A membrane protein-lipid nanoparticle complex, its preparation method and application

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to a membrane protein lipid nanoparticle complex, a preparation method thereof, and an application thereof. The membrane protein lipid nanoparticle complex of the present invention is a nanoparticle liposome complex formed by all or part of the membrane protein antigens expressed on tumor cells and a variety of lipids. This complex can deliver all or part of the membrane protein antigens on tumors to the body through a lipid nanoparticle delivery system, activate the body's immune response, cause a strong humoral and cellular immunity in the body, especially promote the infiltration of CD8-positive T cells and the secretion of IFN-γ in the tumor microenvironment, achieving the effect of preventing or killing tumors, and having no obvious toxic side effects. In addition, when the membrane protein lipid nanoparticle complex of the present invention is used to prepare a tumor vaccine, the carrier delivery efficiency of the membrane protein is high, which can effectively prevent the occurrence of solid tumors or kill cancer cells, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a membrane protein lipid nanoparticle complex, a preparation method thereof, and an application thereof. Background Art

[0002] Cancer remains a major medical problem that urgently needs to be solved globally. According to relevant statistical reports, in 2020, the number of newly diagnosed cancer patients globally exceeded 19 million, and the number of deaths due to cancer exceeded 9.9 million. For the treatment of cancer, especially solid tumors, the current main treatment methods are traditional treatment methods, which mainly include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. Although the current cancer treatment methods have made significant progress and the treatment methods are constantly being innovated, the 5-year survival rate of patients is still relatively low. Therefore, it is urgent to develop new treatment methods. In recent years, driven by the rapid development of tumor immunology and molecular biology, biological immunity has made rapid development in the treatment of colorectal cancer. Malignant tumors such as melanoma, renal cell carcinoma, and non-small cell lung cancer have obtained promising clinical effects from immunotherapy. Despite the overall progress of immunotherapy, this treatment method for cancer patients is still mainly in the experimental stage.

[0003] Tumor vaccines are a promising cancer prevention and treatment strategy. Currently, the research on tumor vaccines mainly focuses on DNA, RNA, and protein vaccines. Among them, DNA vaccines are easily integrated into the patient's genome, increasing the risk of gene mutation. mRNA vaccines, on the other hand, result in transient expression of the encoded protein, thus avoiding complications related to insertional mutagenesis. Moreover, mRNA vaccines can be specifically designed to encode a variety of peptide and protein structures, allowing the expression of the entire antigen. However, although the first and second major histocompatibility complexes (MHC) provide a large number of epitopes, the preparation of RNA vaccines is cumbersome, the cycle for finding new antigens with strong immunogenicity is long, and the preparation cycle of new antigen vaccines is long and the cost is high, making it difficult to apply to ordinary patients. Using membrane proteins as new antigens provides a new idea for the research and development of tumor vaccines and has better development prospects.

[0004] With the outbreak and global pandemic of COVID-19, the pace of vaccines moving from preclinical research to clinical research and application has been accelerated. However, the current progress of vaccines is slow. One of the important reasons is the lack of an efficient delivery system. Commonly used carriers include viral vectors and non-viral vectors. Viral vectors have high transfection efficiency, but they lack targeting, have relatively large safety hazards, have a small vector capacity, and high production costs. Non-viral vectors, on the other hand, have advantages such as high safety factor, easy modification of vector molecules, and suitability for large-scale production, and thus have broad application prospects. Among them, Lipid Nanoparticles (LNP) are the most widely used. LNP generally consists of ionizable amino lipids, phospholipids, cholesterol, and polyethylene glycolated lipids. Structurally, they are all amphiphilic molecules with self-assembling properties. The structures of each component are determined, with good reproducibility, which is conducive to quality management and quality supervision. At the same time, they have advantages such as a long in vivo circulation time and good biocompatibility, and thus have received extensive attention. However, the in vivo delivery efficiency of LNP still needs to be improved. Since phospholipids, cholesterol, and polyethylene glycolated lipids are all mature commercial reagents, the core of LNP delivery system research and development lies in the design and development of highly efficient and low-toxic amino lipids. However, there is currently a lack of key technologies for the development of new amino lipid vaccines.

[0005] After LNP nanoparticles enter the cell, they need to escape from the endosome in order to release antigens in the cytoplasm, enabling DC cells to complete antigen presentation and activate the immune response in the body. However, the current endosomal escape rate of LNP is generally low. Although Lin-MO3-DMA, as the "gold standard" for in vivo evaluation of amino lipids, is currently the most efficient amino lipid and has been approved by the FDA for the first SiRNA therapeutic drug npattro (patigian), only 1%-4% of its RNA escapes from the endosome. It can be seen that endosomal escape has become one of the key factors affecting vaccine delivery. Therefore, designing amino lipids with good encapsulation ability and high endosomal escape ability to solve the delivery problem of LNP nanoparticles has great research significance and practical needs. Summary of the Invention

[0006] In order to overcome the deficiencies of the above-mentioned prior art, the present invention is based on an ionizable amino liposome delivery system, uses cancer cell membrane proteins as antigens, and prepares a protein vaccine for the treatment of cancer, which can effectively inhibit the growth and recurrence of tumors, prolong the survival period of patients, and the vaccine has good safety, simple production process, and is easy to industrialize.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a membrane protein-lipid nanoparticle complex, characterized in that the membrane protein-lipid nanoparticle complex comprises a membrane protein, an ionizable amino lipid, a helper lipid, a structural lipid, and a polymer-conjugated lipid; by molar ratio, the ionizable amino lipid accounts for 30-65% of the total lipids, the helper lipid accounts for 5-25% of the total lipids, the structural lipid accounts for 10-45% of the total lipids, and the polymer-conjugated lipid accounts for 0.5-5% of the total lipids.

[0009] Preferably, the membrane protein is a cell membrane extract of tumor cells, and the cell membrane extract comprises all or part of the protein components of the cell membrane and all or part of the antigens on the cell membrane.

[0010] Further, the membrane protein is a cell membrane protein mixture. The membrane protein is extracted from tumor cells, and the tumor cells include melanoma B16F10 cells, colorectal cancer CT26 cells, pancreatic cancer PAN02 cells, cervical cancer TC-1 cells, breast cancer 4T1 cells, and prostate cancer RM1 cells.

[0011] Preferably, the mass ratio of the ionizable amino lipid to the membrane protein is 1:1 - 50:1.

[0012] Preferably, the structural lipid includes one or more of cholesterol and its derivatives. More preferably, the structural lipid is cholesterol.

[0013] Preferably, the helper lipid includes one or more of DOPC, DSPC, DOPE, DOPG, and DOPS. More preferably, the helper lipid is DSPC.

[0014] Preferably, the polymer-conjugated lipid includes one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified dialkylamine, and PEG-modified dialkylglycerol. More preferably, the polymer-conjugated lipid is selected from PEG2000-DMG, PEG2000-DSPE, C18-PEG2000, and specifically selected from PEG2000-DMG.

[0015] Preferably, the ionizable amino lipid includes, but is not limited to, (2-((4-(dimethylamino)butanoyl)oxy)dodecyl 2-hexyldecanoate).

[0016] The second aspect of the present invention provides a preparation method of the membrane protein-lipid nanoparticle complex described in the first aspect, comprising the following steps:

[0017] S1. Dissolve the ionizable amino lipid, the structural lipid, the helper lipid, and the polymer-conjugated lipid in an organic solvent to obtain an organic phase;

[0018] S2. Dissolve the membrane protein in a buffer to obtain an aqueous phase;

[0019] S3. Thoroughly mix the organic phase and the aqueous phase to obtain a mixture, and prepare a membrane protein lipid nanoparticle complex after replacing the organic solvent and the solvent buffer in the mixture.

