CN114933565A

CN114933565A - Nucleobase derivative nanoparticles and composition thereof

Info

Publication number: CN114933565A
Application number: CN202210516975.4A
Authority: CN
Inventors: 张龙贵; 刘晨; 梁梅桂; 王宇恒
Original assignee: Shenzhen Houcun Nano Pharmaceutical Co ltd
Current assignee: Shenzhen Houcun Nano Pharmaceutical Co ltd
Priority date: 2022-05-12
Filing date: 2022-05-12
Publication date: 2022-08-23

Abstract

A nanometer particle of nucleobase derivative and its composition relates to the field of biological medicine, the nucleobase derivative is a compound shown as formula I or its stereoisomer or tautomer,

the composition includes a compound of formula I, or a stereoisomer or tautomer thereof, and an auxiliary material; the auxiliary material comprises a material selected from: at least one of PEG derivatives, lipids and lipid-like substances.

Description

Nucleobase derivative nanoparticles and composition thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a nucleobase derivative nanoparticle and a composition 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 an exogenous 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 nanoparticle is a main non-viral vector applied to RNA drug development at present, and the first RNAi drug (Patisiran) and the first mRNA drug (BNT162b2, Comirnaty) are listed in the market sequentially at present, so that the clinical application value of the Liposome Nanoparticle (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, there is provided a compound of formula I, or a stereoisomer or tautomer thereof.

In a second aspect, there is provided a nucleobase derivative nanoparticle.

In a third aspect, a nucleic acid nanocomplex is provided.

In a fourth aspect, a pharmaceutical composition is provided.

In a fifth aspect, there is provided a use of the aforementioned compound, nanoparticle, nucleic acid nanocomposite or pharmaceutical composition.

In a sixth aspect, a method of making the foregoing nanoparticle is provided.

In a seventh aspect, there is provided a method of preparing the aforementioned nucleic acid nanoparticle complex.

Detailed Description

In order to solve the above problems, the present invention provides the following technical solutions.

In a first aspect, there is provided a compound of formula I or a stereoisomer or tautomer thereof.

A compound of formula I or a stereoisomer or tautomer thereof,

a second aspect provides a nucleobase derivative nanoparticle comprising: a compound of formula I according to the first aspect or a stereoisomer or tautomer thereof, optionally together with auxiliary materials.

The auxiliary material may include one or more materials selected from: at least one of a PEG derivative, a lipid-like, an alcohol, or an inorganic salt.

The auxiliary material may include one or more materials selected from: PEG derivatives and lipids.

The PEG derivative may include at least one selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, poloxamer, polysorbate, or span. In some embodiments, the PEG derivative comprises a member selected from the group consisting of 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, at least one of PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerol amide, PEG-dipalmitoyl phosphatidylethanolamine, and PEG-1, 2-dimyristoloxyprop-3-amine.

PEG-modified ceramides may include a group selected from PEG-CerC ₁₄ Or PEG-CerC ₂₀ 。

The lipid may comprise a lipid selected from 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. In some embodiments, the lipid comprises at least one selected from the group consisting of cholesterol, lecithin, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-distearoyl-sn-glycero-3-phosphocholine. In some embodiments, the lipid comprises cholesterol and at least one member selected from the group consisting of lecithin, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-distearoyl-sn-glycero-3-phosphocholine.

The lipid may include at least one selected from poloxamers, polysorbates, span, poloxamines, or poloxamine derivatives.

The poloxamines may include compounds selected from

304、

701、

704、

707、

803、

901、

904、

908、

1107、

1301、

1304、

1307、

90R4 or

150R 1.

The poloxamine derivatives may include at least one 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, poloxamine derivatives T704-RT, poloxamine derivatives T704-RC, poloxamine derivatives T904-CR, poloxamine derivatives T904-RC, poloxamine derivatives T904-RT, poloxamine derivatives T90R4-R, and poloxamine derivatives T90R 4-RT.

The poloxamer may include one or more compounds selected from: at least one of poloxamer 188, poloxamer L64, poloxamer 17R4, poloxamer F127, poloxamer F68, poloxamer P123, poloxamer P85, or poloxamer L61.

The polysorbate may include at least one selected from polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

The span may include at least one selected from span 20, span 60, span 65, span 80, or span 85.

The alcohol may comprise 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.

The inorganic salt may include a salt selected from potassium chloride or phosphate.

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 nucleobase derivative nanoparticle. In some embodiments, the compound of formula I is present in an amount of about 31.1 wt%, about 37.6 wt%, about 48.0 wt%, about 59.3 wt%, about 44.8 wt%, about 50.9 wt%, based on the total mass of the nanoparticle.

The lipid content may be 34.9 wt% to 53.5 wt% based on the total mass of the nucleobase derivative nanoparticle. In some embodiments, the lipid is present in an amount of about 34.5 wt%, about 35.4 wt%, about 34.9 wt%, about 43.3 wt%, about 53.5 wt%, based on the total mass of the nanoparticle.

The lipid content may be 0 wt% to 33.5 wt% based on the total mass of the nucleobase derivative nanoparticle. In some embodiments, the lipid is present in an amount of 14.1 wt% to 33.5 wt% based on the total weight of the nanoparticle. In some embodiments, the lipid content is 35.7 wt% based on the total mass of the nanoparticle. In some embodiments, the lipid is present in an amount of 0.0 wt%, 10.0 wt%, 14.0 wt%, 20.0 wt%, 25 wt%, 30 wt%, 34 wt%, based on the total mass of the nanoparticle.

In some embodiments, the nucleobase derivative nanoparticle comprises a compound of formula I, a PEG derivative, and a lipid, wherein the compound of formula I is present in an amount of 37.6 wt% to 59.3 wt% based on the total mass of the nanoparticle; the PEG derivative is present in an amount of about 6.0 wt% to about 8.8 wt%; the content of the lipid is 34.5 wt% -53.5 wt%.

