CN114558127B - Tumor neogenetic antigen DNA nano vaccine capable of being taken by erythrocytes as well as preparation method and application thereof - Google Patents

Tumor neogenetic antigen DNA nano vaccine capable of being taken by erythrocytes as well as preparation method and application thereof Download PDF

Info

Publication number
CN114558127B
CN114558127B CN202210223117.0A CN202210223117A CN114558127B CN 114558127 B CN114558127 B CN 114558127B CN 202210223117 A CN202210223117 A CN 202210223117A CN 114558127 B CN114558127 B CN 114558127B
Authority
CN
China
Prior art keywords
dna
tumor
cells
vaccine
pdna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210223117.0A
Other languages
Chinese (zh)
Other versions
CN114558127A (en
Inventor
刘小龙
吴名
曾永毅
罗自金
蔡志雄
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mengchao Hepatobiliary Hospital Of Fujian Medical University (fuzhou Hospital For Infectious Diseases)
Original Assignee
Mengchao Hepatobiliary Hospital Of Fujian Medical University (fuzhou Hospital For Infectious Diseases)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mengchao Hepatobiliary Hospital Of Fujian Medical University (fuzhou Hospital For Infectious Diseases) filed Critical Mengchao Hepatobiliary Hospital Of Fujian Medical University (fuzhou Hospital For Infectious Diseases)
Priority to CN202210223117.0A priority Critical patent/CN114558127B/en
Publication of CN114558127A publication Critical patent/CN114558127A/en
Application granted granted Critical
Publication of CN114558127B publication Critical patent/CN114558127B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention belongs to the technical field of nano medicine, and particularly relates to a tumor neogenesis antigen DNA nano vaccine capable of taking erythrocytes, a preparation method and application thereof. The DNA nano vaccine is spherical particles with the size ranging from 20 nm to 200nm, a compound formed by DNA plasmids and cationic polymers is encapsulated in PLGA, and the PLGA is used as an excipient to form a stable nano system. The nanosystems can improve transfection efficiency and effectively deliver DNA vaccines to antigen presenting cells. On the basis, the DNA nano vaccine is creatively ridden on the surface of red blood cells, the DNA nano vaccine is ingeniously transported to the spleen of a secondary lymphoid organ through red blood cells, spleen immunity is stimulated, and systemic anti-tumor immune response is induced, so that a good tumor inhibition effect is achieved, and the novel antigen vaccine is high in efficiency and safety and has high clinical application value.

