Hybrid cell membrane modified nano-drug for inhibiting interment of macrophages as well as preparation method and application of nano-drug

Abstract

The invention provides a preparation method of a hybrid cell membrane modified nano-drug for inhibiting interment of macrophages. According to the invention, a multifunctional bionic nano-drug with a'core-shell 'structure is prepared from liver cancer cell membrane and bone marrow-derived macrophage membrane hybrid double-drug-loading carrier nanoparticles. In the application, the bionic function of macrophages is firstly realized, so that the drug is prevented from being swallowed by mononuclear macrophages in vivo, and the side effect of the drug is reduced; secondly, by utilizing the homologous targeting effect of hepatoma carcinoma cells of Hepa1-6, the aggregation of the medicine in tumors is enhanced, and the utilization efficiency of the medicine is improved; finally, the two drugs including sorafenib and Xkr8siRNA are co-delivered, so that the pharmacokinetics is simplified, and the immunotherapy effect of the liver cancer can be promoted by utilizing the inhibition of the Xkr8siRNA on the intercellular effect.

Description

Technical Field

[0001] The invention relates to a hybrid cell membrane modified nano drug for inhibiting macrophage burial, and a preparation method and application thereof. Background Art

[0002] Sorafenib is a first-line anticancer drug used clinically to treat advanced liver cancer, but its hydrophobicity, low bioavailability, side effects and immunosuppression limit its application.

[0003] The reason why sorafenib causes immunosuppression and thus limits its therapeutic effect is mainly due to the upregulation of Xkr8 protein when tumor cells undergo apoptosis, which causes efferocytosis. Xkr8 is a phosphatidylserine (PS) flipping protein. When cells are in a healthy state, cells regulate Xkr8 protein to make PS located on the inner side of the cell membrane, and macrophages cannot recognize and engulf healthy cells. When cells undergo apoptosis, they upregulate Xkr8 protein to flip PS to the outer side of the cell membrane. PS located on the outer side can be recognized, engulfed and cleared by macrophages, thereby maintaining physiological homeostasis. This process is called "efferocytosis". However, in the tumor microenvironment, efferocytosis is conducive to the polarization of macrophages to the M2 phenotype, generating anti-inflammatory and repair signals, creating an immunosuppressive tumor microenvironment, and promoting the occurrence and development of tumors. Therefore, downregulating the expression of Xkr8 in tumor cells can relieve the immunosuppression caused by sorafenib by inhibiting efferocytosis, thereby achieving a better treatment effect for liver cancer.

[0004] In recent years, polymer nanocarriers have shown great potential in improving drug solubility, reducing drug toxicity, passive tumor targeting effects, and biosafety. In addition, the precise targeting and release of drugs can be controlled by modifying the chemical bonds of the carrier in response to the tumor microenvironment, target cell ligands, cell membrane wrapping, etc. Different types of drugs can also be co-delivered through polymer synthesis methods, such as co-delivery of hydrophobic drugs, gene drugs, and antibody drugs. However, the functions of nanomedicines prepared alone are often too single and cannot meet clinical needs. Therefore, it is an important scientific issue to explore an integrated strategy for liver cancer treatment that can escape liver macrophage phagocytosis, target tumor cells, and inhibit efferocytosis in combination with sorafenib.

[0005] The delivery of sorafenib in the existing technology is too single-function, or sorafenib is nano-sized alone, and the nanodrug is modified into a nanodrug that is easier to escape phagocytosis and tumor targeting. However, the delivery of sorafenib alone is likely to cause tumor immunosuppression, limiting its therapeutic effect. Or considering the synergy of sorafenib and other drugs to improve the immunosuppression of single delivery, the design of the carrier is ignored, resulting in low bioavailability of the nanodrug itself and large side effects. This is mainly because the complexity of the function of the nanodrug will inevitably lead to the complexity of the structural design, so it is difficult to design an integrated nanodrug to provide the functions required for the clinical treatment of liver cancer. Summary of the invention

[0006] The purpose of the present invention is to provide a hybrid cell membrane modified nano drug for inhibiting macrophage burial, and a preparation method and application thereof. The nano drug is an integrated nano drug for liver cancer treatment that can escape liver macrophage phagocytosis, target tumor cells, and combine with the burial inhibition effect of sorafenib. The present invention improves the solubility of co-delivered drugs, reduces drug toxicity and side effects, enhances tumor targeting effects, and ensures biosafety, while reversing the immunosuppression caused by sorafenib, thereby achieving a better treatment effect for hepatocellular carcinoma.

[0007] To achieve the above object, the technical solution adopted is: a method for preparing a hybrid cell membrane modified nanomedicine for inhibiting macrophage burial, characterized in that it comprises the following steps:

[0008] (1) The acid-sensitive polymer monomethyl ether polyethylene glycol-polylysine anhydride-polyaspartate aminoethyl piperidine (mPEG-PLLys-Pasp) was mixed with sorafenib and then assembled into single drug-loaded nanodrug NP@SF micelles, and then Xkr8 siRNA and water were added to prepare acid-sensitive nanodrug NP@SF&siRNA loaded with sorafenib and Xkr8 siRNA;

[0009] (2) extracting liver cancer cell membranes and bone marrow-derived macrophage membranes, mixing the extracted liver cancer cell membranes and bone marrow-derived macrophage membranes to obtain a mixed membrane, and then mixing with the nanodrug NP@SF&siRNA prepared in step (1) and extruding and fusing them to obtain a hybrid cell membrane-modified nanodrug HM-NP@SF&siRNA that inhibits macrophage burial.

