A povidone-iodine lipid nanoparticle and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a pyrvinium pamoate lipid nanoparticle and preparation and application thereof. The pyrvinium pamoate lipid nanoparticle comprises pyrvinium pamoate liposomes and / or pyrvinium pamoate lipogels. Phospholipids and cholesterol are selected as membrane materials to assemble and form liposome vesicle structures, and pyrvinium pamoate is encapsulated in the lipid bilayer to obtain the pyrvinium pamoate lipid nanoparticle. The pyrvinium pamoate lipid nanoparticle constructed by the application has excellent tumor targeting enrichment capacity; the loaded pyrvinium pamoate can improve the mechanical hardness of tumor cells, thereby enhancing the T cell-mediated tumor killing effect, and relying on the immune system-dependent pathway to play the tumor inhibition activity. The pyrvinium pamoate lipid nanoparticle can obviously enhance the anti-tumor immune response when combined with an immune checkpoint inhibitor and an adoptive T cell preparation, and realize the tumor immune sensitization combined treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pyravirin chloride lipid nanoparticle, its preparation method, and its application. Background Technology

[0002] Malignant tumors pose a serious threat to human life and health. Traditional treatment methods such as surgery, chemotherapy, and radiotherapy have many limitations, including significant trauma, substantial systemic toxicity, and a high risk of recurrence and metastasis. In recent years, tumor immunotherapy technologies, represented by immune checkpoint inhibitors and adoptive T-cell therapy, have made breakthrough progress, demonstrating significant efficacy in some hematological malignancies and a few solid tumors.

[0003] However, existing tumor immunotherapy systems still face many inherent technical bottlenecks that are difficult to overcome: immune checkpoint inhibitor monotherapy for solid tumors has limited response rates, and most patients develop acquired resistance in the later stages of treatment; when adoptive T-cell therapy is applied to solid tumors, the physical barriers and immunosuppressive properties of the tumor microenvironment severely hinder T-cell tumor infiltration and functional maintenance. Currently, clinically explored combination immunotherapy strategies mainly include the combination of immunotherapies, chemotherapy combined with immunotherapy, and radiotherapy combined with immunotherapy, but these strategies either suffer from systemic toxicity due to a lack of tumor selectivity or fail to effectively reshape the tumor immune microenvironment due to their single mechanism of action, resulting in limited clinical benefits. Currently, there is an urgent need in this field for an immunosensitizing agent that can effectively reverse the tumor immunosuppressive microenvironment and improve the response rate of immunotherapy for solid tumors. Summary of the Invention

[0004] The present invention aims to provide pyravirin chloride lipid nanoparticles, their preparation method, and applications. The lipid nanoparticles constructed by the present invention possess excellent tumor-targeting enrichment capabilities; the loaded pyravirin chloride can enhance the mechanical stiffness of tumor cells, thereby enhancing T cell-mediated tumor killing effects. These lipid nanoparticles can exert tumor-suppressive activity through an immune system-dependent pathway and can significantly improve the anti-tumor therapeutic effects of immune checkpoint inhibitors and adoptive T cell therapy.

[0005] This invention provides pyrethroid chloride lipid nanoparticles, comprising pyrethroid chloride liposomes and / or pyrethroid chloride lipid micelles; The pyrethrum chloride liposomes comprise a mixture of pyrethrum chloride and lipids; the lipid mixture comprises phospholipids, cholesterol, and a targeting ligand-lipid conjugate. The pyravir chloride lipid micelles comprise pyravir chloride, PEG-lipid, and a targeting ligand-lipid conjugate. The targeted ligand-lipid conjugates include DSPE-PEG2000-cRGD and / or DMG-PEG2000-cRGD.

[0006] As a preferred embodiment, in the pyrifos liposomes, the mass ratio of the lipid mixture to pyrifos is 5~20:1; based on the total molar number of the lipid mixture, the molar percentage of the phospholipid is 40%~70%, the molar percentage of the cholesterol is 25%~50%, and the molar percentage of the targeting ligand-lipid conjugate is 5%~10%. The drug loading of the pyrifos chloride lipid micelles is 0.2~1 mg / mL; based on the total molar number of the PEG-lipid and the targeting ligand-lipid conjugate, the molar percentage of the PEG-lipid is 50%~90%, and the molar percentage of the targeting ligand-lipid conjugate is 10%~50%.

[0007] As a preferred embodiment, the phospholipid includes at least one of HSPC, DSPC, and DOTAP; The PEG-lipids include DSPE-PEG2000 and / or DMG-PEG2000.

[0008] The present invention also provides a method for preparing pyravir chloride liposomes in the pyravir chloride lipid nanoparticles described above, including a thin film dispersion method and / or a reverse evaporation method; The thin-film dispersion method includes the following steps: dissolving a mixture of pyrifos chloride and lipids in an organic solvent to obtain a mixture; removing the organic solvent from the mixture to obtain a thin film; adding an aqueous medium to the thin film for hydration to obtain a suspension; and subjecting the suspension to ultrasonication and ultrafiltration concentration to obtain the pyrifos chloride liposomes. The reverse evaporation method includes the following steps: dissolving a mixture of pyrifos chloride and lipids in an organic solvent to obtain an organic phase solution; adding the organic phase solution dropwise to an aqueous medium to perform self-assembly to obtain a self-assembled system; removing the organic solvent from the self-assembled system to obtain a lipid suspension; and sonicating the lipid suspension and concentrating it by ultrafiltration to obtain the pyrifos chloride liposomes. The organic solvent includes at least one of ethanol, chloroform, and pyridine; The ultrafiltration concentration is performed using an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa.

[0009] As a preferred embodiment, the hydration temperature is 42~45℃; the hydration time is 30 min.

[0010] As a preferred embodiment, the power of the ultrasound is 150~300 W; the duration of the ultrasound is 1~5 min.

[0011] This invention also provides a method for preparing pyrifos chloride lipid micelles in the pyrifos chloride lipid nanoparticles described above, comprising the following steps: mixing the PEG-lipid, the targeting ligand-lipid conjugate, and water to obtain a lipid solution; dissolving the pyrifos chloride in an organic solvent to obtain a pyrifos chloride solution; adding the pyrifos chloride solution dropwise to the lipid solution under stirring conditions to form a premix; subjecting the premix to rotary evaporation and sonication to obtain a suspension; and filtering and ultrafiltration to concentrate the suspension to obtain the pyrifos chloride lipid micelles. The volume ratio of the pyravirin chloride solution to the lipid solution is 1:10; the stirring speed is 600~1000 rpm.

[0012] This invention also provides the application of the pyravirin chloride lipid nanoparticles described above in the preparation of antitumor drugs.

[0013] As a preferred embodiment, the drug is used to increase tumor cell rigidity and / or enhance tumor immunity.

[0014] The present invention also provides a pharmaceutical composition comprising the pyravirin chloride lipid nanoparticles described above and an antitumor immunosuppressant; the antitumor immunosuppressant comprising an immune checkpoint inhibitor and / or an adoptive T-cell preparation.

