mRNA vaccine delivery vector, mRNA vaccine, and preparation method and application thereof

Abstract

The present invention discloses an mRNA vaccine delivery vector, an mRNA vaccine, and its preparation method and application. The mRNA vaccine delivery vector is mainly prepared from a polyethylene glycol-polyphosphate block copolymer and a cationic lipid; the polyethylene glycol-polyphosphate block copolymer has a structure represented by formula (I). The mRNA vaccine includes nanoparticles having a vesicle structure prepared from the polyethylene glycol-polyphosphate block copolymer having the structure represented by formula (I) and the cationic lipid, and mRNA having a vaccine effect. The mRNA vaccine delivery vector and the mRNA vaccine can efficiently deliver mRNA into cells, thereby effectively inducing the body to produce a specific immune response for disease treatment and prevention. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of biological carrier materials and mRNA vaccines, and specifically to an mRNA vaccine delivery carrier, an mRNA vaccine, and a preparation method and application thereof. Background Art

[0002] Messenger RNA (mRNA) is a single-stranded RNA synthesized through a transcription process based on complementary base pairing using DNA as a template. mRNA is further translated into polypeptides or proteins by ribosomes in the cytoplasm. Once delivered into the body, mRNA encoding disease-specific antigens can rapidly produce the corresponding antigens and activate a specific immune response, initiating a defense mechanism against foreign substances carrying antigenic determinants. Compared to conventional inactivated pathogen vaccines, using mRNA as a vaccine offers certain advantages. mRNA vaccines are self-adjuvants, stimulating immune response receptors and driving DC maturation, effectively inducing an immune response. This makes mRNA vaccines more immunogenic. The mechanism of action of mRNA dictates that upon entering the cytoplasm, it is rapidly translated into antigenic molecules without genomic integration, effectively and safely inducing an immune response in a short period of time. Compared to traditional vaccines, the production and transcription technology of mRNA vaccines is highly universal. Once the antigen nucleic acid sequence of an mRNA vaccine is determined, production can begin within weeks. This makes it suitable for the prevention and treatment of infectious diseases with high mutation rates and rapidly evolving outbreaks, such as influenza and SARS-CoV-2. The production of mRNA vaccines does not involve the manipulation of potentially infectious virus strains or viral proteins, resulting in a safer production environment. Based on these advantages, mRNA vaccines have become an important tool for targeting Zika virus, influenza virus, SARS-CoV coronavirus, and cancer.

[0003] Although mRNA has certain advantages as the main component that triggers an immune response, as a nucleic acid macromolecule, how to safely and effectively deliver mRNA into the body to exert its effect is a major bottleneck limiting the function of mRNA vaccines. First, mRNA needs to enter the cell and be translated into polypeptides by ribosomes to exert its effect, and mRNA itself is negatively charged, which makes it difficult to pass through the cell membrane that also carries a negative charge. Secondly, as a single-stranded RNA with a large molecular weight (molecular weight is usually 1000-5000bp), mRNA will be degraded by widely existing nucleases and other substances in the body circulation, destroying its integrity. In response to the problem of in vivo delivery of mRNA vaccines, the development and construction of effective mRNA vaccine delivery vectors is currently a hot topic in clinical research. The lipid nanoparticles (LipidNanoparticle) developed by BioNTech and Pfizer use ionizable lipids to encapsulate mRNA vaccines, which trigger an effective immune response after delivery into the body, and have achieved certain results in the SARS-CoV2 new coronavirus pneumonia epidemic. However, the research on using polymer materials as delivery carriers for mRNA vaccines is still in the pioneering stage. It is of great significance to explore the research on how to effectively deliver mRNA vaccines in vivo and effectively activate immune responses based on polymer material delivery carriers.

[0004] Polyphosphoesters are a class of biodegradable polymers that have developed rapidly in recent years. Unlike polyesters with carbonate-linked structures, polyphosphoesters use phosphate bonds as their main chain backbone, have a diverse structure, and side groups can be easily functionalized to produce polyphosphoester materials with different properties. Phosphoester bonds can be slowly degraded by various enzymes in the body under physiological conditions, and have strong potential for biological applications. In recent years, polyphosphoester polymers have attracted much attention in the fields of drug delivery systems, gene transfection tools, tissue engineering materials, and regenerative medicine. Currently, research on polyphosphoesters focuses on the delivery of encapsulated drugs and small molecule nucleic acids for tumor treatment. Due to the large molecular weight of large molecule nucleic acids and their charged hydrophilicity, they are difficult to be encapsulated by conventional hydrophobic interaction carriers. The delivery of large molecule nucleic acids has rarely been explored, and this is an area that needs to be researched and developed. By studying and designing the structure of polyphosphoesters, their application in the delivery system of large molecule nucleic acid mRNA vaccines has broad application prospects. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide an mRNA vaccine delivery vector that can efficiently deliver mRNA vaccines into cells, thereby effectively inducing the body to produce a specific immune response for disease treatment and prevention. It includes the following technical solutions:

[0006] An mRNA vaccine delivery vector is mainly prepared from a polyethylene glycol-polyphosphate block copolymer and a cationic lipid; the polyethylene glycol-polyphosphate block copolymer has a structure shown in formula (I),

[0007]

[0008] Wherein, R is selected from: C1~C 10 Alkyl, C1~C 10 Unsaturated chain hydrocarbon group;

[0009] Both m and n are integers, and 40≦m≦50, 25≦n≦40.

[0010] In some embodiments, R is selected from: C1-C6 alkyl, C1-C6 unsaturated chain hydrocarbon group.

[0011] In some embodiments, R is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, n-hexyl, and allyl.

[0012] In some embodiments, 43≦m≦47, 27≦n≦35.

[0013] In some embodiments, the cationic lipid is (2,3-dioleoyl-propyl)-trimethylammonium chloride and / or BHEM-cholesterol.

[0014] In some embodiments, the molar ratio of the polyethylene glycol-polyphosphate block copolymer to the cationic lipid is 6-10:1, more preferably 7-9:1, and more preferably 7.8-8.2:1.

