Membrane fusion liposome for generating CAR-M in vivo as well as preparation method and application of membrane fusion liposome

Abstract

The invention discloses a membrane fusion liposome for generating CAR-M in vivo as well as a preparation method and application of the membrane fusion liposome, and relates to the technical field of biological medicines. The membrane fusion liposome comprises a lipid membrane and a plasmid which is wrapped by the lipid membrane and is provided with a CAR (Chimeric Antigen Receptor), the lipid membrane is prepared from DOTAP (Dioctyl Terephthalate), DOPE (Dioctyl Polyethylene), DSPE-PEG2000 (Distearoyl Sulfonate Polyethylene Glycol 2000) and According to the invention, the liposome is utilized to directly generate CAR-M in vivo, and a novel, convenient and efficient implementation scheme is provided for the CAR-M therapy to treat solid tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a membrane fusion liposome for producing CAR-M in vivo, a preparation method and application thereof. BACKGROUND

[0002] Tumors seriously endanger people's life and health. Traditional treatment methods face severe challenges: surgical resection is limited by the complex anatomical location of tumors and the difficulty of early diagnosis; radiotherapy and chemotherapy seriously affect the quality of life of patients due to their serious side effects. Chimeric antigen receptor macrophage (CAR-M) therapy opens up a new way for the treatment of solid tumors. Researchers at the University of Pennsylvania used lentiviral gene vector genetic engineering to modify macrophages into CAR-M, which showed excellent therapeutic effect on solid tumors. The HER2 CAR-M developed by the research team has completed an effective phase I clinical experiment.

[0003] In particular, the in situ CAR-M construction technology based on non-viral vectors effectively avoids the cell exhaustion risk and high cost problems brought by in vitro modification, while significantly reducing the potential insertion mutation and immunogenicity risk caused by traditional viral vectors. However, the existing non-viral delivery system faces the core bottleneck of insufficient transfection efficiency. This is mainly due to the fact that most non-viral gene vectors currently enter cells through endocytosis, resulting in lysosomal degradation or exocytosis of the gene drug out of the cell. SUMMARY

[0004] In view of the deficiencies in the above background art, the present application mainly solves the problem of insufficient transfection efficiency of CAR-M non-viral vectors. The present application provides a membrane fusion liposome for producing CAR-M in vivo, a preparation method and application thereof. The present application constructs a liposome containing a cyanine dye aggregate to encapsulate a CAR gene plasmid. After the liposome is diffused to the tumor tissue by intratumoral injection, it can target tumor-associated macrophages (TAM) and deliver the CAR gene plasmid through membrane fusion, so that TAM can express CAR on its surface to target and kill tumor cells, achieving the purpose of treating solid tumors.

[0005] The first object of the present application is to provide a membrane fusion liposome for producing CAR-M in vivo, which comprises a lipid membrane and a CAR-containing plasmid wrapped by the lipid membrane. The raw materials of the lipid membrane include lipids, auxiliary lipids, DSPE-PEG2000 and CyBI7; The lipids are DOTAP or DOPC; The auxiliary lipids are DOPE, DOPC or cholesterol; The plasmid with CAR comprises a single-chain antibody against mesothelin, a transmembrane region, an Fc gamma R, alpha V a beta 3 and an EGFP reporter gene region.

[0006] Preferably, the molar ratio of the lipid, the auxiliary lipid, the DSPE-PEG2000, the CyBI7 is (20-50):(50-80):(1-10):(3-10).

[0007] Preferably, the raw material of the lipid membrane further comprises 16:0 PA-PEG3-mannose. The molar ratio of the lipid, the auxiliary lipid, the DSPE-PEG2000, the 16:0 PA-PEG3-mannose and the CyBI7 is (20-50):(50-80):(1-10):1:(3-10).

[0008] The second object of the present application is to provide a preparation method of the membrane fusion liposome for producing CAR-M in vivo, comprising the following steps: The raw material of the lipid membrane is dissolved in a first solvent as a first oil phase solution, and the first solvent is removed by an evaporator or an electric hair dryer to obtain the lipid membrane; The second solvent is added to the lipid membrane to obtain a second oil phase solution; The CAR plasmid is dissolved in a third solvent as an aqueous phase solution; Based on the microfluidic method, the aqueous phase solution and the second oil phase solution are respectively added to two inlets of a Y-shaped microfluidic chip through injection pumps, and a liposome suspension is obtained at the outlet; The liposome suspension is dialyzed to obtain the membrane fusion liposome for producing CAR-M in vivo.