[0010] Preferably, the preparation method of mPEG-PLLys-PAsp in step (1) comprises the following steps:

[0011] (1a) mPEG-NH2 is weighed, chloroform is added to dissolve it under the protection of nitrogen, and then BCLL-NCA dissolved in N,N-dimethylformamide is added, and the reaction is sealed; after the reaction is completed, the obtained mixed reaction solution is added dropwise to anhydrous ether and refrigerated; then centrifugation is performed to obtain a precipitate, the precipitate is washed with anhydrous ether, and vacuum dried to obtain a white solid monomethyl ether polyethylene glycol-polybenzyloxycarbonyl lysine anhydride copolymer (mPEG-PBCLLys);

[0012] (2a) Weighing the mPEG-PBCLLys prepared in step (1a), adding chloroform under the protection of nitrogen, then adding BLA-NCA dissolved in N,N-dimethylformamide, turning off the nitrogen, and sealing the reaction; after the reaction is completed, adding the obtained mixed solution to anhydrous ether and refrigerating; then centrifuging to obtain a precipitate, washing the precipitate with anhydrous ether, and vacuum drying to obtain a white solid monomethyl ether polyethylene glycol-polybenzyloxycarbonyl lysine anhydride-polybenzyl aspartate copolymer (mPEG-PBCLLys-PBLAsp);

[0013] (3a) taking the mPEG-PBCLLys-PBLAsp prepared in step (2a), dissolving it in anhydrous dimethyl sulfoxide, and then adding 1-(2-aminoethyl)piperidine to react; after the reaction, dialyzing the obtained mixed reaction solution in methanol with a dialysis bag to remove excess 1-(2-aminoethyl)piperidine, and vacuum drying to obtain a light yellow solid monomethyl ether polyethylene glycol-polybenzyloxycarbonyl lysine anhydride-polyaspartic acid aminoethyl piperidine mPEG-PBCLLys-PAsp;

[0014] (4a) mPEG-PBCLLys-PAsp was dissolved with trifluoroacetic acid, and then an acetic acid solution containing hydrogen bromide was added to react. After the reaction, the obtained mixed reaction solution was added to anhydrous ether for precipitation, and the precipitate was obtained by centrifugation to obtain a light yellow solid monomethyl ether polyethylene glycol-polylysine anhydride-polyaspartate aminoethyl piperidine (mPEG-PLLys-PAsp).

[0015] Preferably, in the step (1a), the weight ratio of mPEG-NH2 and BCLL-NCA is 1:2; in the step (2a), the weight ratio of mPEG-PBCLLys and BLA-NCA is 1.74:2.3; in the step (3a), the weight ratio of mPEG-PBCLLys-PBLAsp and 1-(2-aminoethyl)piperidine is 2:3.36; in the step (4a), the weight-to-volume ratio of mPEG-PBCLLys-PAsp and hydrogen bromide is 1.5 g:0.99 mL.

[0016] Preferably, the reaction temperature in step (1a) is 25-37°C and the reaction time is 36-50h; the reaction temperature in step (2a) is 25-37°C and the reaction time is 36-50h; the reaction temperature in step (3a) is 25°C and the reaction time is 24h; the reaction temperature in step (4a) is 25°C and the reaction time is 3h. More preferably, the reaction temperature in step (1a) is 36°C and the reaction time is 48h; the reaction temperature in step (2a) is 35°C and the reaction time is 48h.

[0017] Preferably, in step (1), the weight ratio of mPEG-PLLys-PAsp to sorafenib is 5:1-10:1; the weight molar ratio of the NP@SF micelle to Xkr8 siRNA is 4×10 -2 mg: 2×10 -6 μmol~20×10 -2 mg: 2×10 -6 μmol; The sequence of the Xkr8 siRNA:

[0018] Justice chain: GGCACAAAUUUUAUGCCUGtt;

[0019] Antisense strand: CAGGCAUAAAAUUUGUGCCtt.

[0020] More preferably, in step (1), the weight ratio of mPEG-PLLys-PAsp to sorafenib is 10:1; the weight molar ratio of the NP@SF micelle to Xkr8 siRNA is 8×10 -2 mg: 2×10 -6 μmol.

[0021] Preferably, the method for extracting liver cancer cell membrane in step (2) comprises the following steps:

[0022] Take liver cancer cells, wash the cells with PBS, centrifuge to remove the supernatant, add a hypotonic dissolution buffer to the cell pellet to resuspend, then sonicate on ice to break the cells; place the broken cells on ice, shake violently 2-3 times during the period, discard the pellet after centrifugation, bathe the supernatant at 37°C for 10 minutes, remove the lower layer after centrifugation; add sterile water, place at 4°C for 5 minutes, and then place in a 37°C water bath for 10 minutes; then centrifuge at 25°C and 14000rpm for 10 minutes, remove the lower layer, add sterile water, place at 4°C for 5 minutes, and then place in a 37°C water bath for 10 minutes, centrifuge at 25°C and 14000rpm for 10 minutes, remove the lower layer, and obtain a liver cancer cell membrane protein extract, that is, the liver cancer cell membrane;

