CN112121180B - Composite nanoparticles of poloxamer and or poloxamer and PEG lipids - Google Patents

Composite nanoparticles of poloxamer and or poloxamer and PEG lipids Download PDF

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CN112121180B
CN112121180B CN202011226741.3A CN202011226741A CN112121180B CN 112121180 B CN112121180 B CN 112121180B CN 202011226741 A CN202011226741 A CN 202011226741A CN 112121180 B CN112121180 B CN 112121180B
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CN112121180A (en
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张龙贵
刘晨
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Shenzhen Houcun Nano Pharmaceutical Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • 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
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    • 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

Cationic complex nanoparticles of poloxamers and/or poloxamers and PEG lipids in combination relate to the field of gene therapy, which include poloxamers and/or poloxamers and PEG lipids. The cationic complex nanoparticle comprises One or more of the following; the cationic compound nanoparticle serving as a nucleic acid vector is lower in cost than a viral vector, convenient to control quality, higher in transfection efficiency than a common nano vector, low in toxicity, good in biocompatibility, particularly suitable for transfection of in vitro cells without changing liquid after administration in vitro experiments, effective in vivo and in vitro transfection, simple in preparation and creatively solves the problem that nucleic acid, particularly mRNA, is safely and efficiently delivered in vivo and in vitro.

Description

Composite nanoparticles of poloxamer and or poloxamer and PEG lipids
Technical Field
The invention relates to the field of gene therapy, in particular to a cationic complex nanoparticle for nucleic acid delivery of poloxamer and/or poloxamer and lipid combination, a method for preparing the cationic complex nanoparticle, application of the cationic complex nanoparticle in-vivo and in-vitro cell gene transfection and application of the cationic complex nanoparticle in the field of novel vaccine medicines.
Background
Gene therapy refers to the introduction of exogenous therapeutic genes into target cells to correct or compensate for diseases caused by gene defects and abnormalities, for therapeutic purposes. Gene delivery is the delivery of a biologically active exogenous therapeutic gene into a patient's recipient cells by a delivery technique and the translation of the exogenous therapeutic gene to produce a protein polypeptide to treat a disease. Gene transfection is a technique that transfers or transports nucleic acids with biological functions into cells and maintains the nucleic acids in the cells for their biological functions. In recent years, the era of gene therapy has come formally with the maturation of biotechnology such as in vitro synthesis technology, vector technology, and editing technology of genes. Previously, the U.S. FDA approved two gene therapy drugs, one and Gilvaari, based on RNA interference (RNAi) technology, for the treatment of hereditary thyroxine transporter amyloidosis (hereditary transthyretin amyloidosis hATTR) polyneuropathy and Acute Hepatoporphyria (AHP), respectively, by Alnylam Pharmaceuticals corporation, which has a milestone significance in the new drug development history in the field of gene therapy. However, in another field, with the discovery of more scenes of mRNA application such as protein replacement therapy, protein supplementation therapy, vaccination against cancer and infectious diseases, people have begun to overcome disadvantages of mRNA such as in vivo and in vitro stability, short half-life, and unfavorable immunogenicity, etc., to mine more functions thereof. Compared with DNA gene therapy, IVTmRNA therapy has its own advantages. IVTmRNA can function without entering the nucleus; in contrast, DNA therapies require transcription into RNA into the nucleus. This is mainly because mRNA acts in the cytoplasm and does not need to cross the nuclear membrane, which is difficult to surmount, so mRNA does not integrate into the genome of the target cell, i.e., there is no risk of insertional mutagenesis. mRNA-based therapies and drug development have attracted widespread interest to scientists and investors.
Vaccines are generally divided into two categories: prophylactic vaccines and therapeutic vaccines. The prophylactic vaccine is mainly used for preventing diseases, and the recipient is a healthy individual or a neonate; therapeutic vaccines are mainly used in individuals suffering from a disease, and the recipient is a patient, such as a tumor patient. The prophylactic vaccine is an automatic immune preparation for preventing infectious diseases, which is prepared by artificially attenuating, inactivating or utilizing transgenosis methods of pathogenic microorganisms (such as bacteria, rickettsia, viruses and the like) and metabolites thereof. The vaccine retains the characteristic of pathogenic bacteria for stimulating the immune system of animals. When the animal body contacts the pathogenic bacteria without injury, the immune system can generate certain protective substances such as immune hormone, active physiological substances, special antibodies and the like; when the animal comes into contact with the pathogenic bacteria again, the immune system of the animal body can follow the original memory of the animal body, and more protective substances are manufactured to prevent the pathogenic bacteria from being damaged. The tumor vaccine belongs to therapeutic vaccine, and its working principle is that tumor cell, tumor related protein or polypeptide, gene expressing tumor antigen, etc. are introduced into patient's body to overcome the immunosuppressive state caused by tumor, raise immunogenicity, activate patient's own immune system and induce body's cellular immune and humoral immune response so as to attain the goal of controlling or eliminating tumor.
The DNA vaccine is a novel vaccine developed in the 90 th year of the 20 th century, and is a 3 rd generation vaccine after attenuated vaccine and genetic engineering vaccine. The DNA vaccine has the function of preventing diseases and treating diseases. The DNA vaccine is composed of one or several kinds of exogenous gene inserted plasmid DNA and eukaryotic promoter regulating gene, and the exogenous antigen loaded plasmid DNA can express relevant antigen protein in various cells of mammal under the control of eukaryotic promoter, tail adding signal, relevant enhancer and other gene units. The plasmid recombined with exogenous antigen coding gene is introduced into human or animal cell by a certain gene delivery method, and antigen protein is synthesized in living cell of immune subject body by transcription system of host cell, so as to induce body to produce immune response. The DNA vaccine has the following advantages: the DNA inoculating carrier (such as plasmid) has simple structure, simple and convenient process for purifying plasmid DNA, low production cost and suitability for mass production; the cloning of DNA molecules is easier, so that the DNA vaccine can be updated at any time according to the needs; the DNA molecules are very stable, can be made into DNA vaccine freeze-dried vaccine, can restore the original activity in salt solution when in use, thus being convenient for transportation and preservation; compared with the traditional vaccine, the DNA vaccine has the immunogenicity equivalent to that of the attenuated vaccine, and can activate cytotoxic T lymphocytes to induce cellular immunity, but the DNA sequence codes only a single segment of viral gene, so that the possibility of toxicity reversion is basically avoided, the risk of toxicity reversion of the attenuated vaccine is avoided, and the phenomenon of epitope loss of the DNA vaccine is avoided like the attenuated vaccine or subunit vaccine because the antigen-related epitope of the DNA vaccine in the immune system of the organism is relatively stable. The plasmid can be used as an adjuvant, so that the DNA vaccine is used without adding the adjuvant, thereby reducing the cost and being convenient to use; the multiple plasmid DNA is simply mixed, so that antigens with similar biochemical characteristics (such as different strains derived from the same pathogenic bacteria) or multiple different antigens of one pathogen can be combined together to form the multivalent vaccine, thereby leading one DNA vaccine to be capable of inducing the immune protection effect aiming at multiple antigen epitopes and greatly increasing the flexibility of the production of the DNA vaccine. However, DNA vaccines present the following serious safety issues: whether the exogenous DNA is integrated into the host genome after entering the body, resulting in activation of oncogenes or inactivation of oncogenes; whether the long-term expression of vaccine DNA in vivo can induce the organism to generate immune tolerance or not, in the long term, the immune function of the organism is low; whether vaccine DNA is a foreign substance that causes the body to produce anti-DNA antibodies; whether the CTL response induced by the DNA vaccine would have a killing effect on other cells. These potential risks limit the wide range of DNA vaccines.
