Immune checkpoint-modified vesicle nanoparticles, methods of making and using the same

Abstract

This invention relates to the field of biomedical technology, and particularly to immune checkpoint-modified vesicle nanoparticles, their preparation methods, and applications. Nanovesicles are obtained by constructing engineered mesenchymal stem cells stably expressing PD-L1. These nanovesicles effectively activate immunosuppressive signaling pathways, thereby inhibiting overactivated pathogenic T cell responses, alleviating Th1 and Th17 cell-mediated inflammatory responses, and helping to restore the immunoregulatory function of regulatory T cells, thus achieving the reconstruction of immune homeostasis. Furthermore, the introduction of metal nanozymes with enzyme-like catalytic activity endows the vesicles with the ability to continuously catalyze and scavenge ROS, enabling them to exert sustained antioxidant effects in the inflammatory microenvironment and effectively reduce excessive ROS levels in synovial tissue. In vivo experimental results show that these vesicle nanoparticles not only alleviate joint swelling caused by inflammation but also possess the ability to repair deep pathological damage.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to vesicle nanoparticles based on immune checkpoint modification, their preparation methods, and applications. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia, inflammatory cell infiltration, and progressive joint destruction. If left uncontrolled, it can ultimately lead to irreversible joint deformities and loss of function. The pathogenesis of RA primarily involves the complex interaction between abnormal immune activation, chronic inflammatory response, and oxidative stress damage. Following the breakdown of autotolerance, abnormal activation of lymphocytes in the synovial tissue creates a persistent pro-inflammatory microenvironment, inducing abnormal proliferation of synovial fibroblasts and promoting cartilage erosion. Pathogenic CD4+ T cell subsets, especially Th1 and Th17 cells, extensively infiltrate the synovial tissue, secreting pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-17A (IL-17A), and interferon-γ (IFN-γ), further activating synovial fibroblasts and recruiting macrophages and neutrophils, exacerbating the inflammatory response and tissue destruction. Currently used clinical biologics (such as anti-TNF antibodies and anti-IL-6 receptor antibodies) can partially alleviate symptoms, but they mainly target single inflammatory factors and cannot effectively inhibit pathogenic T cell responses, leading to poor efficacy or relapse in some patients. In addition, systemic immunosuppression significantly increases the risk of infection and tumors.

[0003] In addition to immune abnormalities, significant abnormal accumulation of reactive oxygen species (ROS) is also observed in the synovial tissue of arthritis (RA). ROS primarily originates from activated immune cells and abnormally proliferating synovial cells. Excessive ROS not only directly damages chondrocytes and the extracellular matrix but also activates the NF-κB signaling pathway and the NLRP3 inflammasome, further amplifying the inflammatory response and accelerating joint destruction. Although some antioxidants (such as N-acetylcysteine) have shown some therapeutic effects in animal arthritis models, their clinical application is limited by rapid in vivo clearance, low synovial retention rate, and insufficient cell targeting.

[0004] Furthermore, excessive immune activation and oxidative stress do not exist independently, but rather promote each other through a positive feedback loop: persistent T-cell inflammatory responses drive excessive ROS production, which in turn promotes the secretion of inflammatory cytokines and drives T cells to differentiate into pro-inflammatory phenotypes such as Th1 and Th17, while simultaneously inhibiting the function of regulatory T cells (Tregs), thereby exacerbating immune imbalance. This self-amplifying pathological loop of immune-oxidative stress constitutes a crucial mechanism for the chronicity and refractory nature of rheumatoid arthritis (RA), and has become a key bottleneck preventing current single-target therapies from completely controlling the disease.

[0005] In recent years, mesenchymal stem cells (MSCs) and their derived nanovesicles (NVs) have become a research hotspot in the treatment of rheumatoid arthritis (RA) due to their innate immune-regulating functions. Compared with cell therapy, MSC-derived vesicles have advantages such as higher safety, stronger tissue penetration, and easier engineering. However, the ability of natural vesicles to regulate the complex pathological processes of RA remains limited. The programmed death receptor 1 (PD-1) / programmed death ligand 1 (PD-L1) signaling axis plays a crucial role in maintaining immune tolerance. In autoimmune diseases such as RA, the PD-1 / PD-L1 pathway is impaired, leading to excessive T cell activation. Previous studies have attempted to restore this pathway by delivering the PD-L1 gene via recombinant PD-L1 protein or AAV, but problems such as poor in vivo stability, immunogenicity, and off-target effects exist.

