cells from preparation examples 1-4 in "hydrogel culture systems with different matrix hardness" (containing 4 hardness gradients, i.e., different hydrogel compositions) treated with "without 24(S),25-Epoxycholesterol" and "with 500 nM 24(S),25-Epoxycholesterol" (all adjusted to a uniform concentration of 250 mg / mL) were measured using a commercial cholesterol quantification kit (Abcam Cholesterol Assay Kit) according to the kit instructions. The cholesterol content of each exosome sample was determined by colorimetry, and the results were performed in triplicate and the average value was taken.

[0035] 2. Experimental Results and Analysis In the control group without 24(S),25-Epoxycholesterol, the cholesterol content of exosomes secreted by mesenchymal stem cells gradually increased with the increase of matrix stiffness, and the differences between the stiffness groups were statistically significant. Under the same matrix stiffness conditions, compared with the control group without 24(S),25-Epoxycholesterol, the experimental group with 500 nM 24(S),25-Epoxycholesterol showed a significant increase in exosome cholesterol content, with an average increase of 20%-35%.

[0036] Test Example 2 Detection of the interaction between secreted exosomes and macrophages under different matrix stiffness conditions (Preparation Examples 1-4) This section focuses on the uptake interaction between exosomes secreted by mesenchymal stem cells cultured under different matrix stiffness conditions and macrophages. The specific procedures and results are as follows: 1. DID fluorescent labeling and purification of exosomes Exosomes secreted under different matrix hardness conditions (concentration 1 mg / mL) were mixed with DID dye (concentration 5 μM) and incubated at 37°C in the dark for 20 minutes to achieve fluorescent labeling of exosomes. After incubation, polyethylene glycol 8000 (PEG 8000) was added to the exosome suspension to adjust the final concentration to 5% (w / v), and the mixture was then incubated at 4°C for at least 4 hours. The mixture was then centrifuged at 10,000 rpm for 45 minutes. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in phosphate-buffered saline (PBS) to wash away residual DID dye. To ensure thorough dye washing, this centrifugation-resuspending washing step was repeated 3 times to obtain purified DID-labeled exosomes.

[0037] 2. Co-incubation of exosomes and macrophages and observation of cell staining Purified DID-labeled exosomes were co-incubated with macrophages for 12 hours to construct an exosome-macrophage interaction system. After co-incubation, the culture medium was discarded, and cells were fixed with 4% formaldehyde solution for 15 minutes. After fixation, the cells were washed three times with PBS to remove residual formaldehyde, followed by permeabilization with 0.1% Triton X-100 solution for 5 minutes. After permeabilization, the cells were washed three times again with PBS. Finally, 0.5 μg / mL of 4',6-diamidinyl-2-phenylindole (DAPI) was added to stain the cell nuclei for 5 minutes. After staining, images were captured using a laser confocal scanning microscope (e.g., ...). Figure 4 As shown in the figure, the uptake of exosomes gradually increases with the increase of matrix stiffness, and the overall uptake capacity of exosomes increases after the addition of 24(S),25-Epoxycholesterol.

[0038] Test Example 3 Detection of the immunomodulatory capacity of exosomes secreted under different hardness conditions (preparation examples 1-4) on liver macrophages The immunomodulatory effects of mesenchymal stem cell-derived exosomes cultured in different substrate stiffness on liver macrophages were evaluated using immunofluorescence staining. The influence of 24(S),25-Epoxycholesterol on this regulatory effect was also investigated. Specific procedures and results are as follows: 1. Experimental grouping and co-culture treatment Exosomes secreted by mesenchymal stem cells cultured in hydrogels with four different matrix stiffnesses (1–50 kPa) as described in Examples 1–4 were divided into two groups: 1. Control group (without 24(S),25-Epoxycholesterol); 2. Experimental group (with 500 nM 24(S),25-Epoxycholesterol). The exosomes from each group were co-cultured with liver-derived macrophages for 48 h, with a final exosome concentration of 1 mg / mL. Each group was divided into three replicates.

[0039] 2. Immunofluorescence staining detection After co-culture, the liver macrophages were treated as follows: Fixation and permeabilization: Cells were fixed with 4% paraformaldehyde solution and washed three times with 1×PBS; 0.1% Triton X-100 was added, and permeabilization was performed at room temperature for 15 minutes. Blocking and primary antibody incubation: Cells were blocked with 1% bovine serum albumin (BSA) for 20 minutes; then, specific primary antibodies diluted with blocking buffer, including rabbit iNOS polyclonal antibody (Abcam, catalog number ab15323) or goat liver arginase polyclonal antibody (Abcam, catalog number ab60176), were added, and incubated overnight at 4°C. Secondary antibody incubation: After washing three times with PBS, fluorescent secondary antibody diluted with 1% BSA (1:200 Alexa Fluor 594 labeled goat anti-rabbit IgG, Invitrogen, catalog number A11012; or 1:200 Alexa Fluor 488 labeled donkey anti-goat IgG, Invitrogen, catalog number A11055) was added, and incubated at room temperature in the dark for 60 minutes. Nuclear staining and observation: After washing with PBS, the nuclei were stained with DAPI (Sigma, USA) for 5 minutes, and observed and images were acquired under a laser confocal microscope.

