Liposome STING agonist delivery system based on PD-L1 antibody as well as preparation method and application of liposome STING agonist delivery system

Abstract

The invention provides a lipidosome STING agonist delivery system based on a PD-L1 antibody as well as a preparation method and application of the lipidosome STING agonist delivery system, and belongs to the technical field of medical biology. Preparing lipidosome by adopting an ethanol injection method and an ammonium sulfate gradient technology; the PD-L1 antibody and the STING agonist can be loaded at the same time; the liposome is prepared by adopting an ethanol injection method and an ammonium sulfate gradient technology, and the STING agonist can be wrapped in the liposome in an active drug loading manner; dSPE-PEG2000-NHS and a PD-L1 antibody are mixed and incubated according to a specific proportion by utilizing a post-insertion method to form an antibody conjugated micelle, and the antibody conjugated micelle is fused with a blank liposome to realize antibody modification; secondly, the nano-liposome has good drug carrier characteristics and can effectively protect the loaded drug, reduce the risk of enzymolysis of the drug and improve the stability of the drug in vivo, so that the liposome drug delivery system simultaneously loading the nano-liposome and the nano-liposome is successfully prepared.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medical biotechnology, and particularly relates to a liposome-based STING agonist delivery system based on a PD-L1 antibody and a preparation method and application thereof. BACKGROUND

[0002] As a new treatment method, tumor immunotherapy has higher specificity, smaller side effects, and more durable efficacy than traditional methods such as surgery, radiotherapy, and chemotherapy, and has made a revolutionary breakthrough in cancer treatment. Among them, immune checkpoint inhibitors have achieved remarkable clinical effects, but they also face some challenges such as immune-related adverse reactions, tumor immune escape, etc. STING (stimulator of interferon genes) is a key adaptor protein in the intracellular innate immune signaling pathway, mainly involved in the production of type I interferons and the activation of immune cells. Activation of the STING pathway can promote the maturation and antigen-presenting function of dendritic cells, enhance the activity of cytotoxic T cells, and play an anti-tumor role. In addition, the STING pathway can also regulate the tumor immune microenvironment and inhibit the activity of immune suppressor cells, further enhancing the anti-tumor immune response. STING agonists may cause immune-related adverse reactions when administered systemically, limiting their clinical application. Therefore, developing a STING agonist delivery system that can precisely target tumor tissues is crucial to improving its efficacy and safety. SUMMARY

[0003] The application provides a liposome-based STING agonist delivery system based on a PD-L1 antibody and a preparation method and application thereof to solve the problems in the prior art.

[0004] To achieve the above-mentioned purposes, the technical solution adopted by the present application is as follows: A liposome-based STING agonist delivery system based on a PD-L1 antibody comprises a nano-liposome loaded with a PD-L1 antibody and a STING agonist; the nano-liposome comprises an outer shell and an inner core; the outer shell comprises a closed vesicle formed by a lipid bilayer membrane, a hydrophilic protective layer formed by coupling PEG chains on the outer layer, and a PD-L1 antibody reacted with a NHS ester; the PD-L1 antibody is anchored on the surface of the liposome; and the inner core comprises a STING agonist.

[0005] Preferably, the STING agonist is an amphiphilic weakly basic STING agonist small molecule compound.

[0006] A preparation method of a liposome-based STING agonist delivery system based on a PD-L1 antibody is as follows: Step one: the hydrogenated soybean phosphatidylcholine, cholesterol, methoxy polyethylene glycol distearoyl phosphatidyl ethanolamine is dissolved in an organic solvent, then injected into the preheated ammonium sulfate solution for incubation, to prepare the pretreatment product multilamellar vesicles; Step two: the multilamellar vesicles are pushed by the extruder using the microfiltration membrane group to obtain the blank liposome; Step three: the PD-L1 antibody is mixed with 1,2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine-N-[succinimidyl (polyethylene glycol)-2000] and incubated in 0.1 M phosphate buffer solution at room temperature to react, to prepare the nanobody micelles; Step four: the nanobody micelles are added to the blank liposome, and the antibody-based liposome-like fusion is obtained by incubation; Step five: the antibody-based liposome-like fusion is dialyzed in the phosphate buffer solution to remove ammonium sulfate, and then incubated with the mixture of the STING agonist dissolved in 0.1 M phosphate buffer solution to obtain the nanoliposome.

