Targeting drug-loaded exosome for liver cirrhosis-liver cancer as well as preparation method and application of targeting drug-loaded exosome

Abstract

The invention discloses a targeting drug-loaded exosome for liver cirrhosis-liver cancer and a preparation method and application thereof. The targeting drug-loaded exosome comprises an exosome encapsulated with indocyanine green, and the surface of the drug-loaded exosome is connected with an aptamer of a targeting immune checkpoint and an immune checkpoint inhibitor. According to the targeting drug-loaded exosome provided by the invention, the targeting property and the bioavailability of palbozuril are improved through a nanotechnology and the nucleic acid aptamer, the function of CD8 + T cells is further enhanced, and the malignant transformation of liver cirrhosis-liver cancer is blocked. Experiments prove that the drug-loaded exosome can significantly enhance the cytotoxicity and anti-tumor function of CD8 + T cells in a liver cancer model, and the drug-loaded exosome is compared with a traditional immunotherapy strategy clearly.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical materials, and in particular to a drug-loaded exosome targeted to cirrhosis and liver cancer, and a preparation method and application thereof. Background Art

[0002] Liver cirrhosis and liver cancer are major challenges to global public health. According to the World Health Organization, liver cancer is one of the leading causes of cancer mortality 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. At present, although certain 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 serious side effects. In addition, many patients are already in the late stage at the time of diagnosis and have lost the opportunity for surgical treatment. Therefore, the development of new treatment strategies, especially methods that can accurately target the tumor microenvironment, is of great significance for improving the survival rate of patients with liver cancer.

[0003] In recent years, immune checkpoint inhibitors such as Pembrolizumab have shown significant efficacy in the treatment of various cancers by relieving immunosuppression and enhancing the patient's immune response. However, the application of Pembrolizumab in the treatment of liver cancer still faces many challenges, such as drug instability and nonspecific distribution in the body, which limits its therapeutic potential. In addition, the tumor microenvironment of liver cancer is particularly complex, involving multiple cell types such as immune cells, fibrotic cells, and tumor cells. These cells interact to form an immunosuppressive environment, which greatly reduces the effectiveness of immunotherapy. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a targeted drug-loaded exosome that precisely releases immune checkpoint inhibitors to reshape the tumor immune microenvironment and enhance CD8 + The function of T cells can prevent the malignant transformation of cirrhosis to liver cancer.

[0005] The present invention also provides a method for preparing the above-mentioned targeted drug-loaded exosomes.

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

[0007] The present invention also provides an application.

[0008] According to a first aspect of the present invention, a targeted drug-loaded exosome is proposed, wherein the targeted drug-loaded exosome comprises an exosome encapsulated with indocyanine green, and the surface of the drug-loaded exosome is connected to an aptamer targeting an immune checkpoint and an immune checkpoint inhibitor.

[0009] In some embodiments of the present invention, the immune checkpoint includes 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 linked 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 exosomes is calculated as protein mass.

[0017] According to a second aspect of the present invention, a method for preparing the targeted drug-loaded exosomes according to the first aspect of the present invention is provided, wherein the method comprises the following steps:

[0018] S1: indocyanine green-tetrabutylammonium iodide complex is mixed with exosomes and incubated to obtain exosomes encapsulating indocyanine green;

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

[0020] S3: Use a cross-linker to connect the immune checkpoint-targeting aptamer 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 to tetrabutylammonium iodide in step S1 is 1:(3-10).

[0022] In some embodiments of the present invention, the molar ratio of indocyanine green to 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 linker in step S2 comprises 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 encapsulating 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 encapsulating indocyanine green in step S2 is 1:(25-35).

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

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

[0034] In some embodiments of the present invention, the mass ratio of the immune checkpoint targeting aptamer to the drug-loaded exosomes 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 present invention, a pharmaceutical composition is provided, comprising the targeted drug-loaded exosomes according to the first aspect of the present invention and a pharmaceutically acceptable excipient.

[0037] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a binder, a disintegrant, a lubricant, a coating agent, a suspending agent, a thickening agent, and a surfactant.

[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 liver 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 use of the targeted drug-loaded exosomes described in the first aspect of the present invention in the preparation of tumor-targeted drugs is proposed.

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

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

[0045] In some embodiments of the present invention, the solid tumor comprises at least one of liver cancer, lung cancer, breast cancer, colorectal cancer, gastric 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 the present invention improve the targeting and bioavailability of pambrolizumab through nanotechnology and nucleic acid aptamers, further enhancing the CD8 + The present invention has experimentally confirmed that the drug-loaded exosomes can significantly enhance the CD8 T cell function in the liver cancer model. + The present invention significantly enhances the cytotoxicity and anti-tumor function of T cells, which is in stark contrast to traditional immunotherapy strategies. Furthermore, the present invention significantly improves the distribution and stability of immunomodulators in the body. This combination strategy not only enhances the efficacy of the drug but also reduces the burden of side effects on patients. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0049] Figure 1 Figure 1 is a graph showing the analysis results of scRNA-seq data in Example 1 of the present invention; Figure 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), with darker blue representing higher average expression levels and larger circles representing more cells expressing the gene; Figure B shows 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 difference in cell content between liver cancer tissue and normal liver tissue analyzed by T test in Example 1 of the present invention;

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

[0052] Figure 4 CD8 + The distribution results of T cells and PD-1; A is CD8 + T cells, B is PD-1;

[0053] Figure 5 Schematic diagram of the liver cirrhosis-liver cancer malignant transformation mouse model in Example 2 of the present invention;

[0054] Figure 6 Schematic diagram of blood sample collection for liver function testing in mice in Example 2 of the present invention;