[0040] 3. Results Analysis (1) In the control group, with the increase of matrix stiffness, the fluorescence intensity of iNOS (M1 macrophage marker) of liver macrophage decreased and the fluorescence intensity of arginase (M2 macrophage marker) increased, indicating that the immunomodulatory ability of exosomes on liver macrophages (promoting M2 polarization) gradually increased with the increase of matrix stiffness; (2) In the experimental group (with added 24(S),25-Epoxycholesterol), the immunomodulatory ability of exosomes in each stiffness group was better than that in the control group with the same stiffness. Among them, the regulatory effect of the soft matrix (1-10 kPa) group was the most significant, and the expression level of arginase reached the highest level in each group, suggesting that 24(S),25-Epoxycholesterol can specifically enhance the immunomodulatory activity of exosomes from soft matrix.

[0041] Test Example 4 Detection of the anti-fibrotic ability of exosomes secreted under different hardness conditions (preparation examples 1-4) against corneal epithelial cells The anti-fibrotic effect of exosomes derived from mesenchymal stem cells cultured with different matrix stiffness on corneal epithelial cells was evaluated using immunofluorescence staining, and the influence of 24(S),25-Epoxycholesterol on this effect was investigated. The specific experimental procedures and results are as follows: 1. Experimental grouping and co-culture treatment Exosomes secreted by mesenchymal stem cells cultured in hydrogels with four different matrix hardnesses (1-50 kPa) as described in Examples 1-4 were selected and divided into two groups: A. Control group (exosomes derived from a culture system without 24(S),25-Epoxycholesterol); B. Experimental group (exosomes derived from a culture system supplemented with 500 nM 24(S),25-Epoxycholesterol). Exosomes from each group were co-cultured with corneal epithelial cells for 48 h. The final concentration of exosomes was set at 1 mg / mL for all groups, and three biological replicates were set for each group to ensure the reliability of the results.

[0042] 2. Immunofluorescence staining detection (detection of type I collagen, a marker of fibrosis) After co-culture, corneal epithelial cells were treated as follows to detect the expression level of type I collagen, a key marker of fibrosis: Fixation and permeabilization: The co-culture medium was discarded, and corneal epithelial cells were fixed with 4% paraformaldehyde solution at room temperature. After fixation, the cells were washed three times with 1× phosphate-buffered saline (PBS) for 5 minutes each time. Then, 0.1% Triton X-100 solution was added, and the cells were permeabilized at room temperature for 15 minutes to ensure that the antibody entered the cells and bound the target protein. Blocking and primary antibody incubation: The permeabilization solution was discarded, and 1% bovine serum albumin (BSA) solution was added for blocking at room temperature for 20 minutes to reduce non-specific binding. After blocking, rabbit type I collagen polyclonal antibody (Abcam, catalog number ab34710) diluted with 1% BSA was added, and the cells were incubated overnight at 4°C to allow the primary antibody to fully bind to type I collagen. Secondary antibody incubation: The next day, discard the primary antibody solution and wash the cells three times with 1×PBS for 5 minutes each time; then add Alexa Fluor 594-labeled goat anti-rabbit IgG (H+L) cross-adsorption secondary antibody (Invitrogen, catalog number A11012) diluted 1% BSA at a ratio of 1:200, and incubate at room temperature in the dark for 60 minutes to achieve fluorescent signal labeling. Nuclear staining and observation: Discard the secondary antibody solution and wash the cells three times with 1×PBS; add 4',6-diamidinyl-2-phenylindole (DAPI, Sigma, USA) staining solution and stain at room temperature for 5 minutes to label the cell nuclei; after staining, gently wash with 1×PBS, observe and acquire cell fluorescence images using a laser confocal microscope (e.g., ...). Figure 5 (As shown).

[0043] 3. Experimental Results and Analysis Type I collagen is a core marker of corneal epithelial cell fibrosis; the lower its expression level, the stronger the anti-fibrotic ability of exosomes. Figure 5As can be seen from the results: In the control group, the fluorescence intensity of type I collagen in corneal epithelial cells gradually decreased with increasing matrix hardness, indicating that the anti-fibrotic ability of exosomes against corneal epithelial cells in the control group gradually increased with increasing matrix hardness. In the experimental group, compared with the control group of the same hardness, the anti-fibrotic ability of exosomes was improved in the experimental group with the addition of 24(S),25-Epoxycholesterol. The improvement effect was the most significant in the soft matrix (1-10 kPa) group, and the fluorescence intensity of type I collagen in corneal epithelial cells dropped to the lowest level among all groups, indicating that the anti-fibrotic ability of exosomes from the soft matrix reached the best level after treatment with 24(S),25-Epoxycholesterol.

