CN114869859A - Nucleic acid nanoparticle compound and preparation method thereof - Google Patents

Nucleic acid nanoparticle compound and preparation method thereof Download PDF

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CN114869859A
CN114869859A CN202210655033.4A CN202210655033A CN114869859A CN 114869859 A CN114869859 A CN 114869859A CN 202210655033 A CN202210655033 A CN 202210655033A CN 114869859 A CN114869859 A CN 114869859A
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nucleic acid
peg
nanoparticle
derivative
poloxamine
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张龙贵
刘晨
梁梅桂
张弛
王宇恒
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Shenzhen Houcun Nano Pharmaceutical Co ltd
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Shenzhen Houcun Nano Pharmaceutical Co ltd
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Abstract

The invention relates to a nucleic acid nanoparticle compound and a preparation method thereof, belonging to the field of biological medicine. The nanoparticle comprises a compound shown as a formula I and an auxiliary material. The nanoparticle can encapsulate nucleic acid, and has the advantages of low toxicity, high encapsulation rate, good transfection effect and high bioavailability. The preparation method is simple to operate, low in cost, environment-friendly and beneficial to industrial production.

Description

Nucleic acid nanoparticle compound and preparation method thereof
Technical Field
The invention relates to the field of biological medicine, and particularly relates to a nucleic acid nanoparticle compound and a preparation method thereof.
Background
Gene transfection is a technique by which nucleic acids having a biological function are transferred or transported into a cell and the nucleic acids are maintained in the cell for their biological function. A gene vector refers to a means for introducing a foreign therapeutic gene into a biological cell. At present, the gene vectors with industrial transformation potential internationally are mainly viral vectors and non-viral vectors.
The viral vector is a gene delivery tool for transmitting the genome of a virus into other cells for infection, and has better application prospects such as lentivirus, adenovirus, retrovirus vector, adeno-associated virus vector and the like. However, due to its inherent physicochemical properties and biological activities, viral vectors have serious disadvantages, such as high production cost, limited loading capacity, poor targeting, insertion integration, teratogenic and mutagenic properties, and are not conducive to the development of universal and general therapies.
Non-viral vectors include mainly: liposome nanoparticles, composite nanoparticles, cationic polymer nanoparticles, polypeptide nanoparticles and the like. The liposome nanoparticles are the main non-viral vectors currently applied to RNA drug development, the first RNAi drug (Patisiran) and the first mRNA drug (BNT162b2, Comirnaty) are sequentially listed at present, and the clinical application value of the Liposome Nanoparticles (LNP) is fully verified. Compared with viral vectors, the liposome nanoparticles have the advantages of low production cost, definite chemical structure, convenience for quality control, realization of targeted drug delivery through targeted modification, theoretically unlimited entrapment amount and the like, but most liposome lipid materials are not degradable and have high toxicity, so that the clinical requirement of repeated drug delivery is difficult to meet, and in addition, the problems of poor in vivo transfection effect, metabolism or elimination of nucleic acid in serum, poor bioavailability and the like exist.
Therefore, there is still a need for nanoparticles with low toxicity, good transfection effect and good bioavailability.
Disclosure of Invention
Summary of The Invention
The invention aims to provide a nanoparticle which can encapsulate nucleic acid and has the advantages of low toxicity, high encapsulation rate, good transfection effect and good bioavailability. In order to achieve the purpose, the invention provides the following technical scheme.
In a first aspect, the present invention provides a nanoparticle. The nanoparticle comprises a compound shown as a formula I and an auxiliary material.
In a second aspect, the present invention provides a nucleic acid nanoparticle complex comprising a nucleic acid and a nanoparticle of the first aspect.
In a third aspect, the present invention provides a pharmaceutical composition comprising the nanoparticle complex of the second aspect and a pharmaceutically acceptable excipient.
In a fourth aspect, the present invention provides a method for preparing the nanoparticle of the first aspect.
In a fifth aspect, the present invention provides a use of a nanoparticle according to the first aspect or a nanoparticle complex according to the second aspect.
Detailed Description
In order to solve the above problems, the present invention provides the following technical solutions.
In a first aspect, the present invention provides a nanoparticle.
A nanoparticle, comprising: a compound as shown in formula I and auxiliary materials,
Figure BDA0003689084690000021
wherein n is selected from the group consisting of integers from 5 to 20, preferably from 12 to 18
In some embodiments, the compound of formula I is a compound as shown in NBD003, NBD005, or NBD008,
Figure BDA0003689084690000022
the auxiliary material comprises a material selected from: at least one of a PEG derivative, a lipid-like, an alcohol, or an inorganic salt. In some embodiments, the auxiliary material comprises a material selected from: at least one of a PEG derivative, a lipid, and a lipoid. In some embodiments, the auxiliary material comprises a material selected from: at least two of a PEG derivative, a lipid, and a lipoid. In some embodiments, the adjunct materials include PEG derivatives, lipids, and lipidoids. In some embodiments, the adjunct material comprises a PEG derivative and a lipid. In some embodiments, the adjunct material comprises a lipid and a lipid-like.
The PEG derivative may include at least one selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. In some embodiments, the PEG derivative comprises a PEG selected from 1, 2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (DMG-PEG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), dilauroyl phosphatidylethanolamine-polyethylene glycol (PEG-DLPE), dimyristoyl phosphatidylethanolamine-polyethylene glycol (PEG-DMPE), dipalmitoyl phosphatidylcholine polyethylene glycol (PEG-DPPC), dipalmitoyl phosphatidylethanolamine-polyethylene glycol (PEG-DPPE), PEG-distearoyl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioyl, PEG-distearyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE) or PEG-1, 2-dimyristoyloxypropan-3-amine (PEG-c-DMA).
PEG-modified ceramides may include a group selected from PEG-CerC 14 Or PEG-CerC 20 。
The 1, 2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (DMG-PEG) may comprise a compound selected from 1, 2-dimyristoyl-sn-glycerol methoxypolyethylene glycol 2000(DMG-PEG 2000).
The lipid may comprise a lipid selected from lecithin (PC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-diundecabonyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol (Chol), (2, 3-dioleoyl-propyl) -trimethylamine sulfate (DOTAP), coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, or alpha-tocopherol. In some embodiments, the lipid comprises a lipid selected from the group consisting of lecithin (PC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-diundecapryl-sn-glycero-phosphocholine (DUPC), At least one or at least two or at least three of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol (Chol), (2, 3-dioleoyl-propyl) -trimethylamine sulfate (DOTAP). In some embodiments, the lipid is 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), lecithin (PC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), cholesterol (Chol).
The lipid may comprise a lipid selected from poloxamine or a poloxamine derivative.
The poloxamines may include compounds selected from
Figure BDA0003689084690000031
304、
Figure BDA0003689084690000032
701、
Figure BDA0003689084690000033
704、
Figure BDA0003689084690000034
707、
Figure BDA0003689084690000035
803、
Figure BDA0003689084690000036
901、
Figure BDA0003689084690000037
904、
Figure BDA0003689084690000038
908、
Figure BDA0003689084690000039
1107、
Figure BDA00036890846900000310
1301、
Figure BDA00036890846900000311
1304、
Figure BDA00036890846900000312
1307、
Figure BDA00036890846900000313
90R4 or
Figure BDA00036890846900000314
150R 1.
The poloxamine derivatives may include those selected from the group consisting of poloxamine derivatives T304-T, poloxamine derivatives T304-D, poloxamine derivatives T304-RT, poloxamine derivatives T304-RC, poloxamine derivatives T701-R, poloxamine derivatives T901-C, poloxamine derivatives T803-RT, poloxamine derivatives T304-RT, poloxamine derivatives T704-M, at least one of a poloxamine derivative T704-RT, a poloxamine derivative T704-RC, a poloxamine derivative T901-C, a poloxamine derivative T904-CR, a poloxamine derivative T904-RC, a poloxamine derivative T904-RT, a poloxamine derivative T90R4-R and a poloxamine derivative T90R4-RT (the structural formula of each poloxamine derivative can be seen in CN 111285845B). The alcohol comprises an aqueous solution of alcohol at a concentration greater than 2% vol. In some embodiments, the alcohol comprises an aqueous solution selected from ethanol or ethanol at a concentration greater than 2% vol (e.g., ≧ 10% vol, ≧ 20% vol, ≧ 27% vol, or 20% vol-30% vol).
The compound of formula I may be present in an amount of about 30.0 wt% to about 60.0 wt%, based on the total mass of the nanoparticle. In some embodiments, the compound of formula I can be present in an amount of about 27.5 wt% to about 55.9 wt%, based on the total mass of the nanoparticle; in some embodiments, the compound of formula I is present in an amount of about 30 wt%, 33 wt%, 35 wt%, 38 wt%, 40 wt%, 43 wt%, 45 wt%, 48 wt%, 51 wt%, 54 wt%, 57 wt%, based on the total mass of the nanoparticle.
The content of the PEG derivative may be 0-15.0 wt% calculated on the total mass of the nanoparticle; in some embodiments, the PEG derivative can be present in an amount of 5.0 wt% to 11.2 wt% based on the total mass of the nanoparticle; in some embodiments, the PEG derivative is present in an amount of 0, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, based on the total mass of the nanoparticle.
The lipid content may be 30 wt% to 70 wt% based on the total mass of the nanoparticle. In some embodiments, the lipid may be present in an amount of about 33 wt% to about 60 wt%, based on the total mass of the nanoparticle; in some embodiments, the lipid is present in an amount of 35 wt%, 40 wt%, 41 wt%, 42 wt%, 43 wt%, 44 wt%, 45 wt%, 46 wt%, 47 wt%, 48 wt%, 49 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, based on the total mass of the nanoparticle.
The lipid content may be 0-36 wt% based on the total mass of the nanoparticle. In some embodiments, the lipid-like content may be 18 wt% to 36 wt%, based on the total mass of the nanoparticle; in some embodiments, the lipid-like content can be 0, 5 wt%, 10 wt%, 18 wt%, 20 wt%, 23 wt%, 25 wt%, 27 wt%, 30 wt%, 33 wt%, 35 wt%, based on the total mass of the nanoparticle.
In some embodiments, the compound of formula I is present in an amount of 30 wt% to 60 wt%, the PEG derivative is present in an amount of 0 to 15.0 wt%, the lipid is present in an amount of 30 wt% to 70 wt%, and the lipid is present in an amount of 0 to 36 wt%, based on the total weight of the nanoparticle. In some embodiments, the compound of formula I is present in an amount of about 27.5 wt% to about 55.9 wt%, the PEG derivative is present in an amount of 5.0 wt% to 11.2 wt%, the lipid is present in an amount of about 33 wt% to about 60 wt%, and the lipidoid can be present in an amount of 18 wt% to 36 wt%, based on the total weight of the nanoparticle.
In a second aspect, the present invention provides a nanoparticle composite.
A nanoparticle complex comprising a nucleic acid and a nanoparticle of the first aspect or a compound of formula I.
The weight ratio of the nanoparticle to the nucleic acid can be (1: 2-4: 1): 1. In some embodiments, the weight ratio of the nanoparticle to the nucleic acid is 0.5:1, 0.8:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4: 1. In some embodiments, the mass ratio of the compound of formula I to the nucleic acid is (12-325):100, and in some embodiments, the mass ratio of the compound of formula I to the nucleic acid is (12-45): 100.
The base complementary pairing refers to a phenomenon in which bases of respective nucleotide residues in a nucleic acid molecule are hydrogen-bonded to each other in a corresponding relationship of A and T, A to U and G and C. The compound shown in the formula I can form a base pair with adenine A in nucleic acid, A is connected with T through 2 hydrogen bonds, and double hydrogen bonds are formed between amine and carbonyl of complementary base groups, as shown in a formula II:
Figure BDA0003689084690000041
or the compound shown in the formula I and other conjugated groups in the nucleic acid form an amphiphilic composition through a pi-pi stacking effect, so that the nucleic acid nano-complex is formed by self-assembly under certain conditions. Specifically, the nucleobase derivative disclosed by the invention mainly forms an amphiphilic composition with nucleic acid through base complementary pairing (hydrogen bond) and nucleic acid action, or through a pi-pi stacking effect, a hydrophobic part is arranged in the middle of a nanoparticle in an aqueous solution, and a hydrophilic nucleic acid and a hydrophilic part are arranged on the surface of the nanoparticle and are assembled through hydrophilic and hydrophobic acting forces to form a nucleic acid nano-composite.
The nucleic acid may be chemically modified or non-chemically modified DNA, single or double stranded DNA, coding or non-coding DNA, optionally selected from plasmids, oligodeoxynucleotides, genomic DNA, DNA primers, DNA probes, immunostimulatory DNA, aptamers, or any combination thereof. In some embodiments, the nucleic acid is messenger RNA (mrna), oligoribonucleotides, viral RNA, replicon RNA, transfer RNA (trna), ribosomal RNA (rrna), immunostimulatory RNA (isrna), microrna, small interfering RNA (sirna), small nuclear RNA (snrna), circular RNA (circRNA or oana), small hairpin RNA (shrna) or riboswitches, RNA aptamers, RNA decoys, antisense RNA, ribozymes, or any combination thereof, preferably chemically modified messenger RNA (mrna).
The nucleic acid sequence of the RNA may include all of the nucleic acid sequences listed in patent US9254311B2, as well as all of the sequences listed in the long sequence appendix of that patent. In some embodiments, the RNA sequences of the invention can be obtained by nucleic acid synthesis methods as set forth in patents US9254311B2 or CN 106659803A.
In some embodiments, the nanoparticles can entrap a bioactive to be delivered to the interior of a cell, or optionally can be administered to an animal or human patient who would benefit from administration thereof. In some exemplary but non-limiting embodiments, preferred bioactive molecules suitable for use in the present invention include nucleic acid molecules, such as RNA molecules, preferably mRNA molecules or siRNA molecules.