The mRNA vaccine is a new vaccine developed in recent years, is a new vaccine of the 4 th generation after attenuated vaccine, genetic engineering vaccine and DNA vaccine, and has better safety than the DNA vaccine. The mRNA vaccine is used as a new response scheme, wherein mRNA encoding antigen protein is directly introduced into cells, and the protein is synthesized through an expression system of the cells, so that specific immune response is induced, and the mRNA vaccine has obvious advantages compared with the traditional attenuated live vaccine, inactivated vaccine, antitoxin, subunit vaccine (including polypeptide vaccine), vector vaccine and DNA vaccine.
Firstly, mRNA can be degraded by normal cellular processes, reducing the risk of metabolic toxicity, and in vivo half-life can be modulated by certain modifications and delivery modes, so that mRNA undergoes a natural degradation process in vivo; secondly, mRNA is easy to synthesize, any new antigen can be encoded, mRNA vaccine can be rapidly prepared, and usually at least 6 months are required for producing a traditional influenza vaccine, but due to the high-yield in vitro transcription reaction, the mRNA vaccine can produce the required vaccine within 30 days under the condition of realizing standardized production, and timeliness is particularly important in coping with epidemic situations of novel coronaviruses and avian influenza viruses; in addition, mRNA has better water solubility, is easier to prepare medicine, and can effectively improve the stability of mRNA vaccine by carrying various modification and delivery modes, so as to avoid degradation of mRNA vaccine by in vivo RNase before entering cells; after mRNA enters the cell, enough new antigens are generated by the body through protein translation to rapidly start local T cells, the mRNA vaccine can effectively induce B cell and T cell immune responses, can cause immune memory effect, and can transmit more effective antigens. In terms of immunogenicity, mRNA vaccines can be specifically designed to encode a variety of peptide and protein structures, thereby expressing the entire antigen, and can also be designed to express multiple antigens at once or to contain several or even tens of IVTmRNA sequences in the same nanoformulation for the preparation of multivalent mRNA vaccines. In addition, the mRNA vaccine is non-infectious, belongs to a non-integrated platform, avoids the risks of infection and insertion mutation, and has the advantages of low cost and convenience in storage and transportation.
In addition to application to mRNA vaccine or other malignant immunotherapy development, the IVTmRNA may also be applied to treatment of rare genetic diseases, protein replacement or protein supplementation therapies, multifunctional stem cell therapies, and genome engineering of nucleases, among others. The use of IVTmRNA as a drug either transfers specific genetic information into cells of a patient to alter a disease state. There are two methods: one is to transfer the IVTmRNA into isolated cells and then return these transfected cells to the patient where the IVTmRNA translates the information into a protein that acts in the patient to produce a therapeutic effect. Such methods are commonly used in genome engineering, genetic reprogramming, T cell and Dendritic Cell (DC) based immunotherapy to treat cancer and infectious diseases, and some protein replacement approaches. Another approach is to deliver the IVTmRNA directly into cells in the patient using various means. This approach has application mainly in oncology and infectious diseases, tolerisation protocols for allergy treatment and other protein replacement approaches. The primary site where IVTmRNA exerts pharmacodynamic activity is the cytoplasm, and in contrast to the natural mRNA which is produced in the nucleus and exported into the cytoplasm through the nucleus, IVTmRNA must enter the cytoplasm from the extracellular space. Two key factors determine cytoplasmic bioavailability: one is that high activity RNase ubiquitous in the body rapidly degrades it; the other is the cell membrane, which blocks passive diffusion of negatively charged large mRNA molecules into the cytoplasm. The protein products translated from the IVTmRNA undergo post-translational modification to exert therapeutic effects, and the presence of these two factors may greatly reduce the therapeutic efficacy, and thus efficient gene delivery protocols are particularly important. The half-life of the IVTmRNA template and the protein product are key determinants of pharmacokinetics. The delivery of genes has been a difficulty in transforming genes, especially RNAi, DNA, mRNA and other genetic industries, and is also a pain point in the field of gene therapy.
Gene vectors are a tool for introducing exogenous therapeutic genes into biological cells, and currently, the gene vectors with industrial transformation potential are mainly viral vectors and non-viral vectors. The virus vector is a gene delivery tool for transmitting the genome of the virus into other cells to carry out infection, can be applied to basic research, gene therapy or vaccine, and has good application prospects in the prior art such as lentivirus, adenovirus, retrovirus vector, adeno-associated virus vector and the like. However, viral vectors have serious drawbacks due to their inherent physicochemical properties and biological activities, such as high production cost, limited load, poor targeting, insertion integration, teratogenic mutagenesis, etc., which are disadvantageous for developing general and versatile therapies. Rather than viral vectors include: the direct injection method (direct injection of a solution containing DNA into muscle to cause the expression of adjacent cells into DNA chains, in muscle cells, gene expression may last for several months), the calcium phosphate coprecipitation method (mixing calcium chloride, DNA and phosphate buffer to form calcium phosphate microprecipitation, attaching to cell membranes and entering cytoplasm through endocytosis), receptor-mediated gene transfer (by means of receptor-mediated endocytosis to transfer foreign genes), receptor-mediated gene transfer method (formation of complexes between plasmid DNA and specific polypeptides (ligands) which are recognized by receptors on cell surfaces, if DNA is transported into liver in vivo, DNA and asialoglycoprotein which can be specifically bound to hepatocyte receptors can be selectively taken in order to be ingested by the endocytosis process, the DNA is largely taken up by liver, the expression duration of foreign genes transferred by the method in vivo is short), the injection method (under the condition that DNA is injected into glass by means of receptor-mediated endocytosis to transfer foreign genes in vivo), the method (by means of a direct electron gun, the field strength of a pulse-enhanced gene transfer method is also suitable for the pulse-mediated cell surface of a particle, by means of a pulse-mediated electron gun, the pulse-mediated gene transfer method is also suitable for the pulse-mediated cell surface-specific particle growth by means of a high-impulse pulse-mediated impulse particle-size, such as well as the pulse-mediated cell-mediated particle-size is applied by a pulse-mediated pulse-size-based method, into intact cells, thereby allowing stable transformation of the foreign gene DNA in the target cells and making it possible to obtain expression. The experimental result shows that the target gene can be expressed in skin, muscle, liver, pancreas, stomach, mammary gland and other cells. ) DEAE-dextran and polybrene polycation (positively charged DEAE-dextran or polybrene multimeric complexes and negatively charged DNA molecules allow DNA to bind to the cell surface. The DNA complex is introduced by osmotic shock obtained using DMSO or glycerol. DEAE-dextran is limited to transient transfection. ) DNA can be obtained by sperm cell carrier method (incubation with sperm cells and NDA (pyridine nucleotide coenzyme) -agent). Exogenous genes are introduced into fertilized eggs through the fertilization process, so that the preparation process of transgenic animals is greatly simplified. The transfection method is the latest technology developed in recent years and applied to fish transfer, and has the greatest advantage of simplicity and convenience. ) Etc. However, the above-described non-viral gene delivery method has the following problems: low transfection efficiency, high toxicity, complex operation, adverse targeting modification, etc. The nanoparticle method refers to a method for carrying exogenous genes into cells in vivo or in vitro by nanotechnology, and belongs to a non-viral vector. The nanoparticle method comprises the following steps: liposome nanoparticles, micelle nanoparticles, nano-microspheres, nanoemulsions, dendrimers, copolymer nanoparticles, composite nanoparticles, and the like. Compared with other gene delivery methods, the method has the advantages of low production cost, clear chemical structure, convenience in quality control, capability of realizing local targeted drug delivery through targeted modification, unlimited theoretical inclusion capacity, no risk of integrated induced mutation, capability of being biodegraded through organisms, low cytotoxicity, good biocompatibility, no immunogenicity and capability of being applied to in-vivo and in-vitro gene delivery.
