CN116036308A - Membrane protein lipid nanoparticle compound and preparation method and application thereof - Google Patents

Membrane protein lipid nanoparticle compound and preparation method and application thereof Download PDF

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CN116036308A
CN116036308A CN202310034344.3A CN202310034344A CN116036308A CN 116036308 A CN116036308 A CN 116036308A CN 202310034344 A CN202310034344 A CN 202310034344A CN 116036308 A CN116036308 A CN 116036308A
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cancer
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侍光照
查高峰
张常华
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Shenzhen Hongxin Biotechnology Co ltd
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Seventh Affiliated Hospital Of Sun Yat Sen University Shenzhen
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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a membrane protein lipid nanoparticle compound and a preparation method and application thereof. The membrane protein lipid nanoparticle complex is a nanoparticle liposome complex formed by all or part of membrane protein antigens expressed on tumor cells and various lipids, can deliver all or part of membrane protein antigens on tumors to a body through a lipid nanoparticle delivery system, activates immune response of the body, causes strong humoral immunity and cellular immunity in the body, and especially promotes infiltration of CD8 positive T cells and secretion of IFN-gamma in tumor microenvironment, and has the effect of preventing or killing tumors without obvious toxic and side effects. In addition, the membrane protein lipid nanoparticle composite is used for preparing tumor vaccines, has high delivery efficiency of a membrane protein carrier, can effectively prevent solid tumors or kill cancer cells, and has good application prospect.

Description

Membrane protein lipid nanoparticle compound and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a membrane protein lipid nanoparticle compound and a preparation method and application thereof.
Background
Cancer remains a major medical problem that is urgently needed to be addressed worldwide. According to the related statistics, the global new cancer patients in 2020 are over 1900 ten thousand, and the cancer death number is over 990 ten thousand. For the treatment of cancers, especially solid tumors, traditional treatment methods are mainly adopted at present, and mainly comprise surgery, radiotherapy and chemotherapy, targeted treatment and immunotherapy. Although current cancer treatments have made significant progress and the treatment regimen is continually evolving, the 5-year survival rate of patients is still low, and thus the development of new treatments is urgent. In recent years, biological immunity has been rapidly developed in colorectal cancer treatment, driven by rapid development of tumor immunology and molecular biology. Malignant tumors such as melanoma, renal cell carcinoma and non-small cell lung carcinoma have obtained promising clinical results from immunotherapy. Despite the overall progress of immunotherapy, this treatment for cancer patients is currently largely in the experimental stage.
Tumor vaccines are a promising strategy for cancer prevention and treatment. Current research on tumor vaccines is mainly focused on DNA, RNA and protein vaccines. Among them, DNA vaccines are easily integrated into the patient's genome, increasing the risk of genetic mutation. mRNA vaccines can lead to transient expression of the encoded protein, thus avoiding complications associated with insertional mutagenesis, and can be specifically designed to encode a wide variety of peptide and protein structures, allowing expression of the entire antigen. However, since the Major Histocompatibility Complex (MHC) of the first and second types provides a large number of epitopes, RNA vaccines are cumbersome to prepare, and the period for searching for a new antigen with strong immunogenicity is long, and the preparation period of the new antigen vaccine is long, and the cost is high, so that it is difficult to adapt to common patients. And the membrane protein is used as a new antigen to provide a new thought for the research and development of tumor vaccines, and has better development prospect.
With the outbreak of covd-19 and global pandemic, the pace of vaccine pace from preclinical research to clinical research and application is accelerated, but currently vaccines progress slowly, one of the important reasons for this being the lack of efficient delivery systems. The common vectors comprise viral vectors and non-viral vectors, the viral vectors have high transfection efficiency, but the viral vectors lack targeting, have larger potential safety hazards, and have small vector capacity and higher production cost. The non-viral vector has the advantages of high safety coefficient, easy modification of vector molecules, suitability for mass production and the like, and has wide application prospect. Among them, lipid nanoparticles (Lipid Nanoparticles, LNP) are most used. LNP is generally composed of ionizable amino lipid, phospholipid, cholesterol and polyethylene glycol lipid, structurally is an amphiphilic molecule with self-assembly property, has the advantages of definite structure of each component, good repeatability, convenience for quality management and quality supervision, longer in-vivo circulation time, good biocompatibility and the like, and is widely concerned. However, the in vivo delivery efficiency of LNP remains to be improved, and since phospholipids, cholesterol and pegylated lipids are all mature commercial agents, the heart of LNP delivery system development is to design and develop highly efficient low-toxic amino lipids. However, there is currently no key technology for the development of novel amino lipid vaccines.
After entering the cell, the LNP nanoparticle needs to escape from the endosome to release antigen in the cytoplasm, so that the DC cell finishes antigen presentation and activates immune response in the body. However, the endosome escape rate of LNP is currently generally low, and although Lin-MO3-DMA is currently the most efficient amino lipid as the "gold standard" for amino liposome internal evaluation and is approved by the FDA for the first SiRNA therapeutic drug npattro (patigian), it also escapes endosomes only 1% -4% of the RNA. Therefore, the amino lipid with good wrapping capability and high endosome escape capability is designed to solve the problem of delivery of LNP nano particles, and has great research significance and practical requirements.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention is based on an ionizable amino liposome delivery system, takes cancer cell membrane protein as an antigen, prepares a protein vaccine for treating cancer, can effectively inhibit the growth and recurrence of tumors, prolongs the life cycle of patients, has good safety, simple preparation process and is easy to realize industrialization.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the present invention provides in a first aspect 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 ionizable amino lipid accounts for 30-65% of the total lipid, the auxiliary lipid accounts for 5-25% of the total lipid, the structural lipid accounts for 10-45% of the total lipid, and the polymer conjugated lipid accounts for 0.5-5% of the total lipid.
Preferably, the membrane protein is a cell membrane extract of a tumor cell, the cell membrane extract comprising all or part of the protein component of the cell membrane, and all or part of the antigen on the cell membrane.
Further, the membrane protein is a cell membrane protein mixture. The membrane protein is extracted from tumor cells, wherein the tumor cells comprise melanoma B16F10 cells, colorectal cancer CT26 cells, pancreatic cancer PAN02 cells, cervical cancer TC-1 cells, breast cancer 4T1 cells and prostatic cancer RM1 cells.
Preferably, the mass ratio of the ionizable amino lipid to the membrane protein is 1:1-50:1.
Preferably, the structural lipid comprises one or more of cholesterol and derivatives thereof. More preferably, the structural lipid is cholesterol.
Preferably, the helper lipids comprise one or more of DOPC, DSPC, DOPE, DOPG, DOPS. More preferably, the helper lipid is DSPC.
Preferably, the polymer conjugated lipid comprises one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified dialkylamine, PEG-modified dialkylglycerol. More preferably, the polymer conjugated lipid is selected from the group consisting of PEG2000-DMG, PEG2000-DSPE, C18-PEG2000, in particular from PEG2000-DMG.
Preferably, the ionizable amino lipid includes, but is not limited to, (2- ((4- (dimethylamino) butyryl) oxy) dodecyl 2-hexyl decanoate).