In some embodiments, the biological active is preferably a nucleic acid, such as, for example, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In some embodiments, the preferred biological active may be a DNA molecule. The DNA may be linear DNA or circular DNA, such as DNA in the form of circular plasmids, episomes or expression vectors. In other embodiments, the preferred biological active may be an RNA molecule. The RNA molecule can be any type of RNA molecule (but is not limited to) including, but not limited to, mRNA, siRNA, miRNA, antisense RNA, ribonuclease, or any other type or kind of RNA molecule familiar to those skilled in the art (but not limited to) that will require delivery to the interior of a cell, and in some embodiments, the preferred biological active can be mRNA.
In a third aspect, the present invention provides a pharmaceutical composition.
A pharmaceutical composition comprising the nanoparticle complex of the second aspect and a pharmaceutically acceptable excipient.
The dosage form of the pharmaceutical composition can be injection, suppository, eye drop, tablet, capsule, suspension or inhalant.
In some embodiments, the pharmaceutical composition contains at least one RNA for use in treating or preventing a disease. The RNA-containing composition comprises at least a portion of coding RNA and non-coding RNA; the coding RNA includes at least one coding region encoding at least one therapeutic protein or polypeptide and an immunogenic protein or peptide; the coding RNA is mRNA.
The therapeutic protein or polypeptide may be a cytokine, chemokine, suicide gene product, immunogenic protein or peptide, apoptosis-inducing agent, angiogenesis inhibitor, heat shock protein, tumor antigen, β -catenin inhibitor, STING pathway activator, checkpoint modulator, innate immune activator, antibody, dominant negative receptor and decoy receptor, Myeloid Derived Suppressor Cell (MDSCs) inhibitor, IDO pathway inhibitor, and protein or peptide that binds to an apoptosis inhibitor;
the immunogenic protein or peptide may be a full-length sequence or a partial sequence of at least one protein or peptide from one of the following viruses or bacteria: a novel coronavirus (SARS-CoV-2), a Human Papilloma Virus (HPV), an influenza A or B virus or any other orthomyxovirus (influenza C virus); picornaviruses, such as rhinovirus or hepatitis a virus; togaviruses, such as alphaviruses or rubella viruses, e.g., sindbis virus, semliki forest virus, or measles virus; rubella virus; coronaviruses, in particular of the SARS-CoV-2, HCV-229E or HCV-OC43 subtype; rhabdoviruses, such as rabies virus; paramyxoviruses such as mumps virus; reoviruses, such as A, B or group C rotavirus; hepadnaviruses, such as hepatitis B virus; papovaviruses, such as human papilloma virus of any serotype; adenoviruses, especially types 1 to 47; herpes viruses, such as herpes simplex virus 1,2 or 3; cytomegalovirus, preferably CMVpp 65; EB virus; vaccinia virus; the bacterium Chlamydophila pneumoniae (Chlamydophila pneumoniae); flaviviruses, such as dengue 1 to 4 virus, yellow fever virus, west nile virus, japanese encephalitis virus; hepatitis C virus; a calicivirus virus; filoviruses, such as ebola virus; borna virus; bunyavirus, such as rift valley fever virus; arenaviruses such as lymphocytic choriomeningitis virus or hemorrhagic fever virus; retroviruses, such as HIV; parvovirus.
In a fourth aspect, the present invention provides a method of preparing a nanoparticle according to the first aspect.
A method of making a nanoparticle of the first aspect, comprising: dissolving a compound shown as a formula I in a solvent A to obtain a solution 1, dissolving an auxiliary material in a solvent B to obtain a solution 2, uniformly mixing the solution 1 and the solution 2, performing rotary evaporation at a low temperature, removing the solvent A in a water bath, and filtering to obtain the nanoparticle.
The solvent a may include one selected from ethanol, dichloromethane or chloroform.
The solvent B may include a solvent selected from methanol, ethanol, or chloroform.
The low temperature may be-10 ℃ or less. In some embodiments, the low temperature is-15 ℃ and below.
The water bath may be carried out at 35-65 ℃. In some embodiments, the water bath is a water bath performed at 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ or 65 ℃.
In a fifth aspect, the present invention provides a method of making the nanoparticle composite of the second aspect.
A method of making a nanoparticle composite of the second aspect, comprising: mixing the nanoparticle of the first aspect with a nucleic acid in a solvent C to obtain the nanoparticle complex.
The solvent C may be water.
In a fifth aspect, the present invention provides a use of a nanoparticle according to the first aspect or a nanoparticle complex according to the second aspect.
Use of a nanoparticle of the first aspect or a nanoparticle complex of the second aspect or a pharmaceutical composition of the third aspect in the manufacture of a medicament for in vivo delivery of a nucleic acid.
The invention provides ribonucleic acid vaccines which can safely induce a specific immune system naturally existing in an organism to produce almost any target protein or fragment thereof, take RNA (such as messenger RNA (mRNA)) as a core and take the nanoparticles as a delivery carrier, and the ribonucleic acid vaccines comprise infectious pathogen vaccines such as bacteria and viruses and tumor vaccines. In some embodiments, the RNA is modified. The RNA vaccines disclosed herein can be used to induce immune responses against infectious pathogens or cancers, including cellular immune responses and humoral immune responses, without the risk of, for example, insertional mutagenesis. Depending on the infectious agent and the incidence of cancer, RNA vaccines that use nanoparticles as the delivery vehicle according to the first aspect can be used in a variety of settings. The RNA vaccine can be used for preventing and/or treating infectious pathogens or cancers at various metastatic stages or degrees. The RNA vaccine using the nanoparticle of the first aspect as a delivery vector has superior properties because it has the characteristic property of selective transfection to DC cells, and can achieve higher transfection efficiency and transfection expression amount and generate higher antibody titer when the transfection efficiency is the same or lower.
The present invention provides a ribonucleic acid (RNA) vaccine that is constructed based on the knowledge that RNA (e.g., messenger RNA (mrna)) can safely direct the cellular machinery of the body to produce almost any protein of interest, from native proteins to antibodies and other entirely novel proteins that can have therapeutic activity inside and outside the cell. RNA (e.g., mRNA) vaccines are useful in a variety of contexts depending on the prevalence of infection or the degree or level of unmet medical need.
The nanoparticles according to the first aspect or the nanoparticle complexes according to the second aspect of the invention are used for the prevention, treatment and/or amelioration of a disease selected from the group consisting of: cancer or tumor diseases, infectious diseases, such as (viral, bacterial or protozoal) infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenic diseases, i.e. (genetic) diseases, or genetic diseases in general, diseases which have a genetic background and are typically caused by a defined genetic defect and are inherited according to Mendel's rules, cardiovascular diseases, neuronal diseases, respiratory diseases, digestive diseases, skin diseases, musculoskeletal disorders, connective tissue disorders, neoplasms, immunodeficiency, endocrine, nutritional and metabolic diseases, eye diseases and ear diseases.
The nucleic acid vaccines of the present invention may be administered by any route that produces a therapeutically effective result. Such routes include, but are not limited to, intradermal, subcutaneous, intraperitoneal, oral, intramuscular, intranasal, intraocular, upper respiratory, intravenous, vaginal, rectal administration. In some embodiments, the nucleic acid vaccines of the present invention are administered using injections.
Advantageous effects
Compared with the prior art, one of the technical schemes at least has one of the following beneficial technical effects:
(1) the invention innovatively adopts the compound shown in the formula I for preparing the nanoparticles, the compound shown in the formula I forms an amphiphilic composition with nucleic acid through the action of base complementary pairing (hydrogen bond) or pi-pi stacking effect, a hydrophobic part is arranged in the middle of the nanoparticles in aqueous solution, and the hydrophilic nucleic acid and a hydrophilic part are arranged on the surfaces of the nanoparticles and assembled to form a nucleic acid nano-composite through hydrophilic and hydrophobic acting force. The obtained nanoparticles can be effectively transfected in vivo, can carry mRNA encoding immunogenic peptide or protein to enter cells, effectively release the mRNA, express antigen and effectively achieve the aim of immunotherapy or immunoprophylaxis. The nucleic acid nano-composite can carry mRNA for encoding polypeptide or protein to enter cells, effectively release the mRNA, express the polypeptide and effectively realize the purpose of treating diseases.
(2) The nanoparticle provided by the invention has the particle size ranging from 40 nm to 274nm, has better dispersibility, and the surface charge of the nanoparticle for encapsulating nucleic acid ranges from-41 to 27. .
(3) The nanoparticle provided by the invention has small cytotoxicity and good biocompatibility.
(4) The nanoparticle provided by the invention has the advantages of compression, protection of nucleic acid from degradation, promotion of nucleic acid to penetrate cell membranes, realization of efficient transfection in vivo and in vitro, good biocompatibility and the like.
(5) The obtained nanoparticles are beneficial to transfection of nucleic acid in vivo and in vitro, improvement of serum transformation efficiency and humoral immune activation function, transfection of more cell lines and improvement of in vivo activity of the nanoparticle complex encapsulating the nucleic acid (for example, improvement of anti-tumor effect of the nanoparticle complex encapsulating OVA-mRNA).
(6) The nanoparticles adopt more than two or three lipids (such as DSPC, DOTAP or DOPE) so as to reduce the weight of the carrier during transfection and improve the entrapment capacity of the nanoparticles.
(7) The proportion of the auxiliary materials provided by the invention is beneficial to improving the transfection of the obtained nanoparticles in vivo and in vitro, the seroconversion efficiency and the humoral immune activation function, the transfection of more cell lines and the in vivo activity of the nanoparticle compound for encapsulating nucleic acid.
(8) The preparation method of the nanoparticles is simple to operate, low in cost, environment-friendly and beneficial to industrial production.
Drawings
FIG. 1 illustrates transfection of Fluc-mRNA-loaded nucleic acid nanocomplexes in DC2.4 cells in example four; the abscissa in the figure represents the different prescriptions of nucleic acid nanocomplexes and the ordinate is the relative fluorescence intensity expressed 24h after transfection of nucleic acid nanocomplexes containing the same dose of FLuc-mRNA.
FIG. 2 shows the survival rate of DC2.4 cells after treatment with different prescriptions in example four; the abscissa represents different nucleic acid nanocomplexes and the ordinate represents cell viability, with higher cell viability showing less cytotoxicity.
FIG. 3 illustrates transfection of Luc-pDNA loaded nucleic acid nanocomplexes in DC2.4 cells in example four; the abscissa represents different prescriptions and the ordinate is the relative fluorescence intensity expressed by DC2.4 cells 24h, 48h, 72h, 96h after transfection with the same dose of Luc-pDNA.
FIG. 4 shows transfection of Fluc-mRNA-loaded nucleic acid nanocomplexes in different cells in example four; the abscissa in the figure represents the different prescribed nucleic acid nanocomplexes and the ordinate is the relative fluorescence intensity expressed 24h after transfection of different cells with the same dose of the nucleic acid nanocomplexes containing FLuc-mRNA.
FIG. 5 shows the survival rate of cells treated by different recipes according to the fourth embodiment; the abscissa represents the different nucleic acid nanocomplex formulations, and the ordinate represents cell viability, with higher cell viability showing less cytotoxicity.
FIG. 6 shows transfection of EGFP-siRNA-loaded nucleic acid nanocomplexes into Hela-EGFP cells in example four; the abscissa in the figure represents the different prescribed nucleic acid nanocomplexes, and the ordinate represents the percentage of EGFP-positive cells after 24h transfection of Hela-EGFP transfected nucleic acid nanocomplexes containing the same dose of EGFP-siRNA.
FIG. 7 shows transfection of EGFP-siRNA-loaded nucleic acid nanocomplexes into Hela-EGFP cells in example four; the abscissa in the figure represents the different prescribed nucleic acid nanocomplexes, and the ordinate represents the median fluorescence intensity of the nucleic acid nanocomplexes transfected with the same dose of EGFP-siRNA 24h after Hela-EGFP transfection.
FIGS. 8-10 illustrate IVIS in example five detecting luciferase expression in mice from Fluc-mRNA loaded nucleic acid nanocomplexes.
FIG. 11 shows serum IgG antibody levels of mice immunized with the nucleic acid nanocomplex loaded with neocorona S-mRNA of example six; the abscissa represents the difference between the OD values at two wavelengths of the optical density on the 28 th and 49 th days after the first immunization for different prescriptions, and the OD value is an index for judging the IgG antibody level in serum and reflects the anti-S protein IgG level in serum.
FIG. 12 shows serum IgG antibody titers of mice immunized with the nucleic acid nanocomplexes of example six loaded with the novel corona S-mRNA; the abscissa represents the different dilution of the serum for different prescriptions after 49 days after the first immunization, and the ordinate represents the difference in OD (optical density) values at the two wavelengths. Baseline (twice background) was used as a cut-off to distinguish between positive and negative results, and the maximum dilution at which the OD was higher than this was the titer.
FIG. 13 is a flow chart of the procedure of example seven in which OVA-mRNA-loaded nanoparticle complex vaccine was injected subcutaneously into melanoma mouse mice.
FIG. 14 is a statistical chart showing the effect of the subcutaneous injection of OVA-mRNA loaded nanoparticle complex vaccine formulations on the survival of melanoma mice in example seven.
Figure 15 is a statistical graph showing the effect of different prescribed nanoparticle complex vaccines loaded with OVA-mRNA injected subcutaneously on tumor volume in melanoma mice in example seven.
Definition of terms:
in the invention, "room temperature" means ambient temperature, and may be 20 ℃ to 30 ℃; in some embodiments, from 22 ℃ to 28 ℃; in some embodiments, from 24 ℃ to 26 ℃; and in some embodiments, 25 ℃.
The term "PEG-CerC 14 "or" PEG-CerC 20 "the structural formula is as in patent application CN 107441506A" PEG-CerC 14 "or" PEG-CerC 20 "is said.
