Monocyte-carrying oxygen-producing liposome, and preparation method and application thereof

Abstract

The present application relates to a monocyte-carrying type oxygen-producing liposome and a preparation method and application thereof, wherein the preparation method comprises: synthesizing a lipopeptide CP with a cysteine terminal and a lipid RL containing a sulfur ketal domain through a solid-phase polypeptide synthesis method, extracting a thylakoid membrane TK, dissolving and uniformly mixing the lipid components and the lipopeptide CP using an organic solvent, dissolving and uniformly mixing the lipid RL and a STING agonist DA using an organic solvent, and removing the organic solvent in the mixed solution; adding an aqueous solution to the lipid membrane to hydrate, forming a crude liposome suspension, fusing the thylakoid membrane TK to the liposome using an ultrasonic-extrusion method, and obtaining a liposome product. Compared with the prior art, the liposome prepared by the present application can efficiently deliver DA to the deep part of pancreatic cancer, activate multiple cell STING pathways and immune responses, relieve hypoxia, improve immune suppression caused by hypoxia, and thus enhance the immunotherapy effect of DA.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a mononuclear cell-borne oxygen-producing liposome, its preparation method, and its application. Background Technology

[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 rising annually. Surgery combined with systemic chemotherapy is currently the best treatment to prolong patient survival; however, over 80% of patients are diagnosed at an advanced stage, making surgery unsuitable, and chemotherapy becomes the primary treatment in clinical practice. However, commonly used chemotherapy drugs only extend patient survival by a few months, with very limited efficacy. Therefore, new therapies are urgently needed.

[0003] The core of immunotherapy is to stimulate the patient's anti-tumor adaptive immune response, utilizing the patient's own immune system to eliminate cancer cells and prevent recurrence. Currently, clinically applied immunotherapies mainly focus on overcoming the bottlenecks in T-cell function regulation, especially addressing the immunosuppression problem in the tumor microenvironment. However, clinically used immunotherapies such as immune checkpoint inhibitors (ICIs) 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, with an extreme lack of cytotoxic CD8+ in the pancreatic cancer tumor microenvironment. + T cell infiltration. Therefore, pancreatic cancer is a "cold tumor" lacking T cell infiltration and activity. Immunosuppressive cell populations, such as anti-inflammatory tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), are key factors hindering T cell responses. TAMs are the most abundant non-tumor cells in the pancreatic cancer tumor microenvironment, accounting for approximately 38% of the total infiltrating immune cells. These TAMs are primarily M2 type and can inhibit T cell proliferation and suppress adaptive immunity, making them the main force of immunosuppression.

[0004] Stimulator of interferon genes (STING) agonists have attracted much attention due to their superior immune-activating capabilities. Activation of the STING pathway induces the secretion of type I interferons (IFNs) and pro-inflammatory factors, promoting the maturation of antigen-presenting cells and T-cell anti-tumor immune responses. Activation of the STING signaling pathway has been shown to reshape the immunosuppressive tumor microenvironment, promoting cytotoxic T-cell infiltration in pancreatic cancer and transforming it from a "cold tumor" to a "hot tumor." However, STING agonists are mostly small-molecule drugs, easily cleared by the kidneys after intravenous injection, with short blood half-lives, resulting in insufficient drug accumulation at the tumor site and failing to achieve an effective activation dose. Furthermore, after systemic injection, STING agonists can indiscriminately activate the STING pathway in normal tissues, leading to the production of large amounts of pro-inflammatory cytokines, which can then 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 metastatic tumors. Meanwhile, the characteristics of pancreatic cancer (dense stroma, few blood vessels, and rapid cancer cell proliferation) lead to not only insufficient oxygen supply but also exacerbated oxygen consumption, making it an extremely hypoxic tumor. Hypoxia inhibits the STING pathway and immune cell activation. STING pathway activation combined with hypoxia relief holds promise for reversing the immunosuppressive microenvironment of pancreatic cancer and achieving tumor immunotherapy.

[0005] Patent CN118662445A proposes a STING agonist loaded with ginsenoside liposomes. While this improves the stability and immune activation effect of the STING agonist to some extent, it mainly focuses on specific cancer types such as colorectal cancer and does not fully consider how to overcome the delivery barrier of dense stromal tumors such as pancreatic cancer. It also fails to address the problem of the hypoxic environment in pancreatic cancer, thus limiting its application in the treatment of pancreatic cancer.

[0006] Typically, small molecule drugs have short half-lives, making effective delivery to tumor sites difficult and requiring specific nanocarriers for efficient delivery. However, the unique delivery barriers of pancreatic cancer severely restrict the delivery of nanocarriers. First, pancreatic cancer has a dense fibrous matrix composed of fibroblasts, immune cells, and collagen, occupying >90% of the tumor volume. These fibrous matrix components compress blood vessels, causing them to collapse, resulting in blood perfusion of only 1 / 3 that of a normal pancreas. Studies have shown that only nanoparticles smaller than 50 nm can penetrate the pancreatic cancer tumor matrix barrier, while particles larger than 50 nm are mostly distributed around blood vessels and cannot reach the deep tumor. Furthermore, vascular stenosis and insufficient blood supply further limit the accumulation of ordinary nanocarriers at the pancreatic cancer site. Therefore, to achieve effective delivery and regulation of the STING agonist DA, it is necessary to develop a smart nanodelivery system that can overcome the vascular and matrix barriers of pancreatic cancer and generate oxygen in situ. Summary of the Invention

[0007] The purpose of this invention is to overcome the deficiencies of the prior art by providing a mononuclear cell-carrying oxygen-producing liposome, its preparation method, and its application. It provides a liposome CRM-Lips@DA that can target and deliver 5,6-dimethylxanthenone-4-acetic acid (DA) and alleviate hypoxia. This liposome can efficiently deliver DA to deep sites in pancreatic cancer, activating multiple cellular STING pathways and immune responses. Simultaneously, it can alleviate hypoxia and improve hypoxia-induced immunosuppression, thereby enhancing the immunotherapeutic effect of DA.

[0008] The conceptualization process of this invention posits that pancreatic cancer tissue is highly infiltrated by tumor-associated macrophages (TAMs), with M2-type TAMs being dominant. These TAMs promote tumor growth by releasing growth factors and inhibiting immune surveillance. TAMs primarily originate from peripheral monocytes and are continuously recruited to deeper tumor regions driven by inflammatory factors such as CCL2, CCL5, and CSF-1. Therefore, designing a nanomedicine delivery system capable of "riding" monocytes holds promise for overcoming the blood supply and stromal barriers of pancreatic cancer, enabling drug delivery to deeper tumor regions.