[0015] Beneficial Effects: This invention provides pyrifos chloride lipid nanoparticles, comprising pyrifos chloride liposomes and / or pyrifos chloride lipid micelles; the pyrifos chloride liposomes comprise a mixture of pyrifos chloride and lipids; the lipid mixture comprises phospholipids, cholesterol, and a targeting ligand-lipid conjugate; the pyrifos chloride lipid micelles comprise pyrifos chloride, PEG-lipids, and a targeting ligand-lipid conjugate; the targeting ligand-lipid conjugate comprises DSPE-PEG2000-cRGD and / or DMG-PEG2000-cRGD. This invention uses phospholipids (phospholipids and lipids in the targeting ligand-lipid conjugate) and cholesterol as membrane materials to assemble liposome vesicle structures, achieving tumor-targeted modification of liposomes through targeting ligands, and encapsulating pyrifos chloride in a lipid bilayer to obtain pyrifos chloride lipid nanoparticles. The pyrifos chloride lipid nanoparticles constructed in this invention possess excellent tumor-targeting enrichment capabilities. The loaded pyrifos chloride can enhance the mechanical stiffness of tumor cells, thereby strengthening T cell-mediated tumor killing, and exerting tumor-suppressing activity through an immune system-dependent pathway. When the pyrifos chloride lipid nanoparticles of this invention are used in combination with immune checkpoint inhibitors and adoptive T cell preparations, they can significantly enhance the anti-tumor immune response, achieving combined tumor immunosensitization therapy. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The results of Phalloidin-iFluor 594 immunofluorescence staining to detect intracellular F-actin expression are shown. From left to right, the tumor cells are B16-F10, CT26, A549, and A375. Figure 2 The results of FluidFM cell stiffness testing show that, from left to right, the tumor cells are B16-F10, CT26, A549, and A375. Figure 3 The results of Pyrvinium-enhanced specific T cell killing of tumor cells were shown; where A is the B16-F10 cell model and B is the B16-OVA cell model. Figure 4 The results are physicochemical analysis of pyrethroid chloride lipid nanoparticles; where A represents particle size distribution and B represents potential. Figure 5 The results of transmission electron microscopy for pyravir chloride lipid nanoparticles; Figure 6 Results of pyraclostrobin accumulation in tumor sites at different time points; Figure 7 Results of the immune-dependent antitumor effect mediated by pyrifos chloride lipid nanoparticles: A represents the tumor volume and survival time of C57BL / 6 mice subcutaneously inoculated with B16-F10 cells (n=6); B represents the tumor volume and survival time of BALB / c mice subcutaneously inoculated with CT26 cells (n=8); C represents the tumor volume and survival time of immunodeficient mice (C-NKG) subcutaneously inoculated with B16-F10 cells (n=7); D represents the tumor volume and survival time of immunodeficient mice (C-NKG) subcutaneously inoculated with CT26 cells (n=8). Figure 8 The results show the antitumor effects of pyrifos chloride lipid nanoparticles on immune checkpoint inhibitors. A is a mouse model of melanoma; B is a mouse model of colorectal cancer. From left to right, the results show the dosing regimen, tumor volume, and mouse survival. Figure 9 To enhance the antitumor effect of adoptive T-cell therapy with pyrifos chloride lipid nanoparticles, A shows C57BL / 6 mice subcutaneously inoculated with B16-F10 cells, with the drug regimen, tumor volume, and survival time of each group of mice shown from left to right; B shows C57BL / 6 mice subcutaneously inoculated with B16-OVA cells, with the drug regimen, tumor volume, and survival time of each group of mice shown from left to right. Detailed Implementation

[0018] This invention provides pyrethroid chloride lipid nanoparticles, comprising pyrethroid chloride liposomes and / or pyrethroid chloride lipid micelles; The pyrethrum chloride liposomes comprise a mixture of pyrethrum chloride and lipids; the lipid mixture comprises phospholipids, cholesterol, and a targeting ligand-lipid conjugate. The pyravir chloride lipid micelles comprise pyravir chloride, PEG-lipid, and a targeting ligand-lipid conjugate. The targeted ligand-lipid conjugates include DSPE-PEG2000-cRGD and / or DMG-PEG2000-cRGD.

[0019] Unless otherwise specified, all components involved in this invention can be obtained through conventional commercial methods.

[0020] The pyrvinium chloride lipid nanoparticles of this invention comprise pyrvinium chloride liposomes and / or pyrvinium chloride lipid micelles. In this invention, pyrvinium can indirectly increase tumor cell rigidity by promoting F-actin polymerization, thereby enhancing CD8... + T cell-mediated tumor killing effects exert anti-tumor effects that depend on the immune system.

[0021] The pyrethroid liposomes of this invention comprise a mixture of pyrethroid chloride and lipids; the lipid mixture includes phospholipids, cholesterol, and a targeting ligand-lipid conjugate; the phospholipids of this invention are amphiphilic lipids that can self-assemble to form a lipid bilayer, constituting the liposome vesicle framework. Membrane fluidity is regulated according to the type of phospholipid, enabling the intercalation of hydrophobic drugs and the adsorption of acidic active substances. As the structural framework of lipid nanoparticles, phospholipids directly affect membrane integrity, in vivo escape efficiency, and in vivo circulatory stability.

[0022] Cholesterol acts as a membrane stabilizer, embedding itself in the lipid bilayer to fill lipid gaps, bidirectionally regulating membrane fluidity, reducing vesicle membrane permeability, minimizing leakage of encapsulated drugs, inhibiting liposome aggregation and fusion, and enhancing the in vitro storage stability and long-term in vivo circulation capacity of liposomes.

[0023] The targeted ligand-lipid conjugates of this invention include DSPE-PEG2000-cRGD and / or DMG-PEG2000-cRGD. This invention chemically modifies DSPE-PEG2000 or DMG-PEG2000 to form targeted ligand-lipid conjugates. During lipid nanoparticle assembly, DSPE-PEG2000-cRGD or DMG-PEG2000-cRGD is added as a raw material. Phospholipids (DSPE, DMG) are inserted into the lipid bilayer through hydrophobic interactions, while hydrophilic PEG2000 ensures that the cRGD cyclic peptide is exposed on the surface of the lipid nanoparticles. Direct co-assembly of phospholipids, cholesterol, and the targeted ligand-lipid conjugates forms spatially stable liposomes that target integrins. Furthermore, cRGD can target and bind to tumor cell-specific receptors αvβ3 and NRP1, mediating their passage through tumor angiogenesis and effectively increasing local drug accumulation in tumor tissue. Experimental results showed that the concentration of Pyrvinium delivered by the cRGD-modified lipid nanoparticles in tumor tissues was significantly higher than that in the free drug control group, and it had good controllable release characteristics.

[0024] The piravirin liposomes of this invention utilize phospholipids and cholesterol as membrane materials to assemble liposome vesicle structures. Through targeted ligand modification, tumor-targeted modification of the liposomes is achieved, encapsulating piravirin in a lipid bilayer to obtain piravirin lipid nanoparticles. These piravirin lipid nanoparticles can precisely target tumor tissues; the loaded piravirin can enhance the mechanical stiffness of tumor cells, thereby strengthening T cell-mediated tumor killing, and exerting tumor-suppressive activity through an immune system-dependent pathway. When combined with immune checkpoint inhibitors and adoptive T cell preparations, the piravirin lipid nanoparticles of this invention can more significantly enhance the anti-tumor immune response, achieving combined tumor immunosensitization therapy.

[0025] In one embodiment, based on the total molar percentage of the lipid mixture, the phospholipid molar percentage is 40% to 70%; the cholesterol molar percentage is 25% to 50%; and the targeting ligand-lipid conjugate molar percentage is 5% to 10%. In specific embodiments of the present invention, the molar percentage of the phospholipid can be any value from 40% to 70%, for example, 40%, 45%, 50%, 55%, 60%, 65%, or 70%; the molar percentage of the cholesterol molar percentage can be any value from 25% to 50%, for example, 25%, 30%, 35%, 40%, 45%, or 50%; and the molar percentage of the targeting ligand-lipid conjugate molar percentage can be any value from 5% to 10%, for example, 5%, 6%, 7%, 8%, 9%, or 10%.

[0026] In one embodiment, the phospholipid includes at least one of HSPC (hydrogenated soybean lecithin), DSPC (distearylphosphatidylcholine), and DOTAP (1,2-dioleoyl-3-trimethylammonium propane chloride).

[0027] In one embodiment, the targeting ligand-lipid conjugate includes DSPE-PEG2000-cRGD and / or DMG-PEG2000-cRGD. cRGD is chemically modified onto DSPE-PEG2000 or DMG-PEG2000. During lipid nanoparticle assembly, DSPE-PEG2000-cRGD or DMG-PEG2000-cRGD is incorporated into the liposome formulation, and direct co-assembly can form spatially stable liposomes that target integrins.

[0028] In one embodiment, when the lipid mixture is HSPC, DOTAP, cholesterol and DSPE-PEG2000-cRGD, the molar ratio of HSPC, DOTAP, cholesterol and DSPE-PEG2000-cRGD is 40:30:25:5.

[0029] In one embodiment, when the lipid mixture is DSPC, DOTAP, cholesterol and DSPE-PEG2000-cRGD, the molar ratio of DSPC, DOTAP, cholesterol and DSPE-PEG2000-cRGD is 40:30:25:5.

[0030] In one embodiment, when the lipid mixture is HSPC, DOTAP, cholesterol and DMG-PEG2000-cRGD, the molar ratio of HSPC, DOTAP, cholesterol and DMG-PEG2000-cRGD is 40:30:25:5.

[0031] In one embodiment, when the lipid mixture is DSPC, DOTAP, cholesterol and DMG-PEG2000-cRGD, the molar ratio of DSPC, DOTAP, cholesterol and DMG-PEG2000-cRGD is 40:30:25:5.