[0015] The present invention also provides the use of the polyethylene glycol-polyphosphate block copolymer having the structure shown in formula (I) and the mRNA vaccine delivery vector. The technical solutions include the following:

[0016] The application of the above-mentioned polyethylene glycol-polyphosphate block copolymer having the structure represented by formula (I) in the preparation of mRNA vaccine delivery vectors.

[0017] The application of the above-mentioned mRNA vaccine delivery vector in the preparation of mRNA vaccines.

[0018] The polyethylene glycol-polyphosphate block copolymer provided by the present invention has excellent biocompatibility and high efficiency in delivering nucleic acids, and can be used as a gene transfection tool material. It can be used in combination with cationic lipids to prepare carriers for efficiently delivering mRNA vaccines. It has a stable structure and is not easily degraded, and its preparation method is simple and controllable.

[0019] The present invention also provides an mRNA vaccine, including the following technical solutions:

[0020] An mRNA vaccine comprises nanoparticles prepared from the polyethylene glycol-polyphosphate block copolymer having the structure shown in formula (I), cationic lipids, and mRNA having a vaccine effect. Each of the nanoparticles is composed of an outer shell formed by doping the polyethylene glycol-polyphosphate block copolymer and the cationic lipid, and an aqueous core containing the mRNA. The mRNA vaccine provided by the present invention has high mRNA delivery and transfection efficiency and can effectively induce the body to produce a specific immune response for disease treatment and prevention.

[0021] In some embodiments, the cationic lipid is (2,3-dioleoyl-propyl)-trimethylammonium chloride and / or BHEM-cholesterol.

[0022] In some embodiments, the molar ratio of the polyethylene glycol-polyphosphate block copolymer to the cationic lipid is 6-10:1, more preferably 7-9:1, and more preferably 7.8-8.2:1; the molar ratio of the polyethylene glycol-polyphosphate block copolymer to the mRNA is 80-120:1, preferably 100-110:1, and more preferably 102-106:1.

[0023] In some embodiments, the mRNA with vaccine effect is mRNA related to viral infection or survival, mRNA related to metabolic diseases, mRNA related to tumorigenesis or cell transformation, immunomodulatory mRNA, and mRNA related to inflammation or autoimmunity.

[0024] In some embodiments, the mRNA having a vaccine effect is RBD mRNA.

[0025] The present invention also provides a double emulsion preparation method for the above-mentioned mRNA vaccine, including the following technical solutions:

[0026] A method for preparing an mRNA vaccine comprises the following steps:

[0027] (1) dissolving the polyethylene glycol-polyphosphate block copolymer in an organic solvent to form a liquid A;

[0028] (2) dissolving the cationic lipid in an organic solvent to form liquid B;

[0029] (3) dissolving the mRNA in DEPC water as solution C;

[0030] (4) After the liquid A and the liquid B are mixed, the liquid C is added, and the mixture is preliminarily mixed by ultrasonic treatment to form a colostrum, and then DEPC water is added to the obtained colostrum, and the mixture is mixed by ultrasonic treatment again to form a double emulsion;

[0031] (5) removing the organic solvent from the emulsion to obtain the mRNA vaccine.

[0032] In some embodiments, the concentration of the polyethylene glycol-polyphosphate block copolymer in the solution A in step (1) is 15 μmol / mL-25 μmol / mL.

[0033] In some embodiments, the concentration of the cationic lipid in the second solution in step (2) is 8 μmol / mL-12 μmol / mL.

[0034] In some embodiments, the organic solvent in steps (1) and (2) is ethyl acetate, dichloromethane or chloroform.

[0035] In some embodiments, the concentration of mRNA in the solution C in step (3) is 0.5 μmol / mL-2 μmol / mL.

[0036] In some embodiments, the concentration of mRNA in the solution C in step (3) is 1.5 μmol / mL-2 μmol / mL.

[0037] In some embodiments, the ultrasonic treatment in step (4) lasts for 1 minute to 2 minutes, and the power of the ultrasonic treatment is 50W to 100W.

[0038] In some embodiments, the volume ratio of the DEPC water to the colostrum in step (4) is 5-10:1.

[0039] In some embodiments, the method for removing the organic solvent from the double emulsion in step (5) is a rotary evaporation method, the time is 5 minutes to 60 minutes, and the negative pressure is 0.5 kPa to 1 kPa.

[0040] A microfluidic control preparation method for mRNA vaccines, comprising the following steps:

[0041] (1) dissolving the polyethylene glycol-polyphosphate block copolymer in an organic solvent to form a liquid A;

[0042] (2) dissolving the cationic lipid in an organic solvent to form liquid B;

[0043] (3) dissolving the mRNA in DEPC water as solution C;

[0044] (4) After mixing the liquid A and the liquid B, the mixture is loaded onto the phase A of the microfluidic system; the liquid C is loaded onto the phase B of the microfluidic system, and the mixture is fully mixed in the microfluidic chip using a microfluidic system syringe pump to obtain a colostrum;

[0045] (5) loading the primary emulsion into phase A of the microfluidic system, loading DEPC water into phase B of the microfluidic system, and fully mixing them in the microfluidic chip using a microfluidic system syringe pump to obtain a double emulsion;

[0046] (6) removing the organic solvent from the emulsion to obtain the mRNA vaccine.

[0047] In some embodiments, the concentration of the polyethylene glycol-polyphosphate block copolymer in the solution A in step (1) is 15 μmol / mL-25 μmol / mL.

[0048] In some embodiments, the concentration of the cationic lipid in the second solution in step (2) is 8 μmol / mL-12 μmol / mL.

[0049] In some embodiments, the organic solvent in steps (1) and (2) is ethyl acetate, dichloromethane or chloroform.

[0050] In some embodiments, the concentration of mRNA in the solution C in step (3) is 0.5 μmol / mL-2 μmol / mL.

[0051] In some embodiments, the concentration of mRNA in the solution C in step (3) is 1.5 μmol / mL-2 μmol / mL.

