A drug delivery system of ultrasound-enhanced synergistic immunity and a preparation method and application thereof

Abstract

This invention relates to the field of nanomedicine delivery systems, specifically to an ultrasound-enhanced synergistic immunotherapy drug delivery system, its preparation method, and its applications. The ultrasound-enhanced synergistic immunotherapy drug delivery system includes a lipid shell and a fluorocarbon gas encapsulated within the lipid shell. The lipid shell is made from cancer cell membranes and a lipid membrane formed from synthetic phospholipids. The lipid shell integrates a protein degradation-targeting chimera for degrading PD-L1. This invention combines targeted drug delivery, PD-L1 targeted degradation, and ultrasound enhancement technology to construct an integrated synergistic immunotherapy drug delivery system. Through multi-mechanism synergistic action, it overcomes the bottlenecks of existing PROTAC delivery systems, achieving a highly efficient anti-tumor immune response. This technical solution solves the technical problems of limited tumor tissue penetration and cellular uptake efficiency of PROTAC degrading agents targeting PD-L1, resulting in unsatisfactory delivery effects and promising prospects for widespread application.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine delivery system technology, specifically to an ultrasound-enhanced synergistic immune drug delivery system, its preparation method, and its application. Background Technology

[0002] PD-L1 (Programmed Death-Ligand 1) is a key immune checkpoint molecule in the body's immune system. It primarily transmits immunosuppressive signals by binding to the PD-1 receptor on the surface of T cells, thereby negatively regulating the activation and proliferation of immune cells. In the tumor microenvironment, tumor cells often abnormally overexpress PD-L1, using the PD-1 / PD-L1 signaling pathway to inhibit the killing function of effector T cells, thus achieving tumor immune escape. Therefore, PD-L1 is considered a core molecule in tumor development and immune escape mechanisms, and is also a highly valuable clinical target in the current field of tumor immunotherapy.

[0003] Currently, immune checkpoint inhibitors, represented by anti-PD-L1 monoclonal antibodies, have achieved significant clinical efficacy in various malignant tumors and have become an important means of cancer treatment. However, these antibody-based PD-L1 targeted therapies still have many inherent limitations in clinical application. First, antibody drugs have large molecular weights and strong hydrophilicity, making it difficult to efficiently penetrate dense tumor tissue and reach deep into the tumor, resulting in insufficient drug distribution at the tumor site and difficulty in exerting sufficient inhibitory effects on deep and drug-resistant tumor cells. Second, traditional antibodies only competitively bind to block the interaction between PD-L1 and PD-1, and cannot fundamentally eliminate the intracellular PD-L1 protein, only temporarily blocking its function. In addition, long-term monotherapy with PD-L1 antibody drugs can easily induce adaptive changes in tumor cells, leading to primary or acquired drug resistance, including target mutations, downstream pathway activation, and compensatory upregulation of immunosuppressive molecules, ultimately leading to treatment failure. Therefore, to overcome the bottlenecks of existing therapies, there is an urgent clinical need to develop novel targeting strategies that can efficiently, persistently, and thoroughly regulate PD-L1.

[0004] Proteolysis-targeting chimera (PROTAC) technology, as an emerging protein regulation approach, offers a promising new strategy for addressing the aforementioned shortcomings of PD-L1 targeted therapy. Unlike traditional inhibitor-dependent modes of action, PROTAC molecules are bifunctional small molecules that can simultaneously and specifically bind to both the target protein PD-L1 and the intracellular E3 ubiquitin ligase. By spatially converging these two proteins, PROTAC mediates ubiquitination of the PD-L1 protein, which is then recognized and degraded by the intracellular proteasome. PROTAC technology has several unique advantages: First, its mechanism of action is catalytic degradation, with a single PROTAC molecule capable of cyclically mediating the degradation of multiple target protein molecules, significantly improving its efficacy. Second, PROTAC can directly reduce intracellular PD-L1 protein levels rather than simply blocking its binding activity, maintaining good degradation effects on highly expressed, mutant, or intracellularly localized PD-L1, achieving more thorough target silencing. Third, by eliminating PD-L1 protein at its source, PROTAC can effectively reduce the risk of drug resistance caused by persistent or compensatory expression of the target, potentially delaying or even overcoming acquired drug resistance. Fourth, small PROTAC molecules typically have high degradation activity, achieving significant protein downregulation effects even at low concentrations, possessing potential advantages in drug delivery and therapeutic window.

[0005] Therefore, PROTAC degraders targeting PD-L1 can compensate for the shortcomings of antibody drugs at the mechanism of action level, providing a better solution for tumor immunotherapy. Nevertheless, PD-L1 PROTACs still face a series of key challenges in their clinical translation, including off-target degradation and non-specific effects, insufficient in vivo metabolic stability, and low oral bioavailability. Furthermore, PROTACs also face delivery challenges such as limited tumor tissue penetration and cellular uptake efficiency. These issues, to some extent, limit their in vivo efficacy and clinical application, necessitating improvements through strategies such as structural optimization, targeted delivery system construction, and formulation development to promote the efficient translation of PD-L1 PROTACs from basic research to clinical practice. Summary of the Invention

[0006] This invention aims to provide an ultrasound-enhanced synergistic immunotherapy drug delivery system to address the unsatisfactory delivery effects of existing PD-L1-targeting PROTAC degrading agents, such as limited tumor tissue penetration and cellular uptake efficiency. The ultrasound-enhanced synergistic immunotherapy drug delivery system developed using this approach not only improves the delivery efficiency of PROTAC degrading agents, but also achieves a synergistic effect between PROTAC and the cancer cell membrane-based drug delivery system in terms of immune activation, thereby enhancing the overall drug efficacy in inhibiting tumor growth, metastasis, and recurrence.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: An ultrasound-enhanced synergistic immunotherapy drug delivery system includes a lipid shell and a fluorocarbon gas encapsulated within the lipid shell; the lipid shell is made from cancer cell membranes and lipid membranes formed from synthetic phospholipids; and a protein degradation-targeting chimera for degrading PD-L1 is integrated into the lipid shell.

[0008] This protocol loads a protein degradation-targeting chimeric conjugate (PROTAC) for PD-L1 degradation onto a vector containing cancer cell membranes (TCM-NPs@PRO). This approach aims to induce dendritic cell activation (by inducing the transformation of newly formed dendritic cells into mature dendritic cells) and T cell activation (by increasing CD3+). + CD8 + T cell count, increase CD3 + CD8 + The expression levels of T cell cytotoxicity markers (enhancing the killing effect of T cells on cancer cells) both produced a synergistic effect. That is, PROTAC and TCM-NPs in this regimen produced a synergistic effect in immune activation, thereby enhancing the drug delivery system's efficacy in inhibiting tumor growth, metastasis, and recurrence.

[0009] Furthermore, the mass ratio of the cancer cell membrane to the lipid membrane formed by artificially synthesized phospholipids is 1:9-9:1; preferably, the mass ratio is 9:1.

[0010] The mass ratio of cancer cell membranes to lipid membranes formed by synthetic phospholipids affects the particle size of the prepared nanobubbles. In this scheme, a mass ratio of 9:1 results in the smallest particle size, and smaller particle size enhances the penetration and retention effect of tumor tissue.

[0011] Furthermore, the mass ratio of the cancer cell membrane to the protein degradation targeting chimera for PD-L1 degradation is 40:1-2.5:1; preferably, the mass ratio is 5:1. The mass ratio of the cancer cell membrane to the protein degradation targeting chimera for PD-L1 degradation affects the particle size of the prepared nanobubbles.

[0012] Furthermore, the raw materials for the lipid membrane formed by artificially synthesized phospholipids include dipalmitoylphosphatidylcholine and distearate phosphatidylethanolamine-polyethylene glycol 2000 in a mass ratio of 1:9-9:1. The lipid membrane formed by artificially synthesized phospholipids is obtained by dissolving dipalmitoylphosphatidylcholine and distearate phosphatidylethanolamine-polyethylene glycol 2000 in an organic solvent. After the organic solvent evaporates, the lipid membrane formed by artificially synthesized phospholipids is obtained.

[0013] Furthermore, the cancer cell membrane is provided by a mouse colon cancer cell line; the fluorocarbon gas is perfluoropropane; and the protein degradation targeting chimera used to degrade PD-L1 is PROTAC PD-L1 degrader-1.

[0014] Furthermore, the average particle size of the drug delivery system is 412.7-1216.8 nm; preferably, the average particle size of the drug delivery system is 412.7 nm.

[0015] Furthermore, an ultrasound-enhanced synergistic immune drug delivery system further includes an ultrasound device for applying ultrasound waves to a tumor site to which the drug delivery system is applied; the ultrasound device is used to provide ultrasound waves at 0.173-0.387 MPa.