In some embodiments, the nucleobase derivative nanoparticle comprises a compound of formula I, a lipid, and a lipid, wherein the compound of formula I is present in an amount of 31.0 wt% to 50.9 wt% based on the total weight of the nanoparticle; the content of the lipid is 34.9-35.4 wt%; the content of the lipid is 14.1 wt% -33.5 wt%.

In some embodiments, the nucleobase derivative nanoparticle comprises a compound of formula I, a PEG derivative, and a lipid, the compound of formula I: the PEG derivative is: the lipid mass ratio may be (64-105): (11-15): (61-91). The PEG derivative is selected from DMG-PEG2000, and the lipid is selected from DSPC/PC and cholesterol.

In a third aspect, a nucleic acid nanocomplex is provided.

A nucleic acid nanocomplex, comprising: a nucleic acid and at least one selected from a compound of formula I according to the first aspect or a stereoisomer or tautomer thereof or a nucleobase derivative nanoparticle according to the second aspect.

In some embodiments, a nucleic acid nanoplex comprising a nucleic acid and a compound of formula I according to the first aspect, or a stereoisomer or tautomer thereof, wherein the mass ratio of the nucleic acid to the compound of formula I, or the stereoisomer or tautomer thereof, is 100 (30-300). In some embodiments, a nucleic acid nanoplex comprising a nucleic acid and a compound of formula I according to the first aspect, or a stereoisomer or tautomer thereof, wherein the mass ratio of the nucleic acid to the compound of formula I, or the stereoisomer or tautomer thereof, is 100:30, 100:50, or 100: 300.

In some embodiments, a nucleic acid nanocomplex comprising a nucleic acid and a nucleobase derivative nanoparticle of the third aspect, wherein the mass ratio of the nucleic acid to the nanoparticle of the third aspect is 0.51:1 to 1.47:1, 0.62:1 to 1.12:1, 0.96:1 to 1.10:1, or 0.62:1 to 1.0: 1. In some embodiments, a nucleic acid nanocomplex comprises a nucleic acid and a nanoparticle of the third aspect in a mass ratio of 0.75:1, 0.96:1, 1.12:1, 0.62:1, 0.877:1, 1.02: 1.

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 (such as an NBD011 compound) can form a base pair with guanine G in nucleic acid, C is connected with G through 3 hydrogen bonds, and double hydrogen bonds are formed between amine and carbonyl of complementary base, as shown in a formula II:

or the compound shown in the formula I and other conjugated groups in nucleic acid form an amphiphilic composition through a pi-pi stacking effect, so that the compound is self-assembled to form the nanoparticle under a certain condition. Specifically, the nucleobase derivative disclosed by the invention mainly forms an amphiphilic composition with nucleic acid through base complementary pairing (hydrogen bond) or pi-pi stacking effect, a hydrophobic part is in the middle of a nanoparticle in an aqueous solution, a hydrophilic nucleic acid and a hydrophilic part are on the surface of the nanoparticle, and a nucleobase derivative nanoparticle compound is formed through hydrophilic and hydrophobic acting force assembly.

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 fourth aspect, a pharmaceutical composition is provided.

A pharmaceutical composition comprising the nucleic acid nanocomplex of the third 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 fifth aspect, there is provided a use of the aforementioned compound, nucleobase derivative nanoparticle, nucleic acid nanocomposite or pharmaceutical composition.

Use of a compound of formula I according to the first aspect or a stereoisomer or a tautomer thereof, a nanoparticle according to the third aspect or a nucleic acid nanocomposite according to the fourth aspect or a pharmaceutical composition according to the fifth aspect for the preparation of a product 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 an immune response against an infectious agent or cancer, including cellular and humoral immune responses, without risk that could lead to insertional mutagenesis, for example. The RNA vaccine using the nanoparticle of the first aspect as a delivery vehicle can be used in various environments depending on the incidence of infectious pathogens and cancer. 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 nucleobase derivative nanoparticles according to the first aspect or the nanoparticle complexes according to the third aspect of the invention are useful 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 and ear diseases.

The nucleic acid vaccines of the present invention can be administered by any route that produces therapeutically effective results. 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.

In a sixth aspect, a method of preparing the nucleobase derivative nanoparticles described above is provided.

In some embodiments, a method of making a nanoparticle of the third aspect, comprising: mixing the lipid with a solvent A to obtain a solution i; mixing a compound shown as a formula I or a stereoisomer or tautomer thereof with a solvent B to obtain a solution ii; mixing the lipid and optionally the PEG derivative with solvent C to obtain a solution iii; and (3) mixing the solution ii and the solution iii, removing the solvent B and the solvent C by rotary evaporation, adding the solution i, mixing and filtering to obtain the nanoparticles.

The solvent a may include PBS buffer selected from water or pH 7.4.

The solvent B may include at least one selected from dichloromethane or ethanol.

The solvent C may include at least one selected from ethanol or dichloromethane.

In some embodiments, a method of making a nanoparticle of the third aspect, comprising: mixing the compound shown in the formula I or the stereoisomer or tautomer thereof and the auxiliary material with a solvent D, mixing with water under the conditions of water bath and ultrasound, removing the solvent D by rotary evaporation, and filtering to obtain the nanoparticles.

The solvent D may include at least one selected from ethanol or dichloromethane.

The temperature of the water bath may be 35 ℃ to 55 ℃. In some embodiments, the temperature of the water bath is 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 50 ℃ or 55 ℃.

In some embodiments, a method of making a nanoparticle of the third aspect, comprising: mixing the compound shown in the formula I or the stereoisomer or tautomer thereof with a solvent E, removing the solvent E by rotary evaporation, adding water, performing ultrasonic treatment, and filtering to obtain the nanoparticles.

The solvent E may include at least one selected from dichloromethane or ethanol.

A method of making a nucleic acid nanoparticle complex of the third aspect, comprising: mixing the nanoparticles described in the third aspect or the nanoparticles obtained by the method described in the sixth aspect with nucleic acid in water to obtain the nucleic acid nanoparticle complex.