[0007] As preferred, in step one, the molar ratio of the materials used for preparing the blank liposome, hydrogenated soybean phosphatidylcholine HSPC, cholesterol and methoxy polyethylene glycol distearoyl phosphatidyl ethanolamine DSPE-mPEG2000, is 55-60:39.3:4; the preheating temperature of the ammonium sulfate solution is 55-65℃; the concentration of the ammonium sulfate solution is 200-300 mM; and the incubation time in the preheated ammonium sulfate solution is 28-32 min.

[0008] As preferred, in step two, the microfiltration membrane group is at least two layers of microfiltration membranes with decreasing pore sizes in the extruder pushing direction; and the microfiltration membrane material is polycarbonate membrane, polyethersulfone filter membrane, cellulose membrane or nylon membrane.

[0009] As preferred, the microfiltration membrane pore size includes 50-200 nm; and the extruder pushing frequency is 9-13 times.

[0010] As preferred, in step three, the molar ratio of 1,2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine-N-[succinimidyl (polyethylene glycol)-2000] mixed with the antibody is 5:1-20:1; and the incubation time in the 0.1 M phosphate buffer solution is 1.8-2.4 h.

[0011] As preferred, the molar ratio of the nanobody micelles to the blank liposome is 1:35-40; the incubation temperature is 55-65℃; and the incubation time is 50-70 min.

[0012] Preferably, in step five, the number of dialysis of the antibody-based liposome-like fusion is 2-5 times; the antibody-liposome-STING agonist combination incubation time is 30 min-60 min; and the incubation temperature is 45℃-65℃.

[0013] The application of a PD-L1 antibody-based liposome-like STING agonist delivery system in the preparation of a tumor treatment drug.