In a second aspect, the present invention provides a method for preparing a membrane proteolipid nanoparticle complex according to the first aspect, comprising the steps of:
s1, dissolving ionizable amino lipid, structural lipid, auxiliary lipid and polymer conjugated lipid in an organic solvent to prepare an organic phase;
s2, dissolving membrane protein in a buffer solution to prepare a water phase;
s3, fully and uniformly mixing the organic phase and the water phase to obtain a mixed solution, and replacing an organic solvent and a solvent buffer solution in the mixed solution to obtain the membrane proteolipid nanoparticle composite.
Preferably, the total concentration of the ionizable amino lipid, structural lipid, helper lipid, and polymer conjugated lipid in the organic phase is 1-50mg/mL, and the concentration of the membrane protein in the aqueous phase is 0.1-1.5mg/mL.
Preferably, the organic solvent includes, but is not limited to, ethanol, and the buffer includes, but is not limited to, citrate buffer, acetate solution. More preferably, the buffer is selected from acetic acid-sodium acetate acidic solution or citric acid-sodium citrate acidic solution.
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.
preferably, the specific method for replacing the organic solvent and the solvent buffer solution in the mixed solution is as follows: the mixture is diluted 10-50 times (more preferably 10-20 times) with PSB buffer and then concentrated.
The third aspect of the invention provides an application of the membrane protein lipid nanoparticle complex in preparing tumor vaccines.
The research shows that the membrane protein lipid nanoparticle compound designed by the invention can effectively activate the immune system of organisms, can excite strong humoral immunity and cellular immunity in the bodies, especially promote the CD8 positive T to generate specific IFN-gamma, has the effect of preventing or killing tumors, and has no obvious toxic or side effect.
Preferably, the tumor comprises 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, bile duct cancer. More preferably, the tumor comprises melanoma, colorectal cancer, pancreatic cancer, cervical cancer, breast cancer, prostate cancer.
Preferably, the administration mode of the vaccine comprises nebulization administration, intravenous injection, subcutaneous injection, intramuscular injection, ocular administration.
Compared with the prior art, the invention has the beneficial effects that:
the 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 invention can deliver all membrane protein antigens on tumors to organisms through a lipid nanoparticle delivery system, activate immune response of the organisms, cause strong humoral immunity and cellular immunity in the bodies, especially promote infiltration of CD8 positive T cells and secretion of IFN-gamma in tumor microenvironment, have the effect of preventing or killing the tumors, and have no obvious toxic or side effect. In addition, the membrane protein lipid nanoparticle composite is used for preparing tumor vaccines, the carrier delivery efficiency of the membrane protein is high, the occurrence of solid tumors is effectively prevented, or cancer cells are killed, the application prospect is good, the vaccine safety is good, the manufacturing process is simple, and industrialization is easy to realize.
Drawings
FIG. 1 is a graph showing particle sizes of LNPs prepared from tumor cell membrane proteins B16F10, CT26, PANC02, TC-1, 4T-1, RM 1;
FIG. 2 shows the potential maps of LNPs prepared from B16F10, CT26, PANC02, TC-1, 4T-1, RM1 tumor cell membrane proteins;
FIG. 3 is a graph showing the encapsulation efficiency of LNPs prepared from B16F10, CT26, PANC02, TC-1, 4T-1, RM1 tumor cell membrane proteins;
FIG. 4 is a WB map of B16F10, CT26, PANC02, TC-1, 4T-1, RM1 tumor cell membrane proteins;
FIG. 5 is a graph showing inhibition of tumor growth by LNPs prepared from B16F10, CT26, PANC02, TC-1, 4T-1, RM1 tumor cell membrane proteins;
FIG. 6 is a graph showing that the preparation of LNPs from B16F10, CT26, PANC02, TC-1, 4T-1, RM1 tumor cell membrane proteins activates humoral immunity to produce IgG;
FIG. 7 is a diagram showing that preparation of LNPs from B16F10, CT26, PANC02, TC-1, 4T-1, RM1 tumor cell membrane proteins activates cellular immunity to produce IFN-gamma;
FIG. 8 is a diagram showing that preparation of LNPs from B16F10, CT26, PANC02, TC-1, 4T-1, RM1 tumor cell membrane proteins activates cellular immunity to produce hIFN-V;
FIG. 9 is a graph of safety evaluation of preparation of LNPs from B16F10, CT26, PANC02, TC-1, 4T-1, RM1 tumor cell membrane proteins;
FIG. 10 is a graph showing the evaluation of the efficacy of the LNPs prepared from B16F10, CT26, PANC02, TC-1, 4T-1, RM1 tumor cell membrane proteins in combination with aPD-1 for inhibiting tumor growth;
FIG. 11 is a graph showing the effect of LNPs prepared from B16F10, CT26, PANC02, TC-1, 4T-1, RM1 tumor cell membrane proteins on promoting infiltration of CD 8-positive T cells.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
Example 1B16F10 preparation of Membrane proteolipid nanoparticle Complex vaccine and evaluation of its application Effect
1. Extraction of B16F10 membrane protein antigen
(1) Mouse solid tumor cells (melanoma B16F10 cells) were collected by cell culture and solid tumor excision. Selecting C57/BL mice of 4-6 weeks old for back subcutaneous tumor experiments, planting 100 tens of thousands of B16F10 cells in each mouse, cutting tumor tissues when tumors grow to 9 days, taking about 100mg of the tissues, shearing the tissues by scissors, grinding the tissues to obtain cell tissue fragments, centrifuging (300 g, 5 min), adding 1mL of Biyun tenna protein extraction reagent (product number: P0033) A solution added with PMSF, slightly suspending the tissue fragments, and standing for 10-15min in an ice bath.
(2) And (3) repeatedly freezing and thawing the cell suspension in liquid nitrogen and a water bath kettle at 37 ℃ for 3 times, taking 2-3 mu L, and placing under a microscope for observation until 70-80% of cells are nuclear-free Zhou Yunhuan and complete cell morphology.
(3) Removal of nuclei and unbroken cells: centrifugation at 700g for 10min at 4deg.C was performed, and the supernatant was carefully collected in a fresh centrifuge tube, and the supernatant was not aspirated to the bottom for precipitation, leaving 50. Mu.L of liquid unabsorbed to ensure the purity of the supernatant.
(4) Precipitation of cell membrane fragments: 14000g of supernatant was used for 30min at 4℃to precipitate cell membrane fragments.
(5) Collecting the cytoplasmic protein: the supernatant is sucked to be the cytoplasmic protein.
(6) Extracting cell membrane protein: supernatant fluid is sucked as much as possible at 4 ℃ for 10 seconds with 14000g of supernatant fluid, a small amount of sediment can be sucked, 300 mu L of Biyun Tian membrane protein extraction reagent B solution is added, the solution is vigorously vortexed for 5 seconds at the highest speed, the ice bath is carried out for 5-10 minutes, and the membrane protein is fully extracted by repeating for 3 times. And then centrifuging at 4 ℃ and 14000g for 5 minutes, collecting the supernatant to obtain a membrane protein solution, and preserving at-80 ℃ for later use.