In the context of the present invention, all numbers disclosed herein are approximate values, regardless of whether the word "about" or "approximately" is used. Based on the numbers disclosed, the numerical values of each number may vary by less than + -10% or reasonably as recognized by one of ordinary skill in the art, such as by + -1%, + -2%, + -3%, + -4%, or + -5%.
The terms "optional," "optional," or "optionally" mean that the subsequently described event or circumstance may, but need not, occur. For example, "optional surfactant" means that the surfactant may or may not be present.
The term "weight percent" or "percent by weight" or "wt%" is defined as the weight of an individual component in a composition divided by the total weight of all components of the composition multiplied by 100%.
The terms "above", "below", "within" and the like are to be understood as including the instant numbers, e.g., two or more means ≧ two.
The term "% vol" denotes volume percentage.
The term "and/or" should be understood to mean any one of the options or a combination of any two or more of the options.
As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in the individual undergoing treatment. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis.
The terms "nucleic acid" or "nucleotide" or "polynucleotide" or "nucleic acid sequence" as used herein may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.
By "pharmaceutically acceptable" is meant: a substance or compound which is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
In the present application, a "composition" may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. Generally, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely divided solid carrier, or both.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, some non-limiting examples are further disclosed below to further explain the present invention in detail.
The reagents used in the present invention are either commercially available or can be prepared by the methods described herein.
The term "× g" represents centrifugal acceleration that is more or less times gravitational acceleration, for example, "5000 × g" represents centrifugal acceleration that is 5000 times gravitational acceleration.
DMG-PEG represents 1, 2-dimyristoyl-sn-glyceromethoxypolyethylene glycol; PEG-DMPE means dimyristoyl phosphatidylethanolamine-polyethylene glycol; PEG-DPPC represents dipalmitoylphosphatidylcholine polyethylene glycol; DOTAP stands for (2, 3-dioleoyl-propyl) -trimethylamine sulfate; DOPE represents 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine; DSPC represents 1, 2-distearoyl-sn-glycero-3-phosphocholine; chol represents cholesterol; DOPE represents 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine; DMPC represents 1, 2-dimyristoyl-sn-glycero-phosphocholine; PC represents lecithin;
Figure BDA0003689084690000101
l64 represents poloxamer L64;
Figure BDA0003689084690000102
20 represents tween 20; DPPC represents 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine;
Figure BDA0003689084690000103
80 denotes a span 80; T904-RT represents a loxamine derivative T904-RT; T904-RC represents a loxan amine derivative T904-RC; T90R4-R represents the loxan amine derivative T90R 4-R.
The first embodiment is as follows: synthesis of nucleobase derivatives
The nucleobase derivatives of the invention are produced by any previously known synthetic method known to those of ordinary skill in the art. The simple synthesis method and the specific process of the nucleobase derivative NBD003 are described as follows:
Figure BDA0003689084690000104
thymine-1-acetic acid (184.15mg,1mmol), dodecanol (186.34mg,1mmol), EDCI (249.21mg,1.3mmol) and DMAP (24.5mg,0.2mmol) were mixed and dissolved in 5mL of N-dimethylformamide and 10mL of dichloromethane and stirred at room temperature for 20 h.
After the reaction was completed, TLC (PE: EA ═ 1: 1) showed new dot formation, the reaction solution was transferred to a 250mL separatory funnel, 50mL of dichloromethane and 30mL of water were added, respectively, liquid separation was performed by extraction, the lower organic phase was collected and transferred to a flask, dried by adding anhydrous sodium sulfate, and the above extraction operation was repeated three times. The organic phase obtained is filtered off with suction and spin-dried, and the product, dodecyl thymine-1-acetate, is obtained by column chromatography (100% PE: EA ═ 1: 1) in the form of a white powdery solid with a yield of 88% (309.97mg,0.88 mmol).
Hydrogen spectrum: 1 H NMR(400MHz,CDCl 3 )δ8.34(s,1H),6.93(s,1H),4.43(s,2H),4.18(t,J＝8.0Hz,2H),1.94(s,3H),1.64(d,J＝8.0Hz,2H),1.58(s,2H),1.37-1.26(m,18H),0.88(t,J＝6.8Hz,3H).
mass spectrum: HRMS (ESI) m/z calcd for C 19 H 33 N 2 O 4 + (M+H) + 353.24348,found 353.24347。
The simple synthesis method and the specific process of the nucleobase derivative NBD005 are described as follows:
Figure BDA0003689084690000111
thymine-1-acetic acid (184.15mg,1mmol), hexadecanol (242.44mg,1mmol), EDCI (249.21mg,1.3mmol) and DMAP (24.5mg,0.2mmol) were combined and dissolved in 8mL of N-dimethylformamide and 10mL of dichloromethane and stirred at room temperature for 20 h.
After the reaction was completed, TLC (PE: EA ═ 1: 1) showed new dot formation, the reaction solution was transferred to a 250mL separatory funnel, 50mL of dichloromethane and 30mL of water were added, respectively, liquid separation was performed by extraction, the lower organic phase was collected and transferred to a flask, dried by adding anhydrous sodium sulfate, and the above extraction operation was repeated three times. The organic phase obtained is filtered off with suction and spin-dried, and the product thymine-1-hexadecanoacetate is obtained by column chromatography (100% PE. about.PE: EA. about.1.1) as a white powdery solid in 88% yield (359mg,0.88 mmol).
Hydrogen spectrum: 1 H NMR(500MHz,Chloroform-d)δ9.36(s,1H),7.79(q,J＝1.6Hz,1H),4.49(s,1H),4.09(t,J＝6.2Hz,2H),1.83(d,J＝1.4Hz,3H),1.64(tt,J＝7.5,6.2Hz,2H),1.43–1.35(m,2H),1.35–1.25(m,6H),1.26(s,14H),1.26(d,J＝1.9Hz,2H),0.94–0.85(m,3H).
mass spectrum: HRMS (ESI) m/z calcd for C 23 H 40 N 2 O 4 Na + (M+Na) + 431.28803,found 431.28806。
The simple synthesis method and the specific process of the nucleobase derivative NBD008 are described as follows:
Figure BDA0003689084690000112
thymine-1-acetic acid (184.15mg,1mmol), octadecanol (270.49mg,1mmol), EDCI (249.21mg,1.3mmol) and DMAP (24.5mg,0.2mmol) were combined and dissolved in 8mL of N-dimethylformamide and 10mL of dichloromethane and stirred at room temperature for 20 h.
After the reaction was completed, TLC (PE: EA ═ 1: 1) showed new dot formation, the reaction solution was transferred to a 250mL separatory funnel, 50mL of dichloromethane and 30mL of water were added, respectively, liquid separation was performed by extraction, the lower organic phase was collected and transferred to a flask, dried by adding anhydrous sodium sulfate, and the above extraction operation was repeated three times. The organic phase obtained is filtered off with suction and spin-dried, and the product, octadecyl thymine-1-acetate, is obtained by column chromatography (100% PE. PE: EA. 1.1) as a white powdery solid in 88% yield (384.2mg,0.88 mmol).
Hydrogen spectrum: 1 H NMR(500MHz,Chloroform-d)δ9.36(s,1H),7.79(q,J＝1.6Hz,1H),4.49(s,1H),4.09(t,J＝6.2Hz,2H),1.83(d,J＝1.4Hz,3H),1.64(tt,J＝7.5,6.2Hz,2H),1.43-1.35(m,2H),1.35-1.28(m,2H),1.31-1.25(m,5H),1.26(s,17H),1.26(d,J＝1.9Hz,3H),0.94-0.86(m,3H).
mass spectrum: HRMS (ESI) M/z calcd for C25H44N2O4Na + (M + Na) +459.31933, found 459.31937.
Example two: preparation of nucleic acid nanocomplexes
1) Prescription Rp.01: NBD003 nucleic acid mass ratio of 25:200
Taking the NBD003 out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the NBD003 at the room temperature and adding dichloromethane to dissolve the NBD003, adding the dissolved NBD003 into a round-bottomed flask, reducing the pressure under the condensation circulation condition of-15 ℃, putting the round-bottomed flask containing the sample into a water bath condition at the temperature of 40 ℃ by using a rotary evaporator to carry out rotary evaporation, removing an organic solution in the sample to enable the sample to form a layer of film on the wall of the round-bottomed flask, adding ultrapure water containing the nucleotidase to fully hydrate the film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite of formula Rp.01, and storing in 4 deg.C refrigerator for use.
2) Prescription Rp.02: NBD003 nucleic acid mass ratio of 50:200
Taking the NBD003 out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the NBD003 at the room temperature and adding dichloromethane to dissolve the NBD003, adding the dissolved NBD003 into a round-bottomed flask, reducing the pressure under the condensation circulation condition of-15 ℃, putting the round-bottomed flask containing the sample into a water bath condition at the temperature of 40 ℃ by using a rotary evaporator to carry out rotary evaporation, removing an organic solution in the sample to enable the sample to form a layer of film on the wall of the round-bottomed flask, adding ultrapure water containing the nucleotidase to fully hydrate the film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite of formula Rp.02, and storing in 4 deg.C refrigerator for use.
3) Prescription Rp.03: NBD003 nucleic acid mass ratio of 300:200
Taking the NBD003 out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the NBD003 at the room temperature and adding dichloromethane to dissolve the NBD003, adding the dissolved NBD003 into a round-bottomed flask, reducing the pressure under the condensation circulation condition of-15 ℃, putting the round-bottomed flask containing the sample into a water bath condition at the temperature of 40 ℃ by using a rotary evaporator to carry out rotary evaporation, removing an organic solution in the sample to enable the sample to form a layer of film on the wall of the round-bottomed flask, adding ultrapure water containing the nucleotidase to fully hydrate the film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite of formula Rp.03, and storing in 4 deg.C refrigerator for use.
4) Prescription Rp.04: the mass ratio of NBD003 to DMG-PEG to PC to Chol to nucleic acid is 100:19:40:72:200
Taking NBD003, DMG-PEG, PC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing NBD003 at room temperature and adding dichloromethane for dissolution; respectively weighing DMG-PEG, PC and Chol at room temperature, and dissolving with ethanol; adding the dissolved NBD003, DMG-PEG, PC and Chol into a round-bottomed flask, uniformly mixing, reducing pressure under a condensation circulation condition of-15 ℃, putting the round-bottomed flask containing the sample into a water bath condition of 50 ℃ by using a rotary evaporator for rotary evaporation, removing the organic solution in the sample, forming a layer of lipid film on the wall of the round-bottomed flask, adding ultrapure water for removing the nucleotidase, fully hydrating the lipid film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite of formula Rp.04, and storing in 4 deg.C refrigerator for use.
5) Prescription Rp.05: the mass ratio of NBD003 to DMG-PEG to PC to Chol to nucleic acid is 100:25:38:60:200
Taking NBD003, DMG-PEG, PC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing NBD003 at room temperature and adding dichloromethane for dissolution; respectively weighing DMG-PEG, PC and Chol at room temperature, and dissolving with ethanol; adding the dissolved NBD003, DMG-PEG, PC and Chol into a round-bottomed flask, uniformly mixing, reducing pressure under a condensation cycle condition of-15 ℃, putting the round-bottomed flask containing the sample into a water bath condition of 40 ℃ by using a rotary evaporator for rotary evaporation, removing the organic solution in the sample to enable the sample to form a lipid film on the wall of the round-bottomed flask, adding a 20mM KCl aqueous solution to fully hydrate the lipid film, and adding a stirrer to stir at the rotating speed of 1500rpm after 2 h. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite of formula Rp.05, and storing in 4 deg.C refrigerator for use.
6) Prescription Rp.06: the mass ratio of NBD003 to DMG-PEG to PC to Chol to nucleic acid is 100:22:35:79:200
Taking NBD003, DMG-PEG, PC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing NBD003, DMG-PEG, PC and Chol respectively at the room temperature and adding ethanol for dissolving; adding the dissolved NBD003, DMG-PEG, PC and Chol into a 1.5mL centrifuge tube, uniformly mixing, dropwise adding the mixed solution into a round-bottom flask containing the enucleated enzyme ultrapure water by using an insulin syringe, putting the round-bottom flask into an ultrasonic instrument for ultrasonic treatment in the dropwise adding process, and dropwise adding the mixed solution while ultrasonic treatment under the condition of water bath at 40 ℃. Performing ultrasonic treatment for 20min, performing rotary evaporation with a rotary evaporator at 40 deg.C water bath to remove ethanol, performing rotary evaporation, filtering with 0.22 μm filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano complex of Rp.06 in the prescription, and storing in a refrigerator at 4 deg.C.
7) Prescription Rp.07: the mass ratio of NBD003 to DMG-PEG to PC to Chol to nucleic acid is 100:55:51:36:200
Taking NBD003, DMG-PEG, PC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing NBD003 at room temperature and adding dichloromethane for dissolution; respectively weighing DMG-PEG, PC and Chol at room temperature, and dissolving with ethanol; adding the dissolved NBD003, DMG-PEG, PC and Chol into a round-bottomed flask, uniformly mixing, reducing pressure under a condensation circulation condition of-15 ℃, putting the round-bottomed flask containing the sample into a water bath condition of 50 ℃ by using a rotary evaporator for rotary evaporation, removing the organic solution in the sample, forming a layer of lipid film on the wall of the round-bottomed flask, adding ultrapure water for removing the nucleotidase, fully hydrating the lipid film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite with formula Rp.07, and storing in 4 deg.C refrigerator for use.