In the mRNA field, most cells can spontaneously ingest mRNA, but are inefficient and saturate at low doses. Thus, suitable formulations are needed to protect the IVTmRNA from extracellular RNase-mediated degradation and to facilitate its entry into cells. In the development process of the virus mRNA vaccine and the tumor mRNA vaccine, the IVTmRNA with a specific sequence is delivered to dendritic cells, so that safe, efficient and sufficient expression of the IVTmRNA is crucial for the vaccine to generate drug effect. Currently, nano-delivery technology has become the mainstream delivery technology in RNAi, DNA, mRNA and other gene fields internationally, and the gene with biological activity is delivered to the receptor cells of patients to play a role through intramuscular, intradermal, intranodal, subcutaneous, intravenous, intrathecal, respiratory and digestive routes and other administration routes. The nano-carrier is an irreplaceable delivery system for in-vivo and in-vitro administration of genes in the future, and has infinite potential in clinical application. Currently, global scientists have developed various nano delivery vectors for protecting RNAi, DNA, mRNA and other genes from extracellular DNase or RNase mediated degradation and promoting the genes to enter cells and improving the pharmacokinetic properties of RNAi, DNA, mRNA and other gene preparations, but most of the current delivery systems still have serious instability, short half-life, high in vivo toxicity, low transfection efficiency and other problems.
Disclosure of Invention
It is an object of the present invention to provide cationic complex nanoparticles of poloxamer and/or PEG lipids in combination for delivering nucleic acids with high transfection efficiency; another object of the present invention is to provide cationic complex nanoparticles of poloxamer and/or PEG lipid in combination for delivering nucleic acid with high transfection efficiency and stability; it is still another object of the present invention to provide cationic complex nanoparticles of poloxamer and/or PEG lipids in combination suitable for delivery of mRNA with high transfection efficiency and stability.
In one aspect, the invention provides cationic complex nanoparticles of poloxamers and/or poloxamers in combination with PEG lipids, comprising poloxamers and/or poloxamers in combination with PEG lipids.
In one aspect, the poloxamer (1, 2-ethylenediamine tetraacetic acid and ethylene oxide and methyl ethylene oxide polymers or 1, 2-ethylenediamine tetraisopropanol and methyl ethylene oxide and ethylene oxide polymers of the present invention are commercially available from BASF under the trade name) In some embodiments, the poloxamine is selected from +.>304、/>701、/>704、/>707、/>803、/>901、/>904、/>908、/>1107、/>1301、/>1304、/>1307 or->90R4、/>150R 1; the structural formula of the poloxamine is as follows:
In some embodiments, the poloxamer (poly (propylene glycol) -poly (ethylene glycol) -poly (propylene glycol) copolymer or poly (ethylene glycol) -poly (propylene glycol) -poly (ethylene glycol) copolymer is purchased from sigma aldrich corporation under the trade name) In some embodiments, the poloxamer is selected from +.>17R4、/>P10R5、/>L-121、/>L-31、/>L-64、/>L-85、/>P-10R5、/>L-35、L-61、/>P-123、/>F108、/>F127、/>F68、/>P105、/>P104、/>P-85、/>P103 or->One or more of L122, wherein the poloxamer has the following structural formula:
in certain embodiments, the PEG lipid is a PEGylated amphiphilic polymer, particularly PEG-C-DMG, mPEG-C-CLS, DSPE-PEG-Mal, DSPE-PEG-NH2 or mPEG-DSPE, such as PEG2000-C-DMG, mPEG1000-C-CLS, mPEG2000-C-CLS, mPEG5000-C-CLS, mPEG10000-C-CLS and DSPE-PEG2000-Mal, DSPE-PEG5000-NH 2 mPEG2000/5000-DSPE with the structural formula shown as follows:
the PEGylated amphiphilic polymer compound is beneficial to improving the pharmacokinetic property in the nanoparticle body, stabilizing the nanoparticle and facilitating the targeted modification,
in some embodiments, the poloxamer and/or poloxamer and PEG lipid cationic complex nanoparticle may further comprise at least one lipid having the formula:
In some embodiments, the cationic complex nanoparticle composition comprises904、/>L-64、/>304、/>90R4、/>704、/>701、/>17R4、/>P-85、/>L-31、/>One or more of L-61; in some embodiments, the cationic complex nanoparticle composition consists of L64:P10R5:mPEG 2000-DSPE:DSPC:mPEG 5000-C-CLS in a molar ratio of 50:50:3:20:80. In some embodiments of the present invention, in some embodiments,the cationic complex nanoparticle composition consists of +.>PEG2000-C-DMG, PC, mPEG2000-C-CLS in a molar ratio of 100:3:120:80; in one embodiment, the cationic complex nanoparticle composition consists of +.>904∶/>90R4 to DSPE-PEG200-Mal to PC to mPEG2000-C-CLS molar ratio is 50:50:3:20:160; in one embodiment, the cationic complex nanoparticle composition consists of +.>The molar ratio of L61 to mPEG2000-DSPE to DSPC to mPEG5000-C-CLS is 100:3:20:80. In some embodiments, the cationic complex nanoparticle composition consists of +.>The molar ratio of L85 to DSPE-PEG2000-Mal to DSPC to mPEG2000-C-CLS is 100:2:15:80; in some embodiments, the cationic complex nanoparticle composition consists of L31:DSPE-PEG 200-NH 2 The molar ratio of DOPE to mPEG2000-C-CLS is 40:3:20:80; in some embodiments, the cationic complex nanoparticle composition consists of T304:17R 4:mPEG 2000-DSPE:DOTAP:mPEG 2000-C-CLS in a molar ratio of 100:100:1:30:80; in some embodiments, the cationic complex nanoparticle composition consists of T304:PEG 2000-C-DMG: DSPC: mPEG2000-C-CLS in a molar ratio of 100:3:20:80; in some embodiments, the cationic complex nanoparticle composition consists of T304:PEG 2000-C-DMG: DSPC: mPEG2000-C-CLS in a molar ratio of 100:3:20:80.