[0009] Preferably, the mass ratio of the CAR plasmid in the aqueous phase solution to the solute of the second oil phase solution is 1:(4-30).

[0010] Preferably, the first solvent is one or more of chloroform, methanol, DMSO and ethanol; and the second solvent is ethanol.

[0011] Preferably, the third solvent is pure water or a sodium phosphate solution.

[0012] The third object of the present application is to provide an application of the membrane fusion liposome for producing CAR-M in vivo in the preparation of a drug for preventing or treating tumors.

[0013] Compared with the prior art, the present application has the following beneficial effects: The application provides a membrane fusion liposome for in vivo production of CAR-M and a preparation method and application thereof, the liposome prepared in the application directly produces CAR-M in vivo, intratumor injection directly transforms TAM into CAR-M cells, and provides a novel, convenient and universal implementation scheme for CAR-M therapy.

[0014] The liposome prepared in the application for in vivo production of CAR-M enables the final synthesis of CAR-M to be completed in vivo, and the liposome can efficiently deliver a CAR gene plasmid through membrane fusion, thereby improving the transfection efficiency of existing non-viral vectors. Each component in the lipid membrane of the liposome, especially the cyanine dye capable of being aggregated in the lipid bilayer, is the key to enabling the liposome to achieve efficient membrane fusion. The CAR gene-loaded liposome prepared in the application can deliver the CAR gene to TAM through membrane fusion, and this delivery method can directly deliver the CAR gene plasmid to the cytoplasm, thereby effectively avoiding the lysosome degradation problem existing in most existing non-viral vectors. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is (a) a particle size distribution graph, (b) an average particle size column chart and (c) an average zeta potential column chart of examples 1, 2 and 3; Figure 2 is a membrane fusion efficiency graph of examples 1, 2 and 3; Figure 3 is (a) an agarose gel electrophoresis and (b) a gene loading rate quantification graph of examples 1, 2 and 3; Figure 4 is (a) a flow distribution graph and (b) a transfection efficiency column chart of transfected M2 type macrophages of examples 1, 2, 3 and 4; Figure 5 is the toxicity of examples 1 and 2 on M2 macrophages in vitro; Figure 6 is a cell uptake mechanism research result graph of example 1; Figure 7 is a tumor cell killing effect graph of CAR-M mediated by examples 1 and 2 in vitro; Figure 8 is a non-target tumor cell killing effect graph of CAR-M mediated by examples 1 and 2 in vitro; Figure 9 is (a) a tumor fluorescence quantification column chart and (b) a tumor section fluorescence quantification column chart of tumor expression of CAR mediated by examples 1 and 2; Figure 10 is (a) a tumor growth curve and (b) an H&E staining section graph after treatment of examples 1 and 2; Figure 11 are H&E stained sections of major organs after treatment of Examples 1 and 2. DETAILED DESCRIPTION

[0016] In order to make the skilled in the art better understand the technical solutions of the present application can be implemented, the following specific embodiments and the present application is further described with reference to the drawings, but the examples are not as a limitation of the present application.

[0017] The purpose of the present application is to solve the problem of insufficient transfection efficiency of CAR-M non-viral vectors, and provide a membrane fusion liposome for in vivo production of CAR-M, a preparation method and application thereof, by constructing a liposome containing a car gene plasmid encapsulated by a cyanine dye aggregate, and after diffusing into tumor tissue by intratumoral injection, the liposome can target tumor-associated macrophages (TAM) to deliver CAR gene plasmid through membrane fusion, so that TAM can express CAR on the surface to kill tumor cells, and achieve the purpose of treating solid tumors.

[0018] In order to achieve the above purpose, the first aspect of the present application provides a membrane fusion liposome for in vivo production of CAR-M, the membrane fusion liposome comprising a lipid membrane, and a CAR-containing plasmid wrapped by the lipid membrane; The raw materials of the lipid membrane include lipids, auxiliary lipids, DSPE-PEG2000 and CyBI7; The lipids are DOTAP or DOPC and cholesterol; the auxiliary lipids are DOPE or DOPC; The CAR-containing plasmid comprises a single-chain antibody against mesothelin, a transmembrane region, FcγR, alpha V Beta 3 and EGFP reporter gene region; wherein the EGFP reporter gene is used for flow cytometry to detect the expression of CAR.