8) Prescription Rp.08: the mass ratio of NBD003 to T304-T to DPPC to Chol to nucleic acid is 95:83:55:80:200
Taking T304-T out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing at the room temperature, adding a sodium citrate buffer solution with the pH value of 6.4 for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain a stock solution A; taking the NBD003, the DPPC and the Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the NBD003 at the room temperature and adding dichloromethane to dissolve the NBD 003; respectively weighing DPPC and Chol at room temperature, and dissolving with ethanol; adding the dissolved NBD003, DPPC and Chol into a round-bottomed flask, uniformly mixing, reducing pressure under a condensation cycle condition of-15 ℃, putting the round-bottomed flask containing the sample into a water bath condition of 40 ℃ by using a rotary evaporator, carrying out rotary evaporation, removing the organic solution in the sample, forming a layer of lipid film on the wall of the round-bottomed flask, adding the stock solution A to fully hydrate the lipid film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite of formula Rp.08, and storing in 4 deg.C refrigerator for use.
9) Prescription Rp.09: the mass ratio of NBD003 to T304-T to DPPC to Chol to nucleic acid is 100:85:46:72:200
Taking T304-T out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing at the room temperature, adding a sodium citrate buffer solution with the pH value of 6.4 for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain a stock solution A; taking the NBD003, the DPPC and the Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the NBD003 at the room temperature and adding dichloromethane to dissolve the NBD 003; respectively weighing DPPC and Chol at room temperature, and dissolving with ethanol; adding the dissolved NBD003, DPPC and Chol into a round-bottomed flask, uniformly mixing, reducing pressure under a condensation cycle condition of-15 ℃, putting the round-bottomed flask containing the sample into a water bath condition of 40 ℃ by using a rotary evaporator, carrying out rotary evaporation, removing the organic solution in the sample, forming a layer of lipid film on the wall of the round-bottomed flask, adding the stock solution A to fully hydrate the lipid film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano complex of formula Rp.09, and storing in 4 deg.C refrigerator for use.
10) Prescription Rp.10: the mass ratio of NBD003 to T304-T to DPPC to Chol to nucleic acid is 100:71:69:73:200
Taking T304-T out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing at the room temperature, adding a sodium citrate buffer solution with the pH value of 6.4 for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain a stock solution A; NBD003, DPPC, and Chol were then equilibrated to room temperature from a-20 ℃ freezer, and NBD003, DPPC, and Chol were weighed at room temperature and dissolved in chloroform in a round-bottomed flask, respectively. Removing chloroform by rotary evaporation with a rotary evaporator to make the material mixture cling to the inner wall of the flask, adding the stock solution A into the rotary-dried flask, and performing ultrasonic treatment for 10 min; shearing with a high-speed shearing machine for 5min, continuing to perform ultrasound in a mode of ultrasound 3 seconds and pause for 3 seconds, and continuing to perform ultrasound for 5 min; transferring into dialysis bag with MWCO of 10000, dialyzing with sodium citrate buffer solution with pH of 6.4 for 24 hr, and replacing dialysate every 6 hr. Filtering with 0.22 μm water-based filter membrane after dialysis, mixing with nucleic acid water solution to obtain nucleic acid nano-composite of formula Rp.10, and storing in 4 deg.C refrigerator for use.
11) Prescription Rp.11: the mass ratio of NBD003: T304-T, DPPC: Chol, nucleic acid is 100:380:115:490:80
Taking T304-T out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing at the room temperature, adding a sodium citrate buffer solution with the pH value of 6.4 for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain a stock solution A; taking the NBD003, the DPPC and the Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the NBD003 at the room temperature and adding dichloromethane to dissolve the NBD 003; respectively weighing DPPC and Chol at room temperature, and dissolving with ethanol; adding the dissolved NBD003, DPPC and Chol into a round-bottomed flask, uniformly mixing, reducing pressure under a condensation cycle condition of-15 ℃, putting the round-bottomed flask containing the sample into a water bath condition of 40 ℃ by using a rotary evaporator, carrying out rotary evaporation, removing the organic solution in the sample, forming a layer of lipid film on the wall of the round-bottomed flask, adding the stock solution A to fully hydrate the lipid film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite of formula Rp.11, and storing in 4 deg.C refrigerator for use.
12) Prescription Rp.12: NBD005 nucleic acid mass ratio of 25:100
Taking the NBD005 out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the NBD005 at the temperature of room temperature and adding dichloromethane to dissolve the NBD005, adding the dissolved NBD005 into a round-bottomed flask, reducing pressure under the condensation circulation condition of-15 ℃, putting the round-bottomed flask containing the sample into a water bath condition at the temperature of 40 ℃ by using a rotary evaporator to carry out rotary evaporation, removing an organic solution in the sample, enabling the sample to form a layer of thin film on the wall of the round-bottomed flask, adding ultrapure water containing the nucleotidase to fully hydrate the thin film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2h, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite of formula Rp.12, and storing in 4 deg.C refrigerator for use.
13) Prescription Rp.13: NBD005 nucleic acid mass ratio of 50:100
Taking NBD005 out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing NBD005 at the temperature of-20 ℃, adding dichloromethane to dissolve NBD005, adding the dissolved NBD005 into a round-bottom flask, rotationally evaporating to remove organic solution in a sample under the condition of 40 ℃ water bath by using a rotary evaporator, continuously rotationally evaporating to dry the material after the material is attached to the wall of the flask for 30min, adding denuclease-removing ultrapure water, placing the mixture in an ultrasonic instrument for 50 ℃ intermittent ultrasonic treatment for 40min, filtering by using a 0.22 mu m aqueous filter membrane after ultrasonic treatment, mixing with a nucleic acid aqueous solution to obtain the nucleic acid nanocomposite of Rp.13 in the formula, and storing the nucleic acid nanocomposite in a refrigerator at the temperature of 4 ℃ for later use.
14) Prescription Rp.14: NBD005 nucleic acid mass ratio of 300:100
Taking the NBD005 out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the NBD005 at the temperature of room temperature and adding dichloromethane to dissolve the NBD005, adding the dissolved NBD005 into a round-bottomed flask, reducing pressure under the condensation circulation condition of-15 ℃, putting the round-bottomed flask containing the sample into a water bath condition at the temperature of 40 ℃ by using a rotary evaporator to carry out rotary evaporation, removing an organic solution in the sample, enabling the sample to form a layer of thin film on the wall of the round-bottomed flask, adding ultrapure water containing the nucleotidase to fully hydrate the thin film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2h, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite of formula Rp.14, and storing in 4 deg.C refrigerator for use.
15) Prescription Rp.15: the mass ratio of NBD005 to DMG-PEG to DSPC to Chol to nucleic acid is 43:8:17:31:86
Taking NBD005, DMG-PEG, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, respectively weighing NBD005, DMG-PEG, DSPC and Chol at the room temperature and adding ethanol for dissolving; adding the dissolved NBD005, DMG-PEG, DSPC and Chol into a 1.5mL centrifuge tube, uniformly mixing, dripping the mixed solution into a round-bottom flask containing the enucleated enzyme ultrapure water by using an insulin syringe, putting the round-bottom flask into an ultrasonic instrument for ultrasonic treatment in the dripping process, and dripping the mixed solution while ultrasonic treatment under the condition of water bath at 40 ℃. Performing ultrasonic treatment for 20min, performing rotary evaporation with a rotary evaporator at 40 deg.C water bath to remove ethanol, performing rotary evaporation, filtering with 0.22 μm water-phase filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-complex of formula Rp.15, and storing in 4 deg.C refrigerator.
16) Prescription Rp.16: the mass ratio of NBD005 to DMG-PEG to DSPC to Chol to nucleic acid is 39:12:21:39:80
Taking NBD005, DMG-PEG, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, respectively weighing NBD005, DMG-PEG, DSPC and Chol at the room temperature and adding ethanol for dissolving; adding the dissolved NBD005, DMG-PEG, DSPC and Chol into a 1.5mL centrifuge tube for uniformly mixing, dropwise adding the mixture into a round-bottomed flask containing a sodium citrate buffer solution with the pH value of 6.4 by using an insulin syringe, putting the round-bottomed flask into an ultrasonic instrument for ultrasonic treatment in the dropwise adding process, and dropwise adding the mixed solution while ultrasonic treatment under the condition of water bath at 40 ℃. Performing ultrasonic treatment for 20min, performing rotary evaporation with a rotary evaporator at 40 deg.C water bath to remove ethanol, performing rotary evaporation, filtering with 0.22 μm water phase filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano complex of formula Rp.16, and storing in 4 deg.C refrigerator.
17) Prescription Rp.17: the mass ratio of NBD005 to DMG-PEG to DSPC to Chol to nucleic acid is 65:10:31:36:130
Taking NBD005, DMG-PEG, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, respectively weighing NBD005, DMG-PEG, DSPC and Chol at the room temperature, and adding ethanol for dissolving; adding the dissolved NBD005, DMG-PEG, DSPC and Chol into a 1.5mL centrifuge tube for uniformly mixing, dropwise adding the mixture into a round-bottomed flask containing a sodium citrate buffer solution with the pH value of 6.4 by using an insulin syringe, putting the round-bottomed flask into an ultrasonic instrument for ultrasonic treatment in the dropwise adding process, and dropwise adding the mixed solution while ultrasonic treatment under the condition of water bath at 40 ℃. Performing ultrasonic treatment for 20min, performing rotary evaporation with a rotary evaporator at 40 deg.C water bath to remove ethanol, performing rotary evaporation, filtering with 0.22 μm water-phase filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-complex of formula Rp.17, and storing in 4 deg.C refrigerator.
18) Prescription Rp.18: the mass ratio of NBD005 to DMG-PEG to DSPC to Chol to nucleic acid is 43:10:50:22:86
Taking NBD005, DMG-PEG, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, respectively weighing NBD005, DMG-PEG, DSPC and Chol at the room temperature and adding ethanol for dissolving; adding the dissolved NBD005, DMG-PEG, DSPC and Chol into a 1.5mL centrifuge tube, uniformly mixing, dripping the mixed solution into a round-bottom flask containing the enucleated enzyme ultrapure water by using an insulin syringe, putting the round-bottom flask into an ultrasonic instrument for ultrasonic treatment in the dripping process, and dripping the mixed solution while ultrasonic treatment under the condition of water bath at 40 ℃. Performing ultrasonic treatment for 20min, performing rotary evaporation with a rotary evaporator at 40 deg.C water bath to remove ethanol, performing rotary evaporation, filtering with 0.22 μm water phase filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano complex of formula Rp.18, and storing in 4 deg.C refrigerator.
19) Prescription Rp.19: NBD005 DMG-PEG DSPC Chol nucleic acid mass ratio of 18:19:19:25:36
Taking NBD005, DMG-PEG, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, respectively weighing NBD005, DMG-PEG, DSPC and Chol at the room temperature and adding ethanol for dissolving; adding the dissolved NBD005, DMG-PEG, DSPC and Chol into a 1.5mL centrifuge tube for uniformly mixing, dropwise adding the mixture into a round-bottomed flask containing a sodium citrate buffer solution with the pH value of 6.4 by using an insulin syringe, putting the round-bottomed flask into an ultrasonic instrument for ultrasonic treatment in the dropwise adding process, and dropwise adding the mixed solution while ultrasonic treatment under the condition of water bath at 40 ℃. Performing ultrasonic treatment for 20min, performing rotary evaporation with a rotary evaporator at 40 deg.C water bath to remove ethanol, performing rotary evaporation, filtering with 0.22 μm water phase filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano complex of formula Rp.19, and storing in 4 deg.C refrigerator for use.
20) Prescription Rp.20: NBD005 Pluronic L64, PC, Chol and nucleic acid in the weight ratio of 80:49:57:49:160
Firstly, taking out Pluronic L64 from a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing at the room temperature, adding ultrapure water for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain stock solution A; taking the NBD005, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the NBD005 at the room temperature and adding dichloromethane to dissolve the NBD 005; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD005, PC and Chol into a round-bottom flask, uniformly mixing, reducing pressure under a condensation circulation condition of-15 ℃, putting the round-bottom flask containing the sample into a water bath condition of 40 ℃ by using a rotary evaporator, carrying out rotary evaporation, removing the organic solution in the sample, forming a layer of lipid film on the wall of the round-bottom flask, adding the stock solution A to fully hydrate the lipid film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite with formula Rp.20, and storing in 4 deg.C refrigerator for use.
21) Prescription Rp.21: NBD005 Pluronic L64 PC Chol nucleic acid mass ratio of 100:41:34:45:200
Firstly, taking out Pluronic L64 from a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing at the room temperature, adding ultrapure water for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain stock solution A; taking the NBD005, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the NBD005 at the room temperature and adding dichloromethane to dissolve the NBD 005; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD005, PC and Chol into a round-bottom flask, uniformly mixing, reducing pressure under a condensation circulation condition of-15 ℃, putting the round-bottom flask containing the sample into a water bath condition of 40 ℃ by using a rotary evaporator, carrying out rotary evaporation, removing the organic solution in the sample, forming a layer of lipid film on the wall of the round-bottom flask, adding the stock solution A to fully hydrate the lipid film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite of formula Rp.21, and storing in 4 deg.C refrigerator for use.
22) Prescription Rp.22: NBD005 Pluronic L64 PC Chol nucleic acid mass ratio 65:52:41:50:130
Firstly, taking Pluronic L64 out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing at the room temperature, adding a sodium citrate buffer solution with the pH value of 6.4 to dissolve and mix, and fully oscillating for 5min by using a vortex instrument to obtain a stock solution A; taking the NBD005, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the NBD005 at the room temperature and adding dichloromethane to dissolve the NBD 005; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD005, PC and Chol into a round-bottom flask, uniformly mixing, reducing pressure under a condensation circulation condition of-15 ℃, putting the round-bottom flask containing the sample into a water bath condition of 40 ℃ by using a rotary evaporator, carrying out rotary evaporation, removing the organic solution in the sample, forming a layer of lipid film on the wall of the round-bottom flask, adding the stock solution A to fully hydrate the lipid film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite of formula Rp.22, and storing in 4 deg.C refrigerator for use.
