Drug-loaded exosomes for cirrhosis-liver cancer with targeting property and preparation method and application thereof

Abstract

This invention discloses a targeted drug-loaded exosome for liver cirrhosis-hepatocellular carcinoma, its preparation method, and its applications. The targeted drug-loaded exosome comprises exosomes encapsulated with indocyanine green, and the surface of the drug-loaded exosome is linked to an aptamer targeting immune checkpoints and an immune checkpoint inhibitor. The targeted drug-loaded exosomes provided by this invention improve the targeting and bioavailability of pembrolizumab through nanotechnology and nucleic acid aptamers, further enhancing CD8... + This invention enhances T cell function and blocks the malignant transformation from cirrhosis to liver cancer. Experiments have demonstrated that the aforementioned drug-loaded exosomes significantly enhance CD8+ in a liver cancer model. + The cytotoxicity and anti-tumor function of T cells contrast sharply with traditional immunotherapy strategies.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a targeted drug-loaded exosome for liver cirrhosis and liver cancer, its preparation method, and its application. Background Technology

[0002] Liver cirrhosis and liver cancer pose significant global public health challenges. According to the World Health Organization, liver cancer is one of the leading causes of cancer death worldwide. Patients with long-term cirrhosis have a high probability of developing liver cancer, and the transformation mechanism is complex, involving multiple cell types and signaling pathways. Currently, although some progress has been made in the treatment of liver cancer, including surgical resection, radiotherapy, and chemotherapy, these traditional methods are often accompanied by high recurrence rates and severe side effects. Furthermore, many patients are diagnosed at an advanced stage, losing the opportunity for surgical treatment. Therefore, developing new treatment strategies, especially those that can precisely target the tumor microenvironment, is of great significance for improving the survival rate of liver cancer patients.

[0003] In recent years, immune checkpoint inhibitors such as pembrolizumab have shown significant efficacy in the treatment of various cancers, enhancing patients' immune responses by relieving immunosuppression. However, the application of pembrolizumab in the treatment of liver cancer still faces many challenges, such as the drug's instability and non-specific distribution in the body, which limits its therapeutic potential. Furthermore, the tumor microenvironment of liver cancer is particularly complex, involving multiple cell types including immune cells, fibrotic cells, and tumor cells. The interactions of these cells create an immunosuppressive environment, significantly reducing the effectiveness of immunotherapy. Summary of the Invention

[0004] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a targeted drug-loaded exosome that remodels the tumor immune microenvironment and enhances CD8 expression by precisely releasing immune checkpoint inhibitors. + T cells enhance the function of liver cells, thereby preventing the malignant transformation of cirrhosis into liver cancer.

[0005] This invention also proposes a method for preparing the above-mentioned targeted drug-loaded exosomes.

[0006] The present invention also proposes a pharmaceutical composition.

[0007] The present invention also proposes an application.

[0008] According to a first aspect of the invention, a targeted drug-loaded exosome is provided, the targeted drug-loaded exosome comprising an exosome encapsulated with indocyanine green, the surface of the drug-loaded exosome being linked to an aptamer targeting an immune checkpoint and an immune checkpoint inhibitor.

[0009] In some embodiments of the present invention, the immune checkpoints include at least one of PD-L1 / PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, CD276, CD47, and IDO1.

[0010] In some embodiments of the present invention, the immune checkpoint inhibitor is a PD-L1 / PD-1 inhibitor.

[0011] In some embodiments of the present invention, the PD-L1 / PD-1 inhibitor includes at least one of tislelizumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, and avelumab.

[0012] In some embodiments of the present invention, the aptamer targeting the immune checkpoint targets PD-L1 / PD-1.

[0013] In some embodiments of the present invention, the nucleotide sequence of the aptamer includes SEQ ID NO:1.

[0014] In some embodiments of the present invention, the aptamer targeting the immune checkpoint is covalently attached to the surface of the exosome.

[0015] In some embodiments of the present invention, the immune checkpoint inhibitor is covalently linked to the surface of exosomes.

[0016] In some embodiments of the present invention, the mass ratio of indocyanine green to exosomes is 1:(5-20), and the mass of the exosomes is expressed as protein content.

[0017] According to a second aspect of the present invention, a method for preparing targeted drug-loaded exosomes as described in the first aspect of the present invention is provided, the method comprising the following steps:

[0018] S1: Indocyanine green-tetrabutylammonium iodide complex was mixed with exosomes and incubated to obtain exosomes encapsulated with indocyanine green;

[0019] S2: Use a linker to link the immune checkpoint inhibitor with the indocyanine green-encapsulated exosomes obtained in step S1 to obtain drug-loaded exosomes;

[0020] S3: Use a cross-linking agent to connect the aptamer targeting the immune checkpoint to the drug-loaded exosomes obtained in step S2 to obtain targeted drug-loaded exosomes.

[0021] In some embodiments of the present invention, the molar ratio of indocyanine green and tetrabutylammonium iodide in step S1 is 1:(3-10).

[0022] In some embodiments of the present invention, the molar ratio of indocyanine green and tetrabutylammonium iodide in step S1 is 1:(4-6).

[0023] In some embodiments of the present invention, the mass ratio of indocyanine green to exosomes in step S1 is 1:(5-20).

[0024] In some embodiments of the present invention, the mass ratio of indocyanine green to exosomes in step S1 is 1:(8-13).

[0025] In some embodiments of the present invention, the incubation temperature in step S1 is 35°C-40°C.

[0026] In some embodiments of the present invention, the incubation time in step S1 is 1-3 hours.

[0027] In some embodiments of the present invention, the connector in step S2 includes DSPE-PEG-NHS.

[0028] In some embodiments of the present invention, the mass ratio of the immune checkpoint inhibitor to DSPE-PEG-NHS in step S2 is 1:(20-40).

[0029] In some embodiments of the present invention, the mass ratio of the immune checkpoint inhibitor to DSPE-PEG-NHS in step S2 is 1:(25-35).

[0030] In some embodiments of the present invention, the mass ratio of the immune checkpoint inhibitor to the exosomes encapsulated with indocyanine green in step S2 is 1:(20-40).

[0031] In some embodiments of the present invention, the mass ratio of the immune checkpoint inhibitor to the exosomes encapsulated with indocyanine green in step S2 is 1:(25-35).

[0032] In some embodiments of the present invention, the crosslinking agent in step S3 includes NHS and EDC.

[0033] In some embodiments of the present invention, the mass ratio of the aptamer targeting the immune checkpoint to the drug-loaded exosome in step S3 is 1:(40-60).

[0034] In some embodiments of the present invention, the mass ratio of the aptamer targeting the immune checkpoint to the drug-loaded exosome in step S3 is 1:(45-55).

[0035] In some embodiments of the present invention, the source of the exosomes in step S1 includes 293T cells.

[0036] According to a third aspect of the invention, a pharmaceutical composition is provided comprising the targeted drug-loaded exosomes described in the first aspect of the invention and pharmaceutically acceptable excipients.

[0037] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of binders, disintegrants, lubricants, coating agents, suspending agents, thickeners, and surfactants.

[0038] In some embodiments of the present invention, the pharmaceutical composition can be used to treat and / or prevent liver fibrosis.

[0039] In some embodiments of the present invention, the pharmaceutical composition can be used to treat and / or prevent cirrhosis.

[0040] In some embodiments of the present invention, the pharmaceutical composition can be used to treat and / or prevent tumors.

[0041] In some embodiments of the present invention, the pharmaceutical composition can be used to treat and / or prevent liver cancer.

[0042] According to a fourth aspect of the present invention, the application of the targeted drug-loaded exosomes described in the first aspect of the present invention in the preparation of tumor-targeting drugs is proposed.

[0043] In some embodiments of the present invention, the tumor-targeting drug can be used for the treatment and / or prevention of tumors.

[0044] In some embodiments of the present invention, the tumor includes a solid tumor.

[0045] In some embodiments of the present invention, the solid tumor includes at least one of liver cancer, lung cancer, breast cancer, colorectal cancer, stomach cancer, pancreatic cancer, prostate cancer, cervical cancer, and ovarian cancer.