In some embodiments, the molar ratio of poloxamer or poloxamer to PEG lipid is in the range of (40-200) to (83-163).
In some embodiments, during the preparation of the poloxamer and/or poloxamer-and-lipid combined complexes, the complexes self-assemble into nanoparticles, which are detected as having an average diameter or particle size of less than 1000nm, preferably less than 500nm, for example from about 20 to 300nm. In some embodiments, the complex of poloxamer and/or lipid combination has a polydispersity of 0 to 0.4, in certain embodiments 0.1 to 0.3. In some embodiments, the surface potential of the poloxamer and/or the complex of poloxamer and/or poloxamer and lipid in combination is positively charged, for example, in the range of 5.0mV to 35.0mV, preferably 10.0mV to 25.0mV.
On the other hand, the applicant has found that the poloxamer and/or the poloxamer-lipid combined complex can be used for nucleic acid transfection of in vitro and in vivo cells, and can deliver extracellular nucleic acid into specific cells so as to realize gene expression. Thus, the present invention also provides cationic complex nanoparticles comprising nucleic acids; the entrapment rate of the cationic complex nanoparticle to the nucleic acid is 70% or more, or 85% or more, or 90% or more, for example, about 93.7%, about 90.3%, about 77.6%, about 88.8%, about 89.9%, about 91.4%, about 68.1%, about 88.3%.
In some embodiments, the nucleic acid may be mRNA, RNAi, DNA, or the like, and in one particular embodiment, the nucleic acid is mRNA.
In some embodiments, the nucleic acid is messenger ribonucleic acid (mRNA), which refers to a single-stranded ribonucleic acid (RNA) of the type transcribed from deoxyribonucleic acid (DNA) with genetic information. It serves as a template for protein synthesis on the ribosome, and determines the amino acid sequence of the peptide chain. In vitro transcribed messenger RNA (IVT mRNA) refers to an mRNA transcribed from DNA as a template under in vitro conditions that directs the synthesis of a particular protein to prevent or alter a particular disease.
In a further aspect, the invention provides the use of the poloxamer and/or the poloxamer-mesalamine in combination with lipid complexes for the preparation of an RNA/DNA vaccine drug.
Gene transfection is a technique that transfers or transports nucleic acids with biological functions into cells and maintains the nucleic acids in the cells for their biological functions.
The invention has the advantages that:
according to the invention, poloxamers with different lengths or different polymerization degrees and/or poloxamers with different polymerization degrees and specific PEG lipids are selected to be mixed and freeze-dried in a specific range of proportion, and then nanoparticles can be quickly formed, the obtained nanoparticles have positive electric potential, so that the cationic compound nanoparticles are more stable in and out of the body, the cost of the cationic compound nanoparticles serving as nucleic acid vectors is lower than that of viral vectors, the quality control is convenient, the transfection efficiency is higher than that of a common nano-carrier (lipofectamine 2000 of the industry gold standard thermo Fischer company), the toxicity is low, the biocompatibility is good, no liquid change is needed after the drug is administered in an in vitro experiment, the transfection in vitro and in vivo is particularly suitable, the transfection in vitro is effective, the preparation is simple, and the problem that nucleic acid, especially mRNA is safely and efficiently delivered in the in vitro is creatively solved.
The cationic complex nanoparticle of poloxamer and/or poloxamer and PEG lipid is mainly used for gene transfection of in-vitro and in-vivo cells, and can deliver extracellular genes into specific cells so as to realize gene expression. The poloxamer and/or the poloxamer and lipid combined cationic complex nanoparticle can be used for scientific research, particularly for preparing a gene transfection kit or a cell labelling kit, can also be applied to the field of novel RNA/DNA vaccine medicines, and particularly for the development of mRNA tumor vaccines and mRNA influenza virus vaccines; creatively solves the problem of safe and high-efficiency in-vivo and in-vitro delivery of nucleic acid.
Drawings
FIG. 1 shows the fluorescence expression intensity of transfection in vitro cells after mixing FLuc-mRNA and the compound of formula 1 according to the examples of the present invention in different mass ratios.
FIG. 2 shows the fluorescence expression intensity of transfection in vitro cells after mixing FLuc-mRNA and the prescribed 2 complex according to the example of the present invention at different mass ratios.
FIG. 3 shows the fluorescence expression intensity of transfection in vitro cells after mixing FLuc-mRNA and the prescribed 3 complex according to the example of the present invention at different mass ratios.
FIG. 4 shows the fluorescence expression intensity of transfection in vitro cells after mixing the FLuc-mRNA and the prescribed 4 complex according to the example of the present invention at different mass ratios.
FIG. 5 shows the fluorescence expression intensity of transfection in vitro cells after mixing the FLue-mRNA and the prescribed 5 complex according to the example of the present invention at different mass ratios.
FIG. 6 shows the fluorescence expression intensity of transfection in vitro cells after mixing the FLuc-mRNA and the prescribed 6 complex according to the example of the present invention at different mass ratios.
FIG. 7 shows the fluorescence expression intensity of transfection in vitro cells after mixing the FLuc-mRNA and the compound of formula 7 according to the example of the present invention in different mass ratios.
FIG. 8 shows the fluorescence expression intensity of transfection in vitro cells after mixing FLuc-mRNA and the prescribed 8 complex according to the example of the present invention at different mass ratios.
FIG. 9 shows the fluorescence expression intensity of transfection in DC2.4 cells after mixing with FLuc-mRNA at optimal mass ratio according to different formulations of the examples of the present invention.
FIG. 10 shows the fluorescence expression intensity of transfection in DC2.4 cells after mixing with Luc-pDNA at optimal mass ratios according to the different formulations of the examples of the present invention.
