CN114601922A

CN114601922A - Nanoparticles containing 5-methylpyrimidine-2, 4(1H,3H) -diketone derivatives, preparation method and application thereof

Info

Publication number: CN114601922A
Application number: CN202210248965.7A
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: 2022-03-11
Filing date: 2022-03-11
Publication date: 2022-06-10

Abstract

The invention relates to a nanoparticle and a preparation method and application thereof, belonging to the field of biological medicine. The nanoparticle comprises a 5-methylpyrimidine-2, 4(1H,3H) -diketone derivative 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

Nanoparticles containing 5-methylpyrimidine-2, 4(1H,3H) -diketone derivatives, preparation method and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a nanoparticle and a preparation method and application 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 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, 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 nanoparticle complex comprising a nucleic acid and a nanoparticle according to 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 comprising mixing the nanoparticles of the first aspect.

In a fifth aspect, the present invention provides a method of preparing a nanoparticle composite as described in the second aspect.

In a sixth aspect, the present invention provides a use of a nanoparticle of the first aspect or a nanoparticle complex of the second aspect or a pharmaceutical composition of the third 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,

wherein m is an integer from 0 to 5; n is an integer from 5 to 20. In some embodiments, m is 0, 1, 2, 3, 4, or 5, and n is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

In some embodiments, the compound of formula I is a compound of NBD002,

the compounds of formula I may be synthesized by the michael addition reaction.

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 adjunct material comprises a PEG derivative and a lipid.

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 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) ], dilauroyl phosphatidylethanolamine-polyethylene glycol, dimyristoyl phosphatidylethanolamine-polyethylene glycol (PEG-DMPE), dipalmitoyl phosphatidylcholine polyethylene glycol (PEG-DPPC), dipalmitoyl phosphatidylethanolamine-polyethylene glycol, PEG-distearoyl glycerol, PEG-dipalmitoyl, PEG-dioyl, PEG-distearyl, PEG-diacyl oleamide, PEG-dipalmitoyl phosphatidylethanolamine, or PEG-1, 2-dimyristoyloxypropan-3-amine. In some embodiments, the PEG derivative comprises at least one selected from 1, 2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (DMG-PEG) or dipalmitoylphosphatidylcholine polyethylene glycol (PEG-DPPC).

The 1, 2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (i.e., DMG-PEG) comprises a compound selected from 1, 2-dimyristoyl-sn-glycerol methoxypolyethylene glycol 2000 (i.e., DMG-PEG 2000).

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, 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-diundecenyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or at least one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, or alpha-tocopherol. In some embodiments, the lipid comprises at least one selected from the group consisting of lecithin, 1, 2-distearoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine. In some embodiments, the lipid comprises cholesterol (Chol) and other lipids selected from at least one of 1, 2-distearoyl-sn-glycero-3-phosphocholine, lecithin, and 1, 2-dimyristoyl-sn-glycero-phosphocholine.

The lipid comprises a poloxamer selected from poloxamine or poloxamine derivatives.

Said poloxamine comprises a compound selected from the group consisting of

304、

701、

704、

707、

803、

901、

904、

908、

1107、

1301、

1304、

1307、

90R4 or

150R 1.

The poloxamine derivatives 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.

Calculated by the total mass of the nanoparticles, the content of the compound shown in the formula I is 29-52 wt%. In some embodiments, the compound of formula I is present in an amount of 29 wt%, 30 wt%, 31 wt%, 32 wt%, 33 wt%, 34 wt%, 35 wt%, 36 wt%, 36.9 wt%, 37 wt%, 38 wt%, 39 wt%, 40 wt%, 41 wt%, 42 wt%, 42.4 wt%, 42.5 wt%, 43 wt%, 44 wt%, 45 wt%, 46 wt%, 47 wt%, 48 wt%, 49 wt%, 50 wt%, 51 wt%, or 52 wt%, based on the total mass of the nanoparticle.

Calculated by the total mass of the nanoparticle, the content of the PEG derivative is 6 wt% -12 wt%. In some embodiments, the PEG derivative is present in an amount of 6.0 wt%, 6.5 wt%, 6.9 wt%, 7.0 wt%, 7.5 wt%, 8.0 wt%, 8.5 wt%, 8.9 wt%, 9.0 wt%, 9.5 wt%, 10.0 wt%, 10.2 wt%, 10.5 wt%, 11.0 wt%, 11.4 wt%, 11.5 wt%, or 12.0 wt%, based on the total mass of the nanoparticle.

The total content of the lipid is 40-65 wt% calculated by the total mass of the nanoparticle. In some embodiments, the total content of lipid is 40 wt%, 41 wt%, 41.5 wt%, 42 wt%, 43 wt%, 44 wt%, 45 wt%, 46 wt%, 46.2 wt%, 47 wt%, 48 wt%, 49 wt%, 49.2 wt%, 50 wt%, 50.5 wt%, 50.6 wt%, 51 wt%, 52 wt%, 52.9 wt%, 53 wt%, 54 wt%, 55 wt%, 56 wt%, 57 wt%, 58 wt%, 59 wt%, 60 wt%, 61 wt%, 62 wt%, 63 wt%, 64 wt%, or 65 wt%, calculated on the total mass of the nanoparticle.

The lipid comprises cholesterol in an amount of 23 wt% to 41 wt% (e.g., 23 wt%, 23.5 wt%, 23.8 wt%, 24 wt%, 25 wt%, 26 wt%, 27 wt%, 27.8 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 31.2 wt%, 32 wt%, 32.5 wt%, 32.6 wt%, 33 wt%, 34 wt%, 35 wt%, 36 wt%, 37 wt%, 38 wt%, 39 wt%, 40 wt%, 40.5 wt%, or 41 wt%) and other lipids in an amount of 13 wt% to 24 wt% (e.g., 13 wt%, 23.5 wt%, 23.8 wt%, 24 wt%, 25 wt%, 26 wt%, 27 wt%, 27.8 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 31.2 wt%, 32 wt%, 32.5 wt%, 33 wt%, 34 wt%, 35 wt%, 36 wt%, 37 wt%, 38 wt%, 39 wt%, 40.5 wt%, or 41 wt%) based on the total mass of the nanoparticle, 13.5, 13.6, 14, 15, 16, 16.1, 17, 17.5, 17.7, 18, 19, 20, 21, 21.5, 21.7, 22, 22.5, 22.8, 23, 23.5, 23.7, or 24 wt%).