[0006] Current research largely focuses on single-function treatment strategies, such as simple immunomodulation or simple antioxidant therapy, lacking a comprehensive treatment platform that can synergistically intervene in both immune abnormalities and oxidative stress. Therefore, developing an engineered nanovesicle system that can simultaneously achieve immune checkpoint regulation and highly efficient antioxidant effects at the site of inflamed joints is of great significance for improving the treatment efficacy of rheumatoid arthritis (RA). Summary of the Invention

[0007] This invention aims to at least solve one of the technical problems existing in related technologies. Therefore, the first objective of this invention is to provide a method for preparing vesicle nanoparticles based on immune checkpoint modification; the second objective is to provide vesicle nanoparticles based on immune checkpoint modification; and the third objective is to provide applications of vesicle nanoparticles based on immune checkpoint modification.

[0008] To achieve the first objective, the technical solution adopted by this invention is as follows: The preparation method of immune checkpoint modified vesicle nanoparticles includes the following steps: S100. Mesenchymal stem cells were transfected using a PD-L1 overexpression lentiviral packaging system to obtain MSC-PD-L1 cells with PD-L1 protein anchored on the surface. S200: Nanovesicles expressing PD-L1 on the surface were prepared by membrane extrusion using MSC-PD-L1 cells as raw material. S300, Synthesis of Ruthenium Nanoparticles I using a solvothermal method; S400. In PBS solution, the surface of ruthenium nanoparticle I was modified with DSPE-TK-PEG2000-Mal and DSPE-PEG2000-RGD to obtain a dispersion of ruthenium nanoparticle II with maleimide reactive groups and RGD targeting peptides on the surface. S500: After uniformly mixing the ruthenium nanoparticle II dispersion and the nanovesicles, the PD-L1 on the surface of the nanovesicles reacts with the Mal group on the surface of the ruthenium nanoparticle II to obtain vesicle nanoparticles based on immune checkpoint modification.

[0009] Furthermore, the mesenchymal stem cells are mouse-derived mesenchymal stem cells.

[0010] Furthermore, in step S100, the cells required for transfection are HEK293T cells.

[0011] Furthermore, the PD-L1 overexpression lentivirus packaging system includes psPAX2 packaging plasmid, pMD2.G envelope plasmid, and pCDH-PD-L1 plasmid, with a mass ratio of 5:7.5:10.

[0012] Furthermore, in step S300, the ruthenium source used in the solvothermal method is hydrated ruthenium chloride.

[0013] Further, in step S400, the concentration of the ruthenium nanoparticle II dispersion is 0.2–1 mg / mL, and the protein concentration in the nanovesicles is 0.5–1 mg / mL.

[0014] To achieve the second objective, the technical solution adopted by this invention is as follows: The immune checkpoint-modified vesicle nanoparticles were prepared using any of the methods described above.

[0015] To achieve the third objective, the technical solution adopted by this invention is as follows: Applications of immune checkpoint-modified vesicle nanoparticles, using the immune checkpoint-modified vesicle nanoparticles to prepare drugs for the treatment of one or more of oxidative stress-related diseases, inflammatory diseases, and immune dysregulation diseases.

[0016] Furthermore, the drug includes a pharmaceutically acceptable carrier.

[0017] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: This invention provides immune checkpoint-modified vesicle nanoparticles and their preparation method. The nanovesicles are obtained by constructing engineered mesenchymal stem cells stably expressing PD-L1, and their surface exhibits a high density of the immune checkpoint molecule PD-L1. This structure can effectively activate the PD-1 / PD-L1 immunosuppressive signaling pathway, thereby inhibiting overactivated pathogenic T cell responses, alleviating Th1 and Th17 cell-mediated inflammatory responses, and helping to restore the immunoregulatory function of regulatory T cells (Tregs), thus achieving the reconstruction of immune homeostasis.

[0018] Furthermore, metal nanozymes with enzyme-like catalytic activity are introduced onto the surface of the aforementioned nanovesicles, endowing them with the ability to continuously catalyze the scavenging of reactive oxygen species (ROS). Compared to traditional small-molecule antioxidants, these nanozymes can continuously exert antioxidant effects in the inflammatory microenvironment, effectively reducing excessive ROS levels in synovial tissue, thereby reducing oxidative stress damage to chondrocytes and extracellular matrix, and inhibiting the activation of ROS-mediated inflammatory signaling pathways, thus blocking the progression of inflammation from an antioxidant perspective.

[0019] This invention integrates immune checkpoint regulation and catalytic antioxidant function into the same nanovesicle platform, which can simultaneously intervene in two key pathological processes in the pathogenesis of rheumatoid arthritis (RA): abnormal immune activation and oxidative stress damage. This design can break the positive feedback amplification cycle between the two and achieve a synergistic therapeutic effect.

[0020] In addition, the nanovesicle structure is stable and has good potential for engineering modification, thus possessing excellent scalability and functional integration capabilities. Depending on the treatment needs of different diseases, other functional molecules and regulatory modules can be loaded onto the surface or inside the vesicles, further expanding their application boundaries in the treatment of inflammatory and autoimmune diseases.