[0023] The method for extracting bone marrow-derived macrophage membranes comprises the following steps:

[0024] Take bone marrow-derived macrophages, wash the cells with PBS, centrifuge to remove the supernatant, add a hypotonic lysis buffer to the cell pellet to resuspend, and then sonicate on ice to disrupt the cells; place the disrupted cells on ice, shake violently 2-3 times during the period, discard the precipitate after centrifugation, bathe the supernatant at 37°C for 10 minutes, and take the lower layer after centrifugation; add sterile water, place at 4°C for 5 minutes, and then place in a 37°C water bath for 10 minutes; then centrifuge at 25°C and 14000rpm for 10 minutes, take the lower layer, add sterile water, place at 4°C for 5 minutes, and then place in a 37°C water bath for 10 minutes, and take the lower layer after centrifugation at 25°C and 14000rpm for 10 minutes to obtain a bone marrow-derived macrophage membrane protein extract, that is, the bone marrow-derived macrophage membrane.

[0025] Preferably, the hypotonic dissolution buffer comprises 20 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.4; 10 mM magnesium chloride; 10 mM potassium chloride; 1 wt % protease inhibitor; and 1 wt % 1,4-dimercaptothreitol.

[0026] Preferably, in the step (2), the ratio of the liver cancer cell membrane to the bone marrow-derived macrophage membrane is 3:1-1:1 in terms of protein weight; the weight ratio of the mixed membrane to the nano-drug NP@SF&siRNA is 6:1-1:1; and the liver cancer cell membrane is a Hepa 1-6 liver cancer cell membrane. More preferably, in the step (2), the ratio of the liver cancer cell membrane to the bone marrow-derived macrophage membrane is 1:1 in terms of protein weight; and the weight ratio of the mixed membrane to the nano-drug NP@SF&siRNA is 1:1.

[0027] The present invention provides a hybrid cell membrane-modified nano drug for inhibiting macrophage burial, which is prepared by the above-mentioned preparation method.

[0028] The present invention provides a pharmaceutical composition for treating liver cancer, comprising the hybrid cell membrane modified nanodrug for inhibiting macrophage efferocytosis as described above and an immune checkpoint inhibitor; preferably, the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0029] The present invention provides the use of the hybrid cell membrane modified nano drug for inhibiting macrophage burial or the combined use of the hybrid cell membrane modified nano drug for inhibiting macrophage burial and an immune checkpoint inhibitor in the preparation of a drug for treating liver cancer.

[0030] Beneficial effects:

[0031] The present invention uses liver cancer cell membrane and bone marrow-derived macrophage membrane hybrid dual drug carrier nanoparticles to prepare a "core-shell" structure multifunctional bionic nanomedicine. In terms of application, the present invention first realizes the bionic function of macrophages, so that the drug is prevented from being phagocytosed by mononuclear macrophages in the body, reducing the side effects of the drug; secondly, the homologous targeting effect of Hepa1-6 liver cancer cells is used to enhance the drug aggregation in tumors, thereby improving the utilization efficiency of the drug; finally, the two drugs sorafenib and Xkr8 siRNA co-delivered not only simplify the pharmacokinetics, but also can utilize the inhibition of Xkr8 siRNA on phagocytosis to promote the immunotherapy effect of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 :One-stop delivery of sorafenib (SF) and Xkr8 siRNA using hybrid cell membrane-modified acid-sensitive nanoparticles. (mPEG-PLLys-PAsp(PIP): monomethyl ether polyethylene glycol-polylysine anhydride-polyaspartate aminoethyl piperidine; BMDM: bone marrow-derived macrophages; Hepa 1-6; mouse hepatoma cell line.)

[0033] Figure 2 : H NMR spectra of the reaction products of each step (A) mPEG-PBCLLys, (B) mPEG-PBCLLys-PBLAsp, (C) mPEG-PBCLLys-PAsp and the final product (D) mPEG-PLLys-PAsp (PIP).

[0034] Figure 3 :(A) Transmission electron microscopy (TEM) image of polymer micelles NP@SF loaded with SF. The prepared NP@SF was dropped on a 5μL copper grid and dried, and transmission electron microscopy imaging was performed at a voltage of 120kV. Scale bar: 100nm. (B) Agarose gel electrophoresis experiment evaluated the electrophoretic mobility of micellar siRNA with different N / P. At N / P=2-10, the micelles can completely load Xkr8 siRNA. (C) Absorbance curves of different SF concentrations measured by UV spectrophotometer and (D) the concentration of SF in NP@SF&siRNA.

[0035] Figure 4 .: Transmission electron microscopy image of the nanodrug HM-NP@SF&siRNA, which uses hybrid cell membrane-modified acid-sensitive nanoparticles to deliver sorafenib (SF) and Xkr8 siRNA in one stop. The prepared HM-NP@SF&siRNA was dropped on a 5μL copper grid and dried, and transmission electron microscopy imaging was performed at a voltage of 120kV. Scale bar: 100nm.