[0016] This technical solution also provides a method for preparing an ultrasound-enhanced synergistic immunotherapy drug delivery system, which includes the following steps performed sequentially: S1: The cancer cell membrane is mixed with a protein degradation targeting chimera for degrading PD-L1, and then incubated to obtain a drug-loaded cancer cell membrane; S2: Dispalmitoylphosphatidylcholine and distearate phosphatidylethanolamine-polyethylene glycol 2000 are dissolved in an organic solvent. After the organic solvent evaporates, a lipid membrane formed by artificially synthesized phospholipids is obtained. S3: The drug-loaded cancer cell membrane is mixed with a lipid membrane formed by hydrated synthetic phospholipids, and after ultrasonic treatment, a drug-loaded lipid suspension is obtained; the mass ratio of the cancer cell membrane to the lipid membrane formed by synthetic phospholipids is 1:9-9:1. S4: Add the drug-loaded lipid suspension to the container, then seal the container, evacuate and inject fluorocarbon gas. After shaking, obtain TCM-NBs@PRO nanobubbles.

[0017] Furthermore, in S1, the cancer cell membrane is mixed with a protein degradation-targeting chimera for degrading PD-L1 at a mass ratio of 40:1 to 2.5:1; Furthermore, in S2, the ratio of dipalmitoylphosphatidylcholine, distearylphosphatidylethanolamine-polyethylene glycol 2000, and organic solvent is 1-9 mg: 1-9 mg: 3-7 mL. Furthermore, in S3, the mass ratio of the cancer cell membrane to the lipid membrane formed by artificially synthesized phospholipids is 1:9-9:1; Further, in S4, 0.5 mL of the drug-loaded lipid suspension is transferred to a 2 mL container, the container is sealed, and a vacuum is drawn using a syringe until the syringe plunger can no longer be pulled, maintaining the vacuum state for 10-15 seconds; then excess octafluoropropane is added to the container.

[0018] This technical solution also provides the application of an ultrasound-enhanced synergistic immune drug delivery system in the preparation of immune activators, or drugs that inhibit tumor growth, or drugs that inhibit tumor metastasis, or drugs that inhibit tumor recurrence.

[0019] Preferably, the immune activator is a reagent that activates anti-tumor immunity; more specifically, the reagent that activates anti-tumor immunity is a reagent that degrades PD-L1 protein, or a reagent that induces T cell activation, or a reagent that induces dendritic cell activation.

[0020] The drug delivery system in this protocol serves as a reagent for inducing T cell activation, primarily by enhancing effector T lymphocytes (CD8). + T cells, more specifically CD3 + CD8 + The number of T cells, CD8 + T cells express higher levels of IFN-γ, PD-1, and CD69 (markers of T cell activation and cytotoxicity), and increase the secretion levels of cytokines IFN-γ, granzyme B, and perforin from T lymphocytes. Furthermore, experimental results show that the drug delivery system in this regimen can effectively enhance the secretion of effector T lymphocytes (CD8+). + T cells' ability to kill tumor cells. CD3 is a marker differentiation antigen on the surface of T lymphocytes. + T cells are mature T lymphocytes that express the CD3 molecule. Almost all mature, functional T cells have CD3 on their surface. + T cells ≈ Total number of mature T lymphocytes.

[0021] The drug delivery system in this protocol acts as a reagent to induce dendritic cell activation, primarily by inducing newly formed dendritic cells to transform into mature dendritic cells.

[0022] The drug delivery system in this protocol acts as a reagent for degrading PD-L1 protein, primarily by reducing the number of PD-L1-positive tumor cells, thereby reducing the number of PD-L1-positive dendritic cells.

[0023] Preferably, the tumor is colorectal cancer.

[0024] The drug delivery system in this protocol, acting as a drug to inhibit tumor growth, primarily works by slowing tumor growth and reducing tumor volume and weight. After drug treatment (simultaneously with ultrasound), the tumor tissue exhibits a loose cell arrangement, showing significant apoptosis or necrosis. The PD-L1 protein content in the tumor area decreases (reduced immunosuppression), and CD8... + The number of T cells increases.

[0025] The drug delivery system in this scheme is preferably used to inhibit lung metastasis of colorectal cancer, serving as a drug for inhibiting tumor metastasis.

[0026] The drug delivery system in this protocol acts as a drug to inhibit tumor recurrence. It induces the large-scale expansion and differentiation of central memory T cells (TCM) and effector memory T cells (TEM) to enter local tumor tissue through lymphatic vessels and blood vessels, and ultimately exerts immune effector function to inhibit tumor recurrence.

[0027] The classification of drugs into three categories—those that inhibit tumor growth, those that inhibit tumor metastasis, and those that inhibit tumor recurrence—is not based on their mechanism of action, but rather on the stage of tumor development they intervene in, their clinical treatment objectives, and their clinical administration or treatment guidance value. These three types of drugs act on three independent yet continuous malignant processes: tumor growth, tumor metastasis, and tumor recurrence. The drug delivery system in this protocol possesses all three functions. Drugs that inhibit tumor growth primarily target tumor cell proliferation, survival, and local tumor growth, controlling the increase in primary tumor size and tumor burden to delay disease progression and alleviate clinical symptoms. Drugs that inhibit tumor metastasis primarily intervene in metastasis-related pathways such as tumor invasion, migration, and distant colonization, blocking the spread of tumors from the primary tumor to distant organs. Drugs that inhibit / prevent tumor recurrence primarily target minimal residual disease, dormant tumor cells, and tumor stem cells after treatment, reducing the recurrence of tumors after radical treatment. They are used as adjuvant / maintenance therapy after radical surgery or clinical remission, aiming to prolong recurrence-free survival and increase the likelihood of clinical cure.

[0028] The drug delivery system described in this protocol, as well as the treatment system formed by the drug delivery system and ultrasound equipment, can be used to prepare products for treating tumors, recurrent tumors, and metastatic tumors. More specifically, it can be used to prepare products for treating colorectal cancer, recurrent colorectal cancer, and metastatic colorectal cancer. Tumors, recurrent tumors, and metastatic tumors are all sub-indications with significant clinical guiding significance. Similarly, colorectal cancer, recurrent colorectal cancer, and metastatic colorectal cancer are also sub-indications with significant clinical guiding significance.

[0029] This invention integrates targeted drug delivery, PD-L1 targeted degradation, and ultrasound enhancement technology to construct an integrated synergistic immunotherapy delivery system. Through multi-mechanism synergistic action, it overcomes the current bottleneck in PD-L1 PROTAC delivery, achieving a highly efficient anti-tumor immune response. The specific technical principles of this solution are as follows: (1) Delivery carrier This invention relates to a drug delivery system that uses a lipid membrane formed from cancer cell membranes and synthetic phospholipids as a lipid shell, encapsulating fluorocarbon gas to form nanobubbles (TCM-NBs). PD-L1 PROTAC (a proteolytic targeting chimera) is integrated into the lipid shell to form the TCM-NBs@PRO nanobubble delivery system. The synthetic phospholipids are formulated with a specific ratio of dipalmitoylphosphatidylcholine and distearate phosphatidylethanolamine-polyethylene glycol 2000 to ensure the stability and biocompatibility of the lipid membrane. The cancer cell membrane, as a natural biological membrane, is rich in tumor antigens and adhesion molecules, endowing the system with inherent tumor homing ability, while simultaneously reducing the clearance of the delivery carrier by the reticuloendothelial system, enabling immune escape of the carrier and improving tumor tissue enrichment efficiency.

[0030] In addition, by optimizing the mass ratio of cancer cell membrane to artificially synthesized phospholipids (preferably 9:1) and the mass ratio of cancer cell membrane to PD-L1PROTAC (preferably 5:1), the particle size of nanobubbles (preferably 412.7 nm) can be controlled, and the passive targeted enrichment of the carrier at the tumor site can be enhanced by utilizing the penetration and retention effect (EPR effect) of tumor tissue.

[0031] (2) Ultrasound-assisted precise delivery of PD-L1 PROTAC By applying ultrasound with specific parameters to the tumor site using ultrasound equipment, the cavitation effect induced by ultrasound causes the nanobubbles in the tumor site to burst in a targeted manner. On the one hand, the mechanical force generated by the burst can destroy the dense structure of the tumor tissue, widen the intercellular space of tumor blood vessels, and significantly increase the penetration depth of PD-L1 PROTAC in the tumor tissue, solving the problem of insufficient tumor penetration of PROTAC molecules. On the other hand, the bursting of the bubbles can realize the on-demand and targeted release of PD-L1 PROTAC, with dual spatial and temporal controllability, ensuring that the drug takes effect precisely at the tumor site and reducing drug exposure in non-target tissues.