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

[0056] Figure 8 Figures 2 are the pathological staining results of liver tissues of mice in each group in Example 2 of the present invention; wherein, 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 flow chart of proteomics combined with metabolomics analysis in Example 3 of the present invention;

[0058] Figure 10Figure 3 is a graph showing the results of proteomic analysis in Example 3 of the present invention; wherein, A is a statistical representation of the number of adjacent nodes of the core protein in the gene interaction network diagram, the horizontal axis represents the adjacent node value, and the vertical axis represents the protein name; B is a heat map showing the differential expression of 35 DEPs in the proteomic data, Cirrhosis (N=3) and HC (N=3) represent liver tissue samples from three mice at 14 weeks and three mice at 24 weeks of CCl4 modeling, respectively;

[0059] Figure 11 Figure 3 is a graph showing the enrichment analysis results of differential proteins in Example 3 of the present invention; wherein, A is a GO enrichment bubble diagram of differential proteins in liver tissue samples of 3 mice at 14 weeks and 3 at 24 weeks of CCl4 modeling, BP represents biological process, CC represents cellular component, and MF represents molecular function; B is a KEGG clustering tree diagram of differential genes in liver tissue samples of 3 mice at 14 weeks and 3 at 24 weeks of CCl4 modeling;

[0060] Figure 12 Figure 3 is a functional enrichment analysis result diagram of differential metabolites in Example 3 of the present invention; wherein A is an OPLS-DA score diagram, the horizontal axis represents the predicted component score value, the vertical axis represents the orthogonal component score value, and the percentage represents the component's explanation rate for the data; B is a display of the top 20 differential metabolites between the two groups preliminarily screened in the VIP of the OPLS-DA model; C is the functional enrichment analysis result of the differential metabolites in the SMPDB database;

[0061] Figure 13 Figure 3 shows the results of the combined proteomics and metabolomics analysis in Example 3 of the present invention; A is a volcano plot of differential metabolites screened by the T test algorithm; B is a volcano plot of differential metabolites screened by the P value and fold difference of the univariate analysis; C is a Venn diagram showing the intersection of differential metabolites screened by the OPLS-DA model, the T test algorithm, and the univariate analysis;

[0062] Figure 14 Figure 3 is a functional enrichment analysis result diagram of differential metabolites in Example 3 of the present invention; wherein 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 Schematic diagram of the synthesis of αPG-Exos / Apt in Example 4 of the present invention;

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

[0065] Figure 17 Graph showing the results of characterizing ICG-Exos in Example 4 of the present invention; wherein A represents the size distribution of Exos and ICG-Exos, B represents the hydrodynamic size of ICG-Exos, and C represents 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 irradiation in Example 4 of the present invention;

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

[0068] Figure 20 Graph showing the agarose gel electrophoresis results of ICG-Exos / Apt and αPG-Exos / Apt in Example 4 of the present invention;

[0069] Figure 21 CD8 was observed by laser scanning microscope in Example 4 of the present invention. + Figure 3. T cell uptake of Exos; the scale bar is 25 μm;

[0070] Figure 22 To explore the effect of αPG-Exos / Apt on CD8 + Results of the effect of T cell function; A is the MTT assay for cell activity; B is the CD8 + Diagram of the mechanism of T cells killing tumor cells; C and D are ELISA tests for the levels of IFN-γ and GZMB in the cell culture medium; E and F are Western blot tests for CD8 + GZMB content in T cells; G is immunofluorescence detection of CD8 + Co-localization of CD8 and GZMB in T cells;

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

[0072] Figure 24 This is the confocal microscope detection of CD8 in Example 6 of the present invention. + Results of T cell permeability; scale bar is 25 μm;

[0073] Figure 25 The results of immunofluorescence assays for detecting fibrosis markers α-SMA and E-cadherin in co-cultured cell spheroids in each group in Example 6 of the present invention are shown in FIG. 1 ; wherein 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; wherein A is AFP, B is EpCAM, and the scale bar is 25 μm;

[0076] Figure 28 This is a cell culture image captured in bright field mode using an inverted microscope in Example 6 of the present invention; the cell invasion area is outlined by a white dotted line, and the scale bar is 100 μm;

[0077] Figure 29 This is the fluorescence distribution diagram of each group of tumor-bearing mice observed by NIR fluorescence imaging in Example 7 of the present invention;

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

[0079] Figure 31 Graph showing the liver function test results of each group of mice in Example 7 of the present invention; wherein A represents ALP, B represents AST, and C represents ALT;

[0080] Figure 32 Figures 2 and 3 are the pathological staining results of liver tissues of mice in each group in Example 7 of the present invention; wherein, 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); D is immunofluorescence staining for AFP protein detection (scale bar 25 μm);

[0081] Figure 33 To explore the effect of αPG-Exos / Apt on CD8 + Results of the effects on T cell and liver metabolism; A and B are 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 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; where 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, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all 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 data sets 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 data set were analyzed, and the liver tissue section data of 2 patients in the GSE245908 data set were analyzed. The Seurat package was used to integrate the data in the data set, and the harmony package was used to correct the batch of the sample data after data quality control. Quality control was carried out 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 T-test analysis results showed that CD8 + T cell counts were significantly reduced ( Figure 2 ). Extract CD8 + T cell data, differential analysis revealed that the expression level of PD-1 (PDCD1) in HCC tissues was significantly increased ( Figure 3 ).