Description

Tumor neogenetic antigen DNA nano vaccine capable of being taken by erythrocytes as well as preparation method and application thereof
Technical Field
The invention belongs to the technical field of nano medicine, and relates to a preparation method of a tumor neogenetic antigen DNA nano vaccine capable of taking erythrocytes and application of the tumor neogenetic antigen DNA nano vaccine in tumor immunotherapy.
Background
Liver cancer (hepatocellular carcinoma) is one of the common cancers worldwide. Traditional liver cancer treatment comprises surgery, radiotherapy and chemotherapy and targeted treatment, but the traditional treatment means have the problems of generally poor clinical prognosis, low postoperative survival rate and the like. Whereas cancer immunotherapy has become one of the most promising approaches to cancer treatment by utilizing and enhancing the patient's own immune system to eliminate primary and metastatic tumor cells, currently immune checkpoint inhibitors and CAR-T cell therapies are approved for clinical use. With the development of techniques such as molecular biology, tumor immunology and tumor genomics, tumor neoantigens (neoantigens) are gradually known, and since genetic instability of tumor cells often results in a large number of mutations, expression of nonsensical mutations can produce tumor-specific antigens, and such neoantigens are only specifically present in tumor cells. The Rosenberg team in 2013 discovered neoantigens in tumor cell lines by exon technology and verified that they could trigger an immune response in the body. To date, personalized tumor vaccines based on neoantigens have been a new area of research derived and developed in recent years.
The currently developed neoantigen vaccines mainly comprise DNA vaccines, mRNA vaccines, polypeptide vaccines and cell vaccines. The DNA vaccine (DNA vaccinee) is prepared through injecting the recombinant eukaryotic expression vector encoding protein antigen into animal body to make the antigen produced by expressing exogenous gene in vivo activate organism immunity, and is more stable than mRNA vaccine. However, it is still a challenge to keep the DNA vaccine effective in vivo, and the DNA vaccine rapidly diffuses from the injection site after intramuscular injection or subcutaneous injection of the traditional plasmid DNA, and thus has problems of short circulation time and easy removal. Therefore, direct transfection of a DNA vaccine into host dendritic cells is a more advantageous strategy to improve the efficacy of a DNA vaccine compared to traditional intramuscular and subcutaneous DNA vaccines.
DNA vaccines must enter the nucleus and be transcribed and translated to induce a corresponding immune response, but many cellular barriers limit the nuclear delivery of exogenous DNA, so gene delivery vectors are critical for gene therapy. Currently, widely used gene delivery vectors can be divided into viral and nonviral gene delivery systems. The transfection and expression efficiency of the viral gene delivery system are high, but the potential toxic and side effects such as wild type infection, cancerogenicity and the like exist, and the capacity for loading the target gene is very limited by the limitation of the volume of the virus. The common non-viral vector comprises cationic liposome and cationic high polymer, wherein the liposome DNA complex is the most widely applied vector in the non-viral vector, has the advantages of simple preparation, high biosafety, capacity adjustability of loading genes and the like, so the non-viral vector has obvious advantages in vaccine delivery, and can be used for preparing the neoantigen DNA nano vaccine; in the prior art, the biodegradable PLGA Nano Particles (NPs) are prepared, so that genes can be wrapped inside the nano particles and combined with each other to form a nano particle-gene complex, the genes can be effectively protected from being destroyed in vivo, and the genes are introduced into cells for expression through good cell combination and uptake capacity of the nano particles, so that the therapeutic effect is achieved, however, the PLGA-entrapped genes have the problems of low entrapment efficiency and slow release speed. Because cationic polymers (polymerization) are positively charged and can interact with negatively charged DNA to form complexes (polyplex) for gene transfection, polyethylenimine (PEI) is the most widely studied cationic gene vector at present, and PEI with a large molecular weight has higher gene transfection efficiency but also has higher cytotoxicity, while PEI with a small molecular weight has little cytotoxicity but can hardly mediate gene transfection. Therefore, reduction of cytotoxicity by modifying PEI with large molecular weight is an effective strategy, and the transfection efficiency can be effectively improved in vitro by complexing the positively charged modified PEI into a complex in the prior art, but the problems that the released gene is uncontrollable and easy to be cleared in vivo exist, so that the gene cannot be introduced into cells and expressed are caused.
In order to solve the problems of rapid elimination in NPs and uncontrollable release of erythrocyte carriers, a liposome extrusion method is generally adopted in the prior art, erythrocyte membranes are firstly subjected to ultrasonic treatment, then a liposome extruder is used for sequentially passing through 400nm membranes and 200nm membranes, the prepared erythrocyte membranes are uniformly mixed with PLGA nanoparticles, and then the membranes of 200nm are passed, so that RBC-NP is obtained, and is in a shell-core structure, wherein the core is PLGA, and a monolayer erythrocyte membrane is wrapped outside, but the RBC-NP has no targeting property and has the problem of nonspecific distribution in the body.
And the DNA nano vaccine is directly targeted to immune organs such as spleen and the like, so that the gene delivery can be favorably improved, and the effect of the vaccine is exerted to activate the body immunity.
In summary, it is necessary to construct a DNA nanovaccine that is taken on erythrocytes, to achieve targeting delivery of the DNA nanovaccine, and induce immune response in the body while reducing side effects, which is of great importance in the field of tumor immunotherapy.
At present, the rapid development of tumor molecular biology innovates a new mode of tumor treatment, and tumor-targeted biological treatment gradually becomes a new trend of tumor treatment and makes some breakthrough progress. Tumor targeted biological therapy is a method for selectively delivering drugs or other active substances killing tumor cells to tumor sites by utilizing the specific binding action between antigen-antibody and ligand-receptor, or using the antibody as a therapeutic drug, so that the therapeutic action or drug effect is limited in specific target cells, tissues or organs as far as possible, and the functions of normal cells, tissues or organs are not influenced, thereby improving the curative effect and reducing toxic and side effects. However, this method requires screening a certain kind of molecules specifically expressed by tumor cells or high expression of receptors related to tumor proliferation and invasion, and the difficulty in finding these receptors is high, which affects the implementation of such schemes.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art, and provides a tumor neogenetic antigen DNA nano vaccine capable of taking erythrocytes, a preparation method and application thereof, wherein the DNA nano vaccine comprises a DNA vaccine and a cationic polymer, the cationic polymer can improve transfection efficiency, the DNA nano vaccine is delivered to antigen presenting cells, and the targeting delivery to spleen is realized through erythrocytes, so that spleen immunity is activated, and a body is induced to generate systemic anti-tumor immune response, so that the tumor nano vaccine is a novel tumor vaccine which is safe and efficient and has clinical transformation value.
To achieve the purpose, the invention adopts the following technical scheme:
the particle size of the DNA nano vaccine is 20-200 nm, the DNA nano vaccine is a nano system with a spherical structure, the inner core of the nano system is a compound formed by a cationic polymer PC and the DNA vaccine, the lactide-glycolide copolymer PLGA is wrapped on the surface of the inner core, the DNA vaccine is plasmid pDNA obtained by inserting a nucleotide sequence for expressing tumor cell antigens into an empty plasmid, and the DNA nano vaccine can be ridden on the surface of the red blood cells through electrostatic adsorption.
In a preferred embodiment of the present invention, the cationic polymer PC is formed from polyethylenimine (PEI 25000 ) And 1, 2-epoxytetradecane, wherein the lactide-glycolide copolymer PLGA is PLGA 50:50, more preferably, the cationic polymer is formed from polyethylenimine (PEI 25000 ) And 1, 2-epoxytetradecane in a mass ratio of 1: (0.5-10) reacting at 90 ℃ for 48h to obtain the cationic polymer PC modified by 1, 2-epoxytetradecane, wherein the reaction medium is absolute ethyl alcohol.
Preferably, the PEI 25000 And the mass ratio of the 1, 2-epoxytetradecane is 1: (0.5-5), most preferably 1:0.5.
the synthetic route of the above PC is as follows:
in a preferred embodiment of the present invention, the tumor cells include one or more of Hepa 1-6 (mouse liver cancer cells), B16-F10 (mouse skin melanoma cells), CT26 (mouse colon cancer cells), MC38 (mouse colon cancer cells), RH35 (rat liver cancer cells), 4T1 (mouse breast cancer cells), GL261 (mouse brain glioma) or U87 (human brain astrocytoma).
It is another object of the present invention to provide a novel tumor biotherapy regimen, delivering plasmid pDNA expressing tumor cell antigens to antigen presenting cells, expressing tumor cell antigens, activating MHC I (MHC class I) to present neoantigens to T cells, activating specific T cells, and deactivating specific killing tumors.
Preferably, the tumor cell is a HEpa 1-6 (mouse liver cancer cell), the nucleotide sequence of the expressed tumor cell antigen is shown as SEQ ID No.1, and the nucleotide sequence of the plasmid pDNA is shown as SEQ ID No. 2.
The invention also provides the plasmid pDNA, the plasmid pDNA expresses tumor cell antigen, and the amino acid sequence of the tumor cell antigen is shown as SEQ ID.NO. 3.
Further, the tumor cell antigen comprises 7 immunogenic antigen peptides, wherein the antigen peptides are connected through connecting peptides with the length of 10 amino acids, the 7 antigen peptides are Mapk3 positioned at 11 th to 27 th positions, lmf1 positioned at 38 th to 54 th positions, samd9l positioned at 65 th to 81 th positions, traf7 positioned at 92 th to 108 th positions, dtnb positioned at 119 th to 135 th positions, lbr positioned at 146 th to 162 th positions and Ptpn2 positioned at 173 th to 189 th positions in SEQ ID No.3 respectively, and the amino acid sequence of the connecting peptides is positioned at other positions in SEQ ID No. 3.
The invention also provides the application of the tumor cell antigen in preparing a medicine for treating tumors.
The invention also provides the application of the recombinant plasmid pDNA in preparing medicaments for treating tumors.
The invention also provides a preparation method of the tumor neoantigen DNA nano vaccine, which comprises the following steps: the hydrophilic pDNA is entrapped in PLGA by a double microemulsion method in the presence of hydrophobic excipient PLGA and a gene carrier cationic polymer PC, and the preparation method specifically comprises the following steps:
Dissolving PLGA and cationic polymer PC in an organic solvent, adding DEPC aqueous solution containing DNA vaccine into the organic solvent, forming primary water-oil emulsion after ultrasonic treatment in ice bath, adding DEPC aqueous solution into the aqueous solution, forming oil-water emulsion after ultrasonic treatment in ice bath, and removing the organic solvent to obtain the DNA nano vaccine.
Preferably, the organic solvent is dichloromethane.
Preferably, the mass ratio of the cationic polymer PC to the DNA vaccine is (5-150): 1, most preferably 20:1, a step of;
preferably, the mass ratio of PLGA to DNA vaccine is (1-200): 1, more preferably (1-100): 1, most preferably 100:1, a step of;
the tumor neoantigen DNA nano vaccine prepared by the invention has uniform and stable particle size distribution, good water solubility and biological safety, and can be used for anti-tumor immunity.
Compared with a pure DNA vaccine, the DNA nano vaccine can be effectively delivered to antigen presenting cells, has good transfection efficiency, and can stimulate dendritic cells to mature and regulate and control immune suppression microenvironment.
The invention also provides application of the tumor neoantigen DNA nano vaccine in preparing tumor immunotherapy medicaments.
In the application, after the tumor neoantigen DNA nano vaccine and the red blood cells are uniformly mixed by a mute mixer, the DNA nano vaccine with positive electricity is carried on the red blood cells, and the DNA nano vaccine is transported to the spleen through the targeting effect of the red blood cells.
By utilizing the innate immune function of red blood cells, DNA nanometer vaccine is taken on the red blood cells and then transported to spleen in a targeting way, and antigen is presented in the spleen by utilizing the inherent and unique capacity of the red blood cells, so that the organism immunity is stimulated, the systemic anti-tumor reaction is induced, and the tumor progress is inhibited.
In the present invention, the erythrocytes of the carriage activate the immune response at the spleen, promote the MHCI expression of dendritic cells, and enhance the systemic T cell immune response.
In the invention, the DNA nano vaccine can effectively inhibit the growth of the Hepa 1-6 tumor after inoculation, and prolong the survival period of mice.