[0046] The present invention has at least the following beneficial effects:

[0047] The targeted drug-loaded exosomes provided by this invention improve the targeting and bioavailability of pembrolizumab through nanotechnology and nucleic acid aptamers, further enhancing CD8. + This invention enhances T cell function and blocks the malignant transformation from cirrhosis to liver cancer. Experiments have demonstrated that the aforementioned drug-loaded exosomes significantly enhance CD8+ in a liver cancer model. + The cytotoxic and anti-tumor functions of T cells contrast sharply with traditional immunotherapy strategies. Simultaneously, this invention significantly improves the distribution and stability of immunomodulators in vivo. This combined strategy not only enhances drug efficacy but also reduces the burden of side effects for patients. Attached Figure Description

[0048] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0049] Figure 1 This is a graph showing the analysis results of scRNA-seq data in Example 1 of the present invention; where A shows the expression of some known cell lineage-specific marker genes in different clusters of normal liver tissue (N=3) and liver cancer tissue (N=3), the darker the blue, the higher the average expression level, and the larger the circle, the more cells express the gene; B is a visualization of cell annotation results based on UMAP clustering in normal liver tissue (N=3) and liver cancer tissue (N=3), where each color represents a cell subpopulation;

[0050] Figure 2 This is a graph showing the results of the T-test analysis of the difference in cell content between liver cancer tissue and normal liver tissue in Example 1 of the present invention;

[0051] Figure 3 For the analysis of CD8 in Embodiment 1 of the present invention + Volcano plot of gene expression differences among T cells;

[0052] Figure 4 CD8 on HCC tissue sections in Example 1 of this invention + Distribution results of T cells and PD-1; where A represents CD8. + T cells, B is PD-1;

[0053] Figure 5 This is a schematic diagram of the mouse model for malignant transformation of liver cirrhosis to liver cancer in Example 2 of the present invention;

[0054] Figure 6 This is a schematic diagram of blood sample collection for mouse liver function testing in Example 2 of the present invention;

[0055] Figure 7 The graph shows the liver function test results of mice in each group in Example 2 of the present invention; where A is ALP, B is AST, and C is ALT.

[0056] Figure 8 The images show the pathological staining results of mouse liver tissue in each group in Example 2 of this invention; where A is H&E staining (scale bar 50 μm), B is Sirius red staining (scale bar 100 μm), and C is Masson staining (scale bar 100 μm).

[0057] Figure 9 This is a flowchart of the proteomics-metabolic analysis in Example 3 of the present invention;

[0058] Figure 10The figure shows the results of proteomics analysis in Example 3 of this invention; where A is the statistics of the number of neighboring nodes of the core protein in the gene interaction network diagram, the horizontal axis represents the value of the neighboring nodes, and the vertical axis represents the protein name; B is a heatmap, revealing the differential expression of 35 DEPs in the proteomics data, and Cirrhosis (N=3) and HC (N=3) represent liver tissue samples from 3 mice at 14 weeks and 3 mice at 24 weeks of CCl4 modeling, respectively;

[0059] Figure 11 The graph shows the enrichment analysis results of differentially expressed proteins in Example 3 of this invention; where A is a GO enrichment bubble diagram of differentially expressed proteins in liver tissue samples from 3 mice at 14 weeks and 3 mice at 24 weeks of CCl4 modeling, BP represents biological processes, CC represents cellular components, and MF represents molecular functions; B is a KEGG clustering tree diagram of differentially expressed genes in liver tissue samples from 3 mice at 14 weeks and 3 mice at 24 weeks of CCl4 modeling.

[0060] Figure 12 The graph shows the functional enrichment analysis results of differential metabolites in Example 3 of this invention; where A is the OPLS-DA score graph, the horizontal axis represents the score value of the predicted component, the vertical axis represents the score value of the orthogonal component, and the percentage represents the explanatory power of the component on the data; B is the display of the TOP20 differential metabolites between the two groups initially screened in the VIP of the OPLS-DA model; C is the functional enrichment analysis results of differential metabolites in the SMPDB database.

[0061] Figure 13 The following is a graph showing the results of proteomics combined with metabolomics analysis in Example 3 of this invention; where A is a volcano plot of differentially expressed metabolites selected by the T-test algorithm; B is a volcano plot of differentially expressed metabolites selected by univariate analysis using P-value and fold change; C is the intersection of differentially expressed metabolites selected by Venn using the OPLS-DA model, the T-test algorithm, and univariate analysis.

[0062] Figure 14 The figure shows the functional enrichment analysis results of differential metabolites in Example 3 of the present invention; where A and B are the functional enrichment analysis results of differential metabolites in the SMPDB database, and C and D are the functional enrichment analysis results of differential metabolites in the KEGG database.

[0063] Figure 15 This is a schematic diagram of the synthesis of αPG-Exos / Apt in Embodiment 4 of the present invention;

[0064] Figure 16The image shows the identification results of 293T cell exosomes in Example 4 of this invention; where A is the protein expression of Alix, TSG101, CD81 and calnexin in Exos detected by Western blot, B is the morphology of 293T-Exos observed by TEM, showing the classic structure of disc-shaped vesicles, with a scale bar of 200 μm, and C is the size distribution of 293T-Exos analyzed by nanoparticle tracking.

[0065] Figure 17 The figure shows the results of characterizing ICG-Exos in Embodiment 4 of the present invention; where A is the size distribution of Exos and ICG-Exos, B is the hydrodynamic size of ICG-Exos, and C is the capture efficiency of ICG-Exos.

[0066] Figure 18 This is a graph showing the FI results of ICG and ICG-Exos after incubation in aqueous solution under external light in Example 4 of the present invention;

[0067] Figure 19 This is a fluorescence microscope observation of the carrying effect in Example 4 of the present invention; the scale bar is 10 μm.

[0068] Figure 20 The image shows the agarose gel electrophoresis results of ICG-Exos / Apt and αPG-Exos / Apt in Example 4 of this invention;

[0069] Figure 21 For the observation of CD8 using a laser scanning microscope in Embodiment 4 of the present invention + A diagram showing the uptake of Exos by T cells; the scale bar is 25 μm.

[0070] Figure 22 In Example 5 of this invention, the effect of αPG-Exos / Apt on CD8 was investigated. + The results of the effect on T cell function are shown in the figure; where A represents cell viability as determined by MTT assay; and B represents CD8. + Diagram illustrating the mechanism of T cell killing of tumor cells; C and D show the levels of IFN-γ and GZMB in cell culture medium detected by ELISA; E and F show the levels of CD8 by Western blot. + The content of GZMB in T cells; G represents CD8+ as detected by immunofluorescence. + Co-localization of CD8 and GZMB in T cells;

[0071] Figure 23 For the immunofluorescence detection of CD8 in Example 5 of this invention + Co-localization results of CD8 and GZMB in T cells; scale bar is 25 μm.

[0072] Figure 24 For example, in Embodiment 6 of the present invention, CD8 is detected using a confocal microscope. + The results of T cell permeability are shown in the figure; the scale bar is 25 μm.

[0073] Figure 25 This is a graph showing the results of the immunofluorescence assay in Example 6 of the present invention, detecting the fibrosis markers α-SMA and E-cadherin in co-cultured cell spheroids in each group; the scale bar is 25 μm.

[0074] Figure 26 This is a statistical chart of cell viability detection in Example 6 of the present invention;

[0075] Figure 27 This is a graph showing the effect of αPG-Exos / Apt on the survival rate of liver cancer cells in Example 6 of the present invention; where A is AFP, B is EpCAM, and the scale bar is 25μm.

[0076] Figure 28 The cell culture image is acquired in bright field mode using an inverted microscope in Embodiment 6 of the present invention; wherein the cell invasion area is outlined with a white dashed line, and the scale bar is 100 μm.

[0077] Figure 29 This is a NIR fluorescence imaging diagram of the in vivo fluorescence distribution of tumor-bearing mice in each group, as observed in Example 7 of this invention.

[0078] Figure 30 This is a fluorescence distribution map of the major organs and tumor tissues of mice in each group observed by NIR fluorescence imaging in Example 7 of the present invention;

[0079] Figure 31 The graph shows the liver function test results of mice in each group in Example 7 of the present invention; where A is ALP, B is AST, and C is ALT.

