Engineering modification method for improving repair effect of exosome on liver injury

Abstract

The invention discloses an engineering modification method for improving the repair effect of an exosome on liver injury, which is characterized in that the exosome is subjected to engineering modification, and the exosome is accurately guided to a liver area needing to be treated, so that the repair effect of the exosome on the liver injury is improved. The invention provides a new hope for patients subjected to liver surgery, especially for liver transplantation patients, and the engineered and modified mesenchymal stem cell exosome is expected to assist liver transplantation and improve prognosis of the liver transplantation patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an engineered modification method for enhancing the liver damage repair effect of exosomes. Background Technology

[0002] Exosomes are important paracrine substances of cells. They are small vesicles, 30-150 nm in diameter, that detach from the plasma membrane and bud. Exosomes are characterized by high stability, low immunogenicity, and good tissue compatibility. Exosomes have been shown to exert therapeutic effects in various liver diseases by inhibiting inflammatory responses, reducing hepatic oxidative stress, and inhibiting the activation and proliferation of hepatic stellate cells. A total of 243 exosome-related clinical studies have been reported on www.ClinicalTrials.gov.

[0003] Mesenchymal stem cell exosomes (MSC-Exos), as a rising star in cell-free therapy strategies, have made significant progress in the field of regenerative medicine in recent years. MSC-Exos exert therapeutic effects through mechanisms such as delivering bioactive substances, regulating immune responses, promoting angiogenesis, and inhibiting apoptosis. Studies have shown that miRNAs carried by MSC-Exos, such as miR-21, miR-146a, and miR-181c, play a crucial role in regulating target gene expression and promoting tissue repair. Furthermore, MSC-Exos can also inhibit inflammatory responses and promote cell proliferation and migration by regulating signaling pathways such as NF-κB, MAPK, and PI3K / Akt. MSC-Exos have shown great potential in the treatment of various diseases. For example, in a myocardial infarction model, MSC-Exos can improve cardiac function by promoting angiogenesis and inhibiting cardiomyocyte apoptosis. In an osteoarthritis model, MSC-Exos can promote cartilage repair by regulating chondrocyte metabolism and inhibiting inflammatory responses. In addition, MSC-Exos also show promising applications in neurological diseases, liver diseases, and skin injuries. However, natural (or unmodified) MSC-EXOs have the characteristic of non-specific organ accumulation, which reduces the utilization efficiency of MSC-EXOs.

[0004] Hepatic ischemia-reperfusion injury (IRI) is a significant cause of liver damage during surgical procedures such as hepatectomy and liver transplantation, and a major cause of post-transplant graft dysfunction. Furthermore, the physiological and pathological processes occurring during hepatic ischemia-reperfusion are diverse and complex. The resulting liver dysfunction can further affect distal organs and cause systemic damage. Hepatic ischemia-reperfusion is a major factor influencing liver surgery-related complications and patient survival prognosis. Current clinical treatment options for IRI in liver transplantation include rapid graft acquisition, shortened graft transport, continuous mechanical perfusion, in situ or remote ischemic preconditioning, and ischemia-free transplantation. Targeted exosome therapy for the liver shows great promise, and improving the role of MSC-Exos in liver injury repair is an urgent problem to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide an engineered modification method to enhance the liver damage repair effect of exosomes. By engineering exosomes, the exosomes can be accurately guided to the liver region that needs treatment, thereby enhancing the liver damage repair effect of exosomes.

[0006] The technical solution adopted by this invention to solve its technical problem is: An engineered modification method to enhance the liver injury repair effect of exosomes, comprising the following steps: Mix DSPE-PEG2000 solution and DSPE-PEG2000-Galactose solution, then mix with mesenchymal stem cell exosomes until homogeneous, and heat in a water bath to obtain crude exosomes modified with DSPE-PEG2000 and DSPE-PEG2000-Galactose. The crude exosomes modified with DSPE-PEG2000 and DSPE-PEG2000-Galactose were dialyzed to remove free DSPE-PEG2000 and DSPE-PEG2000-Galactose, ultimately obtaining the finished exosomes modified with DSPE-PEG2000 and DSPE-PEG2000-Galactose. The water bath heating was specifically set to 37±1℃ for 45±1 minutes.