[0036] Figure 5:(A) In vivo aggregation of the co-delivery carrier NP@Cy5.5, H-NP@Cy5.5 before tumor cell membrane modification, and HM-NP@Cy5.5 after tumor cell modification after tail vein injection into an orthotopic liver cancer mouse model. (B) In vitro fluorescence imaging of major organs and tumors 48h after injection. (C) Quantitative statistical graph of fluorescence intensity of major organs and tumors in Figure B (n=3, mean±S). (D) The inhibitory effect of the nanodrug HM-NP@SF&siRNA co-delivered with sorafenib and Xkr8siRNA after tail vein injection (injection once every three days, a total of 5 times) on liver cancer (n=6). (E) Cumulative survival rate of mice after receiving different groups of nanodrug treatment. (F) Magnetic resonance imaging (MRI) statistics of the growth curve of orthotopic liver cancer in mice receiving different treatment groups (n=6). *P<0.05, **P<0.01, ****P<0.0001. DETAILED DESCRIPTION

[0037] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0038] like Figure 1 As shown, the present invention first uses the acid-sensitive polymer mPEG-PLLys-PAsp (PIP) to self-assemble with SF to form the nano drug SF@NP to improve the poor solubility of SF, and then uses the positive adsorption of the primary amine group on the surface of SF@NP to compound with the negatively charged Xkr8 siRNA to form a nano drug that co-delivers SF and siRNA. SF can promote apoptosis of liver cancer cells, but it will cause tumor immunosuppression, limiting the clinical application of SF. However, the delivered Xkr8 siRNA can reverse the immunosuppression of tumors by flipping the phosphatidylserine (PS) of apoptotic tumor cells to the inner side of the cell membrane, so that it is not phagocytosed by macrophages, that is, by inhibiting the phagocytosis of macrophages, it can reverse the immunosuppression of tumors and enhance the therapeutic effect of SF.

[0039] The hybrid bionic technology of cell membrane can, on the one hand, enable the prepared nanomedicine to escape the capture of mononuclear macrophages in the blood through the bionic idea of ​​bone marrow-derived macrophages, enhance the tumor aggregation of nanomedicines and reduce the side effects of drugs; on the other hand, through the specific recognition of CD44 and integrin αvβ3 receptor proteins highly expressed on the liver cancer cell membrane, it is targeted for uptake by tumor cells, thereby improving the targeting and bioavailability of nanomedicines. The present invention aims to utilize the hybrid membrane modification technology of acid-sensitive polymer carriers to construct the nanomedicine HM-NP@SF&siRNA through a "one-stop" delivery strategy to inhibit the cell burial caused by SF and inhibit the growth of hepatocellular carcinoma.

[0040] Example 1

[0041] 1. Synthesis of acid-sensitive polymer monomethyl ether polyethylene glycol-polylysine anhydride-polyaspartate aminoethyl piperidine (mPEG-PLLys-PAsp(PIP)) for co-delivery of small molecule hydrophobic drugs and siRNA.

[0042] (1) The reactants N-ε-benzyloxycarbonyllysine anhydride (BCLL-NCA) and the macromolecular initiator methoxypolyethylene glycol amino (mPEG-NH2) were purchased directly from Ruixi Biotechnology Co., Ltd. 1.0 g of mPEG-NH2 (2000 g / mol, 0.484 mmol) was weighed into a 50 mL reaction bottle, dehydrated at 70 °C in a vacuum for 1 h, and cooled to 36 °C. Under the protection of nitrogen, 5 mL of freshly distilled chloroform was added to dissolve, and then 2.0 g of BCLL-NCA (306.12 g / mol, 6.54 mmol) dissolved in 25 mL of N,N-dimethylformamide was added, and the reaction was sealed at 36 °C for 48 h. After the reaction was completed, the mixed reaction solution was added dropwise to 300 mL of cold anhydrous ether and refrigerated for 12 h. The precipitate was obtained by centrifugation and washed three times with anhydrous ether. After vacuum drying, a white solid monomethyl ether polyethylene glycol-polybenzyloxycarbonyllysine anhydride copolymer (mPEG-PBCLLys) was obtained ( Figure 2 A).

[0043] (2) Benzyloxycarbonyl aspartic acid anhydride (BLA-NCA) was purchased directly from Shanghai MacLean Biochemical Technology Co., Ltd. Weigh 1.74 g mPEG-PBCLLys (0.29 mmol) and add it to a 50 mL reaction bottle. Add 25 mL of freshly distilled chloroform under the protection of nitrogen, and then add 2.3 g BLA-NCA (249.22 g / mol, 9.2 mmol) dissolved in 2 mL of N,N-dimethylformamide. Turn off the nitrogen, seal and stir at 35°C for 48 h. After the reaction is completed, add the mixed solution to 300 mL of cold anhydrous ether for precipitation and refrigerate for 12 h. Centrifuge to obtain the precipitate, wash twice with anhydrous ether, and vacuum dry to obtain a white solid monomethyl ether polyethylene glycol-polybenzyloxycarbonyl lysine anhydride-polyaspartic acid benzyl ester copolymer (mPEG-PBCLLys-PBLAsp) ( Figure 2 B).

[0044] (3) 2.0 g of mPEG-PBCLLys-PBLAsp (0.15 mmol) was placed in a 50 mL reaction bottle, stirred and dissolved with 20 mL of anhydrous dimethyl sulfoxide, and then 3.36 g of 1-(2-aminoethyl)piperidine (128.22 g / mol, 26.2 mmol) was added and reacted at 25° C. for 24 h. After the reaction was completed, the solution was dialyzed in methanol for two days using a 3.5 kDa dialysis bag to remove excess PIP, the dialysate was replaced in time, the methanol was evaporated, and the solution was vacuum dried to obtain a light yellow solid monomethyl ether polyethylene glycol-polybenzyloxycarbonyl lysine anhydride-polyaspartic acid aminoethyl piperidine mPEG-PBCLLys-PAsp ( Figure 2 C).