(7) Taking a small amount of membrane protein solution, measuring the protein concentration by adopting a BCA method, wherein the measured concentration is as follows: about 0.5-1mg/mL.
(8) Western blot verification membrane protein: a proper amount of membrane protein solution is taken, 5 XSDS loading is added for denaturation at 100 ℃ for 5 minutes, and then Western-blot experiment is carried out. ATPA1, sodium potassium atpase protein A1, is a transmembrane protein (see fig. 4) and ATPA1 can be used to verify that the extracted protein mixture is a membrane protein mixture.
2. Preparation and characterization of membrane protein-LNPs lipid nanoparticles
(1) Ionizable amino lipids (2- ((4- (dimethylamino) butanoyl) oxy) dodecyl 2-hexyl decanoate) were combined with structural lipids (cholesterol), helper lipids (DOPC), polymer conjugated lipids (PEG 2000-DMG) at 50:38.5:10:1.5 in absolute ethyl alcohol to prepare the ionizable amino lipid nano-particles, and adding the absolute ethyl alcohol to ensure that the concentration of the ionizable amino lipid is in the range of 0.01M-0.5M after the four components are uniformly mixed. Taking 10mg of ionizable amino lipid, 4mg of cholesterol, 4mg of auxiliary lipid and 2.5mg of surfactant as examples, the amounts of the four components added with absolute ethyl alcohol are 200 mu L, 400 mu L and 100 mu L respectively; further 84.25. Mu.L of ionizable amino lipid, 126.70. Mu.L of cholesterol, 126.49. Mu.L of helper lipid, and 24.1. Mu.L of surfactant were extracted and added to 318.46. Mu.L of absolute ethanol to prepare 680. Mu.L volume of ethanol phase solution. The resulting ethanol phase solution and acetic acid-sodium acetate acid solution (ph=5.0, 25 mm) or citric acid-sodium citrate acid solution (ph=5.0, 25 mm) in which B16F10 membrane protein was dissolved were then mixed in a microfluidic chip at a volume ratio of 1:3 at a flow rate of 12mL/h using a microfluidic preparation system (miana, INano E) to prepare a crude solution of lipid nanoparticles. The crude solution was diluted 10-fold with PBS, ultrafiltered with a 15mL or 50mL ultrafiltration tube (Millipore, 100K) at 4deg.C for 15min at a rotational speed of 1.5 kcf for three times, and finally the mass ratio of ionizable amino lipids to membrane proteins was about 8:1 to obtain membrane protein-LNPs lipid nanoparticles.
(2) Characterization of particle size, PDI and potential: the particle size and PDI of the lipid nanoparticles prepared were determined by Nano-ZSZEN3600 (Malvern). Taking 20 mu L of the lipid nanoparticle solution of the membrane protein-LNPs for particle size or potential measurement, wherein the stabilizing time is 120s, and the method is circulated for three times, and 10 times each time. The results are shown in FIG. 1, and indicate that the particle size of the prepared lipid nanoparticle of the B16F10 membrane protein-LNPs is about 160-210nm, PDI is less than 0.2, and the potential is-5 to +5mV (FIG. 2).
(3) Encapsulation efficiency measurement: measuring by referring to standard procedure of Biyundian BCA protein kit, respectively detecting membrane protein concentration before and after demulsification of x-100, and calculating to obtain LNThe encapsulation efficiency of P is roughly as follows: 2 mu L of ultrafiltered membrane protein-LNPs lipid nanoparticle samples are respectively added into 498 mu L of 1 XPBS and 498 mu L of 2% concentration Triton-100 solution, absorbance is analyzed by an enzyme-labeled instrument after BCA working solution is added, a standard curve is drawn, and finally, the membrane protein concentration is calculated according to the standard curve. The result shows that the encapsulation rate of the B16F10 membrane protein-LNPs sample is more than 90 percent, R 2 Greater than 0.99 (fig. 3).
3. Animal experiment of film protein vaccine action effect
1. Female C57/BL mice are immunized by membrane protein vaccine, and the effect of the vaccine on treating tumors is evaluated
Female C57/BL mice with the age of 4-5 weeks are purchased from the center of experimental animals in Guangdong province, B16F10 cells are inoculated, the seed tumor is marked as day 0, the cell inoculation number is 50 ten thousand/mouse, and the seed tumor part is positioned under the right back skin of the mice; after the tumor is planted, the tumor size is observed, and the tumor volume is 150-200mm when the tumor size is up to 9 days 3 At the time, 9/10 tumor tissues are excised by surgery and used for preparing membrane protein-LNPs, and immunization is carried out by adopting the membrane protein-LNPs on the 10 th day, the 17 th day and the 24 th day, wherein the dosage of the membrane protein is 10 mug/patient; and PBS group is used as a control, the injection site is the right hind limb of the mouse, the injection mode is intramuscular injection, and the injection volume is 100 mu L. Then observing the growth condition of the tumor, measuring the volume of the tumor, and calculating the tumor volume by the following steps: volume v=1/2 length x width. The result shows that the membrane protein-LNPs vaccine can effectively inhibit the growth of tumors. The method comprises the following specific steps:
(1) Murine melanoma cell line B16F10 (purchased from Wuhanprios) was cultured in T25 flasks, when the cell density reached about 80%, the cells were digested with pancreatin for 2min, stopped with DMEM complete medium containing 10% FBS, centrifuged at 1000rpm for 5min, the supernatant was discarded, the cells were resuspended in complete medium and counted, the number of seed tumor cells was 1X 10 6 About 50mm in size for subcutaneous tumor on about day 4/only 3 The mice were then randomly divided into 3 groups of 6 mice each, PBS group, membrane protein-LNPs.
(2) Tumor volume up to 150-200mm on day 9 3 At the time of anesthesia, after fixation, the mice were shaved, skin was disinfected, the back was cut centrally, and the length was about 1cm, free swellingSurgical excision of tumor tissue, excision of tumor tissue with the size of 9/10 of the tumor volume, hemostasis by compression, suturing wound and disinfection. The obtained tumor tissue is prepared into a film-forming protein solution, and the specific method steps are shown in II.
(3) Mice were immunized on days 10, 17, 24, tumor volumes were observed and measured on alternate days.
As shown in FIG. 5, the tumor volume of the B16F10 membrane protein-LNPs group is obviously smaller than that of the PBS group, and the result shows that the B16F10 membrane protein-LNPs vaccine can effectively inhibit the growth of tumors.
