Engineered exosome as well as preparation therefor and use thereof

Abstract

Provided are an engineered exosome, preparation therefor and the use thereof. Introducing a small molecule Sephin1 having the endoplasmic reticulum homeostasis maintenance effect into a mesenchymal stem cell-derived exosome by means of intermittent ultrasond technology effectively reduces the expression of stem cell endoplasmic reticulum disorder related gene proteins in a high-sugar-content environment, promotes stem cell proliferation in a high-sugar-content environment, down-regulates the apoptosis level of stem cells, and promotes osteogenic differentiation of stem cells, thus effectively improving the treatment efficiency of the mesenchymal stem cell-derived exosome.

Description

An engineered exosome and its preparation and application Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to engineered exosomes and their preparation and application. Background Art

[0002] Extracellular vesicles play a crucial role in intercellular communication, acting as transport vehicles for signaling molecules, delivering a variety of functional molecules, such as DNA, RNA, proteins, and lipids, to recipient cells. Mesenchymal stem cell-derived exosomes (MSC-derived exosomes) are MSC-secreted EVs with diameters ranging from approximately 40 to 160 nm. Due to their similar pro-regenerative properties to their parental cells, MSC-derived exosomes have attracted intense interest and been extensively studied. Furthermore, as natural nanovesicles, MSC-derived exosomes offer distinct advantages over artificial nanomaterials, including low immunogenicity, improved biocompatibility, easy uptake by recipient cells, and amenability to engineering. However, the regenerative therapeutic effects of exosomes alone are limited. Therefore, enhancing the functionalization of exosomes to improve therapeutic efficacy is a pressing issue.

[0003] The endoplasmic reticulum (ER) is a key intracellular membrane organelle with numerous essential functions, including protein synthesis, folding, assembly, and transport, regulation of lipid synthesis, ion signaling, and cellular stress regulation. The ER interacts with virtually all other organelles, and maintenance of ER homeostasis is crucial for normal cellular function. Previous studies have demonstrated that diabetic hyperglycemia can induce persistent and robust ER stress in various tissues, leading to impaired cellular function and subsequent pathophysiological changes.

[0004] Therefore, it is crucial to provide an engineered exosome that can restore the regenerative function of stem cells damaged by high-glucose environments. Summary of the Invention

[0005] To address the above issues, the present invention aims to provide engineered exosomes, their preparation, and use. This invention utilizes intermittent ultrasound technology to combine mesenchymal stem cell-derived exosomes with the small molecule Sephin1, which maintains endoplasmic reticulum homeostasis, to construct engineered exosomes. This functionalizes the mesenchymal stem cell-derived exosomes, improving their therapeutic efficacy while ensuring safety.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The first object of the present invention is to provide an engineered exosome, wherein the engineered exosome is an exosome into which Sephin1 (hereinafter referred to as "Sep") is introduced; the engineered exosome has the function of maintaining endoplasmic reticulum homeostasis.

[0008] A second object of the present invention is to provide a method for preparing engineered exosomes, comprising the following steps:

[0009] (S1) placing mesenchymal stem cells in α-MEM medium containing FBS for primary culture and expansion, and then placing them in exosome-free medium for secondary culture to obtain culture fluid;

[0010] (S2) subjecting the culture fluid obtained in step (S1) to continuous centrifugation, collecting the supernatant, and post-processing to obtain mesenchymal stem cell-derived exosomes;

[0011] (S3) Sep is introduced into the mesenchymal stem cell-derived exosomes prepared in step (S2) by intermittent ultrasound, and post-processed to obtain engineered exosomes that maintain the homeostasis of the cellular endoplasmic reticulum: Sep@Exo.

[0012] In one embodiment of the present invention, in step (S1), during the primary culture process, the cells are placed in a 37° C. 5% CO 2 incubator and cultured until the cell confluence reaches 70% to 80%.

[0013] In one embodiment of the present invention, in step (S1), the secondary culture process is carried out in a 5% CO2 incubator at a temperature of 37°C for 40 to 60 hours.