The nanoparticle contains a compound shown in formula I, a PEG derivative and at least one lipid; the compound of formula I: total mass of the lipid: the mass ratio of the PEG derivative is (38-75) to (58-91) to (9-16). In some embodiments, the nanoparticle comprises a compound of formula I and a PEG derivative, and at least one lipid; the mass ratio of the compound shown in the formula I to the total mass of the lipid to the PEG derivative is 50: 58: 10, 38: 84: 9, 56: 61: 15, 75: 61: 11, 58: 83: 16 or 73: 91: 16.

In some embodiments, the nanoparticle comprises a compound of formula I, a PEG derivative, and a lipid; the lipid comprises cholesterol and other lipid, the other lipid is selected from at least one of 1, 2-distearoyl-sn-glycero-3-phosphocholine, lecithin and 1, 2-dimyristoyl-sn-glycero-phosphocholine, and the mass ratio of the compound shown in the formula I, the cholesterol, the other lipid and the PEG derivative is (38-75) to (35-53) to (18-41) to (9-16). In some embodiments, the nanoparticle comprises a compound of formula I, a PEG derivative, and a lipid; the lipid comprises cholesterol and other lipid, the other lipid is selected from at least one of 1, 2-distearoyl-sn-glycero-3-phosphocholine, lecithin and 1, 2-dimyristoyl-sn-glycero-phosphocholine, and the mass ratio of the compound shown in the formula I, the cholesterol, the other lipid and the PEG derivative is 50: 39: 19: 10, 38: 53: 31: 9, 56: 43: 18: 15, 75: 35: 26: 11, 58: 49: 34: 16 or 73: 50: 41: 16.

In a second aspect, the present invention provides a nanoparticle composite.

A nanoparticle complex comprising a nucleic acid and the nanoparticle of the first aspect.

The weight ratio of the nanoparticles to the nucleic acid is 0.90: 1.00-1.80: 1.00. In some embodiments, the weight ratio of the nanoparticle to nucleic acid is 0.90: 1.00, 0.95: 1.00, 0.98: 1.00, 1.00: 1.00, 1.10: 1.00, 1.15: 1.00, 1.17: 1.00, 1.20: 1.00, 1.23: 1.00, 1.25: 1.00, 1.30: 1.00, 1.32: 1.00, 1.35: 1.00, 1.40: 1.00, 1.50: 1.00, 1.60: 1.00, 1.70: 1.00, 1.72: 1.00, 1.75: 1.00, or 1.80: 1.00.

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 (such as NBD002 compound) shown in the formula I can form base pairs with adenine A in nucleic acid, A and T are connected through 2 hydrogen bonds, and double hydrogen bonds are formed between amine and carbonyl of complementary base groups, as shown in the 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 nucleobase derivative nanoparticles are 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) 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 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: novel coronavirus (SARS-CoV-2), human papilloma virus (H.mu.man Papillomavir, HPV), 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 pneumome); 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 some embodiments, a method of preparing a mixture comprising nanoparticles of the first aspect, comprises: mixing a compound shown in a formula I with a solvent A to obtain a solution 1, mixing an auxiliary material with a solvent B to obtain a solution 2, uniformly mixing the solution 1 and the solution 2, mixing with water, and performing post-treatment to obtain a mixed solution containing the nanoparticles. In some embodiments, a method of preparing a mixture comprising nanoparticles of the first aspect, comprises: and (3) mixing the compound shown in the formula I and the auxiliary material with a solvent B, mixing with water, and performing post-treatment to obtain a mixed solution containing the nanoparticles of the first aspect.

The post-treatment comprises filtration or filtration after removing the solvent B and/or the solvent A by water bath rotary evaporation.

The solvent a may include one selected from ethanol or dichloromethane.

The solvent B may include a solvent selected from ethanol.

The water bath may be carried out at 35-50 ℃. In some embodiments, the water bath is a water bath performed at 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃ or 50 ℃.

A method of preparing a mixed liquor containing the nanoparticle composite of the second aspect, comprising: and (3) mixing the mixed solution containing the nanoparticles of the first aspect obtained by the method of the fourth aspect with nucleic acid in a solvent C to obtain a mixed solution containing the nanoparticle composite of the second aspect.

The volume percentage of the solvent B in the mixed solution containing the nano-particle composite of the second aspect is 0-12.5% vol.

The solvent C comprises water.

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 or vaccine 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. 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 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 compound shown in the formula I is innovatively used for preparing the compound of the nanoparticle and the nanoparticle, the compound shown in the formula I forms an amphiphilic composition with nucleic acid through base complementary pairing (hydrogen bond) or pi-pi stacking effect, a hydrophobic part is arranged in the middle of the nanoparticle in aqueous solution, hydrophilic nucleic acid and a hydrophilic part are arranged on the surface of the nanoparticle, and the nucleobase derivative nanoparticle is formed through assembly of hydrophilic and hydrophobic acting force. The obtained nanoparticle and nanoparticle complexes can be effectively transfected in vivo, can carry mRNA encoding immunogenic peptides or proteins into cells, and effectively release the mRNA, express antigens and effectively achieve the purpose of immunotherapy or immunoprophylaxis. The nucleobase derivative 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 nanoparticle composite provided by the invention has the particle size ranging from 84nm to 212nm, has better dispersibility, and the surface charge of the nanoparticle for encapsulating nucleic acid ranges from-1.26 mV to-23 mV.

(3) The nanoparticles and the nanoparticle compound provided by the invention have low cytotoxicity and good biocompatibility.