[0045] (4) 1.5 g of mPEG-PBCLLys-PAsp (0.11 mmol) was dissolved in 20 mL of trifluoroacetic acid, and then 3 mL of hydrogen bromide (33 wt% hydrogen bromide) in acetic acid was added, stirred at 25° C. for 3 h, added to 300 mL of anhydrous ether for precipitation, and centrifuged to obtain the final product, a light yellow solid monomethyl ether polyethylene glycol-polylysine anhydride-polyaspartate aminoethyl piperidine (mPEG-PLLys-PAsp (PIP)) ( Figure 2 D).

[0046] 2. Preparation of nanodrug NP@SF&siRNA for co-delivery of SF and Xkr8 siRNA.

[0047] Take 215 μL of sorafenib solution (10 mM) and place it in a 5 mL centrifuge tube. Take 10 mg of mPEG-PLLys-PAsp (PIP) polymer and dissolve it in 2 mL of dimethyl sulfoxide solvent, and then mix the two evenly. Take 10 mL of PBS buffer in a small beaker and slowly drip the mixed solution under ice bath and ultrasound. Transfer the obtained solution into a dialysis bag (MW = 14 kDa) and dialyze it in PBS buffer overnight to remove dimethyl sulfoxide solvent and free polymer, etc., to obtain NP@SF micelles ( Figure 3 A), and then re-dissolved in DMSO after freeze-drying to determine the drug loading ( Figure 3 C, D).

[0048] The NP@SF micelles were diluted to 10 mg / mL with 10 mM PBS (pH 7.4) buffer, and then deionized water, DEPC solution containing 2.0 μL Xkr8 siRNA (1 μM), and NP@SF micelle solution were added to the centrifuge tube according to the formula in Table 1. After vortexing for 10 seconds, the mixture was allowed to stand for 30 minutes to allow the siRNA to fully complex and obtain nanopharmaceuticals NP@SF&siRNA with different N / P ratios loaded with sorafenib and siRNA ( Figure 3 B) The results are as follows Figure 3B. At N / P = 2-10, the micelles can completely load Xkr8 siRNA. In subsequent experiments, N / P = 4 was selected, that is, 2.0 μL Xkr8 siRNA was combined with 8 μL NP@SF micelle solution and 12 μL deionized water (in N / P, N refers to the molar amount of N element in the PLLys block in the solution, and P refers to the molar amount of P element in the Xkr8 siRNA in the solution. N / P can reflect the performance of polymer micelles complexed with siRNA.).

[0049] Xkr8 siRNA sequence:

[0050] Sense strand (F): GGCACAAAUUUUAUGCCUGtt;

[0051] Antisense strand (R): CAGGCAUAAAAUUUGUGCCtt.

[0052] Table 1 Formulations of NP@SF&siRNA micelles with different N / P ratios

[0053]

[0054] 3. Cell membrane extraction and preparation of HM-NP@SF&siRNA nanodrugs

[0055] The bone marrow-derived macrophage membranes and Hepa 1-6 tumor cell membranes were extracted in the same way. 7 Bone marrow-derived macrophages and 10 7Each Hepa 1-6 tumor cell was washed three times with 1×PBS and centrifuged at 3000rpm for 5min. 1mL of ice-cold hypotonic lysis buffer (20mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.4; 10mM magnesium chloride; 10mM potassium chloride; 1wt% protease inhibitor; 1wt% 1,4-dimercaptothreitol) was added to the cell pellet for resuspending, and then ultrasonicated on ice for 3-4 times (30s each time, 1min interval each time) to disrupt the cells. The disrupted cells were transferred to a 1.5mL pre-cooled centrifuge tube and placed on ice for 10min. During this period, they were shaken vigorously 2-3 times, centrifuged (4℃, 14000rpm, 10min), and the precipitate was discarded. The supernatant was transferred to a 1.5mL pre-cooled centrifuge tube. Then, the supernatant was placed in a 37℃ water bath for 10min, and the lower layer was removed after centrifugation for 10min (25℃, 14000rpm). Add 500 μL of ice-cold sterilized water, place at 4°C for 5 minutes, and then place in a 37°C water bath for 10 minutes. Then centrifuge for 10 minutes (25°C, 14000 rpm) to remove the lower layer, add 500 μL of ice-cold sterilized water, place at 4°C for 5 minutes, place in a 37°C water bath for 10 minutes, centrifuge for 10 minutes (25°C, 14000 rpm), and then remove the lower layer to obtain a pure membrane protein extract without impurities such as the nucleus. Take 100 μL of membrane protein extract, add 0.9 μL of acetone, ice bath for 20 minutes, and centrifuge at 12000 rpm for 20 minutes. The precipitate is dried on ice for 10-30 minutes, and 100 μL of cell lysis buffer (containing protease inhibitors and ethylenediaminetetraacetic acid (EDTA)) is added to dissolve the membrane protein, and the protein concentration is determined by the bicinchoninic acid (BCA) method or the sample buffer is added and vortexed and then boiled and loaded. The rest is stored at -80°C.

[0056] The Hepa1-6 tumor cell membrane and BMDM membrane were ultrasonically mixed in PBS buffer at a protein weight of 1:1 to obtain a mixed membrane solution. Then, 5 mL of 100 μg / mL mixed membrane solution and 5 mL of 100 μg / mL NP@SF&siRNA micelle solution were vortexed and extruded 15-20 times with a polycarbonate membrane with a pore size of 400 nm, and then extruded 15-20 times with a polycarbonate membrane with a pore size of 200 nm. The uncomplexed cell membrane was removed by centrifugation at 4°C and 5000 rpm to obtain the HM-NP@SF&siRNA nanodrug.

[0057] 4. Aggregation effect of HM-NP@SF&siRNA nanodrugs and therapeutic effect on hepatocellular carcinoma

[0058] Preparation of NP@Cy5.5: 10 mg of mPEG-PLLys-PAsp (PIP) polymer was dissolved in 2 mL of dimethyl sulfoxide solvent to obtain PIP solution. 10 mL of PBS buffer was placed in a small beaker and slowly dripped into the above PIP solution under ice bath and ultrasound conditions. The obtained solution was transferred into a dialysis bag (MW = 14 kDa) and dialyzed overnight in PBS buffer to remove dimethyl sulfoxide solvent and free polymers, etc., to obtain NP micelles for use. Then 12 μL of deionized water, DEPC solution containing 2.0 μL Cy5.5-Xkr8 siRNA (xkr8 siRNA coupled to Cy5.5 fluorescence) and 8 μL of NP micelle solution were added to the centrifuge tube respectively, vortexed for 10 seconds and then allowed to stand for 30 minutes to allow siRNA to fully complex to obtain micelle NP@Cy5.5 loaded with Cy5.5-Xkr8 siRNA.

[0059] Preparation of H-NP@Cy5.5: 5 mL of 100 μg / mL Hepa1-6 tumor cell membrane solution and 5 mL of 100 μg / mL NP@Cy5.5 micelle solution were vortexed and then extruded 15-20 times through a polycarbonate membrane with a pore size of 400 nm, and then extruded 15-20 times through a polycarbonate membrane with a pore size of 200 nm. The uncomplexed cell membrane was removed by centrifugation at 4°C and 5000 rpm to obtain H-NP@Cy5.5.

[0060] Preparation of HM-NP@Cy5.5: Hepa1-6 tumor cell membrane and BMDM membrane were ultrasonically mixed in PBS buffer at a protein weight of 1:1 to obtain a mixed membrane solution. Then, 5 mL of 100 μg / mL mixed membrane solution and 5 mL of 100 μg / mL NP@Cy5.5 micelle solution were vortexed and extruded 15-20 times with a polycarbonate membrane with a pore size of 400 nm, and then extruded 15-20 times with a polycarbonate membrane with a pore size of 200 nm. The uncomplexed cell membrane was removed by centrifugation at 4°C, 5000 rpm to obtain HM-NP@Cy5.5.

[0061] In order to detect the effect of hybrid cell membrane modified carrier on drug aggregation in liver cancer, the co-delivery carrier NP@Cy5.5, H-NP@Cy5.5 before tumor cell membrane modification, and HM-NP@Cy5.5 after tumor cell modification were injected into the orthotopic liver cancer mouse model through the tail vein. The fluorescence intensity was detected at different time points using the IVIS imaging system. The live images of mice were captured at 1h, 3h, 6h, 12h, 24h, and 48h after administration. After 48h, the tumor and major organs were taken and analyzed using the IVIS imaging system.

[0062] Figure 5A and B show that the tumor cell membrane modified co-delivery vector has stronger fluorescence accumulation in the tumor than the unmodified one, proving that the liver cancer cell membrane modified co-delivery vector can endow it with tumor targeting ability. It is worth noting that the vector modified with liver cancer cell membrane and bone marrow-derived macrophage (BMDM) membrane hybrid has more fluorescence accumulation than the vector modified with liver cancer cell membrane alone, while there is little fluorescence in other major organs ( Figure 5 C) demonstrates that hybridization with BMDM membranes can further endow the vector with the ability to escape phagocytosis by macrophages in the blood.

[0063] Preparation of NP@SF and HM-NP@SF: First, prepare NP@SF. Take 10 mg and 215 μL of sorafenib solution (10 mM) in a 5 mL centrifuge tube. Dissolve the mPEG-PLLys-PAsp (PIP) polymer in 2 mL of dimethyl sulfoxide solvent, and then mix the two evenly. Take 10 mL of PBS buffer in a small beaker and slowly drip the mixed solution under ice bath and ultrasound. Transfer the obtained solution into a dialysis bag (MW = 14 kDa) and dialyze it in PBS buffer overnight to remove dimethyl sulfoxide solvent and free polymer, etc., to obtain NP@SF for use. Then, the Hepa1-6 tumor cell membrane and BMDM membrane were ultrasonically mixed in PBS buffer at a protein weight of 1:1 to obtain a mixed membrane solution. Then, 5 mL of 100 μg / mL mixed membrane solution and 5 mL of 100 μg / mL NP@SF micelle solution were vortexed and extruded 15-20 times with a polycarbonate membrane with a pore size of 400 nm, and then extruded 15-20 times with a polycarbonate membrane with a pore size of 200 nm. The uncomplexed cell membrane was removed by centrifugation at 4°C and 5000 rpm to obtain HM-NP@SF.

[0064] Preparation of HM-NP@siRNA: First, prepare NP micelles. Take 10mg of mPEG-PLLys-PAsp (PIP) polymer and dissolve it in 2mL of dimethyl sulfoxide solvent to obtain PIP solution. Take 10mL of PBS buffer in a small beaker and slowly drip the above PIP solution under ice bath and ultrasonic conditions. Move the obtained solution into a dialysis bag (MW=14kDa), dialyze it in PBS buffer overnight to remove dimethyl sulfoxide solvent and free polymers, etc., and obtain NP micelles for use. Then add 12μL of deionized water, DEPC solution containing 2.0μL Xkr8 siRNA, and 8μL of NP micelle solution to the centrifuge tube, vortex for 10s and let it stand for 30min to allow siRNA to fully complex to obtain micelle NP@siRNA loaded with Xkr8 siRNA. Then, the Hepa1-6 tumor cell membrane and BMDM membrane were ultrasonically mixed in PBS buffer at a protein weight of 1:1 to obtain a mixed membrane solution. Then, 5 mL of 100 μg / mL mixed membrane solution and 5 mL of 100 μg / mL NP@siRNA micelle solution were vortexed and extruded 15-20 times with a polycarbonate membrane with a pore size of 400 nm, and then extruded 15-20 times with a polycarbonate membrane with a pore size of 200 nm. The uncomplexed cell membrane was removed by centrifugation at 4°C and 5000 rpm to obtain HM-NP@siRNA.

[0065] Figure 5 D, E and F show that the tumor volume was smaller after treatment with hybrid cell membrane-delivered sorafenib HM-NP@SF compared with the control group, indicating that hybrid cell membrane-delivered sorafenib has a certain tumor inhibitory effect, while the tumor inhibitory effect of nanodrugs HM-NP@siRNA and HM-NP@SF that deliver Xkr8siRNA alone is not much different. It is worth noting that mice treated with hybrid cell membrane-modified nanodrugs HM-NP@SF&siRNA that co-deliver sorafenib and Xkr8 siRNA had smaller tumor volume and higher survival rate, indicating that Xkr8 siRNA can synergize with sorafenib to produce a stronger anti-tumor effect. After simultaneous injection of anti-PD-L1 antibody treatment, 80% of liver cancer mice survived, proving that HM-NP@SF&siRNA can be combined with immune checkpoint therapy to further inhibit the growth of liver cancer.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a hybrid cell membrane-modified nanomedicine for inhibiting macrophage burial, characterized in that: The following steps are involved: (1) The acid-sensitive polymer monomethyl ether polyethylene glycol-polylysine anhydride-polyaspartate aminoethyl piperidine (mPEG-PLLys-Pasp) was mixed with sorafenib and then assembled into single drug-loaded nanodrug NP@SF micelles, and then Xkr8 siRNA and water were added to prepare acid-sensitive nanodrug NP@SF&siRNA loaded with sorafenib and Xkr8 siRNA; (2) extracting liver cancer cell membranes and bone marrow-derived macrophage membranes, mixing the extracted liver cancer cell membranes and bone marrow-derived macrophage membranes to obtain a mixed membrane, and then mixing with the nanodrug NP@SF&siRNA prepared in step (1) and extruding and fusing them to obtain a hybrid cell membrane-modified nanodrug HM-NP@SF&siRNA that inhibits macrophage burial.

2. The preparation method according to claim 1, characterized in that: The preparation method of mPEG-PLLys-PAsp in step (1) comprises the following steps: (1a) mPEG-NH2 is weighed, chloroform is added to dissolve it under the protection of nitrogen, and then BCLL-NCA dissolved in N,N-dimethylformamide is added, and the reaction is sealed; after the reaction is completed, the obtained mixed reaction solution is added dropwise to anhydrous ether and refrigerated; then centrifugation is performed to obtain a precipitate, the precipitate is washed with anhydrous ether, and vacuum dried to obtain a white solid monomethyl ether polyethylene glycol-polybenzyloxycarbonyl lysine anhydride copolymer (mPEG-PBCLLys); (2a) Weighing the mPEG-PBCLLys prepared in step (1a), adding chloroform under the protection of nitrogen, then adding BLA-NCA dissolved in N,N-dimethylformamide, turning off the nitrogen, and sealing the reaction; after the reaction is completed, adding the obtained mixed solution to anhydrous ether and refrigerating; then centrifuging to obtain a precipitate, washing the precipitate with anhydrous ether, and vacuum drying to obtain a white solid monomethyl ether polyethylene glycol-polybenzyloxycarbonyl lysine anhydride-polybenzyl aspartate copolymer (mPEG-PBCLLys-PBLAsp); (3a) taking the mPEG-PBCLLys-PBLAsp prepared in step (2a), dissolving it in anhydrous dimethyl sulfoxide, and then adding 1-(2-aminoethyl)piperidine, and reacting at room temperature; after the reaction, dialyzing the obtained mixed reaction solution in methanol with a dialysis bag to remove excess 1-(2-aminoethyl)piperidine, and vacuum drying to obtain a light yellow solid monomethyl ether polyethylene glycol-polybenzyloxycarbonyl lysine anhydride-polyaspartic acid aminoethyl piperidine mPEG-PBCLLys-PAsp; (4a) mPEG-PBCLLys-PAsp was dissolved with trifluoroacetic acid, and then an acetic acid solution containing hydrogen bromide was added to react. After the reaction, the obtained mixed reaction solution was added to anhydrous ether for precipitation, and the precipitate was obtained by centrifugation to obtain a light yellow solid monomethyl ether polyethylene glycol-polylysine anhydride-polyaspartate aminoethyl piperidine (mPEG-PLLys-PAsp).