[0014] In one embodiment of the present invention, in step (S2), the continuous centrifugation is specifically as follows:

[0015] The culture medium was centrifuged at 200-500 g, 1500-2500 g, and 8000-12000 g in sequence. After each centrifugation, the precipitate was discarded and the supernatant was collected.

[0016] In one embodiment of the present invention, in step (S2), the post-treatment is to filter the supernatant and then centrifuge it, then wash it in PBS, and resuspend it in PBS after centrifugation.

[0017] In one embodiment of the present invention, in step (S3), the ratio of Sep to mesenchymal stem cell-derived exosomes is 10-15 μg:1 mg.

[0018] In one embodiment of the present invention, in step (S3), during the intermittent ultrasonic process, the ultrasonic parameters are 10% to 30% amplitude, 5 to 15 seconds on / off, 1 to 3 minutes duration, 4 to 8 cycles, and a 1 to 3 minute cooling period between each cycle.

[0019] In one embodiment of the present invention, in step (S3), the post-treatment is incubation at 37°C followed by purification.

[0020] The third object of the present invention is to provide an application of engineered exosomes in restoring the regenerative function of stem cells damaged in a high-glucose environment.

[0021] In one embodiment of the present invention, the drug is a drug that reduces the expression of gene proteins related to endoplasmic reticulum disorder in stem cells under a high glucose environment.

[0022] In one embodiment of the present invention, the drug is a drug that promotes stem cell proliferation in a high-glucose environment.

[0023] In one embodiment of the present invention, the drug is a drug that downregulates the level of stem cell apoptosis.

[0024] In one embodiment of the present invention, the drug is a drug that promotes osteogenic differentiation of stem cells.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The engineered exosomes prepared by the present invention solve the problems of endoplasmic reticulum homeostasis disorder, impaired stem cell function, and poor bone tissue healing effect in the high-glucose microenvironment of diabetes. The present invention uses intermittent ultrasound technology to introduce the small molecule Sep with the function of maintaining endoplasmic reticulum homeostasis into mesenchymal stem cell-derived exosomes, effectively reducing the expression of gene proteins related to endoplasmic reticulum disorder, promoting stem cell proliferation in a high-glucose environment, downregulating the level of stem cell apoptosis, promoting stem cell osteogenic differentiation, and effectively optimizing the therapeutic efficiency of mesenchymal stem cell-derived exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Scanning electron micrographs of exosomes and engineered exosomes.

[0028] Figure 2 Particle size distribution of exosomes and engineered exosomes.

[0029] Figure 3 Western blot images of exosomes and engineered exosomes.

[0030] Figure 4 Schematic diagram of stem cell proliferation activity after adding exosomes and engineered exosomes to high-glucose culture medium.

[0031] Figure 5 Schematic diagram of stem cell apoptosis levels after adding exosomes and engineered exosomes to high-glucose culture medium.

[0032] Figure 6 Schematic diagram of the expression levels of genes related to endoplasmic reticulum disorder in stem cells after the addition of exosomes and engineered exosomes to high-glucose culture medium.

[0033] Figure 7 Schematic diagram of the expression levels of proteins related to endoplasmic reticulum disorder in stem cells after the addition of exosomes and engineered exosomes to high-glucose culture medium.

[0034] Figure 8 Schematic diagram of alkaline phosphatase staining of stem cell osteogenic induction after addition of exosomes and engineered exosomes to high glucose medium (100 μm).

[0035] Figure 9 Schematic diagram of Alizarin red staining of stem cell osteogenic induction after addition of exosomes and engineered exosomes to high glucose medium (100 μm). DETAILED DESCRIPTION

[0036] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all detection means and methods used are conventional detection means and methods in the art.

[0038] Example 1

[0039] This embodiment provides an engineered exosome (Sep@Exo) and a preparation method thereof.