[0007] The polymer DSPE-PEG2000 (PEG) can prevent the surface aggregation and phagocytosis of nanoparticles, thereby prolonging systemic circulation time. PEG is a safe polymer approved by the US FDA and widely used in drug delivery systems. DSPE-PEG2000-Galactose is a compound grafted with polyethylene glycol (PEG) and galactose onto a phosphatidylethanolamine (DSPE) matrix. DSPE-PEG2000-Galactose is mainly used in the preparation of nanomedicine delivery systems. This invention has found that by co-modifying mesenchymal stem cell exosomes with DSPE-PEG2000 and DSPE-PEG2000-Galactose in a specific ratio, a synergistic effect can be achieved, significantly improving the exosome's repair effect on liver damage.

[0008] In our initial experiments, we found that while DSPE-PEG2000-Galactose alone had a liver-targeting effect, its targeting effect was actually reduced due to its charge, resulting in a counterproductive effect. Further research revealed that only by combining it with a specific ratio of DSPE-PEG2000 could the targeting effect of DSPE-PEG2000-Galactose be fully realized, significantly enhancing the liver repair effect of exosomes.

[0009] The mass ratio of DSPE-PEG2000:DSPE-PEG2000-Galactose:mesenchymal stem cell exosomes is 1:1:20.

[0010] Engineered mesenchymal stem cell exosomes were prepared using the aforementioned engineered modification method to enhance the liver repair effect of exosomes. This invention modifies exosomes with a specific targeting polymer carrier using an improved method, accurately guiding the exosomes to the liver region requiring treatment, thereby enhancing the liver repair effect of exosomes. While targeted polymer carriers are conventionally used for encapsulating nanomedicines, this invention represents a novel application. Those skilled in the art cannot predict the effects of combining polymer carriers with functionally complex exosomes; this invention, through exploratory research, achieves the combination of the two and enhances the liver repair effect of exosomes.

[0011] The liver injury mentioned is liver ischemia-reperfusion injury.

[0012] The beneficial effects of this invention are: by engineering and modifying exosomes, they can be accurately guided to the liver region requiring treatment, thereby enhancing the repair effect of exosomes on liver damage. This invention offers new hope to patients undergoing liver surgery, especially liver transplant patients, as engineered mesenchymal stem cell exosomes are expected to assist in liver transplantation and improve the prognosis of liver transplant patients. Attached Figure Description