[0020] Preferably, the total concentration of the ionizable amino lipid, the structural lipid, the helper lipid, and the polymer-conjugated lipid in the organic phase is 1-50 mg / mL, and the concentration of the membrane protein in the aqueous phase is 0.1-1.5 mg / mL.

[0021] Preferably, the organic solvent includes, but is not limited to, ethanol, and the buffer includes, but is not limited to, citrate buffer and acetate solution. More preferably, the buffer is selected from acetic acid-sodium acetate acidic solution or citric acid-sodium citrate acidic solution.

[0022] Preferably, the volume ratio of the organic phase to the aqueous phase is 1:2-6. More preferably, the volume ratio of the organic phase to the aqueous phase is 1:3.

[0023] Preferably, the specific method for replacing the organic solvent and the solvent buffer in the mixture is: dilute the mixture 10-50 times (more preferably 10-20 times) with PSB buffer and then concentrate it.

[0024] The third aspect of the present invention provides the use of the membrane protein lipid nanoparticle complex described in the first aspect in the preparation of a tumor vaccine.

[0025] Through research, it is found that the membrane protein lipid nanoparticle complex designed by the present invention can effectively activate the body's immune system, can arouse strong humoral immunity and cellular immunity in the body, especially promote CD8-positive T cells to produce specific IFN-γ, and play the role of preventing or killing tumors, and has no obvious toxic and side effects.

[0026] Preferably, the tumors include melanoma, pancreatic cancer, colorectal cancer, gastric cancer, prostate cancer, breast cancer, liver cancer, lung cancer, bladder cancer, kidney cancer, cervical cancer, thyroid cancer, bladder cancer, esophageal cancer, ovarian cancer, oral cancer, nasopharyngeal cancer, cholangiocarcinoma. More preferably, the tumors include melanoma, colorectal cancer, pancreatic cancer, cervical cancer, breast cancer, prostate cancer.

[0027] Preferably, the administration methods of the vaccine include aerosol administration, intravenous injection, subcutaneous injection, intramuscular injection, and ocular administration.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] The present invention discloses a membrane protein lipid nanoparticle complex, which is a liposome complex (membrane protein-LNP) of all membrane protein antigens expressed on tumor cells and ionizable amino lipid nanoparticles. The membrane protein complex designed by the present invention can deliver all membrane protein antigens on tumors to the body through a lipid nanoparticle delivery system, activate the body's immune response, cause a strong humoral and cellular immunity in the body, especially promote the infiltration of CD8-positive T cells and the secretion of IFN-γ in the tumor microenvironment, and achieve the effect of preventing or killing tumors without obvious toxic side effects. In addition, when the membrane protein lipid nanoparticle complex of the present invention is used to prepare a tumor vaccine, the carrier delivery efficiency of the membrane protein is high, which can effectively prevent the occurrence of solid tumors or kill cancer cells, has good application prospects, and the vaccine has good safety, simple production process and is easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Particle size diagrams of LNPs prepared from membrane proteins of B16F10, CT26, PANC02, TC-1, 4T-1, and RM1 tumor cells;

[0031] Figure 2 Potential diagrams of LNPs prepared from membrane proteins of B16F10, CT26, PANC02, TC-1, 4T-1, and RM1 tumor cells;

[0032] Figure 3 Encapsulation efficiency diagrams of LNPs prepared from membrane proteins of B16F10, CT26, PANC02, TC-1, 4T-1, and RM1 tumor cells;

[0033] Figure 4 WB diagrams of membrane proteins of B16F10, CT26, PANC02, TC-1, 4T-1, and RM1 tumor cells;

[0034] Figure 5 Tumor growth inhibition curve diagrams of LNPs prepared from membrane proteins of B16F10, CT26, PANC02, TC-1, 4T-1, and RM1 tumor cells;

[0035] Figure 6 Diagrams of LNPs prepared from membrane proteins of B16F10, CT26, PANC02, TC-1, 4T-1, and RM1 tumor cells activating humoral immunity to produce IgG;

[0036] Figure 7 Diagrams of LNPs prepared from membrane proteins of B16F10, CT26, PANC02, TC-1, 4T-1, and RM1 tumor cells activating cellular immunity to produce IFN-γ;

[0037] Figure 8Preparation of LNPs from B16F10, CT26, PANC02, TC-1, 4T-1, and RM1 tumor cell membrane proteins to activate cellular immunity and generate hIFN-V diagram;

[0038] Figure 9 Safety evaluation diagram of LNPs prepared from B16F10, CT26, PANC02, TC-1, 4T-1, and RM1 tumor cell membrane proteins;

[0039] Figure 10 Efficacy evaluation diagram of LNPs prepared from B16F10, CT26, PANC02, TC-1, 4T-1, and RM1 tumor cell membrane proteins combined with aPD-1 to inhibit tumor growth;

[0040] Figure 11 Effect diagram of LNPs prepared from B16F10, CT26, PANC02, TC-1, 4T-1, and RM1 tumor cell membrane proteins to promote CD8-positive T cell infiltration. Detailed implementation methods

[0041] The following further describes the detailed implementation methods of the present invention. It should be noted here that the description of these implementation methods is used to help understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the following various implementation methods of the present invention can be combined with each other as long as they do not conflict with each other.

[0042] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the test materials used in the following examples can all be obtained through conventional commercial channels unless otherwise specified.

[0043] Example 1 Preparation of B16F10 Membrane Protein Lipid Nanoparticle Complex Vaccine and Evaluation of Its Application Effect

[0044] I. Extraction of B16F10 Membrane Protein Antigen

[0045] (1) Collect mouse solid tumor cells (melanoma B16F10 cells) by cell culture and solid tumor resection methods. Select 4-6 week-old C57 / BL mice for subcutaneous tumor implantation experiments on the back. Implant 1 million B16F10 cells into each mouse. When the tumor grows to the 9th day, excise the tumor tissue, take about 100 mg of the tissue, cut the tissue into pieces with scissors, grind the tissue to obtain cell tissue fragments, and after centrifugation (300 g, 5 min), add 1 mL of Solution A of Beyotime Membrane Protein Extraction Reagent (product number: P0033) added with PMSF, gently suspend the tissue fragments, and place them on ice for 10-15 min.

[0046] (2) Place the cell suspension in liquid nitrogen and a 37 °C water bath and freeze-thaw it three times repeatedly. Take 2 - 3 μL and observe it under a microscope until 70 - 80% of the cells have no perinuclear halos and intact cell morphology.

[0047] (3) Remove the cell nuclei and unbroken cells: Centrifuge at 700 g for 10 min at 4 °C. Carefully collect the supernatant into a new centrifuge tube. Do not aspirate the bottom precipitate when aspirating the supernatant. Leave 50 μL of the liquid unaspirated to ensure the purity of the supernatant.

[0048] (4) Precipitate the cell membrane fragments: Centrifuge the supernatant at 14000 g for 30 min at 4 °C to precipitate the cell membrane fragments.

[0049] (5) Collect the cytoplasmic proteins: Aspirate the supernatant, which is the cytoplasmic protein.

[0050] (6) Extract the cell membrane proteins: Centrifuge the supernatant at 14000 g for 10 s at 4 °C. Aspirate as much supernatant as possible, and a small amount of precipitate can be aspirated. Add 300 μL of Solution B of the Membrane Protein Extraction Reagent from Beyotime, vortex vigorously at the highest speed for 5 s, and incubate on ice for 5 - 10 min. Repeat 3 times to fully extract the membrane proteins. Then centrifuge at 14000 g for 5 min at 4 °C, and collect the supernatant as the membrane protein solution, which is stored at -80 °C for later use.