[0021] In summary, the engineered nanovesicle system constructed in this invention, which combines immune checkpoint regulation and catalytic antioxidant functions, can synergistically intervene in the key pathogenic mechanisms of RA from multiple levels and dimensions. It not only demonstrates potential high-efficiency therapeutic value but also has good prospects for clinical translation, providing a brand-new technical approach and solution for the treatment of RA and other related immune diseases.

[0022] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0023] Figure 1The figure shows the PD-L1 expression level of the MSC-PD-L1 monoclonal cell line provided in Example 1 of this invention; wherein, Figure A is the RT-qPCR verification result, Figure B is the flow cytometry verification result, Figure C is the Western blot verification result, and Figure D is the bar chart of the quantitative analysis of the Western blot verification result.

[0024] Figure 2 The results of particle size and morphology characterization of PD-L1 NV and its control provided in Example 1 of this invention are shown in Figure A, which is the result verified by Western blot, and Figure B is the result of transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis.

[0025] Figure 3 The results are the Ru NPs characterization results provided in Example 1 of this invention; Figure A shows the dynamic light scattering (DLS) results and transmission electron microscope (TEM) images, Figure B shows the X-ray diffraction (XRD) pattern, which shows the diffraction peaks corresponding to metallic Ru and RuO2, and Figure C shows the elemental analysis results of X-ray photoelectron spectroscopy (XPS).

[0026] Figure 4 The figures show the characterization and performance testing results of PD-L1 NV@Ru provided in Example 1 of this invention; Figure A shows the dynamic light scattering (DLS) results and transmission electron microscopy (TEM) images (scale bar 100nm) of PD-L1 NV@Ru; Figure B shows the simulated catalase (CAT) activity results of RuNP, NV, NV@Ru and PD-L1 NV@Ru; and Figure C shows the simulated superoxide dismutase (SOD) activity results of RuNP, NV, NV@Ru and PD-L1NV@Ru.

[0027] Figure 5 The image shows a fluorescence image of the DCF signal in RAW264.7 cells treated with LPS and different nanoparticles, provided in Example 1 of this invention. The scale bar is 100µm.

[0028] Figure 6 This is Example 1 of the present invention, which uses ELISA to detect the concentrations of IL-6, TNF-α and IL-10 in the supernatant of RAW264.7 cells after treatment with different nanoparticles; wherein, Figure A is a bar chart of IL-6 concentration in different treatment groups, Figure B is a bar chart of TNF-α concentration in different treatment groups, and Figure C is a bar chart of IL-10 concentration in different treatment groups.

[0029] Figure 7This is a fluorescence image (scale bar 100µm) of calcein-acetylacetylmethoxyester (Calcein-AM) and propidium iodide (PI) signals in RAW264.7 cells stimulated by H2O2 and treated with different nanoparticles, provided in Example 1 of this invention.

[0030] Figure 8 This is the flow cytometry analysis result provided in Example 2 of the present invention; wherein, Figure A shows the activated CD4 in different treatment groups. + T cell proliferation; Figure B shows the activation of CD8 in different treatment groups. + T cell proliferation, Figure C shows the IFN-γ levels in different treatment groups. + In CD4 + The percentage of T cells, Figure D shows the CD25 content in different treatment groups. + FoxP3 + In CD4 + The percentage of T cells.

[0031] Figure 9 The results are the efficacy evaluation results of the in vivo animal model provided in Example 3 of this invention; wherein, Figure A is a curve of the change of hind paw thickness over time in healthy mice and RA mice in different treatment groups, Figure B is a photograph of the actual appearance of the mouse, and Figure C is an imaging scan of the mouse paw.

[0032] Figure 10 This is a flow cytometry analysis result of mouse spleen provided in Example 3 of this invention; where Figure A shows the CD4 counts in different treatment groups. + FoxP3 + Flow cytometry plots of regulatory T cells (Tregs), Figure B shows CD4 counts in different treatment groups. + IL-17A + Flow cytometry plot of helper T cells 17 (Th17) (Figure C shows CD4). + FoxP3 + Quantitative statistical bar chart, chart D is CD4 + IL-17A + Quantitative statistical bar chart.

[0033] Figure 11 These are histopathological sections of the major organs of the mouse provided in Example 3 of this invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0035] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0036] Example 1 I. Construction of PD-L1 highly expressed mesenchymal stem cells.

[0037] The culture process of mesenchymal stem cells (MSCs) is as follows: MSCs derived from mouse bone marrow are cultured in α-MEM medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) at 37°C and 5% CO2. The medium is changed every 2-3 days. When the cell confluence reaches 80%-90%, the cells are seeded into new culture dishes at a ratio of 1:3. Fresh medium is added, and the cells are cultured at 37°C and 5% CO2 to ensure that they are in good growth condition for subsequent experiments.