[0019] The present application constructs a liposome containing a cyanine dye aggregate to encapsulate a CAR gene plasmid, and each component in the lipid membrane of the liposome, especially the cyanine dye which can aggregate in the lipid bilayer, is the key to the efficient membrane fusion of the liposome. The liposome efficiently delivers CAR gene plasmid through membrane fusion, thereby improving the transfection efficiency of existing non-viral vectors.

[0020] The molar ratio of the lipids, auxiliary lipids, DSPE-PEG2000, and CyBI7 is (20-50):(50-80):(1-10):(3-10).

[0021] In the present application, the raw materials of the lipid membrane also include 16:0 PA-PEG3-mannose. The molar ratio of the lipid, the auxiliary lipid, DSPE-PEG2000, 16:0 PA-PEG3-mannose and CyBI7 is (20-50):(50-80):(1-10):1:(3-10).

[0022] The target protein of the CAR of the plasmid with the CAR is mesothelin (MLSN).

[0023] DOTAP represents 1,2-dioleoyl-3-trimethylammonium-propane; DOPC represents 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE represents dioleoylphosphatidylethanolamine; DSPE-PEG2000 represents 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxypolyethylene glycol-2000); CyBI7 represents a hydrophobic cyanine dye; 16:0 PA-PEG3-mannose represents 1,2-dipalmitoyl-sn-glycero-3-phospho ((ethyl-1',2',3'-triazole) triethylene glycol mannose) ammonium salt, which is connected by a phosphoric acid group to polyethylene glycol (PEG3) from palmitic acid (16:0 PA), and the other end of PEG3 is connected to mannose (mannose).

[0024] The second aspect of the application provides a preparation method of membrane fusion liposomes for in vivo production of CAR-M, comprising the following steps: The raw material of the lipid membrane is dissolved in a first solvent as a first oil phase solution, and the first solvent is removed by an evaporator or an electric hair dryer to obtain a lipid membrane; A second solvent is added to the lipid membrane to obtain a second oil phase solution; A third solvent is used as an aqueous phase solvent to dissolve a CAR plasmid as an aqueous phase solution; Based on a microfluidic method, the aqueous phase solution and the second oil phase solution are respectively added to two inlets of a Y-shaped microfluidic chip through injection pumps, and a liposome suspension is obtained at an outlet; The liposome suspension is dialyzed to obtain the membrane fusion liposomes for in vivo production of CAR-M.

[0025] The mass ratio of the CAR plasmid in the aqueous phase solution to the solute of the second oil phase solution is 1:4-30. The first solvent is one or more of chloroform, methanol, DMSO and ethanol; the second solvent is ethanol; and the third solvent is pure water or a sodium phosphate solution.

[0026] The liposome prepared by the application can deliver the CAR gene to TAM through membrane fusion, which can directly deliver the CAR gene plasmid to the cytoplasm, effectively avoiding the lysosomal degradation problem existing in most existing non-viral vectors. The following technical difficulties are overcome in the preparation process of the application: 1) In order to realize efficient membrane fusion of liposomes and cell membranes, the application needs to screen the ratio of each lipid DOTAP, DOPE, DSPE-PEG2000, 16:0 PA-PEG3-mannose and CyBI7; 2) In order to improve the binding efficiency of liposomes and TAM, the application adds a mannose ligand; 3) In order to maintain the positive charge of the liposome, the application adds DOTAP lipid. It should be noted that the mannose ligand has high affinity and specific binding capacity with the large number of mannose receptors naturally enriched on the surface of macrophages, and is often used in the strategy of targeted drug delivery of macrophages. The 16:0 PA-PEG3-mannose in the application is a phospholipid component that can target macrophages in the liposome by modifying a mannose ligand at the end of PEG. By adding or not adding 16:0 PA-PEG3-mannose during the preparation of the liposome, membrane fusion liposomes with or without mannose ligands on the surface can be generated, thereby generating membrane fusion liposomes with targeting and non-targeting properties.

[0027] For example, a preparation method of a membrane fusion liposome for producing CAR-M in vivo includes the following steps: S1: weigh the raw materials, dissolve them in a first solvent as an oil phase solution, remove the first solvent with an evaporator or an electric hair dryer to obtain a lipid membrane; add a second solvent to the lipid membrane to obtain an oil phase solution.