[0051] (7) Take a small amount of the membrane protein solution and measure the protein concentration using the BCA method. The measured concentration is approximately 0.5 - 1 mg / mL.

[0052] (8) Verify the membrane proteins by Western blot: Take an appropriate amount of the membrane protein solution, add 5×SDS loading buffer, and denature it at 100 °C for 5 min, and then perform the Western-blot experiment. ATPA1, that is, sodium-potassium ATPase protein A1, is a transmembrane protein (see Figure 4 ), and the extracted protein mixture can be verified as a membrane protein mixture using ATPA1.

[0053] II. Preparation and Characterization of Membrane Protein-LNPs Lipid Nanoparticles

[0054] (1) The ionizable amino lipid (2-((4-(dimethylamino)butanoyl)oxy)dodecyl 2-hexyldecanoate) was dissolved in anhydrous ethanol with the structural lipid (cholesterol), co-lipid (DOPC), and polymer-conjugated lipid (PEG2000-DMG) at a molar mass ratio of 50:38.5:10:1.5, respectively, to prepare ionizable amino lipid nanoparticles. After the four components were uniformly mixed, anhydrous ethanol was added to make the concentration of the ionizable amino lipid in the range of 0.01 M - 0.5 M. Taking the example of weighing 10 mg of ionizable amino lipid, 4 mg of cholesterol, 4 mg of co-lipid, and 2.5 mg of surfactant, the amounts of anhydrous ethanol added to the four components were 200 μL, 200 μL, 400 μL, and 100 μL, respectively. Then, 84.25 μL of ionizable amino lipid, 126.70 μL of cholesterol, 126.49 μL of co-lipid, and 24.1 μL of surfactant were taken and added to 318.46 μL of anhydrous ethanol to prepare an ethanol-phase solution with a volume of 680 μL. Then, a microfluidic control preparation system (Myanna, INano E) was used to mix the obtained ethanol-phase solution and an acetic acid-sodium acetate acidic solution (pH = 5.0, 25 mM) or citric acid-sodium citrate acidic solution (pH = 5.0, 25 mM) dissolved with B16F10 membrane protein at a volume ratio of 1:3 in a microfluidic chip at a flow rate of 12 mL / h to prepare a crude solution of lipid nanoparticles. Then, the obtained crude solution was diluted 10 times with PBS solution and ultrafiltered three times at 4 °C at a rotational speed of 1.5 krcf for 15 min using a 15 mL or 50 mL ultrafiltration centrifugal tube (Millipore, 100K), finally making the mass ratio of the ionizable amino lipid to the membrane protein approximately 8:1 to prepare membrane protein-LNPs lipid nanoparticles.

[0055] (2) Characterization of particle size, PDI, and zeta potential: The particle size and PDI of the prepared lipid nanoparticles were measured by Nano-ZS ZEN3600 (Malvern). Take 20 μL of the membrane protein-LNPs lipid nanoparticle solution for particle size or zeta potential measurement, with a stabilization time of 120 s, and repeat three times, 10 times for each cycle. The results are as Figure 1 shown, indicating that the particle size of the prepared B16F10 membrane protein-LNPs lipid nanoparticles is about 160 - 210 nm, the PDI is less than 0.2, and the zeta potential is -5 to +5 mV ( Figure 2 ).

[0056] (3) Encapsulation efficiency determination: The determination was carried out with reference to the standard protocol of the BCA protein kit from Beyotime. The membrane protein concentrations before and after demulsification with Triton X-100 were detected respectively, and the encapsulation efficiency of LNP was calculated. The general process was as follows: 2 μL of the ultrafiltered membrane protein-LNPs lipid nanoparticle sample was added to 498 μL of 1×PBS and 498 μL of 2% Triton X-100 solution respectively. After adding the BCA working solution, the absorbance was analyzed with an enzyme-linked immunosorbent assay (ELISA) reader, and a standard curve was plotted. Finally, the membrane protein concentration was calculated according to the standard curve. The results showed that the encapsulation efficiency of the B16F10 membrane protein-LNPs sample was greater than 90%, R 2 greater than 0.99( Figure 3 ).

[0057] III. Animal experiments on the efficacy of membrane protein vaccines

[0058] 1. Immunize female C57 / BL mice with the membrane protein vaccine to evaluate the effect of the vaccine in treating tumors

[0059] Female C57 / BL mice at 4-5 weeks of age were purchased from the Guangdong Provincial Laboratory Animal Center. B16F10 cells were inoculated, and the tumor inoculation was recorded as day 0. The number of inoculated cells was 500,000 per mouse, and the tumor inoculation site was subcutaneously in the right posterior part of the mouse back. After tumor inoculation, the tumor size was observed. When the tumor volume grew to 150-200 mm 3 at day 9, 9 / 10 of the tumor tissue was surgically removed for the preparation of membrane protein-LNPs, and immunization was carried out with membrane protein-LNPs on days 10, 17, and 24. The dosage of the membrane protein was 10 μg per mouse. The PBS group was used as a control. The immunization injection site was the right hind limb of the mouse, and the injection method was intramuscular injection with a volume of 100 μL. Subsequently, the tumor growth was observed, and the tumor volume was measured. The calculation method was: volume V = 1 / 2 length × width × width. The results showed that the membrane protein-LNPs vaccine of the present invention could effectively inhibit the growth of tumors. The specific steps are as follows:

[0060] (1) The murine melanoma cell line B16F10 (purchased from Wuhan Punosai Co., Ltd.) was cultured in a T25 culture flask. When the cell density reached about 80%, the cells were digested with trypsin for 2 min and the digestion was terminated with DMEM complete medium containing 10% FBS. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended with complete medium and counted. The number of tumor-inoculating cells was 1×10 6 per mouse. At about day 4, the subcutaneous tumor size was about 50 mm 3 , and then the mice were randomly divided into 3 groups: the PBS group, the membrane protein group, and the membrane protein-LNPs group, with 6 mice in each group.

[0061] (2) When the tumor volume grew to 150-200 mm at day 9 3At that time, anesthetize the mice. After fixation, shave the hair, disinfect the skin, make a midline incision on the back, about 1 cm in length, free the tumor tissue, surgically remove the tumor mass tissue, with the excised size being 9 / 10 of the tumor volume, compress to stop bleeding, suture the wound, and disinfect. Prepare the obtained tumor tissue into a membrane protein solution. The specific method steps are shown in Part Two.

[0062] (3) Immunize the mice on the 10th, 17th, and 24th days, and observe and measure the tumor volume every other day.

[0063] The results are as Figure 5 shown. The tumor volume of the B16F10 membrane protein-LNPs group is significantly smaller than that of the PBS group. The results suggest that the B16F10 membrane protein-LNPs vaccine of the present invention can effectively inhibit the growth of tumors.