[0038] The construction of the recombinant lentiviral vector is as follows: construct the pCDH-pdl1 recombinant lentiviral expression vector, fuse the full-length coding sequence of PD-L1 with the transmembrane domain of PDGFR, insert it into the multiple cloning site of the pCDH vector, and the vector simultaneously contains the EGFP fluorescent marker gene and the puromycin resistance gene.

[0039] Lentiviral packaging and transfection were performed as follows: Human embryonic kidney cell line (HEK 293T cells) were seeded into culture flasks, and DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) was added. The cells were cultured at 37°C in a 5% CO2 incubator, with the medium changed every 2-3 days. The cells were then passaged and amplified into HEK cells. When the confluence of 293T cells reached 70%–80%, the old culture medium was discarded, and the cells were washed twice with sterile PBS buffer. Serum-free DMEM culture medium was added, and the cells were incubated in an incubator for 15–20 min. Then, the transfection system (the mass ratio of psPAX2 packaging plasmid, pMD2.G envelope plasmid, and pCDH-PD-L1 plasmid was 5:7.5:10, and PEI was added to prepare the transfection complex according to the mass ratio of the total DNA of the three plasmids to polyethyleneimine (PEI) of 1:3) was added for transfection. After transfection, the cells were incubated for 48 h. The culture medium containing lentivirus was collected, and the collected culture medium was combined and centrifuged at 300g for 10 min to remove cell debris. The collected supernatant was the lentivirus solution. The collected viral supernatant was added to MSCs in good growth condition. After the cells showed fluorescent labeling, stable MSC-PD-L1 cells overexpressing PD-L1 were selected by adding 2 μg / mL puromycin. The selected MSC-PD-L1 cells were digested with trypsin to prepare a single-cell suspension and seeded into 96-well plates with only 1 cell per well. α-MEM complete medium containing puromycin was added and cultured for 10-14 days. Single clones with good growth and uniform fluorescence were selected and transferred to 6-well plates for expansion culture to obtain the MSC-PD-L1 single-clone cell line.

[0040] Three methods were used to examine the expression level of PD-L1 in MSC-PD-L1 monoclonal cell lines, such as... Figure 1 As shown; Figure A shows the RT-qPCR verification results. The verification process is as follows: Total RNA was extracted from wild-type MSCs and MSC-PD-L1 cells, reverse transcribed into cDNA, and RT-qPCR was performed using GAPDH as an internal reference gene. The relative RNA expression level of PD-L1 was calculated. The results showed that PD-L1 RNA expression in MSC-PD-L1 was significantly higher than that in wild-type MSCs. Figure B shows the results of flow cytometry verification. The process is as follows: Two types of cells were collected and incubated with PD-L1-specific fluorescent antibody. The expression intensity of PD-L1 on the cell surface was detected by flow cytometry. The results showed that the expression of PD-L1 on the surface of MSC-PD-L1 cells was significantly higher than that of wild-type MSCs. Figure C shows the results of Western blot validation. The validation process is as follows: Total protein was extracted from the two types of cells, and SDS-PAGE electrophoresis was performed. After transfer to a membrane, the cells were incubated with PD-L1 primary antibody and GAPDH primary antibody, and then incubated with secondary antibody. After development, quantitative analysis was performed. Figure D is a bar chart of the quantitative analysis results of the Western blot validation. The validation results show that the expression level of PD-L1 protein in MSC-PD-L1 is significantly higher than that in wild-type MSC.

[0041] II. Preparation method of PD-L1 nanovesicles.

[0042] Once MSC or MSC-PD-L1 cells reached 80%–90% confluence, they were digested with 0.25% trypsin and collected. After centrifugation at 1000 rpm for 5 minutes, the culture medium was removed, yielding a cell pellet of approximately 0.5 × 10⁻⁶ cells. 7 ~1.5×10 7 The obtained cell pellet was resuspended in PBS buffer (1 mL), and then nanovesicles were prepared by membrane extrusion. The membrane extrusion process was as follows: the cell pellet was repeatedly extruded through polycarbonate membranes with pore sizes of 10 μm, 5 μm, 1 μm and 400 nm 15 to 20 times to obtain PD-L1 nanovesicles with uniform particle size, denoted as PD-L1 NV. The protein concentration of the nanovesicles was determined by the biuret (BCA) method. Vesicles prepared from wild-type MSCs (denoted as NV) were used as a control, and the preparation method was the same as above; The particle size and morphology of PD-L1 NV and NV were characterized, and the results are as follows: Figure 2 As shown; Figure A shows the results verified by Western blot, which indicates that the vesicles retain membrane-bound PD-L1. Figure B shows the results of transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis, which indicate that the particle size of PD-L1 NV is approximately 143 nm.