[0028] S2: dissolve the CAR plasmid with a third solvent as an aqueous phase solvent to obtain an aqueous phase solution, so that the mass ratio of the CAR plasmid to the solute of the oil phase solution is 1: (4-30), and the volume ratio is 1:3.

[0029] S3: prepare the membrane fusion liposome by using a microfluidic method, the aqueous phase and the oil phase are respectively added to two inlets of a Y-shaped microfluidic chip through a syringe pump, and the liposome suspension is obtained at the outlet.

[0030] S4: dialysis is performed on the liposome suspension obtained in S3 to remove free CyBI7 and the second solvent. The molecular weight cut-off of the dialysis is 3.5 kDa-10 kDa.

[0031] The raw materials include DOTAP, DOPE, DSPE-PEG2000 and CyBI7, wherein DOTAP can be replaced by DOPC; DOPE can be replaced by DOPC or cholesterol; DSPE-PEG2000 can be replaced by DMG-PEG; and a part of DSPE-PEG2000, which accounts for 0.5%-1.5% of the total lipid molar amount, can also be replaced by 16:0 PA-PEG3-mannose.

[0032] The third aspect of the present application provides a use of a membrane fusion liposome for in vivo production of CAR-M in the preparation of a drug for preventing or treating tumors.

[0033] The present application first prepares liposomes and performs basic property characterization; then performs in vitro cell level function verification to explore the ability of the liposomes to transfect macrophages into CAR-M. Finally, a mouse subcutaneous tumor model is constructed to evaluate the in vivo effect of the liposomes, and the main evaluation indexes include tumor volume, CAR expression in the tumor, etc.

[0034] It should be noted that the experimental methods used in the present application are conventional methods unless otherwise specified; and the reagents and materials used are commercially available unless otherwise specified.

[0035] In the following examples, DOPE, DOTAP, DSPE-PEG2K and PA-PEG3-mannose are respectively dissolved in chloroform to prepare solutions with corresponding concentrations.

[0036] Example 1 A preparation method of a membrane fusion liposome (CMPIL@pCAR) for in vivo production of CAR-M, comprising: DOPE, DOTAP, DSPE-PEG2K and PA-PEG3-mannose are respectively dissolved in chloroform to prepare solutions with a concentration of 25 mg / mL, and CyBI7 is dissolved in chloroform to prepare a solution with a concentration of 5 mg / mL.

[0037] Then, 29.76 μL (25 mg / mL) of DOPE, 25.13 μL (25 mg / mL) of DOTAP, 8.98 μL (25 mg / mL) of DSPE-PEG2K, 21.1 μL (1 mg / mL) of PA-PEG3-mannose and 17.92 μL (5 mg / mL) of CyBI7 are added to a 2 mL centrifuge tube, and after being blown dry by a hair dryer, 250 μL of ethanol is added to fully dissolve to obtain an organic phase.

[0038] Another 2 mL centrifuge tube was taken, and the plasmid solution containing 62.5 μg of CAR plasmid was added and then diluted with sterile enzyme-free water to obtain 750 μL of an aqueous phase. The total flow rate of the microfluidic device was set to 1 mL, and the flow rate ratio of the organic phase to the aqueous phase was set to 1:3. The preliminary microfluidic work obtained CMPIL@pCAR.

[0039] Subsequently, the CMPIL@pCAR was placed in a dialysis bag with a molecular weight cut-off of 3500 Da and dialyzed in pure water for 3 times, with water changed every 2 h, to finally obtain CMPIL@pCAR.

[0040] The CMPIL@pCAR prepared in this example was characterized, and the results are shown in Figure 1 、 Figure 2 and Figure 3 The particle size of CMPIL@pCAR was 69.6 nm, the Zeta potential was +28.4 mV, the 5-minute membrane fusion efficiency was 79.8%, and the gene loading rate was 99.3%.

[0041] Example 2 A method for preparing a membrane fusion liposome (CPIL@pCAR) for in vivo production of CAR-M, comprising: DOPE, DOTAP, and DSPE-PEG2K were respectively dissolved in chloroform to prepare solutions with a concentration of 25 mg / mL, and CyBI7 was dissolved in chloroform to prepare a solution with a concentration of 5 mg / mL.