2. Membrane protein vaccine for immunizing female C57/BL mice and evaluating effect of vaccine combined with aPD-1 on tumor treatment
Female mice with the age of 4-5 weeks are purchased from the experimental animal center in Guangdong province, the seed tumor is marked as day 0, the cell inoculation number is 100 ten thousand per mouse, and the seed tumor part is positioned under the right back skin of the mice; after the tumor is planted, the tumor size is observed, and the tumor volume is 150-200mm when the tumor size is up to 9 days 3 At this time, 9/10 of tumor tissue was surgically excised for the preparation of membranous protein-LNPs, and the postsurgical mice were divided into 4 groups, PBS group, aPD-1 group alone, membranous protein-LNPs group alone, membranous protein-LNPs+aPD-1 group. And on the 10 th, 17 th and 24 th days, adopting membrane protein-LNPs and aPD-1 for treatment, wherein the dosage of the membrane protein is 10 mug/piece, and the dosage of the aPD-1 is 100 mug/piece; and PBS group is used as a control, the injection site is the right hind limb of the mouse, the injection mode is intramuscular injection, and the injection volume is 100 mu L. Then observing the growth condition of the tumor, measuring the volume of the tumor, and calculating the tumor volume by the following steps: volume v=1/2 length x width. The result shows that the combined use of the membrane protein-LNPs vaccine of the invention and the aPD-1 can effectively inhibit the growth of tumors. The method comprises the following specific steps:
(1) Murine melanoma cell line B16F10 (purchased from Wuhanposai corporation) was cultured in T25 flasks, when the cell density reached about 80%, the cells were digested with pancreatin for 2min, stopped with DMEM complete medium containing 10% FBS, centrifuged at 1000rpm for 5min, the supernatant was discarded, the cells were resuspended in complete medium and counted, the number of neoplastic cells was 1X 10 6 About 50mm in size for subcutaneous tumor on about day 4/only 3 At this time, the mice were then randomly divided into 2 groups, PBS group, aPD-1 group alone, membrane protein-LNPs group alone, membrane eggs White-lnps+agd-1 group, 6 mice each.
(2) Tumor volume up to 150-200mm on day 9 3 When the mice are anesthetized, after fixation, shaving, skin disinfection, back median incision, about 1cm long, free tumor tissue, surgical excision of tumor tissue, excision of 9/10 of tumor volume, hemostasis by compression, wound suturing, disinfection. The obtained tumor tissue is prepared into a film-forming protein solution, and the specific method steps are shown in II.
(3) Mice were immunized on days 10, 17, and 24, with membrane protein at 10 μg/dose, aPD-1 at 100 μg/dose, and tumor volumes were observed and measured on alternate days.
As shown in FIG. 10, the tumor volume of the B16F10 membrane protein-LNPs+aPD-1 group is obviously smaller than that of other groups, which suggests that the combined aPD-1 of the B16F10 membrane protein-LNPs vaccine can effectively inhibit the growth of tumors.
(4) Fresh ex vivo tumor blocks of the control group and the vaccine group were respectively infiltrated in 1×pbs buffer solution of a 24-well plate to ensure cell viability. Secondly, rapidly cutting the tumor into fragments on a sterile culture dish by using a pair of curved ophthalmic surgical scissors, wherein the volume is less than or equal to 1mm 3 Pieces were digested with freshly prepared tissue hydrolysate A (collagenase powder 100mg, deoxyribonuclease I (DNase I) powder 5mg, RMPI-1640 medium to 100 mL) at 220rpm/min on a 37℃constant temperature shaker for 30min. Finally, the remaining fragments were ground with a 5mL syringe handle and filtered through a 70 μm sterile filter, and the tumor single cell suspension was collected with a 15mL centrifuge tube.
Tumor infiltrating lymphocytes are separated (tissue lymphocyte separation kit method: soilebao, cat# P9000): the tumor cell suspension was centrifuged at 500g for 10min to obtain a cell pellet, which was then resuspended and diluted with 3mL of sample diluent. An equal volume of the separation solution was placed in a new 15mL centrifuge tube and the diluted cell suspension was slowly added dropwise over the separation solution. At room temperature, 500g, centrifuging for 30min, separating liquid into 4 layers, carefully sucking the lymphocytes of the 2 nd layer, placing into a new centrifuge tube, re-suspending cell sediment with pre-cooled FBS solution, counting, and adjusting cell concentration to 1×10 7 The sample was kept at one/mL. Finally, the CD8+ T cell ratio was measured by flow cytometry, and the results are shown in FIG. 11, which illustrates the present inventionThe clear B16F10 membrane protein-LNPs vaccine can promote infiltration of CD8 positive T cells.
3. Membrane protein vaccine stimulates powerful humoral immunity and cellular immunity in vivo
Female mice of 4-5 weeks old C57/BL were immunized 1 time with B16F10 membrane protein-LNPs on days 1 and 7, respectively, with 10 μg/PBS group as control. After the mice are anesthetized by isoflurane on day 14, the eyeballs are picked up by forceps and bloodletting is carried out to about 600 mu L of heparin sodium anticoagulation tube, centrifugation is carried out for 800g and 20min at 4 ℃, upper serum is collected, the upper serum is sucked and separated into 2 1.5mL centrifuge tubes by equal volume, and the centrifuge tubes are placed in a refrigerator at-20 ℃ for preservation after labeling. Then the mouse body is soaked in 75% ethanol for 5min, transferred into an ultra clean bench, taken out of the spleen by aseptic dissection, ground by using a 5mL syringe rubber plug and a 200 mesh nylon net under PBS, filtered by a 40 micron filter screen, transferred into a 15mL centrifuge tube for 500g and 5min centrifugation at 4 ℃. After removal of the supernatant, 3mL of red blood cell lysate was added, lysed for 5 minutes, followed by centrifugation at 4℃for 500g, 5 minutes with 6mL of PBS, repeated 2 times of centrifugation washing with PBS, followed by resuspension with 1mL of serum-free 1640 medium.
( 1) Elispot spot assay (IFN- γ assay; the daceae is selected as a kit: mouse IFN-. Gamma. precoated ELISPOT kit Cat #:2210005 )
100. Mu.L of spleen cell fluid was resuspended in 900. Mu.L of 1640 medium containing Australian fetal bovine serum (10%), diluted to 100 Wan cells/mL, and 100. Mu.L was added to the ELISPOT plate, each mouse added with 4 wells, wherein the OVA peptide stimulated 3 wells and the Canavalia protein positive stimulated 1 well. Then 10. Mu.L of 200. Mu.g/mL OVA peptide (complete medium configuration) was added to the peptide wells, 10. Mu.L of 400. Mu.g/mL Canavalia gladiata protein (complete medium configuration) was added to the positive stimulator wells, the cells were ruptured with pre-chilled deionized water at 4℃after incubation in the incubator for 24h, primary antibody (Biotinylated Antibody) was added, the plates were washed, secondary antibody HRP (strepavidin-HRP) was incubated, and incubation was performed at room temperature for 1h on a shaker. PBST washing the plate for 5 times, adding a developing Solution (AEC developing Solution: AEC Solution I (20X), AEC Solution II (20X) and AEC Solution III (200X) in a clean container, uniformly mixing according to the proportion of 180:10:10:1, namely the working Solution), developing for 5-30min in a dark place, stopping with deionized water, and reading the plate for analysis.
As shown in FIG. 7, the B16F10 membrane protein-LNPs vaccine of the invention plays a role in promoting IFN-gamma secretion in tumor microenvironment, thereby activating cellular immunity.