[0086] To characterize the distribution of different cell types in HCC tissue, this example uses a spatial transcriptomics approach to analyze liver tissue slice data from two HCC patients in the GSE245908 dataset. The ST data were first integrated and normalized using the Seurat package. After screening for highly variable genes, the data were subjected to dimensionality reduction and cluster analysis using PCA and UMAP algorithms. By calculating the degree of overlap between genes and cell type-specific genes in scRNA-seq data, the cell types in the ST data were inferred and annotated. The results are shown in Figure 2. Figure 4 As shown. Figure 4 It can be seen that CD8 + T cells are less distributed in HCC tissues, while the expression level of PD-1 is higher.

[0087] Example 2 Construction of a mouse model of liver cirrhosis-liver cancer malignant transformation and model verification

[0088] 1. Construction of a mouse model of liver cirrhosis-hepatocellular carcinoma malignant transformation

[0089] In order to study the development process of liver cirrhosis-liver cancer malignant transformation, this example established a mouse model of liver cirrhosis-liver cancer malignant transformation by oral administration of CCl4. The specific method is as follows:

[0090] SPF-grade male C57BL / 6J mice aged 4-6 weeks and weighing 16-22 g were purchased (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) and housed in separate cages in an SPF-grade animal laboratory. The humidity and temperature in the laboratory were controlled at 60%-65%, 25±2°C, and free food and water were provided under 12-h light and dark alternation conditions. The experiment was started after one week of adaptive feeding, and the health status of the mice was observed before the experiment.

[0091] Each mouse in the control group was gavaged with 100 μL of olive oil (E0505, Selleck), and each mouse in the model group was gavaged with olive oil solution containing 40% CCl4 (C0731530924, Nanjing Reagent) three times a week for 14 weeks. The modeling process was as follows. Figure 5Starting from week 14, tumor growth was observed by in vivo imaging, and then continued to be observed, and changes in tumor volume were recorded every 7 days.

[0092] 2. Validation of the Cirrhosis-HCC Malignant Transformation Mouse Model

[0093] 1) Liver function test:

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

[0095] Depend on Figure 7 Compared with the control group, the levels of ALP, AST, and ALT in the blood samples of the mice in the Model group gradually increased over time, suggesting that the degree of liver function damage was gradually deepened. Notably, serum transaminase levels remained high after discontinuation of CCl4 at week 14, indicating that liver damage had reached an irreversible stage.

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

[0097] ① H&E staining: The tissue samples to be examined were fixed and sectioned in xylene to remove wax. The sections were then rehydrated in 100% ethanol, 95% ethanol, and 70% ethanol, respectively, and washed once with water. The sections were stained in hematoxylin solution (H8070, Solarbio) for 5–10 min at room temperature. The sections were then washed with distilled water, dehydrated in 95% ethanol, and placed in eosin solution (G1100, Solarbio) for 5–10 min. Subsequently, the sections were dehydrated in different concentrations of alcohol (85%, 90%, 95%, and 100%) and finally cleared with dichloromethane.

[0098] ② Sirius red staining: A Sirius red staining kit (50-300-77, Fisher Scientific) was used, and the staining procedure was similar to the aforementioned H&E staining procedure.

[0099] ③Masson staining: Masson staining kit (G1340, Solarbio) was used for staining according to the instructions provided by the kit.

[0100] After the staining was completed, the slides were sealed with neutral resin and observed under an optical microscope after drying. Five fields of view were randomly selected for analysis for each stained section, and at least three sections were examined for each mouse. The percentage of Masson trichrome-positive areas was analyzed using Image-Pro-Plus software (Media Cybernetics, V6.0) and the Ishak scoring method was used for morphometric quantification of collagen. The results are shown in the figure. Figure 8 shown.

[0101] Depend on Figure 8 It can be seen that compared with the Control group, the livers of the mice in the Model group showed signs of CCl4-induced damage, characterized by a light yellow appearance of the liver tissue instead of dark red, a harder texture than normal liver tissue, an irregular surface, nodules of varying sizes, and an increase in the number of nodules with increasing administration time; at 14 weeks, the mice showed obvious liver fibrosis, indicating that the cirrhosis mouse model was successfully established, and at 10 weeks after discontinuation of CCl4 (i.e., 24 weeks), the surface of the mouse liver showed significant nodule formation and disordered tissue structure, and the fibrosis around these nodules increased significantly, which is a typical feature of liver cancer; Sirius red and Masson staining results showed that the fibrosis of the mouse liver gradually worsened over time. The above results all confirm that the mouse model of malignant transformation from cirrhosis to liver cancer was successfully established.

[0102] Example 3 Proteomics combined with metabolomics to detect key proteins and metabolites in the malignant transformation of cirrhosis to liver cancer

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

[0104] 1. Proteomics Sample Preparation and Assay

[0105] Three cases of CCl4 modeling mouse liver tissues at 14 weeks (cirrhosis stage) and three cases at 24 weeks (liver cancer stage) were added to a mortar, and liquid nitrogen was added to the mortar and ground with a pestle. The resulting powder was transferred to a 5 cm 3The cells were then ultrasonically treated 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 cocktail (P6731, Solarbio), and 2 mmol / L EDTA (E1170, Solarbio). The ultrasonic step was repeated 8 times. Next, an equal amount of Tris-saturated phenol (HCC1380, BIOFOUNT) at pH 8.0 was added to the sonicated system, and the mixture was further vortexed for 4 minutes. The mixture was then centrifuged at 5000 g for 10 minutes at 4°C, and the phenol layer on the upper layer 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 allowed 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) and the operation steps were carried out according to the manufacturer's instructions.