(4) The nanoparticle composite provided by the invention has high encapsulation efficiency.

(5) The nanoparticle and nanoparticle compound provided by the invention have the advantages of compressing and protecting nucleic acid from degradation, promoting the nucleic acid to penetrate cell membranes, realizing efficient transfection in vivo and in vitro, having good biocompatibility and the like.

(6) The content of the compound shown in the formula I (such as NBD002 compound) is about 29 wt% -52 wt%, the content of the PEG derivative is about 6 wt% -12 wt%, and the total content of the lipid is about 40 wt% -65 wt%, calculated by the total weight of the nanoparticle (not counting the weight of the nucleic acid), so that the obtained nanoparticle is beneficial to transfection of nucleic acid in vivo and in vitro, is beneficial to improving seroconversion efficiency and humoral immune activation function, is beneficial to transfection of more cell lines, and is beneficial to improving the in vivo activity of the nanoparticle compound carrying the nucleic acid.

(7) When the nanoparticle composite is prepared, the volume percentage of the solvent B in the mixed solution of the nanoparticle composite is 0-12.5% vol, which is beneficial to improving the transfection in vivo and in vitro of nucleic acid, improving the serum conversion efficiency and the humoral immune activation function, transfecting more cell lines and improving the in vivo activity of the nanoparticle composite carrying nucleic acid.

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

Drawings

FIG. 1 is a statistical chart showing the transfection effect of FLUc-mRNA loaded nanoparticle complexes in DC2.4 cells in example four; the abscissa in the figure represents the different prescribed nanoparticle complex compositions and the ordinate is the relative fluorescence intensity expressed 24h after transfection of nanoparticle complex compositions containing the same dose of FLuc-mRNA.

FIG. 2 is a graph showing the survival rate of DC2.4 cells treated with differently prescribed nanoparticle complexes carrying Fluc-mRNA in example four; the abscissa represents the different nanoparticle complex formulations and the ordinate represents cell viability, the higher the cell activity, the less cytotoxic it shows.

FIG. 3 is a histogram showing the transfection effect of the Luc-pDNA-loaded nanoparticle complex 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 is a graph showing the transfection effect of the FLuc-mRNA loaded nanoparticle complex in different cells in example four; the abscissa in the figure represents the different prescribed nanoparticle complex compositions and the ordinate is the relative fluorescence intensity expressed 24h after transfection of different cells with the nanoparticle complex composition containing the same dose of FLuc-mRNA.

FIG. 5 is a graph showing the survival rate of cells treated with different cell types with differently formulated nanoparticle complexes carrying Fluc-mRNA in example four; the abscissa represents the different nanoparticle complex formulations and the ordinate represents cell viability, the higher the cell activity, the less cytotoxic it shows.

FIG. 6 is a graph showing the transfection effect of EGFP-siRNA-loaded nanoparticle complexes in Hela-EGFP cells in example four; the abscissa of the graph represents different prescribed nanoparticle complex compositions, and the ordinate represents the percentage of EGFP positive cells transfected with the nanoparticle complex composition containing the same dose of EGFP-siRNA 24h after Hela-EGFP transfection.

FIG. 7 is a graph showing the transfection effect of EGFP-siRNA-loaded nanoparticle complexes in Hela-EGFP cells in example four; the abscissa of the graph represents the different prescribed nanoparticle complex compositions, and the ordinate represents the median fluorescence intensity of the nanoparticle complex compositions transfected with the same dose of EGFP-siRNA 24h after Hela-EGFP transfection.

Figure 8 shows a whole body in vivo imaging bioluminescence image of luciferase expression in mice using FLuc-mRNA loaded nanoparticle complexes in example five.

FIG. 9 is a statistical plot of serum IgG antibody levels of mice immunized with the nanoparticle complex 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. 10 is a graph showing the results of measurement of serum IgG antibody titer of mice immunized with the nanoparticle complex loaded with the neocorona S-mRNA in 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. 2 × 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. 11 is a statistical graph of the survival rate of C57BL/6J mice vaccinated with B16-OVA melanoma in example seven after subcutaneous injection of different formulations of OVA-mRNA nanoparticle complex vaccine.

FIG. 12 is a statistical plot of the change in tumor volume following subcutaneous injection of different formulations of OVA-mRNA nanoparticle complex vaccine in C57BL/6J mice vaccinated with B16-OVA melanoma, 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 ℃.

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 to include the instant numbers, e.g., two or more means ≧ two.

The term "% vol" or "% (v/v)" means volume percent.

The term "OD" denotes optical density value.

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 of the specification, reference to the description of "one embodiment," "some embodiments," "an example," "a specific example," or "some examples" or the like means 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. "rpm" means the rotational speed "revolutions per minute".

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

The first embodiment is as follows: synthesis of Compound NBD002

Thymine (75.6mg, 0.6mmol), tetradecyl acrylate (241.59mg, 0.9mmol) and triethylamine (240mg, 2.4mmol) were mixed, dissolved in 50mL of methanol, and stirred at room temperature for 20 h.

After the reaction was completed, TLC (PE: EA: 1) showed formation of a new spot, the reaction solution was spin-dried, 50mL of ethyl acetate and 30mL of water were added, liquid separation was performed by extraction, the upper organic phase was collected and transferred to a flask, and the above extraction was repeated three times by adding anhydrous sodium sulfate and drying. The organic phase obtained is filtered off with suction and spin-dried, and by column chromatography (100% PE. PE: EA: 1) 217.64mg of compound NBD002 are obtained (white powdery solid, yield 92%). The obtained compound NBD002 was collected and subjected to hydrogen spectrum and mass spectrum detection. The results are as follows:

hydrogen spectrum: 1H NMR (500MHz, chetororm-d) δ 9.59(s, 1H), 7.73(q, J ═ 1.4Hz, 1H), 4.12(t, J ═ 6.2Hz, 2H), 4.00(t, J ═ 7.1Hz, 2H), 2.69(t, J ═ 7.1Hz, 2H), 1.83(d, J ═ 1.4Hz, 3H), 1.63(tt, J ═ 7.7, 6.1Hz, 2H), 1.43 to 1.32(m, 2H), 1.35 to 1.28(m, 2H), 1.31 to 1.24(m, 7H), 1.26(s, 10H), 0.94 to 0.85(m, 3H).

Mass spectrum: HRMS (ESI) m/z calcd for C₂₂H₃₉N₂O₄+(M+H)+395.29043，found 395.29028。

Example two: preparation of nanoparticle composites of different formulations

1) Prescription Rp.04: the mass ratio of NBD002 to DMG-PEG to DSPC to Chol to nucleic acid is 50: 10: 19: 39: 100

Taking NBD002, DMG-PEG, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, respectively weighing NBD002, DMG-PEG, DSPC and Chol at the room temperature and adding ethanol for dissolving; adding the dissolved NBD002, DMG-PEG, DSPC and Chol into a 1.5mL centrifuge tube, uniformly mixing to obtain a total volume of 1mL, dropwise adding the mixture into a round-bottomed flask containing 15mL of ultra-pure water with the enucleated enzyme by using an insulin syringe, adding a stirrer before dropwise adding, stirring at the rotating speed of 1500rpm, and dropwise adding the mixed solution under the condition of water bath at 40 ℃. Stirring for 20min, filtering with 0.22 μm organic phase filter membrane, adding nucleic acid, blowing, mixing to obtain nanoparticle compound with ethanol concentration of 6.25% (v/v) and formula Rp.04, and storing in 4 deg.C refrigerator for use.

2) Prescription Rp.05: the mass ratio of NBD002 to DMG-PEG to DSPC to Chol to nucleic acid is 50: 10: 19: 39: 100

NBD002, DMG-PEG, DSPC and Chol are taken out from a refrigerator at the temperature of-20 ℃ and balanced to the room temperature, and NBD002, DMG-PEG, DSPC and Chol are respectively weighed at the room temperature and dissolved by adding ethanol; adding the dissolved NBD002, DMG-PEG, DSPC and Chol into a 1.5mL centrifuge tube, uniformly mixing to obtain a total volume of 1mL, dropwise adding the mixture into a round-bottomed flask containing 15mL of the enucleated enzyme ultrapure water by using an insulin syringe, adding a stirrer before dropwise adding, stirring at the rotating speed of 1500rpm, and dropwise adding the mixed solution under the condition of 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, adding nucleic acid, blowing, mixing to obtain nanoparticle compound of formula Rp.05, and storing in 4 deg.C refrigerator.

3) Prescription Rp.06: the mass ratio of NBD002 to DMG-PEG to DSPC to Chol to nucleic acid is 50: 10: 19: 39: 100

Taking NBD002, DMG-PEG, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, respectively weighing NBD002, DMG-PEG, DSPC and Chol at the room temperature and adding ethanol for dissolving; adding the dissolved NBD002, DMG-PEG, DSPC and Chol into a 1.5mL centrifuge tube, uniformly mixing to obtain a total volume of 2mL, dropwise adding the mixture into a round-bottomed flask containing 14mL of the enucleated enzyme ultrapure water by using an insulin syringe, adding a stirrer before dropwise adding, stirring at the rotating speed of 1500rpm, and dropwise adding the mixed solution under the condition of water bath at 40 ℃. Stirring for 20min, filtering with 0.22 μm organic phase filter membrane, adding nucleic acid, blowing, mixing to obtain nanoparticle compound with ethanol concentration of 12.5% (v/v) and Rp.06, and storing in 4 deg.C refrigerator.

4) Prescription Rp.07: the mass ratio of NBD002 to DMG-PEG to DSPC to Chol to nucleic acid is 50: 10: 19: 39: 100

Taking NBD002, DMG-PEG, DSPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, respectively weighing NBD002, DMG-PEG, DSPC and Chol at the room temperature and adding ethanol for dissolving; adding the dissolved NBD002, DMG-PEG, DSPC and Chol into a 1.5mL centrifuge tube, uniformly mixing to obtain a total volume of 3mL, dropwise adding the mixture into a round-bottomed flask containing 13mL of the enucleated enzyme ultrapure water by using an insulin syringe, adding a stirrer before dropwise adding, stirring at the rotating speed of 1500rpm, and dropwise adding the mixed solution under the condition of water bath at 40 ℃. Stirring for 20min, filtering with 0.22 μm organic phase filter membrane, adding nucleic acid, blowing, mixing to obtain nanoparticle compound with ethanol concentration of 18.75% (v/v) and formula Rp.07, and storing in 4 deg.C refrigerator for use.

5) Prescription Rp.08: the mass ratio of NBD002 to DMG-PEG to PC to Chol to nucleic acid is 38: 9: 31: 53: 76

Taking NBD002, DMG-PEG, PC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing NBD002 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 NBD002, DMG-PEG, 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 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 after 2 h. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nanoparticle compound of formula Rp.08, and storing in 4 deg.C refrigerator for use.

6) Prescription Rp.12: the mass ratio of NBD002 to DMG-PEG to PC to Chol to nucleic acid is 56: 15: 18: 43: 112

Taking NBD002, DMG-PEG, PC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing NBD002 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 NBD002, DMG-PEG, 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 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 after 2 h. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nanoparticle compound with Rp.12, and storing in 4 deg.C refrigerator.

7) Prescription Rp.17: the mass ratio of NBD002 to DMG-PEG to PC to Chol to nucleic acid is 75: 11: 26: 35: 150

Taking NBD002, DMG-PEG, PC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing NBD002 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 NBD002, DMG-PEG, 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 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 after 2 h. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nanoparticle compound with Rp.17, and storing in 4 deg.C refrigerator.

8) Prescription Rp.24: the mass ratio of NBD002 to DMG-PEG to PC to Chol to nucleic acid is 83: 5: 14: 25: 166

Taking NBD002, DMG-PEG, PC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing NBD002 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 NBD002, DMG-PEG, 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 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 after 2 h. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nanoparticle compound with Rp.24, and storing in 4 deg.C refrigerator.

9) Prescription Rp.25: the mass ratio of NBD002 to DMG-PEG to PC to Chol to nucleic acid is 19: 16: 37: 61: 38

Taking NBD002, DMG-PEG, PC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, weighing NBD002 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 NBD002, DMG-PEG, 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 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 after 2 h. Stirring for 2 hr, filtering with 0.22 μm water-based filter membrane, adding nucleic acid, blowing, beating, and mixing to obtain nanoparticle compound with Rp.25, and storing in 4 deg.C refrigerator.

10) Prescription Rp.45: the mass ratio of NBD002 to PEG-DPPC to DMPC to Chol to nucleic acid is 58: 16: 34: 49: 116

Taking NBD002, PEG-DPPC, DMPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, respectively weighing NBD002, PEG-DPPC, DMPC and Chol at the room temperature, and adding ethanol for dissolving; adding the dissolved NBD002, PEG-DPPC, DMPC and Chol into a 1.5mL centrifuge tube for uniformly mixing, dripping the mixed solution into a round-bottom flask containing ultrapure water containing the enucleated enzyme 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 nanoparticle compound of formula Rp.45, and storing in 4 deg.C refrigerator.

11) Prescription Rp.51: the mass ratio of NBD002, PEG-DPPC, DMPC, Chol and nucleic acid is 73: 16: 41: 50: 146

Taking NBD002, PEG-DPPC, DMPC and Chol out of a refrigerator at the temperature of-20 ℃ and balancing to room temperature, respectively weighing NBD002, PEG-DPPC, DMPC and Chol at the room temperature, and adding ethanol for dissolving; adding the dissolved NBD002, PEG-DPPC, DMPC and Chol into a 1.5mL centrifuge tube for uniformly mixing, dripping the mixed solution into a round-bottom flask containing ultrapure water containing the enucleated enzyme 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 at 40 deg.C in water bath with rotary evaporator to remove ethanol, filtering with 0.22 μm water phase membrane, adding nucleic acid, blowing, mixing to obtain nanoparticle compound with formula Rp.51, and storing in 4 deg.C refrigerator.

Example three: characterization of the nanoparticle composites of the invention

1) Particle size and potential: nanoparticle composites 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 Nano-sizer (malverm Zetasizer Nano ZSE) at 25 ℃.

As a result: as shown in table 1.

The result shows that the particle size range of the nanoparticle composite is between 80nm and 220nm, the nanoparticle composite has better dispersibility, and the surface charge of the nanoparticle is between-25 mV and 0 mV.

2) Encapsulation efficiency: taking FLuc-mRNA as model mRNA, preparing a nanoparticle compound according to the preparation method described in the example 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 nanoparticle composites

Example four: in vitro cell transfection experiment and cytotoxicity investigation of nanoparticle complex

1) Experiment of DC2.4 cells transfected in vitro by Fluc-mRNA encapsulated by nanoparticle complex: logarithmic growth phase DC2.4 cell suspension at 4X 10⁴The density of each cell per well is divided into 96-well plates, put at 37 ℃ and 5% CO₂And (5) standing and culturing in an incubator. Diluting Fluc-mRNA with concentration of 1 μ g/μ l to 0.1 μ g/μ l with nuclease-free ultrapure water after 24h, preparing nanoparticle complexes from Fluc-mRNA according to the preparation methods of different formulas described in example two, and diluting to 88 μ l with nuclease-free ultrapure water to obtain nanoparticle complex group containing 10ng/μ l Fluc-mRNAThe mixture was allowed to stand for 10min and added to a 96-well plate containing 180. mu.l of opti-MEM medium per well in a volume of 20. mu.l per well, and the wells were repeated 4 times per 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. Among them, KNL was prepared as described in the literature "effective delivery of therapeutic small nucleic acids to positive cancer cells using a key nucleic acid lipid nanoparticles" (Delphine, Luvino et al, Journal of Controlled Release: of the Controlled Release facility, 2013, Vol.3, No. 3-pp.954-61, doi: 10.1016/j.jcontrolled release.2013.09.006).

As a result: as shown in fig. 1.

And (4) conclusion: as shown in FIG. 1, the Fluorogenic expression intensities of FLUC-mRNA of Rp.04, Rp.05, Rp.06, Rp.08, Rp.12, Rp.17, Rp.45 and Rp.51 are all higher than those of KNL control group, wherein Rp.04, Rp.12 and Rp.45 are better than those of other prescriptions.

2) Cytotoxicity experiment of DC2.4 cells transfected in vitro by Fluc-mRNA carried by nanoparticle complex: DC2.4 cell suspension in logarithmic growth phase at 4X 10⁴The density of each cell per well is divided into 96-well plates, put at 37 ℃ and 5% CO₂And (5) standing and culturing in an incubator. After 24h, the Fluc-mRNA was diluted to 0.1. mu.g/. mu.l with nuclease-free ultrapure water at a concentration of 1. mu.g/. mu.l, Fluc-mRNA-loaded nanoparticle complexes were prepared from the Fluc-mRNA by the methods described in example two but with different recipes, then diluted to 88. mu.l with nuclease-free ultrapure water, 10 ng/. mu.l of the mixture of the nanoparticle complex compositions containing Fluc-mRNA was allowed to stand for 10min, and added to 96-well plates containing 180. mu.l of opti-MEM 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. Culturing for 48h, aspirating complete medium, rinsing with PBS for three times, and negative control with non-prescription cell well and cell-freeCCK-8 medium wells were blanked and 90. mu.l serum-free medium and 10. mu.l CCK-8 solution were added to each well and incubation in the incubator continued for 2 h. Absorbance at 450nm was measured using an Omega-Fluostar microplate reader, KNL was used as a control. 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 were 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.

As a result: as 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 prescription of the nanoparticle compound has no obvious cytotoxicity and good biocompatibility, and can be used for subsequent in vivo experiments of animals.

3) Experiment of DC2.4 cells transfected in vitro by Luc-pDNA encapsulated by nanoparticle complex: DC2.4 cell suspension in logarithmic growth phase at 4X 10⁴The density of each cell per well is divided into 96-well plates, put at 37 ℃ and 5% CO₂And (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 Luc-pDNA-entrapped nanoparticle complexes by the preparation methods described in example two but different recipes, diluted to 88. mu.l of a nanoparticle complex composition mixture containing 15 ng/. mu.l Luc-pDNA with nuclease-free ultrapure water, allowed to stand for 30min, and added to 96-well plates containing 180. mu.l of opti-MEM 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. The incubation was continued for 24h, the complete medium was aspirated, 100. mu.l of D-Luciferin solution at a working concentration of 250. mu.g/mL was added to each 96-well plate, incubation was continued for 5min at 37 ℃ and finally imaged with an Omega-Fluostar microplate reader with KNL as the control. Testing Luc-the fluorescence expression intensity of pDNA, tested in duplicate every 24 hours, after each test the medium containing D-Luciferin was aspirated, and after 24 hours of continued culture with fresh complete medium the D-Luciferin test was added and repeated 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

As a result: as shown in fig. 3.

And (4) conclusion: as shown in fig. 3, rp.04, rp.05, rp.06, rp.08, rp.12, rp.17, rp.45, and rp.51 showed better expression at the cellular level, and the fluorescence intensity was higher than that of the KNL control group. The fluorescence intensity of Rp.17 and Rp.51 still has a continuous increasing trend on the third day, and the expression duration is better than that of other prescriptions.

4) The nanoparticle compound is used for carrying Fluc-mRNA in vitro transfection of different cells: the nanoparticle complexes and FLuc-mRNA were administered at 200ng per well in vitro cell transfection manner as described above, and transfection experiments of the encapsulated FLuc-mRNA nanoparticle complexes in 293T (human renal epithelial cells), Hela (human cervical cancer cells), and HL7702 (human hepatic normal cells) cells were tested using KNL as a control group.

As a result: as shown in fig. 4.

And (4) conclusion: as shown in fig. 4, rp.04, rp.05, rp.06, rp.08, rp.12, rp.17, rp.45, and rp.51 showed better expression levels in 293T (human renal epithelial cells), Hela (human cervical cancer cells), and HL7702 (human liver normal cells) cells, and the fluorescence intensities were all higher than those of the KNL control group.

5) The nano-particle compound carries the Fluc-mRNA to transfect different cells in vitro in cytotoxicity experiment: the nanoparticle complexes were administered with FLuc-mRNA at 200ng per well in vitro cell transfection mode as described above, and the cytotoxicity experiments of the nanoparticle complexes in 293T (human renal epithelial cells), Hela (human cervical cancer cells), and HL7702 (human liver normal cells) cell transfection were tested with KNL as a control group. After counting the cells in the logarithmic growth phase on a counting plate, the cell suspension was added to a 96-well plate so that the cell concentration per well was 4X 10⁴And (4) cells. The drug is administered separately in the prescription group, and the non-prescription cell wells are used as negative pairsCCK-8 medium wells without cells were blank as control. After further culturing for 48h, the test solution was aspirated, rinsed three times with PBS, 90ul of serum-free medium and 10ul of CCK-8 solution were added to each well, and incubation was continued in the incubator for 2 h. Absorbance at 450nm was measured using an Omega-Fluostar microplate reader.

As a result: as shown in fig. 5.

6) Experiment of in vitro transfection of Hela-EGFP cells (polyclonal cell line stably expressing EGFP fluorescent protein) with EGFP-siRNA (using EGFP-siRNA as model siRNA) encapsulated by nanoparticle complexes: HeLa cell suspension stably expressing EGFP in logarithmic growth phase at 4X 10⁴The density of each cell per well is divided into 96-well plates, put at 37 ℃ and 5% CO₂And (5) standing and culturing in an incubator. After 24h, EGFP-siRNA with the concentration of 1 μ g/μ l is diluted to 0.1 μ g/μ l by nuclease-free ultrapure water, EGFP-siRNA is taken to prepare EGFP-siRNA-encapsulated nanoparticle complexes according to the preparation methods of Rp.04, Rp.05, Rp.06, Rp.08, Rp.12, Rp.17, Rp.45 and Rp.51 of the formulas described in the second example, KNL is taken as a control group, then the EGFP-siRNA-encapsulated nanoparticle complexes are respectively diluted to 88 μ l of nanoparticle complex mixture containing 10ng/μ l EGFP-siRNA by nuclease-free ultrapure water, after standing for 10min, the EGFP-encapsulated nanoparticle complexes are respectively added to 96-well plates containing 180 μ l opti-MEM culture medium in the volume of 20 μ l per well, and 4 wells are respectively 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.

As a result: as shown in fig. 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 EGFP-siRNA transfected by Rp.04, Rp.05, Rp.06, Rp.08, Rp.12, Rp.17, Rp.45 and Rp.51 shows better gene silencing effect than the KNL control group, wherein Rp.12 is better than other prescriptions.

Example five: small animal fluorescence imaging detection of transfection of nanoparticle complexes in mice

Three female BALB/c mice per group, using FLuc-mRNA as model mRNA, prepared nanoparticle complexes containing FLuc-mRNA according to the preparation method described for formulations rp.04, rp.05, rp.06, rp.07, rp.08, rp.12, rp.17, rp.24, rp.25, rp.45 and rp.51 in example two. Experimental groups 75 μ l of nanoparticle complexes containing 5 μ g FLuc-mRNA were injected into each mouse using an insulin needle. The administration mode is intramuscular injection, and the injection site is the thigh muscle of the mouse. NC represents a blank control group, and 75. mu.l of PBS buffer solution was injected intramuscularly to the insulin needle, while KNL is used as a control group. 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. Mice were subjected to whole body in vivo imaging bioluminescence image detection 5min after substrate injection using a small animal in vivo imaging system (PerkinElmer, IVIS L μmina series iii). A bioluminescent image of the back of the mouse was taken. The results are shown in fig. 8, where one representative mouse was taken from each group, the nanoparticle complexes were formulated to show luciferase expression in whole body in vivo imaging, with the greater the intensity of fluorescence, the greater the luciferase expression.

And (4) conclusion: as shown in fig. 8, rp.04, rp.05, rp.06, rp.08, rp.12, and rp.17 showed fluorescence intensities significantly higher than those of the KNL group, and rp.45 and rp.51 showed fluorescence intensities slightly higher than those of the KNL control group.

Example six: evaluation of humoral immunity effect of nanoparticle complex in mice

New crown S-mRNA was used as model mRNA and was supplied by Shanghai McTech development Co., Ltd (Honngene Biotech Corporation).

The specific information of the S-mRNA stock solution is as follows:

the product name is as follows: COVID-19 Spike Protein, Full Length-mRNA;

product description: 4088 nucleotides in length;

modifications (Modifications): fused 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 8 groups (5 per group) and were each intramuscularly injected with a combination of 75 μ l PBS (blank control), 5 μ g Naked S-mRNA (Naked mRNA) and 5 μ g S Protein (Spike Protein) (positive control, Naked Spike mRNA Protein), KNL control group loaded with 5 μ g S-mRNA and nanoparticle complex loaded with 5 μ g S-mRNA formula Rp.04, Rp.05, Rp.06, Rp.07, Rp.08, Rp.12, Rp.17, Rp.24, Rp.25, Rp.45 and Rp.51 each 75 ul.

Step 2: first serum collection: on day 28, mice were bled at 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 3, step 3: and (3) carrying out secondary immunization on the mice: on day 28, the mice were bled via the outer canthus, and 75 μ l each of PBS (blank control), 5 μ g of Naked S-mRNA and 5 μ g S protein in combination (positive control, Naked Spike mRNAProtein), KNL control group loaded with 5 μ g S-mRNA, and nanoparticle complex formulations loaded with 5 μ g S-mRNA rp.04, rp.05, rp.06, rp.07, rp.08, rp.12, rp.17, rp.24, rp.25, rp.45, and rp.51 were injected intramuscularly. 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 the serum is solidified for 1h at 4 ℃, centrifuging for 5 minutes at 4 ℃ at the rotating speed of 5000 Xg (5000 times of the acceleration of gravity), taking the supernatant, centrifuging for 5 minutes at 4 ℃ at the rotating speed of 10000 Xg, taking the supernatant, adding the supernatant into eight-row 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, 200. mu.l of PBST per well was washed 1 time, 100. mu.l of serum diluted 200-fold with PBS was added, and the mixture was incubated for 2 hours at 25 ℃ in a shaker. The serum was discarded, and after washing the plate 3 times with 200. mu.l PBST per well, 100. mu.l of antibody (antibody diluted 1: 1000 in PBS) was added to each 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, after the second immunization, the OD values of rp.04, rp.05, rp.06, rp.08, rp.12, rp.17, rp.45 and rp.51 are all higher than those of the KNL group, indicating that rp.04, rp.05, rp.06, rp.08, rp.12, rp.17, rp.45 and rp.51 have stronger seroconversion efficiency and humoral immune activation function, wherein the seroconversion efficiency and humoral immune activation function of the prescription rp.12 are optimal.

Step 6: ELISA detection of serum IgG titers: the S Protein (Spike 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 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-, 3906250-fold diluted 1: 3 in 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. The results are shown in table 2 and fig. 10.

And (4) conclusion: as shown in Table 2 and FIG. 10, the mean OD value of PBS group is taken as a baseline, the OD values of Rp.05, Rp.06, Rp.08, Rp.17, Rp.45 and Rp.51 groups are still 2 times higher than the baseline when diluted to 6250 times, and the OD value of Rp.12 group is still 2 times higher than the baseline when diluted to 31250 times, which indicates that the prescriptions of Rp.05, Rp.06, Rp.08, Rp.12, Rp.17, Rp.45 and Rp.51 have stronger seroconversion efficiency and humoral immune activation function.

Table 2: OD value detection results of each group after each prescription treatment

Example seven: evaluation of therapeutic Effect of nanoparticle 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 mouse⁵And (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 nanoparticle complex-OVA-mRNA vaccine: taking OVA-mRNA (purchased from TriLink company in USA), preparing according to the preparation methods of prescriptions Rp.04, Rp.05, Rp.06, Rp.08, Rp.12, Rp.17, Rp.45 and Rp.51 to obtain eight nanoparticle complex-OVA-mRNA vaccines prepared by prescriptions Rp.04, Rp.05, Rp.06, Rp.08, Rp.12, Rp.17, Rp.45 and Rp.51;

3) c57BL/6J mice were vaccinated with the nanoparticle complex-OVA-mRNA vaccine (each injection of nanoparticle vaccine containing 5 μ g of therapeutic agent mRNA-OVA) by foot injection on day 10, day 13 and day 16, respectively, while mice vaccinated with equal volumes of PBS buffer solution and equal volumes of OVA-mRNA solution after dilution and KNL solution loaded with OVA-mRNA 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. The tumor volume was calculated for C57BL/6J mice according to the following formula: v (mm)³)＝x×y²And/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 examination results are shown in fig. 11 and 12: 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. 12 and table 4, the OVA positive control group, the naked mRNA negative control group, the PBS control group, and the nanoparticle complex-OVA-mRNA vaccine group prepared by the prescriptions rp.04, rp.05, rp.06, rp.08, rp.12, rp.17, rp.45, and rp.51 showed tumor growth from day 8 to day 10 of tumor inoculation. From day 18 onward, the nanoparticle complex-OVA-mRNA vaccine groups prepared from rp.04, rp.05, rp.06, rp.08, rp.12, rp.17, rp.4s and rp.51 showed significant tumor growth delays compared to the PBS control group, the naked mRNA negative control group. From day 18 to day 21, the tumor sizes of the nanoparticle complex-OVA-mRNA vaccine groups prepared from rp.04, rp.05, rp.06, rp.08, rp.12, rp.17, rp.45, and rp.51 were all smaller than those of the PBS control group, the naked mRNA negative control group, and the KNL group.

As shown in fig. 11 and table 3, the PBS control group, the naked mRNA negative control group, and the KNL group were all sacrificed at day 36, day 40, and day 41 of the two groups, starting at day 21, day 24, and day 25, respectively, after tumor inoculation. The nanoparticle complex-OVA-mRNA vaccine sets prepared from the prescriptions rp.04, rp.05, rp.06, rp.08, rp.12, rp.17, rp.45, and rp.51 began sacrificing from day 35, day 29, day 31, day 25, day 30, day 26, day 28, and day 26, respectively. Formulas rp.04, rp.05, rp.06, rp.08, rp.12, rp.17, rp.45 and rp.51 all mice in the prepared nanoparticle complex-OVA-mRNA vaccine groups were sacrificed at day 48, day 47, day 44, day 45, day 49, day 45, day 44 and day 44, respectively.

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

And (4) conclusion: compared with the KNL group of the vaccine taking the nanoparticles prepared by the prescription Rp.04, Rp.05, Rp.06, Rp.08, Rp.12, Rp.17, Rp.45 and Rp.51 provided by the invention as carriers, the vaccine can better protect nucleic acid, is beneficial to the delivery of the nucleic acid in vivo, promotes the nucleic acid to penetrate cell membranes, and has the effect of obviously 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 in view of 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

1. A nanoparticle, comprising: a compound as shown in formula I and auxiliary materials,

wherein m is an integer from 0 to 5; n is an integer from 5 to 20.

2. A nanoparticle according to claim 1, wherein the compound of formula I is NBD002,

and/or

The auxiliary material comprises a material selected from: at least one of a PEG derivative, a lipid-like, an alcohol, or an inorganic salt; and/or

The auxiliary material comprises a material selected from: at least one of a PEG derivative, a lipid, and a lipoid; and/or

The auxiliary material comprises PEG derivatives and lipid.

3. 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 1, 2-dimyristoyl-sn-glyceromethoxypolyethylene glycol comprises a polyethylene glycol 2000 selected from 1, 2-dimyristoyl-sn-glyceromethoxypolyethylene glycol; 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 cholesterol and other lipids selected from at least one of 1, 2-distearoyl-sn-glycero-3-phosphocholine, lecithin, and 1, 2-dimyristoyl-sn-glycero-phosphocholine;

the lipid comprises a poloxamer selected from poloxamine or poloxamine derivatives; and/or

Said poloxamine comprises a compound selected from the group consisting of

At least one of; 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.

4. A nanoparticle according to any one of claims 2 to 3, wherein the compound of formula I is present in an amount of from 29% to 52% by weight, 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 6-12 wt%; and/or

The total content of the lipid is 40-65 wt% calculated by the total mass of the nanoparticle; and/or

The lipid comprises cholesterol and other lipid, the other lipid is selected from at least one of 1, 2-distearoyl-sn-glycero-3-phosphocholine, lecithin and 1, 2-dimyristoyl-sn-glycero-phosphocholine, and the content of the cholesterol is 23 wt% -41 wt% and the content of the other lipid is 13 wt% -24 wt% based on the total mass of the nanoparticle;

or

The nanoparticle contains a compound shown in formula I, a PEG derivative and at least one lipid; the compound of formula I: total mass of the lipid: the mass ratio of the PEG derivative is (38-75) to (58-91) to (9-16); or

The nanoparticle contains a compound shown in formula I, a PEG derivative and lipid; the lipids include cholesterol and other lipids selected from at least one of 1, 2-distearoyl-sn-glycero-3-phosphocholine, lecithin, and 1, 2-dimyristoyl-sn-glycero-phosphocholine, the compound of formula I: the mass ratio of the cholesterol to other lipid to the PEG derivative is (38-75) to (35-53) to (18-41) to (9-16).

5. A nanoparticle complex comprising a nucleic acid and the nanoparticle of any one of claims 1-4.

6. The nanoparticle complex of claim 5, wherein the weight ratio of the nanoparticle to the nucleic acid is 0.90: 1.00-1.80: 1.00.

7. A pharmaceutical composition comprising the nanoparticle complex of any one of claims 5-6 and a pharmaceutically acceptable excipient.

8. A method for preparing a mixture comprising nanoparticles according to any one of claims 1 to 4, comprising: mixing a compound shown as a formula I with a solvent A to obtain a solution 1, mixing an auxiliary material with a solvent B to obtain a solution 2, uniformly mixing the solution 1 and the solution 2, mixing with water, and performing post-treatment to obtain a mixed solution containing the nanoparticles of any one of claims 1 to 4; or

It includes: mixing a compound shown in a formula I and an auxiliary material with a solvent B, mixing with water, and performing post-treatment to obtain a mixed solution containing the nanoparticles of any one of claims 1 to 4;

optionally, the post-treatment comprises filtration, or filtration after removal of solvent B and/or solvent a by rotary evaporation in a water bath;

optionally, the solvent a comprises a solvent selected from ethanol or dichloromethane;

optionally, the solvent B comprises a solvent selected from ethanol.

9. A method of preparing a mixed liquor comprising the nanoparticle composite of any one of claims 5-6, comprising: mixing the mixture containing nanoparticles of any one of claims 1 to 4 obtained by the method of claim 8 with a nucleic acid in a solvent C to obtain a mixture containing nanoparticle complexes of any one of claims 5 to 6;

optionally, the volume percentage of the solvent B in the mixed solution containing the nanoparticle composite of any one of claims 5 to 6 is 0 to 12.5% vol;

optionally, the solvent C comprises a solvent selected from water.

10. Use of a nanoparticle according to any one of claims 1 to 4 or a nanoparticle complex according to claims 5 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.