A method of modifying a lipid vesicle with an amphiphilic polymer containing an N-oxidized tertiary amine group

Abstract

This invention discloses a method for modifying lipid vesicles with an amphiphilic polymer containing N-oxide teramine groups. The amphiphilic polymer is OPDEA-PCL. OPDEA-PCL is used to modify synthetic liposomes, yielding amphiphilic polymer-synthetic liposome nanoparticles. OPDEA-PCL is also used to modify natural exosomes, yielding amphiphilic polymer-exosome nanoparticles. The amphiphilic polymer-synthetic liposomes of this invention possess active liver and lung targeting functions. This invention, through the engineered modification of exosomes with OPDEA-PCL, accurately guides exosomes to the liver regions requiring treatment, thereby enhancing the repair effect of exosomes on liver ischemia-reperfusion injury.

Description

Technical Field

[0001] This invention relates to the medical field, and in particular to a method for modifying lipid vesicles with an amphiphilic polymer containing N-oxide teramine groups. Background Technology

[0002] Lipid vesicles include synthetic liposomes, which are lipid bilayers formed by phospholipid molecules aligning their hydrophobic tails inward and hydrophilic heads outward. They possess biomembrane-like properties and are widely used in drug delivery, nanotechnology, and biomimicry. Lipid vesicles also include exosomes and membrane vesicles derived from cell membranes.

[0003] Doxorubicin is an anthracycline antibiotic widely used in cancer treatment, possessing broad-spectrum antitumor activity. However, its clinical application is limited by serious side effects such as dose-dependent cardiotoxicity. To address this issue, researchers developed a novel drug formulation: liposomal doxorubicin (Liposoma). To further prolong the half-life and stability of the liposomes in vivo, polyethylene glycol (PEG) modification was employed. In the late 1990s and early 2000s, the first PEGylated doxorubicin liposome products (such as DoxiL® in the US market and CaeLyx® in Europe) were launched, providing cancer patients with safer and more effective chemotherapy options. However, PEGylation may reduce cellular uptake, thus affecting the efficiency of drug release within tumor cells. Improving the therapeutic efficacy of doxorubicin liposomes has become an urgent problem to solve.

[0004] 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. In addition, 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. Furthermore, 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.

[0005] 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

[0006] The purpose of this invention is to provide a method for modifying lipid vesicles with an amphiphilic polymer containing N-oxide teramine groups. The lipid vesicles include exosomes, synthetic liposomes, and membrane vesicles derived from cell membranes.

[0007] The technical solution adopted by this invention to solve its technical problem is: A method for synthesizing liposomes by modifying an amphiphilic polymer containing N-oxide teramine groups, wherein the amphiphilic polymer containing N-oxide teramine groups is OPDEA-PCL, and the method includes the following steps: (1) Dissolve the amphiphilic polymer containing N-oxidized tertiary amine groups in an organic solvent to obtain an amphiphilic polymer solution; dissolve the synthetic liposomes in physiological saline to obtain a synthetic liposome solution; (2) At 60±1℃, the amphiphilic polymer solution was added to the synthetic liposome solution and shaken for 2 hours to form stable amphiphilic polymer-synthetic liposome nanoparticles. The modified polymer-liposome nanoparticles were dialyzed with physiological saline to remove organic solvents and free synthetic liposomes, and the amphiphilic polymer-synthetic liposome nanoparticle solution was obtained.

[0008] Preferably, the molar ratio of the amphiphilic polymer containing N-oxide teramine groups to liposomes is 1:100-1:5.

[0009] Preferably, the synthetic liposomes are doxorubicin liposomes, paclitaxel liposomes, or irinotecan liposomes.

[0010] Preferably, the organic solvent is selected from acetonitrile, methanol, and DMSO.

[0011] An amphiphilic polymer-synthetic liposome nanoparticle is prepared by a method of modifying liposomes with an amphiphilic polymer containing N-oxide teramine groups, wherein the amphiphilic polymer containing N-oxide teramine groups is used for external modification of the synthesized liposomes. Lung metastasis of liver cancer is a current challenge in the treatment of liver cancer. The OPDEA-PCL-DoxiL (amphiphilic polymer-synthetic liposome nanoparticles) of this invention can inhibit lung metastasis of liver cancer while fighting liver cancer. This invention can treat both liver and lung cancer simultaneously, and can also target and inhibit lung metastasis of in situ liver cancer, while also being effective against breast and ovarian cancer.