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 nucleobase derivative 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, the hydrophobic part is in the middle of the nanoparticles, the hydrophilic nucleic acid and the hydrophilic part are on the surfaces of the nanoparticles in aqueous solution, and the nanoparticles are formed through the assembly of 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 nanoparticle or nanoparticle compound can carry mRNA encoding polypeptide or protein to enter cells, effectively release the mRNA, express the polypeptide and effectively achieve the purpose of treating diseases.

(2) The particle size range of the nucleic acid nano-composite provided by the invention is between 90nm and 240nm, the nucleic acid nano-composite has better dispersibility, and the surface charge of the nucleic acid nano-composite is between 0mV and 35 mV.

(3) The nucleic acid nano-composite provided by the invention has small cytotoxicity and good biocompatibility.

(4) The nucleic acid nano-composite provided by the invention has the advantages of compressing and protecting nucleic acid from being degraded, promoting the nucleic acid to penetrate cell membranes, realizing efficient transfection inside and outside a body, having good biocompatibility and the like.

(5) The nucleic acid is transferred by the nucleobase derivative nanoparticles, so that the in-vivo and in-vitro transfection performance of the nucleic acid, the serum conversion efficiency and the humoral immune activation function are improved, more cell lines are transfected, and the in-vivo activity of the nucleic acid-entrapped nucleic acid nanocomposite is improved.

(6) The proportion of the auxiliary materials provided by the invention is beneficial to improving the transfection of nucleic acid in the obtained nucleic acid nano-composite in vivo and in vitro, improving the seroconversion efficiency and the humoral immune activation function, transfecting more cell lines and improving the activity of the nucleic acid-loaded nano-particle composite in vivo.

(7) The preparation method of the nucleobase derivative nanoparticles and the nucleic acid nanocomposite is simple to operate, low in cost, environment-friendly and beneficial to industrial production.

Drawings

FIG. 1 illustrates transfection of Fluc-mRNA loaded nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles in DC2.4 cells of example four; in the figure, the abscissa represents nucleobase derivative nanoparticle compositions and nucleobase derivative complex nanoparticle compositions of different prescriptions, and the ordinate is the relative fluorescence intensity expressed after transfecting the nucleobase derivative nanoparticle compositions and the nucleobase derivative complex nanoparticle compositions containing the same dose of FLuc-mRNA for 24 hours.

FIG. 2 shows the survival rate of DC2.4 cells after treatment with different prescriptions in example four; the abscissa represents different nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticle formulations, and the ordinate represents cell viability, showing that the higher the cell activity, the lower the cytotoxicity.

FIG. 3 illustrates transfection of Luc-pDNA loaded nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles in DC2.4 cells in example four; the abscissa represents the different prescriptions and the ordinate is the relative fluorescence intensity expressed by DC2.4 cells 24h, 48h, 72h after transfection with the same dose of Luc-pDNA.

FIG. 4 illustrates transfection of Fluc-mRNA loaded nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles in different cells of example four; in the figure, the abscissa represents nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticle compositions of different prescriptions, and the ordinate is the relative fluorescence intensity expressed after different cells are transfected for 24h after the nucleobase derivative complex nanoparticle compositions containing the same dose of FLuc-mRNA are transfected.

FIG. 5 is a graph showing the survival rate of cells treated with different formulations in example four; the abscissa represents different nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticle formulations, and the ordinate represents cell viability, showing that the higher the cell activity, the lower the cytotoxicity.

FIG. 6 shows transfection of EGFP-siRNA-loaded nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles into Hela-EGFP cells in example four; in the figure, the abscissa represents nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticle compositions of different prescriptions, and the ordinate represents the percentage of EGFP positive cells after transfection of nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticle compositions containing the same dose of EGFP-siRNA for 24 hours by Hela-EGFP transfection.

FIG. 7 shows transfection of EGFP-siRNA-loaded nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles into Hela-EGFP cells in example four; in the figure, the abscissa represents nucleobase derivative nanoparticles and nucleobase derivative compound nanoparticle compositions of different prescriptions, and the ordinate represents the fluorescence intensity median of the nucleobase derivative nanoparticles and the nucleobase derivative compound nanoparticle compositions transfected with the same dose of EGFP-siRNA after Hela-EGFP transfection for 24 hours.

FIG. 8 shows the IVIS in example five, which detects the expression of luciferase in mice by the FLuc-mRNA-loaded nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles.

FIG. 9 shows serum IgG antibody levels of mice immunized with nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles bearing the novel crown 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. 10 shows serum IgG antibody titers of mice immunized with nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles bearing the novel crown S-mRNA of example six; 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. 2x 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.

FIGS. 11 and 12 show the results of example seven, in which OVA-mRNA-loaded nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles were injected subcutaneously into C57BL/6J mice to examine the therapeutic effect of the vaccines on melanoma.

Definition of terms:

in the invention, the room temperature refers to the ambient temperature, and can be 10-40 ℃, 15-35 ℃ or 20-30 ℃; 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, whether or not the word "about" or "approximately" is used. Based on the disclosed numbers, it is possible that the numerical value of each number will vary by less than 10% or reasonably as recognized by one of 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, "mixing a lipid and optionally a PEG derivative with solvent C" means "mixing a lipid with solvent C" or "mixing a lipid and a PEG derivative with solvent C".

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 arts. 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.

In the present application, expressions analogous to "compound of formula I", "compound of formula I" and "compound I" all denote the same substance.

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 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-PEG2000 represents 1, 2-dimyristoyl-sn-glyceromethoxypolyethylene glycol 2000; 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-glycerol-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;

l64 represents poloxamer L64;

20 represents tween 20; DPPC represents 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine;

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. EDCI denotes 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. DMAP stands for 4-dimethylaminopyridine. NBD004 represents a NBD004 compound (i.e., a compound of formula C).

Fluc-mRNA represents messenger RNA encoding firefly luciferase; Luc-pDNA represents a plasmid encoding firefly luciferase; EGFP-siRNA represents a small interfering RNA expressed by a silent enhanced green fluorescent protein gene; OVA protein means chicken ovalbumin; mRNA-OVA represents messenger RNA encoding chicken ovalbumin.