[0032] In one embodiment, when the lipid mixture is HSPC, cholesterol, and DSPE-PEG2000-cRGD, the molar ratio of HSPC, cholesterol, and DSPE-PEG2000-cRGD is 56:39:5.

[0033] In one embodiment, when the lipid mixture is HSPC, cholesterol and DMG-PEG2000-cRGD, the molar ratio of HSPC, cholesterol and DMG-PEG2000-cRGD is 56:39:5.

[0034] In one embodiment, when the lipid mixture is DSPC, cholesterol, and DSPE-PEG2000-cRGD, the molar ratio of DSPC, cholesterol, and DSPE-PEG2000-cRGD is 56:39:5.

[0035] In one embodiment, when the lipid mixture is DSPC, cholesterol, and DMG-PEG2000-cRGD, the molar ratio of DSPC, cholesterol, and DMG-PEG2000-cRGD is 56:39:5.

[0036] In one embodiment, the mass ratio of the lipid mixture to pyravirin is 5-20:1, more preferably 10-20:1, for example, 10:1, 12:1, 15:1, or 20:1. This ratio ensures that the pyravirin drug is encapsulated to the maximum extent in the final assembled nanoparticles, thereby improving the drug loading rate and utilization rate.

[0037] The pyrethrum chloride lipid micelles of this invention comprise pyrethrum chloride, PEG-lipids, and targeting ligand-lipid conjugates. As one embodiment, the PEG-lipids include DSPE-PEG2000 (distearylphosphatidylethanolamine-polyethylene glycol 2000) and / or DMG-PEG2000 (dimyristoylglycerol-polyethylene glycol 2000). PEG-lipids are amphiphilic molecules; in the aqueous phase, their hydrophobic tails aggregate to form a core, while their hydrophilic heads face the aqueous phase to form a shell. When the concentration exceeds the critical micelle concentration (CMC), they spontaneously form nanoscale (typically 10-100 nm) spherical structures. The pyrethrum chloride lipid micelles of this invention have a hydrophobic core (encapsulating poorly soluble drugs) and a hydrophilic shell (providing colloidal stability and reducing protein adsorption), and are solid or non-layered aggregates. In this process, hydrophobic drugs are physically encapsulated or dissolved within a hydrophobic micelle core, overcoming the water solubility barrier and significantly improving the apparent solubility and bioavailability of poorly soluble drugs, preventing premature degradation or precipitation in body fluids. Furthermore, the hydrophilic shell (polyethylene glycol PEG) forms a hydration layer that shields surface charges, reduces plasma protein adsorption and mononuclear phagocytic system (MPS) recognition and clearance, prolongs blood half-life, and increases the chance of reaching the target site. Simultaneously, utilizing the high permeability and retention effect (EPR effect) of tumor tissue vessels, pyrifos chloride lipid micelles easily leak out of blood vessels and remain in tumor tissue.

[0038] As one implementation, based on the total molar number of the PEG-lipid and the targeting ligand-lipid conjugate, the molar percentage of the PEG-lipid is 50% to 90%; and the molar percentage of the targeting ligand-lipid conjugate is 10% to 50%. In specific embodiments of the present invention, the molar percentage of the PEG-lipid can be any value from 50% to 90%, such as 50%, 55%, 60%, 65%, 68%, 70%, 75%, 80%, 85%, or 90%; and the molar percentage of the targeting ligand-lipid conjugate can be any value from 10% to 50%, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0039] In one embodiment, the drug loading of the pyraviridine lipid micelles is 0.2~1 mg / mL; more preferably 0.2~0.5 mg / mL. For example, it can be 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mg / mL. The drug loading described in this invention refers to 0.2~1 mg of pyraviridine drug loaded in 1 mL of lipid nanoparticles.

[0040] The present invention also provides a method for preparing the pyravirin liposomes described above, including a thin-film dispersion method and / or a reverse evaporation method; The thin-film dispersion method includes the following steps: dissolving a mixture of pyrifos chloride and lipids in an organic solvent to obtain a mixture; removing the organic solvent from the mixture to obtain a thin film; adding an aqueous medium to the thin film for hydration to obtain a suspension; and subjecting the suspension to ultrasonication and ultrafiltration concentration to obtain the pyrifos chloride liposomes. The reverse evaporation method includes the following steps: dissolving a mixture of pyrifos chloride and lipids in an organic solvent to obtain an organic phase solution; adding the organic phase solution dropwise to an aqueous medium to perform self-assembly to obtain a self-assembled system; removing the organic solvent from the self-assembled system to obtain a lipid suspension; and sonicating the lipid suspension and concentrating it by ultrafiltration to obtain the pyrifos chloride liposomes. The organic solvent includes at least one of ethanol, chloroform, and pyridine; The ultrafiltration concentration is performed using an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa.

[0041] The targeted ligand-lipid conjugate of this invention can be obtained through conventional commercial methods or prepared in-house. Taking DMG-PEG2000-cRGD as an example, the preparation of the targeted ligand includes the following steps: DMG-PEG2000-Mal and cRGD peptide are placed in a reaction flask, and the organic solvent DMF (N,N-dimethylformamide) is added for dispersion to obtain a dispersion; a phospholipid activator is added to the dispersion, dissolved by ultrasonication, and then coupled to obtain a reaction mixture; the reaction mixture is dialyzed and purified to obtain the targeted ligand-lipid conjugate; the molar ratio of DMG-PEG2000-Mal to cRGD peptide is 10:1; the phospholipid activator includes Traut's Reagent (2-iminothionyl hydrochloride); the molar ratio of the phospholipid activator to DMG-PEG2000 is 5:1; the ultrasonic dissolution time is 1 min; the coupling temperature is 25°C; and the coupling time is 2-4 minutes. h; The dialysis includes: placing a dialysis bag containing the reaction mixture in a beaker containing ultrapure water and dialyzing at room temperature for 12-24 h; the molecular weight cutoff of the dialysis bag is 10 kDa; the dialysis of the present invention can remove unreacted small molecule impurities. As one embodiment, the purification includes: centrifuging the liquid in the dialysis bag, collecting the precipitate, washing 2-3 times, and vacuum drying to obtain the purified targeting ligand-lipid conjugate; the centrifugation speed is 10000-12000 rpm, and the time is 5-10 min; the drying temperature is 55-60℃, and the time is 12-24 h. The preparation method of DSPE-PEG2000-cRGD of the present invention is the same as that of DMG-PEG2000-cRGD.

[0042] This invention involves dissolving a mixture of pyrifos chloride and lipids in an organic solvent to obtain a mixture. As one embodiment, the organic solvent includes at least one of ethanol, chloroform, and pyridine. Ethanol has good biocompatibility and meets the requirements of Good Manufacturing Practices (GMP) for pharmaceuticals, making it a preferred solvent for preparing lipid nanoparticles. For certain poorly soluble phospholipid molecules, chloroform or pyridine can be used as the organic solvent. As one embodiment, the amount of pyrifos chloride added is 1 mg per 1 mL of organic solvent; the amount of the lipid mixture added is 5-50 mg; more preferably, the amount of the lipid mixture added is 5-20 mg, and more preferably 5-10 mg. This ratio ensures that the pyrifos chloride drug is encapsulated to the maximum extent within the final assembled nanoparticles, improving drug loading and utilization.

[0043] After obtaining the mixture, the mixture is ultrasonically dispersed to obtain a uniformly dispersed mixture; the ultrasonic dispersion is carried out under water bath conditions; the power of the ultrasound is any value in the range of 150~300 W, for example, it can be 150, 200, 250 or 300 W.

[0044] After obtaining the uniformly dispersed mixture, the organic phase in the uniformly dispersed mixture is removed to obtain a thin film; as one embodiment, the removal method includes rotary evaporation.

[0045] After obtaining the film, an aqueous medium is added to the film for hydration to obtain a suspension. As one embodiment, the aqueous medium includes phosphate-buffered saline (PBS). PBS buffer has a similar ionic composition and osmotic pressure to mammalian body fluids, which can maximize the stability and biocompatibility of lipid nanoparticles in vivo. As one embodiment, the hydration temperature is 42-45°C; the hydration time is 30 min. In specific embodiments of the present invention, the hydration temperature can be any value within the range of 42-45°C, for example, 42, 43, 44, or 45°C.