[0013] Figure 1 Characterization of mesenchymal stem cell exosomes; (A) Transmission electron microscopy image of MSC-EXOs. (B) Western blot analysis of the expression of MSC-Exos-specific markers Alix, TSG101, CD9, CD81, and Calnexin. (C) NTA-based detection of MSC-EXOs particle size and concentration. Scale bar: 200 nm; Figure 2 Characterization of engineered mesenchymal stem cell exosomes; (A) TEM analysis of GPEG-EXOs. Scale bar: 200 nm. (B) NTA detection of GPEG-EXOs. (C) Zeta potential detection of MSC-EXOs and GPEG-EXOs. Scale bar: 200 nm. * p <0.05; Figure 3 The images show the NTA detection results of engineered mesenchymal stem cell exosomes; (A) NTA detection of MSC-EXOs and GPEG-EXOs on day 3; (B) NTA detection of MSC-EXOs and GPEG-EXOs on day 7. Figure 4 This image shows an endocytosis of exosomes within AML12 cells; intracellular fluorescence signals were detected using confocal microscopy; blue fluorescence signals represent AML12 cell nuclei, and red fluorescence signals represent DiD-labeled exosomes. The scale bar is 50 μm. p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 ns, no statistically significant difference; Figure 5 It represents the expression level of Bcl-2 in AML12 cells; Figure 6 The Western blot analysis was used to detect the HRI repair effects of MSC-EXOs and GPEG-EXOs on AML12 cells. Figure 7This study investigated the in vivo distribution of exosomes. Normal mice (n=3) were intravenously injected with PBS, DiD-labeled MSC-EXOs, or GPEG-EXOs, respectively. Fluorescence distribution in the mice was observed and quantitatively analyzed 6 hours post-injection. p <0.05, ** p <0.01; Figure 8 This study describes the in vivo distribution of exosomes after hepatic intrahepatic retinoblastoma (IRI). PBS, DiD-labeled MSC-EXOs, or GPEG-EXOs were intravenously injected into mice with hepatic IRI (n=3). Six hours post-injection, the major organs of the mice were dissected. The fluorescence intensity of the heart, liver, spleen, lungs, and kidneys was measured and quantitatively analyzed. * p<0.05, ns, no statistically significant difference; Figure 9 These are mouse serological markers; serum ALT, AST, and LDH levels in a mouse IRI model after treatment with PBS, MSC-EXOs, and GPEG-EXOs (n=8). p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001; Figure 10 These are the results of HE staining of liver tissue; HE staining analysis was used to study the histopathological changes in mouse livers after treatment with PBS, MSC-EXOs, and GPEG-EXOs. Black dashed lines indicate necrotic areas. The images in the bottom row (scale bar 20μm) are magnified images of the top row (scale bar 200μm). The degree of liver tissue damage was quantitatively analyzed based on the Antelope Scoring (n=3). * p <0.05, ** p <0.01, **** p <0.0001; Figure 11 These are the results of TUNEL staining in liver tissue; TUNEL staining analysis of mouse hepatocyte apoptosis after treatment with PBS, MSC-EXOs, and GPEG-EXOs. Blue represents cell nuclei, and red represents apoptotic cells. Quantitative analysis of fluorescence intensity (n=3). * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001; Figure 12These are the results of routine blood tests in mice; routine blood tests in mice treated with PBS, MSC-EXOs, and GPEG-EXOs. Neu: neutrophil count, Lym: lymphocyte count, Mon: monocyte count, WBC: white blood cell count, PLT: platelet count, RBC: red blood cell count, HGB: hemoglobin. There were no statistically significant differences in the results. Figure 13 These are the results of HE staining of mouse tissues; HE staining of mouse heart, liver, spleen, lung, and kidney tissues after treatment with PBS, MSC-EXOs, and GPEG-EXOs (n=6). Scale bar is 100 μm. Figure 14 This image shows an image of exosomes endocytosis within AML12 cells. Confocal microscopy was used to detect intracellular fluorescence signals in AML12 cells: blue fluorescence signals represent AML12 cell nuclei, and red fluorescence signals represent DiD-labeled exosomes. Scale bar is 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001. There was no statistically significant difference in ns. Detailed Implementation

[0014] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0015] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0016] The exosomes (MSC-EXOs) used in this invention are all derived from umbilical cord-derived mesenchymal stem cells and were purchased from Beijing Saitong Biotechnology Co., Ltd. DSPE-PEG2000 and DSPE-PEG2000-Galactose were both purchased from Ruixi Biotech Co., Ltd.

[0017] Example 1: An engineered modification method to enhance the liver injury repair effect of exosomes, comprising the following steps: Equal volumes of 10 mg / mL DSPE-PEG2000 (prepared with 10 mmol / L, pH 7.4 HEPES buffer) and 10 mg / mL DSPE-PEG2000-Galactose (prepared with 10 mmol / L, pH 7.4 HEPES buffer) were mixed, and then mixed thoroughly with mesenchymal stem cell exosomes. The mixture was heated in a 37°C water bath for 45 minutes to obtain crude exosomes modified with DSPE-PEG2000 and DSPE-PEG2000-Galactose. The mass ratio of DSPE-PEG2000, DSPE-PEG2000-Galactose to mesenchymal stem cell exosomes was 1:1:20. Crude mesenchymal stem cell exosomes modified with DSPE-PEG2000 and DSPE-PEG2000-Galactose were transferred to an activated dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed for 2 hours to remove free DSPE-PEG2000 and DSPE-PEG2000-Galactose. The resulting exosomes modified with DSPE-PEG2000 and DSPE-PEG2000-Galactose were named GPEG-EXOs.