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 NBD011 are described as follows:

synthesis of intermediate 3: compound 1(269.25mg,1mmol), dimethylaminopropanol (103.16mg,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 ═ 1:5) 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 resulting organic phase was suction filtered and spin dried to give product 3 by column chromatography (100% PE to PE: EA 1:5) as a white powdery solid in 89% yield (315.4mg,0.89 mmol).

¹ H NMR(500MHz,Chloroform-d)δ8.08(d,J＝7.1Hz,1H),7.82(s,1H),6.66(d,J＝7.1Hz,1H),4.60(s,1H),4.12(t,J＝6.0Hz,2H),2.64(t,J＝6.5Hz,2H),2.33(s,4H),1.96-1.87(m,1H),1.42(s,6H).

Synthesis of product 4: intermediate 3(177.2mg,0.5mmol) was taken up in 5mL dichloromethane and 1mL trifluoroacetic acid was slowly added dropwise and stirred at room temperature for 2h until the reaction was complete. And (3) spin-drying the solution, adding 30mL of saturated sodium bicarbonate solution to adjust the pH, adding 50mL of ethyl acetate, extracting and separating, collecting an upper organic phase, transferring the upper organic phase into a flask, adding anhydrous sodium sulfate, drying, and repeating the extraction operation for three times. The resulting organic phase was suction filtered and spin dried to give product 4 as a white powdery solid in 96% yield (122.1mg,0.48 mmol).

¹ H NMR(500MHz,Chloroform-d)δ8.00(d,J＝7.0Hz,1H),7.04(s,1H),6.19(d,J＝7.1Hz,1H),4.60(s,1H),4.12(t,J＝6.0Hz,1H),2.64(t,J＝6.5Hz,1H),2.33(s,3H),1.96-1.87(m,1H).

Example two: preparation of nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles

1) Prescription Rp.11: NBD011, DMG-PEG2000, DSPC, Chol and nucleic acid in the weight ratio of 64 to 15 to 31 to 60 to 128

Taking NBD011, DMG-PEG2000, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, firstly weighing NBD011 at the room temperature and adding dichloromethane for dissolving, and then respectively weighing DMG-PEG2000, DSPC and Chol at the room temperature and adding ethanol for dissolving; adding the dissolved NBD011, DMG-PEG2000, DSPC and Chol into a round-bottomed flask and mixing uniformly. Removing ethanol by rotary evaporation at 40 deg.C in water bath to form a lipid film on the wall of the round-bottomed flask, adding ultrapure water containing nucleotidase, fully hydrating the lipid film, and stirring at 1500rpm/min after 2 hr. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nucleobase derivative composite nanoparticle of formula Rp.11, and storing in 4 deg.C refrigerator for use.

2) Prescription Rp.12: NBD011, DMG-PEG2000, DSPC, Chol and nucleic acid in the weight ratio of 82:15:26:48:164

Taking NBD011, DMG-PEG2000, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, firstly weighing NBD011 at the room temperature and adding dichloromethane to dissolve, and then respectively weighing DMG-PEG2000, DSPC and Chol at the room temperature and adding ethanol to dissolve; adding the dissolved NBD011, DMG-PEG2000, DSPC and Chol into a round-bottomed flask and mixing uniformly. Removing ethanol by rotary evaporation at 40 deg.C in water bath to form a lipid film on the wall of the round-bottomed flask, adding ultrapure water containing nucleotidase, fully hydrating the lipid film, and stirring at 1500rpm/min after 2 hr. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nanometer nuclear base derivative compound particle with Rp.12, and storing in 4 deg.C refrigerator.

3) Prescription Rp.13: NBD011, DMG-PEG2000, DSPC, Chol and nucleic acid in the mass ratio of 105:11:26:35:210

Taking NBD011, DMG-PEG2000, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, firstly weighing NBD011 at the room temperature and adding dichloromethane for dissolving, and then respectively weighing DMG-PEG2000, DSPC and Chol at the room temperature and adding ethanol for dissolving; adding the dissolved NBD011, DMG-PEG2000, DSPC and Chol into a round-bottomed flask and mixing uniformly. And (3) rotationally evaporating to remove ethanol by using a rotary evaporator under the condition of water bath at 40 ℃ to form a layer of lipid film on the wall of the round-bottom flask, adding ultrapure water without nucleotidase 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 nanometer nucleobase derivative compound particle with Rp.13 in the prescription, and storing in 4 deg.C refrigerator for use.

4) Prescription Rp.19: NBD011, DMG-PEG2000, DSPC, Chol and nucleic acid in the weight ratio of 138 to 11 to 14 to 25 to 276

Taking NBD011, DMG-PEG2000, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, firstly weighing NBD011 at the room temperature and adding dichloromethane for dissolving, and then respectively weighing DMG-PEG2000, DSPC and Chol at the room temperature and adding ethanol for dissolving; adding the dissolved NBD011, DMG-PEG2000, DSPC and Chol into a round-bottomed flask and mixing uniformly. Removing ethanol by rotary evaporation at 40 deg.C in water bath to form a lipid film on the wall of the round-bottomed flask, adding ultrapure water containing nucleotidase, fully hydrating the lipid film, and stirring at 1500rpm/min after 2 hr. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nucleobase derivative compound nanoparticles of Rp.19, and storing in 4 deg.C refrigerator for use.

5) Prescription Rp.20: NBD011, DMG-PEG2000, DSPC, Chol and nucleic acid in the weight ratio of 32:15:38:53:64

Taking NBD011, DMG-PEG2000, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, firstly weighing NBD011 at the room temperature and adding dichloromethane for dissolving, and then respectively weighing DMG-PEG2000, DSPC and Chol at the room temperature and adding ethanol for dissolving; adding the dissolved NBD011, DMG-PEG2000, DSPC and Chol into a round-bottomed flask and mixing uniformly. Removing ethanol by rotary evaporation at 40 deg.C in water bath to form a lipid film on the wall of the round-bottomed flask, adding ultrapure water containing nucleotidase, fully hydrating the lipid film, and stirring at 1500rpm/min after 2 hr. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nucleobase derivative composite nanoparticle of formula Rp.20, and storing in 4 deg.C refrigerator for use.