[0052] In some embodiments, in step (4), the flow rate ratio of microfluidic phase A to phase B is 5-10:1.

[0053] In some embodiments, in step (5), the flow rate ratio of microfluidic phase A to phase B is 5-10:1.

[0054] In some embodiments, the method for removing the organic solvent from the double emulsion in step (6) is a rotary evaporation method, the time is 5 minutes to 60 minutes, and the negative pressure is 0.5 kPa to 1 kPa.

[0055] The present invention also provides the use of the above-mentioned mRNA vaccine in the preparation of drugs for preventing and / or treating viral pneumonia or related diseases.

[0056] In some embodiments, the viral pneumonia or a related disease thereof is pneumonia or a related disease thereof caused by 2019-nCov infection.

[0057] The present invention also provides the use of the above-mentioned mRNA vaccine in the preparation of drugs for preventing and / or treating tumors.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] In order to solve the problem of delivering macromolecular nucleic acids into cells, the present invention provides a delivery vector capable of effectively delivering mRNA, which is mainly prepared from a polyethylene glycol-polyphosphate block copolymer with a specific structure and a cationic lipid. The polyethylene glycol-polyphosphate block copolymer with a structure of formula (I) provided by the present invention has good biocompatibility and high efficiency in delivering nucleic acids, and can be used as a gene transfection tool material. It can be used in combination with cationic lipids to effectively deliver mRNA vaccines into cells, thereby effectively inducing the body to produce a specific immune response for disease treatment and prevention.

[0060] The present invention further prepares the carrier into an mRNA vaccine composed of nanoparticles with a vesicle structure. The nanoparticles use a polyethylene glycol-polyphosphate block copolymer and a cationic lipid-doped carrier as an outer shell, and an mRNA aqueous solution encapsulated therein as an inner core. The mRNA is stably protected in the aqueous core by the charge interaction with the doped cationic lipids and is not easily dissociated or degraded in the body. At the same time, the nanoparticles in the mRNA vaccine of the present invention have a very small particle size and can easily enter the lymph nodes and contact antigen-presenting cells to activate the immune response. Ultimately, the vaccine of the present invention can induce a strong cellular immune response and humoral immune response in the body, and has a good application prospect.

[0061] Furthermore, the present invention provides a method for preparing an mRNA vaccine. This method, using the specific reaction conditions of the present invention and a double emulsion or microfluidic method, concentrates an aqueous mRNA solution and encapsulates it in nanoparticles formed by doping a polyethylene glycol-polyphosphate block copolymer with a cationic lipid. The method of the present invention is simple and controllable, and can be universally applied to the preparation of mRNA vaccines with different sequences. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is the nuclear magnetic resonance characterization of the polyphosphate polymer skeleton synthesized in Example 3.

[0063] Figure 2 The particle size characterization and scanning electron microscopy images of polyphosphate / mRNA delivery particles with different side groups prepared using the double emulsion method in Example 4 are shown.

[0064] Figure 3 The figure shows the gel electrophoresis results after different polyphosphates are combined with nucleic acids.

[0065] Figure 4 Cytotoxicity test data of polyphosphate / mRNA delivery particles with different side groups.

[0066] Figure 5These are the transfection experimental data of polyphosphates with different side groups encapsulating fluorescent reporter mRNA; Figure a shows the detection results of 293T cells by flow cytometry, and Figure b shows the detection results of DC2.4 cells by flow cytometry.

[0067] Figure 6 The serum neutralizing antibody levels in mice after immunization with polyphosphate / mRNA delivery particles encapsulating the 2019 novel coronavirus receptor spike protein (Spike protein) receptor binding domain RBD mRNA; Figure a shows the IgG neutralizing antibody level in serum; Figure b shows the IgM neutralizing antibody level in serum.

[0068] Figure 7 These are the results of T cell analysis after immunization of mice with polyphosphate mRNA delivery particles encapsulating the 2019 novel coronavirus receptor spike protein (Spike protein) receptor binding domain RBD mRNA; Figure a shows the ELISPOT test results for T cell IFNγ expression; Figure b shows the ELISPOT test results for T cell interleukin-2 expression; Figure c shows the flow cytometry data for the proportion of memory T cells; and Figure d shows the flow cytometry data for the proportion of CD69-positive activated T cells. DETAILED DESCRIPTION

[0069] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0070] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0071] In this application, "plurality" refers to two or more. "And / or" describes the relationship between related objects, indicating that three possible relationships exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, or B exists alone. The character " / " generally indicates that the related objects are in an "or" relationship.

[0072] Unless otherwise specified, the chemical reagents used in the examples are conventional commercially available reagents, and the technical means used in the examples are conventional means familiar to those skilled in the art.

[0073] The present invention provides an mRNA vaccine delivery vector in some embodiments thereof, which is mainly prepared from a polyethylene glycol-polyphosphate block copolymer and a cationic lipid; the polyethylene glycol-polyphosphate block copolymer has a structure shown in formula (I),

[0074]

[0075] Wherein, R is selected from: C1~C 10 Alkyl, C1~C 10 Unsaturated chain hydrocarbon group;

[0076] Both m and n are integers, and 40≦m≦50, 25≦n≦40.

[0077] In some embodiments, R is selected from the group consisting of: C1-C6 alkyl, C1-C6 unsaturated chain hydrocarbon group.

[0078] In some embodiments, R is further preferably selected from the group consisting of: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, n-hexyl, and allyl.

[0079] In some embodiments, 43≦m≦47, 27≦n≦35; further, m is 45.

[0080] In some embodiments, the polyethylene glycol-polyphosphate block copolymer is selected from at least one of the following polymers:

[0081]

[0082] In the polyethylene glycol-polyphosphate block copolymer described herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, including both branched and linear groups, having a certain number of carbon atoms. For example, the term "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a linear or branched arrangement. Specifically, "C1-C6 alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl groups.