[0012] The principle of OPDEA-PCL insertion into lipid vesicles: OPDEA-PCL is an amphiphilic polymer, with OPDEA as the hydrophilic end and PCL as the hydrophobic end. In the bilayer of the liposome, the inner layer is composed of the fatty acid tail of phospholipid, which is also a hydrophobic environment. Therefore, the PCL segment can be inserted and embedded into the phospholipid tail region like an anchor, and maintain stability through hydrophobic interaction.

[0013] The surface of exosomes also has a lipid bilayer structure of lipid vesicles. The principle of OPDEA-PCL modification of exosomes is the same as that of modification and synthesis of liposomes.

[0014] A method for modifying natural exosomes with an amphiphilic polymer containing N-oxide teramine groups, comprising the following steps: A solution of an amphiphilic polymer containing N-oxide teramine groups was mixed evenly with mesenchymal stem cell exosomes and heated in a water bath to obtain crude exosomes modified with an amphiphilic polymer containing N-oxide teramine groups; the amphiphilic polymer containing N-oxide teramine groups was OPDEA-PCL. The crude exosomes modified with an amphiphilic polymer containing N-oxide teramine groups were dialyzed to remove the free amphiphilic polymer containing N-oxide teramine groups, and finally the finished exosomes modified with an amphiphilic polymer containing N-oxide teramine groups were obtained. The modification with the amphiphilic polymer containing N-oxide teramine groups improves the repair effect of exosomes on hepatic ischemia-reperfusion injury.

[0015] Preferably, the concentration of the amphiphilic polymer solution containing N-oxide tertiary amine groups is 10 mg / mL.

[0016] Preferably, the water bath heating is specifically set to: 37±1℃ water bath heating for 45±1 minutes.

[0017] Preferably, the mass ratio of the amphiphilic polymer containing N-oxide tertiary amine groups to mesenchymal stem cell exosomes is 1:20.

[0018] This invention modifies exosomes with OPDEA-PCL using an improved method, accurately guiding the exosomes to the liver region requiring treatment, thereby enhancing the exosomes' repair effect on liver damage. While OPDEA-PCL is conventionally used for encapsulating nanomedicines, this invention represents a novel application. Those skilled in the art could not predict the effects of combining OPDEA-PCL with functionally complex exosomes; this invention, through exploratory research, achieved this combination and enhanced the exosomes' repair effect on liver damage.

[0019] An exosome modified with an amphiphilic polymer containing N-oxide teramine groups is prepared by the method of modifying natural exosomes with the amphiphilic polymer containing N-oxide teramine groups.

[0020] The beneficial effects of this invention are: 1. The hydrophobic segment of PCL is inserted between liposome membranes (liposome vesicles), while the hydrophilic segment of OPDEA is exposed on the liposome membrane surface. This hydrophilic segment ensures good biocompatibility between the amphiphilic polymer-synthetic liposomes and cell membranes, improving bioavailability. The amphiphilic polymer-synthetic liposomes exhibit active liver and lung targeting capabilities and can accumulate in vivo and within organs for extended periods, achieving efficient drug accumulation in tumor tissues. This can enhance the anti-tumor efficacy of drugs and improve the survival rate of patients with advanced cancer.

[0021] 2. The present invention provides amphiphilic polymer-synthetic liposome nanoparticles that exhibit tumor cell selectivity while minimally harming normal cells, thereby significantly reducing the toxicity of chemotherapy drugs to normal cells.

[0022] 3. By engineering exosomes using OPDEA-PCL, the exosomes can be accurately guided to the liver region requiring treatment, thereby enhancing the repair effect of exosomes on liver ischemia-reperfusion injury. This invention offers new hope to patients undergoing liver surgery, especially liver transplant patients. OP-EXOs are expected to assist in liver transplantation and improve the prognosis of liver transplant patients. Attached Figure Description