6) Prescription Rp.32: NBD011:

the mass ratio of F127 to PC to Chol to nucleic acid is 65:70:31:43:130

Firstly, the method is carried out

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 NBD011, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the NBD011 at the room temperature and adding dichloromethane for dissolution; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD011, PC and Chol into a round-bottom flask, uniformly mixing, and rotationally evaporating the organic solvent by using a rotary evaporator under the condition of water bath at 40 ℃ to enable the sample to be roundForming a layer of lipid film on the wall of the 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 nanometer nucleobase derivative compound particle with Rp.32 in the prescription, and storing in 4 deg.C refrigerator for use.

7) Prescription Rp.33: NBD011:

the mass ratio of F127 to PC to Chol to nucleic acid is 93:45:31:43:186

Firstly, the method is carried out

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 NBD011, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the NBD011 at the room temperature and adding dichloromethane for dissolution; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD011, PC and Chol into a round-bottom flask, uniformly mixing, rotationally evaporating the organic solvent by using a rotary evaporator under the condition of 40 ℃ water bath 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 after 2 hours to stir at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nucleobase derivative composite nanoparticle of formula Rp.33, and storing in 4 deg.C refrigerator for use.

8) Prescription Rp.34: NBD011:

the mass ratio of F127 to PC to Chol to nucleic acid is 108:30:31:43:216

Firstly, the method is to

F127 is taken out from a refrigerator at 4 deg.C and balanced to room temperature, and the ultrapure water which is weighed and added with the nucleotidase is dissolved at room temperature, and is fully oscillated for 5min by a vortex instrument and is kept standOvernight to give stock solution A; then taking the NBD011, PC and Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the NBD011 at the room temperature and adding dichloromethane to dissolve the NBD 011; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD011, 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 thin film on the wall of the round-bottom flask, adding the stock solution A to fully hydrate the lipid thin film, and adding a stirrer after 2 hours to stir at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nucleobase derivative compound nanoparticles of Rp.34, and storing in 4 deg.C refrigerator for use.

9) Prescription Rp.39: NBD011:

the mass ratio of F127 to PC to Chol to nucleic acid is 55:85:31:43:110

Firstly, the method is carried out

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; then taking the NBD011, PC and Chol out of a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the NBD011 at the room temperature and adding dichloromethane to dissolve the NBD 011; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD011, PC and Chol into a round-bottom flask, uniformly mixing, rotationally evaporating the organic solvent by using a rotary evaporator under the condition of 40 ℃ water bath 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 after 2 hours to stir at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nucleobase derivative composite nanoparticle of formula Rp.39, and storing in 4 deg.C refrigerator for use.

10) Prescription Rp.40: NBD011:

f127 PC Chol nucleusThe acid mass ratio is 126:15:31:43:252

Firstly, the method is carried out

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 NBD011, the PC and the Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing the NBD011 at the room temperature and adding dichloromethane for dissolution; respectively weighing PC and Chol at room temperature, and dissolving in ethanol; adding the dissolved NBD011, PC and Chol into a round-bottom flask, uniformly mixing, rotationally evaporating the organic solvent by using a rotary evaporator under the condition of 40 ℃ water bath 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 after 2 hours to stir at the rotating speed of 1500 rpm/min. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nanometer nuclear base derivative compound particle with Rp.40, and storing in 4 deg.C refrigerator.

11) Prescription Rp.41: NBD011 nucleic acid mass ratio of 30:100

Weighing 6mg of NBD004 into a 1.5ml of EP tube, dissolving the NBD004 with dichloromethane, transferring the dissolved material to a round-bottom flask, rotationally evaporating the material by using a rotary evaporator under the condition of 40 ℃ water bath to remove organic solution in a sample, continuously evaporating the material to dryness for 30min after the material is attached to the bottle wall, adding enucleating enzyme ultrapure water after 30min, placing the material in an ultrasonic instrument for intermittent ultrasonic treatment at 50 ℃ for 40min, filtering the material by using a 0.22 mu m water-phase filter membrane after ultrasonic treatment, adding nucleic acid, blowing, beating and mixing to obtain the nucleobase derivative nanoparticles with the prescription Rp.41, and storing the nucleobase derivative nanoparticles in a 4 ℃ refrigerator for later use.

12) Prescription Rp.42: NBD011 and nucleic acid with the mass ratio of 50:100

Weighing 10mg of NBD004 into a 1.5ml of 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 evaporating to dryness for 30min after the material is attached to the bottle wall, adding enucleation enzyme 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 nucleobase derivative nanoparticles of the formula Rp.42, and storing in a 4 ℃ refrigerator for later use.

13) Prescription Rp.43: NBD011 nucleic acid mass ratio of 300:100

Weighing 15mg of NBD004 into a 1.5ml of EP tube, dissolving the NBD004 with dichloromethane, transferring the dissolved material to a round-bottom flask, rotationally evaporating the material by using a rotary evaporator under the condition of 40 ℃ water bath to remove organic solution in a sample, continuously evaporating the material to dryness for 30min after the material is attached to the bottle wall, adding enucleating enzyme ultrapure water after 30min, placing the material in an ultrasonic instrument for intermittent ultrasonic treatment at 50 ℃ for 40min, filtering the material by using a 0.22 mu m water-phase filter membrane after ultrasonic treatment, adding nucleic acid, blowing, beating and mixing to obtain the nucleobase derivative nanoparticles with the prescription Rp.43, and storing the nucleobase derivative nanoparticles in a 4 ℃ refrigerator for later use.

Example three: characterization of the nucleobase derivative nanoparticles and nucleobase derivative composite nanoparticles of the invention

1) Particle size and potential: nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles were prepared as described in example two and tested for their dynamic light scattering particle size (size), surface Potential (Zeta Potential) and Polydispersity (PDI) using a Malvern nanosizer (Malvern Zetasizer Nano ZSE) at 25 ℃.