[0044] Test Example 5 The regulatory effect of exosomes with different matrix stiffness and treated with 24(S),25-Epoxycholesterol on cytokine secretion from lung-derived macrophages This study investigated the regulatory effects of exosomes derived from mesenchymal stem cells cultured in different substrates and treated with 24(S),25-Epoxycholesterol on the secretion of inflammation-related cytokines by lung-derived macrophages using enzyme-linked immunosorbent assay (ELISA). The specific experimental design and results are as follows: Exosomes secreted by mesenchymal stem cells cultured in hydrogels with four different matrix hardnesses (1 kPa-50 kPa) as described in Examples 1-4 were used in the experiment. The cells were divided into two groups based on whether 24(S),25-Epoxycholesterol was added to the culture system: a control group (exosomes derived from a culture system without 24(S),25-Epoxycholesterol) and an experimental group (exosomes derived from a culture system with 500 nM 24(S),25-Epoxycholesterol). All exosomes were resuspended in PBS and the concentration was adjusted to 1 mg / mL. Three biological replicates were set up for each group to ensure the reliability of the results. The target cells were lung-derived macrophages in logarithmic growth phase (seeding density 1×10⁶ cells / well). 5 (per hole).

[0045] In the experiment, lung-derived macrophages were seeded into 24-well plates and cultured adherently at 37°C and 5% CO2 for 24 hours. The original culture medium was then discarded, and serum-free culture medium containing exosomes from either the control or experimental groups (final exosome concentration 1 mg / mL) was added. Co-culture continued for another 48 hours. After co-culture, the cell supernatant from each group was collected and centrifuged at 3000 rpm for 20 minutes to remove cell debris. The levels of five cytokines in the supernatant were then detected using a mouse ELISA kit (Thermo Fisher Scientific, Shanghai) according to the manufacturer's instructions. These cytokines included pro-inflammatory factors (IL-1β, IL-6, TNF-α) and anti-inflammatory factors (IL-10, TGF-β1).

[0046] The results showed that in the control group, as the matrix stiffness increased (1 kPa-50 kPa), the levels of pro-inflammatory factors (IL-1β, IL-6, TNF-α) secreted by lung-derived macrophages gradually decreased, while the levels of anti-inflammatory factors (IL-10, TGF-β1) gradually increased, indicating that the anti-inflammatory capacity of exosomes increased with increasing matrix stiffness. Compared with the control group at the same stiffness, the anti-inflammatory capacity of exosomes in the experimental group with the addition of 24(S),25-Epoxycholesterol was significantly improved, with the most significant improvement in the soft matrix (1-10 kPa) group. The levels of pro-inflammatory factors decreased to the lowest among all groups, while the levels of anti-inflammatory factors increased to the highest among all groups, confirming that the anti-inflammatory regulatory effect of soft matrix-derived exosomes reached its best after treatment with 24(S),25-Epoxycholesterol.

[0047] Test Example 6 Therapeutic effects of soft matrix and 24(S),25-Epoxycholesterol-treated mesenchymal stem cell-derived exosomes (Preparation Example 1, i.e., 8+0.05) on an animal model of liver fibrosis. Using a carbon tetrachloride (CCl4)-induced hepatotoxic liver fibrosis model, we verified the therapeutic effect of exosomes derived from mesenchymal stem cells cultured in soft matrix and treated with 24(S),25-Epoxycholesterol. The specific experimental design and results are as follows: The experiment was divided into 4 groups (6 SPF-grade male SD rats, 6-8 weeks old in each group): A. Blank control group (normal feeding, no modeling or intervention); B. Model control group (modeling only, no exosome intervention); C. Conventional exosome group (modeling + conventional mesenchymal stem cell-derived exosome intervention); D. Experimental group (modeling + mesenchymal stem cell-derived exosome intervention cultured in soft matrix and treated with 500 nM 24(S),25-Epoxycholesterol).

[0048] The liver fibrosis model was constructed using the CCl4 chemical induction method: analytical grade CCl4 was mixed with sterile olive oil at a volume ratio of 1:1.5-1:2 to prepare a 40% CCl4 olive oil solution (prepared fresh and stored away from light); rats in the model control group, conventional exosome group, and experimental group were injected intraperitoneally with a dose of 0.2 mL / 100 g calculated based on body weight, twice a week, for 8-12 weeks (preferably 10 weeks); the blank control group was injected intraperitoneally with an equal volume of sterile olive oil during the same period.

[0049] Exosome intervention began in the 4th week of modeling (at which time early fibrosis of the liver had already formed): both the routine exosome group and the experimental group were injected with exosomes via the tail vein at a dose of 100-200 μg / kg body weight, twice a week, until the end of modeling (i.e., simultaneously with the last 4-8 weeks of the modeling cycle); the model control group was injected with an equal volume of sterile PBS at the same time.

[0050] After modeling, all rats were sacrificed, and venous blood was collected from the orbital cavity. After standing at 4°C for 30 minutes, the blood was centrifuged at 3000 rpm for 15 minutes to collect serum. The levels of nitric oxide (NO) and IL-1β in the serum were detected using a mouse ELISA kit (Thermo Fisher Scientific, Shanghai) according to the instructions.

[0051] The results showed that, compared with the blank control group, the serum NO and IL-1β levels in the model control group were significantly increased (P<0.01); compared with the model control group, the above indicators were downregulated in the conventional exosome group (P<0.05); while the downregulation effect of the experimental group (soft matrix + 24(S),25-Epoxycholesterol treated exosomes) was more significant (P<0.01), and significantly better than that of the conventional exosome group (P<0.05), indicating that mesenchymal stem cell-derived exosomes cultured in soft matrix and treated with 24(S),25-Epoxycholesterol have a better therapeutic effect on liver fibrosis.

[0052] Test Example 7 Therapeutic effects of exosomes on animal models of pulmonary fibrosis Using a bleomycin sulfate (BLM)-induced pulmonary fibrosis (PF) model, we verified the therapeutic effect of mesenchymal stem cell-derived exosomes (Preparation Example 1, i.e., 8+0.05) co-treated with soft matrix culture and 24(S),25-Epoxycholesterol. The experimental procedures and results are as follows: 1. Experimental Design and Grouping This experiment strictly followed the regulations of the Biomedical and Animal Ethics Committee of Dalian University of Technology. Forty 8-week-old male C57 / BL6 mice were randomly divided into four groups (n=10 per group): A. Blank control group (normal feeding, no modeling or intervention); B. Model control group (BLM modeling + saline intervention); C. Conventional exosome group (BLM modeling + conventional mesenchymal stem cell-derived exosome intervention); D. Experimental group (BLM modeling + mesenchymal stem cell-derived exosomes cultured in soft matrix and treated with 500 nM 24(S), 25-Epoxycholesterol, preparation example 1, i.e., 8+0.05).

[0053] 2. Construction of a pulmonary fibrosis model and exosome intervention Model Construction: Mice in the model control group, conventional exosome group, and experimental group were lightly anesthetized and pulmonary fibrosis was induced by a single intratracheal injection of 2 mg / kg BLM solution. The blank control group received an equal volume of physiological saline (without BLM) intratracheally during the same period. Exosome Intervention: On day 7 after BLM injection (early stage of pulmonary fibrosis), exosomes (200 μg / mouse, exosome volume determined by BCA protein quantification) were delivered intratracheally to mice in the conventional exosome group and experimental group. The model control group received an equal volume of physiological saline intratracheally during the same period. The intervention frequency was single-time (or multiple interventions may be added as needed based on therapeutic efficacy).

[0054] 3. Detection Indicators and Result Analysis After the intervention (recommended 21 days after BLM injection, typical stage of pulmonary fibrosis), the following tests were performed on mice in each group: Weight monitoring: Mouse weight was recorded weekly. Results showed that the weight of mice in the model control group continued to decrease; the weight loss trend in the conventional exosome group slowed down; the weight of the experimental group significantly rebounded, with a better reversal effect than the conventional exosome group, indicating that it had a more significant effect on improving the overall health of mice. Lung tissue pathological examination: Lung tissue was collected after euthanizing the mice, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and then stained with HE and Masson's trichrome. HE staining showed that the lung tissue of the model control group showed obvious inflammatory infiltration and alveolar structure destruction; the above pathological changes were alleviated in the conventional exosome group; the inflammatory infiltration in the lung tissue of the experimental group was significantly reduced, and the alveolar structure was closer to normal. Masson staining showed that collagen fiber deposition (blue area) in the lung tissue of the model control group was significantly increased; collagen deposition was reduced in the conventional exosome group; the experimental group had the lowest proportion of collagen deposition area, indicating that it had the best effect on inhibiting the progression of pulmonary fibrosis. In summary, mesenchymal stem cell-derived exosomes co-treated with soft matrix and 24(S),25-Epoxycholesterol exhibit superior therapeutic efficacy for pulmonary fibrosis compared to conventional exosomes. They can significantly improve the weight loss trend in model mice and alleviate lung tissue inflammation and fibrosis.

[0055] Comparative Example 1 Comparison of the penetrability of exosomes derived from mesenchymal stem cells modified with soft matrix and 24(S),25-Epoxycholesterol with animal lung tissue cultured on conventional slabs. This comparative study aimed to compare the penetration rate, uptake rate, and retention rate of mesenchymal stem cell-derived exosomes cultured in soft matrix and modified with 24(S),25-Epoxycholesterol (hereinafter referred to as "modified exosomes") with those cultured on conventional plates (hereinafter referred to as "conventional exosomes") in animal lung tissue. The specific experimental design and results are as follows: This experiment strictly followed the regulations of the Biomedical and Animal Ethics Committee of Dalian University of Technology. Thirty 8-week-old male C57 / BL6 mice were randomly divided into three groups (n=10 per group): A. Traditional exosome group (intratracheal injection of DID-labeled traditional exosomes); B. Modified exosome group (intratracheal injection of DID-labeled modified exosomes). Traditional exosomes were derived from the secretory products of mesenchymal stem cells cultured in conventional two-dimensional plates, while modified exosomes were derived from the secretory products of mesenchymal stem cells cultured in 1-10 kPa soft substrate and treated with 500 nM 24(S),25-Epoxycholesterol. Both groups of exosomes were DID fluorescently labeled, and the concentration was adjusted using the BCA protein quantification method to ensure a single injection dose of 200 μg / mouse.

[0056] After injection, mice in each group were randomly selected to undergo euthanasia on days 1, 3, 5, 7, and 14 post-treatment. Intact lung tissue was rapidly isolated, and fluorescence signals were detected in the lung tissue using a live imaging system (model: LB 983 NC100, made in Germany). The following indicators were analyzed using Image-Pro Plus software: A. Fluorescence signal distribution range (reflecting tissue penetration rate; the wider the range, the stronger the penetration); B. Average fluorescence intensity of lung tissue (reflecting tissue uptake rate; the higher the intensity, the greater the uptake); C. Fluorescence signal decay rate at different time points (reflecting tissue retention rate; the slower the decay, the longer the retention time).

[0057] Results: No obvious DID fluorescence signal was detected in the lung tissue of the blank control group; in the traditional exosome group, weak fluorescence was only detected locally in the lung tissue on day 1, with a limited distribution range (low penetration); the fluorescence intensity began to decay significantly on day 3, and was close to the detection limit on day 7 (poor retention rate); while in the modified exosome group, fluorescence signal was widely distributed throughout the lung tissue on day 1 (significantly better penetration than the traditional group), and the average fluorescence intensity was higher than that of the traditional exosome group. Furthermore, the fluorescence signal decayed slowly in this group, maintaining a high fluorescence intensity until day 7, and a clear fluorescence signal was still detectable on day 14 (significantly better retention rate than the traditional group). Figure 6 In summary, compared with traditional exosomes, modified exosomes have better tissue penetration, tissue uptake and tissue retention rates in animal lung tissue, laying the foundation for their long-term effects in the treatment of pulmonary fibrosis.

[0058] Comparative Example 2 Comparison of the immunomodulatory capacity of exosomes derived from mesenchymal stem cells treated with soft matrix and 24(S),25-Epoxycholesterol with that of exosomes cultured on conventional plates. This comparative study used hepatic macrophages as target cells. By detecting the expression levels of macrophage polarization markers (pro-inflammatory M1 marker iNOS and anti-inflammatory M2 marker arginase), the immunomodulatory capacity of mesenchymal stem cell-derived exosomes cultured in soft substrate and treated with 24(S),25-Epoxycholesterol (hereinafter referred to as "modified exosomes") was compared with that of mesenchymal stem cell-derived exosomes cultured in conventional two-dimensional plates (hereinafter referred to as "traditional exosomes"). The specific experimental design and results are as follows: 1. Experimental Materials and Grouping Experimental materials: Traditional exosomes were secreted products of mesenchymal stem cells cultured on conventional two-dimensional plastic plates, purified by ultracentrifugation, and resuspended in 1×PBS; Modified exosomes were secreted products of mesenchymal stem cells cultured in a 1-10 kPa soft substrate and treated with 500 nM 24(S),25-Epoxycholesterol, purified and resuspended in the same manner as traditional exosomes; The concentration of both groups of exosomes was adjusted by BCA protein quantification to ensure that the final concentration was uniformly 1 mg / mL during subsequent co-culture.

[0059] Experimental groups were formed by using logarithmic growth phase liver macrophages (purchased from ATCC) at a rate of 1×10⁻⁶ cells / year. 5 Exosomes were seeded at a density of 1 cell / well in 24-well plates and cultured adherently at 37°C and 5% CO2 for 24 h. The cells were then randomly divided into three groups (each group had three biological replicates to exclude random errors): Blank control group: DMEM medium containing 10% fetal bovine serum was added, without exosome treatment; Modeling group: Macrophages were induced with 100 ng / ml LPS for 24 h, resulting in M1 macrophage polarization; Conventional exosome group: Macrophages were induced with 100 ng / ml LPS for 24 h, resulting in M1 macrophage polarization, and then medium containing conventional exosomes (final exosome concentration 1 mg / mL) was added. The exosome group was modified, and macrophages were induced with 100 ng / ml LPS for 24 h to induce macrophage M1 polarization. Then, culture medium containing modified exosomes (final exosome concentration 1 mg / mL) was added.

[0060] 2. Co-culture of exosomes and hepatic macrophages and detection by immunofluorescence staining Cells in each group were co-cultured at 37℃ and 5% CO2 for 48 h. After co-culture, the expression of iNOS and arginase was detected by immunofluorescence staining. The specific steps are as follows: Fixation and permeabilization: The co-culture medium in each well was discarded, and 4% paraformaldehyde solution was added to fix the cells at room temperature for 20 minutes (to maintain cell morphology). After fixation, the cells were washed three times with 1×PBS for 5 minutes each time. Then, 0.1% Triton X-100 solution was added, and the cells were permeabilized at room temperature for 15 minutes (to disrupt the cell membrane and ensure that the antibody enters the cell to bind the target protein). After permeabilization, the cells were washed three times again with 1×PBS. Blocking and primary antibody incubation: Discard the permeation solution, add 1% bovine serum albumin (BSA) solution, and block at room temperature for 20 minutes (to reduce non-specific antibody binding); after blocking, add rabbit iNOS polyclonal antibody (Abcam, catalog number ab15323) diluted with 1% BSA or goat liver arginase polyclonal antibody (Abcam, catalog number ab60176) to each well, and incubate overnight at 4°C to ensure that the primary antibody binds fully to the target protein. Secondary antibody incubation: The next day, discard the primary antibody solution and wash the cells three times with 1×PBS for 5 minutes each time. Add the corresponding fluorescent secondary antibody (1:200 dilution) to label goat anti-rabbit IgG (Invitrogen, catalog number A11012) with Alexa Fluor 594 (1:200 dilution) according to the primary antibody source. For arginase, add the primary antibody to label donkey anti-goat IgG (1:200 dilution) with Alexa Fluor 488 (Invitrogen, catalog number A11055) with 1% BSA. Incubate at room temperature in the dark for 60 minutes to achieve fluorescent labeling of the target protein. Nuclear staining and observation: Discard the secondary antibody solution and wash the cells three times with 1×PBS. Add DAPI staining solution and stain at room temperature for 5 minutes to label the cell nuclei. After staining, wash once with 1×PBS and observe and acquire cell fluorescence images using a laser confocal microscope.

[0061] 3. Experimental Results and Analysis The immunomodulatory capacity of hepatic macrophages is mainly manifested in "inhibiting M1 type pro-inflammatory polarization and promoting M2 type anti-inflammatory polarization". The lower the expression level of iNOS (M1 marker) and the higher the expression level of arginase (M2 marker), the stronger the immunomodulatory activity of exosomes. Image-Pro Plus software was used to quantitatively analyze the fluorescence intensity of cells in each group. The results showed that: In the blank control group, liver macrophages exhibited the characteristics of "high iNOS and low arginase," with iNOS fluorescence intensity significantly higher than the other two groups (P<0.01) and arginase fluorescence intensity significantly lower than the other two groups (P<0.01), indicating that the cells were in a pro-inflammatory state; In the traditional exosome group, compared with the blank control group, iNOS fluorescence intensity decreased by 30.2%-34.5% (P<0.05), and arginase fluorescence intensity increased by 28.7%-32.1% (P<0.05), indicating that traditional exosomes have a certain immunomodulatory ability, but the effect is limited; In the modified ... Fluorescence intensity decreased by 62.8%-66.3% (P<0.01), while arginase fluorescence intensity increased by 65.4%-68.9% (P<0.01). Furthermore, compared to the conventional exosome group, the modified exosome group showed significantly greater decreases in iNOS and increases in arginase (P<0.05). In conclusion, modified exosomes (soft matrix + 24(S),25-Epoxycholesterol treatment) more significantly regulated the polarization of hepatic macrophages towards the M2 anti-inflammatory phenotype compared to conventionally plate-cultured exosomes, demonstrating stronger immunomodulatory capabilities.

[0062] Comparative Example 3 Comparison of the antifibrotic ability of exosomes derived from mesenchymal stem cells treated with soft matrix and 24(S),25-Epoxycholesterol with conventionally cultured exosomes This comparative study used corneal epithelial cells as the research object. By detecting the expression level of type I collagen, a core marker of fibrosis, the anti-fibrotic ability of mesenchymal stem cell-derived exosomes treated with soft matrix for 24(S),25-Epoxycholesterol (hereinafter referred to as "modified exosomes") was compared with that of mesenchymal stem cell-derived exosomes cultured in conventional two-dimensional plates (hereinafter referred to as "conventional exosomes"). The specific experimental procedures and results are as follows: 1. Experimental Materials and Grouping Experimental materials: Traditional exosomes were secreted products of mesenchymal stem cells cultured in conventional two-dimensional plates (plastic culture dishes), purified by centrifugation, and resuspended in PBS; Modified exosomes were secreted products of mesenchymal stem cells cultured in 1-10 kPa soft substrate and treated with 500 nM 24(S),25-Epoxycholesterol, purified and resuspended in the same manner as traditional exosomes; The concentration of both groups of exosomes was adjusted by BCA protein quantification method to ensure that the final concentration was uniformly 1 mg / mL during subsequent co-culture.

[0063] Experimental grouping: Corneal epithelial cells in the logarithmic growth phase were divided into groups of 1×10⁻⁶. 5 Exosomes were seeded at a density of 1 cell / well in 24-well plates and cultured adherently at 37°C and 5% CO2 for 24 h. The cells were then randomly divided into three groups (each group had three biological replicates): Blank control group: DMEM medium containing 10% fetal bovine serum was added, without exosome treatment; Modeling group: Macrophages were induced with 100 ng / ml LPS for 24 h, resulting in M1 polarization; Conventional exosome group: Macrophages were induced with 100 ng / ml LPS for 24 h, resulting in M1 polarization, followed by the addition of medium containing conventional exosomes (final exosome concentration 1 mg / mL); Modified exosome group: Macrophages were induced with 100 ng / ml LPS for 24 h, resulting in M1 polarization, followed by the addition of medium containing modified exosomes (final exosome concentration 1 mg / mL).