23) Prescription Rp.23: NBD005 Pluronic L64 PC Chol nucleic acid mass ratio of 15:28:24:49:30
Taking Pluronic L64 out of a refrigerator at-20 ℃ and balancing to room temperature, weighing at room temperature, adding sodium citrate buffer solution with pH of 6.4 for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain stock solution A; taking the NBD005, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the NBD005 at the room temperature and adding dichloromethane to dissolve the NBD 005; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD005, PC and Chol into a round-bottom flask, uniformly mixing, reducing pressure under a condensation circulation condition of-15 ℃, putting the round-bottom flask containing the sample into a water bath condition of 40 ℃ by using a rotary evaporator, carrying out rotary evaporation, removing the organic solution in the sample, forming a layer of lipid film on the wall of the round-bottom flask, adding the stock solution A to fully hydrate the lipid film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite of formula Rp.23, and storing in 4 deg.C refrigerator for use.
24) Prescription Rp.24: NBD005 Pluronic L64 PC Chol nucleic acid mass ratio of 48:14:51:21:96
Firstly, taking out Pluronic L64 from a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing at the room temperature, adding ultrapure water for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain stock solution A; taking the NBD005, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the NBD005 at the room temperature and adding dichloromethane to dissolve the NBD 005; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD005, PC and Chol into a round-bottom flask, uniformly mixing, reducing pressure under a condensation circulation condition of-15 ℃, putting the round-bottom flask containing the sample into a water bath condition of 40 ℃ by using a rotary evaporator, carrying out rotary evaporation, removing the organic solution in the sample, forming a layer of lipid film on the wall of the round-bottom flask, adding the stock solution A to fully hydrate the lipid film, adding a stirrer after 2h, and stirring at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-composite with Rp.24, and storing in 4 deg.C refrigerator for use.
25) Prescription Rp.25: NBD005:
Figure BDA0003689084690000181
904: PC: Chol: nucleic acid in a mass ratio of 79:47:51:47:160
Firstly, the method is carried out
Figure BDA0003689084690000182
904, taking out the mixture from a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing at the room temperature, adding ultrapure water for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain stock solution A; taking the NBD005, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the NBD005 at the room temperature and adding dichloromethane to dissolve the NBD 005; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD005, PC and Chol into a 1.5mL centrifuge tube, uniformly mixing, dripping the mixed solution into a round-bottom flask containing the stock solution A by using an insulin syringe, putting the round-bottom flask into an ultrasonic instrument for ultrasonic treatment in the dripping process, and dripping the mixed solution while ultrasonic treatment under the condition of water bath at 40 ℃. Performing ultrasonic treatment for 20min, performing rotary evaporation with a rotary evaporator at 40 deg.C water bath to remove ethanol, performing rotary evaporation, filtering with 0.22 μm water-phase filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-complex of formula Rp.25, and storing in 4 deg.C refrigerator.
26) Prescription Rp.26: NBD005:
Figure BDA0003689084690000191
904: PC: Chol: nucleic acid mass ratio of 95:52:36:49:190
Firstly, the method is carried out
Figure BDA0003689084690000192
904, taking out from a refrigerator at the temperature of-20 ℃, balancing to room temperature, weighing at the room temperature, adding a sodium citrate buffer solution with the pH value of 6.4, dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain a stock solution A; taking the NBD005, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the NBD005 at the room temperature and adding dichloromethane to dissolve the NBD 005; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD005, PC and Chol into a 1.5mL centrifuge tube, uniformly mixing, dripping the mixed solution into a round-bottom flask containing the stock solution A by using an insulin syringe, putting the round-bottom flask into an ultrasonic instrument for ultrasonic treatment in the dripping process, and dripping the mixed solution while ultrasonic treatment under the condition of water bath at 40 ℃. Performing ultrasonic treatment for 20min, performing rotary evaporation with a rotary evaporator at 40 deg.C water bath to remove ethanol, performing rotary evaporation, filtering with 0.22 μm water-phase filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano-complex of formula Rp.26, and storing in 4 deg.C refrigerator.
27) Prescription Rp.27: NBD005:
Figure BDA0003689084690000193
904: PC: Chol: nucleic acid in a mass ratio of 65:55:44:51:130
Firstly, the method is carried out
Figure BDA0003689084690000194
904, taking out the mixture from a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing at the room temperature, adding ultrapure water for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain stock solution A; taking the NBD005, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the NBD005 at the room temperature and adding dichloromethane to dissolve the NBD 005; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD005, PC and Chol into a 1.5mL centrifuge tube, uniformly mixing, dropwise adding the mixture into a round-bottom flask containing the stock solution A by using an insulin syringe, adding a stirrer into the round-bottom flask during dropwise adding, stirring at the speed of 1500rpm/min, and dropwise adding the mixed solution while stirring under the condition of a water bath at 40 ℃. Stirring for 20minAnd (3) rotationally evaporating by using a rotary evaporator under the condition of water bath at 40 ℃ to remove ethanol, filtering by using a 0.22 mu m water-phase filter membrane after the rotational evaporation, mixing with the nucleic acid water solution to obtain the nucleic acid nano-composite of the formula Rp.27, and storing in a refrigerator at 4 ℃ for later use.
28) Prescription Rp.28: NBD005:
Figure BDA0003689084690000195
904: PC: Chol: nucleic acid mass ratio is 18:31:24:49:36
Firstly, the method is carried out
Figure BDA0003689084690000196
904, taking out the mixture from a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing at the room temperature, adding ultrapure water for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain stock solution A; taking the NBD005, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the NBD005 at the room temperature and adding dichloromethane to dissolve the NBD 005; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD005, PC and Chol into a 1.5mL centrifuge tube, uniformly mixing, dripping the mixed solution into a round-bottom flask containing the stock solution A by using an insulin syringe, putting the round-bottom flask into an ultrasonic instrument for ultrasonic treatment in the dripping process, and dripping the mixed solution while ultrasonic treatment under the condition of water bath at 40 ℃. Performing ultrasonic treatment for 20min, performing rotary evaporation with a rotary evaporator at 40 deg.C water bath to remove ethanol, performing rotary evaporation, filtering with 0.22 μm water phase filter membrane, mixing with nucleic acid water solution to obtain nucleic acid nano complex of formula Rp.28, and storing in 4 deg.C refrigerator.
29) Prescription Rp.29: NBD005:
Figure BDA0003689084690000197
904: PC: Chol: nucleic acid in a mass ratio of 42:11:22:20:84
Firstly, the method is carried out
Figure BDA0003689084690000198
904, taking out the mixture from a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing at the room temperature, adding ultrapure water for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain stock solution A; then taking NBD005, PC and Chol out of a refrigerator at-20 ℃ to balanceWeighing NBD005 at room temperature and dissolving with dichloromethane; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD005, PC and Chol into a 1.5mL centrifuge tube, uniformly mixing, dropwise adding the mixture into a round-bottom flask containing the stock solution A by using an insulin syringe, adding a stirrer into the round-bottom flask during dropwise adding, stirring at the speed of 1500rpm/min, and dropwise adding the mixed solution while stirring under the condition of a water bath at 40 ℃. Stirring for 20min, rotary evaporating with rotary evaporator at 40 deg.C water bath to remove ethanol, filtering with 0.22 μm water phase membrane, mixing with nucleic acid water solution to obtain nucleic acid nanometer complex with formula Rp.29, and storing in 4 deg.C refrigerator.
30) Prescription Rp.30: NBD008 nucleic acid mass ratio of 325:100
Weighing NBD008 in a 1.5ml EP tube, dissolving with dichloromethane, transferring to a round bottom flask, rotationally evaporating to remove organic solution in a sample under the condition of 40 ℃ water bath by using a rotary evaporator, continuously rotationally evaporating to dry for 30min after the material is attached to the bottle wall, adding nuclease removal ultrapure water after 30min, placing in an ultrasonic instrument for intermittent ultrasonic treatment at 50 ℃ for 40min, filtering by using a 0.22 mu m water-phase filter membrane after ultrasonic treatment, adding nucleic acid, blowing, beating and mixing to obtain the nucleic acid nanocomposite of the formula Rp.30, and storing in a refrigerator at 4 ℃ for later use.
31) Prescription Rp.31: NBD008 nucleic acid mass ratio of 32:100
Weighing NBD008 in a 1.5ml EP tube, dissolving with dichloromethane, transferring to a round bottom flask, rotationally evaporating to remove organic solution in a sample under the condition of 40 ℃ water bath by using a rotary evaporator, continuously rotationally evaporating to dry for 30min after the material is attached to the bottle wall, adding nuclease removal ultrapure water after 30min, placing in an ultrasonic instrument for intermittent ultrasonic treatment at 50 ℃ for 40min, filtering by using a 0.22 mu m water-phase filter membrane after ultrasonic treatment, adding nucleic acid, blowing, beating and mixing to obtain the nucleic acid nanocomposite of the formula Rp.31, and storing in a refrigerator at 4 ℃ for later use.
32) Prescription Rp.32: NBD008 nucleic acid mass ratio of 45:100
Weighing NBD008 in a 1.5ml EP tube, dissolving with dichloromethane, transferring to a round bottom flask, rotationally evaporating to remove organic solution in a sample under the condition of 40 ℃ water bath by using a rotary evaporator, continuously rotationally evaporating to dry for 30min after the material is attached to the bottle wall, adding nuclease removal ultrapure water after 30min, placing in an ultrasonic instrument for intermittent ultrasonic treatment at 50 ℃ for 40min, filtering by using a 0.22 mu m water-phase filter membrane after ultrasonic treatment, adding nucleic acid, blowing, beating and mixing to obtain the nucleic acid nanocomposite of Rp.32 in the prescription, and storing in a refrigerator at 4 ℃ for later use.
33) Prescription Rp.33: the mass ratio of NBD008: T904: T90R4: DSPC: Chol: nucleic acid is 55:33:33:25:40:72
Taking NBD008, T904, T90R4, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the T904 and the T90R4 at the room temperature, adding ultrapure water of the nucleotidase for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain stock solution A; respectively weighing DSPC and Chol at room temperature, and dissolving with ethanol; NBD008 was weighed out at room temperature and dissolved with dichloromethane. NBD008, DSPC and Chol after dissolution are added into a round-bottom flask and mixed evenly. Removing ethanol by rotary evaporation with a rotary evaporator to make the material tightly attached to the inner wall of the flask, adding the stock solution A into the rotary-dried flask, and performing ultrasonic treatment for 10 min; shearing with a high-speed shearing machine for 5min, continuing to perform ultrasound in a mode of ultrasound 3 seconds and pause for 3 seconds, and continuing to perform ultrasound for 5 min; transferring into dialysis bag with MWCO of 10000, dialyzing with citric acid buffer solution with pH of 6.2-6.8 for 24 hr, and replacing dialysate every 6 hr. Filtering with 0.22 μm water-based filter membrane after dialysis, adding nucleic acid, blowing, beating, and mixing to obtain nucleic acid nanometer complex with Rp.33, and storing in 4 deg.C refrigerator for use.
34) Prescription Rp.34: the mass ratio of NBD008: T904: T90R4: DSPC: Chol: nucleic acid is 78:33:33:38:56:101
Taking NBD008, T904, T90R4, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing T904 and T90R4 at the room temperature, adding ultrapure water of the nucleotidase for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain stock solution A; respectively weighing DSPC and Chol at room temperature, and dissolving with ethanol; NBD008 was weighed out at room temperature and dissolved with dichloromethane. NBD008, DSPC and Chol after dissolution are added into a round-bottom flask and mixed evenly. Removing ethanol by rotary evaporation with a rotary evaporator to make the material cling to the inner wall of the flask, adding the stock solution A into the dried flask, and performing ultrasonic treatment for 10 min; shearing with a high-speed shearing machine for 5min, continuing to perform ultrasound in a mode of ultrasound 3 seconds and pause for 3 seconds, and continuing to perform ultrasound for 5 min; transferring into dialysis bag with MWCO of 10000, dialyzing with citric acid buffer solution with pH of 6.2-6.8 for 24 hr, and replacing dialysate every 6 hr. Filtering with 0.22 μm water-based filter membrane after dialysis, adding nucleic acid, blowing, beating, and mixing to obtain nucleic acid nanometer complex with Rp.34 of the prescription, and storing in 4 deg.C refrigerator for use.
35) Prescription Rp.35: the mass ratio of NBD008: T904: T90R4: DSPC: Chol: nucleic acid is 112:30:30:38:56:146
Taking NBD008, T904, T90R4, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the T904 and the T90R4 at the room temperature, adding ultrapure water of the nucleotidase for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain stock solution A; respectively weighing DSPC and Chol at room temperature, and dissolving with ethanol; NBD008 was weighed out at room temperature and dissolved with dichloromethane. NBD008, DSPC and Chol after dissolution are added into a round-bottom flask and mixed evenly. Removing ethanol by rotary evaporation with a rotary evaporator to make the material tightly attached to the inner wall of the flask, adding the stock solution A into the rotary-dried flask, and performing ultrasonic treatment for 10 min; shearing with a high-speed shearing machine for 5min, continuing to perform ultrasound in a mode of ultrasound 3 seconds and pause for 3 seconds, and continuing to perform ultrasound for 5 min; transferring into dialysis bag with MWCO of 10000, dialyzing with citric acid buffer solution with pH of 6.2-6.8 for 24 hr, and replacing dialysate every 6 hr. Filtering with 0.22 μm water-based filter membrane after dialysis, adding nucleic acid, blowing, beating, and mixing to obtain nucleic acid nanometer complex with Rp.35, and storing in 4 deg.C refrigerator for use.