[0064] 2. Immunize female C57 / BL mice with the membrane protein vaccine to evaluate the effect of the vaccine combined with aPD-1 in treating tumors

[0065] Female mice at 4-5 weeks of age are purchased from the Guangdong Provincial Laboratory Animal Center. The tumor inoculation is recorded as the 0th day, the number of inoculated cells is 1 million per mouse, and the tumor inoculation site is subcutaneously on the right posterior back of the mouse; observe the tumor size after tumor inoculation. When the tumor volume grows to 150-200 mm 3 at the 9th day, surgically remove 9 / 10 of the tumor tissue for preparing membrane protein-LNPs. The postoperative mice are divided into 4 groups: the PBS group, the group treated with aPD-1 alone, the group treated with membrane protein-LNPs alone, and the group treated with membrane protein-LNPs + aPD-1. And on the 10th, 17th, and 24th days, treat with membrane protein-LNPs and aPD-1. The dosage of the membrane protein is 10 μg / mouse, and the dosage of aPD-1 is 100 μg / mouse; use the PBS group as a control. The immunization injection site is the right hind limb of the mouse, the injection method is intramuscular injection, and the injection volume is 100 μL. Then observe the tumor growth, measure the tumor volume, and the calculation method is: volume V = 1 / 2 length × width × width. The results suggest that the membrane protein-LNPs vaccine of the present invention combined with aPD-1 can effectively inhibit the growth of tumors. The specific steps are as follows:

[0066] (1) Culture the murine melanoma cell line B16F10 (purchased from Wuhan Punosai Co., Ltd.) in a T25 culture flask. When the cell density reaches about 80%, digest the cells with trypsin for 2 min, terminate the digestion with DMEM complete medium containing 10% FBS, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells with complete medium and count. The number of tumor-inoculated cells is 1×10 6 / mouse. When the subcutaneous tumor size is about 50 mm 3 at about the 4th day, then randomly divide the mice into 2 groups: the PBS group, the group treated with aPD-1 alone, the group treated with membrane protein-LNPs alone, and the group treated with membrane protein-LNPs + aPD-1, with 6 mice in each group.

[0067] (2) When the tumor volume grows to 150 - 200 mm on the 9th day 3 , anesthetize the mice. After fixation, shave the hair, disinfect the skin, make a midline incision on the back, about 1 cm long, free the tumor tissue, surgically remove the tumor tissue, with the removed size being 9 / 10 of the tumor volume, compress to stop bleeding, suture the wound, and disinfect. Prepare the tumor tissue into a membrane protein solution, and the specific method steps are shown in Part Two.

[0068] (3) Immunize the mice on the 10th, 17th, and 24th days. The dosage of the membrane protein is 10 μg / mouse, and the dosage of aPD - 1 is 100 μg / mouse. Observe and measure the tumor volume every other day.

[0069] The results are as Figure 10 shown. The tumor volume of the B16F10 membrane protein - LNPs + aPD - 1 group is significantly smaller than that of other groups, indicating that the combination of the B16F10 membrane protein - LNPs vaccine and aPD - 1 of the present invention can effectively inhibit tumor growth.

[0070] (4) Immerse the fresh excised tumor blocks of the control group and the vaccine group in 1×PBS buffer in a 24 - well plate to ensure cell viability. Secondly, quickly cut the tumor into pieces with a curved ophthalmic surgical scissors on a sterile culture dish, with the volume ≤ 1 mm 3 , and digest the pieces with freshly prepared tissue enzyme solution A (100 mg of collagenase powder, 5 mg of deoxyribonuclease I (DNaseⅠ) powder, made up to 100 mL with RPMI - 1640 medium) in a 37°C constant temperature shaker at a rotation speed of 220 rpm / min for 30 min. Finally, use a 5 - mL syringe to grind the remaining pieces through a 70 - μm sterile filter, and collect the tumor single - cell suspension in a 15 - mL centrifuge tube.

[0071] Isolation of tumor - infiltrating lymphocytes (using the tissue lymphocyte isolation kit method: Solarbio, product number: P9000): Centrifuge the tumor cell suspension at 500 g for 10 min to obtain a cell pellet, and then resuspend and dilute it with 3 mL of sample diluent. Take an equal volume of separation solution and place it in a new 15 - mL centrifuge tube. Slowly drop the diluted cell suspension on top of the separation solution. At room temperature, centrifuge at 500 g for 30 min. After the liquid is separated into 4 layers, carefully aspirate the lymphocytes in the second layer from the top into a new centrifuge tube, resuspend the cell pellet with pre - cooled FBS solution, count, and adjust the cell concentration to 1×10 7 cells / mL for standby. Finally, use a flow cytometer to detect the ratio of CD8+T cells. The results are as Figure 11 shown, indicating that the B16F10 membrane protein - LNPs vaccine of the present invention can promote the infiltration of CD8 - positive T cells.

[0072] 3. The membrane protein vaccine elicits strong humoral and cellular immunity in vivo

[0073] C57 / BL female mice at 4 - 5 weeks of age were immunized with B16F10 membrane protein - LNPs once on the 1st day and the 7th day respectively, with an immunization dose of 10 μg / mouse each time. The PBS group was used as a control. On the 14th day, the mice were anesthetized with isoflurane, and about 600 μL of blood was collected by puncturing the eyeballs with forceps into heparin - sodium anticoagulant tubes. The samples were centrifuged at 800 g for 20 min at 4°C, and the upper - layer serum was collected. Equal volumes were aspirated and separated into two 1.5 - mL centrifuge tubes, which were labeled and stored in a - 20°C refrigerator. Subsequently, the mouse body was soaked in 75% ethanol for 5 min, transferred to a laminar - flow hood, and the spleen was aseptically dissected. A 5 - mL syringe rubber stopper and a 200 - mesh nylon mesh were used to grind the spleen in PBS, filtered through a 40 - micron filter, transferred to a 15 - mL centrifuge tube, and centrifuged at 500 g for 5 min at 4°C. After removing the supernatant, 3 mL of red - blood - cell lysate was added and lysed for 5 min. Then, 6 mL of PBS was added and centrifuged at 500 g for 5 min at 4°C. The PBS - centrifugation washing was repeated 2 times, and then the cells were resuspended with 1 mL of serum - free 1640 medium.

[0074] (1) Elispot assay (IFN - γ determination; choose the kit from Dakocytomation: Mouse IFN - γ precoated ELISPOT kit Cat#: 2210005)

[0075] Take 100 μL of spleen - cell suspension and resuspend it with 900 μL of 1640 medium containing fetal bovine serum (10%) from Australia, diluted to 1 million cells / mL. Then add 100 μL to the ELISPOT plate, with 4 wells for each mouse. Among them, 3 wells are stimulated with OVA peptide and 1 well is stimulated with concanavalin A as a positive control. Then add 10 μL of 200 μg / mL OVA peptide (prepared with complete medium) to the peptide wells, and add 10 μL of 400 μg / mL concanavalin A (prepared with complete medium) to the positive - stimulation well. After incubation in an incubator for 24 h, the cells are lysed with deionized water pre - cooled at 4°C, add the primary antibody (Biotinylated Antibody), wash the plate, and incubate with the secondary antibody HRP (Streptavidin - HRP) for 1 h at room temperature on a shaker. Wash the plate 5 times with PBST, add the chromogenic solution (AEC chromogenic solution: Mix AEC Dilution, AEC SolutionⅠ(20×), AEC SolutionⅡ(20×), and AEC SolutionⅢ(200×) in a clean container at a ratio of 180∶10∶10∶1 to obtain the working solution), develop color in the dark for 5 - 30 min, terminate with deionized water, and read and analyze the plate.

[0076] The results are as Figure 7As shown, the B16F10 membrane protein-LNPs vaccine of the present invention plays a role in promoting the secretion of IFN-γ in the tumor microenvironment, thereby activating cellular immunity.

[0077] (2) IgG determination: Coating the membrane protein antigen on the plate

[0078] Dilute the membrane protein solution to 100 μg / mL, then transfer it to the sample addition slot, 100 μL per well in the 96-well plate. Seal the plate and incubate overnight at 4°C. Wash the plate 5 times with PBST (Take a 2 L volumetric flask, dissolve the PBS dry powder in ddH2O to make up to 2 L. Mix well and take out 50 mL for standby [i.e., PBS], add 0.05% Tween 20 to the remaining liquid in the volumetric flask and mix well as the washing solution [i.e., PBST]), block with 2.5% BSA for 1 h, wash the plate 5 times with PBST, incubate the diluted serum (take mouse serum in the above animal experiment) (1:10 3 、1:10 4 、1:10 5 、1:10 6 ) for 2 - 4 h; wash the plate 5 times with PBST, incubate the secondary antibody HRP (Goat-Anti-Mouse IgG(H+L)-HRP; Cat: 1036-05), incubate on a shaker at room temperature for 1 h. Wash the plate 5 times with PBST, add TMB and develop color in the dark for 5 - 30 min, add an equal volume (100 μL) of 2 M H2SO4 to terminate the reaction, and analyze the readings with an enzyme-linked immunosorbent assay reader.

[0079] The results are as Figure 6 shown, the B16F10 membrane protein-LNPs vaccine of the present invention plays a role in promoting the secretion of IgG in the tumor microenvironment, thereby activating humoral immunity.

[0080] 4. In vitro killing experiment to evaluate the ability of the membrane protein vaccine to activate effector T cells

[0081] (1) Collect peripheral blood and melanoma tissue specimens from melanoma patients (the samples are from the Seventh Affiliated Hospital of Sun Yat-sen University and have obtained the approval of the relevant ethics committee), and obtain human peripheral blood mononuclear cells and melanoma primary cells. Extract peripheral blood mononuclear cells for standby, as follows:

[0082] 1) Add an appropriate amount of lymphocyte separation medium (Biosharp, BL590) to a short and medium tube.

[0083] 2) Take heparinized venous blood and mix it thoroughly with an equal volume of Hank's solution or RPMI1640, slowly overlay it on the surface of the separation medium along the tube wall with a dropper, pay attention to maintaining a clear interface, and then centrifuge horizontally at 2000 rpm for 20 minutes.

[0084] 3) After centrifugation, the content in the tube is divided into three layers. The upper layer is plasma and Hank's solution, the lower layer mainly consists of red blood cells and granulocytes, and the middle layer is lymphocyte separation solution. There is a narrow white cloudy layer mainly composed of mononuclear cells at the interface between the upper and middle layers. Mononuclear cells include lymphocytes and monocytes. In addition, it also contains platelets.

[0085] 4) Insert a capillary into the cloudy layer, aspirate the mononuclear cells, place them into another short and medium-sized tube, add more than 5 times the volume of Hank's solution or RPMI1640, and centrifuge at 1500 rpm for 10 minutes to wash the cells twice.

[0086] 5) After the last centrifugation, discard the supernatant, add RPMI1640 containing 10% fetal calf serum to resuspend the cells. Take a drop of cell suspension and mix it with a drop of 0.2% trypan blue staining solution. On a hemocytometer, count the total number of cells in four large squares.

[0087] 6) Cell viability detection: Dead cells can be stained blue, while live cells are not stained. Count 200 lymphocytes and calculate the percentage of live cells.

[0088] (2) Preparation of primary melanoma cells: Add 0.25% trypsin or 2000 U / mL collagenase to the melanoma tissue fragments, digest them in a 37°C water bath for more than 30 minutes, centrifuge and take the supernatant, wash it 3 times with 4°C pre-cooled HBSS buffer (Cyagen, HBSS-10001), wash it once with complete DMEM medium, then suspend it with complete DMEM medium, and disperse it by pipetting to make a cell suspension. Count to obtain a cell suspension with a concentration of (5-10)×10 8 cells / L. Finally, inoculate the cells in RPMI-1640 or DMEM containing 10% fetal calf serum and culture them in flasks at 37°C and 5% CO2.

[0089] (3) Preparation of human-derived dendritic cells, T cells and primary melanoma cells to evaluate the activation effect of membrane protein-LNPs on the body's immune system.

[0090] Human IL-4 and GM-CSF (Peprotech) were used to induce the differentiation of peripheral blood-derived monocytes into dendritic cells at a working concentration of 800 U / mL. Suspended and semi-adherent cells were collected after 7 days for subsequent experiments. T lymphocytes were collected from human monocytes by sorting with CD3 (Miltenyi Biotec). Then, membrane protein-LNPs (at a dosage of 10 μg / well) were transfected into dendritic cells for 2 - 12 hours and then co-cultured with T cells for 18 - 24 hours. The ratio of T cells to dendritic cells was 4:1. The cells were cultured in RPMI-1640 medium containing 60 ng / mL IL-21 (Peprotech) and 3000 IU / mL IL-2 (Peprotech). Finally, a dot blot assay was performed using a human IFN-γ kit, with the untreated PBMC group as a control.

[0091] The results are as Figure 8 shown. The B16F10 membrane protein-LNPs vaccine of the present invention plays a role in promoting the secretion of IFN-γ in the tumor microenvironment, thereby activating the immune response of effector T cells.

[0092] 5. Safety evaluation of membrane protein-LNPs on the body

[0093] Serum was collected from tumor-free mice (4 - 5-week-old female C57 / BL mice) on the third day after injecting two doses of the vaccine (injecting B16F10 membrane protein-LNPs, with a membrane protein injection amount of 10 μg / mouse, once a day) to detect liver function, kidney function and other indicators. The control group was injected with PBS. The results indicated that the vaccine had no obvious effect on liver and kidney function and other indicators ( Figure 9 ).

[0094] Example 2 Preparation of CT26 membrane protein lipid nanoparticle complex vaccine and evaluation of its application effect

[0095] I. Extraction of CT26 membrane protein antigen

[0096] The extraction method was the same as that in Example 1, except that the mouse solid tumor cells used were colorectal cancer cells CT26, and the mice selected were 4 - 6-week-old Balb / C mice (Guangdong Laboratory Animal Center).

[0097] Similarly, the protein component of the prepared CT26 membrane protein solution was ATPA1, which is sodium-potassium ATPase protein A1, a transmembrane protein (see Figure 4 ).

[0098] II. Preparation and characterization of membrane protein-LNPs lipid nanoparticles

[0099] The preparation method of the membrane protein-LNPs lipid nanoparticles was the same as that in Example 1, and finally the mass ratio of the ionizable amino lipid to the membrane protein was about 6:1.

[0100] Similarly, the prepared CT26 membrane protein-LNPs lipid nanoparticles had a particle size of about 160-210 nm, a PDI of less than 0.2, and a potential of -5 to +5 mV( Figure 2 ); the encapsulation efficiency of the CT26 membrane protein-LNPs sample was greater than 90%, and the R 2 was greater than 0.99( Figure 3 ).

[0101] III. Animal experiments on the effect of the membrane protein vaccine

[0102] 1. Immunize female Balb / C mice with the membrane protein vaccine to evaluate the effect of the vaccine in treating tumors

[0103] The experimental method was the same as that in Example 1, except that the tumor cells inoculated were the murine colorectal cancer cell line CT26 (purchased from Wuhan Punosai Co., Ltd.), and the number of cells inoculated was: 1 million per mouse.

[0104] The results were also as Figure 5 shown. The tumor volume in the CT26 membrane protein-LNPs group was significantly smaller than that in the PBS group, indicating that the CT26 membrane protein-LNPs vaccine of the present invention could effectively inhibit tumor growth.

[0105] 2. Immunize female Balb / C mice with the membrane protein vaccine to evaluate the effect of the vaccine combined with aPD-1 in treating tumors

[0106] The experimental method was the same as that in Example 1, except that the tumor cells inoculated were the murine colorectal cancer cell line CT26 (purchased from Wuhan Punosai Co., Ltd.).

[0107] The results were also as Figure 10 shown. The tumor volume in the CT26 membrane protein-LNPs + aPD-1 group was significantly smaller than that in other groups, indicating that the CT26 membrane protein-LNPs vaccine of the present invention combined with aPD-1 could effectively inhibit tumor growth.

[0108] 3. The membrane protein vaccine elicits a strong humoral and cellular immunity in vivo

[0109] The experimental method was the same as that in Example 1, except that the mice selected were 4-5-week-old Balb / C mice, and the vaccine used for immunization was CT26 membrane protein-LNPs.

[0110] The results of the Elispot assay (IFN-γ measurement) were also as Figure 7As shown, it demonstrates that the CT26 membrane protein-LNPs vaccine of the present invention plays a role in promoting the secretion of IFN-γ in the tumor microenvironment, thereby activating cellular immunity. The results of IgG determination (membrane protein antigen plating) are also as Figure 6 shown, indicating that the CT26 membrane protein-LNPs vaccine of the present invention plays a role in promoting the secretion of IgG in the tumor microenvironment, thereby activating humoral immunity.

[0111] 4. Evaluation of the ability of the membrane protein vaccine to activate effector T cells by in vitro killing experiment

[0112] The experimental method is the same as that in Example 1, except that: peripheral blood of colorectal cancer patients and their colorectal cancer tissue specimens are collected (the samples are from the Seventh Affiliated Hospital of Sun Yat-sen University and have obtained the approval of the relevant ethics committee), human peripheral blood mononuclear cells and primary colorectal cancer cells are obtained, and peripheral blood mononuclear cells are extracted for standby.

[0113] The results are also as Figure 8 shown, indicating that the CT26 membrane protein-LNPs vaccine of the present invention plays a role in promoting the secretion of IFN-γ in the tumor microenvironment, thereby activating the immune response of effector T cells.

[0114] 5. Safety evaluation of membrane protein-LNPs on the body

[0115] Serum of tumor-free mice (4-5-week-old female Balb / C mice) was collected on the third day after injecting two doses of the vaccine (injecting CT26 membrane protein-LNPs, once a day) to detect indexes such as liver function and kidney function. The control group was injected with PBS. The results suggest that the vaccine has no obvious effect on indexes such as liver and kidney functions ( Figure 9 ).

[0116] Example 3 Preparation of PAN02 membrane protein lipid nanoparticle complex vaccine and evaluation of its application effect

[0117] I. Extraction of PAN02 membrane protein antigen

[0118] The extraction method is the same as that in Example 1, except that: the mouse solid tumor cells used are pancreatic cancer PAN02 cells.

[0119] Similarly, the protein component of the prepared PAN02 membrane protein solution is ATPA1, and ATPA1 is the sodium-potassium ATPase protein A1, which is a transmembrane protein (see Figure 4 ).

[0120] II. Preparation and characterization of membrane protein-LNPs lipid nanoparticles

[0121] The preparation method of membrane protein-LNPs lipid nanoparticles is the same as that in Example 1.

[0122] Similarly, the prepared PAN02 membrane protein-LNPs lipid nanoparticles have a particle size of about 160-210 nm, a PDI of less than 0.2, and a potential of -5 to +5 mV( Figure 2 ); the encapsulation efficiency of the PAN02 membrane protein-LNPs sample is greater than 90%, and R 2 is greater than 0.99( Figure 3 ).

[0123] III. Animal experiments on the effect of membrane protein vaccine

[0124] 1. Immunize female C57 / BL mice with the membrane protein vaccine to evaluate the effect of the vaccine in treating tumors

[0125] The experimental method is the same as that in Example 1, except that the tumor cells inoculated are murine pancreatic cancer cell line PAN02 (purchased from Wuhan Punosai Company).

[0126] The results are also as Figure 5 shown. The tumor volume in the membrane protein-LNPs group is significantly smaller than that in the PBS group, indicating that the PAN02 membrane protein-LNPs vaccine of the present invention can effectively inhibit tumor growth.

[0127] 2. Immunize female C57 / BL mice with the membrane protein vaccine to evaluate the effect of the vaccine combined with aPD-1 in treating tumors

[0128] The experimental method is the same as that in Example 1, except that the tumor cells inoculated are murine pancreatic cancer cell line PAN02 (purchased from Wuhan Punosai Company).

[0129] The results are also as Figure 10 shown. The tumor volume in the PAN02 membrane protein-LNPs + aPD-1 group is significantly smaller than that in other groups, indicating that the PAN02 membrane protein-LNPs vaccine of the present invention combined with aPD-1 can effectively inhibit tumor growth.

[0130] 3. The membrane protein vaccine elicits a strong humoral and cellular immune response in vivo

[0131] The experimental method is the same as that in Example 1, except that the vaccine used for immunization is PAN02 membrane protein-LNPs.

[0132] The results of the Elispot assay (IFN-γ measurement) are also as Figure 7 shown, indicating that the PAN02 membrane protein-LNPs vaccine of the present invention promotes the secretion of IFN-γ in the tumor microenvironment, thereby activating cellular immunity. The results of IgG measurement (membrane protein antigen plating) are also as Figure 6 shown, indicating that the PAN02 membrane protein-LNPs vaccine of the present invention promotes the secretion of IgG in the tumor microenvironment, thereby activating humoral immunity.

[0133] 4. Evaluation of the ability of the membrane protein vaccine to activate effector T cells by in vitro killing experiment

[0134] The experimental method was the same as that in Example 1, except that: peripheral blood and pancreatic cancer tissue specimens of pancreatic cancer patients were collected (the samples were from the Seventh Affiliated Hospital of Sun Yat-sen University and approved by the relevant ethics committee), human peripheral blood mononuclear cells and primary pancreatic cancer cells were obtained, and the peripheral blood mononuclear cells were extracted for standby.

[0135] The results were also as Figure 8 shown, indicating that the PAN02 membrane protein-LNPs vaccine of the present invention played a role in promoting the secretion of IFN-γ in the tumor microenvironment, thereby activating the immune response of effector T cells.

[0136] 5. Safety evaluation of membrane protein-LNPs on the body

[0137] On the third day after two injections of the vaccine (injecting PAN02 membrane protein-LNPs, once a day) into tumor-free mice (4-5 week-old female C57 / BL mice), mouse serum was collected to detect indexes such as liver function and kidney function, and the control group was injected with PBS. The results showed that the vaccine had no obvious effect on indexes such as liver and kidney function( Figure 9 ).

[0138] Example 4 Preparation of TC-1 membrane protein lipid nanoparticle complex vaccine and evaluation of its application effect

[0139] I. Extraction of TC-1 membrane protein antigen

[0140] The extraction method was the same as that in Example 1, except that: the mouse solid tumor cells used were cervical cancer TC-1 cells.

[0141] Similarly, the protein component of the prepared TC-1 membrane protein solution was ATPA1, and ATPA1 was the sodium-potassium ATPase protein A1, which was a transmembrane protein (see Figure 4 ).

[0142] II. Preparation and characterization of membrane protein-LNPs lipid nanoparticles

[0143] The preparation method of the membrane protein-LNPs lipid nanoparticles was the same as that in Example 1.

[0144] Similarly, the particle size of the prepared TC-1 membrane protein-LNPs lipid nanoparticles was about 160-210 nm, the PDI was less than 0.2, and the potential was -5 to +5 mV( Figure 2 ); the encapsulation efficiency of the TC-1 membrane protein-LNPs sample was greater than 90%, and the R 2 was greater than 0.99( Figure 3 ).

[0145] III. Animal Experiments on the Efficacy of Membrane Protein Vaccines

[0146] 1. Female C57 / BL mice were immunized with the membrane protein vaccine to evaluate the effect of the vaccine in treating tumors

[0147] The experimental method was the same as that in Example 1, except that the tumor cells inoculated were murine cervical cancer cell line TC-1 (purchased from Wuhan Punosai Company).

[0148] The results were also as Figure 5 shown. The tumor volume in the TC-1 membrane protein-LNPs group was significantly smaller than that in the PBS group, indicating that the TC-1 membrane protein-LNPs vaccine of the present invention can effectively inhibit tumor growth.

[0149] 2. Female C57 / BL mice were immunized with the membrane protein vaccine to evaluate the effect of the vaccine combined with aPD-1 in treating tumors

[0150] The experimental method was the same as that in Example 1, except that the tumor cells inoculated were murine cervical cancer cell line TC-1 (purchased from Wuhan Punosai Company).

[0151] The results were also as Figure 10 shown. The tumor volume in the TC-1 membrane protein-LNPs + aPD-1 group was significantly smaller than that in other groups, indicating that the combination of the TC-1 membrane protein-LNPs vaccine of the present invention and aPD-1 can effectively inhibit tumor growth.

[0152] 3. The membrane protein vaccine elicits strong humoral and cellular immunity in vivo

[0153] The experimental method was the same as that in Example 1, except that the vaccine used for immunization was TC-1 membrane protein-LNPs.

[0154] The results of the Elispot assay (IFN-γ determination) were also as Figure 7 shown, indicating that the TC-1 membrane protein-LNPs vaccine of the present invention promotes the secretion of IFN-γ in the tumor microenvironment, thereby activating cellular immunity. The results of IgG determination (membrane protein antigen coating) were also as Figure 6 shown, indicating that the TC-1 membrane protein-LNPs vaccine of the present invention promotes the secretion of IgG in the tumor microenvironment, thereby activating humoral immunity.

[0155] 4. In vitro killing experiments were performed to evaluate the ability of the membrane protein vaccine to activate effector T cells

[0156] The experimental method was the same as that in Example 1, except that: peripheral blood of cervical cancer patients and their cervical cancer tissue specimens were collected (the samples were from the Seventh Affiliated Hospital of Sun Yat-sen University and approved by the relevant ethics committee), human peripheral blood mononuclear cells and primary cervical cancer cells were obtained, and the peripheral blood mononuclear cells were extracted for standby.

[0157] The results were also as Figure 8 shown, indicating that the TC-1 membrane protein-LNPs vaccine of the present invention played a role in promoting the secretion of IFN-γ in the tumor microenvironment, thereby activating the immune response of effector T cells.

[0158] 5. Safety evaluation of membrane protein-LNPs on the body

[0159] Serum of tumor-free mice (4-5 week-old female C57 / BL mice) was collected on the third day after two injections of the vaccine (injecting TC-1 membrane protein-LNPs, once a day) to detect indicators such as liver function and kidney function. The control group was injected with PBS. The results showed that the vaccine had no obvious effect on indicators such as liver and kidney functions ( Figure 9 ).

[0160] Example 5 Preparation of 4T1 membrane protein lipid nanoparticle complex vaccine and evaluation of its application effect

[0161] I. Extraction of 4T1 membrane protein antigen

[0162] The extraction method was the same as that in Example 1, except that: the mouse solid tumor cells used were breast cancer 4T1 cells, and the mice selected were 4-6 week-old Balb / C mice (Guangdong Laboratory Animal Center).

[0163] Similarly, the protein component of the prepared 4T1 membrane protein solution was ATPA1, and ATPA1 was sodium-potassium ATPase protein A1, which was a transmembrane protein (see Figure 4 ).

[0164] II. Preparation and characterization of membrane protein-LNPs lipid nanoparticles

[0165] The preparation method of membrane protein-LNPs lipid nanoparticles was the same as that in Example 1.

[0166] Similarly, the particle size of the prepared 4T1 membrane protein-LNPs lipid nanoparticles was about 160-210 nm, the PDI was less than 0.2, and the potential was -5 to +5 mV ( Figure 2 ); the encapsulation efficiency of the 4T1 membrane protein-LNPs sample was greater than 90%, and R 2 was greater than 0.99 ( Figure 3 ).

[0167] III. Animal experiment on the effect of membrane protein vaccine

[0168] 1. Immunize female Balb / C mice with the membrane protein vaccine and evaluate the effect of the vaccine in treating tumors.

[0169] The experimental method is the same as that in Example 1, except that: the tumor cells inoculated are murine breast cancer cell line 4T1 (purchased from Wuhan Punosai Co., Ltd.), and the number of cells inoculated is: 1 million per mouse.

[0170] The results are also as Figure 5 shown. The tumor volume in the 4T1 membrane protein-LNPs group is significantly smaller than that in the PBS group, indicating that the 4T1 membrane protein-LNPs vaccine of the present invention can effectively inhibit tumor growth.

[0171] 2. Immunize female Balb / C mice with the membrane protein vaccine and evaluate the effect of the vaccine combined with aPD-1 in treating tumors.

[0172] The experimental method is the same as that in Example 1, except that: the tumor cells inoculated are murine breast cancer cell line 4T1 (purchased from Wuhan Punosai Co., Ltd.).

[0173] The results are also as Figure 10 shown. The tumor volume in the 4T1 membrane protein-LNPs + aPD-1 group is significantly smaller than that in other groups, indicating that the combination of the 4T1 membrane protein-LNPs vaccine of the present invention and aPD-1 can effectively inhibit tumor growth.

[0174] 3. The membrane protein vaccine elicits strong humoral and cellular immunity in vivo.

[0175] The experimental method is the same as that in Example 1, except that: the selected mice are 4-5-week-old Balb / C mice, and the vaccine used for immunization is 4T1 membrane protein-LNPs.

[0176] The results of the Elispot assay (IFN-γ determination) are also as Figure 7 shown, indicating that the 4T1 membrane protein-LNPs vaccine of the present invention plays a role in promoting the secretion of IFN-γ in the tumor microenvironment, thereby activating cellular immunity. The results of IgG determination (membrane protein antigen coating) are also as Figure 6 shown, indicating that the 4T1 membrane protein-LNPs vaccine of the present invention plays a role in promoting the secretion of IgG in the tumor microenvironment, thereby activating humoral immunity.

[0177] 4. Evaluate the ability of the membrane protein vaccine to activate effector T cells by in vitro killing experiment.

[0178] The experimental method is the same as that in Example 1, except that: collect peripheral blood and breast cancer tissue specimens of breast cancer patients (the samples are from the Seventh Affiliated Hospital of Sun Yat-sen University, and the approval of the relevant ethics committee is obtained), obtain human peripheral blood mononuclear cells and breast cancer primary cells, and extract peripheral blood mononuclear cells for standby.

[0179] The result is also as Figure 8 shown, indicating that the 4T1 membrane protein-LNPs vaccine of the present invention plays a role in promoting the secretion of IFN-γ in the tumor microenvironment, thereby activating the immune response of effector T cells.

[0180] 5. Safety evaluation of membrane protein-LNPs on the organism

[0181] On the third day after two doses of the vaccine (injecting 4T1 membrane protein-LNPs, once a day) were injected into tumor-free mice (4-5-week-old female Balb / C mice), mouse sera were collected to detect indicators such as liver function and kidney function. The control group was injected with PBS. The results suggest that the vaccine has no obvious effect on indicators such as liver and kidney functions ( Figure 9 ).