[0046] After obtaining the suspension, the present invention sonicates the suspension to obtain an ultrasonicated suspension. As one embodiment, the sonication is performed under ice bath conditions; the power of the sonication is 150~300 W; and the sonication time is 1~5 min. In a specific embodiment of the present invention, the power of the sonication can be any value within the range of 150~300 W, for example, 150, 200, 250 or 300 W; and the sonication time can be any value within the range of 1~5 min, for example, 1, 2, 3, 4 or 5 min.

[0047] After obtaining the sonicated suspension, the present invention further concentrates the sonicated suspension by ultrafiltration to obtain ultrafiltered liposomes. The ultrafiltration of the present invention uses an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa for centrifugation. The pyrifos chloride lipid nanoparticles of the present invention have a particle size distribution between 150-200 nm, which can be retained by the 10-30 kDa ultrafiltration membrane. As one embodiment, the centrifugation force is 3000 g; the centrifugation time is 15 min; and the ultrafiltration concentration is performed 3-10 times. The ultrafiltration concentration of the present invention can effectively remove organic solvents, free drugs, and other impurities, thereby achieving the separation and purification of pyrifos chloride lipid nanoparticles, and further adjusting the drug content of the lipid nanosystem. The present invention can increase the encapsulation efficiency of lipid nanoparticles to over 90% through the ultrafiltration concentration step.

[0048] The present invention can also be used to prepare pyravirin chloride liposomes by reverse evaporation.

[0049] This invention involves dissolving a mixture of pyrifos chloride and lipids in an organic solvent to obtain an organic phase solution. As one embodiment, the organic solvent includes ethanol, chloroform, or pyridine. Ethanol has good biocompatibility and meets the requirements of Good Manufacturing Practices (GMP) for pharmaceuticals, making it a preferred solvent for preparing lipid nanoparticles. For certain poorly soluble phospholipid molecules, chloroform or pyridine can be used as the organic solvent. As one embodiment, based on 1 mL of organic solvent, the amount of pyrifos chloride added is 1 mg; the amount of the lipid mixture added is 5-50 mg; more preferably, the amount of the lipid mixture added is 5-20 mg, and more preferably 10-20 mg.

[0050] After obtaining the organic phase solution, the present invention performs ultrasonic dispersion on the organic phase solution to obtain a uniformly pulverized organic phase solution.

[0051] This invention involves adding the organic phase solution dropwise to an aqueous medium to induce self-assembly, thereby obtaining a self-assembled system. As one embodiment, the aqueous medium includes phosphate-buffered saline (PBS). PBS buffer has a similar ionic composition and osmotic pressure to mammalian body fluids, which can maximize the stability and biocompatibility of lipid nanoparticles in vivo. As one embodiment, before the dropwise addition, the aqueous medium is preheated to 37-45°C, for example, 37, 38, 39, 10, 41, 42, 43, 44, or 45°C. As one embodiment, the self-assembly temperature is 37-45°C; the self-assembly rotation speed is 700 rpm; and the self-assembly time is 10-20 min. In specific embodiments of this invention, the self-assembly time can be any value within the range of 10-20 min, for example, 10, 12, 15, 17, 19, or 20 min.

[0052] After obtaining the self-assembled system, the residual organic solvent in the self-assembled system is removed to obtain a lipid suspension. As one embodiment, the removal method includes rotary evaporation or dialysis; as one embodiment, the molecular weight cutoff in the dialysis removal is 10~30 KD, for example, 10 or 30 KD.

[0053] After obtaining the lipid suspension, the lipid suspension is sonicated to obtain an ultrasonicated suspension. As one embodiment, the sonication is performed under ice bath conditions; the power of the sonication is 150-300 W; and the sonication time is 1-5 min. In a specific embodiment of the present invention, the power of the sonication can be any value within the range of 150-300 W, for example, 150, 200, 250, or 300 W; and the sonication time can be any value within the range of 1-5 min, for example, 1, 2, 3, 4, or 5 min.

[0054] The ultrafiltration concentration described in this invention has been discussed above and will not be repeated here.

[0055] The pyrrolidone liposomes prepared by the method described in this invention have an average particle size of 150-200 nm, preferably 170 nm, and a drug encapsulation efficiency of over 90%.

[0056] This invention also provides a method for preparing the pyrifos chloride lipid micelles described above, comprising the following steps: mixing the PEG-lipid, the targeting ligand-lipid conjugate, and water to obtain a lipid solution; dissolving the pyrifos chloride in an organic solvent to obtain a pyrifos chloride solution; adding the pyrifos chloride solution dropwise to the lipid solution under stirring conditions to form a premix; subjecting the premix to rotary evaporation and sonication to obtain a suspension; and filtering and ultrafiltration to concentrate the suspension to obtain the pyrifos chloride lipid micelles. The volume ratio of the pyravirin chloride solution to the lipid solution is 1:10; the stirring speed is 600~1000 rpm.

[0057] This invention involves mixing the PEG-lipid, the targeting ligand-lipid conjugate, and water to obtain a lipid solution. As one embodiment, the concentration of the lipid solution is 1 mg / mL, and the solvent mass is based on the total mass of the PEG-lipid and the targeting ligand-lipid conjugate.

[0058] In this invention, the pyrifos chloride is dissolved in an organic solvent to obtain a pyrifos chloride solution; as one embodiment, the organic solvent includes chloroform; the concentration of the pyrifos chloride solution is 0.2 mg / mL.

[0059] This invention involves adding a pyriproxyfen chloride solution dropwise to a lipid solution under stirring conditions to form a premix. As one embodiment, the volume ratio of the pyriproxyfen chloride solution to the lipid solution is 1:10. As another embodiment, the dropping rate can be 0.5~1 μL / s, for example, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 μL / s. The suitable dropping rate of this invention ensures the uniformity of the lipid nanoparticles. As one embodiment, after the dropping is completed, the process further includes: continuing stirring for 1~5 min. As another embodiment, the stirring speed can be any value from 600~1000 rpm, for example, 600, 700, 800, 900, or 1000 rpm.

[0060] After obtaining the premixed liquid, the present invention performs rotary evaporation on the premixed liquid to obtain a premixed liquid after rotary evaporation. In one embodiment, the rotary evaporation is vacuum rotary evaporation; the vacuum rotary evaporation time can be any value between 20 and 30 minutes, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 minutes. During rotary evaporation, it is necessary to control the vacuum level, which should be controlled to approximately half of the vacuum gauge reading, for example, -0.05 to -0.06 MPa.

[0061] This invention involves sonicating an evaporated premixed liquid to obtain a suspension. As one embodiment, the sonication is probe-type ultrasound (Tip ultrasound). The power of the ultrasound can be any value between 150 and 300 W, for example, 150, 200, 250, or 300 W. As another embodiment, the sonication time can be any value between 1 and 2 minutes. In this invention, ice water is added during the sonication to prevent temperature rise during the process from damaging the lipid nanomicelles.

[0062] After obtaining the suspension, the present invention filters the suspension to obtain a filtered suspension. As one embodiment, the pore size of the filter membrane is 0.22 μm or 0.45 μm. The filtration described in this invention can remove small amounts of aggregated drug.

[0063] The filtered suspension is then concentrated by ultrafiltration to obtain the pyrifos chloride lipid micelles. The ultrafiltration concentration described in this invention has been discussed above and will not be repeated here.

[0064] This invention also provides the application of the pyrvinium chloride lipid nanoparticles described above in the preparation of antitumor drugs. As one embodiment, the drug is used to improve tumor cell rigidity and / or enhance tumor immunity. The pyrvinium described in this invention indirectly improves tumor cell rigidity by promoting F-actin polymerization, thereby enhancing CD8... + T cell-mediated tumor killing effects exert anti-tumor effects that depend on the immune system.

[0065] The present invention also provides a pharmaceutical composition comprising the pyrifos chloride lipid nanoparticles described above and an antitumor immunosuppressant; the antitumor immunosuppressant comprises an immune checkpoint inhibitor and / or an adoptive T-cell preparation. As one embodiment, the immune checkpoint inhibitor comprises at least one of a PD-1 antibody, a PD-L1 antibody, and a CTLA-4 antibody.