FIG. 11 shows the subcutaneous injection of vaccine in C57BL/6J mice after mixing with 5ug 0VA-mRNA according to the optimal mass ratio according to different prescriptions in the embodiment of the invention, and the treatment effect of the vaccine on melanoma is examined.
Detailed Description
Purchased from BASF corporation->Purchased from SigmaAldrich; />904 is abbreviated as T904, >L-64 is abbreviated as L-64, ">304 is abbreviated as T304, te->90R4 is abbreviated as T90R4,704 is abbreviated as T704,>17R4 is abbreviated as 17R4, P->P-85 is abbreviated as P-85, ">701 is abbreviated as T701, (-)>L-61 is abbreviated as L-61, plu->L-31 is abbreviated as L31.
Embodiment one: preparation of cationic Complex nanoparticle LLLRNA1003 prescriptions
Prescription 1: the mole ratio of T304 to PEG2000-C-DMG to DSPC to mPEG2000-C-CLS is 100:3:20:80
Firstly, taking out T304 from a refrigerator at 4 ℃ to be balanced to room temperature, weighing and adding nuclease-removed ultrapure water at the room temperature for dissolution, fully oscillating for 5min by a mediation instrument, and standing overnight to obtain a stock solution A; PEG2000-C-DMG, DSPC and mPEG2000-C-CLS were taken out of the refrigerator at-20deg.C, equilibrated to room temperature and unsealed, weighed at room temperature in a molar ratio of 3:20:80, and dissolved in chloroform in a round bottom flask. Chloroform was removed by evaporation using a rotary evaporator, stock solution a was poured into a round bottom flask, sonicated for 60 minutes at 40 degrees celsius with 2 seconds of sonication for 2 seconds of suspension, transferred into a dialysis bag with MWCO 5000, dialyzed against nuclease-depleted ultrapure water for 12 hours, and the dialysate was changed every 4 hours. Filtering with 0.22um aqueous filter membrane after dialysis to obtain stock solution B, and mixing the stock solution A and the stock solution B according to the mol ratio of 100:3:20:80 to obtain the prescription 1 aqueous solution. The aqueous solution of the prescription 1 is prepared into a freeze-drying agent by a freeze-drying machine and is stored in a refrigerator at the temperature of 4 ℃ for standby.
Prescription 2: the mole ratio of T304:17R 4:mPEG 2000-DSPE to DOTAP to mPEG2000-C-CLS is 100:100:1:30:80
Firstly, taking out T304 and 17R4 from a refrigerator at 4 ℃ to be balanced to room temperature, weighing and adding ultrapure water with nuclease at the room temperature according to the mol ratio of 1:1 for dissolving, fully oscillating for 5min by a mediation instrument, and standing overnight to obtain stock solution A; the mPEG2000-DSPE, DOTAP and mPEG2000-C-CLS were taken out from the refrigerator at-20℃and equilibrated to room temperature and unsealed, weighed at room temperature in a molar ratio of 1:30:80 and dissolved in chloroform in a round bottom flask. Chloroform was removed by evaporation using a rotary evaporator, stock solution a was poured into a round bottom flask, sonicated for 60 minutes at 40 degrees celsius with ultrasound for 3 seconds for 2 seconds, transferred to a dialysis bag with MWCO 5000, dialyzed against nuclease-depleted ultrapure water for 24 hours, and the dialysate was changed every 4 hours. Filtering with 0.22um aqueous filter membrane after dialysis to obtain stock solution B, and mixing the stock solution A and the stock solution B according to the molar ratio of 200:1:30:80 to obtain prescription 2. The prepared aqueous solution of prescription 2. The aqueous solution of prescription 2 is prepared into a freeze-dried agent by a freeze-drying machine and is stored in a refrigerator at 4 ℃ for standby.
Prescription 3: L31:DSPE-PEG 200-NH 2 The molar ratio of DOPE to mPEG2000-C-CLS is 40:3:20:80
Taking L31 out of a refrigerator at 4 ℃ to be balanced to room temperature, weighing and adding nuclease-removed ultrapure water at room temperature for dissolving, fully oscillating for 5min by a mediation instrument, and standing overnight to obtain a stock solution A; DSPE-PEG200-NH2, DOPE and mPEG2000-C-CLS were taken out of the refrigerator at-20deg.C, equilibrated to room temperature and unsealed, weighed at room temperature in a molar ratio of 3:20:80, and dissolved in chloroform in a round bottom flask. Chloroform was removed by evaporation using a rotary evaporator, stock solution a was poured into a round bottom flask, sonicated for 60 minutes at 40 degrees celsius with 2 seconds of sonication for 3 seconds of suspension, transferred into a dialysis bag with MWCO 5000, dialyzed against nuclease-depleted ultrapure water for 12 hours, and the dialysate was changed every 4 hours. Filtering with 0.22um aqueous filter membrane after dialysis to obtain stock solution B, and mixing the stock solution A and the stock solution B according to the mol ratio of 40:3:20:80 to obtain the prescription 3 aqueous solution. The aqueous solution of prescription 3 is prepared into a freeze-dried agent by a freeze-drying machine and is stored in a refrigerator at the temperature of 4 ℃ for standby.
Prescription 4: the molar ratio of L85 to DSPE-PEG2000-Mal to DSPC to mPEG2000-C-CLS is 100:2:15:80
Taking L85 out of a refrigerator at 4 ℃ to be balanced to room temperature, weighing and adding nuclease-removed ultrapure water at room temperature for dissolving, fully oscillating for 5min by a mediation instrument, and standing overnight to obtain a stock solution A; DSPE-PEG2000-Mal, DSPC and mPEG2000-C-CLS were taken out of the refrigerator at-20deg.C, equilibrated to room temperature and unsealed, weighed at room temperature in a molar ratio of 2:15:80, and dissolved in chloroform in a round bottom flask. Chloroform was removed by evaporation using a rotary evaporator, stock solution a was poured into a round bottom flask, sonicated for 60 minutes at 40 degrees celsius with 3 seconds of sonication for 3 seconds of suspension, transferred into a dialysis bag with MWCO 5000, dialyzed against nuclease-depleted ultrapure water for 12 hours, and the dialysate was changed every 4 hours. Filtering with 0.22um aqueous filter membrane after dialysis to obtain stock solution B, and mixing the stock solution A and the stock solution B according to the mol ratio of 100:2:15:80 to obtain the prescription 4 aqueous solution. The aqueous solution of prescription 4 is prepared into a freeze-dried agent by a freeze-drying machine and is stored in a refrigerator at the temperature of 4 ℃ for standby.