[0064] 2. Co-culture of exosomes and corneal epithelial cells and detection by immunofluorescence staining Cells in each group were co-cultured at 37℃ and 5% CO2 for 48 h. After co-culture, the expression of type I collagen was detected by immunofluorescence staining. The specific steps were as follows: The co-culture medium in each well was discarded, and corneal epithelial cells were fixed with 4% paraformaldehyde solution at room temperature for 20 minutes (to maintain cell morphology). After fixation, the cells were washed three times with 1× phosphate-buffered saline (PBS) for 5 minutes each time. Then, 0.1% Triton X-100 solution was added, and the cells were permeabilized at room temperature for 15 minutes (to disrupt the cell membrane and facilitate antibody entry into the cells to bind target proteins). After permeabilization, the cells were washed three times again with 1× PBS. The permeabilization solution was discarded, and 1% bovine serum albumin (BSA) solution was added, and the cells were blocked at room temperature for 20 minutes (to reduce non-specific antibody binding). After blocking, rabbit type I collagen polyclonal antibody (Abcam, catalog number ab34710) diluted with 1% BSA was added, and the cells were incubated overnight at 4℃ to ensure that the primary antibody fully binds to type I collagen. The following day, the primary antibody solution was discarded, and the cells were washed three times with 1×PBS for 5 minutes each time. Then, Alexa Fluor 594-labeled goat anti-rabbit IgG (H+L) cross-adsorption secondary antibody (Invitrogen, catalog number A11012), diluted 1% BSA at a ratio of 1:200, was added and incubated at room temperature in the dark for 60 minutes to achieve fluorescent labeling of type I collagen. After incubation, the secondary antibody solution was discarded, and the cells were washed three times with 1×PBS. DAPI staining solution was added, and the cells were stained at room temperature for 5 minutes to label the nuclei. After staining, the cells were gently washed once with 1×PBS, and the fluorescence images were observed and acquired using a laser confocal microscope.

[0065] 3. Experimental Results and Analysis The expression level of type I collagen directly reflects the degree of fibrosis in corneal epithelial cells; the lower the fluorescence signal intensity, the stronger the ability of exosomes to inhibit fibrosis. Quantitative analysis of the fluorescence intensity of type I collagen in each group of cells was performed using Image-Pro Plus software. The results showed that: strong type I collagen fluorescence signals were visible in the corneal epithelial cells of the blank control group, indicating a basic tendency for fibrosis; the fluorescence intensity of type I collagen in the traditional exosome group was reduced by 28.5%-32.1% compared to the blank control group (P<0.05), indicating that traditional exosomes have a certain anti-fibrotic effect; the fluorescence intensity of type I collagen in the modified exosome group was reduced by 61.3%-65.7% compared to the blank control group (P<0.01), and significantly lower than that in the traditional exosome group (P<0.05). In conclusion, the modified exosomes (soft matrix + 24(S),25-Epoxycholesterol treatment) showed a more significant inhibitory effect on corneal epithelial cell fibrosis compared to traditional exosomes, i.e., a stronger anti-fibrotic ability.

Claims

1. A biomechanically responsive engineered mesenchymal stem cell exosome preparation for treating fibrotic inflammatory diseases, characterized in that, The exosome preparation is an exosome derived from mesenchymal stem cells and containing high cholesterol and high-efficacy proteins. The exosomes are prepared by culturing mesenchymal stem cells on a hydrogel matrix with a modulus hardness range of 1-50 kPa. The secretion volume of the exosomes is (3-5) × 10⁻⁶. 4 The exosomes contain (1-10) cholesterol per 5 mg and have a size of 110-140 nm. The high-efficacy protein is a key functional protein that regulates the biological activity of exosomes and the efficacy of cell action.

2. The engineered mesenchymal stem cell exosome preparation according to claim 1, characterized in that, The fibrotic diseases mentioned include pulmonary fibrosis, liver fibrosis, and ocular fibrosis; The key functional proteins are selected from the ABC superfamily, namely the ATP-binding cassette protein gene family, preferably ABCA family genes. The cholesterol content is (3-5) nM / 5mg.

3. A method for preparing the engineered mesenchymal stem cell exosome preparation according to any one of claims 1-2, characterized in that, The preparation method includes the following steps: Step 1, hydrogel modification treatment: After filtering the hydrogel prepolymer solution through a 0.22um filter membrane, a hydrogel matrix with a mechanical strength of 1-50KPa is obtained by cross-linking polymerization. Then, the surface of the hydrogel matrix is ​​modified to obtain the modified hydrogel. Step 2, stem cell culture: Mesenchymal stem cells were seeded onto the modified hydrogel obtained in Step 1 and cultured to passage 3-6 in αMEM medium containing 10% exosome-free serum and 1% penicillin-streptomycin. After washing with PBS, the cells were cultured in αMEM medium containing 1% penicillin-streptomycin and 100-1000 nM 24(S), 25-Epoxycholesterol for 12-48 hours. 100-300 ml of supernatant was collected and stored. Step 3, exosome purification: Centrifuge the supernatant obtained in Step 2, add exosome precipitation reagent, let stand and centrifuge, discard the supernatant, then add PBS at a volume of 1:5-1:10 of the supernatant obtained in Step 2 to resuspend the precipitate and exosome precipitation reagent, let stand and centrifuge, discard the supernatant, and again add PBS at a volume of 1:50-1:100 of the supernatant obtained in Step 2 to resuspend the precipitate to obtain the exosome preparation; The hydrogel matrix is ​​in the form of a block hydrogel or a microcarrier hydrogel.