[0018] Example 2: Characterization of mesenchymal stem cell exosomes and engineered mesenchymal stem cell exosomes The morphology of MSC-EXOs and GPEG-EXOs was observed using transmission electron microscopy. Exosome samples were diluted with PBS and dropped onto a copper grid. After phosphotungstic acid staining, the grid was air-dried and analyzed by transmission electron microscopy. Western blotting was used to detect exosome protein markers: positive exosome markers TSG101, Alix, CD9, and CD81, and negative exosome marker Calnexin (Western blotting was performed by Beijing Enzekangtai Biotechnology Co., Ltd.). The particle size distribution and concentration of MSC-EXOs and GPEG-EXOs were determined using nanoflow cytometry.

[0019] like Figure 1 As shown in Figure A, MSC-EXOs exhibit a typical double-membrane vesicle structure as observed under transmission electron microscopy (TEM). Figure 1 As shown in Figure B, MSC-EXOs express the exosome surface markers Alix, tumor susceptibility gene 101 (TSG101), CD9, and CD81, but do not express calnexin. Figure 1 As shown in Figure C, the average particle size of MSC-EXOs detected by NTA was 76.31 nm, and the concentration was 4.8 × 10⁻⁶. 9 particles / mL.

[0020] like Figure 2 As shown in Figure A, TEM observation revealed that GPEG-EXOs also possess a typical bilayer membrane structure. Figure 2 As shown in Figure B, the average particle size of GPEG-EXOs is 79.53 nm. The average Zeta potential of MSC-EXOs is -2.86 mV. Figure 2 As shown in C, after engineering modification, although the exosome particle size did not change significantly, GPEG-EXOs decreased to -6.65 mV due to the charge shielding properties of DSPE-PEG2000 and DSPE-PEG2000-Galactose.

[0021] Example 3: Stability of engineered mesenchymal stem cell exosomes To simulate the physiological environment, we placed MSC-EXOs and GPEG-EXOs in cell culture medium containing exosome-free serum at 37°C and incubated them for different times (1 day, 3 days, and 7 days, respectively). Then, we measured and analyzed the particle size distribution of exosomes using nanoflow cytometry.

[0022] We assessed the stability of MSC-EXOs and GPEG-EXOs on days 1, 3, and 7, respectively. Figure 3 As shown, the NTA results indicate that the average particle size of MSC-EXOs and GPEG-EXOs is between 70 and 90 nm, suggesting that the engineered mesenchymal stem cell exosomes remain stable for at least 7 days.

[0023] Example 4: Cell endocytosis of engineered mesenchymal stem cell exosomes MSC-EXOs and GPEG-EXOs were labeled with DiD dye, and free dye was removed by dialysis. AML12 cells (mouse liver cells) were cultured in DMEM containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin, and the culture dishes were placed in an incubator at 37°C with 5% carbon dioxide. To investigate the uptake of MSC-Exos and GPEG-EXOs by AML12 cells, we co-incubated them with MSC-Exos and GPEG-EXOs, and observed the images of AML12 cells under a confocal fluorescence microscope at 1 hour and 3 hours. Thirty minutes before each observation time point, Hoechst 33342 nuclear dye was added to the culture dishes, and the cells were observed after washing.

[0024] like Figure 4 As shown, GPEG-EXOs enter AML12 cells faster than unmodified MSC-EXOs. AML12 cells can engulf more engineered mesenchymal stem cell exosomes in a short period of time.

[0025] Example 5: Study on the in vitro damage repair effect of engineered mesenchymal stem cell exosomes Following the method for constructing a cellular HRI model, AML12 cells were cultured in a hypoxic environment (5% CO2 and 1% O2) at 37°C for 12-24 hours, and then transferred to a reoxygenated environment (5% CO2 and 21% O2) at 37°C for 12-24 hours. Anti-apoptotic B-cell lymphoma 2 (Bcl-2) is an anti-apoptotic protein whose expression is significantly reduced in HRI injury. We used AML12 cells hypoxic for 12 hours and reoxygenated for 24 hours to construct an HRI model. Western blot was used to detect the expression of Bcl-2 protein in the cells, such as... Figure 5 As shown, we found that Bcl-2 protein expression was significantly reduced in AML12 cells after 12 hours of hypoxia and 24 hours of reoxygenation, indicating that the HRI model was successfully constructed.