Prescription 5: the molar ratio of T701 to PEG2000-C-DMG to PC to mPEG2000-C-CLS is 100:3:120:80
Firstly, taking out T701 from a refrigerator at 4 ℃ to be balanced to room temperature, weighing and adding nuclease-removed ultrapure water at room temperature to dissolve, fully oscillating for 5min by a mediation instrument, and standing overnight to obtain a stock solution A; PEG2000-C-DMG, PC and mPEG2000-C-CLS were taken out of the freezer at-20℃and equilibrated to room temperature and unsealed, weighed at room temperature in a molar ratio of 3:120:80 and dissolved in chloroform in a round bottom flask. Chloroform was removed by evaporation using a rotary evaporator, stock solution a was poured into a round bottom flask, sonicated for 60 minutes at 40 degrees celsius with 2 seconds of sonication for 2 seconds of suspension, transferred into a dialysis bag with MWCO 5000, dialyzed against nuclease-depleted ultrapure water for 12 hours, and the dialysate was changed every 4 hours. Filtering with 0.22um aqueous filter membrane after dialysis to obtain stock solution B, and mixing the stock solution A and the stock solution B according to the mol ratio of 100:3:120:80 to obtain the prescription 5 aqueous solution. The aqueous solution of prescription 5 is prepared into a freeze-dried agent by a freeze-drying machine and is stored in a refrigerator at 4 ℃ for standby.
Prescription 6: T904:90R 4:DSPE-PEG 200-Mal:PC:mPEG 2000-C-CLS molar ratio of 50:50:3:20:160
Firstly, taking out T904 and T90R4 from a refrigerator at 4 ℃ to be balanced to room temperature, weighing and adding ultrapure water with nuclease at the room temperature according to the mol ratio of 1:1 for dissolving, fully oscillating for 5min by a rotary instrument, and standing overnight to obtain a stock solution A; DSPE-PEG200-Mal, PC and mPEG2000-C-CLS were taken out of the refrigerator at-20deg.C, equilibrated to room temperature and unsealed, weighed at room temperature in a molar ratio of 3:20:160, and dissolved in chloroform in a round bottom flask. Chloroform was removed by evaporation using a rotary evaporator, stock solution a was poured into a round bottom flask, sonicated for 60 minutes at 40 degrees celsius with 2 seconds of sonication for 3 seconds of suspension, transferred into a dialysis bag with MWCO 5000, dialyzed against nuclease-depleted ultrapure water for 12 hours, and the dialysate was changed every 4 hours. Filtering with 0.22um aqueous filter membrane after dialysis to obtain stock solution B, and mixing the stock solution A and the stock solution B according to the mol ratio of 100:3:20:160 to obtain the prescription 6 aqueous solution. The aqueous solution of prescription 6 is prepared into a freeze-dried agent by a freeze-drying machine and is stored in a refrigerator at the temperature of 4 ℃ for standby.
Prescription 7: the molar ratio of L61 to mPEG2000-DSPE to DSPC to mPEG5000-C-CLS is 100:3:20:80
Taking L61 out from a refrigerator at 4 ℃ to be balanced to room temperature, weighing and adding nuclease-removed ultrapure water at room temperature for dissolving, fully oscillating for 5min by a mediation instrument, and standing overnight to obtain a stock solution A; the mPEG2000-DSPE, DSPC and mPEG5000-C-CLS were taken out from the refrigerator at-20 ℃, equilibrated to room temperature and unsealed, weighed at room temperature in a molar ratio of 3:20:80, and dissolved in chloroform in a round bottom flask. Chloroform was removed by evaporation using a rotary evaporator, stock solution a was poured into a round bottom flask, sonicated for 30 minutes at 40 degrees celsius with 2 seconds of sonication for 2 seconds of suspension, transferred into a dialysis bag with MWCO 5000, dialyzed against nuclease-depleted ultrapure water for 12 hours, and the dialysate was changed every 4 hours. Filtering with 0.22um aqueous filter membrane after dialysis to obtain stock solution B, and mixing the stock solution A and the stock solution B according to the mol ratio of 100:3:20:80 to obtain the aqueous solution of the prescription 7. The aqueous solution of prescription 7 is prepared into a freeze-dried agent by a freeze-drying machine and is stored in a refrigerator at 4 ℃ for standby.
Prescription 8: the molar ratio of L64 to P10R5 to mPEG2000-DSPE to DSPC to mPEG5000-C-CLS is 50:50:3:20:80
Taking out L64 and P10R5 from a refrigerator at 4 ℃ to be balanced to room temperature, weighing and adding nuclease-removed ultrapure water at the room temperature according to a molar ratio for dissolving, fully oscillating for 5min by a cyclone, and standing overnight to obtain a stock solution A; the mPEG2000-DSPE, DSPC and mPEG5000-C-CLS were taken out from the refrigerator at-20 ℃, equilibrated to room temperature and unsealed, weighed at room temperature in a molar ratio of 3:20:80, and dissolved in chloroform in a round bottom flask. Chloroform was removed by evaporation using a rotary evaporator, stock solution a was poured into a round bottom flask, sonicated for 30 minutes at 40 degrees celsius with 3 seconds of sonication for 3 seconds of suspension, transferred into a dialysis bag with MWCO 5000, dialyzed against nuclease-depleted ultrapure water for 12 hours, and the dialysate was changed every 4 hours. Filtering with 0.22um aqueous filter membrane after dialysis to obtain stock solution B, and mixing the stock solution A and the stock solution B according to the mol ratio of 100:3:20:80 to obtain the prescription 8 aqueous solution. The aqueous solution of prescription 8 is prepared into a freeze-dried agent by a freeze-drying machine and is stored in a refrigerator at 4 ℃ for standby.
Embodiment two: characterization of cationic complex nanoparticle formulations of poloxamers and/or poloxamers in combination with lipids
The present invention relates to a method for measuring nanoparticle size and Potential using Malvem Zetasizer Nano ZSE, wherein cationic complex nanoparticles of prescriptions 1, 2, 3, 4, 5, 6, 7 and 8, which do not contain FLuc-mRNA, are prepared into 1ml of a solution to be measured, and the particle size (transmittance Mean), surface Potential (Zeta Potential) and polydispersity (pdi) of the cationic complex nanoparticles of prescriptions 1, 2, 3, 4, 5, 6, 7 and 8, which do not contain FLuc-mRNA, are examined under the condition of 25 ℃, as shown in table 1.
Weighing freeze-dried agents of a formula 1, a formula 2, a formula 3, a formula 4, a formula 5, a formula 6, a formula 7 and a formula 8 which are stored for 6 months at 4 ℃ according to optimal mass ratios of the formula nanoparticles and the FLuc-mRNA (wherein the optimal mass ratio of the formula 1 is 5000, the optimal mass ratio of the formula 2 is 5000, the optimal mass ratio of the formula 3 is 3500, the optimal mass ratio of the formula 4 is 100, the optimal mass ratio of the formula 5 is 1500, the optimal mass ratio of the formula 6 is 5000, the optimal mass ratio of the formula 7 is 2000 and the optimal mass ratio of the formula 8 is 500), adding 500ul of nuclease water for re-dissolution for 10 minutes, and respectively adding and mixing 500ul of the nuclease aqueous solution containing 200ng of the FLuc-mRNA for 10 minutes after blowing for several times to prepare the cationic compound nanoparticle containing the FLuc-mRNA. The size (transmittance Mean), surface Potential (Zeta Potential) and Polydispersity (PDI) of the dynamic light scattering nanoparticles of the cationic complex nanoparticles containing FLuc-mRNA were tested using Malvern Zetasizer Nano ZSE as shown in table 2.