The results are shown in table 1, and the results show that the size range of the nucleobase derivative nanoparticles and the nucleobase derivative composite nanoparticles of the invention is between 95nm and 240nm, the nanoparticles have good dispersibility, and the surface charge of the nanoparticles is between 0mV and 35 mV.

2) The encapsulation efficiency is as follows: taking FLuc-mRNA as model mRNA, preparing nucleobase derivative nanoparticles and nucleobase derivative compound nanoparticles according to the preparation method described in the embodiment II, and measuring the encapsulation rate of each prescription to the mRNA 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 is as follows: 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 package carrying 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 good encapsulation effect on mRNA, and the encapsulation efficiency is above 90%.

Table 1: characterization of nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles

Example four: in vitro cell transfection experiment and cytotoxicity investigation of nucleobase derivative nanoparticles and nucleobase derivative composite nanoparticles

1) Experiment of carrying Fluc-mRNA in vitro transfection DC2.4 cell by nucleobase derivative nanoparticles and nucleobase derivative compound nanoparticles: the logarithmic growth phase DC2.4 cell suspension is divided into 4 × 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 the nucleobase derivative nanoparticles and the nucleobase derivative composite nanoparticles respectively according to the preparation methods of different prescriptions described in example two, then the Fluc-mRNA is diluted to 88 mul by nuclease-free ultrapure water respectively, the mixed liquid of the nucleobase derivative nanoparticle composition and the nucleobase derivative composite nanoparticles containing 10 ng/mul of Fluc-mRNA is kept still for 10min, the volume of 20 mul of each hole is added to a 96-hole plate containing 180 mul of opti-MEM culture medium, and 4 holes are repeated for each sample. After 4h of administration, 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 Omega-Fluostar plate reader.

The results are shown in FIG. 1. And (4) conclusion: as shown in figure 1, the Fluc-mRNA-entrapped nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles prepared by Rp.11, Rp.12, Rp.13, Rp.32, Rp.33, Rp.34, Rp.41, Rp.42 and Rp.43 show better expression in DC2.4 cells, wherein Rp.12 and Rp.33 are better than other prescriptions.

2) Cytotoxicity test of in vitro transfection of DC2.4 cells by Fluc-mRNA carried by the nucleobase derivative nanoparticles and the nucleobase derivative composite nanoparticles: 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 the nucleobase derivative nanoparticles and the nucleobase derivative composite nanoparticles respectively according to the preparation methods of different prescriptions described in example two, then the Fluc-mRNA is diluted to 88 mul by nuclease-free ultrapure water respectively, the mixed liquid of the nucleobase derivative nanoparticle composition and the nucleobase derivative composite nanoparticles containing 10 ng/mul of Fluc-mRNA is kept still for 10min, the volume of 20 mul of each hole is added to a 96-hole plate containing 180 mul of opti-MEM culture medium, and 4 holes 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 indicates that the prescription of the nucleobase derivative nanoparticles and the nucleobase derivative compound nanoparticles has no obvious cytotoxicity and good biocompatibility, and can be used for subsequent in vivo experiments of animals.

3) Experiment of Luc-pDNA in vitro transfection DC2.4 cell carried by nucleobase derivative nanoparticles and nucleobase derivative compound nanoparticles: 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. Preparing the nucleobase derivative nanoparticles and the nucleobase derivative composite nanoparticles by Luc-pDNA according to the preparation methods of different formulas described in example two, diluting the mixture to 88 μ l of mixed solution of the nucleobase derivative nanoparticle composition and the nucleobase derivative composite nanoparticles containing 15ng/μ l of Luc-pDNA by using nuclease-free ultrapure water, standing the mixed solution for 30min, adding the mixed solution to a 96-well plate containing 180 μ l of opti-MEM culture medium in a volume of 20 μ l per well, and repeating 4 wells for each sample. After 4h of dosing, the aspirated 96-well plate was replaced with complete medium. And (3) continuing 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, continuing culturing in an incubator at 37 ℃ for 5min, 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, continuing culturing for 24 hours, adding the D-Luciferin for testing, and repeating for three 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: as shown in FIG. 3, the Luc-pDNA-entrapped and nucleobase derivative nanoparticle and nucleobase derivative complex nanoparticles prepared by the prescriptions Rp.11, Rp.12, Rp.13, Rp.32, Rp.33, Rp.34, Rp.41, Rp.42 and Rp.43 show better expression level at the cellular level, the highest expression level on the next day and decrease from the third day, wherein Rp.11, Rp.12, Rp.32 and Rp.42 are better than other prescriptions.

4) Experiment of carrying Fluc-mRNA in vitro transfection of different cells by the nucleobase derivative nanoparticles and the nucleobase derivative composite nanoparticles: the different formulations described in example two were tested for transfection experiments in 293T (human embryonic kidney cells), Hela (human cervical cancer cells) and HL7702 (human liver normal cells) using in vitro cell transfection with Fluc-mRNA administered at 200ng per well according to the protocol of example four, 1).

The results are shown in FIG. 4. And (4) conclusion: as shown in FIG. 4, the nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles encapsulating Fluc-mRNA in Rp.11, Rp.12, Rp.13, Rp.32, Rp.33, Rp.34, Rp.41, Rp.42 and Rp.43 showed better expression in 293T, Hela and HL7702 cells.

5) Cytotoxicity experiments of different cells transfected in vitro by Fluc-mRNA encapsulated by nucleobase derivative nanoparticles and nucleobase derivative compound nanoparticles: taking FLuc-mRNA-coated nucleobase derivative nanoparticles and nucleobase derivative composite nanoparticles prepared according to different prescriptions in example two, respectively administering FLuc-mRNA-coated nucleic acid nanocomposites of different prescriptions in an amount of 200ng FLuc-mRNA per well according to the in vitro cell transfection mode (namely, prescription group), and testing cytotoxicity experiments of the prescriptions in example two in 293T (human embryonic kidney cell), Hela (human cervical cancer cell) and HL7702 (human liver normal cell) transfection. In reference example four, 4), 293T (human embryonic kidney cells), Hela (human cervical cancer cells) and HL7702 (human liver normal cells) transfection procedures were performed, after replacing the Opti-MEM medium with a complete medium, culturing was continued for 48h, the complete medium was aspirated and rinsed three times with PBS, 90ul of serum-free medium and 10ul of CCK-8 solution were added to each well, with no cell well containing any Fluc-mRNA-loaded nucleic acid nanocomposite as a negative control and a cell-free CCK-8 medium well as a blank control, and incubation was continued for 2h in an incubator. Absorbance at 450nm was measured using an Omega-Fluostar microplate reader.

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 nucleobase derivative nanoparticles and the nucleobase derivative compound nanoparticles has no obvious cytotoxicity and good biocompatibility, and can be used for subsequent in vivo animal experiments.

6) Experiment of in vitro transfection of Hela-EGFP cells (polyclonal cell strain stably expressing EGFP fluorescent protein) with EGFP-siRNA (using EGFP-siRNA as model siRNA) encapsulated by nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles: 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, EGFP-siRNA with the concentration of 1 mug/mul is diluted to 0.1 mug/mul by nuclease-free ultrapure water, the EGFP-siRNA is taken to prepare the nucleobase derivative composite nanoparticles according to the preparation methods of different prescriptions described in the example II, then the EGFP-siRNA is diluted to 88 mul by nuclease-free ultrapure water respectively, the mixed liquid of the nucleobase derivative composite nanoparticles containing 10 ng/mul EGFP-siRNA is kept still for 10min, the mixed liquid is added to a 96-well plate containing 180 mul of opti-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. 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, the better the transfection effect, and the formulas Rp.11, Rp.12, Rp.13, Rp.32, Rp.33, Rp.34, Rp.41, Rp.42 and Rp.43 show better transfection effect.

Example five: fluorescence imaging detection of transfection of nucleobase derivative nanoparticles and nucleobase derivative compound nanoparticles in mice

Each group of three female BALB/c mice was prepared with FLuc-mRNA as model mRNA by the preparation method of the formulation described in example two, including nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles of FLuc-mRNA. Experimental groups 75 μ l of nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles containing 5 μ g FLuc-mRNA was injected to 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, an appropriate amount of substrate D-Luciferin was taken, diluted with PBS to prepare a solution with a concentration of 25mg/mL, kept in the dark for use, 125. mu.l of substrate was injected intraperitoneally into each mouse, the mouse was placed in a small animal anesthesia box, and the ventilation valve was 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 L. mu. min Series III). A bioluminescent image of the back of the mouse was taken. The results are shown in fig. 8, each group was a representative mouse, the experimental group of nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles showed luciferase expression in the whole body in vivo imaging, and the higher the fluorescence intensity, the more luciferase expression.

And (4) conclusion: as shown in FIG. 8, the nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles encapsulating FLUC-mRNA prepared by Rp.11, Rp.12, Rp.13, Rp.32, Rp.33, Rp.34, Rp.41, Rp.42 and Rp.43 have better luciferase expression in mice.

Example six: evaluation of humoral immunity effect of nucleobase derivative nanoparticles and nucleobase derivative complex nanoparticles 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 replaced 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 8 groups (5 per group) and intramuscularly injected with 75 μ l PBS (blank control), 5 μ g naked S-mRNA and 5 μ g S protein combination (positive control) and 75 μ l nucleobase derivative complex nanoparticles loaded with 5 μ g S-mRNA at Rp.11, Rp.12, Rp.13, Rp.32, Rp.33, Rp.34, Rp.41, Rp.42 and Rp.43, respectively

Step 2: first serum collection: on day 28, mice were bled by the outer canthus. After the serum is solidified for 1h at 4 ℃, centrifuging for 5 minutes at 4 ℃ at 5000 Xg rotation speed, taking the supernatant, centrifuging for 5 minutes at 4 ℃ at 10000 Xg rotation speed, taking the supernatant, adding the supernatant into eight rows of PCR tubes, subpackaging and preserving for later use at-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 nucleobase derivative complex nanoparticle formulations rp.11, rp.12, rp.13, rp.32, rp.33, rp.34, rp.41, rp.42 and rp.43, respectively, loaded with 5 μ g S-mRNA. The procedure of the first immunization was 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 the serum is solidified for 1h at 4 ℃, centrifuging for 5 minutes at 4 ℃ at the rotating speed of 5000 Xg (5000 times of gravity acceleration), taking the supernatant, centrifuging for 5 minutes at 4 ℃ at the rotating speed of 10000 Xg, taking the supernatant, adding the supernatant into eight rows of PCR tubes, subpackaging and preserving for later use 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, and after washing the plate 1 time with 200. mu.l of PBST per well, 100. mu.l of serum diluted 200-fold with PBS was added and incubated for 2 hours at 25 ℃ in a shaker. Serum was discarded, and after washing the plate 3 times with 200. mu.l PBST per well, 100. mu.l antibody (antibody diluted 1:1000 in PBS) was added per well and incubated for 1h at 25 ℃ in a shaker. Discarding the antibody, washing the plate for 3 times by 200 mul PBST in each hole, adding 50 mul TMB color development liquid in each hole for reaction in a dark place, adding 50 mul 2M sulfuric acid in each hole to stop the reaction after the positive control hole turns deep 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. 9.

And (4) conclusion: as shown in fig. 9, the OD values of the prescriptions rp.11, rp.12, rp.13, rp.32, rp.33, rp.34, rp.41, rp.42 and rp.43 after the second immunization are significantly higher than those of the PBS blank control group and the naked mRNA negative control group, resulting in significant immune reactions, suggesting that the prescription nanoparticles have strong 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, and after washing the plate 3 times with 200. mu.l PBST per well, 50-, 250-, 1250-, 6250-, 31250-, 156250-, 781250-fold diluted 1: 3-fold with PBS was added, followed by incubation for 2h at 25 ℃ in a shaker. Serum was discarded, and after washing the plate 3 times with 200. mu.l PBST per well, 100. mu.l antibody (antibody diluted 1:1000 in PBS) was added per well and incubated for 1h at 25 ℃ in a shaker. Discarding the antibody, washing the plate with 200. mu.l PBST for 3 times in each well, adding 50. mu.l TMB color development solution in each well for reaction in the dark, adding 50. mu.l 2M sulfuric acid in 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. 10, the present invention uses 2-fold of the average OD value of PBS group as baseline, and the OD values of Rp.11, Rp.12, Rp.34, Rp.41, Rp.42 and Rp.43 groups are still higher than baseline when diluted to 1250-fold, and the OD values of Rp.13, Rp.32 and Rp.33 groups are still higher than baseline when diluted to 6250-fold, 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

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 in the flank, cultured B16-OVA tumor cells were collected, and B16-OVA tumor cells were injected subcutaneously in the flank of the mice to establish a subcutaneous B16-OVA tumor model.