(2) IgG assay: membrane protein antigen plating
The membrane protein solution was diluted to 100. Mu.g/mL and then transferred to a loading well, 100. Mu.L per well in a 96-well plate. Sealing film, and standing overnight at 4deg.C. PBST (taking a 2L volumetric flask, adding ddH2O into PBS dry powder to a volume of 2L, uniformly mixing, taking out 50mL for later use [ namely PBS ], uniformly mixing the rest liquid in the volumetric flask with 0.05% Tween20 as a washing liquid [ namely PBST ]) and washing the plate 5 times, closing the 2.5% BSA for 1h, washing the plate 5 times by the PBST, and incubating diluted serum (mouse serum is taken in the animal experiment) (1:10 3 、1：10 4 、1：10 5 、1：10 6 ) 2-4h; PBST plates were washed 5 times, incubated with secondary antibody HRP (Goat-Anti-Mouse IgG (H+L) -HRP; cat: 1036-05), and incubated with shaking table at room temperature for 1H. PBST plate was washed 5 times, TMB was added to develop color in the dark for 5-30min, and an equal volume (100. Mu.L) of 2M H was added 2 SO 4 The reaction was terminated and the microplate reader read analyzed.
As shown in FIG. 6, the B16F10 membrane protein-LNPs vaccine of the invention plays a role in promoting the secretion of IgG in the tumor microenvironment, thereby activating humoral immunity.
4. In vitro killing experiments to assess the ability of membrane protein vaccines to activate effector T cells
(1) Peripheral blood of a melanoma patient and a melanoma tissue specimen (a sample is obtained from a seventh hospital affiliated with the university of Zhongshan and approved by the relevant ethical committee) were collected, and human peripheral blood mononuclear cells and primary cells of melanoma were obtained. Peripheral blood mononuclear cells were extracted for later use, roughly as follows:
1) An appropriate amount of lymphocyte separation medium (white shark) BL590 was added to the short middle tube.
2) Heparin anticoagulated venous blood is taken and fully and uniformly mixed with an equal amount of Hank's liquid or RPMI1640, slowly overlapped on a separation liquid level along the pipe wall by using a dropper, a clear interface is kept, and then horizontal centrifugation is carried out for 2000rpm multiplied by 20 minutes.
3) The inside of the tube is divided into three layers after centrifugation, the upper layer is blood plasma and Hank's liquid, the lower layer is mainly red blood cells and granulocytes, the middle layer is lymphocyte separating liquid, a white cloud layer narrow band mainly comprising mononuclear cells is arranged at the interface of the upper layer and the middle layer, and the mononuclear cells comprise lymphocytes and monocytes. In addition, platelets are contained.
4) The mononuclear cells were aspirated by inserting capillaries into the cloud layer, placing into another short middle tube, adding more than 5 volumes of Hank's solution or RPMI1640, 1500rpm X10 min, and washing the cells twice.
5) After the last centrifugation, the supernatant was discarded, RPMI1640 resuspended cells containing 10% calf serum were added, one drop of cell suspension was mixed with one drop of 0.2% trypan blue dye, and the total number of cells in four large squares was counted on a hemocytometer plate.
6) Cell viability detection: dead cells can be stained blue, viable cells are not stained, 200 lymphocytes are counted, and the percentage of viable cells is calculated.
(2) Preparation of melanoma primary cells: adding 0.25% trypsin or 2000U/mL collagenase into melanoma crushed tissue block, digesting in water bath at 37deg.C for more than 30min, centrifuging to obtain supernatant, washing with pre-cooled HBSS buffer (Cyagen) at 4deg.C, washing with HBSS-10001 for 3 times, washing with DMEM complete medium for 1 time, suspending with DMEM complete medium, dispersing with suction tube to obtain cell suspension, and counting to obtain concentration (5-10) ×10 8 The individual/L cell suspension was finally inoculated with cells in RPMI-1640 or DMEM containing 10% calf serum at 37℃with 5% CO 2 And (5) culturing in a lower split bottle.
(3) Human dendritic cells, T cells and primary melanoma cells were prepared and the activation of the immune system of the organism by membrane protein-LNPs was evaluated.
Human IL-4 and GM-CSF (Peprotech) are adopted to induce the differentiation of peripheral blood mononuclear cells into dendritic cells, the working concentration is 800U/mL, and suspension and semi-adherent cells are collected after 7 days for subsequent experiments. T lymphocytes were collected from human monocytes by means of CD3 (Miltenyi Biotec) sorting. Then, the membrane protein-LNPs (10 ug/well) were transfected into dendritic cells for 2-12 hours and then co-cultured with T cells for 18-24 hours, the number ratio of T cells to dendritic cells was 4:1 cells were cultured with RPMI-1640 medium containing 60ng/mL IL-21 (Peprotech) and 3000IU/mL IL-2 (Peprotech). Finally, spot experiments were performed using a human IFN-gamma kit, with untreated PBMC as a control.
As shown in FIG. 8, the B16F10 membrane protein-LNPs vaccine of the invention plays a role in promoting IFN-gamma secretion in tumor microenvironment, thereby activating immune response of effector T cells.
5. Evaluation of safety of Membrane protein-LNPs on organism
Tumor-free mice (4-5 week old female C57/BL mice) were injected with two-needle vaccine (B16F 10 membrane protein-LNPs, 10 ug/dose of membrane protein, once daily) and serum from mice was collected for detecting liver function, kidney function, etc. on the third day, and PBS was injected in the control group. The results suggest that the vaccine has no obvious effect on the liver and kidney functions and other indexes (figure 9).
Example 2 preparation of CT26 Membrane proteolipid nanoparticle Complex vaccine and evaluation of its application Effect
1. Extraction of CT26 membrane protein antigen
The extraction method is the same as in example 1, except that: the mouse solid tumor cells used were colorectal cancer cells CT26, and the selected mice were 4-6 week old Balb/C mice (Experimental animal center in Guangdong province).
The protein component of the CT26 membrane protein solution is ATPA1, and ATPA1, namely sodium potassium ATPase protein A1, is transmembrane protein (see figure 4).
2. Preparation and characterization of membrane protein-LNPs lipid nanoparticles
The preparation method of the membrane protein-LNPs lipid nanoparticle is the same as in example 1, and finally the mass ratio of the ionizable amino lipid to the membrane protein is about 6:1.
Also, the particle size of the prepared CT26 membrane protein-LNPs lipid nanoparticle is about 160-210nm, PDI is less than 0.2, and the potential is-5 to +5mV (figure 2); CT26 membrane protein-LNPs sample encapsulation rate is more than 90%, R 2 Greater than 0.99 (fig. 3).
3. Animal experiment of film protein vaccine action effect
1. Membrane protein vaccine for immunizing female Balb/C mice and evaluating effect of vaccine on tumor treatment
The experimental procedure is as in example 1, except that: the inoculated tumor cells were murine colorectal cancer cell line CT26 (purchased from Wuhanplausite Co.) with the following cell inoculation numbers: 100 ten thousand per one.