Table 1: prescription 1, prescription 2, prescription 3, prescription 4, prescription 5, prescription 6, prescription 7, and prescription 8 do not contain the particle size (affinity Mean), surface Potential (Zeta Potential), and Polydispersity (PDI) of the mR NA-free cationic complex nanoparticles.
Table 2: prescription 1, prescription 2, prescription 3, prescription 4, prescription 5, prescription 6, prescription 7, and prescription 8 cationic complex nanoparticles containing mrna have a particle size (affinity Mean), a surface Potential (Zeta Potential), and a Polydispersity (PDI).
Embodiment III: testing the entrapment Rate of cationic Complex nanoparticles of poloxamer and/or Poloxamine in combination with lipid
The encapsulation efficiency of LLLRNA-1003 on FLuc-mRNA was determined using the Quant-iT riboGreen RNA detection kit (ThermoFischer Co.), each of the prescriptions (prescriptions 1, 2, 3, 4, 5, 6, 7, and 8), and the method of the present invention was as follows:
weighing freeze-drying agents of a cation composite nanoparticle which is preserved for 6 months at 4 ℃ and does not contain FLuc-mRNA according to the optimal mass ratio of each prescription nanoparticle to FLuc-mRNA (5000 in the optimal mass ratio of prescription 1, 5000 in the optimal mass ratio of prescription 2, 3500 in the optimal mass ratio of prescription 3, 100 in the optimal mass ratio of prescription 4, 1500 in the optimal mass ratio of prescription 5, 5000 in the optimal mass ratio of prescription 6, 2000 in the optimal mass ratio of prescription 7 and 500 in the optimal mass ratio of prescription 8), adding 500ul of nuclease water for re-dissolution for 10 minutes respectively, and adding and mixing 500ul of nuclease aqueous solution containing 200ng of FLuc-mRNA for 10 minutes after blowing for several times to prepare the cation composite nanoparticle containing FLuc-mRNA. Centrifuging each prescription at 4 ℃ and 16000rpm for 2.5 hours by using a low-temperature high-speed centrifuge, collecting supernatant and quantifying the volume thereof by using a pipette, and recording as V1; measuring the concentration of FLuc-mRNA in the supernatant with the Quant-iT RiboGreen RNA detection kit, designated C1; dissolving the centrifuged precipitate in 1ml of DMSO, taking 100ul of the solution, adding 200ul of heparin sodium solution (6.667 mg/m 1), uniformly mixing, standing at room temperature for 2 hours, recording the replaced volume V2, and measuring the concentration of FLuc-mRNA by using a Quant-iT riboGreen RNA detection kit, and marking as C2; the calculation formula of the entrapment rate of each prescription of LLLRNA-1003 is as follows:
The entrapment rate=100% - (v1c1)/(v1c1+v2c2) ×100%
The inclusion rates of the prescription 1, the prescription 2, the prescription 3, the prescription 4, the prescription 5, the prescription 6, the prescription 7 and the prescription 8 are respectively as follows: 93.7%, 90.3%, 77.6%, 88.8%, 89.9%, 91.4%, 68.1%, 88.3%.
Embodiment four: transfection of different prescriptions in DC2.4 cells after mixing the optimal mass ratio of material to FLuc-mRNA
1) Cell collection
The cells were removed from the incubator, the culture broth was discarded, 10ml of PBS was added, the flask was gently shaken to flow over the cell surface, PBS was discarded, about 2ml of a digestive fluid (0.25% trypsin-0.03% EDTA solution) was added to the flask, and after gentle shaking, the flask was digested for 30 seconds, and observed under a microscope to find cytoplasmic shrinkage, and immediately after increasing the cell gap, the digestion was stopped by adding a serum-containing culture broth. Sucking the culture solution in the bottle by using a suction pipe, repeatedly lightly blowing the bottle wall cells to form single cell suspension, adding the cell suspension into a 15ml centrifuge tube, centrifuging for 5min at 1500 rpm, removing supernatant, re-suspending the cells by using a culture medium, counting on a counting plate, and regulating the concentration of the cell suspension by using the culture medium for later use.
2) Gene transfection of FLuc-mRNA in cells in vitro
The cell suspension was mixed at 4X 10 4 Packing each well to 96-well plate, placing into 37 deg.C and 5% CO 2 And (5) standing and culturing in an incubator. After 24h, FLuc-mRNA at a concentration of 1ug/ul was diluted to 0.1ug/ul with nuclease-free ultrapure water, mixed with the reconstituted and different concentrations of the lyophilized agents of prescription 1, prescription 2, prescription 3, prescription 4, prescription 5, prescription 6, prescription 7 and prescription 8 at different mass ratios of 200ng FLuc-mRNA per well to obtain 88ul of a cationic complex nanoparticle prescription mixture containing FLuc-mRNA, and after standing for 30min, the mixture was added to a 96-well plate containing 180ul of complete medium per well at a volume of 20ul per well, and 4 wells were repeated for each sample using FLuc-mRNA and bare FLuc-mRNA as two positive control groups, each packaged with lipofectamine 2000 from thermo fischer company.
After 24h of administration, a D-Luciferin stock solution with a concentration of 25mg/ml was prepared with DPBS, and the stock solution was used immediately after mixing or stored at-20℃after packaging. D-Luciferin was diluted 1:100 with pre-warmed complete medium to a working concentration of 250ug/ml and the culture broth was aspirated from the 96-well plate. 100ul of D-Luciferin working solution was added to each 96-well plate before imaging, and incubation was continued for 5min in an incubator at 37℃and mixed with prescriptions 1-8 at a concentration of 200ng FLuc-mRNA per well in a mass ratio of 50, 100, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000, respectively, with reference groups of naked mRNA and Lipo 2000, and the fluorescence expression intensity of FLuc-mRNA was tested by imaging with an Omega FLuostar microplate reader. The results are shown in FIGS. 1-8: the abscissa represents the mass ratio of prescription to mRNA, the ordinate is fluorescence intensity, and the ordinate is higher, indicating higher transfection efficiency, and the data indicate that the optimum mass ratio of FLuc-mRNA to prescription 1 is 5000, the optimum mass ratio to prescription 2 is 5000, the optimum mass ratio to prescription 3 is 3500, the optimum mass ratio to prescription 4 is 100, the optimum mass ratio to prescription 5 is 1500, the optimum mass ratio to prescription 6 is 5000, the optimum mass ratio to prescription 7 is 2000, and the optimum mass ratio to prescription 8 is 500.