36) Prescription Rp.36: the mass ratio of NBD008: T904: T90R4: DSPC: Chol: nucleic acid is 55:58:58:12:56:72
Taking NBD008, T904, T90R4, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the T904 and the T90R4 at the room temperature, adding ultrapure water of the nucleotidase for dissolving and mixing, and fully oscillating for 5min by using a vortex instrument to obtain stock solution A; respectively weighing DSPC and Chol at room temperature, and dissolving with ethanol; NBD008 was weighed out at room temperature and dissolved with dichloromethane. NBD008, DSPC and Chol after dissolution are added into a round-bottom flask and mixed evenly. Removing ethanol by rotary evaporation with a rotary evaporator to make the material tightly attached to the inner wall of the flask, adding the stock solution A into the rotary-dried flask, and performing ultrasonic treatment for 10 min; shearing with a high-speed shearing machine for 5min, continuing to perform ultrasound in a mode of ultrasound 3 seconds and pause for 3 seconds, and continuing to perform ultrasound for 5 min; transferring into dialysis bag with MWCO of 10000, dialyzing with citric acid buffer solution with pH of 6.2-6.8 for 24 hr, and replacing dialysate every 6 hr. Filtering with 0.22 μm water-based filter membrane after dialysis, adding nucleic acid, blowing, beating, and mixing to obtain nucleic acid nanometer complex with Rp.36, and storing in 4 deg.C refrigerator for use.
37) Prescription Rp.37: NBD008:
Figure BDA0003689084690000221
the mass ratio of F127 to PC to Chol to nucleic acid is 78:72:30:48:101
Firstly, the method is carried out
Figure BDA0003689084690000222
F127 is taken out from a refrigerator at 4 ℃ and balanced to room temperature, ultrapure water with nucleotidase is weighed and added at room temperature for dissolving, a vortex instrument is used for fully oscillating for 5min, and standing overnight is carried out to obtain stock solution A; taking the NBD008, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the NBD008 at room temperature and adding dichloromethane for dissolution; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD008, PC and Chol into a round-bottom flask, uniformly mixing, rotationally evaporating the organic solvent by using a rotary evaporator under the condition of water bath at 40 ℃ to enable a sample to form a layer of lipid film on the wall of the round-bottom flask, adding the stock solution A to fully hydrate the lipid film, and adding a stirrer to stir at the rotating speed of 1500rpm/min after 2 hours. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nucleic acid nanometer complex of formula Rp.37, and storing in 4 deg.C refrigerator for use.
38) Prescription Rp.38: NBD008:
Figure BDA0003689084690000223
the mass ratio of F127 to PC to Chol to nucleic acid is 55:72:25:48:72
Firstly, the method is to
Figure BDA0003689084690000224
F127 is taken out from a refrigerator at 4 ℃ and is balanced to room temperature, the ultrapure water which is weighed and added with the nucleotidase is dissolved at room temperature and is vortexedFully oscillating the rotary instrument for 5min, and standing overnight to obtain stock solution A; taking the NBD008, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the NBD008 at room temperature and adding dichloromethane for dissolution; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD008, PC and Chol into a round-bottom flask, uniformly mixing, rotationally evaporating the organic solvent by using a rotary evaporator under the condition of water bath at 40 ℃ to enable a sample to form a layer of lipid film on the wall of the round-bottom flask, adding the stock solution A to fully hydrate the lipid film, and adding a stirrer to stir at the rotating speed of 1500rpm/min after 2 hours. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nucleic acid nanometer complex of formula Rp.38, and storing in 4 deg.C refrigerator for use.
39) Prescription Rp.39: NBD008:
Figure BDA0003689084690000225
the mass ratio of F127 to PC to Chol to nucleic acid is 112:45:35:52:146
Firstly, the method is carried out
Figure BDA0003689084690000226
F127 is taken out from a refrigerator at 4 ℃ and balanced to room temperature, ultrapure water with nucleotidase is weighed and added at room temperature for dissolving, a vortex instrument is used for fully oscillating for 5min, and standing overnight is carried out to obtain stock solution A; taking the NBD008, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the NBD008 at room temperature and adding dichloromethane for dissolution; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD008, PC and Chol into a round-bottom flask, uniformly mixing, rotationally evaporating the organic solvent by using a rotary evaporator under the condition of water bath at 40 ℃ to enable a sample to form a layer of lipid film on the wall of the round-bottom flask, adding the stock solution A to fully hydrate the lipid film, and adding a stirrer to stir at the rotating speed of 1500rpm/min after 2 hours. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nucleic acid nanometer complex of formula Rp.39, and storing in 4 deg.C refrigerator for use.
40) Prescription Rp.40: NBD008:
Figure BDA0003689084690000227
F127:PC:Chol:the mass ratio of nucleic acid is 182:25:42:48:237
Firstly, the method is carried out
Figure BDA0003689084690000231
F127 is taken out from a refrigerator at 4 ℃ and balanced to room temperature, ultrapure water with nucleotidase is weighed and added at room temperature for dissolving, a vortex instrument is used for fully oscillating for 5min, and standing overnight is carried out to obtain stock solution A; taking the NBD008, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the NBD008 at room temperature and adding dichloromethane for dissolution; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD008, PC and Chol into a round-bottom flask, uniformly mixing, rotationally evaporating the organic solvent by using a rotary evaporator under the condition of water bath at 40 ℃ to enable a sample to form a layer of lipid film on the wall of the round-bottom flask, adding the stock solution A to fully hydrate the lipid film, and adding a stirrer to stir at the rotating speed of 1500rpm/min after 2 hours. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nucleic acid nanometer complex with Rp.40, and storing in 4 deg.C refrigerator for use.
41) Prescription Rp.41: the mass ratio of NBD008 to DMG-PEG to DSPC to Chol to nucleic acid is 128:15:30:56:166
Taking NBD008, DMG-PEG, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing NBD008, DMG-PEG, DSPC and Chol respectively at the room temperature and adding ethanol for dissolving; adding the dissolved NBD008, DMG-PEG, DSPC and Chol into a 1.5mL centrifuge tube, uniformly mixing, dripping the mixed solution into a round-bottom flask containing the enucleated enzyme ultrapure water by using an insulin syringe, putting the round-bottom flask into an ultrasonic instrument for ultrasonic treatment in the dripping process, and dripping the mixed solution while ultrasonic treatment under the condition of water bath at 40 ℃. Performing ultrasonic treatment for 20min, performing rotary evaporation with a rotary evaporator at 40 deg.C water bath to remove ethanol, performing rotary evaporation, filtering with 0.22 μm water phase filter membrane, adding nucleic acid, blowing, mixing to obtain nucleic acid nanocomposite of formula Rp.41, and storing in 4 deg.C refrigerator.
42) Prescription Rp.42: the mass ratio of NBD008 to DMG-PEG to DSPC to Chol to nucleic acid is 78:15:38:56:101
Taking NBD008, DMG-PEG, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing NBD008, DMG-PEG, DSPC and Chol respectively at the room temperature and adding ethanol for dissolving; adding the dissolved NBD008, DMG-PEG, DSPC and Chol into a 1.5mL centrifuge tube, uniformly mixing, dripping the mixed solution into a round-bottom flask containing the enucleated enzyme ultrapure water by using an insulin syringe, putting the round-bottom flask into an ultrasonic instrument for ultrasonic treatment in the dripping process, and dripping the mixed solution while ultrasonic treatment under the condition of water bath at 40 ℃. Performing ultrasonic treatment for 20min, performing rotary evaporation with a rotary evaporator at 40 deg.C water bath to remove ethanol, performing rotary evaporation, filtering with 0.22 μm water phase filter membrane, adding nucleic acid, blowing, mixing to obtain nucleic acid nanocomposite of formula Rp.42, and storing in 4 deg.C refrigerator.
43) Prescription Rp.43: the mass ratio of NBD008 to DMG-PEG to DSPC to Chol to nucleic acid is 47:15:38:56:61
Taking NBD008, DMG-PEG, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing NBD008, DMG-PEG, DSPC and Chol respectively at the room temperature and adding ethanol for dissolving; adding the dissolved NBD008, DMG-PEG, DSPC and Chol into a 1.5mL centrifuge tube, uniformly mixing, dripping the mixed solution into a round-bottom flask containing the enucleated enzyme ultrapure water by using an insulin syringe, putting the round-bottom flask into an ultrasonic instrument for ultrasonic treatment in the dripping process, and dripping the mixed solution while ultrasonic treatment under the condition of water bath at 40 ℃. Performing ultrasonic treatment for 20min, performing rotary evaporation with a rotary evaporator at 40 deg.C water bath to remove ethanol, performing rotary evaporation, filtering with 0.22 μm water phase filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nucleic acid nano complex of formula Rp.43, and storing in 4 deg.C refrigerator.
44) Prescription Rp.44: the mass ratio of NBD008 to DMG-PEG to DSPC to Chol to nucleic acid is 28:15:38:72:36
Taking NBD008, DMG-PEG, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, respectively weighing NBD008, DMG-PEG, DSPC and Chol at the room temperature, and adding ethanol for dissolving; adding the dissolved NBD008, DMG-PEG, DSPC and Chol into a 1.5mL centrifuge tube, uniformly mixing, dripping the mixed solution into a round-bottom flask containing the enucleated enzyme ultrapure water by using an insulin syringe, putting the round-bottom flask into an ultrasonic instrument for ultrasonic treatment in the dripping process, and dripping the mixed solution while ultrasonic treatment under the condition of water bath at 40 ℃. Performing ultrasonic treatment for 20min, performing rotary evaporation with a rotary evaporator at 40 deg.C water bath to remove ethanol, performing rotary evaporation, filtering with 0.22 μm water phase filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nucleic acid nano complex of formula Rp.44, and storing in 4 deg.C refrigerator.
45) Prescription Rp.45: the mass ratio of NBD008 to DMG-PEG to DSPC to Chol to nucleic acid is 182:9:28:56:237
Taking NBD008, DMG-PEG, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing NBD008, DMG-PEG, DSPC and Chol respectively at the room temperature and adding ethanol for dissolving; adding the dissolved NBD008, DMG-PEG, DSPC and Chol into a 1.5mL centrifuge tube, uniformly mixing, dripping the mixed solution into a round-bottomed flask containing the enucleated enzyme ultrapure water by using an insulin syringe, putting the round-bottomed flask into an ultrasonic instrument for ultrasonic treatment in the dripping process, and dripping the mixed solution while ultrasonic treatment under the condition of water bath at 40 ℃. Performing ultrasonic treatment for 20min, performing rotary evaporation with a rotary evaporator at 40 deg.C water bath to remove ethanol, performing rotary evaporation, filtering with 0.22 μm water phase filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nucleic acid nano complex of formula Rp.45, and storing in 4 deg.C refrigerator.
Example three: characterization of the nucleic acid nanocomplexes of the invention
Particle size and potential: nucleic acid nanocomposites were prepared as described in example two and tested for dynamic light scattering particle size (size), surface Potential (Zeta Potential) and Polydispersity (PDI) using a Malvern Zetasizer Nano ZSE at 25 ℃.
The results are shown in Table 1, and show that the nucleic acid nano-composite of the invention has the particle size ranging from 40 to 274nm, has better dispersibility and the surface charge of the nano-composite ranges from-41 to 27.
Encapsulation efficiency: taking FLuc-mRNA as model mRNA, preparing the nucleic acid nano-composite according to the preparation method described in the embodiment II, and determining the mRNA encapsulation rate of each prescription by using a Quant-iT RiboGreen RNA detection kit (ThermoFische company), wherein the specific method refers to the kit specification, and the brief processing method of the invention comprises the following steps: centrifuging each prescription at 4 deg.C and 20000rpm for 2h with low temperature high speed centrifuge, collecting supernatant, and quantifying the volume with pipette, and recording as V1; measuring the concentration of mRNA in the supernatant by using a Quant-iT RiboGreen RNA detection kit, and marking the concentration as C1; dissolving the centrifuged precipitate in 25ul of chromatographic pure DMSO (dimethylsulfoxide), continuously adding 0.9% physiological saline injection, uniformly mixing, standing at 25 ℃ for 2 hours, recording the total volume V2, and determining the concentration of mRNA (messenger ribonucleic acid) by using a Quant-iT RiboGreen RNA detection kit, wherein the concentration is marked as C2; the packet loading rate calculation formula of each prescription is as follows: the encapsulation efficiency is 100% - (V1C1)/(V1C1+ V2C2) × 100%, and the results are shown in table 1, the formula has a good encapsulation effect on mRNA, and the encapsulation efficiency is 95% or more.
Table 1: characterization of nucleic acid nanocomplexes
Figure BDA0003689084690000251
Example four: in vitro cell transfection experiment and cytotoxicity investigation of nucleic acid nanocomposites
1) Experiment of transfection of nucleic acid nanocomplexes carrying FLuc-mRNA into DC2.4 cells in vitro:
logarithmic growth phase DC2.4 cell suspension at 4X 10 4 The density of each cell per well is divided into 96-well plates, put at 37 ℃ and 5% CO 2 And (5) standing and culturing in an incubator. After 24h, the Fluc-mRNA with the concentration of 1 mug/mul is diluted to 0.1 mug/mul by nuclease-free ultrapure water, the Fluc-mRNA is taken to prepare nucleic acid nano-complexes according to the preparation methods of different prescriptions described in example two, then the nucleic acid nano-complexes are respectively diluted to 88 mul by nuclease-free ultrapure water and mixed solution of the nucleic acid nano-complexes containing 10 ng/mul of the Fluc-mRNA is kept still for 10min, and then the nucleic acid nano-complexes are respectively added to 96-well plates containing 180 mu of lopti-MEM culture medium in the volume of 20 mul per well, and 4 wells are repeated for each sample. After 4h of dosing, the aspirated 96-well plate was replaced with complete medium. The incubation was continued for 24h, the complete medium was aspirated and rinsed once with PBS, 100. mu. l D-Luciferin working solution (working concentration 250. mu.g/mL) was added to each 96-well plate, incubation was continued in an incubator at 37 ℃ for 5min, and the Fluc-mRNA fluorescence expression intensity was measured by imaging with an Omega-Fluostar plate reader.