[0009] The concept of this invention posits that monocytes are the primary phagocytic cells in the blood. Studies have shown that cationic liposomes with a charge of 30-40 mV can specifically target monocytes. Upon entering the cell, the cationic liposomes degrade via the endosome / lysosome pathway. To avoid lysosomal degradation, L-cysteine ​​can be modified on the surface to allow the nanoparticles to enter the endosome-Golgi-endoplasmic reticulum pathway, bypassing lysosomal action. After entering deep into the tumor, the cationic liposomes need to release the drug extracellularly for uptake by tumor cells and myeloid cells. Research shows that after monocytes migrate to tumor tissue, they differentiate into tumor cells (TAMs) under the influence of granulocyte-macrophage-colony stimulating factor (GM-CSF) or macrophage colony stimulating factor (M-CSF), at which point the ROS concentration rapidly increases. ROS has been shown to specifically cleave thioacetate structures. Therefore, incorporating thioaldol domains into liposomes holds promise for achieving ROS-responsive, tumor-specific drug release and rapid exocytosis. Simultaneously, the hypoxic microenvironment of pancreatic cancer is rich in ROS; fusing TK membranes containing catalase with liposomes can decompose ROS to produce oxygen, alleviating tumor hypoxia-induced immunosuppression and improving the efficacy of immunotherapy.

[0010] Based on the above considerations, this invention provides a liposome CRM-Lips@DA that can "hitch a ride" on in vivo monocytes to deliver STING agonists deep into tumors and alleviate hypoxia. This liposome is loaded with the STING agonist DA, and its main lipid components include DOTAP, cholesterol, soybean lecithin, DSPE-PEG2000, lipopeptide CP (terminally cysteine, targeting the Golgi apparatus), and RL (containing a thioketal domain, ROS-responsive cleavage), and is fused with a TK membrane (containing catalase, capable of decomposing ROS and producing oxygen). The liposome CRM-Lips@DA of this invention can target monocytes in the blood and enter the endosome-Golgi-endoplasmic reticulum pathway, evading lysosomal degradation, thereby enabling it to "hitch a ride" on monocytes to reach deep into tumors. Under the stimulation of rapidly elevated ROS in the tumor microenvironment, this liposome rapidly releases DA, which is then exocytotically and utilized by various immune-related cells. This activates the multicellular STING pathway within the tumor, repolarizes TAMs to the M1 type, promotes DC maturation, and consequently 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-induced oxygen production, improving tumor hypoxia and thus alleviating hypoxia-induced immunosuppression, further enhancing the immunoactivating effect of the STING agonist DA.

[0011] The objective of this invention can be achieved through the following technical solutions:

[0012] The first aspect of this invention provides a method for preparing mononuclear cell-borne oxygen-producing liposomes, comprising the following steps:

[0013] S1: A lipopeptide CP with a cysteine ​​terminal and a lipid RL containing a thioketal domain were synthesized by solid-phase polypeptide synthesis. Thylakoid membrane TK was extracted. The thylakoid membrane TK is a biomembrane structure capable of catalyzing ROS oxygen production.

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

[0015] S3: Add an aqueous solution to the lipid membrane for hydration to form a crude liposome suspension. Sonicate the crude liposome suspension to reduce the size of the liposomes and obtain a preliminary liposome structure CR-Lips@DA that can encapsulate the STING agonist DA.

[0016] S4: The thylakoid membrane TK was fused to the liposome CR-Lips@DA using an ultrasonic-extrusion method to obtain the liposome product CRM-Lips@DA, which has mononuclear cell targeting, can release drugs in response to ROS, and has oxygen production 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 cysteine-terminated lipopeptide CP and the thioketal-containing lipid RL via solid-phase polypeptide synthesis includes:

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

[0023] Cysteine ​​residue was introduced at the end of the lipopeptide CP and the cysteine ​​residue was correctly linked to the already constructed amino acid sequence.

[0024] In the synthesis of lipid RL, the starting material TK (thioketal domain) is directly added to the reaction.

[0025] After synthesis, the lipopeptide CP and lipid RL were cleaved from the solid support using a cleavage reagent and then purified to remove unreacted reagents and byproducts, ultimately yielding the lipopeptide CP with a cysteine ​​terminus and the lipid RL containing a thioketal domain.

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

[0027] Spinach leaves were placed in pre-cooled HEPES buffer and homogenized thoroughly in a mortar. After filtration, the filtrate was collected and centrifuged. The intact chloroplasts in the supernatant were collected and resuspended in hypotonic buffer for a preset time. The matrix extract was removed by centrifugation to obtain thylakoid-containing particles. The precipitate was washed with HEPES buffer, sonicated, and centrifuged to remove the lumen extract to obtain the 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 are added to chloroform solvent and vortexed to fully dissolve and mix them evenly to form a homogeneous lipid-lipopeptide CP chloroform solution.

[0030] After accurately weighing the lipid RL and the STING agonist DA, add them to the methanol solvent, vortex to fully dissolve and mix them evenly to form a homogeneous 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 ensure thorough and uniform mixing, ensuring full contact between the components to form a homogeneous 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 from the mixed solution by thin-film hydration to form a uniform lipid film includes:

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

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

[0037] A suitable amount of pure water was slowly added to the lipid membrane formed after the organic solvent was removed by the membrane hydration method, while shaking was performed to allow the lipid membrane to fully contact the water and undergo hydration, gradually forming a crude lipid body suspension.

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

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

[0040] The extracted thylakoid membrane TK and liposome CR-Lips@DA were placed in the same solution and mixed thoroughly. The mixture was then placed in an ice bath and pre-treated with ultrasound to allow the TK membrane and liposomes to initially contact and fuse. The mixture was then extruded through a polycarbonate membrane with a pore size of 400 nm, allowing the mixture to pass through the membrane pores under pressure, thereby promoting the fusion of the TK membrane and liposomes. 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, which has uniform particle size, mononuclear cell targeting, ROS-responsive drug release, and oxygen production function.