[0026] After AML12 cells were hypoxic for 12 hours, MSC-EXOs and GPEG-EXOs were added to the culture medium, followed by reoxygenation for 24 hours. The expression level of Bcl-2 protein in the cells was detected by Western blot.

[0027] Exosomes have been shown to alleviate HRI in AML12 cells. For example... Figure 6 As shown, GPEG-EXOs can significantly increase the expression level of Bcl-2 protein in AML12 cells, thus GPEG-EXOs can significantly alleviate HRI in AML12 cells.

[0028] Example 6: In vivo distribution experiment of engineered mesenchymal stem cell exosomes in mice Methods for constructing a mouse model of liver ischemia-reperfusion injury Mice were randomly divided into four groups: a sham group, a PBS group, an MSC-EXOs group, and a GPEG-EXOs group, with eight mice in each group. Hepatic blood supply was blocked using a non-traumatic vascular clamp, and the clamp was removed 90 minutes after hepatic ischemia. Mice in the Sham group underwent the same procedure, but the blood vessels were not clamped. After closing the peritoneal cavity, PBS, MSC-EXOs, and GPEG-EXOs were injected into the tail vein, with each mouse receiving 55 μg of exosomes. Six hours after restoring hepatic blood supply, serum and major organs were collected from the mice. All procedures were performed by the same surgeon.

[0029] Normal mice were injected with PBS via the tail vein, along with DiD-labeled MSC-EXOs and GPEG-EXOs, and in vivo imaging was performed using a bioluminescence imaging system. Liver IRI model mice were injected with PBS via the tail vein, along with DiD-labeled MSC-EXOs and GPEG-EXOs. Major organs such as the heart, liver, spleen, lungs, and kidneys were then removed and imaged using a bioluminescence imaging system.

[0030] like Figure 7 As shown, the fluorescence intensity in the abdomen of mice in the GPEG-EXOs group was significantly higher than that in the MSC-EXOs group. A mouse liver IRI model was constructed, and PBS, DiD-labeled MSC-EXOs, and GPEG-EXOs were injected via the tail vein. Major organs such as the heart, liver, spleen, lungs, and kidneys were then harvested for imaging. Figure 8 As shown, GPEG-EXOs accumulate more in the liver compared to MSC-EXOs. Therefore, engineered modification improves the liver targeting of MSC-EXOs.

[0031] Example 7: Repair effect of engineered mesenchymal stem cell exosomes on liver injury in mice 1. Preparation of tissue sections (1) Material preparation: After fixing with 4% paraformaldehyde for 1 day, the tissue was trimmed and prepared for dehydration.

[0032] (2) Dehydration: The tissue was dehydrated in a dehydrator in a gradient of alcohol (75%-85%-90%-95%-100%, each for 2 hours), xylene for 2 hours, and paraffin for 12 hours.

[0033] (3) Embedding and sectioning: The fixed tissue is embedded in an embedding machine, the wax block is trimmed and sectioned, and the section thickness is 5μm.

[0034] 2. Procedure for HE staining of tissues (1) Dewaxing to water: Place tissue sections at 65℃ for 1 hour, then rinse with xylene twice for 20 minutes each, followed by a gradient of alcohol (100%-100%-95%-90%-80%-70%, 10 minutes each) and distilled water for 3 minutes.

[0035] (2) Hematoxylin staining of cell nuclei: hematoxylin staining solution, 1% hydrochloric acid alcohol differentiation, 0.6% ammonia water for blueing, and wash with water after each operation.

[0036] (3) Eosin staining of cytoplasm: Eosin staining solution.

[0037] (4) Dehydration and mounting: 2 times with 95% ethanol (5 minutes), 2 times with 100% ethanol (5 minutes), 2 times with xylene (5 minutes), and then mount with neutral resin.

[0038] (5) Use an optical microscope to acquire and analyze images.

[0039] 3. Serological tests in mice Blood was collected from the mouse eye sockets, and serum was collected for blood biochemistry testing.

[0040] When liver cells are damaged, ALT and AST are released from the liver cytoplasm into the bloodstream, resulting in a significant increase in serum ALT and AST levels. Figure 9 As shown, after injecting MSC-EXOs and GPEG-EXOs into the mouse liver IRI model via the tail vein, the serum levels of ALT, AST and LDH in mice decreased, and the decrease was more pronounced in the GPEG-EXOs group.