[0083] In the polyethylene glycol-polyphosphate block copolymer described in the present invention, the term "unsaturated chain hydrocarbon group" refers to a branched and straight-chain unsaturated aliphatic hydrocarbon group having a specific number of carbon atoms, that is, a non-cyclic unsaturated chain hydrocarbon group, and the carbon chain contains one or more carbon-carbon double bonds, or a carbon-carbon triple bond, such as: CH2=CHCH2-, -(CH2)8(CH=CH)CH3, -(CH2)7CH=CH2, -(CH2)8CH=CH2, etc.

[0084] It can be understood that the cationic lipid of the present invention can be (2,3-dioleoyl-propyl)-trimethylammonium chloride and / or BHEM-cholesterol, preferably BHEM-cholesterol.

[0085] In some embodiments, the molar ratio of the polyethylene glycol-polyphosphate block copolymer to the cationic lipid is 6-10:1, more preferably 7-9:1, and more preferably 7.8-8.2:1.

[0086] The polyethylene glycol-polyphosphate block copolymer provided by the present invention has excellent biocompatibility and high efficiency in delivering nucleic acids, and can be used as a gene transfection tool material. It can be used in combination with cationic lipids to prepare carriers for efficiently delivering mRNA vaccines. It has a stable structure and is not easily degraded, and its preparation method is simple and controllable.

[0087] The polyethylene glycol-polyphosphate block copolymer of the present invention can be prepared by conventional methods in the field of polymers.

[0088] In some embodiments of the present invention, an mRNA vaccine is also provided, comprising nanoparticles prepared from the polyethylene glycol-polyphosphate block copolymer having the structure represented by formula (I), a cationic lipid, and mRNA with vaccine activity. Each nanoparticle comprises an outer shell formed by doping the polyethylene glycol-polyphosphate block copolymer and the cationic lipid, and an aqueous core containing the mRNA. The mRNA vaccine provided by the present invention has high mRNA delivery and transfection efficiency and can effectively induce a specific immune response in the body for disease treatment and prevention. The mRNA vaccines in the embodiments of the present invention are also referred to as "mRNA vaccine nanoparticles" or "polyphosphate / mRNA delivery particles" in the embodiments. In some embodiments, the particle size of the nanoparticles is ≤200 nm. Furthermore, the particle size of the nanoparticles is ≤150 nm or ≤100 nm, and can also be 1 nm to 150 nm, 1 nm to 100 nm, or 2 nm to 70 nm. It is generally believed that a small particle size of nanoparticles facilitates their entry into lymph nodes and contact with antigen-presenting cells, thereby better activating the body's cellular immunity.

[0089] In the mRNA vaccine, the molar ratio of the polyethylene glycol-polyphosphate block copolymer and the cationic lipid is preferably 6-10:1, more preferably 7-9:1, and more preferably 7.8-8.2:1; the molar ratio of the polyethylene glycol-polyphosphate block copolymer to the mRNA is preferably 80-120:1, preferably 100-110:1, and more preferably 102-106:1.

[0090] In the present invention, "mRNA with vaccine activity" refers to an mRNA molecule that can be translated into an antigenic protein molecule within a target cell, presented to T cell or B cell receptor epitopes by antigen-presenting cells, and stimulate a response in a subject, particularly a B cell response and / or a T cell response. "Epitope" refers to the region of a protein antigen that interacts with B and / or T cell proteins, namely, the B cell receptor and the T cell receptor.

[0091] It is understood that the type of mRNA antigen is not limited, as long as it can be delivered by the mRNA vaccine delivery vector of the present invention through encapsulation or adsorption. The mRNA antigen used in the embodiments of the present invention can be derived from pathogens, cancer cells, self-antigens or allergens. In certain embodiments, the mRNA is capable of being translated into an antigen molecule related to autoimmunity. In other embodiments, the mRNA antigen is an antigen molecule related to allergies.

[0092] In certain embodiments, the pathogen antigens may be derived from infectious microorganisms such as viruses, bacteria, fungi, and parasites.

[0093] In certain embodiments, the pathogen antigen is from a virus, non-limiting examples of which include coronavirus, vaccinia virus, dengue virus, HSV2, human papillomavirus (HPV), Ebola virus, EBV, hepatitis A virus, HIV, Marburg virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, cytomegalovirus (CMV), HSV1, influenza A virus, West Nile virus, human rhinovirus (HRV), human respiratory syncytial virus (RSV), or Zika virus.

[0094] In certain embodiments, non-limiting examples of the coronavirus include SARS coronavirus (SARS-CoV) or Middle East respiratory syndrome coronavirus (MERS-CoV) or SARS-Cov-2, or 2019 novel coronavirus (2019-nCov).

[0095] In certain embodiments, the pathogen antigen is an antigen related to SARS-Cov-2, such as the S protein, S1 protein of SARS-Cov-2 or the RBD protein of 2019-nCov.

[0096] In certain embodiments, the pathogen antigen is from a parasite, non-limiting examples of which include Trichomonas, Leishmania, Malaria, Cryptosporidium, Trypanosoma, and Schistosoma.

[0097] It is understandable that the mRNA described in the present invention can be natural or artificially synthesized; the structure of the mRNA can be processed and modified by, but not limited to, capping, sugar modification, methylation modification, etc.

[0098] In some embodiments, the mRNA having a vaccine effect is RBD mRNA.

[0099] It is understood that the dosage form of the mRNA vaccine of the present invention is preferably an injection, nasal drops, spray or powder injection. In certain embodiments, the injection is an intramuscular injection or an intravenous injection. In certain embodiments, the mRNA vaccine of the present invention is administered by mucosal inoculation or intravenous injection.

[0100] It can be understood that the mRNA vaccine of the present invention can be prepared by conventional methods in the vaccine field.

[0101] In some embodiments of the present invention, a double emulsion preparation method for an mRNA vaccine is provided, comprising the following steps:

[0102] (1) dissolving the polyethylene glycol-polyphosphate block copolymer in an organic solvent to form a liquid A;

[0103] (2) dissolving the cationic lipid in an organic solvent to form liquid B;

[0104] (3) dissolving the mRNA in DEPC water as solution C;

[0105] (4) After the liquid A and the liquid B are mixed, the liquid C is added, and the mixture is preliminarily mixed by ultrasonic treatment to form a colostrum, and then DEPC water is added to the obtained colostrum, and the mixture is mixed by ultrasonic treatment again to form a double emulsion;

[0106] (5) removing the organic solvent from the emulsion to obtain the mRNA vaccine.

[0107] The organic solvent in steps (1) and (2) can be an organic solvent commonly used in the field of vaccine preparation, and is more preferably ethyl acetate, dichloromethane or chloroform.

[0108] In some embodiments, the concentration of the polyethylene glycol-polyphosphate block copolymer in the solution A in step (1) is preferably 15 μmol / mL-25 μmol / mL, and more preferably 18 μmol / mL-22 μmol / mL; the concentration of the cationic lipid in the solution B in step (2) is preferably 8 μmol / mL-12 μmol / mL; the concentration of the mRNA in the solution C in step (3) is more preferably 0.5 μmol / mL-2 μmol / mL, and more preferably 1.5-2 μmol / mL; the time of the ultrasonic treatment in step (4) is more preferably 1 minute-2 minutes, and the power of the ultrasonic treatment is more preferably 50 W-100 W; the volume ratio of the DEPC water to the colostrum in step (4) is more preferably 5-10:1.

[0109] In some embodiments, the method for removing the organic solvent from the double emulsion in step (5) is a rotary evaporation method, and the time is further preferably 5 minutes to 60 minutes, and the negative pressure is 0.5 kPa to 1 kPa.

[0110] In other embodiments of the present invention, a microfluidic controlled preparation method for an mRNA vaccine is provided, comprising the following steps:

[0111] (1) dissolving the polyethylene glycol-polyphosphate block copolymer in an organic solvent to form a liquid A;

[0112] (2) dissolving the cationic lipid in an organic solvent to form liquid B;

[0113] (3) dissolving the mRNA in DEPC water as solution C;

[0114] (4) After mixing the liquid A and the liquid B, the mixture is loaded onto the phase A of the microfluidic system; the liquid C is loaded onto the phase B of the microfluidic system, and the mixture is fully mixed in the microfluidic chip using a microfluidic system syringe pump to obtain a colostrum;

[0115] (5) loading the primary emulsion into phase A of the microfluidic system, loading DEPC water into phase B of the microfluidic system, and fully mixing them in the microfluidic chip using a microfluidic system syringe pump to obtain a double emulsion;

[0116] (6) removing the organic solvent from the emulsion to obtain the mRNA vaccine.

[0117] The organic solvent in steps (1) and (2) can be an organic solvent commonly used in the field of vaccine preparation, and is more preferably ethyl acetate, dichloromethane or chloroform.

[0118] In some embodiments, the concentration of the polyethylene glycol-polyphosphate block copolymer in the solution A in step (1) is preferably 15 μmol / mL-25 μmol / mL, and more preferably 18 μmol / mL-22 μmol / mL; the concentration of the cationic lipid in the solution B in step (2) is preferably 8 μmol / mL-12 μmol / mL; the concentration of the mRNA in the solution C in step (3) is more preferably 0.5 μmol / mL-2 μmol / mL, and more preferably 1.5 μmol / mL-2 μmol / mL; in step (4), the flow rate ratio of the microfluidic phase A to the phase B is more preferably 5-10:1; in step (5), the flow rate ratio of the microfluidic phase A to the phase B is more preferably 5-10:1.

[0119] In some embodiments, the method for removing the organic solvent from the double emulsion in step (6) is a rotary evaporation method, and the time is further preferably 5 minutes to 60 minutes, and the negative pressure is 0.5 kPa to 1 kPa.

[0120] The present invention is further described in detail below with reference to specific embodiments.

[0121] The experimental equipment used in the following examples include: a glove box; a heat-collecting magnetic stirrer; a low-temperature constant-temperature reaction bath; a double-row tube; a vacuum pump; an enzyme-labeled instrument; an ultraviolet spectrophotometer; a flow cytometer; and a confocal microscope.

[0122] The following experimental materials were used in the following examples: ethylene glycol, N,N-dimethylformamide (Sinopharm Group, Shanghai); phosphorus trichloride (Xiahua Reagent, Chengdu); ethanol, propanol, isopropanol, n-butanol, isobutanol, n-pentanol, and n-hexanol (all purchased from Aladdin Reagent Co., Shanghai); triethylamine, tetrahydrofuran, and chloroform (Sinopharm Group, Shanghai); 1,5,7-triazabicyclo[4.4.0]dec-5-ene and benzyl alcohol (Sigma-Aldrich, St. Louis); and polyethylene glycol monomethyl ether 5000 (Sigma-Aldrich, St. Louis, USA). RBD protein (2019-nCoV receptor binding domain protein) was purchased from Sino Biological. Mouse dendritic cell DC2.4 cell line was purchased from ATCC. Human embryonic kidney 293T cell line was purchased from ATCC.

[0123] Example 1 Synthesis of oxophosphoryl chloride COP:

[0124]

[0125] Before the reaction begins, the reactants are all distilled under reduced pressure, dried, and purified. All reaction devices need to be dried before the reaction, and the reaction must be carried out under anhydrous conditions. The specific reaction is as follows:

[0126] Phosphorus trichloride (275g) is mixed in anhydrous methylene chloride (200mL), constantly stirs under room temperature, slowly drips the methylene chloride (50mL) solution of ethylene glycol (120g) then, and uses anti-backdraft device and tail gas absorption device that reaction generates HCl to be neutralized in sodium hydroxide solution.After reacting 6 hours, utilize rotary vane vacuum pump to remove solvent under anhydrous condition, underpressure distillation purification again, finally obtain cyclic trivalent phosphorus oxychloride (155g, 67%), for the colourless clear liquid with pungent odor.Cyclic trivalent phosphorus oxychloride is dissolved in anhydrous benzene, concentration feeds high purity oxygen after keeping 1.0mol / L, room temperature reaction 2 days, after finishing, underpressure distillation purification obtains pentavalent oxa-phosphoryl chloride (102g, 61%), for the colourless clear liquid with pungent odor.

[0127] Example 2 Synthesis of Phosphate Monomers:

[0128]

[0129] Before the reaction begins, the reactants, solvent, and apparatus are dried. The reaction must be conducted under anhydrous conditions in a three-necked flask containing a constant-pressure dropping funnel. The molar ratio of the reaction materials is 1:1:1 for acyl chloride:alcohol:triethylamine. The small amount of water in the reaction apparatus is removed using heating and vacuum conditions. The specific reaction is as follows:

[0130] First, add anhydrous tetrahydrofuran to a three-necked flask, and then add alkyl alcohols of different chain lengths and structures to a concentration of 1.0 mol / L, then add triethylamine, and then add phosphorus oxychloride COP to the reaction apparatus through a dropping funnel. React at 0°C for 12 hours, and a white precipitate of triethylamine salt will be generated during the reaction. After the reaction is completed, filter and remove the generated triethylamine salt under anhydrous conditions. The collected filtrate is purified by vacuum distillation under anhydrous conditions after removing the solvent, and the boiling point is 109°C (61Pa). The obtained product is a colorless, transparent, clear liquid that can be stored for a long time at -30°C. The yields of each monomer are: EEP (69%), PEP (72%), AEP (65%), BEP (68%), i BEP (53%), PeEP (42%), HEP (37%).

[0131] Example 3 Synthesis of Polyethylene Glycol-Polyphosphate Block Copolymers with Different Side Groups:

[0132]

[0133] Note: The polymers 1-7 prepared in this embodiment are referred to as follows in the present invention: 1 is PEG 45 -b-PEEP 32 , 2 is PEG 45 -b-PPEP 35, 3 is PEG 45 -bP i BEP 31 , 4 is PEG 45 -b-PHEP 28 , 5 is PEG 45 -b-PAEP 33 , 6 is PEG 45 -b-PBEP 30 , 7 is PEG 45 -b-PPeEP 29 .

[0134] The polymerization reaction must be carried out under anhydrous conditions. Before the polymerization reaction, the monomers and solvents are purified by vacuum or atmospheric distillation. The initiator polyethylene glycol monomethyl ether needs to be azeotropically dehydrated with anhydrous toluene. The specific reaction is as follows:

[0135] For example, a polyethylene glycol-polyphosphate block copolymer with a degree of polymerization (DP) of 45 and n=32 and ethyl side groups was prepared. A certain amount of ethyl side-group phosphate monomer (EEP) was weighed into a flask. Tetrahydrofuran was added to a monomer concentration of 1.0 mol / L. The catalyst, TBD (1,5,7-triazabicyclo[4.4.0]dec-5-ene), was added. After thorough mixing, the initiator (polyethylene glycol monomethyl ether with a DP of 45) was added in a molar ratio of 40:1:2. The mixture was reacted at 25°C for 20 minutes. Afterward, benzoic acid, 5 times the molar amount of the catalyst, was added to terminate the reaction. The resulting solution was added dropwise to 0°C diethyl ether, resulting in the formation of a white precipitate. The precipitate was collected, drained, and then re-dissolved in a small amount of tetrahydrofuran. Precipitation with diethyl ether was repeated three times to remove the catalyst and unreacted monomer. The resulting product was dried overnight under vacuum and stored at -30°C after complete drying. According to the method described in this example, polyethylene glycol-polyphosphate block copolymers with different alkyl chains as side groups were prepared. The yields were as follows: PEG 45 -b-PEEP 32 (75%), PEG 45 -b-PPEP 35 (72%), PEG 45 -bP i BEP 31 (61%), PEG 45 -b-PHEP 28 (51%), PEG 45 -b-PAEP 33 (68%), PEG 45 -b-PBEP 30 (65%), PEG 45 -b-PPeEP 29(54%). The degree of polymerization of the phosphate block is calculated from the NMR integral of the polymer and the peak position of the hydrogen atom proton signal, for example, according to Figure 1 The degree of polymerization was calculated by calculating the integrated area ratio of the proton peak b belonging to the polyethylene glycol segment and the proton peak c belonging to the phosphate block.

[0136] Example 4 Preparation of Polyphosphate / mRNA Delivery Particles with Different Side Groups

[0137] (1) Weigh 20 μmol of polyethylene glycol-polyphosphate block copolymer, dissolve it in 1 mL of chloroform or dichloromethane, and mix well to obtain liquid A;

[0138] (2) Weigh 10 μmol of cationic lipid BHEM-cholesterol, dissolve it in 1 mL of chloroform or dichloromethane, and mix well to obtain solution B;

[0139] (3) Weigh 1.55 μmol of S protein mRNA purchased from Hefei Afana Co., Ltd., dissolve it in 1 mL of DEPC water, and mix well to obtain Solution C;

[0140] (4) Mix 400 μL of Solution A with 100 μL of Solution B, then add 50 μL of Solution C, mix thoroughly, and use an ultrasonic cell disruptor at 70 W power for 1 minute to obtain a colostrum;

[0141] (5) Add 5 mL of DEPC water and use an ultrasonic cell disruptor at 70 W power for 1 minute to obtain a complex emulsion;

[0142] (6) The emulsion was subjected to a rotary evaporator to remove the organic solvent component (10 min, 500 Pa), and further concentrated to 1-2 mL of solution to obtain polyphosphate / mRNA delivery particles with different side groups.

[0143] Example 5 Particle Size Characterization of Polyphosphate / mRNA Delivery Particles with Different Side Groups

[0144] The average particle size and distribution of the polyphosphate / mRNA delivery particles in Example 4 were measured using a Malvern Zetasizer dynamic light scattering particle size analyzer. 0.1 mL of the polyphosphate / mRNA delivery particle solution prepared in Example 4 was added to the Malvern microsample detection cell. The detection material was set to polymer, the detection temperature was set to 25°C, the equilibrium time was set to 120 s, and the detection medium was water. The detection results are shown in FIG. Figure 2 shown.