[0066] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0067] The following is some information about the raw materials used in the embodiments of the present invention: Human lung cancer cells A549, human melanoma cells A375, mouse melanoma cells B16-F10, and mouse colorectal cancer cells CT26 were purchased from the American Type Culture Collection (ATCC); catalog numbers: CCL-185, CRL-1619, CRL6475, and CRL-2638, respectively. MC38 cells were donated by Professor Deng Liufu's research group at Shanghai Jiao Tong University. B16-OVA cells were obtained by transfecting B16-F10 cells with OVA lentivirus (purchased from Shanghai Jikai Gene Technology Co., Ltd.), which was custom-made by Shanghai Jikai Gene Technology Co., Ltd.

[0068] Pyrvinium was purchased from Selleck (catalog number: S5816). GP100 peptide and OVA peptide were both purchased from MedChemExpress (catalog numbers: HY-P2506A and HY-P1489A, respectively). CellTrace Violet (CTV) was purchased from Thermo Fisher Scientific (catalog number: C34557).

[0069] Pmel transgenic mice, OT-1 transgenic mice, and immunodeficient mice (C-NKG) were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd. Immunocompliance mice, including C57BL / 6 mice and BALB / c mice, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0070] Example 1 (1) Pyrvinium increases the degree of F-actin polymerization and stiffness of tumor cells. Human lung cancer cells A549, human melanoma cells A375, mouse melanoma cells B16-F10, and mouse colorectal cancer cells CT26 were cultured separately.

[0071] Pyrvinium was dissolved using DMSO to prepare Pyrvinium solutions with concentrations of 10 nM and 100 nM.

[0072] When the tumor cells reached the logarithmic growth phase, Pyrvinium solution was added, with B16-F10 cells, A549 cells, and A375 cells each receiving 100 nM Pyrvinium solution; CT26 cells received 10 nM Pyrvinium solution, and the cells were incubated for 24 h.

[0073] B16-F10 (100 nM), CT26 (10 nM), A549 (100 nM), and A375 (100 nM) cells were treated with Pyrvinium for 24 h. Intracellular F-actin expression was detected using Phalloidin-iFluor 594 (Abbott (Shanghai) Trading Co., Ltd., ab176757) immunofluorescence staining. The results were analyzed using fluorescence microscopy and ImageJ software. The results are as follows: Figure 1 As shown in the figure. The results indicate that Pyrvinium can promote the polymerization of F-actin in B16-F10, CT26, A549 and A375 cells.

[0074] Simultaneously, cell stiffness was measured using a fluid dynamics microscope (FluidFM OMNIUM, Cytosurge, Switzerland), and the results were as follows: Figure 2 As shown in the figure. The results showed that Pyrvinium increased the stiffness of B16-F10, CT26, A549, and A375 cells.

[0075] (2) Pyrvinium enhances the killing effect of specific T cells on tumor cells; A. Spleen cells from Pmel transgenic mice were obtained, stimulated with GP100 peptide in vitro for 48 h, and then activated Pmel CD8 were obtained by magnetic bead sorting. + T cells were pretreated by adding 100 nM Pyrvinium to B16-F10 cells and incubating for 24 h. Pretreated B16-F10 cells were then stained with CTV (v:v = 1:10000 dilution). Activated Pmel CD8 cells were then... + T cells were mixed with pretreated B16-F10 cells at ratios of 1:1, 2:1, and 5:1, and MC38 cells (used as non-target cells, with the same number as pretreated B16-F10 cells) stained with CTV (v:v=1:2000 dilution) were added. The mixture was co-cultured for 4 h and denoted as B16-F10+Pyrvinium.

[0076] Meanwhile, a control group was set up, with the same procedure as the B16-F10+Pyrvinium group, except that the B16-F10 cells were not treated with Pyrvinium.

[0077] After co-culturing for 4 hours, the proportion of surviving cells in each treatment group was measured, and the results are as follows: Figure 3 As shown in Figure A.

[0078] B. Spleen cells from OT-1 transgenic mice were obtained, stimulated with OVA peptide in vitro for 48 h, and then activated OT-1 CD8 cells were obtained by magnetic bead sorting. +T cells. B16-OVA cells were pretreated with 100 nM Pyrvinium for 24 h to obtain pretreated B16-OVA cells, which were then stained with CTV (v:v = 1:10000 dilution). Activated OT-1 CD8... + T cells were mixed with pretreated B16-OVA cells at ratios of 1:1, 2:1, and 5:1, and B16-F10 cells (used as non-target cells, with the same number as pretreated B16-OVA cells) that had been CTV stained (v:v=1:2000 dilution) were added. The mixture was then co-cultured for 4 h and labeled as B16-OVA+Pyrvinium.

[0079] Meanwhile, a control group was set up, and the procedure was the same as B16-OVA+Pyrvinium, except that the B16-OVA cells were not treated with Pyrvinium.

[0080] After co-culturing for 4 hours, the proportion of surviving cells in each treatment group was measured, and the results are as follows: Figure 3 As shown in B.

[0081] The results showed that CD8 + T cells kill more Pyrvinium-pretreated tumor cells.

[0082] Reagents used in Examples 2 and 3 and Comparative Examples 1 and 2: HSPC, DSPC, DOTAP, DSPE-PEG 2000 DMG-PEG 2000 Purchased from Aivit (Shanghai) Pharmaceutical Technology Co., Ltd.; DSPE-PEG 2000 and DSPE-PEG 2000 -cRGD was purchased from Xi'an Ruixi Biotechnology Co., Ltd.; Pyrvinium was purchased from Selleck (catalog number: S5816). All other biochemical reagents were domestically produced analytical grade conventional reagents.

[0083] Example 2 A method for preparing pyrifos chloride lipid nanoparticles, comprising the following steps: (1) Preparation of targeted ligands DMG-PEG2000-Mal and cRGD peptide (molar ratio 10:1) were placed in a reaction flask, and 3-5 mL of organic solvent DMF (N,N-dimethylformamide) was added. After sonication for 1 min to disperse the mixture, Traut's Reagent, an activator for coupling the targeting ligand and phospholipid, was dissolved in the same solvent at a molar ratio of 5:1 to DMG-PEG2000-Mal. After brief sonication (approximately 1 min) to ensure complete dissolution, the mixture was coupled at 25°C for 2 h to obtain a phospholipid DMG-PEG2000-cRGD mixture modified with the cRGD ligand. This mixture was then placed in a dialysis bag (cutoff: 10 kDa) and dialyzed in ultrapure water to remove unreacted small molecule impurities. After dialysis, the dialysate was centrifuged at high speed (10000 rpm, 5... (min), collect the precipitate, wash it 2-3 times with distilled water, and vacuum dry it to obtain the covalently coupled complex of the targeting ligand and phospholipid, DMG-PEG2000-cRGD. (2) 10 mg of a phospholipid mixture constituting the liposome bilayer membrane structure (the molar ratio of HSPC, cholesterol, and DMG-PEG2000-cRGD was 56:39:5) and 1 mg of pyravirin chloride were dissolved together in 1 mL of chloroform; the solution was sonicated in a water bath to disperse it evenly; the organic solvent was removed by rotary evaporation, and the resulting lipid membrane was further dried under vacuum; then 5 mL of pH 7.4 phosphate buffer (PBS) was added and the mixture was mixed and hydrated in a rotary evaporator (400 rpm) at 42°C for 30 min, and the hydrated lipid suspension was collected; the suspension was then subjected to moderate sonication (300 W, sonication time for 1 min) using a probe-type sonicator under ice bath conditions to obtain a sonicated suspension; the sonicated suspension was concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, and centrifuged at 3000 g for 15 min. After centrifugation, the filtrate at the bottom of the ultrafiltration tube was discarded, and the nanoparticle solution inside the ultrafiltration tube was mixed by blowing and mixing, and then ultrafiltration was continued for 15 min. The mixture was subjected to repeated ultrafiltration 3 to 10 times to remove free, unencapsulated drugs, resulting in uniform particle size and surface-targeted modified pyrvinium-loaded liposomes, denoted as LNP-Pyrvinium.