[0041] Pathology is the gold standard for assessing the extent of liver damage. For example... Figure 10 As shown, the areas outlined by the black dashed lines in the HE-stained sections represent the damaged liver tissue. Most of the damaged areas after hepatic intraepithelial neoplasia (IRI) are far from the portal vein. HE staining revealed significant loss of liver tissue structural integrity and marked hepatocyte necrosis in the PBS group, while exosome treatment significantly reversed liver necrosis. After GPEG-EXOs treatment, both the degree of liver damage and the area of ​​necrosis were significantly reduced. The Suzuki score was used to quantitatively assess the degree of liver damage. The scores decreased after exosome treatment, and the score in the PEG-EXOs group was significantly lower than that in the MSC-EXOs group. Therefore, exosomes can effectively alleviate hepatic IRI, and GPEG-EXOs exhibits stronger damage repair capabilities.

[0042] TUNEL can label apoptotic tissue cells. For example... Figure 11 As shown, TUNEL analysis of liver tissue revealed that treatment with MSC-EXOs and GPEG-EXOs reduced apoptosis in mouse liver cells, with GPEG-EXOs exhibiting a more significant anti-apoptotic effect than MSC-EXOs. Therefore, GPEG-EXOs can significantly enhance the repair of liver IRI.

[0043] Example 8: Biosafety Analysis of Engineered Mesenchymal Stem Cell Exosomes To assess the in vivo biocompatibility of engineered exosomes, we intravenously injected healthy mice with PBS, MSC-EXOs, and GPEG-EXOs, respectively. Blood and organ samples were collected from the mice 12 hours after injection. Whole blood was used for routine blood tests, serum was used for blood biochemistry tests, and heart, liver, spleen, lung, and kidney tissues were stained with hematoxylin and eosin (HE).

[0044] like Figure 12 As shown, after injection of MSC-EXOs and GPEG-EXOs, the blood routine indicators of mice remained stable and within the normal reference range, showing no significant difference compared with mice injected with PBS. Figure 13 As shown, HE staining revealed no significant abnormalities in the heart, liver, spleen, lungs, and kidneys of mice after injection of MSC-EXOs and GPEG-EXOs. Therefore, engineered mesenchymal stem cell exosomes are considered to have a high safety profile.

[0045] Example 9: Synergistic effect of DSPE-PEG2000 and DSPE-PEG2000-Galactose-modified exosomes Experimental groups: MSC-EXOs, DSPE-PEG-EXOs, DSPE-GPEG-EXOs, GPEG-EXOs MSC-EXOs group: unmodified mesenchymal stem cell exosomes (named: MSC-EXOs).

[0046] DSPE-PEG-EXOs group: DSPE-PEG2000 solution was mixed thoroughly with mesenchymal stem cell exosomes and heated in a water bath to obtain crude DSPE-PEG2000-modified exosomes. The crude DSPE-PEG2000-modified exosomes were then dialyzed to remove free DSPE-PEG2000, finally obtaining the finished DSPE-PEG2000-modified exosomes. Named: DSPE-PEG-EXOs; DSPE-PEG2000: The mass ratio of mesenchymal stem cell exosomes is 1:20.

[0047] DSPE-GPEG-EXOs group: DSPE-PEG2000-Galactose solution was mixed with mesenchymal stem cell exosomes and heated in a water bath to obtain crude DSPE-PEG2000-Galactose modified exosomes; The crude exosomes modified with DSPE-PEG2000-Galactose were dialyzed to remove free DSPE-PEG2000-Galactose, finally obtaining the finished DSPE-PEG2000 modified exosomes. They were named: DSPE-GPEG-EXOs; The mass ratio of DSPE-PEG2000-Galactose to mesenchymal stem cell exosomes is 1:20.