The DNA nano vaccine can be effectively delivered to antigen presenting cells, has good transfection efficiency, and can stimulate dendritic cells to mature and regulate and control the immune suppression microenvironment.
The PLGA with the ester group end is uncharged, and the existing research shows that the PLGA with the charge can effectively encapsulate the pDNA and release the pDNA, but the problems of overlarge synthesized nano particles, poor nano particle dispersibility and the like exist, and if the uncharged PLGA is directly encapsulated with the pDNA, the encapsulation efficiency is very low and the release is uncontrollable. If only high molecular PEI is introduced, the existing research shows that the high molecular PEI has high transfection efficiency and high toxicity, so that the PEI of the high molecular PEI is further modified, so that the PEI has high transfection efficiency and good biological safety, but only the high molecular PEI is used for complexing pDNA tail vein injection into a mouse body, no targeting exists, the plasmid cannot be introduced into a target organ and the gene cannot be efficiently introduced into cells for expression, and the research designs a method which uses positively charged PC as an inner core to complex pDNA and PLGA as a shell, so that the gene encapsulation efficiency is improved, the toxicity is reduced, the gene is protected from being damaged in vivo, and the gene is introduced into the cells for expression through good cell binding and uptake capacity of nanoparticles.
Compared with the prior art, the invention has the main advantages and beneficial effects that:
(1) The DNA nano vaccine provided by the invention comprises a cationic polymer as a gene vector, has low toxicity and is biodegradable, so that the DNA nano vaccine has good safety and stability. Meanwhile, the transfection efficiency can be improved due to the addition of the cationic polymer, so that the DNA nano vaccine can be effectively delivered to antigen presenting cells, and the effect of the DNA vaccine is improved.
(2) The invention innovatively utilizes the innate immune function of red blood cells, takes DNA nano vaccine on the red blood cells, and then targets and transports the red blood cells to the spleen, and utilizes the inherent and unique capacity of the red blood cells to present antigen in the spleen, thereby stimulating the body immunity and inducing systemic anti-tumor response. In the prevention model, the enhanced immune response effectively slows down the progress of the tumor, which is expected to provide a new dosage form of nano vaccine for tumor immunotherapy.
(3) The preparation method is simple, and the required equipment is conventional equipment.
(4) Spleen and lymph nodes are peripheral lymphoid organs. The primary function of the spleen is to filter pathogens from the blood circulation to produce an immune response to blood-borne antigens, to clear abnormal erythrocytes or to phagocytose senescent erythrocytes in a non-inflammatory pathway. The erythrocytes capture and inactivate microorganisms in the blood and then present the captured pathogens to Antigen Presenting Cells (APCs) in the spleen. This unique function of erythrocytes has been used to carry therapeutic payloads, such as nanoparticle-based targeted transport therapies, which involve the delivery of antigens to spleen APCs. Thus, erythrocytes are ideal candidates for delivery of DNA nanovaccines.
(5) The antigen expressed by the nucleotide sequence of the tumor cell antigen expressed by SEQ ID.NO.1 used in the invention comprises 7 immunogenic antigen peptides, wherein the antigen peptides are connected through connecting peptides with the length of 10 amino acids, and the seven amino acids are mutant amino acids in the neogenesis antigen. Short peptides themselves do not function as the original protein and are presented to the cell surface by the action of the transporter only through binding to the MHC of the histocompatibility complex. Normally, polypeptides formed by degradation of proteins in tumor cells are not immunogenic and cannot be recognized by the immune system as a target. However, partial proteins of tumor cells undergo nonsensical mutation under survival and selection pressure, wherein the mutation site with immunogenicity is presented on the surface of tumor cells as a short peptide (the antigen peptide identified herein belongs to the category), and can be used as a target point for recognition by the immune system. Thus, we can activate the immune system by designing, expressing and delivering these mutant peptides, through the interaction of DC cells with T cells, so that the activated T cells have the function of targeted recognition and killing of tumor cells.
Drawings
FIG. 1 is a cationic polymer PC prepared in example 2 1 H NMR spectrum (300 MHz, CDCl) 3 )。
FIG. 2 is a schematic diagram of the synthetic route for preparing the DNA nanovaccine according to the present invention.
FIG. 3 is a set of characterization graphs of the DNA nanovaccine prepared by the double microemulsion method in example 3. Wherein (fig. 3A) is a Scanning Electron Microscope (SEM) of the DNA nanovaccine, (fig. 3B) is a transmission electron microscope of the DNA nanovaccine, (fig. 3C) is a hydration diameter distribution map of the DNA nanovaccine in DEPC aqueous solution, (fig. 3D) is a gel electrophoresis strip map of the YOYO-3 labeled DNA nanovaccine, (fig. 3E) is a standard curve for detecting yoyoyo-3-pDNA by a fluorescent quantification method, (fig. 3F) is PLGA/pDNA and encapsulation efficiency of pDNA in PPC/pDNA, (fig. 3G) is an in vitro release curve of PPC/pDNA under different environments.
FIG. 4 is a graph showing the antigen presentation of the DNA nanovaccine of examples 4 and 5 after the DNA nanovaccine acts on dendritic cells, wherein (FIG. 4A) is a confocal fluorescence microscope (CLSM) showing transfection after the dendritic cells are incubated with the DNA vaccine and the DNA nanovaccine for 48 hours, (FIG. 4B) is a statistical graph of transfection efficiency of the DNA nanovaccine, and (FIG. 4C) is a graph of flow cytometry to examine the effect of the DNA nanovaccine on dendritic cell maturation.
FIG. 5 shows the experimental results of DNA nanovaccine riding red blood cells in example 7. Wherein (fig. 5A) is the positive rate of erythrocytes assembled in different ratios with DIO-labeled DNA nanovaccine by flow cytometry; (FIG. 5B) is a CLSM diagram of erythrocytes and DNA nanovaccines assembled with erythrocytes, and (FIG. 5C) is a case of observing erythrocytes and the erythrocytes carrying DNA nanovaccine by SEM.
FIG. 6 shows the experimental results of enrichment of DNA nanovaccine of example 8 into various organs after erythrocyte riding. Wherein (FIG. 6A) is an image of fluorescence of organs collected 24h after injection of erythrocytes incubated at different nanoparticle to erythrocyte ratios, and (FIG. 6B) is a statistic of fluorescence intensity for each organ.
FIG. 7 is a graph showing the results of evaluating immune effects of mice on the stimulation of mice after the mice were vaccinated with erythrocytes loaded with DNA nanovaccines in example 9. Wherein (FIG. 7A) is a flow cytometry pattern showing the expression of MHCI of DC in spleen, (FIG. 7B) is a quantitative analysis result of flow cytometry, (FIG. 7C) is a flow cytometry pattern showing CD3 in spleen + CD8 in T cells + Expression of T cells. (FIG. 7D) is the quantitative analysis result, (FIG. 7E) is the flow cytometry map, showing CD8 in spleen + Expression of CD44 in T cells (fig. 7F) is a quantitative analysis result.
FIG. 8 shows the biosafety test results of the DNA nanovaccine in example 10. Wherein (FIG. 8A) is the H & E staining results of each group of liver sections and (FIG. 8B) is the biochemical index of each group of serum.
FIG. 9 shows the results of the experiment for preventing tumor in mice in example 11. Wherein (FIG. 9A) is a graph of tumor growth for each group of mice, (FIG. 9B) is a graph of body weight measurement for each group of mice, and (FIG. 9C) is a graph of survival cycle of mice.
FIG. 10 is a nucleotide sequence of a Hepa 1-6 neoantigen and an amino acid sequence diagram of a Hepa 1-6 neoantigen.
Detailed Description
The invention is further illustrated by the following examples, which are only for further explanation of the technical solutions of the invention and are not to be construed as limiting the scope of the invention, and non-essential modifications or adaptations of the invention according to the foregoing disclosure will be within the scope of the invention.
The experimental materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Materials and equipment:
(1) Polyethyleneimine (PEI) 25000 ) Weight average molecular weight 25000, available from Sigma, usa;
(2) 1, 2-epoxytetradecane is available from TCI company;
(3) Polylactide-glycolide copolymer PLGA (PLGA, 50:50) was purchased from Sigma, PLGA 50:50 means that the polymer consists of 50% lactide and 50% glycolide, and has a molecular weight of 38000-54000;
(4) YOYO-3 (molecular formula C 53 H 62 I 4 N 6 O 2 Molecular weight 1322.4, CAS number 156312-20-8) from Thermo Fisher Scientific, yoyo-3 is a highly sensitive nucleic acid developer, commonly used for nuclear visualization, yoyo-3 is a duplex of YO-PRO-3, belonging to the class of biscyanine (Cyanine duplex) fluorescent probes, which do not fluoresce themselves, but which are 100-1000 fold enhanced in fluorescence after intercalation into DNA;
(4) DiI and DIO are available from Suzhou Yuheng biotechnology Co., ltd;
(5) CD80, CD86, CD11c, MHCI, CD3, CD4, CD8 and CD44 streaming antibodies were purchased from eBioscience, usa;
(6) The nano-particle size potentiometer was purchased from Malvern Instruments company;
(7) Empty plasmid PresentER-Cassette mCherry from addgene company;
(8) The nucleotide sequence of the expressed tumor cell antigen is shown as SEQ ID No.1 (the expressed HEpa 1-6 neoantigen consists of Mapk3, lmf1, samd9l, traf7, dtnb, lbr and Ptpn2 genes).
Example 1: construction of DNA vaccine
Screening out a plurality of HEpa 1-6 (mouse liver cancer cells) neoantigen sequences through second generation sequencing and combining with a bioinformatics calculation method, respectively synthesizing the sequences into corresponding polypeptides, detecting the immunogenicity of the polypeptides in vivo through an Elispot detection method, further screening out one with the strongest immunogenicity, and inserting the polypeptide sequence into a plasmid empty load to form the final pDNA.
The principle of the Elispot detection method is as follows: the cells are stimulated by antigen to locally produce cytokines, which are captured by specific monoclonal antibodies. After cell lysis, the captured cytokine binds to a biotin-labeled secondary antibody, and then to an alkaline phosphatase-labeled avidin. After incubation of BCIP/NBT substrate, the presence of "purple" spots on the PVDF well plate indicates cytokine production by the cells, and the results were obtained after analysis of the spots by the ELISPOT ELISA system.
Construction of DNA vaccine expressing Hepa 1-6 neoantigens:
(1) Mu.l of the digestion system (containing 13. Mu.l of PresentER-Cassette mCherry plasmid, 1. Mu.l of Sfil enzyme, 5. Mu.l of Q.cut Buffer (10X) and 31. Mu.l of double distilled water) were prepared and mixed by shaking, and digested for 1 hour in a dry thermostat at 37 ℃. After completion of the cleavage, calf intestinal alkaline phosphatase (CIP) was added to the mixture, followed by final electrophoresis and recovery of the PresentER-Cassette mCherry plasmid.
(2) The target DNA fragment (nucleotide sequence shown as SEQ ID No.1 and the complementary strand thereof) was artificially synthesized, and PCR amplification was performed using the nucleotide sequence shown as SEQ ID. No.1 as a template and using the upstream/downstream primers.
The upstream primer is 5'-AAGGCCAAGTTGGCCACCATGAAGGCCCGAAAC-3', as shown in SEQ ID. NO. 4;
the downstream primer is 5'-TTGGCCAAGTTGGCCTCATCATGACACAAACCCCAT-3', as shown in SEQ ID. NO. 5;
the PCR amplification reaction system is as follows: 10ng of the synthesized sequence, 2. Mu.L of the upstream primer, 2. Mu.L of the downstream primer and 25. Mu. L Takara PrimerSTAR Max DNA Polymerase (PCR reagent), and finally the system was made up to 50. Mu.L using double distilled water.
(3) The ligation of the DNA fragment of interest (nucleotide sequence shown in SEQ ID No.1 and its complementary strand) and the empty plasmid is then carried out: firstly, 96 mu L of Ethidium Bromide (EB) is added into a target DNA fragment (2 mu L of each nucleotide sequence shown in SEQ ID No.1 and the complementary strand thereof), the mixture is uniformly mixed and then put into hot water, then gradient cooling is carried out until the temperature is reduced to room temperature, 50 mu L of system enzyme digestion target DNA fragment (5 mu L of target DNA fragment, 1 mu L of Sfil enzyme, 5 mu L of Q.cut Buffer (10X) and 39 mu L of double distilled water) are prepared, inactivation is carried out for 10 minutes at 70 ℃, and then 10 mu L of system (3 mu L of PresentER-Cassette mCherry plasmid, 2 mu L of target DNA fragment, 0.5 mu L T ligase, 1 mu L T4 ligase Buffer (10X) and 8.5 mu L of double distilled water) are prepared for connection, and the connection is carried out at room temperature for 1 hour. (4) converting the ligation product: placing competent escherichia coli DH5 alpha on ice, after melting, adding 30 mu L of competent escherichia coli into the connection product of the step (2), gently mixing, incubating on ice for 30 minutes, and then thermally shocking the incubated mixture in a water bath at 42 ℃ for 60 seconds, and incubating on ice for 5 minutes. Next, the above mixture was added to 600. Mu.L of the non-resistant LB medium, and incubated at 37℃for 2 hours on a constant temperature shaking table at 220 rpm/min. Centrifuging 1000g of the cultured bacterial liquid at room temperature for 2min, removing 400 mu L of supernatant, re-selecting the residual liquid, uniformly dripping the residual liquid on an LB solid flat plate which is preheated at 37 ℃ in advance, lightly and uniformly smearing the residual liquid on a heated coating rod, placing the residual liquid on the surface of the flat plate without obvious liquid, and culturing the residual liquid in a bacterial incubator at 37 ℃ for 14 hours in an inverted mode.