As shown in FIG. 5, the tumor volume of the CT26 membrane protein-LNPs group is obviously smaller than that of the PBS group, and the result shows that the CT26 membrane protein-LNPs vaccine can effectively inhibit the growth of tumors.
2. Membrane protein vaccine for immunizing female Balb/C mice and evaluating effect of vaccine combined with aPD-1 on tumor treatment
The experimental procedure is as in example 1, except that: the tumor cells inoculated were murine colorectal cancer cell line CT26 (purchased from Wuhanplaxel Corp.).
As shown in FIG. 10, the tumor volume of the CT26 membrane protein-LNPs+aPD-1 group is obviously smaller than that of other groups, which suggests that the combined use of the CT26 membrane protein-LNPs vaccine of the invention and aPD-1 can effectively inhibit the growth of tumors.
3. Membrane protein vaccine stimulates powerful humoral immunity and cellular immunity in vivo
The experimental procedure is as in example 1, except that: the selected mice are Balb/C mice of 4-5 weeks of age, and the vaccine used for immunization is CT26 membrane protein-LNPs.
The results of the Elispot spot test (IFN-gamma assay) are also shown in FIG. 7, which illustrates that the CT26 membrane protein-LNPs vaccine of the present invention plays a role in promoting secretion of IFN-gamma in tumor microenvironment, thereby activating cellular immunity. The results of the IgG assay (membrane protein antigen plating) are also shown in FIG. 6, which demonstrates that the CT26 membrane protein-LNPs vaccine of the invention acts to promote secretion of IgG in the tumor microenvironment, thereby activating humoral immunity.
4. In vitro killing experiments to assess the ability of membrane protein vaccines to activate effector T cells
The experimental procedure is as in example 1, except that: collecting peripheral blood of colorectal cancer patients and colorectal cancer tissue specimens (samples are from a seventh hospital affiliated with the university of Zhongshan to obtain approval of related ethical committee), obtaining human peripheral blood mononuclear cells and colorectal cancer primary cells, and extracting the peripheral blood mononuclear cells for later use.
As shown in FIG. 8, the CT26 membrane protein-LNPs vaccine of the invention has the effect of promoting IFN-gamma secretion in tumor microenvironment, thereby activating the immune response of effector T cells.
5. Evaluation of safety of Membrane protein-LNPs on organism
Tumor-free mice (4-5 week old female Balb/C mice) were injected with two-needle vaccine (CT 26 membrane protein-LNPs, once daily) and serum from mice was collected for detecting liver function, kidney function, etc. on the third day, and PBS was injected in the control group. The results suggest that the vaccine has no obvious effect on the liver and kidney functions and other indexes (figure 9).
Example 3 preparation of PAN02 Membrane proteolipid nanoparticle Complex vaccine and evaluation of its application Effect
1. Extraction of PAN02 membrane protein antigen
The extraction method is the same as in example 1, except that: the adopted mouse solid tumor cells are pancreatic cancer PAN02 cells.
The protein component of the PAN02 membrane protein solution prepared by the method is ATPA1, wherein ATPA1 is sodium potassium ATPase protein A1, and is transmembrane protein (see figure 4).
2. Preparation and characterization of membrane protein-LNPs lipid nanoparticles
The preparation method of the membrane protein-LNPs lipid nanoparticle is the same as in example 1.
Also, the particle size of the prepared PAN02 membrane protein-LNPs lipid nanoparticle is about 160-210nm, PDI is less than 0.2, and the potential is-5 to +5mV (figure 2); PAN02 membrane protein-LNPs sample encapsulation rate is more than 90%, R 2 Greater than 0.99 (fig. 3).
3. Animal experiment of film protein vaccine action effect
1. Female C57/BL mice are immunized by membrane protein vaccine, and the effect of the vaccine on treating tumors is evaluated
The experimental procedure is as in example 1, except that: the tumor cells inoculated were murine pancreatic cancer cell line PAN02 (purchased from wunpro corporation).
As shown in the figure 5, the tumor volume of the membrane protein-LNPs group is obviously smaller than that of the PBS group, and the result shows that the PAN02 membrane protein-LNPs vaccine can effectively inhibit the growth of tumors.
2. Membrane protein vaccine for immunizing female C57/BL mice and evaluating effect of vaccine combined with aPD-1 on tumor treatment
The experimental procedure is as in example 1, except that: the tumor cells inoculated were murine pancreatic cancer cell line PAN02 (purchased from wunpro corporation).
As shown in the figure 10, the tumor volume of the PAN02 membrane protein-LNPs+aPD-1 group is obviously smaller than that of other groups, which suggests that the PAN02 membrane protein-LNPs vaccine combined with the aPD-1 can effectively inhibit the growth of tumors.
3. Membrane protein vaccine stimulates powerful humoral immunity and cellular immunity in vivo
The experimental procedure is as in example 1, except that: the vaccine used for immunization is PAN02 membrane protein-LNPs.
The results of the Elispot spot test (IFN-gamma assay) are also shown in FIG. 7, which illustrates that the PAN02 membrane protein-LNPs vaccine of the present invention plays a role in promoting IFN-gamma secretion from the tumor microenvironment, thereby activating cellular immunity. The results of the IgG assay (membrane protein antigen plating) are also shown in fig. 6, which demonstrates that the PAN02 membrane protein-LNPs vaccine of the present invention acts to promote IgG secretion in the tumor microenvironment, thereby activating humoral immunity.
4. In vitro killing experiments to assess the ability of membrane protein vaccines to activate effector T cells
The experimental procedure is as in example 1, except that: collecting peripheral blood of pancreatic cancer patients and pancreatic cancer tissue specimens thereof (samples are from a seventh hospital affiliated with the university of Zhongshan to obtain approval of related ethical committee), obtaining human peripheral blood mononuclear cells and pancreatic cancer primary cells, and extracting the peripheral blood mononuclear cells for later use.
The results are also shown in FIG. 8, which illustrates that the PAN02 membrane protein-LNPs vaccine of the present invention plays a role in promoting IFN-gamma secretion from the tumor microenvironment, thereby activating the immune response of effector T cells.
5. Evaluation of safety of Membrane protein-LNPs on organism
Tumor-free mice (4-5 week old female C57/BL mice) were injected with two-needle vaccine (PAN 02 membrane protein-LNPs once daily), and mice serum was collected for detecting liver function, kidney function, etc. on the third day, and PBS was injected in the control group. The results suggest that the vaccine has no obvious effect on the liver and kidney functions and other indexes (figure 9).
Example 4 preparation of TC-1 Membrane proteolipid nanoparticle Complex vaccine and evaluation of its application Effect
1. Extraction of TC-1 membrane protein antigen
The extraction method is the same as in example 1, except that: the adopted mouse solid tumor cells are cervical cancer TC-1 cells.
The protein component of the TC-1 membrane protein solution prepared by the method is ATPA1, wherein ATPA1 is sodium potassium ATPase protein A1, and is transmembrane protein (see figure 4).
2. Preparation and characterization of membrane protein-LNPs lipid nanoparticles
The preparation method of the membrane protein-LNPs lipid nanoparticle is the same as in example 1.