The results are shown in FIG. 1. And (4) conclusion: the results show that the FLuc-mRNA-encapsulating and nucleic acid nanocomplex prepared from Rp.02, Rp.03, Rp.04, Rp.05, Rp.06, Rp.08, Rp.09, Rp.10, Rp.12, Rp.13, Rp.15, Rp.16, Rp.17, Rp.20, Rp.21, Rp.22, Rp.25, Rp.26, Rp.27, Rp.31, Rp.32, Rp.33, Rp.34, Rp.35, Rp.37, Rp.38, Rp.39, Rp.41, Rp.42 and Rp.43 shows better expression in DC2.4 cells, wherein the FLuc-mRNA-encapsulating and nucleic acid nanocomplex prepared from Rp.04, Rp.09, Rp.15, Rp.22, Rp.26, Rp.33, Rp.37 and Rp.42 show the best effect.
2) Cytotoxicity experiments of DC2.4 cells transfected in vitro with FLuc-mRNA-entrapped nucleic acid nanocomplexes:
DC2.4 cell suspension in logarithmic growth phase at 4X 10 4 The density of each cell per well is divided into 96-well plates, put at 37 ℃ and 5% CO 2 And (5) standing and culturing in an incubator. After 24h, the Fluc-mRNA with the concentration of 1 mug/mul is diluted to 0.1 mug/mul by nuclease-free ultrapure water, the Fluc-mRNA is taken to prepare nucleic acid nano-complexes according to the preparation methods of different prescriptions described in example two, then the nucleic acid nano-complexes are respectively diluted to 88 mul by nuclease-free ultrapure water and mixed solution of the nucleic acid nano-complexes containing 10 ng/mul of the Fluc-mRNA is kept still for 10min, and then the nucleic acid nano-complexes are respectively added to 96-well plates containing 180 mu of lopti-MEM culture medium in the volume of 20 mul per well, and 4 wells are repeated for each sample. After 4h of dosing, the aspirated 96-well plate was replaced with complete medium. The culture was continued for 48h, the complete medium was aspirated and rinsed three times with PBS, wells without the prescription cell were used as negative controls and wells with CCK-8 medium without cells were used as blank controls, and 90. mu.l serum-free medium and 10. mu.l CCK-8 solution were added to each well and the incubation was continued in the incubator for 2 h. Absorbance at 450nm was measured using an Omega-Fluostar microplate reader. Cell viability calculation formula:
cell viability = [ a (dosed) -a (blank) ]/[ a (not dosed) -a (blank) ] × 100%;
a (dosing): absorbance of DC2.4 cells, prescription solution and CCK-8 solution added to each well;
a (blank): the absorbance of the CCK-8 solution is added to each well;
a (no drug addition): absorbance of the solution containing DC2.4 cells and CCK-8 was added to each well;
cell viability: cell proliferation activity or cytotoxic activity.
The results are shown in FIG. 2. And (4) conclusion: the results show that the survival rate of the cells is over 90 percent, which shows that the nucleic acid nano-composites with different prescriptions provided by the invention have no obvious cytotoxicity and good biocompatibility, and can be used for subsequent in vivo experiments of animals.
3) Experiments on in vitro transfection of DC2.4 cells with Luc-pDNA-entrapped nucleic acid nanocomposites:
DC2.4 cell suspension in logarithmic growth phase at 4X 10 4 The density of each cell per well is divided into 96-well plates, put at 37 ℃ and 5% CO 2 And (5) standing and culturing in an incubator. After 24h, Luc-pDNA at a concentration of 1. mu.g/. mu.l was diluted to 0.1. mu.g/. mu.l with nuclease-free ultrapure water. Luc-pDNA was used to prepare nucleic acid nanocomposites by the preparation methods described in example two but different recipes, and then diluted to 88. mu.l of nucleic acid nanocomposite mixture containing 15 ng/. mu.l Luc-pDNA with nuclease-free ultrapure water, and left to stand for 30min, and added to 96-well plates containing 180. mu.l of Luc-pDNA medium per well in a volume of 20. mu.l per well, and 4 wells were repeated for each sample. After 4h of dosing, the aspirated 96-well plate was replaced with complete medium. And continuously culturing for 24 hours, sucking out the complete culture medium, adding 100 mu l of D-Luciferin solution with the working concentration of 250 mu g/mL into each 96-well plate, continuously culturing for 5min in an incubator at 37 ℃, imaging by using an Omega-Fluostar enzyme-linked immunosorbent assay, testing the fluorescence expression intensity of the Luc-pDNA, repeating the test once every 24 hours, sucking out the culture medium containing the D-Luciferin after each test is finished, adding a fresh complete culture medium, continuously culturing for 24 hours, adding the D-Luciferin for testing, and continuously detecting for four days. The results are shown in FIG. 3, with the abscissa representing different prescriptions and the ordinate being the relative fluorescence intensity of Luc-pDNA expression at the same dose 24h, 48h, 72h after transfection
The results are shown in FIG. 3. And (4) conclusion: the results show that the prescriptions Rp.02, Rp.03, Rp.04, Rp.06, Rp.08, Rp.09, Rp.12, Rp.15, Rp.20, Rp.21, Rp.22, Rp.25, Rp.26, Rp.27, Rp.31, Rp.32, Rp.33, Rp.34, Rp.35, Rp.37, Rp.38, Rp.39, Rp.41 and Rp.42 show better expression at the cellular level and the highest expression level on the second or third day, wherein the prescriptions Rp.03, Rp.04, Rp.09, Rp.15, Rp.22, Rp.26, Rp.33, Rp.37 and Rp.42 are better than other prescriptions.
4) Different cell experiments were performed with the Fluc-mRNA entrapped nucleic acid nanocomplex in vitro:
referring to the experimental procedure of 1) in example four, transfection experiments were performed by replacing DC2.4 cells with 4T1 (mouse breast cancer cells), Hela (human cervical cancer cells), HL7702 (human hepatic normal cells).
The results are shown in FIG. 4. And (4) conclusion: the results show that the formulas Rp.03, Rp.04, Rp.09, Rp.15, Rp.22, Rp.26, Rp.33, Rp.37, and Rp.42 entrap Fluc-mRNA nucleic acid nanocomplexes show better expression levels in 4T1 cells, Hela cells, and HL7702 cells.
5) Cytotoxicity experiments of Fluc-mRNA-entrapped nucleic acid nanocomposites in vitro transfection of different cells:
referring to the experimental procedure of 2) in example four, transfection experiments were performed by replacing DC2.4 cells with 4T1 (mouse breast cancer cells), Hela (human cervical cancer cells), HL7702 (human hepatic normal cells).
The results are shown in FIG. 5. And (4) conclusion: the results show that the survival rate of the cells is over 90 percent, which indicates that the prescription of the nucleic acid nano-composite has no obvious cytotoxicity and good biocompatibility, and can be used for subsequent in vivo experiments of animals.
6) Experiment of in vitro transfection of Hela-EGFP cells (polyclonal cell line stably expressing EGFP fluorescent protein) with nucleic acid nanocomposite encapsulating EGFP-siRNA (using EGFP-siRNA as model siRNA):
HeLa cell suspension stably expressing EGFP in logarithmic growth phase at 4X 10 4 The density of each cell per well is divided into 96-well plates, put at 37 ℃ and 5% CO 2 And (5) standing and culturing in an incubator. After 24h, 1. mu.g/. mu.l EGFP-siRNA was diluted to 0.1. mu.g/. mu.l with nuclease-free ultrapure water, nucleic acid nanocomposites were prepared from EGFP-siRNA by the method described in example two, then diluted to 88. mu.l nucleic acid nanocomposite mixture containing 10 ng/. mu.l EGFP-siRNA with nuclease-free ultrapure water, and after standing for 10min, the volume of 20. mu.l per well was added to 180. mu.l opti-MEM per wellIn nutrient 96-well plates, 4 wells were repeated for each sample. After 4h of dosing, the aspirated 96-well plate was replaced with complete medium. And (4) continuing culturing for 24h, sucking out complete culture medium, rinsing with PBS once, collecting cells, detecting the fluorescence intensity of the FITC channel of each hole of living cells by using a Bekcman Coulter Cytoflex flow cytometer, and calculating the proportion of the EGFP positive cells in each hole and the median of the fluorescence intensity.
The results are shown in FIGS. 6 and 7. And (4) conclusion: the results show that the lower the EGFP positive cell proportion, the lower the median value of fluorescence intensity indicates better transfection effect, and Rp.03, Rp.04, Rp.09, Rp.15, Rp.26, Rp.33, Rp.37 and Rp.42 show better transfection effect, wherein Rp.15, Rp.33, Rp.37 and Rp.42 are better than other prescriptions.
Example five: mouse transfection of nucleic acid nanocomposite through fluorescence imaging detection of small animals
Three female BALB/c mice per group, using FLuc-mRNA as model mRNA, were prepared with the preparation method of recipe Rp.02, Rp.03, Rp.04, Rp.05, Rp.06, Rp.08, Rp.09, Rp.12, Rp.13, Rp.15, Rp.16, Rp.17, Rp.20, Rp.21, Rp.22, Rp.26, Rp.31, Rp.32, Rp.33, Rp.34, Rp.35, Rp.37, Rp.38, Rp.41 and Rp.42, described in example two, containing FLuc-mRNA. Experimental groups 75 μ l of nucleic acid nanocomplexes containing 5 μ g FLuc-mRNA was injected into each mouse using an insulin needle. The administration mode is intramuscular injection, and the injection site is the thigh muscle of a mouse. Blank control was indicated by NC and insulin needles were injected intramuscularly with 75. mu.l PBS buffer. After 6 hours of administration, a proper amount of substrate D-Luciferin is taken, diluted by PBS to prepare a solution with the concentration of 25mg/mL, kept in the dark for standby, 125 mu l of substrate is injected into the abdominal cavity of each mouse, the mouse is placed in a small animal anesthesia box, and an aeration valve is opened to release isoflurane to anesthetize the mouse. 5min after substrate injection, mice were subjected to whole body in vivo imaging bioluminescence image detection using a small animal in vivo imaging system (Perkinelmer, IVIS lumine Series III). A bioluminescent image of the back of the mouse was taken. The results are shown in FIGS. 8-10, where one representative mouse was taken from each group, and the nucleic acid nanocomposites of the experimental group were formulated to show luciferase expression in whole body in vivo imaging, the higher the fluorescence intensity, the more luciferase expression.
The results are shown in FIGS. 8-10. And (4) conclusion: the nucleic acid nano-composites of each experimental group carrying FLuc-mRNA have better luciferase expression in a mouse body. In the prescription of the experimental group, the luciferase has better Rp.03, Rp.04, Rp.09, Rp.15, Rp.26 and Rp.33 than other prescriptions.
Example six: evaluation of humoral immunity effect of nucleic acid nanocomposite in mice
New crown S-mRNA is taken as model mRNA, the new crown S-mRNA is provided by Shanghai McBiotech Corporation, and the nucleotide sequence of the new crown S-mRNA (cap1 structure, N1-me-pseudo U modified) is shown as S-mRNA in a sequence table.
The specific information of the S-mRNA stock solution is as follows:
the product name is as follows: COVID-19Spike Protein, Full Length-mRNA;
product description: 4088 nucleotides in length;
modifications (Modifications): fully subsampled with N1-Me-pseudo UTP; (all substituted with N1-Me-pseudo UTP);
concentration: 1.0 mg/mL;
storage environment: 1mM sodium citrate pH 6.4;
the storage requirement is as follows: -40 ℃ or below.
The experimental process comprises the following steps:
step 1: first immunization of mice: on day 0, 5-6 weeks female BALB/c mice were divided into 13 groups (5 per group) and intramuscularly injected with 75 μ l PBS (blank control), 5 μ g naked S-mRNA (negative control) and 5 μ g S protein combination (positive control) and 75 μ l nucleic acid nanocomplex formulation loaded with 5 μ g S-mRNA Rp.03, Rp.04, Rp.09, Rp.12, Rp.15, Rp.16, Rp.26, Rp.33, Rp.37 and Rp.42, respectively.
Step 2: first serum collection: on day 28, mice were bled at the outer canthus. After blood is coagulated for 1h at 4 ℃, the blood is centrifuged for 5 minutes at the rotating speed of 5000 Xg and the temperature of 4 ℃, supernatant is taken and then centrifuged for 5 minutes at the rotating speed of 10000 Xg and the temperature of 4 ℃, and the supernatant is taken and added into eight rows of PCR tubes for subpackage and is frozen and stored for standby at the temperature of minus 20 ℃.
And step 3: and (3) carrying out secondary immunization on the mice: on day 28, mice were bled via the outer canthus and injected intramuscularly with 75 μ l PBS (blank control), 5 μ g of a combination of naked S-mRNA and 5 μ g S protein (positive control) and 75 μ l of nucleic acid nanocomplex formulations rp.03, rp.04, rp.09, rp.12, rp.15, rp.16, rp.26, rp.33, rp.37 and rp.42, respectively, loaded with 5 μ g S-mRNA. The process of the first immunization is repeated.
And 4, step 4: and (3) collecting serum for the second time: the mice were bled at the outer canthus 21 days after the second immunization. After blood is coagulated for 1h at 4 ℃, the blood is centrifuged for 5 minutes at the rotating speed of 5000 Xg (5000 times of the acceleration of gravity) and 4 ℃, supernatant is taken and then centrifuged for 5 minutes at the rotating speed of 10000 Xg and 4 ℃, the supernatant is taken and added into eight rows of PCR tubes for subpackaging, and the mixture is frozen and stored for standby at-20 ℃.