[0042] Then, 29.76 μL of DOPE (25 mg / mL), 25.13 μL of DOTAP (25 mg / mL), 8.98 μL of DSPE-PEG2K (25 mg / mL), and 17.92 μL of CyBI7 (5 mg / mL) were added to a 2 mL centrifuge tube, and after blowing dry with a hair dryer, 250 μL of ethanol was added to fully dissolve to obtain an organic phase.

[0043] Another 2 mL centrifuge tube was taken, and the plasmid solution containing 62.5 μg of CAR plasmid was added and then diluted with sterile enzyme-free water to obtain 750 μL of an aqueous phase. The total flow rate of the microfluidic device was set to 1 mL, and the flow rate ratio of the organic phase to the aqueous phase was set to 1:3. The preliminary microfluidic work obtained CMPIL@pCAR.

[0044] Subsequently, the CMPIL@pCAR was placed in a dialysis bag with a molecular weight cut-off of 3500 Da and dialyzed in pure water for 3 times, with water changed every 2 h, to finally obtain CMPIL@pCAR.

[0045] The CMPIL@pCAR prepared in this example was characterized, and the results are shown in Figure 1 、 Figure 2and Figure 3 As shown in FIG. 1, the particle size of the CPIL@pCAR was 42.4 nm, the Zeta potential was +24.7 mV, the 1-minute membrane fusion efficiency was 73.4%, and the gene loading rate was 98.1%.

[0046] Example 3 A method for preparing a membrane fusion liposome (MPIL@pCAR) for in vivo production of CAR-M, comprising: DOPE, DOTAP, DSPE-PEG2K, and PA-PEG3-mannose were respectively dissolved in chloroform to prepare solutions with a concentration of 25 mg / mL. PA-PEG3-mannose was dissolved in chloroform to prepare a solution with a concentration of 1 mg / mL.

[0047] Then, 29.76 μL (25 mg / mL) of DOPE, 25.13 μL (25 mg / mL) of DOTAP, 8.98 μL (25 mg / mL) of DSPE-PEG2K, and 21.1 μL (1 mg / mL) of PA-PEG3-mannose were added to a 2 mL centrifuge tube, and after being blown dry by a hair dryer, 250 μL of ethanol was added to fully dissolve the organic phase.

[0048] Another 2 mL centrifuge tube was taken, 62.5 μg of CAR plasmid solution was weighed and added, and then sterile enzyme-free water was added to dilute to 750 μL of aqueous phase. The total flow rate of the microfluidic device was set to 1 mL, and the flow rate ratio of the organic phase to the aqueous phase was set to 1:3. The preliminary work of the microfluidic device obtained MPIL@pCAR.

[0049] Subsequently, the MPIL@pCAR was placed in a dialysis bag with a molecular weight cut-off of 3500 Da and dialyzed in pure water for 3 times, with water being replaced every 2 h, and finally MPIL@pCAR was obtained.

[0050] The MPIL@pCAR prepared in this example was characterized, and the results are shown in FIG. 1. Figure 1 、 Figure 2 and Figure 3 As shown in FIG. 1, the particle size of the MPIL@pCAR was 42.4 nm, the Zeta potential was +24.7 mV, the 1-minute membrane fusion efficiency was 73.4%, and the gene loading rate was 98.1%.

[0051] Example 4 Preparation of Lipofectamine 3000 / pCAR complex 3 μg CAR plasmid was mixed with 6 μL P3000 reagent, diluted into 750 μL serum-free culture, and then mixed with 750 μL serum-free medium containing 9 μL Lipofectamine 3000 reagent, incubated for 10-15 min, to obtain Lipofectamine 3000 / pCAR (LPF3K / pCAR) complex.

[0052] Example 5 The same as example 1, except that DOTAP is replaced by DOPC, and DOPE is replaced by cholesterol. The molar ratio of DOPC, cholesterol, DSPE-PEG2000, 16:0 PA-PEG3-mannose and CyBI7 is 20:50:1:1:3.

[0053] Example 6 The same as example 1, except that DOTAP is replaced by DOPC, and DOPE is replaced by DOPC. The molar ratio of DOPC, DOPC, DSPE-PEG2000, 16:0 PA-PEG3-mannose and CyBI7 is 50:80:10:1:10.

[0054] Example 7 The same as example 1, except that DOPE is replaced by cholesterol. The molar ratio of DOTAP, cholesterol, DSPE-PEG2000, 16:0 PA-PEG3-mannose and CyBI7 is 35:70:5:1:5.