[0080] Figure 32 The images show the pathological staining results of mouse liver tissues in each group in Example 7 of this invention; where A is H&E staining (scale bar 50 μm), B is Sirius red staining (scale bar 100 μm), C is Masson staining (scale bar 100 μm); and D is immunofluorescence staining for AFP protein detection (scale bar 25 μm).

[0081] Figure 33 In Example 7 of this invention, the effect of αPG-Exos / Apt on CD8 was investigated. + Figure showing the effects of T cells and liver metabolism; where A and B represent flow cytometry analysis of IFN-γ and GZMB-positive CD8+. +T cells; C shows the content of PSMA1 in the liver tissues of mice in each group detected by Western blot; D shows the detection results of the content of sphingolipid metabolites in the sera of mice in each group.

[0082] Figure 34 This is the H&E staining map of the main organs of mice in each group in Example 7 of the present invention; among them, the scale bar is 100 μm. Detailed implementation manners

[0083] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0084] Example 1 Bioinformatics analysis of GEO data

[0085] In order to deeply understand the development process of liver cancer and the changes in the immune microenvironment, in this example, single-cell datasets were obtained from the GEO database, and the immune cell sequencing data sorted from the tumor tissues (GSM7850793, GSM7850794, GSM7850797) of 3 liver cancer patients and the corresponding adjacent normal tissues (GSM7850803, GSM7850804, GSM7850807) in the GSE245906 dataset were analyzed, and the liver tissue section data of 2 patients in the GSE245908 dataset were analyzed. The Seurat package was used to integrate the dataset data, and after data quality control, the harmony package was used to perform batch correction on the sample data. Quality control was performed with the standard of 200 < nFeature_RNA < 6000 & percent.mt < 25, and the top 2000 highly variable genes with the highest expression were screened. The UMAP algorithm was used to perform non-linear dimensionality reduction on the first 30 PCs. The clustering analysis results showed 27 clusters, and the expression of marker genes in each cluster was obtained. According to the search for relevant literature, cell lineage-specific marker genes were obtained and combined with the online website CellMarker to annotate the cells (as Figure 1 shown in A), and a total of 10 cell types were obtained: B Cell, Basophil, CD8 + T Cell, DendriticCell, Helper T Cell, Macrophage, Mast Cell, Monocyte, Natural Killer T (NKT) Cell and Neutrophi (as Figure 1 (As shown in B). T-test analysis results showed that CD8+ in the liver tissue of liver cancer patients... + T cell content was significantly reduced. Figure 2 Extract CD8 + Differential analysis of T cell data revealed a significant increase in PD-1 (PDCD1) expression levels in HCC tissues. Figure 3 ).

[0086] To characterize the distribution of different cell types in HCC tissues, this embodiment uses spatial transcriptomics to analyze liver tissue slice data from two HCC patients in the GSE245908 dataset. First, the ST data were integrated and standardized using the Seurat package. After screening for highly variable genes, dimensionality reduction and cluster analysis were performed using PCA and UMAP algorithms. By calculating the overlap between genes and cell type-specific genes in scRNA-seq data, cell types in the ST data were inferred and annotated. The results are shown below. Figure 4 As shown. By Figure 4 It can be seen that CD8 + T cells are less distributed in HCC tissues, while PD-1 expression levels are higher.

[0087] Example 2: Construction and validation of a mouse model of malignant transformation from cirrhosis to liver cancer.

[0088] 1. Construction of a mouse model of malignant transformation from cirrhosis to hepatocellular carcinoma

[0089] To investigate the malignant transformation process of liver cirrhosis to liver cancer, this embodiment establishes a mouse model of liver cirrhosis-liver cancer malignant transformation by gavage with CCl4. The specific method is as follows:

[0090] Purchase SPF-grade male C57BL / 6J mice aged 4-6 weeks and weighing 16-22g (purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.). Each mouse was housed separately in an SPF-grade animal laboratory with humidity controlled at 60%-65% and temperature controlled at 25±2℃. Free food and water were provided under alternating light and dark conditions for 12 hours. After one week of acclimatization feeding, the experiment began. The health status of the mice was observed before the experiment.

[0091] Control group mice were administered 100 μL of olive oil (E0505, Selleck) by gavage, while model group mice were administered an olive oil solution containing 40% CCl4 (C0731530924, Nanjing Reagent) by gavage three times a week for 14 weeks. The modeling process was as follows. Figure 5As shown. Starting at week 14, tumor growth was observed using in vivo imaging, and this observation continued thereafter, with changes in tumor volume recorded every 7 days.

[0092] 2. Validation of a mouse model of malignant transformation from cirrhosis to hepatocellular carcinoma

[0093] 1) Liver function tests:

[0094] To verify the modeling results, blood samples were collected from mice in the Model and Control groups at 0, 3, 6, 9, 14, and 24 weeks for liver function testing. The sampling time points and methods are as follows: Figure 6 As shown, the blood samples collected above were tested using a Sigma-Aldrich aspartate aminotransferase (AST, MAK055), alanine aminotransferase (ALT, MAK052), and alkaline phosphatase (ALP, MAK447) detection kit. The results are as follows. Figure 7 As shown.

[0095] Depend on Figure 7 It was observed that, compared to the Control group, the levels of ALP, AST, and ALT in the blood samples of the Model group mice gradually increased over time, suggesting a progressively deepening degree of liver damage. Notably, serum transaminase levels remained high even after CCl4 was discontinued at week 14, indicating that liver damage had reached an irreversible stage.

[0096] 2) Liver tissue pathological staining: Liver tissues from mice in the Control group and Model group were collected at 3, 6, 9, 14 and 24 weeks after treatment for observation and pathological staining.

[0097] ①H&E staining: Take the tissue sample to be tested and fix it. After sectioning, place the section in xylene to remove wax, then rehydrate it in 100% ethanol, 95% ethanol, and 70% ethanol respectively, and finally wash it with water once. Place the section in hematoxylin staining solution (H8070, Solarbio) for staining, generally at room temperature for 5-10 minutes. Then wash it with distilled water, dehydrate it in 95% ethanol, and place it in eosin staining solution (G1100, Solarbio) for 5-10 minutes. Then dehydrate it with different concentrations of alcohol (85%, 90%, 95%, 100%), and finally clear it with dichloromethane.

[0098] ②Sirius Red Staining: Use the Sirius Red Staining Kit (50-300-77, Fisher Scientific), and the staining steps are similar to the H&E staining steps described above.

[0099] ③Masson staining: Use the Masson staining kit (G1340, Solarbio) and follow the instructions provided with the kit to perform the staining.

[0100] After staining, slides were mounted with neutral resin, air-dried, and observed under an optical microscope. Five fields of view were randomly selected for analysis for each stained slide, and at least three slides were examined for each mouse. The percentage of Masson's trichrome positive areas was analyzed using Image-Pro-Plus software (Media Cybernetics, V6.0), and collagen was morphometrically quantified using the Ishak scoring method. The results are as follows: Figure 8 As shown.

[0101] Depend on Figure 8 It was observed that, compared to the Control group, the livers of mice in the Model group showed signs of CCl4-induced damage. This damage was characterized by a pale yellow rather than dark red appearance, a firmer texture than normal liver tissue, an irregular surface, and nodules of varying sizes, with the number of nodules increasing with the duration of drug administration. At week 14, mice exhibited significant liver fibrosis, indicating successful establishment of the liver cirrhosis mouse model. At week 24 (week 10 after CCl4 discontinuation), the liver surface showed significant nodule formation and disordered tissue structure, with a marked increase in fibrosis around these nodules, a typical characteristic of hepatocellular carcinoma. Sirius red and Masson staining results showed that liver fibrosis gradually worsened over time. All these results confirm the successful establishment of the liver cirrhosis-hepatocellular carcinoma malignant transformation mouse model.

[0102] Example 3: Detection of key proteins and metabolites in the malignant transformation process of liver cirrhosis to liver cancer using proteomics combined with metabolomics.