[0084] Example 3 A method for preparing pyrifos chloride lipid nanoparticles, comprising the following steps: A mixture of lipids constituting the liposome membrane structure (HSPC, cholesterol, DSPE-PEG2000-cRGD, molar ratio 56:39:5) and pyrifos chloride were dissolved together in anhydrous ethanol. After sonication (300 W, sonication time 1 min) to ensure thorough dispersion, the organic phase solution was slowly added dropwise to PBS buffer preheated to 37-45℃ and pH 7.4 under continuous stirring (700 rpm). Stirring was continued for a certain period of time (700 rpm, 20 min) to promote lipid self-assembly. Residual ethanol was then completely removed by rotary evaporation or ultrafiltration. The resulting lipid suspension was collected and subjected to moderate sonication (300 W, sonication time 1 min) using a probe-type sonicator under ice bath conditions to obtain a sonicated suspension. The sonicated suspension was concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa and centrifuged at 3000 g for 15 minutes. After centrifugation for 15 min, discard the filtrate at the bottom of the ultrafiltration tube, mix the nanoparticle solution inside the ultrafiltration tube by blowing, and continue ultrafiltration for 15 min. Repeat ultrafiltration 3 to 10 times to remove free and unencapsulated drugs and obtain targeted modified pyravir chloride-loaded liposomes, denoted as LNP-Pyr.

[0085] Example 4 A method for preparing pyrethroid chloride lipid nanoparticles, the steps are the same as in Example 2, the difference being that DSPE-PEG is used. 2000 -cRGD was purchased from Xi'an Ruixi Biotechnology Co., Ltd.; the lipid mixture in step (2) consisted of HSPC, DOTAP, cholesterol and DSPE-PEG2000-cRGD, with a molar ratio of 40:30:25:5.

[0086] Example 5 A method for preparing pyrethroid chloride lipid nanoparticles, the steps are the same as in Example 2, the difference being that DSPE-PEG is used. 2000 -cRGD was purchased from Xi'an Ruixi Biotechnology Co., Ltd.; the lipid mixture in step (2) consisted of DSPC, DOTAP, cholesterol and DSPE-PEG2000-cRGD in a molar ratio of 40:30:25:5.

[0087] Example 6 A method for preparing pyravirin chloride lipid nanoparticles, the steps are the same as in Example 2, except that in step (2), the lipid mixture is HSPC, DOTAP, cholesterol and DMG-PEG2000-cRGD, with a molar ratio of 40:30:25:5.

[0088] Example 7 A method for preparing pyravirin chloride lipid nanoparticles, the steps are the same as in Example 2, except that in step (2), the lipid mixture is DSPC, DOTAP, cholesterol and DMG-PEG2000-cRGD, with a molar ratio of 40:30:25:5.

[0089] Example 8 A method for preparing pyravirin chloride lipid nanoparticles, the steps are the same as in Example 3, except that in step (2), the lipid mixture is DSPC, cholesterol and DSPE-PEG2000-cRGD, with a molar ratio of 56:39:5.

[0090] Example 9 A method for preparing pyravirin chloride lipid nanoparticles, the steps are the same as in Example 2, except that in step (2), the lipid mixture is DSPC, cholesterol and DMG-PEG2000-cRGD, with a molar ratio of 56:39:5.

[0091] Example 10 A method for preparing pyrifos chloride lipid micelles comprises the following steps: (1) Dissolving 1.50 mg of DSPE-PEG-2000 and DSPE-PEG2000-cRGD in 50 mL of Milli-Q water to obtain a lipid solution; the molar ratio of DSPE-PEG-2000 to DSPE-PEG2000-cRGD is 50:50; (2) Dissolving 1 mg of pyrifos chloride in 5 mL of chloroform to obtain a pyrifos chloride solution; (3) Placing 50 mL of the lipid solution in a beaker and stirring with a high-speed magnetic force (700 rpm); (4) Using a syringe, adding the pyrifos chloride solution dropwise (0.5~1 μL / s) to the beaker containing the lipid solution. After the addition is complete, continue stirring for 1~5 min to obtain a premix; (5) Transferring the premix to a clean round-bottom flask (50 mL system with 250 ppm water). (6) The premixed solution is rotary evaporated under reduced pressure for 20-30 min on a rotary evaporator (note to control the vacuum degree, about 1 / 2 of the vacuum gauge is sufficient) to obtain the premixed solution after rotary evaporation; (7) The premixed solution after rotary evaporation is placed on a Tip and sonicated for 1-2 min at a power of 150-300 W to obtain a suspension; ice water needs to be added during sonication to prevent the temperature from rising and damaging the lipid micelles; (8) The suspension is transferred to a 50 mL centrifuge tube and filtered with a 0.22 μm syringe filter membrane to remove a small amount of aggregated drug and obtain the filtered suspension; (9) The filtered suspension is concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30 KDa. It is centrifuged at 3000 g for 15 min. After centrifugation, the filtrate at the bottom of the ultrafiltration tube is discarded. The nanoparticle solution inside the ultrafiltration tube is blown and mixed, and then ultrafiltration is continued for 15 min. The solution is concentrated to a final concentration of about 50 mL. mL (concentrated 10 times) was used to obtain pyravir chloride lipid micelles.

[0092] Comparative Example 1: Comparative Example 1 provides a process for preparing blank control lipid nanoparticles using a thin-film dispersion method. Its preparation process is basically the same as in Example 2, except that pyravir chloride is not added in Comparative Example 1. All other components, reagent dosages, operating conditions, and post-processing steps remain exactly the same, denoted as Blank LNP.

[0093] Comparative Example 2: Comparative Example 2 provides a process for preparing blank control lipid nanoparticles using a reverse evaporation method. Its preparation process is basically the same as in Example 3, except that pyrifos chloride is not added in Comparative Example 2. All other components, reagent dosages, operating conditions, and post-processing steps remain exactly the same, denoted as LNP-Control.

[0094] Comparative Example 3 Comparative Example 3 provides a process for preparing blank control lipid nanoparticles using a thin-film dispersion method. Its preparation process is basically the same as in Example 2, except that the ultrafiltration concentration step is omitted.

[0095] Test Example 1 The physicochemical properties of the LNP-Pyrvinium and Blank LNP samples prepared in Example 2 and Comparative Example 1 were characterized.

[0096] The LNP-Pyrvinium and Blank LNP samples prepared in Example 2 and Comparative Example 1 were diluted 5-10 times with ultrapure water, and their average particle size (Z-average) and polydispersity index (PDI) were measured using a dynamic light scattering instrument (Zetasizer Nano ZSE, Malvern Instruments, UK). The particle size distribution results are as follows: Figure 4 As shown in Figure A, the results indicate that the particle size of each sample is mainly concentrated in the range of 150~200 nm, with the dominant particle size peak being around 170 nm.

[0097] The zeta potentials of Blank LNP and LNP-Pyrvinium were simultaneously measured using the same dynamic light scattering instrument. Figure 4 As shown in Figure B, the Zeta potential of both groups of samples is approximately +20 mV, indicating that the loading of pyrifos chloride did not significantly change the surface charge characteristics of the nanoparticles, and the colloidal stability of the system is good.

[0098] The morphology of the nanoparticles was observed using transmission electron microscopy (TEM). A suitable amount of Blank LNP and LNP-Pyrvinium suspension was dropped onto a copper grid coated with a carbon film and allowed to air dry at room temperature for 0.5–1 h; subsequently, field emission transmission electron microscopy (HT7800, Hitachi) was used for imaging observation. The results are as follows: Figure 5 As shown, both types of nanoparticles exhibit a near-spherical structure with complete morphology, clear outline, and uniform distribution.

[0099] Drug loading and encapsulation efficiency determination of lipid nanoparticles: The encapsulation efficiency (EE) of LNP-Pyrvinium in Example 2 and Comparative Example 3 was determined by high performance liquid chromatography (HPLC). The specific procedure was as follows: A quantitatively prepared LNP-Pyrvinium sample was taken, methanol was added to demulsify, and the sample was centrifuged at high speed (15000 × g, 30 min, 4℃). The supernatant was collected. The supernatant was injected into HPLC for analysis, and the content of free pyravirin was quantitatively determined at 250 nm using a UV detector. The encapsulation efficiency (EE) was calculated using the following formula: EE (%) = (actual mass of pyravirin loaded in the nanoparticles / total amount of feed) × 100%. The encapsulation efficiency of LNP-Pyrvinium obtained in Comparative Example 3 was approximately 35%; after optimization (Example 2, introducing an ultrafiltration concentration and purification step), the encapsulation efficiency could be increased to over 90%.

[0100] Meanwhile, the physicochemical properties of the lipid nanoparticles prepared in Example 1 and Examples 1-10 were measured. The results showed that the prepared lipid nanoparticles had complete morphology, clear outline, uniform particle size distribution, and encapsulation efficiency of over 90%.

[0101] Example 11 (1) LNP-Pyrvinium increases the accumulation of Pyrvinium in tumor tissues. Using C57BL / 6 mice as subjects, 1×10⁻⁶ mice were subcutaneously injected. 6 A tumor model was constructed using B16-F10 cells, and the tumor volume was allowed to grow to approximately 100 mm. 3 At that time, the model mice were randomly divided into two groups (n=3): Pyrvinium group: 0.5 mg / kg (mouse body weight) of Pyrvinium was injected via the tail vein; Pyr-LNP group: LNP-Pyrvinium prepared in Example 2 was injected via tail vein at a dose of 0.5 mg / kg (mouse body weight).

[0102] Tumor tissues were collected from each treatment group at 0, 12, 24, 48, 96, and 168 hours after injection. The tumors were weighed, frozen in liquid nitrogen for 1 hour, cryogenically ground, resuspended in an equal volume of PBS buffer, centrifuged at 500G for 5 minutes, and the supernatant was obtained. The concentration of Pyrvinium in the supernatant was detected using high-performance liquid chromatography (TSQ Quantis™ Plus triple quadrupole mass spectrometer). The results are as follows: Figure 6 As shown.

[0103] The results showed that, compared with Pyrvinium alone, the LNP-Pyrvinium delivery system can significantly improve the drug enrichment efficiency in tumor tissues and achieve targeted accumulation.

[0104] (2) LNP-Pyrvinium-mediated immune-dependent antitumor effects A. Using C57BL / 6 mice as subjects, 1×10⁻⁶ mice were subcutaneously injected with 1×10⁻⁶ mice. 6 Mice were randomly divided into three groups (n=6) using B16-F10 cells: PBS group: 200 μL of 1×PBS buffer was injected via tail vein on days 5 and 12 post-vaccination. LNP group: On days 5 and 12 after vaccination, mice were injected via tail vein with 0.5 mg / kg (body weight) of Blank LNP prepared in Comparative Example 1 (injection volume 200 μL). Pyr-LNP group: On days 5 and 12 after inoculation, mice were injected via tail vein with 0.5 mg / kg (body weight of mice) of LNP-Pyrvinium prepared in Example 2 (injection volume 200 μL).

[0105] From day 5 after tumor cell inoculation, tumor volume and survival time of mice in each group were monitored. When the tumor volume of the mouse exceeded 1500 mm, the survival time was recorded. 3 After that, it is considered an ethical death, and the result is as follows: Figure 7 As shown in Figure A.

[0106] B. Using BALB / c mice as subjects, subcutaneously inoculate with 1×10 6 Mice were randomly divided into two groups (n=8) using CT26 cells: PBS group: 200 μL of 1×PBS buffer was injected via tail vein on days 5 and 12 post-vaccination. Pyr-LNP group: On days 5 and 12 after inoculation, mice were injected via tail vein with 0.5 mg / kg (body weight of mice) of LNP-Pyrvinium prepared in Example 2 (injection volume 200 μL).

[0107] From day 5 after tumor cell inoculation, tumor volume and survival time of mice in each group were monitored. When the tumor volume of the mouse exceeded 1500 mm, the survival time was recorded. 3 After that, it is considered an ethical death, and the result is as follows: Figure 7 As shown in B.

[0108] C. Using immunodeficient mice (C-NKG) as subjects, subcutaneously inoculate with 1×10 6 Mice were randomly divided into two groups (n=7) using 10 B16-F10 cells: PBS group: 200 μL of 1×PBS buffer was injected via tail vein on days 5 and 12 post-vaccination. Pyr-LNP group: On days 5 and 12 after inoculation, mice were injected via tail vein with 0.5 mg / kg (body weight of mice) of LNP-Pyrvinium prepared in Example 2 (injection volume 200 μL).

[0109] From day 5 after tumor cell inoculation, tumor volume and survival time of mice in each group were monitored. When the tumor volume of the mouse exceeded 1500 mm, the survival time was recorded. 3 After that, it is considered an ethical death, and the result is as follows: Figure 7 As shown in C.

[0110] D. Immunodeficient mice (C-NKG) were subcutaneously inoculated with 1x10 6 Mice were randomly divided into two groups (n=8) using CT26 cells: PBS group: 200 μL of 1×PBS buffer was injected via tail vein on days 7 and 14 post-vaccination; Pyr-LNP group: On days 7 and 14 after inoculation, mice were injected via tail vein with 0.5 mg / kg (body weight of mice) of LNP-Pyrvinium prepared in Example 2 (injection volume 200 μL).

[0111] From day 7 after tumor cell inoculation, tumor volume and survival time of mice in each group were monitored. When the tumor volume of the mouse exceeded 1500 mm, the survival time was recorded. 3 After that, it is considered an ethical death, and the result is as follows: Figure 7 As shown in D.

[0112] The results showed that LNP-Pyrvinium significantly inhibited tumor growth and prolonged the survival of immunocompetent mice; however, it lost its anti-tumor effect in immunodeficient mice.

[0113] Example 12 Pyr-LNP enhances the antitumor effects of immune checkpoint inhibitors; A. Using C57BL / 6 mice as subjects, 1×10⁻⁶ mice were subcutaneously injected with 1×10⁻⁶ mice. 6 Mice were randomly divided into four groups (n=6) using B16-F10 cells: IgG group: 200 μg of anti-IgG was injected intraperitoneally once every three days for a total of three injections, serving as an isotype control for anti-PD-L1.

[0114] Pyr-LNP group: On day 7 after vaccination, mice were injected via tail vein with 0.5 mg / kg (body weight) of LNP-Pyrvinium prepared in Example 2, once a week for a total of two weeks; Anti-PD-L1 group: On day 8 after vaccination, 200 μg of anti-PD-L1 (purchased from Bio X CellInVivoMAb anti-mouse PD-L1, catalog number #BE0101) was injected intraperitoneally, once every three days, for a total of 3 injections; Pyr-LNP+Anti-PD-L1 group: 7 days after vaccination, mice were injected with 0.5 mg / kg (body weight of mice) of LNP-Pyrvinium prepared in Example 2 via the tail vein once a week for a total of two times; at the same time, on the 8th day after vaccination, mice were injected intraperitoneally with 200 μg of anti-PD-L1 once every three days for a total of 3 injections.

[0115] From day 6 after tumor cell inoculation, tumor volume and survival time in mice were monitored. When the tumor volume of the mouse exceeded 1500 mm, the survival rate was determined. 3 After that, it is considered an ethical death, and the result is as follows: Figure 8 As shown in Figure A. The results indicate that LNP-Pyrvinium can mediate antitumor effects in the B16-F10 model that is unresponsive to anti-PD-L1 treatment, and the combined use of the two has a stronger antitumor effect.

[0116] B. Using BALB / c mice as subjects, subcutaneously inoculate with 1×10 6 Mice were randomly divided into four groups using CT26 cells: IgG group: 200 μg of anti-IgG was injected intraperitoneally once every three days for a total of three injections, serving as an isotype control for anti-PD-1 and anti-CTLA-4 (n=7).

[0117] Pyr-LNP group: On day 4 after vaccination, mice were injected via tail vein with 0.5 mg / kg (body weight) of LNP-Pyrvinium prepared in Example 2, once a week for a total of two times (n=8). Anti-PD-1+anti-CTLA-4 group: On day 5 after vaccination, 100 μg of anti-PD-1 (purchased from Bio X Cell InVivoMAb anti-mouse PD-1, catalog number #BE0146) and anti-CTLA-4 (purchased from Bio X Cell InVivoMAb anti-mouse CTLA-4, catalog number #BE0164) were injected intraperitoneally, once every three days, for a total of 3 injections (n=8); Pyr-LNP+Anti-PD-1+anti-CTLA-4 group: On day 4 after vaccination, mice were injected with 0.5 mg / kg (body weight of mice) of LNP-Pyrvinium prepared in Example 2 via the tail vein, once a week for a total of two times; at the same time, on day 5 after vaccination, mice were injected intraperitoneally with 100 μg of anti-PD-1 and anti-CTLA-4, once every three days for a total of 3 injections (n=8).

[0118] Tumor volume and survival in mice were monitored on day 4 after tumor cell inoculation. Tumors exceeding 1500 mmHg were recorded as tumors. 3 After that, it is considered an ethical death, and the result is as follows: Figure 8 As shown in Figure B. The results showed that the combined use of Pyr-LNP and anti-PD-1 + anti-CTLA-4 significantly enhanced the antitumor effects mediated by either drug alone. Overall, LNP-Pyrvinium can significantly enhance the antitumor effects of immune checkpoint inhibitors.

[0119] Example 14 Pyr-LNP enhances the antitumor effect of adoptive T-cell therapy.

[0120] A. Using C57BL / 6 mice as subjects, 1×10⁻⁶ mice were subcutaneously injected with 1×10⁻⁶ mice. 6 Mice were randomly divided into four groups (n=8) using 10 B16-F10 cells: PBS group: On day 7 after vaccination, 200 μL of 1×PBS buffer was injected via the tail vein.

[0121] Pyr-LNP group: On day 7 after vaccination, mice were injected via tail vein with 0.5 mg / kg (body weight) of LNP-Pyrvinium prepared in Example 2, once a week for a total of two weeks; Pmel group: On day 8 after vaccination, 3×10⁻⁶ cells were intravenously reinfused. 6 Pmel T cells; Pmel T cells were identified using a CD8 T cell sorting kit (Mouse Na) ve CD8 + The T Cell Isolation Kit (company: STEMCELL TECHNOLOGIES, catalog number: 19858A) was obtained from spleen cells of Pmel transgenic mice.

[0122] Pyr-LNP+Pmel group: On day 7 after vaccination, mice were injected via tail vein with 0.5 mg / kg (body weight) of LNP-Pyrvinium prepared in Example 2, once a week for a total of two weeks; simultaneously, on day 8 after vaccination, 3×10 6 Pmel T cells.

[0123] Tumor volume and survival in mice were monitored on day 4 after tumor cell inoculation. Tumors exceeding 1500 mmHg were recorded as tumors. 3 After that, it is considered an ethical death, and the result is as follows: Figure 9 As shown in Figure A. The results indicate that both LNP-Pyrvinium and Pmel T cell infusion can significantly inhibit tumor growth and prolong the survival of mice. More importantly, the combined use of the two has a stronger anti-tumor effect.

[0124] B. Using C57BL / 6 mice as subjects, subcutaneously inoculate with 1×10⁻⁶ 6 Mice were randomly divided into four groups using 100 B16-OVA cells: PBS group: On day 11 after vaccination, 200 μL of 1×PBS buffer was injected via the tail vein (n=9).

[0125] Pyr-LNP group: On day 11 after vaccination, mice were injected with 0.5 mg / kg (body weight of mice) of LNP-Pyrvinium prepared in Example 2 via the tail vein once a week for a total of two weeks (n=8). OT-1 group: On day 12 after vaccination, 3×10⁻⁶ cells were intravenously reinfused. 6 OT-1 T cells; OT-1 T cells are produced using a CD8 T cell sorting kit (Mouse Na...). ve CD8 + T Cell Isolation Kit (company: STEMCELL TECHNOLOGIES, catalog number: 19858A) was obtained from spleen cells of OT-1 transgenic mice (n=8).

[0126] Pyr-LNP+OT-1 group: On day 11 after vaccination, mice were injected via tail vein with 0.5 mg / kg (body weight) of LNP-Pyrvinium prepared in Example 2, once a week for a total of two weeks; simultaneously, on day 12 after vaccination, 3×10 6 OT-1 T cells (n=7).

[0127] From day 11 after tumor cell inoculation, tumor volume and survival time in mice were monitored. When the tumor volume of the mouse exceeded 1500 mm, the survival time was determined. 3 After that, it is considered an ethical death, and the result is as follows: Figure 9 As shown in Figure B. The results indicate that both LNP-Pyrvinium and OT-1 T cell infusion can significantly inhibit tumor growth and prolong the survival of mice. More importantly, the combined use of the two has a stronger anti-tumor effect. Overall, Pyr-LNP can significantly enhance the anti-tumor effect of adoptive T cell therapy.

[0128] In summary, the pyrifos chloride lipid nanoparticles described in this invention can precisely target tumor tissues, improve drug distribution in vivo, and exert anti-tumor effects through an immune system-dependent mechanism. Furthermore, they can significantly enhance the anti-tumor efficacy of immune checkpoint inhibitors and adoptive T-cell therapy. Compared to existing technologies, this invention exhibits significant advantages in tumor targeting, drug delivery efficiency, and immunotherapy sensitization capabilities. It is suitable for combination immunotherapy strategies for drug-resistant tumors and possesses broad prospects for clinical translation and application.

[0129] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A pyrethroid chloride lipid nanoparticle, characterized in that, Including pyrethrum liposomes and / or pyrethrum lipomicelles; The pyrethrum chloride liposomes comprise a mixture of pyrethrum chloride and lipids; the lipid mixture comprises phospholipids, cholesterol, and a targeting ligand-lipid conjugate. The pyravirin chloride lipid micelles comprise pyravirin chloride, PEG-lipid, and a targeting ligand-lipid conjugate. The targeted ligand-lipid conjugates include DSPE-PEG2000-cRGD and / or DMG-PEG2000-cRGD.

2. The pyravirin chloride lipid nanoparticles according to claim 1, characterized in that, In the pyrifos chloride liposomes, the mass ratio of the lipid mixture to pyrifos chloride is 5~20:1; based on the total molar number of the lipid mixture, the molar percentage of the phospholipid is 40%~70%, the molar percentage of the cholesterol is 25%~50%, and the molar percentage of the targeting ligand-lipid conjugate is 5%~10%. The drug loading of the pyrifos chloride lipid micelles is 0.2~1 mg / mL; based on the total molar number of the PEG-lipid and the targeting ligand-lipid conjugate, the molar percentage of the PEG-lipid is 50%~90%, and the molar percentage of the targeting ligand-lipid conjugate is 10%~50%.

3. The pyrethroid chloride lipid nanoparticles according to claim 1, characterized in that, The phospholipids include at least one of HSPC, DSPC, and DOTAP; The PEG-lipids include DSPE-PEG2000 and / or DMG-PEG2000.

4. A method for preparing pyrethroid liposomes in pyrethroid liposomes of any one of claims 1 to 3, characterized in that, This includes thin-film dispersion and / or reverse evaporation methods; The thin-film dispersion method includes the following steps: dissolving a mixture of pyrifos chloride and lipids in an organic solvent to obtain a mixture; removing the organic solvent from the mixture to obtain a thin film; adding an aqueous medium to the thin film for hydration to obtain a suspension; and subjecting the suspension to ultrasonication and ultrafiltration concentration to obtain the pyrifos chloride liposomes. The reverse evaporation method includes the following steps: dissolving a mixture of pyrifos chloride and lipids in an organic solvent to obtain an organic phase solution; adding the organic phase solution dropwise to an aqueous medium to perform self-assembly to obtain a self-assembled system; removing the organic solvent from the self-assembled system to obtain a lipid suspension; and sonicating the lipid suspension and concentrating it by ultrafiltration to obtain the pyrifos chloride liposomes. The organic solvent includes at least one of ethanol, chloroform, and pyridine; The ultrafiltration concentration is performed using an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa.

5. The preparation method according to claim 4, characterized in that, The hydration temperature is 42~45℃; the hydration time is 30 min.

6. The preparation method according to claim 4, characterized in that, The power of the ultrasound is 150~300 W; the duration of the ultrasound is 1~5 min.

7. A method for preparing pyrethrum chloride lipid micelles in pyrethrum chloride lipid nanoparticles according to any one of claims 1 to 3, characterized in that, Includes the following steps: The PEG-lipid, the targeting ligand-lipid conjugate, and water are mixed to obtain a lipid solution; the pyrifos chloride is dissolved in an organic solvent to obtain a pyrifos chloride solution; under stirring conditions, the pyrifos chloride solution is added dropwise to the lipid solution to form a premix. The premixed liquid was subjected to rotary evaporation and sonication to obtain a suspension; the suspension was then filtered and concentrated by ultrafiltration to obtain the pyrifos chloride lipid micelles. The volume ratio of the pyravirin chloride solution to the lipid solution is 1:10; the stirring speed is 600~1000 rpm.

8. The use of the pyravirin chloride lipid nanoparticles according to any one of claims 1 to 3 in the preparation of antitumor drugs.

9. The application according to claim 8, characterized in that, The drug is used to increase the rigidity of tumor cells and / or enhance tumor immunity.

10. A pharmaceutical composition, characterized in that, The invention includes pyravirin chloride lipid nanoparticles as described in any one of claims 1 to 3 and an antitumor immunosuppressant; the antitumor immunosuppressant includes immune checkpoint inhibitors and / or adoptive T-cell preparations.

Images