Transfection experiments in DC2.4 cells were repeated using the above method at the optimal mass ratio for each prescription, and the results of the experiments are shown in FIG. 9.
Fifth embodiment: transfection of different prescriptions in DC2.4 cells after mixing the materials at optimal mass ratio with Luc-pDNA
The cell suspension was mixed at 4X 10 4 The density of each well was packed in 96-well plates, and the plates were placed in a 5% CO2 incubator at 37℃for stationary culture. After 24 hours, the Luc-pDNA having a concentration of 1ug/ul was diluted to 0.1ug/ul with nuclease-free ultrapure water, the mixed solution was mixed with each well of 300ng of Luc-pDNA at a concentration of 300 ul with each well of reconstituted prescription 1 (mass ratio: 5000), prescription 2 (mass ratio: 5000), prescription 3 (mass ratio: 3500), prescription 4 (mass ratio: 100), prescription 5 (mass ratio: 1500), prescription 6 (mass ratio: 5000), prescription 7 (mass ratio: 2000) and prescription 8 (mass ratio: 500) at a mass ratio of the highest protein expression, respectively, to obtain 88ul of a cationic complex nanoparticle prescription mixture containing Luc-pDNA, and after standing for 30 minutes, the mixed solution was added to each well of 96-well plates containing 180ul of complete medium at a volume of 20ul, respectively, and each sample was repeated for 4 wells with lipofectamine 2000 of Thermoscher company and its prescription as positive controls, and wells without Luc-pDNA as negative controls.
After 24h of dosing, 100ul of D-Luciferin solution with working concentration of 250ug/ml was added to each 96-well plate, the culture was continued for 5min in an incubator at 37℃and finally imaged with an Omega-FLuostar, the fluorescence expression intensity of Luc-pDNA was tested, the test was repeated every 24 hours, the medium containing D-Luciferin was aspirated after each test was completed, fresh complete medium was added for 24 hours, and D-Luciferin was added for four days. The results are shown in FIG. 10.
EXAMPLE six treatment of tumor-bearing mouse models with the cationic Complex nanoparticle LLLRNA1003-OVA-mRNA vaccine of the present invention
1) Establishment of B16-OVA melanoma mouse model: amplifying and culturing murine lymphoma cell B16-OVA in vitro to obtain B16-OVA cell line, diluting with DPBS, and beating 5×10 mice each 5 And (3) tumor cells. Female C57BL/6J mice of 7 weeks of age were dehaired on day 0, cultured B16-OVA tumor cells were collected, and B16-OVA tumor cells were subcutaneously injected into the flank of mice to establish a subcutaneous B16-OVA tumor model.
2) Preparation of LLLRNA1003-OVA-mRNA vaccine: the re-dissolved four LLLRNA1003-OVA-mRNA vaccines prepared by the prescription 1, the prescription 2, the prescription 5 and the prescription 6 are respectively obtained after the re-dissolved prescription 1, the prescription 2, the prescription 5 and the prescription 6 are respectively and simply mixed with OVA-mRNA (purchased from TriLink company in the U.S.) lightly for 30 minutes;
3) C57BL/6J mice were vaccinated with LLLRNA1003-OVA-mRNA vaccine (each time with 5ug of nanoparticle vaccine containing therapeutic mRNA-OVA, diluted with 9% physiological saline buffer before injection) by sole injection on day 5, day 8, day 11, day 14, day 17, and day 20, respectively, while mice vaccinated with equal volumes of PBS buffer solution and the same volume of OVA-mRNA solution after dilution were set as control groups, and 5 mice per group were vaccinated.
4) Tumor vertical diameters were measured daily starting on day 10 post tumor inoculation. Tumor volumes of C57BL/6J mice were calculated according to the following formula: v (mm 3) =x×y 2 And/2, in mm, wherein V represents the tumor volume, x represents the tumor major diameter, and y represents the tumor minor diameter. The change in body weight of the C57BL/6J mice was recorded daily with an electronic balance and the survival rate was counted. The examination results are shown in fig. 11: B16-OVA melanoma cells were inoculated subcutaneously on day 0 and vaccinated on days 5, 8, 11, 14, 17 and 20 after tumor inoculation, respectively. LLLRNA1003 vaccine prepared from prescription 1, prescription 2, prescription 5 and prescription 6 on day 11 post-tumor inoculationThe group showed tumor growth starting on day 12, whereas the PBS control group and the naked mRNA group showed sarcoma starting on day 12. From day 13, the LLLRNA1003 vaccine group prepared from prescription 5 and prescription 6 started to see the tumor. From day 14, tumors were seen starting from the LLLRNA1003 vaccine group prepared from prescription 1 and prescription 2. The LLLRNA1003 vaccine group prepared from prescription 1, prescription 2, prescription 5 and prescription 6 showed a significant tumor growth delay compared to the PBS control group and the naked mRNA group. On day 20, the LLLRNA1003 vaccine group prepared from prescription 1, prescription 2, prescription 5 and prescription 6 had smaller tumor sizes than the PBS control group and the naked mRNA group. PBS control and naked mRNA groups started on day 21 and day 25, respectively, after tumor inoculation, all mice were sacrificed at day 30 and day 33, respectively. The LLLRNA1003 vaccine groups prepared from prescription 1, prescription 2, prescription 5 and prescription 6 began to sacrifice from day 39, day 29, day 27 and day 35, respectively. All mice in the LLLRNA1003 vaccine group prepared at prescription 2, prescription 5 and prescription 6 were sacrificed on day 38, day 36 and day 41, respectively. The survival rate of the LLLRNA1003 vaccine group prepared in formulation 1 was continued to be counted until day 44.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (4)

1. The cationic complex nanoparticle combination of poloxamer and/or poloxamer and PEG lipid is characterized in that the cationic complex nanoparticle combination consists of Pluronic L64:Pluronic P-10R5:mPEG2000-DSPE:DSPC:mPEG5000-C-CLS with the molar ratio of 50:50:3:20:80; or the cationic compound nanoparticle composition consists of Tetronic (701:PEG 2000-C-DMG) and PC (PC) in the molar ratio of mPEG2000-C-CLS of 100:3:120:80; or the cationic compound nanoparticle composition consists of Tetronic (g) 304:PEG2000-C-DMG (DSPC) and mPEG2000-C-CLS with the molar ratio of 100:3:20:80; wherein:
。
2. use of cationic complex nanoparticles of poloxamer and/or poloxamine in combination with PEG lipids according to claim 1 for the preparation of a nucleic acid transfection reagent for cells in vitro and in vivo.
3. The use according to claim 2, characterized in that the nucleic acid is mRNA, siRNA, DNA.
4. Use of the cationic complex nanoparticle of claim 1 for the preparation of a medicament for RNA/DNA vaccine.
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