[0014] Compared with the prior art, the application has the advantages and positive effects that: The PD-L1 antibody-based liposome-like STING agonist delivery system, the preparation method and the application thereof adopt the ethanol injection method and the ammonium sulfate gradient technology to prepare liposomes, can simultaneously load the PD-L1 antibody and the STING agonist, adopt the ethanol injection method and the ammonium sulfate gradient technology to prepare liposomes, can wrap the STING agonist in the liposomes by the active drug loading mode, and realizes the antibody modification by mixing and incubating the DSPE-PEG2000-NHS and the PD-L1 antibody at a specific ratio by using the post-insertion method, and then fusing the antibody conjugated micelles with blank liposomes, and secondly, the nanoliposome has good drug carrier characteristics, can effectively protect the loaded drug, reduces the risk of enzymatic hydrolysis of the drug, improves the stability of the drug in the body, and thus successfully prepares the liposome drug delivery system simultaneously loading the two drugs. In the application, the liposome is used as a drug delivery carrier, and the liposome is not only a simple drug carrier in the research, but also realizes the protection of drug activity and the improvement of stability through targeted modification, synergistic delivery and process optimization. After the drug is wrapped by the liposome, the exposure to normal tissues is reduced, and the systemic toxicity is reduced. The liposome wrapping protects the STING agonist from enzymatic hydrolysis or rapid clearance, and promotes the entry of the STING agonist into the cytoplasm through endocytosis or membrane fusion. For hydrophilic or hydrophobic drugs, the liposome can improve the solubility and bioavailability of the drugs. The liposome can simultaneously carry different kinds of therapeutic agents (such as antibodies and small molecule agonists), realize combined treatment, precise delivery and multiple goals of immune microenvironment regulation. In the application, the PD-L1 antibody is mainly used for tumor cells expressing PD-L1, and the damage to normal cells is relatively small, so that the anti-tumor effect can be more accurately exerted, the side effects on other normal tissues and organs of the body are reduced, and the safety and tolerability are good. The STING agonist used in the application is used as an effective payload part, the STING agonist activates the core pathway of innate immunity, enhances adaptive immunity, converts “cold tumors” into “hot tumors”, remodels the tumor microenvironment and establishes systemic anti-tumor immunity, and is a breakthrough strategy for tumor immunotherapy. The liposome STING agonist delivery system based on the PD-L1 antibody has a small particle size (about 135 nm) after the antibody is coupled and the drug is loaded, is uniformly distributed in a solution, has good dispersibility and high stability; the liposome STING agonist delivery system based on the PD-L1 antibody, such as alphaPD-L1-SaLP (liposome in which the PD-L1 antibody is coupled and the STING agonist diABzi is loaded), can specifically bind to MC-38 cells (colon adenocarcinoma cells), induce high expression of PD-L1 after IFN-gamma induction, has high binding specificity and binding efficiency; after the liposome STING agonist delivery system based on the PD-L1 antibody, such as alphaPD-L1-SaLP, targets a tumor, the mRNA levels of IFN-beta and IL-6 in the tumor tissue are significantly increased, the expression of IFN-beta protein is increased, and it is indicated that the STING pathway is activated; the liposome STING agonist delivery system based on the PD-L1 antibody, such as alphaPD-L1-SaLP, can induce polarization of bone marrow-derived macrophages to M1 type (proinflammatory, anti-tumor phenotype), which is manifested as up-regulation of expression of TNF-alpha and iNOS and inhibition of expression of YM1 (M2 marker); the mouse body weight does not significantly decrease during treatment with the liposome STING agonist delivery system based on the PD-L1 antibody, such as alphaPD-L1-SaLP, and it is indicated that the preparation system has low toxicity.

[0015] In summary, the liposome STING agonist delivery system based on the PD-L1 antibody has significant advantages in anti-tumor treatment through innovative combined therapy design, rigorous experimental verification and in-depth mechanism exploration, not only provides a potential new therapy for cellular immunotherapy in anti-tumor treatment, but also provides an important reference for development of immunotherapy strategies for other solid tumors. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0017] Figure 1 The figure is a result graph of determination of antibody concentration before and after coupling by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); Figure 2 The figure is a change graph of liposome particle size, PDI (polydispersity index) and zeta potential of alphaPD-L1-SaLP before and after coupling; Figure 3 The figure is a schematic diagram of cytotoxicity test results; Figure 4Figure showing the results of evaluating the targeting specificity of PD-L1 antibodies after αPD-L1-SaLP acting on tumor cells; Figure 5 Figure showing the immune response in the tumor microenvironment after immunotherapy; Figure 6 Figure showing the change in IL-6 transcription level in the tumor after αPD-L1-SaLP treatment; Figure 7 Figure showing the change in IFN-β transcription level in the tumor after αPD-L1-SaLP treatment; Figure 8 Figure showing the change in IFN-β protein level in the tumor after αPD-L1-SaLP treatment; Figure 9 Figure showing the expression results of YM1, TNF-α, iNOS and ARG-1 in bone marrow-derived macrophages (BMDM) at different stages after αPD-L1-SaLP treatment. DETAILED DESCRIPTION

[0018] In order to enable a clearer understanding of the above-mentioned objects, features and advantages of the present application, the present application will be further described below with reference to the drawings and examples. It should be noted that the examples of the present application and the features in the examples can be combined with each other without conflict.

[0019] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be practiced in a variety of ways beyond the specific details set forth herein, and, accordingly, the present application is not limited to the specific embodiments disclosed below.

[0020] Example 1, a PD-L1 antibody-based liposome STING agonist delivery system and its preparation method and application.