[0055] To illustrate the related performance of the membrane fusion liposome for producing CAR-M in vivo provided by the present application, it is described in conjunction with the accompanying drawings.

[0056] I. Evaluation of the effect of the membrane fusion liposomes provided in examples 1-4 on in vitro transfection of macrophages After the CAR plasmid is wrapped with the liposomes of examples 1, 2, 3 and 4 of the present application, it is incubated with induced M2 type macrophage J774A.1 for 48 h (6 h for liquid change), and the plasmid concentration is 1.5 ng / μL. The proportion of cells expressing EGFP is observed by flow cytometry to prove the in vitro transfection efficiency. From Figure 4It can be seen that the CAR expression efficiency of the membrane fusion liposome group CPIL@pCRA in macrophages is 24.1%, which is much higher than that of the endocytosis type liposome group MPIL@pCAR (1.0%) and the Lipofectamine3000 / pCAR (LPF3K / pCAR) group (6.7%). In addition, the CAR expression efficiency of the CMPIL@pCAR group with the targeting effect based on the membrane fusion liposome is 59.1%, which is 2.45 times that of the non-targeting group CPIL@pCAR. The higher transfection efficiency in Examples 1 and 2 proves the high efficiency of the membrane fusion delivery strategy adopted in the present application in generating CAR-M.

[0057] II. Toxicity evaluation of the membrane fusion liposome provided in Examples 1-2 on macrophages in vitro The CMPIL@pCAR and CPIL@pCAR were added to the pre-prepared DMEM medium containing 2% FBS and 1% P / S (penicillin / streptomycin double antibody) and mixed evenly to prepare different concentration gradients: 0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 64 μM and 80 μM, with 5 repeated holes for each concentration. After 6 h of incubation, the medium was changed, and the cell activity was detected by MTT method after 42 h of continuous incubation. From the cell activity curve shown in FIG. 6, it can be seen that when the concentration of CMPIL and CPIL is 40 μM, the cell activity is maintained at about 80%, indicating that the nanoparticles have no obvious cytotoxicity at this concentration; when the concentration of CMPIL and CPIL is increased to 64 μM, the cell activity is decreased to about 75%. Figure 5 It can be seen that when the concentration of CMPIL and CPIL is 40 μM, the cell activity is maintained at about 80%, indicating that the nanoparticles have no obvious cytotoxicity at this concentration; when the concentration of CMPIL and CPIL is increased to 64 μM, the cell activity is decreased to about 75%.

[0058] III. In vitro macrophage uptake results of the membrane fusion liposome provided in Example 1 under different endocytosis inhibition treatments The J774A.1 cells were seeded in 6-well plates at a density of 2×10 5cells / well in 6-well plates, and at the same time, IL-4 (20 ng / μL) was used to polarize macrophages into M2 type macrophages, and the cells were cultured under normal culture conditions for 24 h, and the cells were allowed to adhere and polarize completely. Chlorpromazine (10 μg / mL), genistein (150 μM), or methyl-β-cyclodextrin (5 mg / mL) was added for 1.5 h, and then the original cell inhibitors in the well plates were washed away, and then CMPIL@pCAR was added to the DMEM medium containing 2% FBS and 1% P / S prepared in advance and mixed well in the well plates (to ensure that the plasmid in each well was 3 μg, and the liposome concentration was 64 μM), and 5 repeated wells were set. After 6 h of culture under normal culture conditions, the culture medium was replaced with 2% FBS DMEM maintenance medium, and the cells were cultured in the incubator for 42 h. In the 4°C experimental group, CMPIL@pCAR complex (concentration as above) was first added, and then the cells were cultured at 4°C for 6 h, and then fresh 2% FBS DMEM maintenance medium was added, and the cells were cultured in the incubator for 42 h. After the culture was completed, the cells were washed with PBS buffer and suspended with PBS solution, filtered with a 70 μm filter, and then the fluorescence intensity of the cells in the FL3 channel was quantified by flow cytometry. Chlorpromazine (CPZ), genistein (Geni), and methyl-β-cyclodextrin (MβCD) were used to inhibit clathrin, caveolin, and lipid raft protein-mediated endocytosis, respectively, and 4°C treatment could inhibit energy-mediated endocytosis. Subsequently, the uptake of CMPIL@pCAR by M2 type macrophages was observed. From Figure 6 It can be seen that there is no significant difference in the flow cytometry fluorescence intensity between the CPZ, Geni, and MβCD inhibitor groups compared with the no endocytosis inhibitor treatment group, indicating that after the endocytosis inhibitor treated cells, CMPIL can still be efficiently taken up by M2 type macrophages. The flow cytometry fluorescence intensity of the 4°C treatment group decreased. It can be seen that the endocytosis inhibitor does not affect the delivery effect of CMPIL on pCAR, indicating that CMPIL@pCAR mainly enters the cells through the membrane fusion pathway.