And 5: ELISA detection of serum IgG content: the S protein was diluted in PBS and the ELISA plate was coated with 100. mu.l of the dilution (containing 1. mu. g S protein) per well and coated for 6h at 4 ℃. The plate was discarded and 200. mu.l PBST was added to each well for 3 washes, followed by 200. mu.l PBS blocking containing 5% BSA in each well and shaking-table blocking at 25 ℃ for 2 h. The blocking solution was discarded, 200. mu.l of PBST was added to each well for 1 wash, 100. mu.l of serum diluted 200-fold with PBS was added, and the mixture was incubated at 25 ℃ for 2 hours in a shaker. The serum was discarded, 200. mu.l of PBST was added to each well for 3 washes, and then 100. mu.l of antibody diluent (antibody diluted 1:1000 in PBS) was added to each well, followed by incubation for 1h at 25 ℃ in a shaker. Discarding the antibody, adding 200 μ l of PBST washing plate into each well for 3 times, adding 50 μ l of TMB color development solution into each well for dark reaction, adding 50 μ l of 2M sulfuric acid into each well for terminating the reaction after the positive control well turns dark blue or reacts for 10 minutes, detecting the optical density at the wavelength of 450nm and 630nm by an enzyme labeling instrument, and calculating the OD value difference to reflect the level of the anti-S protein IgG in the serum.
The results are shown in FIG. 11. And (4) conclusion: the results show that the prescriptions Rp.03, Rp.04, Rp.09, Rp.12, Rp.15, Rp.16, Rp.26, Rp.33, Rp.37 and Rp.42 have OD values obviously higher than those of a blank control group and a naked mRNA negative control group after the second immunization, and cause obvious immune reactions, which indicates that the prescriptions of the nucleic acid nano-complexes have stronger seroconversion efficiency and humoral immune activation function.
Step 6: ELISA detection of serum IgG titers: the S protein was diluted in PBS, and the ELISA plate was coated with 100. mu.l of the dilution (containing 1. mu. g S protein) per well and coated for 6h at 4 ℃. The plate was discarded and 200. mu.l of PBST was added to each well for 1 wash, followed by 200. mu.l of PBS blocking solution containing 5% BSA in each well and shaking-table blocking at 25 ℃ for 2 h. The blocking solution was discarded, 200. mu.l PBST plates were added to each well 3 times, and then 50, 250, 1250, 6250, 31250, 156250, 781250, 3906250 times diluted 1:3 in PBS were added thereto, followed by incubation for 2h at 25 ℃ in a shaker. The serum was discarded, 200. mu.l of PBST was added to each well for 3 washes, and then 100. mu.l of antibody diluent (antibody diluted 1:1000 in PBS) was added to each well, followed by incubation for 1h at 25 ℃ in a shaker. Discarding the antibody, adding 200 μ l PBST washing plate into each well for 3 times, adding 50 μ l TMB color development solution into each well for reaction in a dark place, adding 50 μ l2M sulfuric acid into each well after the positive control well turns dark blue or reacts for 10 minutes to stop the reaction, and detecting the optical density at 450nm and 630nm by an enzyme-labeling instrument.
And (4) conclusion: as shown in table 2 and fig. 12, the present invention uses 2 times of the mean OD value of PBS group as baseline, the OD values of prescriptions rp.04, rp.09, rp.15, rp.16, rp.26, rp.33, rp.37 and rp.42 groups are still higher than baseline when diluted 31250 times, the OD values of prescriptions rp.04, rp.09, rp.15, rp.33 and rp.37 groups are still higher than baseline when diluted 156250 times, and the OD values of prescriptions rp.33 group are still higher than baseline when diluted 781250 times, indicating that these prescriptions have stronger seroconversion efficiency and humoral immune activation function.
Table 2: ELISA detection of serum IgG titer OD value of each prescription
Figure BDA0003689084690000301
Example seven: evaluation of therapeutic Effect of nucleobase derivative Complex-OVA-mRNA vaccine on tumor-bearing mouse model
1) B16-establishment of OVA melanoma mouse model: amplifying and culturing murine lymphoma cell B16-OVA in vitro to obtain B16-OVA cell line, diluting with DPBS, and adding 5 × 10 cells per mouse 5 And (4) tumor cells. 7-week-old female C57BL/6J mice were dehaired on day 0 on flank, cultured B16-OVA tumor cells were collected, B16-OVA tumor cells were injected subcutaneously into flank of mice to establish subcutaneous B cells16-OVA tumor model.
2) Preparation of nucleobase derivative complex-OVA-mRNA vaccine: ten nucleobase derivative complex-OVA-mRNA vaccines prepared from the recipes rp.03, rp.04, rp.09, rp.12, rp.15, rp.16, rp.26, rp.33, rp.37 and rp.42, respectively, were obtained after briefly and gently mixing the recipes with OVA-mRNA (purchased from TriLink, usa) for 30 minutes, respectively;
3) c57BL/6J mice were vaccinated with nucleobase derivative complex-OVA-mRNA vaccine (each injection of nanoparticle vaccine containing 5ug of therapeutic agent mRNA-OVA) by foot injection on day 10, day 13 and day 16, respectively, while mice vaccinated with an equal volume of PBS buffer solution and an equal volume of OVA-mRNA solution after dilution were set as control groups, with 5 mice per group in parallel.
4) Tumor vertical diameter was measured daily starting on day 10 after tumor inoculation. Tumor volume was calculated for C57BL/6J mice according to the following formula: v (mm) 3 )＝x×y 2 And/2 in mm, wherein V represents tumor volume, x represents tumor major diameter, and y represents tumor minor diameter. Meanwhile, the change of the body weight of the C57BL/6J mouse was recorded daily on an electronic balance, and the survival rate was counted.
The results of the examination are shown in fig. 13, 14, and 15: B16-OVA melanoma cells were inoculated subcutaneously on day 0 and vaccinated at day 10, day 13 and day 16, respectively, after tumor inoculation.
And (4) conclusion: as shown in fig. 14 and table 3, the PBS control group and the naked mRNA-OVA group were sacrificed from day 21 and day 26, respectively, after tumor inoculation, and all mice in both groups were sacrificed at day 37 and day 39, respectively. The nucleobase derivative complex-OVA-mRNA vaccine sets prepared from the prescriptions rp.03, rp.04, rp.09, rp.12, rp.15, rp.16, rp.26, rp.33, rp.37 and rp.42 were sacrificed from day 28, day 31, day 29, day 26, day 35, day 27, day 32, day 36, day 35 and day 32, respectively. Prescription rp.03, rp.04, rp.09, rp.12, rp.15, rp.16, rp.26, rp.33, rp.37 and rp.42 all mice of the nucleobase derivative complex-OVA-mRNA vaccine groups prepared were sacrificed at day 41, day 42, day 44, day 43, day 45, day 40, day 45, day 46 and day 44, respectively. The results show that the mice in the experimental group are significantly delayed in all days of sacrifice, i.e., the time to death of the mice, compared to the PBS group and the naked OVA-mRNA group.
As shown in fig. 15 and table 4, the positive control group, naked mRNA-OVA group, and nucleobase derivative complex-OVA-mRNA vaccine groups prepared by the prescriptions rp.03, rp.04, rp.09, rp.12, rp.15, rp.16, rp.26, rp.33, rp.37, and rp.42 showed tumor growth from 8 to 11 days after tumor inoculation. The nucleobase derivative complex-OVA-mRNA vaccine groups prepared from rp.03, rp.04, rp.09, rp.12, rp.15, rp.16, rp.26, rp.33, rp.37 and rp.42 showed significant tumor growth delay compared to the PBS control group and the naked mRNA-OVA group.
Table 3: statistics of sacrifice days after tumor inoculation for each group
Figure BDA0003689084690000311
Table 4: tumor size (mm) after tumor inoculation for each group 3 ) Statistics of changes
Figure BDA0003689084690000321
And (4) conclusion: the vaccine taking the nanoparticles prepared by the prescription Rp.03, Rp.04, Rp.09, Rp.12, Rp.15, Rp.16, Rp.26, Rp.33, Rp.37 and Rp.42 provided by the invention as a carrier shows a good nucleic acid protection effect, is beneficial to delivery of nucleic acid in vivo, promotes the nucleic acid to penetrate cell membranes, and has an obvious effect of improving the activity of mRNA in vivo.
While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention within the context, spirit and scope of the invention. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention.

Claims (10)

1. A nanoparticle, comprising: a compound as shown in formula I and auxiliary materials,
Figure FDA0003689084680000011
wherein n is selected from an integer from 5 to 20, preferably from 12 to 18;
the auxiliary material comprises a material selected from: at least one of a PEG derivative, lipid, lipidoid, alcohol, or inorganic salt; and/or
The auxiliary material comprises a material selected from: at least one of a PEG derivative, a lipid, and a lipoid.
2. A nanoparticle according to claim 2, wherein the PEG derivative comprises at least one selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol; and/or
The PEG derivative comprises a PEG selected from 1, 2-dimyristoyl-sn-glyceromethoxypolyethylene glycol, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ], dilauroyl phosphatidylethanolamine-polyethylene glycol, dimyristoyl phosphatidylethanolamine-polyethylene glycol, dipalmitoyl phosphatidylcholine polyethylene glycol, dipalmitoyl phosphatidylethanolamine-polyethylene glycol, PEG-distearoyl glycerol, PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerol amide, PEG-dipalmitoyl phosphatidylethanolamine, or PEG-1, 2-dimyristol oxypropyl-3-amine; and/or
The DMG-PEG comprises a material selected from DMG-PEG 2000; and/or
The lipid comprises a lipid selected from the group consisting of lecithin, 1, 2-distearoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoleoyl-sn-glycero-3-phosphocholine, 1, 2-dimyristoyl-sn-glycero-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine, 1, 2-diundecabonyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or at least one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, or alpha-tocopherol; and/or
The lipid comprises at least one selected from lecithin, 1, 2-distearoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine; and/or
The lipid comprises a poloxamer selected from poloxamine or poloxamine derivatives; and/or
Said poloxamine comprises a compound selected from the group consisting of
Figure FDA0003689084680000012
304、
Figure FDA0003689084680000013
701、
Figure FDA0003689084680000014
704、
Figure FDA0003689084680000015
707、
Figure FDA0003689084680000016
803、
Figure FDA0003689084680000017
901、
Figure FDA0003689084680000018
904、
Figure FDA0003689084680000019
908、
Figure FDA00036890846800000110
1107、
Figure FDA00036890846800000111
1301、
Figure FDA00036890846800000112
1304、
Figure FDA00036890846800000113
1307、
Figure FDA00036890846800000114
90R4 or
Figure FDA00036890846800000115
150R 1; and/or
The poloxamine derivatives comprise at least one selected from the group consisting of poloxamine derivative T304-T, poloxamine derivative T304-D, poloxamine derivative T304-RT, poloxamine derivative T304-RC, poloxamine derivative T701-R, poloxamine derivative T901-C, poloxamine derivative T803-RT, poloxamine derivative T304-RT, poloxamine derivative T704-M, poloxamine derivative T704-RT, poloxamine derivative T704-RC, poloxamine derivative T904-CR, poloxamine derivative T904-RC, poloxamine derivative T904-RT, poloxamine derivative T90R4-R, and poloxamine derivative T90R 4-RT; and/or
The alcohol comprises an aqueous solution of alcohol at a concentration greater than 2% vol; and/or the alcohol comprises an aqueous solution selected from ethanol or ethanol at a concentration of greater than 2% vol; and/or
The inorganic salt comprises a salt selected from potassium chloride or phosphate.
3. A nanoparticle according to any one of claims 1 to 3, wherein the compound of formula I is present in an amount of 30.0 wt% to 60.0 wt%, based on the total mass of the nanoparticle; and/or
Calculated by the total mass of the nanoparticles, the content of the PEG derivative is 0-15.0 wt%; and/or
The lipid content is 30-70 wt% calculated by the total mass of the nanoparticle; and/or
The content of the lipid is 0-36 wt% calculated by the total mass of the nanoparticle.
4. A nanoparticle complex comprising a nucleic acid and the nanoparticle of any one of claims 1-4.
5. The nanoparticle complex of claim 4, wherein the weight ratio of the nanoparticle to nucleic acid (1: 2-4: 1):1 or the weight ratio of the nanoparticle to the nucleic acid is 0.5:1, 0.8:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4: 1.
6. A nucleic acid nanoparticle complex comprises a compound shown in a formula I and nucleic acid, wherein the mass ratio of the compound shown in the formula I to the nucleic acid is (12-45): 100.
7. A pharmaceutical composition comprising the nanoparticle complex of any one of claims 4-6 and a pharmaceutically acceptable excipient.
8. A method of making a nanoparticle of any one of claims 1-4, comprising: dissolving a compound shown as a formula I in a solvent A to obtain a solution 1, dissolving an auxiliary material in a solvent B to obtain a solution 2, uniformly mixing the solution 1 and the solution 2, performing rotary evaporation at a low temperature, removing the solvent A in a water bath, and filtering to obtain the nanoparticle.
9. A method of preparing the nanoparticle composite of any one of claims 5-6, comprising: mixing the nanoparticle of any one of claims 1-4 with a nucleic acid in solvent C to obtain the nanoparticle complex.
10. Use of a nanoparticle according to any one of claims 1 to 3 or a nanoparticle complex according to claims 4 to 6 or a pharmaceutical composition according to claim 7 for the manufacture of a medicament or vaccine for the in vivo delivery of a nucleic acid.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2021250263A1 (en) * 2020-06-11 2021-12-16 Etherna Immunotherapies Nv Lipid nanoparticles
CN114404391A (en) * 2022-02-16 2022-04-29 深圳厚存纳米药业有限公司 Nano particle, preparation method and application thereof

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Publication number Priority date Publication date Assignee Title
WO2021250263A1 (en) * 2020-06-11 2021-12-16 Etherna Immunotherapies Nv Lipid nanoparticles
CN114404391A (en) * 2022-02-16 2022-04-29 深圳厚存纳米药业有限公司 Nano particle, preparation method and application thereof

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