2) Preparation of nucleobase derivative complex-OVA-mRNA vaccine: briefly gently mixing the prescriptions with OVA-mRNA (purchased from TriLink, usa) for 30 minutes separately to give 9 nucleobase derivative complex-OVA-mRNA vaccines prepared from prescriptions rp.11, rp.12, rp.13, rp.32, rp.33, rp.34, rp.41, rp.42 and rp.43, 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 sole injection on

days

10, 13 and 16, respectively, while mice vaccinated with an equal volume of PBS buffer solution (blank control), 5ug of naked OVA-mRNA solution after dilution (negative control) and 5ug of OVA protein after dilution (positive control) were set as control groups, with 5 mice per group in parallel.

4) Tumor vertical diameter was measured daily starting on day 7 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. Electronic balance for daily useChanges in body weight of C57BL/6J mice were recorded and the survival rate was counted.

And (4) conclusion: as shown in table 3 and fig. 11, the PBS control group and the nude mRNA group were sacrificed in their entirety at day 38 and day 45, respectively, starting at day 21 and day 24 after tumor inoculation. Nucleobase derivative complex-OVA-mRNA vaccine sets prepared from the prescriptions rp.11, rp.12, rp.13, rp.32, rp.33, rp.34, rp.41, rp.42 and rp.43 were sacrificed starting at day 28, day 27, day 30, day 32, day 31, day 29, day 30, day 29 and day 31, respectively, and at all at day 48, day 46, day 49, day 52, day 50, day 49, day 45, day 44 and day 47, 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 table 4 and fig. 12, the OVA positive control group, the naked mRNA group, the PBS control group, and the nucleobase derivative complex-OVA-mRNA vaccine groups prepared by the prescriptions rp.11, rp.12, rp.13, rp.32, rp.33, rp.34, rp.41, rp.42, and rp.43 showed tumor growth from day 8 to day 10 of tumor inoculation. The nucleobase derivative complex-OVA-mRNA vaccine groups prepared from rp.11, rp.12, rp.13, rp.32, rp.33, rp.34, rp.41, rp.42 and rp.43 all had smaller tumor sizes than the PBS control group and the naked mRNA group starting on day 15. The nucleobase derivative complex-OVA-mRNA vaccine groups prepared from rp.11, rp.12, rp.13, rp.32, rp.33, rp.34, rp.41, rp.42 and rp.43 showed significant tumor growth delay compared to PBS control group and naked mRNA group.

Table 3: statistics of sacrifice days after tumor inoculation for each group

Table 4: statistics of tumor size change after tumor inoculation for each group

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound of formula I or a stereoisomer or tautomer thereof,

2. a nucleobase derivative nanoparticle comprising: a compound of formula I according to claim 1 or a stereoisomer or tautomer thereof, and auxiliary materials; the auxiliary material comprises a material selected from: at least one of PEG derivatives, lipids and lipid-like substances.

3. The nucleobase derivative nanoparticle of claim 2, said PEG derivative comprising 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, PEG-modified dialkylglycerol.

4. The nucleobase derivative nanoparticle according to claim 2, wherein said 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-dioleoyl-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.

5. The nucleobase derivative nanoparticle of claim 2, wherein said lipidoid comprises at least one selected from the group consisting of a poloxamer, a polysorbate, a span, a poloxamine, or a poloxamine derivative.

6. The nucleobase derivative nanoparticle according to any one of claims 2-5 comprising a compound of formula I in an amount of 37.6% to 59.3% by weight based on the total mass of the nanoparticle, a PEG derivative and a lipid; the PEG derivative is present in an amount of about 6.0 wt% to about 8.8 wt%; the content of the lipid is 34.5 wt% -53.5 wt%, the PEG derivative is selected from DMG-PEG2000, and the lipid is selected from DSPC and PC or cholesterol.

7. Nucleobase derivative nanoparticles according to any one of claims 2 to 5, comprising a compound of formula I, a lipidoid and a lipid, said compound of formula I being present in an amount of 31.0% to 50.9% by weight, based on the total mass of the nanoparticle; the content of the lipid is 34.9-35.4 wt%; the content of the lipid is 14.1 wt% -33.5 wt%, and the lipid is selected from

F127, said lipid being selected from DSPC, PC or cholesterol.

8. A nucleobase derivative nanoparticle according to any one of claims 2-5 comprising a compound of formula I: the PEG derivative is: the mass ratio of the lipid is (64-105): (11-15): (61-91).

9. A nucleic acid nanocomplex, comprising: a nucleic acid and at least one selected from the group consisting of a compound of formula I according to claim 1 or a stereoisomer or tautomer thereof or a nucleobase derivative nanoparticle according to any one of claims 2-8.

10. The nucleic acid nanoplex according to claim 9, wherein the mass ratio of the nucleic acid to the compound of formula I or the stereoisomer or tautomer thereof is 100 (30-300).

11. A pharmaceutical composition comprising the nucleic acid nanocomplex of any of claims 9 or 10 and a pharmaceutically acceptable excipient.

12. Use of a compound of formula I according to claim 1 or a stereoisomer or a tautomer thereof, a nucleobase derivative nanoparticle according to any one of claims 2 to 8 or a nucleic acid nanocomposite according to any one of claims 9 or 10 or a pharmaceutical composition according to claim 11 for the preparation of a product for the in vivo delivery of a nucleic acid.