[0145] The morphology of the polyphosphate / mRNA delivery particles in Example 4 was observed using a transmission electron microscope. 10 μL of the polyphosphate / mRNA delivery particle solution prepared in Example 4 was placed on a transmission electron microscope sample copper grid, and then allowed to dry in a clean bench at room temperature. After the surface was gold-sprayed, the sample morphology was observed using a scanning electron microscope at 200 kV. The results are shown in FIG. Figure 2 As shown in Table 1, the particle size distribution is consistent with the results of dynamic light scattering particle size analyzer detection, and the particle size of the prepared particles is about 100-300 nm.

[0146] Table 1

[0147]

[0148]

[0149] Example 6 Polyphosphate / mRNA delivery particles with different side groups and nucleic acid binding detection

[0150] Weigh agarose powder, add 0.1% Gelred nucleic acid dye to prepare 1% agarose gel for agarose electrophoresis detection. Prepare polyphosphate / mRNA delivery particles with different side groups according to the method in Example 4, add 5×Loading buffer (loading buffer) to prepare the sample solution for running the gel, and load the sample in the gel strip holes in sequence according to the mRNA amount of 5 μg. After loading, perform electrophoresis at a constant voltage of 120V. At this time, unbound free nucleic acids will enter the pores. Turn off the power after 20 minutes of electrophoresis, and image the gel with ultraviolet light in an agarose electrophoresis gel imager. The experimental detection results are as follows: Figure 3 The results show that the polyphosphate / mRNA delivery particles can effectively block the electrophoresis of mRNA, indicating that the polyphosphate / mRNA delivery particles of the present invention have a strong ability to load mRNA and have good binding force with mRNA.

[0151] Example 7 Cytotoxicity Detection of Polyethylene Glycol-Polyphosphate Block Copolymers with Different Side Groups

[0152] DC2.4 cells were plated at 5×10 5Cells were seeded into 96-well culture plates and cultured in a cell culture incubator at 5% CO2 and 37°C until the cell density reached approximately 70%. Fresh culture medium containing 10% fetal bovine serum (100 μL / well) was then added. 1 to 5.0 μL of the polyethylene glycol-polyphosphate block copolymer material (obtained in Example 3) with different side groups was added to the cell culture medium. After 48 hours of culture, the cell viability was determined using the MTT method. The calculation formula is: [Cell viability (%) = (A1 / A0) x 100, where A1 is the absorption of the treated cell well and A0 is the absorption of the untreated cell well. Each experiment was repeated 6 times. The results are shown in Table 1. Figure 4 As shown, polymer 1, polymer 4, polymer 5, polymer 6, and polymer 7 (their numbers correspond to the numbers in the polymer structural formula in Example 3) have relatively good biocompatibility.

[0153] Example 8 Cell transfection experiment of polyphosphate mRNA delivery particles with different side groups

[0154] Polyphosphate / mRNA delivery particles with different side groups and loaded with 0.1 μg of mCherry mRNA (mCherry mRNA purchased from Hefei Afana Biotechnology Co., Ltd., China) were prepared according to the method in Example 4, wherein the molar ratio of polyethylene glycol-polyphosphate block copolymer to cationic lipid BHEM-cholesterol was 8:1; the molar ratio of the polyethylene glycol-polyphosphate block copolymer to the mRNA was 105:1. 5×10 5 Cell density: DC2.4 cells (or 293T cells) were seeded into 96-well culture plates and cultured in a cell culture incubator at 5% CO2 and 37°C until the cell density reached approximately 70%. Polyphosphate / mRNA delivery particles containing 0.1 μg of mCherry mRNA were added to each well. After incubation for 24 hours, the cells were treated with trypsin to obtain a single-cell suspension. The intracellular mCherry fluorescence signal was detected by flow cytometry. The experimental results are shown in Figure 2. Figure 5 As shown in FIG, a is the detection result of 293T cells by flow cytometry, and FIG b is the detection result of DC2.4 cells by flow cytometry. From the experimental results, it can be seen that the polymers of the present invention (especially polymers 2, 4, and 5) can effectively deliver mRNA to DC2.4 cells with antigen presenting ability. Figure 5 The structural formula of the polyethylene glycol-polyphosphate block copolymer corresponding to the material number in is the same as the structural formula of the polyethylene glycol-polyphosphate block copolymer corresponding to the corresponding number in Example 3.

[0155] Example 9 Neutralizing Antibody Detection in Mice After Immunization with 2019 Novel Coronavirus Receptor Binding Domain (RBD) mRNA Vaccine

[0156] Select material 4 (PEG 45 -b-PHEP 28 ), a novel coronavirus recombinant protein vaccine containing RBD mRNA was prepared according to the method in Example 4 (RBD mRNA was purchased from Hefei Afana Biotechnology Co., Ltd., China), and mice were immunized according to the following immunization protocol: 24 SPF female BALB / c mice (8 weeks old) were randomly divided into 3 groups, with 8 mice in each group. On days 0 and 7, the mice were injected intramuscularly with physiological saline solution, RBD mRNA vaccine (low dose of 0.25 mg / kg) and 40 mg / kg of nasopharyngeal carcinoma. -1 , the highest dose is 0.5mgkg -1 ) were injected twice. Five weeks later, the mice were sacrificed and peripheral blood was collected from the eye sockets. The levels of RBD neutralizing antibodies (IgG and IgM) in the serum were detected by ELISA. The experimental results are shown in the figure. Figure 6 shown.