[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 mononuclear cell-borne oxygen-producing liposome prepared by the method described above, wherein the liposome CRM-Lips@DA has a particle size of 100-200 nm and a surface potential of 30-40 mV;

[0043] The liposome CRM-Lips@DA encapsulates the STING agonist DA with a resolution greater than 90%.

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

[0045] A third aspect of the present invention provides an application of the liposome CR-Lips@DA as described above, wherein the liposome is used to prepare a drug for treating tumors. The liposome CRM-Lips@DA can target and deliver the STING agonist DA to deep tumors, activate the STING pathway within the tumor, repolarize M2 macrophages to M1, promote dendritic cell maturation and cytotoxic T cell infiltration, thereby enhancing the anti-tumor immune response, and alleviate tumor hypoxia through oxygen production, reverse the immunosuppressive tumor microenvironment, and inhibit 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 in this invention are selectively taken up by blood monocytes and enter the endosome-Golgi-endoplasmic reticulum pathway under the action of surface cysteine, avoiding lysosomal degradation. CRM-Lips@DA liposomes "hitch a ride" on monocytes, overcoming the blood supply and matrix barriers of pancreatic cancer to enter the deep tumor. After entering the tumor microenvironment, the ROS concentration in monocytes is sharply increased, destroying the liposome structure and triggering DA release. The released DA is rapidly expelled from the monocytes, and the transmembrane-capable DA is taken up by tumor immune-related cells, activating the intracellular STING pathway. CRM-Lips@DA liposomes retained in monocytes contain a TK membrane, which can catalyze H2O2 production, reversing the immunosuppression caused by hypoxia and synergistically activating the STING pathway. By efficiently delivering dopamine (DA) to various immune-related cells within pancreatic cancer cells and alleviating immunosuppression through in-situ oxygen production, CRM-Lips@DA polarizes pro-tumor M2 macrophages to M1 type, promotes DC maturation and antigen presentation levels, and transforms pancreatic cancer from a "cold tumor" to a "hot tumor," ultimately promoting T cell activation and intratumoral invasion, effectively inhibiting pancreatic cancer growth and metastasis. The CRM-Lips@DA provided by this invention offers a novel immunotherapy option for pancreatic cancer. Attached Figure Description

[0048] Figure 1 .CRM-Lips@DA in vivo delivery process and therapeutic effect diagram.

[0049] Figure 2Characterization of CRM-Lips@DA; 2A: Flowchart of liposome preparation; 2B: Physical mixing and extrusion fluorescence images of DiI-labeled CR-Lips with a self-fluorescent TK membrane; 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: Mononuclear cell targeting of liposomes; 3B: Golgi body targeting of liposomes; 3C: Cell exocytosis of liposome ROS response.

[0051] Figure 4 .CRM-Lips@DA in vitro regulation; 4A-B: M2 polarization rate of macrophages; 4C: Evaluation of oxygen production by 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 curves of liposomes; 5B: Pharmacokinetic parameters of liposomes; 5C: Accumulation of DiD-labeled liposomes in tumor tissue; 5D: Distribution of DiD-labeled liposomes in tumor tissue; 5E: Distribution of C6-labeled liposomes in tissue tissue; 5F: Intratumoral fluorescence distribution of C6-labeled liposomes.

[0053] Figure 6-7 Tumor regulation effect of .CRM-Lips@DA. 6A: Mouse treatment regimen; 6B: Evaluation of intratumoral oxygen production effect; 6C: Evaluation of tumor STING pathway activation; 6D-E: Evaluation of macrophage regulation effect in vivo (M1, M2 macrophages); 7A-B: Evaluation of immune cell regulation effect in vivo (DC cells); 7C-D (T cells).

[0054] Figure 8 Antitumor effects of .CRM-Lips@DA. 8A: Mouse treatment regimen; 8B: Bioluminescent images of mouse tumors during cycle treatment; 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 group after treatment; 8F: Representative H&E staining images of tumor tissues after treatment; 8G / I: Ki67 staining images and quantification of tumor tissues; 8H / J: TUNEL staining fluorescence images and quantification of apoptosis rate of tumor tissues.

[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: Using... Figure 9 Treatment regimen, changes in mouse body weight during treatment; 9C: Figure 9 Post-treatment blood biochemical analysis of mice; 9D: Figure 8 Blood routine analysis of mice after treatment; 9E: representative H&E staining images of major organs of mice after treatment. Detailed Implementation

[0056] Overall, this invention specifically designs a liposome CRM-Lips@DA that delivers the interferon gene stimulator (STING) agonist 5,6-dimethylxanthenone-4-acetic acid (DA) and relieves hypoxia. A lipopeptide CP with a cysteine ​​terminal and a lipid RL containing a thioketal domain were synthesized via solid-phase peptide synthesis. Thylakoid membranes (TK) were extracted. Liposomes CR-Lips@DA were prepared via thin-film hydration, and then the TK membrane was fused to the liposomes using an ultrasonic / extrusion method to obtain CRM-Lips@DA. This liposome has a zeta potential of 31.4 mV, enabling it to be preferentially taken up by monocytes in the blood and "hitch a ride" to deep tumor sites. Under the action of surface cysteine, it enters the endosome-Golgi-endoplasmic reticulum pathway, avoiding lysosomal degradation. Stimulated by reactive oxygen species (ROS), it achieves highly efficient intratumoral delivery of the STING agonist and in situ oxygen production, remodeling pancreatic cancer into a "hot tumor" and activating an adaptive immune response. This effectively inhibits pancreatic cancer growth and metastasis.

[0057] In specific implementation, the CRM-Lips@DA liposome can be delivered to deep tumor sites via monocytes to deliver the stimulator of interferon genes (STING) agonist 5,6-dimethylxanthenone-4-acetic acid (DA) and alleviate hypoxia. Its key feature is that the liposomes can be targeted and taken up by monocytes in the blood, carrying them deep into the tumor. Under the stimulation of reactive oxygen species (ROS), on the one hand, DA can be rapidly released, promoting drug exocytosis and uptake by various immune-related cells, exerting an immune-activating effect; on the other hand, the TK membrane can catalyze ROS to produce oxygen, improving the immunosuppression caused by hypoxia, reshaping pancreatic cancer into a "hot tumor," and activating an adaptive immune response.

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

[0059] In practice, the encapsulation rate of DA by the liposome CRM-Lips@DA is greater than 90%.