Also, the prepared TC-1 membrane protein-LNPs lipid nanoparticle has a particle diameter of about 160-210nm, PDI less than 0.2, and potential of-5 to +5mV (FIG. 2); TC-1 membrane protein-LNPs sample encapsulation rate is more than 90%, R 2 Greater than 0.99 (fig. 3).
3. Animal experiment of film protein vaccine action effect
1. Female C57/BL mice are immunized by membrane protein vaccine, and the effect of the vaccine on treating tumors is evaluated
The experimental procedure is as in example 1, except that: the inoculated tumor cells were murine cervical cancer cell line TC-1 (purchased from Wuhanplausite Corp.).
As shown in FIG. 5, the tumor volume of the TC-1 membrane protein-LNPs group is obviously smaller than that of the PBS group, and the result shows that the TC-1 membrane protein-LNPs vaccine can effectively inhibit the growth of tumors.
2. Membrane protein vaccine for immunizing female C57/BL mice and evaluating effect of vaccine combined with aPD-1 on tumor treatment
The experimental procedure is as in example 1, except that: the inoculated tumor cells were murine cervical cancer cell line TC-1 (purchased from Wuhanplausite Corp.).
As shown in FIG. 10, the tumor volume of the TC-1 membrane protein-LNPs+aPD-1 group is obviously smaller than that of other groups, which suggests that the combination of the TC-1 membrane protein-LNPs vaccine and the aPD-1 can effectively inhibit the growth of tumors.
3. Membrane protein vaccine stimulates powerful humoral immunity and cellular immunity in vivo
The experimental procedure is as in example 1, except that: the vaccine used for immunization is TC-1 membrane protein-LNPs.
The results of the Elispot spot test (IFN-gamma assay) are also shown in FIG. 7, which illustrates that the TC-1 membrane protein-LNPs vaccine of the present invention plays a role in promoting secretion of IFN-gamma in tumor microenvironment, thereby activating cellular immunity. The results of the IgG assay (membrane protein antigen plating) are also shown in FIG. 6, which demonstrates that the TC-1 membrane protein-LNPs vaccine of the present invention acts to promote secretion of IgG in the tumor microenvironment, thereby activating humoral immunity.
4. In vitro killing experiments to assess the ability of membrane protein vaccines to activate effector T cells
The experimental procedure is as in example 1, except that: collecting peripheral blood of cervical cancer patients and cervical cancer tissue specimens (samples are from a seventh hospital affiliated to the university of Zhongshan to obtain approval of related ethics committee), obtaining human peripheral blood mononuclear cells and primary cervical cancer cells, and extracting the peripheral blood mononuclear cells for later use.
As shown in FIG. 8, the TC-1 membrane protein-LNPs vaccine of the invention plays a role in promoting IFN-gamma secretion in tumor microenvironment, thereby activating immune response of effector T cells.
5. Evaluation of safety of Membrane protein-LNPs on organism
Tumor-free mice (4-5 week old female C57/BL mice) were injected with two-needle vaccine (TC-1 membrane protein-LNPs once daily), and mice serum was collected for detecting liver function, kidney function, etc. on the third day after injection, and PBS was injected in the control group. The results suggest that the vaccine has no obvious effect on the liver and kidney functions and other indexes (figure 9).
Example 5 preparation of 4T1 Membrane proteolipid nanoparticle Complex vaccine and evaluation of its application Effect
1. Extraction of 4T1 membrane protein antigen
The extraction method is the same as in example 1, except that: the adopted mouse solid tumor cells are breast cancer 4T1 cells, and the selected mice are Balb/C mice (Experimental animal center in Guangdong province) of 4-6 weeks old.
The protein component of the 4T1 membrane protein solution prepared by the method is ATPA1, wherein ATPA1 is sodium potassium ATPase protein A1, and is transmembrane protein (see figure 4).
2. Preparation and characterization of membrane protein-LNPs lipid nanoparticles
The preparation method of the membrane protein-LNPs lipid nanoparticle is the same as in example 1.
Also, the particle size of the prepared 4T1 membrane protein-LNPs lipid nanoparticle is about 160-210nm, PDI is less than 0.2, and the potential is-5 to +5mV (figure 2); 4T1 membrane protein-LNPs sample encapsulation rate is more than 90%, R 2 Greater than 0.99 (fig. 3).
3. Animal experiment of film protein vaccine action effect
1. Membrane protein vaccine for immunizing female Balb/C mice and evaluating effect of vaccine on tumor treatment
The experimental procedure is as in example 1, except that: the inoculated tumor cells were murine breast cancer cell line 4T1 (purchased from Wuhanplausite Inc.), and the number of cell inoculations was: 100 ten thousand per one.
As shown in FIG. 5, the tumor volume of the 4T1 membrane protein-LNPs group is obviously smaller than that of the PBS group, and the result shows that the 4T1 membrane protein-LNPs vaccine can effectively inhibit the growth of tumors.
2. Membrane protein vaccine for immunizing female Balb/C mice and evaluating effect of vaccine combined with aPD-1 on tumor treatment
The experimental procedure is as in example 1, except that: the tumor cells inoculated were murine breast cancer cell line 4T1 (purchased from Wuhanplausite Corp.).
As shown in FIG. 10, the tumor volume of the 4T1 membrane protein-LNPs+aPD-1 group is obviously smaller than that of other groups, which suggests that the combined use of the 4T1 membrane protein-LNPs vaccine of the invention and aPD-1 can effectively inhibit the growth of tumors.
3. Membrane protein vaccine stimulates powerful humoral immunity and cellular immunity in vivo
The experimental procedure is as in example 1, except that: the selected mice were 4-5 week old Balb/C mice and the vaccine used for immunization was 4T1 membrane protein-LNPs.
The results of the Elispot spot test (IFN-gamma assay) are also shown in FIG. 7, which illustrates that the 4T1 membrane protein-LNPs vaccine of the present invention plays a role in promoting secretion of IFN-gamma in tumor microenvironment, thereby activating cellular immunity. The results of the IgG assay (membrane protein antigen plating) are also shown in FIG. 6, which demonstrates that the 4T1 membrane protein-LNPs vaccine of the invention acts to promote secretion of IgG in the tumor microenvironment, thereby activating humoral immunity.
4. In vitro killing experiments to assess the ability of membrane protein vaccines to activate effector T cells
The experimental procedure is as in example 1, except that: collecting peripheral blood of a breast cancer patient and a breast cancer tissue specimen (a sample is from a seventh hospital affiliated with the university of Zhongshan to obtain approval of related ethical committee), obtaining human peripheral blood mononuclear cells and breast cancer primary cells, and extracting the peripheral blood mononuclear cells for later use.
As shown in FIG. 8, the 4T1 membrane protein-LNPs vaccine of the invention has the effect of promoting IFN-gamma secretion in tumor microenvironment, so as to activate immune response of effector T cells.