[0059] Four, the membrane fusion liposome provided in Examples 1~2 was used to evaluate the killing of tumor cells by CAR-M generated in vitro The killing ability of CAR-M was evaluated by MTT method. The specific experimental procedure is as follows: three effector cell groups were set: CAR-M (CPIL@pCAR), CAR-M (CMPIL@pCAR) and UTD (i.e. macrophages not transduced with CAR carrying vectors), which were treated with CPIL@pCAR, CMPIL@pCAR and PBS for 48 h (6 h for liquid change) respectively. Then, the three groups of effector cells were added to the adherent target cells Panc02-Luc (expressing MSLN), and the effector-to-target ratio (i.e. the number ratio of effector cells to tumor target cells) was set to 1:1 and 3:1, respectively. At the same time, an equal amount of UTD, CAR-M (CPIL@pCAR) and CAR-M (CMPIL@pCAR) macrophages were added to the wells without target cells as control effector cells, and an equal amount of Panc02-Luc cells without effector cells were set as the target cell control group, and a blank control group without any cells was set. Different killing times of 24 h, 30 h and 36 h were set under different effector-to-target ratios. Finally, the cell activity was detected by MTT method. The groups of effector cell group and target cell group in the wells were recorded as interaction group, only effector cell group as effector group, only tumor cell group as target cell group, and no cell group as blank group, and their absorbance values were A 互作组 , A 效应组 , A 靶细胞组 and A 空白组 , respectively. The killing efficiency of CAR-M / UTD on target cells Panc02-Luc was calculated by the formula: (A 互作组 -A 效应组 ) / (A 靶细胞组 -A 空白组 ). As can be seen from Figure 7 , CMPIL@pCAR mediated CAR-M showed the highest killing rate under the conditions of effector-to-target ratio of 3:1 and killing time of 36 h, reaching nearly 85%, which was 2.43 times (35%) of the killing rate of ordinary macrophages UTD, 1.41 times (60%) of the killing rate of non-targeting control group CAR-M (CPIL@pCAR), and the killing effect was obviously enhanced.

[0060] Five, the killing evaluation of CAR-M mediated by the membrane fusion liposomes provided in embodiments 1~2 on non-targeting tumor cells in vitro The experimental method for killing tumor cells by CAR-M was the same, except that the tumor cells were replaced by MDA-MB-231 breast cancer cells which did not express MSLN. From Figure 8It can be seen that there is no significant difference in the killing rate of UTD group and CAR-M (CPIL@pCAR) and CAR-M (CMPIL@pCAR) groups of macrophages to Panc02-Luc, and both are not more than 40%.

[0061] Six, the tissue distribution of CAR in tumor-bearing mice after intratumoral injection of the membrane fusion liposomes provided in embodiments 1-2 C57 / BL6 mice were subcutaneously inoculated with Panc02 tumor cells, and when the tumor grew to 100 mm 3 Intratumoral injection of CMPIL@pCAR and CPIL@pCAR prepared in embodiments 1 and 2 of the application, each group of 3, the dose of administration is 10 μg / one. 48 h later, the mice were dissected, and the expression of CAR in the tumor was observed by small animal imaging system IVIS. It can be seen from Figure 9 a that the CAR expression fluorescence intensity of the CMPIL@pCAR group is 2.5 times that of the CPIL@pCAR group.

[0062] Seven, the intratumoral distribution of CAR in tumor-bearing mice after intratumoral injection of the membrane fusion liposomes provided in embodiments 1-2 The tumor of each experimental group was cut along the largest surface of the tumor for fluorescence scanning to further observe the expression of CAR in the tumor. It can be seen from Figure 9 b that the CAR expression fluorescence intensity of the tumor section of the CMPIL@pCAR group is 3.4 times that of the CPIL@pCAR group.