[0032] (3) Synergistic immune and anti-tumor effects Integrated into a lipid shell, PD-L1 PROTAC enters tumor cells and the tumor microenvironment after being released by bubble bursting. Through its bifunctional structure, it specifically binds to PD-L1 protein and intracellular E3 ubiquitin ligase, mediating PD-L1 protein ubiquitination modification. Subsequently, it is recognized and degraded by the proteasome, thus eliminating PD-L1 protein at its source and relieving the immunosuppressive signals it mediates.

[0033] The degradation of PD-L1 can relieve T cell functional exhaustion and restore CD3. + CD8 +It enhances the proliferation of effector T cells, the secretion of cytokines (IFN-γ, granzyme B, perforin), and their tumor-killing ability; simultaneously, it reduces the immunosuppressive effect of regulatory T cells (Tregs), transforming the immunosuppressive tumor microenvironment into an immune-supportive microenvironment. Furthermore, PD-L1 degradation can interfere with its-mediated tumor metastasis-related signaling pathways, inhibiting tumor cell shedding and invasion; it also induces the expansion and differentiation of central memory T cells (TCMs) and effector memory T cells (TEMs), enabling them to infiltrate tumor tissue through lymphatic vessels and blood vessels, exerting a long-term immune surveillance effect and inhibiting tumor recurrence.

[0034] This innovative approach integrates PD-L1-degradable PROTAC into cancer cell membrane carriers (TCM-NBs), achieving a synergistic effect of "1+1>2" in the immune activation process. This system not only efficiently drives dendritic cell maturation but also significantly enhances CD3 levels. + CD8 + The number and killing activity of T cells. This dual immune enhancement mechanism enables this drug delivery system to exhibit powerful comprehensive efficacy in inhibiting tumor proliferation, metastasis, and recurrence.

[0035] Compared with existing technologies, this invention has significant technical advantages and clinical application value, and its beneficial effects are as follows: (1) Solve the existing PD-L1 PROTAC delivery bottleneck and improve drug delivery efficiency This invention effectively solves the technical challenges of insufficient tumor tissue penetration and low cellular uptake efficiency of PD-L1 PROTAC by combining nanobubble carriers with ultrasound technology. The nanoscale particle size of the nanobubbles can achieve passive enrichment at the tumor site through the EPR effect, and the ultrasound-induced bubble bursting can further enhance the penetration and diffusion of the drug in the tumor tissue, realizing the targeted and efficient release of PD-L1 PROTAC. At the same time, the tumor homing characteristics of the cancer cell membrane further enhance the targeting of the carrier, reduce the distribution of the drug in non-target tissues, reduce systemic toxicity, and solve the problems of insufficient in vivo metabolic stability, low oral bioavailability, and off-target effects of existing PROTACs.

[0036] (2) Achieving a synergistic immune effect This approach successfully achieved a synergistic effect of immune activation by loading PROTAC, which degrades PD-L1, onto a carrier containing a cancer cell membrane. Specifically, this strategy not only promoted the maturation and activation of dendritic cells but also significantly increased CD3 levels. + CD8 +The increased number of T cells and upregulation of their cytotoxic markers significantly enhanced the tumor-clearing ability. Based on this synergistic mechanism of immune regulation between PROTAC and TCM-NBs, this drug delivery system has demonstrated excellent therapeutic effects in inhibiting tumor growth, blocking metastatic pathways, and preventing recurrence.

[0037] (3) Achieve anti-tumor effects in multiple dimensions, covering the entire process of tumor growth, metastasis, and recurrence. The drug delivery system of this invention can exert anti-tumor effects through multiple synergistic mechanisms, achieving effective intervention in the entire process of tumor development: Inhibits tumor growth: It relieves immunosuppression by degrading PD-L1, activates T cells to kill tumor cells, and at the same time, the mechanical force of ultrasonic ablation can directly destroy tumor cells, slow down the growth rate of tumors, and reduce the size and weight of tumors. Inhibiting tumor metastasis: By degrading PD-L1 and interfering with its mediated tumor metastasis signaling pathway, it blocks tumor cell invasion, migration and distant colonization, and is especially suitable for inhibiting lung metastasis of colorectal cancer; Inhibiting tumor recurrence: By inducing the expansion and differentiation of central memory T cells and effector memory T cells, long-term immune surveillance is established to eliminate minimal residual lesions, dormant tumor cells and tumor stem cells after treatment, thereby reducing the risk of tumor recurrence and providing an effective solution for adjuvant therapy after radical tumor resection.

[0038] (4) It has good biocompatibility, high safety, and great potential for clinical translation. The lipid shell of this invention is composed of a cancer cell membrane and synthetic phospholipids. The cancer cell membrane, as a natural biological membrane, exhibits excellent biocompatibility. The synthetic phospholipids are made from clinically commonly used biocompatible materials, further ensuring the safety of the system. Simultaneously, PD-L1 PROTAC possesses catalytic degradation characteristics, achieving efficient target degradation even at low concentrations, thus reducing the dosage and minimizing drug toxicity. Ultrasound-mediated site-specific drug release further enhances drug safety, avoiding the toxic side effects associated with traditional systemic drug administration, and demonstrates promising prospects for clinical translation.

[0039] (5) The preparation process is simple and controllable, and has strong practicality. The preparation method of the drug delivery system of the present invention has clear steps and is simple to operate. It can be completed through conventional experimental operations such as incubation, ultrasound, and shaking, without the need for complex and precision equipment. The proportion of each raw material can be precisely controlled, and nanobubbles with uniform particle size and stable performance can be stably prepared. It can be mass-produced, reducing the cost of clinical application and has strong practicality.

[0040] (6) It has a wide range of indications and is highly targeted. The drug delivery system of this invention can be used to prepare immune activators (activating anti-tumor immunity, inducing T cell activation, inducing dendritic cell activation, and degrading PD-L1 protein), as well as drugs that inhibit tumor growth, metastasis, and recurrence. It is particularly suitable for the treatment of colorectal cancer, recurrent colorectal cancer, and lung metastatic colorectal cancer. It can play an effective role in different stages of tumor disease in clinical practice, providing a new technical solution for tumor immunotherapy. Attached Figure Description

[0041] Figure 1 The encapsulation efficiency and drug loading of TCM-NBs@PRO in Example 1 of the present invention are characterized as follows: (a: UV-Vis spectra of TCM-NBs, TCM-NBs@PRO, and PROTAC; TCM-NBs are untreated nanobubbles, PROTAC is PROTAC PD-L1 degrader-1, and TCM-NBs@PRO are drug-loaded nanobubbles; b: UV-Vis spectra of PROTAC at different concentrations; c: Encapsulation efficiency of cancer cell membrane and PROTAC drug at different mass ratios; d: Drug loading of cancer cell membrane and PROTAC drug at different mass ratios).

[0042] Figure 2 The following are the characterization results of the TCM-NBs@PRO particle size and potential of Example 2 of the present invention: (a: representative images of cancer cell membrane and lipid membrane under optical microscope under different mass ratios; b: representative images of cancer cell membrane and lipid membrane under confocal microscope under a 9:1 mass ratio; c: changes in TCM-NBs@PRO nanobubble particle size of cancer cell membrane and lipid membrane under different mass ratios; d: changes in TCM-NBs@PRO nanobubble potential of cancer cell membrane and lipid membrane under different mass ratios; e: statistical results of TCM-NBs@PRO nanobubble particle size and potential of cancer cell membrane and lipid membrane under different mass ratios; TCM is the CT26 cancer cell membrane; PL is the lipid membrane obtained by rotary evaporation).

[0043] Figure 3 The results of in vitro and in vivo ultrasound imaging of TCM-NBs@PRO in Example 2 of this invention are shown (a: in vitro ultrasound imaging; b: in vivo ultrasound imaging).

[0044] Figure 4 For the analysis of cancer cell membrane proteins of TCM-NBs@PRO in Example 2 of the present invention (a: protein gel electrophoresis showing protein profile; b: proteomics analysis).

[0045] Figure 5The experimental results of the targeting and in vivo distribution of TCM-NBs@PRO in Example 3 of this invention are as follows: (a: representative fluorescence images after co-incubation of nanobubbles with mouse colorectal cancer cells; b: flow cytometry results after co-incubation of nanobubbles with mouse colorectal cancer cells; c: tumor local signals detected by a small animal in vivo fluorescence imaging system at 0, 1, 2, 4 and 6 h after intravenous injection of different nanobubbles; d: signal intensity at 0, 1, 2, 4 and 6 h after intravenous injection of different nanobubbles).

[0046] Figure 6 The experimental results of TCM-NBs@PRO activating dendritic cells in Example 4 of this invention are shown in the following figures: (a: Schematic diagram of experimental method; b: Proportion of mature dendritic cells detected by flow cytometry; c: Statistical results of Figure b; d: Proportion of MHC I positive cells detected by flow cytometry; e: Proportion of MHC II positive cells detected by flow cytometry; f: Statistical results of Figure d; g: Statistical results of Figure e).

[0047] Figure 7 The experimental results of the combined treatment of tumor cells with TCM-NBs@PRO and ultrasound in Example 4 of the present invention are as follows: (a: Schematic diagram of the experimental method and immunofluorescence image of the treated tumor cells; b: Detection results of MGB1 and IL-33 content in the supernatant after combined treatment with TCM-NBs@PRO and ultrasound, or after treatment with TCM-NBs@PRO alone; c: Flow cytometry detection of the activation of dendritic cells by the supernatant of tumor cells treated with TCM-NBs@PRO and ultrasound).

[0048] Figure 8 The experimental results of TCM-NBs@PRO-induced T cell activation in Example 4 of this invention are shown in Figure 4 (a: Schematic diagram of the assay based on dendritic cells and splenic T lymphocytes; bc: CD3 under different treatments). + CD8T + Flow cytometry results of cell proportions; de: Flow cytometry results of T cell activation and proportion of cells positive for cytotoxic markers under different treatments; fh: Levels of cytokines IFN-γ, granzyme B, and perforin in spleen suspension supernatant under different treatments; i: Schematic diagram of the detection method of tumor killing effect of activated spleen single cells under different treatments; jk: Flow cytometry detection results and statistical graphs of tumor killing effect of spleen single cells.

[0049] Figure 9The experimental results of TCM-NBs@PRO degradation of PD-L1 protein in Example 4 of this invention are as follows: (a: Schematic diagram of the mechanism of TCM-NBs@PRO degradation of PD-L1; b: Western blot results of PD-L1 protein expression in CT26 cells after 24 h of treatment with the PD-L1 protein degradation chimeric drug; cf: Western blot results of the effect of different treatments on PD-L1 protein expression in blast cells or CT26 tumor cells; gj: Flow cytometry results of the effect of different treatments on PD-L1 protein expression in blast cells or CT26 tumor cells; k: Immunofluorescence results of the effect of different treatments on PD-L1 protein expression in blast cells or CT26 tumor cells).

[0050] Figure 10 The experimental results of TCM-NBs@PRO activating immunity in vivo in Example 4 of this invention are shown below (a: schematic diagram of animal experiment process; bc: flow cytometry detection and statistical results of the proportion of mature dendritic cells in lymph node tissue; dg: CD8+ in tumor and spleen). + Flow cytometry results of T cell proportions and statistical analysis; hl: Serum levels of TNF-α, IL-6, IFN-γ, granzyme B, and perforin in different experimental groups.

[0051] Figure 11 The experimental results of the in vivo therapeutic effect of TCM-NBs@PRO in Example 5 of this invention are as follows: (a: Schematic diagram of animal experiment process; b: Changes in body weight of mice after treatment in each group; c, d: Photographs of tumor tissue and mice; e: Tumor growth curve; f: H&E staining image of ex vivo tumor; g: CD8) + (Fluorescent immunoassay images of T cells and PD-L1 protein).

[0052] Figure 12 The experimental results of TCM-NBs@PRO inhibiting tumor metastasis in Example 5 of the present invention are as follows: (ab: observation results of lung metastasis lesions on day 20 after treatment; ce: bioinformatics analysis results of TCM-NBs@PRO+US group and control group; f: KEGG analysis results; g: TCM-NBs@PRO treatment group's involvement in the regulation of metastasis-related genes).

[0053] Figure 13 The experimental results of TCM-NBs@PRO inhibiting tumor recurrence in Example 6 of this invention are shown below (a: schematic diagram of animal experiment process; b: photo of experimental mice; c: survival curve; d: body weight change curve; ej: flow cytometry detection and statistical results of TCM-NBs@PRO group and control group). Detailed Implementation

[0054] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0055] Example 1: Preparation of TCM-NBs@PRO To obtain the CT26 cancer cell membrane (mouse colon cancer cell line), a membrane protein extraction kit can be used according to the manufacturer's instructions. Obtaining the cell membrane from cells is a routine method in existing technology. The extraction method is briefly described below: Wash the cells with ice-cold PBS and resuspend them in homogenization buffer in an ice-cold Dounce homogenizer (glass tissue homogenizer). Homogenize the cells 30-50 times on ice, then centrifuge at 700×g for 10 min at 4°C. Collect the supernatant and discard the particles. Next, centrifuge at 14000×g for 30 min at 4°C to obtain the CT26 cell membrane. Wash the obtained CT26 cell membrane twice with PBS. The protein content in the CT26 membrane is determined using a BCA kit. The CT26 membrane is then compared with PROTAC PD-L1 degrader-1 (CAS No.: 2447066-37-5; Chemical Formula: C). 59 H 58 ClN7O 11 Molecular weight: 1076.59; MedChemexpress Biotechnology Co., Ltd. (USA) mixed at a mass ratio of 40:1-2.5:1 (preferably 5:1), and incubated at room temperature for 2 hours (optional range 1.5-2.5 hours) to obtain drug-loaded CT26 cancer cell membranes, named TCM@PRO.

[0056] Add DPPC (dispalmitoylphosphatidylcholine, 8 mg) and DSPE-PEG2000 (distearate phosphatidylethanolamine-polyethylene glycol 2000, 2 mg) to a round-bottom flask (the mass ratio of DPPC to DSPE-PEG2000 can be 1:9-9:1). Add 5 mL of chloroform (optional range 3-7 mL; the ratio of DPPC, DSPE-PEG2000, and chloroform is 1-9 mg:1-9 mg:3-7 mL). Gently shake to completely dissolve the lipids, forming a clear solution. Connect the round-bottom flask to a rotary evaporator and perform conventional rotary evaporation until the chloroform is completely evaporated, forming a uniform lipid film (lipid membrane) on the inner wall of the flask. Add PBS (10 mg:2 mL) to the lipid membrane to hydrate it. Add TCM@PRO to the lipid membrane and mix. The mass ratio of the CT26 cancer cell membrane (TCM) in TCM@PRO to the mass of the lipid membrane obtained by rotary evaporation is 1:9-9:1 (preferably 9:1). Next, the suspension was sonicated until the mixture became clear (power 120W, pulse setting: 5 seconds on, 5 seconds off, frequency 20kHz, duration 1 min). During this step, ice was added to the sonicator, meaning the sonication process was performed in an ice bath to prevent heat-induced protein denaturation. After sonication, a drug-loaded lipid suspension was obtained for subsequent processing.

[0057] Next, transfer 0.5 mL of the suspension to a 2 mL vial, seal the vial, and evacuate it using a syringe. Maintain a uniform evacuation speed until the syringe plunger can no longer be pulled, and keep the vacuum state for 10-15 seconds to ensure that the air inside the vial is fully removed. Immediately afterwards, fill the vial with excess C3F8 (perfluoropropane) gas (filling with inert gas allows the vial to refill from a vacuum state, i.e., replacing the air originally extracted from the vial with inert gas). Finally, mechanically stir the vial at 4000 rpm for 45 seconds using an amalgam shaker (YJT-2, Shanghai, China) to achieve bubble formation (TCM-NBs@PRO).

[0058] Following the above method, without adding PROTAC PD-L1 degrading agent-1 during the preparation process, drug-free nanobubble TCM-NBs can be prepared.

[0059] Example 2: Characterization of TCM-NBs@PRO The ability to load PROTAC drugs onto cancer cell membranes was prioritized for evaluation. (UV-Vis spectroscopy) Figure 1 a) This shows that TCM-NBs@PRO has a significant absorption peak at 341 nm, which is attributed to the characteristic absorption peak of PROTAC. Figure 1 b) confirmed that PROTAC was successfully loaded onto the cancer cell membrane. Figure 1c shows that the encapsulation efficiency (up to 77.5%) is highest when the mass ratio of cancer cell membrane to PROTAC drug is 5:1. At this ratio, the drug loading capacity is approximately 13.4%. Figure 1 (d) This demonstrates that the cancer cell membrane has a good drug loading capacity, and this drug loading ratio was selected for subsequent experimental studies.

[0060] Further analysis was conducted to characterize the self-assembly of gas bubbles formed after mechanical agitation of drug-loaded cancer cell membranes and hydrated phospholipid membranes at different mass ratios. Typical images show that the suspension becomes clearer as the proportion of cancer cell membranes increases. Optical microscopy ( Figure 2 a) This shows that as the proportion of cancer cell membrane increases, the particle size of the formed bubbles decreases significantly. Further observation using confocal microscopy revealed nanobubbles formed when the mass ratio of cancer cell membrane to phospholipid membrane was 9:1. Figure 2 b. Uniformly distributed nanobubbles can be observed. The cell membrane linked to DiI dye exhibits red fluorescence, and the red fluorescence overlaps with the distribution of nanobubbles, proving that the main component of nanobubbles is the cell membrane. Figure 2c and 2d show the particle size variations of self-assembled bubbles at different ratios, illustrating the particle size distribution of nanoparticles at mass ratios of CT26 cancer cell membrane and lipid membrane (CCM:PL = 1:9, 3:7, 5:5, 7:3, 9:1). At CCM:PL = 1:9, 3:7, and 5:5, the obtained nanobubbles exhibit broad particle size distributions, significant tailing, and high polydispersity. Notably, the bubbles formed when the mass ratio of cancer cell membrane to phospholipid membrane is 9:1 have the smallest particle size, 412.7 ± 11.9 nm. In nanobubble-mediated diagnostic and therapeutic applications, reducing bubble size offers multiple benefits: smaller particles enhance the penetration and retention of tumor tissue, facilitate targeted enrichment through vascular endothelial spaces, and avoid rapid clearance by the mononuclear phagocytic system, thus prolonging the blood circulation half-life; in ultrasound imaging, microbubbles are more sensitive to ultrasound, generating stronger nonlinear scattering signals at lower sound pressure levels, improving imaging contrast and resolution, and can penetrate physiological barriers such as the blood-brain barrier, enabling precise imaging of deep tissues and micro-lesions; in drug delivery, the larger surface area of ​​microbubbles allows for the loading of more drugs, and the cavitation effect is milder and more controllable, precisely triggering drug release at the target site, while also being more easily endocytosed for intracellular delivery. The inventors further analyzed that the reason for the higher cancer cell membrane content and smaller particle size may be due to abnormal glycosylation modifications on the cancer cell membrane surface, which typically carries a strong negative charge. As the proportion of tumor membrane increases, the charge density on the mixed membrane surface increases. During vesicle formation, strong electrostatic repulsion prevents adjacent membranes from approaching and fusing, thus limiting vesicle growth and causing the system to tend to maintain a smaller particle size distribution. This is consistent with the results of potential measurements; the higher the cancer cell membrane content, the lower the potential. Furthermore, Figure 2 The data shows that as the proportion of cell membrane increases, the potential of the formed bubbles decreases slightly. When the mass ratio of cancer cell membrane to phospholipid membrane is 9:1, the potential of the nanobubble is -8.38±0.17 mV.

[0061] Next, the in vitro ultrasound enhancement capability of TCM-NBs@PRO was evaluated using a gelatin model. Figure 3 The results showed that TCM-NBs@PRO exhibited good in vitro ultrasound imaging capabilities. The acquired ultrasound image signal showed significant signal attenuation after 30 minutes, possibly due to a substantial increase in surface tension as the bubble radius decreased. Subsequently, when the tumor tissue volume was approximately 150 mm²... 3 At that time, the ultrasound enhancement imaging capability of TCM-NBs@PRO in tumor-bearing mice was evaluated, and ultrasound images were acquired at 0, 3 min, 5 min, and 15 min time points. Figure 3 b).

[0062] Cell adhesion molecules on cancer cell membranes play a crucial role in cell adhesion. To further confirm the successful functionalization of TCM-NBs@PRO with cancer cell adhesion molecules, the content of various proteins on the surface of TCM-NBs@PRO was systematically investigated. Figure 4 Protein gel electrophoresis revealed a protein profile, indicating that TCM-NBs@PRO and cancer cell membranes share similar protein profiles. Further proteomic analysis showed that TCM-NBs@PRO contained abundant amounts of proteins such as EpCAM, N-cadherin, galectin-3, and CDH2, which play important roles in tumor homology targeting. Furthermore, CD47 and CD24 proteins bound to macrophage SIRPα and Siglec-10, respectively, inhibiting their phagocytosis and increasing the in vivo circulation time of the nanoparticles. Notably, proteomic analysis also demonstrated that TCM-NBs@PRO expresses multiple tumor-associated antigens (...). Figure 4 b).

[0063] Example 3: Targeting and in vivo distribution of TCM-NBs@PRO To investigate the cellular uptake rate of TCM-NBs@PROs, cell membrane fluorescent dye DiI was used for labeling. For example... Figure 5 As shown in Figure a, after co-incubating mouse colorectal cancer (CT26) cells with TCM-NBs@PRO and ordinary phospholipid nanoparticles (Lip-NBs) for 1 h, a stronger fluorescence signal was detected inside the cells treated with TCM-NBs@PRO. TCM-NBs@PRO showed higher cellular uptake efficiency compared to ordinary phospholipid nanoparticles. Further flow cytometry analysis showed that the fluorescence intensity of cells treated with TCM-NBs@PRO increased over time, and the positive rate at 1 h was 2.46 times that of the ordinary phospholipid nanoparticle treatment group. Figure 5 b).

[0064] Furthermore, the biodistribution behavior of TCM-NBs@PRO and other nanobubbles (with or without sonication) in CT26 tumor-bearing mice was investigated. Local tumor signals were detected using a small animal fluorescence imaging system at 0, 1, 2, 4, and 6 hours after intravenous injection of TCM-NBs@PRO. Figure 5 As shown in c, fluorescence imaging revealed that the fluorescence intensity of both the TCM-NBs@PRO and ordinary phospholipid nanoparticle-treated groups reached its peak at 4 h, and the fluorescence intensity of the TCM-NBs@PRO group was 4.43 times that of the ordinary phospholipid nanoparticle-treated group. Figure 5(d) This indicates that TCM-NBs@PRO has a greater advantage in accumulation and retention at the tumor site compared to ordinary phospholipid nanobubbles. These results demonstrate the excellent cellular uptake efficiency of the TCM-NBs@PRO nanocarrier and suggest that modification of the cancer cell membrane will further enhance its drug delivery capability. Notably, fluorescence imaging of the TCM-NBs@PRO+US (ultrasound treatment) group showed that the fluorescence intensity in the tumor region reached its peak at 1 hour, and its peak fluorescence intensity was 1.36 times that of the TCM-NBs@PRO group, demonstrating that ultrasound-targeted destruction technology can enhance the tissue penetration of therapeutic drugs and advance the concentration time in the target area.

[0065] Example 4: Immune activation effect of TCM-NBs@PRO (1) The effect of TCM-NBs@PRO on enhancing dendritic cell activation Dendritic cells play a crucial role as antigen-presenting cells in both innate and adaptive immunity. This study found that TCM-NBs@PRO enhances the immune system and promotes personalized immunotherapy by activating dendritic cells, thereby activating T cells and reducing T cell exhaustion. For in vitro dendritic cell activation studies, 5 × 10⁶ cells were used. 5 DC2.4 cells were cultured in 12-well plates and incubated for 24 h before being exposed to four different treatment conditions: ① PBS as a control group; ② free PROTAC (concentration 46.5 μg / mL); ③ TCM-NBs (concentration 300 μg / mL based on TCM); ④ TCM-NBs@PRO (TCM: 300 μg / mL; PROTAC: 46.5 μg / mL). After 24 h of culture, cells were collected and stained with anti-CD86, anti-CD80, anti-MHCI, and anti-MHCII antibodies for flow cytometry analysis.

[0066] Experimental results are as follows Figure 6 As shown in Figure a, dendritic cells were treated with different drug groups, and the expression of dendritic cell activation markers such as MHC class II, CD86, and CD80 was assessed by flow cytometry. Figure 6 b and 6c show that mature dendritic cells (mDCs) treated with TCM-NBs@PRO were significantly more numerous than those in the control group (CD11c). + CD80 + CD86 + The proportion of the group that was significantly increased from 6.37±1.30% to 49.18±2.64%, which was significantly greater than that of the PROTAC and TCM-NBs groups (10.86±1.13% and 21.84±1.09%, respectively). Figure 6 The specific experimental data for c are shown in Table 1.

[0067] Table 1: Percentage of mature dendritic cells after treatment in each group

[0068] Synergistic effects were analyzed using a Bliss-independent model, with the proportion of mature dendritic cells as the effect indicator, and the net effect was calculated as (effect value of each group minus the mean of the control group). To accurately assess drug synergy, the baseline levels observed in the control group were subtracted from all treatment groups before applying the Bliss-independent model to calculate the net effect. Only the actual effects produced by the drugs were calculated, eliminating spontaneous background noise from cells. The theoretical combined effect is calculated using the formula: E exp =E A +E B -(E A ×E B ); where E A (0.1086-0.0637=0.0449), E B (0.2184-0.0637=0.1547) represent the net effects of PROTAC and TCM-NBs treated individually; substituting these values ​​into the formula yields E. exp =0.19265397. E obs The measured net effect of the combined treatment is (0.4918 - 0.0637 = 0.4281). Bliss Score = E obs -E exp A Bliss Score > 0 was considered synergistic. Due to the unequal variances between the two groups (theoretical and experimental combined groups), Welch's independent samples t-test was used to compare the differences between the experimental and theoretical expected values; p < 0.05 was considered statistically significant. After subtracting the baseline level of the control group using the net effect method, the synergistic effect was analyzed using the Bliss independent model. The Bliss Score was approximately 0.2355, and the experimental effect was significantly higher than the theoretical expected value (Welch t-test, t = 19.01, p < 0.001), indicating that the combined application of PROTAC and TCM-NBs can significantly synergistically promote dendritic cell maturation. In other words, in the TCM-NBs@PRO group where PROTAC and TCM-NBs were used in combination, PROTAC and TCM-NBs produced a significant synergistic effect in increasing the proportion of mature dendritic cells.

[0069] The antigen-presenting capacity of dendritic cells was further investigated, and the expression of MHC1 and MHC2 on the surface of treated dendritic cells was detected by flow cytometry. Figure 6 d, e, f, and g demonstrate that the expression levels of MHC1 and MHC2 after TCM-NBs@PRO treatment were 1.63 times and 11.09 times that of the control group, respectively.

[0070] During ultrasound-targeted destruction, cavitation and mechanical effects lead to transient and irreversible cell membrane permeability, causing tumor cells to release damage-associated molecular patterns, thereby promoting dendritic cell maturation. Using 1×10 5 CT26 cells were cultured in 15 mm confocal dishes and incubated for 24 h. Afterward, they were exposed to two different treatment conditions: ① TCM-NBs@PRO; ② TCM-NBs@PRO+US (TCM: 300 μg / mL; PROTAC: 46.5 μg / mL; ultrasound parameters: probe frequency 1 MHz, sound intensity 0.173 MPa, duty cycle 50%, irradiation time 1 min). Immunofluorescence staining was performed after 24 h of culture. The supernatant was collected for ELISA kit analysis. Figure 7 After ultrasound-targeted destruction of tumor cells by nanoparticles, immunofluorescence staining showed a significant increase in CRT fluorescence intensity on the surface of tumor cells. Furthermore, the levels of HMGB1 and IL-33 in the supernatant of the ultrasound-treated group were also significantly increased, being 1.68 times and 5.69 times higher than those in the untreated group, respectively. Figure 7 b). Flow cytometry further detected that the supernatant of tumor cells after ultrasound treatment with TCM-NBs@PRO effectively activated dendritic cells ( Figure 7 c).

[0071] (2) TCM-NBs@PRO induces T cell activation Further evaluation was conducted on the ability of TCM-NBs@PRO to activate anti-tumor immunity. An in vitro assay based on dendritic cells and splenic T lymphocytes was established (see [link to relevant documentation]). Figure 8 a. First, using conventional techniques such as mechanical grinding and filtration, spleen tissue is prepared into a single-cell suspension to obtain a spleen cell suspension containing a large number of T lymphocytes. Then, spleen cells (1 × 10⁶ cells per well) are... 7 Spleen cells and dendritic cells treated 24 hours prior were co-cultured in 6-well plates for 24 hours (dendritic cells:spleen cells = 1:50). Dendritic cell treatments included: ① PBS as a control group; ② free PROTAC (concentration 46.5 μg / mL); ③ TCM-NBs (concentration 300 μg / mL); ④ TCM-NBs@PRO (TCM: 300 μg / mL; PROTAC: 46.5 μg / mL). After co-incubation, flow cytometry was used to detect T cell activation markers, proliferation, and cytokine secretion to comprehensively evaluate the strength of the anti-tumor immune response induced by TCM-NBs@PRO through DC activation.

[0072] Flow cytometry analysis demonstrated that TCM-NBs@PRO treatment effectively activated T lymphocytes and CD3+ from spleen suspension. +CD8 + The number of T cells was 1.38 times that of the control group. Figure 8 bc). Compared with other groups, the TCM-NBs@PRO treatment group showed CD8... + T cells expressed higher levels of IFN-γ and CD69 (markers of T cell activation and cytotoxicity), which were 1.85 and 11.25 times higher than those in the control group, respectively. Figure 8 d, 8e (see Table 2 for specific data). Next, the levels of cytokines IFN-γ, granzyme B, and perforin in the supernatant of spleen suspension after different treatment groups were detected by ELISA. The levels of IFN-γ, granzyme B, and perforin in the TCM-NBs@PRO group were 168.14±9.36 ng / mL, 1.78±0.07 ng / mL, and 30.76±1.45 ng / mL, respectively, which were significantly higher than those in the control group ( Figure 8 (fh), indicating that it has a strong tumor-killing effect.

[0073] Table 2: CD8+ cells expressing IFN-γ or CD69 produced by dendritic cells activating T lymphocytes after treatment in each group + Percentage of T cells

[0074] For IFN-γ, a Bliss independent model combined with the net effect method was used to evaluate the combined effect of PROTAC and TCM-NBs on CD3. + CD8 + Synergistic effect of T cell activation. After deducting the baseline level of the control group (31.96±4.72%), the net effects of PROTAC and TCM-NBs monotherapy were 0.0664 and 0.1502, respectively, and the measured net effect of the combined treatment was 0.3004. The theoretical expected net effect calculated by the Bliss model was 0.2071, and the Bliss Score = 0.0933 > 0, indicating a synergistic effect between the two. Welch independent samples t-test confirmed that the measured net effect of the combined treatment was significantly higher than the theoretical expected value (t = 2.64, p < 0.05).

[0075] Similarly, for CD69, the Bliss independent model combined with the net effect method was used to evaluate the combined effect of PROTAC and TCM-NBs on CD3. + CD8 +The synergistic effect of T cell activation was observed. The net effects of PROTAC and TCM-NBs as single drugs were 0.1399 and 0.4327, respectively, while the measured net effect of the combined treatment was 0.6855. The theoretical expected effect of Bliss was 0.4921, and the Bliss Score (0.1934) > 0, indicating a significant synergistic effect between the two drugs. Welch's independent samples t-test showed that the measured effect of the combined group was significantly higher than the theoretical expected value (t = 14.92, p < 0.001).

[0076] The above analysis results indicate that the combination of PROTAC and TCM-NBs can synergistically enhance CD3 levels in spleen suspension. + CD8 + T cell activation, while significantly increasing CD8 + The expression levels of IFN-γ and CD69 on T cells further enhance T cell activation and killing functions.

[0077] This technical solution also evaluated effector T lymphocytes (CD8). + The effector T cells (T lymphocytes) demonstrated their ability to kill tumor cells. These effector T lymphocytes were activated through the four treatment methods described above. Tumor cells were cultured in vitro and seeded into the lower layer of a Transwell chamber. Simultaneously, a suspension of spleen single cells obtained after treatment was added to the culture system containing tumor cells (the upper layer of the Transwell chamber) for co-incubation. The effector-to-target ratio was 50:1 (spleen single cells:tumor cells), and the incubation time was 24 hours. After treatment, the tumor cell apoptosis rate was measured.

[0078] like Figure 8 As shown in i, spleen suspensions from the different treatment groups were added to transwell chambers of a plate inoculated with tumor cells for 24 hours. The apoptosis rate of tumor cells below the chamber was assessed by annexin V-APC / PI double staining. As expected, the PBS group showed very little apoptosis, and the TCM-NBs@PRO treatment group achieved the highest apoptosis percentage of approximately 23.38%. Figure 8 j, 8k (see Table 3 for specific data), indicating that TCM-NBs@PRO can effectively enhance the ability of dendritic cells to activate T lymphocytes. These results suggest that TCM-NBs@PRO can present antigens to dendritic cells and induce dendritic cell maturation, thereby leading to antigen-specific CD8+ activation. + Activation of T cells enhances the establishment of in vitro anti-tumor immune responses.

[0079] Table 3: Percentage of tumor cells apoptosis after treatment in each group

[0080] The combined activation of CD8 by PROTAC and TCM-NBs was analyzed using the Bliss independent model combined with the net effect method. + The synergistic effect of T cells (spleen single-cell suspension) in tumor cell killing. After deducting the baseline apoptosis rate in the control group, the net effects of PROTAC and TCM-NBs alone were 0.0090 and 0.0600, respectively, and the net effect of the combined treatment was 0.1108. The theoretical expected effect of Bliss was 0.0685, and the Bliss Score = 0.0423 > 0, indicating that the two have a significant synergistic effect. The Welch independent samples t test showed that the measured effect of the combined group was significantly higher than the theoretical expected value (t = 20.12, p < 0.001), indicating that TCM-NBs@PRO, formed by the combination of PROTAC and TCM-NBs, can effectively activate effector T lymphocytes, significantly enhance their ability to kill tumor cells, and produce a synergistic effect.

[0081] (3) TCM-NBs@PRO degrades PD-L1 protein The mechanism of TCM-NBs@PRO in degrading PD-L1 is as follows: Figure 9 As shown in a.

[0082] According to Western blot analysis, after 24 h of treatment with the PD-L1 protein degradation chimeric drug, the expression of PD-L1 protein in CT26 cells decreased in a dose-dependent manner, and a concentration of 20 μg / mL achieved a good protein degradation effect. Figure 9 b). The cells are spaced at 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 μg / mL in 12-well plates and cultured under standard conditions for 24 h. To investigate the specific degradation effect of PROTAC-loaded cell membranes, CT26 cells and dendritic cells were treated as follows: ① saline control group; ② free PROTAC (concentration 46.5 μg / mL); ③ TCM-NBs (concentration 300 μg / mL); and ④ TCM-NBs@PRO (TCM: 300 μg / mL; PROTAC: 46.5 μg / mL). After 24 h of treatment, flow cytometry, immunofluorescence staining, and Western blot were used for analysis.

[0083] Experimental results showed that both TCM-NBs@PRO and PROTAC significantly reduced PD-L1 protein expression, demonstrating that drug loading on tumor cells does not affect the efficacy of PROTAC. Figure 9 These results were further validated by flow cytometry, showing that TCM-NBs@PRO reduced the PD-L1 dendritic cell positivity rate by approximately 46.12% and the PD-L1 CT26 tumor cell positivity rate by approximately 21.56%. Figure 9gj). Immunofluorescence staining of PD-L1 protein also confirmed the effective degradation of PD-L1 protein in CT26 or DC tumor cells treated with TCM-NBs@PRO. Figure 9 In summary, these results indicate that TCM-NBs@PRO can effectively induce the degradation of PD-L1 protein in tumor cells and dendritic cells. The degradation effect on PD-L1 was more significant after TCM-NBs@PRO treatment of dendritic cells, which may be due to the lower PD-L1 protein expression in dendritic cells compared to CT26 tumor cells. Reduced PD-L1 protein expression in dendritic cells can enhance their antigen-presenting capacity and T-cell activation ability.

[0084] (4) TCM-NBs@PRO activates immunity in vivo Based on results obtained at the cellular level, TCM-NBs@PRO significantly enhanced anti-tumor immunity and exerted a significant cytotoxic effect on CT26 tumor cells. Furthermore, ultrasound-targeted nanobubble destruction technology, in addition to enhancing drug penetration, also promoted the release of tumor cell damage-related molecular patterns, further activating the adaptive immune system. To verify the ability of TCM-NBs@PRO +US to activate the immune system in vivo, CT26 tumor cells were subcutaneously implanted into Balb / c mice 7 days before treatment. Tumor-bearing mice (n=5 per group) were randomly divided into 5 groups: ①PBS group; ②Free PROTAC group; ③TCM-NBs+US group; ④TCM-NBs@PRO group; ⑤TCM-NBs@PRO+US group. The dosages of PROTAC and TCM were 0.465 mg / mL and 3 mg / mL, respectively. For the group receiving US treatment, the tumor was irradiated with ultrasound 3 minutes after injection (1 MHz, 0.3 MPa, 50%, 2 min). Figure 10 As shown in Figure a, the mice were treated three times on days 1, 3, and 5, with drug injection followed by ultrasound irradiation of the tumor site. On day 7, the mice were euthanized to assess the activation of the immune system in the tumor-draining lymph nodes and tumor tissue after the same treatment. Tumors, lymph nodes, and spleens were harvested from the mice and processed into single-cell suspensions to detect mature dendritic cells and T cell populations.

[0085] For detailed experimental results, please refer to Figure 10 The TCM-NBs@PRO+US group showed a mature dendritic cell population of 21.6±3.14% in lymph nodes, significantly higher than other groups, indicating enhanced dendritic cell activation in lymph nodes. Figure 10(b, 10c). Furthermore, in the tumor, the percentage of mature dendritic cells in the TCM-NBs@PRO+US treatment group was 27.96±3.2%, exceeding that of the PBS group (5.62±2.06%), PROTAC (15.96±3.71%), TCM-NB+US (20.62±1.56%), and the TCM-NBs@PRO group (17.42±2.16%). These results demonstrate that TCM-NBs@PRO+US effectively stimulates dendritic cell maturation in vivo and promotes recruitment in the tumor region.

[0086] Flow cytometry showed that, compared with the PBS group, PROTAC, TCM-NBs+US, and TCM-NBs@PRO group, the TCM-NBs@PRO+US group had significantly higher levels of CD8+ in tumors. + The percentage of T cells was higher ( Figure 10 d, 10e). Splenic flow cytometry analysis showed a similar trend, with the TCM-NBs@PRO +US treatment group showing CD8... + The percentage of T cells was higher in the PBS group, PROTAC, TCM-NBs+US, and TCM-NBs@PRO group. Figure 10 f, 10g).

[0087] In addition, cytotoxic T lymphocytes secrete granzyme B to induce tumor cell apoptosis. The levels of TNF-α, IL-6, cytokine IFN-γ, granzyme B, and perforin in serum after different treatment groups were detected by ELISA. The levels of IFN-γ, granzyme B, and perforin in the TCM-NBs@PRO+US group were 449.77±30.27 pg / mL, 53.3±4.69 pg / mL, and 5.91±0.34 pg / mL, respectively, significantly higher than those in the control group. The detection of TNF-α and IL-6 also showed the same trend. Figure 10 (hl), indicating that it enhances anti-tumor immune capabilities.

[0088] Example 5: In vivo therapeutic effects of TCM-NBs@PRO After verifying the activation of anti-tumor immunity by TCM-NBs@PRO +US in vivo, the efficacy of TCM-NBs@PRO in treating tumor-bearing mice via low-intensity ultrasound local blasting was further observed and verified to explore the potential of TCM-NBs@PRO in personalized immunotherapy. Throughout the treatment process ( Figure 11 a) Tumor tissue growth in mice was continuously monitored starting from day 1. Tumor volume and mouse weight were measured every two days using calipers. Treatment consisted of intravenous injection of the drug on days 1, 4, and 7, followed by local ultrasound irradiation of the tumor site in CT26 tumor-bearing mice. The experimental grouping, drug dosage, and ultrasound treatment method in this embodiment were exactly the same as in Example 4.

[0089] No significant weight loss was observed in any group throughout the treatment. Tumor growth curves showed that, compared to other groups, the TCM-NBs@PRO +US group had slower tumor growth and the smallest tumor volume and weight. Figure 11 (be). Compared with the control group, all treatment groups showed varying degrees of inhibition of tumor growth in tumor-bearing mice. On day 16 post-injection, the tumor volume in the TCM-NBs@PRO +US group was significantly reduced to 240.2 ± 165.17 mm. 3 It is significantly lower than the Control (1469.98±74.04mm). 3 ), PROTAC (982.33±264.52mm 3 ), TCM-NBs+US (990.63±73.39mm 3 ) and TCM-NBs@PRO (607.44 ±130.06mm) 3 )Group.

[0090] H&E staining of ex vivo tumors showed that the TCM-NBs@PRO +US group exhibited the most loosely arranged tumor cells, the largest nuclear contraction, and the most apoptotic and necrotic cells. Figure 11 f). Compared with the control group, TCM-NBs@PRO +US CD8 + T cell fluorescence intensity was 3 times higher than that of the control group. Figure 11 g). As expected, the PD-L1 fluorescence intensity in the tumor region was significantly increased in the TCM-NBs-only group, 1.5 times that of the control group, further confirming the formation of an immunosuppressive microenvironment. The PD-L1 protein fluorescence intensity in the tumor region was significantly decreased in the TCM-NBs@PRO +US group, 0.5 times that of the control group. These results demonstrate that TCM-NBs@PRO +US can effectively activate CD8+. + T cells are stimulated, and PD-L1 protein expression in tumor cells is reduced. In summary, the TCM-NBs@PRO autotransmitter tumor vaccine can achieve dual targeting of tumors via ultrasound irradiation, further promoting dendritic cell maturation and initiating T cell activation, proliferation, and recruitment, thereby exerting a powerful systemic protective effect.

[0091] Once colorectal cancer metastasizes, subsequent treatment options are limited, and the prognosis is poor. Therefore, this study further investigated whether TCM-NBs@PRO combined with ultrasound therapy could inhibit tumor metastasis. Lung tissue was harvested on day 20 post-treatment for observation. Figure 12Abstract analysis showed that the control group exhibited multiple lung metastases, while the TCM-NBs@PRO +US treatment group showed no significant lung metastases, indicating that this treatment strategy significantly inhibited tumor metastasis. To further explore the mechanism by which TCM-NBs@PRO +US treatment inhibits tumor metastasis, RNA transcriptome sequencing of tumor cells after TCM-NBs@PRO +US treatment was performed. Bioinformatics analysis, using TPM to quantify gene expression distribution in various samples, showed no significant batch effect between the two groups. Figure 12 c). Analysis of tens of thousands of genes in the Control and TCM-NBs@PRO +US groups revealed 652 differentially expressed genes (DDEGs), including 514 upregulated genes and 138 downregulated genes. Figure 12 KEGG analysis showed that DEGs in the TCM-NBs@PRO+US group were enriched with many function-specific BPs (e.g., immune regulation and T cell-mediated immunity), CCs (e.g., extracellular collagen and cell adhesion molecules), and MFs (e.g., cytoskeletal protein binding, growth factor binding, and signal receptor binding). These BPs, CCs, or MFs are guided by several signaling pathways closely related to immune regulation and migration and differentiation, including the PI3K-Akt signaling pathway, mTOR signaling pathway, TNF signaling pathway, and HIF-1 signaling pathway. Figure 12 f). Among these pathways, the PI3K-AKT signaling pathway involved in tumor metastasis is significantly affected by TCM-NBs@PRO. Figure 12 The results showed that the TCM-NBs@PRO treatment group was involved in the regulation of metastasis-related genes, including upregulation of genes such as Cdkn1a, Ppp2r2a, Foxo3, and Pik3r5, and downregulation of genes such as Igf1, Irs1, and Egf. These results indicate that TCM-NBs@PRO combined with ultrasound can modulate tumor metastasis-related signaling pathways and restore immune surveillance in multiple dimensions to inhibit the occurrence of tumor metastasis.

[0092] Example 6: Study on the effect of TCM-NBs@PRO in inhibiting tumor recurrence The foregoing examples have demonstrated that TCM-NBs@PRO combined with ultrasound therapy can effectively activate dendritic cells and T lymphocytes in vivo. This example further evaluates the protective effect of TCM-NBs@PRO+US treatment against tumor recurrence and assesses the impact of TCM-NBs@PRO+US treatment on immune memory in colorectal cancer. The experimental procedure is as follows: Figure 13As shown in Figure a, the primary tumor was surgically removed on day 16 after treatment. Sixty days later, CT26 tumor cells were injected again to verify its immune memory capacity. Changes in tumor volume and body weight in mice were monitored throughout the experiment. Figure 13 bc. There was no significant difference in weight between the two groups. Figure 13 d). Compared with the control group, tumor growth in the TCM-NBs@PRO+US treatment group was significantly inhibited, with an inhibition rate of 100%. The above results confirm that TCM-NBs@PRO+US can specifically enhance the inhibitory effect on tumor recurrence.

[0093] Before the second injection of CT26 tumor cells, spleens and inguinal lymph nodes were collected from mice in the TCM-NBs@PRO+US group and the control group to assess changes in naive T cells, central memory T cells (TCM), and effector memory T cells (TEM). + TEM can rapidly kill tumor cells and provide immediate protection against tumors. In addition, CD8... + TCMs require further differentiation to acquire cytotoxic capabilities, exhibiting greater proliferative potential and survival, strong resistance to apoptosis, sustained delivery of TEMs to the response site, and a more durable immune response. Flow cytometry shows ( Figure 13 ej), compared with the control group, CD4 levels in the spleen and lymph nodes of mice treated with TCM-NBs@PRO+US were significantly higher. + CD8 + An increased proportion of T lymphocytes was observed. Additionally, naïve CD8 cells were also observed. + T and CD4 + T lymphocytes to memory CD8 + T and CD4 + Significant transformation of T lymphocytes. CD8 + TCM and CD8 + The significantly increased proportion of TEM in the spleen indicates an enhanced immune memory effect. However, only CD8 was observed in the lymph nodes. + TCM ratio increased, CD8 + The TEM ratio did not change significantly. This may be due to CD8. + TEMs lack expression of lymphocyte homing receptors, while TCMs express high levels of lymphocyte homing receptors and migrate to secondary lymphoid organs, secreting large amounts of cytokine receptors. Upon re-encountering the same antigen, TCMs and TEMs rapidly exert their effector functions. In summary, TCM-NBs@PRO+US treatment induces large-scale expansion and differentiation of TEMs and TCMs, which then enter local tumor tissue via lymphatic vessels and blood vessels, ultimately exerting immune effector functions to inhibit tumor recurrence.

[0094] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A drug delivery system for ultrasound-enhanced synergistic immunity, characterized in that, It includes a lipid shell and fluorocarbon gas encapsulated within the lipid shell; the raw materials of the lipid shell include cancer cell membranes and lipid membranes formed from artificially synthesized phospholipids; the lipid shell integrates a protein degradation-targeting chimera for degrading PD-L1.

2. The ultrasound-enhanced synergistic immunotherapy drug delivery system according to claim 1, characterized in that, The mass ratio of the cancer cell membrane to the lipid membrane formed by artificially synthesized phospholipids is 1:9-9:1; preferably, the mass ratio is 9:

1.

3. The ultrasound-enhanced synergistic immunotherapy drug delivery system according to claim 1, characterized in that, The mass ratio of the cancer cell membrane to the protein degradation-targeting chimera for PD-L1 degradation is 40:1-2.5:1; preferably, the mass ratio is 5:

1. The mass ratio of the cancer cell membrane to the protein degradation-targeting chimera for PD-L1 degradation affects the particle size of the prepared nanobubbles.

4. The ultrasound-enhanced synergistic immunotherapy drug delivery system according to claim 1, characterized in that, The raw materials for the lipid membrane formed by artificially synthesized phospholipids include dipalmitoylphosphatidylcholine and distearate phosphatidylethanolamine-polyethylene glycol 2000 in a mass ratio of 1:9-9:

1. The lipid membrane formed by artificially synthesized phospholipids is obtained by dissolving dipalmitoylphosphatidylcholine and distearate phosphatidylethanolamine-polyethylene glycol 2000 in an organic solvent. After the organic solvent evaporates, the lipid membrane formed by artificially synthesized phospholipids is obtained.

5. A drug delivery system for ultrasound-enhanced synergistic immunity according to claim 1, characterized in that, The cancer cell membrane was provided by a mouse colon cancer cell line; the fluorocarbon gas was perfluoropropane; and the protein degradation targeting chimera used to degrade PD-L1 was PROTAC PD-L1 degrader-1.

6. A drug delivery system for ultrasound-enhanced synergistic immunization according to any one of claims 1-5, characterized in that, The average particle size of the drug delivery system is 412.7-1216.8 nm; preferably, the average particle size of the drug delivery system is 412.7 nm.

7. A drug delivery system for ultrasound-enhanced synergistic immunization according to any one of claims 1-5, characterized in that, It also includes an ultrasound device for applying ultrasound waves to a tumor site to which the drug delivery system is applied; the ultrasound device is used to provide ultrasound waves at 0.173-0.387 MPa.

8. A method for preparing an ultrasound-enhanced synergistic immunotherapy drug delivery system according to any one of claims 1-5, characterized in that, It includes the following steps performed sequentially: S1: The cancer cell membrane is mixed with a protein degradation targeting chimera for degrading PD-L1, and then incubated to obtain a drug-loaded cancer cell membrane; S2: Dispalmitoylphosphatidylcholine and distearate phosphatidylethanolamine-polyethylene glycol 2000 are dissolved in an organic solvent. After the organic solvent evaporates, a lipid membrane formed by artificially synthesized phospholipids is obtained. S3: The drug-loaded cancer cell membrane is mixed with a lipid membrane formed by hydrated synthetic phospholipids, and after ultrasonic treatment, a drug-loaded lipid suspension is obtained; the mass ratio of the cancer cell membrane to the lipid membrane formed by synthetic phospholipids is 1:9-9:

1. S4: Add the drug-loaded lipid suspension to the container, then seal the container, evacuate and inject fluorocarbon gas. After shaking, obtain TCM-NBs@PRO nanobubbles.

9. A method for preparing an ultrasound-enhanced synergistic immunotherapy drug delivery system according to claim 8, characterized in that, In S1, the cancer cell membrane is mixed with a protein degradation-targeting chimera for degrading PD-L1 at a mass ratio of 40:1 to 2.5:

1. In S2, the ratio of dipalmitoylphosphatidylcholine, distearate phosphatidylethanolamine-polyethylene glycol 2000 and organic solvent is 1-9 mg: 1-9 mg: 3-7 mL. In S3, the mass ratio of cancer cell membrane to lipid membrane formed by artificially synthesized phospholipids is 1:9-9:1; In S4, 0.5 mL of the drug-loaded lipid suspension is transferred to a 2 mL container. After sealing the container, a vacuum is drawn using a syringe until the syringe plunger can no longer be pulled, and the vacuum is maintained for 10-15 seconds. Then, excess perfluoropropane is added to the container.

10. The use of the ultrasound-enhanced synergistic immune drug delivery system according to any one of claims 1-5 in the preparation of immune activators, or in the preparation of drugs that inhibit tumor growth, or in the preparation of drugs that inhibit tumor metastasis, or in the preparation of drugs that inhibit tumor recurrence.

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