[0043] III. Synthesis and Functionalization of Ruthenium Nanoparticles.

[0044] Ruthenium nanoparticles were synthesized using a solvothermal method as follows: 50 mg of hydrated ruthenium chloride was dissolved in 13 mL of distilled water, followed by 0.2 g of oleylamine and 2 mL of cyclohexane. The mixture was sonicated for 10 min and stirred for 1 h. The reaction mixture was then transferred to a high-pressure reactor and reacted at 160 °C for 12 h. After the reaction was completed, the reactor was allowed to cool naturally to room temperature, and the reaction solution was transferred to centrifuge tubes. Add cyclohexane (5 mL), gently shake the centrifuge tube for 5 min to allow the ruthenium nanoparticles to fully dissolve in the cyclohexane phase, let stand for 10 min to allow the system to separate into layers (upper layer is the organic phase containing ruthenium nanoparticles, lower layer is the aqueous phase), aspirate the upper organic phase and transfer it to a new sterile centrifuge tube, add anhydrous ethanol (15 mL) to allow the ruthenium nanoparticles to fully precipitate; then, centrifuge at 11000 rpm for 10 min, collect the precipitate, and wash the precipitate three times with anhydrous ethanol (15 mL) to finally obtain ruthenium nanoparticles, denoted as Ru NPs, and their characterization results are as follows. Figure 3 As shown; Figure A shows the results of Dynamic Light Scattering (DLS) and the image from a Transmission Electron Microscope (TEM). Figure B shows the X-ray diffraction (XRD) pattern, which displays the diffraction peaks corresponding to metallic Ru and RuO2. From this pattern, it can be concluded that Ru NPs have mixed-phase properties. Figure C shows the elemental analysis results of X-ray photoelectron spectroscopy (XPS), which illustrates the elemental composition of Ru-based nanozymes. The figure shows a characteristic peak corresponding to Ru 3p, confirming the successful introduction of ruthenium species.

[0045] A functional lipid molecule blending modification method was used to achieve water-soluble dispersion and surface functionalization of Ru NPs. The process is as follows: Ru NPs (1 mg), DSPE-TK-PEG2000-Mal (5 mg), and DSPE-PEG2000-RGD (5 mg) were mixed and dissolved in chloroform. The mixture was then sonicated for 10 min. Subsequently, the organic solvent in the mixture was removed by rotary evaporation at 40 °C to form a homogeneous lipid film. PBS buffer at pH 7.4 was added to the lipid film for hydration, followed by sonication for 5–10 min to fully disperse the lipid film. Finally, a water-soluble Ru nanozyme dispersion with maleimide (Mal) reactive groups and RGD targeting peptides on the surface was obtained, with a final concentration controlled at approximately 1 mg / mL.

[0046] IV. Preparation of vesicle nanoparticles based on immune checkpoint modification (PD-L1 NV@Ru).

[0047] Using functionalized Ru nanozymes with Mal groups on their surface as a modification carrier, Ru nanozymes were anchored on the surface of PD-L1 NV to construct PD-L1 NV@Ru. The process is as follows: First, PD-L1 NV and functionalized Ru nanozymes were incubated with sonication at room temperature for 30 min to allow Ru nanozymes to be covalently anchored to the vesicle surface. Then, the reaction system was washed with PBS buffer to remove unbound components, and PD-L1 NV was dispersed in PBS buffer at pH 7.2–7.4, adjusting its protein concentration to the range of 0.5–1 mg / mL. At the same time, functionalized Ru nanozyme dispersion (concentration of 0.2–0.5 mg / mL) was added. Under room temperature of 20–25°C, the mixture was gently shaken or sonicated at low power for 30 min to promote the reaction between PD-L1 NV and the Mal groups on the surface of Ru nanozymes, so that Ru nanozymes were stably anchored on the surface of PD-L1 NV, and finally the target product PD-L1 NV@Ru was obtained, which was then dispersed in PBS buffer for later use.

[0048] The preparation process of the reference standard NV@Ru uses NV as the raw material, and the rest of the process is the same as that of PD-L1 NV@Ru.

[0049] Characterization and performance testing results, such as Figure 4 As shown; Figure A shows the dynamic light scattering (DLS) results and transmission electron microscopy (TEM) images (scale bar 100 nm) of PD-L1 NV@Ru. The TEM images show that multiple ruthenium nanoparticles (Ru NP) are anchored on the surface of PD-L1 NV. Figure B shows the catalase (CAT) simulated activity results of Ru NP, NV, NV@Ru and PD-L1 NV@Ru. From this figure, it can be seen that Ru NP, NV, NV@Ru and PD-L1 NV@Ru all have strong CAT-like activity. Figure C shows the simulated superoxide dismutase (SOD) activity results of Ru NP, NV, NV@Ru and PD-L1 NV@Ru. From this figure, it can be seen that Ru NP, NV, NV@Ru and PD-L1 NV@Ru all have strong SOD-like activity.

[0050] Test Example 1 To investigate the antioxidant, anti-inflammatory and immunomodulatory effects of PD-L1 NV@Ru.

[0051] I. The effect of PD-L1 NV@Ru on the oxidative stress level of mouse mononuclear macrophage leukemia cells (RAW264.7) was investigated using the reactive oxygen species fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining method. The groupings are as follows: Control: Blank control group, without LPS or nanoparticle treatment; The experimental group first constructed an inflammatory and highly reactive oxygen species (ROS) pathological environment using 100 ng / mL lipopolysaccharide (LPS). After successful construction of the pathological environment, the antioxidant capacity under this pathological condition was tested using different nanomaterials. The experimental group was divided into five groups: The groups were PBS (negative control), NV (20 μg / mL), Ru NP (20 μg / mL), NV@Ru (20 μg / mL, with a RuNP to vesicle protein concentration ratio of 1:1) and PD-L1 NV@Ru (20 μg / mL, with a RuNP to vesicle protein concentration ratio of 1:1).

[0052] Test results as follows Figure 5 As shown in the figure, stimulation with lipopolysaccharide (LPS) significantly increased the level of reactive oxygen species (ROS) in RAW264.7 cells, resulting in a marked increase in fluorescence signal. However, treatment with PD-L1 NV@Ru significantly inhibited the generation of ROS in cells, leading to a marked decrease in fluorescence signal. These results indicate that PD-L1 NV@Ru can effectively inhibit the oxidative stress response of macrophages.

[0053] The effect of PD-L1 NV@Ru on the secretion of inflammatory factors was investigated using enzyme-linked immunosorbent assay (ELISA). The results are as follows: Figure 6 As shown in the figure; Figure A is a bar chart of IL-6 concentration in different treatment groups, Figure B is a bar chart of TNF-α concentration in different treatment groups, and Figure C is a bar chart of IL-10 concentration in different treatment groups.

[0054] from Figure 6 It can be seen that in LPS-induced macrophages, the levels of TNF-α and IL-6 were significantly higher than those in the normal control group. NV@Ru can partially inhibit the release of cytokines, and PD-L1 NV@Ru treatment reduced the levels of TNF-α and IL-6 most significantly, especially in the NV@Ru and PD-L1 NV@Ru treatment groups. The M2-related anti-inflammatory cytokine IL-10 was significantly increased.

[0055] II. Live and dead cells were stained using Calcein-AM and PI staining to further investigate the cytoprotective effect of PD-L1 NV@Ru against reactive oxygen species-induced oxidative damage. The groupings are as follows: Control: Blank control group, without H2O2 or nanoparticle treatment; The experimental group first treated cells with H2O2 (10 μM) to construct an oxidative stress damage model, simulating the cell-killing effect of a high ROS environment. After successful model construction, the antioxidant capacity of different nanomaterials under this pathological environmental condition was tested. The experimental group was divided into five groups: The groups were PBS (negative control), NV (20 μg / mL), Ru NP (20 μg / mL), NV@Ru (20 μg / mL, with a RuNP to vesicle protein concentration ratio of 1:1) and PD-L1 NV@Ru (20 μg / mL, with a RuNP to vesicle protein concentration ratio of 1:1).

[0056] Test results as follows Figure 7 As shown in the figure, it can be seen that after stimulation with H2O2, the apoptosis of RAW264.7 cells increased significantly, and PD-L1 NV@Ru could significantly inhibit H2O2-induced apoptosis, demonstrating a strong cell protective effect.

[0057] Test Example 2 The inhibition of T cell activation by PD-L1 NV@Ru in vitro was investigated as follows: Mouse spleen T cells were extracted using a mouse spleen lymphocyte isolation kit. The T cells were then incubated with CellTrace™ dye at 37°C for 15 min for staining. After removing residual dye, cells were prepared for subsequent experiments. The dye-labeled T cells were resuspended in complete culture medium containing 3 μg / mL anti-mouse CD3ε antibody, 2 μg / mL anti-mouse CD28 antibody, and 400 U / mL interleukin-2 (IL-2). Cells were incubated with PBS, NV (20 μg / mL), PD-L1 NV (20 μg / mL), or PD-L1 NV@Ru (20 μg / mL, RuNP to vesicle protein mass ratio 1:1) for 48 hours. Cells were then collected, washed, and analyzed by flow cytometry. The results are shown below. Figure 8 As shown; Figure A shows the activation of CD4 in different treatment groups. + T cell proliferation; Figure B shows the activation of CD8 in different treatment groups. + The two graphs show that PD-L1 NV and PD-L1 NV@Ru can significantly inhibit CD4 cell proliferation. + T and CD8 + Proliferation of T cell subsets; Figure C shows the IFN-γ levels in different treatment groups. + In CD4 + The figure shows the proportion of T cells: both PD-L1 NV and PD-L1 NV@Ru significantly inhibited IFN-γ. + In CD4+ The results showed that NV treatment could slightly inhibit T cell proliferation, with PD-L1 NV showing a more significant inhibitory effect. The PD-L1 NV@Ru group showed the most significant inhibitory effect, indicating that the combined effect of immune checkpoint blockade and ROS clearance can synergistically enhance immunosuppressive capacity.

[0058] Figure D shows the different treatment groups CD25. + FoxP3 + In CD4 + The figure shows the proportion of T cells. It can be seen from the figure that the expression of PD-L1 on vesicles enhances the differentiation of Treg (CD4+CD25+Foxp3+) cells. The addition of Ru NPs to PD-L1 NV@Ru only slightly increases the Treg level. This result indicates that PD-L1 signaling plays a dominant role in Treg amplification.

[0059] Test Example 3 I. Investigation of the therapeutic effect of PD-L1 NV@Ru in a mouse model of RA.

[0060] The collagen-induced arthritis mouse model (CIA) highly mimics the key pathological and immunological features of human rheumatoid arthritis, including synovial hyperplasia, inflammatory cell infiltration, cartilage degeneration, bone erosion, and extracellular matrix remodeling. Obvious arthritis symptoms (e.g., erythema and edema of the claws) were observed as early as day 7 after booster immunization. Mice were randomly assigned to five groups based on their initial arthritis scores: PBS, Ru NP (5 mg / kg), NV@Ru (5 mg / kg, RuNP to vesicle protein concentration ratio of 1:1), or PD-L1 NV@Ru (5 mg / kg, RuNP to vesicle protein concentration ratio of 1:1) were administered intravenously every three days until day 43. Healthy DBA / 1 mice were used as controls.

[0061] A standardized clinical scoring system was used to monitor the severity of arthritis, and digital calipers were used to quantitatively measure the thickness of the claws. The results are as follows: Figure 9 As shown; Figure A shows the curves of hind paw thickness changes over time in healthy mice and RA mice in different treatment groups. From this figure, we can see that the condition of mice in the PBS group gradually worsened, and by day 47, the paw thickness increased by about 1.82 times compared with the healthy control group. The PD-L1NV@Ru group showed the fastest and largest decrease in swelling, and its curve was closest to that of the Healthy group, making it the group with the best anti-inflammatory and swelling-reducing effect in vivo.

[0062] Figure B shows a photograph of the actual appearance of the mice. From this figure, it can be seen that the clinical symptoms of the mice in the Ru NP and NV@Ru treatment groups were significantly relieved, as evidenced by reduced swelling and lower arthritis scores. The PD-L1 NV@Ru treatment group showed the most significant therapeutic effect, with joint inflammation almost completely subsiding. The thickness of the paws of the mice in this group was almost restored to the level of healthy mice, indicating that the disease was effectively suppressed. Figure C shows an imaging scan of the mouse paw. This image reveals that the tissue structure of the mice in the PD-L1 NV@Ru group was most similar to that of the healthy group, indicating the most ideal repair effect. Furthermore, the image also shows that the collagen-induced arthritis (CIA) models in both the PBS and Ru NP groups exhibited severe bone erosion and structural damage, indicating significant joint degeneration induced by rheumatoid arthritis (RA). In contrast, the NV@Ru group, especially the PD-L1 NV@Ru group, showed significant preservation of bone morphology and joint structure. The joints in the PD-L1 NV@Ru group maintained a clear trabecular bone structure and a smooth subchondral cartilage surface, similar to the healthy control group. These results demonstrate that PD-L1 NV@Ru not only alleviates surface swelling but also possesses the ability to repair deep pathological damage.

[0063] To assess the restoration of immune homeostasis in mice, this invention further examined the expansion of Treg cells and the balance between Treg cells and Th17 cells, two T cell subsets crucial in the immunopathology of rheumatoid arthritis (RA). Flow cytometry analysis of spleens from different groups of mice yielded the following results: Figure 10 As shown; Figure A shows the CD4 treatment groups. + FoxP3 + Flow cytometry of regulatory T cells (Tregs) shows that CD4+ is present in the PD-L1 NV@Ru group. + FoxP3 + T cells in CD4 + The proportion of T cells was as high as 13.6%, far exceeding that of other control groups. This result indicates that PD-L1 NV@Ru can significantly promote FOXP3 + Treg cell population proliferation; Figure B shows the different treatment groups CD4. + IL-17A + Flow cytometry of helper T cells 17 (Th17) shows that: PD-L1 NV@Ru group CD4 + IL-17A + The proportion of Th17 cells decreased to 2.53%, almost returning to healthy levels, indicating that PD-L1 NV@Ru can significantly inhibit Th17 cells. Figure C is CD4 + FoxP3 + Quantitative statistical bar chart; Figure D is CD4 + IL-17A + Quantitative statistical bar chart.