[0103] This embodiment further investigates the key proteins and metabolites in the malignant transformation process of liver cirrhosis to liver cancer based on the mouse model constructed in Example 2. The experimental procedure is as follows: Figure 9 As shown, the specific experimental methods and results are as follows:

[0104] 1. Proteomics Sample Preparation and Measurement

[0105] Liver tissues from three mice at 14 weeks of CCl4 modeling (cirrhosis stage) and three mice at 24 weeks of CCl4 modeling (hepatocellular carcinoma stage) were added to mortars and pestles. Liquid nitrogen was added to the mortars, and the mixture was ground with a pestle. The resulting powder was then transferred to a 5 cm [material / particle size]. 3The cells were then sonicated in an ice bath using an ultrasonic cell disruptor. The treatment medium was phenol extraction buffer (100206, Sigma-Aldrich) containing 10 mmol / L DTT, 1% protease inhibitor mixture (P6731, Solarbio), and 2 mmol / L EDTA (E1170, Solarbio). The sonication step was repeated 8 times. Next, an equal volume of Tris-saturated phenol (HCC1380, BIOFOUNT) at pH 8.0 was added to the sonicated mixture, and the mixture was vortexed for 4 minutes. Then, the mixture was centrifuged at 5000g for 10 minutes at 4°C, and the supernatant phenol was transferred to a new centrifuge tube. 0.1 mol / L ammonium sulfate-saturated methanol was added to the phenol solution at a volume ratio of 1:5, and the mixture was left to stand overnight to precipitate the protein. Subsequently, the mixture was centrifuged at 4°C for 10 minutes, and the supernatant was removed. Finally, the remaining precipitate was washed once with ice-cold methanol and then three times with ice-cold acetone. The washed protein was redissolved in 8 mol / L urea (U8020, Solarbio), and the protein concentration was determined using a BCA kit (P0012, Beyotime) before use. The procedure was performed according to the manufacturer's instructions.

[0106] 2. Proteolytic digestion, peptide labeling and fractionation, and nano-LC-MS / MS detection and analysis

[0107] 50 μg of protein samples from each group were subjected to enzymatic digestion. The protein solution was mixed with DTT to a concentration of 5 mmol / L and incubated at 56 °C for 30 minutes. Then, acetamide was added to a concentration of 11 mmol / L, and the mixture was incubated at room temperature for 15 minutes. Finally, the urea concentration of the samples was diluted to below 2 mol / L, and trypsin (25200056, Thermo Fisher) was added at a mass ratio of 1:50 (w / w), and enzymatic digestion was carried out overnight at 37 °C. Subsequently, trypsin was added at a mass ratio of 1:10 (trypsin:protein), and enzymatic digestion was continued for 4 hours.

[0108] After trypsin digestion, HyperSep was used. TM Peptides were desalted and vacuum-dried using a C18 purification column (60108-302, Thermo Fisher). The peptides were redissolved in 0.5 mol / L TEAB (90114, Thermo Fisher) and processed according to the manufacturer's instructions for the TMT kit (90064CH, Thermo Fisher). In short, one unit of TMT reagent was thawed and reconstituted in acetonitrile. The peptide mixture was then incubated at room temperature for 2 hours and desalted and dried using a vacuum centrifuge. Equal volumes of each group of labeled peptides were mixed, and the dried peptides were processed using Pierce...TM Fractionation was performed using a high-pH reverse-phase peptide separation kit (84868, Thermo Fisher, USA). The samples were then collected and combined into 15 fractions. The peptides in each fraction were dried and reconstituted with 0.1% formic acid (159002, Sigma-Aldrich, USA).

[0109] Two μg of peptides were taken from each sample and separated using a nano-UPLC system, Easy nLC 1200. Samples were first fed onto a Trap C18 column (100 μm × 20 mm, 5 μm), then subjected to gradient separation using an analytical C18 column (75 μm × 150 mm, 3 μm) at a flow rate of 300 nL / min. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid aqueous solution-acetonitrile solution (containing 95% acetonitrile). The gradient elution program was: 0–2 min, 2%–8% B; 2–71 min, 8%–28% B; 71–79 min, 28%–40% B; 79–81 min, 40%–100% B; 81–90 min, 100% B. The peptides separated by HPLC were analyzed by Q-Exactive HCX mass spectrometry. The analysis time was 60 min, the electrospray voltage was 2.1 kV, the detection mode was positive ion, the precursor ion scan range was 350-1200 m / z, the first-order mass spectrometry resolution was 60000 m / z 200, and the AGC target was 3e. 6 Level 1 Maximum IT: 30ms. Level 2 mass spectrometry resolution: 15000 m / z 200, AGC target: 1e 6 , Level 2 MaximumIT: 25ms, MS2Activation Type: HCD, Isolation window: 20Th, Normalized collisionenergy: 32.

[0110] The obtained LC-MS / MS data were processed using MaxQuant software, including peptide identification and protein quantification. Tandem mass spectrometry searches were performed using the UniProt 14.1 (2009) database (https: / / www.uniprot.org / ) and a reverse bait database. Trypsin / P was designated as the cleavage enzyme, allowing up to two missing cleavages. The initial search was 20 ppm, the major search was 5 ppm, and the fragment ion mass tolerance was 0.02 Da. Screening criteria included a peptide false discovery rate (FDR) ≤ 0.01, a protein FDR ≤ 0.01, and peptide score distribution. Differentially expressed proteins (DEPs) between liver tissue samples were screened using the R software "limma" package (with thresholds of |log2FC|>1 and P.value<0.05).

[0111] In mouse HCC tissue samples, 10,508 protein molecules were identified, and 1,132 significantly differentially expressed proteins were found, of which 583 were upregulated and 549 were downregulated. Subsequently, the top 100 DEPs by |log2FC| were extracted, and a PPI network was constructed. The number of neighboring nodes for each gene in the network graph was counted, and proteins with no neighboring nodes were removed. The results yielded 35 key proteins (…). Figure 10 A) CD8A, CD8B, IFNG, and GZMB, etc. + T cell-related protein expression was significantly downregulated, while PSMA1 protein expression was significantly upregulated. Figure 10 B). Plasma proteasome (PSMA1) levels are a reliable early marker of malignant transformation in liver cirrhosis (PMID: 19201777). Pathway enrichment analysis was performed on the differentially expressed proteins identified through screening, and the results are as follows: Figure 11 As shown. Figure 11 The results showed that the differentially expressed proteins were mainly enriched in lipid, proteasome, and T cell-related pathways.

[0112] 3. Metabolomics analysis

[0113] In this embodiment, plasma samples were collected from 6 mice at 14 weeks of CCl4 modeling (cirrhosis stage) and 6 mice at 24 weeks of CCl4 modeling (hepatocellular carcinoma stage). Non-targeted metabolomics analysis was performed using liquid chromatography-mass spectrometry. Specific experimental methods and results are as follows:

[0114] Serum samples were thawed at room temperature, and 300 μL of each sample was transferred to a 1.5 mL polypropylene tube, mixed with 900 μL of 80% methanol and 0.1% formic acid, vortexed for 2 min, and then centrifuged at 12000 g for 10 min. The supernatant was transferred to an autosampler vial. Plasma metabolomics analysis was performed using an LC20 ultra-high performance liquid chromatograph (Shimadzu, Japan) combined with a Triple TOF-6600 mass spectrometer (ABSciex). A Waters ACQUITY UPLC HSS T3C18 (100 × 2.1 mm, 1.8 μm) column was selected for chromatographic analysis. The column temperature was maintained at 40 °C, and elution was performed at a flow rate of 0.4 mL / min. The mobile phase consisted of an aqueous solution of acetonitrile containing 0.1% formic acid. The gradient elution program for mobile phase B was as follows: 5%, 0.0–11.0 min; 90%, 11.0–12.0 min; 5%, 12.1–14 min. The eluent is introduced directly into the mass spectrometer without splitting.

