Mononuclear cell carrying type oxygen-producing liposome as well as preparation method and application thereof

Abstract

The invention relates to a mononuclear cell carrying type oxygen-producing liposome and a preparation method and application thereof.The preparation method comprises the steps that lipopeptide CP with cysteine at the tail end and lipid RL containing a thioketal structural domain are synthesized through a solid-phase polypeptide synthesis method, a thylakoid membrane TK is extracted, the lipid component and the lipopeptide CP are dissolved with an organic solvent and mixed to be uniform, and the monocyte carrying type oxygen-producing liposome is obtained. The preparation method comprises the following steps: dissolving lipid RL and STING agonist DA by using an organic solvent, uniformly mixing, and removing the organic solvent in the mixed solution; and adding the aqueous phase solution into the lipid membrane for hydration to form a crude liposome suspension, and fusing the thylakoid membrane TK onto the liposome by using an ultrasonic-extrusion method to obtain a liposome product. Compared with the prior art, the lipidosome prepared by the invention can efficiently deliver DA to the deep part of pancreatic cancer, activate multiple cell STING pathways and immune response, relieve hypoxia and improve immunosuppression caused by hypoxia, thereby enhancing the immunotherapy effect of DA.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a monocyte-carrying oxygen-producing liposome, a preparation method thereof, and an application thereof. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal digestive system tumor, accounting for over 90% of pancreatic tumors. Its global incidence and mortality rates are increasing annually. Surgery combined with systemic chemotherapy is currently the best treatment option for prolonging patient survival. However, over 80% of patients are diagnosed with advanced disease, making surgery inappropriate and the primary treatment option. Commonly used chemotherapy drugs only extend patient survival by a few months, with limited efficacy. Therefore, new therapies are urgently needed.

[0003] The core of immunotherapy is to stimulate the patient's anti-tumor adaptive immune response, use the patient's own immune system to eliminate cancer cells, and block their recurrence. The current clinical immunotherapy mainly focuses on breaking through the bottleneck of T cell function regulation, especially solving the problem of immunosuppression in the tumor microenvironment. However, clinically used immunotherapies such as immune checkpoint inhibitors (ICI) and adoptive cell transfer therapy (ACT) have not achieved ideal results in the treatment of pancreatic cancer. This is mainly because pancreatic cancer has an immunosuppressive microenvironment, and there is an extreme lack of cytotoxic CD8 in the pancreatic cancer tumor microenvironment. + T cell infiltration. Therefore, pancreatic cancer is a "cold tumor" that lacks T cell infiltration and activity. Among them, immunosuppressive cell populations, such as anti-inflammatory tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), are key factors that hinder T cell responses. Among them, TAMs are the non-tumor cells with the highest content in the pancreatic cancer tumor microenvironment, accounting for ~38% of the total infiltrating immune cells. These TAMs are mainly M2 type, which can inhibit T cell proliferation and inhibit the body's adaptive immunity, and are the main force of immunosuppression.

[0004] Stimulator of interferon genes (STING) agonists have attracted considerable attention for their superior immune-stimulating properties. Upon activation of the STING pathway, the secretion of type I interferons (IFNs) and proinflammatory cytokines promotes antigen-presenting cell maturation and T cell anti-tumor immune responses. Activation of the STING signaling pathway has been shown to reshape the immunosuppressive tumor microenvironment, promoting the infiltration of cytotoxic T cells in pancreatic cancer, and transforming pancreatic cancer from a "cold" to a "hot" tumor. However, STING agonists are mostly small molecules that are easily cleared by the kidneys after intravenous injection, have a short half-life in the blood, and lack sufficient drug accumulation in tumors, preventing them from achieving an effective activating dose. Furthermore, after systemic injection, STING agonists can indiscriminately activate the STING pathway in normal tissues, leading to the production of large amounts of proinflammatory cytokines, which can trigger inflammation, organ failure, and even death. Therefore, STING agonists are currently mostly administered via intratumoral injection. However, intratumoral injection is not practical for primary visceral tumors such as pancreatic cancer, or for metastatic tumors. Furthermore, the characteristics of pancreatic cancer (dense stroma, few blood vessels, and rapid cancer cell proliferation) result in insufficient oxygen supply and increased oxygen consumption, making it an extremely hypoxic tumor. Hypoxia inhibits the STING pathway and immune cell activation. Activating the STING pathway, combined with alleviating hypoxia, has the potential to reverse the immunosuppressive microenvironment of pancreatic cancer and enable tumor immunotherapy.

[0005] Patent CN118662445A proposes a STING agonist carried by ginsenoside liposomes. Although it improves the stability and immune activation effect of the STING agonist to a certain extent, it mainly focuses on specific cancer types such as colorectal cancer, and does not fully consider how to break through the delivery barrier of dense matrix tumors such as pancreatic cancer. It does not solve the hypoxic environment problem of pancreatic cancer, which limits its application effect in the treatment of pancreatic cancer.

[0006] Typically, active small molecule drugs have a short half-life, making them difficult to effectively deliver to tumors. This requires specialized nanocarriers for effective delivery. However, pancreatic cancer presents unique delivery barriers that severely restrict nanocarrier delivery. First, pancreatic cancer cells possess a dense fibrous stroma composed of fibroblasts, immune cells, and collagen, which occupies >90% of the tumor volume. This fibrous stroma compresses blood vessels, causing them to collapse, resulting in blood perfusion that is only one-third that of a normal pancreas. Studies have shown that only nanoparticles smaller than 50 nm can penetrate the pancreatic cancer tumor stroma barrier, while particles larger than 50 nm are mostly distributed around blood vessels and unable to reach deep into the tumor. Furthermore, vascular constriction and insufficient blood supply further limit the accumulation of conventional nanocarriers in pancreatic cancer. Therefore, to achieve effective delivery and regulatory effects of the STING agonist DA, it is necessary to develop intelligent nanodelivery systems that can penetrate the vascular and stroma barriers of pancreatic cancer cells and generate oxygen in situ. Summary of the Invention

[0007] The purpose of the present invention is to overcome the defects of the above-mentioned prior art and provide a monocyte-carrying oxygen-producing liposome and its preparation method and application, and provide a liposome CRM-Lips@DA that can target the delivery of 5,6-dimethylxanthenone-4-acetic acid (DA) and relieve hypoxia. The liposome can efficiently deliver DA deep into pancreatic cancer, activate the STING pathway and immune response of multiple cells, and at the same time, relieve hypoxia and improve the immunosuppression caused by hypoxia, thereby enhancing the immunotherapeutic effect of DA.

[0008] During the conception of this invention, it was believed that pancreatic cancer tissue is highly infiltrated by TAMs, with M2 TAMs predominating. These TAMs promote tumor growth by releasing growth factors and suppressing immune surveillance. TAMs are primarily derived from peripheral monocytes and are continuously recruited deep into the tumor, driven by inflammatory factors such as CCL2, CCL5, and CSF-1. Therefore, designing a nano-drug delivery system that can "hitch a ride" on monocytes has the potential to overcome the blood supply and matrix barriers of pancreatic cancer and deliver drugs deep into the tumor.

[0009] During the conception of the present invention, it was believed that monocytes are the main phagocytes in the blood. Studies have shown that cationic liposomes with a charge of 30-40mV can specifically target monocytes. After entering the cell, the cationic liposomes are degraded through the endosomal / lysosomal pathway. To avoid lysosomal degradation, the surface can be modified with L-cysteine to allow the nanoparticles to enter the endosomal-Golgi apparatus-endoplasmic reticulum pathway, thereby avoiding the action of the lysosomes. After the cationic liposomes enter deep into the tumor, they need to release the drug to the extracellular space before they can be taken up by tumor cells and myeloid cells. Studies have shown that after monocytes migrate to tumor tissue, they will differentiate into TAMs under the action of Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) or Macrophage Colony Stimulating Factor (M-CSF), at which time the ROS concentration increases rapidly. ROS has been shown to specifically cleave thioketal structures. Therefore, incorporating a thioketal domain into liposomes is expected to achieve ROS-responsive tumor-site selective drug release and rapid exocytosis. Furthermore, the hypoxic microenvironment of pancreatic cancer is rich in ROS. Fusion of a catalase-containing TK membrane with liposomes can decompose ROS to produce oxygen, alleviating immunosuppression caused by tumor hypoxia and improving the efficacy of immunotherapy.

[0010] Based on the above considerations, the present invention provides a liposome CRM-Lips@DA that can "ride" on monocytes in the body to achieve deep tumor STING agonist delivery and hypoxia relief. The liposome is loaded with STING agonist DA, and the main lipid components include DOTAP, cholesterol, soy lecithin, DSPE-PEG2000, lipopeptide CP (terminal cysteine, targeting the Golgi apparatus) and RL (containing thioketal domain, ROS response cleavage), and is fused with TK membrane (containing catalase, which can decompose ROS and produce oxygen). The liposome CRM-Lips@DA described in the present invention can target monocytes in the blood and enter the endosome-Golgi apparatus-endoplasmic reticulum pathway, escaping lysosomal degradation, so that it can "ride" monocytes into the deep tumor. Under the stimulation of the rapidly elevated ROS in the tumor microenvironment, the liposomes rapidly release DA, which in turn promotes its exocytosis to the extracellular space. Utilized by various immune-related cells, this activates the STING pathway in multiple cells within the tumor, repolarizing TAMs to the M1 type and promoting the maturation of DCs. This in turn enhances the infiltration and activity of cytotoxic T cells within the tumor, activating the adaptive immune response. Simultaneously, CRM-Lips@DA, retained within monocytes, utilizes the TK membrane to catalyze ROS to produce oxygen, improving tumor hypoxia and thereby alleviating hypoxia-induced immunosuppression and promoting the immune-activating effects of the STING agonist DA.

[0011] The purpose of the present invention can be achieved by the following technical solutions:

[0012] The first aspect of the present invention provides a method for preparing monocyte-carrying oxygen-producing liposomes, comprising the following steps:

[0013] S1: Synthesize lipopeptide CP with a terminal cysteine and lipid RL containing a thioketal domain by solid-phase peptide synthesis, and extract thylakoid membrane TK, which is a biological membrane structure capable of catalyzing ROS oxygen production;

[0014] S2: The lipid component and lipopeptide CP are dissolved and mixed uniformly in a first organic solvent, and the lipid RL and STING agonist DA are dissolved and mixed uniformly in a second organic solvent. The two solutions are mixed uniformly to obtain a mixed solution, and then the organic solvent in the mixed solution is removed by a thin film hydration method to form a uniform lipid film;

[0015] S3: adding an aqueous solution to the lipid film for hydration to form a crude liposome suspension, which is then sonicated to reduce the liposome size, thereby obtaining a preliminary liposome structure, CR-Lips@DA, capable of encapsulating the STING agonist DA.

[0016] S4: The thylakoid membrane TK was fused to the liposome CR-Lips@DA using the ultrasound-extrusion method to obtain the liposome product CRM-Lips@DA, which has monocyte targeting, can respond to ROS to release drugs, and has oxygen-producing function.

[0017] Furthermore, in S1, the amino acid sequence of the lipopeptide CP is SEQ ID NO.1;

[0018] Specifically, it is Fmoc-K(Fmoc)-HHG-acp-GRSSRSSRSSSC.

[0019] The amino acid sequence of the lipid RL is SEQ ID NO.2.

[0020] Specifically, it is Fmoc-K(Fmoc)-GGG-TK-PEG6.

[0021] Furthermore, in S1, the specific process of synthesizing the lipopeptide CP with a terminal cysteine and the lipid RL containing a thioketal domain by solid phase peptide synthesis includes:

[0022] The Fmoc-protected amino acids are sequentially linked to the solid support, and the Fmoc protecting groups of the amino acids are removed one by one using a deprotection reagent. The next amino acid is then added in sequence, and the operation is repeated until the sequence construction of the target lipopeptide CP and lipid RL is completed;

[0023] Introducing cysteine at the synthetic end of the lipopeptide CP and correctly connecting the cysteine to the constructed amino acid sequence;

[0024] During the synthesis of lipid RL, the raw material TK (thioketal domain) is directly put into the reaction;

[0025] After the synthesis is completed, the lipopeptide CP and lipid RL are cut off from the solid phase support using a cutting reagent and purified to remove unreacted reagents and by-products, ultimately obtaining a lipopeptide CP with a cysteine terminal and a lipid RL containing a thioketal domain.

[0026] Furthermore, in S1, the specific process of extracting thylakoid membrane TK includes:

[0027] Place the spinach leaves in pre-cooled HEPES buffer, fully homogenize in a mortar, filter and collect the filtrate, centrifuge the filtrate, collect the intact chloroplasts in the supernatant, resuspend the chloroplasts in hypotonic buffer, leave for a preset time, and then centrifuge to remove the matrix extract to obtain particles containing thylakoids. Wash the precipitate with HEPES buffer, ultrasonicate, and centrifuge to remove the lumen extract to obtain green thylakoid membrane TK.

[0028] Furthermore, in S2, the specific preparation process of the mixed solution includes:

[0029] The lipid components containing DOTAP, cholesterol, soybean lecithin, DSPE-PEG2000 and lipopeptide CP were added to the chloroform solvent, and the mixture was fully dissolved and mixed under vortexing to form a uniform lipid-lipopeptide CP chloroform solution;

[0030] After accurately weighing lipid RL and STING agonist DA, add them to methanol solvent and vortex to fully dissolve and mix them evenly to form a uniform lipid RL-DA methanol solution;

[0031] The prepared lipid-lipopeptide CP chloroform solution and lipid RL-DA methanol solution were mixed and vortexed on a vortex shaker to fully mix them and ensure full contact between the components to form a uniform mixed solution;

[0032] The molar ratio of DOTAP, cholesterol, soybean lecithin, DSPE-PEG2000, lipopeptide CP, and lipid RL is 40-60:15-25:10-20:1-3:1-10:5-15;

[0033] The mass ratio of the STING agonist DA to the lipid component is 1-30:170.

[0034] Furthermore, in S2, the specific process of removing the organic solvent in the mixed solution by thin film hydration method to form a uniform lipid film includes:

[0035] The uniformly mixed solution was transferred to a container, placed on a rotary evaporator, and subjected to rotary evaporation at 38° C. and a vacuum degree of -0.1 MPa to evaporate and remove the organic solvent, and the solution gradually formed a uniform lipid film.

[0036] Furthermore, in S3, an aqueous solution is added to the lipid film for hydration to form a crude liposome suspension, and the crude liposome suspension is subjected to ultrasonic treatment to reduce the size of the liposomes. The specific process includes:

[0037] Slowly add an appropriate amount of pure water to the lipid film formed after removing the organic solvent by the thin film hydration method, shaking while adding, so that the lipid film is fully exposed to water and hydrated, gradually forming a coarse liposome suspension;

[0038] The obtained crude liposome suspension was placed in an ice bath and ultrasonically treated using a cell disruptor to uniformly disperse the liposome particles and reduce their size, ultimately obtaining a liposome CR-Lips@DA suspension with relatively uniform particle size.

[0039] Furthermore, in S4, the specific process of fusing the thylakoid membrane TK to the liposome CR-Lips@DA using the ultrasound-extrusion method includes:

[0040] The extracted thylakoid membrane TK and liposome CR-Lips@DA were placed in the same solution and mixed evenly. The mixture was placed in an ice bath and preliminarily treated with ultrasound to allow initial contact and fusion of the TK membrane and liposome. The mixture was then extruded through a polycarbonate membrane with a pore size of 400 nm. The mixture was forced through the membrane pores under pressure to promote the fusion of the TK membrane and liposome. The mixture was then extruded again through a polycarbonate membrane with a pore size of 200 nm to obtain the final liposome product CRM-Lips@DA with uniform particle size, monocyte targeting, the ability to release drugs in response to ROS, and the ability to produce oxygen.

[0041] The mass ratio of the liposome CR-Lips@DA to the thylakoid membrane TK is 1 to 10:1.

[0042] The second aspect of the present invention provides a monocyte-carrying oxygen-producing liposome prepared by the above method, wherein the particle size of the liposome CRM-Lips@DA is 100-200 nm and the surface potential is 30-40 mV;

[0043] The liposome CRM-Lips@DA has an encapsulation efficiency of greater than 90% for the STING agonist DA;

[0044] The liposome CRM-Lips@DA can rapidly release the drug DA under ROS stimulation and catalyze ROS to produce oxygen through the thylakoid membrane TK.

[0045] The third aspect of the present invention provides an application of the above-mentioned liposome CR-Lips@DA, which is used to prepare drugs for treating tumors. The liposome CRM-Lips@DA can activate the STING pathway in the tumor by targeted delivery of the STING agonist DA deep into the tumor, repolarize M2 macrophages to M1, promote dendritic cell maturation and cytotoxic T cell infiltration, thereby enhancing the anti-tumor immune response, and relieve tumor hypoxia by producing oxygen, reversing the immunosuppressive tumor microenvironment, and inhibiting tumor growth and metastasis.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The CRM-Lips@DA liposomes prepared by the present invention are selectively taken up by monocytes in the blood, and under the action of surface cysteine, enter the endosome-Golgi apparatus-endoplasmic reticulum pathway, avoiding lysosomal degradation; CRM-Lips@DA liposomes "ride" on monocytes, overcome the blood supply and matrix barriers of pancreatic cancer, and enter deep into the tumor; after entering the tumor microenvironment, the ROS concentration in monocytes increases sharply, destroying the liposome structure and triggering DA release; the released DA is rapidly excreted to the extracellular space by monocytes, and the DA with transmembrane ability is taken up by immune-related cells in the tumor, activating the intracellular STING pathway; the CRM-Lips@DA liposomes retained in monocytes contain TK membranes, which can catalyze H2O2, produce oxygen, reverse the immunosuppressive effects caused by hypoxia, and synergistically activate the STING pathway. By efficiently delivering DA to various immune-related cells within pancreatic cancer and alleviating immunosuppression through in situ oxygen production, CRM-Lips@DA polarizes tumor-promoting M2 macrophages to M1 macrophages, promoting DC maturation and antigen presentation, and regulating pancreatic cancer from a "cold tumor" to a "hot tumor." Ultimately, it promotes T cell activation and intratumoral infiltration, effectively inhibiting pancreatic cancer growth and metastasis. The CRM-Lips@DA provided by this invention provides a new alternative immunotherapy option for pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 .Schematic diagram of the in vivo delivery process and efficacy of CRM-Lips@DA.

[0049] Figure 2.Characterization of CRM-Lips@DA; 2A: Flow chart of liposome preparation; 2B: Physical mixing of DiI-labeled CR-Lips and self-fluorescent TK membrane and fluorescence image after extrusion; 2C: Fluorescence spectra (left) and UV absorption spectra (right) of TK membrane, CR-Lips (without TK membrane) and CRM-Lips@DA; 2D: Liposome particle size and zeta potential diagram; 2E: Transmission electron microscopy image of liposomes; 2F: In vitro stability of liposomes; 2G: Oxygen production capacity of liposomes; 2H: Drug release curve of liposomes.

[0050] Figure 3 .In vitro delivery of CRM-Lips@DA; 3A: Monocyte targeting of liposomes; 3B: Golgi targeting of liposomes; 3C: Investigation of cell exocytosis in response to ROS by liposomes.

[0051] Figure 4 .CRM-Lips@DA regulation in vitro; 4A-B: M2 polarization rate of macrophages; 4C: Evaluation of oxygen production of liposomes in macrophages; 4D: Macrophage repolarization effect of liposomes; 4E: Activation effect of liposomes on the STING pathway of macrophages; 4F: Activation effect of liposomes on the STING pathway of tumor cells.

[0052] Figure 5 .In vivo delivery and distribution of CRM-Lips@DA; 5A: Pharmacokinetic curve of liposomes; 5B: Pharmacokinetic parameters of liposomes; 5C: Tumor tissue accumulation of DiD-labeled liposomes; 5D: Tumor distribution of DiD-labeled liposomes; 5E: Tissue distribution of C6-labeled liposomes; 5F: Intratumoral fluorescence distribution of C6-labeled liposomes.

[0053] Figure 6-7 CRM-Lips@DA's tumor-modulating effects. 6A: Mouse treatment protocol; 6B: Intratumoral oxygen production assessment; 6C: Evaluation of tumor STING pathway activation; 6D-E: In vivo macrophage regulation (M1 and M2 macrophages); 7A-B: In vivo immune cell regulation (DCs); 7C-D (T cells).

[0054] Figure 8 Anti-tumor effect of CRM-Lips@DA. 8A: Mouse treatment schedule; 8B: Bioluminescence images of mouse tumors during treatment cycles; 8C: Quantitative curve of bioluminescence intensity of mouse tumors; 8D: Representative images of tumors removed after treatment; 8E: Ex vivo imaging of major organs of mice in each treatment group; 8F: Representative H&E staining images of tumor tissues after treatment; 8G / I: Images and quantification of Ki67 staining of tumor tissues; 8H / J: Fluorescence images of TUNEL staining of tumor tissues and quantification of apoptosis rates.

[0055] Figure 9 Safety evaluation of CRM-Lips@DA. 9A: Expression of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) cytokines in the blood of healthy mice after a single dose; 9B: Figure 9 Treatment plan, changes in mouse body weight during treatment; 9C: Figure 9 Blood biochemical analysis of mice after treatment; 9D: Figure 8 Routine blood analysis of mice after treatment; 9E: Representative H&E staining images of major organs of mice after treatment. DETAILED DESCRIPTION

[0056] Overall, the present invention specifically designs a liposome-based drug, CRM-Lips@DA, for delivering the interferon gene stimulator (STING) agonist 5,6-dimethylxanthenone-4-acetic acid (DA) and hypoxia relief. A cysteine-terminated lipopeptide (CP) and a thioketal domain-containing lipid (RL) were synthesized via solid-phase peptide synthesis. Thylakoid (TK) membranes were extracted, and CR-Lips@DA liposomes were prepared via thin-film hydration. The TK membranes were then fused to the liposomes using ultrasound / extrusion to yield CRM-Lips@DA. With a zeta potential of 31.4 mV, the liposomes are preferentially taken up by monocytes in the blood and "ride" on them deep into tumors. Driven by surface cysteine, they enter the endosome-Golgi apparatus-endoplasmic reticulum pathway, avoiding lysosomal degradation. Stimulated by reactive oxygen species (ROS), they achieve efficient intratumoral delivery of the STING agonist and in situ oxygen production, reshaping pancreatic cancer into a "hot tumor" and activating the adaptive immune response, effectively inhibiting pancreatic cancer growth and metastasis.

[0057] In specific implementation, the liposome CRM-Lips@DA can "ride" on monocytes in the body to achieve deep tumor delivery of the interferon gene stimulator (STING) agonist 5,6-dimethylxanthenone-4-acetic acid (DA) and hypoxia relief. Its characteristics are that the liposome can be targeted and taken up by monocytes in the blood and carried deep into the tumor by monocytes; under the stimulation of reactive oxygen species (ROS), on the one hand, it can quickly release DA, promote drug exocytosis, and be taken up by various immune-related cells to exert an immune activation effect; on the other hand, the TK membrane can catalyze ROS to produce oxygen, improve the immunosuppression caused by hypoxia, reshape pancreatic cancer into a "hot tumor", and activate the adaptive immune response.

[0058] In a specific implementation, the particle size of the liposome CRM-Lips@DA is 100-200 nm, and the surface potential is 30-40 mV.

[0059] In a specific implementation, the encapsulation efficiency of the liposome CRM-Lips@DA for DA is greater than 90%.

[0060] In specific implementation, the preparation method of the liposome CRM-Lips@DA is as follows: Figure 1 , including the following steps:

[0061] 1) Synthesize lipopeptide CP and lipid RL by solid-phase peptide synthesis;

[0062] 2) Extraction of TK membrane;

[0063] 3) The lipid components and lipopeptide CP were dissolved in chloroform, and the lipid RL and STING agonist DA were dissolved in methanol, and CR-Lips@DA was prepared by thin film hydration method;

[0064] 4) The TK membrane was fused with CR-Lips@DA by ultrasound / extrusion method to obtain CRM-Lips@DA.

[0065] 5) The preparation method according to claim 4, characterized in that in step 1, the lipopeptide CP is (Fmoc-K(Fmoc)-HHG-acp-GRSSRSSRSSSC); the lipid RL is Fmoc-K(Fmoc)-GGG-TK-PEG6. Fmoc is a fluorenylmethyloxycarbonyl protecting group used to protect the amino groups of subsequent amino acids; K(Fmoc) is Fmoc-protected lysine (K); histidine (H); glycine (G); 6-aminocaproic acid (acp); arginine (R); serine (S); cysteine (C); thioketal (TK); and hexaethylene glycol (PEG6).

[0066] Specifically, the TK membrane extraction process in step 2 includes homogenizing the spinach leaves in a mortar with pre-chilled HEPES buffer, filtering and collecting the homogenate. The intact chloroplasts are then collected by centrifugation at 8,000 × g for 10 minutes. The chloroplasts are then resuspended in hypotonic buffer (10 mM HEPES, pH 8.00) for 2 hours and centrifuged at 12,000 × g for 30 minutes to remove the stromal extract, thereby obtaining thylakoid-containing particles. The particles are then washed with 10 mM HEPES buffer, sonicated at 4°C for 15 minutes, and centrifuged at 15,000 × g for 15 minutes to remove the lumen extract, resulting in the green TK membrane.

[0067] In a specific implementation, the molar ratio of DOTAP, cholesterol, soybean lecithin, DSPE-PEG2000, CP, and RL in the lipid components in step (3) is 40-60:15-25:10-20:1-3:1-10:5-15.

[0068] In a specific implementation, it is preferred that the molar ratio of DOTAP, cholesterol, soybean lecithin, DSPE-PEG2000, CP, and RL in the lipid components is 50:20:13:2:5:10.

[0069] In a specific implementation, the mass ratio of the drug DA to the lipid component in step (3) is 1-30:170.

[0070] In a specific implementation, it is preferred that the mass ratio of the drug DA to the lipid component is 0.05:1.

[0071] In a specific implementation, the liposome preparation process in step (3) includes: dissolving each lipid component DOTAP, cholesterol, soybean lecithin, DSPE-PEG2000, CP, RL, and the drug DA in chloroform / methanol, mixing them, and removing the organic solvent using a rotary evaporator to obtain a lipid film. Pure water is added for hydration, and the resulting crude liposome suspension is sonicated to reduce its size to obtain liposome CR-Lips@DA.

[0072] In a specific implementation, the mass ratio of the lipids of CR-Lips@DA and the TK membrane fusion in step (4) is 1 to 10:1.

[0073] In a specific implementation, it is preferred that the mass ratio of the lipids of the CR-Lips@DA to the TK membrane fusion is 4:1.

[0074] In specific implementation, the preparation process of liposomes in step (4) includes: adding TK membrane to CR-Lips@DA, mixing, and subjecting the suspension to ultrasonic and extrusion treatment to obtain CRM-Lips@DA liposomes with uniform particle size.

[0075] In specific implementation, the application of liposome CRM-Lips@DA in the preparation of drugs for treating diseases can target monocytes in the blood, achieve Golgi apparatus targeting, avoid lysosome-related pathway degradation, and have ROS-responsive drug release and cell exocytosis.

[0076] In specific implementation, liposome CRM-Lips@DA can be internalized by monocytes and enter the endosome-Golgi apparatus-endoplasmic reticulum pathway, avoiding lysosomal degradation, thereby "riding" monocytes across the pancreatic cancer delivery barrier.

[0077] In specific implementation, the drug DA encapsulated in the liposome CRM-Lips@DA can repolarize M2 macrophages to M1 type. At the same time, after being exocytosed to the extracellular space, it effectively activates the STING pathway and related immune responses of tumor cells and dendritic cells.

[0078] In specific implementation, immune "cold tumors" can be reshaped into "hot tumors".

[0079] In a specific implementation, the liposome CRM-Lips@DA can repolarize M2 macrophages to M1 macrophages. At the same time, the exocytotic DA can activate the STING pathway of dendritic cells and tumor cells.

[0080] In specific implementation, the liposome CRM-Lips@DA can produce oxygen in a ROS-enriched environment and alleviate immunosuppression caused by hypoxia.

[0081] In specific implementation, the liposome CRM-Lips@DA can increase the intratumoral CD8+ T cell infiltration and activity.

[0082] In specific implementation, CRM-Lips@DA combined with PD-1 antibody showed good anti-tumor effect, significantly inhibiting tumor growth and metastasis in pancreatic cancer-bearing mice.

[0083] During specific implementation, the liposome has good biocompatibility and safety.

[0084] In practice, the liposome CRM-Lips@DA exhibited negligible immune, systemic, blood, and organ toxicity.

[0085] In specific implementation, liposome CRM-Lips@DA can also be used to deliver other immune activators, including cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), imiquimod, and resiquimod.

[0086] In specific implementation, liposome CRM-Lips@DA is suitable for immunotherapy of tumors other than pancreatic cancer.

[0087] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. Any features, such as preparation methods, materials, biological components, structures, and composition ratios, not explicitly described in this technical solution are considered to be common technical features disclosed in the prior art. The various reagents and raw materials used in the present invention are all commercially available products or products that can be prepared by known methods.

[0088] Example 1

[0089] Lipopeptide CP Synthesis: First, select a 2-CL resin and employ the FMOC solid-phase synthesis method. The synthesis yield is 1.5 mmol. Amino acids are represented by FMOC-AAn-OH (where n is the amino acid position number at the C-terminus of the polypeptide chain). Then, starting from the C-terminus of the peptide, FMOC-AA1-OH is dehydrated and condensed with the resin using the condensation reagent DIEA and the solvent DCM to obtain FMOC-AA1-2-CL resin. Next, the FMOC group is removed using a DMF solution containing 20% piperidine for 20 minutes. After removal, the resin is washed with DMF four times for 1 minute each to obtain NH2-AA1-2-CL resin. Next, FMOC-AA2-OH is dehydrated and condensed with the resin using the condensation reagents DIC and HOBT in DMF as the solvent to obtain FMOC-AA2-AA1-2-CL resin. Repeat steps 3 and 4 above to sequentially condense the remaining amino acids until peptide synthesis is complete. Finally, the peptide was cleaved using a lysate containing 95% TFA, 1% H2O, 2% EDT, and 2% TIS for 2 hours. After cleavage, the lysate was filtered into ice ether and centrifuged to obtain a crude product, which was then separated and prepared by HPLC using an acetonitrile gradient of 15-40% for 40 minutes. Finally, a sample with an HPLC analysis purity greater than 95% was obtained and lyophilized. The sequence is Fmoc-K(Fmoc)-HHG-acp-GRSSRSSRSSSC.

[0090] Lipid RL synthesis: 2-CL resin was selected, with a synthesis amount of 1.5 mmol. Amino acids were represented by FMOC-AAn-OH (n represents the amino acid position from the C-terminus). Synthesis began at the C-terminus of the peptide. FMOC-AA1-OH was dehydrated and condensed with the resin using the condensation reagent DIEA / DCM as the solvent to produce FMOC-AA1-2-CL resin. FMOC was then removed using a DMF solution containing 20% piperidine for 20 minutes. After removal, the resin was washed with DMF four times for 1 minute each to produce NH2-AA1-2-CL resin. Next, FMOC-AA2-OH was condensed with the resin using DIC and HOBT to produce FMOC-AA2-AA1-2-CL resin. This process was repeated for the remaining amino acids. Finally, the peptide was cleaved using a 20% TFE / 80% DCM solution for 2 hours. After cleavage, the lysate was rotary evaporated and subsequently purified by HPLC using a 15-40% acetonitrile gradient over 40 minutes. A peptide sample with a purity greater than 95% was obtained and lyophilized. The sequence was Fmoc-K(Fmoc)-GGG-TK-PEG6. Purification was performed by preparative HPLC chromatography, and successful synthesis was confirmed by HPLC-MS.

[0091] TK membrane extraction: Homogenize 100 g of spinach leaves in pre-chilled HEPES buffer in a mortar and pestle, filter, and collect the filtrate. Centrifuge at 8,000 × g for 10 minutes to collect intact chloroplasts. Resuspend them in hypotonic buffer (10 mM HEPES, pH 8.0) for 2 hours and centrifuge at 12,000 × g for 30 minutes to remove the stromal extract and obtain thylakoid-containing particles. Wash with 10 mM HEPES buffer, sonicate at 4°C for 15 minutes, and centrifuge at 15,000 × g for 15 minutes to remove the lumen extract, resulting in green TK membranes. Successful TK membrane extraction was confirmed by UV and fluorescence spectroscopy, and TK membrane concentration was determined using a BCA protein quantification kit.

[0092] Liposome synthesis: DOTAP, cholesterol, soy lecithin, DSPE-PEG2000, and CP were dissolved in chloroform, while RL and DA were dissolved in methanol. The lipid components DOTAP, cholesterol, soy lecithin, DSPE-PEG2000, CP, RL, and the drug DA were added to a 50 mL eggplant-shaped flask and mixed thoroughly. The molar ratio of DOTAP, cholesterol, soy lecithin, DSPE-PEG2000, CP, and RL was 50:20:13:2:5:10, and the mass ratio of drug DA to lipid components was 0.05:1. Organic solvents, including chloroform and methanol, were removed by rotary evaporation under vacuum at 38°C to obtain a lipid film. Pure water was added and hydrated for 30 minutes. The resulting crude liposome suspension was collected in a centrifuge tube and sonicated five times in a cell disruptor while in an ice bath to obtain CR-Lips@DA liposomes. The TK membrane was added at a TK:liposome mass ratio of 1:4, placed on ice, and sonicated intermittently for 2 minutes using a cell ultrasonic disruptor. Finally, the membrane was extruded using polycarbonate membranes with pore sizes of 400 nm and 200 nm, respectively, to obtain CRM-Lips@DA liposomes with uniform particle size. Finally, glucose solution was used to adjust the liposome solution to a physiological isotonic solution ( Figure 2 A).

[0093] When only physically mixed, DiI (cell membrane red fluorescent probe) labeled CR-Lips (red) and TK membrane (green) basically do not overlap; in contrast, after sonication / extrusion, the red fluorescence on CR-Lips and the green fluorescence of TK membrane mostly overlap, showing yellow fluorescence, indicating that the TK membrane has been successfully hybridized to the liposomes ( Figure 2 B).

[0094] Comparison of the infrared absorption and fluorescence spectra of TK membrane alone, CR-Lips (without TK membrane), and CRM-Lips@DA showed that CRM-Lips@DA had the same characteristic peaks as TK membrane, while CR-Lips had no corresponding peaks, indicating that TK membrane and liposomes were successfully fused ( Figure 2 C).

[0095] The average particle size of CM-Lips is 169.6 nm, and the average particle size of CRM-Lips@DA liposomes is 169.8 nm, indicating that the addition of RL has no effect on the liposome particle size at this ratio ( Figure 2 D left). The average potential of CRM-Lips@DA is 31.4 mV, indicating that CRM-Lips@DA has potential for monocyte targeting ( Figure 2 D right).

[0096] Since the liposomes contain RL (thioketal domain, which can be specifically destroyed by ROS) lipids, transmission electron microscopy analysis was performed on CRM-Lips@DA and CRM-Lips@DA after incubation with ROS. The results showed that CRM-Lips@DA has a clear spherical morphology ( Figure 2 E left), while the liposome structure was cleaved after incubation with ROS, losing its spherical shape ( Figure 2 E right), indicating that ROS can destroy the liposome structure.

[0097] In order to explore the serum stability of CRM-Lips@DA, its fluorescence spectrum changes were measured by the fluorescence resonance energy transfer (FRET) method. The fluorescent probes DiI and DiO (molar ratio 1:1) were simultaneously encapsulated in CRM-Lips@DA and incubated in PBS containing 10% fetal bovine serum (FBS). The changes in fluorescence intensity at different times were detected by a microplate reader (λex / λem=450 / 480-725nm). After incubation for 12 hours, the fluorescence spectrum of CRM-Lips@DA hardly changed, and a significant FRET effect could still be observed. However, after the addition of acetonitrile (ACN) to destroy the liposome structure, DiI showed an obvious emission peak at 480nm, that is, the FRET effect was weakened, indicating that the liposome structure of CRM-Lips@DA was destroyed. Therefore, these results show that CRM-Lips@DA maintains structural integrity in the blood for at least 12 hours and has good stability in the blood ( Figure 2 F).

[0098] CRM-Lips@DA containing TK membrane rapidly produced oxygen upon contact with H2O2, reaching ~2 mg / L within 5 minutes. However, CR-Lips@DA without TK membrane and the blank solvent group produced almost no oxygen. This result further demonstrates that the TK membrane has successfully fused with the liposomes and retained the oxygen production capacity of the TK membrane. Figure 2 G).

[0099] The release rate of CRM-Lips@DA in the release medium with H2O2 was significantly higher than that in the medium without H2O2. This result shows that CRM-Lips@DA can release drugs rapidly under the stimulation of ROS in vivo ( Figure 2 H).

[0100] Verification Example 1

[0101] Liposomes labeled with DiD (a far-infrared fluorescent probe for cell membranes, representing 1% of the lipid composition) were constructed using the aforementioned method. After incubation with fresh mouse whole blood (anticoagulated) at a 100 μM liposome concentration for 1 hour, DiD fluorescence intensity in monocytes and B cells was measured using a flow cytometer (CytoFLEX LX, Beckman, USA).

[0102] RAW 264.7 cells were seeded in 12-well plates and incubated overnight. RM-Lips@DiO labeled with DiO (a cell membrane green fluorescent probe) was added and incubated for 2 hours. After washing with PBS, Golgi-Tracker Red (150 μg / mL) and LysoTracker Red (50 nM) were added to the cells to stain the Golgi apparatus and lysosomes, respectively. The cells were incubated for 30 minutes, washed with PBS, and fixed with paraformaldehyde for 10 minutes. Cell nuclei were stained with DAPI, and anti-fluorescence quenching mounting medium was added. Colocalization of liposomes with the Golgi apparatus or lysosomes was observed using a laser confocal microscope (FV3000, Olympus, Japan).

[0103] RAW 264.7 cells were seeded in 12-well plates and incubated overnight. C6 (coumarin 6)-labeled liposomes CRM-Lips@C6 were prepared to facilitate observation of drug retention in cells. CRM-Lips@C6 and macrophages were incubated in a hypoxic incubator for 2 hours. After removing the liposomes, one group was added with culture medium containing inducing factors (IL-4, IL-10, and M-CSF) and ROS (1mM H2O2), while the other group was replaced with ordinary culture medium. Cells were subsequently collected at different time points (0, 2, 6, and 12 hours) and the intracellular C6 fluorescence intensity was measured using a flow cytometer (Cytoflex LX, 23Beckman, USA) to evaluate exocytosis.

[0104] Flow cytometry results showed that the uptake efficiency of CRM-Lips@DiD by monocytes was 6.06 times that of B cells ( Figure 3 A) shows that liposomes can be targeted and taken up by monocytes in mouse blood.

[0105] The distribution of CRM-Lips@DiO in the Golgi apparatus was significantly enhanced compared to RM-Lips@DiO, and correspondingly, the distribution in the lysosome was reduced ( Figure 3 B), demonstrating cysteine-dependent Golgi targeting of liposomes.

[0106] From the flow cytometry quantitative curve, it can be seen that the residual amount of C6 in the cells of the group with ROS addition (1mM H2O2 to simulate the tumor microenvironment) is 15.58%, while the amount of C6 in the control group without ROS addition is 35.54%, indicating that under the stimulation of ROS, liposomes can quickly release drugs, allowing cells to expel drugs out of the cell faster ( Figure 3 C).

[0107] Verification Example 2

[0108] To verify the in vitro regulatory effect of liposomes, RAW 264.7 macrophages were induced into the M2 phenotype using IL-4, IL-10, and M-CSF (1 μg / mL, ratio = 10:5:5). The expression of CD206 (a marker of M2 macrophages) was analyzed by flow cytometry (Cytoflex LX, Beckman, USA). Phalloidin was used to label the cytoskeleton, and cell morphology was observed under a laser confocal microscope (FV3000, Olympus, Japan) before and after induction.

[0109] RAW 264.7 cells were seeded into 12-well plates and induced to the M2 phenotype as described above. The cells were cultured overnight in a hypoxic incubator for 12 hours. The probe Ru(dpp)3]Cl2 (an oxygen-indicating probe, destructible by molecular oxygen) (1.5 μL, 6 mg / mL) was added to each well according to the grouping, mixed thoroughly, and then the corresponding liposomes were added. The cells were incubated in the hypoxic incubator for another 2 hours. After the incubation period, the probe fluorescence intensity was observed using a multimodal in vitro imager (λex / λem = 470 / 600 nm).

[0110] RAW 264.7 cells were seeded in 12-well plates and induced into the M2 phenotype using the three induction factors described above. PBS, free DA, Lips@DA, M-Lips@DA, CM-Lips@DA, and CRM-Lips@DA (final DA concentration: 10 μg / mL) were then added to the plates. The plates were incubated in a hypoxic incubator for 12 hours, and the cells were harvested and CD86 expression was determined by flow cytometry. RNA was extracted from each group and analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR).

[0111] To simulate the interaction between macrophages and tumor cells in vivo, a 0.4 μm Transwell plate was used for co-incubation experiments. RAW 264.7 cells were seeded in the upper chamber and induced into M2 macrophages as described above. PBS, free DA, Lips@DA, M-Lips@DA, CM-Lips@DA, and CRM-Lips@DA (DA final concentration: 10 μg / mL) were added and incubated in a hypoxic incubator for 2 hours. The drug-containing culture medium was then discarded and new culture medium was added. Panc02 pancreatic cancer cells were seeded in the lower chamber and cultured in a hypoxic incubator for 24 hours. Tumor cells in the lower chamber were collected, RNA was extracted, quantified, and reverse transcribed. RT-qPCR was used to detect the mRNA expression of the corresponding STING pathway (INF-β, CXCL10, and CCL5) to evaluate the effect of STING pathway activation in tumor cells.

[0112] Flow cytometry results showed that after incubation with three induction factors, IL-4, IL-10, and M-CSF (1 μg / mL, ratio = 10:5:5), the expression of CD206 in macrophages increased by 9.17 times compared with the uninduced group ( Figure 4 A). Confocal images show that the induced macrophages are spindle-shaped, which is consistent with the morphological characteristics of M2 macrophages, indicating that M2 macrophages were successfully induced ( Figure 4 B).

[0113] In vitro imaging results showed that the fluorescence intensity of the PBS+Ru(dpp)3]Cl2 and CR-Lips+Ru(dpp)3]Cl2 groups did not change significantly, while the fluorescence intensity of the CRM-Lips+Ru(dpp)3]Cl2 group was significantly reduced compared with the PBS+Ru(dpp)3]Cl2 group, indicating that the CRM-Lips liposomes fused with TK membrane can produce oxygen in an environment rich in ROS, thereby effectively alleviating hypoxia ( Figure 4 C).

[0114] The expression of M1 macrophage-related proteins (CD86) was detected by flow cytometry to evaluate the repolarization effect of the liposomes. Compared with the control group, the expression of CD86 (M1 macrophage marker) in macrophages in the CRM-Lips@DA treatment group was significantly increased, indicating that M2 macrophages were effectively repolarized to M1 ( Figure 4 D).

[0115] The expression of STING pathway-related factors (INF-β, CCL10, CCL5) in macrophages after treatment with different liposomes was detected by RT-qPCR. The results showed that the mRNA expression levels of STING pathway-related factors in the CRM-Lips@DA group were significantly increased compared with the control group, indicating that CRM-Lips@DA can effectively activate the STING pathway in macrophages ( Figure 4 E).

[0116] The expression of STING pathway-related factors (INF-β, CCL10, CCL5) in Panc02 pancreatic cancer cells co-incubated with macrophages using Transwell chambers was detected by RT-qPCR technology. The results showed that the CRM-Lips@DA group had the most significant activation effect on Panc02 pancreatic cancer cells, and the mRNA expression of STING pathway-related factors (INF-β, CCL10, CCL5) was significantly upregulated, indicating that after CRM-Lips@DA was taken up by macrophages, the structure was cleaved due to ROS stimulation, and a sufficient concentration of DA was exocytosed, thereby effectively activating the STING pathway of tumor cells ( Figure 4 F).

[0117] Verification Example 3

[0118] The delivery and biodistribution of CRM-Lips@DA liposomes in mice were investigated. First, the pharmacokinetic behavior of the liposomes in vivo was investigated in SD rats.

[0119] First, liposomes were labeled with DiI (a red fluorescent probe for the cell membrane). SD rats were randomly divided into four groups (n=3) and injected with RM-Lips@DiI, CM-Lips@DiI, CRM-Lip@DiI, and CLP (clodronate liposomes, which can eliminate monocytes / macrophages in the body) + CRM-Lips@DiI through the tail vein at a dose of 0.3 mg / kg DiI. The CLP+CRM-Lips@DiI group was intraperitoneally injected with CLP (concentration: 5 mg / mL; injection volume: 1 mL) 24 hours in advance to deplete macrophages in the rats. At the preset time point, blood was collected from the retinal venous plexus of the rats and centrifuged at 3500 rpm for 10 minutes to obtain plasma. The plasma DiI fluorescence intensity (λex / λem=549 / 565 nm) was detected using an enzyme-linked microplate reader. The DiI plasma concentration-time curve was drawn, and non-compartmental model analysis was performed using DAS2.0 software to calculate PK parameters.

[0120] The half-life of the CLP+CRM-Lips@DiI group was 19.376 h, which was significantly prolonged compared with 9.448 h of CRM-Lips@DiI, indicating that the liposomes were mainly taken up and cleared by monocytes in the blood. Figure 5AB).

[0121] Verification Example 4

[0122] The biodistribution of CRM-Lips@DA liposomes in mice was investigated. An orthotopic pancreatic cancer-bearing mouse model was established. Following tail vein injection of CRM-Lips@DiD or CRM-Lips@C6, the ability of the formulation to cross the pancreatic cancer delivery barrier and whether the liposomes could promote drug exocytosis in the ROS-rich tumor microenvironment were investigated.

[0123] Panc02-Luc cells were cultured at a rate of 5 × 10 6 / mL was suspended in a solution of PBS:Matrigel = 1:1, and 1×10 5 The cells were inoculated into the pancreatic tail of mice to establish an orthotopic pancreatic cancer tumor-bearing mouse model. This model mimics key pathological features of pancreatic cancer, such as hypoxia, lack of blood vessels, and dense fibrous stroma.

[0124] Ten days after modeling, mice were randomly divided into CM-Lips@DiD, CRM-Lips@DiD, and CLP+CRM-Lips@DiD groups (n=3). The CLP+CRM-Lips@DiD group received an intraperitoneal injection of CLP (concentration: 5 mg / mL; injection volume: 200 μL) 24 hours prior to treatment to deplete monocytes and macrophages. Four, 12, and 24 hours after tail vein administration, mice were euthanized, and tumors were harvested. The distribution of liposomes in tumors was observed using a small animal imaging system.

[0125] Tumor tissues collected 12 h after injection were embedded in Tissue-Tek OCT compound, quickly frozen, and cut into 5 μm sections. They were immunofluorescently stained for CD68 (a marker of macrophages), blocked with a fluorescent blocking agent containing DAPI, and observed under a laser confocal microscope (FV3000, Olympus, Japan).

[0126] Orthotopic pancreatic cancer-bearing mouse models were established as described above. Ten days after modeling, mice were randomly divided into CM-Lips@C6, CRM-Lips@C6, and CLP+CRM-Lips@C6 groups (n=3). Similarly, CLP+CRM-Lips@C6 was pre-injected with CLP (concentration: 5 mg / mL; injection volume: 200 μL) intraperitoneally to deplete macrophages in mice. Twelve hours after tail vein administration, mice were euthanized and tumors were removed. To observe intratumoral exocytosis of CRM-Lips@C6, tumor tissues were embedded in Tissue-Tek OCT composite, snap-frozen, and sectioned into 5 μm sections. Immunofluorescence staining for CD68 (a marker of macrophages) was performed, followed by blocking with a fluorescent blocker containing DAPI and observation under a laser confocal microscope (FV3000, Olympus, Japan).

[0127] The tumor targeting ability of liposomes was investigated by in vivo imaging. Tumor fluorescence images and corresponding quantitative results 4, 12, and 24 hours after tail vein injection showed that the intratumoral accumulation of CRM-Lips@DiD was significantly higher than that of the CLP+CRM-Lips@DiD group. This indicates that CRM-Lips@DiD can "ride" monocytes in the body into tumor tissue and has a stronger distribution in the tumor site ( Figure 5 C).

[0128] The co-localization of DiD (green) and CD68 (red) in tumor sections was analyzed. The results showed that CRM-Lips@DiD (green) co-localized well with macrophages (red) in the tumor site, and the tumor distribution was much higher than that of the CLP+CRM-Lips group, indicating that CRM-Lips can "ride" on monocytes, penetrate deep into pancreatic cancer tumors, and improve intratumoral accumulation ( Figure 5 D).

[0129] Because CRM-Lips@DA can only be further taken up and utilized by key immune-related cells in the tumor microenvironment after exocytosis of the STING agonist DA within the tumor. The tumor exocytosis of the drug can be determined by examining the co-localization of C6 (green) and CD68 (red). The fluorescence intensity of C6 (green) of CLP+CRM-Lips@C6 was significantly lower than that of the other two groups, indicating that the liposomes rely on monocytes to enter the tumor tissue; in the non-ROS-responsive CM-Lips@C6 liposome group, most of the C6 fluorescence signals co-localized with CD68-labeled TAMs, indicating a lower degree of C6 exocytosis; while in the ROS-responsive CRM-Lips@C6 liposome group, most of the C6 signals were distributed around CD68, indicating a large amount of exocytosis. These results indicate that after CRM-Lips@C6 "rides" monocytes to reach the tumor microenvironment, it can release C6 under ROS stimulation, thereby promoting the exocytosis of C6 from monocytes to the extracellular space and being utilized by other cells in the microenvironment ( Figure 5 EF).

[0130] Verification Example 5

[0131] Pancreatic cancer orthotopic tumor-bearing mouse models were established as described above. Mice were randomly divided into PBS, free DA, and CRM-Lips@DA (n=3). DA was administered 3 times via the tail vein at 2 mg / kg ( Figure 6 A) Tumors were removed, embedded in Tissue-Tek OCT compound, rapidly frozen, and sectioned into 7 μm sections. The sections were fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.2% Triton X-100 for 10 minutes, and blocked with goat serum for 1 hour. Immunofluorescence staining for HIF-1α was performed and observed using a laser confocal microscope (FV3000, Olympus, Japan).

[0132] An orthotopic pancreatic cancer tumor-bearing mouse model was established as described above. Mice were randomly divided into PBS, free DA, CRM-Lips@DA, PD-1 antibody, and CRM-Lips@DA + PD-1 antibody groups (n = 6). Each DA formulation was administered via the tail vein at a dose of 2 mg / kg DA on days 7, 11, and 15, and 5 mg / kg PD-1 antibody was administered intraperitoneally on days 8, 12, and 16. After three cycles of treatment, mice were sacrificed by cervical dislocation, and tumors were harvested. The mRNA expression of STING pathway-related cytokines (CXCL9, CCL5, CXCL1, CXCL10, and IFN-β) was analyzed by RT-qPCR.

[0133] An orthotopic pancreatic cancer tumor-bearing mouse model was established as described above. After treatment, the tumors were removed and grouped and stained according to M1 / M2 macrophages, DCs, and CD4 / 8 T cells. The distribution of various immune cell subsets within the tumor was analyzed by flow cytometry (CytoFLEX LX, Beckman, USA).

[0134] Pancreatic cancer is an extremely hypoxic tumor, and hypoxia inhibits the activation of the STING pathway and immune cells. HIF-1α is a factor closely related to tumor hypoxia. Compared with the control group and the free DA treatment group, CRM-Lips@DA significantly reduced the expression of HIF-1α in the tumor, indicating that CRM-Lips@DA can alleviate tumor hypoxia and is expected to improve the immunosuppression caused by hypoxia ( Figure 6 B).

[0135] Liposome CRM-Lips@DA can deliver the STING agonist DA to pancreatic cancer sites, so its activation effect on the intratumoral STING pathway was investigated. RT-qPCR results showed that after the treatment cycle, the CRM-Lips@DA group significantly increased the expression of STING-related genes in the tumor compared with the PBS group or the DA monotherapy group, indicating that the intratumoral STING pathway was effectively activated ( Figure 6 C).

[0136] STING agonists can reshape the tumor immune microenvironment and restore tumor immune responses. There is a high proportion of M2 (tumor-promoting) macrophages in pancreatic cancer, and there is a lack of T cell infiltration. Therefore, the changes in cell subtypes in tumor tissue after cyclical treatment were examined to investigate the in vivo immune activation effect of CRM-Lips@DA. After cyclical CRM-Lips@DA treatment, the expression of CD86 in pancreatic cancer mouse tumors increased by 14.4 times compared with the PBS control group, indicating that the liposome cyclic treatment significantly promoted the expression of M1 macrophages; at the same time, the expression of CD206 was significantly reduced, indicating that the liposome cyclic treatment can significantly reduce the number of M2 macrophages ( Figure 6 D / E).

[0137] DC cells are a type of "bridge" cell that connects innate immunity and adaptive immunity. However, DC cells are lacking in the microenvironment of solid tumors such as pancreatic cancer. After cyclic treatment, the expression of mature DC cells in the CRM-Lips@DA group increased by 2.11 times compared to the PBS group, and the CRM-Lips@DA+PD-1 antibody treatment increased it by 2.73 times. There was no significant improvement in the free DA treatment group ( Figure 7A / B). This indicates that systemic DA injection cannot effectively deliver the drug to the tumor site and produce a regulatory effect, while CRM-Lips@DA can significantly promote DC cell maturation in the tumor microenvironment through efficient DA delivery.

[0138] Activated CD8 + T cells, also known as cytotoxic T cells, are the body's core anti-tumor effector cells that can exert immune surveillance functions by directly killing tumor cells. CRM-Lips@DA can increase the expression of STING-related genes in tumor sites, effectively repolarize tumor M2 macrophages, and at the same time, activate DC cells. These key factors have an important impact on improving T cell infiltration in pancreatic cancer. Compared with the PBS control group, T cell activation in the CRM-Lips@DA and CRM-Lips@DA+PD-1 antibody groups increased by 11.18 and 15.76 times, respectively ( Figure 7 C / D). These results indicate that CRM-Lips@DA can improve T cell infiltration in the tumor microenvironment of pancreatic cancer and reverse it to a “hot tumor”.

[0139] Verification Example 6

[0140] Orthotopic pancreatic cancer tumor-bearing mouse models were established as described above. Mice were randomly divided into PBS, free DA, CRM-Lips@DA, PD-1 antibody, and CRM-Lips@DA+PD-1 antibody groups (n=6). DA was administered via tail vein at a dose of 2 mg / kg on days 9, 13, 17, 21, and 25, and PD-1 antibody was administered intraperitoneally at a dose of 5 mg / kg on days 10, 14, 18, 22, and 26, for a total of 5 cycles ( Figure 8 A). To visually observe the therapeutic effects in mice, D-luciferin potassium salt (150 mg / kg) was intraperitoneally injected every five days. Mice were anesthetized with isoflurane, and in situ tumor growth was recorded using a small animal in vivo imaging device. The day after the end of each dosing cycle, organs (heart, liver, spleen, lung, kidney, and intestine) and tumor tissues were removed from each group of mice. Fluorescence signals from each tissue were observed using an in vivo imaging device to investigate the presence of suspected metastatic lesions. In addition, the removed tumors were photographed and their size recorded to evaluate the therapeutic effect.

[0141] Tumor tissues were fixed in 4% paraformaldehyde overnight, dehydrated for 2 days, embedded in paraffin, and sectioned. To determine tumor cell morphology, apoptosis rate, and proliferation activity, sections were subjected to H&E staining, TUNEL analysis, and Ki67 immunohistochemistry, and observed under a microscope.

[0142] Since systemic injection of STING agonists can potentially distribute to normal tissues, triggering the secretion of large amounts of proinflammatory cytokines and producing severe side effects, we investigated whether liposome CRM-Lips@DA could reduce the drug's immunotoxicity through targeted delivery of the STING agonist DA. Healthy C57 mice were administered the drug once as described above. Blood was collected from each group, serum separated, and IL-6 and TNF-α levels were measured using an enzyme-linked immunosorbent assay (ELISA) in each of the five treatment groups (PBS, free DA, CRM-Lips@DA, PD-1 antibody, and CRM-Lips@DA + PD-1 antibody).

[0143] To assess systemic toxicity, mouse body weights were recorded every four days during the treatment period. Following treatment, blood samples were collected from the mice, and plasma was separated for routine blood analysis and biochemical analysis to assess hematologic toxicity. Major organs (heart, liver, spleen, lungs, and kidneys) were removed and stained with H&E. The morphology of each organ was observed under a microscope to assess organ toxicity.

[0144] From the tumor in vivo fluorescence and corresponding fluorescence quantitative curves taken during the treatment, it can be seen that the tumors in the PBS and DA alone groups grew rapidly, and the PD-1 antibody group had a certain inhibitory effect on tumor growth. In contrast, the tumor fluorescence intensity and tumor fluorescence curves in the CRM-Lips@DA and CRM-Lips@DA+PD-1 antibody groups were significantly lower than those in the other groups, indicating that CRM-Lips@DA can significantly inhibit pancreatic cancer growth by activating tumor immune response ( Figure 8 B / C). In order to observe the tumor treatment effect more intuitively, the mouse tumors were removed and photographed. The results further confirmed that CRM-Lips@DA and CRM-Lips@DA+PD-1 antibody had smaller tumors and better treatment effects than other groups ( Figure 8 D). Except for the CRM-Lips@DA and CRM-Lips@DA+PD-1 antibody combined treatment groups, other groups showed high-intensity metastatic signals, especially in the mesentery ( Figure 8 E). These results indicate that CRM-Lips@DA and CRM-Lips@DA+PD-1 antibody combination therapy can not only inhibit orthotopic tumor growth but also significantly suppress tumor metastasis.

[0145] H&E staining images of the tumors showed that the CRM-Lips@DA combined with PD-1 antibody treatment group caused more nuclear damage and cytoplasmic degradation than the other groups ( Figure 7F). In addition, TUNEL staining and Ki67 staining results showed that the CRM-Lips@DA combined with PD-1 antibody treatment group had the highest level of cell apoptosis and the lowest proliferation activity, confirming that the combined treatment has a better anti-tumor effect ( Figure 8 GJ).

[0146] The results of TNF-α and IL-6 tests confirmed that the injection of free DA led to increased secretion of these two factors, while liposome encapsulation could significantly reduce the abnormal secretion of inflammatory factors caused by DA and reduce the immune stimulation ( Figure 9 A). After cyclic treatment with CRM-Lips@DA or CRM-Lips@DA + PD-1 antibody, the body weight of mice did not change significantly, indicating that the liposome and combination therapy have negligible systemic toxicity ( Figure 9 B). At the same time, the results of blood routine / blood biochemistry analysis were all within the normal range, indicating that the liposome and combined therapy did not cause blood toxicity ( Figure 9 C / D). H&E staining results of organs of mice in each treatment group showed that there were no obvious abnormalities in the cell morphology of all organs in the treatment group, indicating that the liposome and combination therapy had negligible organ toxicity ( Figure 9 E).

[0147] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A method for preparing monocyte-carrying oxygen-producing liposomes, characterized in that: The following steps are involved: S1: Synthesize lipopeptide CP with a terminal cysteine and lipid RL containing a thioketal domain by solid-phase peptide synthesis, and extract thylakoid membrane TK, which is a biological membrane structure capable of catalyzing ROS oxygen production; S2: The lipid component and lipopeptide CP are dissolved and mixed uniformly in a first organic solvent, and the lipid RL and STING agonist DA are dissolved and mixed uniformly in a second organic solvent. The two solutions are mixed uniformly to obtain a mixed solution, and then the organic solvent in the mixed solution is removed by a thin film hydration method to form a uniform lipid film; S3: adding an aqueous solution to the lipid film for hydration to form a crude liposome suspension, which is then sonicated to reduce the liposome size, thereby obtaining a preliminary liposome structure, CR-Lips@DA, capable of encapsulating the STING agonist DA. S4: The thylakoid membrane TK was fused to the liposome CR-Lips@DA using the ultrasound-extrusion method to obtain the liposome product CRM-Lips@DA, which has monocyte targeting, can respond to ROS to release drugs, and has oxygen-producing function.

2. The method for preparing a monocyte-carrying oxygen-producing liposome according to claim 1, characterized in that: In S1, the amino acid sequence of the lipopeptide CP is SEQ ID NO. 1; The amino acid sequence of the lipid RL is SEQ ID NO.

2.

3. The method for preparing a monocyte-carrying oxygen-producing liposome according to claim 1, characterized in that: In S1, the specific process of synthesizing the cysteine-terminated lipopeptide CP and the thioketal domain-containing lipid RL by solid-phase peptide synthesis includes: The Fmoc-protected amino acids are sequentially linked to the solid support, and the Fmoc protecting groups of the amino acids are removed one by one using a deprotection reagent. The next amino acid is then added in sequence, and the operation is repeated until the sequence construction of the target lipopeptide CP and lipid RL is completed; Introducing cysteine at the synthetic end of the lipopeptide CP and correctly connecting the cysteine to the constructed amino acid sequence; During the synthesis of lipid RLs, the thioketal domain was directly put into the reaction; After the synthesis is completed, the lipopeptide CP and lipid RL are cut off from the solid phase support using a cutting reagent and purified to remove unreacted reagents and by-products, ultimately obtaining a lipopeptide CP with a cysteine terminal and a lipid RL containing a thioketal domain.

4. The method for preparing a monocyte-carrying oxygen-producing liposome according to claim 1, characterized in that: In S1, the specific process of extracting thylakoid membrane TK includes: Place the spinach leaves in pre-cooled HEPES buffer, fully homogenize in a mortar, filter and collect the filtrate, centrifuge the filtrate, collect the intact chloroplasts in the supernatant, resuspend the chloroplasts in hypotonic buffer, leave for a preset time, and then centrifuge to remove the matrix extract to obtain particles containing thylakoids. Wash the precipitate with HEPES buffer, ultrasonicate, and centrifuge to remove the lumen extract to obtain green thylakoid membrane TK.

5. The method for preparing a monocyte-carrying oxygen-producing liposome according to claim 1, characterized in that: In S2, the specific preparation process of the mixed solution includes: The lipid components containing DOTAP, cholesterol, soybean lecithin, DSPE-PEG2000 and lipopeptide CP were added to the chloroform solvent, and the mixture was fully dissolved and mixed under vortexing to form a uniform lipid-lipopeptide CP chloroform solution; After accurately weighing lipid RL and STING agonist DA, add them to methanol solvent and vortex to fully dissolve and mix them evenly to form a uniform lipid RL-DA methanol solution; The prepared lipid-lipopeptide CP chloroform solution and lipid RL-DA methanol solution were mixed and vortexed on a vortex shaker to fully mix them and ensure full contact between the components to form a uniform mixed solution; The molar ratio of DOTAP, cholesterol, soybean lecithin, DSPE-PEG2000, lipopeptide CP, and lipid RL is 40-60:15-25:10-20:1-3:1-10:5-15; The mass ratio of the STING agonist DA to the lipid component is 1-30:

170.

6. The method for preparing a monocyte-carrying oxygen-producing liposome according to claim 5, characterized in that: In S2, the specific process of removing the organic solvent in the mixed solution by thin film hydration to form a uniform lipid film includes: The uniformly mixed solution was transferred to a container, placed on a rotary evaporator, and subjected to rotary evaporation at 38° C. and a vacuum degree of -0.1 MPa to evaporate and remove the organic solvent, and the solution gradually formed a uniform lipid film.

7. The method for preparing a monocyte-carrying oxygen-producing liposome according to claim 1, characterized in that: In S3, an aqueous solution is added to the lipid film for hydration to form a crude liposome suspension, and the crude liposome suspension is subjected to ultrasonic treatment to reduce the size of the liposomes. The specific process includes: Slowly add an appropriate amount of pure water to the lipid film formed after removing the organic solvent by the thin film hydration method, shaking while adding, so that the lipid film is fully exposed to water and hydrated, gradually forming a coarse liposome suspension; The obtained crude liposome suspension was placed in an ice bath and ultrasonically treated using a cell disruptor to uniformly disperse the liposome particles and reduce their size, ultimately obtaining a liposome CR-Lips@DA suspension with relatively uniform particle size.

8. The method for preparing a monocyte-carrying oxygen-producing liposome according to claim 1, characterized in that: In S4, the specific process of fusing the thylakoid membrane TK to the liposome CR-Lips@DA using the ultrasound-extrusion method includes: The extracted thylakoid membrane TK and liposome CR-Lips@DA were placed in the same solution and mixed evenly. The mixture was placed in an ice bath and preliminarily treated with ultrasound to allow initial contact and fusion of the TK membrane and liposome. The mixture was then extruded through a polycarbonate membrane with a pore size of 400 nm. The mixture was forced through the membrane pores under pressure to promote the fusion of the TK membrane and liposome. The mixture was then extruded again through a polycarbonate membrane with a pore size of 200 nm to obtain the final liposome product CRM-Lips@DA with uniform particle size, monocyte targeting, the ability to release drugs in response to ROS, and the ability to produce oxygen. The mass ratio of the liposome CR-Lips@DA to the thylakoid membrane TK is 1 to 10:

1.

9. A monocyte-carrying oxygen-generating liposome prepared by the method according to any one of claims 1 to 8, characterized in that: The particle size of liposome CRM-Lips@DA is 100-200 nm, and the surface potential is 30-40 mV; The liposome CRM-Lips@DA has an encapsulation efficiency of greater than 90% for the STING agonist DA; The liposome CRM-Lips@DA can rapidly release the drug DA under ROS stimulation and catalyze ROS to produce oxygen through the thylakoid membrane TK.

10. A use of the liposome as claimed in claim 9, characterized in that: The liposomes are used to prepare drugs for treating tumors. The liposome CRM-Lips@DA can activate the STING pathway in the tumor by targeted delivery of the STING agonist DA deep into the tumor, repolarize M2 macrophages to M1 macrophages, promote dendritic cell maturation and cytotoxic T cell infiltration, thereby enhancing the anti-tumor immune response, and relieve tumor hypoxia by producing oxygen, reversing the immunosuppressive tumor microenvironment, and inhibiting tumor growth and metastasis.

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