[0157] Example 10 Detection of Neutralizing Antibodies in Mice After Immunization with the 2019 Novel Coronavirus Receptor Binding Domain (RBD) mRNA Vaccine

[0158] SPF female BALB / c mice (8 weeks old) were immunized with a high dose of RBD mRNA according to the protocol in Example 9. The mice were sacrificed, and the lungs, spleens, and inguinal lymph nodes were removed. After grinding to a single cell suspension, the immune cells were labeled with fluorescent flow cytometry antibodies. The cells were washed twice with phosphate-buffered saline, resuspended in 200 μL phosphate-buffered saline, and detected using a flow cytometer. The experimental results are shown in Figure 2. Figure 7 As shown, Figure a is the ELISPOT test result of T cell IFNγ expression; Figure b is the ELISPOT test result of T cell interleukin 2 expression; Figure c is the flow cytometry detection data of memory T cell proportion; Figure d is the flow cytometry detection data of CD69 positive activated T cell proportion.

[0159] From the experimental results of Examples 9 and 10, it can be found that the polyphosphate / mRNA delivery particles of the present invention can significantly induce cellular immune responses and humoral immune responses specific to the 2019 novel coronavirus receptor binding domain (RBD) in mice.

[0160] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the following embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0161] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. An mRNA vaccine delivery vector, characterized in that: It is mainly prepared from polyethylene glycol-polyphosphate block copolymer and cationic lipid; the polyethylene glycol-polyphosphate block copolymer has the structure shown in formula (I), Wherein, R is selected from: C1~C 10 Alkyl, C1~C 10 Unsaturated chain hydrocarbon group; m and n are both integers, and 40≦m≦50, 25≦n≦40; The cationic lipid is BHEM-cholesterol; The molar ratio of the polyethylene glycol-polyphosphate block copolymer to the cationic lipid is 6-10:

1.

2. The mRNA vaccine delivery vector according to claim 1, characterized in that R is selected from: C1-C6 alkyl, C1-C6 unsaturated chain hydrocarbon group containing one carbon-carbon double bond; and / or, 43≦m≦47, 27≦n≦35.

3. The mRNA vaccine delivery vector according to claim 2, characterized in that R is selected from the group consisting of: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, n-hexyl, and allyl.

4. The mRNA vaccine delivery vector according to claim 1, characterized in that The polyethylene glycol-polyphosphate block copolymer is selected from at least one of the following polymers:

5. The mRNA vaccine delivery vector according to any one of claims 1 to 4, characterized in that The molar ratio of the polyethylene glycol-polyphosphate block copolymer to the cationic lipid is 7-9:

1.

6. The mRNA vaccine delivery vector according to claim 5, characterized in that The molar ratio of the polyethylene glycol-polyphosphate block copolymer to the cationic lipid is 7.8-8.2:

1.

7. Use of the mRNA vaccine delivery vector according to any one of claims 1 to 5 in the preparation of mRNA vaccines.

8. An mRNA vaccine, characterized in that Nanoparticles comprising a polyethylene glycol-polyphosphate block copolymer having a structure represented by formula (I) as described in any one of claims 1 to 4, a cationic lipid, and mRNA having a vaccine effect; the molar ratio of the polyethylene glycol-polyphosphate block copolymer to the cationic lipid is 6-10:

1.

9. The mRNA vaccine according to claim 8, characterized in that The molar ratio of the polyethylene glycol-polyphosphate block copolymer to the cationic lipid is 7-9:1; the molar ratio of the polyethylene glycol-polyphosphate block copolymer to the mRNA is 80-120:

1.

10. The mRNA vaccine according to claim 9, characterized in that The molar ratio of the polyethylene glycol-polyphosphate block copolymer to the cationic lipid is 7.8-8.2:

1.

11. The mRNA vaccine according to claim 9, characterized in that The molar ratio of the polyethylene glycol-polyphosphate block copolymer to the mRNA is 100-110:

1.

12. The mRNA vaccine according to claim 11, characterized in that The molar ratio of the polyethylene glycol-polyphosphate block copolymer to the mRNA is 102-106:

1.

13. The mRNA vaccine according to any one of claims 8 to 12, characterized in that The mRNA with vaccine effect is mRNA related to viral infection or survival, mRNA related to metabolic diseases, mRNA related to tumor occurrence or cell transformation, immunomodulatory mRNA, and mRNA related to inflammation or autoimmunity.

14. The mRNA vaccine according to claim 13, characterized in that The mRNA with vaccine effect is RBDmRNA.

15. A method for preparing the mRNA vaccine according to any one of claims 8 to 14, characterized in that: The following steps are involved: (1) dissolving the polyethylene glycol-polyphosphate block copolymer in an organic solvent to form a liquid A; (2) dissolving the cationic lipid in an organic solvent to form liquid B; (3) dissolving the mRNA in DEPC water as solution C; (4) After the liquid A and the liquid B are mixed, the liquid C is added, and the mixture is preliminarily mixed by ultrasonic treatment to form a colostrum, and then DEPC water is added to the obtained colostrum, and the mixture is mixed by ultrasonic treatment again to form a double emulsion; (5) removing the organic solvent from the emulsion to obtain the mRNA vaccine.

16. The method for preparing the mRNA vaccine according to claim 15, characterized in that: The concentration of the polyethylene glycol-polyphosphate block copolymer in the solution A in step (1) is 15 μmol / mL-25 μmol / mL; and / or, The concentration of the cationic lipid in the solution B in step (2) is 8 μmol / mL-12 μmol / mL; and / or, The organic solvent in steps (1) and (2) is ethyl acetate, dichloromethane or chloroform; and / or, The concentration of mRNA in the solution C in step (3) is 0.5 μmol / mL-2 μmol / mL; and / or, The ultrasonic treatment in step (4) lasts for 1 minute to 2 minutes and has a power of 50W to 100W; and / or, The volume ratio of the DEPC water to the colostrum in step (4) is 5-10:1; and / or, The method for removing the organic solvent in the double emulsion in step (5) is a rotary evaporation method, the time is 5 minutes to 60 minutes, and the negative pressure is 0.5 kPa to 1 kPa.

17. Use of the mRNA vaccine according to any one of claims 8 to 14 in the preparation of a medicament for preventing and / or treating tumors or viral pneumonia.

18. The use according to claim 17, characterized in that The viral pneumonia is pneumonia caused by 2019-nCov infection.

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