3. The preparation method according to claim 2, characterized in that: In the step (1a), the weight ratio of mPEG-NH2 and BCLL-NCA is 1:2; in the step (2a), the weight ratio of mPEG-PBCLLys and BLA-NCA is 1.74:2.3; in the step (3a), the weight ratio of mPEG-PBCLLys-PBLAsp and 1-(2-aminoethyl)piperidine is 2:3.36; in the step (4a), the weight-to-volume ratio of mPEG-PBCLLys-PAsp and hydrogen bromide is 1.5 g:0.99 mL.

4. The preparation method according to claim 2, characterized in that: In the step (1a), the reaction temperature is 25-37°C and the reaction time is 36-50h; in the step (2a), the reaction temperature is 25-37°C and the reaction time is 36-50h; in the step (3a), the reaction temperature is 25°C and the reaction time is 24h; in the step (4a), the reaction temperature is 25°C and the reaction time is 3h.

5. The preparation method according to claim 1, characterized in that: The weight ratio of mPEG-PLLys-PAsp to sorafenib in step (1) is 5:1-10:1; the weight molar ratio of NP@SF micelle to Xkr8siRNA is 4×10 -2 mg: 2×10 -6 μmol~20×10 -2 mg: 2×10 -6 μmol; The sequence of the Xkr8 siRNA: Justice chain: GGCACAAAUUUUAUGCCUGtt; Antisense strand: CAGGCAUAAAAUUUGUGCCtt; Preferably, in step (1), the weight ratio of mPEG-PLLys-PAsp to sorafenib is 10:1; the weight molar ratio of NP@SF micelle to Xkr8 siRNA is 8×10 -2 mg: 2×10 -6 μmol.

6. The preparation method according to claim 1, characterized in that: The method for extracting liver cancer cell membrane in step (2) comprises the following steps: Take liver cancer cells, wash the cells with PBS, centrifuge to remove the supernatant, add a hypotonic dissolution buffer to the cell precipitate to resuspend, then sonicate on ice to break the cells; place the broken cells on ice, shake violently 2-3 times during the period, discard the precipitate after centrifugation, bathe the supernatant at 37°C for 10 minutes, remove the lower layer after centrifugation; add sterile water, place at 4°C for 5 minutes, and then place in a 37°C water bath for 10 minutes; then centrifuge at 25°C and 14000rpm for 10 minutes, remove the lower layer, add sterile water, place at 4°C for 5 minutes, and then place in a 37°C water bath for 10 minutes, centrifuge at 25°C and 14000rpm for 10 minutes, remove the lower layer, and obtain a liver cancer cell membrane protein extract, namely the liver cancer cell membrane; The method for extracting bone marrow-derived macrophage membranes comprises the following steps: Take bone marrow-derived macrophages, wash the cells with PBS, centrifuge to remove the supernatant, add a hypotonic lysis buffer to the cell pellet to resuspend, and then sonicate on ice to disrupt the cells; place the disrupted cells on ice, shake violently 2-3 times during the period, discard the pellet after centrifugation, bathe the supernatant at 37°C for 10 minutes, and remove the lower layer after centrifugation; add sterile water, place at 4°C for 5 minutes, and then place in a 37°C water bath for 10 minutes; then centrifuge at 25°C and 14000rpm for 10 minutes, remove the lower layer, add sterile water, place at 4°C for 5 minutes, and then place in a 37°C water bath for 10 minutes, centrifuge at 25°C and 14000rpm for 10 minutes, and then remove the lower layer to obtain a bone marrow-derived macrophage membrane protein extract, that is, the bone marrow-derived macrophage membrane; Preferably, the hypotonic dissolution buffer comprises 20 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.4; 10 mM magnesium chloride; 10 mM potassium chloride; 1 wt % protease inhibitor; and 1 wt % 1,4-dimercaptothreitol.

7. The preparation method according to claim 1, characterized in that: In the step (2), the usage ratio of liver cancer cell membrane and bone marrow-derived macrophage membrane is 3:1-1:1 according to the protein weight; the weight ratio of the mixed membrane to the nano drug NP@SF&siRNA is 6:1-1:1; and the liver cancer cell membrane is Hepa 1-6 liver cancer cell membrane.

8. A hybrid cell membrane-modified nanomedicine for inhibiting macrophage efferocytosis prepared by the preparation method according to any one of claims 1 to 7.

9. A pharmaceutical composition for treating liver cancer, characterized in that: It comprises the hybrid cell membrane modified nanodrug for inhibiting macrophage burial as claimed in claim 8 and an immune checkpoint inhibitor; preferably, the immune checkpoint inhibitor is an anti-PD-L1 antibody.

10. Use of the hybrid cell membrane modified nanodrug for inhibiting macrophage burial as claimed in claim 8 or the hybrid cell membrane modified nanodrug for inhibiting macrophage burial as claimed in claim 8 and an immune checkpoint inhibitor in the preparation of a drug for treating liver cancer.

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