[0182] Example 6 Preparation of RM1 membrane protein lipid nanoparticle complex vaccine and evaluation of its application effect

[0183] I. Extraction of RM1 membrane protein antigen

[0184] The extraction method is the same as that in Example 1, except that: the mouse solid tumor cells used are prostate cancer RM1 cells.

[0185] Similarly, the protein component of the prepared RM1 membrane protein solution is ATPA1, and ATPA1 is the sodium-potassium ATPase protein A1, which is a transmembrane protein (see Figure 4 ).

[0186] II. Preparation and characterization of membrane protein-LNPs lipid nanoparticles

[0187] The preparation method of membrane protein-LNPs lipid nanoparticles is the same as that in Example 1.

[0188] Similarly, the particle size of the prepared RM1 membrane protein-LNPs lipid nanoparticles is about 160-210 nm, the PDI is less than 0.2, and the potential is -5 to +5 mV ( Figure 2 ); the encapsulation efficiency of the RM1 membrane protein-LNPs sample is greater than 90%, and R 2 is greater than 0.99 ( Figure 3 ).

[0189] III. Animal experiment on the effect of membrane protein vaccine

[0190] 1. Immunize female C57 / BL mice with the membrane protein vaccine to evaluate the effect of the vaccine in treating tumors

[0191] The experimental method is the same as that in Example 1, except that: the tumor cells inoculated are the murine prostate cancer cell line RM1 (purchased from Wuhan Punosai Co., Ltd.).

[0192] The result is also asFigure 5 As shown, the tumor volume of the RM1 membrane protein-LNPs group was significantly smaller than that of the PBS group, suggesting that the RM1 membrane protein-LNPs vaccine of the present invention can effectively inhibit tumor growth.

[0193] 2. Immunize female C57 / BL mice with the membrane protein vaccine to evaluate the effect of combining the vaccine with aPD-1 in treating tumors

[0194] The experimental method was the same as that in Example 1, except that the inoculated tumor cells were the murine prostate cancer cell line RM1 (purchased from Wuhan Punosai Co., Ltd.).

[0195] The results were also as Figure 10 shown. The tumor volume of the RM1 membrane protein-LNPs + aPD-1 group was significantly smaller than that of other groups, suggesting that the combination of the RM1 membrane protein-LNPs vaccine of the present invention and aPD-1 can effectively inhibit tumor growth.

[0196] 3. The membrane protein vaccine elicits strong humoral and cellular immunity in vivo

[0197] The experimental method was the same as that in Example 1, except that the vaccine used for immunization was RM1 membrane protein-LNPs.

[0198] The results of the Elispot assay (IFN-γ determination) were also as Figure 7 shown, indicating that the RM1 membrane protein-LNPs vaccine of the present invention promotes the secretion of IFN-γ in the tumor microenvironment, thereby activating cellular immunity. The results of IgG determination (membrane protein antigen coating) were also as Figure 6 shown, indicating that the RM1 membrane protein-LNPs vaccine of the present invention promotes the secretion of IgG in the tumor microenvironment, thereby activating humoral immunity.

[0199] 4. In vitro killing experiment to evaluate the ability of the membrane protein vaccine to activate effector T cells

[0200] The experimental method was the same as that in Example 1, except that peripheral blood and prostate cancer tissue specimens of prostate cancer patients were collected (the samples were from the Seventh Affiliated Hospital of Sun Yat-sen University and obtained the approval of the relevant ethics committee), human peripheral blood mononuclear cells and primary prostate cancer cells were obtained, and peripheral blood mononuclear cells were extracted for standby.

[0201] The results were also as Figure 8 shown, indicating that the RM1 membrane protein-LNPs vaccine of the present invention promotes the secretion of IFN-γ in the tumor microenvironment, thereby activating the immune response of effector T cells.

[0202] 5. Safety evaluation of membrane protein-LNPs on the body

[0203] Serum samples were collected from tumor-free mice (4-5 week-old female C57 / BL mice) on the third day after two doses of the vaccine were injected (RM1 membrane protein-LNPs were injected once a day), and liver and kidney function and other indicators were detected. The control group was injected with PBS. The results showed that the vaccine had no significant effect on liver and kidney function and other indicators( Figure 9 ).

[0204] Based on Examples 1-6, the membrane protein vaccine provided by the present invention can deliver tumor membrane protein-related antigens to the antigen-presenting cells (APCs) of the body through the lipid nanoparticle delivery system, enhance specific and non-specific antigen presentation, activate the body's immune response, promote T cell proliferation and activation, and have the effect of preventing or killing tumors, and has good safety. It can be seen that the membrane protein-LNPs designed by the present invention can effectively activate the body's immune system, arouse strong cellular and humoral immunity, and have no obvious toxic and side effects, and have good application prospects.

[0205] The above detailed description of the embodiments of the present invention, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions and variations of these embodiments still fall within the protection scope of the present invention.

Claims

1. A membrane protein-lipid nanoparticle complex, characterized in that, The membrane protein-lipid nanoparticle complex comprises a membrane protein, an ionizable amino lipid, a helper lipid, a structural lipid, and a polymer-conjugated lipid; the structural lipid is cholesterol, the helper lipid is DSPC, the polymer-conjugated lipid is PEG2000-DMG, and the ionizable amino lipid is (2-((4-(dimethylamino)butanoyl)oxy)dodecyl 2-hexyldecanoate); By molar ratio, the ionizable amino lipid accounts for 30-65% of the total lipids, the helper lipid accounts for 5-25% of the total lipids, the structural lipid accounts for 10-45% of the total lipids, and the polymer-conjugated lipid accounts for 0.5-5% of the total lipids; the membrane protein is a cell membrane extract of tumor cells, and the cell membrane extract comprises all or part of the protein components of the cell membrane and all or part of the antigens on the cell membrane.

2. The membrane protein-lipid nanoparticle complex according to claim 1, wherein The mass ratio of the ionizable amino lipid to the membrane protein is 1:1 to 50:

1.

3. The preparation method of the membrane protein-lipid nanoparticle complex according to claim 1 or 2, characterized in that, Comprising the following steps: S1. Dissolve the ionizable amino lipid, the structural lipid, the helper lipid, and the polymer-conjugated lipid in an organic solvent to obtain an organic phase; S2. Dissolve the membrane protein in a buffer solution to obtain an aqueous phase; S3. Mix the organic phase and the aqueous phase thoroughly to obtain a mixture, and prepare the membrane protein-lipid nanoparticle complex after replacing the organic solvent and the solvent buffer in the mixture.

4. The preparation method of the membrane protein-lipid nanoparticle complex according to claim 3, characterized in that, The total concentration of the ionizable amino lipid, the structural lipid, the helper lipid, and the polymer-conjugated lipid in the organic phase is 1-50 mg / mL, and the concentration of the membrane protein in the aqueous phase is 0.1-1.5 mg / mL.

5. Use of the membrane protein-lipid nanoparticle complex according to claim 1 or 2 in the preparation of a tumor vaccine or a tumor treatment preparation.

6. The application according to claim 5, characterized in that, The tumors include melanoma, pancreatic cancer, colorectal cancer, gastric cancer, prostate cancer, breast cancer, liver cancer, lung cancer, bladder cancer, kidney cancer, cervical cancer, thyroid cancer, bladder cancer, esophageal cancer, ovarian cancer, oral cancer, nasopharyngeal cancer, cholangiocarcinoma.

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