[0063] Eight, tumor growth curve and H&E staining results after intratumoral injection of the membrane fusion liposomes provided in embodiments 1-2 C57 / BL6 mice were subcutaneously inoculated with Pan02 tumor cells, and when the tumor grew to 100 mm 3 Intratumoral injection of CMPIL@pCAR and CPIL@pCAR prepared in embodiments 1 and 2 of the application, each group of 5, the dose of administration is 10 μg / one. Treatment once every five days, a total of five times. Record the length and width of the mouse tumor every five days, and calculate the tumor volume. After 40 days of treatment, the mice were dissected, and the tumor sections were stained with H&E, and observed under a microscope. It can be seen from Figure 10 a that after the treatment, the average tumor size of PBS, CPIL@pCAR, CMPIL@pCAR groups is 597.4 mm 3 , 481.6 mm 3 and 335.2 mm 3 , respectively, and the tumor inhibition efficiency of CPIL@pCAR and CMPIL@pCAR is 20% and 44%, respectively. It can be seen from Figure 10It can be seen that the tumor density of the CMPIL@pCAR experimental group is the sparsest, and the tumor cell nuclei are obviously dissociated, while the CPIL@pCAR experimental group is the second, and the tumor tissue of the PBS group still presents a dense condition.

[0064] Nine, the H&E staining results of main organs after intratumoral injection of the membrane fusion liposome provided in embodiments 1~2 After the treatment, the heart, liver, spleen, lung and kidney tissues of the mice were dissected, and the tissue sections were stained with H&E, and observed under a microscope. Figure 11 It can be seen that the morphologies of the heart, liver, spleen, lung and kidney tissues of each group have no obvious changes, and there is no obvious organic damage.

[0065] The preferred embodiments and their effects are described in the present application. However, once the basic creative concept is known, those skilled in the art can make additional changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0066] Although the embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A membrane-fused liposome for in vivo CAR-M production, characterized in that, The membrane-fused liposome includes a lipid membrane and a CAR-containing plasmid encapsulated in the lipid membrane. The raw materials of the lipid membrane include lipids, auxiliary lipids, DSPE-PEG2000 and CyBI7; The lipid is DOTAP or DOPC; The auxiliary lipid is DOPE, DOPC, or cholesterol; The CAR-containing plasmid contains a single-chain antibody against mesothelin, a transmembrane region, FcγR, and α. V β3 and EGFP reporter gene regions.

2. The membrane-fused liposome for in vivo CAR-M generation according to claim 1, characterized in that, The molar ratio of the lipid, auxiliary lipid, DSPE-PEG2000, and CyBI7 is (20~50):(50~80):(1~10):(3~10).

3. The membrane-fused liposome for in vivo CAR-M generation according to claim 1, characterized in that, The raw material of the lipid membrane also includes 16:0 PA-PEG3-mannose; The molar ratio of the lipid, auxiliary lipid, DSPE-PEG2000, 16:0 PA-PEG3-mannose and CyBI7 is (20~50):(50~80):(1~10):1:(3~10).

4. A method for preparing membrane-fused liposomes for in vivo CAR-M production according to any one of claims 1 to 3, characterized in that, Includes the following steps: The raw material for the lipid membrane is dissolved in a first solvent to form a first oil phase solution. The first solvent is removed by an evaporator or a hair dryer to obtain the lipid membrane. A second solvent is added to the lipid membrane to obtain a second oil phase solution; The CAR plasmid was dissolved in a third solvent as the aqueous phase solvent to form an aqueous solution. Based on microfluidics, an aqueous solution and a second oil-phase solution are added to the two inlets of a Y-shaped microfluidic chip via injection pumps, and a liposome suspension is obtained at the outlet. The liposome suspension is dialyzed to obtain membrane-fused liposomes for in vivo CAR-M production.

5. The method for preparing membrane-fused liposomes for in vivo CAR-M production according to claim 4, characterized in that, The mass ratio of the CAR plasmid in the aqueous solution to the solute in the second oil phase solution is 1:(4~30).

6. The method for preparing membrane-fused liposomes for in vivo CAR-M production according to claim 4, characterized in that, The first solvent is one or more of chloroform, methanol, DMSO, and ethanol; the second solvent is ethanol.

7. The method for preparing membrane-fused liposomes for in vivo CAR-M production according to claim 4, characterized in that, The third solvent is pure water or sodium phosphate solution.

8. The use of a membrane-fused liposome for in vivo CAR-M generation as described in any one of claims 1 to 3 in the preparation of a drug for the prevention or treatment of tumors.

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