[0115] The mass spectrometry conditions were as follows: ionization voltage, 5500 V; capillary temperature, 550 °C; spray gas flow rate, 50 psi; auxiliary heating gas flow rate, 60 psi. Orthogonal partial least squares-discriminant analysis (OPLS-DA) and permutation tests (100 permutations) were used to analyze preprocessed data to prevent overfitting. Metabolites with a VIP score >1 and p-value <0.05 in the OPLS-DA model were identified as differential metabolites (DMs). Combined with univariate analysis, metabolites with a fold change ≥2 and fold change ≤0.5, and a p-value <0.05 in Student's t-test, were selected as the final differential metabolites. MetaboAnalyst (Version 5.0) was used to identify relevant metabolic pathways. Simultaneously, changes in sphingomyelin levels in mouse serum were detected using the Sigma-Aldrich phospholipid quantification kit (MAK262). In this embodiment, the metabolomics data were standardized and subjected to PCA cluster analysis to construct the OPLS-DA model, and score plots for each group were generated. Figure 12 A), by Figure 12 As shown in A, the metabolic profiles of the cirrhosis group and the liver cancer group were significantly separated.

[0116] The OPLS-DA evaluation model was further validated. Based on the OPLS-DA results, differentially expressed metabolites between the two groups were initially screened from the variable importance projections of the obtained multivariate analysis OPLS-DA model. The results are as follows: Figure 12 B and Figure 12As shown in C. Further screening of differentially metabolites was conducted using the t-test algorithm, along with the p-value and fold change from univariate analysis. The results are shown in... Figure 13 A and Figure 13 As shown in B, a total of 71 overlapping differential metabolites were obtained. Figure 13 C). The differentially metabolites were mainly lipid metabolites, especially sphingomyelin compounds, which mostly showed an increasing trend in the hepatocellular carcinoma group.

[0117] To further explore the potential functions and roles of differentially metabolites, this embodiment uses the MetaboAnalyst 5.0 database to perform functional enrichment analysis on the obtained differentially metabolites. The results are as follows: Figure 14 As shown. By Figure 14 Analysis of the SMPDB and KEGG databases shows that differentially metabolites are mainly enriched in lipid metabolism and related pathways.

[0118] In summary, this embodiment reveals that CD8+ is involved in the malignant transformation process from cirrhosis to hepatocellular carcinoma. + The expression of T cell-related proteins was significantly downregulated, while the expression of PSMA1 protein was significantly upregulated, and the differential metabolites were mainly enriched in lipid metabolism and other related pathways.

[0119] Example 4: Construction of a targeted drug-loaded exosome

[0120] To address the significant activation of the PD-1 / PD-L1 pathway during the malignant transformation from cirrhosis to hepatocellular carcinoma, this embodiment provides a method for targeted delivery of CD8 to tumor tissue. + T cells carrying drug-loaded exosomes that enhance the efficacy of anti-PD-1 antibodies; the preparation process is as follows: Figure 15 As shown, the specific preparation method is as follows:

[0121] 1. Isolation of exosomes

[0122] When 293T cells reached 80%-90% confluence, the supernatant was discarded and the cells were washed with 2×PBS. Then, 25 mL of serum-free IMDM medium (12440053, Gibco) was added to each culture flask, and the cells were incubated for 48 hours under humidified conditions of 37°C and 5% CO2. The cell supernatant was collected into a 50 mL centrifuge tube and centrifuged at 300 g for 10 minutes at 4°C to remove cell debris. The supernatant was again collected into another 50 mL centrifuge tube. After collecting the supernatant, it was centrifuged at 2000 g for 20 minutes at 4°C. The supernatant was then transferred to a sterile tube for a high-speed centrifuge and centrifuged at 16500 g for 30 minutes at 4°C. The supernatant was then transferred to an ultracentrifuge tube and centrifuged at 120000 g for at least 70 minutes at 4°C using a fixed-angle rotor. The supernatant was then completely discarded. Add 1 mL of 4°C PBS to each ultracentrifuge tube and resuspend the precipitate using a micropipette. Mix the solutions from the same group into ultracentrifuge tubes, then add 4°C PBS until the tubes are more than 3 / 4 full. Centrifuge at 120,000 g for 60 minutes at 4°C, removing as much supernatant as possible, and resuspend the precipitate again with sterile PBS to obtain 293T cell-derived exosomes (293T-Exos).

[0123] Western blot was used to detect the expression of classic exosome markers (Alix, TSG101, CD81) in the above exosomes. The results are as follows: Figure 16 As shown in Figure A, the results indicate that all of the above-mentioned exosome markers were expressed in the exosomes prepared in this embodiment. Transmission electron microscopy and NTA analysis also confirmed the typical disc-shaped morphology of the exosomes, with a size ranging from 50 to 300 nm. Figure 16 B and Figure 16 C).

[0124] 2. Preparation of drug-loaded exosomes (αPG-Exos / Apt)

[0125] 1) Encapsulating indocyanine green (ICG) in a 293T-Exos:

[0126] 16 mg ICG (HY-D0711, MedChemExpress) and 46 mg tetrabutylammonium iodide (TBAI, HY-Y0791, MedChemExpress) were dissolved in methanol and mixed thoroughly. The methanol was evaporated, and the ICG-TBAI complex was dissolved in DMSO for later use. 40 μg of exosomes (based on protein content) was mixed with the ICG-TBAI complex containing 4 μg of ICG (i.e., the protein content ratio of ICG to exosomes was 1:10), and incubated at 37°C for 2 hours. After incubation, the free ICG-TBAI complex was removed using a desalting column (MWCO, 40 kDa) to obtain the successfully encapsulated ICG Exos (ICG-Exos).

[0127] 2) Drug loading treatment of ICG-Exos:

[0128] The FITC-tagged pembrolizumab (HY-P9902, MedChemExpress) was diluted with PBS, and then centrifuged at 2000g for 5 min using a 10kDa ultrafiltration centrifuge tube to remove the solvent. The DSPE-PEG-NHS (HY-W441014, MedChemExpress) adapter was dissolved in DMSO. Excess adapter was mixed with the antibody solution, and the mixture was continuously stirred at 4°C for 24 hours, with a pembrolizumab to adapter mass ratio of 1:30. Subsequently, the mixture was centrifuged again at 2000g for 5 min using a 10kDa ultrafiltration centrifuge tube to remove unlinked adapters. The resulting DSPE-PEG-NHS-Pembrolizumab solution was reacted with the purified ICG-Exos from step 1) at 4°C for 3 hours, with a pembrolizumab to ICG-Exos mass ratio of 1:30. When the end of the connector, DSPE, was inserted into the membrane of ICG-Exos, antibody-functionalized drug-loaded exosomes αPG-Exos were obtained by centrifugation at 100,000g for 2 hours in an ultracentrifuge at 4°C.

[0129] 3) Constructing targeted drug-loaded exosomes:

[0130] A 50 μL solution of 10 μM PA9-1 aptamer (PA9-1-Apt, Sangon Biotech) was added to a solution containing 46 mg EDC (0.3 mmol, HY-D0178, MedChemExpress) and 35 mg NHS (0.3 mmol / L, HY-Y0623, MedChemExpress), and reacted for 1 hour to activate the aptamer carboxyl group. Subsequently, the activated aptamer solution was incubated with 1 mL of αPG-Exos (80 mg / mL) suspension at a mass ratio of 1:50. At this point, the PA9-1 aptamer was covalently modified onto the surface of the exosome, yielding αPG-Exos / Apt. The nucleotide sequence of the above PA9-1 aptamer is: 5'-AAGGCCTTGGTAAAGGGGCTTGGATTAAAACCTACTTTCCCGGGGFFFGGACCCG-3' (SEQ ID NO:1).

[0131] 3. Testing and characterization of exosomes

[0132] To test the various performance characteristics of αPG-Exos / Apt, the following exosome groups were also constructed for comparison in this embodiment:

[0133] ①Exos group (wild-type 293T cell-derived exosomes); ②ICG-Exos group (293T cell-derived exosomes encapsulating ICG); ③αPG-Exos group (293T cell-derived exosomes encapsulating ICG and conjugated with Pembrolizumab); ④ICG-Exos / Apt group (293T cell-derived exosomes encapsulating ICG and modified with PA9-1 aptamer); ⑤αPG-Exos / Apt group (293T cell-derived exosomes encapsulating ICG and conjugated with Pembrolizumab and modified with PA9-1 aptamer).