[0048] GPEG-EXOs group: DSPE-PEG2000 solution and DSPE-PEG2000-Galactose solution were mixed, and then mixed evenly with mesenchymal stem cell exosomes. The mixture was heated in a water bath to obtain crude exosomes modified with DSPE-PEG2000 and DSPE-PEG2000-Galactose. The crude exosomes modified with DSPE-PEG2000 and DSPE-PEG2000-Galactose were dialyzed to remove free DSPE-PEG2000 and DSPE-PEG2000-Galactose, finally obtaining the finished exosomes modified with DSPE-PEG2000 and DSPE-PEG2000-Galactose. They were named GPEG-EXOs. The mass ratio of DSPE-PEG2000:DSPE-PEG2000-Galactose:mesenchymal stem cell exosomes is 1:1:20.

[0049] The specific water bath heating settings for the above groups were: 37℃ water bath heating for 45 minutes. The DSPE-PEG2000 was 10 mg / mL, prepared using 10 mmol / L, pH 7.4 HEPES buffer. The DSPE-PEG2000-Galactose was 10 mg / mL, prepared using 10 mmol / L, pH 7.4 HEPES buffer. The dialysis bag was an activated dialysis bag with a molecular weight cutoff of 3.5 kDa.

[0050] MSC-EXOs, DSPE-PEG-EXOs, DSPE-GPEG-EXOs, and GPEG-EXOs were labeled with DiD dye, and free dye was removed by dialysis. AML12 cells (mouse liver cells) were cultured in DMEM containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin, and the culture dishes were placed in an incubator at 37°C with 5% carbon dioxide. To investigate the uptake of MSC-EXOs, DSPE-PEG-EXOs, DSPE-GPEG-EXOs, and GPEG-EXOs by AML12 cells, we co-incubated them with AML12 cells and observed the images of AML12 cells under a confocal fluorescence microscope at 1 hour and 3 hours. Thirty minutes before each observation time point, Hoechst 33342 nuclear dye was added to the culture dishes, and the cells were observed after washing.

[0051] like Figure 14 The results show that DSPE-PEG-EXOs entered AML12 cells the slowest, while GPEG-EXOs entered AML12 cells faster than both DSPE-PEG-EXOs and DSPE-GPEG-EXOs. Specific fluorescence detection data are shown in the table below: Mean fluorescence intensity during endocytosis .

[0052] The synergistic effect was calculated using the Bliss independent model. AB (Theoretical) = E A+E B -E A *E B If E AB (Actual) > E AB (Theoretical): A synergistic effect has been demonstrated. Calculations showed that for the GPEG-EXOs group, the predicted effect at 1 hour was 0.984, while the actual effect was 1.088, indicating a greater actual effect than the theoretical effect, thus demonstrating a synergistic effect. Similarly, the predicted effect at 3 hours was 0.999, while the actual effect was 1.080, again indicating a synergistic effect. Therefore, the 1:1 mixture of DSPE-PEG2000 and DSPE-PEG2000-Galactose for engineering mesenchymal stem cell exosomes exhibits a synergistic effect, potentially improving the endocytosis efficiency of engineered mesenchymal stem cell exosomes by AML12 cells.

[0053] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. An engineered modification method for improving the liver injury repair effect of exosomes, characterized in that, The steps are as follows: Mix the DSPE-PEG2000 solution and the DSPE-PEG2000-Galactose solution, then mix them with the mesenchymal stem cell exosomes uniformly, and heat in a water bath to obtain the DSPE-PEG2000 and DSPE-PEG2000-Galactose modified exosome crude product; Dialyze the DSPE-PEG2000 and DSPE-PEG2000-Galactose modified exosome crude product to remove free DSPE-PEG2000 and DSPE-PEG2000-Galactose, and finally obtain the finished product DSPE-PEG2000 and DSPE-PEG2000-Galactose modified exosome.

2. The method of engineered modification of claim 1, wherein, The water bath heating is specifically set as 37±1℃ water bath heating for 45±1 minutes.

3. The method of engineered modification of claim 1, wherein, The mass ratio of DSPE-PEG2000: DSPE-PEG2000-Galactose: mesenchymal stem cell exosome = 1:1:

20.

4. The engineered modified mesenchymal stem cell exosome prepared by the method for improving the repair effect of exosomes on liver injury according to claim 1.

5. The method of engineered modification of claim 1, wherein, The liver injury is liver ischemia-reperfusion injury.

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