[0023] Figure 1 This is a particle size diagram of nanoparticles in Example 1 of the present invention; Figure 2 This is the particle size diagram of nanoparticles in Example 2 of the present invention; Figure 3 This is the particle size diagram of nanoparticles in Example 3 of the present invention; Figure 4 This is the particle size diagram of nanoparticles in Example 4 of the present invention; Figure 5 This is a morphology diagram of nanoparticles in Example 3 of the present invention; Figure 6 These are the results of a cell CCK experiment; Figure 7 These are the results of a cell endocytosis experiment, with time points from left to right: 1 hour and 3 hours. Figure 8 These are experimental results regarding the distribution of drugs in animals; Figure 9 These are experimental results regarding the drug accumulation concentration in the liver and lungs; Figure 10 These are the results of pharmacokinetic experiments; Figure 11 This is a comparison of fluorescence intensity in orthotopic liver tumors in mice under different treatment groups; Figure 12 This is a quantitative fluorescence signal map of the tumor at the endpoint of the liver orthotopic tumor assay; Figure 13 This is a comparison of the fluorescence intensity of lung metastases in mice from different treatment groups; Figure 14 This is a quantitative fluorescence signal map of the tumor as the experimental endpoint for lung metastases; Figure 15 This is a confocal fluorescence microscopy and flow cytometry result of a tumor cell selectivity assay; Figure 16 This is a schematic diagram illustrating the modification principle of the amphiphilic polymer-doxorubicin liposome nanoparticles; Figure 17 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, ns, no statistically significant difference; Figure 18 This study investigated the in vivo distribution of exosomes. PBS-labeled, DiD-labeled MSC-EXOs, and OP-EXOs were intravenously injected into normal mice (n=3). Fluorescence distribution in the mice was observed and quantitatively analyzed 6 hours post-injection. p <0.05, ** p <0.01; Figure 19 This study investigated the in vivo distribution of exosomes after hepatic intrahepatic retinoblastoma (IRI). PBS, DiD-labeled MSC-EXOs, and OP-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 20 These are mouse serological markers; serum ALT, AST, and LDH levels in a mouse IRI model after treatment with PBS, MSC-EXOs, and OP-EXOs (n=8). p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001; Figure 21 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, GPEG-EXOs, and OP-EXOs. Black dashed lines indicate necrotic areas. The images in the bottom row (scale bar 20μm) are magnified versions of the images in 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 22 This shows the TUNEL staining results of liver tissue; TUNEL staining analysis of mouse hepatocyte apoptosis after treatment with PBS, MSC-EXOs, and OP-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 23These 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, GPEG-EXOs, and OP-EXOs (n=6). Scale bar is 100 μm. Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0025] 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.

[0026] 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. The preparation method of OPDEA-PCL is described in Example 1 of patent application No. 202310210058.8.

[0027] Methods for synthesizing liposomes by modifying amphiphilic polymers containing N-oxide teramine groups Example 1: Preparation of amphiphilic polymer-doxorubicin liposome nanoparticles by intercalation 20 mg of OPDEA-PCL was dissolved in 0.2 mL of acetonitrile, an organic solvent, and dispersed evenly. Doxorubicin liposomes were diluted with physiological saline to 0.5 mg / mL. The OPDEA-PCL solution and the doxorubicin liposome solution were mixed at a molar ratio of 1:100. The mixture was shaken at 60 °C and 800 rpm for 2 h to form nanoparticles. The formed amphiphilic polymer-doxorubicin liposomes were dialyzed with physiological saline (dialysis bag 3.5 kDa) to remove the organic solvent and free drug, thus obtaining the amphiphilic polymer-doxorubicin liposome (OPDEA-PCL-DoxiL) nanoparticle solution.

[0028] Nanoparticle size analysis (PDI) was used to determine the particle size of amphiphilic polymer-doxorubicin liposome nanoparticles. Figure 1 The insertion process principle of OPDEA-PCL-DoxiL is explained in [link to OPDEA-PCL-DoxiL insertion process]. Figure 16 .

[0029] Example 2: The difference between this embodiment and Example 1 is that: the molar ratio of OPDEA-PCL to doxorubicin liposomes is 3:100; the particle size of the amphiphilic polymer-doxorubicin liposome nanoparticles was measured using a nanoparticle size analyzer, PDI (… Figure 2 ).

[0030] Example 3: The difference between this embodiment and Example 1 is that: the molar ratio of OPDEA-PCL to doxorubicin liposomes is 1:20; the particle size of the amphiphilic polymer-doxorubicin liposome nanoparticles was measured using a nanoparticle size analyzer, PDI (… Figure 3 ). The morphology of amphiphilic polymer-doxorubicin liposome nanoparticles was observed using transmission electron microscopy. Figure 5 ).

[0031] Example 4: The difference between this embodiment and Example 1 is that: the molar ratio of OPDEA-PCL to doxorubicin liposomes is 1:10; the particle size of the amphiphilic polymer-doxorubicin liposome nanoparticles was measured using a nanoparticle size analyzer, PDI (… Figure 4 ).

[0032] Example 5: The difference between this embodiment and Example 1 is that the molar ratio of OPDEA-PCL to doxorubicin liposomes is 1:5, and the organic solvent is DMSO.

[0033] Experiment 1: Cytotoxicity assays of OPDEA-PCL-DoxiL and DoxiL After co-incubating the nanoparticles from Example 3 and doxorubicin liposomes with H22 cells (mouse liver cancer cells) for 24 hours, the cytotoxicity of the two was compared. The experimental results showed that the cytotoxicity of the OPDEA-PCL-DoxiL nanoparticles was twice that of the doxorubicin liposomes. Figure 6 ) Experiment 2: OPDEA-PCL-DoxiL and DoxiL endocytosis assay Example 3: OPDEA-PCL-DoxiL and DoxiL were diluted to 10 µg / mL and incubated with H22 cells for 1 h or 3 h. Cells were then harvested and flow cytometry was used to detect the positive cell rate. The results showed that OPDEA-PCL-DoxiL could be taken up by liver cancer cells more quickly and efficiently. Figure 7 ).

[0034] Experiment 3: In vivo distribution of OPDEA-PCL / DiD-DoxiL, DiD-DoxiL (DiD is a fluorescent dye added for in vivo tracking) in mice. In Example 3, OPDEA-PCL / DiD-DoxiL and DiD-DoxiL were prepared to a DOX equivalent of 0.5 mg / mL and injected into mice via the tail vein. The distribution of the dye in the mice was monitored at different time points using a small animal imaging system.

[0035] Mice were sacrificed at different time points, and their hearts, livers, spleens, lungs, and kidneys were harvested. Fluorescence intensities of the corresponding organs were imaged using a mouse imaging system. The experimental results showed that OPDEA-PCL-modified nanoparticles accumulated more effectively in the liver and lungs. Figure 8 ).

[0036] Experiment 4: Detection of DOX drug concentrations in mouse liver and lungs In Example 3, OPDEA-PCL-DoxiL was prepared to a DOX equivalent of 0.5 mg / mL and injected into mice via the tail vein. Mice were sacrificed 48 hours after administration, and drugs were extracted from liver and lung tissues. The drug concentration in the organs was determined by HPLC. The experimental results showed that the OPDEA-PCL-modified nanoparticles achieved higher drug concentrations in the liver and lungs. Figure 9 ).

[0037] Experiment 5: Pharmacokinetics of DOX in Mice In Example 3, OPDEA-PCL-DoxiL and DoxiL were diluted to a DOX equivalent of 0.5 mg / mL and injected into ICR mice via the tail vein. After a certain period, blood samples were collected from the mice's eyeballs, and the concentration of DOX in the plasma was determined by HPLC. The experimental results showed that OPDEA-PCL-DoxiL had a longer half-life and a longer blood circulation time. Figure 10 ).

[0038] Experiment 6: In vivo animal model experiment This invention uses BALB / c mice to undergo orthotopic liver inoculation with H22. Luc Liver cancer cells or lung metastases 4T1 Luc A breast cancer model was used, and the drug was administered via tail vein to verify its efficacy at the endpoint.

[0039] H22 vaccination Luc Specific process of liver cancer cell line formation: 15 male BALB / c mice were selected and injected orally with 5*10 mmol / L of the liver. 5 H22 Luc Cells / 25uL / animal, after inoculation wait until the fluorescence intensity reaches 10 6 Mice were then divided into three groups and administered the drug once via tail vein. At the experimental endpoint, the abdominal fluorescence signal of each mouse was captured using a small animal imaging system. The results showed that OPDEA-PCL-Doxil significantly reduced the abdominal tumor burden in mice. Figures 11-12 ).

[0040] 4T1 inoculation for lung metastasis Luc Specific procedure for establishing a breast cancer model: 21 female BALB / c mice were selected and injected with 1*10 g of urea solution via the tail vein. 6 4T1 LucCells / 100µL PBS / animal, after inoculation wait until the fluorescence intensity reaches 10 4 Mice were then divided into three groups and administered the drug once via tail vein. At the experimental endpoint, fluorescence signals in each mouse were captured using a small animal imaging system. The results showed that OPDEA-PCL-Doxil significantly reduced the lung tumor burden in mice. Figures 13-14 ).

[0041] Experiment 6: Tumor Cell Selectivity Experiment Experimental steps: 1*10 6 NIH3T3 cells and 1*10 6 H22 cells were seeded into well plates, and after NIH3T3 cells adhered, nanoparticles (OPDEA-PCL / DoxiL) from Example 3 were added and incubated for another 3 hours. Cell endocytosis was observed using a confocal fluorescence microscope or the positive cell rate was detected by flow cytometry.

[0042] Explanation of tumor selection results ( Figure 15 In a co-incubation system of tumor cells (H22) and normal cells (NIH3T3), after 3 hours of drug administration, the polymer-liposome nanoparticles exhibited a significant uptake advantage by tumor cells. Confocal microscopy revealed that the nanoparticles were mainly distributed within the cytoplasm of tumor cells, while only a small amount were distributed within normal cells. Further quantitative analysis by flow cytometry showed that 88.6% of the nanoparticles entered H22 tumor cells, while only 13.7% entered NIH3T3 normal cells. These results suggest that the nanoparticles of this invention exhibit tumor cell selectivity and minimally harm normal cells, thus significantly reducing the toxicity of chemotherapy drugs to normal cells. This phenomenon may be related to the difference in endocytic pathways between tumor cells and normal cells: normal cells have relatively intact cell membrane structures and mainly rely on lipid raft-mediated endocytosis; while tumor cells have more fluid membranes and tend to take up exogenous nanoparticles through micropinocytosis or macropinocytosis.

[0043] Methods for modifying natural exosomes with amphiphilic polymers containing N-oxide teramine groups Example 6 10 mg / ml of OPDEA-PCL (prepared with 10 mmol / L, pH 7.4 HEPES buffer) was incubated with MSC-EXOs and heated in a 37°C water bath for 45 min to obtain crude OPDEA-PCL-modified MSC-EXOs. The mass ratio of OPDEA-PCL to MSC-EXOs was 1:20. The crude OPDEA-PCL-modified MSC-EXOs was transferred to an activated dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed for 2 hours to remove free OPDEA-PCL. The final product was OPDEA-PCL-modified exosomes (named: OP-EXOs).

[0044] endocytosis of OP-EXOs MSC-EXOs and OP-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 OP-EXOs by AML12 cells, we co-incubated them with MSC-Exos and OP-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.

[0045] like Figure 17 As shown, OP-EXOs enter AML12 cells faster than unmodified MSC-EXOs. AML12 cells can engulf more OP-EXOs in a short period of time.

[0046] In vivo distribution of OP-EXOs 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 an OP-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 OP-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.

[0047] Normal mice were injected with PBS, DiD-labeled MSC-EXOs, or OP-EXOs via the tail vein, and in vivo imaging was performed using a bioluminescence imaging system. Liver IRI model mice were injected with PBS, DiD-labeled MSC-EXOs, or OP-EXOs via the tail vein, and major organs such as the heart, liver, spleen, lungs, and kidneys were harvested and imaged using a bioluminescence imaging system.

[0048] like Figure 18 As shown, the fluorescence intensity in the abdomen of mice in the OP-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 or OP-EXOs were injected via the tail vein. Major organs such as the heart, liver, spleen, lungs, and kidneys were then harvested for imaging. Figure 19 As shown, OP-EXOs accumulate more in the liver compared to MSC-EXOs. Therefore, engineered modifications improve the liver targeting of MSC-EXOs.

[0049] Repairing effect of OP-EXOs on mouse liver injury 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 20 As shown, after injecting MSC-EXOs and OP-EXOs into the mouse liver IRI model via the tail vein, the serum levels of ALT, AST, and LDH in mice decreased, with the OP-EXOs group showing a more significant decrease.

[0050] Pathology is the gold standard for assessing the extent of liver damage. For example... Figure 21 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 OP-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 scores in the OP-EXOs group were significantly lower than those in the MSC-EXOs group. Therefore, exosomes can effectively alleviate hepatic IRI, and OP-EXOs exhibits stronger damage repair capabilities.