[0021] STING (stimulator of interferon genes) is a key adaptor protein in the intracellular innate immune signaling pathway, mainly involved in the production of type I interferon and the activation of immune cells. Activation of the STING pathway can promote the maturation and antigen presentation function of dendritic cells, enhance the activity of cytotoxic T cells and play an anti-tumor role. In addition, the STING pathway can also regulate the tumor immune microenvironment, inhibit the activity of immune suppressor cells, and further enhance the anti-tumor immune response. STING agonists may cause immune-related adverse reactions when administered systemically, limiting their clinical application. Therefore, the development of a STING agonist delivery system that can precisely target tumor tissues has become a key to improving its efficacy and safety.

[0022] PD-L1 antibody precisely targets tumor cell PD-L1 protein, blocks PD-1 / PD-L1 immune inhibition pathway, releases T cell function inhibition; activates adaptive immunity, induces persistent anti-tumor response; is suitable for a variety of PD-L1 high expression solid tumors, and can be combined with chemotherapy, radiotherapy or other immunotherapy to enhance efficacy; guided by biomarkers (such as PD-L1 expression, TMB), improve precision, while having controllable safety.

[0023] Liposomes are nanoscale vesicles composed of phospholipid bilayers, which have good biocompatibility and drug loading capacity.

[0024] The combination of PD-L1 antibody-liposome technology and STING agonist can construct PD-L1 antibody liposome STING agonist conjugate, which is expected to overcome the limitations of STING agonist in systemic administration. Through the targeting of antibodies, STING agonists can be precisely delivered to tumor tissues, reducing distribution in normal tissues and reducing the risk of immune-related adverse reactions. At the same time, after the release of STING agonists in tumor tissues, immune cells can be activated, and anti-tumor immune response can be enhanced, and the tumor immune microenvironment can be reshaped. Therefore, the present application proposes a kind of liposome-based STING agonist delivery system based on PD-L1 antibody and its preparation method and application.