[0134] 1) ICG encapsulation efficiency and stability testing:

[0135] To determine encapsulation efficiency, dynamic light scattering (DLS; Malvern Instruments Ltd.) was used to test the hydrodynamic dimensions of Exos and ICG-Exos. The relative encapsulation efficiency was calculated using the following formula: (Amount of ICG in Exos) / (Amount of ICG added) × 100%. The results are shown below. Figure 17 As shown. By Figure 17It can be seen that Exos exhibits a narrower size distribution, with a size of approximately 35 nm, while ICG-Exos shows large aggregation and a wide size distribution. Compared to Exos, the hydrodynamic size of ICG-Exos is much larger, and the encapsulation efficiency of ICG within Exos is approximately 15%. ICG is highly susceptible to external physicochemical conditions such as light. After incubation at 37°C for predetermined times (0, 3, 6, 20 h) under illumination, the relative fibrillation (FI) of ICG and ICG-Exos was examined using an IVIS imaging system (Lumina II; Caliper Life Sciences). The excitation and emission wavelengths were 780 nm and 831 nm, respectively. The relative FI was calculated using the following formula: (FI after incubation) / (initial FI at 0 hours) × 100%. The results are shown below. Figure 18 As shown. By Figure 18 It can be seen that although both free ICG and ICG-TBAI showed severe FI loss, the FI of ICG-Exos did not decrease during incubation, indicating that incorporating ICG into Exos can protect ICG from physicochemical damage caused by light exposure in solution.

[0136] 2) Fluorescence microscopy observation of Pembrolizumab and aptamers carried by exosomes:

[0137] Fluorescence microscopy revealed that FITC-labeled pembrolizumab was detected in both the αPG-Exos and αPG-Exos / Apt groups. Figure 19 To confirm whether the PA9-1 aptamer was successfully coupled to the exosome, agarose gel electrophoresis was performed, and the results are as follows: Figure 20 As shown. By Figure 20 It can be seen that in the ICG-Exos / Apt group and the αPG-Exos / Apt group, the mobility of the aptamers was hindered after the introduction of exosomes, which confirms that the aptamers successfully connected to the surface of the exosomes.

[0138] 3) Immunofluorescence staining to observe CD8 + T cell uptake of Exos

[0139] Exos derived from 293T cells were seeded into 24-well plates. Dil dye (C1036, Beyotime) was added to 40 μg of Exos to achieve a final concentration of 25 μM. The mixture was then incubated at room temperature for 30 minutes, followed by reaction with CD8+. +T cells were co-incubated for 24 hours. After incubation, unbound dye was removed by ultracentrifugation. Next, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 30 minutes. Finally, the cell nuclei were stained with DAPI (C1005, Beyotime) for 30 minutes, and then photographed at 400× using a BX53 fluorescence microscope (Olympus) equipped with a camera. The images were analyzed using ImageJ Pro Plus 6.0 software, and the results are as follows: Figure 21 As shown. By Figure 21 It can be seen that CD8 in the PBS group and the αPD1 (Pembrolizumab) group + No red fluorescence signal was observed in T cells, while significant red fluorescence signals were observed in the other four cell groups, with the strongest red fluorescence signals observed in the αPG-Exos group and the αPG-Exos / Apt group; indicating that Exos in each group can be converted by CD8. + T cells successfully took up the drug. In summary, this embodiment successfully constructed a drug-loaded exosome αPG-Exos / Apt that is modified with a nucleic acid aptamer and precisely releases Pembrolizumab.

[0140] Example 5: Drug-loaded exosomes αPG-Exos / Apt on CD8 + The impact of T cell function

[0141] 1. MTT assay for CD8 + T cell activity

[0142] CD8 + T cells were produced at a rate of 1 × 10 per well. 4 Cells were seeded at a density of 1,000 cells per well in 96-well plates, and CD3 / CD28 was added to stimulate their activation (this step is required for all subsequent experiments). The cells were incubated for 24 hours, co-cultured with liver cancer cells for 48 hours, and then the CD8+ cells from the lower chamber were isolated. + T cells. Cells from different treatment groups were mixed with 0.01 mL of MTT (CT02, Sigma Aldrich) solution and incubated in an incubator for 4 h. Then, 0.1 mL of isopropanol containing 0.04 mol / L HCl was added to each well. The mixture was repeatedly pipetted using a multichannel pipette to ensure thorough mixing. HCl converted phenol red in the tissue culture medium to a yellow color that did not interfere with the MTT assay. Cell viability was determined by measuring absorbance at 570 nm, calculated as (ΔA_sample - ΔA_blank) / (ΔA_control - ΔA_blank). Where ΔA_sample is the absorbance difference of the sample, ΔA_blank is the absorbance difference of the blank, and ΔA_control is the absorbance difference of the control group. The results are shown below. Figure 22 As shown in Figure A.

[0143] Depend on Figure 22 As shown in Figure A, compared with the PBS group, the other groups did not show significant cytotoxicity; compared with the Exos group and the ICG-Exos group, the CD8+ of the αPD1 group, αPG-Exos group, ICG-Exos / Apt group, and αPG-Exos / Apt group was significantly higher. + T cell activity was significantly enhanced, with the αPG-Exos / Apt group showing the most significant enhancement.

[0144] 2. ELISA detection of CD8 + T cells' cytotoxic ability

[0145] CD8 + T cells secrete granzyme B (GZMB) and interferon-γ (IFN-γ), which makes it easier for T cells to recognize and kill tumor cells (mechanisms such as...). Figure 22 (As shown in B). Cell culture supernatant was collected and analyzed using the IFN-γ ELISA kit (BMS606-2, Invitrogen) and the GZMB ELISA kit (BMS6029, Invitrogen). First, the antigen was diluted to an appropriate concentration with coating diluent. 5% fetal bovine serum was used to block the enzyme-labeled reaction wells at 37°C for 40 min. The diluted sample was added to the enzyme-labeled reaction wells, followed by the enzyme-labeled antibody, substrate solution, and finally 50 μL of stop solution to terminate the reaction. The experimental results were measured within 20 min. The plate was read at 450 nm using a microplate reader, a standard curve was plotted, and the data were analyzed. The results are shown below. Figure 22 C and Figure 22 As shown in D.

[0146] Depend on Figure 22 C and Figure 22 As shown in D, compared with the Exos group and the ICG-Exos group, the IFN-γ and GZMB in the supernatant of the αPD1 group, αPG-Exos group, ICG-Exos / Apt group and αPG-Exos / Apt group were significantly increased, and the IFN-γ and GZMB content in the fine supernatant of the αPG-Exos / Apt group was the highest.

[0147] 3. Western blot detection of CD8 + T cells' cytotoxic ability

[0148] First, cells were collected and lysed using enhanced RIPA lysis buffer (P0013B, Beyotime) containing protease inhibitors. Protein concentration was then determined using a BCA protein quantification kit (P0012, Beyotime). Proteins were separated using 10% SDS-PAGE. The separated proteins were electrotransferred to a PVDF membrane and blocked with 5% BSA at room temperature for 2 hours to prevent non-specific binding. Diluted primary antibodies (rabbit anti-human) were added and the membrane was incubated at room temperature for 1 hour. After washing, HRP-labeled goat anti-rabbit secondary antibody was added and incubated at room temperature for 1 hour. Equal volumes of solutions A and B in ECL developing solution (32209, Thermo Scientific) were mixed in a dark room and dropped onto the membrane. The membrane was then exposed for imaging using a gel imaging system. Images were taken using a Bio-Rad image analysis system, and the bands in the Western blot images were quantified using ImageJ analysis software. β-actin was used as an internal control. Each experiment was repeated three times. The results are shown below. Figure 22 E and Figure 22 As shown in F, all antibodies used were purchased from Invitrogen, with the following details: IFN-γ (PA5-95560), dilution 1:1000; GZMB (PA5-17457), dilution 1:2500; PSMA1 (PA1-963), dilution 1:1000; β-actin (MA1-140), dilution 1:5000. Figure 22 E and Figure 22 As shown in F, compared with the Exos group and the ICG-Exos group, the levels of IFN-γ and GZMB in the cells of the αPD1 group, αPG-Exos group, ICG-Exos / Apt group and αPG-Exos / Apt group were significantly increased, with the highest levels of IFN-γ and GZMB in the cells of the αPG-Exos / Apt group.