[0051] TUNEL can label apoptotic tissue cells. For example... Figure 22 As shown, TUNEL analysis of liver tissue revealed that treatment with MSC-EXO and OP-EXOs reduced apoptosis in mouse liver tissue cells, with OP-EXOs exhibiting a more significant anti-apoptotic effect than MSC-EXOs. Therefore, OP-EXOs can significantly enhance the repair of hepatic IRI.

[0052] Biosafety analysis of OP-EXOs To assess the in vivo biocompatibility of engineered exosomes, we intravenously injected healthy mice with PBS, MSC-EXOs, or OP-EXOs, respectively. Organ specimens were collected from the mice 12 hours after injection, and the heart, liver, spleen, lung, and kidney tissues were stained with hematoxylin and eosin (HE).

[0053] like Figure 23 As shown, HE staining revealed no significant abnormalities in the heart, liver, spleen, lungs, and kidneys of mice after injection of MSC-EXOs and OP-EXOs. Therefore, OP-EXOs are considered to have a high safety profile.

[0054] 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. A method for synthesizing liposomes by modifying an amphiphilic polymer containing an N-oxide teramine group, characterized in that, The amphiphilic polymer containing N-oxide tertiary amine groups is OPDEA-PCL, and the method includes the following steps: (1) Dissolve the amphiphilic polymer containing N-oxidized tertiary amine groups in an organic solvent to obtain an amphiphilic polymer solution; dissolve the synthetic liposomes in physiological saline to obtain a synthetic liposome solution; (2) At 60±1℃, the amphiphilic polymer solution was added to the synthetic liposome solution and shaken for 2 hours to form stable amphiphilic polymer-synthetic liposome nanoparticles. The modified polymer-liposome nanoparticles were dialyzed with physiological saline to remove organic solvents and free synthetic liposomes, and the amphiphilic polymer-synthetic liposome nanoparticle solution was obtained.

2. The method according to claim 1, characterized in that, The molar ratio of amphiphilic polymers containing N-oxidized teramine groups to liposomes is 1:100-1:

5.

3. The method according to claim 1, characterized in that, The synthetic liposomes are doxorubicin liposomes, paclitaxel liposomes, or irinotecan liposomes.

4. The method according to claim 1, characterized in that, The organic solvent is selected from acetonitrile, methanol, and DMSO.

5. An amphiphilic polymer-synthetic liposome nanoparticle, characterized in that, The liposomes are prepared by the method of modifying and synthesizing liposomes with an amphiphilic polymer containing an N-oxide teramine group as described in claim 1, wherein the liposomes are synthesized by intercalation modification with an amphiphilic polymer containing an N-oxide teramine group.

6. A method for modifying natural exosomes with an amphiphilic polymer containing N-oxide teramine groups, characterized in that, Includes the following steps: A solution of an amphiphilic polymer containing N-oxide teramine groups was mixed evenly with mesenchymal stem cell exosomes and heated in a water bath to obtain crude exosomes modified with an amphiphilic polymer containing N-oxide teramine groups; the amphiphilic polymer containing N-oxide teramine groups was OPDEA-PCL. The crude exosomes modified with an amphiphilic polymer containing N-oxide teramine groups were dialyzed to remove the free amphiphilic polymer containing N-oxide teramine groups, and finally the finished exosomes modified with an amphiphilic polymer containing N-oxide teramine groups were obtained. The modification with the amphiphilic polymer containing N-oxide teramine groups improves the repair effect of exosomes on hepatic ischemia-reperfusion injury.

7. The method according to claim 6, characterized in that, The concentration of the amphiphilic polymer solution containing N-oxide tertiary amine groups is 10 mg / mL.

8. The method according to claim 6, characterized in that, The water bath heating is specifically set as follows: 37±1℃ water bath heating for 45±1 minutes.

9. The method according to claim 6, characterized in that, The mass ratio of amphiphilic polymers containing N-oxidized tertiary amine groups to mesenchymal stem cell exosomes is 1:

20.

10. An exosome modified with an amphiphilic polymer containing an N-oxide teramine group, characterized in that, It is prepared by modifying natural exosomes with an amphiphilic polymer containing N-oxide teramine groups as described in claim 6.

Images