[0040] (S1) Mesenchymal stem cells were cultured in α-MEM medium containing 10% FBS (37°C, 5% CO2 incubator). When the cells reached 70-80% confluence, the original medium was replaced with α-MEM medium without exosomes (10% FBS). After 48 hours of culture (37°C, 5% CO2 incubator), the supernatant was collected.

[0041] (S2) isolating exosomes from the collected supernatant by continuous centrifugation; specifically, the collected supernatant is centrifuged at 300 g for 10 minutes at 4°C, the precipitate is discarded, the supernatant is collected, the supernatant is centrifuged at 2000 g for 20 minutes, the precipitate is discarded, the supernatant is collected, and the supernatant is centrifuged at 10,000 g for 30 minutes, the precipitate is discarded, and the supernatant is collected to remove cells and debris; next, the supernatant is filtered using a 0.22 μm filter (sterile), and then centrifuged at 100,000 g for 90 minutes at 4°C using an ultracentrifuge. The exosome pellet is washed in PBS and ultracentrifuged again at 100,000 g for 90 minutes; the exosome pellet is then resuspended in PBS to obtain the exosome product;

[0042] (S3) Sep was introduced into Exosomes via intermittent sonication. Specifically, 12 μg of Sep was mixed with 1 mg of exosome product in 1 mL of PBS to obtain a suspension. The suspension was then subjected to intermittent and gentle sonication using a sonicator with 20% amplitude, 10-second on / off, 2-minute duration, and 6 cycles, with a 2-minute cool-down period between each cycle. Following sonication, the exosomes were incubated at 37°C for 2 hours to restore the exosome membrane structure. Finally, unloaded Sep molecules were removed using ultrafiltration to obtain purified Sep@Exo.

[0043] Performance Analysis:

[0044] (1) Characterization of Exo (exosome product obtained in step (S2) above) and Sep@Exo: For morphological analysis, exosomes were fixed with 2.5% glutaraldehyde and placed on an electron microscope grid, then stained with 1% uranyl acetate solution for 2 minutes. After washing with distilled water and drying, images were captured using a transmission electron microscope at 120 kV. As shown in Figure 1, Exo and Sep@Exo exhibited the typical cup-shaped or spherical morphology of exosomes. Exosomes were suspended in PBS, and the size distribution of exosomes was measured using a particle size analyzer. As shown in Figure 2, Exo and Sep@Exo had similar particle size distributions, concentrated in the range of 40 to 160 nm. Exosome marker proteins were detected using Western blot (WB). As shown in Figure 3, Exo and Sep@Exo specifically contained exosome marker proteins CD9, CD81, Alix, and TSG101, and almost no negative marker protein Calnexin was expressed.

[0045] (2) Cell culture: Mesenchymal stem cells were isolated from the femur and tibia of 3-week-old male Sprague Dawley (SD) rats. Specifically, after removing both ends of the femur and tibia, the cells were washed from the bone marrow and cultured in normal glucose medium (α-MEM containing 5.5 mM D-glucose) containing 10% FBS and 1% penicillin-streptomycin at 37°C in an incubator containing 5% CO2. Mesenchymal stem cells at passages 2-4 and with a growth density of 80-90% confluence were used for subsequent experiments. To simulate hyperglycemia, a high glucose (HG) concentration of 35 mM was used, and HG medium was prepared by adding 29.5 mM D-glucose to normal glucose medium. Commercial Cyagen osteogenic medium was used for osteogenic induction (OI). Exo and Sep@Exo were both used at a concentration of 50 μg / mL.

[0046] (3) Cell proliferation activity detection: After 1, 4, and 7 days of cell culture, cell viability was assessed using a cell counting kit. Specifically, the culture medium was discarded, 110 μL of CCK-8 working solution was added, and the cells were incubated at 37°C for 2 h. The OD value was detected at a wavelength of 450 nm using a microplate reader. The results are shown in Figure 4 (CON is the control group, HG is the high glucose concentration group, HG-Exo is the Exo group under high glucose concentration, and HG-Sep@Exo is the Sep@Exo group under high glucose concentration). The high glucose environment damaged the cell proliferation activity; compared with the HG group, the HG-Exo group showed increased cell viability, and the HG-Sep@Exo group showed better therapeutic effect.