[0025] A kind of liposome-based STING agonist delivery system based on PD-L1 antibody, including nano-liposome loaded with PD-L1 antibody and STING agonist;The nano-liposome includes shell and core;The shell includes closed vesicle formed by lipid bilayer membrane, hydrophilic protective layer formed by outer layer coupling PEG chain and PD-L1 antibody and NHS ester reaction;The anchor is on the surface of liposome;The core includes STING agonist;The STING agonist is amphiphilic weak basic STING agonist small molecule compound; A preparation method of a liposome-based STING agonist delivery system based on PD-L1 antibody, the specific preparation method is as follows: Step one: the hydrogenated soybean phosphatidylcholine, cholesterol, methoxy polyethylene glycol distearoylphosphatidyl ethanolamine is dissolved in an organic solvent, then injected into the preheated ammonium sulfate solution for incubation, to prepare the pretreatment product multilamellar vesicles; the molar ratio of the materials used in the preparation of blank liposomes, hydrogenated soybean phosphatidylcholine HSPC, cholesterol and methoxy polyethylene glycol distearoylphosphatidyl ethanolamine DSPE-mPEG2000 is 55-60:39.3:4; preferably, such as 55:39.3:4, 55.2:39.3:4, 55.5:39.3:4, 58:39.3:4, 60:39.3:4; in this application, the most preferred is 55.5:39.3:4; the preheating temperature of the ammonium sulfate solution is controlled at 55-65℃; the concentration of the ammonium sulfate solution is 200-300mM; preferably, such as 200mM, 220mM, 250mM, 280mM, 300mM; in this application, the most preferred is 250mM; the incubation time in the preheated ammonium sulfate solution is 28-32min; preferably, 28min, 30min, 32min; in this application, the most preferred is 30min; Step two: the multilamellar vesicles are pushed and extruded by a microfilter membrane group through an extruder to obtain blank liposomes; the microfilter membrane group is at least two layers of microfilter membranes with decreasing pore size in the extrusion direction of the extruder; it aims to separate multilamellar vesicles of different sizes to obtain a uniform size vesicle population; the material of the microfilter membrane is polycarbonate membrane, polyethersulfone filter membrane, cellulose membrane or nylon membrane; the pore size of the microfilter membrane includes 50-200nm; in this application, the microfilter membrane is three, from large to small, 200nm, 100nm and 50nm; the extrusion frequency of the extruder is 9-13 times; preferably, 7-11 times; in this application, 8 times; Step three: the PD-L1 antibody is mixed with 1,2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine-N-[succinimidyl (polyethylene glycol)-2000] and incubated at room temperature in 0.1M phosphate buffer at pH=0.7 to undergo addition reaction to prepare nanobody micelles; the molar ratio of 1,2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine-N-[succinimidyl (polyethylene glycol)-2000] mixed with the antibody is 5:1-20:1; preferably, such as 5:1, 10:1, 20:1, in this application, the most preferred is 10:1; the incubation time in 0.1M phosphate buffer is 1.8-2.4h; preferably, 1.8h, 2h, 2.2h, 2.4h, in this application, the most preferred is 2h; Step four: adding the nanobody micelles into the blank liposomes to obtain the antibody-based liposome-like fusion by incubation; the molar ratio of the nanobody micelles to the blank liposomes is 1:35-40; the incubation temperature is 55-65°C; preferably, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, and most preferably, 60°C in this application; the incubation time is 50-70 min; preferably, 50 min, 60 min, 70 min, and most preferably, 60 min in this application; Step five: removing the ammonium sulfate from the antibody-based liposome-like fusion by dialysis in a phosphate buffer, and then incubating the mixture with the STING agonist dissolved in the 0.1 M phosphate buffer to obtain the nanoliposomes; the nanoliposomes are the antibody-coupled STING agonist-loaded liposome-like drug delivery system; the dialysis frequency of the antibody-based liposome-like fusion is 2-5 times; preferably, 3 times in this application; the antibody-liposome-STING agonist combination incubation time is 30-60 min; preferably, 30 min, 40 min, 50 min, 60 min; most preferably, 40 min in this application; the incubation temperature is 45-65°C; preferably, 60°C in this application.

[0026] Experiment one: the physical and chemical properties and the antibody and drug loading efficiency of the nanoliposomes of Example 1 were determined as shown in Table 1. Figure 1 As shown in Table 1, the dynamic light scattering measured the particle size of the blank liposomes (LP) without antibody connection as 89.45±0.26 nm, PDI=0.14, and zeta potential=-23.40 mV. After the antibody connection (aPD-L1-LP), the particle size increased to 92.66±0.34 nm, and the zeta potential rose to-17.73 mV, indicating that the antibody was successfully connected. After loading diABzi (aPD-L1-SaLP), the particle size increased to 136.63±1.89 nm, PDI=0.48, and zeta potential=-20.80 mV; SDS-PAGE showed that the antibody stably existed on the surface of the liposomes.

[0027] Experiment two: cytotoxicity test of the nanoliposomes of Example 1 (aPD-L1-SaLP: PD-L1 antibody-coupled and STING agonist diABzi-loaded liposomes), LP: blank liposomes, aPD-L1-LP: PD-L1 antibody-coupled liposomes, SaLP: STING agonist diABzi-loaded liposomes, and free drugs: MC-38 (colon adenocarcinoma cells) and 4T1 (breast cancer cells) were inoculated into 96-well plates, respectively; after incubation with the CCK-8 reagent, the 450 nm absorbance was measured and the relative cell survival rate (RCV) was calculated. The results are shown in Table 2. Figure 2 , Figure 3Figure 1 shows the results of the experiment, wherein Figure 2 Figure 1 shows the results of the experiment, wherein The results show that LP and aPD-Ll-LP at 1 mg / ml show significant toxicity on 4T1 cells (RCV ~ 70%). aPD-Ll-SaLP at 5 μg / ml significantly inhibits cell activity (RCV ~ 50%), indicating that the liposome is safe at a reasonable dose, but 4T1 is more sensitive to the liposome.