(5) Monoclonal colonies were picked and subjected to a small shake validation: adding 8mL of LB liquid medium (ampicillin) into a 15mL centrifuge tube, picking a monoclonal colony from the LB plate cultured in the step (3) in an ultra-clean bench, placing the monoclonal colony into the LB liquid medium, culturing for 13h on a constant temperature shaking table at 220rpm/min at 37 ℃ until bacterial liquid becomes turbid, and extracting the target plasmid.
(6) mu.L of the cleavage system (5. Mu.L of the target plasmid (500 ng), 0.5. Mu.L of Q.cut EcoRI, 0.5. Mu.L of Q.cut HindIII, 2. Mu.L of Q.cut Buffer (10X) and 12. Mu.L of double distilled water) were prepared, and after shaking and mixing, the mixture was cleaved for half an hour in a dry thermostat at 37 ℃. After the enzyme digestion is finished, the plasmid structure is verified by running gel and sequencing.
The nucleotide sequence of the HEpa 1-6 neoantigen shown in SEQ ID No.1 is inserted into a presentER-Cassette mCherry empty plasmid to obtain a plasmid pDNA for encapsulating DNA, and the plasmid structure is determined by gene sequencing, wherein the nucleotide sequence is shown in SEQ ID No. 2.
The nucleotide sequence of the expression Hepa 1-6 neoantigen shown in FIG. 10 and SEQ ID No.1 consists of: 7 DNA fragments for expressing the immunogenic antigen peptide and DNA fragments for expressing the connecting peptide, wherein the 7 DNA fragments for expressing the antigen peptide comprise nucleotide sequences for expressing Mapk3 at 31 to 81 positions, nucleotide sequences for expressing Lmf1 at 112 to 162 positions, nucleotide sequences for expressing Samd9l at 193 to 243 positions, nucleotide sequences for expressing Traf7 at 275 to 324 positions, nucleotide sequences for expressing Dtnb at 355 to 405 positions, nucleotide sequences for expressing Lbr at 436 to 486 positions and nucleotide sequences for expressing Pt2 at 517 to 567 positions in SEQ ID.NO.1, and the DNA fragments for expressing the connecting peptide are positioned at other positions in SEQ ID.NO. 1.
The expressed HEpa 1-6 neoantigen has an amino acid sequence shown as SEQ ID No.3 and comprises 7 immunogenic antigen peptides, wherein the antigen peptides are connected through connecting peptides with the length of 10 amino acids, as shown in figure 10, the 7 antigen peptides comprise Mapk3 positioned at 11 th to 27 th positions, lmf1 positioned at 38 th to 54 th positions, samd9l positioned at 65 th to 81 th positions, traf7 positioned at 92 th to 108 th positions, dtnb positioned at 119 th to 135 th positions, lbr positioned at 146 th to 162 th positions and Ptpn2 positioned at 173 th to 189 th positions in the SEQ ID No.3, and the amino acid sequence of the connecting peptides is positioned at other positions in the SEQ ID No. 3.
Mapk3: mitogen activated protein kinase 3, mapk3, is a protein encoding gene. Plays a role in the signaling cascade, regulating various cellular processes, such as proliferation, differentiation, and cell cycle progression, in response to various extracellular signals. Mapk-mediated cell adhesion movement, degradation of extracellular matrix, generation of new blood vessels, and apoptosis are closely related to occurrence, development and metastasis of tumors.
LMF1: the lipase maturation factor 1 recombinant protein is involved in the maturation of a specific protein in the endoplasmic reticulum. Maturation and transport of active lipoprotein lipase (LPL) by the secretory pathway is required.
Samd9l: linker proteins, are involved in endosomal fusion. Down-regulation of growth factor signaling is mediated by internalization of growth factor receptors.
Traf7: tumor necrosis factor receptor-related factors, the associated pathways of which include ubiquitin-proteasome dependent proteolysis and class I MHC mediated antigen processing and presentation. Several studies have demonstrated that Traf7 is involved in a variety of vital activities such as immunization and tumorigenesis.
Dtnb: beta-estrogen is a protein coding gene. Diseases associated with DTNB include muscular dystrophy and Hermansky-Pudlak syndrome. Related pathways include muscular dystrophy and dystrophy protein-glycoprotein complexes.
Lbr: laminin B receptors are involved in cholesterol biosynthesis and neutrophil differentiation. The gene entity (GO) associated with this gene includes RNA binding and oxidoreductase activity, acting as a donor to the CH-CH group and NAD or NADP as an acceptor. Malignant tumor cells bind more Lbr than non-malignant cell surfaces, and laminin receptors can promote adhesion of cancer cells to extracellular matrix, especially basal lamina, induce expression and secretion of proteolytic enzymes, especially collagenase, promote migration and proliferation of cancer cells, promote proliferation of vascular endothelial cells, angiogenesis, and promote accumulation of cancer cells and platelets to form cancer plugs in cancer metastasis.
Ptpn2: tyrosine phosphatase, a protein-encoding gene. Related pathways include interferon gamma signaling and activation of cAMP dependent PKA. Studies have shown that tumor cells may balance deregulated protein kinase activity by upregulating tyrosine phosphatases, which may affect tumor genesis and metastasis.
The tumor antigen peptide is a short peptide formed by degrading tumor intracellular proteins through a proteasome, and is usually 9 to 25 amino acids in length, and the short peptide has no function of the original proteins, and is only presented on the cell surface under the action of a transport protein by combining with a tissue compatible complex MHC. Normally, polypeptides formed by degradation of proteins in tumor cells are not immunogenic and cannot be recognized by the immune system as a target. However, partial proteins of tumor cells undergo nonsensical mutation under survival and selection pressure, wherein the mutation site with immunogenicity is presented on the surface of tumor cells as a short peptide (the antigen peptide identified herein belongs to the category), and can be used as a target point for recognition by the immune system. Thus, we can activate the immune system by designing, expressing and delivering these mutant peptides, through the interaction of DC cells with T cells, so that the activated T cells have the function of targeted recognition and killing of tumor cells.
The neoantigen has no broad spectrum because the neoantigen has tumor cell specificity, i.e. different tumor cells can generate different protein mutations under different environments. However, we have identified tumor neoantigens that obtained Hepa 1-6 (mouse hepatoma cells) and activated the immune system via the neoantigens, thereby performing precise immune killing on tumors.
MHC class I (MHC I): on the surface of a normal cell, conditions in the normal cell can be provided, for example, when the cell is infected by virus, the amino acid chain (peptide) of the outer membrane fragment of the virus is presented outside the cell through MHC, so that the cell can be identified by a killer CD8+ T cell and the like for killing.
Example 2: synthesis of cationic Polymer PC
0.5g of 1, 2-epoxytetradecane is dissolved in 20mL of absolute ethanol and then added into a 50mL round bottom flask, after the temperature is raised to 90 ℃, 5mL of a solution containing 1g of PEI is slowly added dropwise into the round bottom flask 25000 Then reacting for 48 hours under the reflux condition of 90 ℃, and cooling to room temperature after the reaction is finished to obtain the required target product cationic polymer PC, wherein the structural characterization result is shown in figure 1, and the result shows that: the cationic polymer PC consisting of 47 PEI and 11 1, 2-epoxytetradecane was successfully synthesized.
The above-described synthetic route for PC is shown below:
the PEI molecule has one protonizable nitrogen atom corresponding to every two carbon atoms, and the nitrogen atoms form primary ammonia, secondary ammonia and tertiary ammonia, so that PEI has the capability of absorbing protons under almost any pH condition, PEI can absorb H in the acidic environment of an endosome, the osmotic pressure of the PEI is increased, the membrane is unstable and even breaks, DNA in a phagocytized compound can escape, DNA degradation is avoided, the epoxy bond of the epoxy cross-linking agent is utilized to react with the primary amino group, the epoxy bond is opened to generate hydroxyl groups to realize cross-linking, the hydroxyl groups in a cross-linking product can increase the water solubility of the polymer, but the epoxy and amine react more than the double bond, if the control is poor, the PEI and the epoxy cross-linking agent are generally prepared into a very dilute solution, and the cross-linking agent solution is slowly dripped into the PEI solution. Cationic polymers are important as gene vectors, cytotoxicity and transfection efficiency are important, and general strategies for designing low-toxicity cationic polymers enable cationic polymers with shorter connecting chain lengths, cationic polymers with longer molecular chains have greater toxicity, and cationic polymers with longer chain lengths mediate higher transfection efficiency.
Example 3: preparation and characterization of DNA nanovaccines
Preparing the DNA nanometer vaccine by adopting a double microemulsion method:
(1) 400. Mu.L of DEPC aqueous solution containing 100. Mu.g of DNA (i.e., pDNA constructed in example 1) was added to 1mL of methylene chloride containing 2mg of PC prepared in example 2 and 10mg of PLGA to give a mixture. The above mixture was treated in an ice bath at 60W ultrasonic power for 2 minutes to form a primary aqueous oil emulsion.
(2) Then adding 3mLDEPC water into the primary water-oil emulsion, performing ultrasonic treatment for 2 minutes under the power of 60W to further emulsify to form an oil-water emulsion, and then removing dichloromethane by a rotary evaporator to finally obtain the DNA nano vaccine (PPC/pDNA), wherein the synthetic route diagram of the DNA nano vaccine is shown in figure 2.
(3) Preparation of particles not encapsulating pDNA (hereinafter abbreviated to PPC): repeating the steps (1) and (2) except that: the DEPC aqueous solution of step (1) was not added with pDNA.
(4) Preparation of PLGA-coated pDNA particles (hereinafter referred to as PLGA/pDNA) the above steps (1) and (2) were repeated except that: the dichloromethane of the step (1) contains no PC.
The DNA nanovaccine obtained by scanning electron microscope, transmission electron microscope and Dynamic Light Scattering (DLS) characterization, see fig. 3A to 3C, in which the PPC group in fig. 3C is an unencapsulated DNA plasmid group, and the PPC/pDNA group is a DNA nanovaccine group, as follows: the embodiment obtains the nano particles with spherical structure, uniform and stable morphology and particle size in the range of 50-100 nm. According to the method, hydrophobic PLGA and PC are dissolved in dichloromethane, hydrophilic pDNA is entrapped in the PLGA by a double-microemulsion method, a spherical structure of the DNA nano vaccine can be observed from a transmission electron microscope result, and particle SEM results before and after entrapment of the pDNA are similar, which indicates that the pDNA is not adhered to the surface of nanoparticles, but is inside the nanoparticles, and further the dispersion and morphology of the nanoparticles are not affected.
The binding capacity of the cationic polymer PC to DNA was determined by gel electrophoresis migration experiments:
200. Mu.g of the pDNA constructed in example 1 (at a concentration of 1. Mu.g/. Mu.L) and 50. Mu.L of YOYO-3 (at a concentration of 10. Mu.M) were taken, and the pDNA and YOYO-3 were mixed at a ratio of 4:1, and oscillating for 2 hours at 4 ℃ to obtain a YOYO-3-pDNA mixed solution (the concentration is 0.5 mug/mu L), and synthesizing PPC/YOYO-3-pDNA according to the steps (1) and (2) (except that the step (1) is that the YOYO-3-pDNA mixed solution is used for replacing pDNA and other reaction conditions are the same), and judging the entrapment condition by using the gel electrophoresis strips of the pDNA and the products. The results are shown in fig. 3D, which shows successful entrapment of DNA vaccine.
The prepared YOYO-3-pDNA (concentration: 0.5. Mu.g/. Mu.L) was used as a standard solution, and diluted into YOYO-3-pDNA standard solutions (concentrations: 20, 10, 5, 2.5, 1.25, 0.625 and 0.3125. Mu.g/mL, respectively) of different concentrations. The fluorescence intensity (excitation wavelength: 550nm, emission wavelength: 551-700 nm) of each concentration of the YOYO-3-pDNA standard solution was measured using a fluorescence spectrophotometer, a standard curve of YOYO-3-pDNA was drawn, and the drawn standard curve of YOYO-3-pDNA was shown in fig. 3E. In a certain concentration range, the linear regression equation is y=32.45x+43.12, and the correlation coefficient (R 2 ) =0.9973, the linear relationship correlation is relatively high.
According to the standard curve of YOYO-3-pDNA, the encapsulation rate of PLGA/pDNA and PPC/YOYO-3-pDNA is measured, 100 mu L of PLGA/pDNA and PPC/YOYO-3-pDNA synthesized by the synthesis method is taken, the PLGA/pDNA and the PPC/YOYO-3-pDNA are centrifuged for 10min at 10000rpm and 4 ℃, the supernatant is taken, the fluorescence intensity of YOYO-3-pDNA in the supernatant is analyzed, finally the concentration of YOYO-3-pDNA in the supernatant is calculated by a regression equation, and the encapsulation rate of the DNA nano vaccine is calculated according to the following equation:
total DNA content (μg) =pdna concentration x volume (a)
Unwrapped DNA content (μg) =pdna concentration in supernatant x volume (b)
Encapsulation efficiency (%) = (total pDNA content-unwrapped pDNA content)/total DNA content x 100% (c)