[0047] (4) Detection of cell apoptosis level: After 7 days of cell culture, the Annexin V-FITC / PI apoptosis detection kit was used to analyze the apoptosis rate of cells in each group. Specifically, the cells were digested and collected, washed once with PBS buffer, washed once with binding buffer, and then resuspended in 195 μL binding buffer supplemented with 5 μL Annexin V-FITC and 10 μL PI staining solution. Incubated in the dark at room temperature for 15 minutes, washed with buffer, and analyzed by flow cytometry. The results are shown in Figure 5 (CON is the control group, HG is the high glucose concentration group, Exo is the Exo group under high glucose concentration, and HG-Sep@Exo is the Sep@Exo group under high glucose concentration). The high glucose environment increased the level of cell apoptosis. Compared with the HG group, the HG-Exo group showed a downregulated apoptosis rate, while the apoptosis rate of the HG-Sep@Exo group was almost restored to normal.

[0048] (5) RT-PCR detection of gene expression: After 7 days of cell culture, total cell RNA was extracted using RNAiso Plus and then reverse transcribed into cDNA using the PrimeScript First-Strand cDNA Synthesis Kit. Real-time PCR was prepared with the Green Master Mix kit and performed in a LightCycler 480 PCR system. The target genes were GRP78, CHOP, GRP94, PERK, ATF6, and ENR1. GAPDH was used as an internal reference gene, and 2 -ΔΔCT Methods: Relative gene expression levels were analyzed. As shown in Figure 6 (CON: control group, HG: high glucose group, HG-Exo: Exo group under high glucose, and HG-Sep@Exo: Sep@Exo group under high glucose), the expression levels of genes associated with endoplasmic reticulum dysregulation increased under high glucose conditions. Compared with the HG group, the HG-Exo group showed a downward trend, while the HG-Sep@Exo group exhibited a more significant effect.

[0049] (6) Western blot detection of protein expression: Cells or exosomes were lysed with RIPA lysis buffer supplemented with protease inhibitors and sonicated in an ice bath for 30 minutes. The protein concentration was quantified using a BCA protein detection kit and adjusted to be consistent. The samples were placed in a 100°C metal bath for 7 minutes. Each group of protein samples was separated by 4%-20% gradient SDS-PAGE gel and then transferred to a polyvinylidene difluoride membrane. Next, the membrane was incubated with 5% BSA for 1 hour, incubated with a specific primary antibody at 4°C overnight, washed 3 times with PBS on a shaker, incubated with a secondary antibody for 1 hour, and washed 3 times with PBS on a shaker. After the membrane reacted with the ECL reagent, the signal was captured using a membrane imaging system. The results are shown in Figure 7 (CON is the control group, HG is the high glucose concentration group, HG-Exo is the Exo group under high glucose concentration, and HG-Sep@Exo is the Sep@Exo group under high glucose concentration). The expression level of proteins related to endoplasmic reticulum dysregulation increased under high glucose conditions. Compared with the HG group, the HG-Exo group showed a downward trend, and the HG-Sep@Exo group showed a more significant downward trend, which is consistent with the results in Figure 6 ; this indicates that Exo itself has a certain role in maintaining endoplasmic reticulum homeostasis, and the engineered exosomes Sep@Exo enhance its role due to the introduction of Sep.

[0050] (7) Alkaline phosphatase staining: After 7 days of osteogenic induction (OI), the samples were stained using a BCIP / NBT alkaline phosphatase colorimetric kit. Specifically, the culture medium was discarded, the cells were washed 3 times with PBS, fixed in 4% PFA for 20 minutes, washed 3 times with PBS, incubated with alkaline phosphatase colorimetric working solution for 30 minutes, washed 3 times with PBS, and images were collected using a scanner and microscope. The results are shown in Figure 8. Alkaline phosphatase is an important marker for osteogenic differentiation. The results showed that compared with the OI group, the alkaline phosphatase staining in the OI-HG group was very light. The high sugar environment destroyed the osteogenic differentiation function of stem cells, which was improved in the OI-HG-Exo group, while the staining in the OI-HG-Sep@Exo group was darker.