[0060] For the specific implementation, the preparation method of the liposome CRM-Lips@DA is described in [reference needed]. Figure 1 This includes the following steps:

[0061] 1) Lipopeptide CP and lipid RL were synthesized via solid-phase polypeptide synthesis.

[0062] 2) Extract the 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. CR-Lips@DA was prepared by thin-film hydration method.

[0064] 4) The TK membrane is fused with CR-Lips@DA by ultrasonic / 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. Wherein, Fmoc is a fluorene methoxycarbonyl protecting group used to protect the amino group of the subsequent amino acid; K(Fmoc) is lysine (K); histidine (H); glycine (G); 6-aminocaproic acid (acp); arginine (R); serine (S); cysteine ​​(C); thioketal (TK); and hexaethylene glycol (PEG6).

[0066] In specific implementation, the extraction process of the TK membrane in step 2 includes: homogenizing spinach leaves in a mortar with pre-cooled HEPES buffer, filtering and collecting the homogenate. Then, centrifuging at 8000×g for 10 min yields intact chloroplasts. The chloroplasts are then resuspended in hypotonic buffer (10 mM HEPES, pH 8.00) for 2 h, centrifuged at 12000×g for 30 min to remove matrix extracts, obtaining particles containing thylakoids. The particles are washed with 10 mM HEPES buffer, sonicated at 4°C for 15 min, and centrifuged at 15000×g for 15 min to remove lumen extracts, yielding a green TK membrane.

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

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

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

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

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

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

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

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

[0075] In practical applications, the use of liposome CRM-Lips@DA in the preparation of drugs for treating diseases can target monocytes in the blood, achieve Golgi targeting, avoid degradation via lysosomal pathways, and have ROS-responsive drug release and exocytosis.

[0076] In practice, the liposome CRM-Lips@DA can be endocytosed by monocytes and enter the endosome-Golgi-endoplasmic reticulum pathway, avoiding lysosomal degradation, thus "riding" monocytes to cross the pancreatic cancer delivery barrier.

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

[0078] In practice, the immune "cold tumor" can be reshaped into a "hot tumor".

[0079] In practice, the liposome CRM-Lips@DA can repolarize M2 macrophages into M1 macrophages, while the exocytotic DA can activate the STING pathway in dendritic cells and tumor cells.

[0080] In practice, the liposome CRM-Lips@DA can produce oxygen in a ROS-rich environment, alleviating immunosuppression caused by hypoxia.

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

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

[0083] In practice, the liposomes exhibit good biocompatibility and safety.

[0084] In practice, liposome CRM-Lips@DA exhibits negligible immunotoxicity, systemic toxicity, hematologic toxicity, and organ toxicity.

[0085] In practice, liposome CRM-Lips@DA can also be used for the delivery of other immune activators, including cyclic guanylate-adenosine monophosphate (cGAMP), imiquimod, and resiquimod.

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

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

[0088] Example 1

[0089] Lipopeptide (CP) synthesis: First, 2-CL resin was selected as the resin type, and FMOC solid-phase synthesis was employed with a synthesis amount of 1.5 mmol. Amino acids were represented as FMOC-AAn-OH (where n is the site number of the amino acid at the C-terminus of the polypeptide chain). Then, starting from the C-terminus of the polypeptide, FMOC-AA1-OH was 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 was removed using a DMF solution containing 20% ​​piperidine for 20 min. After removal, the resin was washed four times with DMF for 1 min each time to obtain NH2-AA1-2-CL resin. Then, using the condensation reagents DIC and HOBT, along with DMF as solvent, FMOC-AA2-OH was dehydrated and condensed with the resin to obtain FMOC-AA2-AA1-2-CL resin. Steps 3 and 4 were repeated to sequentially condense the remaining amino acids until the polypeptide synthesis was complete. Finally, peptide lysis was performed using a lysis buffer of 95% TFA, 1% H₂O, 2% EDT, and 2% TIS for 2 hours. After lysis, the lysis buffer was filtered into ice-cold ether and centrifuged to obtain a crude product. This crude product was then separated by HPLC under acetonitrile gradient conditions of 15-40% for 40 minutes. The final sample, with an HPLC purity greater than 95%, was lyophilized. Its sequence is Fmoc-K(Fmoc)-HHG-acp-GRSSRSSRSSSC.

[0090] Lipid RL synthesis: 2-CL resin was selected, and the synthesis amount was 1.5 mmol. Amino acids were represented as FMOC-AAn-OH (n represents the amino acid position starting from the C-terminus). Synthesis began from the C-terminus of the peptide. Using the condensation reagent DIEA / DCM as a solvent, FMOC-AA1-OH was dehydrated and condensed with the resin to obtain FMOC-AA1-2-CL resin. Then, FMOC was removed using a DMF solution containing 20% ​​piperidine for 20 min. After removal, the resin was washed four times with DMF for 1 min each time to obtain NH2-AA1-2-CL resin. Next, FMOC-AA2-OH was condensed with the resin using DIC and HOBT to obtain FMOC-AA2-AA1-2-CL resin. This process was repeated to synthesize the remaining amino acids sequentially. Finally, peptide lysis was performed using a 20% TFE / 80% DCM lysis buffer for 2 h. After lysis, the lysate was rotary evaporated, followed by HPLC separation and purification using an acetonitrile gradient of 15-40% for 40 min, ultimately yielding a peptide sample with a purity greater than 95%, which was then lyophilized. The sequence was Fmoc-K(Fmoc)-GGG-TK-PEG6. The product was purified by preparative HPLC, and successful synthesis was confirmed by HPLC-MS.

[0091] TK membrane extraction: 100g of spinach leaves were homogenized in a mortar with pre-cooled HEPES buffer, filtered, and the filtrate was collected. The filtrate was centrifuged at 8000×g for 10 min to collect intact chloroplasts. The chloroplasts were resuspended in hypotonic buffer (10mM HEPES, pH 8.0) for 2 h, and centrifuged at 12000×g for 30 min to remove the matrix extract, yielding thylakoid-containing particles. The particles were washed with 10mM HEPES buffer, sonicated at 4℃ for 15 min, and centrifuged at 15000×g for 15 min to remove the lumen extract, yielding a green TK membrane. Successful TK membrane extraction was confirmed by UV and fluorescence spectroscopy, and the 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 drug DA were added to a 50 mL round-bottom 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. The chloroform / methanol and other organic solvents were removed by vacuum rotary evaporation at 38°C to obtain a lipid film. Pure water was added, and the mixture was hydrated for 30 min. The obtained crude liposome suspension was collected in centrifuge tubes and sonicated 5 times in a cell disruptor under ice bath conditions to obtain CR-Lips@DA liposomes. Add the corresponding TK membrane at a TK:liposome mass ratio of 1:4, place on ice, and intermittently sonicate for 2 minutes using a cell sonicator. Finally, extrude through polycarbonate membranes with pore sizes of 400 nm and 200 nm respectively to obtain CRM-Lips@DA liposomes with uniform particle size. Finally, adjust the liposome solution to a physiologically isotonic state using glucose solution. Figure 2 A).

[0093] When physically mixed only, the DiI (red fluorescent probe for cell membranes) labeled CR-Lips (red) and TK membrane (green) showed little overlap. In contrast, after sonication / extrusion, the red fluorescence on CR-Lips and the green fluorescence on the TK membrane largely overlapped, exhibiting yellow fluorescence, indicating that the TK membrane had been successfully hybridized to the liposomes. Figure 2 B).

[0094] Comparing the infrared absorption and fluorescence spectra of TK membrane alone, CR-Lips (without TK membrane), and CRM-Lips@DA, the results showed that CRM-Lips@DA had the same characteristic peaks as the TK membrane, while CR-Lips did not show corresponding peaks, indicating that the TK membrane was successfully fused with the liposomes. Figure 2 C).

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

[0096] Because the liposomes contain RL (thioacetal domain, which can be specifically destroyed by ROS) lipids, transmission electron microscopy was performed on CRM-Lips@DA and CRM-Lips@DA after incubation with ROS. The results showed that CRM-Lips@DA has a clearly defined spherical morphology. Figure 2 E left), while the liposomes incubated with ROS have cleaved their structure and lost their spherical shape ( Figure 2 E (right) indicates that ROS can disrupt the structure of liposomes.

[0097] To investigate the serum stability of CRM-Lips@DA, fluorescence spectral changes were measured using fluorescence resonance energy transfer (FRET). 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). Fluorescence intensity changes at different time points were detected using a microplate reader (λex / λem = 450 / 480-725 nm). After 12 h of incubation, the fluorescence spectrum of CRM-Lips@DA showed almost no change, and a significant FRET effect was still observed. However, after adding acetonitrile (ACN) to disrupt the liposome structure, DiI showed a significant emission peak at 480 nm, indicating a weakened FRET effect and suggesting that the liposome structure of CRM-Lips@DA was disrupted. Therefore, these results indicate that CRM-Lips@DA maintains its structural integrity in the blood for at least 12 h, demonstrating good serum stability. Figure 2 F).

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

[0099] The release rate of CRM-Lips@DA in the presence of H2O2 was significantly greater than that in the absence of H2O2. This result indicates that CRM-Lips@DA can release rapidly in vivo under ROS stimulation. Figure 2 H).

[0100] Verification Example 1

[0101] First, construct DiD-labeled liposomes CRM-Lips@DiD (a far-infrared fluorescent probe for cell membranes, comprising 1% of the lipid component) using the method described above. After incubating with fresh mouse whole blood at a concentration of 100 μM (anticoagulated) for 1 h, the fluorescence intensity of DiD in monocytes and B cells was measured using flow cytometry (CytoFLEX LX, Beckman, USA).

[0102] RAW 264.7 cells were seeded in 12-well plates and incubated overnight. RM-Lips@DiO (a green fluorescent probe for cell membranes) labeled with DiO was added, and incubation was continued for 2 hours. Cells were washed with PBS, and Golgi-Tracker Red (150 μg / mL) and LysoTracker Red (50 nM) were added to stain the Golgi apparatus and lysosomes, respectively. 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. The co-localization 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 within cells. CRM-Lips@C6 were incubated with macrophages in a hypoxic incubator for 2 hours. After removing the liposomes, one group was supplemented with medium containing inducing factors (IL-4, IL-10, and M-CSF) and ROS (1 mM H2O2), while the other group was replaced with normal medium. Cells were subsequently collected at different time points (0, 2, 6, and 12 hours), and intracellular C6 fluorescence intensity was measured using flow cytometry (Cytoflex LX, 23 Beckman, USA) to assess 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) indicates that liposomes can be targeted and taken up by monocytes in mouse blood.

[0105] CRM-Lips@DiO showed significantly enhanced distribution in the Golgi apparatus compared to RM-Lips@DiO, while its distribution in lysosomes decreased. Figure 3 B) indicates that liposomes exhibit cysteine-dependent Golgi apparatus targeting.

[0106] Flow cytometry data showed that the residual C6 level in cells with ROS (1 mM H2O2, to simulate the tumor microenvironment) was 15.58%, while the C6 level in the control group without ROS was 35.54%. This indicates that under ROS stimulation, liposomes can rapidly release drugs, allowing cells to expel the drugs more quickly. 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 type using three inducing factors: IL-4, IL-10, and M-CSF (1 μg / mL, ratio = 10:5:5). CD206 expression (a marker of M2 macrophages) was analyzed by flow cytometry (Cytoflex LX, Beckman, USA). Cytoskeleton markers were used to label the cytoskeleton, and morphological changes in uninduced and induced cells were observed under a laser confocal microscope (FV3000, Olympus, Japan).

[0109] RAW 264.7 cells were seeded in 12-well plates and induced to the M2 type as described above. The cells were then cultured overnight for 12 hours in a hypoxic incubator. According to the groups, probe Ru(dpp)3]Cl2 (an oxygen indicator probe, which can be destroyed by molecular oxygen) (1.5 μL, 6 mg / mL) was added first, mixed well, and then the corresponding liposomes were added. The cells were then incubated in a hypoxic incubator for another 2 hours. After incubation, changes in probe fluorescence intensity were observed using a multi-mode in vitro imaging system (λex / λem = 470 / 600 nm).

[0110] RAW 264.7 cells were seeded in 12-well plates and induced to the M2 phenotype using the three inducing factors mentioned 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 wells, and the plates were incubated in a hypoxic incubator for 12 h. Cells were collected, and CD86 levels were detected by flow cytometry. Simultaneously, 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, co-incubation experiments were performed using 0.4 μm Transwell plates. RAW 264.7 cells were seeded in the upper chamber and induced into M2 macrophages as described above. Then, PBS, free DA, Lips@DA, M-Lips@DA, CM-Lips@DA, and CRM-Lips@DA (final DA concentration: 10 μg / mL) were added, and the cells were incubated in a hypoxic incubator for 2 h. The drug-containing medium was then discarded, and fresh medium was added. Panc02 pancreatic cancer cells were seeded in the lower chamber and cultured in a hypoxic incubator for 24 h. Tumor cells from the lower chamber were collected, RNA was extracted, and after quantification and reverse transcription, the mRNA expression of the corresponding STING pathways (INF-β, CXCL10, and CCL5) was detected by RT-qPCR to assess the STING pathway activation effect of tumor cells.

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

[0113] In vitro imaging results showed no significant change in fluorescence intensity between the PBS+Ru(dpp)3]Cl2 and CR-Lips+Ru(dpp)3]Cl2 groups, while the fluorescence intensity of the CRM-Lips+Ru(dpp)3]Cl2 group was significantly lower than that of the PBS+Ru(dpp)3]Cl2 group. This indicates that CRM-Lips liposomes fused with a TK membrane can produce oxygen in a ROS-rich environment, thereby effectively alleviating hypoxia. Figure 4 C).

[0114] The repolarization effect of this liposome was evaluated by detecting the expression of M1 macrophage-related protein (CD86) using flow cytometry. Compared with the control group, the expression of CD86 (a marker of M1 macrophages) in macrophages treated with CRM-Lips@DA was significantly increased, indicating that M2 macrophages were effectively repolarized to M1 type (CD86). Figure 4 D).

[0115] The expression of STING pathway-related factors (INF-β, CCL10, CCL5) in macrophages after different liposome treatments 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 in Transwell chambers was detected by RT-qPCR. The results showed that the CRM-Lips@DA group had the most significant activation effect on Panc02 pancreatic cancer cells, with significantly upregulated mRNA expression of STING pathway-related factors (INF-β, CCL10, CCL5). This indicates that after macrophages took up CRM-Lips@DA, ROS-stimulated structural cleavage led to the exocytosis of sufficient DA, thus effectively activating the STING pathway in 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 was examined in SD rats.

[0119] First, liposomes were labeled with DiI (a red fluorescent probe for cell membranes). SD rats were randomly divided into four groups (n=3), and each group was injected intravenously via tail vein with RM-Lips@DiI, CM-Lips@DiI, CRM-Lip@DiI, or CLP (clophosphonate liposomes, which can deplete monocytes / macrophages in vivo) + CRM-Lips@DiI at a dose of 0.3 mg / kg DiI. The CLP + CRM-Lips@DiI group was pre-injected intraperitoneally with CLP (concentration: 5 mg / mL; injection volume: 1 mL) 24 h prior to depletion of macrophages in the rats. At predetermined time points, blood was collected from the retinal venous plexus of rats and centrifuged at 3500 rpm for 10 min to obtain plasma. The fluorescence intensity of DiI in plasma was detected using an ELISA reader (λex / λem = 549 / 565 nm). DiI plasma concentration-time curves were plotted, 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 longer than that of CRM-Lips@DiI (9.448 h), indicating a statistically significant difference. This suggests that liposomes are 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. After tail vein injection of CRM-Lips@DiD or CRM-Lips@C6, the ability of the formulation to cross the pancreatic cancer delivery barrier was examined, as well as whether the liposomes could promote drug exocytosis in the ROS-enriched tumor microenvironment.

[0123] Panc02-Luc cells were loaded at 5 × 10 6 The solution was suspended at a concentration of 1 × 10⁹ / mL in a PBS:Matrigel solution of 1:1, with each mouse receiving 1 × 10⁹ / mL. 5 A specific amount of cells was inoculated into the tail of the mouse pancreas to establish an orthotopic pancreatic cancer-bearing mouse model. This model can simulate key pathological features of pancreatic cancer, such as hypoxia, hypovascularity, and dense fibrous matrix.

[0124] Ten days after modeling, mice were randomly divided into three groups: CM-Lips@DiD, CRM-Lips@DiD, and CLP+CRM-Lips@DiD (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 the injection to deplete the mice's monocytes / macrophages. Mice were euthanized at 4, 12, and 24 hours after tail vein administration, and tumors were collected. The distribution of liposomes in the tumors was observed using a small animal imaging system.

[0125] Tumor tissue collected 12 hours after injection was embedded in a Tissue-Tek OCT complex, rapidly frozen, and cut into 5 μm sections. The sections were then subjected to CD68 (labeled macrophages) immunofluorescence staining, blocked with a DAPI-containing fluorescent blocking agent, and observed under a laser confocal microscope (FV3000, Olympus, Japan).

[0126] An orthotopic pancreatic cancer-bearing mouse model was established using the method described above. Ten days after modeling, the mice were randomly divided into three groups: CM-Lips@C6, CRM-Lips@C6, and CLP+CRM-Lips@C6 (n=3). Similarly, the CLP+CRM-Lips@C6 group was pre-administered via intraperitoneal injection of CLP (concentration: 5 mg / mL; injection volume: 200 μL) to deplete the macrophages in the mice. Twelve hours after tail vein administration, the mice were euthanized, and the tumors were removed. To observe the intratumoral exocytosis of CRM-Lips@C6, the tumor tissue was embedded in a Tissue-Tek OCT complex, rapidly frozen, and sectioned into 5 μm sections. These sections were then subjected to CD68 (labeled macrophages) immunofluorescence staining, blocked with a DAPI-containing fluorescent blocking agent, and observed under a laser confocal microscope (FV3000, Olympus, Japan).

[0127] The tumor-targeting ability of liposomes was examined using 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 "hitch a ride" on monocytes in vivo to enter tumor tissue and has a stronger distribution at the tumor site. Figure 5 C).

[0128] Co-localization analysis of DiD (green) and CD68 (red) in tumor sections showed that CRM-Lips@DiD (green) had excellent co-localization with macrophages (red) in the tumor site, and the tumor distribution was much higher than in the CLP+CRM-Lips group. This indicates that CRM-Lips can "hitch a ride" on monocytes to penetrate deep into pancreatic cancer tumors and improve intratumoral accumulation. Figure 5 D).

[0129] Because CRM-Lips@DA can only be taken up and utilized by key immune-related cells in the tumor microenvironment after intratumoral exocytosis of the STING agonist DA, the tumor exocytosis of the drug can be determined by examining the co-localization of C6 (green) and CD68 (red). The C6 (green) fluorescence intensity of CLP+CRM-Lips@C6 was significantly lower than that of the other two groups, indicating that liposomes rely on monocytes to enter the tumor tissue. In the non-ROS-responsive CM-Lips@C6 liposome group, most of the C6 fluorescence signal 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 signal was distributed around CD68, indicating a large amount of exocytosis. These results indicate that after CRM-Lips@C6 "hitchhiks" with 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, where it can be utilized by other cells in the microenvironment. Figure 5 EF).

[0130] Verification Example 5

[0131] A mouse model of orthotopic pancreatic cancer was established using the method described above. Mice were randomly divided into PBS, free DA, and CRM-Lips@DA (n=3). DA was administered three times via tail vein at a dose of 2 mg / kg. Figure 6 A). The tumor was removed, embedded in a Tissue-Tek OCT complex, rapidly frozen, and sectioned into 7μm sections. It was fixed with 4% paraformaldehyde at room temperature for 15 min, permeated with 0.2% Triton X-100 for 10 min, blocked with goat serum for 1 h, and subjected to HIF-1α immunofluorescence staining. The sections were then observed using a laser confocal microscope (FV3000, Olympus, Japan).

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

[0133] The above method was used to construct an orthotopic pancreatic cancer mouse model. After treatment, the tumor was removed and stained according to M1 / M2 macrophages, DC cells, 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 a severely 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 tumor HIF-1α expression, indicating that CRM-Lips@DA can alleviate tumor hypoxia and may improve hypoxia-induced immunosuppression. Figure 6 B).

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

[0136] STING agonists can remodel the tumor immune microenvironment and restore the tumor immune response. Pancreatic cancer contains a high proportion of M2 (pro-tumor) macrophages, while lacking T cell infiltration. Therefore, this study investigated changes in cell subtypes within tumor tissue after cyclical treatment to assess the in vivo immune activation effect of CRM-Lips@DA. After cyclical CRM-Lips@DA treatment, compared to the PBS control group, CD86 expression in pancreatic cancer mouse tumors increased 14.4-fold, indicating that cyclical liposome treatment significantly promoted M1 macrophage expression; simultaneously, CD206 expression significantly decreased, indicating that cyclical liposome treatment significantly reduced the number of M2 macrophages. Figure 6 D / E).

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

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

[0139] Verification Example 6

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

[0141] Tumor tissue was fixed overnight in 4% paraformaldehyde, dehydrated for 2 days, embedded in paraffin, and sectioned. To determine the morphological changes, apoptosis rate, and proliferative activity of tumor cells, the sections were stained with H&E, analyzed by TUNEL, and immunohistochemically stained with Ki67, and observed under a microscope.

[0142] Systemic injection of STING agonists may distribute to normal tissues, causing the secretion of large amounts of pro-inflammatory cytokines and resulting in severe toxic side effects. Therefore, this study investigated whether liposome CRM-Lips@DA could target and deliver the STING agonist DA, thereby reducing the drug's immunotoxicity. Healthy C57 mice were administered the drug once as described above. Blood was collected from each group of mice, and serum was separated. The levels of IL-6 and TNF-α in the five treatment groups (PBS, free DA, CRM-Lips@DA, PD-1 antibody group, and CRM-Lips@DA+PD-1 antibody) were detected by enzyme-linked immunosorbent assay (ELISA).

[0143] To assess systemic toxicity, mouse body weight was recorded every 4 days during the treatment period. After treatment, blood samples were collected from mice, plasma was separated, and complete blood count and blood biochemistry analyses were performed to assess blood toxicity. Simultaneously, major organs (heart, liver, spleen, lungs, and kidneys) were removed from the mice, stained with H&E, and their morphology was observed under a microscope to assess organ toxicity.

[0144] The in vivo fluorescence and corresponding quantitative fluorescence curves of the tumors captured during treatment showed that the tumors grew rapidly in the PBS and DA monotherapy groups, while the PD-1 antibody group exhibited 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 other groups, indicating that CRM-Lips@DA can significantly inhibit pancreatic cancer growth by activating the tumor immune response. Figure 8 B / C). To more intuitively observe the tumor treatment effect, mouse tumors were removed and photographed. The results further confirmed that CRM-Lips@DA and CRM-Lips@DA+PD-1 antibodies had smaller tumor volumes and better therapeutic effects compared to other groups. Figure 8 D). Except for the CRM-Lips@DA and CRM-Lips@DA+PD-1 antibody combination therapy groups, all other groups showed high-intensity metastatic signal, especially in the mesentery. Figure 8 E). These results indicate that the combination therapy of CRM-Lips@DA and CRM-Lips@DA+PD-1 antibody can not only inhibit the growth of tumors in situ, but also significantly inhibit tumor metastasis.

[0145] H&E staining images of the tumors showed that the CRM-Lips@DA combined with PD-1 antibody treatment group resulted in more nuclear damage and cytoplasmic degradation than other groups. Figure 7F). Furthermore, TUNEL and Ki67 staining results showed that the CRM-Lips@DA combined with PD-1 antibody treatment group had the highest level of apoptosis and the lowest proliferative activity, confirming that the combination therapy had superior anti-tumor efficacy. Figure 8 GJ).

[0146] TNF-α and IL-6 levels confirmed that injection of free dopamine (DA) led to increased secretion of both, while liposome encapsulation significantly reduced the abnormal secretion of inflammatory factors induced by DA and decreased immunostimulation. Figure 9 A). Mice showed no significant change in body weight after cyclical treatment with CRM-Lips@DA or CRM-Lips@DA+PD-1 antibody, indicating that the liposomes and combination therapy had negligible systemic toxicity. Figure 9 B). Meanwhile, the results of routine blood tests and blood biochemistry analyses were all within the normal range, indicating that the liposomes and combination therapy did not cause blood toxicity. Figure 9 C / D). H&E staining results of organs in mice from each treatment group showed no significant abnormalities in cell morphology across all organs in the treatment groups, indicating that the liposomes and combination therapy have negligible organ toxicity. Figure 9 E).

[0147] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any 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 invention should be within the protection scope of the present invention.

Claims

1. A method for preparing mononuclear cell-borne oxygen-producing liposomes, characterized in that, Includes the following steps: S1: A lipopeptide CP with a cysteine ​​terminal and a lipid RL containing a thioketal domain were synthesized by solid-phase polypeptide synthesis. Thylakoid membrane TK was extracted. The thylakoid membrane TK is a biomembrane structure capable of catalyzing ROS oxygen production. S2: The lipid components and lipopeptide CP are dissolved and mixed evenly using a first organic solvent. The lipid RL and STING agonist DA are dissolved and mixed evenly using a second organic solvent. The two solutions are mixed evenly to obtain a mixed solution. Then, the organic solvent in the mixed solution is removed by a thin-film hydration method to form a uniform lipid film. The DA is 5,6-dimethylxanthine-4-acetic acid. S3: Add an aqueous solution to the lipid membrane for hydration to form a crude liposome suspension. Sonicate the crude liposome suspension to reduce the size of the liposomes and obtain a preliminary liposome structure CR-Lips@DA that can encapsulate the STING agonist DA. S4: The thylakoid membrane TK was fused to the liposome CR-Lips@DA using an ultrasonic-extrusion method to obtain the liposome product CRM-Lips@DA, which has mononuclear cell targeting, can release drugs in response to ROS, and has oxygen production function. The mass ratio of the liposome CR-Lips@DA to the thylakoid membrane TK fusion is 1~10:1; 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; In S2, the specific preparation process of the mixed solution includes: The lipid components containing DOTAP, cholesterol, soybean lecithin, DSPE-PEG2000 and lipopeptide CP are added to chloroform solvent and vortexed to fully dissolve and mix them evenly to form a homogeneous lipid-lipopeptide CP chloroform solution. After accurately weighing the lipid RL and the STING agonist DA, add them to the methanol solvent, vortex to fully dissolve and mix them evenly to form a homogeneous 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 ensure thorough and uniform mixing, ensuring full contact between the components to form a homogeneous 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.

2. The method for preparing a mononuclear cell-loaded 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-containing lipid RL via solid-phase polypeptide synthesis includes: Fmoc-protected amino acids were sequentially linked to a solid support. The Fmoc protecting groups of each amino acid were removed one by one using a deprotection reagent. Then, the next amino acid was added sequentially. The operation was repeated until the sequence construction of the target lipopeptide CP and lipid RL was completed. Cysteine ​​residue was introduced at the end of the lipopeptide CP and the cysteine ​​residue was correctly linked to the already constructed amino acid sequence. In the synthesis of lipid RL, the thioketal domain is directly introduced into the reaction. After synthesis, the lipopeptide CP and lipid RL were cleaved from the solid support using a cleavage reagent and then purified to remove unreacted reagents and byproducts, ultimately yielding the lipopeptide CP with a cysteine ​​terminus and the lipid RL containing a thioketal domain.

3. The method for preparing a mononuclear cell-borne oxygen-producing liposome according to claim 1, characterized in that, In S1, the specific process for extracting TK from the thylakoid membrane includes: Spinach leaves were placed in pre-cooled HEPES buffer and homogenized thoroughly in a mortar. After filtration, the filtrate was collected and centrifuged. The intact chloroplasts in the supernatant were collected and resuspended in hypotonic buffer for a preset time. The matrix extract was removed by centrifugation to obtain thylakoid-containing particles. The precipitate was washed with HEPES buffer, sonicated, and centrifuged to remove the lumen extract to obtain the green thylakoid membrane TK.

4. The method for preparing a mononuclear cell-borne oxygen-producing liposome according to claim 3, characterized in that, In S2, the specific process of removing organic solvents from the mixed solution and forming a uniform lipid membrane by thin-film hydration includes: The well-mixed solution was transferred to a container and placed on a rotary evaporator. Rotary evaporation was carried out at 38°C and a vacuum of -0.1 MPa to evaporate and remove the organic solvent, and the solution gradually formed a uniform lipid film.

5. The method for preparing a mononuclear cell-borne oxygen-producing liposome according to claim 1, characterized in that, In S3, an aqueous solution is added to the lipid membrane for hydration to form a crude liposome suspension. The crude liposome suspension is then subjected to ultrasonic treatment to reduce the liposome size. The specific process includes: A suitable amount of pure water was slowly added to the lipid membrane formed after the organic solvent was removed by the membrane hydration method, while shaking was performed to allow the lipid membrane to fully contact the water and undergo hydration, gradually forming a crude lipid body suspension. The obtained crude liposome suspension was placed in an ice bath and sonicated using a cell disruptor to uniformly disperse the liposome particles and reduce their size, ultimately yielding a liposome CR-Lips@DA suspension with relatively uniform particle size.

6. The method for preparing a mononuclear cell-borne oxygen-producing liposome according to claim 1, characterized in that, In S4, the specific process of fusing thylakoid membrane TK to liposomes CR-Lips@DA using the ultrasonic-extrusion method includes: The extracted thylakoid membrane TK and liposome CR-Lips@DA were placed in the same solution and mixed thoroughly. The mixture was then placed in an ice bath and pre-treated with ultrasound to allow the TK membrane and liposomes to initially contact and fuse. The mixture was then extruded through a polycarbonate membrane with a pore size of 400 nm, allowing the mixture to pass through the membrane pores under pressure, thereby promoting the fusion of the TK membrane and liposomes. 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, which has uniform particle size, mononuclear cell targeting, ROS-responsive drug release, and oxygen production function.

7. A mononuclear cell-borne oxygen-producing liposome prepared by the method according to any one of claims 1 to 6, characterized in that, The liposomes CRM-Lips@DA have a particle size of 100~200 nm and a surface potential of 30~40 mV; The liposome CRM-Lips@DA encapsulates the STING agonist DA with a success rate of greater than 90%. The liposome CRM-Lips@DA can rapidly release the drug DA under ROS stimulation and produce oxygen from ROS through TK catalysis of the thylakoid membrane.

8. An application of the liposomes as described in claim 7, characterized in that, The liposomes are used to prepare drugs for treating tumors. The liposome CRM-Lips@DA can deliver the STING agonist DA to the deep part of the tumor, activate the STING pathway in the tumor, repolarize M2 macrophages to M1, promote dendritic cell maturation and cytotoxic T cell infiltration, thereby enhancing the anti-tumor immune response. It can also relieve tumor hypoxia by producing oxygen, reverse the immunosuppressive tumor microenvironment, and inhibit tumor growth and metastasis. The tumor is pancreatic cancer.

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