The encapsulation efficiency of PLGA/pDNA is calculated to be 7.13+/-1.22% by the formulas (a), (b) and (c), and the encapsulation efficiency of PPC/YOYO-3-pDNA is calculated to be 73.08 +/-0.86%. The results are shown in FIG. 3F, which shows that PPC/pDNA can payload pDNA.
The DNA nano vaccine PPC/pDNA prepared by the embodiment has better encapsulation efficiency, and the in-vitro release of the DNA nano vaccine PPC/pDNA under different environments is detected by a Nanodrop spectrophotometer. mu.L of PPC/pDNA synthesized by the above synthesis method was dispersed in 500. Mu.L of PBS buffer at pH5.0 and pH7.4, respectively, and incubated on a shaker at 37 ℃. After centrifugation at 10000rpm for 10 minutes at a predetermined time point, 0.5mL of the supernatant was extracted, and 0.5mL of PBS buffer corresponding to pH was added to keep the volume constant. The supernatants collected at different time points were checked for plasmid release by nanomap spectrophotometry. The results refer to FIG. 3G, and the results show that the DNA nanovaccine in the acid environment can effectively release plasmid at 216h, and the release amount reaches 74.95+/-0.06% of the total amount, which is probably because PLGA at the ester group end is uncharged, and the PLGA is taken as a carrier to protect the genes from being destroyed for a long time.
Labelling DNA nanovaccines with DIO or DiI: after 100 mug of DIO or DiI is additionally added into 1mL of dichloromethane in the step (1), the steps (1) and (2) are carried out, the obtained product is placed in a regenerated fiber dialysis bag (MWCO 3500) after nano particles are synthesized, and then a large amount of ultrapure water is used for separating out the unencapsulated DIO or DiI in the solution, so that the target product-DIO marked DNA nano vaccine or DiI marked DNA nano vaccine is obtained for standby.
DEPC water is ultrapure water (primary water) treated with DEPC (diethyl pyrocarbonate ) and autoclaved at high temperature, colorless liquid, and is free of RNA, DNA and protein impurities.
Example 4: uptake and intracellular localization of DNA nanovaccines by dendritic cells
After incubation for 48h with 0.2 μg of pDNA obtained in example 1 and PPC/pDNA synthesized in example 3 (containing 0.2 μg of pDNA, the encapsulated DNA mass can be controlled according to the encapsulation efficiency and total DNA content of the plasmid measured in example 3) respectively added to mouse bone marrow derived dendritic cells (BMDCs), the original drug-loaded medium was aspirated and the cells were washed 3 times with phosphate buffer, treated with 4% paraformaldehyde fixative for 20min at room temperature, after washing all cells in the same manner, the BMDCs were stained with DAPI (nuclear stain), and finally the transfection status of each group of DNA vaccines was observed with confocal microscopy, each group of fluorescence imaging pictures see fig. 4A. The results showed that the PPC-containing vector harbored pDNA groups can observe a distinct mCherry fluorescent signal, then the pDNA groups did not observe a fluorescent signal, confirming that PPC this vector can effectively promote more efficient uptake and delivery of pDNA by BMDCs. Fig. 4A is a graph showing the transfection of pDNA with nanocarriers on BMDCs cells for 48h after encapsulation of pDNA, which shows that: the transfection efficiency of pDNA alone in BMDCs cells was almost 0, and the transfection efficiency of pDNA in BMDCs cells was more than 90% as the nanocarriers were effective to introduce and express genes into the body after being entrapped by the nanocarriers, as can be seen in FIG. 4B. Because PLGA at the ester base end is uncharged, PLGA is taken as a carrier to protect genes from being destroyed, and cationic polymers in the nano carrier provide good transfection efficiency for the nano carrier.
mCherry is a red fluorescent protein derived from mushroom coral (mushroom coral), and the sequence of the pDNA gene in this example contains mCherry, and when the pDNA is expressed, it can carry mCherry fluorescence, and in this example, it is used to determine the expression of the pDNA. DAPI, 4', 6-diamidino-2-phenylindole, is a fluorescent dye capable of binding strongly to DNA, which in this example is used to label the nucleus.
Example 5: dendritic cell activation assay
According to 5X 10 5 Immature BMDCs were seeded in 24 well plates and treated with PPC/pDNA (containing 0.2. Mu.g pDNA), 25. Mu.g PPC and 0.2. Mu.g pDNA, respectively, at 37℃for 24 hours (Ctrl group is a group without any addition of substances). Centrifugation at 800g for 5 min, followed by labeling of BMDCs with CD11C-APC, CD80-PE and CD 86-PE-Cy7 fluorescent antibodies by incubation in the dark for 30min, washing the cells three times with phosphate buffer, and analysis of CD11C on BMDCs by flow cytometry + CD80 + And CD11C + CD86 + The expression level of (3) is shown in FIG. 4C. As can be seen from the results of fig. 4A and 4B described above, PPC/pDNA has a stronger immunostimulatory function due to the delivery of PPC vector, inducing maturation of BMDCs.
CD11c is the adhesion protein leukocyte integrin, and CD11c is expressed in normal tissues, mainly in myeloid lineage cells, such as myeloblasts, promyelocytes, late promyelocytes, leaf-free and leaf-free neutrophils, at high levels in tissue macrophages and monocytes, and at low levels in granulocytes. It is also expressed in NK cells, activated T cells, lymphocyte lines.
CD80 is a membrane antigen necessary for T lymphocyte activation. Expressed in dendritic cells. He belongs to the immunoglobulin superfamily, whose receptors are CD28 and CD152 (CTLA 4).
CD86, expressed on dendritic cells. He belongs to the immunoglobulin superfamily, whose ligands are CD28 and CD152 (CTLA 4). CD86 interacts with inducer CD28 and inhibitor CTLA4 and is the main cofactor for inducing T lymphocyte proliferation and IL-2 production.
Example 6: blood collection and processing
Whole blood from male C57BL/6 mice was collected with a heparin sodium anticoagulation tube, the collected whole blood was centrifuged at 1000g for 10 minutes at 4℃at 7 up-speed and 1 down-speed, the buffy coat containing white blood cells at the interface of plasma and red blood cells was carefully aspirated and discarded, then red blood cells were separated by gentle resuspension in cold 0.01M PBS, the above procedure was repeated 2 times, and finally the separated red blood cells were washed and slowly resuspended in 10mL cold 0.01M PBS and then stored at 4℃to give a solution called a red blood cell stock solution.
Example 7: DNA nanovaccine riding on erythrocytes
The DNA nanovaccine PPC/pDNA prepared in example 3 was centrifuged at 10000rpm at 4℃for 10min and resuspended in cold PBS, and then mixed with an equal volume containing 1X 10 8 Erythrocyte stock of individual cells was mixed in a 2.0mL EP tube. The EP tube was placed on a silent mixer and uniformly rotated at 5rpm for 1 hour. The riding red blood cells were then centrifuged at 100g at 4℃for 5min, the supernatant carefully removed, and washed twice with 1mL of cold 0.01 MPBS. The riding red blood cells were finally resuspended in 500 μl cold 0.01M PBS for further characterization.
The DIO-labeled DNA nanovaccine prepared in example 3 was mounted on erythrocytes in various ratios as described above, and then the percentage of erythrocytes carrying the DNA nanovaccine was determined by flow cytometry (see fig. 5A, where Ctrl group is a group without DNA nanovaccine and the abscissa represents 10, 50, 100 and 250 μg of DIO-labeled DNA nanovaccine, respectively, mounted on a DNA-containing cell 1×10 8 Red blood cell stock of individual cells). The results show that: with the increase of the proportion of the DNA nano vaccine, the positive rate of the red blood cells is also increased.
Subsequently, the DiI-labeled DNA nanovaccine prepared in example 3 was assembled with erythrocytes in the above ratio at 100: 1 and red blood cells before and after carrying the DNA nanovaccine were observed using CLSM (see fig. 5B). Meanwhile, the DNA nanovaccine was further demonstrated to ride on erythrocytes by scanning electron microscopy (see fig. 5C). The result of the CLSM image and the scanning electron microscope can prove that the DNA nano vaccine is successfully ridden on the red blood cells.
Example 8: targeting erythrocytes of a stool vehicle to the spleen
The DiI-labeled DNA nanovaccine prepared in example 3 was mounted on erythrocytes in different ratios as in example 7 above,the obtained DiI-labeled DNA nanovaccine of 10, 50, 100 and 250. Mu.g was loaded with a DNA nanovaccine containing 1X 10 8 And (3) carrying out biological distribution analysis on the mixed solution of different riding ratios and 100 mug DiI marked DNA nano vaccine by injecting the mixed solution into a male C57BL/6 mouse through tail vein after 24 hours, euthanizing the mouse, taking out main organs of the mouse, and analyzing the fluorescence intensities of different organs to evaluate the influence of the red blood cells of different ratios of the riding cart on the in-vivo distribution. FIG. 6A is a fluorescence image of each organ, FIG. 6B is a 100. Mu.g DiI-labeled DNA nanovaccine riding on a DNA containing 1X 10 8 The fluorescence intensity of each organ was counted and compared in the erythrocyte stock of each cell. The results show that: when the ratio of the DNA nano vaccine to the red blood cells reaches 100: the fluorescence intensity of the spleen is strongest and the enrichment effect is best.
Example 9: assessment of immune response of DNA nanovaccine elicited at spleen
The 6-8 week male C57BL/6 mice were selected, and the mice were divided into four groups, 200. Mu.L of 0.01M PBS, NPs (DNA nanovaccine PPC/pDNA prepared in example 3) and RBC-NPs (I) (I is once DNA nanovaccine for tail intravenous injection) were injected tail vein, and RBC-NPs (II) (II is twice DNA nanovaccine for tail vein injection, this time the first needle of two vaccinations) (NPs contains 10. Mu.g pDNA), on day 8 equal amounts of PBS, NPs and RBC-NPs (II) (this time the second needle of two vaccinations) were injected tail vein-wise, respectively, and the RBC-NPs (I) group (this time no substance was injected), and four days later the mice were euthanized, and the spleens were collected. In the above experiments, 100. Mu.g of DNA nanovaccine for RBC-NPs was loaded onto a DNA nanovaccine containing 1X 10 8 Erythrocyte stock of individual cells.
Freshly harvested spleens were added to six-well plates containing 1mL PMI 1640 complete medium, and the medium was homogenized with a sterile syringe until the medium was cloudy, using Ficoll-Paque TM Centrifuging PREMUM sterile solution at 800g and 4deg.C for 30min to obtain single cell suspension, washing the single cell suspension with cold 0.01M PBS for 3 times, and adding relative antibodies (CD 11C-APC, MHCI-FITC, CD3-FITC, CD4-Percp, CD8-PE and CD44-PE-C respectively) to the single cell suspensiony 7) for 30min. After the incubation, the cells were washed 3 times and resuspended with 200 μl of 0.01M PBS, and finally analyzed by flow cytometry for MHC I expression of dendritic cells in the spleen after vaccination (see 7A and 7B), CD8 in the spleen + Expression of T cells (see 7C and 7D) and expression of CD44 in memory T cells in spleen (see 7E and 7F). The results show that: the erythrocytes of the stool vehicle promote the high expression of MHCI of DC in spleen and increase CD8 in mice + The content of T cells and memory T cells shows that the antigen can be presented to APCs after the erythrocytes of the stool vehicle are efficiently enriched to the spleen, so that the spleen immunity of the mice is further stimulated to enhance the systemic T cell immune response.
MHC class I (MHC I): on the surface of a normal cell, conditions in the normal cell can be provided, for example, when the cell is infected by virus, the amino acid chain (peptide) of the outer membrane fragment of the virus is presented outside the cell through MHC, so that the cell can be identified by a killer CD8+ T cell and the like for killing.
The co-receptor for CD3 (cluster of differentiation 3) T cells is a protein complex. The transmembrane region of the CD3 molecule is connected with the transmembrane regions of two peptide chains of the TCR through a salt bridge to form a TCR-CD3 complex, and the TCR-CD3 complex is jointly involved in the recognition of the antigen by the T cells. Activation signals generated by TCR recognition of antigen are transduced into T cells by CD 3.
CD4 is expressed primarily by helper T (Th) cells, which are receptors for recognition of antigens by Th cell TCRs, and is associated with non-polypeptide regions of MHC class ii molecules, involved in Th cell TCRs recognizing antigen processes.
The CD8 molecule is a leukocyte differentiation antigen, a glycoprotein on part of the T cell surface that aids in T Cell Receptor (TCR) recognition of the antigen and is involved in T cell activation signaling, also known as co-receptor for TCR. T cells expressing CD8 (cd8+ T cells) typically differentiate into cytotoxic T Cells (CTLs) upon activation, capable of specifically killing target cells.
CD44 is a complex transmembrane adhesion glycoprotein expressed on a variety of human cells including embryonic stem cells, differentiated cells and cancer cells. CD44 is a putative marker of tumor stem cells, a key regulator of Epithelial Mesenchymal Transition (EMT), involved in tumor development, progression and metastasis.
Example 10: DNA nanovaccine biosafety
Selecting 6-8 week male C57BL/6 mice, dividing into two groups, one group not vaccinated, and one group injected with RBC-NPs (RBC-NPs 100 μg DNA nanovaccine on day 1 and day 8 respectively) by tail vein once (1×10) 8 Erythrocyte stock of individual cells) (10. Mu.g pDNA in NPs used above) was used to treat healthy unvaccinated mice after four daysGroup) and RBC-NPs vaccinated mice were euthanized, and the liver and serum were collected.