[0051] (8) Alizarin red staining: After 21 days of osteogenic induction (OI), the culture medium was discarded, the cells were washed 3 times with PBS, fixed in 4% PFA for 20 minutes, and washed 3 times with double-distilled water. 1% alizarin red was used for staining for 20 minutes, and images were collected using a scanner and microscope. The results are shown in Figure 9. Alizarin red staining was used to detect calcium salt deposition during osteogenic differentiation. The results showed that compared with the OI group, calcium salt deposition was less common in the OI-HG group, which was improved in the OI-HG-Exo group, while the alizarin red staining in the OI-HG-Sep@Exo group was darker. This indicates that engineered exosomes effectively rescued the osteogenic differentiation ability of stem cells while maintaining endoplasmic reticulum homeostasis.

[0052] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the explanations of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. An engineered exosome, characterized in that, The engineered exosomes are exosomes into which Sephin1 is introduced; the engineered exosomes have the function of maintaining endoplasmic reticulum homeostasis.

2. A method for preparing the engineered exosomes as described in claim 1, characterized in that, It includes the following steps: (S1) The mesenchymal stem cells are placed in α-MEM medium containing FBS for primary culture and amplification, and then placed in exosome-free medium for secondary culture to obtain a culture solution. (S2) The culture solution obtained in step (S1) is centrifuged continuously, and the supernatant is collected, and then processed to obtain mesenchymal stem cell-derived exosomes. (S3) Sephin1 is introduced into the mesenchymal stem cell-derived exosomes prepared in step (S2) by intermittent ultrasound, and then processed to obtain engineered exosomes that maintain endoplasmic reticulum homeostasis in cells: Sep@Exo.

3. The preparation method of an engineered exosome according to claim 2, wherein, In step (S2), the continuous centrifugation is specifically as follows: The culture solution is centrifuged at 200-500 g, 1500-2500 g, and 8000-12000 g in sequence. After each centrifugation, the precipitate is discarded and the supernatant is collected.

4. The preparation method of an engineered exosome according to claim 2, wherein, In step (S3), the dosage ratio of Sephin1 to mesenchymal stem cell-derived exosomes is 10-15 μg: 1 mg.

5. The preparation method of an engineered exosome according to claim 2, characterized in that, In step (S3), during the intermittent ultrasound process, the ultrasound parameters are 10%-30% amplitude, 5-15 seconds on / off, 1-3 minutes duration, 4-8 cycles, and there is a 1-3 minute cooling period between each cycle.

6. Use of the engineered exosomes as described in claim 1 in the preparation of a drug for restoring the regenerative function of stem cells damaged in a high-glucose environment.

7. Use of an engineered exosome according to claim 6 in the preparation of a drug for restoring the regenerative function of stem cells damaged in a hyperglycemic environment, characterized in that, The drug is a drug that reduces the expression of proteins of genes related to endoplasmic reticulum dysregulation in stem cells under a high-glucose environment.

8. Use of an engineered exosome according to claim 6 in the preparation of a drug for restoring the regenerative function of stem cells damaged in a hyperglycemic environment, characterized in that, The drug is a drug that promotes the proliferation of stem cells under a high-glucose environment.

9. Use of an engineered exosome according to claim 6 in the preparation of a drug for restoring the regenerative function of stem cells damaged in a hyperglycemic environment, characterized in that, The drug is a drug that downregulates the apoptosis level of stem cells.

10. Use of an engineered exosome in the preparation of a drug for restoring the regenerative function of stem cells damaged in a hyperglycemic environment, characterized in that, The drug is a drug that promotes the osteogenic differentiation of stem cells.

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