[0028] Experiment Three: Detection of whether the PD-L1 antibody specifically binds to the PD-L1 receptor on the surface of MC-38 cells, wherein the MC-38 cells are pretreated with IFN-γ for 16 hours to induce PD-L1 expression; the cells are treated with a PD-L1 antibody or a HER2 antibody (isotype control), respectively; the fluorescence signal after antibody binding is observed by immunofluorescence microscopy; the results are shown in Figure 4 Figure 2, wherein Figure 4 Figure 2 shows the results of the experiment, wherein

[0029] Experiment Four: Detection of the anti-tumor effect of the target aPD-Ll-SaLP on the MC-38 colon cancer model: C57BL / 6 mice are subcutaneously inoculated with MC-38 cells, and when the tumor volume reaches 90 mm 3 After 3 days, the mice are grouped for treatment.

[0030] The groups are PBS, free diABzi, LP, free combination (diABzi + aPD-Ll), aPD-Ll-LP, free aPD-Ll, SaLP, and aPD-Ll-SaLP. The results are shown in Figure 5As shown, the αPD-L1-SaLP group had significantly lower levels than other groups. No significant decrease in body weight was observed in any of the treatment groups, indicating low toxicity of the formulation; this suggests that αPD-L1-SaLP exhibits the strongest antitumor activity by synergistically activating innate and adaptive immunity.

[0031] Experiment 5: Detection of expression levels of immune-related genes (IFN-β, IL-6) in tumor tissue: RNA and protein were extracted from tumor tissue after treatment; the mRNA expression of IFN-β and IL-6 was detected by qPCR; the IFN-β protein level was measured by ELISA; the results are as follows. Figure 6 , Figure 7 , Figure 8 As shown, the mRNA expression of IFN-β and IL-6 was significantly upregulated in the αPD-L1-SaLP group. The IFN-β protein concentration was significantly increased in the αPD-L1-SaLP group, indicating that αPD-L1-SaLP promotes the secretion of type I interferon and pro-inflammatory factors by activating the STING pathway, thereby enhancing anti-tumor immunity.

[0032] Experiment 6: Detecting the effect of target αPD-L1-SaLP on macrophage polarization (M1 / M2 phenotype): Mononuclear cells were isolated from mouse bone marrow and differentiated into M0, M1 (LPS+IFN-γ induced) or M2 (IL-4 induced); BMDM were treated with αPD-L1-SaLP. The results are as follows Figure 9 As shown, αPD-L1-SaLP upregulates TNF-α and ARG-1 (M1 markers) and inhibits YM1 (M2 markers); indicating that αPD-L1-SaLP induces macrophage polarization towards the pro-inflammatory M1 type, enhancing the anti-tumor activity of the tumor microenvironment. Based on the results of Experiments 1-6 above, six core experimental systems verified that the liposome-antibody-STING agonist produced using the liposome drug delivery system method of this invention possesses low toxicity, high targeting, and drug protection capabilities. Its anti-tumor effect stems from the synergistic effect of PD-L1 antibody blocking immune checkpoints and STING agonist activating innate immunity. By inducing type I interferon secretion and macrophage M1 polarization, the tumor microenvironment is reshaped, reversing "cold tumors" into "hot tumors," thus confirming the application of the PD-L1 antibody-based liposome-based STING agonist delivery system of this application in the preparation of therapeutic tumor drugs.

[0033] The above merely describes preferred embodiments of the present application, but does not limit the present application to other forms, and any person skilled in the art can make changes or modifications to the above disclosed technical contents into equivalent embodiments with equivalent changes, and apply to other fields. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application without departing from the technical solution content of the present application still belongs to the protection scope of the technical solution of the present application.