The liver was immersed in formalin to fix the liver tissue, followed by embedding the liver tissue with paraffin. The embedded liver tissue was sectioned and the sectioned tissue was hematoxylin-eosin stained. With specific results, see FIG. 8A, which is then collectedThe serum of the group and RBC-NPs (II) was subjected to biochemical index analysis (see FIG. 8B). The results show that: although it can be seen in fig. 6A that the DNA nanovaccine is enriched in the liver, it has no effect on the liver, and the serum biochemical index also confirms that the biosafety and cytotoxicity of the DNA nanovaccine applied in vivo are low.
Example 11: mouse tumor cell challenge experiments
Selecting 6-8 week male C57BL/6 mice, dividing the mice into four groups (7 mice in each group), and injecting 200 mu L of 0.01M PBS, NPs (DNA nanovaccine PPC/pDNA prepared in example 3), RBC-NPs (I) (the ratio is 100:1, I is one DNA nanovaccine injected into the tail vein) and RBC-NPs (II) (II is two DNA nanovaccines injected into the tail vein, this time is the first needle for two vaccinations) (10 mu g pDNA is contained in the NPs), subcutaneously injecting liver cancer cells Hepa 1-6 (1×10) into the right inguinal of the mice for preventive treatment after seven days 6 /just) a subcutaneous tumor model was constructed. Mice were injected tail vein equivalent amount on day 8And RBC-NPs (II) (this time the second needle of the two vaccinations). In the above experiments, 100. Mu.g of DNA nanovaccine for RBC-NPs was loaded onto a DNA nanovaccine containing 1X 10 8 Erythrocyte stock of individual cells. The tumor size of the mice was measured every 2 days throughout the treatment, and the tumor volume formula was: (Long) Tumor(s) X width of 2 Tumor(s) ) And/2, observing for 39 days. Tumor volume exceeding 1500mm 3 Mice were euthanized soon thereafter. During observation, the tumor growth curve, weight change and life cycle of the mice are shown in fig. 9A to 9C, and the results show that: the tumor growth of the red blood cell group mice inoculated with the excrement car for many times is obviously inhibited, and meanwhile, the survival time of the mice is prolonged, so that the DNA nano vaccine for riding the red blood cells has good tumor growth prevention capability.
Sequence listing
<110> Fujian medical university Meng Chao liver and gall Hospital (Fuzhou infectious disease Hospital)
<120> a tumor neogenetic antigen DNA nano vaccine capable of being taken by erythrocytes, and preparation method and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 597
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ggaggcagcg gaggcggagg gagtggcggc atgaaggccc gaaactacct gcagtttctg 60
ccctcgaaaa ccaaggtggc tggaggcagc ggaggcggag ggagtggcgg ccgaggagaa 120
cactaccggt acaaggtcag cctccccggg ggccagcacg ccggaggcag cggaggcgga 180
gggagtggcg gccatgttct ctgggactta aagcagatgt ttcggtgtgc tgtcttgaaa 240
aacggaggca gcggaggcgg agggagtggc ggctgggaca cttgtaccac ttacaagtgg 300
caaaagacac tggaaggtca tgatggaggc agcggaggcg gagggagtgg cggcctatca 360
acgtacagaa cagcttgcac gttacgattt gtacagaagc gatgcggagg cagcggaggc 420
ggagggagtg gcggcctgta cactcacttc ctgcagttgc cactggcagc caccgggttc 480
tccgtgggag gcagcggagg cggagggagt ggcggcaaaa ggtggttata ttggcaacct 540
actctcacta agatggggtt tgtgtcaggc ggcagtctgg gaggaggggg gagtggc 597
<210> 2
<211> 9065
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 60
agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 120
cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 180
ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 240
gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 300
cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 360
cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 420
ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 480
catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 540
tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 600
ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 660
catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 720
cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 780
cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 840
acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 900
ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 960
tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac 1020
attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga 1080
cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga 1140
tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg 1200
gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat 1260
accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg 1320
caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 1380
gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 1440
taaaacgacg gcgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc 1500
acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc 1560
cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg 1620
gtgatgccgg ccacgatgcg tccggcgtag aggcgattag tccaatttgt taaagacagg 1680
atatcagtgg tccaggctct agttttgact caacaatatc accagctgaa gcctatagag 1740
tacgagccat agataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa 1800
gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa 1860
atacataact gagaatagag aagttcagat caaggttagg aacagagaga cagcagaata 1920
tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga 1980
tggtccccag atgcggtccc gccctcagca gtttctagag aaccatcaga tgtttccagg 2040
gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc 2100
tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac aacccctcac 2160
tcggcgcgcc agtcctccga tagactgcgt cgcccgggta cccgtattcc caataaagcc 2220
tcttgctgtt tgcatccgaa tcgtggactc gctgatcctt gggagggtct cctcagattg 2280
attgactgcc cacctcgggg gtctttcatt tggaggttcc accgagattt ggagacccct 2340
gcccagggac caccgacccc cccgccggga ggtaagctgg ccagcggtcg tttcgtgtct 2400
gtctctgtct ttgtgcgtgt ttgtgccggc atctaatgtt tgcgcctgcg tctgtactag 2460
ttagctaact agctctgtat ctggcggacc cgtggtggaa ctgacgagtt ctgaacaccc 2520
ggccgcaacc ctgggagacg tcccagggac tttgggggcc gtttttgtgg cccgacctga 2580
ggaagggagt cgatgtggaa tccgaccccg tcaggatatg tggttctggt aggagacgag 2640
aacctaaaac agttcccgcc tccgtctgaa tttttgcttt cggtttggaa ccgaagccgc 2700
gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct gactgtgttt 2760
ctgtatttgt ctgaaaatta gggccagact gttaccactc ccttaagttt gaccttaggt 2820
cactggaaag atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa gaagagacgt 2880
tgggttacct tctgctctgc agaatggcca acctttaacg tcggatggcc gcgagacggc 2940
acctttaacc gagacctcat cacccaggtt aagatcaagg tcttttcacc tggcccgcat 3000
ggacacccag accaggtccc ctacatcgtg acctgggaag ccttggcttt tgacccccct 3060
ccctgggtca agccctttgt acaccctaag cctccgcctc ctcttcctcc atccgccccg 3120
tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta tccagccctc 3180
actccttctc taggcgccgg aattagatct ctcgactagg gataacaggg taattgtttg 3240
aatgaggctt cagtacttta cagaatcgtt gcctgcacat cttggaaaca cttgctggga 3300
ttacttcttc aggttaaccc aacagaaggc tcgaggtgac tggagttcag acgtgtgctc 3360
ttccgatcgc caccatgcct aatcatcagt ccgggtcacc taccggcagt tcagacctgc 3420
tccttgatgg caagaaacaa cgagcccatc tggcgctgag gagaaaacgg cgacgggaaa 3480
tgcgcaagat taaccgaaag gtgagaagaa tgaatctcgc acccattaaa gaaaaaacag 3540
cctggcagca cctgcaagct ctgatcttcg aggcggaaga agtgttgaag acttctcaaa 3600
ctccacagac ctccctcacg ctgtttctag cactcttggc cggaggcagc ggaggcggag 3660
ggagtggcgg catgaaggcc cgaaactacc tgcagtttct gccctcgaaa accaaggtgg 3720
ctggaggcag cggaggcgga gggagtggcg gccgaggaga acactaccgg tacaaggtca 3780
gcctccccgg gggccagcac gccggaggca gcggaggcgg agggagtggc ggccatgttc 3840
tctgggactt aaagcagatg tttcggtgtg ctgtcttgaa aaacggaggc agcggaggcg 3900
gagggagtgg cggctgggac acttgtacca cttacaagtg gcaaaagaca ctggaaggtc 3960
atgatggagg cagcggaggc ggagggagtg gcggcctatc aacgtacaga acagcttgca 4020
cgttacgatt tgtacagaag cgatgcggag gcagcggagg cggagggagt ggcggcctgt 4080
acactcactt cctgcagttg ccactggcag ccaccgggtt ctccgtggga ggcagcggag 4140
gcggagggag tggcggcaaa aggtggttat attggcaacc tactctcact aagatggggt 4200
ttgtgtcagg cggcagtctg ggaggagggg ggagtggcgg ccacatttgc ttctgacaca 4260
actgtgttca ctagcaacct caaacagaca ccatggtgca tctgactcct gaggagaagt 4320
ctgccgttac tgccctgtgg ggcaaggtga acgtggatga agttggtggt gaggccctgg 4380
gcaggttggt atcaaggtta caagacaggt ttaaggagac caatagaaac tgggcatgtg 4440
gagacagaga agggccaaac aggccaaaga tcggaagagc gtcgtgtagg gaaagagtgt 4500
agatctcggt ggtcgccgta tcattgaatt caaggggcta ctttaggagc aattatcttg 4560
tttactaaaa ctgaatacct tgctatctct ttgatacatt tttacaaagc tgaattaaaa 4620
tggtataaat taaatcactt ttttcaattc taccgggtag gggaggcgct tttcccaagg 4680
cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc 4740
tggcctcgca cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc 4800
cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc 4860
gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac 4920
agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc 4980
tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc 5040
gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt 5100
caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct ttcgacctgc 5160
agcccaagct taccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg 5220
tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca 5280
ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc 5340
gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct 5400
ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg 5460
ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc 5520
accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg 5580
gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg 5640
tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct 5700
tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca 5760
agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac 5820
cccatcatcc aattccgccc ccccccccta acgttactgg ccgaagccgc ttggaataag 5880
gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga 5940
gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg 6000
ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt 6060
gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca 6120
ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc 6180
agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat 6240
tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc 6300
ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaacgtctag gccccccgaa 6360
ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca accatggtga 6420
gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca 6480
tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct 6540
acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct 6600
gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg 6660
acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga 6720
acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt 6780
tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga 6840
agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga 6900
agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca 6960
agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca 7020
agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg 7080
agggccgcca ctccaccggc ggcatggacg agctgtacaa gtaattaatt aagaattatc 7140
aagcttatcg ataccgtcga cctgcagcca agcttatcga taaaataaaa gattttattt 7200
agtctccaga aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa 7260
gtaacgccat tttgcaaggc atggaaaata cataactgag aatagagaag ttcagatcaa 7320
ggttaggaac agagagacag cagaatatgg gccaaacagg atatctgtgg taagcagttc 7380
ctgccccggc tcagggccaa gaacagatgg tccccagatg cggtcccgcc ctcagcagtt 7440
tctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt 7500
atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc 7560
tcaataaaag agcccacaac ccctcactcg gcgcgccagt cctccgatag actgcgtcgc 7620
ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg 7680
ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcatgggt 7740
aacagtttct tgaagttgga gaacaacatt ctgagggtag gagtcgaata ttaagtaatc 7800
ctgactcaat tagccactgt tttgaatcca catactccaa tactcctgaa atagttcatt 7860
atggacagcg cagaagagct ggggagaatt aattcgtaat catggtcata gctgtttcct 7920
gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 7980
aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 8040
gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 8100
agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 8160
gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 8220
gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 8280
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 8340
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 8400
tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 8460
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 8520
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 8580
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 8640
ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 8700
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 8760
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 8820
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 8880
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 8940
gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 9000
cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 9060
gacag 9065
<210> 3
<211> 199
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Met Lys Ala Arg Asn Tyr
1 5 10 15
Leu Gln Phe Leu Pro Ser Lys Thr Lys Val Ala Gly Gly Ser Gly Gly
20 25 30
Gly Gly Ser Gly Gly Arg Gly Glu His Tyr Arg Tyr Lys Val Ser Leu
35 40 45
Pro Gly Gly Gln His Ala Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
50 55 60
His Val Leu Trp Asp Leu Lys Gln Met Phe Arg Cys Ala Val Leu Lys
65 70 75 80
Asn Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Trp Asp Thr Cys Thr
85 90 95
Thr Tyr Lys Trp Gln Lys Thr Leu Glu Gly His Asp Gly Gly Ser Gly
100 105 110
Gly Gly Gly Ser Gly Gly Leu Ser Thr Tyr Arg Thr Ala Cys Thr Leu
115 120 125
Arg Phe Val Gln Lys Arg Cys Gly Gly Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Leu Tyr Thr His Phe Leu Gln Leu Pro Leu Ala Ala Thr Gly Phe
145 150 155 160
Ser Val Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Arg Trp Leu
165 170 175
Tyr Trp Gln Pro Thr Leu Thr Lys Met Gly Phe Val Ser Gly Gly Ser
180 185 190
Leu Gly Gly Gly Gly Ser Gly
195
<210> 4
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
aaggccaagt tggccaccat gaaggcccga aac 33
<210> 5
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
ttggccaagt tggcctcatc atgacacaaa ccccat 36