[0064] II. In vivo safety assessment of PD-L1 NV@Ru.

[0065] This invention involves injecting healthy mice via the tail vein with PD-L1 NV@Ru (5 mg / kg, RuNP to vesicle protein mass concentration ratio of 1:1) or PBS. The heart, liver, spleen, lungs, and kidneys of each group of mice were then stained with hematoxylin and eosin (HE) to observe for tissue damage. The results are as follows: Figure 11 As shown in the figure, there is no obvious histological damage to any organ, which indicates that PD-L1 NV@Ru has good biocompatibility.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing vesicle nanoparticles based on immune checkpoint modification, characterized in that, Includes the following steps: S100. Mesenchymal stem cells were transfected using a PD-L1 overexpression lentiviral packaging system to obtain MSC-PD-L1 cells with PD-L1 protein anchored on the surface. S200: Nanovesicles expressing PD-L1 on the surface were prepared by membrane extrusion using MSC-PD-L1 cells as raw material. S300. Ruthenium nanoparticles I were synthesized using a solvothermal method as follows: 50 mg of hydrated ruthenium chloride was dissolved in 13 mL of distilled water, followed by 0.2 g of oleylamine and 2 mL of cyclohexane. The mixture was sonicated for 10 min and stirred for 1 h. The reaction mixture was then transferred to a high-pressure reactor and reacted at 160 °C for 12 h. After the reaction was completed, the reactor was allowed to cool naturally to room temperature. The reaction solution was then transferred to a centrifuge tube, and 5 mL of cyclohexane was added. The centrifuge tube was gently shaken for 5 min to allow the ruthenium nanoparticles to fully dissolve in the cyclohexane phase. The mixture was allowed to stand for 10 min to allow the system to separate into two phases: an upper organic phase containing ruthenium nanoparticles and a lower aqueous phase. The upper organic phase was aspirated and transferred to a new sterile centrifuge tube. 15 mL of anhydrous ethanol was added to allow the ruthenium nanoparticles to precipitate fully. The mixture was then centrifuged at 11000 rpm for 10 min, and the precipitate was collected. The precipitate was washed three times with 15 mL of anhydrous ethanol to finally obtain ruthenium nanoparticles I. S400. In PBS solution, the surface of ruthenium nanoparticle I was modified with DSPE-TK-PEG2000-Mal and DSPE-PEG2000-RGD to obtain a dispersion of ruthenium nanoparticle II with maleimide reactive groups and RGD targeting peptides on the surface. S500: After uniformly mixing the ruthenium nanoparticle II dispersion with the nanovesicles, the PD-L1 on the surface of the nanovesicles reacts with the Mal group on the surface of the ruthenium nanoparticle II to obtain vesicle nanoparticles based on immune checkpoint modification.

2. The method for preparing vesicle nanoparticles based on immune checkpoint modification as described in claim 1, characterized in that, The mesenchymal stem cells mentioned are mouse-derived mesenchymal stem cells.

3. The method for preparing vesicle nanoparticles based on immune checkpoint modification as described in claim 1, characterized in that, In step S100, the cells required for transfection are HEK293T cells.

4. The method for preparing vesicle nanoparticles based on immune checkpoint modification as described in claim 1, characterized in that, The PD-L1 overexpression lentivirus packaging system includes psPAX2 packaging plasmid, pMD2.G envelope plasmid, and pCDH-PD-L1 plasmid, with a mass ratio of 5:7.5:

10.

5. The method for preparing vesicle nanoparticles based on immune checkpoint modification as described in claim 1, characterized in that, In step S400, the concentration of the ruthenium nanoparticle II dispersion is 0.2–1 mg / mL, and the protein concentration in the nanovesicles is 0.5–1 mg / mL.

6. Vesicle nanoparticles based on immune checkpoint modification, characterized in that, It was prepared using the method for preparing immune checkpoint modified vesicle nanoparticles as described in any one of claims 1 to 5.

7. The application of vesicle nanoparticles based on immune checkpoint modification, characterized in that, A drug is prepared using the immune checkpoint-modified vesicle nanoparticles as described in claim 6, wherein the drug is a drug for treating rheumatoid arthritis.

8. The application of the immune checkpoint-modified vesicle nanoparticles as described in claim 7, characterized in that, The drug includes a pharmaceutically acceptable carrier.

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