[0106] 2. Proteolysis, Peptide Labeling and Fractionation, and Nano-LC-MS / MS Detection and Analysis

[0107] 50 μg of each protein sample was enzymatically treated. The protein solution was mixed with DTT to a concentration of 5 mmol / L and allowed to stand at 56°C for 30 minutes. Acetamide was then added to a concentration of 11 mmol / L and allowed to stand at room temperature for 15 minutes. Finally, the urea concentration of the sample was diluted to less than 2 mol / L, and trypsin (25200056, Thermo Fisher) was added at a mass ratio of 1:50 (w / w) and enzymatically digested at 37°C overnight. Trypsin was then added at a mass ratio of 1:10 (trypsin: protein) and enzymatic digestion was continued for 4 hours.

[0108] After trypsin digestion, use HyperSep TM The peptides were desalted using a C18 purification column (60108-302, Thermo Fisher) and dried under vacuum. The peptides were redissolved in 0.5 mol / L TEAB (90114, Thermo Fisher) and the manufacturer's instructions for the TMT kit (90064CH, Thermo Fisher) were followed. Briefly, 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 by vacuum centrifugation. Equal amounts of each group of labeled peptides were mixed and the dried peptides were purified using PierceTM The peptides were fractionated using a high pH reversed-phase peptide separation kit (84868, Thermo Fisher, USA). The samples were then collected and combined into 15 fractions, and the peptides in each fraction were dried and reconstituted with 0.1% formic acid (159002, Sigma-Aldrich, USA).

[0109] 2 μg of peptides were collected from each sample and separated using the nano-UPLC Easy nLC 1200 system. The sample was first loaded onto a Trap C18 column (100 μm × 20 mm, 5 μm) and then gradient separated on a C18 analytical column (75 μm × 150 mm, 3 μm) at a flow rate of 300 nL / min. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in water in acetonitrile (containing 95% acetonitrile). The gradient elution program was as follows: 2% to 8% B (0-2 min); 8% to 28% B (2-71 min); 28% to 40% B (71-79 min); 40% to 100% B (79-81 min); and 100% B (81-90 min). The peptides separated by liquid phase were analyzed by mass spectrometry on a Q-Exactive HCX mass spectrometer. The analysis time was 60 min, the electrospray voltage was 2.1 kV, the detection mode was positive ion, the parent ion scan range was 350-1200 m / z, the primary mass spectrometry resolution was 60000 @ m / z 200, and the AGC target was 3e 6 , Maximum IT: 30ms. Secondary 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 resulting 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 / ) with a reverse decoy database. Trypsin / P was designated as the cleavage enzyme, and up to two missed cleavages were allowed. The primary search was performed at 20 ppm, the primary search at 5 ppm, and the fragment ion mass tolerance was 0.02 Da. The thresholds for peptide false discovery rate (FDR) ≤ 0.01 and protein FDR ≤ 0.01 for library identification, as well as peptide score distribution, were used as screening criteria. 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. The top 100 DEPs ranked by |log2FC| were then extracted, and a PPI network was further constructed. The number of adjacent nodes for each gene in the network graph was counted, and proteins with no adjacent nodes were removed. As a result, 35 key proteins ( Figure 10 A), CD8A, CD8B, IFNG and GZMB + The expression of T cell-related proteins was significantly downregulated, and PSMA1 protein was significantly upregulated ( Figure 10 B). Plasma proteasome (PSMA1) levels are a reliable early marker for malignant transformation of cirrhosis (PMID:19201777). Pathway enrichment analysis was performed on the screened differentially expressed proteins, and the results were as follows: Figure 11 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 example, plasma samples were collected from 6 mice at 14 weeks of CCl4 modeling (cirrhosis stage) and 6 mice at 24 weeks of CCl4 modeling (liver cancer stage), and non-targeted metabolomics analysis was performed by liquid chromatography-mass spectrometry. The 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 minutes, and centrifuged at 12,000 g for 10 minutes. The supernatant was transferred to an autosampler vial. Plasma metabolomics analysis was performed using an LC20 ultra-high performance liquid chromatograph (Shimadzu, Japan) coupled with a Triple TOF-6600 mass spectrometer (ABSciex). Chromatographic analysis was performed using a Waters ACQUITY UPLC HSS T3C18 (100 × 2.1 mm, 1.8 μm) column. 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 acetonitrile in water containing 0.1% formic acid. The gradient elution program for mobile phase B was as follows: 5% for 0.0–11.0 minutes; 90% for 11.0–12.0 minutes; and 5% for 12.1–14 minutes. The eluent is introduced directly into the mass spectrometer without fragmentation.

[0115] Mass spectrometry conditions were as follows: ionization voltage, 5500 V; capillary temperature, 550°C; nebulizer gas flow rate, 50 psi; and auxiliary heating gas flow rate, 60 psi. Preprocessed data were analyzed using orthogonal partial least squares-discriminant analysis (OPLS-DA) and permutation tests (100 permutations) to prevent overfitting. Metabolites with a VIP score >1 and a p-value <0.05 in the OPLS-DA model were identified as differential metabolites (DMs). Univariate analysis was combined with metabolites with a fold difference ≥2 and a fold difference ≤0.5, and a p-value <0.05 in the Student's t test, to be considered 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 determined using the Sigma-Aldrich Phospholipid Quantification Kit (MAK262). In this example, metabolomics data were normalized and PCA clustered, OPLS-DA model was constructed, and score graphs of each group were drawn ( Figure 12 A) by Figure 12 A shows that the metabolic profiles of the cirrhosis group and the liver cancer group were significantly separated.

[0116] The OPLS-DA evaluation model was further verified. Based on the OPLS-DA results, the differential metabolites between the two groups were preliminarily screened from the variable importance projection of the multivariate analysis OPLS-DA model. The results are as follows Figure 12 B and Figure 12C. We further combined the T test algorithm with the P value and difference fold of univariate analysis to screen differential metabolites. The results are shown in Figure 13 A and Figure 13 As shown in B, a total of 71 intersection differential metabolites were obtained ( Figure 13 C) The differential metabolites were mainly lipid metabolites, especially sphingomyelin compounds, most of which showed an increasing trend in the liver cancer group.

[0117] To further explore the possible functions and effects of differential metabolites, this example used MetaboAnalyst5.0 database to perform functional enrichment analysis on the obtained differential metabolites. The results are as follows: Figure 14 As shown. Figure 14 It can be seen that the analysis of SMPDB database and KEGG database showed that the differential metabolites were mainly enriched in lipid metabolism and other related pathways.

[0118] In summary, this example found that CD8 + The expression of T cell-related proteins was significantly downregulated, 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] In order to solve the problem of significant activation of the PD-1 / PD-L1 pathway during the malignant transformation of cirrhosis to liver cancer, this embodiment provides a method for targeted delivery of CD8 + T cells, drug-loaded exosomes that carry and 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 (Exos)

[0122] When 293T cell fusion reaches 80%-90%, discard the supernatant and wash the cells with 2×PBS. Then add 25mL serum-free IMDM medium (12440053, Gibco) to each culture flask and continue to incubate for 48 hours in a humidified condition of 37°C and 5% CO2. The cell supernatant is collected in a 50mL centrifuge tube and centrifuged at 300g for 10 minutes at 4°C to remove cell debris. The supernatant is collected in another 50mL centrifuge tube again. After collecting the supernatant, the supernatant is centrifuged at 2000g for 20 minutes at 4°C, transferred to a sterile tube for a high-speed centrifuge, and centrifuged at 16500g for 30 minutes at 4°C. The supernatant is transferred to an ultracentrifuge tube again and centrifuged at 120000g for at least 70 minutes at 4°C using a fixed-angle rotor, and the supernatant is completely discarded. Add 1 mL of 4°C PBS to each ultracentrifuge tube and resuspend the pellet with a micropipette. Mix the solutions from the same group into the ultracentrifuge tube and then add 4°C PBS to a volume exceeding 3 / 4 of the tube. Centrifuge at 120,000 g for 60 minutes at 4°C, remove as much supernatant as possible, and resuspend the pellet in sterile PBS to obtain 293T cell-derived exosomes (293T-Exos).

[0123] WB was used to detect the expression of classic exosome markers (Alix, TSG101, CD81) in the above exosomes, and the results were as follows Figure 16 As shown in A, the results showed that the above exosome markers were expressed in the exosomes prepared in this example. Transmission electron microscopy and NTA analysis also confirmed the typical disc-shaped morphology of exosomes, with a size range of 50-300nm ( Figure 16 B and Figure 16 C).

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

[0125] 1) Encapsulation of indocyanine green (ICG) in 293T-Exos:

[0126] 16 mg of ICG (HY-D0711, MedChemExpress) and 46 mg of 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 mass) were mixed with the ICG-TBAI complex containing 4 μg of ICG (i.e., the protein mass 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 Exos that successfully encapsulated ICG (ICG-Exos).

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

[0128] FITC-labeled pembrolizumab (Pembrolizumab, HY-P9902, MedChemExpress) was diluted with PBS, and then the solvent was removed by centrifugation at 2000g for 5 minutes using a 10kDa ultrafiltration centrifuge tube. The DSPE-PEG-NHS (HY-W441014, MedChemExpress) linker was dissolved in DMSO, the excess linker was mixed with the above antibody solution, and then the mixed system was continuously stirred at 4°C for 24 hours. The mass ratio of pembrolizumab to linker was 1:30. Subsequently, repeated centrifugation at 2000g for 5 minutes using a 10kDa ultrafiltration centrifuge tube was used to remove the unconnected linker. The obtained DSPE-PEG-NHS-Pembrolizumab solution was reacted with the ICG-Exos purified in step 1) at 4°C for 3 hours. The mass ratio of pembrolizumab to ICG-Exos was 1:30. When the terminal DSPE of the linker was inserted into the membrane of ICG-Exos, antibody-functionalized drug-loaded exosomes αPG-Exos were obtained by centrifugation at 100,000 g for 2 h in an ultracentrifuge at 4 °C.

[0129] 3) Construction of targeted drug-loaded exosomes:

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

[0131] 3. Testing and Characterization of Exosomes

[0132] In order to test the various properties of αPG-Exos / Apt, the following exosome groups were also constructed for comparison:

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

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

[0135] To determine the encapsulation efficiency, dynamic light scattering (DLS; Malvern Instruments Ltd.) was used to measure the hydrodynamic size of Exos and ICG-Exos. The relative encapsulation efficiency was calculated as follows: (ICG amount in Exos) / (ICG addition amount) × 100%. The results are shown in Figure 17 As shown. Figure 17It can be seen that Exos showed a narrow size distribution of about 35 nm, while large aggregation and wide size distribution were observed in ICG-Exos; compared with Exos, the hydrodynamic size of ICG-Exos was much larger, and the encapsulation efficiency of ICG in Exos was about 15%. ICG is highly susceptible to external physicochemical conditions such as light. After incubation at 37°C for a predetermined time (0, 3, 6, 20 h), the relative 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 as follows: (FI after incubation) / (initial FI at 0 h) × 100%. The results are shown in Figure 2. Figure 18 As shown. 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 the incubation process, indicating that the incorporation of ICG into Exos can protect ICG from the physicochemical damage of light irradiation 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 group and the αPG-Exos / Apt group. Figure 19 ), in order to confirm whether the PA9-1 aptamer was successfully coupled to the exosomes, agarose gel electrophoresis was performed, and the results were as follows Figure 20 As shown. Figure 20 It can be seen that in the ICG-Exos / Apt group and the αPG-Exos / Apt group, the mobility of the aptamer was hindered after the introduction of exosomes, confirming that the aptamer was 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, and Dil dye (C1036, Beyotime) was added to 40 μg of Exos to a final concentration of 25 μM. The cells were then reacted at room temperature for 30 minutes to allow the cells to react with CD8 +T cells were co-incubated for 24 h, and unbound dye was removed by ultracentrifugation after the incubation period. Next, the cells were rinsed three times with PBS and fixed with 4% paraformaldehyde for 30 min. Finally, the nuclei were stained with DAPI (C1005, Beyotime) for 30 min, and the cells were 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 shown in Figure 2. Figure 21 As shown. Figure 21 It can be seen that the CD8 + No red fluorescence signal was observed in T cells, while obvious red fluorescence signals were observed in cells of the other four groups, and the red fluorescence signals in the αPG-Exos group and the αPG-Exos / Apt group were the strongest, indicating that Exos in each group can be targeted by CD8 + In summary, this example successfully constructed aptamer-modified drug-loaded exosomes αPG-Exos / Apt that precisely released Pembrolizumab.

[0140] Example 5 Drug-loaded exosomes αPG-Exos / Apt on CD8 + Effects on T cell function

[0141] 1. MTT detection of CD8 + T cell viability

[0142] CD8 + T cells were expressed at 1 × 10 4 The cells were seeded in a 96-well plate at a density of 100 cells / well, and CD3 / CD28 was added to stimulate their activation (this step is required for the following experiments) and incubated for 24 hours. After co-culture with liver cancer cells for 48 hours, the CD8 + T cells. The cells in different treatment groups were mixed with 0.01 mL of MTT (CT02, Sigma Aldrich) solution and incubated in the incubator for 4 hours. Then, 0.1 mL of isopropanol containing 0.04 mol / L HCl was added to each well. Use a multichannel pipette to repeatedly pipette and mix thoroughly. HCl converts the phenol red in the tissue culture medium into a yellow color that does not interfere with the MTT formazan measurement. Cell viability is determined by measuring the absorbance at 570 nm. Cell viability = (ΔA_sample-ΔA_blank) / (ΔA_control-ΔA_blank). Among them, 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 in the figure. Figure 22 As shown in A.

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

[0144] 2.ELISA detection of CD8 + T cell killing 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). Take the cell culture supernatant and use the IFN-γ ELISA kit (BMS606-2, Invitrogen) and GZMB ELISA kit (BMS6029, Invitrogen) respectively. First, dilute the antigen to an appropriate concentration with the coating diluent, block the enzyme-labeled reaction wells with 5% calf serum at 37°C for 40 minutes, add the diluted sample to the enzyme-labeled reaction wells, then add the enzyme-labeled antibody, add the substrate solution, and finally add 50 μL of the stop solution to each well to terminate the reaction. Measure the experimental results within 20 minutes. Read the plate under the conditions of 450nm on the microplate reader, draw the standard curve, and analyze the data; the results are shown in Figure 22 C and Figure 22 As shown in D.

[0146] Depend on Figure 22 C and Figure 22 D showed that compared with the Exos group and ICG-Exos group, the IFN-γ and GZMB levels 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 levels in the supernatant of the αPG-Exos / Apt group were the highest.

[0147] 3. Western blot detection of CD8 + T cell killing ability

[0148] First, cells were collected and lysed with an enhanced RIPA lysis buffer (P0013B, Beyotime) containing protease inhibitors, and then the protein concentration was determined using a BCA protein quantification kit (P0012, Beyotime). Proteins were separated using 10% SDS-PAGE, and the separated proteins were electrotransferred to the surface of a PVDF membrane. 5% BSA was blocked at room temperature for 2 hours to block nonspecific binding, and diluted primary antibodies were added and incubated at room temperature for 1 hour. The primary antibodies were all rabbit anti-human. After washing the membrane, HRP-labeled goat anti-rabbit secondary antibodies were added and incubated at room temperature for 1 hour. Equal amounts of A and B solutions of ECL developer (32209, Thermo Scientific) were mixed in a dark room, added dropwise to the membrane, and placed in a gel imager for exposure and imaging. The Bio-Rad image analysis system was used to take pictures, and the grayscale of each group of bands in the Western blot image was quantified using Image J analysis software. The internal reference was β-actin. Each experiment was repeated 3 times, and the results were as follows: 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 ratio 1:1000; GZMB (PA5-17457), dilution ratio 1:2500; PSMA1 (PA1-963), dilution ratio 1:1000; β-actin (MA1-140), dilution ratio 1:5000. Figure 22 E and Figure 22 F showed that compared with the Exos group and ICG-Exos group, the IFN-γ and GZMB levels in the cells 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 levels in the cells of the αPG-Exos / Apt group were the highest.

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

[0150] CD8 + T cells were fixed on slides and washed twice with DPBS containing 0.05% Tween 20, permeabilized with 0.1% Triton X-100 for 3 minutes, and washed twice more with DPBS containing Tween 20. The samples were blocked for 1 hour with 5% goat serum and 0.3 mol / L glycine in DPBS. After this blocking step, sections were incubated with the primary antibody for 2 hours. The sections were then incubated with the secondary antibody overnight at 4°C. The antibodies used are listed in Table 1.