5. Evaluation of safety of Membrane protein-LNPs on organism
Tumor-free mice (4-5 week old female Balb/C mice) were injected with two-needle vaccine (4T 1 membrane protein-LNPs once daily), and mice serum was collected for detecting liver function, kidney function, etc. on the third day after injection, and PBS was injected in the control group. The results suggest that the vaccine has no obvious effect on the liver and kidney functions and other indexes (figure 9).
Example 6 preparation of RM1 Membrane proteolipid nanoparticle Complex vaccine and evaluation of its application Effect
1. Extraction of RM1 membrane protein antigen
The extraction method is the same as in example 1, except that: the adopted mouse solid tumor cells are prostatic cancer RM1 cells.
The protein component of the RM1 membrane protein solution is ATPA1, and ATPA1, namely sodium potassium ATPase protein A1, is transmembrane protein (see FIG. 4).
2. Preparation and characterization of membrane protein-LNPs lipid nanoparticles
The preparation method of the membrane protein-LNPs lipid nanoparticle is the same as in example 1.
Also, the particle size of the prepared RM1 membrane protein-LNPs lipid nanoparticle is about 160-210nm, PDI is less than 0.2, and the potential is-5 to +5mV (FIG. 2); RM1 membrane protein-LNPs sample encapsulation rate is more than 90%, R 2 Greater than 0.99 (fig. 3).
3. Animal experiment of film protein vaccine action effect
1. Female C57/BL mice are immunized by membrane protein vaccine, and the effect of the vaccine on treating tumors is evaluated
The experimental procedure is as in example 1, except that: the tumor cells inoculated were murine prostate cancer cell line RM1 (purchased from Wohplaxel).
As shown in FIG. 5, the tumor volume of the RM1 membrane protein-LNPs group is obviously smaller than that of the PBS group, and the result shows that the RM1 membrane protein-LNPs vaccine of the invention can effectively inhibit the growth of tumors.
2. Membrane protein vaccine for immunizing female C57/BL mice and evaluating effect of vaccine combined with aPD-1 on tumor treatment
The experimental procedure is as in example 1, except that: the tumor cells inoculated were murine prostate cancer cell line RM1 (purchased from Wohplaxel).
As shown in FIG. 10, the tumor volume of the RM1 membrane protein-LNPs+aPD-1 group is obviously smaller than that of other groups, which suggests that the combination of the RM1 membrane protein-LNPs vaccine of the invention and aPD-1 can effectively inhibit the growth of tumors.
3. Membrane protein vaccine stimulates powerful humoral immunity and cellular immunity in vivo
The experimental procedure is as in example 1, except that: the vaccine used for immunization is RM1 membrane protein-LNPs.
The results of the Elispot spot test (IFN-gamma assay) are also shown in FIG. 7, which illustrates that the RM1 membrane protein-LNPs vaccine of the present invention plays a role in promoting secretion of IFN-gamma by the tumor microenvironment, thereby activating cellular immunity. The results of the IgG assay (membrane protein antigen plating) are also shown in FIG. 6, which demonstrates that the RM1 membrane protein-LNPs vaccine of the invention acts to promote secretion of IgG in the tumor microenvironment, thereby activating humoral immunity.
4. In vitro killing experiments to assess the ability of membrane protein vaccines to activate effector T cells
The experimental procedure is as in example 1, except that: and collecting peripheral blood of a prostate cancer patient and a prostate cancer tissue sample thereof (a sample is obtained from a seventh hospital affiliated with the university of Zhongshan and approved by the related ethical committee), obtaining human peripheral blood mononuclear cells and primary cells of the prostate cancer, and extracting the peripheral blood mononuclear cells for later use.
As shown in FIG. 8, the RM1 membrane protein-LNPs vaccine of the invention has the effect of promoting IFN-gamma secretion in tumor microenvironment, thereby activating the immune response of effector T cells.
5. Evaluation of safety of Membrane protein-LNPs on organism
Mice without tumor (4-5 week old female C57/BL mice) were injected with two-needle vaccine (RM 1 membrane protein-LNPs once daily), and mice serum was collected for detecting liver function, kidney function, etc. on the third day, and PBS was injected in the control group. The results suggest that the vaccine has no obvious effect on the liver and kidney functions and other indexes (figure 9).
According to the comprehensive examples 1-6, the membrane protein vaccine provided by the invention can deliver the tumor membrane protein related antigen to Antigen Presenting Cells (APC) of an organism through a lipid nanoparticle delivery system, enhance specific and non-specific antigen presentation, activate immune response of the organism, promote proliferation and activation of T cells, and has the effects of preventing or killing tumors, and good safety. Therefore, the membrane protein-LNPs designed by the invention can effectively activate the immune system of organisms, excite strong cellular immunity and humoral immunity, have no obvious toxic or side effect and have good application prospect.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.

Claims (10)

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 ionizable amino lipid accounts for 30-65% of the total lipid, the auxiliary lipid accounts for 5-25% of the total lipid, the structural lipid accounts for 10-45% of the total lipid, and the polymer conjugated lipid accounts for 0.5-5% of the total lipid.
2. The membrane protein lipid nanoparticle complex of claim 1, wherein the membrane protein is a cell membrane extract of tumor cells, the cell membrane extract comprising all or part of the protein components of the cell membrane and all or part of the antigens on the cell membrane.
3. The membrane protein lipid nanoparticle complex of claim 1, wherein the mass ratio of the ionizable amino lipid to membrane protein is 1:1-50:1.
4. A membrane proteolipid nanoparticle complex according to claim 1, wherein the structural lipids comprise one or more of cholesterol and derivatives thereof.
5. A membrane proteolipid nanoparticle complex according to claim 1, wherein the helper lipids comprise one or more of DOPC, DSPC, DOPE, DOPG, DOPS.
6. The membrane protein lipid nanoparticle complex of claim 1, wherein the polymer conjugated lipid comprises one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified dialkylamine, PEG-modified dialkylglycerol.
7. A method for preparing a membrane proteolipid nanoparticle complex according to any one of claims 1 to 6, comprising the steps of:
s1, dissolving ionizable amino lipid, structural lipid, auxiliary lipid and polymer conjugated lipid in an organic solvent to prepare an organic phase;
s2, dissolving membrane protein in a buffer solution to prepare a water phase;
s3, fully and uniformly mixing the organic phase and the water phase to obtain a mixed solution, and replacing an organic solvent and a solvent buffer solution in the mixed solution to obtain the membrane proteolipid nanoparticle composite.
8. The method of preparing a membrane protein lipid nanoparticle complex according to claim 7, wherein the total concentration of the ionizable amino lipid, structural lipid, auxiliary lipid, and polymer conjugated lipid in the organic phase is 1-50mg/mL, and the concentration of the membrane protein in the aqueous phase is 0.1-1.5mg/mL.
9. Use of the membrane protein lipid nanoparticle complex of any one of claims 1-6 in the preparation of a tumor vaccine.
10. The use according to claim 9, wherein the tumor comprises 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.
CN202310034344.3A 2023-01-10 2023-01-10 Membrane protein lipid nanoparticle compound and preparation method and application thereof Pending CN116036308A (en)

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