4. The preparation method according to claim 3, characterized in that, The polyacrylamide hydrogel prepolymer contains 0.1-1 w / v of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, 5-10 w / v of acrylamide, 0.01-1 w / v of N,N'-methylenebisacrylamide, and 0.01-1 w / v of N-acryloyloxysuccinimide.

5. The preparation method according to claim 3, characterized in that, The modified hydrogels include polyacrylamide hydrogel, gelatin hydrogel, methacrylamide gelatin hydrogel, sodium alginate hydrogel, methacrylamide sodium alginate hydrogel, hyaluronic acid hydrogel, and methacrylamide hyaluronic acid hydrogel. When the modified hydrogel is a polyacrylamide hydrogel, the composition of the hydrogel prepolymer is as follows: acrylamide concentration of 6-9 w / v%, N,N'-methylenebisacrylamide concentration of 0.05-0.8 w / v%, N-acryloyloxysuccinimide concentration of 0.05-0.8 w / v%, and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone concentration of 0.3-0.8 w / v%. When the modified hydrogel is a gelatin hydrogel, the concentration of gelatin in the hydrogel prepolymer solution is 5-20 w / v%. When the modified hydrogel is a methacrylamide gelatin hydrogel, the composition of the hydrogel prepolymer is as follows: the concentration of methacrylamide gelatin is 5-20 w / v, and the concentration of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone is 0.1-1 w / v. When the modified hydrogel is a sodium alginate hydrogel, the concentration of sodium alginate in the hydrogel prepolymer solution is 1-8 w / v%. When the modified hydrogel is a sodium alginate hydrogel with methacrylamide, the composition of the hydrogel prepolymer is as follows: the concentration of sodium alginate with methacrylamide is 1-8 w / v, and the concentration of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone is 0.1-1 w / v. When the modified hydrogel is a hyaluronic acid hydrogel, the concentration of hyaluronic acid in the hydrogel prepolymer solution is 1-8 w / v%. When the modified hydrogel is a methacrylamide hyaluronic acid hydrogel, the composition of the hydrogel prepolymer is as follows: the concentration of methacrylamide hyaluronic acid is 1-8 w / v, and the concentration of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone is 0.1-1 w / v.

6. The preparation method according to claim 3, characterized in that, Step 1 is selected from at least one of chemical crosslinking, photocrosslinking, thermal crosslinking, enzyme crosslinking, and physical crosslinking; The surface modification agent used in step 1 is selected from at least one of gelatin, collagen, fibrinogen and polylysine, with a concentration of 0.1-1 mg / ml; The modified hydrogel described in step 1 has a coating containing cell adhesion sites, the coating being selected from at least one of gelatin, collagen, fibrinogen, and polylysine.

7. The preparation method according to claim 3, characterized in that, In step 2, the cells are cultured in αMEM medium containing 1% penicillin-streptomycin and 100-1000 nM24(S),25-Epoxycholesterol for 24-48 hours. The supernatant is collected, stored at -80 to -4℃, and then centrifuged at 4000-10000 rpm for 15-60 minutes to remove cells, apoptotic bodies, and cell debris. Preferably, the concentration of 24(S),25-Epoxycholesterol is 300-500 nM; The exosome precipitation reagent in step 2 consists of 5-10 w / v% polyethylene glycol phase and 0.1-0.5 mM salt ions, wherein the polyethylene glycol phase is selected from at least one of PEG6000, PEG8000, and PEG10000, and the salt ions are selected from at least one of magnesium chloride, calcium chloride, and sodium chloride. The conditions for centrifugation in step 2 are as follows: The settling time in the centrifugation process is 8-12 hours. The centrifugation speed during static centrifugation is 8000-12000 rpm; The centrifugation time in the static centrifugation is 30-60 minutes.

8. The use of any one of the engineered mesenchymal stem cell exosome preparations according to claims 1-2 in the preparation of drugs for treating fibrotic diseases, characterized in that, The medication for treating fibrosis can be administered via intravenous injection, intratracheal injection, or local injection; local injection includes ocular injection and local liver injection.

9. The application according to claim 8, characterized in that, The fibrosis treatment drug reduces the level of pro-fibrotic factor TGF-β1 in the blood of fibrosis model animals to within the mean ± 2 SD range of the healthy control group, or decreases by more than 25% compared with before treatment.

10. The application according to claim 8, characterized in that, The fibrotic disease treatment drug regulates the polarization of macrophages towards the M2 anti-inflammatory phenotype, increasing the expression level of the M2 marker arginase by 65.4-68.9%, decreasing the levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α by 30-50%, and increasing the levels of anti-inflammatory factors IL-10 and TGF-β1 by 40-60%.

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