[0151] Table 1 Antibody information used for immunofluorescence staining

[0152]

[0153] On the second day, wash 3 times with DPBS containing Tween20, add secondary antibody and incubate at room temperature for 1 hour, rinse 3 times with DPBS, use DAPI to stain the nucleus for 5 minutes, soak the slide in PBS 3 times (5 minutes / time), and wash away the excess DAPI. Carefully remove the cell slide from the cell culture plate with a curved fine syringe needle and tweezers and place it on a slide with anti-fluorescence quenching mounting medium (cell side down). Observe and photograph under a fluorescence microscope. The quantitative method is the fluorescence coverage area under a fixed field of view with a 40× objective lens. Take 6 fields of view for each group and take the average value. The results are as follows Figure 22 G and Figure 23 As shown. Figure 22 G and Figure 23 It can be seen that compared with the Exos group and ICG-Exos group, the number of CD8 / GZMB double-positive cells in the αPD1 group, αPG-Exos group, ICG-Exos / Apt group and αPG-Exos / Apt group increased significantly, and the number of double-positive cells in the αPG-Exos / Apt group was the highest. In summary, this example confirms 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] In order to further explore the effect of the prepared αPG-Exos / Apt on the malignant transformation of liver fibrosis to liver cancer, this example constructed a carboxyfluorescein succinimidyl ester (CFSE)-labeled CD8 + T cells, liver fibrosis cells, and liver cancer cells were co-cultured into 3D spheres, and each group of Exos was added for co-incubation. The specific experimental methods and results are as follows:

[0156] 1. Construction of 3D cell spheroids

[0157] 1000 Hepa1-6 liver cancer cells / spheroids were seeded into 35- or 81-microwell agarose models created using 3D Petri dishes, a Microtissues spheroid formation device. One minute after cell seeding, 1 mL (for 35-microwell dishes) or 2 mL (for 81-microwell dishes) of cell culture medium were added, and 3D spheroids were formed at 37°C and 5% CO2.

[0158] 2. Treat 3D cell spheroids with different Exos:

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

[0160] 3. Detection of CD8 + T cell penetration:

[0161] On the second day of culture, CD8 + T cells, cirrhotic cells (hepatocytes isolated from the liver of mice modeled for 14 weeks) and tumor cells were added to the 3D cell spheres constructed in step 1 at a ratio of 5:1:1 and further co-cultured in DMEM medium supplemented with 10% fetal bovine serum and 100U / mL penicillin / streptomycin. After culturing at 37°C for 24 hours, the cell spheres were washed and fixed in 4% paraformaldehyde and imaged using a ZEN 710 confocal microscope. Images were collected at the middle height of the microspheres, and surface maps were obtained using Image J software. The results are shown in Figure 2. Figure 24 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 + The T cell penetration rate increased significantly, among which the αPG-Exos / Apt group had the highest cell penetration rate.

[0163] 4. Immunofluorescence Detection of Liver Fibrosis

[0164] Immunofluorescence assay was used to detect the fibrosis level (α-SMA and E-cadherin) of hepatocytes in 3D co-cultured cell spheres and evaluate the effect on the progression of liver cirrhosis. Figure 25 shown by Figure 25 It can be seen that compared with the PBS group, Exos group and ICG-Exos group, the fibrosis levels of hepatocytes in the αPD1 group, αPG-Exos group, ICG-Exos / Apt group and αPG-Exos / Apt group were significantly reduced, and the fibrosis level of hepatocytes in the αPG-Exos / Apt group was the lowest.

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

[0166] Adding CD8 to 3D cell spheroids + After 24 h of T cell co-culture, the cell spheres were washed, stained using a cell viability / cytotoxicity assay kit (30002, Biotium), and fixed. Images were taken using a confocal microscope, and Z-stacks were scanned from the top to the middle projection of the cell sphere at 5 μm intervals and then presented as maximum intensity projections. To quantify live / dead cells, the total cell area of each dye was measured using Image J software, and the results were shown in Figure 2. Figure 26 shown.

[0167] Depend on Figure 26 It can be seen 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, and the αPG-Exos / Apt group had the most tumor cell death. Immunofluorescence detection of liver cancer cell markers AFP and EpCAM further verified this result ( Figure 27 ).

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

[0169] After 2 days of co-culture, type I collagen (CC050, Sigma-Aldrich) was injected. First, the collagen was neutralized to a pH of 7.0-8.0 and added to the cell spheroids in the 35-well agarose model. After incubation for 4 minutes, the entire culture system was inverted and incubated for 1 hour. Thereafter, the culture system was flipped and RPMI medium containing 5% FBS and 1% penicillin-streptomycin was added. The invasion assay was performed for 2 days and the images were taken using an inverted microscope (XDS-900, Caikon). The results are shown in Figure 2. Figure 28 As shown. Figure 28It can be seen that compared with the PBS group, Exos group and ICG-Exos group, the invasive and spreading 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 and spreading ability of spheroids in the αPG-Exos / Apt group was the worst.

[0170] The above results indicate that αPG-Exos / Apt can prevent the fibrosis and malignant transformation of hepatocytes.

[0171] Example 7 Effects of drug-loaded exosomes αPG-Exos / Apt on tumor microenvironment and liver fibrosis-liver cancer malignant transformation

[0172] The in vitro experiments in Example 6 have confirmed that αPG-Exos / Apt can prevent the fibrosis and malignant transformation of liver cells, and play a role in preventing and treating liver cancer. To further verify whether αPG-Exos / Apt also has a therapeutic effect in vivo, this example uses the cirrhosis-liver cancer malignant transformation mouse model provided in Example 2 for verification, and the drug administration groups include: ①Blank (untreated group); ②Exos group (wild 293T cell-derived exosome treatment group); ③ICG-Exos (293T cell-derived exosome treatment group with successful ICG encapsulation); ④αPG-Exos group (293T cell-derived exosome treatment group with successful ICG encapsulation and Pembrolizumab conjugated); ⑤ICG-Exos / Apt group (293T cell-derived exosome treatment group with successful ICG encapsulation and PA9-1 aptamer modification); ⑥αPG-Exos / Apt group (293T cell-derived exosome treatment group with successful ICG encapsulation and Pembrolizumab conjugated PA9-1 aptamer modification); ⑦αPD1+ICG group (1 mg / mL Pembrolizumab+ICG treatment group). At 14 weeks of modeling, mice were injected with 100 μg of Exos (50 μg / 100 μL PBS) via the tail vein three times a week. Samples were collected after administration for the following tests:

[0173] 1. In vivo live imaging

[0174] The biodistribution of Exos in each group was evaluated by in vivo imaging. The in vivo imaging was observed within 24 hours after the tail vein injection of Exos in each group. After 24 hours, the main organs and tumors were dissected out for in vitro imaging. The results were as follows: Figure 29 and Figure 30 shown.

[0175] Depend on Figure 29 and Figure 30It can be seen that within 24 hours of administration, the fluorescence signals of the αPG-Exos group, ICG-Exos / Apt group and αPG-Exos / Apt group were mainly located in the kidneys, liver and tumor tissues. The Exos group, ICG-Exos group and αPD1+ICG group were quickly metabolized from the kidneys due to their non-targeted nature or smaller particle size, and the accumulation in the tumor site gradually decreased. However, the fluorescence signals in the tumor site of the αPG-Exos group, ICG-Exos / Apt group and αPG-Exos / Apt group lasted longer, and the tumor fluorescence signal intensity of 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 in the tumor site.

[0176] 2. Liver function tests

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

[0178] 3. Pathological Staining

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

[0180] 4. Flow Cytometry Detection of CD8 + T cell cytotoxicity

[0181] Flow cytometry was used to detect CD8 + The expression of IFN-γ and GZMB in T cells was analyzed. 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 the other groups, and the number of positive cells in the αPG-Exos / Apt group was the highest.

[0182] 5. Western blot detection of PSMA1 expression level

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

[0184] 6. Detection of changes in sphingomyelin metabolites in serum

[0185] The kit was used to detect the changes in the levels of sphingomyelin metabolites in the serum of mice after administration. The results were as follows: Figure 33 As shown in D. Figure 33 D shows that compared with the other groups, the content 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 was significantly reduced.

[0186] 7. H&E Staining of Major Organs

[0187] After the treatment, the main organs were removed for H&E staining, and no obvious pathological changes were found in the main organs ( Figure 34), indicating that αPG-Exos / Apt has good biocompatibility. The above results indicate that αPG-Exos / Apt can reshape the tumor microenvironment and prevent the malignant transformation of liver fibrosis to liver cancer.

[0188] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A targeted drug-loaded exosome, characterized in that: The targeted drug-loaded exosomes include exosomes encapsulating indocyanine green, and the surfaces of the drug-loaded exosomes are connected to aptamers targeting immune checkpoints and immune checkpoint inhibitors.

2. The targeted drug-loaded exosomes according to claim 1, characterized in that The immune checkpoints include at least one of PD-L1 / PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, CD276, CD47, and IDO1; Preferably, the immune checkpoint inhibitor is a PD-L1 / PD-1 inhibitor; More preferably, the PD-L1 / PD-1 inhibitor includes at least one of tislelizumab, nivolumab, pembrolizumab, atezolizumab, durvalumab and avelumab.

3. The targeted drug-loaded exosomes according to claim 2, characterized in that The aptamer targeting the immune checkpoint targets PD-L1 / PD-1; Preferably, the nucleotide sequence of the aptamer comprises SEQ ID NO:

1.

4. The method for preparing targeted drug-loaded exosomes according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: S1: indocyanine green-tetrabutylammonium iodide complex is mixed with exosomes and incubated to obtain exosomes encapsulating indocyanine green; S2: Using a linker, the immune checkpoint inhibitor is connected to the exosomes encapsulated with indocyanine green obtained in step S1 to obtain drug-loaded exosomes; S3: Use a cross-linker to connect the immune checkpoint-targeting aptamer to the drug-loaded exosomes obtained in step S2 to obtain targeted drug-loaded exosomes.

5. The preparation method according to claim 4, characterized in that The molar ratio of indocyanine green to tetrabutylammonium iodide in step S1 is 1:(3-10); Preferably, the mass ratio of indocyanine green to exosomes is 1:(5-20); Preferably, the incubation temperature is 35°C-40°C; Preferably, the incubation time is 1-3 hours.

6. The preparation method according to claim 4, characterized in that The linker in step S2 includes DSPE-PEG-NHS; Preferably, the mass ratio of the immune checkpoint inhibitor to DSPE-PEG-NHS in step S2 is 1:(20-40); Preferably, in step S2, the mass ratio of the immune checkpoint inhibitor to the exosomes encapsulating indocyanine green is 1:(20-40).

7. The preparation method according to claim 4, characterized in that The cross-linking agent in step S3 includes NHS and EDC; Preferably, in step S3, the mass ratio of the aptamer targeting the immune checkpoint to the drug-loaded exosomes is 1:(40-60).

8. The preparation method according to claim 4, characterized in that The source of the exosomes in step S1 includes 293T cells.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the targeted drug-loaded exosomes described in any one of claims 1 to 3 and pharmaceutically acceptable excipients.

10. Use of the targeted drug-loaded exosomes according to any one of claims 1 to 3 in the preparation of tumor-targeted drugs.

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