Claims

1. A liposomal STING agonist delivery system based on PD-L1 antibody, characterized in that, The invention comprises nanoliposomes loaded with PD-L1 antibody and STING agonist; the nanoliposomes include a shell and a core; the shell comprises a closed vesicle formed by a lipid bilayer membrane, a hydrophilic protective layer formed by an outer layer coupled with PEG chains, and a layer formed by the reaction of PD-L1 antibody with NHS ester; the core is anchored on the surface of the liposome; the core comprises STING agonist.

2. The liposome-based STING agonist delivery system based on PD-L1 antibody according to claim 1, characterized in that, The STING agonist is an amphiphilic, weakly basic, small molecule compound of STING agonist.

3. The method for preparing a liposomal STING agonist delivery system based on a PD-L1 antibody according to claim 2, characterized in that, The specific preparation method is as follows: Step 1: Hydrogenated soybean phosphatidylcholine, cholesterol, and methoxy polyethylene glycol distearate phosphatidylethanolamine are dissolved in an organic solvent and then injected into a preheated ammonium sulfate solution for incubation to obtain a pretreated multilayer vesicle. Step 2: Use a microfiltration membrane module to push multiple layers of vesicles through a pusher to obtain blank liposomes; Step 3: Mix PD-L1 antibody with 1,2-distearate-SN-glycerol-3-phosphatidylethanolamine-N-[succinimide (polyethylene glycol)-2000] and incubate at room temperature in 0.1 M phosphate buffer to prepare nanobody micelles; Step 4: Add nano-antibody micelles to blank liposomes and incubate to obtain antibody-based liposome fusion products; Step 5: Dialyze the antibody-based liposome fusion in phosphate buffer to remove ammonium sulfate, and then incubate it with a mixture of STING agonist dissolved in 0.1 M phosphate buffer to obtain nanoliposomes.

4. The method for preparing a liposomal STING agonist delivery system based on a PD-L1 antibody according to claim 3, characterized in that, In step one, the molar ratio of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, and methoxy polyethylene glycol distearate acylphosphatidylethanolamine (DSPE-mPEG2000) used in preparing blank liposomes was 55-60:39.3:4; the preheating temperature of the ammonium sulfate solution was 55℃-65℃; the concentration of the ammonium sulfate solution was 200mM-300mM; and the incubation time in the preheated ammonium sulfate solution was 28 min-32 min.

5. The method for preparing a liposomal STING agonist delivery system based on a PD-L1 antibody according to claim 3, characterized in that, In step two, the microfiltration membrane assembly consists of at least two layers of microfiltration membranes with pore sizes decreasing sequentially along the extrusion direction of the extruder; the microfiltration membrane material is polycarbonate membrane, polyethersulfone membrane, cellulose membrane, or nylon membrane.

6. A method for preparing a liposomal STING agonist delivery system based on a PD-L1 antibody according to claim 5, characterized in that, The microfiltration membrane has a pore size of 50 nm to 200 nm; the extruder pushes the membrane 9 to 13 times.

7. A method for preparing a liposomal STING agonist delivery system based on a PD-L1 antibody according to claim 3, characterized in that, In step three, the molar ratio of 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine-N-[succinimide (polyethylene glycol)-2000] to the antibody is 5:1 to 20:1; and the incubation time in 0.1 M phosphate buffer is 1.8 h to 2.4 h.

8. A method for preparing a liposomal STING agonist delivery system based on a PD-L1 antibody according to claim 3, characterized in that, In step four, the molar ratio of nanobody micelles to blank liposomes is 1:35~40; the incubation temperature is 55℃-65℃; and the incubation time is 50 min-70 min.

9. A method for preparing a liposomal STING agonist delivery system based on a PD-L1 antibody according to claim 1, characterized in that, In step five, the number of dialysis cycles for the antibody-based liposome fusion is 2-5; the antibody-liposome-STING agonist binding incubation time is 30-60 min; and the incubation temperature is 45℃-65℃.

10. The use of a PD-L1 antibody-based liposomal STING agonist delivery system according to any one of claims 1-2 in the preparation of a tumor therapeutic drug.

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