[0149] 4. Immunofluorescence staining to detect co-localization of CD8 and GZMB

[0150] The CD8 sample was tested using 4% paraformaldehyde. + T cells were fixed on a glass slide and washed twice with DPBS containing 0.05% Tween 20, infiltrated with 0.1% Triton X-100 for 3 minutes, and then washed twice more with DPBS containing Tween 20. The samples were blocked for 1 hour with DPBS containing 5% goat serum and 0.3 mol / L glycine. After this blocking step, the first antibody was added and incubated. The sections were then blocked with 5% goat serum in PBS for 2 hours, and then incubated overnight at 4°C with the second antibody. Information on the antibodies used is shown in Table 1.

[0151] Table 1 Information on antibodies used for immunofluorescence staining

[0152]

[0153] On the second day, the cells were washed three times with DPBS containing Tween 20, incubated with secondary antibody at room temperature for 1 hour, rinsed three times with DPBS, and then stained with DAPI for 5 minutes. The cell slides were then rinsed three times with PBS (5 minutes each time) to remove excess DAPI. The cell slides were carefully removed from the cell culture plate using a curved fine syringe needle and small forceps and placed on a glass slide with anti-fluorescence quenching mounting medium (cell side down). The slides were observed and photographed under a fluorescence microscope. Quantification was performed by taking the fluorescence coverage area under a fixed field of view with a 40× objective lens. Six fields of view were taken from each group, and the average value was calculated. The results are shown below. Figure 22 G and Figure 23 As shown. By Figure 22 G and Figure 23 It was found that, compared with the Exos group and the ICG-Exos group, the number of CD8 / GZMB double-positive cells was significantly increased in the αPD1 group, αPG-Exos group, ICG-Exos / Apt group, and αPG-Exos / Apt group, with the αPG-Exos / Apt group having the highest number of double-positive cells. In summary, this example demonstrates that αPG-Exos / Apt can significantly increase CD8... + T cell activity and cytotoxicity.

[0154] Example 6: Effects of drug-loaded exosomes αPG-Exos / Apt on hepatocyte fibrosis and malignant transformation

[0155] To further investigate the effect of the prepared αPG-Exos / Apt on the malignant transformation process of liver fibrosis to hepatocellular carcinoma, this example constructs CD8 labeled with carboxyfluorescein succinimide (CFSE). + T cells, liver fibrosis cells, and liver cancer cells were co-cultured in 3D spheres, and Exos from each group were added for co-incubation. The specific experimental methods and results are as follows:

[0156] 1. Construction of 3D cell spheres

[0157] 1000 Hepa1-6 liver cancer cells / spheroids were seeded in 35- or 81-well agarose gel models prepared using 3D Petri dishes, which are spheroid-forming devices for microtissues. One minute after seeding, 1 mL (for 35-well tubing) or 2 mL (for 81-well tubing) of cell culture medium was added, and the mixture was incubated at 37°C and 5% CO2 to form 3D cell spheroids.

[0158] 2. Treatment of 3D cell spheres with different Exos:

[0159] The specific groupings include: (1) PBS group (untreated group); (2) Exos group (treated group of wild-type 293T cell-derived exosomes); (3) ICG-Exos group (treated group of 293T cell-derived exosomes successfully encapsulated with ICG); (4) αPG-Exos group (treated group of 293T cell-derived exosomes successfully encapsulated with ICG and conjugated with Pembrolizumab); (5) ICG-Exos / Apt group (treated group of 293T cell-derived exosomes successfully encapsulated with ICG and modified with PA9-1 aptamer); (6) αPG-Exos / Apt group (treated group of 293T cell-derived exosomes successfully encapsulated with ICG and conjugated with Pembrolizumab and modified with PA9-1 aptamer); (7) αPD1 group (treated group of Pembrolizumab). After constructing 3D co-cultured cell spheres, 20 μg / mL of the above-mentioned Exos groups or 20 μg / mL of Pembrolizumab were added to the cell culture medium for co-incubation for 24 h.

[0160] 3. Detect CD8 + T-cell penetration rate:

[0161] On day 2 of culture, CD8 labeled with CFSE (150347-59-4, Sigma-Aldrich) will be... + T cells, along with cirrhotic cells (hepatocytes isolated from the livers of mice at 14 weeks of gestation) and tumor cells, were added in a 5:1:1 ratio to the 3D cell spheres constructed in step 1, and further co-cultured in DMEM medium supplemented with 10% fetal bovine serum and 100 U / mL penicillin / streptomycin. After culturing at 37°C for 24 h, the cell spheres were washed and fixed in 4% paraformaldehyde for imaging using a ZEN 710 confocal microscope. Images were acquired at the mid-height of the microspheres, and surface maps were obtained using ImageJ software. The results are shown below. Figure 24 As shown.

[0162] Depend on Figure 24 It can be seen that, compared with the PBS group, Exos group, ICG-Exos group, and ICG-Exos / Apt group, the CD8 concentration in cells and supernatant of the αPD1 group, αPG-Exos group, and αPG-Exos / Apt group was significantly higher. + T cell penetration increased significantly, with the αPG-Exos / Apt group showing the highest cell penetration.

[0163] 4. Immunofluorescence assay for liver fibrosis level

[0164] The levels of fibrosis (α-SMA and E-cadherin) in hepatocytes from 3D co-cultured cell spheres were detected by immunofluorescence assay to assess their impact on the progression of cirrhosis. The results are as follows: Figure 25 As shown; by Figure 25 It can be seen that, compared with the PBS group, Exos group and ICG-Exos group, the fibrosis level of hepatocytes in the αPD1 group, αPG-Exos group, ICG-Exos / Apt group and αPG-Exos / Apt group was significantly reduced, and the αPG-Exos / Apt group had the lowest level of hepatocyte fibrosis.

[0165] 5. Detect CD8 + T cell cytotoxicity against 3D cell spheroids

[0166] Add CD8 to 3D cell spheres + After T cell co-culturing for 24 h, the cell spheres were washed, stained, and fixed using a cell viability / cytotoxicity assay kit (30002, Biotium). Images were captured using a confocal microscope, with the Z-stack projected from the top of the cell sphere to the center at 5 μm intervals, then rendered as the maximum intensity projection. To quantify the live / dead cell ratio, the total cell area for each stain was measured using ImageJ software. The results are shown below. Figure 26 As shown.

[0167] Depend on Figure 26 It was found that the survival rates of tumor cells in the αPD1 group, αPG-Exos group, ICG-Exos / Apt group, and αPG-Exos / Apt group were significantly lower than those in the PBS group, Exos group, and ICG-Exos group, with the αPG-Exos / Apt group showing the highest tumor cell death rate. Immunofluorescence assays of liver cancer cell markers AFP and EpCAM further validated this result. Figure 27 ).

[0168] 6. Detect the invasive ability of liver cancer cells in each group:

[0169] After two days of co-culturing, type I collagen (CC050, Sigma-Aldrich) was injected. First, the collagen was neutralized to pH 7.0-8.0, and then added to cell spheres in a 35-well agarose gel model. After incubation for 4 minutes, the entire culture system was inverted and incubated for 1 hour. Subsequently, the culture system was inverted and RPMI medium containing 5% FBS and 1% penicillin-streptomycin was added. Invasion assays were performed for two days, and images were taken using an inverted microscope (XDS-900, Caikon). The results are shown below. Figure 28 As shown. By Figure 28It can be seen that, compared with the PBS group, Exos group and ICG-Exos group, the invasive diffusion ability of spheroids in the αPD1 group, αPG-Exos group, ICG-Exos / Apt group and αPG-Exos / Apt group was significantly reduced, and the invasive diffusion ability of spheroids in the αPG-Exos / Apt group was the worst.

[0170] These results indicate that αPG-Exos / Apt can prevent hepatocyte fibrosis and malignant transformation.

[0171] Example 7: Effects of drug-loaded exosomes αPG-Exos / Apt on the tumor microenvironment and liver fibrosis-hepatocellular carcinoma malignant transformation.

[0172] The in vitro experiments in Example 6 have confirmed that αPG-Exos / Apt can prevent hepatocyte fibrosis and malignant transformation, thus playing a role in the prevention and treatment of liver cancer. To further verify whether αPG-Exos / Apt also has a therapeutic effect in vivo, this embodiment uses the cirrhosis-hepatocellular carcinoma malignant transformation mouse model provided in Example 2 for verification. The drug administration groups include: ① Blank (untreated group); ② Exos group (wild-type 293T cell-derived exosomes treatment group); ③ ICG-Exos (293T cell-derived exosomes treatment group with successfully encapsulated ICG); ④ αPG-Exos group (293T cell-derived exosomes treatment group with successfully encapsulated ICG and conjugated with Pembrolizumab); ⑤ ICG-Exos / Apt group (293T cell-derived exosomes treatment group with successfully encapsulated ICG and conjugated with Pembrolizumab and conjugated with Pembrolizumab); ⑦ αPD1+ICG group (1 mg / mL Pembrolizumab+ICG treatment group). At week 14 of modeling, 100 μg Exos (50 μg / 100 μL PBS) was injected intravenously into mice three times a week. Samples were collected after the administration period for the following tests:

[0173] 1. In vivo imaging

[0174] The biodistribution of Exos in each group was assessed using in vivo imaging. In vivo imaging was performed within 24 hours after tail vein injection of Exos. After 24 hours, major organs and tumors were dissected and removed for in vitro imaging. The results are as follows: Figure 29 and Figure 30 As shown.

[0175] Depend on Figure 29 and Figure 30It was found that within 24 hours of drug administration, the fluorescence signals of the αPG-Exos group, ICG-Exos / Apt group, and αPG-Exos / Apt group were mainly located in the kidney, liver, and tumor tissue. Due to their non-targeting or small particle size, the Exos group, ICG-Exos group, and αPD1+ICG group were quickly metabolized by the kidney, and their accumulation at the tumor site gradually decreased. However, the fluorescence signal at the tumor site of the αPG-Exos group, ICG-Exos / Apt group, and αPG-Exos / Apt group had a longer duration, and the fluorescence signal intensity of the tumor in the αPG-Exos / Apt group was the strongest. This is because the dual targeting ability of Pembrolizumab and Apt leads to a prolonged accumulation time at the tumor site.

[0176] 2. Liver function test

[0177] Liver function tests were performed on mice in each group, and the results are as follows: Figure 31 A- Figure 31 As shown in C. Figure 31 A- Figure 31 As shown in C, compared with the Blank group, Exos group, and ICG-Exos group, the levels of ALP, AST, and ALT in the blood samples of mice in the αPD1+ICG group, αPG-Exos group, ICG-Exos / Apt group, and αPG-Exos / Apt group were significantly reduced at 24 weeks. This may be due to the influence of the degree of accumulation in tumor tissue. The liver function damage relief effect was poor in the αPD1+ICG group mice, while the liver function damage relief was the most obvious in the αPG-Exos / Apt group mice.

[0178] 3. Pathological staining

[0179] Liver tissues were collected from mice in each group at 24 weeks of age for observation and pathological staining. The results are as follows: Figure 31 D and Figure 32 As shown. By Figure 31 D and Figure 32 As shown in Figure A, compared with the Blank group, Exos group, and ICG-Exos group, the number of liver nodules in mice in the αPD1+ICG group, αPG-Exos group, ICG-Exos / Apt group, and αPG-Exos / Apt group was significantly reduced, with the αPG-Exos / Apt group having the fewest liver nodules. Figure 32 B and Figure 32 C indicates that liver fibrosis was reduced in mice in the αPD1+ICG group, αPG-Exos group, ICG-Exos / Apt group, and αPG-Exos / Apt group, with the least liver fibrosis in the αPG-Exos / Apt group. Figure 32D indicates that AFP expression was significantly reduced in the livers of mice in the αPD1+ICG group, αPG-Exos group, ICG-Exos / Apt group, and αPG-Exos / Apt group, with the lowest AFP expression in the αPG-Exos / Apt group.

[0180] 4. Flow cytometry detection of CD8 + T cell cytotoxicity

[0181] CD8 counts in mice after drug administration were detected by flow cytometry. + The expression of IFN-γ and GZMB in T cells was analyzed, and the results are as follows: Figure 33 A and Figure 33 As shown in B. Figure 33 A and Figure 33 B shows that the number of IFN-γ and GZMB positive cells in the αPD1+ICG, αPG-Exos, ICG-Exos / Apt, and αPG-Exos / Apt groups was significantly increased compared with other groups, with the αPG-Exos / Apt group having the highest number of positive cells.

[0182] 5. Western blot analysis of PSMA1 expression levels

[0183] The expression level of PSMA1 in mouse liver tissue after drug administration was detected by Western blot, and the results are as follows: Figure 33 As shown in C. Figure 33 As shown in C, compared with other groups, the expression level of PSMA1 in the liver tissue of mice in the αPD1+ICG group, αPG-Exos group, ICG-Exos / Apt group and αPG-Exos / Apt group was significantly reduced.

[0184] 6. Detect changes in sphingomyelin metabolites in serum.

[0185] The changes in the levels of sphingomyelin metabolites in mouse serum after drug administration were detected using a kit, and the results are as follows: Figure 33 As shown in D. Figure 33 As shown in D, compared with other groups, the levels of sphingomyelin metabolites (SM) in the serum of mice in the αPD1+ICG group, αPG-Exos group, ICG-Exos / Apt group and αPG-Exos / Apt group were significantly reduced.

[0186] 7. H&E staining of major organs

[0187] After treatment, major organs were removed and subjected to H&E staining. No obvious pathological changes were found in the major organs. Figure 34The results indicate that αPG-Exos / Apt has good biocompatibility. All of these results suggest that αPG-Exos / Apt can remodel the tumor microenvironment and prevent the malignant transformation from liver fibrosis to hepatocellular carcinoma.

[0188] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A targeted drug-loaded exosome, characterized in that, The targeted drug-loaded exosomes include exosomes encapsulated with indocyanine green, and the surface of the drug-loaded exosomes is attached with aptamers targeting immune checkpoints and immune checkpoint inhibitors. The immune checkpoint inhibitor is a PD-1 inhibitor; the PD-1 inhibitor includes at least one of tislelizumab, nivolumab, and pembrolizumab. The aptamer targeting the immune checkpoint targets PD-L1; the nucleotide sequence of the aptamer is SEQ ID NO:1; The targeted drug-loaded exosomes are prepared by a method comprising the following steps: S1: Indocyanine green-tetrabutylammonium iodide complex was mixed with exosomes and incubated to obtain exosomes encapsulated with indocyanine green; S2: Use a linker to link the immune checkpoint inhibitor with the indocyanine green-encapsulated exosomes obtained in step S1 to obtain drug-loaded exosomes; S3: Use a cross-linking agent to connect the aptamer targeting the immune checkpoint to the drug-loaded exosomes obtained in step S2 to obtain targeted drug-loaded exosomes; The source of the exosomes in step S1 includes 293T cells; the linker in step S2 includes DSPE-PEG-NHS; and the cross-linking agent in step S3 includes NHS and EDC.

2. The targeted drug-loaded exosome according to claim 1, characterized in that, The molar ratio of indocyanine green and tetrabutylammonium iodide in step S1 is 1:(3-10).

3. The targeted drug-loaded exosome according to claim 2, characterized in that, The mass ratio of indocyanine green to exosomes is 1:(5-20).

4. The targeted drug-loaded exosome according to claim 2, characterized in that, The incubation temperature is 35℃-40℃.

5. The targeted drug-loaded exosome according to claim 2, characterized in that, The incubation time is 1-3 hours.

6. The targeted drug-loaded exosome according to claim 1, characterized in that, The mass ratio of the immune checkpoint inhibitor to DSPE-PEG-NHS in step S2 is 1:(20-40).

7. The targeted drug-loaded exosome according to claim 6, characterized in that, In step S2, the mass ratio of the immune checkpoint inhibitor to the exosomes encapsulated with indocyanine green is 1:(20-40).

8. The targeted drug-loaded exosome according to claim 1, characterized in that, In step S3, the mass ratio of the aptamer targeting the immune checkpoint to the drug-loaded exosome is 1:(40-60).

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises targeted drug-loaded exosomes as described in any one of claims 1-8 and pharmaceutically acceptable excipients.

10. The use of the targeted drug-loaded exosomes as described in any one of claims 1-8 in the preparation of tumor-targeting drugs.

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