Claims (12)

1. The particle size of the DNA nano vaccine is 20-200 nm, the DNA nano vaccine is a nano system with a spherical structure, the inner core of the nano system is a compound formed by a cationic polymer PC and the DNA vaccine, the surface of the inner core is wrapped by lactide-glycolide copolymer PLGA, the DNA vaccine is plasmid pDNA obtained by inserting a nucleotide sequence for expressing tumor cell antigens into an empty plasmid, and the DNA nano vaccine can be ridden on the surface of the red blood cells through electrostatic adsorption;
the cationic polymer is composed of polyethyleneimine PEI 25000 And 1, 2-epoxytetradecane in a mass ratio of 1: (0.5-10) reacting at 90 ℃ for 48 hours to obtain a cationic polymer PC modified by 1, 2-epoxytetradecane, wherein the reaction medium is absolute ethyl alcohol, and the lactide-glycolide copolymer PLGA is PLGA 50:50.
2. The tumor neoantigen DNA nanovaccine of claim 1, wherein the tumor cells comprise one or more of Hepa 1-6 (mouse hepatoma cells), B16-F10 (mouse skin melanoma cells), CT26 (mouse colon carcinoma cells), MC38 (mouse colon carcinoma cells), RH35 (rat hepatoma cells), 4T1 (mouse breast cancer cells), GL261 (mouse brain glioma), or U87 (human brain astrocytoma).
3. The tumor neoantigen DNA nano vaccine according to claim 2, wherein the nucleotide sequence of the expressed tumor cell antigen is shown as SEQ ID. NO.1, the tumor cell is a Hepa 1-6 cell, and the nucleotide sequence of the plasmid pDNA is shown as SEQ ID. NO. 2.
4. The tumor neoantigen DNA nanovaccine of claim 3, wherein the plasmid pDNA expresses a tumor cell antigen having an amino acid sequence as shown in SEQ ID. No. 3.
5. The tumor neoantigen DNA nanovaccine of claim 3, wherein the tumor cell antigen comprises 7 immunogenic antigen peptides, wherein the antigen peptides are linked by a 10 amino acid-long linker peptide, and wherein the 7 antigen peptides are each at positions 11-27 in SEQ ID. NO.3 Mapk3Located at positions 38-54Lmf1、At positions 65-81Samd9lLocated at 92 th to 108 th positionsTraf7At positions 119-135DtnbAt positions 146-162LbrLocated at 173 th to 189 th positionsPtpn2The amino acid sequence of the linker peptide is located elsewhere in SEQ ID. NO. 3.
6. The use of a tumor cell antigen according to any one of claims 4-5 in the preparation of a medicament for the treatment of tumors.
7. Use of a recombinant plasmid pDNA as claimed in claim 3 for the preparation of a medicament for the treatment of tumors.
8. A method for preparing the tumor neoantigen DNA nanovaccine of any of claims 1-3, comprising the steps of: hydrophilic pDNA obtained according to any one of claims 1-3 is entrapped inside PLGA by a double microemulsion method in the presence of hydrophobic excipient PLGA and a gene carrier cationic polymer PC.
9. The preparation method according to claim 8, characterized in that it comprises in particular the following steps:
dissolving PLGA and cationic polymer PC in an organic solvent, adding DEPC aqueous solution containing DNA vaccine into the organic solvent, forming primary water-oil emulsion after ultrasonic treatment in ice bath, adding DEPC aqueous solution into the aqueous solution, forming oil-water emulsion after ultrasonic treatment in ice bath, and removing the organic solvent to obtain the DNA nano vaccine.
10. The method of claim 9, wherein the organic solvent is methylene chloride; the mass ratio of the cationic polymer PC to the DNA vaccine is (5-150): 1, a step of; the mass ratio of PLGA to DNA vaccine is (1-200): 1.
11. use of a tumor neoantigen DNA nanovaccine according to any one of claims 1-3 in the preparation of a tumor immunotherapeutic medicament.
12. The use according to claim 11, characterized in that the use is: after the tumor neoantigen DNA nano vaccine is uniformly mixed with red blood cells, the DNA nano vaccine with positive electricity is taken on the red blood cells, and the DNA nano vaccine is transported to the spleen through the targeting effect of the red blood cells.
CN202210223117.0A 2022-03-09 2022-03-09 Tumor neogenetic antigen DNA nano vaccine capable of being taken by erythrocytes as well as preparation method and application thereof Active CN114558127B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210223117.0A CN114558127B (en) 2022-03-09 2022-03-09 Tumor neogenetic antigen DNA nano vaccine capable of being taken by erythrocytes as well as preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210223117.0A CN114558127B (en) 2022-03-09 2022-03-09 Tumor neogenetic antigen DNA nano vaccine capable of being taken by erythrocytes as well as preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN114558127A CN114558127A (en) 2022-05-31
CN114558127B true CN114558127B (en) 2023-08-22

Family

ID=81717614

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210223117.0A Active CN114558127B (en) 2022-03-09 2022-03-09 Tumor neogenetic antigen DNA nano vaccine capable of being taken by erythrocytes as well as preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN114558127B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115364244B (en) * 2022-09-06 2024-04-12 中国医学科学院生物医学工程研究所 Drug delivery system for taking and placing neutrophils as well as preparation method and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113559271A (en) * 2020-04-10 2021-10-29 上海交通大学 Components of macrophage targeting vector system, preparation method and application of macrophage targeting vector system in drug and nucleic acid delivery

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10874621B2 (en) * 2013-10-17 2020-12-29 The Brigham And Women's Hospital, Inc. Cationic nanoparticles for co-delivery of nucleic acids and therapeutic agents
KR20160121584A (en) * 2014-02-26 2016-10-19 에트리스 게엠베하 Compositions for gastrointestinal administration of RNA
WO2016065306A1 (en) * 2014-10-23 2016-04-28 The Brigham And Women's Hospital, Inc. Amphiphile-polymer particles
WO2018089688A1 (en) * 2016-11-09 2018-05-17 Jinjun Shi Restoration of tumor suppression using mrna-based delivery system

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113559271A (en) * 2020-04-10 2021-10-29 上海交通大学 Components of macrophage targeting vector system, preparation method and application of macrophage targeting vector system in drug and nucleic acid delivery

Also Published As

Publication number Publication date
CN114558127A (en) 2022-05-31

Similar Documents

Publication Publication Date Title
KR102533061B1 (en) Modified NK-92 haNK003 cells for clinical use
ES2819976T5 (en) Compositions and medical uses for TCR reprogramming with fusion proteins
CN114558127B (en) Tumor neogenetic antigen DNA nano vaccine capable of being taken by erythrocytes as well as preparation method and application thereof
CN1271277A (en) Biodegradable macromers fro the controlled release of biologically active substances
EA028988B1 (en) Method of in vitro analyzing genetically modified t cell culture to detect a contaminant
CN108409840A (en) The Chimeric antigen receptor of anti-CD123 single-chain antibodies and combinations thereof and application
CN109111525B (en) HLA-G chimeric antigen receptor, coding sequence, expression vector and application
CN113402590B (en) KHL polypeptide and application thereof in preparation of TABP-EIC cells
CN106754723A (en) A kind of immunocyte and its application with anti-tumor function
AU2016378480A1 (en) Endothelium-specific nucleic acid regulatory elements and methods and use thereof
CN111450098A (en) Application of lamivudine in delaying senescence and preparation of cell model for screening anti-senescence drugs
CN114601923B (en) Neoantigen composition and application thereof in preparation of medicines for treating tumors
CN115968300A (en) Vectors and methods for in vivo transduction
CN111154791B (en) Recombinant CD6 gene, T cell modified by same, preparation method and application
US20040131591A1 (en) Vector system
AU2017313806B2 (en) Tumor-targeting bead vectors and methods of using the same
EP4257676A1 (en) Modified immune cell and use thereof
CN113355288B (en) Preparation method and application of universal chimeric antigen receptor T cell for treating COVID-19
CN114616239B (en) Chimeric antigen receptor and T cell expressing chimeric antigen receptor therein
JP2006525351A (en) Methods for diagnosing and treating cancer
CN109957017A (en) It is a kind of to target the single-chain antibody of OX40, Chimeric antigen receptor T cell and its preparation method and application
US20230270860A1 (en) Modified mrna for multicell transformation
CN113717951A (en) Equine encephalitis virus-based oncolytic virus and application thereof
KR20230138781A (en) Oncolytic virus for colorectal cancer treatment based on a recombinant newcastle virus vector containing a TRAIL gene and colorectal cancer treatment composition using the same
CN113710268A (en) Drug delivery compositions

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant